HEXA-, HEPTA- ET OCTAPEPTIDES A ACTIVITE ANTI-ANGIOGENIQUE

HEXA-, HEPTA-, AND OCTAPEPTIDES HAVING ANTIANGIOGENIC ACTIVITY

Abrégé français

L'invention concerne des composés représentés par la formule (SEQ ID NO:1), lesquels sont utiles pour le traitement d'affections résultant de l'angiogenèse ou exacerbées par elle. La présente invention concerne également des compositions pharmaceutiques comprenant de tels composés; des méthodes de traitement consistant à utiliser ces composés, et des méthodes permettant d'inhiber l'angiogenèse.

Abrégé anglais

Compounds of formula (SEQ ID NO:1), which are useful for treating conditions
that arise from or are exacerbated by angiogenesis, are described. Also
disclosed are pharmaceutical compositions comprising these compounds, methods
of treatment using these compounds, and methods of inhibiting angiogenesis.

Revendications

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A compound which is N-Ac-Gly-Val-D-aIle-Ser-Gln-Ile-Arg-ProNHCH2CH3.
2. A pharmaceutical composition comprising a compound of claim 1, or a
therapeutically acceptable salt thereof, in combination with a therapeutically
acceptable
carrier.
3. Use of a compound of claim 1 or a therapeutically acceptable salt thereof
for
inhibiting angiogenesis.
4. Use of a compound of claim 1 or a therapeutically acceptable salt thereof
for treating
cancer.
5. Use of a compound of claim 1 or a therapeutically acceptable salt thereof
for the
manufacture of a medicament for inhibiting angiogenesis.
6. Use of a compound of claim 1 or a therapeutically acceptable salt thereof
for the
manufacture of a medicament for treating cancer.
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Description

CA 02466170 2010-05-18
HEXA-, HEPTA-, AND OCTAPEPTIDES HAVING ANTIANGIOGENIC ACTIVITY
Technical Field
The present invention relates to methods of inhibiting angiogenesis, methods
of treating
cancer, and compounds having activity useful for treating conditions which
arise from or are
exacerbated by angiogenesis. Also disclosed are pharmaceutical compositions
comprising the
compounds and methods of treatment using the compounds.
Background of the Invention
Angiogenesis is the fundamental process by which new blood vessels are formed
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 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.
Although angiogenesis is a highly regulated process under normal conditions,
many
diseases (characterized as "angiogenic diseases") are driven by persistent
unregulated angio-
genesis. Otherwise stated, unregulated angiogenesis may either cause a
particular disease directly
or exacerbate an existing pathological condition. For example, the growth and
metastasis of solid
tumors have been shown to be angiogenesis-dependent. Based on these findings,
there is a
continuing need for compounds which demonstrate antiangiogenic activity due to
their potential
use in the treatment of various diseases such as cancer. Peptides having
angiogenesis inhibiting
properties have been described in commonly-owned WO01/38397, WO01/38347,
W099/61476,
and U.S. Patent Publication No. 2003/0050246. However, it would be desirable
to prepare
antiangiogenic compounds having improved profiles of activity and smaller
size.
Summary of the Invention
In its principle embodiment, the present invention provides a compound of
formula (1)
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaalo (SEQ ID NO: 1)
or a therapeutically acceptable salt thereof, wherein
Xaal is selected from the group consisting of hydrogen and R-(CH2)n-C(O)-,
wherein n is
an integer from 0 to 8 and R is selected from the group consisting of alkoxy,
alkyl, amino, aryl,
carboxyl, cycloalkenyl, cycloalkyl, and heterocycle;
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Xaa2 is selected from the group consisting of alanyl, D-alanyl, (I S,3R)-1-
aminocyclopentane-3-carbonyl, (1 S,4R)-1-aminocyclopent-2-ene-4-carbonyl, (1
R,4S)-1-
aminocyclopent-2-ene-4-carbonyl, asparaginyl, 3-cyanophenylalanyl, 4-
cyanophenylalanyl, 3,4-
dimethoxyphenylalanyl, 4-fluorophenylalanyl, 3-(2-fury l)alany1, glutaminyl, D-
glutaminyl, glycyl,
lysyl(N-epsilon acetyl), 4-methylphenylalanyl, norvalyl, and sarcosyl;
Xaa3 is selected from the group consisting of alanyl, (I R,4S)- I -
aminocyclopent-2-ene-4-
carbonyl, arginyl, asparaginyl, D-asparaginyl, t-butylglycyl, citrullyl,
cyclohexylglycyl, glutaminyl,
D-glutaminyl, glutamyl, glycyl, histidyl, isoleucyl, leucyl, lysyl(N -epsilon-
acetyl), methionyl,
norvalyl, phenylalanyl, N-methylphenylalanyl, prolyl, seryl, 3-(2-
thienylalanyl), threonyl, valyl, and
N-methylvalyl;
Xaa4 is selected from the group consisting of D-alanyl, D-alloisoleucyl, D-
allylglycyl, D-4-
chlorophenylalanyl, D-citrullyl, D-3-cyanophenylalanyl, D-homophenylalanyl, D-
homoseryl,
isoleucyl, D-isoleucyl, D-leucyl, N-methyl-D-leucyl, D-norleucyl, D-norvalyl,
D-penicillaminyl, D-
phenylalanyl, D-prolyl, D-seryl, D-thienylalanyl, and D-threonyl;
Xaa5 is selected from the group consisting ofallothreonyl, aspartyl,
glutaminyl, D-
glutaminyl, N-methylglutaminyl, N-methylglutamyl, glycyl, histidyl, homoseryl,
isoleucyl, lysyl(N-
epsilon-acetyl), methionyl, seryl, N-methylseryl, threonyl, D-threonyl,
tryptyl, tyrosyl, and
tyrosyl(O-methyl);
Xaa6 is selected from the group consisting of alanyl, N-methylalanyl,
allothreonyl,
glutaminyl, glycyl, homoseryl, leucyl, lysyl(N-epsilon-acetyl), norleucyl,
norvalyl, D-norvalyl, N-
methylnorvalyl, octylglycyl, ornithyl(N-delta-acetyl), 3-(3-pyridyl)alanyl,
sarcosyl, seryl, N-
methylseryl, threonyl, tryptyl, valyl, and N-methylvalyl;
Xaa7 is selected from the group consisting of alanyl, alloisoleucyl, aspartyl,
citrullyl,
isoleucyl, D-isoleucyl, leucyl, D-leucyl, lysyl(N-epsilon-acetyl), D-lysyl(N-
epsilon-acetyl), N-
methylisoleucyl, norvalyl, phenylalanyl, prolyl, and D-prolyl;
Xaa8 is selected from the group consisting of arginyl, D-arginyl, citrullyl,
glutaminyl,
histidyl, homoarginyl, lysyl, lysyl(N-epsilon-isopropyl), ornithyl, and 3{3-
pyridyl)alanyl;
Xaag is absent or selected from the group consisting of N-methyl-D-alanyl, 2-
aminobutyryl,
D-glutaminyl, homoprolyl, hydroxyprolyl, leucyl, prolyl, D-prolyl, and D-
valyl; and
Xaa1O is selected from the group consisting of D-alanylamide, azaglycylamide,
glycylamide,
D-lysyl(N-epsilon-acetyl)amide, a group represented by the formula NH-(CH2)n-
CHRIR2; and a
group represented by the formula -NHR3, wherein n is an integer from 0 to 8;
RI is selected from
the group consisting of hydrogen, alkyl, cycloalkenyl, and cycloalkyl; R2 is
selected from the group
consisting of hydrogen, alkoxy, alkyl, aryl, cycloalkenyl, cycloalkyl,
heterocycle, and hydroxyl,
with the proviso that when n is 0, R2 is other than alkoxy or hydroxyl; and R3
is selected from the
group consisting of hydrogen, cycloalkenyl, cycloalkyl, and hydroxyl.
In a preferred embodiment, the present invention provides a compound of
formula (I), or a
therapeutically acceptable salt thereof, wherein Xaa2 is selected from the
group consisting of alanyl,
D-alanyl, asparaginyl, 4-cyanophenylalanyl, 4-methylphenylalanyl, and
norvalyl; and Xaa1, Xaa3,
Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaag, and Xaa10 are as described for formula
(1).
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In another preferred embodiment, the present invention provides compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is selected from
the group consisting of
glutaminyl and D-glutaminyl, and Xaal, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8,
Xaag, and Xaa10 are
as described for formula (1).
In another preferred embodiment, the present invention provides a compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is glycyl; Xaa3 is
selected from the group
consisting of arginyl, asparaginyl, D-asparaginyl, citrullyl, lysyl(N-epsilon-
acetyl), and histidyl; and
Xaa1, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaag, and Xaa10 are as described for
formula (1).
In another preferred embodiment, the present invention provides a compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is glycyl; Xaa3 is
selected from the group
consisting of valyl and N-methylvalyl, Xaa6 is selected from the group
consisting of norvalyl and N-
methylnorvalyl; and Xaa1, Xaa4, Xaa5, Xaa7, Xaa8, Xaag, and Xaa10 are as
described for formula (I).
In another preferred embodiment, the present invention provides a compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is glycyl; Xaa3 is
selected from the group
consisting of valyl and N-methylvalyl, Xaa6 is selected from the group of
glutaminyl, seryl, and
threonyl; and Xaa1, Xaa4, Xaa5, Xaa7, Xaa8, Xaag, and Xaa10 are as described
for formula (1).
In another preferred embodiment, the present invention provides a compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is glycyl; Xaa3 is
selected from the group
consisting of glutaminyl, D-glutaminyl, phenylalanyl, and N-
methylphenylalanyl, Xaa7 is isoleucyl;
and Xaa1, Xaa4, Xaa5, Xaa6, Xaa8, Xaag, and Xaa10 are as described for formula
(I).
In another preferred embodiment, the present invention provides a compound of
formula (I),
or a therapeutically acceptable salt thereof, wherein Xaa2 is glycyl; Xaa3 is
selected from the group
consisting of glutaminyl, D-glutaminyl, and phenylalanyl; Xaa7 is selected
from the group
consisting of D-isoleucyl, lysyl(N-epsilon acetyl), and D-prolyl; and Xaa1,
Xaa4, Xaa5, Xaa6, Xaa8,
Xaag, and Xaa10 are as described for formula (I).
In another embodiment, the present invention provides a pharmaceutical
composition
comprising a compound of formula (I), or a therapeutically acceptable salt
thereof, in combination
with a therapeutically acceptable carrier.
In another embodiment, the present invention provides a method of inhibiting
angiogenesis
in a mammal in recognized need of such treatment comprising administering to
the mammal a
therapeutically acceptable amount of a compound of formula (1) or a
therapeutically acceptable salt
thereof.
In another embodiment, the present invention provides a method of treating
cancer in a
mammal in recognized need of such treatment comprising administering to the
mammal a
therapeutically acceptable amount of a compound of formula (1) or a
therapeutically acceptable salt
thereof.
Detailed Description of the Invention
As used herein, the singular forms "a", "an", and "the" include plural
reference unless the
context clearly dictates otherwise.
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As used in the present specification the following terms have the meanings
indicated:
The term "alkoxy," as used herein, represents an alkyl group attached to the
parent molecular
moiety through an oxygen atom.
The term "alkyl," as used herein, represents 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 six
carbon atoms (Cr-C6
alkyl). Alkyl groups of one to three carbon atoms (Cr-C3 alkyl) are more
preferred for the present
invention.
The term "alkylcarbonyl," as used herein, represents an alkyl group attached
to the parent
to molecular moiety through a carbonyl group.
The term "amino," as used herein, represents-NRaRb, wherein Ra and Rb are
independently
selected from the group consisting of hydrogen, alkyl, and alkylcarbonyl.
The term "aryl," as used herein, represents a phenyl group, or a bicyclic or
tricyclic fused
ring system wherein one or more of the fused rings is a phenyl group. Bicyclic
fused ring systems
are exemplified by a phenyl group fused to a cycloalkenyl group, as defined
herein, a cycloalkyl
group, as defined herein, or another phenyl group. Tricyclic fused ring
systems are exemplified by a
bicyclic fused ring system fused to a cycloalkenyl group, as defined herein, a
cycloalkyl group, as
defined herein or another phenyl group. Representative examples of aryl
include, but are not limited
to, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, phenyl, and
tetrahydronaphthyl.
The aryl 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, carboxyl, halo,
and hydroxyl.
The term "carbonyl," as used herein, represents-C(O)-.
The term "carboxyl," as used herein, represents -C02H.
The term "cycloalkenyl," as used herein, refers to a non-aromatic cyclic or
bicyclic 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,
3o 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, carboxyl, halo, and hydroxyl.
The term "cycloalkyl," as used herein, refers to a saturated monocyclic,
bicyclic, or 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, carboxyl, halo, and
hydroxyl.
The term "halo," as used herein, represents F, Cl, Br, or I.
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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. The term
"heterocycle" also includes
bicyclic groups in which the heterocycle ring is fused to an aryl group, as
defined herein. 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, 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 ofalkoxy, alkyl,
carboxyl, halo, and hydroxyl.
The term "hydroxyl," as used herein, represents -0H.
The term "therapeutically acceptable salt," as used herein, represents salts
or zwitterionic
forms of the compounds of the present invention which are water or oil-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, citrate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate,
camphorate, camphorsulfonate, digluconate, glycerophosphate, hemisulfate,
heptanoate, hexanoate,
formate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-
hydroxyethansulfonate, 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 be
3o employed to form therapeutically acceptable addition salts include
inorganic acids such as
hydrochloric, hydrobromic, sulfuric, and phosphoric, and organic acids such as
oxalic, maleic,
succinic, and citric.
Unless indicated otherwise by a "D" prefix, e.g., D-Ala or NMe-D-lle, 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- Ingo ld-
Prelog "R" and "S"
designations are used to specify the stereochemistry of chiral centers in
certain acylsubstituents 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, el al., Angew. Chem Int. Ed. Engl., 5, 385-415 (1966).
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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-terminus" refers to the free a-amino group of an amino
acid in a peptide, and
the term "a-C-terminus" 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 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
io aminoacyl residues employed in this 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 I
Abbreviation Definition
Ala alanyl
AIaNH2 alanylamide
alle alloisoleucyl
alloThr allothreonyl
alloThr(t-Bu) allothreonyl(O-t-butyl)
Arg arginyl
Arg(Pmc) arginyl(NG-2,2,5,7,8-pentamethylchroman-
6-sulfonyl)
Fmoc-Arg(Pbf)-OH N-Fmoc-NG-(2,2,4,6,7-
pentamethyldihydrobenzofuran-5-
sulfonyl)arginine
Asn asparaginyl
Asn(Trt) asparaginyl(trityl)
Asp aspartyl
Asp(Ot-Bu) aspartyl(O-t-butyl)
Cit citrullyl
Fmoc 9-fl uorenylmethyloxycarbonyl
Gin glutaminyl
Gln(Trt) glutaminyl(trityl)
Glu glutamyl
NMeGIu N-methylglutamyl
NMeGIu(t-Bu) N-methylglutamyl(t-butyl)
Gly glycyl
His histidyl
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His(Trt) histidyl(trityl)
Hser homoseryl
Ile isoleucyl
Leu leucyl
Lys(Ac) lysyl(N-epsilon-acetyl)
Met methionyl
6-Me-nicotinyl 6-methylnicotinyl
Nle norleucyl
Nva norvalyl
NMeNva N-methylnorvalyl
Orn(Ac) ornithyl(N-delta-acetyl)
Pen penicillaminyl
Phe phenylalanyl
(4-CH3)Phe 4-methylphenylalanyl
(4-CN)Phe 4-cyanophenylalanyl
NMePhe N-methylphenylalanyl
Pro prolyl
ProNHCH2CH3 prolylethylamide
3-Pal 3-(3-pyridyl)alanyl
Sar sarcosyl
Ser seryl
Ser(t-Bu) seryl(O-t-butyl)
Thr threonyl
Thr(t-Bu) threonyl(O-t-butyl)
Trp tryptyl
Trp(Boc) tryptyl(t-butoxycarbonyl)
Tyr tyrosyl
Tyr(t-Bu) tyrosyl(O-t-butyl)
Val valyl
NMeVa1 N-methylvalyl
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-lmpex International, Inc. Tools for Peptide & Solid Phase
Synthesis 1998-1999
Catalogue.
Compositions
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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, 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) 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 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
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; 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, 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 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
(Helicobacterpylori). 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 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
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excipients, and optionally sustained-release matrices, such as biodegradable
polymers, to form
therapeutic compositions.
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), 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,
io 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 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 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 benefit/risk
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 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 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
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, ntravaginally,
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, as well as sterile
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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),
carboxymethyIcellulose 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
microorganisms may be
1o 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),
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 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. Suitable inert carriers include sugars such
as lactose. Desirably,
3o at least 95% by weight of the particles of the active ingredient have an
effective particle size in the
range of 0.01 to 10 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 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.
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
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penetrate the corneal and internal regions of the eye, as for example the
anterior chamber, posterior
chamber, vitreous body, aqueous humor, vitreous 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
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 wax which
are solid at room
temperature liquid at body temperature and therefore melt in the rectum or
vaginal cavity and
to 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 multi-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 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.
33et seq.
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 antiangiogenic agents to enhance their
effectiveness, or combined
with other antiangiogenic agents and administered together 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 I to 300 mg/kg body weight.
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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 nct limited to those
listed above, include in principle any agents useful for the treatment or
prophylaxis of angiogenic
diseases.
Determination of Biological Activity
In Vitro Assay for Angiogenic Activity
The human microvascular endothelial (HMVEC) migration assay was run according
to the
procedure of S. S. Tolsma, 0. V. Volpert, D. J. Good, W. F. Frazier, P. J.
Polverini and N. Bouck, J.
1 o Cell Biol. 1993, 122, 497-511.
The HMVEC migration assay was carried out using Human Microvascular
Endothelial
Cells-Dermal (single donor) and Human Microvascular Endothelial Cells,
(neonatal). The HMVEC
cells were starved overnight in DME containing 0.01% bovine serum albuminutes
(BSA). Cells
were then harvested with trypsin and resuspended in DME with 0.0 1% BSA a 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, MD). The chamber was assembled and
inverted, and cells
were allowed to attach for 2 hours at 37 C to polycarbonate chemotaxis
membranes (5 m pore
size) that had been soaked in 0.01% gelatin overnight and dried. The chamber
was then reinverted,
and test substances (total volume of 50 L), including activators, 15 ng/mL
bFGF/VEGF, were
added to the wells of the upper chamber. The apparatus was incubated for 4
hours at 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 positive control.
Representative compounds inhibited human endothelial cell migration in the
above assay by
at least 50% when tested at a concentration of I nM. Preferred compounds
inhibited human
endothelial cell migration by approximately 65% to 90% when tested at a
concentration of I nM and
most preferred compounds inhibited human endothelial cell migration by
approximately 50% to
95% at a concentration of 0.1 nM. As shown by these results, the compounds of
the present
invention demonstate enhanced potency.
Synthesis of the Peptides
This invention is intended to encompass compounds having formula (I) when
prepared by
synthetic processes or by metabolic processes. Preparation of the compounds of
the invention by
metabolic processes include those occurring in the human or animal body (in
vivo) or processes
occurring in vitro.
The polypeptides of the present invention may be synthesized by many
techniques 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.
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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. 1, Academic Press (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 Calbiocherr}
Novabiochem Corp. (San Diego, CA, U.S.A.) unless otherwise noted herein.
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 carboxyl
group of the first amino acid is protected by a suitable protecting group. The
protected or
to 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 complimentary (amino or
carboxyl) 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 allthe desired amino acids have
been linked in the
proper sequence, any remaining protecting groups (and any solid support) are
removed sequentially
or concurrently, to 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 involves
solid phase peptide synthesis. 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 contained
therein. Suitable protecting groups are 9-fluorenylmethyloxycarbonyl (Fmoc), t-
butoxycarbonyl
(Boc), benzyloxycarbonyl (Cbz), biphenylisopropyl-oxycarbonyl, t-
amyloxycarbonyl,
isobornyloxycarbonyl, (a,(x)-dimethyl-3,5-dimethoxybenzyloxycarbonyl, 0-
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:
2,2,5,7,8-
pentamethylchroman-6-sulfonyl (Pmc), and 2,2,4,6,7-
pentamethyldihydrobenzofuran-S-sulfonyl
(Pbf); for asparagine: trityl (Trt); for aspartic acid: t-buyl (t Bu); for
glutamine: trityl (Trt); for N-
methylglutamic acid: t-butyl (t-Bu); for histidine: trityl (Trt); for lysine:
t-butoxycarbonyl (Boc); for
seryl: t-butyl (t-Bu); for threonine and allothreonine: t-butyl (t-Bu); for
tryptophan: t-
butoxycarbonyl (Boc); and for tyrosine: t-butyl (t-Bu).
In the solid phase peptide synthesis method, the C4erminal 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
preferred solid support for
synthesis of C-terminal carboxyl peptides is Sieber amide resin or Sieber
ethylamide resin. The
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preferred solid support for C-terminal amide peptides is Sieber ethylamide
resin available from
Novabiochem Corporation.
The C-terminal amino acid is coupled to the resin by means of a coupling
mediated by N,N'-
dicyclohexylcarbodiimide (DCC), N,N'-di isopropylcarbodiimide (DIC), [O-(7-
azabenzotriazol-l-
yl)-1,1,3,3-tetramethyluronium hexafluorophosphate] (HATU), or O-benzotriazol-
l-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate (HBTU), with or without 4-
dimethylaminopyridine
(DMAP), 1-hydroxybenzotriazole (HOBT), N-methylmorpholine (NMM), benzotriazol-
I-yloxy-
tris(dimethylamino)phosphonium-hexafluorophosphate (BOP) or bis(2-oxo-3-
oxazolidinyl)phosphine chloride (BOPCI), for about I to about 24 hours at a
temperature of between
10 C and 50 C in a solvent such as dichloromethane or DMF.
When the solid support is Sieber amide or Sieber ethylamide 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.) with 1-
hydroxybenzotriazole
(HOBT, I equiv.), or [O-(7-azabenzotriazol-l-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate]
(HATU, I equiv.) with N-methylmorpholine (1 equiv.) in DMF.
The coupling of successive protected amino acids can be carried out in an
automatic
polypeptide synthesizer as is well known in the art. In a preferred
embodiment, thea-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 preferably carried
out in DMF. The
coupling agent is normally O-benzotriazol-l-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate
(HBTU, I equiv.) or [O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate]
(HATU, I equiv.) in the presence of N-methylmorpholine (NMM, 1 equiv.).
At the end of the solid phase synthesis, the polypeptide is removed from the
resin 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
3o a cleavage reagent, for example trifluoroacetic acid containing thianisole,
water, or ethanedithiol.
In cases where the C-terminus of the polypeptide is an alkylamide, the resin
is 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.
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
carboxymethylcelIulose; partition chromatography, e.g., on SEPHADEX G-25, LH-
20 or
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countercurrent distribution; high 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: DMF for N,N-
dimethylformamide; HBTU for O-benzotriazol-l-yl-N,N,N',N'-
tetramethyluroniumhexafluorophosphate; NMM for N-methylmorpholine; and TFA for
trifluoroacetic acid.
to Example I
N-Ac-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3
In the reaction vessel of a Rainin peptide synthesizer was placed Fmoc-Pro-
Sieber
ethylamide resin (0.25 g, 0.4 mmol/g loading). The resin was solvated with DMF
and amino acids
were coupled sequentially according to the following synthetic cycle:
(1) 3 x 1.5 minute washes with DMF;
(2) 2 x 15 minute deprotection using 20% piperidine;
(3) 6 x 3 minute washes with DMF;
(4) addition of amino acid;
(5) activation of amino acid with 0.4 M HBTU/NMM and coupling;
(6) 3 x 1.5 minute washes with DMF.
The protected amino acids were coupled to the resin in the following order:
Protected Amino Acid Coupling time
Fmoc-Arg(Pmc) 30 minutes
Fmoc-Ile 30 minutes
Fmoc-Nva 30 minutes
Fmoc-Thr(t-Bu) 30 minutes
Fmoc-D-Ile 30 minutes
Fmoc-Val 30 minutes
Fmoc-Gly 30 minutes
acetic acid 30 minutes -
Upon completion of the synthesis the peptide was cleaved from the resin using
a mixture of
(95:2.5:2.5) TFA/anisole/water for 3 hours. The peptide solution was
concentrated under vacuum
and then precipitated with diethyl ether and filtered. The crude peptide was
purified by HPLC using
a C-18 column and a solvent system varying over 50 minutes in a gradient of 5%
to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Val-D-lle-Thr-Nva-lle-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt=
3.16 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
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(ESI) m/e 923 (M+H)+; Amino Acid Anal.: 0.96 Gly; 1.01 Val; 1.98 Ile; 0.46
Thr; 0.94 Nva; 1.03
Arg; 0.98 Pro.
Example 2
N-Ac-Gly-Val-D-alle-Thr-Nva-Ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-alle for Fmoc-D-Ile in
Example
1. After cleavage of the peptide from the resin and workup the crude product
was purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.0 1% TFA. The pure fractions were lyophilized
to provideN-Ac-
1o Gly-Val-D-alle-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt
= 2.97 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01 % TFA); MS
(ESI) m/e 923.7 (M+H)+; Amino Acid Anal.: 0.94 Gly; 0.98 Val; 2.06 Ile; 0.51
Thr; 1.04 Nva; 1.00
Arg; 0.97 Pro.
Example 3
N-Ac-Gly-Val-D-Ile-al loThr-Nva-l le-Arg-ProNHCHZCH3
The desired product was prepared by substituting Fmoc-alloThr(t-Bu) for Fmoc-
Thr(t-Bu) in
Example 1. After cleavage of the peptide from the resin and workup the crude
product was purified
by HPLC using a C-18 column and a solvent system varying over 50 minutes in a
gradient of 5% to
100% acetonitrile/water containing 0.01% TFA. The pure fractions were
lyophilized to provide N-
Ac-Gly-Val-D-Ile-alloThr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt:
Rt = 2.95
minutes (gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01%
TFA); MS (ESI) m/e 923.7 (M+H)+; Amino Acid Anal.: 1.01 Gly; 0.92 Val; 2.03
Ile; 0.58 Thr; 0.99
Nva; 1.05 Arg; 0.97 Pro.
Example 4
N-Ac-Gly-Val-D-Ile-Thr-Gln-ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Gln(Trt) for Fmoc-Nva in
Example
1. After cleavage of the peptide from the resin and workup the crude product
was purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
GIy-Val-D-lle-Thr-Gln-fle-Arg-ProNHCHZCH3 as the trifluoroacetate salt: Rt=
2.48 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 952.7 (M+H)+; Amino Acid Anal.: 1.03 Gly; 1.00 Val; 2.10 Ile; 0.53
Thr; 0.90 Glu; 0.95
Arg; 1.03 Pro.
Example 5
N-6-Me-nicotinyl-Gly-Val-D-I le-Thr-Nva-Ile-Arg-ProNHCHZCH3
The desired product was prepared by substituting 6-methylnicotinic acid for
acetic acid in
Example 1. After cleavage of the peptide from the resin and workup the crude
product was purified
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WO 03/037268 PCT/US02/34811
by HPLC using a C-18 column and a solvent system varying over 50 minutes in a
gradient of 5% to
100% acetonitrile/water containing 0.01% TFA. The pure fractions were
lyophilized to provide N-
6-Me-nicotinyl-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the
trifluoroacetate salt: Rt =
2.62 minutes (gradient varying over 10 minutes from 20% to 80%
acetonitrile/water containing
0.01% TFA); MS (ESI) m/e 1000.6 (M+H)+; Amino Acid Anal.: 1.01 Gly; 0.94 Val;
2.13 Ile; 0.55
Thr; 1.00 Nva; 1.01 Arg; 1.04 Pro.
Example 6
N-Ac-G ly-Phe-D-l le-Thr-Nva-Ile-Arg-Pro-D-A I aNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Phe for Fmoc-Val, and adding a coupling with Fmoo-
Pro before the
coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide from
the resin and
workup the crude product was purified by HPLC using a C-18 column and a
solvent system varying
over 50 minutes in a gradient of 5% to 100% acetonitrile/water containing
0.01% TFA. The pure
fractions were lyophilized to provide N-Ac-Gly-Phe-D-Ile-Thr-Nva-Ile-Arg-Pro-D-
AlaNH2 as the
trifluoroacetate salt: Rt = 3.15 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 1014.6 (M+H)+; Amino
Acid Anal.: 1.01
Gly; 0.97 Phe; 2.03 Ile; 0.43 Thr; 1.03 Nva; 1.11 Arg; 0.99 Pro; 0.93 Ala.
Example 7
N-Ac-Gly-Val-D-alle-Ser-Ser-Ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-alle for Fmoc-D-Ile
and Fmoo-
Ser(t-Bu) for both Fmoc-Thr(t-Bu) and Fmoc-Nva in Example 1. After cleavage of
the peptide from
the resin and workup the crude product was purified by HPLC using a C-18
column and a solvent
system varying over 50 minutes in a gradient of 5% to 100% acetonitrile/water
containing 0.01%
TFA. The pure fractions were lyophilized to provide N-Ac-Gly-Val-D-aIle-Ser-
Ser-Ile-Arg-
ProNHCH2CH3 as the trifluoroacetate salt: Rt = 2.32 minutes (gradient varying
over 10 minutes
from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ESI) m/e 897.5
(M+H)+; Amino
Acid Anal.: 0.96 Gly; 0.91 Val; 2.11 Ile; 0.59 Ser; 1.06 Arg; 1.04 Pro.
Example 8
N-Ac-Gly-Val-D-alle-Thr-Ser-1 le-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-alle for Fmoc-D-Ile
and Fmoo-
Ser(t-Bu) for Fmoc-Nva in Example 1. After cleavage of the peptide from the
resin and workup the
crude product was purified by HPLC using a C-I8 column and a solvent system
varying over 50
minutes in a gradient of 5% to 100% acetonitrile/water containing 0.01 % TFA.
The pure fractions
were lyophilized to provide N-Ac-Gly-Val-D-alle-Thr-Ser-Ile-Arg-ProNHCH2CH3 as
the
trifluoroacetate salt: Rt = 2.35 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 911.5 (M+H)+; Amino
Acid Anal.: 0.98
4o Gly; 1.03 Val; 2.09 Ile; 0.48 Thr; 0.27 Ser; 1.05 Arg; 1.01 Pro.
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Example 9
N-Ac-Gly-Val-D-aile-Ser-Thr-Ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-alle for Fmoc-D-Ile,
Fmoc-Ser(t-
Bu) for Fmoc-Thr(t-Bu) and Fmoc-Thr(t-Bu) for Fmoc-Nva in Example 1. After
cleavage of the
peptide from the resin and workup the crude product was purified by HPLC using
a C-18 column
and a solvent system varying over 50 minutes in a gradient of 5% to 100%
acetonitrile/water
containing 0.01% TFA. The pure fractions were lyophilized to provide N-Ac-Gly-
Val-D-alle-Ser-
Thr-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt = 2.36 minutes
(gradient varying over 10
to minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI)
m/e 911.5 (M+H)+;
Amino Acid Anal.: 0.96 Gly; 0.93 Val; 2.04 Ile; 0.31 Ser; 0.50 Thr; 1.04 Arg;
0.99 Pro.
Example 10
N-Ac-Gly-Val-D-alle-Ser-Gin-Ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-alle for Fmoc-D-Ile,
Fmoc-Ser(t-
Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva in Example 1. After
cleavage of the
peptide from the resin and workup the crude product was purified by HPLC using
a C-18 column
and a solvent system varying over 50 minutes in a gradient of 5% to 100%
acetonitrile/water
containing 0.01% TFA. The pure fractions were lyophilized to provideN-Ac-Gly-
Val-D-alle-Ser-
Gln-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt= 2.39 minutes
(gradient varying over 10
minutes from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ESI)
m/e 938.5 (M+H)+;
Amino Acid Anal.: 1.00 Gly; 0.95 Val; 2.10 Ile; 0.33 Ser; 1.04 Glu; 1.02 Arg;
1.04 Pro.
Example I]
N-Ac-Gly-Gln-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using a C-18 column and a
solvent system varying
over 50 minutes in a gradient of 5% to 100% acetonitrile/water containing
0.01% TFA. The pure
fractions were lyophilized to provide N-Ac-Gly-Gin-D-Ile-Thr-Nva-Ile-Arg-Pro-D-
AlaNH2 as the
trifluoroacetate salt: Rt = 1.42 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 995.5 (M+H)+;Amino Acid
Anal.: 1.01
Gly; 1.03 Glu; 2.03 Ile; 0.51 Thr; 1.01 Nva; 1.05 Arg; 0.97 Pro; 1.04 Ala.
Example 12
N-Ac-GIy-GIn-D-I le-Thr-Nva-D-lle-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Gln(Trt) for Fmoc-Val
and Fmoc-D-
Ile for Fmoc-Ile in Example 1. After cleavage of the peptide from the resin
and workup the crude
product was purified by HPLC using a C-I 8 column and a solvent system varying
over 50 minutes
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in a gradient of 5% to 100% acetonitrile/water containing 0.01% TFA. The pure
fractions were
lyophilized to provide N-Ac-Gly-Gln-D-Ile-Thr-Nva-D-Ile-Arg-ProNHCH2CH3 as the
trifluoroacetate salt: Rt = 1.98 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01 % TFA); MS (ESI) m/e 952.5 (M+H)+; Amino
Acid Anal.: 1.03
Gly; 0.99 Glu; 2.09 lie; 0.53 Thr; 0.98 Nva; 1.03 Arg; 0.98 Pro.
Example 13
N-Ac-Gly-Val-D-Ile-Thr-Nva-D-I le-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-D-Ile for Fmoc-Ile in
Example 1.
l0 After cleavage of the peptide from the resin and workup the crude product
was purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Val-D-Ile-Thr-Nva-D-lle-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt =
3.04 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01 % TFA); MS
(ESI) m/e 923.5 (M+H)+; Amino Acid Anal.: 0.99 Gly; 1.02 Val; 2.12 lie; 0.51
Thr; 0.98 Nva; 1.04
Arg; 1.07 Pro.
Example 14
N-Ac-Gly-Val-I le-Thr-Nva-D-Ile-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Ile for Fmoc-D-Ile and
Fmoc-D-Ile
for Fmoc-Ile in Example 1. After cleavage of the peptide from the resin and
workup the crude
product was purified by HPLC using a C-I8 column and a solvent system varying
over 50 minutes
in a gradient of 5% to 100% acetonitrile/water containing 0.01 % TFA. The pure
fractions were
lyophilized to provide N-Ac-Gly-Val-Ile-Thr-Nva-D-Ile-Arg-ProNHCH2CH3 as the
trifluoroacetate
salt: Rt = 2.71 minutes (gradient varying over 10 minutes from 20% to 80%
acetonitrile/water
containing 0.01% TFA); MS (ESI) m/e 923.5 (M+H)+; Amino Acid Anal.: 0.97 Gly;
1.03 Val; 2.10
Ile; 0.55 Thr; 0.93 Nva; 1.02 Arg; 0.95 Pro.
Example 15
N-Ac-Gly-Val-D-Ile-Thr-Nva-Pro-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Pro for Fmoc-Ile in
Example 1.
After cleavage of the peptide from the resin and workup the crude product was
purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Val-D-Ile-Thr-Nva-Pro-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt =
2.45 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.0 1% TFA); MS
(ESI) m/e 907.5 (M+H)+; Amino Acid Anal.: 1.05 Gly; 1.00 Val; 1.10 lie; 0.49
Thr; 1.01 Nva; 1.04
Arg; 2.12 Pro.
Example 16
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N-Ac-Gly-Val-D-l le-Thr-Nva-Lys(Ac)-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Lys(Ac) for Fmoc-Ile in
Example 1.
After cleavage of the peptide from the resin and workup the crude product was
purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Val-D-Ile-Thr-Nva-Lys(Ac)-Arg-ProNHCH2CH3 as the trifluoroacetate salt:
Rt= 2.39 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 980.5 (M+H)+; Amino Acid Anal.: 0.97 Gly; 1.02 Val; 1.08 Ile; 0.49
Thr; 1.04 Nva; 0.89
Lys; 1.01 Arg; 1.03 Pro.
Example 17
N-Ac-G Iy-G l n-D-I le-Thr-Nva-lle-Arg-ProNHCH2CH3
The desired product was prepared by substituting Fmoc-Gln(Trt) for Fmoo-Val in
Example
1. After cleavage of the peptide from the resin and workup the crude product
was purified by HPLC
using a C-18 column and a solvent system varying over 50 minutes in a gradient
of 5% to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gln-D-lle-Thr-Nva-lle-Arg-ProNHCH2CH3 as the trifluoroacetate salt: Rt=
2.02 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 952.5 (M+H)+; Amino Acid Anal.: 0.94 Gly; 1.04 Glu; 2.07 Ile; 0.43
Thr; 1.01 Nva; 1.10
Arg; 0.97 Pro.
Example 18
N-Ac-G ly-G ln-D-alle-Thr-Nva-Ile-Arg-Pro-D-A laNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-alle for Fmoc-D-Ile,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using a G18
column and a solvent system varying over 50 minutes in a gradient of 5% to
100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gln-D-alle-Thr-Nva-Ile-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt =
1.20 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 995.5 (M+H)+.
Example 19
N-Ac-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide and adding a coupling with Fmoc-Pro before the coupling
with Fmoc-
Arg(Pmc) in Example 1 After cleavage of the peptide from the resin and workup
the crude product
was purified by HPLC using a C-18 column and a solvent system varying over 50
minutes in a
gradient of 5% to 100% acetonitrile/water containing 0.01 % TFA. The pure
fractions were
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lyophilized to provide N-Ac-Gly-Val-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AIaNH2 as the
trifluoroacetate
salt: Rt = 2.34 minutes (gradient varying over 10 minutes from 20% to 80%
acetonitrile/water
containing 0.0 1% TFA); MS (ESI) m/e 966.7 (M+H)+.
Example 20
N-Ac-Gly-Gin-D-l le-Thr-Nva-D-Pro-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-Pro for Fmoc-Ile,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
1o the peptide from the resin and workup the crude product was purified by
HPLC using a C-18
column and a solvent system varying over 50 minutes in a gradient of 5% to
100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gln-D-Ile-Thr-Nva-D-Pro-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt
= 1.05 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 979.6 (M+H)+.
Example 21
N-Ac-Gly-Val-D-Ile-Thr-G l n-lie-Arg-Pro-D-A laN H2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Nva, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using a C-18 column and a
solvent system varying
over 50 minutes in a gradient of 5% to 100% acetonitrile/water containing 0.0
1% TFA. The pure
fractions were lyophilized to provide N-Ac-GIy-Val-D-Ile-Thr-Gln-I le-Arg-Pro-
D-AIaNH2 as the
trifluoroacetate salt: Rt = 1.65 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.0 1% TFA); MS (ESI) m/e 995.7 (M+H+.
Example 22
N-Ac-Gly-G In-D-Ile-al loThr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-alloThr(t-Bu) for Fmoc-
Thr(t-Bu), and
adding a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in
Example 1. After
cleavage of the peptide from the resin and workup the crude product was
purified by HPLC using a
C-18 column and a solvent system varying over 50 minutes in a gradient of 5%
to 100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
GIy-GIn-D-lle-alloThr-Nva-lle-Arg-Pro-D-AlaNH2 as the trifluoroacetate salt:
Rt = 1.24 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 995.7 (M+H)+.
Example 23
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N-Ac-Gly-Gin-D-I le-Thr-Nva-Lys(AC)-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Lys(Ac) for Fmoc-lle,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using a C 18
column and a solvent system varying over 50 minutes in a gradient of 5% to
100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gln-D-lle-Thr-Nva-Lys(Ac)-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt:
Rt = 0.94 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01 % TFA); MS
to (ESI) m/e 1052.7 (M+H)+.
Example 24
N-Ac-G ly-G ln-D-Ile-Thr-Ser-1 le-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Ser(t-Bu) for Fmoc-
Nva, and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using a C 18
column and a solvent system varying over 50 minutes in a gradient of 5% to
100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gln-D-lle-Thr-Ser-Ile-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt =
0.92 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01 % TFA); MS
(ESI) m/e 983.6 (M+H)+.
Example 25
N-Ac-Gly-Gln-D-I le-Thr-Nva-D-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-Ile for Fmoc-Ile,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using a G18
column and a solvent system varying over 50 minutes in a gradient of 5% to
100%
acetonitrile/water containing 0.01% TFA. The pure fractions were lyophilized
to provideN-Ac-
Gly-Gin-D-Ile-Thr-Nva-D-Ile-Arg-Pro-D-AlaNH2 as the trifluoroacetate salt: Rt
= 1.41 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 995.7 (M+H)+.
Example 26
N-Ac-G ly-D-Gln-D-lle-Thr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-D-Gln(Trt) for Fmoc-Val, and adding a coupling
with Fmoc-Pro
before the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the
peptide from the
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resin and workup the crude product was purified by HPLC using a C-I 8 column
and a solvent
system varying over 50 minutes in a gradient of 5% to 100% acetonitrile/water
containing 0.01%
TFA. The pure fractions were lyophilized to provide N-Ac-Gly-D-Gln-D-Ile-Thr-
Nva-Ile-Arg-Pro-
D-AIaNH2 as the trifluoroacetate salt: Rt = 1.14 minutes (gradient varying
over 10 minutes from
20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 995.5
(M+H)+.
Example 27
N-Ac-Gln-Val-D-I le-Thr-Nva-l le-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
lo Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Gly, and adding a coupling
with Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions were lyophilized to give N-Ac-Gin-Val-D-Ile-Thr-Nva-lie-Arg-
Pro-D-AIaNH2 as
the trifluoroacetate salt: Rt = 2.71 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01 % TFA); MS (ESI) m/e 1037.6 (M+H)+; Amino
Acid Anal.: 0.89
Glu; 1.01 Val; 2.05 lie; 0.54 Thr; 0.98 Nva; 0.99 Arg; 1.01 Pro; 1.01 Ala.
Example 28
N-Ac-Gln-Val-D-1le-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example 1 was used but substituting Fmoo-Gln(Trt)
for Fmoc-
Gly. After cleavage of the peptide from the resin and workup the crude product
was purified by
HPLC using C-I 8 column and with a solvent mixture varying over 50 minutes in
a gradient from
5% to 100% acetonitrile-water containing 0.01 % TFA. The pure fractions were
lyophilized to give
N-Ac-Gln-Val-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt:
Rt= 2.86 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 994.6 (M+H)+; Amino Acid Anal.: 0.96 Glu; 1.02 Val; 1.98 Ile; 0.59
Thr; 1.01 Nva; 1.06
Arg; 0.99 Pro.
Example 29
N-Ac-(4-CH3)Phe-G In-D-I le-Thr-Nva-lle-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-(4-CH3)Phe for Fmoc-Gly, Fmoc-Gln(Trt) for Fmoc-
Val, and adding
a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage
of the peptide from the resin and workup the crude product was purified by
HPLC using C-18
column and with a solvent mixture varying over 50 minutes in a gradient from
5% to 100%
acetonitrile-water containing 0.01 % TFA. The pure fractions were lyophilized
to give N-Ac (4-
CH3)Phe-Gln-D-Ile-Thr-Nva-lle-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt:
Rt = 3.19 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
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(ESI) m/e 1099.7 (M+H)+; Amino Acid Anal.: 1.00 Glu; 2.03 Ile; 0.51 Thr; 1.03
Nva; 1.02 Arg;
1.10 Pro; 1.02 Ala.
Example 30
N-Ac-(4-CN)Phe-Gin-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-(4-CN)Phe for Fmoc-Gly, Fmoc-Gln(Trt) for Fmoc-
Val, and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile=
water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
(4-CN)Phe-Gln-D-
Ile-Thr-Nva-Ile-Arg-Pro-D-AlaNH2as the trifluoroacetate salt: Rt= 2.88 minutes
(gradient varying
over 10 minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS
(ESI) m/e 1110.6
(M+H)+; Amino Acid Anal.: 0.97 Glu; 2.11 Ile; 0.49 Thr; 1.01 Nva; 0.95 Arg;
1.04 Pro; 1.01 Ala.
Example 31
N-Ac-G ly-A sn-D-Ile-Thr-Nva-I Ie-Arg-Pro-D-A laN H2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Asn(Trt) for Fmoc-Val, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.0 1% TFA.
The pure fractions were lyophilized to give N-Ac-Gly-Asn-D-Ile-Thr-Nva-Ile-Arg-
Pro-D-AlaNH2as
the trifluoroacetate salt: Rt = 1.75 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 981.6 (M+H)+; Amino
Acid Anal.: 0.99
Gly; 0.96 Asp; 2.05 Ile; 0.55 Thr; 1.02 Nva; 1.01 Arg; 1.00 Pro; 1.02 Ala.
Example 32
N-Ac-G ly-C it-D-I le-Thr-Nva-i le-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Cit for Fmoc-Val, and adding a coupling with Fmoc-
Pro before the
coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide from
the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions were lyophilized to give N-Ac-GIy-Cit-D-Ile-Thr-Nva-Ile-Arg-
Pro-D-AlaNH2as
the trifluoroacetate salt: Rt = 4.08 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.01 % TFA); MS (ESI) m/e 1024.6 (M+H)+; Amino
Acid Anal.: 1.03
Gly; 0.94 Cit; 2.07 Ile; 0.53 Thr; 1.00 Nva; 0.99 Arg; 0.97 Pro; 1.01 Ala.
Example 33
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N-Ac-Gly-Lys(AC)-D-Ile-Thr-Nva-Ile-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Lys(Ac) for Fmoc-Val, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.0 1% TFA.
The pure fractions were lyophilized to give N-Ac-Gly-Lys(Ac)-D-Ile-Thr-Nva-Ile-
Arg-Pro-D-
AlaNH2as the trifluoroacetate salt: Rt = 4.16 minutes (gradient varying over
10 minutes from 20%
to 80% acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 1037.7 (M+H)+.
Example 34
N-Ac-Gly-His-D-I le-Thr-Nva-I le-Arg-ProNHCH2CH3
The procedure described in Example 1 was used but substituting Fmoo-His(Trt)
for Fmoc-
Val. After cleavage of the peptide from the resin and workup the crude product
was purified by
HPLC using C-I 8 column and with a solvent mixture varying over 50 minutes in
a gradient from
5% to 100% acetonitrile-water containing 0.0 1% TFA. The pure fractions were
lyophilized to give
N-Ac-Gly-His-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt:
Rt = 3.88 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 961.6 (M+H)+.
Example 35
N-Ac-G ly-H is-D-I le-Thr-Nva-l le-Arg-Pro-D-A IaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-His(Trt) for Fmoc-Val, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions were lyophilized to give N-Ac-Gly-His-D-Ile-Thr-Nva-lle-Arg-
Pro-D-AlaNH2as
the trifluoroacetate salt: Rt = 3.70 minutes (gradient varying over 10 minutes
from 20% to 80%
3o acetonitrile/water containing 0.01 % TFA); MS (ESI) m/e 1004.6 (M+H)+.
Example 36
N-Ac-Gly-Asn-D-I le-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I was used but substituting Fmoo-Asn(Trt)
for Fmoc-
Val. After cleavage of the peptide from the resin and workup the crude product
was purified by
HPLC using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from
5% to 100% acetonitrile-water containing 0.01% TFA. The pure fractions were
lyophilized to give
N-Ac-Gly-Asn-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt:
Rt = 3.88 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) We 938.7 (M+H)+.
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Example 37
N-Ac-Gly-D-Asn-D-Ile-Thr-Nva-Lys(Ac)-Arg-ProNHCH2CH3
The procedure described in Example 1 was used but substituting Fmoc-D-Asn(Trt)
for
Fmoc-Val and Fmoc-Lys(Ac) for Fmoc-Ile. After cleavage of the peptide from the
resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01 % TFA.
The pure fractions were lyophilized to give N-Ac-Gly-D-Asn-D-Ile-Thr-Nva-
Lys(Ac)-Arg-
ProNHCH2CH3 as the trifluoroacetate salt: Rt = 3.65 minutes (gradient varying
over 10 minutes
i o from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ES1) m/e
995.6 (M+H)
Example 38
N-Ac-Gly-Gin-D-l le-Tyr-Nva-lle-Arg-ProNHCH2CH3
The procedure described in Example 1 was used but substituting FmooGln(Trt)
for Fmoc-
Val and Fmoc-Tyr(t-Bu) for Fmoc-Thr(t-Bu). After cleavage of the peptide from
the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions were lyophilized to give N-Ac-Gly-Gln-D-Ile-Tyr-Nva-Ile-Arg-
ProNHCH2CH3
as the trifluoroacetate salt: Rt = 4.43 minutes (gradient varying over 10
minutes from 20% to 80%
acetonitrile/water containing 0.01% TFA); MS (ESI) m/e 1014.5 (M+H)+.
Example 39
N-Ac-G ly-Gln-D-I le-Thr-Nva-Pro-Arg-Pro-D-A laNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Pro for Fmoc-Ile, and
adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile
water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
Gly-Gln-D-Ile-
Thr-Nva-Pro-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 3.74 minutes
(gradient varying
over 10 minutes from 20% to 80%acetonitrile/water containing 0.01% TFA); MS
(ES!) m/e 979.5
(M+H)+.
Example 40
N-Ac-Gly-Gln-D-Ile-Met-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Met for Fmoc-Thr(t-
Bu), and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
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water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
Gly-Gln-D-Ile-
Met-Nva-Ile-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 4.48 minutes
(gradient varying
over 10 minutes from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS
(ESI) m/e 1025.5
(M+H)+.
Example 41
N-Ac-Gly-Gin-D-Ile-Thr-Gln-Ile-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val and Fmoc-Nva, and adding a
coupling with
1o Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1. After
cleavage of the peptide
from the resin and workup the crude product was purified by HPLC using C- 18
column and with a
solvent mixture varying over 50 minutes in a gradient from 5% to 100%
acetonitrile-water
containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-Gly-Gln-
D-Ile-Thr-Gln-
Ile-Arg- Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 3.75 minutes
(gradient varying over 10
minutes from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ESI)
m/e 1024.6
(M+H)+.
Example 42
N-Ac-Gly-Arg-D-Ile-Thr-Nva-lie-Gln-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Arg(Pmc) for Fmoc-Val, Fmoc-Gln(Trt) for Fmoc-
Arg(Pmc), and
adding a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in
Example 1. After
cleavage of the peptide from the resin and workup the crude product was
purified by HPLC using C-
18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions were lyophilized
to give N-Ao-Gly-
Arg-D-Ile-Thr-Nva-Ile-Gln-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 3.96
minutes (gradient
varying over 10 minutes from 20% to 80% acetonitrile/water containing 0.01%
TFA); MS (ESI) m/e
995.6 (M+H)+.
Example 43
N-Ac-Gly-Gln-D-Ile-Tyr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Tyr(t-Bu) for Fmoc-
Thr(t-Bu), and
adding a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in
Example 1. After
cleavage of the peptide from the resin and workup the crude product was
purified by HPLC using C-
18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01%TFA. The pure fractions were lyophilized to
give N-Ao-Gly-
GIn-D-lle-Tyr-Nva-Ile-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 4.41
minutes (gradient
varying over 10 minutes from 20% to 80% acetonitrile/water containing 0.01%
TFA); MS (ESI) m/e
1057.5 (M+H)+.
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Example 44
N-Ac-Gly-Gin-D-Leu-Thr-Nva-l le-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-Leu for Fmoc-D-Ile,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile
water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
Gly-Gln-D-Leu-
Thr-Nva-Ile-Arg-Pro-D-AlaNH2 as the trifluoroacetate salt: Rt = 4.00 minutes
(gradient varying
over 10 minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS
(ESI) m/e 995.6
(M+H)+.
Example 45
N-Ac-Gly-Gin-D-Leu-Ser-Nva-Ile-Arg-Pro-D-AlaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-Leu for Fmoc-D-Ile,
Fmoc-Ser(t-Bu)
for Fmoc-Thr(t-Bu), and adding a coupling with Fmoc-Pro before the coupling
with Fmoc-
Arg(Pmc) in Example 1. After cleavage of the peptide from the resin and workup
the crude product
was purified by HPLC using C-18 column and with a solvent mixture varying over
50 minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions were
lyophilized to give N-Ac-Gly-Gin-D-Leu-Ser-Nva-Ile-Arg-Pro-D-AlaNH2 as the
trifluoroacetate
salt: Rt = 4.05 minutes (gradient varying over 10 minutes from 20% to 80%
acetonitrile/water
containing 0.01 % TFA); MS (ESI) m/e 981.5 (M+H)+.
Example 46
N-Ac-Gly-G In-D-al le-Thr-Ser-Ile-Arg-Pro-D-AIaN H2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-alle for Fmoc-D-Ile,
Fmoc-Ser(t-Bu)
for Fmoc-Nva, and adding a coupling with Fmoc-Pro before the coupling with
Fmoc-Arg(Pmc) in
Example 1. After cleavage of the peptide from the resin and workup the crude
product was purified
by HPLC using C-18 column and with a solvent mixture varying over 50 minutes
in a gradient from
5% to 100% acetonitrile-water containing 0.01 % TFA. The pure fractions were
lyophilized to give
N-Ac-Gly-Gin-D-alle-Thr-Ser-lle-Arg-Pro-D-AlaNH2 as the trifluoroacetate salt:
Rt = 3.55 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01% TFA); MS
(ESI) m/e 983.5 (M+H)+.
Example 47
N-Ac-Gly-Gin-D-al le-Thr-Nva-Lys(Ac)-Arg-Pro-D-AlaNH2
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The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-D-alle for Fmoc-D-Ile,
Fmoc-Lys(Ac)
for Fmoc-Ile, and adding a coupling with Fmoc-Pro before the coupling with
Fmoc-Arg(Pmc) in
Example 1. After cleavage of the peptide from the resin and workup the crude
product was purified
by HPLC using C-1 8 column and with a solvent mixture varying over 50 minutes
in a gradient from
5% to 100% acetonitrile-water containing 0.01 % TFA. The pure fractions were
lyophilized to give
N-Ac-Gly-Gln-D-al le-Thr-Nva-Lys(Ac)-Arg-Pro-D-AIaNH2 as the trifluoroacetate
salt: Rt = 3.70
minutes (gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.01%
TFA); MS (ESI) We 1009.6 (M+H)+.
Example 48
N-Ac-Gly-Gin-D-Ile-Asp-Nva-lie-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Asp(Ot-Bu) for Fmoc-
Thr(t-Bu), and
adding a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in
Example 1. After
cleavage of the peptide from the resin and workup the crude product was
purified by HPLC using C-
18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions were lyophilized
to give N-Ao-Gly-
Gln-D-Ile-Asp-Nva-Ile-Arg-Pro-D-AlaNH2 as the trifluoroacetate salt: Rt = 4.00
minutes (gradient
varying over 10 minutes from 20% to 80% acetonitrile/water containing 0.01%
TFA); MS (ESI) m/e
1009.5 (M+H)+.
Example 49
N-Ac-G ly-G l n-D-I le-Thr-Trp-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Trp(Boc) for Fmoc-Nva,
and adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrilo-
water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
Gly-Gln-D-Ile-
Thr-Trp-Ile-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt: Rt = 4.46 minutes
(gradient varying over
10 minutes from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ESI)
m/e 1082.5
(M+H)+.
Example 50
N-Ac-Gin-Gin-D-Ile-Thr-Nva-Lys(Ac)-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Gln(Trt) for Fmoc-Gly and Fmoc-Val, Fmoc-Lys(Ac)
for Fmoc-Ile,
and adding a coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in
Example 1.
After cleavage of the peptide from the resin and workup the crude product was
purified by HPLC
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using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
100% acetonitrile-water containing 0.01% TFA. The pure fractions were
lyophilized to give N-Ac-
Gln-Gin-D-Ile-Thr-Nva-Lys(Ac)-Arg-Pro-D-AIaNH2 as the trifluoroacetate salt:
Rt = 3.965 minutes
(gradient varying over 10 minutes from 20% to 80% acetonitrile/water
containing 0.0 1% TFA); MS
(ESI) m/e 1067.8 (M+H)+.
Example 51
N-Ac-Ala-Gln-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example 1 was used but substituting Fmoo-Gln(Trt)
for Fmoc-
Val and Fmoc-Ala for Fmoc-Gly. After cleavage of the peptide from the resin
and workup the
crude product was purified by HPLC using C-18 column and with a solvent
mixture varying over 50
minutes in a gradient from 5% to 100% acetonitrile-water containing 0.01% TFA.
The pure
fractions were lyophilized to give N-Ac-Ala-Gln-D-Ile-Thr-Nva-Ile-Arg-
ProNHCH2CH3 as the
trifluoroacetate salt: Rt = 4.215 minutes (gradient varying over 10 minutes
from 20% to 80%
acetonitrile/water containing 0.0 1% TFA); MS (ESI) m/e 966.6 (M+H)+.
Example 52
N-Ac-A sn-Val-D-Ile-Thr-Nva-1le-Arg-Pro-D-AIaNHZ
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Asn(Trt) for Fmoc-Gly, and adding a coupling with
Fmoc-Pro before
the coupling with Fmoc-Arg(Pmc) in Example 1. After cleavage of the peptide
from the resin and
workup the crude product was purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions were lyophilized to give N-Ac-Asn-Val-D-lie-Thr-Nva-Ile-Arg-
Pro-D-AIaNHZ
as the trifluoroacetate salt: Ft = 4.4155 minutes (gradient varying over 10
minutes from 20% to 80%
acetonitrile/water containing 0.01 % TFA); MS (ESI) m/e 1023.6 (M+H)+.
Example 53
N-Ac-Ala-Gin-D-lie-Thr-Nva-Ile-Arg-Pro-D-AIaNH2
The desired product was prepared by substituting Fmoc-D-Ala-Sieber amide resin
for Fmoc-
Pro-Sieber ethylamide, Fmoc-Ala for Fmoc-Gly, Fmoc-Gln(Trt) for Fmoc-Val, and
adding a
coupling with Fmoc-Pro before the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product was purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions were lyophilized to give N-Ac-
Ala-GIn-D-Ile-Thr-
Nva-Ile-Arg-Pro-D-AIaNHZ as the trifluoroacetate salt: Rt = 3.995 minutes
(gradient varying over
10 minutes from 20% to 80% acetonitrile/water containing 0.01% TFA); MS (ESI)
m/e 1009.6
(M+H)+.
Example 54
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N-Ac-Asn-Val-D-I le-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I was used but substituting Fmoc-Asn(Trt)
for Fmoc-
Gly. After cleavage of the peptide from the resin and workup the crude product
was purified by
HPLC using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from
5% to 100% acetonitrile-water containing 0.01% TFA. The pure fractions were
lyophilized to give
N-Ac-Asn-Val-D-lie-Thr-Nva-l le-Arg-ProNHCH2CH3
as the trifluoroacetate salt: Rt = 4.62 minutes (gradient varying over 10
minutes from 20% to 80%
acetonitrile/water containing 0.0 1% TFA); MS (ESI) m/e 980.7 (M+H)+.
Example 55
N-Ac-Gly-Val-D-I le-Ser-Gln-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-Ser(t-
Bu) for
Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva. After cleavage of the peptide
from the resin and
workup the crude product can be purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions can be lyophilized to give N-Ac-Gly-Val-D-Ile-Ser-Gln-Ile-
Arg-ProNHCH2CH3
as the trifluoroacetate salt.
Example 56
N-Ac-Gly-Val-D-Leu-Ser-Gln-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting FmocrD-Leu
for Fmoc-
D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva. After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
Gly-Val-D-Leu-
Ser-Gln-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 57
N-Ac-Gly-Phe-D-Ile-Ser-Gin-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-Phe for
Fmoc-Val,
Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva. After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
Gly-Phe-D-l le-
Ser-Gln-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 58
N-Ac-G ly-Val-D-al le-Ser-G l n-Lys(Ac)-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting FmocD-alle
for Fmoc-
D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva and
Fmoc-Lys(Ac)
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for Fmoc-Ile. After cleavage of the peptide from the resin and workup the
crude product can be
purified by HPLC using C-18 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-GIy-Val-D-alle-Ser-Gin-Lys(Ac)-Arg-ProNHCH2CH3 as the
trifluoroacetate salt.
Example 59
N-Ac-Gly-Val-D-alle-Ser-G In-lie-Arg-ProNHCH(CH3)2
The procedure described in Example 1 can be used but substituting Fmoo-D-alle
for Fmoc-
D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva and
Fmoc-Pro-[4-(4-
N-i sopropy lam i no)methyl-3 -methoxyphenoxy] butyry I AM resin instead of
Fmoc-Pro Sieber
ethylamide resin. After cleavage of the peptide from the resin and workup the
crude product can be
purified by HPLC using C-18 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-GIy-Val-D-alle-Ser-GIn-lie-Arg-ProNHCH(CH3)2 as the
trifluoroacetate
salt.
Example 60
N-Ac-GIy-Val-D-al le-Tyr-Gln-Ile-Arg-ProNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo-D-alle
for Fmoc-
D-Ile, Fmoc-Tyr(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Gln(Trt) for Fmoc-Nva. After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
Gly-Val-D-alle-
Tyr-Gln-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 61
N-Ac-Gly-Gln-D-alle-Ser-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Gln(Trt) for
3o Fmoc-Val, Fmoc-D-alle for Fmoc-D-Ile and Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu).
After cleavage of
the peptide from the resin and workup the crude product can be purified by
HPLC using C-18
column and with a solvent mixture varying over 50 minutes in a gradient from
5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
GIn-D-alle-Ser-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 62
N-Ac-GIy-G In-D-al le-Ser-GIn-IIe-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-
Gln(Trt) for
Fmoc-Val and Fmoc-Nva, Fmoc-D-alle for Fmoc-D-Ile and Fmoc-Ser(t-Bu) for Fmoc-
Thr(t-Bu).
After cleavage of the peptide from the resin and workup the crude product can
be purified by HPLC
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using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
100% acetonitrile-water containing 0.01% TFA. The pure fractions can be
lyophilized to give N-
Ac-Gly-G ln-D-aIle-Ser-Gln-Ile-Arg-ProNHCHZCH3 as the trifluoroacetate salt.
Example 63
N-Ac-GIy-Val-D-aIle-Ser-Gln-l le-Arg-Pro-D-A IaNH2
The desired product can be prepared by substituting Fmoc-D-Ala-Sieber amide
resin for
Fmoc-Pro-Sieber ethylamide, D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-
Thr(t-Bu), Fmoc-
Gln(Trt) for Fmoc-Nva, and adding a coupling with Fmoc-Pro before the coupling
with Fmoc-
lo Arg(Pmc) in Example 1. After cleavage of the peptide from the resin and
workup the crude product
was purified by HPLC using C-I 8 column and with a solvent mixture varying
over 50 minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions were
lyophilized to give N-Ac-Gly-Val-D-alle-Ser-Gln-Ile-Arg-Pro-D-AIaNH2 as the
trifluoroacetate
salt.
Example 64
N-Ac-Gly-Val-D-aIle-Thr-Gln-Ile-Arg-ProNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo D-alle
for Fmoc-
D-Ile and Fmoc-Gln(Trt) for Fmoc-Nva. After cleavage of the peptide from the
resin and workup
the crude product can be purified by HPLC using C-18 column and with a solvent
mixturevarying
over 50 minutes in a gradient from 5% to 100% acetonitrile-water containing
0.01% TFA. The pure
fractions can be lyophilized to give N-Ac-Gly-Val-D-alle-Thr-Gln-Ile-Arg-
ProNHCH2CH3 as the
trifluoroacetate salt.
Example 65
N-Ac-Gly-His-D-alle-Ser-Gin-Ile-Arg-ProNHCHZCH3
The procedure described in Example 1 can be used but substituting Fmoo-
His(Trt) for Fmoc-
Val, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-
Gln(Trt) for
Fmoc-Nva. After cleavage of the peptide from the resin and workup the crude
product can be
purified by HPLC using C-18 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-Gly-His-D-alle-Ser-Gln-Ile-Arg-ProNHCH2CH3 as the
trifluoroacetate
salt.
Example 66
N-(6-Me-nicotinyl)-GIy-Val-D-alle-Ser-Gin-I le-Arg-ProNHCHZCH3
The procedure described in Example I can be used but substituting 6-methyl-
nicotinic acid
for acetic acid, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu)
and Fmoc-
Gln(Trt) for Fmoc-Nva. After cleavage of the peptide from the resin and workup
the crude product
can be purified by HPLC using C-18 column and with a solvent mixture varying
over 50 minutes in
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a gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-(6-Me-nicotinyl)-Gly-Val-D-alle-Ser-Gln-Ile-Arg-
ProNHCH2CH3 as the
trifluoroacetate salt.
Example 67
N-Ac-G ly-NMeVal-D-I le-Thr-Nva-I le-Arg-ProNHCH2CH 3
The procedure described in Example 1 can be used but substituting Fmoo-NMeVaI
for
Fmoc-Val and using HATU instead of HBTU in the coupling of the N-methylamino
acid. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
io C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
NMeVal-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 68
N-Ac-Gly-NMePhe-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-NMePhe
for
Fmoc-Val and using HATU instead of HBTU in the coupling of the N-methylamino
acid. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give 114-Ac-Gly-
NMePhe-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 69
N-Ac-Gly-Val-D-Ile-Thr-N MeNva-I le-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-NMeNva
for
Fmoc-Nva and using HATU instead of HBTU in the coupling of the N-methylamino
acid. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
Val-D-Ile-Thr-NMeNva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 70
N-Ac-Gly-Val-D-I le-NMeG lu-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
NMeGlu(t-Bu) for
Fmoc-Thr(t-Bu) and using HATU instead of HBTU in the coupling of the N-
methylamino acid.
After cleavage of the peptide from the resin and workup the crude productcan
be purified by HPLC
using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
100% acetonitrile-water containing 0.01% TFA. The pure fractions can be
lyophilized to give N-
Ac-Gly-Val-D-Ile-NMeGlu-Nva-lle-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
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Example 71
N-Ac-Gly-Val-D-al le-Ser-Gln-l le-ArgNHCH2CH3
The procedure described in Example I was used but substituting Fmoo-Arg(Pbf)-
[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Nva, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu), Fmoc-D-alle for Fmoc-
D-Ile, and
omitting the coupling with Fmoc-Arg(Pmc) in example 1. After cleavage of the
peptide from the
resin and workup the crude product was purified by HPLC usingC-18 column and
with a solvent
mixture varying over 50 minutes in a gradient from 5% to 100% acetonitrile-
water containing
0.01% TFA. The pure fractions were lyophilized to give N-Ac-Gly-Val-D-aIle-Ser-
Gln-Ile-
1o ArgNHCH2CH3 as the trifluoroacetate salt. Rt= 0.83 minutes (gradient
varying over 10 minutes
from 20% to 80% acetonitrile/water containing 0.0 1% TFA); MS (ESI) m/e 841.6
(M+H)+
Example 72
N-Ac-Gly-Val-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin and
omitting the coupling with Fmoc-Arg(Pmc) in example 1. After cleavage of the
peptide from the
resin and workup the crude product can be purified by HPLC using C-18 column
and with a solvent
mixture varying over 50 minutes in a gradient from 5% to 100% acetonitrile-
water containing
0.01% TFA. The pure fractions can be lyophilized to give N Ac-Gly-Val-D-Ile-
Thr-Nva-Ile-
ArgNHCH2CH3 as the trifluoroacetate salt.
Example 73
N-(6-Me-nicotinyl)-Gly-Val-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, 6-
methylnicotinic acid for acetic acid and omitting the coupling with Fmoc-
Arg(Pmc) in example 1.
After cleavage of the peptide from the resin and workup the crude product can
be purified by HPLC
using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
100% acetonitrile-water containing 0.01% TFA. The pure fractions can be
lyophilized to give N.(6-
Me-nicotinyl)-Gly-Val-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate
salt.
Example 74
N-Ac-Gly-Val-D-I Ie-alloThr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
alloThr(t-Bu) for Fmoc-Thr(t-Bu) and omitting the coupling with Fmoc-Arg(Pmc)
in example 1.
After cleavage of the peptide from the resin and workup the crude product can
be purified by HPLC
using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
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100% acetonitrile-water containing 0.01 %TFA. The pure fractions can be
lyophilized to give N-
Ac-Gly-Val-D-Ile-alloThr-Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 75
N-Ac-Gly-Gin-D-l le-Thr-Nva-Ile-ArgNHCHzCH3
The procedure described in Example 1 can be used but substituting Fmoc-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Val and omitting the coupling with Fmoc-Arg(Pmc) in example
1. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
to C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
GIn-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 76
N-Ac-GIy-Val-D-alle-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-D-
alle for Fmoc-D-Ile and omitting the coupling with Fmoc-Arg(Pmc) in example 1.
After cleavage
of the peptide from the resin and workup the crude product can be purified by
HPLC using C-18
column and with a solvent mixture varying over 50 minutes in a gradient from
5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
Val-D-alle-Thr-Nva-lle-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 77
N-Ac-GIy-Val-D-alle-Ser-Ser-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-D-
aIle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-Nva and
omitting the coupling
with Fmoc-Arg(Pmc) in example 1. After cleavage of the peptide from the resin
and workup the
crude product can be purified by HPLC using C-l 8 column and with a solvent
mixture varying over
50 minutes in a gradient from 5% to 100%acetonitrile-water containing 0.01%
TFA. The pure
fractions can be lyophilized to give N-Ac-GIy-Val-D-alle-Ser-Ser-Ile-
ArgNHCH2CH3 as the
trifluoroacetate salt.
Example 78
N-Ac-GIy-Val-D-Ile-Thr-Gln-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Nva and omitting the coupling with Fmoc-Arg(Pmc) in example
1. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
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C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
Val-D-Ile-Thr-Gln-lle-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 79
N-Ac-Gly-Val-D-Ile-Thr-Ser-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethyla-riide
resin, Fmoc-
Ser(t-Bu) for Fmoc-Nva and omitting the coupling with Fmoc-Arg(Pmc) in example
1. After
io cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
Val-D-Ile-Thr-Ser-lle-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 80
N-Ac-Gly-Val-D-Ile-Thr-Nva-D-l le-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-D-
lie for Fmoc-Ile and omitting the coupling with Fmoc-Arg(Pmc) in Example 1.
After cleavage of
the peptide from the resin and workup the crude product can be purified by
HPLC using C-18
column and with a solvent mixture varying over 50 minutes in a gradient from
5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gly-
Val-D-Ile-Thr-Nva-D-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 81
N-Ac-Gln-Val-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Gly and omitting the coupling with Fmoc-Arg(Pmc). After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
Gln-Val-D-lle-
Thr-Nva-lle-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 82
N-Ac-Nva-Val-D-Ile-Thr-Nva-I le-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-Nva
for Fmoc-Gly and omitting the coupling with Fmoc-Arg(Pmc). After cleavage of
the peptide from
the resin and workup the crude product can be purified by HPLC using C-I 8
column and with a
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solvent mixture varying over 50 minutes in a gradient from 5%to 1000/o
ac"efo'nmile-water"
containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-Nva-
Val-D-Ile-Thr-
Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 83
N-Ac-Nva-Val-D-lie-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-Nva for
Fmoc-
Gly. After cleavage of the peptide from the resin and workup the crude product
can be purified by
HPLC using C-l 8 column and with a solvent mixture varying over 50 minutes in
a gradient from
to 5% to 100% acetonitrile-water containing 0.01% TFA. The pure fractions can
be lyophilized to
give N-Ac-Nva-Val-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate
salt.
Example 84
N-Ac-D-Gin-Val-D-Ile-Thr-Nva-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-D-
Gln(Trt) for
Fmoc-Gly. After cleavage of the peptide from the resin and workup the crude
product can be
purified by HPLC using C-18 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-D-Gln-Val-D-Ile-Thr-Nva-lie-Arg-ProNHCH2CH3 as the
trifluoroacetate
salt.
Example 85
N-Ac-D-Gin-Val-D-I le-Thr-Gln-lle-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-D-
Gln(Trt) for
Fmoc-Gly and Fmoc-Gln(Trt) for Fmoc-Nva. After cleavage of the peptide from
the resin and
workup the crude product can be purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions can be lyophilized to give N-Ac-D-Gln-Val-D-Ile-Thr-Gln-Ile-
Arg-
ProNHCH2CH3 as the trifluoroacetate salt.
Example 86
N-Ac-Gin-Val-D-al le-Ser-G In-I le-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Gln(Trt) for
Fmoc-Gly, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and
Fmoc-Gln(Trt)
for Fmoc-Nva. After cleavage of the peptide from the resin and workup the
crude product can be
purified by HPLC using C-l 8 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-Gin-Val-D-alle-Ser-Gin-Ile-Arg-ProNHCH2CH3 as the
trifluoroacetate
salt.
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Example 87
N-Ac-Gin-Val-D-alle-Ser-Ser-Ile-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Gln(Trt) for
Fmoc-Gly, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and
Fmoc-Nva. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Gln-
Val-D-alle-Ser-Ser-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 88
N-Ac-D-Gln-Val-D-alle-Ser-Ser-I le-Arg-ProNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo-D-
Gln(Trt) for
Fmoc-Gly, Fmoc-D-alle for Fmoc-D-lle, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and
Fmoc-Nva. After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01 % TFA. The pure fractions can be
lyophilized to give N-Ac-D-
Gln-Val-D-alle-Ser-Ser-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 89
N-Ac-Gln-Val-D-al le-Ser-Gln-l le-ArgNHCH2CH3
The procedure described in Example I can be used but substituting
Fmoo.Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Gly, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-
Thr(t-Bu), Fmoc-
Gln(Trt) for Fmoc-Nva and omitting the coupling with Fmoc-Arg(Pmc). After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
Gln-Val-D-aIle-
Ser-Gln-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 90
N-Ac-D-Gin-Val-D-alle-Ser-Gln-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting
Fmoo.Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-D-
Gln(Trt) for Fmoc-Gly, Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-
Thr(t-Bu), Fmoc-
Gln(Trt) for Fmoc-Nva and omitting the coupling with Fmoc-Arg(Pmc). After
cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
D.GIn-Val-D-
alle-Ser-Gln-lle-ArgNHCH2CH3 as the trifluoroacetate salt.
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Example 91
N-Ac-Gin-Val-D-I le-Thr-Nva-Pro-ArgNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
Gln(Trt) for Fmoc-Gly, Fmoc-Pro for Fmoc-lle and omitting the coupling with
Fmoc-Arg(Pmc).
After cleavage of the peptide from the resin and workup the crude product can
be purified by HPLC
using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from 5% to
100% acetonitrile-water containing 0.01%TFA. The pure fractions can be
lyophilized to give N-
Ac-Gln-Val-D-Ile-Thr-Nva-Pro-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 92
N-Ac-Ala-Gln-D-I le-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-Ala
for Fmoc-Gly, Fmoc-Gln(Trt) for Fmoc-Val and omitting the coupling with Fmoc-
Arg(Pmc). After
cleavage of the peptide from the resin and workup the crude product can be
purified by HPLC using
C-18 column and with a solvent mixture varying over 50 minutes in a gradient
from 5% to 100%
acetonitrile-water containing 0.01% TFA. The pure fractions can be lyophilized
to give N-Ac-Ala-
Gln-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
Example 93
N-Ac-D-Ala-Val-D-I le-Thr-Gln-I le-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-D-Ala
for Fmoc-
Gly and Fmoc-Gln(Trt) for Fmoc-Nva. After cleavage of the peptide from the
resin and workup the
crude product can be purified by HPLC using C-18 column and with a solvent
mixture varying over
50 minutes in a gradient from 5% to 100% acetonitrile-water containing 0.01%
TFA. The pure
fractions can be lyophilized to give N-Ac-D-Ala-Val-D-Ile-Thr-Gin-Ile-Arg-
ProNHCH2CH3 as the
trifluoroacetate salt.
Example 94
N-Ac-A la-Gln-D-I le-Thr-Ser-I le-Arg-ProNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoo-Ala for
Fmoc-Gly
and Fmoc-Gln(Trt) for Fmoc-Val, Fmoc-Ser(t-Bu) for Fmoc-Nva. After cleavage of
the peptide
from the resin and workup the crude product can be purified by HPLC using C-18
column and with
a solvent mixture varying over 50 minutes in a gradient from 5% to 100%
acetonitrile-water
containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-Ala-
Gln-D-lle-Thr-Ser-
lle-Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 95
N-Ac-Ala-Val-D-al le-Ser-G In-I le-Arg-ProNHCH2CH3
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CA 02466170 2004-04-30
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The procedure described in Example I can be used but substituting Fmoc-Ala for
Fmoc-Gly,
Fmoc-D-alle for Fmoc-D-Ile, Fmoc-Ser(t-Bu) for Fmoc-Thr(t-Bu) and Fmoc-
Gln(Trt) for Fmoc-
Nva. After cleavage of the peptide from the resin and workup the crude product
can be purified by
HPLC using C-18 column and with a solvent mixture varying over 50 minutes in a
gradient from
5% to 100% acetonitrile-water containing 0.01 % TFA. The pure fractions can be
lyophilized to
give N-Ac-Ala-Val-D-alle-Ser-Gln-Ile-Arg-ProNHCH2CH3 as the trifluoroacetate
salt.
Example 96
N-Ac-Ala-Val-D-al le-Ser-Gln-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting
Fmoo.Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-Ala
for Fmoc-Gly, Fmoc-D-alle for Fmoc-DIle, Fmoc-Ser(t-Bu) for Fmoc-Thr-(t-Bu),
Fmoc-Gln(Trt)
for Fmoc-Nva and omitting the coupling with Fmoc-Arg(Pmc). After cleavage of
the peptide from
the resin and workup the crude product can be purified by HPLC using C-18
column and with a
solvent mixture varying over 50 minutes in a gradient from 5% to 100%
acetonitrile-water
containing 0.01% TFA. The pure fractions can be lyophilized to give N -Ac-Ala-
Val-D-alle-Ser-
Gln-Ile-ArgNHCH2CH3 as the'trifluoroacetate salt.
Example 97
N-Ac-(4CH3)Phe-Gin-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example 1 can be used but substituting Fmoc-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, Fmoc-
(4CH3)Phe for Fmoc-Gly, Fmoc-Gln(Trt) for Fmoc-Val and omitting the coupling
with Fmoc-
Arg(Pmc). After cleavage of the peptide from the resin and workup the crude
product can be
purified by HPLC using C-18 column and with a solvent mixture varying over 50
minutes in a
gradient from 5% to 100% acetonitrile-water containing 0.01% TFA. The pure
fractions can be
lyophilized to give N-Ac-(4CH3)Phe-Gln-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3 as the
trifluoroacetate
salt.
Example 98
N-Ac-(4C H3)Phe-Gln-D-Ile-Thr-Gln-I le-Arg-ProNHCH2CH3
The procedure described in Example I can be used but substituting Fmoc-
(4CH3)Phe for
Fmoc-Gly and Fmoc-Gln(Trt) for Fmoc-Val and Fmoc-Nva. After cleavage of the
peptide from the
resin and workup the crude product can be purified by HPLC using C-18 column
and with a solvent
mixture varying over 50 minutes in a gradient from 5% to 100% acetonitrile-
water containing
0.01% TFA. The pure fractions can be lyophilized to give N Ac-(4CH3)Phe-Gln-D-
Ile-Thr-Gln-Ile-
Arg-ProNHCH2CH3 as the trifluoroacetate salt.
Example 99
N-Ac-Gin-Val-D-Ile-Thr-Nva-Lys(Ac)-Arg-ProNHCH2CH3
-41-

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The procedure described in Example I can be used but substituting Fmoo-
Gln(Trt) for
Fmoc-Gly and Fmoc-Lys(Ac) for Fmoc-Ile. After cleavage of the peptide from the
resin and
workup the crude product can be purified by HPLC using C-18 column and with a
solvent mixture
varying over 50 minutes in a gradient from 5% to 100% acetonitrile-water
containing 0.01% TFA.
The pure fractions can be lyophilized to give N-Ac-Gln-Val-D-lle-Thr-Nva-
Lys(Ac)-Arg-
ProNHCH2CH3 as the trifluoroacetate salt.
Example 100
N-Ac-(6-Me-nicotinyl)-Gly-Val-D-Ile-Thr-Nva-Ile-ArgNHCH2CH3
The procedure described in Example I can be used but substituting Fmoo-
Arg(Pbf)-[4-(4-N-
ethyl)methyl-3-methoxyphenoxy]butryl AM resin for Fmoc-Pro Sieber ethylamide
resin, 6-methyl-
nicotinic acid for acetic acid and omitting the coupling with Fmoc-Arg(Pmc).
After cleavage of the
peptide from the resin and workup the crude product can be purified by HPLC
using C-18 column
and with a solvent mixture varying over 50 minutes in a gradient from 5% to
100% acetonitrile-
water containing 0.01% TFA. The pure fractions can be lyophilized to give N-Ac-
(6.Me-nicotinyl)-
Gly-Val-D-lle-Thr-Nva-Ile-ArgNHCH2CH3 as the trifluoroacetate salt.
It will be evident to one skilled in the art that the present invention is not
limited to the
foregoing 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 made to the appended
claims, rather than to the foregoing examples, and all changes which come
within the meaning and
range of equivalency of the claims are therefore intended to be embraced
therein.
-42-

CA 02466170 2004-04-30
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1/3
SEQUENCE LISTING
<110> Abbott Laboratories
Haviv, Fortuna
Bradley, Michael F.
<120> HEPTA-, OCTA- AND NONAPEPTIDES HAVING
ANTIANGIOGENIC ACTIVITY
<130> 6853.WO.01
<140> Not Yet Assigned
<141> 2002-10-30
<150> 10/263,812
<151> 2002-10-04
<150> 10/000,681
<151> 2001-10-31
<160> 1
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Antiangiogenic Peptide
<221> VARIANT
<222> (1) ... (1)
<223> Xaa = hydrogen or R-(CH2)n-C(O)-, wherein n is an
integer from 0 to 8, R is alkoxy, alkyl, amino,
aryl, carboxyl, cycloalkenyl, cycloalkyl, and
heterocycle at position 1
<221> VARIANT
<222> (2) ... (2)
<223> Xaa = Ala, (1S,3R)-1-aminocyclopentane-3-carbonyl,
(1S,4R)-1-aminocyclopent-2-ene-4-carbonyl,
(1R,4S)-1-aminocyclopent-2-ene-4-carbonyl and Asn
at position 2
<221> VARIANT
<222> (2) ... (2)
<223> 2 (Continued)
Xaa = 3-cyanophenylalanyl, 4-cyanophenylalanyl,
3,4-dimethoxyphenylalanyl, and
4-fluorophenylalanyl at position 2
<221> VARIANT
<222> (2) ... (2)

CA 02466170 2004-04-30
WO 03/037268 PCT/US02/34811
2/3
<223> 2 (Continued)
Xaa = 3-(2-furyl)alanyl, Gln, Gly, Lys(Ac),
4-methylphenylalanyl, Nva and Sar at position 2
<221> VARIANT
<222> (3)...(3)
<223> Xaa = Ala,
(1R,4S)-1-aminocyclopent-2-ene-4-carbonyl, Arg,
Asn, t-butylglycyl, Cit, and cyclohexylglycyl, at
position 3
<221> VARIANT
<222> (3)...(3)
<223> 3 (Continued)
Xaa = Gln, Glu, Gly, His, Ile, Leu, Lys(Ac), Met,
Nva, Phe, NMePhe, Pro, Ser, 3-(2-thienylalanyl),
Thr, Val and N-methylvalyl at position 3
<221> VARIANT
<222> (4)...(4)
<223> Xaa = Ile at position 4
<221> VARIANT
<222> (5) ... (5)
<223> Xaa = alloThr, Asp, Gln, N-methylglutaminyl,
NMeGlu, Gly, His, Hser, Ile, Lys(Ac), Met, Ser,
N-methylseryl, Thr, Trp, Tyr, or tyrosyl(0-methyl)
at position 5
<221> VARIANT
<222> (6)...(6)
<223> Xaa = Ala, N-methylalanyl, alloThr, Gln, Gly,
Hser, Leu, Lys(Ac), Nle, Nva, NMeNva, octylglycyl,
Orn(Ac), 3-Pal, Sar, Ser, N-methylseryl, Thr, Trp,
Val and N-methylvalyl at position 6
<221> VARIANT
<222> (7)...(7)
<223> Xaa = Ala, alle, Asp, Cit. Ile, Leu, Lys(Ac),
N-methylisoleucyl, Nva, Phe, and Pro at position 7
<221> VARIANT
<222> (8)...(8)
<223> Xaa = Arg, Cit, Gln, His, homoarginyl, Lys,
lysyl(N-epsilon-isopropyl), Orn, and 3-Pal at
position 8
<221> VARIANT
<222> (9)...(9)
<223> Xaa = 2-aminobutyryl, homoprolyl, hydroxyprolyl,
Leu, and Pro at position 9
<221> VARIANT
<222> (10)...(10)
<223> Xaa = azaglycylamide, glycylamide,
-NH-(CH2)n-CHR1R2, -NHR3, wherein n is an integer
from 0 to 8, R1 is hydrogen, alkyl, cycloalkenyl,

CA 02466170 2004-04-30
WO 03/037268 PCT/US02/34811
3/3
and cycloalkyl at position 10
<221> VARIANT
<222> (10)...(10)
<223> 10 (Continued)
Xaa = R2 is hydrogen, alkoxy, alkyl, aryl,
cycloalkenyl, cycloalkyl, heterocycle, and
hydroxyl at position 10
<221> VARIANT
<222> (10)...(10)
<223> 10 (Continued)
Xaa = with the proviso that when n is 0. R2 is
other than alkoxy or hydroxyl at position 10
<221> VARIANT
<222> (10)...(10)
<223> 10 (Continued)
Xaa = R3 is hydrogen, cycloalkenyl, cycloalkyl, or
hydroxyl at position 10
<400> 1
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10