Title of Invention

TAMANDARIN ANALOGS AND FRAGMENTS THEREOF AND METHODS OF MAKING AND USING

Abstract The present invention is directed to a compound of Formula I wherein R1, R2, R3, R4, R5, R6, W, X, Y, and Z are defined herein. The compounds of the present invention are useful as anticancer agents. Specifically, the compounds are useful for treating or preventing cancer and tumor growth. The present invention is also directed to compositions comprising a compound according to the above formula. The present invention is also directed to methods of using a compound according to the above formula
Full Text TAMANDARIN ANALOGS AND FRAGMENTS THEREOF AND
METHODS OF MAKING AND USING
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0001] The present invention relates to macrocyclic depsipeptides, including
didemnin and tamandarin analogs and fragments thereof, which are useful as
anti-cancer agents and for other purposes. Methods of using these compounds
as inhibitors of protein synthesis, cell growth, tumorigenesis, and viral
infection, and for immunosuppresive therapy, and as enhancers of apoptosis
are also provided Methods of making the tamandarin analogs are also
provided.
BACKGROUND ART
[0002] Tamandarins A and B are naturally occurring didemnin analogs which
have been isolated from a marine tunicate. Tamandarins A and B belong to a
family of compounds (didemnins—from organisms of the Didemnidae family)
which potently inhibit protein synthesis and cell cycle progression and induce
rapid apoptosis (Grubb et at, Biochem. Biophys, Res. Commun. 275:1130-
1136 (1995); Johnson et al, FEBS Lett. 383:1-5 (1996); Johnson et al,
Immunol Cell Biol 77:242-248 (1999); Johnson et ah, J. Cell. Biochem.
72:269-278 (1999)). Other didemnins, including didemnin B, didemnin M,
and dehydrodidemnin B, exhibit cytotoxic and cytostatic effects as well.
[0003] Tamandarins A and B exhibit biological activity which is similar to the
activities exhibited by didemnin B. See, e.g., Liang, et al., J. Am. Chem. Soc.
223:4469-4474 (2001). For example, tamandarin A is a potent inhibitor of
protein synthesis, cell growth, and tumorigenesis. Tamandarin A exhibits
significant in vitro activity against pancreatic carcinoma (Liang et al, Org.
Lett 7:1319-1322(1999)).
[0004] A significant limitation on the use of the tamandarins and didemnins,
either for research or for other applications, is the limited supply of

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tamandarins that is available from natural sources and the difficulty and
expense of isolating these compounds. A need exists for a method of
synthesizing tamandarins A and B and other didemnin analogs.
[0005] Furthermore, very few derivatives or analogs of tamandarins A and B
have been prepared and examined. Additional derivatives and analogs of
tamandarins A and B and didemnins are needed. Additionally, compound
which are useful in the synthesis of tamandarins and didemnins are needed.
Additional compounds that are useful as inhibitors of protein synthesis, cell
growth, tumorigenesis, and viral infection, and for immunosuppresive therapy,
and/or as enhancers of apoptosis are also needed.
SUMMARY OF THE INVENTION
[0006] A first aspect of the present invention is directed to a compound of
Formula I, IA, or II.
[0007] A second aspect of the present invention is directed to a composition,
for example a pharmaceutical composition, comprising a compound of
Formula I, IA, or II and a suitable carrier or excipient.
[0008] A third aspect of the present invention is directed to a method of
inhibiting or preventing the growth of a cancer cell or tumor, said method
comprising contacting a cancer cell with an effective amount of a compound
of Formula I, IA, or II. In one embodiment, the method is directed to treating
or preventing cancer in a subject in need of such treatment, comprising
administering to said subject an effective amount of Formula I, IA, or II.
[0009] A fourth aspect of the present invention is directed to a method of
inhibiting or preventing tumorigenesis, said method comprising contacting a
cell or cellular component with an effective amount of a compound of
Formula I, IA, or II. In one embodiment, the method is directed to inhibiting
or preventing tumorigenesis in a subject in need of such treatment, comprising
administering to said subject an effective amount of Formula I, IA, or II.
[0010] A fifth aspect of the present invention is directed to a method of
inhibiting or preventing protein synthesis, said method comprising contacting

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a cell or cellular component with an effective amount of a compound of
Formula I, IA, or II. In one embodiment, the method is directed to inhibiting
or preventing protein synthesis in a subject in need of such treatment,
comprising administering to said subject an effective amount of Formula I,
IA,orII.
[0011] A sixth aspect of the present invention is directed to a method of
enhancing apoptosis, said method comprising contacting a cell or cellular
component with an effective amount of a compound of Formula I, IA, or II.
In one embodiment, the method is directed to enhancing apoptosis in a subject
in need of such treatment, comprising administering to said subject an
effective amount of Formula I, IA, or II.
[0012] A seventh aspect of the present invention is directed to a method of
providing immunosuppresive therapy, said method comprising contacting a
cell or cellular component with an effective amount of a compound of
Formula I, IA, or II. In one embodiment, the method is direct to providing
immunosuppresive therapy to a subject in need of such treatment, comprising
administering to said subject an effective amount of Formula I, IA, or IL
[0013] A eighth aspect of the present invention is directed to the use of a
compound according to Formula I, IA, or II in the preparation of a
pharmaceutical composition useful for inhibiting or preventing the growth of a
cancer cell or tumor.
[0014] A ninth aspect of the present invention is directed to a method of
preventing or treating a viral infection, said method comprising administering
to a subject in need of such treatment an effective amount of a compound of
Formula I, IA, or II.
[0015] A tenth aspect of the present invention is directed to the use of a
compound according to Formula I, IA, or II in the preparation of a
pharmaceutical composition useful for one or more of the methods described
herein.
[0016] An eleventh aspect of the present invention is directed to a method of
preparing a compound according to Formula I, IA, or II.

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BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 is the structure of Tamandarin A illustrating the numbering
convention used herein and in Sakai et al, J. Med. Chem. 59:2819-2834
(1996).
DETAILED DESCRIPTION OF THE INVENTION
[0018] A first aspect of the present invention is directed to a compound of
Formula I:

wherein
R1 and R2 are independently H or CH alkyl, or R1 and R2 together
form the alkyl ring of a proline or homoproline residue;
R3 is selected from the group consisting of a side chain of an amino
acid, a naphthylmethyl group, and a first fluorophore;
R4isHorCH3;
R5 is H, an amine protecting group, an amino acid residue, a
polypeptide, a peptide which contains a second fluorophore, a chemical
moiety bound to a solid support, or a moiety containing from about 1 to about
50 non-hydrogen atoms;
R6 is an isoleucine side chain or a valine side chain;
WisOorNH;
XisOorNH;
Y is H or a hydroxyl protecting group; and

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Z is C(O) or C(O)-CH(CH3>C(O);
provided that if R1 and R2 together form the alkyl ring of a proline residue, R4
is methyl, W is O, X is O, and Z is C(O), then R3 is not an isoleucine side
chain, a valine side chain, an alanine side chain, a norleucine side chain, a
norvaline side chain, leucine side chain, a histidine side chain, a tryptophan
side chain, an arginine side chain, a lysine side chain, a second fluorophore, or
a benzyl optionally substituted with OH, -OCH3, -CO(C6H5), -Br, -I, -F, -Cl,
-CH3, and -C2H5; or
■I rt
alternatively provided that provided that if R and R together form the
alkyl ring of a proline residue, R4 is methyl, and X is O, then R3 is
naphthylmethyl.
[0019] In one embodiment, R and R are H. In another embodiment, R and
R are C^ alkyl, for example methyl. In another embodiment, R is H and R
is methyl. In another embodiment, R1 is methyl and R2 is H. In a further
embodiment, R1 and R2 together form the alkyl ring of a proline residue. That
is, in one embodiment, R and R together form a 1,3-propanediyI group.
[0020] In one embodiment, R3 is side chain of an arnino acid, in particular a
naturally occurring arnino acid. In one embodiment, R is amino acid side
chain such as an isoleucine side chain, i.e., a 2-butyl moiety, preferably having
(R) stereochemistry, a valine side chain, i.e., a 2-propyl moiety, an alanine side
chain, i.e., a methyl moiety, a norleucine side chain, i.e., a 1-butyl moiety, a
norvaline side chain, i.e., a 1-propyl moiety, a leucine side chain, i.e., an
isobutyl moiety, preferably having (S) stereochemistry, a phenylalanine side
chain, i.e., a benzyl moiety, a histidine side chain, i.e., a 4-imidazolylmethyl
moiety, a tryptophan side chain, i.e., a 3-indolylmethyl moiety, a tyrosine side
chain, i.e., a 4-hydroxybenzyl moiety, an arginine side chain, i.e., a
4-guanidinylbutyl moiety, and a lysine side chain, i.e., a 4-aminobutyl moiety.
[0021] In another embodiment, R3 is a side chain of a non-naturally occurring
amino acid. Non-naturally occurring amino acids are known in the art. In one
embodiment, R3 is a naphthylmethyl group. Alternatively, R3 is a benzyl
group substituted with OH, Q_4 alkoxy such as OCH3 and OCH^CHs,
CO(C6H5), F, Cl, Br, I, or CM alkyl such as CH3 and CH2CH3.

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[0022} In another embodiment, R3 is a first fluorophore. Moieties which are
fluorophores are known in the art. In one embodiment, the fluorophore is an
amino acid side chain as described above further containing a fluorophore
moiety (e.g., a fluorophore linked with one of the amino acid side chains
described above). Such a fluorophore includes

[0023] In one embodiment, R5 is H, an amine protecting group, an amino acid
residue, a polypeptide, or a peptide which contains a second fluorophore.
[0024] In one embodiment, the invention comprises a compound of Formula I
wherein R5 is a hydrogen atom or an amine protecting group suitable for
protection of amino acids. Such protecting groups are known in the art and
referred to throughout this disclosure. A suitable protecting group for R5
includes, for example, terr-butoxycarbonyl. Examples of suitable protecting
groups can be found in references such as Green and Wuts (1991, Protective
Groups in Organic Synthesis, Wiley, New York, 2nd Edition) and Bodansky
(1993, Principles of Peptide Synthesis, Springer, Berlin).
[0025] In one embodiment, R5 is an amino acid residue, e.g., a leucine residue,
or a polypeptide comprising two or more amino acid residues. In certain
embodiments, the R5 group contains 1-10 amino acid residues. In another
embodiment, the R5 group contains 1, 2, 3, 4, or 5 amino acid residues. In
certain instances, the amino acid residue or polypeptide contains one or more
suitable protecting groups. Examples of such amino acid residues and
polypeptides include:
-(JV-methyl)leucine;
- (AT-methyl)leucine-proline;
-(iV-CBz-JV-methyl)leucine;
-(JV-methyl)leucine-proline-lactate;
-(iV-methyl)leucine-proline-pyruvate;
-(iV"-methyl)leucine-proline-lactate-g]utamine-pyroglutamate;

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-(AT-methyl)leucine-proHne4actate-glutamine-cyclopentanoate;
-(iV-methyOleucine-proline-lactate-leucine-pyroglutamate;
-(N-methyl)leucine-proline~lactate-glutamine-cyclopentanoate;
~(iV-methyl)leucine-proline-alanine4eucme-pyroglutarnate; and
-(N-methyl)leucine-proline-(A^-methyI)alanine-leucine-pyrogIutamate.
[0026] In another embodiment, R5 contains an JV-glycidyl-L-proIine residue.
A suitable value of R5 is -(JV-methyl-i?-leucine)-S-proline-glycidate.
[0027] Other suitable R5 groups include those having the structure shown
below.


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[0028] In another embodiment, R5 is a terminal, non-ammo acid moiety
having from about 1 to about 15 non-hydrogen atoms, e.g., carbon, oxygen,
nitrogen, sulfur, halogens, and combinations thereof. Exemplary groups
include those having the following structure:


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[0032] wherein Rr is H, Boc, isobutyryl, pyruvyl, or acryloyl.
[0033] In another embodiment, R5 is a peptide comprising a fluorophore.
Another suitable group for R5 includes ~(JV-methyl)leucine-proline-lactate-
(fluorophore). Other suitable R5 groups having a fluorophore include those
having the structure shown below.

[0034] In another embodiment, R5 is an amino acid residue, a polypeptide, or
a chemical moiety, or linker, bound, e.g., covalently attached, with a support,
e.g., a glass or silica plate, an agarose bead, or other polymeric bead, etc. In
one embodiment, R5 is a group having the formula:

wherein SS represents a solid support. In another embodiment, the chemical
linker or moiety is an organic linking group, such as an alkylene or alkenylene

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group, for example, comprising 1-20 carbon atoms. In another embodiment,
R5 can be represented as L-SS, wherein L is a bivalent organic linking group
and SS is a solid support. Solid supports are known in the art. For example,
see MacLean etal, PureAppl. Chem. 7i(12):2349-2365 (1999).
[0035] When R5 comprises an JV-methyl-leucine residue, the alpha-carbon
atom of that residue can have either (7?) or (5) stereochemistry. Other amino
acid residues within R5 can have either (R) or (5) stereochemistry, but, in one
embodiment, they have (5) stereochemistry at their alpha-carbon atom. When
R5 comprises a lactate residue, the lactate residue is preferably an (S)-lactate
residue. In another embodiment, every amino acid residue within R5 other
than the leucine (or iV-methyl-leucine) residue (if present) attached directly to
the nitrogen atom of the ring of Formula I has (S) stereochemistry.
[0036] In another embodiment, R3 contains a fluorophore moiety, e.g., a
fluorophore linked with one of the amino acid side chains described above.
[0037] In one embodiment, Y is H. In another embodiment, Y is a hydroxyl
protecting group. Examples of hydroxyl protecting groups which can be
present at Y include an alkyl-substituted silyl moiety, an aryl-substituted silyl
moiety, or a silane substituted with both alkyl and aryl moieties. An example
of a useful hydroxyl protecting group is a triisopropylsilyl moiety (TIPS).
Another suitable protecting group includes trimethylsilyl. Other hydroxyl
protecting groups which can be used at Y in Formula I are known in the art
and are described in references such as Green and Wuts (1991, Protective
Groups in Organic Synthesis, 2nd Edition, Wiley, New York).
[0038] A first subclass of compounds is a compound according to Formula IA
wherein R5 is a moiety having the structure:


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wherein
R1 and R2 are independently H or CM alkyl, or R1 and R2 together
form the alkyl ring of a proline residue;
R is selected from the group consisting of a side chain of an amino
acid and a first fluorophore;
R4isHorCH3;
R5 is H, an amine protecting group, an amino acid residue, a
polypeptide, a peptide which contains a second fluorophore, a chemical
moiety bound to a solid support, or a moiety containing from about 1 to about
30 non-hydrogen atoms;
R is an isoleucine side chain or a valine side chain;
XisOorNH;and
Y is H or a hydroxyl protecting group;
provided that if R and R together form the alkyl ring of a proline residue, R
is methyl, W is O, X is O> and Z is C(O), then R3 is not an isoleucine side
chain, a valine side chain, an alanine side chain, a norleucine side chain, a
norvaline side chain, leucine side chain, a histidine side chain, a tryptophan
side chain, an arginine side chain, a Iysine side chain, a second fluorophore, or
a benzyl optionally substituted with OH, -OCH3, -CO(C6H5), -Br, -I, -F, -Cl,
-CH3,and~C2H5;or
1 1
alternatively provided that provided that if R andR together form the
alkyl ring of a proline residue, R4 is methyl, and X is O, then R3 is
naphthylmethyl.
[0039] In one embodiment in this first subclass, R1 and R2 are H. In another
embodiment, R and R are CM alkyl, for example methyl. In another
embodiment, R1 is H and R2 is methyl. In another embodiment, R1 is methyl
and R2 is H. In a further embodiment, R1 and R2 together form the alkyl ring
of a proline residue. That is, in one embodiment, R and R together form a
1,3-propanediyl group.
[0040] In one embodiment within this first subclass, R3 is side chain of an
amino acid, in particular a naturally occurring amino acid. In one
embodiment, R3 is amino acid side chain such as an isoleucine side chain, i.e.,

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a 2-butyl moiety, preferably having (R) stereochemistry, a valine side chain,
i.e., a 2-propyl moiety, an alanine side chain, i.e., a methyl moiety, a
norleucine side chain, i.e.t a 1-butyl moiety, a norvaline side chain, i.e., a
1-propyl moiety, a leucine side chain, i.e., an isobutyl moiety, preferably
having (5) stereochemistry, a phenylalanine side chain, i.e., a benzyl moiety, a
histidine side chain, Le.t a 4-imidazolylmethyl moiety, a tryptophan side chain,
i.e., a 3-indolylmethyl moiety, a tyrosine side chain, i.e., a 4-hydroxybenzyl
moiety, an arginine side chain, i.e., a 4-guanidinylbutyl moiety, and a lysine
side chain, i.e., a 4-aminobutyl moiety.
[0041] In another embodiment within this first subclass, R is a side chain of a
non-naturally occurring amino acid. Non-naturally occuring amino acids are
known in the art. For example, suitable non-naturally occuring amino acids
include, but are not limited to, cycloleucine, homocycloleucine, 9-anthracenyl-
alanine, 3-(3-benzothienyl)alanine, 3-cyclohexylalanine, 3,3-diphenylalanine,
3-pyridyIalanine, 3-(2-furyl)alanine, and 3-sytrylalanine, 3-(2-thienyl)alanine,
2-phenylglycine, allylglycine, 2-cyclohexylglycine, propargylglycine,
2-(trifluormethyI)phenylalanine, the like. In one embodiment, R is a
naphthylmethyl group. Alternatively, R is a benzyl group substituted with
OH, OCH3 and OCH2CH3, CO(C6H5), F, Cl, Br, I, or CH3 and CH2CH3.
[0042] In another embodiment within this first subclass, R3 is a first
fluorophore. In another embodiment, R3 contains a fluorophore. Moieties
which are fluorophores are known in the art. In one embodiment, the
fluorophore is an amino acid side chain as described above further containing
a fluorophore moiety (e.g., a fluorophore linked with one of the amino acid
side chains described above). Such a fluorophore includes

[0043] In one embodiment within this first subclass, R is H, an amine
protecting group, an amino acid residue, a polypeptide, or a peptide which
contains a second fluorophore.

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[0044] In one embodiment within this first subclass, the invention comprises a
compound of Formula IA wherein R5 is a hydrogen atom or an amine
protecting group suitable for protection of amino acids. Such protecting
groups are known in the art and referred to throughout this disclosure. A
suitable protecting group for R5 includes, for example, te/t-butoxycarbonyl.
Examples of suitable protecting groups can be found in references such as
Green and Wuts (1991, Protective Groups in Organic Synthesis, Wiley, New
York, 2nd Edition) and Bodansky (1993, Principles of Peptide Synthesis,
Springer, Berlin).
[0045] In one embodiment within this first subclass, R5 is an amino acid
residue, e.g., a leucine residue, or a polypeptide comprising two or more
amino acid residues. In certain embodiments, the R5 group contains 1-10
amino acid residues. In another embodiment, the R5 group contains 1,2, 3,4,
or 5 amino acid residues. In certain instances, the amino acid residue or
polypeptide contains one or more suitable protecting groups. Examples of
such amino acid residues and polypeptides include:
-(iV-methyl)leucine;
-(/V-methyl)Ieucine-proline;
-(iV-CBz-JV-methyl)leucine;
-(/V-methyl^eucine-proIine-lactate;
-(iV-methyl)leucine-proline-pyruvate;
-(TV-methyl)leucine-proline-lactate-glutamine-pyroglutamate;
-(Af-niethyl)leucine-proline-lactate-glutarnine-cyclopentanoate;
-(TV-methyOleucine-proline-lactate-leucine-pyroglutamate;
-(//-methyOleucine-proline-lactate-glutamine-cyclopentanoate;
-(A^-methyl)leucine-proIine-alanine-leucine-pyroglutamate;and
-(A?'-methyl)leucine-proline-(A^methyl)alanine-leucine-pyroglutamate.
[0046] In another embodiment within this first subclass, R5 contains an N-
glycidyl-L-proline residue. A suitable value of R5 is -(A^-methyl-^-leucine)-^-
proline-glycidate.
[0047] Other suitable R5 groups include those having the structure shown
below.


[0048] Other suitable groups for R5 include those having an amino acid
residue other than one of the common twenty amino acids. Suitable examples
of such amino acids are known in the art and include, for example,
hydroxyproline, dehydroproline, 4-carboxyglutamic acid, hydroxylysine,
A^N-dimethylargmine, iodotyrosine, (3-alanine, and the like. In another
embodiment, R5 is a peptide comprising a fluorophore. Another suitable group
for R5 includes -(iV-methyl)leucine-proline-lactate-(fluorophore). Other
suitable R5 groups having a fluorophore include those having the structure
shown below.


[0051] wherein R10 is H, C1-4 alkyl, or phenyl. In one example of this
subclass, R10 is H. In this embodiment, the moiety is bound directly to the
amine group of the macrocycle forming an amide linkage, i.e., R5 is the group
shown above. In another embodiment, the moiety is in the terminal position
of the R5 group. That is, the moiety is connected to the amine group by
another moiety, for example, an amino acid residue, a peptide residue, or a
linker. In a further embodiment, the moiety shown above is connected to the
rest of the R5 group by an ester linkage. For example, JV-glycidyl-L-proIine is
used to prepare suitable R5 moieties to prepare a compound according to
Formula I, IA, or II, or alternatively, Af-glycidyl-L-proline is coupled directly
with the free amine of the macrocycle, e.g., compound 35, deprotected 61,

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deprotected 74, deprotected 85, or deprotected 92. In another embodiment,
iV-glycidyl-L-proline is reacted with the terminus of the R5 group, for
example, reacted with deprotected free amine of compound 63 shown in
Scheme 17.
[0052] In another embodiment within this first subclass, R5 is an amino acid
residue, a polypeptide, or a chemical moiety, or linker, bound, e.g., covalently
attached, with a support, e.g.y a glass or silica plate, an agarose bead, or other
polymeric bead, etc. In one embodiment, R5 is a group having the formula:
II H II H
O n O "
wherein SS represents a solid support. In another embodiment, the chemical
linker or moiety is an organic linking group, such as an alkylene or alkenylene
group, for example, comprising 1-20 carbon atoms. In another embodiment,
R5 can be represented as L-SS, wherein L is a bivalent organic linking group
and SS is a solid support. Solid supports are known in the art. For example,
see MacLean et at, PureAppl. Chetn. 7i(12):2349-2365 (1999).
[0053] When R5 comprises an Af-methyl-leucine residue, the alpha-carbon
atom of that residue can have either (R) or (5) stereochemistry. Other amino
acid residues within R5 can have either (R) or (S) stereochemistry, but, in one
embodiment, they have (5) stereochemistry at their alpha-carbon atom. When
R5 comprises a lactate residue, the lactate residue is preferably an (>S)-lactate
residue. In another embodiment, every amino acid residue within R5 other
than the leucine (or iV-methyl-leucine) residue (if present) attached directly to
the nitrogen atom of the ring of Formula I has (S) stereochemistry.
[0054] In another embodiment within this first subclass, R5 contains a
fluorophore moiety, e.g., a fluorophore linked with one of the amino acid side
chains described above,
[0055] In another embodiment within this first subclass, R5 is a moiety having
from about 1 to about 50 non-hydrogen atoms, e.g., carbon, nitrogen, oxygen,
sulfur, halogens, and mixtures thereof. In another embodiment within this first

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subclass, R is a moiety having from about 1 to about 40 non-hydrogen atoms.
In another embodiment within this first subclass, R5 is a moiety having from
about 1 to about 30 non-hydrogen atoms. In another embodiment within this
first subclass, R5 is a moiety having from about 1 to about 20 non-hydrogen
atoms. In another embodiment within this first subclass, R5 is a moiety having
from about 1 to about 10 non-hydrogen atoms. Suitable moieties include, but
are not limited to, peptides, carbohydrates, saccharides, oligosaccharides,
steroids, bioactive molecules, lipids, nucleotides, nucleosides, vitamins, and
the like. Such moieties are bound directly to the amine of the macrocycle, or
are connected to the macrocycle amine by an organic linker. Other suitable
moieties containing from about 1 to about 50 non-hydrogen atoms include, but
are not limited to, moieties having one or more of the following groups: alkyl,
alkenyl, alkynyl, cycloalkyl, alkoxy, cycloalkoxy, halogen, amino, thio, keto,
aryl, heteroaryl, halo, haloalkyl, aryl, heterocycle, cycloalkyl, heteroaryl,
alkyl, alkenyl, alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl,
heteroarylalkenyl, heteroaryl alkynyl, cycloalkylalkyl, heterocycloalkyl,
hydroxyalkyl, aminoalkyl, carboxyalkyl, alkoxyalkyl, nitro, amino, ureido,
cyano, acylamino, hydroxy, thiol, acyloxy, azido, alkoxy, carboxy,
aminocarbonyl, alkylthiol groups, and combinations thereor.
[00561 In one embodiment within this first subclass, Y is H. In another
embodiment, Y is a hydroxyl protecting group. Examples of hydroxyl
protecting groups which can be present at Y include an alkyl-substituted silyl
moiety, an aryl-substituted silyl moiety, or a silane substituted with both alkyl
and aryl moieties. An example of a useful hydroxyl protecting group is a
triisopropylsilyl moiety (TIPS). Another suitable protecting group includes
trimethylsilyl. Other hydroxyl protecting groups which can be used at Y in
Formula IA are known in the art and are described in references such as Green
and Wuts (1991, Protective Groups in Organic Synthesis, 2nd Edition, Wiley,
New York).
[00571 A second subclass of compounds is a compound according to Formula
IA wherein R5 is a moiety such that the compound has the structure


wherein R is hydrogen or a chemical moiety which can be enzymatically
cleavable (i.e., an enzyme-cleavable moiety). As used herein, an enzyme-
cleavable moiety can include any chemical moiety which can be cleaved (i.e.,
chemically detached from) in the presence of a specific enzyme. Examples of
enzymes capable of chemically detaching an enzyme-cleavable moiety include
carboxypeptidases, (3-lactamase, |3-galactosidase, penicillin V-amidase,
cytosine deaminase, nitroreductase, alkaline phosphatase, beta-glucuronidase,
and catalytic antibodies. Examples of enzyme-cleavable moieties which can
be incorporated in a compound described herein include cephalosporins, beta-
glucosides, phosphate, pyrophosphate, p-D-galactosides, nitrobenzamidine,
cytosine, carbamates, peptides, and amino acids.
[0058] In another embodiment within this subclass, R7 is an enzyme-cleavable
moiety such as a dipeptide linked with glutamine-pyroglutamate, or a moiety
having the structure of one of the following


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carbohydrate moiety above, for example, is, in one embodiment, in the form of
a sodium or potassium salt.
[0060] After cleavage 'of an enzyme-cleavable moiety by an enzyme, the
resulting tamandarin analog exhibits one or more of the physiological
activities described herein. A tamandarin analog of the present invention
having an enzyme-cleavable moiety can, optionally, exhibit these activities
before the cleavage of the enzyme-cleavable moiety. However, in a preferred
embodiment, the analog exhibits therapeutic activity only following cleavage
of the enzyme-cleavable moiety therefrom.
[0061] As described above, a tamandarin analog having the structure of
Formula I can be bound with a support. The identity of the support is not
critical. The support can be substantially any material with which such an
analog can be bound, e.g., by covalent attachment through the R5 moiety.
Examples of support materials include bonded silicates, cross-linked agarose,
polyacrylamide, dextran, and allyl dextran. Such support materials can be
chemically modified using reactive chemical moieties in order to facilitate
covalent attachment of the analog with the support. Chemical modifications of
this type are known in the art, and can, for example, include modification of a
support with cyanogen bromide groups, epoxide groups, tresyl groups, and
carboxyhexyl groups. Protocols for preparation of a support and subsequent
attachment of a compound to the support are available in the art, and can be
modified by one skilled in the art for use with a tamandarin analog described
herein.
[0062] A subclass of useful compounds according to Formula IA are
compounds comprising a fluorescent substituent, a photoreactive moiety, such
as a moiety having the structure

or a moiety bound with a support, e.g., at R or R . Compounds having a such
a photoreactive moiety are useful or labeling proteins, for example.

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Fluorescent substituents and photoreactive moieties are known in the art.
Other suitable photoreactive R5 groups include

[0063] Another subclass of useful compounds is a compound according to
Formula IA, wherein R1 and R2 together form the alkyl ring of a proline
residue; R is a benzyl group optionally substituted with one or more selected
from the group consisting of OH, OCH3, CO(C6H5), F, Cl, Br, I, CH3, and
C2H5, preferably OCH3; R4 is H; R5 is anyone of the embodiments defined
above; R6 is a valine side chain; X is O; and Y is H.
[0064] Another subclass of useful compounds is a compound according to
Formula IA, wherein R1 is H; R2 is CH3; R3 is a benzyl group optionally
substituted with one or more selected from the group consisting of OH, OCH3,
CO(C6H5), F, Cl, Br, I, CH3, and CH2CH3, and preferably is OCH3; R4 is CH3;
R5 is is anyone of the embodiments defined above; R6 is a valine side chain; X
is O; and Y is H.
[0065] Another subclass of useful compounds is a compound according to
Formula IA, wherein R1 is CH3; R2 is CH3; R3 is a benzyl group optionally
substituted with one or more selected from the group consisting of OH, OCH3,
CO(C6H5), F, Cl, Br, I, and CH3, and CH2CH3, and preferably is OCH3; R4 is
CH3; R5 is is anyone of the embodiments defined above; R6 is a valine side
chain; X is O; and Y is H.

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[0066] Another subclass of useful compounds is a compound according to
Formula IA, wherein R1 and R2 together form the alkyi ring of a proline
residue; R3 is a naphthylmethyl group; R4 is CH3; R5 is is anyone of the
embodiments defined above; R6 is a valine side chain; X is O; and Y is H.
[0067] Another subclass of useful compounds of Formula IA is a compound
having the formula

wherein R8 is defined as for R5. In one embodiment, R8 is H, an amine
protecting group, or -(iV~methyl)leucine~proline-lactate.
[0071] Examples of suitable, novel compounds, which are useful in the
methods and compositions disclosed herein, include a compound according to
Formula I, IA, or II wherein:
R1 and R2 together form the alkyl ring of a proline residue, R3 is
benzyl, R4 is CH3, R5 is (iV-methyl-i?-leucine)-5-pro3ine-5-lactate, R6 is a
valine side chain, X is O, and Y is H (compound 36);

WO 2004/084812 PCT/US2004/008275
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R and R together form the alkyl ring of a proline residue, R is
benzyl, R4 is CH3, R5 is H, R6 is a valine side chain, X is 0, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
benzyl, R4 is CH3, R5 is H-HC1, R6 is a valine side chain, X is O, and Y is H
(compound 35);
R and R together form the alkyl ring of a proline residue, R is
benzyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is O, and Y is TIPS
(compound 34);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
benzyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is O, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyI, R4 is H, R5 is (Ar-methyl-J?-leucine)-S-proline-S--lactate, R6
is a valine side chain, X is O, and Y is H (compound 62);
R and R together form the alkyl ring of a proline residue, R is
4-methoxybenzyl, R4 is H, R5 is Boc, R6 is a valine side chain, X is O, and Y
is H (compound 61);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is H, R6 is a valine side chain, X is O, and Y is
H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is (A^-methyI-7?-Ieucine)-lS'-proline-5-pyruvate,
R6 is a valine side chain, X is O, and Y is H (compound 64);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is (iV-methyl-i?-leucine), R6 is a valine side
chain, X is O, and Y is H;
R and R together form the alkyl ring of a proline residue, K is
4-methoxybenzyl, R4 is H, R5 is A^-Cbz-A'-methyl-^-leucine, R6 is a valine side
chain, X is O, and Y is H (compound 63);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is H-HC1, R6 is a valine side chain, X is 0, and
YisH;

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R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is (iV-methyl-
iMeucine)-S-proline-S'-lactate, R6 is a valine side chain, X is O, and Y is H
(compound 75);
R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is Boc, R6 is
a valine side chain, X is O, and Y is H (compound 74);
R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is H, R6 is a
valine side chain, X is O, and Y is H;
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is
(7^methyl-i?-leucine)-S-proline-S-lactate, R6 is a valine side chain, X is O, and
Y is H (compound 150);
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is Boc, R6
is a valine side chain, X is O, and Y is H (compound 85);
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is H, R6 is
a valine side chain, X is O, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
2-naphthylmethyl, R4 is CH3, R5 is (Ar-methyl-i?-leucine)-S-proIine-,S-lactate,
R6 is a valine side chain, X is O, and Y is H (compound 93);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
2-naphthylmethyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is O, and
Y is H (compound 92);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
2-naphthylmethyl, R4 is CH3, R5 is H, R6 is a valine side chain, X is O, and Y
isH;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is CH3, R5 is (Af-methyl-i?-Ieucine)-S-proline-S-lactate,
R6 is a valine side chain, X is NH, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is CH3, R5 is H, R6 is a valine side chain, X is NH, and Y
isH;
R and R together form the alkyl ring of a proline residue, R is
4-methoxybenzyl, R4 is CH3) R5 is Boc, R6 is a valine side chain, X is NH, and
YisH;

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and pharmaceutically acceptable salts thereof.
[0072] The present invention also includes a salt of a compound according to
Formula I, IA, or II. The term salt refers to an acid- and/or base-addition salt
of a compound according to Formula I, IA, or II. Acid-addition salts can be
formed by adding an appropriate acid to the compound according to Formula
I, IA, or II. Base-addition salts can be formed by adding an appropriate base
to the compound according to Formula I, IA, or II. Said acid or base does not
substantially degrade, decompose, or destroy said compound according to
Formula I, IA, or II. Examples of suitable salts include hydrochloride,
hydrobromide, acetate, furmate, maleate, oxalate, and succinate salts. Other
suitable salts include sodium, potassium, carbonate, and tromethamine salts.
[0073] It is further understood that the present invention encompasses
tautomers of a compound of Formula I, IA, or II. Tautomers are well-known
in the art and include keto-enol tautomers.
[0074] The compounds of Formula I, IA, or II may also be solvated,
including hydrated. Hydration may occur during manufacturing of the
compounds or compositions comprising the compounds, or the hydration may
occur over time due to the hygroscopic nature of the compounds.
[0075] Certain compounds within the scope of Formula I, IA, or II may be
derivatives referred to as "prodrugs." The expression "prodrug" denotes a
derivative of a known direct acting drug, which derivative has enhanced
delivery characteristics and therapeutic value as compared to the drug, and is
transformed into the active drug by an enzymatic or chemical process.
Prodrugs are derivatives of the compounds of the invention which have
metabolically cleavable groups and become by solvolysis or under
physiological conditions the compounds of the invention which are
pharmaceutically active in vivo. An acid derivative form may offer
advantages of solubility, tissue compatibility, or delayed release in the
mammalian organism (see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24,
Elsevier, Amsterdam 1985). Prodrugs include acid derivatives well known to
practitioners of the art, such as, for example, esters prepared by reaction of the

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PCT/US2004/008275

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parent acid with a suitable alcohol, or amides prepared by reaction of the
parent acid compound with an amine.
[0076] When any variable occurs more than one time in any constituent or in
Formula I, its definition on each occurrence is independent of its definition at
every other occurrence, unless otherwise indicated. Also, combinations of
substituents and/or variables are permissible only if such combinations result
in stable compounds.
[0077] It is further understood that compounds disclosed in U.S. Patent
No. 6,509,315 are excluded from the present invention as described in the
above provisos.
Definitions
[007S] As used herein, each of the following terms has the meaning associated
with it in this section.
[0079] As used herein, certain amino acid residues (in particular the twenty
common amino acids) are represented by the full name thereof, by the three
letter code corresponding thereto, or by the one-letter code corresponding
thereto, as indicated by the following:

Full Name Three-Letter Code One-Letter Code
Aspartic Acid Asp D
Glutamic Acid Glu E
Lysine Lys K
Arginine Arg R
Histidine His H
Tyrosine Tyr y
Cysteine Cys c
Asparagine Asn N
Glutamine Gin O
Serine Ser s
Threonine Thr T
Glycine Gly G
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine He I
Methionine Met M

WO 2004/084812 PCT/US2004/008275
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Proline Pro P
Phenylalanine Phe F
Tryptophan Trp N
[0080] As used herein, the term "amino acid side chain" refers to a moiety
comprising all of the atoms of an amino acid excluding the alpha-carbon atom,
a hydrogen atom bound with the alpha-carbon, the atoms of the alpha-carboxyl
moiety and the alpha-amine moiety. By way of example, an "alanine side
chain" refers to a methyl group, a "valine side chain" refers to a 2-propyl (or
isopropyl) group, and a 3-cyclohexylalanine side chain refers to a
cyclohexylmethyl group (i.e., C6Hn-CH2-).
[0081] "Inhibition" of a process in a cell (e.g., inhibition of protein synthesis,
inhibition of cell growth, inhibition of cell cycle progression, inhibition of cell
proliferation, or inhibition of tumorigenesis) means reduction (e.g., by at least
10%, 25%, 50%, 75%, 90%, 95%, or even 100%) of the rate at which the
process proceeds, reduction (e.g., by at least 10%, 25%, 50%, 75%, 90%,
95%, or even 100%) of the rate at which the process is initiated, or both.
[0082] "Enhancement" of a process in a cell (e.g., enhancement of apoptosis)
means increasing (e.g. by at least 10%, 25%, 50%, 75%, 90%, 95%, or even
100%) the rate at which the process proceeds, increasing (e.g., by at least 10%,
25%, 50%, 75%, 90%, 95%, or even 100%) the rate at which the process is
initiated, or both.
[0083] As used herein, the term "pharmaceutically acceptable carrier" means a
chemical composition with which a tamandarin analog or fragment, as
described herein, can be combined and which, following the combination, can
be administered to a subject (e.g., a human or other animal).
[0084] As used herein, the term "physiologically acceptable" ester or salt
means an ester or salt form of a tamandarin analog or fragment, as described
herein, which is compatible with other ingredients of a pharmaceutical
composition and which is not deleterious to a subject to which the composition
is to be administered.
[0085] As used herein, "parenteral administration" of a pharmaceutical
composition includes any route of administration characterized by physical

WO 2004/084812 PCT/US2004/008275
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breaching of a tissue of a subject and administration of the pharmaceutical
composition through the breach in the tissue. Parenteral administration thus
includes, but is not limited to, administration of a pharmaceutical composition
by injection of the composition, by application of the composition through a
surgical incision, by application of the composition through a tissue-
penetrating non-surgical wound, and the like. In particular, parenteral
administration can include, but is not limited to, subcutaneous, intraperitoneal,
intramuscular, intrastemal injection, and kidney dialytic infusion techniques.
[0086] As used herein, the term "anti-viral activity" means preventing
replication of a virus in the cell, preventing infection of the cell by a virus, or
reversing a physiological effect of infection of the cell by a virus. An anti-viral
agent is an composition of matter which, when delivered to a cell, exhibits
anti-viral activities. Anti-viral agents are well known and described in the
literature. By way of example, AZT (zidovudine) is an anti-viral agent which
is thought to prevent replication of HTV in human cells.
[0087] The terms describin different chemical moieties used herein are terms
of the art and are understood by one of ordinary skill in the art.
[0088] The term "homoproline," as used herein, refers to a proline moiety
containing an additional methylene group in the ring, Le., having a
6-membered ring.
[0089] The term ""non-amino acid moiety" as used herein refers to a moiety
that does not contain both an amino group and a carboxy group. Such a
moiety is exemplified above for R5. A non-amino acid moiety may be a
moiety at the terminus of the R5 group, for example, those shown above.
[0090] The term "protecting group," as used herein by itself or as part of
another group, refers to a chemical moiety that can readily be attached to a
particular functional group (and forming a protected functional group) when
desired to protect said functional group from undesired chemical reactions and
then at a later point be removed from said protected functional group to reveal
the original functional group. See, for example, Green and Wuts (1991,
Protective Groups in Organic Synthesis, Wiley, New York, 2nd Edition,
pages 1-9), which is hereby incorporated by reference in its entirety.

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Examples of protecting groups are well known in the art. Examples of
suitable protecting groups can be found in references such as Green and Wuts
(1991, Protective Groups in Organic Synthesis, Wiley, New York, 2nd Edition)
and Bodansky (1993, Principles of Peptide Synthesis, Springer, Berlin).
Specific, non-limiting examples of protecting groups include acetyl, benzyl,
f-butoxycarbonyl (Boc), benzoyl, dimethylisopropylsilyl, f-butyldimethylsilyl,
methoxymethyl, and benzyloxycarbonyl. Protecting groups attached to a solid
support are also suitable.
[0091] The term "amine protecting group," as used herein by itself or as part
of another group, refers to a chemical moiety that can readily be attached to an
amine group (and forming a protected amine) when desired to protect said
amine from undesired chemical reactions and then at a later point be removed
from said protected amine to reveal the original amine. Examples of amine
protecting groups are well known in the art. Examples of suitable amine
protecting groups can be found in references such as Green and Wuts (1991,
Protective Groups in Organic Synthesis, Wiley, New York, 2nd Edition) and
Bodansky (1993, Principles of Peptide Synthesis, Springer, Berlin). Specific,
non-limiting examples of amine protecting groups include carbarnates such as
f-butoxycarbonyl, methoxycarbonyl, and benzyloxycarbonyl, amides such as
formamide and trifluoroacetamide, and cyclic imides such as M-phthalimido.
[0092] The term "hydroxy protecting group," as used herein by itself or as part
of another group, refers to a chemical moiety that can readily be attached to an
hydroxy group (and forming a protected hydroxy) when desired to protect said
amine from undesired chemical reactions and then at a later point be removed
from said protected hydroxy to reveal the original hydroxy group. Examples
of hydroxy protecting groups are well known in the art. Examples of suitable
hydroxy protecting groups can be found in references such as Green and Wuts
(1991, Protective Groups in Organic Synthesis, Wiley, New York, 2nd Edition)
and Bodansky (1993, Principles of Peptide Synthesis, Springer, Berlin).
Specific, non-limiting examples of amine protecting groups include ethers
such as methoxymethyl, tetrahydropyranyl, and benzyl, silyl ethers such as

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trimethyl silyl and triisopropylsilyl, and carbonates such as 9-fluorenylmethyl
and benzyl.
[0093] Although detailed definitions have not been provided for every term
used herein, each term is understood by one of ordinary skill in the art.
Compositions
[0094] A composition according to the present invention includes a
pharmaceutical composition comprising a novel compound of Formula I, IA,
or II, as defined above, and one or more pharmaceutically acceptable
excipients. Preferred compositions of the present invention are
pharmaceutical compositions comprising a compound selected from one or
more embodiments listed above, and one or more pharmaceutically acceptable
excipients. Pharmaceutical compositions that comprise one or more
compounds of Formula I, IA, or II may be formulated, as is well known in the
prior art, such as by reference to known compilations as Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., USA.
[0095] In one embodiment of the invention, the composition comprises a
compound selected from one or more of the individual embodiments listed
above.
[0096] The invention encompasses pharmaceutical compositions comprising
at least one of the tamandarin analogs and the physiologically active fragments
described herein. Such compositions can comprise the analog/fragment and a
pharmaceutically acceptable carrier. By way of example, a pharmaceutical
composition according to the invention comprises a pharmaceutically
acceptable carrier and a tamandarin analog having the structure of either
Formula I, IA, or II as an active agent. As a further example, a
pharmaceutical composition according to the invention comprises a
pharmaceutically-acceptable carrier and one or more of the following
compounds:
[0097] In another embodiment, a pharmaceutical composition of the invention
further comprises one or more additional pharmaceutically active agents such
as, other tumor therapy agents, other anti-infective agents, and the like.

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[0098] Such pharmaceutical compositions can be used, for example, in the
methods described herein and for inhibiting one or more of protein synthesis,
cell cycle progression, tumorigenesis, growth, and proliferation in a cell. In
addition, such compositions can be used in the methods described herein for
enhancing apoptosis in a cell.
[0099] Pharmaceutical compositions that are useful in the methods of the
invention can be administered systemically in oral solid formulations,
ophthalmic, suppository, aerosol, topical, or other similar formulations. In
addition to the active agent, such pharmaceutical compositions can contain
pharmaceutically-acceptable carriers and other ingredients known to enhance
and facilitate drug administration. Other possible formulations, such as
nanoparticles, liposomes, resealed erythrocytes, and imrnunologically based
systems, can also be used to administer the active agent according to the
methods of the invention.
[00100] The invention encompasses pharmaceutical compositions which
consist of the active agent, in a form suitable for administration to a subject, or
the pharmaceutical composition comprises the active agent and one or more
pharmaceutically acceptable carriers, one or more additional ingredients, or
some combination of these. The active agent can be present in the
pharmaceutical composition in the form of a physiologically acceptable ester
or salt, such as in combination with a physiologically acceptable cation or
anion, as is well known in the art.
[00101] The formulations of the pharmaceutical compositions described herein
can be prepared by any method known or hereafter developed. In general,
such preparatory methods include the step of bringing the active agent into
association with a carrier or one or more other accessory ingredients, and then,
if necessary or desirable, shaping or packaging the product into a desired
single- or multi-dose unit.
[00102] Although the descriptions of pharmaceutical compositions provided
herein are principally directed to pharmaceutical compositions which are
suitable for ethical administration to humans, it is understood by the skilled
artisan that such compositions are generally suitable for administration to

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animals of all sorts. Modification of pharmaceutical compositions suitable for
administration to humans in order to render the compositions suitable for
administration to various animals is well understood, and the ordinarily skilled
artisan can design and perform such modification with merely ordinary, if any,
experimentation. Subjects to which administration of the pharmaceutical
compositions of the invention is contemplated include, but are not limited to,
humans and other primates, and mammals including commercially relevant
mammals such as cattle, pigs, horses, sheep, cats, and dogs.
[00103] Pharmaceutical compositions that are useful in the methods of the
invention can be prepared, packaged, or sold in formulations suitable for oral,
rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic,
or another route of administration. Other contemplated formulations include
projected nanoparticles, liposomal preparations, resealed erythrocytes
containing the active agent, and immunologically-based formulations.
[00104] A pharmaceutical composition of the invention can be prepared,
packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit
doses. As used herein, a "unit dose" is a discrete amount of the
pharmaceutical composition comprising a predetermined amount of the active
agent. The amount of the active agent is generally equal to the dosage of the
active agent which would be administered to a subject or a convenient fraction
of such a dosage such as, for example, one-half or one-third of such a dosage.
[00105] Controlled- or sustained-release formulations of a pharmaceutical
composition of the invention can be made using conventional technology.
[00106] A formulation of a pharmaceutical composition of the invention
suitable for oral administration can be prepared, packaged, or sold in the form
of a discrete solid dose unit including, but not limited to, a tablet, a hard or
soft capsule, a cachet, a troche, or a lozenge, each containing a predetermined
amount of the active agent. Other formulations suitable for oral administration
include, but are not limited to, a powdered or granular formulation, an aqueous
or oily suspension, an aqueous or oily solution, or an emulsion.

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[00107] As used herein, an "oily" liquid is one which comprises a carbon-
containing liquid molecule and which exhibits a less polar character than
water.
[00108] A tablet comprising the active agent may, for example, be made by
compressing or molding the active agent, optionally with one or more
additional ingredients. Compressed tablets can be prepared by compressing,
in a suitable device, the active agent in a free-flowing form such as a powder
or granular preparation, optionally mixed with one or more of a binder, a
lubricant, an excipient, a surface active agent, and a dispersing agent. Molded
tablets can be made by molding, in a suitable device, a mixture of the active
agent, a pharmaceutically acceptable carrier, and at least sufficient liquid to
moisten the mixture. Pharmaceutically acceptable excipients used in the
manufacture of tablets include, but are not limited to, inert diluents,
granulating and disintegrating agents, binding agents, and lubricating agents.
Known dispersing agents include, but are not limited to, potato starch and
sodium starch glycollate. Known surface active agents include, but are not
limited to, sodium lauryl sulfate. Known diluents include, but are not limited
to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose,
calcium phosphate, calcium hydrogen phosphate, and sodium phosphate.
Known granulating and disintegrating agents include, but are not limited to,
corn starch and alginate. Known binding agents include, but are not limited to,
gelatin, acacia, pre-gelatinized maize starch, polyvinylpyrrolidone, and
hydroxypropyl methylcellulose. Known lubricating agents include, but are not
limited to, magnesium stearate, stearate, silica, and talc.
[00109] Tablets can be non-coated or they can be coated using known methods
to achieve delayed disintegration in the gastrointestinal tract of a subject,
thereby providing sustained release and absorption of the active agent. By way
of example, a material such as glyceryl monostearate or glyceryl distearate can
be used to coat tablets. Further by way of example, tablets can be coated using
methods described in U.S. Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to
form osmotically-controlled release tablets. Tablets can further comprise a
sweetening agent, a flavoring agent, a coloring agent, a preservative, or some

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combination of these in order to provide pharmaceutically elegant and
palatable preparation.
[00110] Hard capsules comprising the active agent can be made using a
physiologically degradable composition, such as gelatin. Such hard capsules
comprise the active agent, and can further comprise additional ingredients
including, for example, an inert solid diluent such as calcium carbonate,
calcium phosphate, or kaolin.
[00111] Soft gelatin capsules comprising the active agent can be made using a
physiologically degradable composition, such as gelatin. Such soft capsules
comprise the active agent, which can be mixed with water or an oil medium
such as peanut oil, liquid paraffin, or olive oil.
[00112] Liquid formulations of a pharmaceutical composition of the invention
which are suitable for oral administration can be prepared, packaged, and sold
either in liquid form or in the form of a dry product intended for reconstitution
with water or another suitable vehicle prior to use.
[00113] Liquid suspensions can be prepared using conventional methods to
achieve suspension of the active agent in an aqueous or oily vehicle. Aqueous
vehicles include, for example, water and isotonic saline. Oily vehicles include,
for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as
arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral
oils such as liquid paraffin. Liquid suspensions can further comprise one or
more additional ingredients including, but not limited to, suspending agents,
dispersing or wetting agents, emulsifying agents, demulcents, preservatives,
buffers, salts, flavorings, coloring agents, and sweetening agents. Oily
suspensions can further comprise a thickening agent. Known suspending
agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats,
sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and
cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose,
hydroxypropylmethylcellulose. Known dispersing or wetting agents include,
but are not limited to, naturally-occurring phosphatides such as lecithin,
condensation products of an alkylene oxide with a fatty acid, with a long chain
aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or

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with a partial ester derived from a fatty acid and a hexitol anhydride (e.g.,
polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene
sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively).
Known emulsifying agents include, but are not limited to, lecithin and acacia.
Known preservatives include, but are not limited to, methyl, ethyl, or
n-propyl-/?ara-hydroxybenzoates, ascorbate, and sorbate. Known sweetening
agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and
saccharin. Known thickening agents for oily suspensions include, for example,
beeswax, hard paraffin, and cetyl alcohol.
[00114] Liquid solutions of the active agent in aqueous or oily solvents can be
prepared in substantially the same manner as liquid suspensions, the primary
difference being that the active agent is dissolved, rather than suspended, in
the solvent. Liquid solutions of the pharmaceutical composition of the
invention can comprise each of the components described with regard to liquid
suspensions, it being understood that suspending agents will not necessarily
aid dissolution of the active agent in the solvent. Aqueous solvents include,
for example, water and isotonic saline. Oily solvents include, for example,
almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive,
sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as
liquid paraffin.
[00115] Powdered and granular formulations of a pharmaceutical preparation
of the invention can be prepared using known methods. Such formulations
can be administered directly to a subject, used, for example, to form tablets, to
fill capsules, or to prepare an aqueous or oily suspension or solution by
addition of an aqueous or oily vehicle thereto. Each of these formulations can
further comprise one or more of dispersing or wetting agent, a suspending
agent, and a preservative. Additional excipients, such as fillers and
sweetening, flavoring, or coloring agents, can also be included in these
formulations.
[00116] In another embodiment, a pharmaceutical composition of the invention
is prepared, packaged, or sold in the form of oil-in-water emulsion or a waters
in-oil emulsion. The oily phase can be a vegetable oil such as olive or arachis

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oil, a mineral oil such as liquid paraffin, or a combination of these. Such
, compositions can further comprise one or more emulsifying agents such as
naturally occurring gums such as gum acacia or gum tragacanth, naturally-
occurring phosphatides such as soybean or lecithin phosphatide, esters or
partial esters derived from combinations of fatty acids and hexitol anhydrides
such as sorbitan monooleate, and condensation products of such partial esters
with ethylene oxide such as polyoxyethylene sorbitan monooleate. These
emulsions can also contain additional ingredients including, for example,
sweetening or flavoring agents.
[00117] In another embodiment, a pharmaceutical composition of the invention
is prepared, packaged, or sold in a formulation suitable for rectal
administration. Such a composition can be in the form of, for example, a
suppository, a retention enema preparation, and a solution for rectal or colonic
irrigation.
[00118] Suppository formulations can be made by combining the active agent
with a non-irritating pharmaceutically acceptable excipient which is solid at
ordinary room temperature (i.e., about 20°C) and which is liquid at the rectal
temperature of the subject (i.e., about 37°C in a healthy human). Suitable
pharmaceutically acceptable excipients include, but are not limited to, cocoa
butter, polyethylene glycols, and various glycerides. Suppository formulations
can further comprise various additional ingredients including, but not limited
to, antioxidants and preservatives.
[00119] Retention enema preparations or solutions for rectal or colonic
irrigation can be made by combining the active agent with a pharmaceutically
acceptable liquid carrier. As is well known in the art, enema preparations can
be administered using, and can be packaged within, a delivery device adapted
to the rectal anatomy of the subject. Enema preparations can further comprise
various additional ingredients including, but not limited to, antioxidants and
preservatives.
[00120] A pharmaceutical composition of the invention can be prepared,
packaged, or sold in a formulation suitable for vaginal administration. Such a
composition can be in the form of, for example, a suppository, an impregnated

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or coated vaginally-insertable material such as a tampon, a douche
preparation, or a solution for vaginal irrigation.
[00121] Methods for impregnating or coating a material with a chemical
composition are known in the art, and include, but are not limited to methods
of depositing or binding a chemical composition onto a surface, methods of
incorporating a chemical composition into the structure of a material during
the synthesis of the material (i.e., such as with a physiologically degradable
material), and methods of absorbing an aqueous or oily solution or suspension
into an absorbent material, with or without subsequent drying.
[00122] Douche preparations or solutions for vaginal irrigation can be made by
combining the active agent with a pharmaceutically acceptable liquid carrier.
As is well known in the art, douche preparations can be administered using,
and can be packaged within, a delivery device adapted to the vaginal anatomy
of the subject. Douche preparations can further comprise various additional
ingredients including, but not limited to, antioxidants, antibiotics, anti-fungal
agents, and preservatives.
[00123] Formulations of a pharmaceutical composition suitable for parenteral
administration can comprise the active agent combined with a
pharmaceutically acceptable carrier, such as sterile water or sterile isotonic
saline. Such formulations can be prepared, packaged, or sold in a form suitable
for bolus administration or for continuous administration. Injectable
formulations can be prepared, packaged, or sold in unit dosage form, such as
in ampules or in multi-dose containers containing a preservative. Formulations
for parenteral administration include, but are not limited to, suspensions,
solutions, emulsions in oily or aqueous vehicles, pastes, and implantable
sustained-release or biodegradable formulations. Such formulations can
further comprise one or more additional ingredients including, but not limited
to, suspending, stabilizing, or dispersing agents. In one embodiment of a
formulation for parenteral administration, the active agent is provided in dry
(i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g.,
sterile pyrogen-free water) prior to parenteral administration of the
reconstituted composition.

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[00124] In another embodiment, the pharmaceutical composition is prepared,
packaged, or sold in the form of a sterile injectable aqueous or oily suspension
or solution. This suspension or solution can be formulated according to the
known art, and can comprise, in addition to the active agent, additional
ingredients such as the dispersing agents, wetting agents, or suspending agents
described herein. Such sterile injectable formulations can be prepared using a
non-toxic parenterally-acceptable diluent or solvent, such as water or
1,3-butanediol, for example. Other acceptable diluents and solvents include,
but are not limited to, Ringer's solution, isotonic sodium chloride solution, and
fixed oils such as synthetic mono- or di-glycerides. Other parentally-
administrable formulations which are useful include those which comprise the
active agent in microcrystalline form, in a liposomal preparation, or as a
component of a biodegradable polymer systems. Compositions for sustained
release or implantation can comprise pharmaceutically acceptable polymeric
or hydrophobic materials such as an emulsion, an ion exchange resin, a
sparingly soluble polymer, or a sparingly soluble salt.
[00125] Formulations suitable for topical administration include, but are not
limited to, liquid or semi-liquid preparations such as liniments, lotions, oil-in-
water or water-in-oil emulsions such as creams, ointments or pastes, and
solutions or suspensions. Topically-administrable formulations may, for
example, comprise from about 1% to about 10% (w/w) active agent, although
the concentration of the active agent can be as high as the solubility limit of
the active agent in the solvent. Formulations for topical administration can
further comprise one or more of the additional ingredients described herein.
[00126] A pharmaceutical composition of the invention can be prepared,
packaged, or sold in a formulation suitable for pulmonary administration via
the buccal cavity. Such a formulation can comprise dry particles which
comprise the active agent and which have a diameter in the range from about
0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers.
Such compositions are conveniently in the form of dry powders for
administration using a device comprising a dry powder reservoir to which a
stream of propellant can be directed to disperse the powder or using a self-

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propelling solvent/powder-dispensing container such as a device comprising
the active agent dissolved or suspended in a low-boiling propellant in a sealed
container. In one embodiment, such powders comprise particles wherein at
least 98% of the particles by weight have a diameter greater than 0.5
nanometers and at least 95% of the particles by number have a diameter less
than 7 nanometers. In another embodiment, at least 95% of the particles by
weight have a diameter greater than 1 nanometer and at least 90% of the
particles by number have a diameter less than 6 nanometers. Dry powder
compositions, in one embodiment, include a solid fine powder diluent such as
sugar and are conveniently provided in a unit dose form.
[00127] Low boiling propellants generally include liquid propellants having a
boiling point of below 65°F. at atmospheric pressure. In one embodiment, the
propellant can constitute 50 to 99,9% (w/w) of the composition, and the active
agent can constitute 0.1 to 20% (w/w) of the composition. The propellant can
further comprise additional ingredients such as a liquid non-ionic or solid
anionic surfactant or a solid diluent (preferably having a particle size of the
same order as particles comprising the active agent).
[00128] Pharmaceutical compositions of the invention formulated for
pulmonary delivery can also provide the active agent in the form of droplets of
a solution or suspension. Such formulations can be prepared, packaged, or sold
as aqueous or dilute alcoholic solutions or suspensions, optionally sterile,
comprising the active agent, and can conveniently be administered using any
nebulization or atomization device. Such formulations can further comprise
one or more additional ingredients including, but not limited to, a flavoring
agent such as saccharin sodium, a volatile oil, a buffering agent, a surface
active agent, or a preservative such as methylhydroxybenzoate. In one
embodiment, the droplets provided by this route of administration have an
average diameter in the range from about 0.1 to about 200 nanometers.
[00129] The formulations described herein as being useful for pulmonary
delivery are also useful for intranasal delivery of a pharmaceutical
composition of the invention.

WO 2004/084812 PCT/US2004/008275
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[00130] Another formulation suitable for intranasal administration is a coarse
powder comprising the active agent and having an average particle from about
0.2 to 500 micrometers. Such a formulation is administered in the manner in
which snuff is taken, i.e., by rapid inhalation through the nasal passage from a
container of the powder held close to the nares.
[0100] Formulations suitable for nasal administration may, for example,
comprise from about 0.1% (w/w) to about 100% (w/w) of the active agent, and
can further comprise one or more of the additional ingredients described
herein.
[0101] A pharmaceutical composition of the invention can be prepared,
packaged, or sold in a formulation suitable for buccal administration. Such
formulations may, for example, be in the form of tablets or lozenges made
using conventional methods, and may, for example, contain 0.1 to 20% (w/w)
active agent, the balance comprising an orally dissolvable or degradable
composition and, optionally, one or more of the additional ingredients
described herein. Alternatively, formulations suitable for buccal
administration can comprise a powder or an aerosolized or atomized solution
or suspension comprising the active agent. Such powdered, aerosolized, or
aerosolized formulations, when dispersed, have an average particle or droplet
size, in one embodiment, in the range from about 0.1 to about 200 nanometers,
and can further comprise one or more of the additional ingredients described
herein.
[0102] In another embodiment, a pharmaceutical composition of the invention
is prepared, packaged, or sold in a formulation suitable for ophthalmic
administration. Such formulations may, for example, be in the form of eye
drops including, for example, a 0.1-1.0% (w/w) solution or suspension of the
active agent in an aqueous or oily liquid carrier. Such drops can further
comprise buffering agents, salts, or one or more other of the additional
ingredients described herein. Other ophthalmically-administrable formulations
which are useful include those which comprise the active agent in
microcrystalline form or in a liposomal preparation.

WO 2004/084812 PCT/US20 04/0082 75
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[0103] As used herein, "additional ingredients" include, but are not limited to,
one or more of the following: excipients; surface active agents; dispersing
agents; inert diluents; granulating and disintegrating agents; binding agents;
lubricating agents; sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as gelatin;
aqueous vehicles and solvents; oily vehicles and solvents; suspending agents;
dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts;
thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; anti-
fungal agents; stabilizing agents; and pharmaceutically acceptable polymeric
or hydrophobic materials. Other "additional ingredients" which can be
included in the pharmaceutical compositions of the invention are known in the
art and described, for example in Genaro, ed., 1985, Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., which is
incorporated herein by reference.
[0104] The relative amounts of the active agent, the pharmaceutically
acceptable Carrier, and any additional ingredients in a pharmaceutical
composition of the invention will vary, depending upon the identity, size, and
the type and severity of condition of the subject treated and further depending
upon the route by which the composition is to be administered. By way of
example, the composition can comprise between 0.1% and 100% (w/w) active
agent.
[0105] In another embodiment, the present invention is directed to a
composition comprising a compound of Formula I, IA, or II and a carrier,
wherein said carrier is suitable for an assay. Such carriers may include solid
carriers and liquid carriers. A composition suitable for an assay may, but not
necessarily, be sterile. Examples of suitable carriers for assays include
dimethylsulfoxide, ethanol, dichloromethane, methanol, chloroform,
AfJV-dimethylformamide, and the like. In one embodiment, the present
invention is directed to a composition consisting essentially of a compound of
Formula I, IA, or II and a carrier, wherein said carrier is suitable for an assay.

WO 2004/084812 PCT/US2004/008275
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Uses of the Compounds and Compositions
[0106] Tamandarin analogs and physiologically active fragments thereof, as
disclosed herein, such as compounds having the structure of Formula I, IA, or
II, can be used to affect a variety of physiological processes. Each of these
compounds can be used to inhibit protein synthesis. Furthermore, the
compounds can be used to inhibit progression of a cell through the cell cycle.
While not being bound by any particular theory of operation, it is believed that
the cell cycle-inhibiting activity of the compounds can be attributed to
inhibition of protein synthesis and possibly also to inhibition of other cellular
activities associated with DNA replication or cell division. Tamandarin
analogs and their active fragments also induce apoptosis in cells. The
physiological activities attributable to tamandarin analogs and fragments make
these compounds useful for alleviating a variety of disorders in which one or
more of cell growth, proliferation, and survival are aberrant. Examples of
such disorders include cancers at various stages, e.g., tumorigenesis, tumor
growth, and metastasis, and viral infections at various stages, e.g., infection of
cells with virus particles, production of virus particles within a cell, and
survival of vims-infected cells. Furthermore, the compounds of the present
invention can be used to treat cancers (e.g., breast cancer), viral, fungal,
parasitic, and bacterial infections, autoimmune disorders, allergies, other
hyper-immune disorders, and atherosclerosis.
[0107] The subject of the method disclosed herein is preferably an animal,
including, but not limited, a cow, horse, sheep, pig, chicken, turkey, quail, cat,
dog, mouse, rat, rabbit, and guinea pig, and is more preferably a mammal, and
most preferably a human.
[0108] The tamandarin analogs and fragments described herein can be used
for anti-proliferative, anti-tumor, anti-viral, and immunosuppressive purposes.
Accordingly, a further aspect of the present invention is directed to a method
of inhibiting or preventing the growth of a cancer cell, comprising contacting
said cancer cell with an effective amount of a compound of Formula I, IA, or
II. For example, these compounds can be used in a pharmaceutical

WO 2004/084812 PCT/US2004/008275
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preparation or medicament to be administered to a patient afflicted with a
disorder in which one or more of protein synthesis, cell growth, proliferation,
and survival are aberrant Such medicaments can be used to treat disorders
such as cancers (e.g., breast cancer). Examples of anti-tumor activities that
can be exhibited by the compounds described herein include inhibition of
tumorigenesis, inhibition of metastasis, inhibition of tumor cell growth,
inhibition of tumor cell proliferation, and enhancement of tumor cell
apoptosis. Dehydrodidemnin exhibits activity against cell lines derived from
several human solid tumor types, including non-small cell lung cancer and
colon tumor cell lines, and exhibits selective anti-tumor activity against non-
small cell lung cancer, melanomas, ovarian cancer, and colorectal cancer
(Depenbrock et al., Brit. J. Cancer 75:739-744 (1998)). The tamandarin
analogs and fragments described herein exhibit anti-tumor activities in cells of
one or more of these lines, as well as in cells of the corresponding tumor type
in vivo. Determination of the effectiveness of any particular tamandarin
analog or fragment described herein against any particular tumor type can be
made using standard methods involving, for example, one or more of the 60
standard tumor cell lines maintained in the U.S. National Cancer Institute drug
screening program.
[01091 A further aspect of the present invention is directed to a method of
inhibiting, treating, or preventing a viral infection comprising administering a
compound of Formula I, IA, or II to a subject in need of such treatment.
Examples of anti-viral activities that can be exhibited by the tamandarin
analogs and fragments described herein include inhibition of binding of a virus
with a cellular target, inhibition of infection of a cell by a virus, inhibition of
cellular synthesis of virus components, inhibition of intracellular assembly of
virus particles, inhibition of release of virus particles from an infected cell,
inhibition of growth of a cell infected by a virus, inhibition of proliferation of
a cell infected by a virus, and induction of death (i.e., apoptosis) of a cell
infected by a virus. The anti-viral activity of the compounds described herein
can, for example, be used to treat or prevent viral infections of mammals and
associated symptoms. By way of illustration, a didemnin analog or fragment

WO 2004/084812 PCT/US2004/008275
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described herein can be used to treat or prevent infections by viruses such as
Rift Valley Fever virus, Dengue virus, or any of the equine encephalitis
viruses.
[0110] In a further embodiment, the present invention is directed to method of
providing immunosuppressive therapy to a subject in need thereof, comprising
administering an effective amount of a compound of Formula I, IA, or II to
said subject. Examples of immunosuppressive activities that can be exhibited
by the tamandarin analogs and fragments described herein include inhibition
of a cellular immune response to an immunogen (e.g., an infectious agent, or a
transplanted cell or tissue) and inhibition of a humoral immune response to an
immunogen. Examples of disorders in which immunosupression can be
desirable include autoimmune disorders, transplant rejection disorders (e.g.,
rejection of a solid tissue or bone marrow transplant), development of an
immune response to an implanted device (e.g., a stent or a heart valve),
immune hypersensitivity, and anaphylaxis.
[0111] In another embodiment, a compound according to the present invention
is used to treat a tumor, including solid tumors such as bladder, breast, colon,
liver, ovary, gastric, pancreas, prostate, renal, retinoblastomas, melanoma,
fibrosarcoma, or osteosarcoma. In another embodiment, a compound of the
present invention is used to treat a leukemia or lymphoma, such as
promyelocytic leukemia, acute Iymphoblastic, chronic myelogenous leukemia,
T cell lymphoma, cutaneous T cell lymphoma, Burkitt's B cell lymphoma, and
B cell lymphoma. In another embodiment, a compound of the present
invention is used to treat a cancer or tumor represented by a cell line shown to
be inhibited by the compound.
[0112] The tamandarin analogs and fragments described herein can be
administered in vitro to a cell or tissue (e.g., a cultured cell or tissue, or a cell
or tissue harvested from one animal prior to introduction into the same or a
different animal). Alternatively, the agents can be administered to the cell or
tissue in vivo by administering the agent or a pharmaceutical composition
comprising the agent to an animal (e.g., a mammal such as a human) that
comprises the cell or tissue.

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[0113] In one embodiment of the methods described herein, a tamandarin
analog described herein and having an enzyme-cleavable group attached
thereto is administered to an animal. In one embodiment, upon cleavage of
the enzyme-cleavable group, the compound is transformed from an inactive
(or less active) form to an active (or more active) form. Thus, the tamandarin
analog can be selectively activated at a body location at which the enzyme
activity occurs.
[0114] The enzyme which is used to cleave a tamandarin analog having an
enzyme-cleavable moiety attached can be an enzyme which naturally occurs at
a body location in an animal. Alternatively, the enzyme can be provided to the
animal, for example as a composition comprising the enzyme or a nucleic acid
which encodes the enzyme. As another example, the enzyme can be coupled
(e.g., covalently, using a cross-linking agent or by expression as an enzyme-
antibody fusion protein) with an antibody that specifically binds with a tissue
{e.g., cancerous cells such as leukemic cells or cells of a solid tumor) at a body
location in the animal, and the antibody-enzyme complex can be administered
to an animal. Administration of a tamandarin analog having an attached
enzyme-cleavable group to the same animal results in preferential activation of
the compound at the tissue or body location. The physiological effect of the
compound can thereby be localized at the tissue or body location, and any side
effect attributable to the activated compound can thereby be reduced or
minimized.
[0115] The compounds of the present invention may be administered in an
effective amount within the dosage range of about 0.01 mg/kg to about 300
mg/kg, preferably between 0.1 mg/kg to 100 mg/kg body weight, more
preferably from 0.1 mg/kg to 10 mg/kg body weight. A compound of the
present invention may be administered in a single daily dose, or the total daily
dosage may be administered in divided doses of two, three, or four times daily.
The exact dosage and frequency of administration depends on the particular
compound of Formula I, IA, or II used, the particular condition being treated,
the severity of the condition being treated, the age, weight, and general
physical condition of the particular patient as well as other medication the

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individual may be taking, as is well known to those skilled in the art. The
dosages may be varied depending upon the requirements of the patient, the
severity of the condition being treated, and the compound being employed.
[0116] In all cases of administration, it is understood that the compound of
Formula I, IA, or II can be administered as a pharmaceutical composition
comprising said compound and a pharmaceutically acceptable excipient, as
described herein. Alternatively, the compound of Formula I, IA, or II may be
administered as a pure material if appropriate.
[0117] In an additional aspect of the present invention, a compound of
Formula I, IA, or II may be used alone or in combination with one or more
additional pharmaceutical active agents (e.g., an anticancer agent, an antiviral
agent.
[0118] In another embodiment, a compound of the invention is also useful in a
drug discovery assay. A compound of Formula I, IA, or II may be used in an
assay to determine the efficacy and/or potency of other compounds as anti-
cancer, anti-viral, or immunosuppresive agents. These assays include in vivo
and in vitro assays. The compounds of the present invention can be used as
controls or can be used as lead compounds to discover new, useful compounds
and drugs.
[0119] While still not being bound by any particular theory of operation, it is
believed that the physiological activities attributable to the tamandarin analogs
and fragments described herein result from one or more interactions between
such analogs or fragments and at least one cellular component. This
interaction(s) leads, directly or indirectly, to the observed cellular response.
Accordingly, the invention encompasses use of a compound having the
structure of Formula I, IA, or II to identify one or more cellular components
which contributes to a disorder phenotype in an individual. Identification of
such a cellular component can indicate an effective course of treatment for
alleviating the disorder. Examples of compounds useful for this purpose
include tamandarin analogs and fragments which have the structure of
Formula I, IA, or II and which comprise a fluorescent substituent (e.g., part of
R5), photoreactive chemical moiety, or a moiety bound with a support.

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[0120] Fluorescent and other detectably labeled tamandarin analogs described
herein (as well as their physiologically active fragments) can be used to
identify cells in which those analogs and fragments can exert their
physiological effects. For example, cells which absorb or bind with a
fluorescent compound having the structure of one of Formula I, IA, or II can
be identified or isolated. Identification or isolation of such cells can be used to
diagnose a disorder associated with the presence of such cells. Identification
or isolation of these cells can also indicate which of the tamandarin analogs
and fragments are efficacious for treating a disorder involving the cells.
[0121] Compounds of the present invention having a photoreactive moiety are
useful as photoaffinity probes. Photoaffinity probes are useful for identifying,
labeling, and/or modifying target proteins. Identification of the target protein
is useful for understanding of disease and cellular processes. A photoaffinity
probe of the present invention is for such a goal. Additionally, in another
embodiment, a photoaffinity probe of the present invention is used to
modulate or deactivate a target protein.
[0122] A support-bound tamandarin analog (or a support-bound fragment of a
tamandarin analog which exhibits a corresponding physiological activity) can
be used to identify cells which comprise, on their surfaces or elsewhere,
receptor proteins, glycoproteins, and the like, which are capable of interacting
or binding with the analog. As an example, a tamandarin analog having the
structure of Formula I, IA, or II and attached to a support can, by virtue of its
interaction with a particular cellular receptor, be used to identify or physically
isolate cells of a particular type (e.g., tumor cells) which are characterized by
the presence of the particular receptor.
[0123] In one embodiment, a method according to the present invention uses a
compound selected from one or more of the individual embodiments listed
above. In another embodiment, the method uses a compound is selected from
the group consisting of a compound according to Formula I, IA, or II
wherein.

WO 2004/084812 PCT/US2004/008275
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R1 and R2 together fonn the alkyl ring of a proline residue, R3 is
benzyl, R4 is CH3, R5 is (A^methyl-7?-Ieucine)-5'-proIine-5-lactate, R6 is a
valine side chain, X is O, and Y is H (compound 36);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
benzyl, R4 is CH3, R5 is H, R6 is a valine side chain, X is 0, and Y is H;
R and R together form the alkyl ring of a proline residue, R is
benzyl, R4 is CH3, R5 is H-HC1, R6 is a valine side chain, X is 0, and Y is H
(compound 35);
R and R together form the alkyl ring of a proline residue, R is
benzyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is O, and Y is TIPS
(compound 34);
R and R together form the alkyl ring of a proline residue, R is
benzyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is O, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is (7V-methyl-iMeucine)-S'-proline-1S~Iactate, R6
is a valine side chain, X is O, and Y is H (compound 62);
R and R together form the alkyl ring of a proline residue, R is
4-methoxybenzyl, R4 is H, R5 is Boc, R6 is a valine side chain, X is O, and Y
is H (compound 61);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is H, R6 is a valine side chain, X is O, and Y is
H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is (iV-methyl-7?-leucine)-5-proline-5-pyruvate,
R6 is a valine side chain, X is 0, and Y is H (compound 64);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is (N-methyl-iMeucine), R6 is a valine side
chain, X is O, and Y is H;
4-methoxybenzyl, R4 is H, R5 is iV-Cbz-7V-methyl-7?-leucine, R6 is a valine side
R and R together form the alkyl ring of a proline residue, R is
oxybenzyl, R4 is H, R5 is N-Cbz-N
chain, X is O, and Y is H (compound 63);

WO 2004/084812 PCT/US2004/008275
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R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is H, R5 is H-HC1, R6 is a valine side chain, X is 0, and
YisH;
R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3) R5 is (JV-methyl-
/Meucine)-1Sr-proline-5'-lactate, R6 is a valine side chain, X is 0, and Y is H
(compound 75);
R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is Boc, R6 is
a valine side chain, X is O, and Y is H (compound 74);
R1 is H, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is H, R6 is a
valine side chain, X is O, and Y is H;
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3j R5 is (N-
methyl-??-leucine)-1S"proline-5r-lactate, R6 is a valine side chain, X is O, and Y
is H (compound 150);
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3) R5 is Boc, R6
is a valine side chain, X is O, and Y is H (compound 85);
R1 is CH3, R2 is CH3, R3 is 4-methoxybenzyl, R4 is CH3, R5 is H, R6 is
a valine side chain, X is O, and Y is H;
R and R~ together form the alkyl ring of a proline residue, R is
2-naphthylmethyl, R4 is CH3, R5 is (7V-methyI-iMeucine)-5r-proline-,SF-lactate,
R6 is a valine side chain, X is O, and Y is H (compound 93);
R1 and R2 together form the alkyl ring of a proline residue, R3 is
2-naphthylmethyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is 0, and
Y is H (compound 92);
R and R together form the alkyl ring of a proline residue, R is
2-naphthylmethyl, R4 is CKb, R5 is H, R6 is a valine side chain, X is O, and Y
isH;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is CHs, R5 is (iV-methyl-tf-Ieucine^S-proline-S-Iactate,
R6 is a valine side chain, X is NH, and Y is H;
R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is CH3, R5 is H, R6 is a valine side chain, X is NH, and Y
is H; and

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R1 and R2 together form the alkyl ring of a proline residue, R3 is
4-methoxybenzyl, R4 is CH3, R5 is Boc, R6 is a valine side chain, X is NH, and
YisH;
and pharmaceutically acceptable salts thereof.
Methods of Preparation of Compounds
[0124] A further aspect of the present invention is a method of synthesizing a
compound according to Formula I, IA, or II. The compound of Formula I,
IA, or II can be synthesized according to the general method outlined in the
following schemes and descriptions. Specific, non-limiting examples of the
synthesis are provided in the Examples section below.
[0125] In reference to methods of making the analogs and fragments described
herein, the substituents R1, R2, R3, R4, R5, R6, W, X, Y, and Z have the same
meanings as used above.
[0126] As used in the present disclosure, a protection reaction can include any
reaction whereby one or more chemical moieties are covalently (but
reversibly) attached to one of a nitrogen atom, an oxygen atom, and a sulfur
atom of a molecule. Such attachment prevents the atom or atoms from
participating in non-desired chemical reactions, i.e., becoming covalently
attached to other chemical moieties, and donating or accepting either of
protons and electrons to other chemical moieties. A chemical moiety thus
attached is referred to as "a protecting group." By way of example, the
nitrogen atom of a compound such as D-valine, can be protected using a
reagent such as carbobenzyloxy-succinimide (CBZ-succinimide). Use of this
reagent in a standard protocol yields a protected D-valine, i.e., D-N-Cbz-
valine. In this compound, the CBZ moiety acts as an amine protecting group,
and the nitrogen atom to which it is attached cannot readily undergo additional
chemical reactions. As an alternative example, the hydroxyl moiety of
compound 11 can be protected using a reagent such as triisopropylsilyltriflate
(TTPSOTf) to yield compound 13 (Example 1). In this compound, Y is a
triisopropylsilyl (TIPS) moiety and acts as a hydroxyl protecting group,

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preventing chemical reactions with the oxygen atom to which this moiety is
attached.
[0127] Protocols for performing protection reactions and comprehensive
information about chemical moieties that can be used as protecting groups is
known in the art and is found in references such as Green and Wuts (1991,
Protective Groups in Organic Synthesis, Wiley, New York) or Bodansky
(1993, Principles of Peptide Synthesis, Springer, Berlin).
[0128] Certain tamandarin analogs and fragments of the invention can be
made by the synthetic pathway shown in Schemes 1 and 2.
Scheme 1



[0129] In the process shown, a compound having the structure of Formula IV
is converted to a compound having the structure of Formula V.
[0130] Such a series of reactions can include, but is not limited to, a protection
reaction, an activation reaction, an esterification reaction, and an ester
hydrolysis reaction. The amine group of Formula IV is preferably protected
prior to performing the esterification and hydrolysis reactions. A specific
example of making a compound having the structure of Formula V is given in
Example 1. "APG" refers to an amine protecting group, such as, but not
limited to, carbobenzyloxy (CBZ) moiety or tert-butyloxycarbonyl (BOC)
moiety. Alternative amine protecting groups can also be used, as described
herein and in the art.

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[0131] An example of an activation reaction included in the method of making
a compound having the structure of Formula V is depicted in Example 1. The
activation step can involve a reagent such as pentafluorophenol (PFPOH).
Esterification reactions which do not require an activated intermediate can also
be employed to make a compound having the structure of Formula V.
[0132] Any method of ester hydrolysis known in the art that does not
comprise harsh conditions which favor racemization can be used to make a
compound having the structure of Formula V. By way of example, a
compound having the structure of Formula V wherein the carboxy group is
esterified to form the alkyl ester, e.g., methyl ester, can be hydrolyzed using a
strong base in a solvent mixture. Reagents and conditions suitable for ester
hydrolysis under milder conditions (i.e., including conditions which do not
favor racemization) can be readily selected by one skilled in the art.
[0133] A compound having the structure of Formula VI can be esterified with,
for example, allyl bromide (e.g., as described in Example 1), to yield a
compound having the structure of Formula VII.
[0134] A compound having the structure of Formula V and a compound
having the structure of Formula VH can be coupled (e.g., esterified) to yield a
compound having the structure of Formula VIII (e.g., Step 3 of Scheme 1).
Optionally, such a reaction can be performed using a catalyst, a coupling
reagent, or an esterification reagent. Reagents and conditions useful for this
type of reaction are known in the art and exemplified in Example 1.
Tamandarin fragments having the structure of Formula VIII exhibit one or
more of the pharmacological activities described herein.
[0135] A compound having the structure of Formula VIII can be hydrolyzed
to yield a compound having the structure of Formula IX.
[0136] As described elsewhere herein, reaction conditions and reagents
suitable for ester hydrolysis are known in the art, and can be readily applied by
a skilled artisan. An example of this type of hydrolysis is depicted in
Example 1. Tamandarin fragments having the structure of Formula IX exhibit
one or more of the pharmacological activities described herein.

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[0137] The carboxyl group of a compound having the structure of Formula IX
can be activated to yield a compound having the structure of Formula X.
[0138] In Formula X, "ACT" refers to an activating group, such as a
pentafluorophenyl (PFP) moiety. Another example of an activating group is
an JV-hydroxysuccinimide moiety. Chemical activation can be performed
using reagents such as an activating reagent, a catalyst, an activating group
donor, or the like. By way of example, compound 8, depicted in Example 2, is
activated by covalent attachment of a PFP group to yield compound 18.
Protocols for activating a compound in the manner disclosed herein are known
in the art. Tamandarin fragments having the structure of Formula X exhibit
one or more of the pharmacological activities described herein.
[0139] The activated compound having the structure of Formula X can be
coupled with a third reactant having the structure of Formula XI to yield a
compound having the structure of Formula XII. In Formulas XI and XII,
SEM refers to 2-(trimethylsilyl)ethoxycarbonyl. An example of this reaction
is depicted in Example 1, in which compound 24 is coupled with
compound 18 to yield compound 25. The reagents and conditions necessary
for preparation of a protected peptide such as compound 18 are described, for
example, in Li et al.t J. Am. Chem. Soc. 112: 7659-7672 (1990). Tamandarin
analogs having the structure of Formula XII exhibit one or more of the
pharmacological activities described herein.
[0140] A tamandarin analog having one or more of the pharmacological
activities described herein can be made by removing one of the amino
protecting groups and the carbonyl hydroxyl protecting group of a compound
having the structure of Formula XII and cyclizing the compound to yield a
compound having the structure of Formula XIII. This process is shown in
Step 7 of Scheme 2.
[0141] An example of reactions of this type is shown in Example 1.
Chemically deprotecting a compound such as one having the structure of
Formula XII can be accomplished by reacting the compound with one or more
reagents to remove a protecting group of the compound. Exemplary
deprotection reactions are disclosed herein. Other protocols for deprotecting a

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compound are known in the art, and can be readily applied by a skilled artisan
to deprotection of a compound having the structure of Formula XII.
Cyclization of a deprotected compound otherwise having the structure of the
Formula XII can be accomplished using methods known in the art for
macrocyclization of peptides. For example, the macrocyclization conditions
can be similar or identical to those used in the cyclization that yields
compound 34, described in Example 2. Tamandarin analogs having the
structure of Formula XIII exhibit one or more of the therapeutic activities
described herein.
[0142] One or more of the protecting groups of a compound having the
structure of the Formula XIII can be removed to yield a compound having the
structure of Formula XIV. This deprotection is shown in Step 8 of Scheme 2.
Tamandarin analogs having the structure of Formula XIV exhibit one or more
of the therapeutic activities described herein.
[0143] Yet another active compound can be made by coupling a compound
having the structure of Formula XIV and a reagent having the moiety of R5.
This reaction is exemplified in Examples 1-4. The R5 substituent group can
comprise an enzyme cleavable moiety, preferably at or near the distal end
thereof (relative to the macrocycle). Such a moiety can be cleavable by an
enzyme, for example, a carboxypeptidase, a fMactamase, a (3-galactosidase, a
penicillin V-amidase, a cytosine deaminase, a nitroreductase, an alkaline
phosphatase, a |3-glucuronidase, and a catalytic antibody. An example of an
R5 moiety which comprises an enzyme-cleavable moiety , is
-(A^-methyl)leucine-(5)proline-(5)lactate-(5)gIutamine-(5')pyroglutamate.
Other examples of enzyme-cleavable moieties are described herein. Other
suitable reagents include those having he specific R5 groups shown above
wherein the open valence is replaced by a functional group, e.g., OH, such that
the moiety contains a functional group, e.g., COOH, suitable for coupling with
the amine of the macrocycle.
[0144] Other tamandarin analogs and fragments of the invention can be made
by the synthetic pathway shown in Scheme 3.


[0145] In Scheme 3, a compound of Formula XV is reacted with a compound
of Formula XVI to form a compound of Formula XVII. Step 10 can be
performed using suitable esterification conditions to form the compound of
Formula XVII. The compound of Formula XVII is then condensed with a
compound of Formula XVIII to form a compound of Formula XIX. The
compound of Formula XIX is then reacted to form the macrocycle of Formula
Xin. Step 12 comprises the deprotection of the amine and the carboxylate
groups, and further comprises the macrocyclization reaction. The macrocycle
of Formula XIII is deprotected (wherein R5 is H) and then reacted with a
suitable moiety to form a compound according to Formula I. It is noted that a
compound of Formula XIII is within the scope of a Formula I.

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[0146] When X is NH, such that the ester bond is replaced by an amide bond,
the amide fragment containing R3, R4, and R5 can be prepared as shown in
Scheme 4.

[0147J In Scheme 4, a compound of Formula XX is first reacted with a
suitable amine protecting group to form a compound of Formula XXI. For
example, reaction 14 can use (Boc)2O and NaOH in dioxane. A compound of
Formula XXI is then esterified to form a suitable ester of Formula XXII. A
suitable ester includes an alkyl ester, such as a methyl ester. The ester may be
formed by reacting the compound of Formula XXI with an alkyl halide, such
as methyl iodide, and a suitable base, such as KHCO3. The ester is then
reduced to form an alcohol of Formula XXIII. The reduction can be carried
out using standard conditions and reagents, such as NaBEU. The compound of
Formula XXIII is then converted to a compound of Formula XXIV, wherein
PG is a suitable protecting group, such as tert-butyldimethylsilyl,
trimethylsilyl, or triisopropylsilyl, and LG is an appropriate leaving group,
such as mesyl or tosyl group. Preferably, Step 16 comprises two steps,'
wherein the first step is protecting the primary alcohol and the second step is

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converting the secondary alcohol to a suitable leaving group. The compound
of Formula XXIV is then reacted with an azide compound, such as NaN3, to
form a compound of Formula XXV. The compound of Formula XXV is then
reduced under suitable conditions, e.g., H2 and Pd on carbon in ethanol, to
yield an amine of Formula XXVI. Condensation of the amine of Formula
XXVI with an acid for Formula XXVH under suitable conditions yields an
amide of Formula XXVIII. The formation of the amide bond may be
catalyzed with a suitable coupling reagent(s), for example, DCC, HOBt, and
NMM. The amide of Formula XXVIII is then deprotected to yield an alcohol
of Formula XXIX, which is oxidized to form the acid of Formula XXX.
Suitable reagents and conditions for performing Steps 20 and 21 are well
known in the art. The compound of Formula XXX is then used in place of the
compound of Formula XVIII shown in Scheme 3 to prepare a compound
according to Formula IA wherein X is NH.
[0148] A compound according to Formula I is prepared using a method
analogous to that used for preparing the compounds of Formula IA described
herein. When Z is C(O)-CH(CH3)-C(O) and W is O or NH, a suitable HIP-
isostatine moiety can be used in place of a compound of Formula X in
Scheme 1. Suitable HIP-isostatine moieties are known. See, e.g.,
Pfizenmeyer et al, Bioorg. Med. Chem. Lett. 5:3653-3656 (1998).
[0149] Variation of the substituents of the tamandarin analogs and fragments
may require slight modifications in the general methods described herein. It is
understood that the invention includes such modifications, as they could be
readily designed by one of ordinary skill in the art of synthetic chemistry.
[0150] 'H-NMR, I3C-NMR, IR, and MS spectra were recorded according to
standard procedures. Significant peaks are tabulated in the order: number of
protons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br
s, broad singlet) and coupling constant(s) in Hertz.

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EXAMPLES
EXAMPLE 1
Total Synthesis of Tamandarin B
Scheme 5





[0151] D-W-Cbz-Valine pentafluorophenyl (PFP) Ester (10): D-iV-Cbz-Valine
(9.8 g, 39.0 nsmol) was dissolved in CH2C12 (100 mL) and cooled to 0° C,
followed by the addition of pyridine (3.4 mL, 42.9 mmol,) and PFP-
trifluoroacetate (4.08 mL, 46.8 mmol). The solution was stirred lh at room
temperature and quenched with NH4CI (50 mL, sat). The organic layer was

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washed with 5% HC1 (100 mL), NaHCO3 (100 mL, sat), dried (Na2SO4),
filtered and the solvent evaporated. The resulting PFP ester 10 was obtained as
a colorless oil (15.8 g, 92%) and used directly in the next step. This compound
showed identical te-NMR as that reported in ref 23. 'H-NMR (500 MHz,
CDC13): 1.01 (d, J = 6.9 Hz, 3H), 1.08 (d, J = 6.9 Hz, 3H) 2.33-2.41 (m, 1H),
4.63-4.70 (m, 1H), 5.15 (s, 2H), 5.22 (d, J = 7.8 Hz, 1H), 7.30-7.41 (m, 5H).
[0152] (4i?)-4-BenzyIoxycarbonylan7ino-5-methyl-3-oxo-hex.anoic Acid
Methyl Ester (11): To a solution of PFP ester 10 (6 g, 14.4 mmol) in
anhydrous THF (30 mL), cooled to -78QC, a solution of lithium enolate of
methyl acetate was added. The enolate was prepared by addition of methyl
acetate (4.12 mL, 50.3 mmol) to a solution of LDA (51.8 mmol in 50 mL of
anhydrous THF) at -78 °C and the resulting solution was stirred for lh. The
reaction mixture was stirred for 45 min more at the same temperature, and
quenched with NH4CI (50 mL) at -78 °C. After warming to room temperature,
the solution was diluted with EtOAc (mL). The organic layer was separated
and washed with 10% HC1 (100 mL), 5% NaHCO3 (100 mL) and NaCl
(lOOmL, sat), dried (Na2SO4), filtered and concentrated. The crude oil was
purified by column chromatography (silica gel, EtOAc/Hexanes 1/9) to yield
the methyl ester 11 (2.8 g, 64%) as colorless oil. ^-NMR (500 MHz, CDC13):
0.80 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 2.20-2.29 (m, 1H), 3.51 (d, J
= 11.8 Hz), 3.55 (d, J = 17.7 Hz), 3.70 (s, 3H), 4.41 (q, J = 4.4 Hz, 1H), 5.09
(s, 2H), 5.32 (d, J = 12.0 Hz, 1H), 7.29-7.39 (m, 5H).
[01531 (3S,4i?)4-Benzyloxycarbonylaniino-3-hydroxy-5-methylhexanoic
Acid Methyl Ester (12): To a solution of methyl ester 11 (2.8 g, 9.15 mmol) in
HPLC MeOH (30 mL), cooled to -78 QC, potassium borohydxide (1.72 g, 32
mmol) was added in portions. The reaction mixture was stirred at -78 °C for 10
min, warmed to -20 °C for 30 min and then to 0 °C for 10 min. The reaction
was quenched with acetic acid to pH 7 and extracted with CH2C12 (3 x 50 mL).
The organic layer was washed with NaHCO3 (50 mL, sat), NaCl (50 mL, sat),
dried (Na2SO4), filtered and evaporated. The alcohol 12 was obtained as a
colorless oil (2.7 g, 98%) and used directly in the next step. !H-NMR (500
MHz, CDC13): 0.87 (d, J = 6.7 Hz, 3H), 0.94 (d, J = 6.7 Hz, 3H), 2.10-2.19 (m,

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1H), 2.47 (dd, J = 16.7 and 9.1 Hz), 2.58 (dd, J = 16.7 and 2.9 Hz, 1H), 3.11-
3.20 (m, 1H), 3.56-3.63 (m, 1H), 3.68 (s, 3H), 3.90-3.98 (m, 1H), 4.64 (d, J =
7.9 Hz, 1H), 5.09 (s, 2H), 7.30-7.39 (m, 5H).
[0154] (SS^iQ^-BenzyloxycarbonylanTino-S-methyl-S^triisopropyl-
silanyloxy)hexanoic Acid Methyl Ester (13): To a solution of the crude
alcohol 12 (2.7 g, 9.1 romol) in CH2C12 (10 mL) under argon, cooled to 0 °C,
2,6-lutidine (2.6 mL, 22.7mmol) and triisopropylsilyl triflate (3.6 mL, 13.6
mmol) were added. The reaction mixture was stirred at 0 °C for 30 min and
then at room temperature for 2h and diluted with CH2CI2 (25 mL). The organic
layer was separated and washed with 10% HC1 (50 mL), NaHCO3 (50 mL,
sat) and NaCl (50 mL, sat), dried (Na2SO4), filtered and concentrated. The
crude oil was purified by column chromatography (silica gel, diethyl
ether/hexanes 2:98 to 15:85) to yield 13 (2.9 g, 70%) as colorless oil. 'H-NMR
(500 MHz, CDCI3): 0.88 (d, J = 6.8 Hz, 3H), 0.92 (d, J = 6.7 Hz, 3H), 1.01-
1.07 (m, 21H), 1.97-2.04 (m, 1H), 2.53 (dd, J = 15.3 and 4.7 Hz, 1H), 2.60
(dd, J = 15.2 and 7.5 Hz, 1H), 3.57 (s, 3H), 3.59-3.65 (m, 1H), 4.34-4.40 (m,
1H), 4.78 (d, J = 10.3 Hz, 1H), 5.06-5.09 (m, 2H), 121-135 (m, 5H).
[0155] (3S,4i?)-4-Benzyloxycarbonylamino~5-methyl-3-(triisopropyI-
silanyloxy)hexanoic Acid (14): To a solution of 13 (2.2 g, 4.7 mmol) in 50 mL
of THF/MeOH (1:1), cooled to 0 °C, IN NaOH solution (55 mL, 32 mmol)
was added. The reaction was stirred at 0 °C for 2h and then overnight at rt. The
reaction mixture was concentrated and diluted with H2O (20 mL), cooled to 0
°C, acidified to pH 2 with IN KHSO4 solution and extracted with EtOAc (3x
20 mL). The combined organic layers were washed with NaCl (50 mL, sat),
dried (Na2SO4), filtered and evaporated. The resulting acid 14 was obtained as
a colorless oil (1.76g, 86%) and used directly in the next step. !H-NMR (500
MHz, CDCI3): 0.86 (mf 3H), 0.95 (d, J = 6.7 Hz, 3H), 1.04-1.14 (m, 21H),
1.90-2.00 (m, 1H), 2.52-2.72 (m, 2H), 3.60-3.62 (m, 1H), 4.28-4.36 (m, 1H),
5.02 (d, J = 10.4 Hz, 1H), 5.07-5.13 (m, 3H), 7.25-7.35 (m, 5H).
[0156] (2»S)-{l-(25,3i?)-[3-BenzyIoxycarbonylamino-2-(triisopropyl-
silaniloxy)hexyl]vinyloxy}-3-methylbutyric acid Allyl Ester (16): To a
solution of acid 14 (1.76 g, 3.89 mmol) in CH2C12 (20mL), cooled to 0 °C,

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a-hydroxyisovaleric acid ally] ester 15 (615 mg, 3.89 mmol), EDAC HC1 (820
mg, 4.20 mmol) and DMAP (94 mg, 0.77 mmol) were added sequentially. The
reaction mixture was stirred at 0 °C for lh and then overnight at room
temperature. The reaction was diluted with CH2CI2 and the organic layer was
washed with 10% HC1 (50 mL), NaHCO3 (50 mL, sat) and NaCl (50mL, sat),
dried (Na2SO4), filtered and concentrated. The crude oil was purified by
column chromatography (silica gel, EtOAc/hexanes 9:1) to yield the allyl ester
16 (1.2 g, 52%) as colorless oil. aH-NMR (500 MHz, CDC13): 0.88-0.99 (m,
12H), 1.00-1.10 (m, 21H), 1.96-2.05 (m, 1H), 2.17-2.25 (m, 1H), 2.65 (dd, J =
15.8 and 5.3 Hz, 1H), 2.71 (dd, J = 15.8 and 7.3 Hz, 1H), 3.62-3.72 (m, 1H),
4.40-4.45 (m, 1H), 4.57-4.63 (m, 2H), 4.79 (d, J = 4.8 Hz, 1H), 4.88 (d, J =
10.6 Hz, 1H), 5.07 (s, 2H), 5.20-5.23 (m, 1H), 5.28-5.32 (m, 1H), 5.83-5.91
(m,lB), 7.26-738 (m,5H).
[0157] (25)-{l~(2^3i?)-[3-BenzyIoxycarbonylamino-2-(triisopropyl-
silaniloxy)hexyI]vinyloxy}-3-methylbutyric Acid Pentafluorophenyl Ester
(18): To a solution of allyl ester 16 (1.2 g, 2.02 mmol) in dry THF (15 mL)
Pd(Pfi3)4 (231 mg, 0.20 mmol) and morpholine (1.7 mL, 20 mmol) were
added, at room temperature. The mixture was stirred overnight and the
reaction mixture was concentrated, diluted with CH2CI2 (20 mL) and washed
with IN HC1 (20 mL) and H2O (20 mL). The organic layer was dried
(Na2SO4) filtered and concentrated to afford acid 17. The residue was
dissolved in CH2CI2 (5 mL), cooled to 0 °C, followed by the sequential
addition of PEPOH (384 mg, 2.09 mmol), EDAC.HC1 (45 mg, 72.38 mmol)
and DMAP (49 mg, 0.39 mmol). The reaction mixture was stirred at 0 °C for
l/2h and then 4h at room temperature. The reaction was diluted with CH2CI2
and the organic layer was washed with 10% HCI (50 mL), 5% NaHCO3 (50
mL) and NaCl (50 mL, sat), dried (Na2SC>4), filtered and concentrated. The
crude oil was purified by column chromatography (silica gel, EtOAc/hexanes
9/1) to yield pentafluorophenyl ester 18 (0.71 g, 50% two steps overall) as a
colorless oil. !H-NMR (500 MHz, CDC13): 0.89 (d, J = 7.0 Hz, 3H), 0.92 (d, J
= 7.0 Hz, 3H), 1.02-1.06 (m, 21H), 1.06-1.11 (m, 6H), 2.00-2.08 (m, 1H),
2.31-2.40 (m, 1H), 2.60-268 (dd, J = 15.8 and 4.8 Hz, 1H), 2.77 (dd, J = 15.8

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and 7.9 Hz, 1H), 3.63-3.70 (m, 1H), 4.40-4.45 (m, 1H), 4.79 9d, J = 103Hz,
1H), 4.98-5.03 (m, 2H), 5.04-5.08 9m, 1H), 7.26-7.34 (m, 5H).
[0158] L-Cbz-JV-Leucyl-L-prolirie Methyl Ester (19): To a solution of L-Cbz-
leucine (902 mg, 3.40 mmol) and L-proKne methyl ester hydrochloride (563
mg, 3.40 mmol) in dry CH2C12 (10 mL), cooled to 0 °C, dicyclohexyl
carbodiimide (DCC) (912 mg, 4.42 mmol), 1-hydroxybenzotriazole (HOBt)
(551 mg, 4.08 mmol) and 7V-methylmorpholine (NMM) (2.6 mL, 13.6 mmol)
were added. After lh the reaction was warmed to room temperature and stirred
overnight. The mixture was diluted with ether (10 mL) and filtered. The
filtrate was diluted with EtOAc (25 mL) and washed with 10% HCI (100 mL),
NaHCO3 (100 mL, sat) and NaCl (lOOmL, sat) solutions. The organic layer
was dried (Na2SCU) and the solvent evaporated. The crude oil was purified by
column chromatography (silica gel, EtOAc/Hexanes 2/8) to yield L-Cbz-W-
leucyl-L-proline methyl ester (1.24 g, 98%) as white foam. ^-NMR (500
MHz, CDC13): 0.94 (d, J=6.6 Hz, 3H), 0.99 (d, J=6.5 Hz, 3H), 1.50-1.65 (d,
J=6.5 Hz, 2H), 1.734.79 (m, 1H), 1.94-2.22 (m, 4H), 3.57-3.78 (m, 2H), 3.70
(s, 3H), 4.50-4.55 (m, 2H), 5.03-5.09 (m, 2H), 5.33 (d, J= 8.8 Hz, 2H), 7.28-
7.34 (m, 15H).
[0159J L-Cbz-AT-Leucyl-L-proline (20): To a solution of dipeptide ester 19
(887 mg, 2.36 mmol) in THF (20 mL), cooled to 0 °C, a solution of LiOH (198
mg, 4.72 mmol) in water (20 mL) was added dropwise. The reaction was
warmed to room temperature and stirred overnight. The resulting solution was
concentrated under reduced pressure and extracted with ether. The aqueous
layer was acidified to pH 2 with 50% HCI and extracted with EtOAc (3x20
mL). The organic layer was dried (Na2SO4) and the solvent evaporated to
yield L-Cbz-iV-Leucyl-L-proline 20 (774 mg, 90%) as a white foam. This
compound showed identical :H-NMR as that reported in ref 24. !H-NMR
(500 MHz, CDCI3): 0.92 (d, J=6.5 Hz, 3H), 0.96 (d, J=6.5 Hz, 3H), 1.43-1.58
(m, 2H), 1.71-1.76 (m, 1H), 1.99-2.23 (m, 4H), 3.55-3.58 (m, 1H), 3.75-3.78
(m> 1H), 4.51-4.58 (m, 2H), 5.03-5.09 (m, 2H), 7.28-7.34 (m, 15H).
[0160] L-Cbz-Ar-O-Dimethyltyrosine (21): To a stirred solution of L-Cbz-Tyr
(5.0 g, 15.8 mmol) in THF (84 mL), at 0 °C, finely powered KOH (8.86g, 158

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mol) was added in portions, followed by the addition of tetrabutylammonium
hydrogen sulfate (0.5 g, 10% by weight). Then, dimethyl sulfate (9.74 mL,
103 mmol) was added dropwise over 15 min. The reaction was stirred for an
additional 30 min and H2O (50 mL) was added. After stirring 5h at room
temperature, 20% aqueous ammonium hydroxide solution was added (20 mL).
The reaction was diluted with ether (100 mL), the aqueous layer was separated
and the organic layer was extracted with saturated aq NaHCO3 (2x40 mL).
The combined aqueous layers were acidified to pH 1 with 1M KHSO4 and
extracted with EtOAc (2x200 mL). The organic layers were combined, dried
(Na2SO4), filtered and concentrated. The resulting acid 21 (4.3 g, 85%) was
obtained as a yellow oil and used in the next step without purification.
!H-NMR (500 MHz, CDC13): 2.78 and 2.84 (s, 3H), 2.92-3.06 (m, 1H), 3.21-
3.30 (m, 1H), 3.76 (s, 3H), 4.80-4.90 (m, 1H), 5.00-5.10 (m, 2H), 6.65-6.79
(m, 2H), 7.00-7.09 (m, 2H), 7.24-7.33 (m, 5H).
[0161] L-Cbz-W, O-Dimethyltyrosine-O-L-iV-Boc-threonine-SEM ester: To a
solution of L-Cbz-AT-O-dimethyltyrosine (21) (188 mg, 0.54mmol) in CH2C12
(2 mL), cooled to 0 °C, Boc-Thr-O-SEM (100 mg, 0.45mmo3) was added. To
the resulting solution Et3N (165 (xL, 1.18 mmol), DMAP (14 mg, 0.11 mmol)
and isopropenyl chloroformate (60 \ih, 0.54 mmol) were added. The reaction
was stirred at 0 °C for lh, diluted with Et2O (10 mL) and washed with 10%
HC1 (10 mL), 5% NaHCO3 (lOmL) and saturatedNaCl (10 mL) solutions. The
organic layer was dried (Na2SO4) and the solvent evaporated. The crude oil
was purified by column chromatography (silica gel, EtOAc/Hexanes 10:1) to
yield the dipeptide 22 (131 mg, 43%). !H-NMR (200 MHz, CDC13): 0.01 (m,
9H), 0.92-0.96 (m, 2H), 1.23-1.29 (m, 3H), L44 and 1.43 (s, 9H), 2.72 and
2,79 (s, 3H), 2,81-2.98 and 3.17-3.21 (m, 2H), 3.67-3.72 (m, 2H), 3.76 (s, 3H),
4.20-4.50 (m, 2H), 4.60-4.78 (m, 1H), 5.03-5.11 (m, 2H), 5.25-5.43 (m, 2H),
6.74-6.78 (m, 2H), 6.97-7.06 (m, 2H), 7.22-7.33 (m, 5H).
[0162] L-N, O-Dimethyltyrosine-O-L-Boc-threonine-OSEM (22): To a
solution of L-Cbz-//-methylphenylalanine-O-Boc-threonine-OSEM (231 mg.
0.34 mmol) in MeOH under argon, Pd(OH)2 (23 mg) was added. The reaction
was purged with H2 and stirred overnight under a hydrogen atmosphere

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(1 atm). The mixture was filtered through Celite®. The filtrate was
concentrated to yield compound 22 (174 mg, 94%) as a yellow oil. 'H-NMR
(500 MHz, CDC13): 0.0 (s, 9H), 0.91 (t, J= 8.2, 2H), 1.23 and 1,25 (d, J=s 5.6
Hz, 3H), 1.41 (s, 9H), 2.31 (s, 3H), 2,79 (s, 3H), 2.82 and 2.83 (ddf J= 14.3
and 6.4 Hz), 3.45 (t, J=6.1 Hz, 1H), 3.64-3.67 (m, 2H), 4.48 and 5.23 (d, J=9-7
Hz), 5.02 (d, J=5.2 Hz, 2H), 6.75-6.78 (m, 2H), 6.92-7.05 (m, 2H).
[0163] L-Cbz-Leucyl-L-prolyl-L-A^O-Dimethyltyrosine-O-L-Boc-threonine-
0-SEM (23): To a solution of L-Cbz-Leu~L-Pro-OH (98.4 mg, 0.27 mmol) in
CH2CI2 (2 mL), cooled to -15 °C, BOP-C1 (69 mg, 0.27 mmol) was added,
followed by the dropwise addition of NMM (30 |^L, 0.27 mmol). A separate
solution of L-A^O-dimethyltyrosine-O-L-Boc-threonine-O-SEM (147 mg,
0.27 mmol) in CH2C12 (2 mL) was cooled to -15 °C and added to the reaction
followed by the addition of 7V-methylmorpholine (NMM) (30 ^L, 0.54 mmol).
The mixture was stirred 15 min at -15 °C, lh at 0 °C and overnight at room
temperature. The reaction mixture was diluted with EtOAc (15 mL) and
washed with 10% HC1 (10 mL), NaHCO3 (10 mL, sat) and NaCl (10 mL, sat).
The organic layer was dried (Na2SC>4) and the solvent evaporated. The crude
oil was purified by column chromatography (silica gel, EtOAc/Hexanes 3/7)
to yield the tetrapeptide 23 (130 g, 55%) as pale yellow oil. *H-NMR (500
MHz, CDCI3): 0.00 (s, 9H), 0.67-0.87 (m, 2H), 0.89-1.00 (m, 6H), 1.19-1.21
(d, J= 6.8 Hz), 1.36 (d, J= 6.8 Hz, 3H), 1.46 (s, 9H), 1.54-1.99 (m, 7H), 2.72
(s), 2.73 (s, 3H), 2.91-3.00 (m, 1H), 3.05-3.15 (m), 3.50-3.55 (m, 1H), 3.66-
3.89 (m, 4H), 3.75 (s, 3H), 4.25-4.33 (m, 1H), 4.41-4.61 (m, 2H), 4.67-4.68
(m, 1H), 4.62-4.75 (m, 1H), 4.95-5.01 (dd, J = 8.5 and 3.2 Hz), 5.01-5.17 (m,
2H), 5.27-5.62 (m, 3H), 7.16-7.42 (m, 10H), 7.81 (m, 1H), 8.00 (m).
[0164] L-Leucyl-L-prolyl-L-A^6>-Dimethyltyrosine-L-A^-Boc-threonine-O-
SEM (24): To a solution of L-Cbz-leucyl-L-prolyl-L-Af-methylphenylalanine-
O-L-Boc-threonine-OSEM (665 mg, 0.74 mmol) in MeOH (10 mL) under
argon, Pd(OH)2 (119 mg) was added. The reaction was purged with H2 and
stirred overnight under H2 atmosphere (1 atm). The mixture was filtered
through Celite. The filtrate was concentrated to yield the free amino
tetrapeptide 24 (547 mg, 94%) as a colorless oil which was used in the next

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step without further purification. !H-NMR (500 MHz, CDC13): 0.001 (s, 9H),
0.64-0.86 (m, 2H), 0.88-0.96 (m, 6H), 1.19-1.21 (d, J= 6.3 Hz), 1.33-1.34 (d,
J= 6.3 Hz, 3H), 1.44 (s, 9H), 1.63-1.92 (m, 3H), 1.92-2.01 and 2.08-2.24 (m,
4H), 2.73 (s), 2.86-2.96 and 3.10-3.19 (m, 2H), 3.45-3.72 (m, 4H), 4.34-4.69
(m, 3H), 4.78-4.81 (dd, J= 8.0 and 3.3 Hz, 1H), 5.01-5.10 (m, 1H), 5.17-5.52
(m) and 5.58-7.60 (m, 3H) 6.77-6.83 (m, 2H), 7.00-7.10 (m, 2H)
[0165] Tamandarin B Protected Linear Precursor (25): To a solution of the
PFP ester 18 (285 mg, 0.39 mmol) in CH2C12 (1.5 mL), cooled to 0 °C? was
added D1EA (170 uL, 0.98 mmol) and the solution was stirred for 20 min,
followed by the addition of amine 24 (292 mg, 0.39 mmol) in CH2C12 (1.5
mL) and DMAP (9.5 mg, 0.078 mmol). The reaction mixture was stirred at 0
°C for lh and then at rt for an additional lh. The reaction was quenched with
NH4CI (3mL) and diluted with CH2C12 (10 mL). The organic layer was washed
with 10% HC1 (10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL, sat), dried
(Na2SC>4), filtered and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 9:1) to yield 25 (285 mg, 57%) as
a colorless oil. ]H-NMR (500 MHz, CDC13): 0.008.(s, 9H), 0.73-0.83 (m, 2H),
0.85-0.92 (m, 9H), 0.92-1.08 (m, 21H), 1.45 (s, 4H), 1.13-2.19 (m, 11H), 2.43-
2.46 (m, 1H) and 2,54-2.58 (m. 1H), 2.64 (s, 3H), 2.88 (s, 3H), 3.09-3.17 (m,
2H), 3.44-3.73 (m, 4H), 3.75 (s, 3H), 3.79-3.89 (m, 1H), 4.38-4.55 (m, 4H),
4.69-4.81 (m, 1H), 4.96-5.05 (m, 3H), 5.18-5.43 (m, 3H), 5.46-5.48 (d, J = 6.0
Hz, 1H), 6.83 (m, 2H), 6.95-7.11 (m, 2H), 7.25-7.38 (m, 5H), 7.55-7.77 (m)
and 8.85-8.87 (m,2H).
[0166] Tamandarin B Protected Macrocycle (26): To a solution of the fully
protected linear precursor 25 (350 mg, 0.27 mmol) dissolved in CH2C12 (5
mL), at 0 °C, MgBr2.Et20 (210 mg, 0.81 mmol) was added. The reaction was
stirred at 0 °C for 2h and overnight at rt. The reaction was diluted with CH2C12
and the organic layer was washed with 10% HC1 (10 mL) and NaCl (10 mL,
sat), dried (Na2SO4), filtered and concentrated. The resulting acid (319 mg)
was obtained as a white foam and used directly in the next step. To a solution
of crude acid (319 mg) in MeOH (5 mL) under argon, Pd(OH)2 (28 mg) was
added. The reaction was purged with H2 and stirred overnight under H2

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atmosphere (1 atm). The mixture was filtered through Celite. The filtrate was
concentrated to yield the linear precursor (296 mg) as a white foam which was
used directly in the next step. The crude amino acid linear precursor was
dissolved in DMF (27 mL) and cooled to 0 °C HATU (123 mg, 0.32 mmol)
was added followed by the dropwise addition of DEEA (141 \iL, 0.81 mmol).
The reaction mixture was stirred at 0 °C for 1 h and then overnight. The
reaction mixture was concentrated in vacuo, diluted with EtOAc (10 mL) and
washed 10% HCl (10 mL), 5% NaHCO3 (10 mL) and NaCI (10 mL, sat), dried
(Na2SO4), filtered and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 5/1) to yield protected
macrocycle 26 (50 mg, 19% three steps overall) as white foam. This
compound showed identical JH-NMR as that reported in ref 23. ]H-NMR (500
MHz, CDC13): 0.78-1.07 (m, 39H), 1.21-1.48 (m, 17H), 1.56-1.92 (m, 4H),
1.73-1.81 (m, IB), 1.95-2.12 (m, 2H), 2.43-2.44 (m, 1H) and 3.11-3.17 (m,
1H), 2.52 (s, 3H), 2.89-3.00 (m, 1H), 3.30-3.32 (m, 1H), 3.49-3.52 (m, 1H),
3.60-3.62 (m, 1H), 3.66-3.70 (m, 1H), 3.77 (s, 3H), 4.14-4.19 (m, 1H), 4.37-
4.43 (m, 1H), 4.46-4.48 (d, J= 7.6 Hz, 1H), 4.55-4.86 (m, 1H), 4.88-4.92 (m,
1H), 6.82-6.83 (d, J= 8.5 Hz, 2H), 7.06-7.07 (d, J= 8.5 Hz, 2H), 7.41-7.48 (m,
2H).
[0167] Tamandarin B (2): To a solution of Boc protected macrocycle 26 (10
mg, 0.01 mmol) in HPLC EtOAc was added a solution of HCl in EtOAc. The
resulting solution was stirred at room temperature for 2h. The solution was
concentrated and the residue diluted with CH2CI2 and concentrated again to
yield the hydrochloride salt (quantitative yield) as a white solid, which was
used directly in the next step. To a mixture of the macrocycle amine salt (9
mg, 0.01 mmol) and side chain (6.1 mg, 0.015 mmol) in CH2C12 (0.50 mL) at
0 °C was added BOP (8.4 mg, 0.015 mmol) and NMM (6 uL, 0.05 mmol).
After 30 min at 0 °C, the reaction was stirred at rt overnight The reaction was
treated with NaCI solution (2 mL, sat) and extracted with EtOAc (2x10 mL)
The organic layers were washed with 10% HCl (5 mL), 5% NaHCO3 (5 mL)
and NaCI (5 mL, sat), dried (Na2SO4), filtered and concentrated. The crude oil
(8 mg, 61%) was purified by HPLC .

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EXAMPLE2
Synthesis of Phe5 Tamandarin B
[0168] A phenylalanine analog of tamandarin B (Phe5 Tamandarin B) was
synthesized according to the method shown in Schemes 9 and 10.
Scheme 10



[0169] L-Cbz-iV-Methylphenylalanine (28): To a solution stirred solution of
L-Cbz-Phe (5.1 g, 17.8 nunol) in THF (90 mL), at 0 °C, finely powered KOH
(6.7 g, 0.12 mol) was added in portions, followed by the addition of
tetrabutylammonium hydrogen sulfate (0.51 g, 10% by weight). Then,
dimethyl sulfate (6.4 mL, 71.2 mmol) was added dropwise over 15 min. The
reaction was stirred for an additional 30 min and H2O (50 mL) was added.
After stirring 5h at room temperature, 20% aqueous ammonium hydroxide
solution was added (20 mL). The reaction was diluted with ether (100 mL),
the aqueous layer was separated and the organic layer was extracted with
saturated aq NaHCC>3 (2x40 mL). The combined aqueous layers were acidified
to pH 1 with 1M KHSO4 and extracted with EtOAc (2x200 mL). The organic
layers were combined, dried (Na2SO4), filtered and concentrated. The resulting

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acid 28 (4.5 g, 85%) was obtained as a yellow oil and used in the next step
without purification. 'H-NMR (500 MHz, CDCI3): 2.71 and 2.78 (s, 3H), 2.94-
3.06 (m, 1H), 3.17-3.32 (m, 1H), 4.79-4.89 (m, 2H), 4.95 and 5.03 (s, 2H),
7.04-7.28 (m, 10H).
[0170] L-Cbz-TV-Methylphenylalanine-O-L-Boc-threonine-OSEM (29): To a
solution of Cbz-iV-methylphenylalanine (2g, 6.56 mmol) in CH2CI2 (40 mL),
cooled to 0°C, Boc-Thr-OSEM (2.08 g, 5.96 mmol) was added. To the
resulting solution Et3N (1.9 mL, 13 mmol), DMAP (146 mg, 1.19 mmol) and
isopropenyl chloroformate (0.71 mL, 6.56 mmol) were added. The reaction
was stirred at 0° C for lh, diluted with Et2O (150 mL) and washed with 10%
HC1 (50 mL), 5% NaHCO3 (50mL) and saturated NaCl (50 mL) solutions. The
organic layer was dried (Na2SC>4) and the solvent evaporated. The crude oil
was purified by column chromatography (silica gel, EtOAc/Hexanes 10:1) to
yield the dipeptide 29 (1.79 g, 60%). *H-NMR (200 MHz, CDCI3): 0.0 (s,
9H), 0.96 (m, 2H), 1.25 and 1.30 (d, J=1.6 Hz, 3H), 1.46 (s, 9H), 2.75 and
2.83 (s, 3H), 2.92-3.06 (m, 1H), 3.20-3.40 (m, 1H), 3.75-3.85 (m, 2H), 4.44
(m, 2H), 4.83-4.90 (m, 1H), 5.01-5.15 (m, 3H),5.25-5.40 (m, 2H), 5.46 (m,
1H), 7.09-7.35 (m,10H).
[0171] L-iV-Methylphenylalanine-O-L-Boc-threonine-OSEM (30): To a
solution of L-Cbz-iV-methylphenylalanine-O-Boc~threonine-OSEM (1.7 g. 2.6
mmol) in MeOH under argon, Pd(OH)2 (170 mg) was added. The reaction was
purged with H2 and stirred overnight under a hydrogen atmosphere (1 atm).
The mixture was filtered through Celite. The filtrate was concentrated to yield
the compound 30 (1.32 g, 98%) as yellow oil. 'H-NMR (500 MHz, CDCI3):
0.0 (s, 9H), 0.93 (t, J= 8.2, 2H), 1.04 and 1,28 (d, J= 5.6 Hz, 3H), 1.44 (s, 9H),
2.29 (s, 3H), 2.83 and 2.93 (dd, J= 14.3 and 6.4 Hz), 3.40 (t, 3=6.1 Hz, 1H),
3.66-3.68 (m, 2H), 4.48 and 5.23 (d, J=9.7 Hz), 5.02(d, J=5.2 Hz, 2H), 7.08-
7.28 (m, 5H).
[0172] L-Cbz-Leucyl-L-prolyl-L-A^-methylphenylalanine-O-L-Boc-threonine-
O-SBM (31): To a solution of L-Cbz-Leu-L-Pro-OH (1.0 g, 2.84 mmol) in
CH2CI2 (25 mL), cooled to -15 °C, BOP-C1 (0.72 g, 2.84 mmol) was added,
followed by the dropwise addition of NMM (318 \xL, 2.84 mmol). A separate

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solution of 30 (1.32g, 2,58 mmol) in CH2C12 (25 mL) was cooled to -15 °C
and added to the reaction followed by the addition of JV-methylmorpholine
(NMM) (0.58 mL, 5.16 mmol). The mixture was stirred 15 min at -15 °C, lh
at 0 °C and overnight at room temperature. The reaction mixture was diluted
with EtOAc and washed with 10% HC1 (100 mL), NaHCO3 (100 mL, sat) and
NaCl (lOOmL, sat). The organic layer was dried (Na2SC>4) and the solvent
evaporated. The crude oil was purified by column chromatography (silica gel,
EtOAc/Hexanes 3/7) to yield the tetrapeptide 31 (1.02 g, 55%) as pale yellow
oil. Rf 0.19 (AcOEt/Hexanes 3:7). [afc20 = -38.5 (c=l, CH2C12). 'H-NMR
(500 MHz, CDC13): 0.0 (s, 9H), 0.73-0.77 (m, 2H), 0.92-1.00 (m, 6H), 1.21 (d,
J= 6.7 Hz), 1.36 (d, J= 6.7 Hz, 3H), 1.46 (s, 9H), 1.54-1.99 (m, 7H), 2.72 (s),
2.78 (s, 3H), 2.93-3.01 (m, 1H), 3.05-3.15 (m), 3.50-3.55 (m, 1H), 3.66-3.89
(m, 4H), 4.29-4.35 (m, 1H), 4.41-4.61 (m, 2H), 4.63-4.65 (m, 1H), 4.72-4.74
(dd, J= 8.1 and 3.1 Hz, 1H), 4.95-5,01 (dd, J = 8.1 and 3.1 Hz), 5.05-5.17 (m,
2H), 5.27-5.62 (m, 3H), 7.16-7.42 (m, 10H), 7.71 (d, J = 10 Hz, 1H), 8.01 (d, J
= 9.2 Hz). I3C-NMR (125 MHz, CDC13): -1.46, 16.2, 16.6, 16.9, 17.9, 21.4,
23.4, 24.5, 24.7, 25.1, 28.2, 29.2, 34.5, 39.5, 40.7, 46.8, 51.1, 53.3, 55.1, 57.6,
58.8, 62.1, 65.8, 62.7, 67.9, 68.2, 71.2, 72.5, 80.1, 89.8, 90.2, 126.6, 127.3,
127.9, 128.4, 128.8, 129.4, 156.0, 169.1, 172.7, 173.8. IR (neat) 3278, 2954,
1703,1640,1521,1435,1365. HRMS m/z cald for C44H66N4O11SiNa (M+Na):
877.4395 found 877.4404.
[0173] L-Leucyl-L-prolyl-L-A'-methylpheny]alamne-(9-L-Boc-threonine-
0SEM (32): To a solution of 31 (223 mg, 0.26 mmol) in MeOH under argon,
Pd(OH)2 (50 mg) was added. The reaction was purged with H2 and stirred
overnight under H2 atmosphere (1 atm). The mixture was filtered through
Celite. The filtrate was concentrated to yield the free amino tetrapeptide 32
(192 mg, 97%) as yellow oil, which was used in the next step without
purification. ]H-NMR (500 MHz, CDC13): 0.0 (s, 9H), 0.72-0.76 (m, 2H),
0.90-0.99 (m, 6H), 1.22 (d, J= 6.7 Hz), 1.35 (d, J= 6.7 Hz, 3H), 1.45 (s, 9H),
1.47-1.53 9m, 2H), 1.81-2.01 (m, 5H), 2.71 (s), 2.79 (s, 3H), 2.92-3.00 (m,
1H), 3.05-3.15 (m), 3.51-3.56 (m, 1H), 3.67-3.90 (m, 4H), 4.29-4.35 (m, 1H),
4.42-4.63 (m, 2H), 4.63-4.65 (m, 1H), 4.72-4.74 (dd, J= 8.0 and 3.2 Hz, 1H),

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5.01 (d, J = 6.0 Hz, 1H); 5.21-5.59 (m, 3H), 7.05-7.29 (m, 5H), 7.53 (d, J = 10
Hz),7.68(d,J = 9.1Hz).
[0174] [iV-MePhe5]-Tarnandarin B Protected Linear Precursor (33): To a
solution of the PFP ester 18 (191 mg, 0.26 mmol) in CH2CI2 (5 mL), cooled to
0 °C was added DEBA (116 uX, 0.65 mmol) and the solution was stirred for 20
min, followed by the addition of amine 32 (192 mg, 0,27 mmol) in CH2C12
(5 mL) and DMAP (0.6 mg, 0.006 mmol). The reaction mixture was stirred at
0°C for lh and then at rt for an additional In. The reaction was quenched with
NH4CI and diluted with CH2CI2. The organic layer was washed with 10% HC1
(100 mL), 5% NaHCO3 (100 mL) and NaCl (lOOmL, sat), dried (Na2SO4),
filtered and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 9:1) to yield 33 (153 g, 44%) as
colorless oil. Rf 0.20 (AcOEt/Hexanes 3/1). [a]D2° = -44.0 (c = 1, CHCfe).
'H-NMR (500 MHz, CDC13): 0.0 (s, 9H), 0.87-0.89 (m, 2H), 0.90-0.98 (m,
18H), 1.02-1.10 (m, 21H), 1.43 (s, 4H), 1.11-2.10 (m, 9H), 2.49-2.51 (m, 3H),
2.68 (s, 3H), 2.89 (s, 3H), 3.09-3.17 (m, 2H), 3.65-3.67 (m, 1H), 3.68-3.72 (m,
5H), 4.31-4.45 (m, 1H), 4.20-4.33 (m, 3H), 4.69-4.79 (m, 2H), 4.96-5.05 (m,
3H), 5.18-5.43 (m, 3H), 5.47 (d, J = 6.0 Hz, 1H), 6.83 (d, J = 7.9 Hz, IK),
7.11-7.41 (m, 11H), 7.82 (d, J = 6.5 Hz, 1H), 8.88 (d, J = 8.5 Hz, 1H). I3C-
NMR (125 MHz, CDCh): 13.1, 17.3, 18.1, 18.5, 19.3, 20.9, 21.6, 23.9, 25.4,
28.6, 29.6, 30.5, 34.8, 35.0, 39.9, 47.3, 49.4, 55.3, 57.3, 59.3, 60.7, 62.6, 66.7,
68.6, 79.1, 80.6, 90.6, 127,1, 128.2, 129.3, 137.4, 156.8, 157.1, 169.5, 170.4,
171.0, 172.1, 174.2. IR (neat) 3446, 2956, 2868, 1738, 1703, 1636 1455.
HRMS m/z cald for C65Hio7N5Oi5Si2Na (M+Na): 1276.7199 found 1276.7273.
[0175] [Ar-MePhe5]-Tamandarin B Protected Macrocycle (34): To a solution
of the fully protected linear precursor 33 (140 mg, 0.11 mmol) dissolved in
CH2CI2 (5 mL), at 0°C, MgBr2.Et20 (85 mg, 0.33 mmol) was added. The
reaction was stirred at 0°C for 2h and overnight at rt. The reaction was diluted
with CH2C12 and the organic layer was washed with 10% HC1 (100 mL) and
NaCl (lOOmL, sat), dried (Na2SC>4), filtered and concentrated. The resulting
acid (130 mg) was obtained as a white foam and use directly in the next step.
To a solution of crude acid (130 mg, 0.30 mmol) in MeOH (5 mL) under

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argon, Pd(OH)2 (28 mg) was added. The reaction was purged with H2 and
stirred overnight under H2 atmosphere (1 atm). The mixture was filtered
through Celite. The filtrate was concentrated to yield the linear precursor
(87 mg, 78%) as a white foam which was used directly in the next step. The
crude amino acid linear precursor (87 mg, 0.08 mmol) was dissolved in DMF
(9 rnL) and cooled to 0 °C. HATU (40 mg, 0.10 mmol) was added followed by
the dropwise addition of DIEA (46 piL, 0.26 mmol). The reaction mixture was
stirred at 0 °C for lh and then overnight. The reaction mixture was
concentrated in vacuo, diluted with EtOAc (10 mL) and washed with 10%
HC1 (10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL, sat), dried (Na2SO4),
filtered and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 5/1) to yield protected
macrocycle 34 (10 mg, 15% three steps overall) as white foam. Rf 0.40
(AcOEt/Hexanes 3/7). [a]D20 = -36.1 (c = 1, CHC13). !H-NMR (500 MHz,
CDCI3): 0.79-1.09 (m, 18H), 1.09-1.10 (s, 21H), 1.27 (d, J = 6.4 Hz, 3H), 1.44
(s, 9H), 1.56-1.70 (m, 3H), L73-1.81 (m, 1H), 1.99-2.23 (m, 4H), 2.27 (dd, J =
17.0 and 2.9 Hz, 1H), 2.42 (dd, J = 17.0 and 3.0 Hz, 1H), 2.50 (s, 3H), 2.61
(dd, J = 17.1 and 6.5 Hz, 1H), 2.98-3.00 (m, 1H), 3.19-3.23 (m, 1H), 3.30-3.32
(m, 1H), 3.42 (dd, J = 9.8 and 4.9 Hz, 1H), 3.57 (dd, J = 10 and 4.4 Hz, 1H),
3.60-3.62 (m, 1H), 3.63-3.64 (m, 1H), 4.03-4.21 (m, 1H), 4.32-4.36 (m, 1H),
4.37-4.45 (m, 1H), 4.57-4.60 (m, 1H), 4.60-4.69 (m, 1H), 4.88-4.92 (m, 1H),
4.94 (d, J = 6.0 Hz, 1H), 5.80 (m, 1H), 7.12-7.17 (m, 2H), 7.28-7.31 (m, 3H),
7.48-7.51 (m, 2H). i3C-NMR (125 MHz, CDC13): 12.2,12.6,15.1, 16.5, 17.5,
18.0, 18.2, 18.8,19.6, 20.7, 21.0, 23.5, 24.9, 27.9, 28.1, 30.1, 31.0, 35.1, 38.5,
39.7, 40.9, 47.7, 48.1, 55.5, 56.9, 593, 65.9, 68.8, 69.9, 71.8, 80.1, 81.2,
126.9, 128.7, 129.5, 138.1, 155.9, 168.7, 170.9, 171.0, 172.2. IR (neat) 3331,
2926,1743,1636,1513,1456.
[0176] [#-MePhe5]-Tamandarin B (36): To a solution of Boc protected
macrocycle 34 (10 mg, 0.01 mmol) in HPLC EtOAc was added a solution of
HC1 in EtOAc. The resulting solution was stirred at room temperature for 2h.
The solution was concentrated, and the residue diluted with CH2CI2 and
concentrated again to yield the hydrochloride salt 35 (9 mg, quantitative yield)

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as a white solid, which was used directly in the next step. To a mixture of the
macrocycle amine salt (9 mg, 0.01 mmol) and side chain (6.1 mg, 0.015
mmol) in CH2C12 (0.50 mL) at 0 °C was added BOP (8.4 mg, 0.015 mmol) and
NMM (6 \iL, 0.05 mmol). After 30 min at 0°C, the reaction was stirred at rt
overnight The reaction was treated with NaCl solution (2 mL, sat) and
extracted with EtOAc (2x10 mL) The organic layers were washed with 10%
HC1 (5 mL), 5% NaHCO3 (5 mL) and NaCl (5 mL, sat), dried (Na2SO4),
filtered and concentrated. The crude oil (8 mg, 61%) was purified by HPLC.
EXAMPLE 3
Attempted Synthesis of Leu5 Tamandarin B
[0177] The attempted synthesis of a Ieucine analog of tamandarin B (Leu5
Tamandarin B) is shown in schemes shown below.



[0178] The tetrapeptide unit consisting of Leu3, Pro4, Leu5, and Thr6
(compound 41) was prepared from L-leucine using the same known synthetic
sequence starting from Cbz-L-Leu (37) instead of Cbz-L-Phe or Cbz-L-Tyr
(Scheme 9).
[0179] Compound 41 was deprotected using hydrogen and palladium
hydroxide to yield amine 42. Compound 42 was reacted with compound 18
(used as a mixture of isomers) under conditions similar to those used above,
e.g., for preparing compond 33. This reaction produced compound 43.
Cyclization of compound 43 was attempted under conditions shown above,
e.g., for preparing compound 34. However, the cyclization reaction under
these conditions was not successful.
EXAMPLE 4
Synthesis of Ser6 Tamandarin B
[0180] A serine analog of tamandarin B (Ser6 Tamandarin B) was synthesized
as shown in Schemes 12-16.






[0181] D-AT-Boc-Valine pentafluophenyl (PFP) Ester (44): D-iV-Boc-Valine (5
g, 23 mmol) was dissolved in CH2C12 (60 mL) and cooled to 0°C, followed by
the addition of pyridine (1.7 mL, 25.3 mmol,) and PHP-trifluoroacetate (4.08
mL, 27.6 mmol). The solution was stirred lh at room temperature and
quenched with NH4CI (50 mL, sat). The organic layer was washed with 5%
HC1 (100 mL), NaHCO3 (100 mL, sat), dried (Na2SO4), filtered and the
solvent evaporated. The resulting PFP ester 44 was obtained as a colorless oil
(8.6 g, 98%) and used directly in the next step. JH-NMR (500 MHz, CDCI3):
1,03 (d, J=6.7 Hz, 3H), 1,09 (d, J=6.7 Hz, 3H), 1.47 (s, 9H), 2,30-2.38 (m,
1H), 4.57-4.58 (m, 1H), 5.01-5.02 (m, 1H).
[0182] (4y?)~4-te??-Butoxycarbonylanuno-5-methyl-3-oxohexanoic Acid
Methyl Ester (45): To a solution of PFP ester 44 (7 g, 18.3 mmol) in
anhydrous THF (30 mL), cooled to -78°C, a solution of lithium enolate of
methyl acetate was added. The enolate was prepared by addition of methyl
acetate (5.4 mL, 65.0 mmol) to a solution of LDA (65.0 mmol in 50 mL of
anhydrous THF) at -78°C and the resulting solution was stirred for lh. The
reaction mixture was stirred for 45 min more at the same temperature, and
quenched with NH4CI (50 mL) at -78°C. After warming to room temperature,
the solution was diluted with EtOAc (150 mL). The organic layer was
separated and washed with 10% HC1 (100 mL), 5% NaHCO3 (100 mL) and
NaCl (100 mL, sat), dried (Na2SC>4), filtered and concentrated. The crude oil
was purified by column chromatography (silica gel, EtOAc/Hexanes 1/9) to

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yield the methyl ester 45 (3.0 g, 61%) as colorless oil. Rf 0.14
(EtOAc/Hexanes 1/9). [a]D20 = -13.2 (c=0.5, CHC33). !H-NMR (500 MHz,
CDC13): 0.81 (d, J= 6.8 Hz) and 0.83-0.92 (m, 2H), 0.99 (d, J= 6.8 Hz, 2H),
1.44 (s, 9H), 2.20-2.25 (m, 1H), 3.53 (m, 2H), 3.71 (s, 3H). 4.29 (m, 1H), 5.09
(m, 1H). 13C-NMR (125 MHz, CDC13): 16.6,19.7, 28.2, 29.5,46.8, 52.3, 64.3,
80.08, 155.9, 167.1, 202.0. IR (neat) 3442.3, 2966.2, 1750.0, 1715.9, 1515.0,
1508.3, 1366.9, 1165.6, 1014.0. HRMS m/z cald for Ci3H24NO5 (M+H):
274.1654 found 274.1654.
[0183] (35r,4i?)-4-/e7t-Butoxycarbonylamino-3-hydroxy-5-methylhexanoic
Acid Methyl Ester (46): To a solution of methyl ester 45 (2.4 g, 8.70 mmol) in
HPLC MeOH (100 mL), cooled to -78°C, potassium borohydride (1.4 g,
26.1 mmol) was added in portions. The reaction mixture was stirred at -78°C
for 10 min, wanned to -20°C for 30 min and then to 0°C for 10 min. The
reaction was quenched with acetic acid to pH = 7 and extracted with CH2CI2
(3 x 50 mL). The organic layer was washed with NaHCO3 (50 mL, sat), NaCI
(50 mL, sat), dried (Na2SO4), filtered, and evaporated. The alcohol 46 was
obtained as a colorless oil (2.2 g, 92%) and used directly in the next step.
'H-NMR (500 MHz, CDCI3): 0.88 (d, J=6.8 Hz, 3H), 0.93 (d, J=6.8 Hz, 3H),
1.44 (s, 9H), 2.03-2.17 (m, 1H), 2.41-2.52 (m, 1H), 2.58-2.63 (m,lH),
3.50-3.55 (m, 1H), 3.71 (s, 3H), 3.89-3.93 (m, 1H), 3.38-3.41(m, 1H).
[0184] (35,4i?)-4-?err-Butoxycarbonylamino-5-methyl-3-(^rt-butyldimethyl-
silanyloxy)hexanoic Acid Methyl Ester (47): To a solution of the crude
alcohol 46 (2.2 g, 7.9 mmol) in DMF (12 mL), under argon, cooled to 0 °C,
imidazole (1.61 g, 23.7mmol) and te??-butyldimethylsilyl chloride (3.57 mL,
23.7 mmol) were added. The reaction mixture was stirred at 0°C for 30 rnin
and then at room temperature for 18h and diluted with Et2O (50 mL). The
organic layer was separated and washed with 10% HC1 (50 mL), NaHCO3 (50
mL, sat) and NaCI (50 mL, sat), dried (Na2SO4), filtered, and concentrated.
The crude oil was purified by column chromatography (silica gel,
EtOAc/Hexanes 1/9) to yield 47 (2.59 g, 98%) as colorless oil. Rf 0.31
(EtOAc/Hexanes 1/9). [afo20 = +5.30 (c=l, CH2C12). *H-NMR (500 MHz,
CDCI3): 0.07 (s, 6H), 0.84-0.91 (m, 15H), 1.42 (s, 9H), 1.98-2.01 (m, 1H),

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2.43 (dd, J=6.0 and 19Hz, 1H), 2.52 (dd, 1=5.9 and 18.5Hz, 1H), 3.47-3.48 (m,
1H), 3.65 (s, 3H), 4,17-4.19 (m, 1H), 4,41-4.44 (m, 1H). I3C-NMR (125
MHz, CDCl3):-3.6, 16.6, 17.60, 18.9, 25.7, 27.8, 28.2, 39.9, 51.5, 51.9, 59.5,
70.19,155.9,172.2. IR (neat) 3373.1, 2960.7, 1717.0, 1504.6,1366.1, 1173.0,
837.0. HRMS m/z cald for Ci9H39NO5Si (M+Na): 390.2675 found 390.2676.
[0185] (3,S,4i2)-4-fert-Butoxycarbonylamino-5-methyl-3-(^rt-butyldimethyl-
silanyloxy)hexanoic Acid (48): To a solution of 47 (500 nig, 1.28 mmol) in 25
mL of THF/MeOH (1:1), cooled to 0°C, IN NaOH solution (25 mL) was
added. The reaction was stirred at 0°C for 2h and then overnight at rt. The
reaction mixture was concentrated and diluted with H2O (20 mL), cooled to
0°C, acidified to pH 2 with IN KHSO4 solution, and extracted with EtOAc (3x
20 mL). The combined organic layers were washed with NaCl (50 mL, sat),
dried (Na2SC>4), filtered and evaporated. The resulting acid 48 was obtained as
a colorless oil (1.76g, 86%) and used directly in the next step. ]H-NMR (500
MHz, CDCI3): 0.06 (s, 6H), 0.84-0.95 (m, 15H), 1.43 (s, 9H), 2,01-2.11 (m,
1H), 2.53 (m, 1H), 2.64 (m, 1H), 3,53-3,58 (m, 1H), 4.10-4.14 (m, 1H), 3.65
(s, 3H), 4,17-4.19 (m, 1H), 4,51-4.54 and 6.29-6.31 (m, 1H).
[0186] iV-a~Hydroxyvaleryl-iV-Leucyl-Proline Acid Benzyl Ester (51): To a
solution of hydrochloride salt 49 (1.56 mg, 4.23 mmol) in CH2CI2 (2 mL),
cooled to 0°C, NMM (522 \XL, 4.65 mmol) was added. After 15 min
a-hydroxyvaleric acid (50) (500 mg, 4.23 mmol) and DCC (959 mg, 4.65
mmol) were added in portions. The reaction mixture was stirred 14h at room
temperature diluted with CH2C12 (25 mL) and washed with HC1 IN (20 mL),
NaHCO3 (20 mL, sat) and brine (20 mL), dried (Na2SO4), filtered and
concentrated. The crude oil was purified by column chromatography (silica
gel, grad EtOAc/Hexanes 1/1 to 3/1) to yield 51 (1.47 g, 84%) as a white
solid. Rf 0.46 (EtOAc/Hexanes 1/2). !H-NMR (500 MHz, CDC13): 0.81 (d,
J=7.0 Hz, 3H), 0.92 (m, 6H), 0.97 (d, J=7.0 Hz, 3H), 1.42 (m, 1H), 1.63 (m,
2H), 2.00 (m, 3H), 2.19 (m, 2H), 3.60 (m, 1H), 3.85 (m, 1H), 3.88 (d, J=4.8
Hz, 1H), 4.66 (m, 1H), 4.80 (m, 1H), 5.06 (d, J=12.3 Hz, 1H), 5.14 (d, J=12.3
Hz, 3H), 7.32 (m, 5H), 7.41 (d, 8.4, 1H). 13C-NMR (125 MHz, CDCI3): 15.3,

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19.1, 21.3, 23.1, 24.4, 24.7, 28.8, 31.3, 40.5, 46.8, 48.2, 58.2, 66.7, 75.8,
127.9,128.1,128.3,135.3,117.4,117.9,173.9.
[0187] (35,4i?)-4-tert-Butoxycarbonylamino-5-methyI-3-(fert-butyldiniethyl"
silany3oxy)hexanoic Acid ^a-Hydroxyvaleryl-JV-Leucyl-O-Benzyl-Prolyl
Ester (52): To a solution of alcohol 51 (400 mg, 0.96 mmol) in CH2C12
(8 mL), cooled to -5 °C, DMAP (35 mg, 0.28 mmol) acid 48 (425 mg,
1.15 mmol) and DCC (247 mg, 1.15 mmol) were added in portions. The
reaction mixture was stirred 7 h at -5°C, filtered, and evaporated. The residue
was dissolved in CH3CN (10 mL), filtered again, and evaporated. The crude
oil was dissolved in EtOAc (20 mL) and washed with 10% KHSO4 (15 mL),
NaHCO3 (15 mL, sat) and NaCl (15 mL, sat). The organic layer was dried
(Na2SO4) and the solvent evaporated. The crude oil was purified by column
chromatography (silica gel, grad EtOAc/Hexanes 1/4) to yield 52 (110 mg,
60%). Rf 0.47 (AcOEt/Hexanes 1:1). [cc]D20 = - 69.0 (c=l, CH2C12).
!H-NMR (500 MHz, CDC13): rotamers 0.07 (s, 6H), 0.75-1.10 (m, 27H), 1.49
(s, 9H), 1.50-1.71 (m, 2H), 1.75-2.20 (m, 3H), 2.12-2.21 (m, 2H), 2.41-2.63
(m, 2H), 3.12-3.15 (m, 1H), 3.41-3.49 (m, 2H), 3.53-3.62 (m, 2H), 3.68-3.72
(m, 1H), 3.81-3.88 (m, 1H), 4.05-4.10 (m, 1H), 4.21 (d, J "8.0 Hz, 1H), 4.49-
4.51 (m, 1H), 4.60-4.69 (m, 2H), 4.77 (d, J =10.0 Hz, 1H), 4.90-5.01 (m, 2H),
5.11-5.22 (m, 2H), 6.88 (d, J = 9.0 Hz, 1H), 7.22-7.33 (m, 5H), 8.33 (d, J =
6.3 Hz, 1H). 13C~NMR (125 MHz, CDCI3): -5.1, -4.8, 14.7, 17.8, 18.0, 18.9,
20.4, 21.2, 23.2, 24.6, 24.7, 24.9, 25.4, 25.7, 28.4, 28.8, 30.2, 33.9, 38.9, 43.0,
48.4, 58.8, 63.2, 66.8, 71.5, 79.3, 82.6, 128.1, 128.3, 128.5, 135.5, 157.4,
170.0, 170.9, 171.2, 171.8, 172.1. IR (neat) 3265.8, 2937.7, 2903.6, 2362.0,
1740.4,1692.3,1643.4,1451.6,1386.8,1365.4,1167.2,1091.1, 837.3. HRMS
ni/z cald for C^EbNsOgSiNa (M+Na): 796.4700 found 798.4735.
[0188] L-A^-Boc-Serine phenacyl ester (55): To a stirred suspension of
L-Af-Boc-serine (2 g, 9.7 mmol) in EtOAc (20 mL), cooled to 0 °C, Et3N
(1.39 mL, 9.7 mmol) and bromoacetophenone (1.9 g, 9.7 mmol) were added.
The resulting mixture was stirred at room temperature for 2 days, diluted with
EtOAc (50 mL) and washed with 10% HC1 (20 mL), 5% NaHCO3 (20mL) and
saturated NaCl (20 mL) solutions. The organic layer was dried (Na2S(>4) and

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the solvent evaporated. The residue was triturated with ether and filtered to
afford 55 (3.4 g, 98%) as a yellow foam. Rf 0.50 (AcOEt/Hexanes 1/1). [a]D20
= -19.1 (c = 1, CHC13). 2H-NMR (500 MHz, CDCI3): 1,43 and 1,46 (s, 3H),
3.70-373 (m, 1H), 3.84-3.88 (m, 1H), 4.22 -4.24 (m, 1H), 4.51-4.52 (m, 1H),
5.28-5.36 (m, 1H), 5.61-5.66 (m, 2H), 7.49 (t, J= 6.5 Hz, 2H), 7.57 (t, J=
6.3 Hz, 1H), 7.87 (dd, J= 1.9 and 6.3 Hz, 2H). I3C-NMR (125 MHz, CDCI3):
27.2, 28.1, 56.0, 63.7, 66.3, 79.8, 85.0, 127.6, 128.8, 133.3, 134.3, 146.6,
155.5,170.1, 192.8. JR (neat) 3439.8, 2978.7, 2935.6, 1807.8,1756.6,1704.8,
1506.6,1369.7,1162.9.
[0189] O-(LrN-Cbz-Nt 0-Dimemyltyrosyl)~L-JV-Boc-serine Phenacyl Ester
(56): To a solution of L-iV-Boc-serine phenacyl ester 55 (1.42 g. 3.96 mmol) in
CH2CI2 (20 mL), cooled to 0 °C, DMAP (145 mg, 1.18 mmol) and L-tf-Cbz-
N, 0-dimethyltyrosine (21) (1.36 g, 3.96 mmol) were added. After stirring 10
min at 0°C, DCC (897 mg, 4.35 mmol) was added. The reaction mixture was
stirred overnight at room temperature. The mixture was filtered and the filtrate
concentrated to dryness. The residue was dissolved in CH3CN (25 mL),
filtered again and evaporated. The crude oil was dissolved in EtOAc (50 mL)
and washed with 10% KHSO4 (30 mL), NaHCO3 (50 mL, sat) and NaCl
(50 mL, sat). The organic layer was dried (Na2SO4) and the solvent
evaporated. The crude oil was purified by column chromatography (silica gel,
grad EtOAc/Hexanes 1/4 to 1/2) to yield the dipeptide 56 (1.69 g, 63%) as a
pale white solid. Rf 0.12 (AcOEt/Hexanes 1:4). mp = 64-65°C. [afo20 = -12.0
(c=l, CH2CI2). ]H-NMR (500 MHz, CDC13):1.49 (s, 3H), 2.85 and 3.01 (s,
3H), 3.04-3.11 (m, 1H), 3.27-3.37 (m, 1H), 3.80 (s, 3H), 4.53-4.67 (m, 1H),
4.70 (dd, J= 3-9 and 11.7Hz, 1H), 4.81-4.82 (m, 1H), 4.94-5.01 (m, 1H), 5.08-
5.14 (m, 2H), 5.30-5.35 (m, 1H), 5.48-5.52 (m, 1H), 6.76-6.83 (m, 2H), 7.05-
7.24 (m, 2H), 7.27-7.34 (m, 5H), 7.51 (t, J=7.3 Hz, 2H), 7.64 (t, J=7.2 Hz,
1H), 7.89 (d, J=7.5 Hz, 2H). I3C-NMR (125 MHz, CDCI3): 28.3, 32.2, 33.8,
34.2, 52.9, 55.1, 60.9, 64.9, 67.2, 67.3, 113.9, 127.4, 127.7, 127.8, 127, 9,
128.3,128.9,129.1,129.8,133.9,136.6,158.3,170 .4,170.6,190.8. IR(neat)
3404.1, 2932.5, 1703.2, 1513.7, 1453.1, 1402.5, 1248.0, 1162.6, 1033.0,
754.8.

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[0190] O-(LrN-Cbz-N, 0-DimethyItyrosyl)-L-A^-Boc-serine (57): To a
solution of serine phenacyl ester 56 (500 mg, 0.73 mmol) in aq. AcOH (6 mL,
90%), cooled to 0°C, powered Zn (381 mg, 5.48 mmol) was added. The
resulting mixture was stirred 3h at 0°C, filtered over Celite® and the Celite®
washed with EtOAc (25 mL). The filtrate was washed with 10% KHSO4
(20 mL), NaHCO3 (20 mL, sat), and NaCl (20 mL, sat). The organic layer
was dried (Na2SC>4), and the solvent evaporated. The crude oil was purified by
column chromatography (silica gel, MeOH/CHkCfe 1/9) to yield the acid 57
(290 mg, 75%) as a white solid. Rf 0.35 (MeOH/CH2Cl2 1/9). mp = 71-72 °C.
[cc]D20 =-2.5 (c=l, CH2CI2). ^-NMR (500 MHz, CDC13): 1.43 (m, 9H), 2.78
(s, 3H), 2.81-3.01 (m, 1H), 3.15-3.26 (m, 1H), 3.79 (s, 3H), 4.31-4.45 (m, 2H),
3.65-3.70 (m, 1H), 4.89-5.05 (m, 2H), 5.55-5.61 (m, 1H), 6.71-6.80 and 6.93-
7.05 (m, 2H), 7.26-7.33 (m, 5H). 13C-KMR (125 MHz, CDC13): 28.3, 33.4,
33.7, 53.1, 55.1, 60.8, 67.5, 68.0, 80.0, 113.9, 127.4, 127.8, 128.3, 128.8,
129.6, 129.7, 136.5, 137.0, 158.3, 170.1, 174.3. IR (neat) 3354.1, 2933.3,
1706.0, 1513.7, 1247.5, 1163.3, 1032. HRMS m/z cald for CaTHs^CWa
553.2162 (M+Na): found 553.2176.
[0191] [Ser^-Tamandarin B Protected Linear Precursor (59): To a solution of
52 (117 mg, 0.18 mmol) in dioxane (5 mL), cooled to 0°C, was added a
solution of HC1 in dioxane (5 mL). The resulting solution was stirred at room
temperature for 2h. The solution was concentrated, and the residue diluted
with CH2C12 and concentrated again to yield the hydrochloride salt 58 (90 mg,
quantitative yield) as a white solid, which was used directly in the next step.
To a mixture of hydrochloride salt 58 (90 mg, 0.15 mmol) HBTU (61 mg,
0.16 mmol), HOBt (20 mg, 0.15 mmol) and Tyr-ser acid 57 (80 mg,
0.15 mmol), cooled to -5°C a solution of CH2CI2/DMF 2/1 (3 mL) was added,
the reaction was stirred 5 min and DIPEA (106 uL, 0.60 mmol) was added.
The resulting solution was stirred at -5°C overnight, diluted with 'BuOMe
(10 mL), washed with 10% KHSO4 (10 mL), NaHCO3 (10 mL, sat), and NaCl
(10 mL, sat), dried (Na2SC>4), filtered, and concentrated. The crude oil was
purified by column chromatography (silica gel, grad EtOAc/Hexanes 1/1) to
yield 59 (105 mg, 65%). Rf 0.34 (AcOEt/Hexanes 1:1). [a]D20 = - 59.1 (c=l,

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CH2CI2). JH-NMR (500 MHz, CDC13): 0.88-1.00 (m, 18H), 1.47 (s, 9H),
1.49-1.52 (m, 4H), 2.03-2.06 (m, 5H), 2.22-2.25 (m, 3H), 2.55-2.60 (m, 1H),
2.70-2.84 (m, 3H), 2.98-3.03 (m, 1H), 3.27 (dt, J= 5.8 and 20.0 Hz, 1H), 3.80
(s, 3H), 4.12-4.15 (m, 1H), 4.50-4.53 (m, 3H), 4.53-4.59 (m, 2H), 5.00-5.23
(m, 6H), 5.80-5.83 (m, 1H), 6.78-6.80 (m, 3H), 7.06-7.08 (m, 3H), 7.29-7.38
(m, 11H). 13C-NMR (125 -MHz, CDCI3):14.5, 17.3, 18.6, 20.2, 21.0, 23.3,
24.6, 24.8,27.8,28.2,28.8,30.3,32.5,33.8,38.8,46.8,48.4,55.1,58.9,60.3,
60.5, 61.0, 64.3. 66.9, 67.2, 68.5, 78.1, 78.5, 79.3, 113.8, 127.5, 127.9,128.1,
128.3, 128.5, 128.8, 129.7, 135.3, 136.5, 156.7, 157.5, 158.2, 167.3, 168.0,
169.6,170.7,171.3. IR (neat) 3315.5, 2968.3,1744.0,1670.0,1637.7,1513.9,
1447.3, 1170.6. HRMS in/z cald for CsT^NsOigNa 1096.5470 (M+Na):
found 1096.5498.
[0192] [Ser6]-Tamandarin B macrocycle (61): To a solution of protected linear
precursor 59 (206 mg, 0.19 mmol) in MeOH (15 mL) under argon, Pd(OH)2
(81 mg) was added. The reaction was purged with H2 and stirred overnight
under H2 atmosphere (1 arm). The mixture was filtered through Celite®. The
filtrate was concentrated to yield the free linear precursor 60 (150 mg, 91%) as
a yellow oil, which was used in the next step without purification. The crude
amino acid linear precursor 60 (150 mg, 0.17 mmol) was dissolved in CH3CN
(30 mL) and cooled to 0 °C. HATU (160 mg, 0.42 mmol) was added followed
by the dropwise addition of NNM (38 pL, 0.34 mmol). The reaction mixture
was stirred at 0 °C for 1 h and then overnight. The reaction mixture was
concentrated in vacuo, diluted with EtOAc (15 mL), washed with 10% KHSO4
(10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL, sat), dried (Na2SO4),
filtered, and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 2/1) to yield protected
macrocycle 61 (130 mg, 89 %) as a white foam. Rf 0.23 (AcOEt/Hexanes
■ 1/2). [afo20 = -56.1 (c = 1, CHCI3). *H-NMR (500 MHz, CDCI3): 0.89-1.05
(m, 18H), 1.45 (m, 9H), 1.62-1.74 (m, 1H), 2.00-2.20 (m, 4H), 2.55 (m, 3H),
2.81 (dd, 1H), 2.99(dd, 1H), 3.15 (dd, 1H), 3.36 (dd, 1H), 3.53 (m, 1H), 3.67
(m, 1H), 3.73 (m, 1H), 3.80 (s, 3H), 3,91 (m, 1H), 4.10 (m, 1H), 4.19 (m, 1H),
4.42 (td, J= and , 1H), 4.48 (m, 1H), 4.51 (dd, J= and , 1H), 4.59 (m, 1H), 4,91

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(m, 1H), 5.06 (m, 2H), 6.84 (d, 2H), 7.01 (d, 2H), 7.77 (d, 2H), 8.55 (d, 2H).
13C-NMR (125 MHz, CDC13): 17.4, 17.8, 18.7, 20.2, 20.6, 21.4, 23.5, 24.9,
25.1,26.0, 28.0, 28.1,28.3,28.6, 28.8, 30.2, 30.7, 34.1, 38.6,46.1,47.0,48.7,
48.9, 52.4, 55.3, 57.2, 62.9, 63.7, 65.6, 68.7, 69.3, 78.7, 80.5, 114.1, 129.8,
130.3, 155.6, 158.6, 169.1, 170.8, 171.1, 171.2. IR (neat) 3314.2, 2924.3,
2359.9, 1742.6, 1630.7. HRMS m/z cald for CgyHTgNsO^Na 1096.5470
(M+Na): found 1096.5498.
[0193] [iV-Ser6]-Tamandarin B (62): To a solution of Boc protected
macrocycle 61 (20 mg, 0.024 mmol) in HPLC dioxane (10 mL) was added a
solution of HC1 in dioxane (10 mL). The resulting solution was stirred at room
temperature for 2h. The solution was concentrated and the residue diluted with
CH2C12 and concentrated again to yield the hydrochloride salt (18 mg,
quantitative yield) as a white solid, which was used directly in the next step.
To a mixture of the macrocycle amine salt (18 mg, 0.023 mmol) and side
chain (11 mg, 0.035 mmol) in CH2C12 (3 mL) at 0 °C was added BOP (15 mg,
0.035 mmol) and NMM (10 \\L, 0.092 mmol). After 30 min at 0 °C, the
mixture was stirred at rt overnight The reaction was treated with sat NaCl
solution (2 mL) and extracted with EtOAc (2x10 mL) The organic layers were
washed with 10% HC1 (5 mL), 5% NaHCO3 (5 mL) and NaCI (5 mL, sat),
dried (Na2SO4), filtered and concentrated. The crude oil (24 mg) was purified
by HPLC. :H-NMR (300 MHz, CDC13): 8 0.82-0.96 (m, 24H), 1.09-1.28 (m,
14H), 1.394.43 (m, 3H), 1.64 (m, 2H), 1.88-1.98 (m, 2H), 2.10-2.15 (m, 2H),
2.58 (s, 3H), 2.90-2.95 (m, 1H), 3.05 (s, 3H), 3.09-3.12 (m, 1H), 3.35-3.43 (m,
IE), 3.43-3.82 (m, 5H), 3.79 (s, 3H), 3.98-4.05 (m, 1H), 4.12-4.17 (m, 1H),
4.45-4.50 (m, 1H), 4.62-4.66 (m, 1H), 4.67-4.70 (m, 2H), 5.05 (d, J= 5.3,1H),
5.29-5.32 (m, 2H), 6.80 (d, J=8.7 Hz, 2H), 7.02 (d, J=8.6 Hz, 2H), 7.65 (d,
J=6.7 Hz, 1H), 8.09 (d, J=9.5 Hz, 1H), 8.45 (d, J=8.9 Hz, 1H). HRMS m/z cald
for C52H8iN7Oi4Na (M+Na): 1050.5739 found 1050.5731.

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EXAMPLE5
Synthesis of Ser6 Pyr Tamandarin B
[0194] A serine-pyruvate analog of tamandarin (Ser6 Pyr Tamandarin B) was
prepared as shown in Scheme 17.
Scheme 17

[0195] The 7V-Boc group of the Ser6 tamandarin B macrocycle was removed
using HC1 (gaseous) in dioxane. Subsequent coupling with D-Cbz-iV-Me-Leu
using HATU and NMM in DMF/DCM afforded the depsipeptide 63 in 50%
yield. Hydrogenolysis of the CBz group and coupling with the prolyl-pyruvyl
fragment, using conditions as described herein, afforded the desired analog 64.




[0197] JV-Boc-L-Leucyl-JV-L-Alanine Acid Benzyl Ester (67): To a solution of
JV-Boc-L-alanine acid benzyl ester (66) (660 mg, 2.36 mmol) in HPLC EtOAc
(10 mL) a saturated solution of HC1 in EtOAc (5 mL) was added. The
resulting solution was stirred at room temperature for 2h. The solution was

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concentrated and the residue diluted with CH2CI2 and concentrated again to
yield the hydrochloride salt (quantitative yield) as a white solid, which was
used directly in the next step. To a solution of hydrochloride salt (500 mg,
2.78 mmol) in CH2C12 (6 mL), cooled to 0°C, NMM (423 pL, 4.17 rnmol) was
added. After 15 min, AT-Boc-L-leucine monohydrate (693 mg, 2.78 mmol),
HOBt (425 mg, 2.78 mmol) and DCC (630 mg, 3-05 mmol) were added. The
reaction mixture was stirred 14h at room temperature diluted with CH2CI2
(25 mL) and filtered. The resulting solution was washed with HC1 IN (20
mL), NaHCO3 (20 mL, sat) and brine (20 mL), dried (Na2SO4), filtered and
concentrated. The crude oil was purified by column chromatography (silica
gel, grad EtOAc/Hexanes 1/6) to yield 67 (650 mg, 78%). XH-NMR (500
MHz, CDCI3): 0.99-1.01 (m, 6H), 1.43 (d, J= 6.8 Hz, 3H), 1.44 (s, 9H), 1.45-
1.75 (m, 3H), 4.15-4.25 (m, 1H), 4.55-4.66 (m, 1H), 5.00-5.25 (m, 2H), 5.45-
5.53 (m, 1H), 7.29-1.34 (m, 5H). 13C-NMR (125 MHz, CDC13): 21.5, 24.1,
28.05, 41.2, 47.7, 52.5, 66.6, 793, 82.2, 127.8, 128.1, 128.5, 135.2, 155.6,
172.2,172.6.
[0198] iV-Hydroxyvaleryl-Ar-Leucyl-Alanine Acid Benzyl Ester (69): To a
solution of 67 (625 mg) in HPLC AcOEt (10 mL) a saturated solution of HC1
in AcOEt (5 mL) was added. The resulting solution was stirred at room
temperature for 2h. The solution was concentrated and the residue diluted with
CH2CI2 and concentrated again to afford the hydrochloride salt 68
(quantitative yield) as a white solid, which was used directly in the next step.
To a solution of the hydrochloride salt 68 (522 mg, 1.59 mmol) in CH2CI2
(6 mL), cooled to 0 DC, NMM (197 \xL, 1.75 mmol) was added. After 15 min
cc-hydroxyvaleric acid (187 mg, 1.59 mmol) and DCC (360 mg, 1.75 mmol)
were added in portions. The reaction mixture was stirred 14h at room
temperature diluted with CH2C12 (25 mL) and washed with HC1 IN (20 mL),
NaHCO3 (20 mL, sat) and brine (20 mL), dried (Na2SO4), filtered and
concentrated. The crude oil was purified by column chromatography (silica
gel, EtOAc/Hexanes 1/1) to yield 69 (480 mg, 77%) as a white solid.
Rf = 0.31 (EtOAc/Hexanes 1/1). [ajD20 = -45.3 (c=l, CH2C12). 1H-NMR(500
MHz, CDCI3): 0.82 (d, J= 6.8 Hz, 3H), 0.86 (d, J= 6.1 Hz, 3H), 0.90 (d, J= 6.1

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Hz, 3H), 0.99 (d, J= 6.8 Hz, 3H), 1.36 (d, J= 7.1 Hz, 3H), 1,57-1.63 (m, 3H),
1.89-1.93 (m, 1H), 2.02-2.16 (m, 1H), 3.95-3.97 (m, 1H), 4.49-4.96 (m, 2H),
5.13 (c, J= 12.3, 2H), 7.03 (d, J= 7.0 Hz, 1H), 7.18 (d, J= 8.5 Hz, 1H), 7.30-
7.35 (m, 5H). I3C-NMR (125 MHz, CDC13): 15.5,17.8,19.1,21.7,22.8,24.6,
31.6, 33.8, 40.9, 51.1, 67.1, 76.2, 128.1, 128.3, 168.6, 135.3, 172.1, 172.8,
173.8.
[0199] (31S,4i?)-4-fer?-Butoxycarbonylamino-5-methyl-3-(/e7t-butyl-
dimethylsilanyloxy)-hexanoic Acid JV-Hydroxyvaleryl-iV-Leucyl-
O-Benzylalanine Ester (70): To a solution of peptide 69 (200 rag, 0.51 mmol)
in CH2C12 (4 mL), cooled to -5 °C, DMAP (18.4 mg, 0.15 mmol) acid 48
(229 mg, 0.61 mmol) and DCC (157 mg, 0.76 mmol) were added in portions.
The reaction mixture was stirred 7h at -5 °C, filtered and evaporated. The
residue was dissolved in CH3CN (7 mL), filtered again, and evaporated. The
crude oil was dissolved in EtOAc (10 mL) and washed with 10% KHSO4
(10 mL), NaHCO3 (10 mL, sat) and NaCl (10 mL, sat). The organic layer was
dried (Na2SO4), and the solvent evaporated. The crude oil was purified by
column chromatography (silica gel, grad EtOAc/Hexanes 1/4) to yield 70
(340 mg, 89%) as white foam. Rf 0.72 (AcOEt/Hexanes 1:1). [a]D20 = -50.4
(c=l, CH2C12). :H-NMR (500 MHz, CDC13): mixture of rotamers 0.05 (s,
6H), 0.77-1.04 (m, 27H), 1.30 and 1.39 (d, J=6.7 Hz, 3H), 1.42 and 1.52 (s,
9H), 1.30-1.70 (m, 5H), 2.56-2,70 (m, 2H), 3.19-3.22 (m, 1H), 3.41-3.47 (m,
1H), 3.83-3.85 (m ,1H), 3.99-4.03 (m, 1H), 4.30-4.32 (m ,2H), 4.35-4.38 (m,
1H), 4.48-4.53 (m, 1H), 4.67-4.70 (m, 1H), 4.73-4.75 (m, 1H), 4.82-4.85 (m,
1H), 4.99-5.13 (m, 2H), 5.15-5.21 (m, 2H), 6.33 (d, J= 7.2 Hz, 1H), 7.18-7.30
(m, 5H), 8.39 (d, J= 8.0 Hz, 1H). 13C-NMR (125 MHz, CDC13): -5.1, -4.8,
14.6, 17.2,17.6, 17.9,18.6, 20.3, 21.2, 22.8, 22.9, 24.5, 24.6, 24.8, 25.3, 25.6,
28.1, 28.2, 34.8, 39.6,48.0,48.3, 50.8, 50.9, 55.5, 63.1, 66.6, 70.1, 71.3, 78.9,
79.5, 82.1, 127.9, 128.0, 128.3, 135.4, 156.1, 157.3, 169.6, 170.0, 170.5,
170.9, 171.5, 172.2, 172.6, 172.9. HRMS m/z cald for Cs^NsOgSiNa
(M+Na): 772.4544 found 772.4551.
[0200] [Ala^-Tamandarin B Protected Linear Precursor (73): To a solution of
70 (80 mg, 0.10 mmol) in dioxane (2 mL), cooled to 0°C, was added a solution

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of HC1 in dioxane (2 mL). The resulting solution was stirred at room
temperature for 2h. The solution was concentrated and the residue diluted with
CH2CI2 and concentrated again to yield the hydrochloride salt 71 (61 mg,
quantitative yield) as a white solid, which was used directly in the next step.
To a mixture of hydrochloride salt (61 nig, 0.10 mmol) HBTU (41.7 mg, 0.11
nunol), HOBt (13.5 mg, 0.10 mmol) and Tyr-Thr acid 72 (54 mg, 0.10 mmol),
cooled to -5 °C, a solution of CH2CI2/DMF 2/1 (3 mL) was added, the reaction
was stirred 5 min and DIPEA (71 uX, 0.40 mmol) was added. The resulting
solution was stirred at -5 °C overnight, diluted with *BuOMe (10 mL) and
washed with 10% KHSO4 (10 mL), NaHCO3 (10 mL, sat) and NaCl (10 mL,
sat), dried (NaaSOiO, filtered and concentrated. The crude oil was purified by
column chromatography (silica gel, grad EtOAc/Hexanes 1/1) to yield 73
(98 mg, 86%). Rf 0.38 (AcOEt/Hexanes 1:1). [a]D20 = -34.4 (c=l, CH2C12).
'H-NMR (500 MHz, CDC13): 0.88-1.02 (m, 18H), 1.19 (d, J=6.9 Hz, 3H), 1.26
(d, J=6.9 Hz, 3H), 1.48 (s, 9H), 1.55-1.62 (m, 4H), 1.87-1.95 (m, 2H), 2.22-
2.45 (m, 2H), 2.55-2.70 (m, 3H), 2.84 (s, 3H), 2.97-3.05 (m, 1H), 3.11-3.19
(m, 1H), 3.81 (s, 3H), 4.12-4.35 (m, 2H), 4.41-4.48 (m, 1H), 4.55-4.58 (m,
1H), 4.63-4.66 (m, 1H), 5.00-5.20 (m, 6H), 5.30-5.33 (m, 1H), 5.62 (d, J= 7.3
Hz, 1H), 6.77 (d, J= 8.3 Hz, 2H), 6.98 (m, 1H), 7.06 (d, J= 7.3 Hz, 1H), 7.19
(m, 1H), 7.29-7.42 (m, 10H), 7.60-7.66 (m, 1H). l3C-NMR (125 MHz,
CDCI3): 16.2, 16.9, 17.6, 18.7, 20.1, 23.1, 24.5, 24.8, 25.5, 26.9, 28.1, 28.2,
30.2, 32.3, 33.8, 34.0, 37,9, 39.8, 48.2, 51.1, 55.2, 57.5, 60.6, 61.1, 66.9, 67.5,
69.1, 70.0, 78.1, 80.3, 113.9, 127.5, 128.0, 128.3, 128.4, 128.5, 129.8, 1315.3,
136.2, 156.9, 158.4, 170.1, 170.9, 171.2, 171.9, 172.4. IR (neat) 3297.6,
2962.0, 1744.0, 1658.8, 1514.2, 1166.6. HRMS m/z cald for Csv^NsO^Na
1096.5470 (M+Na): found 1096.5498.
[0201] [Ala4]-Tamandarin B macrocycle (74): To a solution of protected
linear precursor 73 (102 mg, 0.09 mmol) in MeOH (88 mL), under argon,
Pd(OH)2 (44 mg) was added. The reaction was purged with H2 and stirred
overnight under H2 atmosphere (1 atm). The mixture was filtered through
Celite® and the filtrate was concentrated to yield the free liner precursor 14
(77 mg, 96%) as a yellow oil. The crude amino acid linear precursor (77 mg,

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0.91 mmol) was dissolved in CH3CN (20 mL) and cooled to 0 °C. HATU
(815 mg, 0.22 mmol) was added followed by the dropwise addition of NNM
(20 ^L, 1.82 mmol). The reaction was stirred at 0 °C for 1 h and then
overnight. The reaction mixture was concentrated in vacuo, diluted with
EtOAc (15 mL), and washed 10% KHSO4 (15 mL), 5% NaHCO3 (15 mL) and
NaCl (15 mL, sat), dried CN^SO,*), filtered, and concentrated. The crude oil
was purified by column chromatography (silica gel, EtOAc/Hexanes 2/1) to
yield protected macrocycle 74 (45 mg, 60%) as white foam. Rf 0.24
(AcOEt/Hexanes 1/2). [a]D20 = -33.09 (c = 1, CHC13). 3H-NMR (500 MHz,
CDCI3): 0.92-1.04 (m, 18H), 1.16 (d, J= 6.8 Hz, 3H), 1.17-1.30 (m, 3H), 1.49
(s, 9H), 1.51-1.61 (m, 1H), 1.73-1.75 (m, 1H), 1.83-1.86 (m, 1H), 1.93-1.97
(m, 1H), 2.30-2.40 (m, 1H), 2.57-2.39 (m, 1H), 3.00-3.05 (m, 1H), 3.08 (s,
3H); 3.19-3.21 (m, 1H), 3.49-3.51 (m, 1H), 3.82 (s, 3H), 3.98-4.00 (m, 1H),
5.01-5.06 (m, 2H), 5.27-5.30 (m, 1H), 5.56-5.66 (m, 1H), 6.67-6.69 (m, 1H),
6.88 (d, J= 7.9Hz, 2H), 7.00-7.09 (m, 1H), 7.12 (d, 2H). 13C-NMR (125 MHz,
CDCI3): 15.1, 18.4, 18.9, 20.9, 21.1, 23.2, 24.0, 24.8, 24.9, 25.5, 28.0, 28.1,
30.1, 33.7, 34.4, 39.01, 39.3, 45.4, 47.1, 51.2, 55.3, 69.3, 71.9, 79.5, 80.6,
114.1, 114.2,129.9, 132.3, 156.1, 169.01,170.9, 171.5, 172.3, 173.2. HRMS
m/z cald for C^HfeNsOizNa 842.4527(M+Na): found 842.4545.
[0202] [7V-Ala4]-Tamandarin B (75): To a solution of Boc protected
macrocycle 74 (11 mg, 0.013 mmol) in HPLC dioxane (5 mL) was added a
solution of HC1 in dioxane (5 mL). The resulting solution was stirred at room
temperature for 4h. The solution was concentrated, and the residue diluted
with CH2CI2 and concentrated again to yield the hydrochloride salt
(quantitative yield) as a white solid, which was used directly in the next step.
To a mixture of the macrocycle amine salt (10 mg, 0.013 mmol) and side
chain (6.2 mg, 0.019 mmol) in CH2C12 (2 mL) at 0 °C was added BOP
(8.4 mg, 0.019 mmol) and NMM (6 \xL, 0.052 mmol). After 30 min at 0 °C,
the reaction was stirred at rt overnight. The reaction was treated with NaCl
solution (5 mL, sat) and extracted with EtOAc (2x10 mL). The organic layers
were washed with 10% HC1 (5 mL), 5% NaHCO3 (5 mL) and NaCl (5 mL,
sat), dried (Na2SC>4), filtered and concentrated. The crude oil (10 mg, 77 %)

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was purified by HPLC. *H-NMR (300 MHz, CDC13): 5 0.82-1.46 (m, 32H),
1.46-2.10 (m, 12H), 2.16 (m, 4H), 2.65 (m, 1H), 2.82-3.01 (m, 1H), 3.05 (s,
3H), 3.20 (s, 3H), 3.49-3.55 (m, 2H), 3.63-3.92 (m, 5H), 3.85 (s, 3H), 4.09 (m,
1H), 4.32-4.43 (m, 2H), 4.60-4.65 (m, 1H), 4.75 (t, J= 6.7, 1H), 4.88 (t, J=8.0,
1H), 5.01 (d, J= 5.3, 1H), 5.24 (s, 2H), 6.64-6.66 (m, 1H), 6.85 (d, J=8.7 Hz,
2H), 7.14 (d, J=8.8 Hz, 2H), 7.22-7.23 (m, 1H). MS Calcd for C51H81N7O14:
1016.2. Found: 1016.4 (M+H)+.
EXAMPLE7
Synthesis of L-Me-Ala4 Tamandarin B
[0203] A L-methylalanine analog of tamandarin B (L-Me-Ala4 Tamandarin B)
was synthesized as shown in Schemes 21 and 22. Compound 85 was then
reacted to form the L-methylalaninine analog as described below.



[0204] Boc-L-iV-Methylalanine (76): To a solution stirred solution of L-Boc-
alanine (5 g, 26.4 mmol) in THF (80 mL), at 0 °C, finely powered KOH
(10.4 g, 187 mmol) was added in portions, followed by the addition of
tetrabutylammonium hydrogen sulfate (0.5 g, 10% by weight). Then, dimethyl
sulfate (10 mL, 105 mmol) was added dropwise over 15 min. The reaction was
stirred for an additional 30 min, and H2O (50 mL) was added. After stirring 5h
at room temperature, 20% aqueous ammonium hydroxide solution was added
(20 mL). The reaction was diluted with ether (100 mL), the aqueous layer was
separated, and the organic layer was extracted with saturated aqueous
NaHCO3 (2 x 40 mL). The combined aqueous layers were acidified to a pH of
1 with 1M KHSO4 and extracted with EtOAc (2x200 mL). The organic layers

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were combined, dried QXsdSOt), filtered, and concentrated. The resulting acid
76 (4.3 g, 80%) was obtained as a yellow oil and used in the next step without
purification. ^-NMR (500 MHz, CDCI3): 1.43-1.55 (m, 12H), 2.91 (br s,
3H)? 4.54-4.58 (m, IK), 4.89-4.93 (m, 1H).
[0205] JV-Boc-L-TV-Methylalanine Benzyl Ester (77): To a solution of L-Boc-
iV-methylalanine 76 (2.1 g, 10.3 mmol) in THF (10 mL), cooled to 0 °C, Et3N
(1.6 mL, 11.3 mmol) and benzyl bromide (1.3 mL, 11.3 mmol) were added.
The mixture was stirred 18h at room temperature, and the solvent was
evaporated at reduced pressure. The residue was evaporated, diluted with
CH2CI2 (20 mL) and washed with HC1 IN (15 mL), NaHCO3 sat (15 mL), and
H2O (15 mL). The organic layers were combined, dried (Na2SO4), filtered and
concentrated. The resulting ester 77 (2.25 g, 95%) was obtained as a yellow oil
and used in the next step without purification. !H-NMR (500 MHz, CDCI3):
1.444.54 (m, 12H), 2.81 and 2.90 (br s, 3H), 4.49-4.53 (m, 1H), 4.83-4.89
(m, 1H), 5.18 (s, 2H), 1.21-1 A3 (m, 5H).
[0206] A^-Boc-L-Leucyl-L-A^-Methylalanine Acid Benzyl Ester (79): To a
solution of L-iV-Boc-JV-methylalanine benzyl ester (77) (320 mg, 1.09 mmol)
in HPLC dioxane (8 mL) a saturated solution of HC1 in dioxane (5 mL) was
added. The resulting solution was stirred at room temperature for 2h. The
solution was concentrated and the residue diluted with CH2CI2 and
concentrated again to afford the hydrochloride salt (quantitative yield) as a
white solid, which was used directly in the next step. To a solution of
hydrochloride salt (250 mg, 1.09 mmol) in CH2C12 (10 mL) and DMF (5 mL),
cooled to 0 °C, NMM (221 pL, 2.08 mmol) was added. After 15 min,
W-Boc-L-Ieucine monohydrate (246 mg, 0.99 mmol) and HATU (779 mg,
2.08 mmol) were added. The reaction mixture was stirred 14h at room
temperature and CH2C12 was evaporated. The resulting residue was diluted
with ethyl ether (25 mL) and washed with HC1 IN (20 mL), NaHCO3 sat
(20 mL,) and brine (20 mL), dried (Na2SC>4), filtered and concentrated. The
crude oil was used without purification in the next step. ^-NMR (500 MHz,
CDCI3): 0.91 (d, J = 6.7 Hz, 3H), 0.99 (d, J = 6.7 Hz, 3H), 1.48 (m, 13H),
1.69-1.78 (m, 2H), 3.01 (s, 3H), 4.60-4.71 (m, 1H), 5.10-5.29 (m, 3H), 5.35-

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5.48 (m, 1H), 7.31-7.42 (m, 5H). HRMS m/z cald for C22H34N2O5Na (M+Na):
429.2365 found 429.2385.
[0207] A^-Hydroxyvaleryl-A^L-Leucyl-//-L-Methylalanine Acid Benzyl Ester
(81): To a solution of dipeptide 79 (360 mg) in HPLC dioxane (10 mL) a
saturated solution of HC1 in dioxane (6 mL) was added. The resulting solution
was stirred at room temperature for 2h. The solution was concentrated and the
residue diluted with CH2CI2 and concentrated again to afford the
hydrochloride salt 80 (330 mg, quantitative yield) as a white solid, which was
used directly in the next step. To a solution of the hydrochloride salt 80
(303 mg, 0.88 mmol) in CH2C12 (2 mL), cooled to 0 °C, NMM (108 ML,
0.96 mmol) was added. After 15 min hydroxyvaleric acid (104 mg,
0.88 mmol) and DCC (200 mg, 0.96 mmol) were added in portions. The
reaction mixture was stirred 14h at room temperature, diluted with CH2CI2
(20 mL), washed with HC1 IN (20 mL), NaHCO3 (20 mL, sat), and brine
(20 mL), dried (Na2SO4), filtered, and concentrated. The crude oil was
purified by column chromatography (silica gel, EtOAc/Hexanes 2/1) to yield
81 (258 mg, 72%) as colorless oil. Rf 0.30 (EtOAc/Hexanes 2/1). [cc]D20 =
-37.2 (c=l, CH2CI2). 'H-NMR (500 MHz, CDCI3): 0.87 (d, J= 6.8 Hz, 3H),
0.89 (d, J= 6.1 Hz, 3H), 0.90 (d, J= 6.1 Hz, 3H), 1.03 (d, J= 6.8 Hz, 3H), 1.42
(d, J= 7.3 Hz, 3H), 1.55-1.66 (m, 1H), L66-.1.76 (m, 1H), 2.14-2.20 (m, 1H),
3.02 (s, 3H), 3.98 (d, J=lHz, 1H), 4.98-5.02 (m, 1H), 5.17 (m, 2H), 5.37 (c, J=
6.9 Hz, 2H), 7.29-7.40 (m, 5H). 13C-NMR (125 MHz, CDC13): 14.07, 15.5,
19.12, 21.5, 21.6, 23.1, 23.3, 24.6, 24.7, 25.4, 31.0, 31.7, 33.4, 41.3, 47.2.
52.3. 67.0. 76.1. 128.2. 128.4. 128.6. 135.3. 171.3. 173.3. 173.5. IR (neat)
3378.8, 2958.9, 1740.3, 1652.0, 1635.5, 1558.0, 1506.2, 1456.2, 1196.1,
1086.6. HRMS m/z cald for C22H34N2O5Na (M+Na): 429.2365 found
429.2370.
[0208] (35,4i?)-4-rert-Butoxycarbonylamino-5-methyl-3-(re?t-butyldimethyl-
silanyloxy)hexanoic Acid A'-Hydroxyvaleryl-A'-L-leucyl-A^-L-methylalanine
Benzyl Ester (82): To a solution of peptide 81 (245 mg, 0.61 mmol) in CH2C12
(4 mL), cooled to -5°C, DMAP (22.4 mg, 0.15 mmol) acid 48 (271 mg,
0.72 mmol) and DCC (157 mg, 0.18 mmol) were added in portions. The

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reaction mixture was stirred 7 h at -5°C, filtered, and evaporated. The residue
was dissolved in CH3CN (8 mL), filtered again, and evaporated. The crude oil
was dissolved in EtOAc (15 mL) and washed with 10% KHSO4 (15 mL),
NaHCO3 (15 mL, sat) and NaCl (15 mL, sat). The organic layer was dried
(Na2SO4), and the solvent was evaporated. The crude oil was purified by
column chromatography (silica gel, EtOAc/Hexanes 1/4) to yield 70 (325 mg,
70%) as white foam. Rf 0.22 (AcOEt/Hexanes 4:1). [a]D20 = -33.6 (c=l,
CH2C12). JH-NMR (500 MHz, CDC13): mixture of rotamers 0.01 (s, 6H),
0.85-1.00 (m, 27H), 1.39 (d, J=6.7 Hz, 3H), 1.44 and 1.51 (s, 9H), 1.29-1.40
(m, 1H), 1.56-1,70 (m, 2H), 1.89-1.92 (m, 1H), 2.00-2.09 (m, 2H), 2.43-2.46
(m, 1H), 2.66-2.70 (m, 1H), 2.69-2.70 (m ,1H), 2.94 and 3.01 (s, 3H), 3.43-
3.46 (m, 1H), 3.60-3.65 (m, 1H), 3.98-4.02 (m, 4H), 4.09-4.14 (m, 2H), 4.31
(d, J=7.8Hz, 1H), 4.81-4.82 (m, 1H), 4.99-5.13 (m, 2H), 5.15-5.21 (m, 1H),
6.63 (d, J= 7.2 Hz, 1H), 6.74 (d, J= 5.2 Hz, 1H), 7.18-7.30 (m, 5H), 8.39 (d, J=
6.5 Hz, 1H). 13C-NMR (125 MHz, CDC13): -5.0, -4.5, 13.9, 14.1, 14.7, 16.5,
17.6,18.1, 18.5,18.8, 20.4, 21.3, 21.5, 23.2, 24.6, 24.7, 25.7, 26.9, 27.7, 28.4,
30.1, 30.4, 30.7, 30.9, 39.3, 39.9, 41.8, 42.8, 47.0, 51.9, 52.3, 59.4, 60.2, 63.0,
66.7, 66.9, 70.1, 71.4, 78.9, 79.2, 79.4, 82.4, 128.08, 128.2, 128.3, 128.5,
135.4, 135.5, 156.2, 157.3, 169.1, 169.7, 170.8, 171.0, 171.2, 171.6, 172.5,
173.6. IR (neat) 2959.1, 1740.5, 1694.0, 1644.4, 1521.2, 1471.5, 1387.0,
1252.7 1166.6, 1086.1. HRMS m/z cald for C4oH69N309SiNa (M+Na):
786.4700 found 786.4707.
[0209] [iV-Me-Ala4]-Tamandarin B Protected Linear Precursor (84): To a
solution of 82 (285 mg, 0.37 mmol) in dioxane (5 mL), cooled to 0°C, was
added a solution of HC1 in dioxane (5 mL). The resulting solution was stirred
at room temperature for 2h. The solution was concentrated, and the residue
was diluted with CH2CI2 and concentrated again to yield the hydrochloride salt
83 (218 mg, quantitative yield) as a white solid, which was used directly in the
next step. To a mixture of hydrochloride salt 83 (218 mg, 0.37 mmol) HBTU
(147.3 mg, 0.38 mmol), HOBt (50 mg, 0.37 mmol) and Tyr-Thr acid 72
(201 mg, 0.37 mmol), cooled to -5°C, a solution of CH2CI2/DMF 2/1 (10 mL)
was added, the reaction was stirred 5 min, and DIPEA (264 fiL, 1.48 mmol)

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was added. The resulting solution was stirred at -5°C overnight, diluted with
f-BuOMe (25 mL), washed with 10% KHSO4 (20 mL), NaHCO3 (20 mL, sat)
and NaCl (20 mL, sat), dried (Na2SO4), filtered and concentrated. The crude
oil was purified by column chromatography (silica gel, grad EtOAc/Hexanes
1/1) to yield 84 (280 mg, 69%). Rf 0.22 (AcOEt/Hexanes 1:1). [afo20 =-45.8
(c=l, CH2C12). XH-NMR (500 MHz, CDCI3): 0.87-1.01 (m, 18H), 1.00-1.16
(m, 4H), 1.28 (d, J=6.9 Hz, 3H), 1.47 (s, 9H), 1.68-1.69 (m, 2H), 1.70-1.77
(m, 1H), 2.07-2.09 (m, 1H), 2.22-2.25 (m, 1H), 2.75-2.77 (in, 1H), 2.85, 2.87
(m, 1H), 2.88 (s, 3H), 2.98 (s, 3H), 3.23 (dd, J=7.3 and 14.3 Hz, 1H), 3.77 (s,
3H), 4.10-4.15 (m, 1H), 4.21-4.28 (m, 2H), 4.35-4.38 (m, 1H), 4.60-4.66 (m,
1H), 5.00-5.12 (m, 2H), 5.20-5.33 (m, 4H), 5.25-5.30 (m, 1H), 5.58 (d, J= 7.3
Hz, 1H), 6.79 (d, J= 8.3 Hz, 2H), 7.02 (m, 1H), 7.09 (d, J= 7.3 Hz, 1H), 7.20
(m, 1H), 7.29-7.37(m, 10H). 13C-NMR (125 MHz, CDC13): 14.4, 16.8, 17.8,
18.6, 19.0, 20.7,21.7, 23.7, 25.1, 28.5, 28.6, 30.9, 31.3,32.3, 34.5, 39.0,41.1,
41.2, 47.6, 52.7, 55.6, 58.1, 61.2, 67.9, 69.2, 70.5, 71.0, 78.6, 81.1, 114.3,
128.05, 128.4, 128.6, 128.8, 129.0, 130.3, 135.7, 136.8, 156.1, 157.3, 158.8,
169.9, 170.4, 171.4, 171.6, 173.7. IR (neat) 3318.3, 2961.1, 2993.3, 1741.8,
1683.1, 1636.1, 1514.0, 1455.7, 1306.1, 1247.8, 1175.8. HRMS m/z cald for
C57HsiN5Oi5Na 1098.2756 (M+Na): found 1098.5657.
[0210] [iV-Me-Ala4]-Tamandarin B macrocycle (85): To a solution of
protected linear precursor 84 (280 mg, 0.33 mmol) in MeOH (20 mL), under
argon, Pd(OH)2 (100 mg) was added. The reaction was purged with H2 and
stirred overnight under H2 atmosphere (1 atm). The mixture was filtered
through Celite®, and the filtrate was concentrated to yield the free liner
precursor (208 mg, 94%) as a yellow oil. The crude amino acid linear
precursor (208 mg, 0.24 rnmol) was dissolved in CH3CN (50 mL) and cooled
to 0 °C. HATU (219 mg, 0.57 mmol) was added followed by the dropwise
addition of NNM (51 \iL, 0.48 mmol). The reaction mixture was stirred at 0 °C
for 1 h and then overnight. The reaction mixture was concentrated in vacuo,
diluted with EtOAc (30 mL), washed 10% KHSO4 (25 mL), 5% NaHCO3
(25 mL) and NaCl (25 mL, sat), dried (Na2SO4), filtered, and concentrated.
The crude oil was purified by column chromatography (silica gel,





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diluted with EtOAc (50 mL) and washed with 10% HC1 (20 mL), 5% NaHCO3
(20inL) and saturated NaCl (20 mL) solutions. The organic layer was dried
(Na2SO4), and the solvent was evaporated. The residue was triturated with
ether and filtered to afford 86 (2.8 g, 83%) as yellow foam. Rf =0.55
(AcOEt/Hexanes 1/1). [ocfo20 = -29.4 (c = 2, AcOEt). :H-NMR (300 MHz,
CDC13): 1.31 (d, J= 6.6 Hz, 3H), 1,46 (s, 9H), 3.77 (s, 1H), 4,44 (m, 1H), 4.60
(m, 1H), 5.34 (d, J=16.5 Hz, 1H), 5.37 (m, 1H), 5,68 (d, J= 16.7 Hz, 1H), 7.51
(m, 2H), 7.65 (m, 1H), 7.92 (m, 1H).
[0214] L-W-Cbz-iV-Methyl-2-naphthylalanine (87): To a stirred solution of
L-iV-Cbz~2-naphthylalanine (0.80 g, 2.29 mmol) in THF (40 mL), at 0 °C,
finely powered KOH (0.89 g, 16.0 mmol) was added in portions, followed by
the addition of tetrabutylammonium hydrogen sulfate (80 mg, 10% by
weight). Then, dimethyl sulfate (0,86 mL, 9.16 mmol) was added dropwise
over 15 min. The reaction was stirred for an additional 30 min and H2O
(50 mL) was added. After stirring 5h at room temperature, 20% aqueous
ammonium hydroxide solution was added (10 mL). The reaction was diluted
with ether (50 mL), the aqueous layer was separated and the organic layer was
extracted with saturated aq NaHCO3 (2x20 mL). The combined aqueous layers
were acidified to pH 1 with 1M KHSO4 and extracted with EtOAc
(2x100 mL). The organic layers were combined, dried (Na2SO4), filtered and
concentrated. The resulting acid 87 (832 mg, 90%) was obtained as a yellow
oil and used in the next step without purification. 'H-NMR (500 MHz,
CDCI3): 2,89 and 2.94 (s, 3H), 2.27-3.32 (m, 1H), 3.46-3.55 (m, 1H), 4.97-
5.09 (m, 3H), 7.12-7.52 (m, 12H).
[0215] O-(L-^-Cbz-A^-Methyl-2-naphthylalanine)-L-iV-Boc-threonine
Phenacyl Ester (88): To a solution of L-iV-Boc-threonine phenacyl ester 86
(852 mg. 2.29 mmol) in CH2C12 (15 mL), cooled to 0 °C, DMAP (84 mg, 0.68
mmol) and L-Af-Cbz-N-methylnaphthylalanine 87 (832 mg, 2.29 mmol) were
added. After stirring 10 min at 0 °C, DCC (519 mg, 2.52 mmol) was added.
The reaction mixture was stirred overnight at room temperature. The mixture
- was filtered, and the filtrate was concentrated to dryness. The residue was
dissolved in CH3CN (15 mL), filtered again, and evaporated. The crude oil

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was dissolved in EtOAc (50 mL) and washed with 10% KHSO4 (50 mL),
NaHCO3 (50 mL, sat) and NaCI (50 mL, sat). The organic layer was dried
' (Na2SO4), and the solvent was evaporated. The crude oil was purified by
column chromatography (silica gel, grad EtOAc/Hexanes 3/1) to yield the
dipeptide 88 (1.02 g, 68%) as a pale yellow oil. Rf 0.25 (AcOEt/Hexanes 1:3).
[a]D20 = -27.6 (c = 1, CH2C12). JH-NMR (500 MHz, CDC13): 1.22 (d, J= 6.9
Hz, 3H), 1.49 (s, 9H), 2.77 and 2.92 (mf 3H), 3.21-3.31 (m, 1H), 3.50-3.56 (m,
1H), 3.75 (m, 1H), 4.46-4.48 (m, 1H), 4.67-4.69 (m, 1H), 5.00-5.71 (m, 4H),
7.16-7.46 (m ,3H), 7.47-7.64 (m, 4H), 7.51-7.94 (m, 5H). 13C-NMR (125
MHz, CDCI3): 17.0,17.3, 28.7, 35.3, 31.7, 32.0, 35.38, 39.8, 50.1, 57.5, 59.8,
66.9, 67.3, 67.6, 68.7, 80.0, 125.9, 126.4, 127.5, 127.8, 127.9, 128.0, 128.2,
128.4, 128.8, 129.3, 129.5, 132.7, 133.8, 133.9, 134.4, 135.0, 169.3, 169.7,
171.4, 193.6. IR (neat) 3433.6, 2977.9, 1753.9, 1707.6, 1598.4, 1510.7,
1450.0,1367.5,1314.5,1161.2.
[02161 O-(L-^-Cbz-//-Methylnaphthylalanine)-L-A^-Boc-threonine (89): To a
solution of serine phenacyl ester 88 (500 mg, 0.75 mmol) in aq. AcOH (7 mL,
90%), cooled to 0 °C, powdered Zn (383 mg, 5.48 mmol) was added. The
resulting mixture was stirred 3h at 0 °C, filtered over Celite®, and the Celite®
washed with EtOAc (25 mL). The filtrate was washed with 10% KHSO4
(20 mL), NaHCO3 (20 mL, sat), and NaCI (20 mL, sat). The organic layer was
dried (Na2SO4), and the solvent was evaporated. The crude oil was purified by
column chromatography (silica gel, MeOH/CBfeCk 1/9) to yield the acid 89
(248 mg, 60%) as white foam. Rf 0.35 (MeOH/CH2Cl2 1/9). [a]D20 = +7.10
(c=0.7, CH2C12). ^-NMR (500 MHz, CDC13): 1.29-1.32 (m, 3H), 1.48 (s,
9H), 2.78-2.80 (m, 3H), 3.17-3.22 (m, 1H), 3.25-3.54 (m, 1H), 4.52-4.53 (m,
1H), 5.03-5.15 (m, 2H), 5.44-5.64 (m, 2H), 7.19-7.36 (m, 5H), 7.50-7.54 (m,
3H), 7.71-7.89 (m, 4H). 13C-NMR (125 MHz, CDCI3): 16.4,16.7, 20.7,28.1,
31.7, 32.0, 34.6, 34.9, 39.8, 50,1, 57.3, 60.6, 66.4, 72.2, 80.0, 125.4, 125.9,
126.7, 127.5, 127.9, 128.2, 128.8, 132.2, 133.4, 134.2, 136.1, 156.1, 156.9,
169.3,169.7,169.8. IR (neat) 3435.1,2978.4,1743.4,1709.6,1499.9,1402.2,
1315.6,1217.1,1162.0,1060.6,752.2.

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[0217] [2-Naphthylala5]-Tamandarin B Protected Linear Precursor (90): To a
solution of 56 (110 mg, 0.14 mmol) in dioxane (5 mL), cooled to 0 °C, was
added a solution of HC1 in dioxane (5 mL). The resulting solution was stirred
at room temperature for 2h. The solution was concentrated, and the residue
was diluted with CH2CI2 and concentrated again to yield the hydrochloride salt
(85 mg, quantitative yield) as a white solid, which was used directly in the
next step. To a mixture of hydrochloride salt (85 mg, 0.14 mmol) HBTU
(53 mg, 0.14 mmol), HOBt (19 mg, 0.14 mmol) and acid 89 (79 mg,
0.14 mmol), cooled to -5°C, a solution of CH2CI2/DMF 2/1 (4 mL) was added.
The reaction was stirred 5 min, and DIPEA (72 ]}L, 0.56 mmol) was added.
The resulting solution was stirred at -5°C overnight, diluted with f-BuOMe
(10 mL), washed with 10% KHSO4 (10 mL), NaHCO3 (10 mL, sat) and NaCl
(10 mL, sat), dried (Na2SO4), filtered, and concentrated. The crude oil was
purified by column chromatography (silica gel, grad EtOAc/Hex 1/1) to yield
90 (60 mg, 54%). Rf 0.23 (AcOEt/Hexanes 1:1). [a]D20 = -34.6 (c=l,
CH2CI2). !H-NMR (500 MHz, CDC13): 0.89-1.01 (m, 18H), 1.26-1.29 (m,
5H), 1.48 (s, 9H), 1.70-1.62 (m, 2H), 2.03-2.22 (m, 5H), 2.21-2.25 (m, 3H),
2.55-2.60 (m, 1H), 2.82-2.93 (m, 3H), 3.03-3.18 (m, 1H)? 3.45-3.49 (m, 1H),
3.60-3.79 (m, 2H), 4.12-4.15 (m, 1H), 4.50-4.51 (m, 1EQ, 4.92-5.34 (m, 6H),
5.34-5.47 (m, 1H), 7.16-7.35 (m, 9H), 7.44-7.85 (m, 8H). 13C-NMR (125
MHz, CDCI3): 14.1, 16.9, 17.9, 18.4, 19.0, 18.7, 20.2, 20.9, 21.6, 23.7, 24.5,
24.7, 25.5, 28.1, 28.2, 28.9, 29.6, 30.4, 30.6, 33.9, 38.7, 41.0, 43.0, 44.1, 46.8,
48.4, 57.9, 60.3, 60.5, 66.9, 67.0, 67.3, 68.6, 70.3, 77.9, 78.2, 79.1, 125.5,
125.9, 127.0, 127.2, 127.5, 127.7, 127.8, 128.0, 128.3, 128.5, 130.1, 132.2,
133.4, 135.1, 135.2, 136.9, 142.0, 157.0, 157.2, 157.3, 169.9, 171.3, 171.9,
180.8. IR (neat) 3326.4, 3062.0, 2961.1, 2873.6, 2248.6, 1743.7, 1681.9,
1635.0, 1537.2, 1453.5,1367.4, 1170.2. HRMS m/z cald for C6iH81N5Oi4Na
(M+Na): 130.5677 found 130.5634.
[0218] [2-Naphthylala5]-Tamandarin B macrocycle (92): To a solution of
protected linear precursor 90 (60 mg, 0.054 mmol) in MeOH under argon,
Pd(OH)2 (21 mg) was added. The reaction was purged with H2 and stirred
overnight under H2 atmosphere (1 atm). The mixture was filtered through

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Celite®. The filtrate was concentrated to yield the free linear precursor 91
(45 mg, 96%) as a yellow oil, which was used in the next step without
purification.
[0219] The crude amino acid linear precursor (45 mg, 0.050 mmol) was
dissolved in CH3CN (10 mL) and cooled to 0 °C. HATU (46 mg, 0.12 mmol)
was added followed by the dropwise addition of NNM (11 uJL, 0.10 mmol).
The reaction mixture was stirred at 0 °C for 1 h and then overnight. The
reaction mixture was concentrated in vacuo, diluted with EtOAc (10 mL),
washed with 10% KHSO4 (10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL,
sat), dried (Na2SO4), filtered, and concentrated. The crude oil was purified by
column chromatography (silica gel, EtOAc/Hexanes 2/1) to yield protected
macrocycle 92 (28 mg, 65%) as white foam. Rf 0.26 (AcOEt/Hexanes 1/2).
[ob20 = -55.2 (c = 1, CHC13). 'H-NMR (500 MHz, CDC13): 0.90-1.02 (m,
24H), 1.29 (d, 6.6Hz, 3H), 1.48 (s, 9H), 1.71-1.85 (m, 2H), 2.05-2.09 (m, 2H),
2.19-2.23 (m .2H), 2.52 (s, 3H), 2.96 (dd, J= 4.7 and 16.9 Hz, 1H), 3.46 (dd,
J= 10.7 and 14.0 Hz, 1H), 3.67 (dd, J= 4.3 and 13.9 Hz, 1H), 3.73-3.77 (m,
2H), 3.78-3.83 (m, 1H), 3.99-4.05 (m, 1H), 4.39 (dd, J= 3.1 and 10.3 Hz, 1H),
4.56-4.59 (m, 1H), 4.92 (t, J= 12.0 Hz, 1H), 4.97-5.00 (m, 2H), 5.00-5.02 (m,
1H), 7.36-7.37 (m, 1H), 7.49-7.52 (m, 2H), 7.56-7.58 (m, 2H), 7.72-7.74 (m,
1H), 7.76-7.79 (m, 2H), 7.98-8.00 (m, 1H). I3C-NMR (125 MHz, CDC13):
15.6, 18.4, 189, 18.9, 20.6, 21.2, 23.9, 25.3, 25.4, 28.0, 28.4, 28.5, 30.8, 35.6,
39.0, 39.3, 39.5, 47.2, 48.8, 56.3, 57.5, 60.7, 66.0, 69.3, 71.9, 79.6, 80.7,
126.3, 126.9 127.5, 127.8, 128.2, 128.5, 128.9, 132.8, 133.9, 135.9, 156.2,
169.0, 170.1, 170.9, 171.7, 172.0 173.2. IR (neat) 3339.5, 2962.0, 2872.0,
2248.4,1742.0,1665.7,1635.5,1529.6,1451.1,1167.4, 851.3.
[0220] [2-Naphthylala5]-Tamandarin B (93): To a solution of Boc protected
macrocycle 92 (28 mg, 0.032 mmol) in HPLC dioxane (3 mL) was added a
solution of HC1 in dioxane (3 mL). The resulting solution was stirred at room
temperature for 2h. The solution was concentrated and the residue diluted with
CH2CI2 and concentrated again to yield the hydrochloride salt (quantitative
yield) as a white solid, which was used directly in the next step. To a mixture
of the macrocycle amine salt (25 mg, 0.031 mmol) and side chain (14.6 mg,

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0.046 mmol) in CH2C12 (4 mL) at 0 °C was added BOP (20.3 mg, 0.046
mmol) and NMM (14 \iL, 0.12 mmol). After 30 min at 0 °C, the reaction was
stirred at it overnight. The reaction was treated with NaCl solution (2 mL, sat)
and extracted with EtOAc (2x10 mL) The organic layers were washed with
10% HC1 (5 mL), 5% NaHCO3 (5 mL), and NaCl (5 mL, sat), dried (Na2SO4),
filtered, and concentrated. The crude oil (8 mg, 61%) was purified by HPLC.
!H-NMR (300 MHz, CDC13): 8 0.82-0.96 (m, 24H), 1.09-1.28 (in, 14H), 1.31
(d, J=6.8, 3H), 1.39 (d, J=6.8, 3H), 1.64 (m, 3H), 2.10 (m, 4H), 2.48 (s, 3H),
3.10 (s, 3H), 3.09-3.15 (m, 2H), 3.35-3.41 (m, 2H), 3.43-3.82 (m, 5H), 4.29-
4.33 (m, 1H), 4.42-4.47 (m, 1H), 4.65-4.70 (m, 1H), 4.72 (t, J= 6.8,1H), 4.87
(t, 1=8.3, 1H), 5.02 (d, fe 5.3, 1H), 5.29-5.32 (m, 2H), 7.24-7.27 (m, 1H),
7.45-7.63 (m, 4H), 7.67-7.83 (m, 4H). HRMS tn/z calcd for C56H83N7Oi3Na
(M+Na): 1084.5946 found 1084.5987.
EXAMPLE 9
Replacement of an Ester Bond by an Amide Bond in the Tamandarin
Macrocycle
[0221] The preparation of a compound according to Formula XXX was
accomplished as shown in Schemes 27 and 28.



[0222} M-Boc-L-allo-Threonine Methyl Ester (106): To a solution of 7V-Boc-
L-atfo-threonine (500 mg, 2.28 mmol) in DMF (5 mL), KHCO3 (637 mg,
4.56 mmol) and Mel (227 (il, 3.65 mmol) were added and the reaction was
stirred 5h at rt. Water (20 mL) was added and the mixture was extracted with
ether (3x20 mL), dried (Na2SO4) and evaporated to afford the methyl ester 106
(494 mg, 93%) that was used without purification in the next step. ^-NMR
(500 MHz, CDCI3): 1.19-1.25 (m, 3H), 1.48 (s, 9H), 3.78 (s, 3H), 4.05-4.10
(m, 1H), 4.32-4.40 (m, 1H), 4.45-4.54 (m, 1H).
[0223] 2-tert-Butoxycarbonylamino-l,3-butanodiol (107): To a solution of
methyl ester 106 (450 mg, 1.93 mmol) in HPLC EtOH (7 mL), cooled to 0 °C,
sodium borohydride (146 mg, 3.86 mmol) was added in portions. The reaction
mixture was stirred at 0 °C for 2 h and at room temperature for 2h. The
reaction was quenched with NH4CI (sat, 15 mL), extracted with AcOEt (3 x
10 mL), dried (Na2SO4) and evaporated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 2/1) to yield the diol 107
(347 mg, 87%) as colorless oil. Rf 0.21 (AcOEt/Hexanes 2/1). [a]D20 = -10.7
(c = 1, CHCI3). JH-NMR (500 MHz, CDCI3): 1.28 (d, J= 6.9 Hz, 3H), 1.48 (s,

WO 2004/084812 PCT/US2004/008275
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9H), 3.43-3.54 (m, 1H), 3.73 (dd, J=4.0 nd 14.1 Hz, 1H), 3.98 (dd, J=5.9 and
13.1 Hz, 1H), 4.00-4.05 (m, 1H), 5.33-5.40 (m, 1H). 13C-NMR (125 MHz,
CDC13): 20.3, 28.2, 28.3, 56.1, 62.4, 69.9, 79.8, 153.3. IR (neat) 3343.8,
2975.1 ,2931.7,1686.4, 1520.0,1366.6,1248.9,1171.1,1046.3.
[0224] 2-?e?t-ButoxycarbonyIamino-l-(ferr-butyldimethylsilanyloxy)-
3-butanol: To a solution of diol 107 (340 mg, 1.65 mmol) in CH2C12 (4 mL),
cooled to 0 °C, Et3N (274 \iL, 1.98 mmol) and BMAP (8 mg, 0.04 mmol)
were added. The solution was stirred 5 min, and TBSCI (248 mg, 1.65 mmol)
was added. The reaction was stirred 18 h at room temperature quenched with
NH4CI (sat, 15 mL) and extracted with CH2C12 (3 x 10 mL), dried (Na2SO4)
and evaporated. The crude oil was purified by column chromatography (sib'ca
gel, EtOAc/Hexanes 1/9) to yield the TBS alcohol (300 mg, 56%) as colorless
oil. Rf 0.37 (AcOEt/Hexanes 1/9). [a]D20 = -24.7 (c = 1, CHC13). ^-NMR
(500 MHz, CDC13): 0.05 (s, 6H), 0.86 (s, 9H), 1.30 (d, J= 6.8 Hz, 3H), 1.47 (s,
9H), 3.46-3.51 (m, 1H), 4.75-4.80 (m, 1H), 4.89-4.92 (m ,1H), 3.98 (dd, J=1.9
and 11.5 Hz, 1H), 5.22-5.30 (m, 1H). IR (neat) 3447.8, 2954.8, 2857.1,
2362.1,1699.0,1498.3,1254.3,1173.8, 1108.1, 836.9, 777.5.
[0225J 2-^enl-Butoxycarbonylamino-l-(^/t-butyIdimethylsilanyloxy)-3-
methylsulphonyloxybutane (108): To a solution of alcohol (100 mg, 0.31
mmol) in CH2C12 (10 mL), cooled to 0 °C, Et3N (64 yL, 0.46 mmol) and MsCl
(31 \\L, 0.40 mmol) were added. The solution was stirred lh at room
temperature quenched with NH4CI (sat, 15 mL) and extracted with AcOEt(3 x
10 mL), dried (Na2SO4) and evaporated to afford 108 (113 mg, 91%). The
crude residue was used with further purification in the next step. JH-NMR
(500 MHz, CDCI3): 0.04 (s, 6H), 0.88 (s, 9H), 1.45-1.50 (m, 12H), 2.99 (s,
3H), 3.66-3.71 (m, 1H), 4.85-4.90 (m, 1H), 4.80-4.92 (m ,1H).
[0226] 3-Azido-2-te^butoxycarbonylan±io-l-(tert-butyIdimethyl-
silanyloxy)butane (109): To a solution of mesylate 108 (500 rng, 1.26 mmol)
in HMPA (2 mL), NaN3 (409 mg, 6.29 mmol) and 15-crown-5 ether (252 mg,
1.26 mmol) were added. The solution was stirred at 55 °C for 3 h. The reaction
mixture was added to EtOAc (30 mL), washed with brine (10 mL), dried
(Na2SO4) and evaporated. The crude oil was purified by column

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chromatography (silica gel, EtOAc/Hexanes 1/15) to afford azide 109 (160
mg, 34%) as a colorless oil. Rf 0.33 (AcOEt/Hexanes 1/15). [afo20 = -46.6
(c= 0.4, CHC13). *H-NMR (500 MHz, CDCI3): 0.04 (s, 6H), 0.88 (s, 9H),
1.32 (d, J= 6.7 Hz, 3H), 1.48 (s, 9H), 3.51-3.61 (m, 1H), 4.75-4.90 (m, 2H),
4.50-4.62 (m ,1H). I3C-NMR (125 MHz, CDCI3): -5.5, 14.1, 16.5, 25.8, 28.3,
31.6, 55.2, 57.2, 62.4, 174.3. IR (neat) 3326.1, 2954.0, 2931.1, 2857.9,
2359.9, 2107.2, 1718.2, 1005.7, 1471.2, 1366.0, 1254.5, 1167.7, 1098.0,
838.1.
[0227] 3-Amino-2-fert-Butoxycarbonylamino-l-(^rt-butyldimethyl-
silanyloxy)butane (110): To a solution of azide 109 (35 mg, 0.10 mmol) in
EtOH (1 mL), under argon, 10%Pd/C (7 mg) was added. The reaction was
purged with H2 and stirred overnight under H2 atmosphere (1 arm). The
mixture was filtered through celite®. The Filtrate was concentrated to yield the
amine 110 (29 mg, 90%). The crude residue was used without further
purification in the next step. ^-NMR (500 MHz, CDCI3): 0.05 (s, 6H), 0.90
(s, 9H), 1.12 (d, J= 6.7 Hz, 3H), 1.47 (s, 9H), 3.21-3.31 (m, 1H), 3.42-3.48 (m,
1H), 3.63-3.82 (m ,2H), 5.01-5.10 (m, 1H). !3C-NMR (125 MHz, CDC13): -
5.1,19.1, 21.3, 26.2, 28.8,47.1, 56.9, 64.5,176.5.
[0228] 3-(N-Cbz-iV,0-Dimethyltyrosyl)amino-2-fe7t-butoxycarbonylamino-
l~(te/t-butyl-dimethylsilanyloxy)butane (111): To a solution of amine 110
(20 mg, 0.062 mmol) in CH2C12 (1 mL), cooled to 0 °C, NMM (7 uX,
0.069 mmol) and HOBt (25 mg, 0.18 mmol) were added. The solution was
stirred at 0 °C for 15 minutes, and a solution of JV-Cbz-iV,C>-dimethyltyrosine
(21) (21 mg, 0.062 mmol) in CH2C12 (1 mL) was added. Finally a solution of
DCC (14.2 mg, 0.069 mmol) in CH2C12 (1 mL) was added. The mixture was
stirred 16 h at room temperature and filtered to remove the precipitate. The
filtrate was diluted with CH2C12 (10 mL) and washed with 10% KHSO4.
(10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL, sat), dried (Na2SO4),
filtered, and concentrated. The crude oil was purified by column
chromatography (silica gel, EtOAc/Hexanes 1/3) to yield 111 (26 mg, 70%) as
1
a white foam. Rf 0.36 (AcOEt/Hexanes 1/3). [a]D20 = -40.1 (c = 0.5, CHCI3).
^-NMR (500 MHz, CDCI3): 0.09 (s, 6H), 0.95 (s, 9H), 1.14-1.17 (m, 3H),

WO 2004/084812 PCT/US2004/008275
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1.43 (s, 9H), 2.86 (s, 3H), 3.29-3.32 (m, 1H), 3.42-3.48 (m, 1H), 3.60-3.68 (m,
2H), 3.82 (s, 3H), 4.17-4.18 (m, 1H), 4.87-4.89 (m, 1H), 4.95-4.96 (m, 1H),
5.02-5.14 (m, 2H), 5.21-5.22 (m, 2H), 6.34-6.37 (m, 1H), 6.79-6.83 (m, 2H),
6.98-7.00 (m, 1H), 7.01-7.17 (m, 2H), 7.20-7.36 (m, 5H). I3C-NMR (125
MHz, CDC13): -5.5, 17.9, 18.2, 25.8, 28.3, 33.2, 44.5, 55.12, 62.6, 67.3, 57.1,
66.1,113.8,127.5,127.9,128.4,129.8,158.2,171.5. R(neat) 3328.5,2930.3,
2856.3, 2361.0, 2346.6, 2249.0, 1708.8, 1513.7, 1452.5, 1449.5. HRMS m/z
cald for C34H53N5O7Na (M+Na): 666.3550 found 666.3571.
[0229] 3-(Ar-Cbz-A?',O-Diinethyltyrosyl)amino-2-^r^--butoxycarbonylaraino-
1-butanol (112): A solution of 111 (15 mg, 0.086 mmol) in AcOH (1 mL) was
stirred 15 h. at room temperature. The reaction was concentrated to yield 112
(11 mg, 90%) as a colorless oil. The crude residue was used with further
purification in the next step. 'H-NMR (500 MHz, CDCI3): 1.04 (d, J= 6.9 Hz,
3H), 1.45 (s, 9H), 2.81 (s, 3H), 2.88-2.92 (m, 1H), 3.02-3.09 (m, 1H), 3.29-
3.33 (m, 1H), 3.51-3.62 (m, 1H), 3.70-3.79 (m, 1H), 3.78 (s, 3H), 4.02-4.08
(m, 1H), 4.43-4.47 (m, 1H), 4.51-4.55 (m, 1H), 5.05-5.18 (m, 2H), 5.22 (s,
2H), 5.98-5.99 (m, 1H), 6.72 ((d, J= 7.9 Hz, 2H), 6.90-7.01 (m, 2H), 7.22-7.38
(m, 5H).
EXAMPLE 10
Modification to the Lac9 unit
[0230] A compound according to Formula I, wherein R5 contains a modified
lactate unit is prepared as described here.
[0231] (#)-glycidic acid (117) was prepared from (S)-serine following the
procedure described by Petit and Larcheveque, Org. Synth. 75:31-44 (1998).


[0235] The benzyl ester is removed without opening of the epoxide moiety
using LiOH in MeOH. Ar~GlycidyI-L-proline is then used to prepare suitable
R5 moieties to prepare a compound according to Formula I, IA, or II, or
alternatively, N-Glycidyl-L-proline is coupled directly with the free amine of
the macrocycle, e.g., compound 35, deprotected 61,
[0236] 7V-Glycidyl-L-Proline Benzyl Ester (120): To a solution of L-proline
benzyl ester (156 mg, 0.49 mmol) in DMF (2 mL), cooled to 0 °C, NMM (56
(XL, 0.53 mmol) and HOBt (198 mg, 1.47 mmol) were added. The solution
was stirred at 0 °C for 15 minutes and a solution of glycidic acid21 (100 mg,
1.13 mmol) in CH2C12 (2 mL) was added. Finally a solution of DCC (253 mg,
1.23 mmol) in CH2CI2 (2 mL) was added. The mixture was stirred 16 h at
room temperature and filtered to remove the precipitate. The residue was
diluted with CH2C12 (10 mL) and washed with 10% KHSO4 (10 mL), 5%
NaHCO3 (10 mL) and NaCl (10 mL, sat), dried (Na2SO4), filtered and

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concentrated. The crude oil was purified by column chromatography (silica
gel, EtOAc/Hexanes 1/1) to yield 120 (95 mg, 70%) as white foam. Rf 0. 26
(AcOEt/Hexanes 1/1). JH-NMR (500 MHz, CDC13): mixture of rotamers
1.87-1.92 (m, 2H), 1.91-1.98 (m, 2H), 1.99-2.03 (m, 1H), 2.19-2.20 (m, 2H),
2.20-2,22 (m, 1H), 2.66-2,68 (m, 1H), 2,80-2,82 (m, 1H), 2.94-2,95 (m, 2H),
3.30-3,31 (m, 1H), 3.56-3.57 (m, 2H), 357-370 (m, 2H), 3.72-3.85 (m, 1H),
4.57-4.59 (m, 1H), 4.70-4.72 (m, 1H), 5.16 (s, 2H), 5.22-5.29 (m, 2H), 7.25-
7.38 (m, 10H). I3C-NMR (125 MHz, CDC13): 22.2, 24.9, 28.7, 30.9, 31.5,
45.9, 46.3, 46.4, 47.2 ,48.0, 48.5, 58.7, 59.3, 66,9, 61 A, 128.1, 128.2, 128.4,
128.5, 128.7, 170.0, 182.3. HRMS m/z cald for C15HnN04a (M+Na):
298.1055 found 298.1047.
[0237] iV-Glycidyl-L-proline is prepared from compound 120 by hydrolyzing
the benzyl ester with L1OH/H2O in methanol.
EXAMPLE 11
Biological Activity of Tamandarin Analogs
[0238] The experiments described in this example demonstrate that the
tamandarin analogs have the desired bioactivity.
[0239] As used herein, "GI50 " refers to the dose of a compound which is
capable of producing 50% inhibition of cell growth. GI50 is assessed by
comparing growth of cells to which a compound has been administered with
growth of the same cells to which the compound has not been administered.
[0240] As used herein, "LC50" refers to the dose of a compound which is
capable of producing 50% lethality in cells. LC50 is assessed by comparing
death of cells in a population of cells to which a compound has been
administered with the death of cells in a population of the same cells to which
the compound has not been administered.
[0241] tpNCI-60" refers to a 60 rumor cell line panel which from the National
Cancer Institute (NCI, Frederick, Md.). "NCI-60 Mean" is the average GI50 or
LC50 for the panel treated with the selected compound.
[0242] The data for the compounds are disclosed in the following tables.



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HeLa CCL-2 human cervix cervix epitheloid carcinoma
HeLa-APL CCL-3 human cervix cervix epitheloid carcinoma,
characterized as aplidine resistant
cells
PANC1 CRL-1469 human pancreas pancreatic epitheloid carcinoma
[0244] INHIBITION OF CELLS GROWTH BY COLORIMETRIC ASSAY.
[0245] A colorimetric type of assay, using sulforhodamine B (SRB) reaction
has been adapted for a quantitative measurement of cell growth and viability
[following the technique described by Philip Skehan, et al. (1990), New
colorimetric cytotoxicity assay for anticancer drug screening, /. Nail. Cancer
hist. 52:1107-1112].
[0246] This form of assay employs 96 well cell culture microplates of 9 mm
diameter (Faircloth, 1988; Mosmann, 1983). Most of the cell lines are
obtained from American Type Culture Collection (ATCC) derived from
different human cancer types.
[0247] Cells are maintained in RPMI 1640 10% FBS, supplemented with 0.1
g/1 penicillin and 0.1 g/1 streptomycin sulfate and then incubated at 37°C, 5%
CO2 and 98% humidity. For the experiments, cells were harvested from
subconfluent cultures using trypsin and resuspended in fresh medium before
plating.
[0248] Cells are seeded in 96 well microtiter plates, at 5 x 103 cells per well in
aliquots of 195 pi medium, and they are allowed to attach to the plate surface
by growing in drug free medium for 18 hours. Afterward, samples are added
in aliquots of 5 fx\ in a ranging from 10 to 10-8 figfm\, dissolved in
DMSO/EtOH/PBS (0.5:0.5:99). After 48 hours exposure, the antitumor effect
are measured by the SRB methodology: cells are fixed by adding 50 /tl of cold
50% (wt/vol) trichloroacetic acid (TCA) and incubated for 60 minutes at 4°C.
Plates are washed with deionised water and dried. One hundred fii of SRB
solution (0.4% wt/vol in 1% acetic acid) is added to each microtiter well and
incubated for 10 minutes at room temperature. Unbound SRB is removed by

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washing with 1% acetic acid. Plates are air dried and bound stain is solubilized
with Tris buffer. Optical densities are read on a automated spectrophotometric
plate reader at a single wavelength of 490 run.
[0249] The values for mean +/- SD of data from triplicate wells are calculated.
Some parameters for cellular responses can be calculated: GI = growth
inhibition, TGI = total growth inhibition (cytostatic effect) and LC = cell
killing (cytotoxic effect).
[0250] Table 1 illustrates data on the biological activity of the compounds (in
Molar units) of the present invention.
[0251]

Compound
62 Compound 75 Compound 93
DU-145 GI50 6.79E-09 1.81E-06 2.17E-09
TGI 2.49E-08 4.95E-06 4.39E-09
LC50 8.26E-08 9.84E-06 8.83E-09
LN-caP GI50 2.71E-09 5.48E-07 1.98E-09
TGI 7.24E-09 2.97E-06 4.33E-09
LC50 5.84E-08 9.84E-06 9.70E-09
IGROV GI50 1.99E-06 1.99E-06 2.09E-09
TGI 5.29E-06 5.29E-06 1.28E-08
LC50 9.84E-06 9.84E-06 6.66E-07
IGROV-ET GI50 4.56E-07 6.86E-06 1.59E-07
TGI 1.90E-06 9.84E-06 6.54E-07
LC50 6.95E-06 9.84E-06 2.81E-06
SK-BR-3 GI50 3.74E-09 7.89E-07 1.83E-09
TGI 4.05E-08 3.39E-06 1.89E-08
LC50 9.73E-06 9.84E-06 4.65E-06
MEL-28 GI50 1.66E-08 1.50E-06 4.14E-09
TGI 8.24E-08 3.46E-06 1.52E-08
LC50 6.75E-07 8.00E-06 8.79E-08
A-549 GI50 5.15E-08 5.85E-O6 6.96E-09
TGI 3.51E-07 9.84E-06 4.86E-08
LC50 2.90E-06 9.84E-06 5.95E-07
K-562 GI50 7.80E-08 5.87E-06 2.00E-08
TGI 3.03E-07 9.46E-06 4.73E-08
LC50 2.39E-06 9.84E-06 1.98E-07
PANC-1 GI50 3.49E-08 3.75E-06 6.24E-09
TGI 8.82E-07 9.84E-06 9.00E-08
LC50 9.73E-06 9.84E-06 9.41E-06
HT-29 GI50 2.37E-08 4.22E-06 4.60E-09
TGI 2.03E-07 9.84E-06 6.98E-08

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PCT/US2004/008275

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LC50 9.73E-06 9.84E-06 5.58E-06
LOVO GI50
TGI
LC50 3.89E-08
2.31E-07
3.66E-06 3.55E-06
9.84E-06
9.84E-06 6.84E-09
4.27E-08
2.49E-06
LOVO-
DOX GI50
TGI
LC50 2.39E-06
8.40E-06
9.73E-06 9.84E-06
9.84E-06
9.84E-06 4.29E-07
2.33E-06
9.41E-06
HELA GI50
TGI
LC50 3.76E-09
2.61E-08
3.68E-07 1.18B-06
3.60E-06
9.84E-06 2.81E-09
9.79E-09
5.49E-08
HELA-
APL GI50
TGI
LC50 4.11E-07
5.41E-06
9.73E-06 9.84E-06
9.84E-06
9.84E-06 3.64E-O8
1.68E-O7
2.28E-06
[0252] Having now fully described this invention, it will be understood by
those of ordinary skill in the art that the same can be performed within a wide
and equivalent range of conditions, formulations and other parameters without
affecting the scope of the invention or any embodiment thereof. All patents
and publications cited herein are fully incorporated by reference herein in their
entirety.

WO 2004/084812 PCT/US2004/008275
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WHAT IS CLAIMED IS:

I
or a pharmaceutically acceptable salt thereof, wherein
R1 and R2 are independently H or CM alkyl, or R1 and R2 together
form the alkyl ring of a proline or homoproline residue;
R3 is selected from the group consisting of a side chain of an amino
acid and a first fluorophore;'
R4 is H or CH3;
R5 is H, an amine protecting group, an amino acid residue, a
polypeptide, a peptide which contains a second fluorophore, a chemical
moiety bound to a solid support, or a moiety containing from about 1 to about
50 non-hydrogen atoms;
R6 is an isoleucine side chain or a valine side chain;
WisOorNH;
XisOorNH;and
Y is H or a hydroxyl protecting group;
Z is C(O) or C(O)-CH(CH3)-C(O);
provided that if R1 and R2 together form the alkyl ring of a proline
residue, R4 is methyl, and X is O, then R3 is naphthylmethyl.
2. The compound according to claim 1 having the formula


or a pharmaceutically acceptable salt thereof, wherein
10 1*7'
R and R are independently H or C1-4 alkyl, or R and R together
form the alkyl ring of a proline residue;
R3 is selected from the group consisting of a side chain of an amino
acid and a first fluorophore;
R4isHorCH3;
R5 is H, an amine protecting group, an amino acid residue, a
polypeptide, a peptide which contains a second fluorophore, a chemical
moiety bound to a solid support, or a moiety containing from about 1 to about
50 non-hydrogen atoms;
R6 is an isoleucine side chain or a valine side chain;
XisOorNH;and
Y is H or a hydroxyl protecting group;
provided that if R1 and R2 together form the alkyl ring of a proline
residue, R4 is methyl, and X is O, then R3 is naphthylmethyl.
3. The compound according to claim 2, wherein R1 is H and R2 is methyl.
4. The compound according to claim 2, wherein R1 and R2 are methyl.
5. The compound according to claim 2, wherein R1 and R2 together form
the alkyl ring of a proline residue.

WO 2004/084812 PCT/US2004/008275
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6. The compound according to claim 2, wherein R is a side chain of an
ammo acid.
7. The compound according to claim 2, wherein R3 is naphtylmethyl.
8. The compound according to claim 2, wherein R is a benzyl group
optionally substituted with OH, OCH3, CO(C6H5), F, Cl, Br, I, CH3, or C2H5.
9. The compound according to claim 2, wherein R3 contains a fluorophore.
10. The compound according to claim 2, wherein R4 is CH3.
11. The compound according to claim 2, wherein R4 is H.
12. The compound according to claim 2, wherein R5 is H.
13. The compound according to claim 2, wherein R5 is an amine protecting
group.
14. The compound according to claim 2, wherein R5 is an amino acid
residue or a polypeptide.
15. The compound according to claim 2, wherein R5 contains a fluorophore.
16. The compound according to claim 2, wherein R5 is selected from the
group consisting of -(iV-methyl)leucine;
-(Af-methyl)leucine-proline;
-(iV-CBz-N-methyl)leucine;
-(JV-methyl)leucine-prolme-lactate;
-(iV-methyl)leucine-proline-pyruvate;
-(i^-methyljleucine-proline-lactate-glutainine-pyroglutamate;
-(iV-methyl)leucine-proline-lactate-glutamine-cyclopentanoate;

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-(A^-methyl)leucine-proline-Iactate-leucine-pyroglutamate;
-(iV-methyl)leucine-proline-lactate-glutamine-cyclopentanoate;
-(Jv"-methyl)leucine-proline-alanine-leucine-pyroglutamate, and
-(A^-methyl)leucine-proline-(iV-methyl)aIanine-leucine-pyroglutamate.
17. The compound according to claim 2, wherein R6 is a valine side chain.
18. The compound according to claim 2, wherein R6 is a leucine side chain.
19. The compound according to claim 2, wherein Y is H.
20. The compound according to claim 2, wherein Y is a hydroxyl protecting
group.
21. The compound according to claim 2, wherein X is O.
22. The compound according to claim 2, wherein X is NH.
23. The compound according to claim 2, wherein R1 and R2 together form
the alkyl ring of a proline residue; R is a benzyl group optionally substituted
with one or more selected from the group consisting of OH, OCH3, CCKCeHs),
F, Cl, Br, I, CH3, and C2H5; R4 is H; R6 is a valine side chain; X is O; and Y is
H.
24. The compound according to claim 2, wherein R1 is H; R2 is CH3; R^is a
benzyl group optionally substituted with one or more selected from the group
consisting of OH, OCH3, CO(C6H5), F, Cl, Br, I, CH3, and C2H5; R4 is CH3;
R5 is as defined above; R6 is a valine side chain; X is O; and Y is H.
25. The compound according to claim 2, wherein R1 is CH3; R2 is CH3; R3
is a benzyl group optionally substituted with one or more selected from the

WO 2004/084812 PCT/US2004/008275
-122-
group consisting of OH, OCH3, CO(QH5), F, Cl> Br, I, CH3, and C£t5,
preferably OCH3; R4 is CH3; R6 is a valine side chain; X is O; and Y is H.
26. The compound according to claim 2, wherein R1 and R2 together form
the alkyl ring of a proline residue; R3 is a naphthylmethyl group; R4 is CH3; R6
is a valine side chain; X is O; and Y is H.
27. The compound according to claim 2, wherein R5 consists of 1-5 amino
acid residues.





34. A composition comprising a compound according to any of one of
claims 1-33 and a pharmaceutically compatible excipient or carrier.
35. A method of inhibiting, treating, or preventing tumorigenesis,
comprising contacting a cell with an effective amount of a compound
according to any one of claims 1-33..
36. A method of preventing or inhibiting the growth of a cancer cell,
comprising contacting a cancer cell with an effective amount of a compound
according to any one of claims 1-33.
37. A method of inhibiting or preventing protein synthesis, comprising
contacting a cell or cellular component with an effective amount of a
compound of any one of claims 1-33.
38. A method of enhancing' apoptosis, comprising contacting a cell or
cellular component with an effective amount of a compound according to any
one of claims 1-33.
39. A method of providing immunosuppresive therapy, comprising
administering to a subject in need thereof an effective amount of a compound
according to any one of claims 1-33.

WO 2004/084812 PCT/US2004/008275
-100-
EtOAc/Hexanes 2/1) to yield protected macrocycle 85 (94 mg, 48%) as a
white foam. Rf 0.30 (AcOEt/Hexanes 1/2). [a]D20 = -28.77 (c = 1, CHC13).
^-NMR (500 MHz, CDC13): 0.88-1.00 (m, 17H), 1.22-1.33 (m, 3H), 1.17-
1.30 (m, 4H), 1.49 (s, 9H), 1.48-1.52 (m, 1H), 1.97-2.05 (m, 2H), 2.09-2.14
(m, 1H), 2.30-2.40 (m, 1H), 2.51 (dd, J= 7.0 and 13.2 Hz, 1H), 2.59-2.61 (m,
2H), 2.80 and 2.91 (s, 3H), 2.88-2.91 (m, 1H), 3.09 and 3.15 (s, 3H), 3.10-3.15
(m, 1H), 3.28-3.33 (m, 1H), 3.49-3.51 (m, 1H), 3.75 and 3.78 (s, 3H), 4.12-
4.13 (m, 1H), 4.22-4.23 (m, 1H), 4.51-4.52 (m, 1H), 4.92-4.93 (m, 1H), 4.94
(d, J=5.8 Hz, 1H), 4.98-5.05 (m, 2H), 5.19-5.21 (m, 1H), 5.25-5.27 (m, 1H),
6.80 (t, J= 6.5 Hz, 2H), 7.03 (dd, J= 8.5 and 11.7 Hz, 2H), 7.40 (d, J= 8.6 Hz,
1H), 7.71 (d, J= 9.2 Hz, 1H), 7.89 (d, J= 8.1 Hz, 1H). 13C-NMR (125 MHz,
CDCI3): 14.1, 14.8, 15.5, 18.3, 18.8, 19.0, 19.4, 20,6, 23.8, 23.9, 25.0, 25.3,
28.4, 30.1, 30.2, 30.8, 30.9, 33.9, 34.8, 38.9, 39.3, 47.7, 48.2, 51.3, 55.6, 56.5,
62.0 66.6, 69.3, 69.5, 70.3, 71.5, 79.3, 80.9, 81.0, 114.4, 114.7, 128.4, 129.7,
130.2, 130.4, 130.8 ,156.2, 156.4, 159.0, 159.2, 169.1, 170.0, 170.2, 171.4,
171.8 ,172.0, 172.9, 174.1, 174.3. IR (neat) 3334.2, 2960.5, 1745.1, 1658.6,
1961.2, 1514.1, 1248.5, 1164.1, 1082.6. HRMS m/z cald for C42H67N5Oi2Na
856.4683 (M+Na): found 856.4617.
[0211] [iV-Me-Ala^-Tamandarin B (120): To a solution of Boc protected
macrocycle 85 (15 mg, 0.018 mmol) in HPLC dioxane (5 mL) was added a
solution of HC1 in dioxane (5 mL). The resulting solution was stirred at room
temperature for 4h. The solution was concentrated, and the residue was diluted
with CH2CI2 and concentrated again to yield the hydrochloride salt (14 mg,
quantitative yield) as a white solid, which was used directly in the next step.
To a mixture of the macrocycle amine salt (14 mg, 0.018 mmol) and side
chain (10 mg, 0.031 mmol) in CH2C12 (3 mL) at 0 °C was added BOP (14 mg,
0.031 mmol) and NMM (9 pL, 0.72 mmol). After 30 min at 0 °C, the reaction
was stirred at rt overnight The reaction was treated with NaCl solution
(10 mL, sat) and extracted with EtOAc (3x10 mL) The organic layers were
washed with 10% HC1 (10 mL), 5% NaHCO3 (10 mL) and NaCl (10 mL, sat),
dried (Na2SO4), filtered and concentrated. The crude oil (13 mg) was purified
by HPLC.


The present invention is directed to a compound of Formula I wherein R1, R2, R3, R4, R5, R6, W, X, Y, and Z are defined herein. The compounds of the present invention are useful as anticancer agents. Specifically, the compounds are useful for treating or preventing cancer and tumor growth. The present invention is also directed to compositions comprising a compound according to the above formula. The present invention is also directed to methods of using a compound according to the above formula

Documents:

02072-kolnp-2005-abstract.pdf

02072-kolnp-2005-claims.pdf

02072-kolnp-2005-description complete.pdf

02072-kolnp-2005-drawings.pdf

02072-kolnp-2005-form 1.pdf

02072-kolnp-2005-form 3.pdf

02072-kolnp-2005-form 5.pdf

02072-kolnp-2005-international publication.pdf

2072-kolnp-2005-granted-abstract.pdf

2072-kolnp-2005-granted-claims.pdf

2072-kolnp-2005-granted-correspondence.pdf

2072-kolnp-2005-granted-description (complete).pdf

2072-kolnp-2005-granted-drawings.pdf

2072-kolnp-2005-granted-examination report.pdf

2072-kolnp-2005-granted-form 1.pdf

2072-kolnp-2005-granted-form 18.pdf

2072-kolnp-2005-granted-form 3.pdf

2072-kolnp-2005-granted-form 5.pdf

2072-kolnp-2005-granted-gpa.pdf

2072-kolnp-2005-granted-reply to examination report.pdf

2072-kolnp-2005-granted-specification.pdf


Patent Number 236057
Indian Patent Application Number 2072/KOLNP/2005
PG Journal Number 38/2009
Publication Date 18-Sep-2009
Grant Date 17-Sep-2009
Date of Filing 21-Oct-2005
Name of Patentee JOULLIE MADELEINE M
Applicant Address 288 ST.JAMES PLACE, PHILADELPHIA, PENNSYLVANIA
Inventors:
# Inventor's Name Inventor's Address
1 JOULLIE MADELEINE M 288 ST.JAMES PLACE, PHILADELPHIA, PENNSYLVANIA 19106
PCT International Classification Number A61K
PCT International Application Number PCT/US2004/008275
PCT International Filing date 2004-03-19
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/456,967 2003-03-21 U.S.A.