Note: Descriptions are shown in the official language in which they were submitted.
t~ ~ rr oc re rCGC r~ ~Ge.-
C ~ t ~ ~ I` C C
e r r e r r r ~ r r ~ t~ C e
r 2 1 5 3 2 2 8
PEPTIDE INHIBITORS bF CELL ADHES ION
CROSS-REFERENCE TQ RELATED APPLICATIONS
This application is related to a PCT application
published as WO 92/00995, published January 23, 1992 and
hereby incorporated in its entirety by referencè. WO
92/00995 discloses cyclic peptides containing arginine- -
glycine-aspartic acid (RGD) sequences that are useful for
modulating cell adhesion mediated by the fibronectin
receptor. WO 92/00995 also discloses that peptides
containing arginine-cysteine-aspartic acid (RCD? sequences
are also effective.
sACKGROUND OF THE INVEN~ION
Field of Invention
The present invention relates to novel cyclic
peptides and peptidomimetic compounds which are
characterized by cell adhesion modulation activity.
Description of Related Art
The extracellular matrix (ECM) is the major component
of connective tissue which provides for structural
integrity, promotes cell migration and differentiation. A:s
part of these functions, extracellular matrix molecules such
as fibronectin, collagen, laminin, von Willebrand factor,
thrombospondin, fibrinogen, and tenascin have been shown to
support adhesion of cells in vitro. This adhesive
interaction is critical for a number of biological processes
SUBS'l'l'l'U'l'~ SHEET
c c e c G rt ccc c cc cc~c
o c o c ~ 1 5 3 2 2 8 ~ r ~ ~ r ~ ~ ~
including hemostasis, thrombosis, wound healing, tumor
metastasis, immunity and inflammatio~.
Fibronectin (FN) is the prototype ECM molecule. The
major cell attachment site in the fibronectin molecule has
been reproduced synthetically with the amino acid sequence
arginine-glycine-aspartic acid, or RGD using single letter
nomenclature. Peptides containing the RGD sequence which
either inhibit or promote cell adhesion have been described
(US Patent Nos. 4,589,881j4,661,111; 4,517,686; 4,683,291,
4,578,079; 4,614,517; and 4,792,525). Changes in the
peptide as small as the exchange of alanine ~or glycine or
glutamic acids ~or aspartic acid, which constitute the
addition of a single methyl or methylene group to the
tripeptide, eliminates these activities (Pierschbacher et
. 15 al, PNAS, 81:5985 (1984)). Recently, a second FN cell
binding domain has been identified within the alternatively
spliced region of the A chain of the molecule. A ten amino
acid recognition sequence (GPEILDVPST) (SEQ. ID. NO.:1) in
FN has been shown to be the site which interacts with cells
(Wayner et al., J. Cell Biol., 109:1321 (1989); Guan et al.,
Cell, 60:53 (1990)).
The receptors which recognize these sites on FN belong
to a gene superfamily called integrins which consist o~
heterodimeric complexes of non-covalently associated alpha
and beta subunits. A common ~ subunit combines with unique
subunits to form an adhesion receptor o~ de~ined
~ specificity. Eight beta subunits have been cloned and
sequenced to date. The ~1 subfamily, also known as the VLA
family (Very Late Activation Antigens), binds to ECM
molecules such as FN, collagen and laminin. For reviews,
see, Hynes, Cell, 48:549 (1987); Hemler, Annu. Rev.
. Immunol., 8:365 (1990). Leukocyte interaction with FN at
the two spatially separate binding domains is mediated by
two distinct integrins. The RGD site is recognized by the
integrin ~5~1, while, EILDV (SEQ. ID. NO.:2) is recognized
by ~4~1 (Pytela et al., Cell, 40:191 (1985); Wayner et al.,
J. Cell Biol. 109:1321 (1989); Guan et al, Cell 60:53
SUBS'l'l'l'U'll~ SHEET
O O Q r C ~1 e ~, e e c c e ~
O ~ n ~ r ~ O ~ r- ~ ~ e
o ~ C ~ C C C C
r ~ r
215~228
2A
(1990)). Kumagai et al. have shown that a cyclic peptide
containing an RGD sequence can inhibit the attachment of
cells of a few tumor cell lines to plate coated with
fibronectin (H. Kumagai et al, Biochem. Biophys. Res. Comm.
177:74 (1991)).
Eyal et al, in European Patent Application 0 ~78 101
A2, disclose a number of peptide ~ragments of thrombospondin
that retain some of the activities of the parent protein.
They also show that similar peptides, differing by one or
two amino acids, are present in a number of proteins, not
having obvious cell-adhesion functions, from various
organisms. Bachem AG, a company which sells a number of
protein and peptide products, has available a peptide having
the amino acid sequence cyclo-(~-Ala-Gly-~-Ala-Gly) (cat.
no. H-2~75), which is similar to compounds encompassed by
the present invention, but does not fall within the scope of
the presently claimed compounds.
Vascular endotheiial cells form the interface between
blood and tissues and control the passage of leukocytes as
well as plasma fluid into tissues. A variety of signals
generated at the site of inflammation can activate both
endotheIial cells as well as circulating leukocytes so that
they become more adhesive to one another. Following this
'. ,
SUBS'l'l'l'U'l'~ SHEET
= ~
~ WO94/15958 215 ~ 2 ~ ~ PCT/~94/00026
inltia~ adhesion the ieukocy~es migrate into the tissues to
~erform host defense functions. Several adhesion molecules
have been identif iea which are involved in leukocyte--
endothelial interact ons. On the leukocytes, members of the
~2 integrin subfamily, which includes CDlla/CD18,
CDllb/CD18, and CDllc/CD18, have been shown to play an
important role in this process. In the ~1 subfamily, in
addition to binding to fibronectin, ~4~1 interacts with a
cytokine inducible molecule on endothelial cells termed
vascular cell adhesion molecule (VCAM). Other molecules on
endothelial cells which bind to the leukocytes include
ICAM-1, ICAM-2, E-selectin and P-selectin (Carlos and
Harlan, Immunol. Rev., 114:1 (1990); Osborn, L., Cell, 62:3
(1990); Springer T., Nature, 346:425 (1990); Geng et al.,
Nature, 347:757 (1990); Stoolman, L. Cell, 56:907 (1989)).
Recent data supports an important role for the integrin
~4~1 in inflammation. In vitro data show that antibodies to
~4 block adhesion of lymphocytes to synovial endothelial
cells; this adhesion plays a potential role in rheumatoid
arthritis (van Dinther-Janssen et al, J. Immunol., 147:4207
(1991)). Studies in which monoclonal antib~dies to ~4 block
adhesion of basophils and eosinophils to cytokine activated
endothelial cells (Walsh et al, J. Immunol., 146:3419
(1991); Bochner et al, J. Exp. Med., 173:1553 (1991)) imply
a potential role of ~4 in allergy and asthma. Additionally,
in ~ivo studies have shown that experimental autoimmune
encephalomyelitis can be blocked by anti-~4 monoclonal
antibodies (Yednock et al., Nature, 356:63 (1992)).
Migration of leukocytes to an inflammatory site can also be
blocked by anti-~4 monoclonal antibodies (Issekutz et al.,
J. Immunol., 147:4178 (1991)). Lastly, in a model of
contact hypersensitivity, peptides GRGDSP (SEQ. ID. NO.:3)
or EILDV (SEQ. ID. NO.:2) block ear swelling when
administered with sensitized cells into a challenged naive
recipient mouse suggesting that both ~4~1 and possibly ~5~1
are involved in this inflammatory response (Ferguson et al.,
Proc. Natl. Acad. Sci. USA, 88:8072 (1991)). Thus, ~4~1 and
21~ 3 2 2 ~ PCT/~94/0002 ~
~5~1 are -mportan~ recepto~ taraets ~or contro~ of
inflammatorv diseases.
SUM~RY O F THE I NVENT I ON -
The present invention relates to compounds having
activity as cell adhesion modulators. The compounds do not
contain the amino acid sequence arginine-glycine-aspartic
acid (Arg-Gly-Asp or RGD), i.e., the RGD tripeptide epitope.
In fact, some of the compounds do not have any of the three
amino acids of the RGD epitope.
The compounds, in one aspect, sufficiently mimic extra-
cellular matrix ligands or other cell adhesion ligands so as
to bind to cell surface receptors. Such receptors include
integrin receptors, in general, including the fibronectin,
collagen, l~min;n, LFA-1, MAC-1, pl50, p95, vitronectin and
gpIIb/IIIa receptors. The novel compounds have been found
to modulate cell adhesion by competing, for example, with
ligands containing the appropriate amino acid sequence and
by binding to ligand-directed receptors on cell surfaces.
The cell adhesive protein, such as (but not limited to)
fibronectin, is sufficiently inhibited from binding to the
cell's receptor so as to prevent or reduce cell adhesion.
Other uses include enh~ncing cell adhesion by using the
compounds to attach cells to a surface, or by other
promotion of cell adhesion. The useful compounds herein
described function as cell-adhesion modulators.
One objective of the present invention is to provide
novel compounds which act to modulate cell adhesion.
Another objective of the present invention is to
provide novel non-RGD-containing compounds which are capable
of binding to a cellular receptor which modulates cell
adhesion.
Another objective of the present invention is to
provide a novel method for modulating cell adhesion using
novel compounds.
094/l5958 ~ 1~ 3 ~ 2 ~ PCT/~94/000~6
Anothe~ objective of the present invention is to
provide compounds which bind to a cellular adhesion molecule
or integrin recepcor.
Another objective of the present invention is to
provide compounds having extraordinarily high potencies in
modulating cell adhesion to integrin receptors, including
inhibition of cell adhesion to the fibronectin receptor.
Thus, in one regard, the present invention includes
compounds having an ICso of less than about 500 ~M as
established in a U937-fibronectin adhesion assay; and in
another regard, the invention includes compounds having an
IC50 of less than about 100 ~M in such assay. The invention
also includes methods for obtaining (either in vi tro or in
vivo) such fibronectin receptor adhesion inhibition, and
integrin receptor adhesion inhibition. The compounds of the
present invention accomplish strong inhibition, at low
concentrations, with an IC50 of less than about 500 ~M, or
alternatively less than about 100 ~M.
Another objective of the present invention is to
provide compounds having high potencies in modulating
leukocyte adhesion to endothelial cells. Thus, in one
regard, the present invention includes compounds havina an
ICso of less than about 200 ~M as established in a ,Jurkat-
endothelial cell adhesion assay; and in another regard, the
invention includes compounds having an IC50 of less than
about 10 ~M in such assay. Compounds with activity below lo
~M are most preferred, below 100 ~M are not as preferred,
below 500 ~M lesser preferred and above 500 ~M least
preferred. The invention also includes methods for
obtaining (either in vitro or in vivo) such leukocyte
receptor adhesion inhibition. The compounds of the present
invention accomplish strong inhibition at low
concentrations, with an IC50 of less than about 250~M, or
alternatively less than about 50 ~M.
Another object of the present invention is to provide
novel compounds which modulate cell adhesion by binding to
cell adhesion molecules or integrin receptors wherein said
WO94/15958 ; PCTnB9410002
~ i53~ 28 ~ 5
ccmpound is resis~ant tc degrada~ion in vivo due to
_nclusior of peptidomimetic residues, modified amino acids
or D-isomers Ot amino acids.
Another objective of the present invention is to
provide novel compounds, formulations, and methods whlch may
be used in the study, diagnosis, treatment or prevention of
diseases and conditions which involve or relate to cell
adhesion, including but not limited to rheumatoid arthritis,
asthma, allergies, adult respiratory distress syndrome
(ARDS), cardiovascular disease, thrombosis or harmful
platelet aggregation, reocclusion following thrombolysis,
allograft rejection, graft versus host disease, organ
transplantation, septic shock, reperfusion injury,
psoriasis, eczema, contact dermatitis and other skin
inflammatory diseases, osteoporosis, osteoarthritis,
atherosclerosis, neoplastic disease including metastasis of
neoplastic or cancerous growth, wound healing enhancement,
treatment of certain eye diseases such as detaching retina,
Type I diabetes, multiple sclerosis, systemic lupus
erythematosus (SLE), inflammatory and immunoinflammatory
conditions including ophthalmic inflammatory conditions and
inflammatory bowel disease (e.g., ulcerative colitis and
regional enteritis), and other autoimmune diseases.
The cell adhesion protein fibronectin (FN) has been
implicated in the binding of capacitated sperm to oocytes
(Fusi and Bronson, J. Androl., 13:28-35 (1992)). Thus,
another object of the present invention is to provide
compounds which may be used as contraceptives by inhibiting
the binding of sperm to oocytes. The present invention also
provides a possible means of diagnosing infertility
resulting from defective adhesion of sperm to oocytes.
Another objective is to provide derivative compounds,
such as, but not limited to, antibodies and anti-idiotype
antibodies to the compounds disclosed in order to study,
diagnose, treat or prevent the above-described diseases and
conditions which relate to cell adhesion.
~ WO94/15958 215 3 22 8 PCT~B94/00026
Ano~her objective of t~e present ilvention is to
proviae a matrix which can be used to purify proteins,
polysaccharides or other compounds which specifically bind
to the cyclic peptides of the present invention with high
affinity.
.
DETAILED DESCRIPTION OF TH~ INVENTION
While cell adhesion is required for certain normal
physiological functions, there are situations in which cell
adhesion is undesirable, or in which modulated cell adhesion
is desirable.
Altered leukocyte-endothelial interactions are
implicated in a number of inflammatory diseases where
inappropriate attachment of leukocytes leads to further
injury of affected tissue. In vi tro results show that such
detrimental attachment, in which the leukocyte adheres to
endothelial cells or to the extracellular matrix, is
mediated by integrin receptors on the leukocyte. In this
situation, peptides or other compounds with a binding
affinity to integrin receptors are desirable as competitive
antagonists and should be useful in treating inflammatory
diseases including ARDS, asthma and rheumatoid arthritis.
Cell adhesion also contributes to metastasis of
cancerous tumors. Metastasis has been called "the major
underlying cause of death from cancer." Welch, et al.,
Intern. J. Cancer, 43:449 (1989). An RGD-containing peptide
which would prevent cell adhesion to basement membrane
components may be useful to prevent or eliminate metastasis.
See, Humphries et al, Science, 223:469 tl986)i Liotta,
Cancer Res., 46:1 (1986); Roose, Biochem. Biophys. Acta.,
7 :263 (1986). A peptide or other compound with suitable
affinity for RGD receptors should likewise have anti-
metastasis utility.
Harmful blood clotting is also caused by increased cell
adhesion. The attachment, spreading and aggregation of
platelets on extracellular matrices are central events in
thrombus formation. These events can be regulated by the
wo 94/15958 ~ ~5 3 ~ ~ PCT/~94/00026 ~
family of platelet adhesive glyco roteins, fibrinoae~,
fibronectin, and von Willebrand factor. ~ibrinoge~
functions as a cofactor for platelet aggregation, while
fibronectin supports platelet attachment and spreading
reactions. Von Willebrand factor is important in platelet
attachment to and spreading on subendothelial matrices, Plow
et al., PNAS-USA, 82:8057 (1985). A peptide or other
compound which would function as an antagonist and bind to
cell receptors which recognize the matrix glycoprotein RGD
site would be beneficial as an anti-thrombotic.
Other physiological conditions may be treated by
stimulatory modulation of cell adhesion. Wound healing, for
example, is undesirably prolonged when insufficient cell
adhesion occurs. A peptide or other compound with suitable
affinity for integrin receptors when attached, for example,
to a suitably positioned matrix or surface, may be able to
promote beneficial cell adhesion and resultant wound healing
by binding cells with the appropriate RGD-recognizing
receptor.
Also, in prosthetic implantation, such peptides or
other compounds coating the prosthesis would provide a
biocompatible surface to the prosthesis. Implantation of a
prosthesis coated with a compound of the present invention
would result in the prosthesis acquiring a covering of
cells. This cell layer bound to the prosthesis would
minimize rejection that might otherwise occur due to
stimulation of the immune system by the prosthesis itself.
As another example, coating of prosthetic devices which are
used in connection with the circulatory system with a
compound of the present invention which stimulates
endothelial cell adhesion, especially on a surface exposed
to blood flow, would enhance seeding of endothelial cells to
form a layer on the blood-exposed surface of the device.
When completely formed, the endothelial layer would prevent
damage to blood cells often observed to be caused by non-
endothelialized prostheses.
~ WO94/15958 21 5 3 ~ 2 8 PCT/~94/00026
The cell adhesion modulation compounas o~ the present
lnvention are represented in part by aminc acid seauence
formulas wherein the individual amino acids are represented
by their standard three-letter, or alternatively, one-letter
abbreviations.
Where such abbreviations for amino acids are used
without an indication of enantiomeric structure, either the
l- or d-enantiomers may suitably be utilized.
Additional abbreviations used herein include:
1,1-ACC: l-Amino-l-cyclohexanecarboxylic
acid
Ada: 1-A~m~ntaneacetic acid
(Ada)-Ala: ~-A~m~ntylalanine
Ada-CA: 1-A~m~ntanecarboxylic acid
Aib: ~-Aminoisobutyric acid (2-
methylalanine)
~-Ala: ~-Alanine (3-aminopropionic acid)
~-Asp: ~-Aspartic acid
(~-CN)A: ~-cyano-alanine
AMBA: 4-(Aminomethyl)benzoic acid
AnB: 4-Aminobutyric acid
AnC: 6-Aminocaproic acid
(AMP): 2-aminomethylpyridine
ARDS: Adult respiratory distress syndrome
BOC: tert-butyloxycarbonyl
[(3-Br)Tyr]: 3-bromo-tyrosine
BOP: benzotriazol-1-yl-tris(dimethylamino)-
phosphonium hexafluorophosphate
BSA: Bovine serum albumin
CBO:cis-Bicyclo[3,3,0]octane-2-carboxylic
acid
Cbz: Benzyloxycarbonyl
CHA: 3-(Cyclohexyl)-Alanine
CHAc: 3-Cyclohexylacetic acid
Chx: Cyclohexyl ester
Cl-Ala:chloroalanine
CPA: Cyclohexylphenylacetic acid
dA: D-alanine
DCC: dicyclohexylcarbodiimide
DCM: Dichloromethane
Dhp: 3,4-Dehydro-proline
DIEA: Diisopropylethylamine
DMEM: Dulbecco's Modified Eagle's Medium
DMF: Dimethylformamide
d-Nal: D-3-(2'-naphthyl)alanine
Dpr: diaminopropane
DTC L-5,5-dimethylthiazoline-4-carboxylic
acid
dV: D-valine
1-FCA:1-fluorenecarboxylic acid
9-FCA: 9-fluorenecarboxylic acid
WO94/15958 ~l~ 3 ~ ~ 8 PCT~B94/00026 -
;O
9-FA:9-fluoreneacetic acid
5-FINC:5- luoroindole carboxylic acid
Fm: Fluorenylme~hyl ester
FMOC: Fluorenylmethyloxycarbonyl
FN: Fibronectin
GAC: Guanidine-acetic acid
3-Glu: Gamma-aminopentane-1,5-dioic acid
HCA: Hydroci nn~m; C acid
HOBt: l-hydroxybenzotriazole
HomoC: Homocysteine
HomoP: homoproline
HomoR: homoarginine
HomoS: Homoserine
Hyp: 4-Hydroxyproline
ICAM-1: Intercellular adhesion molecule l
IC50: Inhibitory concentration, concentration
at which adhesion is inhibited to 50~ of
control level
l-Nal: 1-3-(2'-Naphthyl)alanine
IPA: isopropyl alcohol
Isonipecotic acid: 4-piperidinecarboxylic
acld
3-Me-Ada: 3-Methyl-1-adamantaneacetic acid
mono MeR: N-methyl-arginine
Mpr: 3-Mercaptopropionic acid
(des-~-amino cysteine)
MTC: L-2-methylthiazolidine-4-carboxylic
acid
NACA: 3-Noradamantanecarboxylic acid
Naph-Ac: 1-Naphthylacetic acid
NB-Ac: 2-Norbornaneacetic acid
Nic-Lys: Nicotinyl lysine
Nle: Norleucine
[(N-Me)R~:N-methyl arginine
norAda-CA: 3-Noradamantanecarboxylic acid
norArg: Norarginine
(H~NC(=NH)NH(CH2)2CH(NH2)CO~H)
Orn: Ornithine
O-Cys: Cysteic acid
Pen: Penicillamine
~ -dimethylcysteine)
PhAc: Phenylacetic acid
PMP: 1-(~-Mercapto-~
cyclopentamethylene)propinonic acid
PyE: Pyroglutamic acid
pyroGlu: Pyroglutamic acid
QC: Quinaldic acid
R.T.: Room Temperature (about 24C)
Sar: Sarcosine
SLE: Systemic lupus erythematosus
TA: 3-~-Thienyl-Alanine
TC: DL-thiazolidine-2-carboxylic acid
TCA: 1,4-thiazene-3-carboxylic acid
TEA: Triethylamine
TFA: Trifiuoroacetic acid
(thiop): 3-thioproline or
WO94/15958 PCT/~94/00026
11 2I~228
l-thiazolidine-~-carboxylic acid)
TIC: 1,2,3,4-tetrahydroisoauinoline-3-
carboxylic acid
tlc: thin layer chromatography
TTC: L-tetrahydrothiazine-4-carboxylic acid
VLA: Very late activat on antigens
Definitions
As used herein, the following words and/or
phrases have the following meanings:
~Analog~ means a compound which is a
derivative of a parent compound in which chemical
substituents are appended to a backbone
recognizable as the parent compound.
Furthermore, the parent compound is derivatized
in a manner such that it retains its basic
chemical function. Thus, an "amino acid analog~
is an amino acid which is derivatized at a side
chain carbon or nitrogen, N-derivatized at the
nitrogen bonded to the ~-carbon and the like, but
which retains the ability to form peptide bonds.
It is noted that D-enantiomers of amino acids are
thus encompassed by this definition. As further
examples, "arginine analog" is a compound which
consists of an arginine backbone and substituents
appended thereto. Thus, the genus of arginine
analogs includes, but is not limited to, the
compounds N-methylArg, N-lower alkyl-Arg, N,N-
dimethyl-Arg, N,N-di-lower alkyl-Arg, homoArg,
norArg, side-chain guanidinyl substituted N-
nitro-Arg, N~-nitro-Arg, N,N'-dimethyl-Arg, N,N'-
di-lower alkyl Arg, Arginine derivatized at the
~, ~ or ~ carbon with nitro-, alkyl-, aryl-,
nitroalkyl- or nitroaryl- groups, and the like.
"Phenylalanine analogs" include those
compounds which have halogen, methyl or lower
alkyl, nitro or hydroxyl substituents attached to
the phenyl ring; non-exclusive examples being p-
nitro-Phe, p-halo-Phe, p-amino-Phe and
WO94/15958 2 ~ 3 ~ æ ~ PCT/~94/00026
12
pentafluoro-Phe. Di-substituted analogc, e.g.
dichlorophenylalanine, o,m-dimethylphenylalanine
and the like are encompassed as phenylalanine
analogs, as are heterodisubstituted analogs, e.g.
S o-methyl-m-chloro-phenylalanine. As noted
above, N-alkyl substituted compounds such as N-
methyl-Phe are encompassed.
"Tyrosine analogs" would be homologous to
phenylalanine analogs, for instance, 3-bromo-Tyr,
3,5-dibromo-Tyr and 3,5-diiodo-Tyr, and also
encompass derivatives of the ring hydroxyl such
as O-methyl-tyrosine, O-lower alkyl-tyrosine,
etc.
"Proline analogs" include sulfur-containing
compounds such as 3-thioproline and also
compounds such as homoproline, hydroxyproline,
3,4-dihydroxyproline, DL-thiazolidine-2-
carboxylic acid, 1~4-tetrahydrothiazine-3-
carboxylic acid, L-5,5-dimethylthiazoline-4-
carboxylic acid and 1/3-tetrahydrothiazine-4
carboxylic acid, L-tetrahydrothiazine-4-
carboxylic acid and 1,3-, 1,4- and 1,5-
thiazepine-carboxylic acids.
"Aspartate analogs" and "glutamate analogsll
include esters of the ~-carboxylic acid function
of these amino acids.
"Lysine analogs" include amides of the ~-
amino group and of the ~-amino group and alkyl
derivatives of the ~-amino group and the ~-amino
group. Also encompassed as lysine analogs are
DpR, ornithine, homolysine and similar analogs of
these and related amino acids.
A. Descri~tion of the Com~ounds
In one aspect, the present invention is
directed to a compound of the formula
~ WO94/15958 PCT/~94/00026
12153228
X--(XI)-LI-1-2-3-4-5-6-L-(-YI)-Y-
- (I)
In the above structure I, a bridge is formed
via the cyclizing moiety Z between Ll and L' such
that the compound is cyclized. It will be
appreciated that in structures depicted below in
this disclosure, "~HN" represents an ~ amino
group of the amino terminal amino acid in a
sequence. Similarly, terminal ~-carboxyls are
denoted in structural representations as "~C=O".
Side chain functional groups are indicated the
structural representations in parenthesis.
Ll and L2 are chosen so that each contains a
functional group which contributes to the
formation of the cyclizing bridge moiety Z.
Thus, Z is formed from functional groups
contributed by L' and L2 and may also contain
additional atoms and spacer groups. As is
discussed in more detail below, preferred
functional groups include thiol, amino and
carboxyl groups. Such functional groups may be
borne on the side chain of amino acids or amino
acid analogs, or may constitute the ~-amino group
(in Ll) or ~-carboxy group (in L-) thereof.
Alternatively, the functional group contributing
to the cyclization may be provided by a non-
peptide cyclizing linker moiety which is
covalently linked to residues 1 and/or 6.
In preferred embodiments of the invention,
the bridging residues Ll and L2 are each selected
from the residues Cys, Pen, and homoC. For Ll,
additional preferred residues are Mpr and PMP.
All of these residues contain a sulfhydryl group.
For L- an additional preferred residue is
WO9~/15958 ~lS 3 ~ PCT/~94/0002 ~
1~
mercapto-eLhylamine (MEA). If MEA is usea, Yland
Y- is absent. Thus, the bridsing cyclization can
be accomplished~ by oxidative coupli~g of the
sulfhydryls~ to ~orm a disulfide bond between
residues Ll and L-. In such a case, the cyclizing
moiety Z is a covalent bond between the two
sulfur atoms. This may also be depicted
generally for compounds wherein, for example,
both Ll and L2 are Cys residues as follows:
(S) (S)
X2-(XI-)Cys - l - 2 - 3 - 4 - 5 - 6 - Cys(-Y~)_y2
wherein (as in other similar depictions used
herein) the side chain functional group portion
(here a sulfur a~om in both instances) appears in
parentheses above the residue having the side
chaln .
Particularly preferred embodiments are those
such that Ll is Cys or Mpr and L2 is Cys.
The cyclizing bridge may also be formed by
a hydrocarbon moiety, for example a
(poly)methylene bridge moiety of the form -(CH2) n~
where n is an integer of from 1 to 8, preferably
1 to about 4. One type of such bridge is
represented below, wherein a cyclic compound with
three methylene residues (representing Z) between
two cysteine side-chain sulfur atoms
(representing Ll and L2) is depicted:
(S) (CH2) 3 (S)
1 l
X2-(X~-)Cys-1-2-3-4-5-6-Cys(-Y~) _y2
(See, L. Fieser et al., "Reagents for Organic
Synthesis", Vol. 1, pp. 356-357, J. Wiley and
~ WO94/15958 PCT/~94/00026
1~ 2I5322~
Sons: (1967); Fieser, J . Amer. Chem. Soc.,
95:1945 (1959)).
In another preferred embodiment, Ll and L'
may be chosen from other amino acids or anaiogs
or amino acid mimetics which provide, as
functional groups suitable for the formation of
a cyclizing moiety, a side chain or the amino- or
carboxyl-terminus of an amino acid or analog
residue. For example, L- may be selected from
Asp, Glu, or other amino acids or analogs which
provide a suitable side chain carboxyl group for
cyclic linkage, through formation of an amide
bond in a condensation reaction, with an amino
group (e.g., an N~-amino group, or a side chain
amino group as on, for example, Lys or Orn) on Ll,
provided, however, that the structure
~HN (C=O)
Gly-Arg-Gly-Asp-Ser-Pro-Asp-Gly
2G is not included. The cyclizing moiety Z will in
such cases be a simple bond between Ll and L2.
Likewise, an amino acid residue L2 may
provide a carboxyl group from its carboxyl
terminus for amide linkage with either a side
chain amino or ~-amino group on an amino acid
residue or analog Ll; or the direction of the
amide linkage may be reversed where Ll provides a
side chain carboxyl group and L2 provides a side
chain amino group. Such structures may be
exemplified as follows:
(HN) (C=O)
X--(XI-)Lys-1-2-3-~-5-6-Asp(-YI)-Y'
W094/15958 ~53~ - PCT~B94/00026 ~
whe~e~n the side chain amino and carbonvl groups
of Ll (Lys).a~d ~- (Asp) are directly bonded;
(O=C) (NH)
X2-(XI-)Asp-l-2-3-4-5-6-Orn(-Y~) _y2
wherein amide bond direction (from side chains of
Ll and L2) is reversed;
~HN (C=O)
Ll-1-2-3-4-5-6-GlU (_yl) _y2
or
(HN) ~C=O
X2-(X~-)Orn-1-2-3-4-5-6-L2
wherein the depicted amino terminus of Ll is
directly bonded to the side chain carboxyl group
~f Glu (L2), or the depicted carboxyl terminus of
L2 is directly bonded to the side chain amino
group of Orn (Ll);
~HN ~C=O
Ll-1-2-3-4-5-6-L2
wherein the depicted ~-amino terminus at Ll is
directly bonded to the depicted carboxyl terminus
at L-, such that an amide bond is formed in the
peptide "backbone" of the compound.
In other preferred embodiments of the
invention, diketo and diamino linking moieties Z
such as those o~ the form
~ WO91/15958215 3 2 2 8 PCT/~94/00026
C
Il 11
-C-(CH.) n -C~
and
5-NH-(CH.) n - NH-
wherein n is GS defined above, may also be used.
Diketo linkers can be used to join, for
example, the c-amino groups of lysine residues,
while diamino linkers are conveniently employed
to cyclize the ~-carboxy groups of glutamic acid
or aspartic acid residues. Such examples yield
compounds having the structures exemplified by
O O
Il 11
(HN) - C-(CH.)n-C-(NH)
X,- (Xl) -Ll-1-2-3-4-5-6-L2- (_y~) _y2
and
( O=C ) -NH - ( CH2) n-NH- ( C=O )
l l
X--(XI-)Ll-1-2-3-4-5-6- L-(_yl)-Y'
Here, as elsewhere, the side chain functional
groups (amino and carbonyl) on Ll and L- are
depicted in parentheses above the residue
abbreviation.
The foregoing are but examples of suitable
hydrocarbon-containing bridges, and other forms
will also be apparent to those skilled in the
art. Where the cyclizing moiety Z includes a
~ 30 portion with such a hydrocarbon form, it may be
branched and may, where of a size appropriate to
form a stable structure (particularly, where Z
comprises two o~ more methylere moie_ies), also
WO9~/15958 2~3`~ - & PCT/~94/00026 ~
inc~uae one o- more hete~oasom-cc-~airi-c
substi~uen~s inc_ucina hydroxyl, amino, ritr-,
alkoxyl and halo subst~tuents. Such subs~i~uen~s
may be used to a'fe~t the solubility anc/or
biodistribution c~aracteristics of the subjec;
compounds. Aroma~ic or cycloalkyl hydrocarbon-
containing bridge groups may also ~e utilized in
the Z position, as for example diketo or diaminc
structures such as
0 0
Il 11
-C-(C6H4)-C- or -HN-(C6Hg)-NH-
Simple hydrocarbon moieties of from 1 to about 4
carbons are preferred for hydrocarbon portions of
Z-moieties.
It is of course possible for the bridging
moiety to be heterobifunctional, that is, to have
a keto group at one end and an amino group at the
other. Thus, one may employ as a bridging moiety
a structure which includes many of the elements
previously discussed, as drawn below:
-C-(C6HIo)~(cH~)n-N
The cyclizing bridge between Ll and L- can
also be formed via a monosulfide (thioether)
linkage, as exemplified below. One method for
making such a linkage is to use cysteine at L' or
L2 and to use a residue providing a bromo-acetic
O
Il
acid [or Br-CH,-(C~,)nC- where (n=0-4) -n genera'l
func.ional group G_ the other linking si.e (See,
~ WO94/15958 21 S 3 2 Z 8 PCT/~94/00026
B~rker e_ a ., J. Med. ~hem., ~:2î40-2~48
( 1992 ) ) .
(H2C) (S)
"Ala"-1-2-3-4-5-6-Cys-~l-Y-
Alternatively, Llcan be an ~,~ dehydroalanine and
L~ can be a cysteine residue. Reaction of the two
yields a lanthionine-like thioether linkage.
The cyclizing bridge between Ll and L2 may
also be formed via a monosulfide (thioether)
linkage, as exemplified below.
(S) (CH2)
X2-(XI-)Cys-1-2-3-4-5-6-''Ala''(-Yl)-Y-
In this regard, see, Palmer et al., in
~Peptides--Chemistry, Structure, Biology", pp.
616-618, Rivier & Marshall, Ed., Escom. Leider
(1990); and Jung, op. cit., pp. 865-869.
Analogs of amino acid residues may also be
utilized for Ll and/or L2, as for example homologs
(wherein a side chain is lengthened or shortened
while still providing a carboxyl, amino or other
reactive precursor functional group for
cyclization), d-enantiomers of amino acids,
analogs having variant side chains with
appropriate functional groups (as for example ~-
cyanoalanine, canavanine, djenkolic acid, ~-
azaphenylalanine or 2-amino-hexanedioic acid) or
other amino acid analogs (See, for example, the
amino acid analogs described above).
Amino acid-mimetic structures that are
capable of being covalently bonde throush an
.
WO9~/15958 ~ ~-^ PCT/~9~100026 ~
2~j32~
_~ide kond to a carboxvl andjor am~no term-nus o~
,he ~esidue sequence 1-2-3-~-5-6, ana which
~rovide a suitable precursor furc-ional a~oup ror
cyciiza~ion (through Z~, may also be e~plovec in
~ositions Ll and~or L-. Such amino ac~d-mimetic
s~ructures include organic species contain ng one
cr more heteroatoms including at least one
functional group (preferably a heteroatom-
containing functional group) which can
participate in cyclization. Examples include
residues of the form
-NH-(CH,)n-C-
wherein n ranges from 1 to about 8, and
preferably from 1 to 4, as for example residues
of ~-alanine and gamma-aminobutyric acid. (Where
n is l, the amino acid glycine, rather than an ~-
amino acid mimetic, results.) Such a structure
may, similar to the amino acids and ~mino acid
analogs discussed above, be utilized as L~
(wherein the carbonyl group depicted above,
formed for example from a carboxyl precursor,
conveniently forms an amide linkage with the
amino terminus of residue 2 or, if present,
residue 1), or it may be utilized as Ll (wherein
the depicted amino group may engage in an amide
linkage with the carboxyl terminus of the
terminal residue 4, 5 or 6). If only one such
linking residue L is used, it may serve as both
Ll and L2 (and thereby include Z) in that
cyclization can be achieved through formation of
two amide bonds, one at each terminus of the
sequence 1-2-3-4-5-6. Such structures result in
3_ the exemplifying form
~ WO 94/15958 21 ~ ~ ~ 2 8 PCT/IB94/00026
2 _
o
Il
C --( CH~ ) n NH
NH-1-2-3-4-5-6-C
,~ 11
O
where the N~-terminus and the carboxyl terminus
of the sequence 1-2-3-4-5-6 are bonded directly
to, respectively, the carbonyl residue and the
amino residue of the amino acid mimetic-linking
group depicted immediately above to form two
peptide-mimetic amide bonds. Likewise,
cyclization can be achieved with such an amino
acid mimetic-linking moiety wherein a side chain
functional group on a second linking moiety
appended (as Ll or L7) to one terminus of the
numbered sequence 1-2-3-4-5-6 (as for example an
amino or carboxyl side chain group) engages in
bonding to the mimetic moiety, and the mimetic
moiety (as L7 or Ll) cyclizes the compound to the
remaining terminal residue of the number~d
sequence. This may be exemplified by structures
of the form
O
Il
C (CH,)n-NH-(C=0)
~HN-1-2-3-4-5-6-L-(-Y-)_yl
wherein L7 (as for example Asp) provides the side
chain carbonyl aroup depicted in parenthesis,
residue 1 provides the depicted N~-terminal amino
group, and the amino acid mimetic linking moiety
~S ~ PCTl~94/00026 ~
Il
C-(CH,) n~
serves as Ll.
Amino acid~mimetic structures conta_n ng
aromatic, cycl~alkyl or other linking portions
can also be utilized as Ll and/or L-, such as
structures of the form
O O
-C-(C6H4)-NH- or -C-(C6HIo)-NH-
Similarly, the heterobifunctional (keto-amino)
structures depicted above may also serve as a Z-
group in linking complementary side chain
functional groups on Ll and L2 (e.g., a side chain
amino group on Ll and a side chain carboxyl group
on L') through two amide bond structures.
Other means of cyclization through
appropriate choices of Ll, L' and Z will be
recognized by those skilled in the art and are
included in the scope of the present invention.
It is also specifically contemplated that
the foregoing discussion of cyclizing moieties
(Z), bridging residues (Ll and L2), substituents,
amino acid analogs, amino acid mimetics,
cyclization methods, and the like are applicable,
mutatis mutandis, to the other structural
formulas discussed hereinafter. Residue 1 in
structure I is most preferably absenti resiaue 2
is most preferably Arg; residue 3 is most
preferably Ala; residue 4 is most preferably Asp;
residue 5 is most preferably 3-thioproline
(thiop); and residue 6 is preferably absent. The
sequence Arg-Ala-Asp-(thiop) (SE~. I3. NC.:C`,
~ WO94/1~958 21~ ~ 2 2 ~ PCT/~94/00026
resi~ues '- ) i5 mos~ p~e~erred for res--~es ~-2-
3_4_C_5
Also particularly preferred is G sequence
wherein residue l is absenl, residue 2 is absent,
residue 3 is Asp, residue 4 is (thio?) and
residues 5 and 6 are both absent. Thus, the
sequence Asp-(thiop) is also preferred for
residues 1-2-3-4-5-6.
A third particularly preferred seauence is
one in which Xl is Gly, residue l and residue 2
are both absent, residue 3 is Asp, residue 4 is
(thiop) and residues 5 and 6 are both absent.
A fourth particularly preferred seauence is
one in which x2 is (l-FCA), Xl and residues l and
2 are all absent, residue 3 is Asp, residue 4 is
(thiop) and residues 5 and 6 are both absent.
A fifth particularly preferred compound is
one wherein XZ is Fmoc, Xl is Arg, residues l and
2 are both absent, residue 3 is AnB, residue 4 is
(thiop) and residues 5 and 6 are both absent.
A sixth particularly preferred compound is
one wherein XZ is absent, X~ is Arg, residues l
and 2 are both absent, residue 3 is Ala, residue
4 is (thiop) and residues 5 and 6 are both
absent.
A seventh particularly preferred compound is
one wherein Xl and X~ are absent, residue l is
absent, residue 2 is Arg, residue 3 is d-Ala,
residue 4 is Asp, residue 5 is absent and residue
6 is (thiop).
An eighth particularly preferred compound is
one wherein residue l is absent, residue 2 is
Arg, residue 3 is Ala, residue 4 is Leu, residue
5 is absent and residue 6 is (thiop).
X~ and Y~ are each optional in structure I.
Where present, they are preferably each
nde?endently selected so as to e~hance the
WO9~/15958 21 5 3 2 ~ PCT/IB94/00026
GC_~ vitv o- the resultan. compound a ~cr ,~
pr~serve the compound agains~ me~abolism in, for
example, the in vivo environment and to ~ncreas2
the effective harf-life of the compouna. ln this
regard, the use of one or more d-amino acids,
most preferably at one or more terminal residue
position in the compound (i.e., at the amino-most
and/or carboxyl-most residue position, or in X~or
yl) are believed to stabilize the compouna against
metabolism by proteolytic or other enzymes in the
body. Specific preferred residues for position
X~ include Gly, Phe, Leu, Asn, Val, Tyr, Ala, Arg,
His, 1- or 2-naphthylalanine, cyclohexyl-Ala-,
AMBA, AnC, AnB and ~-amino-lower alkyl carboxylic
acids, Aib-, Ser-Tyr-Asn-, Ala-Thr-Val-, and p-
chloro-Phe-. Preferred residues for position yl
include Ala, -Ala-Ser, -Ala-Ser-Ser, -Ala-Ser-
Ser-Lys, -Ala-Ser-Ser-Lys-Pro, Thr,
-Thr-Phe, -Aib, -p-chloro-Phe, AMBA, AnC, AnB, ~-
amino-lower alkyl carboxylic acids, 1- or 2-
naphythlalanine, and -(cyclohexyl)Ala. Such Xland
yI groups are preferred also in the corresponding
positions given in the structural formulas
described hereinafter.
Where a substituent X~ or y2 incorporating R'
other than hydrogen is used, e.g., for X'
including acyl groups R'CO, especially formic
acid, acetic acid and other lower alkyl
carboxylic acids, including linear mixed-function
carboxylic acids which contain nitrogen and
sulfur (e.g. 3-mercaptopropionic acia) are
preferred. For y2 including amino groups of the
form R'NH, especially lower alkyl amines are
especially preferred. Additional preferred
substituents for X~ include those derived from
bulky compounds such as adamantaneacet~c acid,
aaamantanecarboxylic acid, 1- or 2-naphthylace~c
~ WO94/15958 21 ~ 3 2 2 8 PCT/~94/00026
ac~a, 2-ncr cr~aneace~ic ~cia, 3-noraàama~._a.e-
carboxvlic acid, 3-me~h.yladamataneace_ic acia.
Additional F~e~erred subslituents for v- include
lower alkyl amines, aryl amines, 1- or 2-
adamantvlamine and amino acids having the ~-
carboxylic acid replaced by a tetrazole group.
Each R' is individually a pharmaceutically
suitable substituent group, preferably one
selected from the group consisting of hydrogen,
linear and branched, unsubstituted and
substituted Cl-C8 lower alkyls, C~-Cg alkenyls, C2-
C8 alkynyls, C6-CI, aryls, C7-C~4 alkaryls, C7-CI4
cycloalkaryls and C3-CI, cycloalkyls, and, in the
case of -NR'2, from cycljzed groups forming, in an
attachment with the nitrogen atom, a 5-8 membered
heterocyclic ring optionally containing oxygen,
nitrogen or sulfur as a further ring heteroatom,
formic acid, acetic acid, heterocyclic carboxylic
acids, aryl carboxylic acids, heteroaromatic
carboxylic acids, alkyl carboxylic acids, alkenyl
carboxylic acids, alkynyl carboxylic acids,
other mixed-function sulfur and nitrogen
contcining linear carboxylic acids, adamantyl,
fluorenyl, l-FCA, 9-FCA, 9-FA, FMOC, Ada, Ada-CA,
NACA, 3-Me-Ada, (NB)-Ac, PhAc, Naph-Ac, HCA, QC,
CPA, DTC, TCA, AMBA, other multi-ring aromatic
and heteroaromatic carboxylic and acetic acids,
QC, CPA, BOC, 5-FINC, and CBO.
Zl53~g 25 PCT/~94/0002
Struc_ure- e~_mpl~r}ed bv the form~
-Tyr- ana -Thr- -Ser-
'l I
(OR') (OR') (OR')
S O
Il
-NH-CH-C-
I
(CH.) m ~ (OR') where m = 2, 3 or 4
such as those set forth with respect to residue
5 in Structure I, represent derivatives of amino
acid residues wherein the side chain hydroxyl
group (shown in parentheses) is optionally
substituted with a group of the form R' which can
be other than hydrogen as defined above.
Where such substituted residues are employed
in position 5 of structure I, R' is preferably
selected from hydroqen and C~ through C8 lower
alkyls, particularly methyl and ethyl alkyl
moieties.
A particularly preferred compound within the
scope of structure I includes:
(S) (S)
(l-FCA)-Cys-Asp-(thiop)-Cys
(SEQ. ID. NO.:4) wherein the shorthand structure
-Cys-, consistent with similar usage elsewhere in
this description, represents a cysteine residue
with its side chain sulfur atom separately
depicted, and likewise the structure (S)--(S)
represents a aisul~ide bond. The compound
-
O9~/15958 ~1 5 3 ~ ~ 8 PCT/~94/00026
aep-c.ea has ~een shown .~ be ~c=:ve
inhibiting cell adhesion to r-ibronectin.
Other preferred compounds are thcse wnerein
residue numDer 1 is absent o~- Leu; resi~e 2 is
Arg; residue Ll is Cys; residue 4 is Asp; ~-esiaue
5 is absent or Ser; residue 6 is absent, Pro or
(thiop); residue L- is Cys. The compounas in the
following Table 1 are particularly prefe~red.
Selected compounds in Table 1 are l-sted in
the Sequence Listing as follows: cmpd. 2, (SEQ.
ID. NO.:13); cmpd. 3, (SEQ. ID. NO.:5); cmpds.
23, 33, 48, 49, 51, 54, 55, 62 and 92, (SEQ. ID.
NO.:4); cmpd. 29, (SEQ. ID. NO.:6); cmpd. 42,
(SEQ. ID. NO.:7); cmpd. 50, (SEQ. ID. NO.:8);
cmpds. 65 and 66, (SEQ. ID. NO.:9); cmpd. 67,
(SEQ. ID. NO.:10); cmpd. 68, (SEQ. ID. NO.:11);
cmpd. 69, (SEQ. ID. NO.:12); cmpd. 71, (SEQ. ID.
NO.:14); cmpd. 75, (SEQ. ID. NO.:15); cmpd. 77,
(SEQ. ID. NO.:16); cmpds. 78 and 79, (SEQ. ID.
NO.:17); cmpd. 80, (SEQ. ID. NO.:18); cmpd. 81,
(SEQ. ID. NO.:19); cmpd. 82, (SEQ. ID. NO.:20);
cmpd. 84, (SEQ. ID. NO.:21); cmpd. 85, (SEQ. ID.
NO.:22); cmpd. 86, (SEQ. ID. NO.:23); cmpd. 87,
(SEQ. ID. NO.:24); cmpd. 89, (SEQ. ID. NO.:25).
PCllt~ 9 4 / o o ~ 2 6
21.~322~ 3 l. 03. 94
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SUBSrl lrl IJ l ~ SE~EET
PCI~I~ 94/oo 6
2~5~-2283 1. o~.294
31
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SUBSTlTUrE SHEET (RIJLE 26)
~ _ _ _ _ _
PCTIIB ~ 4 / O O 0 2 6
3 1. 03. 4
21~322&
32
1~ 0 r ~1 0 c~ (~i ~<~1 ~D o c G~ ~ C ~ ~ ~ G~
, .. . ... . . . . . . . . . . . . . . . . D r G~ 1 r 7 r G~ r c~ ~
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CD C O ~1 Ul ~~ ~ ~(~1G~Ul ~~i~1G~ D O 1
~ . . . . . .. . . . . . . . . . . . . . .
a) a) o ~ r o 1 G~ ~ G~ O O CO ~ r ~ o
JJ F ~ ~ ~i
a) -~ -
_ ln Ul ~ O O O O O ~ O u~ u~ Ul Ln ~ O ~ O ~ ~ o
~,~0
A A A A A A A A A A A A A A A A A A A A A A
S~ ~` ` ` ` `--``-------- -- -- -- -- _ _ _ _ _
a)-~oooooooooooo.ooooooooo
I I II I I I ' I I I I ~ I I I I I I I
o o o o o ~ o m u~ ~ o c~ o ~ ~ o
~-- G~ ~
" ~ r
~n ~ . . _ .
ul a) r- o ~ ~ a~
Q. ~ ~ a ~ ul ~
* ~ ~ ~ a
a ~ * * Q.
*~ ~ * a a * o
a * -- ~ a ~ _ v -~
a _ -- _ Q, ~ ~ 5:
*Q. ^ ~ ^ ~ O . O Q~ ~- 2 0 ~ G a o -,1 * ~ O --
* ._-~ O -~ ~ -.1 ~ * a * ~ -~ a
a Q.S --I ~ -- S v a ~ a v 5: ~
O V ~ ' ~ V -- -- Q, ~ ~ 1~
Q, -.1-- -- .¢ -- a Q. O Q. a -- _ o
o s * a -- a ~ a * _ o * -~ ~ o * ~ ~ ,_
a~ v a ~ a ~ -- ~ a ~ -~1 a s ~ -,~ a- c~
{J S .-- ^ ~ ~ * -- ^ -- ~ S Cl. V ^ ~ _ _ :~ ~ ~ V
.a Q. ~ * -- * * Q. Z v a -- Q, v ~ Q, ~ ~
a, -- ~ o -- ~ x x x o ~ a o -- o o -- -- ~ a
a * -,1 * ~ a a * * -~ a -,~ ~ ~ h ~ ,:s;
:> ~ s x o -- -- -- s -- ~ x ~ ~ ~: s s m m Q~ ~:
~ 0~ _ V ~ ~ ~ ~ _ V ~ ~ _ _~ ~ ~ V V I I I *
U~ * ~ * ~ ~ -- * -- -- ~
~ v a v v v v v a -- _ v v ~ _ a a -- -- -- ~
Q ~ ¢ C~ Q Q ~ ~, ~¢ Q ~, * ~
~¢ ~1 * ~ ~ ~ --I ~ * ~¢ ¢ ~1 ~ * ~ * V * * * ~5
-- -- V -- -- -- -- -- V -- -- -- -- V -- V ~ C~ V V
~ '
Q. O 1-- (~ G~ O ~ ~ ~ ~ U~ ~ G~ O ~ ~ ~ r
v z ~ r ~ r c~ r ~ r ~ 0 ~ a~
SU8STITUTE SHEET (RULE 26)
PCT/IB 9 4 / 0 ~ 09~ 6
3 3 2 ~ 2 ~
~ ~ ~ o o~
.,~ . . . .
~,
s~
o -
~.~ ~
D ~ ~ ~ r
_ o ~ U~ o
0
/\ J~ ~ A
1 ~ I I I I
a ~ o o o o o
I . ~ I
~r
~ ~ o C~ Ul O U~
V ~
o o
* ~
*
V -~ ~ .
V ~ -~1
a) ~ * _ * ~
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* o ._ o ~
V ~ ~ -~ V
U -- -- V
~ * -- V
-- , V '~
~ O ~ o
V Z
~1 IR~T1TI ~r ~ ~tDI 11 C
WO9/15958 21~ 3 2 ~ 8 PCT/IB94/00026
34
As discussea above ~n con~unc,ior -wi~n
S~ruc~ure I, 'inking residues Ll and L- ot`n-r than
Cvs, and Z ~roups other than simpie bonas, may
also be use'ully employed. It will be seen in
this regard that the presence of resiaues on
either side of LI will typically require
cyclization (through Z) to L- through a siae chain
or other functional group on LI that is not
engaged in bonding to the adjacent residues.
Residue Ll may more generally be engaged in
cyclization through either a terminal (typically,
carboxyl) functional group or a side chain
functional group.
Preferred residues for position XI include
Gly-, Phe-, Leu-, Asn-, Val-, Tyr, 1- or 2-
naphthylalanine, cyclohexylAla-, AMBA, AnC, AnB,
~-amino lower alkylcarboxylic acids, Aib-, Ser-
Tyr-Asn-, Ala-Thr-Val-, and p-chloro-Phe-.
Preferred residues for position Ylinclude -Ala, -
Ala-Ser, -Ala-Ser-Ser, -Ala-Ser-Ser-Lys, -Ala-
Ser-Ser-Lys-Pro, -Thr, -Thr-Phe, -Aib, -p-chloro-
Phe, AMBA, AnC, AnB, ~-amino-lower alkyl
carboxylic acids, 1- or 2-naphthylalanine, and
-(cyclohexylAla).
Where a substituent x2 or y2 incorporating R'
other than hydrogen is used, e.g., acyl groups
R'CO or amino groups of the form R'NH, preferred
substituents include those derived from bulky
compounds such as adamantaneacetic acid,
adamantanecarboxylic acid, 1- or 2-naphthylacetic
acid, 2-norbornaneacetic acid, 3-noradamantane-
carboxylic acid, 3-methyladamataneacetic acid for
Xl, and 1- or 2-adamantylamine for Y-. Other
suitable R' groups are those derived from lower
alkyl amines, lower arylamines or acids such as
from 9-fluoreneacetic acid, 1-fluorenecarboxylic
acid, 4-fluorenecarboxylic ac G, 2 -
WO94/15958 2 1~ ~ 2 2 ~ PCT/~94/00026
_'uorenecGrboxvl-- ac-d, q- l-orere- CGrbOXV1
acid, phenylace~ic, hydroxy-innamic acid,
quinaidic acid, formic acid, acetic acid,
trifluoroacetic ac_d, cvclohexyl acetic ac d, and
3-mercaptopro3ionic acid. In general, one would
choose an acid moie~y for X- and a basic moiety
for Y~.
Derivatives of the compounds of Struc~ure I
may be useful in the generation of antigens
which, in turn, may be useful to generate
antibodies. These antibodies will, in some
cases, themselves be effective in inhibiting cell
adhesion or modulating immune activity by acting
as receptors for matrix proteins or other ligands
or, if anti-idiotypic, by acting to block
cellular receptors.
2. Therapeutic Utility
In the practice of the therapeutic methods
of the present invention, an effective amount of
the active compound, including derivatives or
salts thereof, or a pharmaceutical composition
containing the same, as described below, is
administered via any of the usual and acceptable
methods known in the art, either singly or in
combination with another compound or compounds of
the present invention or other pharmaceutical
agents such as immunosuppressants,
antihistamines, corticosteroids, and the like.
These compounds or compositions can thus be
administered orally, sublingually, topically
(e.g., on the skin or in the eyes), by inhalation
or by suppository, parenterally (e.g.,
intramuscularly, intravenously, subcutaneously or
intradermally), or by inhalation, and in the form
of either solid or liquid dosage including
tablets, suspensions, and aerosols, as is
W094/l5958 2~3~ PCT/~94/0002 ~
-scussea ir -- mor~ aetail ~elow. ~ne
aàministration can be conducted in sinc_e uni,
cosage form wi.h continuous therapy or i- single
àose therapy ad libitum. A unit dose is ce~ined
as 1 to 3000 ms for a human patient.
Useful pharmaceutical carriers for the
preparation of the pharmaceutical compositions
hereof can be solids, liquids or mixtures
thereof; thus, the compositions can take the form
of tablets, pills, capsules, powders, enterically
coated or other protected formulations (such as
binding on ion exchange resins or other carriers,
or packaging in lipid or lipoprotein vesicles or
adding additional terminal amino acids),
sustained release formulations, erodable
formulations, implantable devices or components
thereof, microsphere formulations, solutions
(e.g., ophthalmic drops), suspensions, elixirs,
aerosols, and the like.
Water, saline, aqueous dextrose, and glycols
are preferred liquid carriers, particularly (when
isotonic) for injectable solutions. The carrier
can be selected from various oils including those
of petroleum, animal, vegetable or synthetic
origin, for example, peanut oil, soybean oil,
mineral oil, sesame oil, and the like. Suitable
pharmaceutical excipients include starch,
cellulose, talc, glucose, lactose, sucrose,
gelatin, malt, rice, flour, chalk, silica gel,
magnesium stearate, sodium stearate, glycerol
monostearate, sodium chloride, dried skim milk,
glycerol, propylene glycol, water, ethanol, and
the like The compositions may be subjected to
conventional pharmaceutical expedients such as
sterilization and may contain conventional
pharmaceutical additives such as preservatives,
stabilizing agents, wetting o~ emulsifying
2 ~
~ ~094/15958 PCT/~94/00026
_ _
aaents, sai~s fcY aaius.ing osmG~ic pressure,
bu~fers, ana the 1 ke. Suitable pharmaceuticai
carriers and their formulations are described in
Martin, "Remington's Pharmaceutical Ssiences",
15th Ed.; Mack Publishing Co., Easto~ (1975);
see, e.g., pp. 1405-1412 and pp. 1461-la87. Such
compositions will, in general, contain an
effective amount of the active compound together
with a suitable amount of carrier so as to
prepare the proper dosage form for proper
administration to the host.
In one preferred embodiment, the tnerapeutic
methods of the present invention are practiced
when the relief of symptoms is specifically
required or perhaps imminent; in another
preferred embodiment, the method hereof is
effectively practiced as continuous or
prophylactic treatment.
In the practice of the therapeutic methods
of the invention, the particular dosage of
pharmaceutical composition to be administered to
the subject will depend on a variety of
considerations including the nature of the
disease, the severity thereof, the schedule of
administration, the age and physical
characteristics of the subject, and so forth.
Proper dosages may be established using clinical
approaches familiar to the medicinal arts. It is
presently believed that dosages in the range of
0.1 to 100 mg of compound per kilogram of subject
body weight will be useful, and a range of 1 to
100 mg per kg generally preferred, where
administration is by injection or ingestion.
Topical dosages may utilize formulations
containing generally as low as 0.1 mg of compound
per ml of liquid carrier or excipient, with
multiple daily applications being appropriate.
WO9~/15958 ~ PCT/~9~/00026 -
~he ,~-..~ounds anG therape-_~ c cr
pharmace~tica compositions of the -nvention
might be use~u' ir~ the studv or trea~mer.t of
diseases or c'her conditions which are ~,ediated
by the bindinc of integrin receptors to ligands,
including conditions involving ina~ropriate
(e.g., excessive or insufficient) binding of
cells to natural or other ligands. Such diseases
and conditions might include in l~mmatory
diseases such as rheumatoid arthritis, asthma,
allergy conditions, adult respiratory distress
syndrome, inflammatory bowel diseases (e.g.,
ulcerative colitis and regional enteritis) and
ophthalmic inflammatory diseases; autoimmune
diseases; thrombosis or inappropriate platelet
aggregation conditions, and cardiovascular
disease; prevention of occlusion following
thrombolysis; neoplastic disease including
metastasis conditions; contraception through
inhibition of fertilization and embryo
implantation; as well as conditions wherein
increased cell binding is desired, as in wound
healing or prosthetic implantation situations as
discussed in more detail above.
The compounds of the present invention might
find use in the diagnosis of diseases which
result from abnormal cell adhesion. For example,
excessive adhesion of leukocytes to endothelial
cells or to exposed extracellular matrix in blood
vessels has been implicated in early s.ages of
atherosclerosis. Thus, a person demonstrating
excessive binding of leukocytes to endothelial
cells might be at risk for developing occluded
arteries. One might detect this risk '~actor by
determining which species of the compound of the
present invention is able to inhibit the binding
of leukocytes to endothelial cells, then
WO9~/15958 ~ 2 8 PCT/~94/00026
measuring the binding cf tha~ compcur.a .3
enaothelial cells of the patient thought to be at
risk.
- Furthe~more, the compounds of the present
invention might find use in the diagnosis of
autoimmune diseases caused by antibodies which
bind to cell adhesion molecules or whicr bind to
receptors for cell adhesion molecules. For
example, if a disease is caused by antibodies
binding to a cell adhesion molecule mimicked by
a compound of structure I, then a diagnostic test
for the presence of such antibodies is easily
performed by immunoassay of blood or serum from
a patient using the compound of structure I bound
to a substrate so as to capture the antibodies.
The bound antibody can be detected by the means
typical of the art such as a labelled second
antibody directed to the Fc portion of human
antibodies or using labelled Fc-binding proteins
from bacteria (protein A or protein G). In the
alternative situation, where the compound of
structure I binds to the same receptor as the
disease-causing antibody, a competitive
immunoassay format can be used. In this format,
the compound I is labelled and competition for
binding to receptor protein attached to the
substrate can be measured.
In addition, derivatives of the present
compounds might be useful in the generation of
antigens which are prepared by coupling the
peptides to a carrier protein. Animals are then
immunized with this complex thereby generating
antibodies to the peptides. These antibodies
- will, in some cases, themselves be effective in
inhibiting cell adhesion or modulating immune
activity by acting as receptors fo- matrix
proteins or other cell adhesion ligands, or, if
WO9~/15958 53~ PCT/~91/0002
an.l-_d~oty~ic,~ bv acz ' ~ O block ce_'~ia~
recep~ors.
Fur~hermore, the c~mpounas o~ the present
invention might be usea to produce matrices for
purifying substances whicn bind to the compounds
of the present invention with hish affinity.
Such a matrix could be produced, for example, by
covalently attaching a compound of the present
invention to a derivatized chromatographic
support. In one embodiment of this aspect of the
invention, a cyclic peptide listed in Table 1
which contains a free amino group can be coupled
to a cyanogen bromide activated chromatography
resin, such as that available from Pharmacia,
(Uppsala, Sweden, cat. no. 52-1153-00-AK). If
neccessary, an amino group can be introduced into
the desired peptide, either by addition of a
lysine residue, or by addition of another amine-
containing residue. Alternatively, of course,
carbodiimide-activated resin can be used in
conjunction with cyclic peptides bearing free
carboxyl functions.
The peptide is coupled using the protocol
essentially as provided by the manufacturer. The
cyclic peptide-derivatized resin can then be used
to purify proteins, polysaccharides or the like
which may bind the cyclic peptide with high
affinity. Such a purification would be
accomplished by contacting the cyclic peptide-
derivatized resin with a sample containing thecompound to be affinity purified under conditions
which allow formation of the specific complex,
washing of the complex bound to the resin with a
solution which removes unwanted substances, but
leaves the complex intact, and then eluting the
substance to be purified by washing the resin
with a solution which disrupts the complex.
WO9~/15958 PCT/~94/00026
41 2 1 5 3~
EX~PLr C
Although any me~hods and materia a sim lar
or equivalent to those describea here--L can be
used in the prac'ice or testing of the present
invention, the pre~erred methods and materials
are now described. As set forth above, all
publications to which reference is made are
incorporated herein by reference.
EXAMPLE 1
SYNTHESIS AND FORMULATION OF COMPOUNDS
The "backbones," i.e., the peptide-bond
linked portions of the cyclic compounds of the
invention were generally synthesized using solid
phase peptide synthesis, and then cyclized using
;5 a procedure which, where necessary, selectively
removed protective groups from only the residues
involved in cyclization. In this way, the
peptide sequence in the compound was not changed
or lengthened, but the peptide was properly
cyclized. Other methods for synthesis and
cyclization are known in the art and may be
employed in the preparation of the cyclic
compounds and formulations disclosed herein.
Unless otherwise noted, the methods described in
PCT International Publication No. WO 92/00995,
published 23 January 1992, are generally
applicable to synthesis of the peptides of the
present invention.
Thus, peptide sequences in the compounds of
this invention may be synthesized by the solid
phase peptide synthesis (for example, BOC or
FMOC) method, by solution phase synthesis, or by
other techniques known in the art including
WO94/1~958 ~ PCT/~9~/0002 -
3~ ~2
com~i~a~lon~ c the ~oregoin~ me-hoa~. ~he BOC
and rMOC met~ods, which are esta~lished and
widely used, are described in the ~ollowing
references:
Merrifield, J. Am. Chem. Soc., 88:2149
(1963);
Meienhofer, "Hormonal Proteins and
Peptides", pp. 48-267, C.H. Li, Ed., Academic
Press (1983);
Barany et al, in ~'The Peptides", pp. 3-285,
E. Gross and J. Meienhofer, Eds., Academic Press,
New York (1980).
Experimental:
SPECIFIC SYNTHESIS EXAMPLES
N,N-dialkvl-arqinines
N,N-dimethyl-arginine, N,N'-dimethyl-
arginine and N,N'-diethyl-arginine were
synthesized using the general procedure set forth
in PCT International Publication No. WO 92/00995,
published on January 23, 1992 and herein
incorporated by reference.
Protection of the modified arqinine: The material
from above is suitable for BOC-protection without
crystallization or other purification. One
equivalent of the amino acid is dissolved in 1
eq. of 1 N NaOH and an equal volume of dioxane.
BOC-Anhydride (1.1 eq.) was dissolved in dioxane
and stirred at R.T. for 4 hrs while maintaining
pH 9 by addition of 1 N NaOH when necessary. The
reaction is followed by tlc for the
disappearance of the starting material
(visualized with ninhydrin spray). When the
reaction was complete acetic acid was added to pH
WO91/15958 Z 5 3 ~ 2 ~ PCT/~94/00026
~3
-. A te~ s.i~r~ ~ ~o- 15 minu~es .he pr-d_-_ was
isola.ed followira lyophilization.
~lc System -- me~hancl/ammonium hydrcx d~
Dimethyl-Arginine at ~f 0.6, Diethyl-Ar~in ne a~
Rf 0.8.
Proline Analoas
The syntheses of (D,L)-thiazolidine-2-
carboxylic acid, N-BOC-(D,L)-thiazolidine-2-
carboxylic acid, N-BOC-(L)-5,5-aimethyl-
thiazolidine-4-carboxylic acid, (L)-2-
methylthiazolidine-4-carboxylic acid and N-BOC-
(L)-2-methylthiazolidine-4-carboxylic acid are
set forth in PCT International Publication No. WO
92/00995, published on January 23, 1992 and
previously incorporated by reference.
L-l,3-Tetrahydrothiazine-4-carboxvlic acid
Procedure:
To 5 g homocysteine thiolactone HCl (Sigma) in 75
ml of water 3 ml of lN HCl and 16 ml of 37~
aqueous formaldenyde solution (Aldrich) were
added. After stirring for 2 days at room
temperature the solution was concentrated to
dryness (bath temperature 40 - 50 C). The
product was dissolved in l00 ml of 3A ethanol,
filtered and concentrated to 25 ml in vacuo. An
equal volume of ethyl acetate was added dropwise
and the mixture was allowed to sit in the freezer
overnight. The off-white or tan crys.als were
washed with ethyl acetate and dried unde~ vacuum
to afford about 3.0 g (50.4 ~ yield). The crude
product was dissolved in a minimum amount of hot
3A ethanol and cooled in ice-bath. After
crystallization began an additional volume of
ethyl acetate was stirred in and the mix.ure was
WO9~/15958 ~ . PCT/~94/0002 ~
2t,ra3,~
_~ole~ in an ice-ba~h for ~ nrs. ~he ~r-auc~
-~vs_als were ccllecred, washed wi~n elhyl
aceta~e and dried in vacuo to afford 2 am L-1,3-
tetrahydrothiazrne-4-cGrboxylic acid.
Analytical:
Silica gel tlc in System A (Butanol/Acetic
acid~Water/Pyridine 4:1:2:1): product Rf o.~.
melting point: 208-210 C dec.
Rotation [water~ -14.03 d=1.
N-BOC-(L)-1,3-Tetrahydrothiazine-4-carboxylic
acld
Procedure:
To 3 gm of L-ll3-tetrahydrothiazine-4-carboxylic
acid 16.3 mmol) in 50 ml water, 17 ml of lN NaOH
and 50 ml of dioxane (peroxide free~ were added
3.8 gm (BOC)2O (17.4 mmol) in 10 ml of dioxane.
The pH was of the reaction was maintained at 8-9
with lN NaOH and the reaction was stirred
overnight. Overnight reaction was found to be
incomplete. Additional (BOC) 2 (O . 38 gm) was
added and the mixture was stirred for an
additional 4 hrs to complete the reaction. The
reaction mixture was concentrated in vacuo to
half volume and extracted with hexane (2x50 ml).
The hexane layers were discarded. The aqueous
layer was cooled with an ice-bath, acidified to
pH 3 with lN NaHSO4 and extracted with three 50 ml
portions of ethyl acetate. The pooled ethyl
acetate layers were back washed with water (3x50
ml), dried over anhydrous MgSO~ and filtered. The
solvent was removed in vacuo to give a
spontaneously crystallizing light amber oil. A
small amount of hexane was used to break up and
facilitate collection of the solids. The produc~
WO94/15958 2 1 5 3 2 ~ 8 PCT/~94/00026
~_
was aried u-ae~ ~acuum ~o cive 3.6 Gm c an e---
wr.ite powàe~
Ana'y~ical:
Silica gel tlc,system A; product Rf 0.8.
M.P. 112-113C. Rotation in acetone, -162.4, d=1.
Anal. Calcd. for C,oHI7N~O~Sl; F.W. 247: C, 48.58; H,
6.88; N, 5.67. Found: C, 48.52; H, 6.94; N,
5.67.
(L~-1,4-Tetrahydrothiazine-3-carboxYlic acid
Ste~ 1: To a solution of sodium (16.8 gm) in 1.5
1 liquid ammonia L-cystine (Sigma, 38 gm) was
added in small portions over 1/2 hr until the
blue color was permanently discharged.
Bromoethanol (Aldrich, 56 gm) was added slowly
over 45 min and stirred overnight as the ammonia
was allowed to evaporate. Ste~ 2: The residue
from step 1 was dissolved in 1500 ml of conc. HCl
and heated to 90-95C for 7 hr. The soluticn was
concentrated in vacuo and the solid collected.
The solid was slurried in 1200 ml of isopropyl
alcohol and filtered. The mother liquor was
concentrated to a slush, filtered and the filter
cake air dried. Step 3: The step 2 solid t45 gm)
was dissolved in 1 l DMF and 750 ml of
triethylamine was added. The mixture was heated
for 2.5 hr at 90-95C and then concentrated to
dryness on a rotary evaporator. The solids were
dissolved in 1.5 l water and applied to an 800 ml
column of Amberlite IR-120 H+ resin. After
washing until neutral the product was eluted with
1.5 N aqueous ammonium hydroxide. The product
was concentrated to dryness, taken up in water,
treated with carbon, filtered through a celite
WO9~/15958 ~ PCT/~94/0002
paâ ana di~~_ed slowly with a~out ~ V2_ mes c
acetone. T:~e c~ys~als we~e collec;ed, washed
with ace_one and air dried to afford l~.2 gm of
the aesired (L)~ -te-rahydrothiazine-3-
carboxylic acid.
N-BOC-(h)-l,4-Tetrahvdrothiazine-3-
carboxvlic acid
To a mixture of (L)-1,4-tetrahydrothiazine-
3-carboxylic acid (16 gm) dissolved in l:l
dioxane/lN NaOH was added (BOC) 2 (24 gm)
dissolved in l0 ml dioxane. The pH was
maintained 5~ 9 and allowed to stand at room
temperature overnight. The solution was
concentrated to l/2 volume and extracted in
succession with 3 portions of 50 ml each of
hexane. The aqueous layer was diluted to 300 ml
with water, cooled and acidified to pH 3 with
NaHSO4. The solids were collected, washed with
water and dried over P2O5 to afford 22.4 gm of a
white powder of the desired N-BOC-(L)-1,4-
Tetrahydrothiazine-3-carboxylic acid.
Amino acid precursors were purchased from
BACHEM (Torrance, California). DCC was from
Sigma Co. (St. Louis, Mo); TFA was from
Halocarbon Co. (New York, NY). Triethylamine was
from Fisher Scientific (Fairlane, NJ). ~-
Methylbenzhydrylamine resin was from CBA Inc.
(Boulder. CO). Other reagents were of analytical
grade or better.
All peptides were synthesized by the solid
phase method (Barany et al, The Peptides, E.
Gross and J. Meienhofer eds., Volume 2, Part A,
Chapter l, Academic Press, Inc., 1-28~ (1979))
with a Beckman Automated peptide syn~hesizer
(Sys~em 99~, Beckman Instruments, Inc., Palo
WO94/15958 21 5 ~ 2 2 ~ ~ PCT/~9~/00026
Alto, Cali crniaj usinc BOC/DCC cnemis.ry (Baranv
et a', The Pep_ides, E. Gross and J. Meienho-er
eds., Volume " Par~ A, Chap~er 1, Academic
Press, Inc., --284 (1979)). Other ac~ivating
agents include DCC/HOBt (D.L. Nyugen and B.
Castro, "Peptide Chemistry", pp. 231-238 Protein
Research Foundation Press, Osaka (1987)) or BOP
or BOP/HOBt (D. Hudson, J. Org. Chem., 53:617-624
(1988)).
Attachment of N-BOC-S-(4-methylbenzyl)-
Cysteine (BOC-Cys-(4-MeBzl) to the chloromethyl
polystyrene resin (Merrifield resin) was done in
the presence of potassium fluoride (Horiki, Chem.
Lett. (~2):166-168 (1978)).
General Procedures for Synthesis of Cyclic
Peptides
Peptide Svnthesis: BOC-Cys(4-MeBzl)-polystyrene
resin (for C-terminal carboxylic acids) or 4-
methylbenzhydrylamine resin (for C-terminal
carboxamides) was used for the stepwise assembly
of the product peptides using the BOC amino acid
procedure in Table 2. Related procedures can be
found in the published International Patent
Application WO 92/00995, which was previously
incorporated by reference. Following coupling of
the last amino acid the N-terminal BOC protecting
group was removed by mixing the resin with
TFA:DCM (1:1) for 20 minutes. Following rinsing
in order with DCM (3X), MeOH (2X), DCM (3X) the
resin was air dried.
Cleavaae: The BOC-deprotected peptides on-resin
were cleaved by stirring at -5 to 0C with a
cocktail of distilled anhydrous HF (10 ml/g
resin), anisole (l ml/g resin) and dimethyl
sulfide (0.5 ml/g resin). After one hour the HF
was evaporated under reduced pressure. The
cleaved peptide/resin mixture was washed three
WO9~/15958 . ~ PCT/~9~/0002
.imes w th àieth~' et~er ard ~h-- e~ra~ wit~
80~ aq. acetic acid. The combi~^d extracts (200
ml/g resin) were pooled and ca~ried on ta the
cyciizat-on step.
Cvclization: The formation of t:~e intramolecular
disulfide bridge was accomplished by usirg the
iodine oxidation method (Wunch et al, In~. J.
Peptide Protein Res., 32:358-383 (1988),
Bodanszky, Int. J. Peptide Protein Res., 25:449-
474 (1985~). Saturated I~in glacial acetic acid
was added dropwise to the stirred crude peptide
in 80~ aqueous acetic acid until the solution
turned light brown. After stirring for 1 hr at
room temperature, the excess ioaine was quenched
by adding saturated aqueous ascorbic acid. The
cyclized peptide was concentrated in vacuo,
resuspended in water and lyophilized.
Purification: The cyclic peptide was purified
using a Waters Delta Prep 3000 system (Waters,
Milford, Massachusetts) equipped with Vydac C~g
column (15-20 mm, 5X30 cm ID), using a linear
gradient of increasing acetonitrile concentration
in 1~ triethylammonium phosphate (TEAP, pH 2.3)
as mobile phase. The appropriate fractions were
pooled to give the pure peptide as a phosphate
salt. The peptide salt was applied again to the
column and eluted with a 0.5~ aqueous HOAc ln
acetonitrile to afford the desired acetate salt
form.
Analysis: The purified peptides were analyzed
using a Beckman System 126 (Detector module 166),
equipped with a Beckman Cl8 column (RPC18
Ultrasphere, 5 ~m, 4.6 x 150 mm ID). Elution was
performed with buffer A = 0.1 M sodium phosphate
in water, pH 4.4 - 4.5, buffer B = 60
acetonitrile in buffer A, using a linear gradient
of buffer B and a flow rate of 1 ml/minute. The
WO9~/~5958 2 1 ~ ~2 2 8 PCTI~94/00026
. me c the gradient was adjus.eG -~r each
peptide, beins 20 or 30 minutes, to provide
e ulior. o- the peptide near the midd'e of the
- g~adier.t. Retention times of each of the
preferred peptides are shown in the table of
preferred compounds (Table 1).
Fmoc Removal: Piperidine (6 ml) is added to 24 ml
o~ DMF solution containing 1 g FMOC peptide to
give a 20~ piperidine in DMF. The solution is
stirred for twenty minutes at room temperature
and then evaporated in vacuo until dry. The
residue is dissolved in ethyl acetate/water (1:1)
and the water layer is collected. The water layer
is back extracted twice with ethyl acetate,
filtered and lyophilized.
WO 91/1~,958 PCT/IB9~/00026--
?.,~,S~
~BL~ 2
Cchedule ~ S~ d Phase PeDtide SyrLt:~sis
( T ~ A dep-o~ c _ ion/ DCC coup 1 ing )
Ste~ Rea~ent Vol~(ml) T~me(min~
l DCM wash (3X) 20 0.5
TFA-DCM (l:l) 20
3 TFA-DCM (1:1) 20 20
4 DCM wash (3X) 20 0.5
MeOH wash (2X) 20 0. 5
6 DCM wash (3X) 20 0.5
7 TE~-DCM (1:9) 20
8 TEA-DCM (1:9) 20 5
9 DCM wash (3X) 20 0.5
MeOH wash (2X) 20 0. 5
11 DCM wash (3X) 20 0.5
12A BOC-AA ( 3. 2 mM,
2 equiv.) in DCM
(DMF)*~ 10
12B DCC in DCM (0.5M) 6.4 120***
13 DCM wash (2X) 20 0. 5
14 DCM-MeOH (1:1) (2X) 20 0. 5
TEA-DCM (1:9) 20 0.5
16 MeOH wash (2X) 20 0. 5
17 DCM wash (3X) 20 0.5
18 Ac20 in DCM (1:3) 20 20
19 )CM wash (3X) 20 0.5
MeO~ wash (2X) 20 0. 5
21 DCM wash (3X) 20 0.5
* The volume given is for the synthesis using 2 g of resin with
the substitution of 0.8 mM/g of resin.
** DMF added where BOC-AA is insoluble in DCM alone.
*** When the coupling is deterrnined incomplete by the
ninhydrin test, recoupling is necessary with a mixture
consisting of amino acid, BOP reagent in DMF and DIEA in
a 1:1:3 ratio for one hour. After recoupling, the resin is
filtered and the ninhydrin test is performed to determine
whether coupling is complete. If not the procedure is
repeated until ~he test shows the coupling is complete.
~ W094/15958 PCT/~94/00026
322~
Synthes s o~ Amide Link~d Cycl~c
Com~ounds !C~de Chain-Cide C`-ain Linkace)
In this example, the following compound was
synthesized:
(HN) (C=O)
(AdaCA)-R-K-D-(thiop)-D
All amino acids and amino acid derivatives
were purchased from BACHEM (Torrance,
California~. 9-Fluorenylmethanol and DDC were
obtained from Sigma Chemical Co. (St. Louis,
Missouri~. Diisopropylethylamine and 4-
(dimethylamino~-pyridine were obtained from
Aldrich (Milwaukee, Wisconsin~. Unless otherwise
noted, other reagents were of analytical grade
and used without further purification.
All residues were linked by the solid phase
method using BOC protection. The side chain
carboxyl groups of Asp and Glu were protected as
fluorenylmethyl esters and the ~-amino group of
Lys and ~-amino group of Gly were protected as N-
FMOC. The amide bridge between the two side
chains (on Asp and Lys~ was synthesized while the
peptide was bound on the resin. This procedure
is represented by Figure la.
(a~ Preparation of N-BOC-O-9-
fluorenylmethyl omega-esters of
aspartic and glutamic acids.
The N-BOC-O-9-fluorenylmethyl omega-esters
of aspartic and glutamic acids were prepared
following the procedure as generally described by
Bolin, (Bolin et al., Organic Preparations and
Procedures Intern., 21:67-74 (1989)) with certain
modifications.
Z ~ 2 g PCT/~94/0002 ~
C-^~-9-fluore~vlmethvl as~a~are.
8.3_ g (2~.7 mmol) of ".-BOC-^~-
~_nzylaspartate anG 4.80 g (24.5 mmol) of ~-
: orenylmethanol were dissolved in 150 ml DCM.
5 T:-e solution was chilled in an ice bat~. 30 mg
(~.24 mmol) of 4-(dimethylamino)pyridin~ was
a~ded to the solution followed by adcition of
5.31 g (25.7 mmol) DCC in portions, over 10
minutes. The resulting mixture was stirred for
o^.e hour with continued cooling. The
precipitated N,N~-dicyclohexylurea was removed by
f_ltration and the filtrate was diluted with 250
m DCM. This solution was extracted with (in
order) 10~ citric acid (2 x 50 ml), H.O (1 x 50
ml), 25~ NaHCO3 (2X50 ml), H.O (1 x 50 ml), brine
(1 x 50 ml). The solution was then dried over
MgSO4, and concentrated to an oily residue.
Recrystallization from methanol/ether/petroleum
ether (1:3:10) yielded 10.85 g (84~) N-BOC-0-
benzyl-O~-fluorenylmethyl-aspartate, with a
melting point of 74 -77C . 5.5 g (10.9 mmol) o~
the above product was then dissolved in 150 ml
warmed methanol, and hydrogenated over 300 mg of
2C~ Pd(OH)~/C for 1. 5 hr at room temperature and
a pressure of 35-40 pSi. The catalyst was
f ltered off and the solvent was evaporated in
vacuo. The residual oil was redissolved in 200
mi diethyl ether and extracted with (in order) 1~
NaHCO3 (3 x 50 ml), H2O (1 x 50 ml), 5~ citric
acid (2 x 50 ml), and brine (1 x 50 ml). The
erner layer was dried over MgSO~and concentrated.
~e-rystallization from diethyl ether/petroleum
e-her yielded 3.53 g of N-~OC-O~-9-
fluorenylmethyl aspartate, with a melting point
o. 135-137C.
2-1~53228
WO94/15958 PCT/~94100026
53
N-BOC-O-fluorenvlmethvl-qlutamate (~amma ester).
N-BOC-O~-benzylglutamate (4.5 g, 13.3 mol)
and 9-fluorenyl-methanol (2.5 g, 12.5 mmol) were
dissolved in 100 ml DCM. The solution was
stirred and chilled in an ice bath. To the
solution, 15.5 mg (0.13 mmol) of 4-
(dimethylamino)-pyridine and 2.75 g (13.3 mmol)
of DCC were added, and the resulting mixture was
stirred for 4 hr with continued cooling.
Precipitated N,N'-dicyclohexylurea was filtered
off and filtrate was diluted with 200 ml DCM.
The solution was extracted and treated in the
same manner as was described above for the
aspartate. This yielded N-BOC-O~-benzyl-O-
fluorenylmethylglutamate (gamma ester) (4.2 g),
with a melting point of 97-99.5C. 4.0 g (7.75
mmol) of the foregoing produce was hydrogenated
over 125 mg of 10~ Pd/C in 200 ml mixture of
MeOH/EtOH/IPA (2:1:1) for 2 hr at room
temperature at 40 psi. The reaction mixture was
filtered to remove the catalyst, and concentrated
to an oily residue. The residue was then mixed
with 150 ml diethyl ether and combined aqueous
layers were back-extracted with diethyl ether (2
x 40 ml). The combined ether layers were dried
over MgSO4, filtered, and concentrated to a white
form. N-BOC-O-fluorenylmethylglutamate (gamma
ester) (2.3 g) was obtained by recrystallizing
the crude residue from diethyl ether/petroleum
ether (1:10), melting point 123.5-126C.
(b) Synthesis of protected RK*D(thiop)D*
peptide sequence.
Synthesis of the above peptide was performed
using, in conjunction, an automated peptide
synthesizer (System 990, Beckman Instruments,
Inc., Palo Alto, California) and a manual peptide
synthesis apparatus (S.C. Glass Tech, Bonica,
WO 9~115958 7,~2~ ~ PCT/IB94100026
Ca I ~ rcr-- a~ . _vF'-As? (O~m) CC~ AM r~s- n (' . O g,
0.75 mmol) from Applied Biosys.ems (ros~er City,
Cali.o~nia) was used as the s~artinc resin. The
following amino acids were used in the synthesis:
BOC-(3-thiop), BOC-Asp(O-benzyl), BOC-Lys(N~-
FMOC), and BOC-Arg(N~-tos). Excess amino acid (2-
3 fold) was used for each coupling. The peptide
chain was constructed on the Beckman peptide
synthesizer using BOC chemistry with the stepwise
addition of each amino acid following the
standardized cycle similar to that presented in
Table 2, with adjustments for scale. 50~ TFA in
DCM, 5~ DIEA in DCM, and 0.5 M of DCC in DCM were
used as deprotecting agent, neutralizer, and
activating agent, respectively, for each
coupling.
(c) Capping of peptide sequence.
Following the removal of the BOC group from
the N-terminal Arg with 50~ TFA in DCM, and
neutralization with 5~ DIEA in DCM, the protected
peptide on resin was reacted with activated
AdaCA. The N-terminal deprotected, side chain
protected peptide on resin was washed with MeOH
(2 x 1 min), DCM (3 x 1 min); neutralized with 5~
DIEA again in DCM (1 x 1 min, 1 x 20 min); washed
with DCM (3 x 1 min); and capped with (AdaCA),
DCC and HOBt in DMF. The peptide then was
cyclized by forming an amide linkage between the
~-carboxyl group of Asp and the ~-amino group of
Lys by the general procedure below.
(d) General cyclization procedure for
formation of the amide bridge.
After the construction cf the peptide chain,
the amidating cyclization was carried out
according to the following protocol. Filtering
was performed between each step: (1) MeOH (2 x 1
WO94/15958 2 I ~ 3 2 2 8 PCTl~94/00026
min); (2 ) DCM (3 x 1 min); (3 ) 20~ piperidine in
DMF, wash for l min, and deprotection for 20 min;
(4) DMF (2 x 1 min); (5) MeOH (2 x l min); (6)
DCM (3 x 1 min); (7) BOP reagent (4 equiv.) in
DMF (20 ml/gram of resin), stir for 2 min. and
add DIEA (2~ of DMF volume), stir for 4 hrs (the
completion of the cyclization reaction was
monitored by the ninhydrin test; if the reaction
was judged incomplete at 4 hrs, the reaction was
continued until the ninhydrin test was negative);
(8) DMF (2 x l min); (9) DCM (2 x l min); (l0)
MeOH (2 x l min).
The final cyclic compound was removed from
the resin by treatment with HF in the presence of
l0~ anisole for l hr at 0C. After evaporation of
the HF, the residue was washed with diethyl ether
and extracted from the resin with 5~ HOAc in H~O.
The aqueous extract was lyophilized to yield the
crude peptide.
(e) Purification
The compound was purified using a Waters
Delta Prep 3000 system (Waters, Milford, MA)
equipped with a Cl8 column, using a linear
gradient of increasing acetonitrile concentration
in TEAP (pH 2 . 2 to 2 . 4 ) as the mobile phase. The
collected fractions of the pure compound were
pooled and applied again to the Cl8 column. This
time the sample was eluted with 0. 5~ HOAc to
convert the phosphate salt form of the peptide to
the desired acetate form. The pure peptide
fractions were pooled, concentrated in vacuo,
redissolved in water and lyophilized to give 92 . 9
mg of peptide, 98 . 7~ HPLC purity, white powder.
Wo9~/15958 ~ 2~ PCT/IB91/0002 4
,
rrh_sis c Amide-Lin.~eQ Cycl~c Com~o~rds
(Bac.~bor.e-Side Chain Linkage!
In th~s example, the following compound was
synthesized:
1 l
G - R - v - 3 (TTC) - D - NH.
The manual synthesis cf the above compound began
with 4-methylbenzhydrylamine resin (2.0 g, 1.4
mmol) from CBA, Inc. (Boulder, Colorado). The
peptide chain was assembled by using the BOC
procedure described in the synthesis of the
compound of Example 4 above. BOC-Asp(Fm), BOC-
1,4-TTC, BOC-Asp(O-benzyl), BOC-Val, BOC-Arg(Ng-
tosyl) and N-FMOC-Gly were used in the synthesis.
The cyclization between the terminal amino
group of Gly and the ~-carboxyl group of Asp6 was
performed according to the general amide
cyclization procedure described above.
The cyclic compound then was cleaved from
the resin by HF and 10~ anisole for 1 hr at 0C.
Following evaporation to the HF, the mixture was
washed with diethyl ether (ether layer discarded)
and extracted with lN HOAc. The aqueous extract
was lyophilized to yield 1.23 g of the crude
compound.
Purification of the compound was achieved
using a Waters preparative HPLC system with a C~8
column, following the method described in the
preceding example. Yield was 678 mg pure produce
compound, HPLC purity of 99.7~, white powder.
~ 094/15958 21` 5 3 2 2 ~ PCTl~94/00026
Svnthesis c- Amide L-nked Cyclic Com~cunas
(Cvcliza~ion Pr~or to Com~l te rhain Assemblv)
In this example, the following compound is
synthesized (SEQ. I3. NO.:18)
5(HN) (C=O)
G-R-A-L-(thiop)-D
The compound is made using the procedure
described in PCT Application WO 92/00995,
l0published January 23, 1992.
Synthesis of Cyclic Disulfide Com~ounds
In this example, the following compound was
prepared (SEQ. ID. NO.:4):
(S) (S)
l l
(l-FCA)-Cys-D-(3-thiop)-Cys
l-FCA was purchased from Alrich Chemical
Company (Milwaukee, WI). All amino acids, amino
20acid derivatives and analogs and unnatural amino
acids were purchased from BACHEM (Torrance,
California). DCC was from Sigma Chemical Co.
(St. Louis, Missouri). Trifluoroacetic acid was
from Halocarbon Co. (New York, New York).
25Triethylamine was from Fisher Scientific (Fair
Lawn, New Jersey). Other reagents were obtained
from conventional sources and of analytical
grade.
All peptides were synthesized by the solid
30phase method with a Beckman automated peptide
synthesizer (System 990, Beckman Instruments,
Inc., Palo Alto, California) using BOC chemistry.
Attachment of N-BOC-S-p-methylbenzyl-
cysteine (BOC-Cys~4-MeBzl)) to the
35chlcromethylpolystyrene resin (Merrifieid resinj
WO9~/15958 ~ PCT/~94/00026 -
3~
was dore in the p~esence 5_ p .as ium -~ loriae.
BOC-Cys(4-MeBzl) (Q.q molar ec.) was reac~ed w th
sweiled Merrifield resin (Bio-~ad lab., ~ichmond,
California) (1.0 molar eq.) in DMF in presence o,
K~ (1.8 molar eq.) at 80C for 15 hr. The resin
then was filtered, washed, ana dried. The molar
substitution of the resin was determined by
weight. The sequential elaboration of the
peptide chain on the BOC-Cys(~-MeBzl) resin was
carried out stepwise using the BOC procedure
according to the procedure in Table 2 above. At
the end of the synthesis the N-terminal BOC
protecting group was removed using TFA:DCM (1:1)
for 30 min.
The compound was then cleaved from the resin
with HF, the disulfide was formed as described
earlier and purified as usual.
Svnthesis of Crclic PeDtides Containinq
Carboxy-terminal NHNR' (es~ecially AMP) or
Tetrazole
In general, peptides having the carboxy-
terminus modi~ied by amidation to NHNR', in
particular, those compounds having AMP
derivatives of the carboxyl-terminus, or those
peptides in which the carboxyl-terminus has been
replaced by tetrazole, are synthesized by a
slight variation of the technique by which the
other peptides of the present invention are made.
To make these compounds, that portion of the
cyclic peptide except for the carboxy-terminal
amino acid (i.e. except yl) is synthesized by the
solid-phase method described above and cleaved
from the resin. The peptide is then coupled to
the appropriately derivatized carboxyl-terminal
amino acid, using solution-phase techniques.
This procedure is described in the published PCT
WO94/15958 Z1 5 3 2 ~ ~ PCT/~94/00026
_aten~ app~-_atior WO 92/00C995, prev- ousiy
ncorporated ky reference. Details cf ~o;utior
?nase peptiae synthesis techniques can be founa
n ~odanszky, "Peptide Synthesis", Sp~in~er-
Verlag, New Vork (1984).
- Synthesis of amino acids having the ~-
carboxylic acid replaced by tetrazole can be
performed according to Langry (Langry, K.C., J.
Org. Chem. 56:2400-2404 (l99l)).
EXAMPLF 2
CELL ADHESION INHIBITION ASSAYS:
U937 Cell Fibronectin Adhesion Assav
The following assay established the activity
of the present compounds in inhibiting cell
adhesion in a representative in vitro system.
This assay is a competition assay in which both
fibronectin and a test compound are present.
Microtiter plates were first precoated with
fibronectin. The test peptide was then added in
increasing concentrations with cells known to
contain _he fibronectin receptor. The plates
were incubated, washed and stained for
quantitation of attached cells. The present
assay directly demonstrates the anti-cell
adhesion activity and adhesion modulatory
activity of the present compounds.
The cell line U937 was purchased from
American Type Culture Collection (Rockville, MD).
The cells are cultured in RPMI media (J.R.
Scientific Company, Woodland Hills, CA)
containing l0~ fetal calf serum. Fibronectin was
purified from human plasma according to the
procedures of Engvall et al., Int. J. Cancer,
20:l-4 (l977).
Microtiter plates (96-well, Falcon) were
coated overnight at 4C with 0.l ml c- a 5 ~g/m'
WO 9~/15958 PCT/IB94/00026~
?,3 53~?~8
r _nec~n 'n pnos~nate bu,~ e~ea sa i-3 ~P3S).
As a c~n~rol, 5 ~g/ml bovine sevum album-n (BSA)
was aaded to wells. Unbound ~roteins were
removea f~om plates by washing with ~3S. To
block non-reacted sites, the plates are coated
with 100 ~l of a 2.5 mg/ml BSA solutior in PBS
for one hour at 37C. U937 cells were collected
and washed twice in Hanks' Balanced Salt Solution
(HBSS). The cells were counted and adjusted to
2.5 x 1o6 cells/ml in Dulbecco's Moaified Eagle's
Medium (DMEM) plus BSA (2.5 mg/ml) for the cell
attachment assay. The test compounds were
dissolved in DMEM-BSA and the pH adjusted to 7.4
with 7.5~ sodium bicarbonate. The compounds (100
,1l1) were generally added to the FN-coated wells,
at 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9,
1.95 and 0.98 ~g/ml final concentration. The
concentrations used for testing were adjusted
depending on the potency of the peptide. U937
cells (100 ~Ll) were added to the wells and the
plates were incubated for one hour at 37C.
Following this incubation the plates were washed
once with PBS and the attached cells were fixed
with 3~ paraformaldehyde in PBS and stained with
0.5~ toluidine blue in 3.7~ formaldehyde. The
cells were stained overnight at room temperature
and the optical density at 590 nm of toluidine
blue stained cells was determined using a
vertical pathway spectrophotometer to quantitate
attachment (VMAX Kinetic Microplate Reader,
Molecular Devices, Menlo Park, CA). This
procedure is a modification of previously
published method, Cardarelli et al., PNAS-USA
_:2647-2651 (1986).
Jurka_-CS-1 Adhesion Assav (Jurkat-cY,, assay)
WO94/lS958 ~ I ~ 3 ~ 2 ~ PCT/~94/00026
~1
The CS-_ derivea pep~ides C~r;~ LDVPST
(S_~. D. NO.:26) and CLHGPIELVSDPT (SFQ. ID.
NG.:27) were immobilized onto microtiter plates
- us ng the heterobifunctional crosslinker 3-
_ ~2 -p yrid yl d it hio) p rop i o n ic aci d
N-hydroxysuccinimide ester (SPDP) (Sigma, St.
Louis, M0) according to publishec methods
(Pierschbacher, PNAS-USA 80:1224 (1983)).
Briefly, microtiter plates were coated at room
lC temperature with 20 ~g/ml human serum albumin
(HSA) for 2 hours then derivatized with 10 ~g/ml
SPDP for 1 hour. After removal of excess unbound
reagents the cysteine containing peptides were
added and allowed to crosslink overnight at 4 C.
The following day the cell adhesion assay was
carried out as specified in the "U937 Cell
Fibronectin Adhesion Assay", except that the
human T-lymphocyte cell line, Jurkat, was used.
Jurkat-Endothelial Cell Adhesion Assay
The following assay established the activity
of the present compounds in inhibiting cell-cell
adhesion in a representative in vitro system.
This assay measures the adhesive interactions of
a T-cell line, Jurkat, to endothelial cell
monolayers in the presence of test compounds.
The test compounds are added in increasing
concentrations with T-cells and this is added to
endothelial cell monolayers. The plates are
incubated, washed and the percentage of attached
3Q cells is quantitated. The present assay directly
demonstrates the cell adhesion inhibitory
activity and adhesion modulatory activity of the
present compounds.
Human umbilical vein endothelial cells were
purchased from Clonetics (San Diego, CA) a~
passage number 2. The cells were growth on O. 5
WO91/15958 ~ PCT/IB9~/0002
~orc~ne s~in cela_in p~-e-coa~ea _las~s (cicma,
S~. Louis, MO) in EGM-W media (Clore~ics, San
~ieao, CA) supplemented with l0~ etal bovine
serum. Cells are re~ed every 2-3 days, reac~ing
confluence by day 4 to 6. The cells are
monitored for facto~~ vIII antigen and our results
show that at passage 12, the cells are positive
for this antigen. The endothelial cells are not
used following passage 12.
The T-cell line Jurkat was obtained from
American Type Culture Collection and cultured in
RPMI containing l0~ fetal calf serum. The cells
are washed twice in HBSS and resuspended in
Dulbecco's Minimal Eagle's Media (DMEM)
containing 2.5 mg/ml Human Serum Albumin (HSA).
Jurkat cells (lxl06 cells/ml) are stained with l0
~g/ml fluorescein diacetate (Sigma, St. Louis,
MO) in HSSS containing 5~ fetal calf serum. The
cells are stained for 15 minutes in the dark at
room temperature, washed 2 times, and resuspended
in DMEM-HSA solution.
Confluent endothelial monolayers grown in
96-well tissue culture plates are stimulated for
4 hours at 37C with 0.l ng/ml ( 50 U/ml)
recombinant IL-l (Amgen, Thousand Oaks, CA).
Following this incubation, the monolayers are
washed twice with HBSS and 0.l ml of DMEM~HSA
solution are added. Jurkat cells (5 x 105 cells)
are combined with the appropriate concentration
of peptide and 0.l ml of the Jurkat cell-peptide
mixture are added to the endothelial cell
monolayers. Generally, 250, 50, l0 and 2 ~M
peptide concentrations are tested. With several
potent peptides the ICso is determined by testing
the peptides at 50, l0, 2 and 0.4 ~M. The plates
are placed on ice for 5 minutes to allow for
Jurkat cell settlina and the plates are i.cubated
WO91/1:958 21 5 3 Z 2 8 PCT11B9J/00026
a~ 37`'C -or 20 minutes. Following th,-
incuba~i~-, the monolayers are washed twice with
PBS cor.-~_.ing l mM calcium chioriae and ; mM
magnesiu.. cnloriae and the plates are read usinc
a Pandex Fluorescence Concentration Analyze~
(Baxter, .~.'undelein, IL). Fluorescence in each
well is measured as Arbitrary Fluorescence Units
and percen~ adhesion in the absence of peptide is
adjusted to lO0~ and the ~ adhesion in the
presence c- peptides is calculated. Monolayers
are also fixed in 3~ paraformaldehyde and
evaluated microscopically to verify the adhesion.
The cell adhesion assay was performed as
outlined a~ove in "U937-FN assay" except that the
human T lymphocyte cell line, Jurkat, was used in
place of the U937 cells.
Results of U937 Cell Adhesion to Fibronectin
Potency is expressed in ~M units. Activity
is defined in this assay as an IC50 below 500 ~M.
This should be taken to mean that compounds that
require a higher molarlty to inhibit adhesion by
50 ~ are s~ill active and of interest hut are of
overall lesser interest because of the high dose
expected to be required when given in vivo to
humans. Compounds with activity below lO ~M are
most preferred, below lO0 ~M are not as
preferred, below 500 ~M lesser preferred and
above 500 ~M least preferred.
Thus an aspect of the present invention is
to provide compounds having extraordinarily hish
potencies in modulating cell adhesion, includinc
but not limited to inhibition of cell adhesion to
fibronectin. Data are also provided to show that
compounds not containing an RGD sequence are
effective inhibitors of adhesion to FN.
W094/l5958 ~ i5 ~ ~ 2 8 54 PCT/~94/0002
?.~su'.~ c- J~ka~ cth~li~ 5 ~ ~ C ' ~ -
ResuI tS 0. the nhlbition o~ uur~a_ cell
adhesion tO IL-l s.imulated endot:~e~a_ cells.
Actlvity (A) i~ this assay ls defined ar~ ar~ly
as an IC~o below 250 ~M; and inactivity (I) as IC50
> 250 ~M. As above, this does not mean that ~he
compounds wit an ICso > 250 ~M a~e ac~ually
inactive but rather they are not potent enough to
be as practical for human use as those wlth lower
IC50.
Thus an aspect of the present invention is
to provide compounds having extraordinarily high
potencies in modulating cell adhesion, including
but not limited to inhibition of T-cell adhesion
to endothelial cells. The exact receptors
involved in this interaction and the specific
receptors targeted by the test compounds include,
but are not limited to, ~4~ 4~7, on the
leukocyte and VCAM-1 on the endothelial cells.
Lastly, data are provided to show that compounds
not containing an RGD sequence are effective
inhibitors of cell-cell adhesion.
The following Table 3 shows results from
three of the above assays using data from various
compounds of the present invention.
Selected compounds in Table 3 are listed in
the Sequence Listing as follows: cmpd. 2, (SEQ.
ID. NO.:13); cmpd. 3, (SEQ. ID. NO. :5)i cmpds.
23, 33, 48, 49, 51, 54, 55, 62 and 92, (SEQ. ID.
NO.:4); cmpd. 29, (SEQ. ID. NO.:6); cmpd. 42,
(SEQ. ID. NO.:7); cmpd. 50, (SEQ. ID. NO. :8);
cmpds. 65 and 66, (SEQ. ID. NO.:9); cmpd. 67,
(SEQ. ID. NO.:10); cmpd. 68, (SEQ. ID. NO.:11);
cmpd. 69, (SEQ. ID. NO.:12); cmpd. 71, (SEQ. ID.
NO.:14); cmpd. 75, (SEQ. ID. NO. :15); cmpd. 77,
~SEQ. ID. NO. :16)i cmpds. 78 and 79, (SEQ. ID.
NO.:i7); cmpd. 80, (SEQ. ID. NO.:18); cmpc. 81,
WO9~/15958 2 1 5 3 2 2 8 PCT/~94/00026
(SEQ/~ ID. NO.:l9); cmpc. 8G, (SEQ. I~. ~.:20);
cmpd. 84, (SEQ. ID. NO.:21); cmpd. 85, (SEQ. ID.
NO.:22); cmpd. 86, (SEQ. ID. NO.:23); cmpd. 87,
(SEQ. ID. NO.:24)i cmpd. 89, ~SEQ. ID. NO.:25).
~, ~ PCT~18 94/ooo26
3 1. 03. 94
66 21!~322~
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SUBSTIT,JTE SHEET (RULE 26)
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SUBSTITUTE SHEET (RULE 26)
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SUBSTITLITE SHEET (RULE 26)
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SU8STITUTE SHEET (RULE 26)
~ WO9~/15958 21~ 3 2 2 ~ PCT/~94/00026
77
SEQUENCE LISTING
(1) GrNERAL INFORMATION:
(i) APPLICANT: Chang, Shiu-Lan N.
Carderelli, Pina M.
Lobl, Thomas J.
(ii) TITLE OF INVENTION: Peptide Inhibitors
of Cell Adhesion
(iii) NUMBER OF SEQUENCES: 28
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Birch, Stewart, Kolasch
& Birch
(B) STREET: P.O. Box 747
(C) CITY: Falls Church
(D) STATE: Virginia
(E) COUNTRY: USA
(F) ZIP: 22040-3487
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0,
Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/001,773
(B) FILING DATE: 08-JAN-1993
(C) CLASSIFICATION:
(viii~ ATTORNEY/AGENT INFORMATION:
(A) NAME: Murphy Jr., Gerald M.
(B) REGISTRATION NUMBER: 28,977
(C) REFERENCE/DOCKET NUM3ER: 485-103P
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 703-241-1300
(B) TELEFAX: 703-241-2848
(C) TELEX: 248345
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
WO 9~/15958 21 5 3 2 2 ~ PCT/IB9~/00026 ~IY
/ _
(ii- ) ~iYPC"'r:ETICAL. P~O
~ v ) r ~AGM ~ NT TYPE: internal
(ix) F-ATURE~
(A) N~E/KEY: Pep~iae
(B) LOCATION: 1..10
(D) OTHER INFORMATION: /label= peptide
/note= "Cell recognition site in
Fibronectin"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Gly Pro Glu Ile Leu Asp Val Pro Ser Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: 1..5
(D) OTHER INFORMATION: /label= peptide
/note= "alpha-4-beta-1 integrin
recognition
peptide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Glu Ile Leu Asp Val
1 5
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
~ ~O9~/l5958 215 3 2 ~ 8 PCT/~94/00026
(v) F~AGMENT TYPE: in~ernal
(ix) F r ATURE:
r (A) NAME/KEY: Pep.ide
(B) LOCATION: 1..6
(D) OTHER INFORMATION: /label= peptide
/note= "blocking peptide in
contacthypersensitivity
challenge experiment."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Gly Arg Gly Asp Ser Pro
1 5
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) ~ENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:
/label=modified_aa
/note= 'Iderivatized with 1-FCA,
Ada, GAC, DTC, Fmoc, 5-FINC,
CBO or sarcosine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION:
/label=modified_aa
/note= "thioproline"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "can be c-terminal
amidated, e.g. cmpd. 92
in Tables 1 and 3, or
c-terminal derivatized
with AMP as in claim 8."
WO9~ 958 PCT/~94/00026
74
(x-) SEOUE~C_ D~SCRIPTION: SEQ ID ~T~ 4
Cys Asp Xa~ Cys
'2) INFORMATION FOR SEQ I3 NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l.. 7
(D) OTHER INFORMATION: /label= peptide
/note= "cmpd. 3 in Tables l and
3"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Cys Arg Gly Asn Ser Pro Cys
l 5
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Peptide
(B) LOCATION: l.. 8
(D) OTHER INFORMATION: /label= peptide
/note= "cmpd. 29 in Tables l and
3."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Cys Gly Lys Gly Glu Ser Pro Cys
l 5
5,
~ WO9~/15958 215 3 ~ 2 8 PCT/~94/00026
,5
(2) INFCR~TION FOR SEQ IJ NO:7:
(i) SEQUENCE C~ARACTE~ISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
/label= modified aa
/note= "p-Cl phenylalanine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 6
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Cys Ala Gly Asp Phe Xaa Cys
l 5
;2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 7
(D) OTHER INFORMATION:
/label= modified aa
/note= "p-Cl phenylalanine~
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 8
(D) OTHER INFORMATION:
/label= modified_aa
WO94/15958 PCT/~94/00026
2~ 8. 7~
,~o~e= "thioproline"
(x-) SEQU~NCE DESCRIPTION: SEQ ID NO:8:
Cys Glv Arg Ala Gly Ala Phe Xaa Cvs
l 5
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:
/label= modified_aa
/note= "can be derivatized with
Anb"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Arg Ala Asp Xaa Asp
l 5
(2) INFORMATION FOR SEQ ID NO:l0:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: l
(D) OTHER INFORMATION:
/label= modified_aa
W094/l5958 ,7 21~ ~2 ~ ~ PCTl~94/00026
/note= "de~iva~ized wi~ b"
(ix) F_ATURE:
(A) NAME/KEY: Modi ied-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Arg Val Asp Xaa Asp
1 5
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Lys Ala Asp Xaa Asp
1 5
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
WO9~/15958 PCT/IB9~/00026
2 ~S ~ ' 7
jiabe'~= moaif.ed_a~
~note= "_hio~rQline"
(xi) SEQUENCE DESCRIPTTON: SEQ ID NO:12:
Gly Arg Ala Asp Xaa As~
1 5
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION:
/label= modified_aa
/note= "sarcosine"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Cys Arg Xaa Asp Ser Pro Cys
1 5
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:
/label= modified_aa
/note= "ornithine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: ~
WO9~/15958 215 3 ~ 2 ~ PCT/~94/00026
?~
;~) OTHER INFORMATION:
~label= modified_aa
/note= "thioproline"
(xi) S_QUENCE DESCRIPTION: SEQ ID NO:14:
- Xaa A_a Asp Xaa Asp
l 5
(2) INFORMATION FOR SEQ ID NO:l5:
(i) Sr.QUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Gly Arg Val Asp Xaa Asp
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: l
(D) OTHER INFORMATION:
/label= modified_aa
/note= "derivatized with Anb"
(ix) FEATURE:
(A) l~AME/KEY: Mod fied-site
WO9~/l5958 PCT/~94/00026
~) LOCATION: ~
(D) OTHER INFORMATION:
~labeI= moa ified_aa
/note= "~hioproline"
(xi) SEQUENCE DESCRI?~ION: SEQ ID NO:16:
Arg Phe Asp Xaa Asp
1 5
(2) INFORMATION FOR SEQ ID NO:17:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:
/label= modified_aa
/note= "derivatized with l-FCA"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label- modified_aa
/note= "can be thioproline, see
cmpd. 79"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Lys Ala Asp Pro Asp
1 5
WO94/15958 ~53~8 PCT/~94/00026
(2) INFOR~TION F3R SEQ 3 NO:18:
( ) C-QUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D~ OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Gly Arg Ala Leu Xaa Asp
(2) INFORMATION FOR SEQ ID.NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1
(D) OTHER INFORMATION:
/label= modified_aa
/note= "derivatized with Aib"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
WO91/15958 z~ 82 PCT/~9~/00026
(x-,~ S_~UENCr DESCRIP~IO~-: SE~ ID .~-~:l9:
Arg Ala Asp Xaa Asp
l 5
(2) INFORMATION FOR SEQ ID NO.20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/ÆY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Gly Arg Phe Asp Xaa Asp
(2) INFORMATION FOR SEQ ID NO:2l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3 .
(D) OTHER INFORMATION:
/label= modified_aa
/note= "3-Br tyrosine"
(ix) FEAiluRE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 5
(D) OTHER INFORMATION:
/label= modified_aa
/note= ''thioproline'
WO91/15958 Z1 5 3 2 ~8 PCT/IB91/00026
~3
(x ) C-U-~C~ ~ESC~TDTION: S~Q ID
G y Arg Tv~ As~ Xaa Asp
l 5
(2) INFORMATION FOR S r Q I3 NO:22:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) F~AGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION:
/label= modified_aa
/note= "3-Br tyrosine"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Gly Arg Tyr Xaa Cys
l 5
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 3
(D) OTHER INFORMATION:
/label= modified_aa
/no~e= "3-pyridyl alanine"
WO9~/15958 2~ C4 PCT/IB94/00026
~ix) F~A~F:
(A) NAME~-~EY: Modifieà-si~e
(~) LOCA~ION: 5
(3) CTHER IN~ORMATION:
~iabel= modified_aa
/nc~e= l~thioproline~
(xi) SEQUENCE DESCRIPTION: .~EQ I~ NO:23:
Gly Arg Ala Asp Xaa Asp
l 5
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: l
(D) OTHER INFORMATION:
/label= modified_aa
/note= "derivatized with AMBA"
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Arg Ala Asp Xaa Asp
l 5
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: pept ide
(v) FRAGMENT TYPE: intern,al
~ WO9~/15958 PCT/~94/00026
S5 2 1 ~ 3~
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(3) LOCATION: 4
(D) OTHER INFORMATION:
/labei= modified_aa
/no~e= "thioproline"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Gly Met Asp Xaa Asp
1 5
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: peptide
(B) LOCATION: 1...13
(D) OTHER INFORMATION: /label= peptide
/note= "CS-1 derived peptide"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Cys Leu His Pro Gly Glu Ile Leu Asp Val Pro
1 5 10
Ser Thr
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: pep~ide
(B) LOCATION: 1...13
WO9~/15958 PCT/IB94/00026
(~) C~ER I~FGRM~ ~N: ~Ia~e~= pep~
/rote= "C~-_ de~ived pep_ide"
~x~) SEQUENCE DES~R-PTICN: SEQ I3 NO:27:
Cys Leu His Gly Pro Ile Glu Leu Val Ser Asp
l 5 l0
Pro Thr
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: circular
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: l
(D) OTHER INFORMATION:
/label= modified_aa
/note= "derivatized with l-FCA
or acetylated."
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 4
(D) OTHER INFORMATION:
/label= modified_aa
/note= ~'c-terminal amidated."
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Cys Asp Val Cys
