Note: Descriptions are shown in the official language in which they were submitted.
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
N-ALKYLATED PEPTIDES HAVING ANTIANGIOGENIC ACTIVITY
Technical Field
The invention relates to novel compounds having activity useful for treating
conditions which arise or are exacerbated by angiogenesis, pharmaceutical
compositions
~ o comprising the compounds, methods of treatment using the compounds, and
methods of
inhibiting angiogenesis.
Background of the Invention
Angiogenesis is the fundamental process by which new blood vessels are formed
~ 5 and is essential to a variety of normal body activities (such as
reproduction, development
and wound repair). Although the process is not completely understood, it is
believed to
involve a complex interplay of molecules which both stimulate and inhibit the
growth of
endothelial cells, the primary cells of the capillary blood vessels. Under
normal conditions
these molecules appear to maintain the microvasculature in a quiescent state
(i.e., one of
2o no capillary growth) for prolonged periods that may last for weeks, or in
some cases,
decades. However, when necessary, such as during wound repair, these same
cells can
undergo rapid proliferation and turnover within as little as five days
(Folkman, J. and
Shing, Y., The Journal of Biological Chemistry, 267(16): 10931-10934, and
Folkman, J.
and Klagsbrun, M., Science, 235: 442-447 ( 1987)).
25 Although angiogenesis is a highly regulated process under normal
conditions,
many diseases (characterized as "angiogenic diseases") are driven by
persistent
unregulated angiogenesis. Otherwise stated, unregulated angiogenesis may
either cause a
particular disease directly or exacerbate an existing pathological condition.
For example,
ocular neovascularization has been implicated as the most common cause of
blindness. In
3o certain existing conditions such as arthritis, newly formed capillary blood
vessels invade
the joints and destroy cartilage. In diabetes; new capillaries formed in the
retina invade the
vitreous, bleed, and cause blindness. Growth and metastasis of solid tumors
are also
angiogenesis-dependent (Folkman, J., Cancer Research, 46: 467-473 (1986),
Folkman, J.,
Journal of the National Cancer Institute, 82: 4-6 (1989)). It has been shown,
for example,
35 that tumors which enlarge to greater than 2 mm, must obtain their own blood
supply and
do so by inducing the growth of new capillary blood vessels. Once these new
blood
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
vessels become embedded in the tumor, they provide a means for tumor cells to
enter the
circulation and metastasize to distant sites, such as the liver, the lung, and
the bones
(Weidner, N., et. al., The New England Journal ofMedicine, 324(1): 1-8
(1991)).
Several angiogenesis inhibitors are currently under development for use in
treating
angiogenic diseases (Gasparini, G. and Harris, A.L., J Clin Oncol 13(3): 765-
782, (1995)).
A number of disadvantages have been associated with many of these compounds. A
potent angiogenesis inhibitor, for example suramin, can cause severe systemic
toxicity in
humans at doses required to reach antitumor activity. Other compounds, such as
retinoids,
interferons, and antiestrogens are safe for human use, but have only a weak
anti-
~ o angiogenic effect.
A new class of compounds having particularly effective in vitro and in vivo
angiogenesis inhibiting properties, as well as a promising toxicity profile,
has been
described in commonly-owned U.S. Patent Application No. 09/316,888, filed May
21,
1999. Novel changes in position 3 of the new angiogenesis inhibitors is
described in
~5 copending provisional U.S. Patent Application Ser. No. 60/166,791, filed
November 22,
1999. Although the compounds have demonstrated satisfactory enzymatic
stability and
bioavailability, it would be desirable to prepare analogs of the
antiangiogenic peptides
having enhanced stability against in vivo enzymatic cleavage, improved
pharmacokinetics,
increased water solubility, and potentially better oral bioavailability.
Summary of the Invention
The present invention relates to a novel class of compounds having
angiogenesis-
inhibiting properties. The invention provides nona- and decapeptides wherein
the nitrogen
atom of at least one of the amide bonds of an amino acid residue in positions
2 through 9
of the peptide is N-alkylated. Compounds of the invention exhibit enhanced
metabolic
stability, improved pharmacokinetics, increased water solubility, and
potentially better oral
bioavailability.
In one aspect, the present invention provides a compound of formula (I)
Xaal-Xaa2-Xaa3-Xaa4-XaaS-Xaa6-Xaa~-Xaag-Xaa9-Xaa~o-Xaa~~ (I), (SEQ ID NO:1)
I 2 3 4 5 6 7 8 9
or a pharmaceutically acceptable salt, prodrug, ester, or solvate thereof,
wherein
at least one amide bond of an amino acid residue represented by Xaa3, Xaa4,
XaaS,
Xaab, Xaa,, Xaag, Xaa~, and Xaa,o is N-alkylated;
2
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Xaa, is absent or Xaa, is selected from the group consisting of hydrogen, N-
methylprolyl, and an acyl group, wherein the acyl group is selected from the
group
consisting of
R'-(CHZ)~-C(O)-, wherein n is an integer from 0 to 8 and R' is selected from
the group consisting of N-acetylamino, alkoxy, alkyl, aryl, carboxy,
cycloalkenyl, cycloalkyl, heterocycle, and hydroxy; and
RZ-CHzCHz-O-(CHZCHZO)P CHz-C(O)-, wherein p is an integer from 1 to 8
and RZ is selected from the group consisting of hydrogen, N-acetylamino,
and alkyl;
provided that Xaa, is absent only when Xaaz is N-(R3)-prolyl;
XaaZ is an N-alkylated amino acid selected from the group consisting of N-(R3)-
alanyl, N-(R3)-glycyl, N-(R3)-norvalyl, and N-(R3)-prolyl, wherein R3 is C,-CS-
alkyl; or XaaZ is an N-unalkylated amino acid selected from the group
consisting of
(3-alanyl,
D-alanyl,
4-aminobutyryl,
( 1 R,3 S)-1-aminocyclopentane-3-carbonyl,
( 1 S,3R)-1-aminocyclopentane-3-carbonyl,
( 1 R,4S)-1-aminocyclopent-2-ene-4-carbonyl,
( 1 S,4R)-1-aminocyclopent-2-ene-4-carbonyl,
asparaginyl,
3-(4-chlorophenyl)alanyl,
3-(4-cyanophenyl)alanyl,
glutaminyl,
glutamyl,
glycyl,
4-hydroxyprolyl,
3-(4-methylphenyl)alanyl,
3o prolyl,
seryl, and
threonyl;
Xaa3 is an N-alkylated amino acid selected from the group consisting of N-(R3)-
alanyl, N-(R3)-glycyl, N-(R3)-leucyl, and N-(R3)-phenylalanyl, wherein R3 is
as
3
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
defined above; or Xaa3 is an N-unalkylated amino acid selected from the group
consisting of
alanyl,
(1 S,3R)-1-aminocyclopentane-3-carbonyl,
( 1 S,4R)-1-aminocyclopent-2-ene-4-carbonyl,
asparaginyl,
aspartyl,
3-(3-cyanophenyl)alanyl,
3-(4-cyanophenyl)alanyl,
1 o glutaminyl,
glycyl,
leucyl,
lysyl(N-epsilon-acetyl),
3-(4-methylphenyl)alanyl,
norvalyl,
prolyl, and
phenylalanyl;
Xaa4 is an N-alkylated amino acid selected from the group consisting of N-(R3)-
2o alanyl, N-(R3)-glycyl, N-(R3)-homophenylalanyl, N-(R3)-isoleucyl, N-(R3)-
leucyl,
N-(R3)-norvalyl, N-(R3)-phenylalanyl, N-(R3)-D-phenylalanyl, N-(R3)-Beryl, N-
(R3)-tyrosyl, N-(R3)-valyl, and N-(R3)-D-valyl, wherein R3 is as defined
above; or
Xaa4 is an N-unalkylated amino acid selected from the group consisting of
alanyl,
alloisoleucyl,
allylglycyl,
2-aminobutyryl,
( 1 R,4S)-aminocyclopent-2-ene-4-carbonyl,
asparaginyl,
3o aspartyl,
3-[2-(5-bromothienyl)]alanyl,
3-(3-chlorophenyl)alanyl,
3-(4-chlorophenyl)alanyl,
3-(3-cyanophenyl)alanyl,
cyclohexylalanyl,
3-(3,4-dimethoxyphenyl)alanyl,
4
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
3-(3-fluorophenyl)alanyl,
3-(4-fluorophenyl)alanyl,
glutaminyl,
glycyl,
histidyl,
homophenylalanyl,
homoseryl,
isoleucyl,
leucyl,
lysyl(N-epsilon-acetyl),
methionyl,
methionyl(sulfone),
3-(4-methylphenyl)alanyl,
3-(naphth-1-yl)alanyl,
~ 5 3-(naphth-2-yl)alanyl,
norornithyl,
norvalyl,
phenyalanyl,
phenylglycyl,
2o prolyl,
3-(3-pyridyl)alanyl,
3-(4-thiazolyl)alanyl,
3-(2-thienyl)alanyl,
Beryl,
25 seryl(O-benzyl),
styrylalanyl,
tryptyl,
tyrosyl,
valyl, and
3o D-valyl;
XaaS is an N-alkylated amino acid selected from the group consisting of N-(R3)-
D-
homophenylalanyl, N-(R3)-D-isoleucyl, N-(R3)-D-leucyl, and N-(R3)-D-
phenylalanyl, wherein R3 is as defined above; or XaaS is an N-unalkylated
amino
35 acid selected from the group consisting of
D-alanyl,
5
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
alloisoleucyl,
D-alloisoleucyl,
D-2-aminobutyryl,
D-3-(4-aminophenyl)alanyl,
D-asparaginyl,
D-3-(3-benzothienyl)alanyl,
D-t-butylglycyl,
D-(chlorophenyl)alanyl,
D-citrullyl,
D-3-(3-cyanophenyl)alanyl,
D-cyclohexylalanyl,
cyclohexylglycyl,
D-cysteinyl(S-acetamidomethyl),
D-cysteinyl(S-t-butyl),
~ 5 D-3-(3,4-difluorophenyl)alanyl,
D-(3,4-dimethoxyphenyl)alanyl,
D-glutaminyl,
glycyl,
D-homophenylalanyl,
2o D-homoseryl,
isoleucyl,
D-isoleucyl,
D-leucyl,
D-lysyl(N-epsilon-nicotinyl),
25 D-lysyl,
D-methionyl,
D-3-(4-methylphenyl)alanyl,
D-3-(naphth-1-yl)alanyl,
D-3-(naphth-2-yl)alanyl,
3o D-3-(4-nitrophenyl)alanyl,
D-norleucyl,
D-ornithyl,
D-penicillaminyl(S-acetamidomethyl),
D-penicillaminyl(S-benzyl),
35 D-penicillaminyl(S-methyl),
D-penicillaminyl,
6
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
D-3-(pentafluorophenyl)alanyl,
D-phenylalanyl,
D-prolyl,
D-seryl(O-benzyl),
D-seryl,
D-(2-thienyl)alanyl,
D-threonyl(O-benzyl),
D-threonyl,
D-3-(3-trifluoromethylphenyl)alanyl,
D-(3,4,5-trifluorophenyl)alanyl,
D-tryptyl,
D-tyrosyl(O-ethyl),
D-tyrosyl, and
D-valyl;
Xaab is an N-alkylated amino acid selected from the group consisting of N-(R3)-
aspartyl, N-(R3)-glutamyl, N-(R3)-glycyl, N-(R3)-Beryl, N-(R3)-threonyl, N-
(R3)-
threonyl(O-benzyl), and N-(R3)-tyrosyl, wherein R3 is as defined above; or
Xaab is
an N-unalkylated amino acid selected from the group consisting of
2o alanyl,
allothreonyl,
D-allothreonyl,
allylglycyl,
asparaginyl,
aspartyl,
glutaminyl,
glycyl,
histidyl,
homoseryl,
D-homoseryl,
3-(4-hydroxymethylphenyl)alanyl,
isoleucyl,
lysyl(N-epsilon-acetyl),
methionyl,
3-(naphth-2-yl)alanyl,
norvalyl,
7
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
octylglycyl,
prolyl,
3-(3-pyridyl)alanyl,
seryl,
D-Beryl,
threonyl,
D-threonyl,
tryptyl,
tyrosyl, and
tyrosyl(O-methyl);
Xaa, is an N-alkylated amino acid selected from the group consisting of N-(R3)-
alanyl, N-(R3)-glycyl, N-(R3)-isoleucyl, N-(R3)-leucyl, N-(R3)-D-leucyl, N-
(R3)-
norleucyl, N-(R3)-norvalyl, N-(R3)-Beryl, N-(R3)-threonyl, and N-(R3)-valyl,
~ 5 wherein R3 is as defined above; or Xaa, is an N-unalkylated amino acid
selected
from the group consisting of
alanyl,
allothreonyl,
allylglycyl,
20 3-(4-amidophenyl)alanyl,
2-aminobutyryl,
arginyl,
asparaginyl,
cyclohexylalanyl,
25 glutaminyl,
D-glutaminyl,
glycyl,
homoalanyl,
homoseryl,
30 4-hydroxyprolyl,
leucyl,
D-leucyl,
lysyl(N-epsilon-acetyl),
methionyl sulfone,
35 methionyl sulfoxide,
methionyl,
8
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
norleucyl,
norvalyl,
D-norvalyl,
octylglycyl,
ornithyl(N-delta-acetyl),
phenylalanyl,
propargylglycyl,
Beryl,
D-seryl,
threonyl,
tryptyl,
tyrosyl, and
valyl;
~ 5 XaaB is an N-alkylated amino acid selected from the group consisting of N-
(R3)-
alanyl, N-(R3)-D-alanyl, N-(R3)-isoleucyl, and N-(R3)-leucyl, wherein R3 is as
defined above; or Xaa$ is an N-unalkylated amino acid selected from the group
consisting of
alanyl,
2o alloisoleucyl,
D-alloisoleucyl,
allylglycyl,
citrullyl,
glycyl,
25 isoleucyl,
D-isoleucyl,
leucyl,
D-leucyl,
lysyl(N-epsilon-acetyl),
3o D-lysyl(N-epsilon-acetyl),
methionyl,
3-(naphth-1-yl)alanyl,
norvalyl,
prolyl,
35 D-prolyl, and
valyl;
9
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Xaaq is the N-alkylated amino acid N-(R3)-arginyl, wherein R3 is as defined
above;
or Xaag is an N-unalkylated amino acid selected from the group consisting of
[(4-amino-N-isopropyl)cyclohexyl]alanyl,
3-(4-amino-N-isopropylphenyl)alanyl,
arginyl(NGN~~diethyl),
arginyl,
D-arginyl,
citrullyl,
glutaminyl,
3-(4-guanidinophenyl)alanyl,
histidyl,
homoarginyl,
lysyl(N-epsilon-isopropyl),
~ 5 lysyl(N-epsilon-nicotinyl),
lysyl,
norarginyl,
ornithyl,
ornithyl[N-delta-(2-imidazolinyl)],
20 ornithyl(N-delta-isopropyl), and
3-(3-pyridyl)alanyl;
Xaa,o is an N-alkylated amino acid selected from the group consisting of N-
(R3)-
alanyl, N-(R3)-D-alanyl, N-(R3)-glycyl, N-(R3)-homoalanyl, and N-(R3)-
norvalyl,
25 wherein R3 is as defined above; or Xaa,o is an N-unalkylated amino acid
selected
from the group consisting of
D-alanyl,
2-aminobutyryl,
D-2-aminobutyryl,
30 2-aminoisobutyryl,
3,4-dehydroprolyl,
4-hydroxyprolyl,
phenylalanyl,
prolyl,
35 D-prolyl,
1,2,3,4-tetrahydroisoquinoline-3-carbonyl, and
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
D-valyl; and
Xaa" is a hydroxy group or an amino acid amide selected from the group
consisting of:
alanylamide,
D-alanylamide,
alanylethylamide,
D-alanylethylamide,
azaglycylamide,
1 o glycylamide,
glycylethylamide,
lysyl(N-epsilon-acetyl),
D-lysyl(N-epsilon-acetyl),
N-methyl-D-alanylamide,
sarcosylamide,
serylamide,
D-serylamide,
a residue represented by the formula
R4
-NH-(CH2)S-CHRS
and
2o a group represented by the formula -NH-R6; wherein
s is an integer from 0 to 8;
R4 is selected from the group consisting of hydrogen, alkyl, and a 5-
to 6-membered cycloalkyl ring;
RS is selected from the group consisting of hydrogen, alkoxy, alkyl,
aryl, cycloalkenyl, cycloalkyl, heterocycle, and hydroxy;
provided that s is not zero when RS is hydroxy or alkoxy; and
R6 is selected from hydrogen and hydroxy.
In another aspect, the present invention provides a composition for treating a
3o patient in need of anti-angiogenesis therapy comprising a compound of
formula (I) in
combination with a pharmaceutically acceptable carrier.
Yet another aspect of the present invention provides a method for treating a
patient
in need of anti-angiogenesis therapy comprising administering to the patient a
therapeutically effective amount of a compound of formula (I).
11
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Still yet another aspect of the present invention provides a composition for
the
treatment of a disease selected from cancer, arthritis, psoriasis,
angiogenesis of the eye
associated with infection or surgical intervention, macular degeneration, and
diabetic
retinopathy comprising a compound of formula (I) in combination with a
pharmaceutically
acceptable carrier.
In yet another aspect, the present invention provides a method of isolating a
receptor from an endothelial cell comprising binding a compound of formula (I)
to the
receptor to form a peptide receptor complex, isolating the peptide receptor
complex, and
purifying the receptor.
Detailed Description of the Invention
Definition of Terms
As used herein, the singular forms "a", "an", and "the" include plural
reference
unless the context clearly dictates otherwise.
As used in the present specification the following terms have the meanings
indicated:
The term "N-acetylamino," as used herein, refers to -NHC(O)CH3.
The term "acyl," as used herein, refers to an alkyl group attached to the
parent
molecular moiety through a carbonyl group.
2o The term "alkoxy," as used herein, refers to an alkyl group attached to the
parent
molecular moiety through an oxygen atom.
The term "alkyl," as used herein, refers to a monovalent group derived from a
straight or branched chain saturated hydrocarbon by the removal of a hydrogen
atom.
Preferred alkyl groups for the present invention invention are alkyl groups
having from
one to five carbon atoms (C,-CS alkyl). Alkyl groups of one to three carbon
atoms (C,-C3
alkyl) are more preferred for the present invention.
The term "amino," as used herein, refers to -NHZ.
The term "aryl," as used herein, represents a mono- or bicyclic carbocyclic
ring
system having one or two aromatic rings and is exemplified by phenyl,
naphthyl, 1,2-
3o dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, fluorenyl, indanyl, indenyl,
and the like. The
aryl groups of the present invention can be optionally substituted with one,
two, three,
four, or five substitutents independently selected from the group consisting
of alkoxy,
alkyl, carboxy, and halo.
The term "carbonyl," as used herein, refers to -C(O)-.
The term "carboxy," as used herein, refers to COZH.
The term "cycloalkenyl," as used herein, refers to a non-aromatic cyclic or
bicyclic
12
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
ring system having three to ten carbon atoms and one to three rings, wherein
each five-
membered ring has one double bond, each six-membered ring has one or two
double
bonds, each seven- and eight-membered ring has one to three double bonds, and
each nine-
to ten-membered ring has one to four double bonds. Examples of cycloalkenyl
groups
include cyclohexenyl, octahydronaphthalenyl, norbornylenyl, and the like. The
cycloalkenyl groups of the present invention can be optionally substituted
with one, two,
three, four, or five substituents independently selected from the group
consisting of
alkoxy, alkyl, carboxy, halo, and hydroxy.
The term "cycloalkyl," as used herein, refers to a saturated monocyclic,
bicyclic, or
o tricyclic hydrocarbon ring system having three to twelve carbon atoms.
Examples of
cycloalkyl groups include cyclopropyl, cyclopentyl, bicyclo[3.1.1]heptyl,
adamantyl, and
the like. The cycloalkyl groups of the present invention can be optionally
substituted with
one, two, three, four, or five substituents independently selected from the
group consisting
of alkoxy, alkyl, carboxy, halo, and hydroxy.
~ 5 The term "halo," as used herein, refers to F, Cl, Br, or I.
The term "heterocycle," as used herein, refers to a five-, six-, or seven-
membered
ring containing one, two, or three heteroatoms independently selected from the
group
consisting of nitrogen, oxygen, and sulfur. The five-membered ring has zero to
two
double bonds and the six- and seven-membered rings have zero to three double
bonds.
2o The term "heterocycle" also includes bicyclic groups in which the
heterocycle ring is fused
to an aryl group. The heterocycle groups of the present invention can be
attached through
a carbon atom or a nitrogen atom in the group. Examples of heterocycles
include, but are
not limited to, furyl, thienyl, pyrrolyl, pyrrolidinyl, oxazolyl, thiazolyl,
imidazolyl,
imidazolinyl, pyrazolyl, isoxazolyl, isothiazolyl, piperidinyl, morpholinyl,
25 thiomorpholinyl, piperazinyl, pyridinyl, indolyl, indolinyl, benzothienyl,
and the like. The
heterocycle groups of the present invention can be optionally substituted with
one, two,
three, or four substituents independently selected from the group consisting
of alkoxy,
alkyl, carboxy, halo, and hydroxy.
The term "hydroxy," as used herein, refers to -OH.
3o The term "nicotinyl," as used herein, refers to the acyl group derived from
nicotinic
acid, i.e. pyridine-3-carboxylic acid. The term "2-Me-nicotinyl" or "2-
methylnicotinyl"
refers to a nicotinyl moiety substituted with a methyl group at the carbon
adjacent to the
nitrogen atom in the 2-position.
The term "nitrogen protecting group" or "N-protecting group," as used herein,
35 refers to an easily removable group which is known in the art to protect an
amino group
against undesirable reaction during synthetic procedures and to be selectively
removable.
13
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
The use of nitrogen protecting groups is well known in the art for protecting
groups
against undesirable reactions during a synthetic procedure and many such
protecting
groups are known (see, for example, T.H. Greene and P.G.M. Wuts, Protective
Groups in
Organic Synthesis, 2nd edition, John Wiley & Sons, New York (1991). Examples
ofN-
protecting groups include, are not limited to, acyl groups including acetyl,
trifluoroacetyl,
acylisothiocyanate, aminocaproyl, benzoyl and the like, and acyloxy groups,
including t-
butyloxycarbonyl (Boc) and carbobenzyloxy (Cbz), 9-fluorenylmethoxycarbonyl
(Fmoc),
and the like.
The term "pharmaceutically acceptable ester," as used herein, refers to esters
which
o hydrolyze in vivo and include those that break down readily in the human
body to leave
the parent compound or a salt thereof. Suitable ester groups include, for
example, those
derived from pharmaceutically acceptable aliphatic carboxylic acids,
particularly alkanoic,
alkenoic, cycloalkanoic and alkanedioic acids, in which each alkyl or alkenyl
moiety
advantageously has not more than six carbon atoms. Examples of particular
esters include
15 formates, acetates, propionates, butyrates, acrylates and ethylsuccinates.
The term "pharmaceutically acceptable prodrugs," as used herein, refers to
those
prodrugs of the compounds of the present invention which are, within the scope
of sound
medical judgement, suitable for use in contact with the tissues of humans and
lower
animals with undue toxicity, irritation, allergic response, and the like,
commensurate with
2o a reasonable benefit/risk ratio, and effective for their intended use, as
well as the
zwitterionic forms, where possible, of the compounds of the invention. The
term
"prodrug" refers to compounds that are rapidly transformed in vivo to yield
the parent
compound of the above formula, for example by hydrolysis in blood. A thorough
discussion is provided in T. Higuchi and V. Stella, Pro-drugs as Novel
Delivery Systems,
2s Vol. 14 of the A.C.S. Symposium Series, and in Edward B. Roche, ed.,
Bioreversible
Carriers in Drug Design, American Pharmaceutical Association and Pergamon
Press,
1987, both of which are incorporated herein by reference.
The term "pharmaceutically acceptable salt," as used herein, represents salts
or
zwitterionic forms of the compounds of the present invention which are water
or oil-
3o soluble or dispersible, which are suitable for treatment of diseases
without undue toxicity,
irritation, and allergic response; which are commensurate with a reasonable
benefit/risk
ratio, and which are effective for their intended use. The salts can be
prepared during the
final isolation and purification of the compounds or separately by reacting an
amino group
with a suitable acid. Representative acid addition salts include acetate,
adipate, alginate,
3s citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
camphorate,
camphorsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate,
hexanoate,
14
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-
hydroxyethansulfonate
(isethionate), lactate, maleate, mesitylenesulfonate, methanesulfonate,
naphthylenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate,
pectinate,
persulfate, 3-phenylproprionate, picrate, pivalate, propionate, succinate,
tartrate,
trichloroacetate,trifluoroacetate, phosphate, glutamate, bicarbonate, para-
toluenesulfonate,
and undecanoate. Also, amino groups in the compounds of the present invention
can be
quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and
iodides;
dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl, myristyl, and
steryl chlorides,
bromides, and iodides; and benzyl and phenethyl bromides. Examples of acids
which can
~ o be employed to form therapeutically acceptable addition salts include
inorganic acids such
as hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such
as oxalic,
malefic, succinic, and citric.
Basic addition salts can be prepared during the final isolation and
purification of
the compounds by reacting a carboxy group with a suitable base such as the
hydroxide,
~ 5 carbonate, or bicarbonate of a metal cation or with ammonia or an organic
primary,
secondary, or tertiary amine. The cations of pharmaceutically acceptable salts
include
lithium, sodium, potassium, calcium, magnesium, and aluminum, as well as
nontoxic
quaternary amine cations such as ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine,
20 diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-
methylpiperidine, N-methylmorpholine, dicyclohexylamine, procaine,
dibenzylamine,
N,N-dibenzylphenethylamine, 1-ephenamine, and N,N'-dibenzylethylenediamine.
Other
representative organic amines useful for the formation of base addition salts
include
ethylenediamine, ethanolamine, diethanolamine, piperidine, and piperazine.
25 The term "pharmaceutically acceptable solvate," as used herein, refers to
an
aggregate that comprises one or more molecules of the solute, such as a
compound of
formula (I), with one or more molecules of solvent.
The term "receptor," as used herein, refers to chemical groups or molecules on
the
cell surface or in the cell interior that have an affinity for a specific
chemical group or
3o molecule. Isolation of receptors relevant to the antiangiogenic activity of
the peptide of
the invention can provide useful diagnostic tools.
The term "shikimyl," as used herein, refers to the acyl residue derived from
shikimic acid or [3R-(3a,4a,5(3)-3,4,5-trihydroxy]-1-cyclohexene-1-carboxylic
acid. A
"dihydroshikimyl" group denotes the fully saturated analog of shikimic acid.
35 The term "succinyl," as used herein, refers to the acyl residue derived
from
succinic acid or (1,4-dioxobutyl)-1-carboxylic acid.
is
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Unless indicated otherwise by a "D-" prefix, e.g. D-Ala or N-Me-D-Ile, the
stereochemistry of the a-carbon of the amino acids and aminoacyl residues in
peptides
described in this specification and the appended claims is the natural or "L"
configuration.
The Cahn-Ingold-Prelog "R" and "S" designations are used to specify the
stereochemistry
of chiral centers in certain acyl substituents at the N-terminus of the
peptides of this
invention. The designation "R,S" is meant to indicate a racemic mixture of the
two
enantiomeric forms. This nomenclature follows that described in R.S. Cahn, et
al., Angew.
Chem. Int. Ed. Engl., 5, 385-415 (1966).
All peptide sequences are written according to the generally accepted
convention
whereby the a-N-terminal amino acid residue is on the left and the a-C-
terminal is on the
right. As used herein, the term "a-N-terminal" refers to the free a-amino
group of an
amino acid in a peptide, and the term "a-C-terminal" refers to the free a-
carboxylic acid
terminus of an amino acid in a peptide.
For the most part, the names on naturally occurring and non-naturally
occurring
~ s aminoacyl residues used herein follow the naming conventions suggested by
the IUPAC
Commission on the Nomenclature of Organic Chemistry and the IUPAC-IUB
Commission on Biochemical Nomenclature as set out in "Nomenclature of a-Amino
Acids (Recommendations, 1974) " Biochemistry, 14(2), (1975). To the extent
that the
names and abbreviations of amino acids and aminoacyl residues employed in this
2o specification and appended claims differ from those suggestions, they will
be made clear
to the reader. Some abbreviations useful in describing the invention are
defined below in
the following Table 1.
Table 1
Abbreviation Definition
N-Ac-Sar N-acetylsarcosyl
Ala alanyl
(3-Ala (3-alanyl
AlaNH2 alanylamide
AIaNH-ethyl alanylethylamide
alloIle alloisoleucyl
alloThr allothreonyl
alloThr(t-Bu) allothreonyl(O-t-butyl)
Allylgly allyglycyl
4-AmdPheAla 3-(4-amidophenyl)alanyl
2-Ambut 2-aminobutyryl
16
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
4-Ambut 4-aminobutyryl
( 1 R,3 S)-AmCyCO ( 1 R,3 S)-1-aminocyclopentane-3-carbonyl
( 1 S,3R)-AmCyCO ( 1 S,3R)-1-aminocyclopentane-3-carbonyl
( 1 R,4S)-AmCyeCO ( 1 R,4S)-1-aminocyclopent-2-ene-4-
carbonyl
( 1 S,4R)-AmCyeCO ( 1 S,4R)-1-aminocyclopent-2-ene-4-
carbonyl
2-Amisobut 2-aminoisobutyryl
4-AmIspCha [(4-amino-N-isopropyl)cyclohexyl]alanyl
4-AmIspPheAla 3-(4-amino-N-isopropylphenyl)alanyl
4-AmPheAla 3-(4-aminophenyl)alanyl
Arg arginyl
Arg(diethyl) arginyl(N~'N~~ diethyl)
Asn asparaginyl
Asn(Trt) asparaginyl(trityl)
AzaGIyNH2 azaglycylamide
3-BzlThiAla 3-(3-benzothienyl)alanyl
5-BrThiAla 3-[2-(5-bromothienyl)]alanyl
Gly(t-Bu) t-butylglycyl
3-CIPheAla 3-(3-chlorophenyl)alanyl
4-CIPheAla 3-(4-chlorophenyl)alanyl
Cit citrullyl
3-CNPheAla 3-(3-cyanophenyl)alanyl
4-CNPheAla 3-(4-cyanophenyl)alanyl
Cha cyclohexylalanyl
Chg cyclohexylglycyl
Cys cysteinyl
Cys(acme) cysteinyl(S-acetamidomethyl)
Cyst-Bu) cysteinyl(S-t-butyl)
dePro 3,4-dehydroprolyl
3,4-diFPheAla 3-(3,4-difluorophenyl)alanyl
3,4-diOMe-PheAla 3-(3,4-dimethoxyphenyl)alanyl
Fmoc 9-fluorenylmethyloxycarbonyl
3-FPheAla 3-(3-fluorophenyl)alanyl
4-FPheAla 3-(4-fluorophenyl)alanyl
17
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Gln glutaminyl
Gln(Trt) glutaminyl(trityl)
GlyNH2 glycylamide
GIyNH-ethyl glycylethylamide
4-GuPheAla 3-(4-guanidinophenyl)alanyl
Hala homoalanyl
Harg homoarginine
His histidyl
HpheAla homophenylalanyl
Hser homoseryl
4-OHMePheAla 3-(4-hydroxymethylphenyl)alanyl
4-OHPro 4-hydroxyprolyl
Ile isoleucyl
Leu leucyl
Lys lysyl
Lys(Ac) lysyl(N-epsilon-acetyl)
Lys(Isp) lysyl(N-epsilon-isopropyl)
Lys(Nic) lysyl(N-epsilon-nicotinyl)
Met methionyl
Met(OZ) methionyl(sulfone)
Met(O) methionyl(sulfoxide)
N-MeArg(Mtr) (NG-4-methoxy-2,3,6-
trimethylbenzenesulfonyl)arginyl
N-MeAla N-methylalanyl
N-MeAlaNH2 N-methylalanylamide
N-MeAIaNH-ethyl N-methylalanyl ethylamide
N-MeAsp N-methylaspartyl
N-MeAsp(t-Bu) N-methylaspartyl(S-t-butyl)
N-MeGlu N-methylglutamyl
N-MeGlu(t-Bu) N-methylglutamyl(S-t-butyl)
N-MeIle N-methylisoleucyl
N-MeLeu N-methylleucyl
N-MeNle N-methylnorleucyl
N-MeNva N-methylnorvalyl
N-MeNvaNH-ethyl N-methylnorvalyl ethylamide
1s
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
4-MePheAla 3-(4-methylphenyl)alanyl
N-MePheAla N-methylphenylalanyl
N-MePro N-methylprolyl
N-MeSer(t-Bu) N-methylseryl(O-t-butyl)
N-MeThr(Bzl) N-methylthreonyl(O-benzyl)
N-MeThr(t-Bu) N-methylthreonyl(O-t-butyl)
N-MeTyr N-methyltyrosyl
N-MeTyr(t-Bu) N-methyltyrosyl(O-t-butyl)
N-MeVal N-methylvalyl
1-Nal 3-(naphth-1-yl)alanyl
2-Nal 3-(naphth-2-yl)alanyl
4-NOzPheAla 3-(4-nitrophenyl)alanyl
Narg norarginyl
Nle norleucyl
Nor norornithyl
Nva norvalyl
octylgly octylglycyl
Orn ornithyl
Orn(Ac) ornityl(N-delta-acetyl)
Orn(Im) ornithyl[N-delta-(2-imidazolinyl)]
Orn(Isp) ornithyl(N-delta-isopropyl)
Pen penicillaminyl
Pen(Sacme) penicillaminyl(S-acetamidomethyl)
Pen(SBzI) penicillaminyl(S-benzyl)
Pen(SMe) penicillaminyl(S-methyl)
3-PentaFPheAla 3-(pentafluorophenyl)alanyl
Arg(Pmc) (NG-2,2,5,7,8-pentamethylchroman-6-
sulfonyl)arginyl
PheAla phenylalanyl
PheGly phenylglycyl
Pro prolyl
ProNH-ethyl prolyl ethylamide
Propgly propargylglycyl
3-PyrAla 3-(3-pyridyl)alanyl
Sar sarcosyl
19
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
SarNHz sarcosylamide
SarNH-ethyl sarcosyl ethylamide
Ser seryl
SerNH2 serylamide
Ser(Bzl) seryl(O-benzyl)
Ser(t-Bu) seryl(O-t-butyl)
StyAla styrylalanyl
Tic 1,2,3,4-tetrahydroisoquinoline-3-carbonyl
4-ThzAla 3-(4-thiazolyl)alanyl
2-ThiAla 3-(2-thienyl)alanyl
Thr threonyl
Thr(Bzl) threonyl(O-benzyl)
Thr(t-Bu) threonyl(O-t-butyl)
3-CF3PheAla 3-(3-trifluoromethylphenyl)alanyl
3,4,5-TriFPheAla 3-(3,4,5-trifluorophenyl)alanyl
Tyr tyrosyl
Tyr(t-Bu) tyrosyl(O-t-butyl)
Tyr(Et) tyrosyl(O-ethyl)
Tyr(Me) tyrosyl(O-methyl)
Val valyl
When not found in the table above, nomenclature and abbreviations may be
further
clarified by reference to the Calbiochem-Novabiochem Corp. 1999 Catalog and
Peptide
Synthesis Handbook or the Chem-Impex International, Inc. Tools for Peptide &
Solid
Phase Synthesis 1998-1999 Catalogue.
In one aspect, the present invention relates to compounds of formula (I),
wherein
Xaa2-Xaa,o each represent an amino acyl residue; Xaa, may be absent or Xaa, is
hydrogen,
N-methylprolyl, or an acyl group; and Xaa" is a hydroxy group, an amino acid
amide, or
an amino residue. The amino acyl residues represented by Xaa2- Xaa,o can have
an N-
o alkylated or an N-unalkylated amide bond. At least one of the amide bonds on
a residue
represented by Xaa3, Xaa4, Xaas, Xaab, Xaa,, Xaag, Xaag, or Xaa,o is N-
alkylated.
Xaa, is absent or is selected from the group consisting of hydrogen; N-
methylprolyl; R'-(CHZ)~-C(O)-, wherein n is an integer from 0 to 8 and R' is
selected from
the group consisting of N-acetylamino, alkoxy, alkyl, aryl, carboxy,
cycloalkenyl,
~ 5 cycloalkyl, heterocycle, and hydroxy; and RZ-CHzCHz-O-(CHZCHzO)p CHZ-C(O)-
,
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
wherein p is an integer from 1 to 8 and RZ is selected from the group
consisting of
hydrogen, N-acetylamino, and alkyl. Preferably, Xaa, is absent or is selected
from the
group consisting of acetyl, N-methylprolyl, and succinyl.
Amino acyl residues suitable for the XaaZ position include N-methylalanyl,
sarcosyl, N-ethylglycyl, N-methylnorvalyl, N-methylprolyl, (3-alanyl, D-
alanyl, 4-
aminobutyryl, (1R,3S)-1-aminocyclopentane-3-carbonyl, (1S,3R)-1-
aminocyclopentane-3-
carbonyl, (1R,4S)-1-aminocyclopent-2-ene-4-carbonyl, (1S,4R)-1-aminocyclopent-
2-ene-
4-carbonyl, asparaginyl, 3-(4-chlorophenyl)alanyl, 3-(4-cyanophenyl)alanyl,
glutaminyl,
glutamyl, glycyl, 4-hydroxyprolyl, 3-(4-methylphenyl)alanyl, prolyl, Beryl,
and threonyl.
When Xaaz is an N-alkylated prolyl residue, Xaa, is absent. Preferably, Xaa2
is an amino
acyl residue selected from the group consisting of sarcosyl and N-
methylprolyl.
Suitable amino acyl residues for Xaa3 include N-methylalanyl, sarcosyl, N-
methylleucyl, N-methylphenylalanyl, alanyl, (1S,3R)-1-aminocyclopentane-3-
carbonyl,
(1S,4R)-1-aminocyclopent-2-ene-4-carbonyl, asparaginyl, aspartyl, 3-(3-
~5 cyanophenyl)alanyl, 3-(4-cyanophenyl)alanyl, glutaminyl, glycyl, leucyl,
lysyl(N-epsilon
acetyl), 3-(4-methylphenyl)alanyl, norvalyl, prolyl, and phenylalanyl. The
preferred
amino acid residues for Xaa3 are N-methylalanyl and glycyl.
The N-alkylated residues suitable for Xaa4 include N-methylalanyl, sarcosyl, N-
methylhomophenylalanyl, N-methylisoleucyl, N-methylleucyl, N-methylnorvalyl, N-
2o methylphenylalanyl, N-methyl-D-phenylalanyl, N-methylseryl, N-
methyltyrosyl, N-
methylvalyl, and N-methyl-D-valyl. N-Unalkylated amino acyl residues suitable
for Xaa4
are alanyl, alloisoleucyl, allylglycyl, 2-aminobutyryl, (1R,4S)-aminocyclopent-
2-ene-4-
carbonyl, asparaginyl, aspartyl, 3-[2-(5-bromothienyl)]alanyl, 3-(3-
chlorophenyl)alanyl, 3-
(4-chlorophenylalanyl), 3-(3-cyanophenyl)alanyl, cyclohexylalanyl, 3-(3,4-
25 dimethoxyphenyl)alanyl, 3-(3-fluorophenyl)alanyl, 3-(4-fluorophenylalanyl),
glutaminyl,
glycyl, histidyl, homophenylalanyl, homoseryl, isoleucyl, leucyl, lysyl(N-
epsilon-acetyl),
methionyl, methionyl(sulfone), 3-(4-methylphenyl)alanyl, 3-(naphth-1-
yl)alanyl, 3-
(naphth-2-yl)alanyl, norornithyl, norvalyl, phenylalanyl, phenylglycyl,
prolyl, 3-(3-
pyridyl)alanyl, 3-(4-thiazolyl)alanyl, 3-(2-thienyl)alanyl, Beryl, seryl(O-
benzyl),
3o styrylalanyl, tryptyl, tyrosyl, valyl, and D-valyl. Preferred amino acyl
residues for Xaa4
are N-methylalanyl, N-methylisoleucyl, N-methylleucyl, N-methylnorvalyl, N-
methylphenylalanyl, N-methyl-D-phenylalanyl, N-methylvalyl, N-methyl-D-valyl,
asparaginyl, glutaminyl, isoleucyl, phenylalanyl, and valyl.
The N-alkylated amino acyl residues suitable for Xaas are N-methyl-D-
35 homophenylalanyl, N-methyl-D-isoleucyl, N-methyl-D-leucyl, and N-(R3)-D-
phenylalanyl. N-unalkylated amino acyl residues suitable for XaaS are D-
alanyl,
21
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
alloisoleucyl, D-alloisoleucyl, D-2-aminobutyryl, D-3-(4-aminophenyl)alanyl, D-
asparaginyl, D-3-(3-benzothienyl)alanyl, D-t-butylglycyl, D-(4-
chlorophenyl)alanyl, D-
citrullyl, D-3-(3-cyanophenyl)alanyl, D-cyclohexylalanyl, cyclohexylglycyl, D-
cysteinyl(S-acetamidomethyl), D-cysteinyl(S-t-butyl), D-3-(3,4-
difluorophenyl)alanyl, D-
(3,4-dimethoxyphenyl)alanyl, D-glutaminyl, glycyl, D-homophenylalanyl, D-
homoseryl,
isoleucyl, D-isoleucyl, D-leucyl, D-lysyl, D-lysyl(N-epsilon-nicotinyl), D-
methionyl, D-3-
(4-methylphenyl)alanyl, D-3-(naphth-1-yl)alanyl, D-3-(naphth-2-yl), D-3-(4-
nitrophenyl)alanyl, D-norleucyl, D-ornithyl, D-penicillaminyl, D-
penicillaminyl(S-
acetamidomethyl), D-penicillaminyl(O-benzyl), D-penicillaminyl(S-methyl), D-3-
~o (pentafluorophenyl)alanyl, D-phenylalanyl, D-prolyl, D-Beryl, D-seryl(O-
benzyl), D-(2-
thienyl)alanyl, D-threonyl, D-threonyl(O-benzyl), D-3-(3-
trifluoromethylphenyl)alanyl, D-
(3,4,5-trifluorophenyl)alanyl, D-tryptyl, D-tyrosyl, D-tyrosyl(ethyl), and D-
valyl. The
amino acyl residues preferred for XaaS are N-methyl-D-leucyl, D-alloisoleucyl,
D-
isoleucyl, D-leucyl, D-homophenylalanyl, and D-penacillaminyl(S-methyl).
~ 5 Examples of N-alkylated amino acids suitable for Xaab are N-
methylaspartyl, N-
methylglutamyl, sarcosyl, N-methylseryl, N-methylthreonyl, N-methylthreonyl(O-
benzyl),
and N-methyltyrosyl. N-unalkylated amino acyl residues suitable for Xaa6 are
alanyl,
allothreonyl, D-allothreonyl, allylglycyl, glutaminyl, glycyl, histidyl,
homoseryl, D-
homoseryl, 3-(4-hydroxymethylphenyl)alanyl, isoleucyl, lysyl(N-epsilon-
acetyl),
2o methionyl, 3-(naphth-2-yl)alanyl, norvalyl, octylglycyl, prolyl, 3-(3-
pyridyl)alanyl, Beryl,
D-Beryl, threonyl, D-threonyl, tryptyl, tyrosyl, and tyrosyl(O-methyl). The
preferred
amino acyl residues for Xaab are N-methylaspartyl, N-methylglutamyl, sarcosyl,
N-
methylseryl, N-methyltyrosyl, N-methylthreonyl(O-benzyl), allothreonyl, seryl,
threonyl,
and tyrosyl.
25 N-Alkylated amino acyl residues suitable for Xaa~ are N-methylalanyl,
sarcosyl, N-
methylisoleucyl, N-methyleucyl, N-methyl-D-leucyl, N-methylnorleucyl, N-
methylnorvalyl, N-methylseryl, N-methylthreonyl, and N-methylvalyl. The N-
unalkylated
amino acyl residues suitable for Xaa, are alanyl, allothreonyl, allylglycyl, 3-
(4-
amidophenyl)alanyl, 2-aminobutyryl, arginyl, asparaginyl, cyclohexylalanyl,
glutaminyl,
3o D-glutaminyl, glycyl, homoalanyl, homoseryl, 4-hydroxyprolyl, leucyl, D-
leucyl, lysyl(N-
epsilon-acetyl), methionyl, methionyl sulfone, methionyl sulfoxide, norleucyl,
norvalyl, D-
norvalyl, octylglycyl, ornithyl(N-delta-acetyl), phenylalanyl,
propargylglycyl, Beryl, D-
seryl, threonyl, tryptyl, tyrosyl, and valyl. Preferably, the amino acyl
residue for Xaa, is
selected from the group consisting of N-methylalanyl, sarcosyl, N-
methylisoleucyl, N-
s5 methylleucyl, N-methyl-D-leucyl, N-methylnorleucyl, N-methylnorvalyl, N-
methylseryl,
N-methylthreonyl, N-methylvalyl, norleucyl, norvalyl, and seryl.
22
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Suitable N-alkyl amino acyl residues for XaaB include N-methylalanyl, N-methyl-
D-alanyl, N-methylisoleucyl, and N-methylleucyl. N-Unalkylated amino acyl
residues
suitable for Xaag include alanyl, alloisoleucyl, D-alloisoleucyl, allylglycyl,
citrullyl,
glycyl, isoleucyl, D-isoleucyl, leucyl, D-leucyl, lysyl(N-epsilon-acetyl), D-
lysyl(N-
epsilon-acetyl), methionyl, 3-(naphth-1-yl)alanyl, norvalyl, prolyl, D-prolyl,
and valyl.
Preferably, the amino acyl residue for Xaag is selected from the group
consisting of N-
methylalanyl, N-methyl-D-alanyl, N-methylisoleucyl, N-methylleucyl, isoleucyl,
D-
isoleucyl, and D-lysyl(N-epsilon-acetyl).
One of the N-alkylated amino acyl residues suitable for Xaa, is N-
methylarginyl.
~ o N-Unalkylated amino acyl residues for Xaag are selected from the group
consisting of [(4-
amino-N-isopropyl)cyclohexyl]alanyl, 3-(4-amino-N-isopropylphenyl)alanyl,
arginyl,
arginyl(N°N°~diethyl), citrullyl, glutaminyl, 3-(4-
guanidinophenyl)alanyl, histidyl,
homoarginyl, lysyl(N-epsilon-isopropyl), lysyl(N-epsilon-nicotinyl), lysyl,
norarginyl,
ornithyl, ornithyl(N-delta-imidazolinyl), ornithyl(N-delta-isopropyl), and 3-
(3-
15 pyridyl)alanyl. Preferred amino acyl residues for Xaa9 are arginyl and N-
methylarginyl.
N-Alkylated amino acids suitable for the Xaa,o position include N-
methylalanyl,
N-methyl-D-alanyl, sarcosyl, N-methylhomoalanyl, and N-methylnorvalyl. Other
residues
suitable for Xaa,o include D-alanyl, 2-aminoburyryl, D-2-aminobutyryl, 2-
aminoisobutyryl, 3,4-dehydroprolyl, 4-hydroxyprolyl, phenylalanyl, prolyl, D-
prolyl,
20 1,2,3,4-tetrahydroisoquinoline-3-carbonyl, and D-valyl. The amino acyl
residues preferred
for Xaa,o are N-methylalanyl, sarcosyl, N-methylnorvalyl, and prolyl.
Preferably, one or two residues selected from Xaa3, Xaa4, Xaas, Xaa~, Xaa,,
XaaB,
Xaa~ and Xaa,o has an N-alkylated amino acyl residue. The more preferred
compounds of
the invention have one N-alkylated amide bond on an amino acyl residue not
including
25 Xaa,, as represented by Xaa3, Xaa4, XaaS, Xaab, Xaa,, XaaB, Xaa, or Xaa,o.
Xaa" is a hydroxy group or an amino acid amide selected from the group
consisting of alanylamide, D-alanylamide, alanylethylamide, D-
alanylethylamide,
azaglycylamide, glycylamide, glycylethylamide, lysyl(N-epsilon-acetyl), D-
lysyl(N-
epsilon-acetyl), N-methyl-D-alanylamide, sarcosylamide, serylamide, and D-
serylamide;
30 or Xaa, ~ is a group represented by the formula
Ra
-NH-(CHZ)S-CHRS
or a group represented by the formula -NH-R6, wherein s is an integer selected
from 0 to
8; R4 is selected from hydrogen, alkyl, and a 5- to 6-membered cycloalkyl
ring; RS is
selected from the group consisting of hydrogen, alkoxy, alkyl, aryl,
cycloalkenyl,
35 cycloalkyl, heterocycle, and hydroxy, provided that s is not zero when RS
is hydroxy or
23
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
alkoxy; and R6 is selected from hydrogen and hydroxy. The preferred Xaa"
groups for
modifying the C-terminus of the invention are NH-ethyl and D-alanylamide.
Compositions
The compounds of the invention, including not limited to those specified in
the
examples, possess anti-angiogenic activity. As angiogenesis inhibitors, such
compounds
are useful in the treatment of both primary and metastatic solid tumors,
including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx, esophagus,
stomach,
pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract
(including kidney,
~ o bladder and urothelium), female genital tract (including cervix, uterus,
and ovaries as well
as choriocarcinoma and gestational trophoblastic disease), male genital tract
(including
prostate, seminal vesicles, testes and germ cell tumors), endocrine glands
(including the
thyroid, adrenal, and pituitary glands), and skin, as well as hemangiomas,
melanomas,
sarcomas (including those arising from bone and soft tissues as well as
Kaposi's sarcoma)
~ 5 and tumors of the brain, nerves, eyes, and meninges (including
astrocytomas, gliomas,
glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas, and
meningiomas). Such compounds may also be useful in treating solid tumors
arising from
hematopoietic malignancies such as leukemias (i.e. chloromas, plasmacytomas
and the
plaques and tumors of mycosis fungosides and cutaneous T-cell
lymphoma/leukemia) as
2o well as in the treatment of lymphomas (both Hodgkin's and non-Hodgkin's
lymphomas).
In addition, these compounds may be useful in the prevention of metastases
from the
tumors described above either when used alone or in combination with
radiotherapy and/or
other chemotherapeutic agents.
Further uses include the treatment and prophylaxis of autoimmune diseases such
as
25 rheumatoid, immune and degenerative arthritis; various ocular diseases such
as diabetic
retinopathy, retinopathy of prematurity, corneal graft rejection, retrolental
fibroplasia,
neovascular glaucoma, rubeosis, retinal neovascularization due to macular
degeneration,
hypoxia, angiogenesis in the eye associated with infection or surgical
intervention, and
other abnormal neovascularization conditions of the eye; skin diseases such as
psoriasis;
3o blood vessel diseases such as hemagiomas, and capillary proliferation
within
atherosclerotic plaques; Osler-Webber Syndrome; myocardial angiogenesis;
plaque
neovascularization; telangiectasia; hemophiliac joints; angiofibroma; and
wound
granulation. Other uses include the treatment of diseases characterized by
excessive or
abnormal stimulation of endothelial cells, including not limited to intestinal
adhesions,
35 Crohn's disease, atherosclerosis, scleroderma, and hypertrophic scars, i.e.
keloids.
Another use is as a birth control agent, by inhibiting ovulation and
establishment of the
24
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
placenta. The compounds of the invention are also useful in the treatment of
diseases that
have angiogenesis as a pathologic consequence such as cat scratch disease
(Rochele
minutesalia quintosa) and ulcers (Helicobacter pylori). The compounds of the
invention
are also useful to reduce bleeding by administration prior to surgery,
especially for the
treatment of resectable tumors.
The compounds of the invention may be used in combination with other
compositions and procedures for the treatment of diseases. For example, a
tumor may be
treated conventionally with surgery, radiation or chemotherapy combined with a
peptide of
the present invention and then a peptide of the present invention may be
subsequently
~ o administered to the patient to extend the dormancy of micrometastases and
to stabilize and
inhibit the growth of any residual primary tumor. Additionally, the compounds
of the
invention may be combined with pharmaceutically acceptable excipients, and
optionally
sustained-release matrices, such as biodegradable polymers, to form
therapeutic
compositions.
~ 5 A sustained-release matrix, as used herein, is a matrix made of materials,
usually
polymers, which are degradable by enzymatic or acid-base hydrolysis or by
dissolution.
Once inserted into the body, the matrix is acted upon by enzymes and body
fluids. A
sustained-release matrix desirably is chosen from biocompatible materials such
as
liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic
acid),
2o polylactide co-glycolide (copolymers of lactic acid and glycolic acid)
polyanhydrides,
poly(ortho)esters, polypeptides, hyaluronic acid, collagen, chondroitin
sulfate, carboxylic
acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino
acids, amino
acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl
propylene,
polyvinylpyrrolidone and silicone. A preferred biodegradable matrix is a
matrix of one of
25 either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers
of lactic acid
and glycolic acid).
When used in the above or other treatments, a therapeutically effective amount
of
one of the compounds of the present invention may be employed in pure form or,
where
such forms exist, in pharmaceutically acceptable salt form. By a
"therapeutically effective
3o amount" of the compound of the invention is meant a sufficient amount of
the compound
to treat an angiogenic disease, (for example, to limit tumor growth or to slow
or block
tumor metastasis) at a reasonable benefitlrisk ratio applicable to any medical
treatment. It
will be understood, however, that the total daily usage of the compounds and
compositions
of the present invention will be decided by the attending physician within the
scope of
35 sound medical judgment. The specific therapeutically effective dose level
for any
particular patient will depend upon a variety of factors including the
disorder being treated
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
and the severity of the disorder; activity of the specific compound employed;
the specific
composition employed, the age, body weight, general health, sex and diet of
the patient;
the time of administration, route of administration, and rate of excretion of
the specific
compound employed; the duration of the treatment; drugs used in combination or
coincidential with the specific compound employed; and like factors well known
in the
medical arts. For example, it is well within the skill of the art to start
doses of the
compound at levels lower than those required to achieve the desired
therapeutic effect and
to gradually increase the dosage until the desired effect is achieved.
Alternatively, a compound of the present invention may be administered as
~ o pharmaceutical compositions containing the compound of interest in
combination with one
or more pharmaceutically acceptable excipients. A pharmaceutically acceptable
carrier or
excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent,
encapsulating
material or formulation auxiliary of any type. The compositions may be
administered
parenterally, intracisternally, intravaginally, intraperitoneally, topically
(as by powders,
ointments, drops or transdermal patch), rectally, or bucally. The term
"parenteral" as used
herein refers to modes of administration which include intravenous,
intramuscular,
intraperitoneal, intrasternal, subcutaneous and intraarticular injection and
infusion.
Pharmaceutical compositions for parenteral injection comprise pharmaceutically-
acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions
or emulsions,
2o as well as sterile powders for reconstitution into sterile injectable
solutions or dispersions
just prior to use. Examples of suitable aqueous and nonaqueous carriers,
diluents, solvents
or vehicles include water, ethanol, polyols (such as glycerol, propylene
glycol,
polyethylene glycol, and the like), carboxymethylcellulose and suitable
mixtures thereof,
vegetable oils (such as olive oil), and injectable organic esters such as
ethyl oleate. Proper
fluidity may be maintained, for example, by the use of coating materials such
as lecithin,
by the maintenance of the required particle size in the case of dispersions,
and by the use
of surfactants.
These compositions may also contain adjuvants such as preservative, wetting
agents, emulsifying agents, and dispersing agents. Prevention of the action of
3o microorganisms may be ensured by the inclusion of various antibacterial and
antifungal
agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
It may also
be desirable to include isotonic agents such as sugars, sodium chloride, and
the like.
Prolonged absorption of the injectable pharmaceutical form may be brought
about by the
inclusion of agents which delay absorption, such as aluminum monostearate and
gelatin.
Injectable depot forms are made by forming microencapsule matrices of the drug
in
biodegradable polymers such as polylactide-polyglycolide, poly(orthoesters),
26
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
poly(anhydrides), and (poly)glycols, such as PEG. Depending upon the ratio of
drug to
polymer and the nature of the particular polymer employed, the rate of drug
release can be
controlled. Depot injectable formulations are also prepared by entrapping the
drug in
liposomes or microemulsions which are compatible with body tissues.
The injectable formulations may be sterilized, for example, by filtration
through a
bacterial-retaining filter, or by incorporating sterilizing agents in the form
of sterile solid
compositions which can be dissolved or dispersed in sterile water or other
sterile injectable
medium just prior to use.
Topical administration includes administration to the skin or mucosa,
including
~ o surfaces of the lung and eye. Compositions for topical administration,
including those for
inhalation, may be prepared as a dry powder which may be pressurized or non-
pressurized.
In non-pressurized powder compositions, the active ingredient in finely
divided form may
be used in admixture with a larger-sized pharmaceutically-acceptable inert
carrier
comprising particles having a size, for example, of up to 100 micrometers in
diameter.
15 Suitable inert carriers include sugars such as lactose. Desirably, at least
95% by weight of
the particles of the active ingredient have an effective particle size in the
range of 0.01 to
micrometers.
Alternatively, the composition may be pressurized and contain a compressed
gas,
such as nitrogen or a liquified gas propellant. The liquified propellant
medium and indeed
2o the total composition is preferably such that the active ingredient does
not dissolve therein
to any substantial extent. The pressurized composition may also contain a
surface active
agent, such as a liquid or solid non-ionic surface active agent or may be a
solid anionic
surface active agent. It is preferred to use the solid anionic surface active
agent in the form
of a sodium salt.
25 A further form of topical administration is to the eye. A compound of the
invention is delivered in a pharmaceutically acceptable ophthalmic vehicle,
such that the
compound is maintained in contact with the ocular surface for a sufficient
time period to
allow the compound to penetrate the corneal and internal regions of the eye,
as for
example the anterior chamber, posterior chamber, vitreous body, aqueous humor,
vitreous
3o humor, cornea, iris/ciliary, lens, choroid/retina and sclera. The
pharmaceutically-
acceptable ophthalmic vehicle may, for example, be an ointment, vegetable oil
or an
encapsulating material. Alternatively, the compounds of the invention may be
injected
directly into the vitreous and aqueous humour.
Compositions for rectal or vaginal administration are preferably suppositories
35 which may be prepared by mixing the compounds of this invention with
suitable non-
irritating excipients or carriers such as cocoa butter, polyethylene glycol or
a suppository
27
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
wax which are solid at room temperature liquid at body temperature and
therefore melt in
the rectum or vaginal cavity and release the active compound.
Compounds of the present invention may also be administered in the form of
liposomes. As is known in the art, liposomes are generally derived from
phospholipids or
other lipid substances. Liposomes are formed by mono- or mufti-lamellar
hydrated liquid
crystals that are dispersed in an aqueous medium. Any non-toxic,
physiologically-
acceptable and metabolizable lipid capable of forming liposomes can be used.
The present
compositions in liposome form can contain, in addition to a compound of the
present
invention, stabilizers, preservatives, excipients, and the like. The preferred
lipids are the
~o phospholipids and the phosphatidyl cholines (lecithins), both natural and
synthetic.
Methods to form liposomes are known in the art. See, for example, Prescott,
Ed., Methods
in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et
seg.
While the compounds of the invention can be administered as the sole active
pharmaceutical agent, they may also be used in combination with one or more
agents
which are conventionally administered to patients for treating angiogenic
diseases. For
example, the compounds of the invention are effective over the short term to
make tumors
more sensitive to traditional cytotoxic therapies such as chemicals and
radiation. The
compounds of the invention also enhance the effectiveness of existing
cytotoxic adjuvant
anti-cancer therapies. The compounds of the invention may also be combined
with other
2o antiangiogenic agents to enhance their effectiveness, or combined with
other
antiangiogenic agents and administered togdiethyl ether with other cytotoxic
agents. In
particular, when used in the treatment of solid tumors, compounds of the
invention may be
administered with IL-12, retinoids, interferons, angiostatin, endostatin,
thalidomide,
thrombospondin-1, thrombospondin-2, captopryl, angioinhibins, TNP-470,
pentosan
polysulfate, platelet factor 4, LM-609, SU-5416, CM-101, Tecogalan,
plasminogen-K-5,
vasostatin, vitaxin, vasculostatin, squalamine, marimastat or other MMP
inhibitors, anti-
neoplastic agents such as alpha inteferon, COMP (cyclophosphamide,
vincristine,
methotrexate and prednisone), etoposide, mBACOD (methortrexate, bleomycin,
doxorubicin, cyclophosphamide, vincristine and dexamethasone), PRO-MACE/MOPP
(prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide,
cisplatin,
taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine),
vincristine,
vinblastine, and the like as well as with radiation.
Total daily dose of the compositions of the invention to be administered to a
human or other mammal host in single or divided doses may be in amounts, for
example,
from 0.0001 to 300 mg/kg body weight daily and more usually 1 to 300 mg/kg
body
weight.
28
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
It will be understood that agents which can be combined with the compound of
the
present invention for the inhibition, treatment or prophylaxis of angiogenic
diseases are
not limited to those listed above, include in principle any agents useful for
the treatment or
prophylaxis of angiogenic diseases.
The peptides of the invention may be used for the development of affinity
columns
for isolation of receptors relevant to the antiangiogenic activity of the
peptide of the
invention, e.g. TSP-1 receptor, in, for example, cultured endothelial cells.
As is known in
the art, isolation and purification of the receptor may be followed by amino
acid
sequencing to identify and isolate polynucleotides which encode the receptor.
o Recombinant expression of this receptor would allow greater amounts of
receptor to be
produced, e.g. to produce a sufficient quantity for use in high throughput
screening assays
to identify other angiogenesis inhibitors.
Determination of Biological Activity
~5 In Vitro Assay for An~io~enic Activity
The human microvascular endothelial (HMVEC) migration assay was run
according to the procedure of S. S. Tolsma, O. V. Volpert, D. J. Good, W. F.
Frazier, P. J.
Polverini and N. Bouck, J. Cell Biol. 122, 497-511 (1993).
The HMVEC migration assay was carried out using Human Microvascular
2o Endothelial Cells-Dermal (single donor) and Human Microvascular Endothelial
Cells,
(neonatal). The BCE or HMVEC cells were starved overnight in DME containing
0.1
bovine serum albumin (BSA). Cells were then harvested with trypsin and
resuspended in
DME with 0.1% BSA at a concentration of 1.5 X 106 cells per mL. Cells were
added to
the bottom of a 48 well modified Boyden chamber (Nucleopore Corporation, Cabin
John,
25 MD). The chamber was assembled and inverted, and cells were allowed to
attach for 2
hours at 37 °C to polycarbonate chemotaxis membranes (5 pm pore size)
that had been
soaked in 0.1 % gelatin overnight and dried. The chamber was then reinverted,
and test
substances (total volume of 50 pL), including activators, 15 ng/mL bFGF/VEGF,
were
added to the wells of the upper chamber. The apparatus was incubated for 4
hours at
30 37 °C. Membranes were recovered, fixed and stained (Diff Quick,
Fisher Scientific) and
the number of cells that had migrated to the upper chamber per 3 high power
fields
counted. Background migration to DME + 0.1 BSA was subtracted and the data
reported
as the number of cells migrated per 10 high power fields (400X) or, when
results from
multiple experiments were combined, as the percent inhibition of migration
compared to a
35 positive control.
29
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Representative compounds described in Examples 1 to 109 inhibited human
endothelial cell migration in the above assay by at least 50% inhibition when
tested at
concentrations of 100 nM. Preferred compounds inhibited human endothelial cell
migration by at least 51 % when tested at concentrations of 10 nM, and more
preferred
compounds inhibited human endothelial cell migration by at least 51 % at
concentrations of
1 nM.
Synthesis of the Peptides
The polypeptides of the present invention may be synthesized by many
techniques
~ o that are known to those skilled in the art. For solid phase peptide
synthesis, a summary of
the many techniques may be found in J.M. Stewart and J.D. Young, Solid Phase
Peptide
Synthesis, W.H. Freeman Co. (San Francisco), 1963 and J. Meienhofer, Hormonal
Proteins and Peptides, vol. 2, p. 46, Academic Press (New York), 1973. For
classical
solution synthesis see G. Schroder and K. Lupke, The Peptides, vol. l,
Academic Press
~ 5 (New York), 1965.
Reagents, resins, amino acids, and amino acid derivatives are commercially
available and can be purchased from Chem-Impex International, Inc. (Wood Dale,
IL,
U.S.A.) or Calbiochem-Novabiochem Corp. (San Diego, CA, U.S.A.) unless
otherwise
noted herein.
2o In general, these methods comprise the sequential addition of one or more
amino
acids or suitably protected amino acids to a growing peptide chain. Normally,
either the
amino or carboxy group of the first amino acid is protected by a suitable
protecting group.
The protected or derivatized amino acid can then be either attached to an
inert solid
support or utilized in solution by adding the next amino acid in the sequence
having the
2s complimentary (amino or carboxy) group suitably protected, under conditions
suitable for
forming the amide linkage. The protecting group is then removed from this
newly added
amino acid residue and the next amino acid (suitably protected) is then added,
and so forth.
After all the desired amino acids have been linked in the proper sequence, any
remaining
protecting groups (and any solid support) are removed sequentially or
concurrently, to
3o afford the final polypeptide. By simple modification of this general
procedure, it is
possible to add more than one amino acid at a time to a growing chain, for
example, by
coupling (under conditions which do not racemize chiral centers) a protected
tripeptide
with a properly protected dipeptide to form, after deprotection, a
pentapeptide.
A particularly preferred method of preparing compounds of the present
invention
35 involves solid phase peptide synthesis.
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
In this particularly preferred method the a-amino function is protected by an
acid
or base sensitive group. Such protecting groups should have the properties of
being stable
to the conditions of peptide linkage formation, while being readily removable
without
destruction of the growing peptide chain or racemization of any of the chiral
centers
s contained therein. Suitable protecting groups are 9-
fluorenylmethyloxycarbonyl (Fmoc),
t-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl-
oxycarbonyl, t-
amyloxycarbonyl, isobornyloxycarbonyl, (a,a)-dimethyl-3,5-
dimethoxybenzyloxycarbonyl, O-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl,
and the
like. The 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group is preferred.
Particularly preferred side chain protecting groups are: for arginine and
lysine:
acetyl (Ac), adamantyloxycarbonyl, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl
(Boc),
4-methoxybenzenesulfonyl, N°-4-methoxy-2,3,6-trimethylbenzenesulfonyl
(Mtr), nitro,
2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), and p-toluenesulfonyl; for
asparagine:
trityl (Trt); for aspartyl: t-butyl (t-Bu); for glutamyl: t-butyl (t-Bu); for
glutaminyl: trityl
~ 5 (Trt); for histidine: trityl (Trt), benzyl, benzyloxycarbonyl (Cbz), p-
toluenesulfonyl and
2,4-dinitrophenyl; for penicillamine: methyl; for serine: t-butyl (t-Bu),
benzyl and
tetrahydropyranyl; for threonine: benzyl, and t-butyl (t-Bu); for tryptophan:
formyl and t-
butyloxycarbonyl (Boc); and for tyrosine: acetyl (Ac), benzyl, O-
bromobenzyloxycarbonyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl, 2,6-
dichlorobenzyl, and
2o isopropyl.
In the solid phase peptide synthesis method, the C-terminal amino acid is
attached
to a suitable solid support or resin. Suitable solid supports useful for the
above synthesis
are those materials which are inert to the reagents and reaction conditions of
the stepwise
condensation-deprotection reactions, as well as being insoluble in the media
used. The
2s preferred solid support for synthesis of C-terminal carboxy peptides is 4-
hydroxymethyl-
phenoxymethyl-copoly(styrene-1% divinylbenzene). The preferred solid support
for C-
terminal amide peptides is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-
acetamidoethyl resin available from Applied Biosystems.
The C-terminal amino acid is coupled to the resin by means of a coupling
mediated
30 by N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC),
[O-(7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophoshpate] (HATU),
or O-
benzotriazol-1-yl-N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU), with
or
without 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT),
benzotriazol-1-yloxy-tris(dimethylamino)phosphoniumhexafluorophosphate (BOP)
or
3s bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCI), for about 1 to about
24 hours at a
temperature of between 10 °C and 50 °C in a solvent such as
dichloromethane or DMF.
31
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
When the solid support is 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)-
phenoxyacetamidoethyl resin, the Fmoc group is cleaved with a secondary amine,
preferably piperidine, prior to coupling with the C-terminal amino acid as
described above.
The preferred reagents used in the coupling to the deprotected 4-(2',4'-
dimethoxyphenyl-
Fmoc-aminomethyl)phenoxyacetamidoethyl resin are O-benzotriazol-1-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-
hydroxybenzotriazole
(HOBT, 1 equiv.), or [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophoshpate] (HATU, 1 equiv.), in DMF.
The coupling of successive protected amino acids can be carried out in an
~o automatic polypeptide synthesizer as is well known in the art. In a
preferred embodiment,
the a-amino function in the amino acids of the growing peptide chain are
protected with
Fmoc. The removal of the Fmoc protecting group from the N-terminal side of the
growing
peptide is accomplished by treatment with a secondary amine, preferably
piperidine. Each
protected amino acid is then introduced in about 3-fold molar excess and the
coupling is
~5 preferably carried out in DMF. The coupling agent is normally O-
benzotriazol-1-yl-
N,N,N',N'-tetramethyluroniumhexafluorophosphate (HBTU, 1 equiv.) and 1-hydroxy-
benzotriazole (HOBT, 1 equiv.) or [O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophoshpate] (HATU, 1 equiv.).
At the end of the solid phase synthesis, the polypeptide is removed from the
resin
2o and deprotected, either in succession or in a single operation. Removal of
the polypeptide
and deprotection can be accomplished in a single operation by treating the
resin-bound
polypeptide with a cleavage reagent, for example trifluoroacetic acid
containing thianisole,
water, or ethanedithiol.
In cases wherein the C-terminus of the polypeptide is an alkylamide, the resin
is
25 cleaved by aminolysis with an alkylamine. Alternatively, the peptide may be
removed by
transesterification, e.g. with methanol, followed by aminolysis or by direct
transamidation.
The protected peptide may be purified at this point or taken to the next step
directly. The
removal of the side chain protecting groups is accomplished using the cleavage
cocktail
described above.
3o The fully deprotected peptide is purified by a sequence of chromatographic
steps
employing any or all of the following types: ion exchange on a weakly basic
resin in the
acetate form; hydrophobic adsorption chromatography on underivitized
polystyrene-
divinylbenzene (for example, AMBERLITE~ XAD); silica gel adsorption
chromatography; ion exchange chromatography on carboxymethylcellulose;
partition
35 chromatography, e.g. on SEPHADEX~ G-25, LH-20 or countercurrent
distribution; high
32
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
performance liquid chromatography (HPLC), especially reverse-phase HPLC on
octyl- or
octadecylsilyl-silica bonded phase column packing.
The foregoing may be better understood in light of the examples which are
meant
to describe compounds and process which can be carried out in accordance with
the
invention and are not intended as a limitation on the scope of the invention
in any way.
Abbreviations which have been used the following examples are: NMP for N-
methylpyrrolidinone; HATU for [O-(7-azabenzotriazol-1-yl)-1,1,3,3-
tetramethyluronium
hexafluorophosphate]; DMF for N,N-dimethylformamide, and TFA for
trifluoroacetic
acid.
EXAMPLE 1
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeNva-Ile-Arg-ProNH-ether
In the reaction vessel of an Applied Biosystems 433A peptide synthesizer was
placed 0.1 mM of Fmoc-Pro-Sieber ethylamide resin. Cartridges of 1 mM amino
acids
~ 5 were sequentially loaded. The Fastmoc 0.1 with previous peak monitoring
protocol was
used with the following synthetic cycle:
1. Resin solvated with NMP for about 5 minutes;
2. Resin washed with NMP for about 5 minutes;
3. Fmoc group removed using 50% piperidine solution in NMP for 5 minutes,
2o resin washed, and sequence repeated 3 to 4 times;
4. Fmoc-amino acid activated with 1mM of O.SM HATU in DMF;
5. Activated Fmoc-amino acid added to the reaction vessel followed by addition
of 1 mM of 2M diisopropylamine in NMP solution;
6. Fmoc-amino acid coupled for 20 minutes;
2s 7. Resin washed and Fmoc-group removed using 50% piperidine in NMP.
The following protected amino acids were sequentially coupled to the resin
using the
above protocol:
Amino acid Coupling time
1. Fmoc-Arg(Pmc) 20 minutes
2. Fmoc-Ile 20 minutes
3. Fmoc-NMeNva 20 minutes
4. Fmoc-Thr(t-Bu) 20 minutes
5. Fmoc-D-Ile 20 minutes
6. Fmoc-Val 20 minutes
7. Fmoc-Gly 20 minutes
8. Fmoc-Sar 20 minutes
33
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
9. acetic acid 20 minutes
Upon completion of the synthesis the resin-bound peptide was washed with
methanol three times and dried in vacuo, then treated with a (95:5) TFA/water
solution (3
mL) at room temperature overnight. The resin was filtered and washed 3 times
with
methanol. The filtrates and the washes were combined and concentrated in
vacuo. The
s residue was treated with diethyl ether and the precipitate was filtered to
provide the crude
peptide as an amorphous powder. This was purified by preparative HPLC using a
C-18
column with a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
containing 0.01% TFA over a period of 50 minutes. The pure fractions were
lyophilized
to provide N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeNva-Ile-Arg-ProNH-ethyl as a
~o trifluoroacetate salt; R, = 4.259 minutes (using a C-18 column and a
solvent system
increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 1008 (M+H); Amino Acid
Anal.: 1.14
Pro; 1.70 Arg; 1.96 Ile; 0.47 Thr; 0.97 Val; 0.90 Gly; 0.98 Sar.
15 EXAMPLE 2
N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-NMeIIe-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeIIe for Fmoc-Ile in Example 1. Upon completion of the synthesis,
cleavage of the peptide from the resin, removal of the protecting groups, and
precipitation
2o with diethyl ether, the crude peptide was obtained. This was purified by
preparative HPLC
using a C-18 column with a solvent system increasing in gradient from 5% to
100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-NMeIIe-Arg-ProNH-
ethyl
as a trifluoroacetate salt; R, = 4.416 minutes (using a C-18 column and a
solvent system
2s increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 1008 (M+H); Amino Acid
Anal.: 1.05
Pro; 1.26 Arg; 1.0 Ile; 0.54 Thr; 1.29 Nva; 1.02; Val; 0.94 Gly; 1.01 Sar.
FXAMPT F ~
3o N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeAIa-Ile-Are-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeAIa for Fmoc-
NMeNva in Example 1. Upon completion of the synthesis, cleavage of the peptide
from
the resin, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column with a
35 solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
34
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
0.01 % TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeAIa-Ile-Arg-ProNH-ethyl as a trifluoroacetate
salt; R, _
2.84 minutes (using a C-18 column and a solvent system increasing in gradient
from 20%
to 95% acetonitrile/water containing IOmM ammonium acetate over a period of 10
minutes); MS (ESI) m/e 980 (M+H); Amino Acid Anal.: 0.99 Pro; 1.54 Arg; 2.10
Ile; 0.50
Thr; 0.95 Val; 0.96 Gly; 0.98 Sar.
EXAMPLE 4
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeVaI-Ile-Are-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeVaI for Fmoc
NMeNva in Example 1. Upon completion of the synthesis, cleavage of the peptide
from
the resin, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column with a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeVaI-Ile-Arg-ProNH-ethyl as a trifluoroacetate
salt; Rt =
3.11 minutes (using a C-18 column and a solvent system increasing in gradient
from 20%
to 95% acetonitrile/water containing l OmM ammonium acetate over a period of
10
minutes); MS (ESI) m/e 1008 (M+H); Amino Acid Anal.: 1.03 Pro; 1.47 Arg; 2.06
Ile;
0.49 Thr; 0.96 Val; 1.01 Gly; 0.99 Sar.
EXAMPLE 5
N-Ac-Sar-Glv-Val-D-Ile-Thr-Nva-Ile-Are-NMeAIaNH-ethvl
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeAIa for Fmoc-Pro in Example 1. Upon completion of the synthesis,
cleavage of the peptide from the resin, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column with a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
3o were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-NMeAIaNH-
ethyl
as a trifluoroacetate salt; R, = 2.84 minutes (using a C-18 column and a
solvent system
increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 982 (M+H); Amino Acid
Anal.: 1.46
Arg; 2.02 Ile; 1.04 Nva; 0.47 Thr; 0.98 Val; 0.96 Gly; 1.03 Sar.
EXAMPLE 6
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Art-SarNH-ethyl
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-Sar for Fmoc-Pro in Example 1. Upon completion of the synthesis,
cleavage of
the peptide from the resin, removal of the protecting groups, and
precipitation with diethyl
ether, the crude peptide was obtained. This was purified by preparative HPLC
using a C-
18 column with a solvent system increasing in gradient from 5% to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-SarNH-ethyl
as a
trifluoroacetate salt; R, = 2.92 minutes (using a C-18 column and a solvent
system
~o increasing in gradient from 20% to 95% acetonitrile/water containing IOmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 968 (M+H); Amino Acid
Anal.: 1.49
Arg; 2.07 Ile; 1.05 Nva; 0.55 Thr; 0.98 Val; 0.96 Gly; 1.96 Sar.
EXAMPLE 7
~ 5 N-Succinyl-Sar-Gly-Val-D-Ile-Thr-NMeNva-Ile-Arg-ProNH-ethyl
The desired product was prepared by the procedure described in Example 1 with
the following modification: coupling with acetic acid at the end of the
synthesis was
replaced with treatment of the peptide resin overnight with a 10-fold excess
of succinic
anhydride/pyridine in NMP. Upon completion of the synthesis, washing of the
resin-
20 bound peptide, cleavage of the peptide from the resin, removal of the
protecting groups,
and precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column with a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01 % TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide N-Succinyl-Sar-Gly-Val-D-Ile-Thr-
NMeNva-
25 Ile-Arg-ProNH-ethyl as a trifluoroacetate salt; R, = 2.61 minutes (using a
C-18 column and
a solvent system increasing in gradient from 20% to 95% acetonitrile/water
containing
l OmM ammonium acetate over a period of 10 minutes); MS (ESI) m/e 1066 (M+H);
Amino Acid Anal.: 1.59 Arg; 2.23 Ile; 0.50 Thr; 1.01 Val; 1.00 Gly; 1.02 Sar;
0.99 Pro.
3o EXAMPLE 8
N-Succinyl-Sar-Gly-V al-D-Leu-Thr-NMeNva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-D-Leu for Fmoc-D-Ile in
Example 7. Upon completion of the synthesis, washing of the resin-bound
peptide,
cleavage of the peptide from the resin, removal of the protecting groups, and
precipitation
35 with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column with a solvent system increasing in gradient from 5%
to 100%
36
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-succinyl-Sar-Gly-Val-D-Leu-Thr-NMeNva-Ile-Arg-
ProNH-
ethyl as a trifluoroacetate salt; R, = 2.67 minutes (using a C-18 column and a
solvent
system increasing in gradient from 20% to 95% acetonitrile/water containing l
OmM
ammonium acetate over a period of 10 minutes); MS (ESI) m/e 1066 (M+H); Amino
Acid
Anal.: 1.61 Arg; 1.01 Ile; 1.10 Leu; 0.45 Thr; 0.95 Val; 0.95 Gly; 1.01 Sar;
0.93 Pro.
EXAMPLE 9
N-Ac-Sar-NMeAIa-Val-D-Ile-Thr-Nva-Ile-Are-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeAIa for Fmoc-Gly
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative HPLC
using a C-18 column with a solvent system increasing in gradient from 5% to
100%
~5 acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The
pure fractions
were lyophilized to provide N-Ac-Sar-NMeAIa-Val-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl
as a trifluoroacetate salt; R, = 3.43 minutes (using a C-18 column and a
solvent system
increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 1022 (M+H).
EXAMPLE 10
N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-NMeAr~-ProNH-ethyl
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeArg(Mtr) for Fmoc-Arg(Pmc) in Example 1. Upon completion of the
synthesis, cleavage of the peptide from the resin, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column with a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-
NMeArg-
3o ProNH-ethyl as a trifluoroacetate salt; R, = 3.43 minutes (using a C-18
column and a
solvent system increasing in gradient from 20% to 95% acetonitrile/water
containing
l OmM ammonium acetate over a period of 10 minutes); MS (ESI) m/e 1008 (M+H).
EXAMPLE 11
N-Ac-S ar-Gly-NMe V al-D-Ile-Thr-Nva-Ile-Ark-ProNH-ethyl
37
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
The desired product was prepared by substituting Fmoc-NMeVaI for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column with a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-Ac-Sar-Gly-NMeVaI-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl
as a trifluoroacetate salt; Rt = 3.42 minutes (using a C-18 column and a
solvent system
increasing in gradient from 20% to 95% acetonitrile/water containing lOmM
ammonium
~o acetate over a period of 10 minutes); MS (ESI) m/e 1008 (M+H).
EXAMPLE 12
N-Ac-Sar-Gly-NMeIIe-D-Ile-Thr-Nva-Ile-Are-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeIIe for Fmoc-Val and
15 Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage
of the resin-bound peptide, removal of the protecting groups, and
precipitation with diethyl
ether, the crude peptide was obtained. This was purified by preparative HPLC
using a C-
18 column with a solvent system increasing in gradient from 5% to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
2o were lyophilized to provide N-Ac-Sar-Gly-NMeIIe-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl as
a trifluoroacetate salt; R, = 3.83 minutes (using a C-18 column and a solvent
system
increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 1022 (M+H).
25 EXAMPLE 13
N-Ac-Sar-Gly-NMePhe-D-Ile-Thr-Nva-Ile-Are-ProNH-ethXl
The desired product was prepared by substituting Fmoc-NMePheAla for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
3o with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column with a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-Ac-Sar-Gly-NMePhe-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl
as a trifluoroacetate salt; R, = 4.00 minutes (using a C-18 column and a
solvent system
35 increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 minutes); MS (ESI) m/e 1056 (M+H).
38
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 14
N-Ac-Sar-Gl~-NMeNva-D-I le-Thr-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeNva for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide from the resin, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column with a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
~ o pure fractions were lyophilized to provide N-Ac-Sar-Gly-NMeNva-D-Ile-Thr-
Nva-Ile-
Arg-ProNH-ethyl as a trifluoroacetate salt; Rt = 3.55 minutes (using a C-18
column and a
solvent system increasing in gradient from 20% to 95% acetonitrile/water
containing
l OmM ammonium acetate over a period of 10 minutes); MS (ESI) m/e 1008 (M+H).
EXAMPLE 17
N-Ac-Sar-Gly-NMeAIa-D-Ile-Thr-Nva-Ile-Arg-ProNH-etl~l
The procedure described in Example 1 was used substituting Fmoc-NMeAIa for
Fmoc-Val and Fmoc-Nva for Fmoc-NMeNva. Upon completion of the synthesis,
cleavage
of the resin-bound peptide, removal of the protecting groups, and
precipitation with diethyl
2o ether, the crude peptide was obtained. This was purified by preparative
HPLC using a C-
18 column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
containing 0.01 % TFA over a period of 50 minutes. The pure fractions were
lyophilized
to provide N-Ac-Sar-Gly-NMeAIa-D-Ile-Thr-Nva-Ile-Arg-ProNH-ethyl as a
trifluoroacetate salt; MS (ESI) m/e 978 (M+H).
EXAMPLE 18
N-MePro-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting NMePro for acetic acid and
Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage
of the resin-bound peptide, removal of the protecting groups, and
precipitation with diethyl
ether, the crude peptide was obtained. This was purified by preparative HPLC
using a C-
18 column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
containing 0.01% TFA over a period of 50 minutes. The pure fractions were
lyophilized
to provide N-MePro-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-ProNH-ethyl as a
trifluoroacetate
salt; MS (ESI) m/e 1063 (M+H).
39
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 21
N-Ac-Sar-Gly-Val-D-Ile-NMeThr~Bzl)-Nva-Ile-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeThr(Bzl) for Fmoc-
Thr(t-Bu) and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-NMeThr(Bzl)-
Nva-
~ o Ile-Arg-ProNH-ethyl as a trifluoroacetate salt; MS (ESI) m/e I 084 (M+H).
EXAMPLE 22
N-Ac-Sar-Gly-Val-D-Ile-Thr-Sar-Ile-Arg-ProNH-ether
The desired product was prepared by substituting Fmoc-Sar for Fmoc-NMeNva in
~ 5 Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01% TFA
over a period of 50 minutes. The pure fractions were lyophilized to provide N-
Ac-Sar-
2o Gly-Val-D-Ile-Thr-Sar-Ile-Arg-ProNH-ethyl as a trifluoroacetate salt; R, =
2.68 minutes
(using a C-18 column and a solvent system increasing in gradient from 20% to
95%
acetonitrile/water containing l OmM ammonium acetate over a period of 10
minutes); MS
(ESI) m/e 966 (M+H); Amino Acid Anal.: 1.07 Pro; 1.21 Arg; 2.11 Ile; 0.47 Thr;
1.01;
Val; 0.97 Gly; 2.07 Sar.
EXAMPLE 23
N-Ac-Sar-Gly-Val-D-Leu-Sar-Nva-Ile-Art-ProNH-et~l
The desired product was prepared by substituting Fmoc-D-Leu for Fmoc-D-Ile,
Fmoc-Sar for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon
3o completion of the synthesis, cleavage of the resin-bound peptide, removal
of the protecting
groups, and precipitation with diethyl ether, the crude peptide was obtained.
This was
purified by preparative HPLC using a C-18 column and a solvent system
increasing in
gradient from 5% to 100% acetonitrile/water containing 0.01% TFA over a period
of 50
minutes. The pure fractions were lyophilized to provide N-Ac-Sar-Gly-Val-D-Leu-
Sar-
Nva-Ile-Arg-ProNH-ethyl as a trifluoroacetate salt; MS (ESI) m/e 964 (M+H).
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 24
N-Ac-Sar-Gly-NMeLeu-D-Ile-Thr-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeLeu for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide N-Ac-Sar-Gly-NMeLeu-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl
~ o as a trifluoroacetate salt; MS (ESI) m/e 1022 (M+H).
EXAMPLE 25
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeLeu-Ile-Ar~~ProNH-ether
The desired product was prepared by substituting Fmoc-NMeLeu for Fmoc-
15 NMeNva in Example 1. Upon completion of the synthesis, cleavage of the
resin-bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
2o N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeLeu-Ile-Arg-ProNH-ethyl as a trifluoroacetate
salt; MS
(ESI) m/e 1022 (M+H).
EXAMPLE 26
N-Ac-Sar-Gly-Val-D-alloIle-Thr-NMeNva-Ile-Arg-ProNH-ethyl
25 The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile
in Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01% TFA
30 over a period of 50 minutes. The pure fractions were lyophilized to provide
N-Ac-Sar-
Gly-Val-D-alloIle-Thr-NMeNva-Ile-Arg-ProNH-ethyl as a trifluoroacetate salt;
MS (ESI)
m/e 1008 (M+H).
EXAMPLE 27
35 N-Ac-Sar-Gly-V al-D-al loI le-Thr-NMe V al-I le-Ark-ProNH-ethyl
41
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile and Fmoc-NMeVaI for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide can be obtained. This can
be purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions can be lyophilized to provide N-Ac-Sar-Gly-Val-D-alloIle-Thr-
NMeVaI-Ile-
Arg-ProNH-ethyl as a trifluoroacetate salt.
EXAMPLE 28
N-Ac-Sar-Gly-V al-D-Ile-Thr-NMeNva-Ile-Art-Pro-D-AIaNH,
The desired product was prepared using the procedure in Example 1 with the
following modifications: Fmoc-D-Ala-Sieber amide resin was substituted for
Fmoc-Pro-
Sieber ethylamide resin, and a coupling with Fmoc-Pro prior was added prior to
the
~5 coupling with Fmoc-Arg(Pmc). Upon completion of the synthesis, cleavage of
the resin-
bound peptide, removal of the protecting groups, and precipitation with
diethyl ether, the
crude peptide was obtained. This was purified by preparative HPLC using a C-18
column
and a solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01% TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
2o N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeNva-Ile-Arg-Pro-D-AlaNH2 as a
trifluoroacetate salt;
MS (ESI) m/e 1051 (M+H).
EXAMPLE 29
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeVaI-Ile-Ark-Pro-D-AIaNH~
25 The desired product was prepared by substituting Fmoc-NMeVaI for Fmoc-
NMeNva in Example 28. Upon completion of the synthesis, cleavage of the resin-
bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column and a
solvent system increasing in gradient from S% to 100% acetonitrile/water
containing
30 0.01 % TFA over a period of 50 minutes. The pure fractions were lyophilized
to provide
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeVaI-Ile-Arg-Pro-D-AIaNHz as a trifluoroacetate
salt; R,
= 3.54 minutes (using a C-18 column and a solvent system increasing in
gradient from
20% to 95% acetonitrile/water containing l OmM ammonium acetate over a period
of 10
minutes); MS (ESI) m/e 1051 (M+H); Amino Acid Anal.: 1.00 Pro; 1.13 Arg; 2.07
Ile;
35 0.53 Thr; 1.05 Ala; 1.03 Val; 1.05 Gly; 1.03 Sar.
42
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 30
N-Ac-Sar-Gly-Val-D-Ile-NMeThr-Nva-Ile-Arg-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-NMeThr(t-Bu) for
Fmoc-Thr(t-Bu) and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of
the synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide can be obtained. This can
be purified
purified by preparative HPLC using a C-18 column and a solvent system
increasing in
gradient from 5% to 100% acetonitrile/water containing 0.01% TFA over a period
of 50
minutes. The pure fractions can then be lyophilized to provide N-Ac-Sar-Gly-
Val-D-Ile-
~ o NMeThr-Nva-Ile-Arg-ProNH-ethyl as a trifluoroacetate salt.
EXAMPLE 31
N-Ac-Sar-Gly-Val-D-Ile-NMeSer-Nva-Ile-Arg-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeSer(t-Bu) for Fmoc-
15 Thr(t-Bu) and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. Thiswas
purified by
preparative HPLC using a C-I8 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
2o pure fractions were lyophilized to provide N-Ac-Sar-Gly-Val-D-Ile-NMeSer-
Nva-Ile-Arg-
ProNH-ethyl as a trifluoroacetate salt; Rt = 3.01 minutes (using a C-18 column
and a
solvent mixture increasing in gradient from 10% to 95% acetonitrile/water
containing
0.01% TFA over a period of 10 minutes); MS (ESI) m/e 994.7 (M+H).
25 EXAMPLE 32
N-Ac-Sar-Gly-Val-D-Leu-NMeSer-Nva-Ile-Arg-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-Leu for Fmoc-D-Ile,
Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in Example
1. Upon completion of the synthesis, cleavage of the resin-bound peptide,
removal of the
3o protecting groups, and precipitation with diethyl ether, the crude peptide
can be obtained.
This can be purified by preparative HPLC using a C-18 column and a solvent
system
increasing in gradient from 5% to 100% acetonitrile/water containing O.OI% TFA
over a
period of 50 minutes. The pure fractions can be lyophilized to provide N-Ac-
Sar-Gly-Val-
D-Leu-NMeSer-Nva-Ile-Arg-ProNH-ethyl as a trifluoroacetate salt.
EXAMPLE 33
43
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
N-Ac-S ar-Gly-V al-D-Leu-S er-NMeNva-I le-Are-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-Leu for Fmoc-D-Ile
and Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) in Example 1. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide can be obtained. This can be purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
can be lyophilized to provide N-Ac-Sar-Gly-Val-D-Leu-Ser-NMeNva-Ile-Arg-ProNH-
ethyl as a trifluoroacetate salt.
EXAMPLE 34
N-Ac-Sar-Gly-Val-D-alloIle-Ser-NMeSer-Ile-Ark-ProNH-ether
The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile,
Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-NMeSer(t-Bu) for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01% TFA
over a period of 50 minutes. The pure fractions were lyophilized to provide N-
Ac-Sar-
2o Gly-Val-D-alloIle-Ser-NMeSer-Ile-Arg-ProNH-ethyl as a trifluoroacetate
salt; R, = 3.21
minutes (using a C-18 column and a solvent system increasing in gradient from
20% to
95% acetonitrile/water containing l OmM ammonium acetate over a period of 10
minutes);
MS (ESI) m/e 982 (M+H); Amino Acid Anal.: 1.02 Pro; 1.32 Arg; 2.12 Ile; 0.31
Ser; 1.01
Val; 1.03 Gly; 0.94 Sar.
EXAMPLE 35
N-Ac-Sar-Gly-Val-D-alloIle-Thr-NMeSer-Ile-Art-ProNH-eth~
The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-Ile
3o and Fmoc-NMeSer(t-Bu) for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01 % TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide N-Ac-Sar-Gly-Val-D-alloIle-Thr-
NMeSer-Ile-
Arg-ProNH-ethyl as a trifluoroacetate salt; R, = 2.82 minutes (using a C-18
column and a
44
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
solvent mixture increasing in gradient from 10% to 95% acetonitrile/water
containing
0.01% TFA over a period of 10 minutes); MS (ESI) m/e 996.7 (M+H).
EXAMPLE 36
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeSer-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeSer(t-Bu) for Fmoc-
NMeNva in Example 1. Upon completion of the synthesis, cleavage of the resin-
bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column and a
~o solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01% TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeSer-Ile-Arg-ProNH-ethyl as a trifluoroacetate
salt; R~ _
2.77 minutes (using a C-18 column and a solvent system increasing in gradient
from 10%
to 95% acetonitrile/water containing 0.01% TFA over a period of 10 minutes);
MS (ESI)
~ 5 m/e 996.6 (M+H).
EXAMPLE 37
N-Ac-Sar-Gly-Val-D-alloIle-Thr-NMeSer-Ile-Ark-Pro-D-AIaNH2
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-
DIIe
2o and Fmoc-NMeSer(t-Bu) for Fmoc-NMeNva in Example 28. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide can be obtained. This can
be purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
25 pure fractions can be lyophilized to provide N-Ac-Sar-Gly-Val-D-alloIle-Thr-
NMeSer-Ile-
Arg-Pro-D-AlaNH2 as a trifluoroacetate salt.
EXAMPLE 38
N-Ac-Sar-Gly-Phe-D-Ile-Thr-NMeVaI-Ile-Ark-Pro-D-AIaNH~
3o The desired product can be prepared by substituting Fmoc-PheAla for Fmoc-
Val
and Fmoc-NMeVaI for Fmoc-Nva in Example 28. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide can be obtained. This can be purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
35 acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The
pure fractions
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
can be lyophilized to provide N-Ac-Sar-Gly-Phe-D-Ile-Thr-NMeVaI-Ile-Arg-Pro-D-
AlaNH2 as a trifluoroacetate salt.
EXAMPLE 3
N-Ac-Sar-Gly-V al-D-alloI le-T~-NMeNva-I le-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-
DIIe
and Fmoc-Tyr(t-Bu) for Fmoc-Thr(t-Bu) in Example 1. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide can be obtained. This can be purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
can be lyophilized to provide N-Ac-Sar-Gly-Val-D-alloIle-Tyr-NMeNva-Ile-Arg-
ProNH-
ethyl as a trifluoroacetate salt.
15 EXAMPLE 40
N-Ac-Sar-Gly-Val-D-alloIle-Tyr-NMeVaI-Ile-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile, Fmoc-Tyr(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-NMeVaI for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
2o removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Val-D-alloIle-Tyr-NMeVaI-Ile-Arg-ProNH-ethyl as a
trifluoroacetate salt.
EXAMPLE 41
N-Ac-Sar-Gly-Gln-D-Ile-Thr-NMeNva-Ile-Art-Pro-D-AlaNH2
The desired product can be prepared by substituting Fmoc-Gln(Trt) for Fmoc-Val
in Example 28. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
3o removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Gln-D-Ile-Thr-NMeNva-Ile-Arg-Pro-D-AlaNH2 as a trifluoroacetate
salt.
EXAMPLE 43
46
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
N-Ac-Sar-Gly-Val-D-alloIle-NMeThr-Nva-Ile-Are-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile, Fmoc-NMeThr(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01% TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Val-D-alloIle-NMeThr-Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 44
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeSer-Ile-Arg-Pro-D-AIaNHz
The desired product can be prepared by Fmoc-NMeSer(t-Bu) for Fmoc-NMeNva
in Example 28. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01% TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Val-D-Ile-Thr-NMeSer-Ile-Arg-Pro-D-AIaNHz as trifluoroacetate
salt.
EXAMPLE 45
N-Ac-Sar-Gly-NMeVaI-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product can be prepared by substituting Fmoc-NMeVaI for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 28. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide can be obtained. This can be purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
can be lyophilized to provide N-Ac-Sar-Gly-NMeVaI-D-Ile-Thr-Nva-Ile-Arg-Pro-D-
3o AIaNHz as trifluoroacetate salt.
EXAMPLE 46
N-Ac-Sar-GI~NMeVa1-D-alloIle-Thr-Nva-Ile-Ark-ProNH-ether
The desired product can be prepared by substituting Fmoc-NMeVaI for Fmoc-Val,
Fmoc-D-alloIle for Fmoc-D-Ile, and Fmoc-Nva for Fmoc NMeNva in Example 1. Upon
completion of the synthesis, cleavage of the resin-bound peptide, removal of
the protecting
47
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
groups, and precipitation with diethyl ether, the crude peptide can be
obtained. This can
be purified by preparative HPLC using a C-18 column and a solvent system
increasing in
gradient from 5% to 100% acetonitrile/water containing 0.01 % TFA over a
period of 50
minutes. The pure fractions can be lyophilized to provide N-Ac-Sar-Gly-NMeVaI-
D-
alloIle-Thr-Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate salt.
EXAMPLE 47
N-Ac-Sar-Gl~Va1-D-HpheAla-Thr-NMeNva-Ile-Arg-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-HpheAla for Fmoc-
D-Ile in Example 1. Upon completion of the synthesis, cleavage of the resin-
bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide can be obtained. This can be purified by preparative HPLC using a C-18
column
and a solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Val-D-HpheAla-Thr-NMeNva-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 48
N-Ac-Sar-Gl~-Val-D-HpheAla-Thr-NMeVaI-Ile-Art-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-HpheAla for Fmoc-
2o D-Ile and Fmoc-NMeVaI for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide can be obtained. This can
be purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions can be lyophilized to provide N-Ac-Sar-Gly-Val-D-HpheAla-Thr-
NMeVaI
Ile-Arg-ProNH-ethyl as trifluoroacetate salt.
EXAMPLE 49
N-Ac-Sar-Gly-Val-D-Pen(SMeI-Thr-NMeNva-Ile-Art-ProNH-ethyl
3o The desired product can be prepared by substituting Fmoc-D-Pen(SMe) for
Fmoc-
D-Ile in Example 1. Upon completion of the synthesis, cleavage of the resin-
bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide can be obtained. This can be purified by preparative HPLC using a C-18
column
and a solvent system increasing in gradient from S% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
48
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
N-Ac-Sar-Gly-Val-D-Pen(SMe)-Thr-NMeNva-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 50
N-Ac-Sar-Gl~Val-D-Pen(SMe)-Thr-NMeVaI-Ile-Arg-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-Pen(SMe) for Fmoc-
D-Ile and Fmoc-NMeVaI for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide can be obtained. This can
be purified by
~o preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01 % TFA over a period of 50
minutes. The
pure fractions can be lyophilized to provide N-Ac-Sar-Gly-Val-D-Pen(SMe)-Thr-
NMeVaI-Ile-Arg-ProNH-ethyl as trifluoroacetate salt.
~5 EXAMPLE 51
N-Ac-Sar-Gly-Val-D-alloIle-NMeSer-Ser-Ile-Are-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile, Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Ser(t-Bu) for Fmoc-NMeNva
in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
2o removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
N-Ac-Sar-Gly-Val-D-alloIle-NMeSer-Ser-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt; Rt
25 = 3.21 minutes (using a C-18 column and a solvent system increasing in
gradient from
20% to 95% acetonitrile/water containing I OmM ammonium acetate over a period
of 10
minutes); MS (ESI) m/e 982 (M+H); Amino Acid Anal.: 1.05 Pro; 0.93 Arg; 0.35
Ser;
2.01 Ile; 0.96 Val; 1.01 Gly; 0.99 Sar.
3o EXAMPLE 54
NAc-Sar-Gly-Val-NMe-D-Leu-Thr-Nva-Ile-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-NMe-D-Leu for Fmoc-D-
Ile and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
35 with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
49
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
acetonitrile/water containing 0.01% TFA over a period of 50 min. The pure
fractions were
lyophilized to provide NAc-Sar-Gly-Val-NMe-D-Leu-Thr-Nva-Ile-Arg-ProNH-ethyl
as
trifluoroacetate salt, R, = 3.87 minutes (using a C-18 column and a solvent
system
increasing in gradient from 20% to 95% acetonitrile/water containing l OmM
ammonium
acetate over a period of 10 min); MS (ESI) m/e 1008 (M+H); Amino Acid Anal.:
0.98 Pro;
0.99 Arg; 1.04 Ile; 0.99 Nva; 0.55 Thr; 1.06 Val; 0.98 Gly; 1.00 Sar.
EXAMPLE 55
NAc-Sar-Gly-Val-D-Ile-Thr-Nva-Ile-Art-NMeNvaNH-ether
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeNva-Sieber ethylamide resin for Fmoc-Pro-Sieber ethylamide resin
in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
~ 5 system increasing in gradient from 5% to 100% acetonitrile/water
containing 0.01 % TFA
over a period of 50 min. The pure fractions were lyophilized to provide NAc-
Sar-Gly-
Val-D-Ile-Thr-Nva-Ile-Arg-NMeNvaNH-ethyl as trifluoroacetate salt; Rt = 4.21
minutes
(using a C-18 column and a solvent system increasing in gradient from 20% to
95%
acetonitrile/water containing l OmM ammonium acetate over a period of 10
minutes); MS
20 (ESI) m/e 1010 (M+H); Amino Acid Anal.: 0.99 Arg; 2.04 Ile; 1.03 Nva; 0.55
Thr; 1.0
Val; 0.97 Gly; 0.98 Sar.
EXAMPLE 56
NAc-Sar-Gly-Val-NMe-D-Leu-Ser-Nva-Ile-Art-ProNH-ether
25 The desired product was prepared by substituting Fmoc-NMe-D-Leu for Fmoc-D-
Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in
Example 1.
Upon completion of the synthesis, cleavage of the resin-bound peptide, removal
of the
protecting groups, and precipitation with diethyl ether, the crude peptide was
obtained.
This was purified by preparative HPLC using a C-18 column and a solvent system
30 increasing in gradient from 5% to 100% acetonitrile/water containing 0.01%
TFA over a
period of 50 minutes. The pure fractions were lyophilized to provide NAc-Sar-
Gly-Val-
NMe-D-Leu-Ser-Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate salt; R, = 3.36
minutes
(using a C-18 column and a solvent system increasing in gradient from 10% to
95%
acetonitrile/water containing 0.01 % TFA over a period of 10 minutes); MS
(ESI) m/e
35 994.7 (M+H).
so
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 57
NAc-Sar-Gly-Asn-NMe-D-Leu-Ser-Nva-Ile-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-Asn(Trt) for Fmoc-Val,
Fmoc-NMe-D-Leu for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva
for Fmoc-NMeNva in Example 1. Upon completion of the synthesis, cleavage of
the
resin-bound peptide, removal of the protecting groups, and precipitation with
diethyl ether,
the crude peptide was obtained. This was purified by preparative HPLC using a
C-18
column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
containing 0.01% TFA over a period of 50 minutes. The pure fractions were
lyophilized
~ o to provide NAc-Sar-Gly-Asn-NMe-D-Leu-Ser-Nva-Ile-Arg-ProNH-ethyl as
trifluoroacetate salt; R, = 2.39 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01 %
TFA over a
period of 10 minutes); MS (ESI) m/e 1009.7 (M+H).
EXAMPLE 58
NAc-Sar-Gly-NMeNva-D-alloIle-Thr-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeNva for Fmoc-Val,
Fmoc-D-alloIle for Fmoc-D-Ile, and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon
completion of the synthesis, cleavage of the resin-bound peptide, removal of
the protecting
2o groups, and precipitation with diethyl ether, the crude peptide was
obtained. This was
purified by preparative HPLC using a C-18 column and a solvent system
increasing in
gradient from 5% to 100% acetonitrile/water containing 0.01 % TFA over a
period of 50
minutes. The pure fractions were lyophilized to provide NAc-Sar-Gly-NMeNva-D-
alloIle-
Thr-Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate salt; Rt = 3.31 minutes (using
a C-18
2s column and a solvent system increasing in gradient from 10% to 95%
acetonitrile/water
containing 0.01 % TFA over a period of 10 minutes); MS (ESI) m/e 1008.7 (M+H);
Amino
acid Anal.: 0.99 Pro; 0.99 Arg; 0.91 Nva; 0.49 Thr; 2.14 Ile; 0.97 Gly; 1.00
Sar.
EXAMPLE 59
3o NAc-Sar-Gly-Asn-D-Leu-NMeSer-Nva-Ile-Are-ProNH-ether
The desired product was prepared by substituting Fmoc-Asn(Trt) for Fmoc-Val ,
Fmoc-D-Leu for Fmoc-D-Ile, Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva
for Fmoc-NMeNva in Example 1. Upon completion of the synthesis, cleavage of
the
resin-bound peptide, removal of the protecting groups, and precipitation with
diethyl ether,
a5 the crude peptide was obtained. This was purified by preparative HPLC using
a C-18
column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
51
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
containing 0.01 % TFA over a period of 50 minutes. The pure fractions were
lyophilized
to provide NAc-Sar-Gly-Asn-D-Leu-NMeSer-Nva-Ile-Arg-ProNH-ethyl as
trifluoroacetate salt; R, = 2.28 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01% TFA
over a
period of 10 minutes); MS (ESI) m/e 1009.7 (M+H).
EXAMPLE 60
NAc-Sar-Gly-NMePhe-D-Ile-Thr-Nva-Ile-Are-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-NMePheAla for Fmoc-Val
~ o and Fmoc-Nva for Fmoc-NMeNva in Example 28. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
~ 5 were lyophilized to provide NAc-Sar-Gly-NMePhe-D-Ile-Thr-Nva-Ile-Arg-Pro-D-
AlaNH2
as trifluoroacetate salt; Rt = 3.45 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01 %
TFA over a
period of 10 minutes); MS (ESI) m/e 1099.7 (M+H).
2o EXAMPLE 61
NAc-Sar-Gly-Val-D-alloIle-NMeSer-Nva-Ile-Arg-ProNH-ether
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile, Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
25 removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Val-D-alloIle-NMeSer-Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 80
NAc-Sar-Gly-V al-D-I le-Thr-NMeNIe-I le-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeNIe for Fmoc-
NMeNva in Example 1. Upon completion of the synthesis, cleavage of the resin-
bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide was obtained. This was purified by preparative HPLC using a C-18
column and a
52
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions were lyophilized to
provide
NAc-Sar-Gly-Val-D-Ile-Thr-NMeNIe-Ile-Arg-ProNH-ethyl as trifluoroacetate salt;
R, _
3.45 minutes (using a C-18 column and a solvent system increasing in gradient
from 10%
to 95% acetonitrile/water containing 0.01 % TFA over a period of 10 minutes);
MS (ESI)
m/e 1022 (M+H); Amino Acid Anal.: 1.03 Pro; 1.09 Arg; 0.45 Thr; 1.81 Ile; 1.07
Val;
1.01 Gly; 1.04 Sar.
EXAMPLE 81
NAc-Sar-Gly-Val-D-Ile-Sar-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-Sar for Fmoc-Thr(t-Bu)
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-Sar-Nva-Ile-Arg-ProNH-ethyl
as
trifluoroacetate salt; Rt = 3.16 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01% TFA
over a
2o period of 10 minutes); MS (ESI) m/e 964.7 (M+H); Amino Acid Anal.: 1.01
Pro; 1.00
Arg; 0.92 Nva; 2.04 Ile; 1.04 Val; 0.99 Gly; 2.01 Sar.
EXAMPLE 82
NAc-Sar-Gly-Val-D-alloIle-Sar-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile,
Fmoc-Sar for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon
completion of the synthesis, cleavage of the resin-bound peptide, removal of
the protecting
groups, and precipitation with diethyl ether, the crude peptide was obtained.
This was
purified by preparative HPLC using a C-18 column and a solvent system
increasing in
3o gradient from 5% to 100% acetonitrile/water containing 0.01% TFA over a
period of 50
minutes. The pure fractions were lyophilized to provide NAc-Sar-Gly-Val-D-
alloIle-Sar-
Nva-Ile-Arg-ProNH-ethyl as trifluoroacetate salt; R, = 3.15 minutes (using a C-
18 column
and a solvent system increasing in gradient from 10% to 95% acetonitrile/water
containing
0.01 % TFA over a period of 10 minutes); MS (ESI) m/e 964.7 (M+H); Amino Acid
Anal.:
1.00 Pro; 0.99 Arg; 0.90 Nva; 2.11 Ile; 1.03 Val; 0.96 Gly; 1.96 Sar.
53
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
EXAMPLE 83
NAc-Sar-Gly-Val-D-Ile-Thr-Nva-NMeAIa-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeAIa for Fmoc-Ile in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a'C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-Thr-Nva-NMeAIa-Arg-ProNH-
ethyl
~o as trifluoroacetate salt; Rt = 2.86 minutes (using a C-18 column and a
solvent system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01% TFA
over a
period of 10 minutes); MS (ESI) m/e 966.7 (M+H); Amino Acid Anal.: 0.97 Pro;
0.98
Arg; 0.97 Nva; 0.51 Thr; 1.09 Ile; 1.02 Val; 0.96 Gly; 1.10 Sar.
EXAMPLE 84
NAc-Sar-Gly-Val-D-Ile-NMeAsp-Nva-Ile-Arg_-ProNH-ether
The desired product was prepared by substituting Fmoc-NMeAsp(t-Bu) for Fmoc-
Thr(t-Bu) and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
2o precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01 % TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-NMeAsp-Nva-
Ile-Arg-
ProNH-ethyl as trifluoroacetate salt; R, = 2.89 minutes (using a C-18 column
and a solvent
system increasing in gradient from 10% to 95% acetonitrile/water containing
0.01% TFA
over a period of 10 minutes); MS (ESI) m/e 1022.7 (M+H); Amino Acid Anal.:
1.02 Pro;
1.02 Arg; 0.92 Nva; 2.01 Ile; 1.04 Val; 1.00 Gly; 0.98 Sar.
EXAMPLE 85
3o NAc-Sar-Gly-Val-D-Ile-Thr-NMe-D-Leu-Ile-Art-ProNH-ether
The desired product was prepared by substituting Fmoc-NMe-D-Leu for NMeNva
in Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01 % TFA
over a period of SO minutes. The pure fractions were lyophilized to provide
NAc-Sar-Gly-
54
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
Val-D-Ile-Thr-NMe-D-Leu-Ile-Arg-ProNH-ethyl as trifluoroacetate salt; R~ =
3.58 minutes
(using a C-18 column and a solvent system increasing in gradient from 10% to
95%
acetonitrile/water containing 0.01% TFA over a period of 10 minutes); MS (ESI)
m/e
1022.8 (M+H); Amino Acid Anal.: 1.04 Pro; 1.03 Arg; 0.47 Thr; 1.87 Ile; 1.06
Val; 1.01
Gly; 1.05 Sar.
EXAMPLE 86
NAc-Sar-Gly-Val-D-Ile-NMeGIu-Nva-Ile-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMeGIu(t-Bu) for Fmoc-
~ o Thr(t-Bu) and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of
the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
~5 pure fractions were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-NMeGIu-Nva-
Ile-Arg-
ProNH-ethyl as trifluoroacetate salt; RI = 3.12 minutes (using a C-18 column
and a solvent
system increasing in gradient from 10% to 95% acetonitrile/water containing
0.01 % TFA
over a period of 10 minutes); MS (ESI) m/e 1036.7 (M+H); Amino Acid Anal.:
1.01 Pro;
1.0 Arg; 0.93 Nva; 2.04 Ile; 1.04 Val; 0.98 Gly; 1.0 Sar.
EXAMPLE 87
NAc-Sar-Gly-NMe-D-Val-D-Ile-Thr-Nva-Ile-Arg-ProNH-ethyl
The desired product was prepared by substituting Fmoc-NMe-D-Val for Fmoc-Val
and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide NAc-Sar-Gly-NMe-D-Val-D-Ile-Thr-Nva-Ile-Arg-ProNH-
3o ethyl as trifluoroacetate salt; Rt = 3.12 minutes (using a C-18 column and
a solvent system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01 %
TFA over a
period of 10 minutes); MS (ESI) m/e 1008.7 (M+H); Amino Acid Anal.: 0.99 Pro;
1.02
Arg; 0.97 Nva; 0.43 Thr; 2.06 Ile; 0.96 Gly; 0.99 Sar.
EXAMPLE 88
NAc-Sar-Gly-Val-D-Ile-alloThr-NMeNIe-Ile-Art-ProNH-ethyl
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
The desired product was prepared by substituting Fmoc-alloThr(t-Bu) for Fmoc-
Thr(t-Bu) and Fmoc-NMeNIe for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
precipitation with diethyl ether, the crude peptide was obtained. This was
purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-alloThr-
NMeNIe-Ile-
Arg-ProNH-ethyl as trifluoroacetate salt; R, = 3.59 minutes (using a C-18
column and a
solvent system increasing in gradient from 10% to 95% acetonitrile/water
containing
0 0.01 % TFA over a period of 10 minutes); MS (ESI) m/e 1022.8 (M+H); Amino
Acid
Anal.: 1.05 Pro; 0.97 Arg; 0.52 Thr; 1.88 Ile; 1.01 Gly; 1.03 Sar.
EXAMPLE 89
NAc-Sar-Gly-NMe-D-Phe-D-Ile-Thr-Nva-Ile-Arg-ProNH-ether
~ 5 The desired product was prepared by substituting Fmoc-NMe-D-PheAla for
Fmoc-
Val and Fmoc-Nva for Fmoc-NMeNva in Example 1. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
2o acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The
pure fractions
were lyophilized to provide NAc-Sar-Gly-NMe-D-Phe-D-Ile-Thr-Nva-Ile-Arg-ProNH-
ethyl as trifluoroacetate salt; Rt = 3.32 minutes (using a C-18 column and a
solvent system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01% TFA
over a
period of 10 minutes); MS (ESI) m/e 1056.7 (M+H).
EXAMPLE 90
NAc-Sar-Glv-NMe-D-Phe-D-Ile-Thr-Nva-Ile-Art-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-NMe-D-PheAla for Fmoc-
Val and Fmoc-Nva for Fmoc-NMeNva in Example 28. Upon completion of the
synthesis,
3o cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01% TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide NAc-Sar-Gly-NMe-D-Phe-D-Ile-Thr-Nva-Ile-Arg-Pro-D-
AIaNHz as trifluoroacetate salt; Rt = 3.18 minutes (using a C-18 column and a
solvent
56
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
system increasing in gradient from 10% to 95% acetonitrile/water containing
0.01% TFA
over a period of 10 minutes); MS (ESI) m/e 1099.7 (M+H).
EXAMPLE 91
NAc-Sar-Gly-Val-D-Ile-Thr-Nva-NMeLeu-Art-ProNH-ethyl
The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMeLeu for Fmoc-Ile in Example 1. Upon completion of the synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-Thr-Nva-NMeLeu-Arg-ProNH-
ethyl
as trifluoroacetate salt; R, = 3.39 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01 %
TFA over a
15 period of 10 minutes); MS (ESI) m/e 1008.7 (M+H).
EXAMPLE 92
NAc-Sar-Gly-Asn-D-Leu-NMeSer-Nva-Ile-A~-ProNH-ether
The desired product was prepared by substituting Fmoc-Asn(Trt) for Fmoc-Val,
2o Fmoc-D-Leu for Fmoc-D-Ile, Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-
Nva
for Fmoc-NMeNva in Example 1. Upon completion of the synthesis, cleavage of
the
resin-bound peptide, removal of the protecting groups, and precipitation with
diethyl ether,
the crude peptide was obtained. This was purified by preparative HPLC using a
C-18
column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
25 containing 0.01 % TFA over a period of 50 minutes. The pure fractions were
lyophilized
to provide NAc-Sar-Gly-Asn-D-Leu-NMeSer-Nva-Ile-Arg-ProNH-ethyl as
trifluoroacetate salt; Rt = 2.28 minutes (using a C-18 column and a solvent
system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01% TFA
over a
period of 10 minutes); MS (ESI) m/e 1009.7 (M+H).
EXAMPLE 93
NAc-Sar-Gly-Val-D-alloIle-Ser-NMeSer-Ile-Art-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile,
Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-NMeSer(t-Bu) for Fmoc-NMeNva in
Example 28. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
57
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
was obtained. This was purified by preparative HPLC using a C-18 column and a
solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01% TFA
over a period of 50 minutes. The pure fractions were lyophilized to provide
NAc-Sar-Gly-
Val-D-alloIle-Ser-NMeSer-Ile-Arg-Pro-D-AlaNH2 as trifluoroacetate salt; Rt =
2.74
minutes (using a C-18 column and a solvent system increasing in gradient from
10% to
95% acetonitrile/water containing 0.01 % TFA over a period of 10 minutes); MS
(ESI) m/e
1025.7 (M+H).
EXAMPLE 94
NAc-Sar-Gly-Val-D-alloIle-NMeSer-Ser-Ile-Arg-ProNH-ether
The desired product was prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile,
Fmoc-NMeSer(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Ser(t-Bu) for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
~5 was obtained. This was purified by preparative HPLC using a C-18 column and
a solvent
system increasing in gradient from 5% to 100% acetonitrile/water containing
0.01 % TFA
over a period of 50 minutes. The pure fractions were lyophilized to provide
NAc-Sar-Gly-
Val-D-alloIle-NMeSer-Ser-Ile-Arg-ProNH-ethyl as trifluoroacetate salt; R, =
2.53 minutes
(using a C-18 column and a solvent system increasing in gradient from 10% to
95%
2o acetonitrile/water containing 0.01 % TFA over a period of 10 minutes); MS
(ESI) m/e
982.6 (M+H).
EXAMPLE 95
NAc-S ar-Gly-V al-D-I le-Thr-Nva-NMe-D-A1 a-Art-ProNH-ether
25 The desired product was prepared by substituting Fmoc-Nva for Fmoc-NMeNva
and Fmoc-NMe-D-Ala for Fmoc-Ile in Example 1. Upon completion of the
synthesis,
cleavage of the resin-bound peptide, removal of the protecting groups, and
precipitation
with diethyl ether, the crude peptide was obtained. This was purified by
preparative
HPLC using a C-18 column and a solvent system increasing in gradient from 5%
to 100%
3o acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The
pure fractions
were lyophilized to provide NAc-Sar-Gly-Val-D-Ile-Thr-Nva-NMe-D-Ala-Arg-ProNH-
ethyl as trifluoroacetate salt; Rt = 2.53 minutes (using a C-18 column and a
solvent system
increasing in gradient from 10% to 95% acetonitrile/water containing 0.01 %
TFA over a
period of 10 minutes); MS (ESI) m/e 966.7 (M+H).
EXAMPLE 97
ss
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
NAc-Sar-Gly-Val-D-alloIle-NMeTyr-Nva-Ile-Art-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-alloIle for Fmoc-D-
Ile, Fmoc-NMeTyr(t-Bu) for Fmoc-Thr(t-Bu), and Fmoc-Nva for Fmoc-NMeNva in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Val-D-alloIle-NMeTyr-Nva-Ile-Arg-ProHEt as trifluoroacetate salt.
EXAMPLE 99
NAc-Sar-Gly-V al-D-Ile-Thr-NMeNva-D-Ile-Art-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-Ile for Fmoc-Ile in
Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Val-D-Ile-Thr-NMeNva-D-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 100
NAc-Sar-Gly-Val-D-Ile-alloThr-NMeNva-Ile-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-alloThr(t-Bu) for
Fmoc-
Thr(t-Bu) in Example 1. Upon completion of the synthesis, cleavage of the
resin-bound
peptide, removal of the protecting groups, and precipitation with diethyl
ether, the crude
peptide can be obtained. This can be purified by preparative HPLC using a C-18
column
and a solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Val-D-Ile-alloThr-NMeNva-Ile-Arg-ProNH-ethyl as trifluoroacetate
salt.
EXAMPLE 101
NAc-Sar-Gly-Gln-D-Ile-Thr-NMeNva-D-Ile-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-Gln(Trt) for Fmoc-Val
and Fmoc-D-Ile for Fmoc-Ile in Example 1. Upon completion of the synthesis,
cleavage
of the resin-bound peptide, removal of the protecting groups, and
precipitation with diethyl
ether, the crude peptide can be obtained. This can be purified by preparative
HPLC using
59
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
a C-18 column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water containing 0.01 % TFA over a period of 50 minutes. The pure
fractions
can be lyophilized to provide NAc-Sar-Gly-Gln-D-Ile-Thr-NMeNva-D-Ile-Arg-ProNH-
ethyl as trifluoroacetate salt.
EXAMPLE 102
NAc-Sar-Gly-Val-D-Ile-Thr-NMeNva-D-Lys(Ac)-Art-ProNH-ethyl
The desired product can be prepared by substituting Fmoc-D-Lys(Ac) for Fmoc-
Ile
in Example 1. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
~ o removal of the protecting groups, and precipitation with diethyl ether,
the crude peptide
can be obtained. This can be purified by preparative HPLC using a C-18 column
and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01 % TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Val-D-Ile-Thr-NMeNva-D-Lys(Ac)-Arg-ProNH-ethyl as trifluoroacetate
~ 5 salt.
EXAMPLE 103
NAc-Sar-Gly-Gln-D-alloIle-NMeTyr-Nva-Ile-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-Gln(Trt) for Fmoc-
Val,
2o Fmoc-D-alloIle for Fmoc-D-Ile, Fmoc-NMeTyr(t-Bu) for Fmoc-Thr(t-Bu), and
Fmoc-Nva
for Fmoc-NMeNva in Example 1. Upon completion of the synthesis, cleavage of
the
resin-bound peptide, removal of the protecting groups, and precipitation with
diethyl ether,
the crude peptide can be obtained. This can be purified by preparative HPLC
using a C-18
column and a solvent system increasing in gradient from 5% to 100%
acetonitrile/water
25 containing 0.01 % TFA over a period of 50 minutes. The pure fractions can
be lyophilized
to provide NAc-Sar-Gly-Gln-D-alloIle-NMeTyr-Nva-Ile-Arg-ProNH-ethyl as
trifluoroacetate salt.
EXAMPLE 104
3o NAc-Sar-Gly-Gln-D-alloIle-NMeTyr-Nva-D-Ile-Art-ProNH-ether
The desired product can be prepared by substituting Fmoc-Gln(Trt) for Fmoc-
Val,
Fmoc-D-alloIle for Fmoc-D-Ile, Fmoc-NMeTyr(t-Bu) for Fmoc-Thr(t-Bu), Fmoc-Nva
for
Fmoc-NMeNva, and Fmoc-D-Ile for Fmoc-Ile in Example 1. Upon completion of the
synthesis, cleavage of the resin-bound peptide, removal of the protecting
groups, and
35 precipitation with diethyl ether, the crude peptide can be obtained. This
can be purified by
preparative HPLC using a C-18 column and a solvent system increasing in
gradient from
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
5% to 100% acetonitrile/water containing 0.01% TFA over a period of 50
minutes. The
pure fractions can be lyophilized to provide NAc-Sar-Gly-Gln-D-alloIle-NMeTyr-
Nva-D-
Ile-Arg-ProNH-ethyl as trifluoroacetate salt.
EXAMPLE 105
NAc-Sar-Gly-Phe-D-Ile-Thr-NMeNva-Ile-Ar~Pro-D-AIaNHz
The desired product can be prepared by substituting Fmoc-PheAla for Fmoc-Val
in
Example 28. Upon completion of the synthesis, cleavage of the resin-bound
peptide,
removal of the protecting groups, and precipitation with diethyl ether, the
crude peptide
~o can be obtained. This can be purified by preparative HPLC using a C-18
column and a
solvent system increasing in gradient from 5% to 100% acetonitrile/water
containing
0.01% TFA over a period of 50 minutes. The pure fractions can be lyophilized
to provide
NAc-Sar-Gly-Phe-D-Ile-Thr-NMeNva-Ile-Arg-Pro-D-AlaNH2 as trifluoroacetate
salt.
15 EXAMPLE 109
NMePro-Gly-Ile-D-Ile-Thr-NMeNva-Ile-Ark-ProNH-ether
The desired product can be prepared by substituting NMePro for Fmoc-Sar and
Fmoc-Ile for Fmoc-Val in Example 1, and omitting the final coupling with
acetic acid.
Upon completion of the synthesis, cleavage of the resin-bound peptide, removal
of the
2o protecting groups, and precipitation with diethyl ether, the crude peptide
can be obtained.
This can be purified by preparative HPLC using a C-18 column and a solvent
system
increasing in gradient from 5% to 100% acetonitrile/water containing 0.01% TFA
over a
period of SO minutes. The pure fractions can be lyophilized to NMePro-Gly-Ile-
D-Ile-Thr-
NMeNva-Ile-Arg-ProNH-ethyl as trifluoroacetate salt.
It will be evident to one skilled in the art that the instant invention is not
limited to
the forgoing illustrative examples, and that it can be embodied in other
specific forms
without departing from the essential attributes thereof. It is therefore
desired that the
examples be considered in all respects as illustrative and not restrictive,
reference being
3o made to the appended claims, and all changes which come within the meaning
and range
of equivalency of the claims and therefore intended to be embraced therein.
61
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
SEQUENCE LISTING
<110> Abbott Laboratories
Haviv, Fortuna
Henkin, Jack
Kalvin, Douglas M.
Bradley, Michael F.
<120> N-ALKYLATED PEPTIDES HAVING
ANTIANGIOGENIC ACTIVITY
<130> 6632.PC.01
<140> Not Yet Assigned
<141>
<150> Unknown
<151> 2000-10-31
<150> US 09/447,099
<151> 1999-11-22
<160> 1
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Antiangiogenetic Peptide
<221> VARIANT
<222> (1) . . . (1)
<223> Xaa = N-methylprolyl at position 1
<221> VARIANT
<222> (2) . . . (2)
<223> Xaa = N-(R3)Ala, N-(R3)Gly, N-(R3)Nva, N-(R3)Pro,
B-Ala, Asn, 4-ClPheAla, 4-CNPheAla, Gln, Glu, Gly,
4-OHPro, 4-MePheAla, Pro, Ser, or Thr at position
2
<221> VARIANT
<222> (3) . . . (3)
<223> Xaa = N-(R3)Ala, N-(R3)Gly, N-(R3)Leu,
N-(R3)PheAla, Ala, Asn, Asp, 3-CNPheAla,
4-CNPheAla, Gln, Gly, Leu, Lys(Ac), 4-MePheAla,
Nva, Pro, and PheAla as position 3
<221> VARIANT
<222> (4) . . . (4)
<223> Xaa = N-(R3)Ala, N-(R3)Gly, N-(R3)HpheAla,
N-(R3)Ile, N-(R3)Leu, N-(R3)Nva, N-(R3)PheAla,
N-(R3)Ser, N-(R3)Tyr, N-(R3)Val, Ala, AlloIle,
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
2
Allylgly, 2-Ambut, Asn, Asp at position 4
<221> VARIANT
<222> (4) . . . (4)
<223> Xaa = 5-BrThiAla, 3-ClPheAla, 4-ClPheAla,
3-CNPheAla, Cha, 3,4-diOMe-PheAla, 3-FpheAla,
4-FpheAla, Gln, Gly, His, HpheAla, Hser, Ile, Leu,
Lys(Ac), Met, Met(02), 4-MePheAla at position 4
<221> VARIANT
<222> (4)...(4)
<223> Xaa = 1-Nal, 2-Nal, Nor, Nva, PheAla, PheGly,
Pro, 3-PyrAla, 4-ThzAla, 2-ThiAla, Ser, Ser(Bzl),
StyAla, Trp, Tyr, Val at position 4
<221> VARIANT
<222> (5)...(5)
<223> Xaa = AlloIle, Chg, Gly, Ile at position 5
<221> VARIANT
<222> (6)...(6)
<223> Xaa = N-(R3)Asp, N-(R3)Glu, N-(R3)Gly, N-(R3)Ser,
N-(R3)Thr, N-(R3)Thr(Bzl), N-(R3)Tyr, Ala,
AlloThr, Allylgly, Asn, Asp, Gln, Gly, His, Hser,
4-OHMePheAla, Ile, Lys(Ac) at position 6
<221> VARIANT
<222> (6)...(6)
<223> Xaa = Met, 2-Nal, Nva, Octylgly, Pro,3-PyrAla,
Ser, Thr, Trp, Tyr, Tyr(Me) at position 6
<221> VARIANT
<222> (7)...(7)
<223> Xaa = N-(R3)Ala, N-(R3)Gly, N-(R3)Ile, N-(R3)Leu,
N-(R3)Nle, N-(R3)Nva, N-(R3)Ser, N-(R3)Thr,
N-(R3)Val, Ala, AlloThr, Allylgly, 4-AmdPheAla,
2-Ambut, Arg, Asn at position 7
<221> VARIANT
<222> (7)...(7)
<223> Xaa = Cha, Gln, Gly, Hala, Hser, 4-OHPro, Leu,
Lys(Ac), Met(02), Met(O), Met, Nle, Nva,
Octylgly, Orn(Isp), PheAla, ProGly, Ser, Thr, Trp,
Tyr, and Val at position 7
<221> VARIANT
<222> (8)...(8)
<223> N-(R3)Ala, N-(R3)Ile, N-(R3)Leu, Ala, AlloIle,
Allylgly, Cit, Gly, Ile, Leu, Lys(Ac), Met, 1-Nal,
Nva, Pro, and Val at position 8
<221> VARIANT
<222> (9) . . . (9)
<223> Xaa = N-(R3)Arg, 4-AmIspCha, 4-AmIspPheAla,
Arg(diethyl), Arg, Cit, Gln, 4-GuPheAla, His,
Harg, Lys(Isp), Lys(Nic), Lys, Nor, Orn, Orn(Im),
Orn(Isp), and 3-PyrAla at position 9
CA 02386893 2002-04-08
WO 01/38397 PCT/US00/32105
3
<221> VARIANT
<222> (10)...(10)
<223> Xaa = N-(R3)Ala, N-(R3)Gly, N-(R3)Hala, N-(R3)Nva,
2-Ambut, 2-Amisobut, dePro, 4-OHPro, PheAla, Pro,
and Tic at position 10
<221> VARIANT
<222> (11)...(11)
<223> Xaa = AlaNH2, AlaNH-ethyl, AzaGIyNH2, GlyNH2,
GlyNH-ethyl, Lys(Ac), SarNH2, and SerNH2 at
position 11
<400> 1
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
