CA2153228A1 - Peptide inhibitors of cell adhesion - Google Patents
Peptide inhibitors of cell adhesionInfo
- Publication number
- CA2153228A1 CA2153228A1 CA002153228A CA2153228A CA2153228A1 CA 2153228 A1 CA2153228 A1 CA 2153228A1 CA 002153228 A CA002153228 A CA 002153228A CA 2153228 A CA2153228 A CA 2153228A CA 2153228 A1 CA2153228 A1 CA 2153228A1
- Authority
- CA
- Canada
- Prior art keywords
- compound
- asp
- arg
- phe
- ala
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Abstract
Cyclized integrin receptor antagonists useful in modulating cell adhesion, related to fibronectin, as well as leukocyte adhesion to endothelial cells, are disclosed. Methods for synthesizing, testing, formulating, and using the compounds as therapeutic agents are also disclosed.
Description
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 (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 2 ~ 3 ~ æ ~ PCT/~94/00026 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 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 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
,~ 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).
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WO9/15958 21~ 3 2 ~ 8 PCT/IB94/00026 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.
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 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)).
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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SUBSTITUTE SHEET (RULE 26) PCTIIB ~ 4 / O O 0 2 3 ~. a3.94 21~32~
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o ~ ~3 ~ ~, S 5~ _ C ,_ SU8STITUTE SHEET (RULE 26) ~ WO9~/15958 21~ 3 2 2 ~ PCT/~94/00026 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 (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 (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 (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 (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 (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 (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:
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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~
(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 (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:
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/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 (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:
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/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 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:
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/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:
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(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:
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/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 (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 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
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 (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 2 ~ 3 ~ æ ~ PCT/~94/00026 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 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 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
,~ 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).
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WO9/15958 21~ 3 2 ~ 8 PCT/IB94/00026 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.
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 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)).
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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SUBSTITUTE SHEET (RULE 26) PCTIIB ~ 4 / O O 0 2 3 ~. a3.94 21~32~
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o ~ ~3 ~ ~, S 5~ _ C ,_ SU8STITUTE SHEET (RULE 26) ~ WO9~/15958 21~ 3 2 2 ~ PCT/~94/00026 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 (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 (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 (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 (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 (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 (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~
(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 (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 (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 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 (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 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
Claims (28)
What is claimed is:
1. A compound of the formula:
or a pharmaceutically acceptable salt thereof, wherein L1 and L2 are each selected from the group consisting of Cys, Pen, Mpr, AnB, AnC, .beta.-Ala, Lys, Orn, Dpr, Asp, Glu, a residue of an amino acid, an amino acid analog and an amino acid mimetic having a functional group suitable for the formation of a cyclizing bridge between L1 and L2;
Z is a cyclizing moiety or bond between L1 and L2;
1 is optional and, when present, is selected from the group consisting of Leu, Tyr, Phe, Ile, Pro, a Pro analog, Gly, Ala, Val, norLeu, norVal, Trp, D-Nal, Sar, (Ada)-Ala, AnC, AnB, Lys, .omega.-amino-lower alkyl carboxylic acid, Gly, Ala, Gly-Gly, Ala-Ala, AnC-AnC, AnB-AnB, .beta.-Ala, .beta.-Ala-.beta.-Ala, 1-Nal, TTC, TCA, DTC and MTC;
2 is optional and, when present, is selected from the group consisting of Arg, an Arg analog, Lys, a lysine analog, His, Ala, Gly, Sar, Leu, AnB, Phe, an analog of Phe, Pro, a Pro analog, TCA, TTC, DTC and Dpr;
3 is selected from the group consisting of Gly, Sar, Ala, an alanine analog, d-Ala, Ile, Val, d-Val, d-Nal, Phe, Lys, Arg, Asp, .beta.-Asp, Asn, AiB, AnB, Glu, Met, Leu, Tyr, 3Br-Tyr, cysteic acid, 3,5-dibromo-Tyr, and 3,5-diiodo-Tyr;
4 is selected from the group consisting of Asp, d-Asp, Glu, Asn, Gln, Fm-esters of Asp and Glu, other substituted alkyl, aryl and alkaryl esters of Asp and Glu, alkyl, aryl and alkaryl amides of Asn and Gln, Gly, Ala, Leu, Val, AnB, Phe, o, m, p-halo-Phe, p-nitro-Phe, Arg, Cys, TIC, Pro, and thiop;
5 is optional and, when present, is selected from the group consisting of Trp, Ala, Val, Phe, o, m, p-halo-Phe, p-nitro-Phe, p-fluoro-Phe, p-chloro-Phe, 3,5-dibromo-Tyr, p-methoxy-Tyr, , , , and where m is 2, 3 or 4;
6 is optional and, when present, is selected from Pro, d-Pro, thiop, 1,3-dithiazepine, 1,4-dithiazepine and 1,5-dithiazepine, a Pro analog, 1,1-ACC, Dhp, Hyp, homoPro, Phe, DTC, TTC, TC, MTC, TCA, o-halo-Phe, m-halo-Phe, p-halo-Phe, p-nitro-Phe, isonipecotic acid, N-methylalanine and TIC;
X1 is optional and, when present, is selected from the group consisting of sequences of from 1 to 4 D- or L-amino acids and amino acid analogs, Ada, AnC, AnB, CBO, .omega.-amino-lower alkyl-carboxylic acid and AMBA, lower alkyl, aromatic carboxylic acid, alkylaromatic carboxylic acid and SAR;
y1 is optional and, when present, is selected from the group consisting of sequences of from 1 to 4 D- or L-amino acids and amino acid analogs, Ada, AnC, AnB, CBO, .omega.-amino-lower alkyl-carboxylic acid, AMBA, aminomethylpyridine, lower alkyl amine, aromatic amine, alkylaromatic amine and SAR;
X2 is an optional N.alpha.-substituent R' or R'CO-, 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 containing linear carboxylic acids, adamantyl, fluorenyl, 1-FCA, 9-FCA, 9-FA, FMOC, Ada, Ada-CA, NAcA, 3-Me-Ada, (NB)-Ac, PhAc, Naph-Ac, HCA, QC, CPA, DTC, TCA, AMBA, a multi-ring aromatic carboxylic acid, a heteroaromatic carboxylic acid, acetic acid, QC, CPA, BOC, 5-FINC, and CBO;
Y2 is an optional carboxyl-terminal substituent selected from the group consisting of -OR', NHR', NR'NH2, NHNHR', -NR'2, -NHNH2, -SR', aminomethylpyridine, and an amino acid having the .alpha.-carboxylic acid moiety replaced by a tetrazole;
and wherein each R' is individually a pharmaceutically suitable substituent group, preferably one selected from the group consisting of hydrogen, linear and branched, unsubstituted and substituted C1-C8 lower alkyls, C2-C8 alkenyls, C2-C8 alkynyls, C6-C14 aryls, C7-C14 alkaryls, C7-C14 cycloalkaryls and C3-C14 cycloalkyls, and, in the case of -NR'2, from cyclized 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, with the provisos that (1) when 3 is Gly and when 2 is Arg or an analog of Arg, then 4 is not ASP, an analog of Asp, Glu or an analog of Glu;
(2) when 3 is Gly and 4 is Asp, an analog of Asp, Glu or an analog of Glu, then 2 is not Arg or an analog of Arg;
(3) when 1 and 2 are absent and 3 is Asp or Glu, X1 is not Arg or a derivative of Arg or an amino acid sequence having a carboxy-terminal Arg; and (4) the peptide has no sequence R-G-D.
or a pharmaceutically acceptable salt thereof, wherein L1 and L2 are each selected from the group consisting of Cys, Pen, Mpr, AnB, AnC, .beta.-Ala, Lys, Orn, Dpr, Asp, Glu, a residue of an amino acid, an amino acid analog and an amino acid mimetic having a functional group suitable for the formation of a cyclizing bridge between L1 and L2;
Z is a cyclizing moiety or bond between L1 and L2;
1 is optional and, when present, is selected from the group consisting of Leu, Tyr, Phe, Ile, Pro, a Pro analog, Gly, Ala, Val, norLeu, norVal, Trp, D-Nal, Sar, (Ada)-Ala, AnC, AnB, Lys, .omega.-amino-lower alkyl carboxylic acid, Gly, Ala, Gly-Gly, Ala-Ala, AnC-AnC, AnB-AnB, .beta.-Ala, .beta.-Ala-.beta.-Ala, 1-Nal, TTC, TCA, DTC and MTC;
2 is optional and, when present, is selected from the group consisting of Arg, an Arg analog, Lys, a lysine analog, His, Ala, Gly, Sar, Leu, AnB, Phe, an analog of Phe, Pro, a Pro analog, TCA, TTC, DTC and Dpr;
3 is selected from the group consisting of Gly, Sar, Ala, an alanine analog, d-Ala, Ile, Val, d-Val, d-Nal, Phe, Lys, Arg, Asp, .beta.-Asp, Asn, AiB, AnB, Glu, Met, Leu, Tyr, 3Br-Tyr, cysteic acid, 3,5-dibromo-Tyr, and 3,5-diiodo-Tyr;
4 is selected from the group consisting of Asp, d-Asp, Glu, Asn, Gln, Fm-esters of Asp and Glu, other substituted alkyl, aryl and alkaryl esters of Asp and Glu, alkyl, aryl and alkaryl amides of Asn and Gln, Gly, Ala, Leu, Val, AnB, Phe, o, m, p-halo-Phe, p-nitro-Phe, Arg, Cys, TIC, Pro, and thiop;
5 is optional and, when present, is selected from the group consisting of Trp, Ala, Val, Phe, o, m, p-halo-Phe, p-nitro-Phe, p-fluoro-Phe, p-chloro-Phe, 3,5-dibromo-Tyr, p-methoxy-Tyr, , , , and where m is 2, 3 or 4;
6 is optional and, when present, is selected from Pro, d-Pro, thiop, 1,3-dithiazepine, 1,4-dithiazepine and 1,5-dithiazepine, a Pro analog, 1,1-ACC, Dhp, Hyp, homoPro, Phe, DTC, TTC, TC, MTC, TCA, o-halo-Phe, m-halo-Phe, p-halo-Phe, p-nitro-Phe, isonipecotic acid, N-methylalanine and TIC;
X1 is optional and, when present, is selected from the group consisting of sequences of from 1 to 4 D- or L-amino acids and amino acid analogs, Ada, AnC, AnB, CBO, .omega.-amino-lower alkyl-carboxylic acid and AMBA, lower alkyl, aromatic carboxylic acid, alkylaromatic carboxylic acid and SAR;
y1 is optional and, when present, is selected from the group consisting of sequences of from 1 to 4 D- or L-amino acids and amino acid analogs, Ada, AnC, AnB, CBO, .omega.-amino-lower alkyl-carboxylic acid, AMBA, aminomethylpyridine, lower alkyl amine, aromatic amine, alkylaromatic amine and SAR;
X2 is an optional N.alpha.-substituent R' or R'CO-, 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 containing linear carboxylic acids, adamantyl, fluorenyl, 1-FCA, 9-FCA, 9-FA, FMOC, Ada, Ada-CA, NAcA, 3-Me-Ada, (NB)-Ac, PhAc, Naph-Ac, HCA, QC, CPA, DTC, TCA, AMBA, a multi-ring aromatic carboxylic acid, a heteroaromatic carboxylic acid, acetic acid, QC, CPA, BOC, 5-FINC, and CBO;
Y2 is an optional carboxyl-terminal substituent selected from the group consisting of -OR', NHR', NR'NH2, NHNHR', -NR'2, -NHNH2, -SR', aminomethylpyridine, and an amino acid having the .alpha.-carboxylic acid moiety replaced by a tetrazole;
and wherein each R' is individually a pharmaceutically suitable substituent group, preferably one selected from the group consisting of hydrogen, linear and branched, unsubstituted and substituted C1-C8 lower alkyls, C2-C8 alkenyls, C2-C8 alkynyls, C6-C14 aryls, C7-C14 alkaryls, C7-C14 cycloalkaryls and C3-C14 cycloalkyls, and, in the case of -NR'2, from cyclized 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, with the provisos that (1) when 3 is Gly and when 2 is Arg or an analog of Arg, then 4 is not ASP, an analog of Asp, Glu or an analog of Glu;
(2) when 3 is Gly and 4 is Asp, an analog of Asp, Glu or an analog of Glu, then 2 is not Arg or an analog of Arg;
(3) when 1 and 2 are absent and 3 is Asp or Glu, X1 is not Arg or a derivative of Arg or an amino acid sequence having a carboxy-terminal Arg; and (4) the peptide has no sequence R-G-D.
2. The compound of claim 1, wherein L1 and L2 are both Cys, and cyclization is achieved through a disulfide bond, lanthionine-like monosulfide, dithioether, or other linkage completing the cyclizing bridge.
3. A compound of claim 1, wherein said compound has a structure selected from the group consisting of:
, , , , , , and
, , , , , , and
4. A compound of claim 1, wherein said compound has a structure selected from the group consisting of:
, , , and .
, , , and .
5. A compound of the formula , wherein X1, X2, 1, 2, L1, 4, 5, 6, L2, y1, and y2 are defined as in claim 1;
with the proviso that (1) if 1 and 2 are absent and 4 is Asp or Glu, then X1 is not Arg or a derivative of Arg or an amino acid sequence having a carboxy-terminal Arg; and (2) if 2 is Arg, then 4 is not Asp or Glu.
with the proviso that (1) if 1 and 2 are absent and 4 is Asp or Glu, then X1 is not Arg or a derivative of Arg or an amino acid sequence having a carboxy-terminal Arg; and (2) if 2 is Arg, then 4 is not Asp or Glu.
6. A compound of the formula wherein X2 is a protecting group and 6 is Pro or a derivative of Pro.
7. The compound according to claim 6 which has the formula or
8. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier.
9. A pharmaceutical composition comprising the compound of claim 5 and a pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising the compound of claim 6 and a pharmaceutically acceptable carrier.
11. Use of the compound of any one of claims 1-7 in a composition for the prevention or treatment of a disease resulting from inappropriate cell adhesion to extracellular matrix, wherein said compound is present in an amount per dose that is effective in restoring appropriate cell adhesion to extracellular matrix.
12. Use of the compound of any one of claims 1-7 in a composition for the restoration of normal adhesion of cells to extracellular matrix, wherein said compound is present in an amount per dose providing 0.1 to 100 mg/kg in a subject.
13. Use of the compound of any one of claims 1-7 in a composition for inhibiting adhesion of leukocytes to extracellular matrix, wherein said compound is present in an amount per dose effective for inhibiting adhesion of leukocytes to extracellular matrix.
14. An antibody which specifically binds to the compound of claim 1.
15. Use of a compound of any one of claims 1-7 in a method of diagnosing disease resulting from the presence of antibodies which bind to endothelial cells.
16. Use of a compound of any one of claims 1-7 in a method of diagnosing disease resulting from the presence of antibodies which bind to extracellular matrix.
17. Use of a compound of any one of claims 1-7 in a method for diagnosing disease resulting from excessive adhesion of cells to endothelial cells.
18. Use of a compound of any one of claims 1-7 in a method for producing a biocompatible surface for a prosthetic device, wherein said compound is applied to said device prior to implantation of said device.
19. Use of the compound of any one of claims 1-7 in a composition for treating a disease selected from the group consisting of rheumatoid arthritis, asthma, allergies, adult respiratory distress syndrome, 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, 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, ulcerative colitis, regional enteritis, and other autoimmune diseases, wherein said compound is present in said composition in an amount effective for treating said disease.
20. Use of the compound of any one of claims 1-7 in a composition for contraception, inhibition of fertilization, inhibition of sperm maturation, or inhibition of sperm capacitation, wherein said compound is present in an amount effective for inhibiting fertilization, inhibiting sperm maturation or inhibiting sperm capacitation.
21. Use of a compound of claim 1 in a matrix for the purification of macromolecules having affinity for a compound of claim 1.
22. Use of a compound of claim 1 in a method for purifying macromolecules which specifically bind with high affinity to a compound of claim 1.
23. Use of the compound of any one of claims 1-7 in a composition for inhibiting adhesion of leukocytes to extracellular matrix, wherein said compound is present in an amount per dose providing 0.1 to 100 mg/kg in a subject.
24. The use of claim 19, wherein said compound is present in an amount per dose providing 0.1 to 100 mg/kg in a subject.
25. The use of claim 20, wherein said compound is present in an amount per dose providing 0.1 to 100 mg/kg in a subject.
26. The compound of claim 1, wherein Z is a bifunctional linking group selected from the set consisting of diketo-, diamino- and heterobifunctional linking groups.
27. A compound of claim 5, having the structure or .
28. A compound of claim 1 , having the structure
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US177393A | 1993-01-08 | 1993-01-08 | |
US08/001,773 | 1993-01-08 | ||
CN94115995A CN1114961A (en) | 1993-01-08 | 1994-07-11 | Peptide inhibitors of cell adhesion |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2153228A1 true CA2153228A1 (en) | 1994-07-21 |
Family
ID=37075853
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002153228A Abandoned CA2153228A1 (en) | 1993-01-08 | 1994-01-07 | Peptide inhibitors of cell adhesion |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP0677060A1 (en) |
JP (1) | JPH08505628A (en) |
CN (1) | CN1114961A (en) |
CA (1) | CA2153228A1 (en) |
SG (1) | SG52262A1 (en) |
WO (1) | WO1994015958A2 (en) |
Families Citing this family (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ263455A (en) * | 1993-03-29 | 1997-10-24 | Du Pont Merck Pharma | Synthesis of platelet glycoprotein iib/iiia inhibitors |
US5811391A (en) * | 1994-08-25 | 1998-09-22 | Cytel Corporation | Cyclic CS-1 peptidomimetics, compositions and methods of using same |
US6056958A (en) * | 1994-12-09 | 2000-05-02 | Dupont Pharmaceuticals | Method of treatment of arterial and venous thromboembolic disorders |
GB9524630D0 (en) * | 1994-12-24 | 1996-01-31 | Zeneca Ltd | Chemical compounds |
US7001921B1 (en) | 1995-01-23 | 2006-02-21 | Biogen Idec Ma Inc. | Cell adhesion inhibitors |
US6306840B1 (en) | 1995-01-23 | 2001-10-23 | Biogen, Inc. | Cell adhesion inhibitors |
US5821329A (en) * | 1996-06-06 | 1998-10-13 | Tanabe Seiyaku Co., Ltd. | Cyclic peptide inhibitors of β1 and β2 integrin-mediated adhesion |
WO1996040781A1 (en) * | 1995-06-07 | 1996-12-19 | Tanabe Seiyaku Co., Ltd. | CYCLIC PEPTIDE INHIBITORS OF β1 AND β2 INTEGRIN-MEDIATED ADHESION |
AU6311996A (en) * | 1995-07-06 | 1997-02-05 | Zeneca Limited | Peptide inhibitors of fibronectine |
US6248713B1 (en) | 1995-07-11 | 2001-06-19 | Biogen, Inc. | Cell adhesion inhibitors |
US6037324A (en) * | 1996-01-04 | 2000-03-14 | Leukosite, Inc. | Inhibitors of MAdCAM-1-mediated interactions and methods of use therefor |
GB9613112D0 (en) | 1996-06-21 | 1996-08-28 | Zeneca Ltd | Chemical compounds |
US6239108B1 (en) | 1996-07-11 | 2001-05-29 | Biogen, Inc. | Cell adhesion inhibitors |
US6686350B1 (en) | 1996-07-25 | 2004-02-03 | Biogen, Inc. | Cell adhesion inhibitors |
EP0914605B1 (en) | 1996-07-25 | 2007-05-30 | Biogen Idec MA Inc. | Molecular model for vla-4 inhibitors |
DE19647382A1 (en) | 1996-11-15 | 1998-05-20 | Hoechst Ag | Heterocycles as inhibitors of leukocyte adhesion and VLA-4 antagonists |
DE19647381A1 (en) | 1996-11-15 | 1998-05-20 | Hoechst Ag | New heterocycles as leukocyte adhesion inhibitors and VLA-4 antagonists |
EP1015479A4 (en) * | 1997-04-11 | 2002-07-24 | Lilly Co Eli | Combinatorial libraries of peptidomimetic macrocycles and processes therefor |
DE19741235A1 (en) | 1997-09-18 | 1999-03-25 | Hoechst Marion Roussel De Gmbh | Novel imidazolidine derivatives, their preparation, their use and pharmaceutical compositions containing them |
DE19741873A1 (en) * | 1997-09-23 | 1999-03-25 | Hoechst Marion Roussel De Gmbh | New 5-ring heterocycles, their preparation, their use and pharmaceutical preparations containing them |
EP0970965A4 (en) | 1997-11-13 | 2002-07-17 | Toray Industries | Cyclic peptides and medicinal use thereof |
DE19751251A1 (en) | 1997-11-19 | 1999-05-20 | Hoechst Marion Roussel De Gmbh | Substituted imidazolidine derivatives, their manufacture, their use and pharmaceutical preparations containing them |
DE19821483A1 (en) | 1998-05-14 | 1999-11-18 | Hoechst Marion Roussel De Gmbh | New imidazolidine derivatives useful as leukocyte adhesion and migration inhibitors and/or VLA-4 receptor antagonists for treating E.G. inflammatory and allergic disorders |
CZ298413B6 (en) | 1998-05-28 | 2007-09-26 | Biogen Idec Ma Inc. | Beta-alanine derivative, pharmaceutical composition in which the derivative is comprised and use of the derivative for the preparation of a medicament |
WO1999065944A1 (en) * | 1998-06-15 | 1999-12-23 | Ixsys Incorporated | PEPTIDE INHIBITORS OF αVβ3 AND αVβ¿5? |
PL203114B1 (en) | 1998-09-14 | 2009-08-31 | Regents Board Of | Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists |
US6608027B1 (en) | 1999-04-06 | 2003-08-19 | Boehringer Ingelheim (Canada) Ltd | Macrocyclic peptides active against the hepatitis C virus |
DE19922462A1 (en) | 1999-05-17 | 2000-11-23 | Aventis Pharma Gmbh | New phenylureidobenzyl-substituted spiro-imidazolidinedione derivatives, are inhibitors of leukocyte adhesion or migration or VLA-4 receptors, useful e.g. for treating inflammatory or allergic disease |
EP1741428A3 (en) | 1999-08-13 | 2007-05-09 | Biogen Idec MA, Inc. | Cell adhesion inhibitors |
ES2415708T3 (en) | 1999-12-16 | 2013-07-26 | Biogen Idec Ma Inc. | Methods of treatment of ischemic or hemorrhagic lesion of the central nervous system through the use of anti-integrin alpha4 antagonists |
US6875743B1 (en) | 2000-11-28 | 2005-04-05 | Biogen, Inc. | Cell adhesion inhibitors |
DE10111877A1 (en) | 2001-03-10 | 2002-09-12 | Aventis Pharma Gmbh | Novel imidazolidine derivatives, their preparation, their use and pharmaceutical compositions containing them |
DE10137595A1 (en) | 2001-08-01 | 2003-02-13 | Aventis Pharma Gmbh | New 3-alkylaminoalkyl-imdazolidin-4-one derivatives, are VLA-4 receptor and leukocyte adhesion and/or migration inhibitors, useful e.g. for treating inflammatory, allergic, autoimmune or cardiovascular disease |
WO2003070709A1 (en) | 2002-02-20 | 2003-08-28 | Ajinomoto Co.,Inc. | Novel phenylalanine derivative |
US7570871B2 (en) | 2002-11-12 | 2009-08-04 | Koninklijke Philips Electronics N.V. | Continue recording channel feature for personal video recorder |
US7642235B2 (en) | 2003-09-22 | 2010-01-05 | Boehringer Ingelheim International Gmbh | Macrocyclic peptides active against the hepatitis C virus |
BRPI0418026A (en) | 2003-12-22 | 2007-04-17 | Ajinomoto Kk | phenylalanine derivatives or pharmaceutically acceptable salts thereof, pharmaceutical composition, alpha4 integrin antagonist, and therapeutic or preventative agent for diseases |
EP1730167B1 (en) | 2004-01-21 | 2011-01-12 | Boehringer Ingelheim International GmbH | Macrocyclic peptides active against the hepatitis c virus |
US7196112B2 (en) | 2004-07-16 | 2007-03-27 | Biogen Idec Ma Inc. | Cell adhesion inhibitors |
WO2008103378A2 (en) | 2007-02-20 | 2008-08-28 | Merrimack Pharmaceuticals, Inc. | Methods of treating multiple sclerosis by administration of alpha-fetoprotein in combination with an integrin antagonist |
CN102057054B (en) | 2008-04-11 | 2015-06-10 | 梅里麦克制药股份有限公司 | Human serum albumin linkers and conjugates thereof |
EP2671889B1 (en) | 2010-04-02 | 2018-09-12 | The Regents Of The University Of Michigan | RF amide-related peptides and methods thereof |
WO2018140510A1 (en) | 2017-01-25 | 2018-08-02 | Biogen Ma Inc. | Compositions and methods for treatment of stroke and other cns disorders |
CN110423265A (en) * | 2019-04-03 | 2019-11-08 | 中国药科大学 | A kind of preparation method and purposes of cyclic peptide TRF2 inhibitor |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5306620A (en) * | 1987-07-08 | 1994-04-26 | The Scripps Research Institute | Antibodies that bind to a ligand-induced binding site on integrin and induce integrin activation |
WO1992008476A1 (en) * | 1990-11-07 | 1992-05-29 | The Scripps Research Institute | Peptides that inhibit platelet binding of adhesion molecules |
CA2078817A1 (en) * | 1991-10-18 | 1993-04-19 | Beat A. Imhof | Anti-.alpha.6-integrin-antibodies |
US5578704A (en) * | 1992-04-03 | 1996-11-26 | Genentech, Inc. | Antibody to osteoclast alphavbeta3 ntegrin |
US5225531A (en) * | 1992-04-09 | 1993-07-06 | Washington University | Hexapeptide Lys Gly Ala Gly Asp Val |
-
1994
- 1994-01-07 WO PCT/IB1994/000026 patent/WO1994015958A2/en not_active Application Discontinuation
- 1994-01-07 CA CA002153228A patent/CA2153228A1/en not_active Abandoned
- 1994-01-07 EP EP94906984A patent/EP0677060A1/en not_active Withdrawn
- 1994-01-07 JP JP6516421A patent/JPH08505628A/en active Pending
- 1994-01-07 SG SG1996001553A patent/SG52262A1/en unknown
- 1994-07-11 CN CN94115995A patent/CN1114961A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
CN1114961A (en) | 1996-01-17 |
EP0677060A1 (en) | 1995-10-18 |
JPH08505628A (en) | 1996-06-18 |
SG52262A1 (en) | 1998-09-28 |
WO1994015958A2 (en) | 1994-07-21 |
WO1994015958A3 (en) | 1994-09-29 |
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