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EP0677060A1 - Die Zell-Adhäsion hemmende Peptide - Google Patents

Die Zell-Adhäsion hemmende Peptide

Info

Publication number
EP0677060A1
EP0677060A1 EP94906984A EP94906984A EP0677060A1 EP 0677060 A1 EP0677060 A1 EP 0677060A1 EP 94906984 A EP94906984 A EP 94906984A EP 94906984 A EP94906984 A EP 94906984A EP 0677060 A1 EP0677060 A1 EP 0677060A1
Authority
EP
European Patent Office
Prior art keywords
cys
thiop
compound
asp
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.)
Withdrawn
Application number
EP94906984A
Other languages
English (en)
French (fr)
Inventor
Shiu-Lang Ng Chiang
Pina M. Cardarelli
Thomas J. Lobl
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tanabe Seiyaku Co Ltd
Original Assignee
Tanabe Seiyaku Co Ltd
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Filing date
Publication date
Application filed by Tanabe Seiyaku Co Ltd filed Critical Tanabe Seiyaku Co Ltd
Publication of EP0677060A1 publication Critical patent/EP0677060A1/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/02—Linear peptides containing at least one abnormal peptide link
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel cyclic peptides and peptidomimetic compounds which are characterized by cell adhesion modulation activity.
  • the extracellular matrix is the major component of connective tissue which provides for structural integrity, promotes cell migration and differentiation. As 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 including hemostasis, thrombosis, wound healing, tumor metastasis, immunity and inflammation.
  • Fibronectin 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 cr promote ceil adhesion have been described (US Patent Nos. 4 , 589, 881;4 , 661, 111; 4,517,686; 4,683,291; 4,578,079; 4,614,517; and 4,792,525) .
  • the receptors which recognize these sites on FN belong to a gene superfamily called integrins which consist of heterodimeric complexes of non-covalently associated alpha and beta subunits.
  • a common ⁇ subunit combines with unique ⁇ ; subunits to form an adhesion receptor of defined specificity. Eight beta subunits have been cloned and sequenced to date.
  • the ⁇ l subfamily also known as the VLA family (Very Late Activation Antigens) , binds to ECM molecules such as FN, collagen and laminin.
  • Vascular endothelial 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 endothelial cells as well as circulating leukocytes so that they become more adhesive to one another.
  • the leukocyces migrate into the tissues to perform host defense functions .
  • adhesion molecules have been identified which are involved in leukocyte-- endothelial interactions.
  • 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.
  • VCAM vascular cell adhesion molecule
  • ⁇ 40l and ⁇ .5/31 are important receptor targets for control cf inflammatory diseases.
  • 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, laminin, 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 enhancing 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 .
  • Another objective of the present invention is to provide compounds which bind to a cellular adhesion molecule or integrin receptor.
  • 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.
  • the present invention includes compounds having an IC 50 of less than about 500 ⁇ M as established in a U937-fibronectin adhesion assay; and in another regard, the invention includes compounds having an
  • 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 IC 50 of less than about 500 ⁇ M, or alternatively less than about 100 ⁇ M.
  • the present invention includes compounds having an IC 50 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 IC 50 of less than about 10 ⁇ M in such assay.
  • Compounds with activity below 10 ⁇ 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 vi tro or in vivo) such leukocyte receptor adhesion inhibition.
  • the compounds of the present invention accomplish strong inhibition at low concentrations, with an IC 50 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 ⁇ compound is resistant tc degradation in vi vo due to inclusion of peptidomimetic residues, modified amino acids or D-isomers of amino acids.
  • Another objective of the present invention is to provide novel compounds, formulations, and methods which 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
  • the cell adhesion protein fibronectin (FN) has been implicated in the binding of capacitated sperm to oocytes
  • 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.
  • Another objective of the present invention is to provide 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.
  • 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 family of platelet adhesive giycoproteins, fibrinogen, fibronectin, and von Willebrand factor.
  • Fibrinogen 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, 12:8057 (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.
  • 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.
  • 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.
  • the cell adhesion modulation compounds of the present invention are represented in part by amino acid sequence formulas wherein the individual amino acids are represented by their standard three-letter, or alternatively, one-letter abbreviations.
  • DMEM Dulbecco's Modified Eagle's Medium
  • 9-FCA 9-fluorenecarboxylic acid
  • 9-FA -fluoreneacetic acid
  • GAC Guanidine-acetic acid
  • Hyp 4-Hydroxyproline ICAM-1: Intercellular adhesion molecule 1
  • IC 50 Inhibitory concentration, concentration at which adhesion is inhibited to 50% of control level
  • Isonipecotic acid 4-piperidinecarboxylic acid 3-Me-Ada: 3-Methyl-1-adamantaneacetic acid mono MeR: N-methyl-arginine Mpr: 3-Mercaptopropionic acid
  • Nle Norleucine [ (N-Me)R] :N-methyl arginine norAda-CA: 3-Noradamantanecarboxylic acid norArg: Norarginine
  • PhAc Phenylacetic acid
  • PMP 1- (/3-Mercapto- ⁇ , ?- cyclopentamethylene)propinonic acid
  • PyE Pyroglutamic acid
  • pyroGlu Pyroglutamic acid
  • Sar Sarcosine SLE: Systemic lupus erythematosus
  • 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.
  • 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.
  • arginine analog is a compound which consists of an arginine backbone and substituents appended thereto.
  • 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 ⁇ , y 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 pentafluoro-Phe .
  • Di-substituted analogs e.g. dichlorophenylalanine, o,m-dimethylphenylalanine and the like are encompassed as phenylalanine analogs, as are heterodisubstituted analogs, e.g. o-methyl-m-chloro-phenylalanine .
  • 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 .
  • Protein 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.
  • esters of the ⁇ -carboxylic acid function of these amino acids include esters of the ⁇ -carboxylic acid function of these amino acids.
  • Lysine analogs include amides of the a- amino group and of the e-amino group and alkyl derivatives of the ⁇ -amino group and the e-amino group. Also encompassed as lysine analogs are
  • the present invention is directed to a compound of the formula X 2 - (X 1 ) -L 1 - l - 2 - 3 - 4 - 5 - 6 -L : ( -Y 1 ) -Y :
  • ⁇ _HN represents an amino group of the amino terminal amino acid in a sequence.
  • Side chain functional groups are indicated the structural representations in parenthesis.
  • L 1 and L 2 are chosen so that each contains a functional group which contributes to the formation of the cyclizing bridge moiety Z.
  • Z is formed from functional groups contributed by L 1 and L 2 and may also contain additional atoms and spacer groups.
  • 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 L 1 ) or o;-carboxy group (in L 2 ) thereof.
  • 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.
  • the bridging residues L 1 and L 2 are each selected from the residues Cys, Pen, and homoC.
  • additional preferred residues are Mpr and PMP. All of these residues contain a sulfhydryl group.
  • an additional preferred residue is mercapto-ethyiamine (MEA) . If MEA is used, Y 1 and Y : is absent.
  • MEA mercapto-ethyiamine
  • Y 1 and Y is absent.
  • the bridging cyclization can be accomplished by oxidative coupling of the sulfhydryls to form a disulfide bond between residues L 1 and L 2 .
  • 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 L 1 and L 2 are Cys residues as follows:
  • L 1 is Cys or Mpr and L 2 is Cys .
  • the cyclizing bridge may also be formed by a hydrocarbon moiety, for example a
  • L 1 and L 2 may be chosen from other amino acids or analogs 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.
  • L 2 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 L 1 , provided, however, that the structure
  • the cyclizing moiety Z will in such cases be a simple bond between L 1 and L 2 .
  • an amino acid residue L 2 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 L 1 ; or the direction of the amide linkage may be reversed where L 1 provides a side chain carboxyl group and L 2 provides a side chain amino group.
  • Such structures may be exemplified as follows:
  • n is as defined above, may also be used.
  • Diketo linkers can be used to join, for example, the e-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
  • cyclizing moiety Z includes a 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 or more methylene moieties) , also include one cr more heteroatcm-containing substituents including hydroxyl, amino, nitro, alkoxyl and halo substituents. Such substituents may be used to affect the solubility and/cr biodistribution characteristics of the subject compounds.
  • Aromatic or cycloalkyl hydrocarbon- containing bridge groups may also be utilized in the Z position, as for example diketo or diamino structures such as
  • Simple hydrocarbon moieties of from 1 to about 4 carbons are preferred for hydrocarbon portions of Z-moieties.
  • bridging moiety 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.
  • a bridging moiety may employ as a bridging moiety a structure which includes many of the elements previously discussed, as drawn below:
  • the cyclizing bridge between L 1 and L 2 can also be formed via a monosulfide (thioether) linkage, as exemplified below.
  • thioether monosulfide linkage
  • One method for making such a linkage is to use cysteine at L 1 or L 2 and to use a residue providing a bromo-acetic
  • L 1 can be an , ⁇ dehydroalanine and L 2 can be a cysteine residue. Reaction of the two yields a lanthionine-like thioether linkage.
  • the cyclizing bridge between L 1 and L 2 may also be formed via a monosulfide (thioether). linkage, as exemplified below.
  • Analogs of amino acid residues may also be utilized for L 1 and/or L 2 , as for example homologs
  • Amino acid-mimetic structures that are capable of bein ⁇ covalentlv bonded throu ⁇ h an amide bond to a carboxyl and/or amino terminus of the residue sequence 1-2-3-4-5-6, and which provide a suitable precursor functional group for cyciization (through Z) , may also be employed in positions L 1 and/or L 2 .
  • Such amino acid-mimetic structures include organic species containing one or more heteroatoms including at least one functional group (preferably a heteroatom- containing functional group) which can participate in cyciization. Examples include residues of the form
  • n ranges from 1 to about 8, and preferably from 1 to 4, as for example residues of /3-alanine and gamma-aminobutyric acid.
  • n is 1, the amino acid glycine, rather than an - amino acid mimetic, results.
  • Such a structure may, similar to the amino acids and amino acid analogs discussed above, be utilized as L 2 (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 L 1 (wherein the depicted amino group may engage in an amide linkage with the carboxyl terminus of the terminal residue 4, 5 or 6) .
  • linking residue L may serve as both L 1 and L 2 (and thereby include Z) in that cyciization 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 the exemplifying form 0
  • 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.
  • cyciization can be achieved with such an amino acid mimetic-linking moiety wherein a side chain functional group on a second linking moiety appended (as L 1 or L 2 ) 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 L 2 or L 1 ) cyclizes the compound to the remaining terminal residue of the numbered sequence.
  • a side chain functional group on a second linking moiety appended (as L 1 or L 2 ) 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 L 2 or L 1 ) cyclizes the compound to the remaining terminal residue of the numbered sequence.
  • L 2 (as for example Asp) provides the side chain carbonyl group depicted in parenthesis
  • residue 1 provides the depicted N ⁇ -terminal amino group
  • Amino acid mimetic structures containing aromatic, cycloalkyl or other linking portions can also be utilized as L 1 and/or L 2 , such as structures of the form
  • heterobifunctional (keto-amino) structures depicted above may also serve as a Z- group in linking complementary side chain functional groups on L 1 and L 2 (e.g., a side chain amino group on L 1 and a side chain carboxyl group on L 2 ) through two amide bond structures .
  • Residue 1 in structure I is most preferably absent; residue 2 is most preferably Arg; residue 3 is most preferably Ala; residue 4 is most preferably Asp; residue 5 is most preferably 3-thioproline
  • residue 6 is preferably absent.
  • the sequence Arg-Ala-Asp- (thiop) (SEQ- ID. NO. : ) , residues 1-4) is most preferred for residues 1-2- 3-4-5-6.
  • residue 1 is absent
  • residue 2 is absent
  • residue 3 is Asp
  • residue 4 is (thiop)
  • residues 5 and 6 are both absent.
  • sequence Asp- (thiop) is also preferred for residues 1-2-3-4-5-6.
  • a third particularly preferred sequence is one in which X 1 is Gly, residue 1 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 sequence is one in which X 2 is (1-FCA) , X 1 and residues 1 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 X 2 is Fmoc, X 1 is Arg, residues 1 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 X 2 is absent, X 1 is Arg, residues 1 and 2 are both absent, residue 3 is Ala, residue
  • a seventh particularly preferred compound is one wherein X 1 and X 2 are absent, residue 1 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 1 is absent, residue 2 is Arg, residue 3 is Ala, residue 4 is Leu, residue
  • X 1 and Y 1 are each optional in structure I.
  • d-amino acids are believed to stabilize the compound against metabolism by proteolytic or other enzymes in the body.
  • residues for position X 1 include Gly, Phe, Leu, Asn, Val, Tyr, Ala, Arg, His, 1- or 2-naphthylalanine, cyclohexyl-Ala- , AMBA, AnC, AnB and b-amino-lower alkyl carboxylic acids, Aib- , Ser-Tyr-Asn-, Ala-Thr-Val- , and p- chloro-Phe-.
  • Preferred residues for position Y 1 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, fo- amino-lower alkyl carboxylic acids, 1- or 2- naphythlalanine, and - (cyclohexyl)Ala.
  • Such X 1 and Y 1 groups are preferred also in the corresponding positions given in the structural formulas described hereinafter.
  • substituent X 2 or Y 2 incorporating R' other than hydrogen is used, e.g., for X 2 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 acid) are preferred.
  • X 2 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 acid) are preferred.
  • Y 2 including amino groups of the form R'NH especially lower alkyl amines are especially preferred.
  • Additional preferred substituents for X 2 include those derived from bulky compounds such as adamantaneacetic acid, adamantanecarboxylic acid, 1- or 2-naphthylac ⁇ tic aci ⁇ , _-r.crccmaneacetic aci ⁇ , J -nora ⁇ amantane- carboxyiic acid, 3-methyladamataneacet ⁇ c acid.
  • Additional preferred substituents for Y 2 include lower alkyl amines, aryl amines, 1- or 2- adamantylamin ⁇ and amino acids having the a- 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 C : - C s lower alkyls, C 2 -C 8 alkenyls, C 2 - C g alkynyls, C 6 -C 14 aryls, C 7 -C 14 alkaryls, C 7 -C 14 cycloalkaryls and C 3 -C 14 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, formic acid, acetic acid, heterocyclic carboxylic acids, aryl carboxylic acids, heteroaromatic carboxylic acids, alkyl carboxylic acids, alkenyl carboxylic acids, alkynyl carboxylic acids, other mixed
  • 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.
  • R' is preferably selected from hydrogen and through C 8 lower alkyls, particularly methyl and ethyl alkyl moieties.
  • a particularly preferred compound within the scope of structure I includes :
  • residue number 1 is absent or Leu; residue 2 is Arg; residue L 1 is Cys; residue 4 is Asp; residue
  • residue 6 is absent, Pro or
  • residues L 1 and L 2 other than Cys, and Z groups other than simple bonds may also be usefully employed. It will be seen in this regard that the presence of residues on either side of L 1 will typically require cyciization (through Z) to L 2 through a side chain or other functional group on L 1 that is not engaged in bonding to the adjacent residues. Residue L 2 may more generally be engaged in cyciization through either a terminal (typically, carboxyl) functional group or a side chain functional group.
  • Preferred residues for position X 1 include Gly-, Phe-, Leu-, Asn- , Val-, Tyr, 1- or 2- naphthylalanine, cyclohexylAla- , AMBA, AnC, AnB, fo-amino lower alkylcarboxylic acids, Aib-, Ser- Tyr-Asn-, Ala-Thr-Val- , and p-chloro-Phe- .
  • Preferred residues for position Y 1 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, b-amino-lower alkyl carboxylic acids, 1- or 2-naphthylalanine, and - (cyclohexylAla) .
  • substituent X 2 or Y 2 incorporating R' other than hydrogen 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 X 2 , and 1- or 2-adamantylamine for Y 2 .
  • R' groups are those derived from lower alkyl amines, lower arylamines or acids such as from 9-fluoreneacetic acid, 1-fluorenecarboxylic acid, 4 - fluorenecarboxylic acid, 2- fluorenecarboxylic acid, 9-fluorene- carboxylic acid, phenylacetic, hydroxycinnamic acid, quinaidic acid, formic acid, acetic acid, trifluoroacetic acid, cyclohexyl acetic acid, and 3-mercaptopropionic acid.
  • 9-fluoreneacetic acid 1-fluorenecarboxylic acid
  • 4 - fluorenecarboxylic acid 2- fluorenecarboxylic acid
  • 9-fluorene- carboxylic acid phenylacetic, hydroxycinnamic acid, quinaidic acid, formic acid, acetic acid, trifluoroacetic acid, cyclohexyl acetic acid, and 3-
  • Derivatives of the compounds of Structure 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.
  • 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
  • a unit dose is defined as 1 to 3000 mg 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.
  • compositions may be subjected to conventional pharmaceutical expedients such as sterilization and may contain conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts fcr adjusting osmotic pressure, buffers, and the like.
  • suitable pharmaceutical carriers and their formulations are described in Martin, "Remington's Pharmaceutical Sciences", 15th Ed. ; Mack Publishing Co., Easton (1975) ; see, e.g., pp. 1405-1412 and pp. 1461-1487.
  • 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.
  • the therapeutic 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.
  • 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.
  • the compounds and therapeutic or pharmaceutical compositions of the invention might be useful in the study or treatment of diseases or ether conditions which are mediated by the binding of integrin receptors to ligands, including conditions involving inappropriate (e.g., excessive or insufficient) binding of cells to natural or other ligands.
  • Such diseases and conditions might include inflammatory 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.
  • 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 which bind to receptors for cell adhesion molecules.
  • a disease is caused by antibodies binding to a cell adhesion molecule mimicked by a compound of structure I
  • 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) .
  • 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.
  • 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 for matrix proteins or other cell adhesion ligands, or, if anti-idiotypic, by acting to block cellular receptors .
  • the compounds of the present invention might be used to produce matrices for purifying substances which bind to the compounds of the present invention with high affinity.
  • a matrix could be produced, for example, by covalently attaching a compound of the present invention to a derivatized chromatographic support.
  • 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) .
  • a cyanogen bromide activated chromatography resin such as that available from Pharmacia, (Uppsala, Sweden, cat. no. 52-1153-00-AK) .
  • 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.
  • carbodiimide-activated resin can be used in conjunction with cyclic peptides bearing free carboxyl
  • 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 the compound 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.
  • EXAMPL EXAMPL
  • 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 a procedure which, where necessary, selectively removed protective groups from only the residues involved in cyciization. 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 cyciization 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.
  • 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 combinations cf the foregoing methods.
  • BOC and FMOC methods which are established and widely used, are described in the following references : Merrifield, J. Am. Chem. Soc, 88 :2149
  • 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.
  • 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 tic for the disappearance of the starting material (visualized with ninhydrin spray) .
  • acetic acid was added to pH 5.
  • Af er stirring for 15 minutes the product was isolated following lyophilization.
  • Tic System methane1/ammonium hydroxide, 1:1 Dimethyl-Arginine at Rf 0.6, Diethyl-Arginine at Rf 0.8.
  • the pH was of the reaction was maintained at 8-9 with IN NaOH and the reaction was stirred overnight. Overnight reaction was found to be incomplete. Additional (BOC) 2 0 (0.38 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 IN NaHS0 4 and extracted with three 50 ml portions of ethyl acetate.
  • Step 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.
  • Step 2 The residue from step 1 was dissolved in 1500 ml of cone. HCl and heated to 90-95°C for 7 hr. The solution 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 (45 gm) was dissolved in 1 1 DMF and 750 ml of triethylamine was added. The mixture was heated for 2.5 hr at 90-95°C and then concentrated to dryness on a rotary evaporator. The solids were dissolved in 1.5 1 water and applied to an 800 ml column of Amberlite IR-120 H+ resin. After washing until neutral the product was eluted with
  • 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.
  • activating 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., 52:617-624 (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) ) .
  • Cleava ⁇ e The BOC-deprotected peptides on-resin were cleaved by stirring at -5 to 0°C with a cocktail of distilled anhydrous HF (10 ml/g resin) , anisole (1 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 times with diethyl ether and then extracted with 80% aq. acetic acid. The combined extracts (200 ml/g resin) were pooled and carried on to the cyciization step.
  • Cyciization The formation of the intramolecular disulfide bridge was accomplished by using the iodine oxidation method (Wunch et al, Int. J. Peptide Protein Res., 12:368-383 (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 iodine 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 18 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 in acetonitrile to afford the desired acetate salt form.
  • TEAP triethylammonium phosphate
  • 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. 9 - luorenylmethvi asoarate
  • the residual oil was redissolved in 200 ml diethyl ether and extracted with (in order) 1% NaHC0 3 (3 x 50 ml) , H 2 0 (1 x 50 ml) , 5% citric acid (2 x 50 ml) , and brine (1 x 50 ml) .
  • the ether layer was dried over MgS0 4 and concentrated. Recrystallization from diethyl ether/petroleum ether yielded 3.53 g of N-BOC-O" 3 ⁇ - fluorenylmethyl aspartate, with a melting point of 135-137°C. N-BOC-O-fluorenylmethyl- lutamate (gamma 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-
  • 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 123.5-126°C.
  • BCC-Asp (OF ) OCH ; -PAM resm (1.0 g, 0.75 mmol) from Applied Biosystems (Foster City, California) was used as the starting 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 s -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.
  • AdaCA The N-terminal deprotected, side chain protected peptide on resin was washed with MeOH
  • the amidating cyciization was carried out according to the following protocol. Filtering was performed between each step: (1) MeOH (2 x 1 min) ; (2) DCM (3 x 1 min) ; (3) 20% piperidine in DMF, wash for 1 min, and deprotection for 20 min; (4) DMF (2 x 1 min) ; (5) MeOH (2 x 1 min) ; (6) DCM (3 x 1 min) ; (7) BOP reagent (4 equiv.) in DMF (20 ml/gram of resin) , stir for 2 min.
  • the final cyclic compound was removed from the resin by treatment with HF in the presence of 10% anisole for 1 hr at 0°C. After evaporation of the HF, the residue was washed with diethyl ether and extracted from the resin with 5% HOAc in H 2 0. The aqueous extract was lyophilized to yield the crude peptide.
  • the compound was purified using a Waters Delta Prep 3000 system (Waters, Milford, MA) equipped with a Cig 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 C 18 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.
  • the cyclic compound then was cleaved from the resin by HF and 10% anisole for 1 hr at 0°C. Following evaporation to the HF, the mixture was washed with diethyl ether (ether layer discarded) and extracted with IN HOAc. The aqueous extract was lyophilized to yield 1.23 g of the crude compound.
  • 1-FCA was purchased from Alrich Chemical Company (Milwaukee, WI) . All amino acids, amino acid 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) . Triethylamine was from Fisher Scientific (Fair Lawn, New Jersey) . Other reagents were obtained from conventional sources and of analytical grade .
  • peptides having the carboxy- terminus modified by amidation to NHNR' are synthesized by a slight variation of the technique by which the other peptides of the present invention are made.
  • that portion of the cyclic peptide except for the carboxy-terminal amino acid i.e. except Y 1
  • the peptide is then coupled to the appropriately derivatized carboxyl-terminal amino acid, using solution-phase techniques.
  • the following assay established the activity of the present compounds in inhibiting cell adhesion in a representative in vi tro 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 the 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 10% fetal calf serum.
  • Fibronectin was purified from human plasma according to the procedures of Engvall et al . , Int. J. Cancer, 2_0:l-4 (1977) .
  • Microtiter plates (96-well, Falcon) were coated overnight at 4°C with 0.1 ml cf a 5 ⁇ g/mi fibronectin m phosphate buffered saline (PBS) .
  • PBS buffered saline
  • BSA bovine serum albumin
  • Unbound proteins were removed from plates by washing with PBS. To block non-reacted sites, the plates are coated with 100 ⁇ l of a 2.5 mg/ml BSA solution in PBS for one hour at 37°C. U937 cells were collected and washed twice in Hanks' Balanced Salt Solution (HBSS) . The cells were counted and adjusted to 2.5 x 10 6 cells/ml in Dulbecco's Modified 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.
  • DMEM Dulbecco's Modified Eagle's Medium
  • BSA 2.5 mg/ml
  • the compounds (100 ⁇ l) 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 ⁇ l) were added to the wells and the plates were incubated for one hour at 37°C. 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) .
  • VMAX Kinetic Microplate Reader Molecular Devices, Menlo Park, CA
  • Jurkat-CS-1 Adhesion Assay Jurkat- ⁇ assay
  • the following assay established the activity of the present compounds in inhibiting cell-cell adhesion in a representative in vi tro 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 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) at passage number 2. The cells were growth on 0.5% porcine skin gelatin pre-coated flasks (Sigma, St. Louis, MO) in EGM-UV media (Clonetics, San Diego, CA) supplemented with 10% fetal bovine serum. Cells are refed every 2-3 days, reaching confluence by day 4 to 6. The cells are monitored for factor 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 Clonetics (San Diego, CA) at passage number 2. The cells were growth on 0.5% porcine skin gelatin pre-coated flasks (Sigma, St. Louis, MO) in EGM-UV media (Clonetics, San Diego, CA) supplemented with 10% fetal bovine serum. Cells are refed every 2-3 days, reaching confluence by day 4 to 6. The cells are monitored for factor VIII
  • DMEM Dulbecco's Minimal Eagle's Media
  • HSA Human Serum Albumin
  • Jurkat cells lxlO 6 cells/ml are stained with 10 ⁇ 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 37°C with 0.1 ng/ml ( 50 U/ml) recombinant IL-1 (Amgen, Thousand Oaks, CA) . Following this incubation, the monolayers are washed twice with HBSS and 0.1 ml of DMEM-HSA solution are added.
  • Jurkat cells (5 x 10 5 cells) are combined with the appropriate concentration of peptide and 0.1 ml of the Jurkat cell-peptide mixture are added to the endothelial cell monolayers. Generally, 250, 50, 10 and 2 ⁇ M peptide concentrations are tested.
  • the IC S0 is determined by testing the peptides at 50, 10, 2 and 0.4 ⁇ M. The plates are placed on ice for 5 minutes to allow for Jurkat ceil settling and the plates are incubated at 37°C for 20 minutes. Following this incubation, the monolayers are washed twice with PBS containing 1 mM calcium chloride and I mM magnesium chloride and the plates are read using a Pandex Fluorescence Concentration Analyzer (Baxter, Mundelein, IL) . Fluorescence in each well is measured as Arbitrary Fluorescence Units and percent adhesion in the absence of peptide is adjusted to 100% and the % adhesion in the presence cf 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 above in "U937-FN assay" except that the human T lymphocyte cell line, Jurkat, was used in place of the U937 cells.
  • Potency is expressed in ⁇ M units. Activity is defined in this assay as an IC 50 below 500 ⁇ M. This should be taken to mean that compounds that require a higher molarity to inhibit adhesion by 50 % are still active and of interest but are of overall lesser interest because of the high dose expected to be required when given in vivo to humans. Compounds with activity below 10 ⁇ M are most preferred, below 100 ⁇ M are not as preferred, below 500 ⁇ M lesser preferred and above 500 ⁇ M least preferred.
  • 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 cell adhesion to fibronectin. Data are also provided to show that compounds not containing an RGD sequence are effective inhibitors of adhesion to FN. Results cf Jurkat-Endothelial Cell Adhesion
  • Results cf the inhibition of Jurkat ceil adhesion to IL-1 stimulated endothelial cells.
  • Activity (A) in this assay is defined arbitrarily as an IC 50 below 250 ⁇ M; and inactivity (I) as IC 50 > 250 ⁇ M. As above, this does not mean that the compounds with an IC 50 > 250 ⁇ M are actually inactive but rather they are not potent enough to be as practical for human use as those with lower IC 50 .
  • 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, ⁇ i i ⁇ l , ⁇ _ 4 /3 7 , on the leukocyte and VCAM-1 on the endothelial cells.
  • data are provided to show that compounds not containing an RGD sequence are effective inhibitors of cell-cell adhesion.
  • O b >250(d) detached endothelial cell monolayers at a concentration of 250 ⁇ M or Z O greate .
  • Ada Ada, GAC, DTC, Fmoc, 5-FINC, CBO or sarcosine

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EP94906984A 1993-01-08 1994-01-07 Die Zell-Adhäsion hemmende Peptide Withdrawn EP0677060A1 (de)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US177393A 1993-01-08 1993-01-08
US1773 1993-01-08
PCT/IB1994/000026 WO1994015958A2 (en) 1993-01-08 1994-01-07 Peptide inhibitors of cell adhesion
CN94115995A CN1114961A (zh) 1993-01-08 1994-07-11 细胞粘连肽抑制剂

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EP (1) EP0677060A1 (de)
JP (1) JPH08505628A (de)
CN (1) CN1114961A (de)
CA (1) CA2153228A1 (de)
SG (1) SG52262A1 (de)
WO (1) WO1994015958A2 (de)

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JPH08505628A (ja) 1996-06-18
WO1994015958A2 (en) 1994-07-21
WO1994015958A3 (en) 1994-09-29
CA2153228A1 (en) 1994-07-21
SG52262A1 (en) 1998-09-28
CN1114961A (zh) 1996-01-17

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