CN1114961A - Peptide inhibitors of cell adhesion - Google Patents

Peptide inhibitors of cell adhesion Download PDF

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Publication number
CN1114961A
CN1114961A CN94115995A CN94115995A CN1114961A CN 1114961 A CN1114961 A CN 1114961A CN 94115995 A CN94115995 A CN 94115995A CN 94115995 A CN94115995 A CN 94115995A CN 1114961 A CN1114961 A CN 1114961A
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compound
acid
ala
thiop
phe
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S-L·N·彻昂
P·M·卡德雷里
T·J·洛勃
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Tanabe Seiyaku Co Ltd
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Tanabe Seiyaku Co Ltd
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Priority to SG1996001553A priority Critical patent/SG52262A1/en
Priority to EP94906984A priority patent/EP0677060A1/en
Priority to PCT/IB1994/000026 priority patent/WO1994015958A2/en
Priority to CA002153228A priority patent/CA2153228A1/en
Priority to JP6516421A priority patent/JPH08505628A/en
Application filed by Tanabe Seiyaku Co Ltd filed Critical Tanabe Seiyaku Co Ltd
Priority to CN94115995A priority patent/CN1114961A/en
Publication of CN1114961A publication Critical patent/CN1114961A/en
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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
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    • 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, 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

Abstract

An annulating integrator-component receptor antagon ist for regulating cell adhesion, including the adhesions relative to fibronectin and between leucocyte and endotheliocyte, and its synthesis, test, formulation and application method are disclosed.

Description

Peptide inhibitors of cell adhesion
The application with on January 23rd, 1992 disclosed wo92/00995 PCT application relevant, and at this as the full text reference.
The present invention relates to have cytoadherence and regulate active new cyclic peptide and peptide simulated compound.
Extracellular matrix (ECM) is the main component of reticular tissue, and this reticular tissue can make structural integrity, promotes cell migration and differentiation.As the part of these functions, extracellular matrix molecule such as Zeta protein, collagen protein, kelp amino acid (laminin), the VonWillebrand factor, thrombospondin, Fibrinogen and tenascin have been found in the external cytoadherence that promotes.This adhesion can play key effect for many bioprocesss, and this process comprises hemostasis, thrombosis, wound healing, metastases, immunity and inflammation.
Zeta protein (FN) is a prototype ECM molecule.Main cell combining site is with the acid of aminoacid sequence arginine-glycine-aspartic acid or synthesize method with the RGD warp of single letter designation and copy in the Zeta protein molecule.The peptide that contains the RGD sequence that can suppress also can to promote cytoadherence is disclosed (United States Patent (USP) 4,589,881; 4,661,111; 4,517,686; 4,683,291; 4,578,079; 4,614,517 and 4,792,525), when substituting glycine with L-Ala or substitute aspartic acid, when in the tripeptides structure, increasing a methyl or methylene radical with L-glutamic acid, the like this little variation of peptide all can be eliminated these activity (people such as Pierschbacher, PNAS, 81:5985 (1984)).Recently, sew up at another of molecule A chain and identified second FN cell land in district.Have now found that ten amino acid recognition sequences (GPEILDVPST) among the FN are position (people such as Wayner, J.Cell Biol., 109:1321 (1089) with cell response (SEQ.ID.NO.:1); People such as Guan.Cell，60：53(1990)。
The acceptor that can discern last these combining sites of FN belongs to a genoid Superfamily that is made of α and the β subunit different dimeric complexes of non covalent bond bonded, and it is called integrates plain (integrin).Common β subunit combines with specific alpha subunit and can form the adhesion receptor with above-mentioned characteristic.So far clone eight β subunits and determined sequence.β 1Subfamily, also being referred to as VLA section (the slowest active antigen) can combine with ECM molecule and FN, collagen protein and kelp amino acid, referring to Hynes.Cell, 48:549 (1987); Hemler, Anna, Rev.Immunol., 8:365 (1990).White corpuscle and the FN effect between different land, two gaps can independently be integrated plain (integrin) by two and regulate.The RGD position can be integrated plain α 5 β 1Identification, and EILDV (SEQ.ID.NO.:2) can be by α 4β 1Identification people such as (, Cell, 40:191 (1985)) Pytela; People such as Wayner, J.CellBiol.109:1321 (1989); People such as Guan, Cell 60:53 (1990)).
Vascular endothelial cell has constituted the interface between blood and tissue and has controlled white corpuscle and blood plasma inflow tissue.The various signals that produce at inflammation part can activate endotheliocyte and mobile hemocyte and make their easier each other adhesions that becomes, and after this initial adhesion, white corpuscle enters tissue to finish the host defense function.Several adhesion molecules relevant with reaction between white corpuscle-endotheliocyte are identified.On white corpuscle, have now found that β 2 integrates plain subfamily member, comprises CD11a/CD18, CD11b/CD18 and CD11c/CD18 play crucial effect in this process.At β 1In the subfamily, except can be with Zeta protein combines, α 4 β 1 also can with endotheliocyte on a kind of be called vascular cell adhesion molecule (VCAM) but the reaction of cytokinin inducing molecule.Can comprise ICAM-1 with other molecule of white corpuscle bonded on the endotheliocyte, 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); Genget al., Nature, 347:757 (1990); Stoolman, L.Cell, 56:907 (1989)).
Nearest data show that integrin alpha 4 β 1 play an important role in inflammation, anti-α 4 antibody of in vitro tests data presentation can block lymphocyte and the adhesion of synovia endotheliocyte; This adhesion acts on and plays crucial effect people such as (Van Dinther-Janssen), J Immunol., 147:4207 (1991) in the wind dominance sacroiliitis.Studies show that of antagonism α 4 monoclonal antibodies retardance basophilic leukocyte and oxyphie and the adhesion of cytokinin activated endothelial cells, α 4 plays crucial effect (people such as Walsh in transformation reactions and asthma, J.Immunol., 146:3419 (1991); People such as Bocher, J.Exp.Med., 173:1553 (1991).In addition, discover in the body that tentative autoimmunity encephalomyelitis can be by anti--α 4 monoclonal antibodies retardance (people such as Yednock, Nature, 356:63 (1992).White corpuscle also can be by anti--α 4 monoclonal antibodies retardance people such as (, J.Immunol., 147:4178 (1991)) Issikutz to the migration of inflammation part.In the contact hypersensitivity model, find recently, when using sensitized cell for the pure lines acceptor mouse that excites, peptide GRGDSP (SEQ.ID.NO:3) or EILDV (SEQ.ID.NO:2) can block ear swelling, this shows α 4 β 1, also may be α 5 β 1, both are relevant (people such as Ferguson, Proc.Natl.Acad.Sci.USA, 88:8072 (1991) of inflammatory response therewith all.Thereby α 4 β 1 and α 5 β 1 are the important acceptor target molecules of disease of controlling inflammation.
The present invention relates to have the active compound of cytoadherence conditioning agent, this compound does not contain aminoacid sequence arginine-glycine-aspartic acid acid (Arg-Gly-Asp or RGD), i.e. RGD three peptide epitopes.In fact, some these compounds do not have three of RGD epi-positions amino acid whose any one.
On the one hand, The compounds of this invention can be enough to analog cell epimatrix part or other cytoadherence part combines with cell surface receptor.This receptoroid comprises integrin receptor, generally comprises Zeta protein, collagen protein, kelp amino acid, LFA-1, MAC-1, P150, P95, vitronectin and gpIIb/IIIa acceptor.Have now found that this new compound can be by for example striving with the part that contains suitable aminoacid sequence and by regulating cytoadherence with the direct bonded receptors bind of part with cell surface unexpectedly.By with cell on receptors bind suppress for example (but being not limited only to) Zeta protein of cell adhesion protein effectively, to prevent or to reduce cytoadherence.The application of others also comprises by because of The compounds of this invention cell surface being adhered to or promoting the method for cytoadherence to strengthen the cytoadherence effect by other.This compound with practical value at this as the cytoadherence conditioning agent.
An object of the present invention is to provide a kind of new compound of regulating the cytoadherence effect that has.
Another object of the present invention provide a kind of can with the new compound that does not contain RGD of receptors bind on the cell of adjustable ganglion cell's adhesion.
Another object of the present invention provides the novel method that the new compound of a kind of usefulness is regulated cytoadherence.
Another object of the present invention provide a kind of can with cell adhesion molecule or integrin receptor bonded compound.
Another object of the present invention provides a kind of in adjusting cell and integrin receptor adhesion, comprises the compound that has remarkable effectiveness in inhibition cell and the Zeta protein adhesion.On the one hand, present invention resides in the IC that measures in the U937-Zeta protein adhesion test 50Compound less than about 100 μ M; On the other hand, present invention resides in the IC that measures in this test 50Compound less than about 100 μ M.The present invention also comprises this Zeta protein acceptor adhesion restraining effect of realization (in external or body) and the inhibiting method of integrin receptor adhesion.Under low concentration, use IC 50The The compounds of this invention that is lower than about 500 μ M or is lower than about 100 μ M can reach stronger restraining effect.
A present invention also purpose provides a kind of compound that has more potent power in regulating white corpuscle and endotheliocyte adhesion.Therefore, on the one hand, present invention resides in the IC that measures in the Jurkat-endotheliocyte adhesion test 50Less than the compound of 200 μ M, on the other hand, present invention resides in the IC that measures in this test 50Compound less than about 10 μ M.The active compound that is lower than 10 μ M is most preferred, is lower than 100 μ M and is as preferred, is lower than 500 μ M and is preferred and be more not preferred greater than 500 μ M.The present invention also comprises the inhibiting method of this leukocyte receptors adhesion of realization (in external or body).Under low concentration, use IC 50The The compounds of this invention that is lower than about 250 μ M or is lower than about 50 μ M can reach stronger restraining effect.
A further object of the invention provides a kind of by combine the new compound of regulating cytoadherence with cell adhesion molecule or integrin receptor, and wherein said compound is owing to the amino acid or the amino acid D-isomer that comprise peptide simulation residue, modification tolerate the vivo degradation effect.
A further object of the invention provide can be used for relating to or with the research of cytoadherence diseases associated, diagnosis, the new compound of treatment or prevention, preparation and method, described disease comprises, but be not limited only to rheumatic arthritis, asthma, transformation reactions, adult respiratory distress syndrome (ARDS), cardiovascular diseases, thrombosis or deleterious platelet aggregation, inaccessible again after the thrombolysis, the homotransplantation rejection, graft is to host's rejection (graftversus host disease), organ transplantation, septic shock, perfusion property damage again, psoriasis, eczema, contact dermatitis and other dermatitis, osteoporosis, osteoarthritis, atherosclerosis, tumor disease comprises tumour or growth of cancers metastatic tumor, wound healing is strong excessively, the treatment of some illness in eye such as retinal detachment, type i diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), inflammation and immune inflammation comprise ophthalmia and enteritis (as ulcerative colitis and limitation joint enteritis) and other autoimmune disorder.
Have now found that cell adhesion protein Zeta protein (FN) relevant with the combination of capacitation sperm and ovocyte (Fusi and Broson, J.Androl., 13:28-35 (1992)).Like this, another object of the present invention also relates to and can suppress the compound that sperm and ovocyte bonded can be used as contraceptive bian.The present invention also provides a kind of possible diagnostic method of the infertility that sperm and the incomplete adhesion of ovocyte are caused.
Another object of the present invention also provides the derivative compound that is used for the diseases related research of above-mentioned cytoadherence, diagnosis, treatment or prevention, for example, but is not limited only to the antibody of anti-described compound and anti--genotype antibody.
Another object of the present invention provides a kind of matrix, and it can be used for purifying with high-affinity, and specificity is in conjunction with protein, polysaccharide or other compound of cyclic peptide of the present invention.
Although cytoadherence is the needs of some normal physiological function, do not need cytoadherence in some cases, perhaps need to regulate cytoadherence in some cases.
Reaction in many diseases associated with inflammation between white corpuscle-endotheliocyte can change, and wherein unfavorable adhering to of white corpuscle can cause being subjected to the invasion and attack tissue to produce further infringement.In vitro tests is the result show, this kind is deleterious to be adhered to, wherein white corpuscle and endotheliocyte or and extracellular matrix between adhesion, can regulate by the integrin receptor on the white corpuscle.In the case, can be required competitive antagonist, and can be used for treating inflammation that this inflammation comprises ARDS, asthma and rheumatic arthritis with integrin receptor affinity bonded peptide or other compound.
Cytoadherence also can cause the transfer of tumour, and metastases is known as " the lethal main potential cause of tumour ".People such as Welch, Intern.J.Cancer, 43:449 (1989).The peptide that contains RGD that can block with basement membrane component generation cytoadherence can be used for retardance or eliminates metastases.See people such as Humphries, Science, 223:469 (1986); Liotta, Cancer Res., 46:1 (1986); Roose, Biochem.Biophys.Acta., 738:263 (1986).Equally, peptide or other compound that the RGD acceptor is had a suitable avidity also should have the anti-metastasis effect.
Strengthen the formation of the deleterious blood clot of cytoadherence effect also can causing.Thrombocyte adhering on extracellular matrix, distribution and cohesion are thrombotic main processes, and this reaction can be regulated by waiting with glycoprotein, Fibrinogen, Zeta protein and the thrombin of PA.When Zeta protein promoted thrombocyte to adhere to and scatters reaction, Fibrinogen became the common factor of platelet aggregation.Thrombin plays crucial effect in thrombocyte adheres to and scatters on interior subcutaneous matrix, people such as Plow, PNAS-USA, 82:8057 (1985).Having the antagonist function can will be useful antithrombotic agent with the cell receptor bonded peptide that can discern matrix glycoprotein RGD site or other compound also.
Other physiological maladies can be regulated the cytoadherence treatment by stimulating.For example, when deficiency takes place cytoadherence, can prolong wound healing time, this is undesirable.For example when with suitable location matrix or surface attachment, the peptide or other compound that integrin receptor are had suitable avidity can promote cytoadherence and wound healing by cell is combined with the RGD-identification receptor that suits.
For example, in the prosthese transplantation, with this type of peptide or other compound the prosthese dressing can be formed a biocompatible surface on this prosthese, when implanting the prosthese of using the The compounds of this invention dressing, this prosthese can be by cell institute clad, and the cellular layer of this Periprosthetic can lower the rejection that excites immunity system to occur by prosthese itself.Another example is, when using a kind of stimulating endothelial cell, when the The compounds of this invention dressing that particularly is exposed to the endotheliocyte adhesion on blood flow surface connects the prosthetic appliance of the recycle system, it can promote the inoculation of endotheliocyte and form an endodermis on the surface that this device is exposed to blood flow, after being completed into, the hemocyte infringement that this endodermis can stop the prosthese by not healed by endothelium examined through routine to cause.
A part has the The compounds of this invention of regulating the cytoadherence effect can be represented with the aminoacid sequence formula, and wherein each amino acid can perhaps also can be represented with a letter abbreviations with three conventional letter representations.
For used amino acid abbreviations, when not indicating its enantiomer-specific structure, be to use its l-or d-enantiomorph aptly.
Other abbreviation used herein comprises:
1,1-ACC:1-amino-1-naphthenic acid
The Ada:1-adamantane acetic acid
(Ada)-Ala: β-adamantyl L-Ala
The Ada-CA:1-adamantanecarboxylic acid
Aib: α-An Jiyidingsuan (2-methylalanine)
β-Ala: Beta-alanine (3-alanine)
β-Asp: β-aspartic acid
(the A of β-CN): beta-cyano-L-Ala
AMBA:4-(amino methyl) phenylformic acid
The AnB:4-aminobutyric acid
The AnC:6-hexosamine
(AMP): the 2-aminomethyl-pyridine
ARDS: adult respiratory distress syndrome
BOC: tertbutyloxycarbonyl
[(3-Br) Tyr]: 3-bromo-tyrosine
BOP: benzotriazole-1-base-three (dimethylamino) Phosphonium phosphofluoric acid ester
BSA: bovine serum albumin
CBO: cis-two ring is [3,3,0] octane-2-carboxylic acid also
Cb2: carbobenzoxy-(Cbz)
CHA:3-(cyclohexyl) L-Ala
The CHAc:3-cyclohexyl acetic acid
Chx: cyclohexyl ester
Cl-Ala: chloro L-Ala
CPA: cyclohexylphenylacetic acid
The dA:D-L-Ala
DCC: dicyclohexylcarbodiimide
DCM: methylene dichloride
Dhp:3,4-dehydrogenation-proline(Pro)
DIEA: diisopropyl ethyl amine
The improved Eagle substratum of DMEM:Dulbecco
DMF: dimethyl formamide
D-Nal:D-3-(2 '-Nai Ji) L-Ala
Dpr: diaminopropanes
DTC:L-5,5-dimethylthiazole quinoline-4-carboxylic acid
The dv:D-Xie Ansuan
1-FCA:1-fluorenes carboxylic acid
9-FCA:9-fluorenes carboxylic acid
The 9-FA:9-fluorene acetic acid
5-FINC:5-fluoro indole carboxylic acid
Fm: fluorenyl methyl esters
FMOC: fluorenylmethyloxycarbonyl
FN: Zeta protein
GAC: guanidine-acetate
3-Glu:r-aminopentane-1, the 5-diacid
HCA: phenylpropionic acid
The HOBt:1-hydroxybenzotriazole
HomoC: homocysteine
HomoP: high proline(Pro)
HomoR: homoarginine
HomoS: high tryptophan
The Hyp:4-oxyproline
ICAM-1: iuntercellular adhesion molecule 1
IC50: inhibition concentration, adhesion are suppressed to the concentration of control level 50%
1-Nal:1-3-(2 '-naphthyl) L-Ala
IPA: isopropyl alcohol
Isoni pecotic acid:4-piperidine carboxylic acid
3-Me-Ada:3-methyl isophthalic acid-adamantane acetic acid
Mono MeR:N-methyl-arginine
Mpr:3-thiohydracrylic acid (going-the alpha-amino group halfcystine)
MTC:L-2-methylthiazol quinoline-4-carboxylic acid
Adamantanecarboxylic acid falls in NACA:3-
The Naph-Ac:1-naphthyl acetic acid
NB-Ac:2-norcamphane acetate
Nic-Lys: nicotinyl Methionin
Nle: fall leucine
[(N-Me) R]: N-methylarginine
Adamantanecarboxylic acid falls in norAda-CA:3-
NorArg: fall arginine
(H 2NC(＝NH)NH(CH 2) 2CH(NH 2)CO 2H)
Orn: ornithine
O-Cys: cysteic acid
Pen: Trolovol
(β, Beta-Dimethylcysteine)
PhAc: toluylic acid
PMP:1-(β-sulfydryl-β, β-ring penta methylene radical) propionic acid
PyE: Pyrrolidonecarboxylic acid
Pyro Glu: Pyrrolidonecarboxylic acid
QC: quinardinic acid
R.T. room temperature (about 24 ℃)
Sar: sarkosine
SLE: systemic lupus erythematosus
TA:3-β-thienyl-L-Ala
TC:DL-thiazolidine-2-carboxylic acid
TCA:1,4-thiazine-3-carboxylic acid
TEA: triethylamine
TFA: trifluoroacetic acid
(thiop): the 3-Thioproline or
1-thiazolidine-4-carboxylic acid
TIC:1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid
Tlc: thin-layer chromatography
TTC:L-tetrahydrochysene thiazine-4-carboxylic acid
VLA: the slowest active antigen
Following word used herein and/or short speech have following implication:
" analogue " meaning i.e. the derivative of the substituent parent compound of affix chemistry on discernible parent compound skeleton, and parent compound should keep its basic chemical functional with the back gained analogue of deriving someway.Like this, " amino acid analogue " is side chain carbon or nitrogen by derivation, and the nitrogen that links to each other with alpha-carbon is by the amino acid of N-derivation, but it should remain with the ability that forms peptide bond.Should be noted that amino acid whose D-enantiomorph is also included within this definition.Another example is that " arginine analog " refers to contain the arginine skeleton and be connected with substituent compound thereon.Therefore this type of arginine analog include but not limited to, compound N-methyl-Arg, and N-low alkyl group-Arg, N, N-dimethyl-Arg, N, N-two low alkyl groups-Arg, Arg falls in high Arg, and side chain is N-nitro-Arg that guanidine radicals replaces, N ω-nitro-Arg, N, N '-dimethyl-Arg, N, N '-two low alkyl groups-Arg, beta, gamma or δ carbon by nitro-, alkyl-, aryl-, the arginine derivative that replaces of 4-nitro alkyl-or nitro aryl-group etc.
" phenylalanine analogues " comprises the compound that is connected with halogen, methyl or low alkyl group, nitro or hydroxyl substituent on the phenyl ring; The example of not getting rid of has couple nitro-Phe, to halo-Phe, to amino-Phe and five fluoro-Phe.Phenylalanine analogues also comprises dibasic analogue such as dichlorobenzene L-Ala, o, m-dimethyl benzene L-Ala etc., the dibasic analogue of different substituents such as o-methyl-m-chloro-phenylalanine.The compound such as the N-methyl-Phe of the replacement of N-alkyl also should be included as mentioned above.
" tyrosine analogue " is similar to phenylalanine analogues, for example: 3-bromo-Tyr, 3,5-two bromos-Tyr and 3,5-two iodos-Tyr, and also comprise ring hydroxy derivatives such as o-methyl-tyrosine, o-low alkyl group-tyrosine etc.
" proline analogs " comprises sulfocompound such as 3-Thioproline and following compounds: high proline(Pro), oxyproline, 3,4-dihydroxyl proline(Pro), DL-thiazolidine-2-carboxylic acid, 1,4-tetrahydrochysene thiazine-3-carboxylic acid, L-5,5-dimethylthiazole quinoline-4-carboxylic acid and 1,3-tetrahydrochysene thiazine-4-carboxylic acid, L-tetrahydrochysene thiazine-4-carboxylic acid and 1,3-, 1,4-and 1,5-sulphur azatropylidene.
" aspartic acid ester analogs " and " glutamate analogue " comprises the ester of this amino acid omega-carboxylic acid function's base.
" lysine analogues " comprises the acid amides of alpha-amino group and epsilon-amino and the alkyl derivative of alpha-amino group and epsilon-amino.Lysine analogues also comprises DpR, ornithine, high-lysine and these amino acid and relevant amino acid whose similitude.
On the one hand, the present invention relates to following formula: compound:
Figure A9411599500241
In the said structure formula 1, at L 1And L 2Between through becoming bridge of the Z-shaped one-tenth of cyclic group, compound is by cyclization.In the structure that further describes hereinafter, the alpha-amino group of amino terminal amino acid in " α HN " expression sequence.Equally, α-carboxyl is represented with " α C=o " in structural formula.Side chain functional group is represented with the bracket inner structure.
To L 1And L 2Selection should make its each all contain one and can help to encircle the functional group that abutment group forms.Like this, Z can be by L 1And L 2The functional group of contributing constitutes and can also can contain other atom and spacer groups.More particularly, preferred functional group comprises thiol, amino and carboxyl.This functional group can be generated by the side chain of amino acid or amino acid analogue, perhaps can be by the alpha-amino group (L of itself 1In) or α-carboxyl (L 2In) constitute.In addition, provide becoming functional group that ring contributes also can become to encircle linking group by the non-peptide that is connected with residue 1 and/or 6 covalent linkage.
In preferred version of the present invention, bridge residue L 1And L 2Be selected from residue Cys, Pen and homoC respectively.For L 1, other preferred residue also has Mpr and PMP, and all these residues all contain sulfydryl.For L 2, other preferred residue also has mercapto-ethylamine (MEA).If use MEA, Y then 1And Y 2To not exist.Like this, the sulfydryl by oxidative coupling is with at residue L 1And L 2Between form a disulfide linkage and can realize that bridged ring closes.In the case, one-tenth cyclic group Z is a covalent linkage between two sulphur atoms.It can also be expressed as wherein for example L 1And L 2Both are the following formula: compound of Cys residue
Figure A9411599500251
Wherein (other similar statement method as used herein) functional side chain base section (being the two ends sulphur atom herein) is listed in the bracket that has above the side chain residue.
Preferred scheme is L 1Be Cys or Mpr L 2Be Cys.
The ring bridge also can be formed by alkyl, for example by formula-(CH 2) n-wherein n be the integer of 1-8, (many) methylene radical abutment that is preferably among the 1-4 forms.A kind of this type of bridge can followingly represent that wherein ring compound (is represented L at two cysteine side chain sulphur atoms 1And L 2) between three methylene radical residues (representing Z) are arranged:
Figure A9411599500252
(seeing L.Fieser et al., " Reagents for Organic Synthesis ", Vol.1, pp.356-357, J.Wiley and Sons:(1967); Fieser, J.Amer.Chem.Soc., 96:1945 (1959)).
In another preferred version, L 1And L 2Can be selected from other amino acid or analogue or amino acid dummy, this amino acid or analogue or amino acid dummy can provide the side chain of amino acid or similar residue or amino-or carboxyl-end as the functional group that is suitable for being formed into cyclic group.For example, L 2Can be selected from Asp, Glu or other amino acid or analogue, above-mentioned amino acid or analogue can improve a suitable side chain carboxyl group, by in condensation reaction with L 1On amino (N for example a-amino, or go up side chain amino) form amido linkage connecting into ring, but the following formula structure is not included as Lys or Orn. Becoming cyclic group Z in the case is L 1And L 2Between singly-bound.
Equally, amino-acid residue L 21 carboxyl on the C-terminal can be provided, with amino-acid residue or analogue L 1Form amido linkage between last side chain amino or the alpha-amino group; Perhaps the direction of this amido linkage also can be changed, wherein L 1Provide 1 side chain carboxyl group, L 21 side chain amino is provided.This structure can be in the following example shown in:
Figure A9411599500262
L wherein 1(Lys) and L 2(Aps) side chain amino and the direct chain of carbonyl close;
Figure A9411599500263
Direction (the L of acid amides chain wherein 1And L 2Side switch) be converted;
Figure A9411599500271
Or Wherein said L 1N-terminal and Glu (L 2) the direct chain of side chain carboxyl group closes perhaps described L 2C-terminal and Orn (L 1) the amino Direct Bonding of side chain;
Figure A9411599500273
Wherein said L 1Terminal and the described L of last alpha-amino group 2Last C-terminal Direct Bonding so forms 1 acid amides chain in The compounds of this invention peptide " skeleton ".
In another preferred version of the present invention, also can use diketone as follows and diamino linking group Z, as shown in the formula
Figure A9411599500281
With-NH-(CH 2) n-NH-wherein n as above defines.
For example, the diketone linking group can be used for being connected with the epsilon-amino of lysine residue, and the diamino linking group is generally used for the δ-carboxyl Cheng Huan with L-glutamic acid or asparagicacid residue.This example gained compound is that following structure is arranged
Figure A9411599500282
With
Figure A9411599500283
L wherein 1And L 2Last side chain functional group (amino and carboxyl) is seen upward square brackets of residue abbreviation symbol.
The example that above only relates to suitable hydrocarbonaceous bridge, and other form is conspicuous for those skilled in the art.When including the hydrocarbon part as cyclic group Z, this hydrocarbon can be branched-chain hydrocarbon, but when this hydrocarbon size is suitable for forming a rock steady structure (particularly Z comprises two or more methylene radical), it can comprise that also containing one or more heteroatomic substituting groups comprises hydroxyl, amino, nitro, alkoxyl group and halogenic substituent.This type of substituting group can be used to change the solubleness and/or the bio distribution of purpose compound.The abutment group of containing aryl or cycloalkyl hydrocarbon also can be used on the Z position, as following diketone or diamino structure
Figure A9411599500291
Or-HN-(C 6H 8)-NH-is for the hydrocarbyl portion in the Z group, and the simple alkyl that contains 1 to 4 carbon is preferred
Certainly, becoming abutment group is that the situation of two different functional groups also is possible, that is to say, an end is that ketone group and the other end are for amino.The structure as follows that like this, wherein includes the elementary cell of many above-mentioned discussion also can serve as abutment group:
L 1And L 2Between the cyclization bridge can also form through a monosulfidic bond that is shown below (thioether).A kind of formation method of this key is at L 1And L 2The last use on another link position with halfcystine can provide bromo-acetate [or general formula N=0-4 wherein] residue of functional group.(seeing Barker et al., J.Med.Chem., 35:2040-2048 (1992)). In addition, L 1Can be α, β dehydroalanine, L 2It can be cysteine residues.The two reaction can produce a L-lanthionine thioether linking group.
L 1And L 2Between the cyclization bridge also can form through a monosulfidic bond that is shown below (thioether).
Figure A9411599500302
About this referring to
Palmer?et?al.，in“Peptides--Chemistry，Structure，Biology”，pp.616-618，Rivier?&?Marshall，Ed.，Escom.Leider(1990)；and?Jung，op.cit.，pp.865-869.
The amino-acid residue analogue also can be used as L 1And/or L 2For example (wherein side chain is elongated or when shortening for homologue, still can be provided as carboxyl, amino or other active precursor functional group of ring usefulness), contain different side-chain analogs (for example beta-cyano L-Ala, canavanine, djenkolic acid, α-diazobenzene L-Ala or 2-aminoadipic acid) or other amino acid analogue (referring to for example amino acid analogue mentioned above) of the functional group that suits in the amino acid d-enantiomorph, side chain.
At L 1And/or L 2Can also use the amino acid analog structure on the position, this amino acid analog structure should be able to at the carboxyl of residue sequence 1-2-3-4-5-6 and/or N-terminal through an acid amides chain covalent bonding; And should be able to be provided as the suitable precursor functional group of ring (through Z) usefulness.This amino acid analog structure comprises and contains one or more heteroatomic organic fragments that it should include at least one functional group that participates in into ring (preferably containing heteroatomic functional group).Example comprises the following formula residue Wherein n is 1 to about 8, is preferably 1-4, for example the residue of Beta-alanine and r-aminobutyric acid.(when n=1, being amino acid glycine rather than a-amino acid stand-in).This class formation and above-mentioned amino acid and amino acid analogue are similar, can be used as L 2(wherein the described carbonyl that is for example formed by carboxyl precursor usually can with residue 2/ or if possible, the N-terminal of residue 1 forms the acid amides chain), perhaps it also can be used as L 1(wherein said amino can form the acid amides chain with the C-terminal of terminal residue 4,5 or 6).If during only with this type of connection residue L, it can represent L 1And L 2Both (and also comprise Z at this), annulation can be finished by forming two amido linkages, and an amido linkage is arranged on each end of sequence 1-2-3-4-5-6, and the example of this class formation is as shown in the formula structure Sequence 1-2-3-4-5-6N wherein α-terminal and C-terminal respectively with the carbonyl residue of above-mentioned simulation amino acid linking group and the amido linkage of two simulating peptide of amino residue Direct Bonding formation.Also can realize annulation with this amino acid analog linking group equally, wherein be connected second linking group on the Serial No. end (as L 1And L 2) on functional side chain group (as amino or carbonyl functional side chain group) with the simulation group be connected, and this simulation group (as L 2Or L 1) with this compound cyclisation to the residue terminal residue of Serial No..The step example can be as shown in the formula shown in the structure L wherein 2(for example Asp) provides the described side chain carbonyl in the bracket, and residue 1 provides described N α-terminal amino group, simulation amino acid linking group is L 1
The amino acid analog structure that contains aryl, cycloalkyl or other linking group also can be used as L 1And/or L 2, following formula structure for example
Figure A9411599500323
Or
Figure A9411599500324
Equally, above-mentioned two difference in functionality bases (ketone-amino) structure also can be used as Z group, wherein L 1And L 2Go up complementary side chain functional group (as L 1Last side chain amino and L 2Last side chain carboxyl group) is bonded to the Z group through two amido link structures.
By selecting L aptly 1, L 2Other method that becomes ring with Z be those skilled in the art of the present technique expect obtain and be included in the scope of the invention.
Be above-mentioned relevant one-tenth cyclic group (Z), bridging residue (L with being appreciated that 1And L 2), the discussion of substituting group, amino acid analogue, amino acid analog thing, cyclization method etc., other structural formula of having done to be applicable to hereinafter under the situation of necessary correction in detail and being discussed.Residue 1 most preferably is not exist among the structural formula I; Residue 2 most preferably is Arg; Residue 3 most preferably is Ala; Residue 4 most preferably is Asp; Residue 5 most preferably is that 3-Thioproline (thiop) and residue 6 preferably do not exist.For residue 1-2-3-4-5-6 sequence A rg-Ala-Asp-(thiop) (SEQ.ID.NO.:9) residue 1-4 be most preferred.More preferably wherein residue 1 does not exist, and residue 2 does not exist, and residue 3 is Asp, and residue 4 is (thiop) and residue 5 and 6 both all non-existent sequences.Like this, also be preferred for residue 1-2-3-4-5-6 sequence A sp-(thiop).
The third preferred sequence is X wherein 1Be Gly, both all exist residue 1 and residue 2, and residue 3 is Asp, and residue 4 is (thiop) and residue 5 and 6 both all non-existent sequences.
The 4th kind of preferred sequence is X wherein 2Be (1-FCA), X 1All do not exist with residue 1 and 2, residue 3 is Asp, and residue 4 is (thiop) and residue 5 and 6 both all non-existent sequences.
The 5th kind of preferred compound is X wherein 2Be Fmoc, X 1Be Arg, residue 1 and 2 both all do not exist, residue 3 is AnB, residue 4 is (thiop) and residue 5 and 6 both all non-existent compounds,
The 6th kind of preferred compound is X wherein 2There is not X 1Be Arg, residue 1 and 2 both all do not exist, residue 3 is Ala, residue 4 is (thiop) and residue 5 and 6 both all non-existent compounds.
The 7th kind of preferred compound is X wherein 1And X 2Do not exist, residue 1 does not exist, and residue 2 is Arg, and residue 3 is d-Ala, and residue 4 is Asp, and residue 5 does not exist and the compound of residue 6 for (thiop).
The 8th kind of preferred compound is that wherein residue 1 does not exist, and residue 2 is Arg, and residue 3 is Ala, and residue 4 is Leu, and residue 5 does not exist and the compound of residue 6 for (thiop).
X in structural formula I 1And Y 1Each all is optional.When they existed, preferably they independently selected to each other, purpose be for improve the active of gained compound and/or for example in vivo environment down the protection compound exert oneself and suffer metabolism and improve the effective transformation period of compound.For this reason, more preferably on one or more terminal residues position of compound (promptly N-terminal and or carboxyl terminal residue position on, or X 1Or Y 1In) use one or more d-amino acid, to improve compound in vivo to the stability of proteolysis or other enzymes metabolism.X 1Preferred more specifically residue comprises Gly, Phe, Leu, Asn, Val, Tyr, Ala, Arg, His, 1-or 2-naphthylalanine, cyclohexyl-Ala-, AMBA, AnC, AnB and omega-amino--lower alkanols alkanoic acid, Aib, Ser-Tyr-Asn, Ala-Thr-Val-and to chloro-Phe-on the position.Y 1On the position preferred residue comprise Ala ,-Ala-Ser ,-Ala-Ser-Ser ,-Ala-Ser-Ser-Lys ,-Ala-Ser-Ser-Lys-Pro, Thr,-Thr-Phe ,-Aib, to chloro-Phe, AMBA, AnC, AnB, omega-amino--lower alkanols alkanoic acid, 1-or 2-naphthylalanine and-(cyclohexyl) Ala is corresponding to position that structural formula is given hereinafter described, this X 1And Y 1Also be preferred.
When using R 1It is not the substituent X of hydrogen 2Or Y 2The time, X for example 2Preferably comprise acyl group R ' CO, particularly formic acid, acetate or other lower alkanols alkanoic acid, also comprise the straight chain mixing functions yl carboxylic acid (for example 3-dredges basic propionic acid) that contains nitrogen and sulphur.For Y 2The amino that preferably comprises formula R ' NH, particularly low-grade alkylamine.For X 2Other preferred substituted comprises by compound such as adamantane acetic acid, adamantanecarboxylic acid, 1-or 2-naphthyl acetic acid, 2-norcamphane acetate, 3-falls adamantanecarboxylic acid, 3-methyl adamantane acetate deutero-substituting group.For Y 2Other preferred substituted comprises low-grade alkylamine, arylamines, 1-or 2-amantadine and the amino acid that contains α-carboxylic acid that is replaced by tetrazyl.Each R 1Be respectively substituting group suitable on the medicine, the C that preferably is selected from hydrogen, straight chain and side chain, does not replace and replaces 1-C 8Low alkyl group, C 2-C 8Alkenyl, C 2-C 8Alkynyl group, C 6-C 14Aryl, C 7-C 14Alkaryl, C 7-C 14Cycloalkaryl and C 3-C 14Cycloalkyl, for-NR ' 2It can be formed into cyclic group with institute's azine atom, promptly contain aerobic arbitrarily, nitrogen or sulphur are as the 5-8 unit heterocycle of other ring hetero atom, formic acid, acetate, heterocyclic carboxylic acid, aryl carboxylic acid, the hetero-aromatic ring carboxylic acid, paraffinic acid, alkenoic acid, the chain acetylenic acid, other mixing functions base straight-chain carboxylic acid of sulfur-bearing and nitrogen, 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, other many cyclophanes ring and hetero-aromatic ring carboxylic acid and acetate, QC, CPA, BOC, 5-FINC and CBO.Below cited structure
Figure A9411599500361
Figure A9411599500362
M=2 wherein, 3 or 4 be as residue 5 among the structural formula I, represents amino-acid residue that pendant hydroxyl group (shown in the bracket) is wherein at random replaced by formula R ' the group thing that spreads out, and this R ' is not except that be as above-mentioned definition the hydrogen.
When using the residue of this type of replacement on the structural formula I5 position, preferably R ' is selected from hydrogen and C 1-C 8Low alkyl group, particularly methyl and ethyl.
Particularly preferred compound comprises among the structural formula I: (SEQ.ID.NO.:4) structure-Cys-that wherein abridges to other is local used similar, represents the cysteine residues that its side chain sulphur atom is described respectively herein, and is same, structure (S) ... (S) table disulfide bond.Described compound has the adhesion effect that suppresses cell and Zeta protein.
Other preferred compound does not exist for residue 1 wherein or is Leu; Residue 2 is Arg; Residue L 1Be Cys; Residue 4 is Asp; Residue 5 does not exist or is Ser; Residue 6 does not exist, for Pro or (thiop); Residue L 2Compound for Cys.Under the 1 listed compound of tabulating be particularly preferred.
The sequence table of selected compounds is as follows in the table 1:
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).
Table 1
Keep
Gradient compound number sequence mass spectrum (time (min) % purity 1 C of %A-min-＞%B) ★(NiC-Lys) GDSPC ★8-20 '-＞28 9.9 99.9 2 C ★R (Sar) DSPC ★0-20 '-＞20 10.9 96.9 3 C ★RGNSPC ★0-20 '-＞20 9.2 98.1 4 RC ★NPC ★0-20 '-＞20 7.5 97.8 5 FmocKC ★DPC ★40-20 '-＞60 8.9 97.3 6 KC ★DPC ★2-30 '-＞32 3.9 82.9 7 FmocAC ★D (thiop) C ★50-20 '-＞70 12.5 99.7 8 FmocFC ★D (thiop) C ★70-20 '-＞90 8.1 99.4 9 FmocHC ★D (thiop) C ★55-20 '-＞75 9.3 ★69.2 10 FmocLC ★D (thiop) C ★55-20 '-＞75 11.0 99.0 11 FmocRC ★A (thiop) C ★50-20 '-＞70 7.6 97.4 12 FmoCRC ★L (thiop) C ★55-20 '-＞75 10.2 95.3 13 FmocRC ★F (thiop) C ★55-20 '-＞75 10.3 98.6 14 AC ★D (thiop) C ★0-30 '-＞30 5.2 88.5 15 RC ★A (thiop) C ★0-20 '-＞20 10.1 ★64.3
Gradient retention time compound number sequence mass spectrum
% purity
(％A-min-＞％B) Time(min) 16 LC ★D(thiop)C ★ 2-30′-＞32 13.9 87.4 17 FC ★D(thiop)C ★ 2-20′-＞22 17.1 88.1 18 RC ★F(thiop)C ★ 2-20′-＞32 27.1 77.7 19 HC ★D(thiop)C ★ 2-30′-＞32 21.0 ★61.2 20 Rc ★L(thiop)C ★ 2-30′-＞32 22.9 ★54.6 21 FmocGC ★D(thiop)C ★ 50-30′-＞70 6.5 99.2 22 FmocPC ★D(thiop)C ★ 45-20′-＞65 10.8 99.7 23 AdaC ★D(thiop)C ★ 45-20′-＞65 6.3 99.7 24 FmocAC ★A(thiop)C ★ 60-20′-＞80 6.2 96.4 25 (1-FCA)AC ★D(thiop)C ★?715.90 45-20′-＞65 7.1 98.1 26 (1-FCA)C ★D(thiop)C ★ 645 45-20′-＞65 8.7 99.0 27 (1-FCA)RC ★[(p-Cl)Phe](thiop)C ★ 60-20′-＞80 7.1 98.5 28 GC ★D(thiop)C ★ 40-20′-＞60 8.0 95.7 29 C ★GKGESPC ★ 2-20′-＞22 6.4 96.8 30 (1FCA)(Sar)C ★D(thiop)C ★ 45-20′-＞65 6.3 99.1 31 (thiop)C ★D(thiop)C ★ 2-20′-＞22 15.5 99.5 32 PC ★D(thiop)C ★ 2-20′-＞22 7.0 88.9 33 (Sar)C ★D(thiop)C ★ 2-20′-＞22 6.6 93.5
Gradient retention time compound number sequence mass spectrum
% purity
(％A-min-＞％B) (min) 34 FmocRC ★(thiop)C ★ 55-20′-＞75 6.9 99.1 35 (1-FCA)(thiop)C ★D(thiop)C ★ 50-20′-＞70 5.9 86.7 36 FmocK-AdaC ★D(thiop)C ★ 980 75-20′-＞95 6.8 97.7 37 FmocRC ★(BD)(thiop)C ★ 831 45-20′-＞65 12.2 96.7 38 FmocK(AnB)C ★D(thiop)C ★ 45-20′-＞65 6.8 98.5 39 [(N-Me)R]C ★D(thiop)C ★ 2-20′-＞22 8.2 92.9 40 C ★RGA[(p-Cl)Phe](thiop)C ★ 35-20′-＞55 6.5 98.1 41 AdaGGC ★RGA[(p-Cl)Phe](thiop)C ★ 60-20′-＞80 8.0 96.6 42 C ★AGD[(p-Cl)Phe](thiop)C ★ 30-20′-＞50 6.2 97.2 43 AdaGGC ★AGD[(p-Cl)Phe](thiop)C ★ 55-20′-＞75 5.4 97.2 44 FmocRC ★(AnB)(thiop)C ★ 800.89 50-20′-＞70 7.0 99.3 45 FmocRC ★(AiB)(thiop)C ★ 45-20′-＞65 7.9 96.6 46 FmocRC ★V(thiop)C ★ 60-20′-＞80 6.7 98.1 47 FmocRC ★(β-CN)A(thiop)C ★ 75-20′-＞95 10.3 89.0 48 (GAC)C ★D(thiop)C ★ 2-20′-＞22 13.6 96.1 49 (DTC)C ★D(thiop)C ★ 15-20′-＞35 10.0 89.8
Gradient retention time compound number sequence mass spectrum
% purity
(％A-min-＞％B) (min) 50 C ★GRGA[(p-Cl)Phe](thiop)C ★ 30-20′-＞50 7.5 99.2 51 FmocC ★D(thiop)C ★ 50-20′-＞70 8.9 89.5 52 (TCA)C ★D(thiop)C ★ 2-20′-＞22 9.1 98.1 53 (1-FCA)GC ★D(thiop)C ★ 701.94 45-20′-＞65 6.4 97.8 54 (5-FINC)C ★D(thiop)C ★ 35-20′-＞55 9.7 98.6 55 (CBO)C ★D(thiop)C ★ 35-20′-＞55 8.8 95.7 56 FmocKPC ★D(thiop)C ★ 45-20′-＞65 8.7 98.3 57 RC ★(AnB)(thiop)C ★ 2-20′-＞22 12.3 91.1 58 DC ★R(thiop)C ★ 2-20′-＞22 9.9 89.6 59 KPC ★D(thiop)C ★ 2-20′-＞22 10.7 88.7 60 (1-FCA)KC ★E(thiop)C ★ 45-20′-＞65 6.3 99.7 61 RC ★(O-Cys)(thiop)C ★ 2-20′-＞22 19.1 99.6 62 (1-FCA)C ★(thiop)DC ★ 55-20′-＞75 5.8 86.9 63 FmocRC ★N(thiop)C ★ 40-20′-＞60 11.0 99.0 64 RC ★(β-Ala)(thiop)C ★ 20-20′-＞40 10.1 84.7 65 R ★AD(thiop)D ★ 2-20′-＞22 12.8 89.4 66 (Anb) ★RAD(thiop)D ★ 658.25 2-20′-＞22 9.9 92.9
Gradient retention time compound number sequence mass spectrum
% purity
(％A-min-＞％B) (min) 67 (Anb) ★RVD(thiop)D ★ 5-20′-＞25 10.8 99.8 68 (1-FCA)K ★AD(thiop)D ★ 737.27 45-20′-＞65 6.4 94.7 69 G ★RAD(thiop)D ★ 630.19 2-20′-＞22 7.0 96.1 70 (1-FCA)K ★(dA)D(thiop)D ★ 40-20′-＞60 10.1 98.0 71 (1-FCA)Orn ★AD(thiop)D ★ 40-20′-＞60 8.5 98.8 72 (1-FCA)(dK) ★AD(thiop)(dD) ★ 40-20′-＞60 9.3 95.2 73 (1-FCA)(dK ★)(dA)(dD)(dP)(dD ★) 40-20′-＞60 8.1 96.4 74 (1-FCA)(dK ★)AD(thiop)D ★ 40-20′-＞60 8.5 97.2 75 G ★RVD(thiop)D ★ 2-20′-＞22 12.1 99.6 76 (Anb) ★R(D-Nal)D(thiop)D ★ 30-20′-＞50 9.3 96.0 77 (Anb) ★RFD(thiop)D ★ 15-20′-＞35 6.9 98.4 78 (1-FCA)K ★ADPD ★ 45-20′-＞65 6.5 99.9 79 (1-FCA)K ★D(thiop)D ★ 666.19 45-20′-＞65 6.3 98.1 80 G ★RAL(thiop)D ★ 15-20′-＞35 12.1 83.1 81 (Aib) ★RAD(thiop)D ★ 658.7 2-20′-＞22 10.3 97.4 82 G ★RFD(thiop)D ★ 10-20′-＞30 10.9 98.6 83 RGC ★D(thiop)C ★ 2-20′-＞22 8.4 97.8 84 G ★R[(3-Br)Tyr]D(thiop)D ★ 10-20′-＞30 12.2 99.6 85 G ★R[(3-Br)Tyr](thiop)C ★ 2-20′-＞22 7.6 97.6 86 G ★R[(3-pyridyl)A]D(thiop)D ★ 2-20′-＞22 12.0 98.9 87 (AMBA) ★RAD(thiop)D ★ 10-20′-＞30 10.8 92.2
Gradient retention time compound number sequence mass spectrum
% purity
(％A-min-＞％B) (min)?88 (1-FCA)C ★D(TIC)C ★ 689.06 50-20′-＞70 8.4 98.8?89 G ★MD(thiop)D ★ 2-20′-＞22 8.1 91.1?90 [(N-Me)R]C ★L(thiop)C ★ 621.05 15-20′-＞3?5 11.7 99.2?91 RC ★Y(thiop)C ★ 10-20′-＞30 12.6 98.0?92 (1-FCA)C ★D(thiop)C ★-NH2?644 55-20′-＞75 7.8 99.9
Said structure formula I wherein connects residue L 1And L 2Be not Cys, Z can not use for single bonded this structure yet.This moment, we found, L 1The residue that last either side exists can be through side chain or L 1Other functional group and the L that are not adjacent residue Cheng Jian of last existence 2Cheng Huan (through Z).Common residue L 2Through terminal functional group (being typically carboxyl) or side chain functional group Cheng Huan.
X 1The preferred residue in position comprises Gly, Phe, Leu, Ash, Val-, Tyr, 1-or 2-naphthalene third formic acid, cyclohexyl Ala-, AMBA, AnC, AnB, omega-amino-lower alkanols alkanoic acid, Aib-, Ser-, Tyr-Ash-, Ala-Thr-Val-and to chloro-Phe-.Y 1The preferred residue in position comprises-Ala ,-Ala-Ser ,-Ala-Ser-Ser ,-Ala-Ser-Ser-Lys ,-Ala-Ser-Ser-Lys-Pro ,-Thr ,-Thr-Phe ,-Aib ,-to chloro-Phe, AMBA, AnC, AnB, omega-amino--lower alkanols alkanoic acid, 1-or 2-naphthylalanine and-(cyclohexyl Ala).
When using R ' not to be the substituent X of hydrogen 2Or Y 2The time, during as acyl group R ' CO or formula R ' NH amino, to X 2Preferred substituted comprises by compound falls adamantanecarboxylic acid, the replacement of 3-methyl adamantane acetate deutero-as adamantane acetic acid, adamantanecarboxylic acid, 1-or 2-naphthyl acetic acid, 2-norcamphane acetate, 3-, and to Y 2Preferred substituted comprises 1-or 2-amantadine.Other suitable R ' group is as 9-fluorene acetic acid, 1-fluorenes formic acid, 4-fluorenes formic acid, 2-fluorenes formic acid, 9-fluorenes formic acid, benzene second, hydroxy succinic acid, quinardinic acid, formic acid, acetate, trifluoroacetic acid, cyclohexyl acetic acid and 3-thiohydracrylic acid deutero-group by rudimentary alkanamine, lower aryl amine or acid.Usually, X 2Select sour residue and Y 2Select basic group.
The derivative of formula I compound can be used for preparing antigen, thereby also can be used for preparing antibody.In many cases, this antibody itself can perhaps, during as anti-id AB, suppress cytoadherence or regulate immunocompetence by the retardance cell receptor effectively by the acceptor as stromatin or other part.
When implementing methods of treatment of the present invention, can be by any routine well known in the art and acceptable method, perhaps use separately or combine the active compound of using significant quantity and comprise its derivative or salt with another kind of or several The compounds of this invention or other medicament such as immunosuppressor, antihistaminic agent, corticosteroid etc., or the pharmaceutical composition that contains them as described below.These compounds or composition can by oral, hypogloeeis, part (as on skin or intraocular), suction or suppository, non-enteron aisle (as intramuscular, intravenously, subcutaneous, intracutaneous) or inhalation and use with solid or liquid dosage form such as tablet, suspension and aerosol form, hereinafter also will do more detailed discussion to this.When using, can use the single dose that arbitrarily adds in the single unit dosage of continuous treatment usefulness or the treatment for patient, unitary dose is at 1-3000mg.
But the pharmaceutical carrier solid, liquid or its mixture that are suitable in the preparation of pharmaceutical compositions of the present invention; Like this; this composition can be tablet, granula, capsule, pulvis, enteric-coated or have other protection preparation (as be attached on ion exchange resin or other carrier, or be wrapped in lipid or the lipoprotein capsule or add other end amino acid), continue releasing agent, easily being etched property preparation, implanting device or implant medicine, microspheres agent, solution (as the eye dropping liquid), suspension, elixir, aerosol etc.
Water, salt solution, D/W and ethylene glycol is preferred liquid vehicle, particularly to injection liquid (equity is oozed injection liquid).This carrier can be selected from following various oil, it comprises from oil, animal, plant or synthetic oil, for example peanut oil, soya-bean oil, mineral oil, sesame wet goods, appropriate drug vehicle comprise starch, Mierocrystalline cellulose, talcum, glucose, lactose, sucrose, gelatin, Fructus Hordei Germinatus, rice, flour, chalk, silica gel, Magnesium Stearate, sodium stearate ,-stearin, sodium-chlor, dried skimmed milk, glycerine, propylene glycol, water, ethanol etc.This composition can carry out the conventional medicine processing as sterilization also can contain conventional medicine additive such as sanitas, stablizer, wetting agent or emulsifying agent, the isotonic salt of adjusting, damping fluid etc.Appropriate drug carrier and preparation such as Martin, " Remington ' s Pharmaceutical Sciences ", 15th Ed.; Mack Publishing Co., Easton (1975); Referring to, pp.1405-1412 and pp.1461-1487.Described in.Usually, this composition should contain the active compound of significant quantity and the carrier of sufficient quantity, is suitable for the dosage forms that the host uses to make.
A preferred scheme is, when needs maybe may be when in case of emergency needing mitigation symptoms, to use methods of treatment of the present invention, another preferred scheme is when continuous treatment or prophylactic treatment, to use present method effectively.
When implementing methods of treatment of the present invention, depend on various situations for the concrete dosage of host's drug administration composition, it comprises kind, its severity, therapeutic regimen, host's age and physical appearance of disease or the like.Appropriate dosage can be determined according to the clinical consumption that field of medicaments is known.When through injection or during oral medication, operable dosage is per kilogram host body weight 0.1-100mg compound, generally per kilogram of body weight 1-100mg preferably.Local using dosage is the ingredients of repeatedly using every day every milliliters of liquid carrier or the minimum 0.1mg of the containing compound of vehicle.
The compounds of this invention and treatment or medicinal compositions can be used for combining disease or other studies on some in dications of and the treatment of regulating with part by integrin receptor, and this disease or symptom comprise and combine undesired (strong or not enough as crossing) diseases associated with cell and natural or other part.This type of disease or symptom can comprise diseases associated with inflammation such as rheumatic arthritis, asthma, transformation reactions, adult respiratory distress syndrome, enteritis (as ulcerative colitis and limitation joint enteritis) and ophthalmia; Autoimmune disorder, thrombosis or thrombocyte are not suitable for moving the disease that causes, and cardiovascular disorder; Inaccessible prevention after the thrombolysis; Tumor disease comprises metastatic tumor; Sterile and the embryo transfer that the fertilization inhibition causes; And when need strengthening cytoadherence, and aforesaid wound healing or prosthese are transplanted.
The compounds of this invention is found the diagnosis that also can be used for owing to the undesired disease that causes of cytoadherence, for example, white corpuscle and endotheliocyte or and blood vessel in adhesion between the extracellular matrix that exposes excessively all relevant with early atherosclerosis, like this, can there be the danger that causes vascular occlusion in adhesion over-drastic people between white corpuscle and endotheliocyte.When this hazardous condition is detected, at first can suppress combining between white corpuscle and endotheliocyte, determine whether to exist such danger by measuring combining between this compound and host's endotheliocyte then by measuring which kind of compound of the present invention.
In addition, the present invention also is found the diagnosis that can be used for the autoimmune disorder that caused by antibody, described antibody can combine with cell adhesion molecule or can with the receptors bind of cell adhesion molecule, for example, if a kind of disease is caused by a kind of antibody, and this antibody can be in conjunction with the cell adhesion molecule by the compound simulation of structure I, can carry out immunoassay catching this antibody to patient blood or serum by the structure I compound that use is attached on a kind of substrate so, thereby easily realize detecting the diagnostic test that this antibody exists.Fc conjugated protein (albumin A or Protein G) with the careful bacterium in source of the second antibody of anti-people's antibody Fc part of ability city ordinary method such as mark or applying marking can detect bonded antibody.Another kind of situation is, when structural formula I compound can be with the receptors bind of the antibody that causes disease, can use and strive immunoassay unexpectedly.In the case, with formula I compound mark and can measure on itself and the substrate and strive sexual reaction unexpectedly between receptor protein.
In addition, the derivative of The compounds of this invention also can be used for antigen and generates, this antigen can generate by peptide of the present invention and carrier proteins coupling, with this mixture with animal immune after, can produce the antibody of anti-this peptide, in some cases, these antibody itself are by the acceptor as stromatin or other cytoadherence part, perhaps, if anti-id AB also can suppress cytoadherence effectively or regulate immunocompetence by the retardance cell receptor.
In addition, The compounds of this invention also can be used for production matrix, can combine and have material than strong affinity with The compounds of this invention to be used for purifying.For example, this type of matrix can prepare by The compounds of this invention and deutero-chromosorb covalent bonds.To this concrete scheme of the present invention be, table 1 is listed contain free amine group cyclic peptide can with bromination nitrile activatory chromatography resin (by Pharmacia, Uppsala, Sweden, Cat.no.52-1153-00-AK) coupling.If necessary, can amino be incorporated in the required peptide by adding lysine residue or containing amino different base by adding another kind.In addition, certain available carbodiimide activatory resin combines with the cyclic peptide that has the carboxyl function base.
The coupled reaction of peptide can be carried out with the method that producer provided basically.And cyclic peptide-deutero-resin can be used for purifying can be with than high affinity and cyclic peptide bonded protein, polysaccharide or other material.This purifying can be realized with following process, being about to cyclic peptide-deutero-resin combines with the sample that contains the compound of desiring the purifying affinity, purification condition is to make it form specific complex, be attached to mixture on the resin with removing the solution washing that does not need material but do not damage mixture, with the solution washing resin that can dissociate this mixture, wash-out obtains desiring the material of purifying then.
Although can be used for still having provided preferable methods and raw material at this in the invention process or the test with similar or identical any method described herein and raw material, as indicated above, all disclosed contents all can be used as the reference of this paper in the reference.
Embodiment 1
Synthetic and the general formula of compound
" main chain " is that the part that the peptide bond of ring compound of the present invention connects is synthesized with the solid phase method of peptide synthesis usually; then; if necessary; only optionally remove the method Cheng Huan of protecting group in the residue when being used in into ring; by this method; peptide sequence can not be changed in the compound, perhaps increased, but this peptide can be aptly by Cheng Huan.Known other in field, end synthesizes and the cyclization method also can be used for preparing ring compound described herein and general formula compound.Except as otherwise noted, method generally can be used for the synthetic of peptide of the present invention described in the international open WO92/00995 (1992.1.23 is open) of PCT.
Therefore, peptide sequence can pass through the solid phase method of peptide synthesis (as BOC or FMOC method) in the The compounds of this invention, and liquid phase synthesizing method or other method well known in the art comprise that the combining method of aforesaid method is synthetic.Known and be widely used BOC and the visible following reference of FMOC method:
Merrifield，J.Am.Chem.Soc.，88：2149(1963)；
Meienhofer，“Hormonal?Proteins?andPeptides”，pp.48-267，C.H.Li，Ed.，AcademicPress(1983)；
Barany?et?al，in“The?Peptides”，pp.3-285，E.Gross?and?J.Meienhofer，Eds.，Academic?Press，New?York(1980).
Concrete synthetic embodiment
N, N-dialkyl group-arginine
With the synthetic N of ordinary method among the international open WO92/00995 (1992.1.23 is open) of this paper reference PCT, N-dimethyl-arginine, N, N '-dimethyl-arginine and N, N '-diethyl-arginine.
Modified arginic protection: above-mentioned gained raw material is suitable for the BOC-protection, and need not crystallization or other purge process.1 equivalent amino acid is dissolved in 1 equivalent 1N NaOH and the isopyknic diox.BOC-acid intoxicated (1.1eq) is dissolved in diox and in stirring at room 4 hours, this moment if necessary can to keep PH be 9 by adding 1N NaOH.Reaction is monitored with tlc, up to raw material disappearance (with triketohydrindene hydrate spraying colour developing).When reaction is finished, add acetate until PH to 5.Stir after 15 minutes, isolate product, with postlyophilization.Tlc system-methyl alcohol/ammonium hydroxide, dimethyl-arginic Rf was 0.6 in 1: 1, diethyl-arginic Rf is 0.8.
Proline analogs
(D, L)-thiazolidine-2-carboxylic acid, N-BOC-(D, L)-thiazolidine-2-carboxylic acid, N-BOC-(L)-5,5-dimethyl-thiazolidine-4-carboxylic acid, (L)-the synthetic of 2-methylthiazol alkane-4-carboxylic acid and N-BOC (L)-2-methylthiazol alkane-4-carboxylic acid be disclosed among the international open WO92/00995 (1992.1.23 is open) of this paper reference PCT.L-1,3-tetrahydrochysene thiazine-4-carboxylic acid method:
In the 5g homocysteine thiolactone hydrochloride (Sigma) in 75ml water, add 3ml 1N Hcl and 16ml 37% formalin (Aldrich).After 2 days, solution concentration is extremely done (bath temperature 40-50 ℃) in stirring at room.Product is dissolved in the 100ml 3 ethanol, and filtering also, vacuum concentration drips isopyknic ethyl acetate and makes mixture standing over night in refrigerator to 25ml.Canescence or brown crystallization obtain about 3.0g (productive rate 50.4%) with ethyl acetate washing and vacuum-drying.This crude product is dissolved in the heat 3 ethanol of minimum and cools off in ice bath, add isopyknic ethyl acetate behind the beginning crystallization, mixture cooled off in cryostat 4 hours.Collect the crystallization that generates, obtain 2gm L-1,3-tetrahydrochysene thiazine-4-carboxylic acid with ethyl acetate washing and vacuum-drying.Analyze: (butanols/acetic acid/water/pyridine 4: 1: 2: silica gel tlc 1): the Rf of product is 0.4 to system A.Fusing point: 208-210 ℃ decomposition. specific rotation [water]-14.03 ° d=1.N-BOC-(L)-1,3-tetrahydrochysene thiazine-4-carboxylic acid method:
To in 50ml water, add 3.8gm (BOC) among the 3gm L-1 in 17ml 1N NaOH and the 50ml diox (not containing amide), 3-tetrahydrochysene thiazine-4 carboxylic acid (16.3mmol) 2The 10ml dioxane solution of O (17.4mmol), with 1N NaOH make the PH of reaction remain on 8-9 and will react the stirring spend the night.The back discovery reaction of spending the night is incomplete, adds (BOC) again 2O (0.38gm) and with mixture restir 4 hours to reacting completely.With the reaction mixture vacuum concentration to half volume and with hexane extraction (2 * 50ml), remove hexane layer, water layer cools off with ice bath, uses 1N NaHSO 4Be acidified to PH3 also with 3 parts of 50ml ethyl acetate extractions.The refrigerative ethyl acetate layer wash with water (3 * 50ml), anhydrous MgSO 4Dry also filtration.Solvent removed in vacuo obtains the bright amber oily thing of spontaneous crystallization.Handle and the collection solid with a small amount of hexane,, obtain the 3.6gm pale solid product vacuum-drying.Analyze: silica gel tlc, system A; The Rf of product is 0.8.M.P.112°-113℃。Specific rotation in the acetone-162.4 °, d=1.Ultimate analysis C 10H 17N 1O 4S 1F.W.247; Calculated value: C, 48.58; H, 6.88; N 15.67.Measured value: C, 48.52; H 16.94; N 15.67.(L)-1,4-tetrahydrochysene thiazine-3-carboxylic acid
Step 1: in 1/2 hour, in 1.51 liquid ammonia solutions of sodium (16.8gm), add in batches the L-halfcystine (Sigma, 38gm), until blue look completely dissolve.(Aldrich, 56gm) also stirring is spent the night, and makes the ammonia evaporation slowly to add bromo ethanol in 45 minutes.Step 2: step 1 gained resistates is dissolved in the dense Hcl of 1500ml and is heated to 90-95 ℃, heated 7 hours.Solution for vacuum concentration is collected solid.Solid is made slurry and is filtered in the 1200ml Virahol, mother liquor is condensed into cream, filters and puts down filter cake in air drying.Step 3: step 2 gained solid (45gm) is dissolved in 1L DMF and adds the 750ml triethylamine.Mixture is concentrated into dried on rotatory evaporator in 90-95 ℃ of heating 2.5 hours then.Solid is dissolved in 1.5L water and handles on 800ml Amberlite IR-120 H+ resin column, and wash-out is until going out neutral products with 1.5N ammonium hydroxide aqueous solution wash-out.Product is concentrated into dried, water-soluble, handles, filter and slowly dilute with about 2ml acetone through Celite pad with carbon.Collect crystallization, use washing with acetone, dry air obtains required (L)-1 of 16.2gm, 4-tetrahydrochysene thiazine-3-carboxylic acid.
N-BOC-(L)-1,4-tetrahydrochysene thiazine-3-carboxylic acid
To being dissolved in 1: 1 (L)-1 among diox/1N NaOH, add (BOC) that is dissolved in the 10ml diox in 4-tetrahydrochysene thiazine-3-carboxylic acid (16gm) mixture 2O (24gm) makes PH remain on 9 and in the room temperature standing over night.With solution concentration to 1/2 volume and in suction filter with three parts of 50ml hexane extractions.Water layer is diluted with water to 300ml, cools off and uses NaHSO 4Be acidified to PH3.Collect solid, wash and use P with water 2O 5Drying gets the required N-BOC-(L)-1 of 22.4gm white powder, 4-tetrahydrochysene thiazine-3-carboxylic acid.
(Torrance California) has bought amino acid precursor by BACHEN.(st.Louis Mo) obtains DCC by Sigma company; (New York NY) obtains TFA by Halocarton company.(Fairlane NJ) obtains triethylamine by Fisher Scientific.4-methyldiphenyl methylamine resin is obtained by CBA Inc (Boulder.CO).Other reagent is the reagent of analytical pure or higher category.
The all available BOC/DCL chemical method of all peptides
(Baranyet al, The Peptides, E.Gross and J.Meienhofereds., Volume 2, Part A, Chapter 1, AcademicPress, Inc., 1-284 (1979)). (System 990, Beckman Instruments, Inc. by Beckman automatic peptide synthesizer, Palo Alto California) passes through solid phase method
(Barany et al, The Peptides, E.Gross and J.Meienhofer eds., Volume 2, Part A, Chapter 1, Academic Press, Inc., 1-284 (1979)) synthetic.Other activator also comprises DCC/HOBT
(D.L.Nyugen?and?B.Castro，＂Peptide?Chemistry＂，pp.231-238?ProteinResearch?Foundation?Press，Osaka(1987))or?BOPor?BOP/HOBt(D.Hudson，J.Org.Chem.，53：617-624(1988)).
The absorption reaction of N-BOC-S-(4-methyl-benzyl) halfcystine (BOC-Cys-(4-MeBzl)) on chloromethyl polystyrene resin (Merrifield resin) should be carried out general method peptide synthetic of (Horiki, Chem.Lett. (#2): 166-168 (1978)) synthesis of cyclic peptide in the presence of Potassium monofluoride: as described in Table 2 at BOC-Cys (4-MeBzl)-polystyrene resin that all uses in steps for preparing peptide with BOC amino acid (to C-end carboxylic acid) or 4-methyldiphenyl methylamine resin (to C-end methane amide).Relevant process can be referring to this paper reference International Patent Application WO 92/00995.In the end an amino acid by coupling after, can remove the terminal BOC protecting group of N-in 20 minutes by resin and TFA: DCM (1: 1) is mixed.Subsequently, resin is used DCM (3X), MeOH (2X), DCM (3X), washing successively, and resin is in air drying.Dissociate: by dissociating in stirring the peptide that takes off the BOC protection on can be with resin under-5-0 ℃ with the mixture of new steaming anhydrous HF (10ml/g resin), methyl-phenoxide (1ml/g resin) and dimethyl sulfide (0.5ml/g resin).After 1 hour, remove HF under reduced pressure, dissociated peptide/resin compound extracts with 80% acetic acid aqueous solution then with ether washing three times.The extraction liquid (200ml/g resin) that merges cooled off and proceed into the ring step.Cheng Huan: can form the intramolecular disulfide bridge with the iodine oxidation style
(Wunch et al, Int.J.Peptide Protein Res., 32:368-383 (1988), Bodanszky, Int.J.Peptide Protein Res., 25:449-474 (1985)). stir down and in the crude product peptide in 80% acetic acid aqueous solution, drip I 2Saturated glacial acetic acid solution, be fulvescent until solution.After 1 hour, add saturated aqueous ascorbic acid in stirring at room to remove excessive iodine.To become the peptide vacuum concentration of ring, resuspending is also lyophilize in water.Purifying: cyclic peptide can be with Vydac C is housed 18Post (15-20mm, 5 * 30cm ID) Waters Delta Prep 3000 systems (Waters, Milford, Massachwetts) purifying, (TEAP, PH2.3) the concentration gained linear gradient liquid in is made moving phase at 1% 3 second ammonium phosphoric acid salt with continuous increase acetonitrile.Collect suitable component, obtain the pure peptide of phos-phate forms.Peptide salt is handled with pillar once more and with the acetonitrile solution wash-out of 0.5% moisture HOAc, is obtained required acetate.Analyze: with Beckman C is housed 18(4.6 * 150mmID) Beckman System 126 (166 type detector) analyzes the peptide of purifying post for the extraordinary ball of RPC18,5 μ m.With the linear gradient liquid wash-out of buffer B, flow velocity is 1ml/min, (buffer A=0.1M sodium phosphate aqueous solution, PH4.4-4.5, the buffer A solution of buffer B=60% acetonitrile).Regulate the gradient elution time of every kind of peptide, should peptide be gone out when the gradient of nearly centre by wash-out at 20 or 30 minutes.The retention time of every kind of preferred peptide is as shown in preferred compound table (table 1).Fmoc removes: add piperidines (6ml) in the 24ml DMF solution that contains 1g FMOC peptide, obtain the DMF solution of 20% piperidines.Solution is in stirring at room 20 minutes, evaporated in vacuo then.Be dissolved in resistates in ethyl acetate/water (1: 1) and collect water layer, water layer is used ethyl acetate extraction twice again, filters and lyophilize.
Table 2
Solid phase method of peptide synthesis flow process (TFA deprotection/DCC coupling) step reagent volume ★(ml) time (min) 1 DCM wash (3x) 20 0.5 2 TFA-DCM (1: 1) 20 13 TFA-DCM (1: 1) 20 20 4 DCM wash (3X) 20 0.5 5 MeOH wash (2X) 20 0.5 6 DCM wash (3X) 20 0.5 7 TEA-DCM (1: 9) 20 18 TEA-DCM (1: 9) 20 59 DCM wash (3X) 20 0.510 MeOH wash (2X) 20 0.511 DCM wash (3X) 20 0.512A BOC-AA (3.2mM
2?equiv.)in?DCM
(DMF) ★ ★1012B DCC in DCM (0.5M) 6.4 120 ★ ★ ★13 DCM wash (2X) 20 0.514 DCM-MeOH (1∶1) (2X) 20 0.515 TEA-DCM (1∶9) 20 0.516 MeOH wash (2X) 20 0.517 DCM wash (3X) 20 0.518 Ac20 in DCM (1∶3) 20 2019 DCM wash (3X) 20 0.520 MeOH wash (2X) 20 0.521 DCM wash (3X) 20 0.5 *To the volume of volume when replacing the 0.8mM/g resin synthetic with the 2g resin. *When BOC-AA is insoluble in pure DCM, add DMF. * *When showing that through ninhydrin reaction coupled reaction is incomplete, be necessary with 1: 1: 3
Amino acid, the bop reagent in DMF and DIEA mixture again
Inferiorly carried out coupled reaction 1 hour.After the coupled reaction, resin filter is also used indenes three
Whether the ketone test detects coupled reaction complete.If not exclusively, then repeat this process,
Show that until test coupled reaction is complete.
The ring compound that amido linkage connects
In the synthetic present embodiment of (side chain-side chain key connects), will synthesize following formula: compound:
Figure A9411599500571
All amino acid and amino acid derivative all buy from BACHEM (Torrance, California).9-fluorenyl methyl alcohol and DDC derive from Sigma Chemical Co. (St.Louis, Missouri), diisopropyl ethyl amine and 4-(dimethylamino) pyridine derive from Aldrich (Milwaukee, Wisconsin).Except as otherwise noted, other reagent is analytical reagent, and need not to be further purified.
All residues all are with connecting through solid phase method in the BOC protection.The side chain carboxyl group of Asp and Glu can be protected becomes the fluorenyl methyl esters and the alpha-amino group of the epsilon-amino of Lys and Gly can the protected N-FMOC of one-tenth.Lactam bridge between two side chains (on Asp and the Lys) can synthesize when peptide is adsorbed in resin.This method is shown in accompanying drawing 1a.
(a) the N-BOC-O-9-fluorenyl methyl ε of aspartic acid and L-glutamic acid-
The preparation of ester
The N-BOG-O-9-fluorenyl methyl ε-ester of aspartic acid and L-glutamic acid can be according to the described general method of the Bolin that has done some modification preparation (people such as Bolin, Organic Preparations and Procedures (Intern., 21:67-74 (1989)).N-BOC-O β-9-fluorene methyl aspartate
With 8.31g (25.7mmol) N-BOC-O α-benzyl aspartate and 4.80g (25.4mmol) 9-fluorenyl methyl alcohol is dissolved among the 150ml DCM.Solution cools off in ice bath, in batches to wherein adding 30mg (0.24mmol) 4-(dimethylamino) pyridine, adds 5.31g (25.7mmol) DCC subsequently in 10 minutes.The gained mixture stirred 1 hour under the continuation cooling, removed by filter the N that is settled out, N '-dihexyl urea, and filtrate is diluted with 250ml DCM.This solution with (using successively) 10% citric acid (2 * 50ml), H 2O (1 * 50ml), 25%NaHCO 3(2 * 50ml), H 2O (1 * 50ml), salt solution (1 * 50ml) extraction, solution MgSO then 4Drying, concentrate an oily resistates.Recrystallization in methanol/sherwood oil (1: 3: 10) obtains 10.85g (84%) N-BOC-O α-benzyl-O β-fluorenyl methylaspartic acid ester, fusing point are 74-77 ℃.The above-mentioned product of 5.5g (10.9mmol) is dissolved in the 150ml hot methanol, uses 300mg 20%pd (OH) down in room temperature 35-40psi pressure 2/ C hydrogenation 1.5 hours, filtering catalysis is sharp and solution for vacuum evaporated.Remaining oily matter is dissolved in the 200ml diethyl ether again, with (using successively) 1%NaHCO 3(3 * 50ml), H 2O (1 * 50ml), 5% citric acid (2 * 50ml) and salt solution (1 * 50ml) extraction, ether layer MgSO 4Dry and concentrated.By recrystallization in diethyl ether/sherwood oil, obtain 3.53g N-BOC-O β-9-fluorene methyl aspartate, fusing point are 135-137 ℃.N-BOC-O-fluorene methyl-glutamate (γ ester)
With N-BOC-O α(4.5g, 13.3mmol) (2.5g 12.5mmol) is dissolved in 100ml DCM to-benzyl glutamate, and solution stirs cooling in ice bath with 9-fluorenyl methyl alcohol.In solution, add 15.5mg (0.13mmol) 4-(dimethylamino) pyridine and 2.75g (13.3mmol) DCC, the gained mixture was stirred 4 hours under the continuation cooling.The N that filtering is settled out, N '-dicyclohexylurea (DCU), filtrate is diluted with 200ml DCM.Solution is used with method identical described in above-mentioned aspartate extraction and is handled, and obtains N-BOC-O α-benzyl-O-fluorene methyl glutamate (γ ester) (4.2g), fusing point is 97-99.5 ℃.Room temperature and 40psi in 200ml MeOH/EtOH/IPA (2: 1: 1) mixture filter reaction mixture to remove catalyzer down with 125mg 10%pd/C hydrogenation 2 hours with the above-mentioned product of 4.0g (7.75mmol), concentrate the oily resistates.Resistates mixes with the 150ml ether, and the water layer that merges is used extracted with diethyl ether (2 * 40ml) again.The ether layer MgSO that merges 4Drying, filter and concentrate a white thing.With crude product resistates recrystallization in ether/sherwood oil (1: 10), obtain N-BOC-fluorene methyl glutamate (γ ester) (2.3g), fusing point is 123.5-126 ℃.
(b) Bao Hu RK*D (thiop) D* peptide sequence is synthetic
Above-mentioned peptide synthetic can by unite use the automatic peptide synthesizer (System 990, Beckman Instruments, Inc., Palo Alto, California) and manually (S.C.Glass Tech, Bonica California) carry out the peptide synthesizer.By AppliedBiosystems (Foster City, the BOC-Asp that California) obtains (OFm) OCH 2(1.0g is 0.75mmol) as material resin for-PAM resin.Use following amino acid: BOC-(3-thiop), BOC-Asp (O-benzyl), BOC-Lys (N in synthetic ε-FMOC) and BOC-Ary (N δ-tos).All use excess of ammonia base acid (2-3 doubly) in per step coupled reaction.Peptide chain can be in the Beckman peptide synthesizer constitutes with the BOC raw material, is doing under the situation of suitable adjustment according to progressively adding every seed amino acid to standard rating cycle similar shown in the table 2.The DCM solution of the DCM solution of the DCM solution of 50%TFA, 5%DIEA and 0.5M DCC is used separately as deprotection agent, neutralizing agent and activator in per step coupled reaction.
(c) sealing of peptide sequence
After the DCM solution with 50%TFA has removed the BOC group of Arg N-end, with the DCM solution neutralization of 5%DIEA, with protected peptide on the resin and activatory AdaCA reaction.On the resin N-end by deprotection, the protected peptide of side chain with MeOH (2 * 1min), (3 * 1min) washings are with 5%DIEA and DCM (1 * 1min, 1 * 20min) neutralization for DCM; (3 * 1min) washings are also used (AdaCA), DCC and HoBt end-blocking in DMF with DCM.Then, according to this peptide of following general method by between the epsilon-amino of the β-carboxyl of Asp and Lys, forming amido linkage by cyclization.
(d) the general one-tenth ring method of formation lactam bridge
Behind the peptide chain formation, the amidation annulation can carry out as follows, all filters between following per step: (1) MeOH (2 * 1min); (2) DCM (3 * 1min); The DMF solution of (3) 20% piperidines washed 1 minute and deprotection 20 minutes; (4) DMF (2 * 1min); (5) MeOH (2 * 1min); (6) DCM (3 * 1min); (7) DMF (20ml/g resin) solution of bop reagent (4 equivalent) stirred 2 minutes and adds DIEA (20%DMF volume), stir 4 hours (whether complete with the ninhydrin reaction monitoring reaction; If judge in the time of 4 hours that reaction is incomplete, then reaction is proceeded, and is negative until ninhydrin reaction); (8) DMF (2 * 1min); (9) DCM (2 * 1min); (10) MeOH (2 * 1min).
In the presence of 10% methyl-phenoxide, handled 1 hour with HF in 0 ℃, with final ring compound by dissociateing on the resin.After steam removing HF, resistates is with the ether washing and with the H of 5%HOAc 2The O extracting resin.With the water extract lyophilize, obtain the crude product peptide.
(e) purifying
With C is housed 18(MA) purifying compounds is made moving phase with constantly increasing the concentration gained linear gradient liquid (PH2.2-2.4) of acetonitrile in TEAP for Waters, Milford in Waters Delta Prep 3000 systems of post.Compile the component of collecting that contains pure compound and use C once more 18Post is handled, and use the 0.5%HOAc wash-out this moment, and sample is transformed into required acetate form by the phos-phate forms of peptide.The component that will contain pure peptide is compiled, vacuum concentration, and soluble in water again and lyophilize obtains 92.9mg peptide white powder, and HPLC purity is 98.7%.
Synthesizing of the ring compound that amido linkage connects
(major key-side chain key connects)
In this embodiment, will synthesize following formula: compound:
Figure A9411599500611
Above-claimed cpd is by CBA, and (Boulder, Colorado) (2.0g, 1.4mmol) operation is synthetic by hand in beginning for gained 4-methyldiphenyl methylamine resin for Inc..With the foregoing description 4 compounds synthetic described in the synthetic peptide chain of BOC method, use BOC-Asp (Fm), BOC-1.4-TTC, BOC-Asp (O-benzyl), BOC-Val, BOC-Arg (N-tosyl) and N-FMOC-Gly in synthetic.
Gly terminal amino group and Asp 6Annulation between terminal β-carboxyl can become the ring method to carry out by above-mentioned general acid amides.
Handle with HF and 10% methyl-phenoxide in 0 ℃ and ring compound to be dissociateed on by resin in 1 hour.Subsequently, steam and remove HF, mixture is with ether washing (removing ether layer) and use 1N HOAc extraction.With the water extract lyophilize, obtain 1.23g crude product compound.
According to the described method of the foregoing description, with C is housed 18The Waters of post prepares HPLC system this compound of purifying, obtains the 678mg pure compound, white powder, and HPLC purity is 99.7%.
Synthesizing of the ring compound that amido linkage connects
(Cheng Huan before chain has synthesized)
In the present embodiment, will synthesize following formula: compound (SEQ.ID.NO.:18):
This compound can be applied for the preparation of method described in the WO92/00995 (1992.1.23 is open) with PCT.
Synthesizing of ring-type disulfide
In this embodiment, will prepare following formula: compound (SEQ.ID.NO.:4):
(Milwaukee WI) has bought 1-FCA by Alrich Chemical Company.(Torrance Califoraia) has bought by BACHEM for all amino acid, amino acid derivative and analogue and alpha-non-natural amino acid.DCC must in Sigma Chemical Co. (St.Louis, Missouri), trifluoroacetic acid must be in Halocarbon Co. (NewYork, New York), triethylamine must be in Fisher Scientific (Fair Lawn, New Jersey).Other reagent all can be obtained and is analytical reagent by routine source.
The all available BOC raw material of all peptides Beckman automatic peptide synthesizer (System 990, Beckman Instruments, and Inc., Palo Alto, California) synthetic through solid phase method.
The attachment reaction of N-BOC-S-P methyl-benzyl benzyl Gelucystine (BOC-Cys (4-MeBzl)) on chloromethyl polystyrene resin (Merrifield resin) should be carried out in the presence of Potassium monofluoride.In the presence of KF (1.8 molar equivalent) in 80 ℃ with BOC-Cys (4-MeBzl) (0.9 molar equivalent) and swollen Merrifield resin (Bio-Radlab., Richmond, California) (1.0 molar equivalent) reacted 16 hours in DMF, then with resin filter, washing and dry.By gravimetry than the mole number that records substituent on the resin.Peptide chain on BOC-Cys (4-MeBzl) resin is handled with BOC method substep successively according to the method in the above-mentioned table 2, last, remove the terminal BOC protecting group of N-30 minutes with TFA:DCM (1: 1).
Then with HF with compound by dissociateing on the resin, make disulphide as mentioned above and use the ordinary method purifying.
Contain C-terminal NHNR ' (particularly AMP) or
Synthesizing of the cyclic peptide of tetrazolium
Usually C-terminal is changed into NHNR ' by acid amides and the peptide modified, particularly has the compound of C-terminal AMP derivative, or wherein the peptide that replaced by tetrazolium of C-terminal can be used the method for preparing other peptide of the present invention is done to change to prepare slightly.In preparation during this compounds, in the cyclic peptide except C-terminal amino acid (promptly except Y 1Part in addition) is can be with above-mentioned solid phase method synthetic and by dissociateing on the resin, then with liquid phase method with this peptide and the coupling of suitable deutero-C-terminal amino acid.This method is referring to this paper reference-disclosed PCT patent application W92/000995.The details of liquid phase method of peptide synthesis can be referring to Boolanszky, " Peptide Synthesis ", Springer-Verlag, New York (1984).
Wherein α-carboxylic acid can be carried out according to the described method of Langry (Langry, K.C., J.Org.Chem.56:2400-2404 (1991)) by amino acid whose the synthesizing that tetrazolium replaces.
Embodiment 2 cytoadherence inhibition tests: U937 cell Zeta protein adhesion test
Following test is in representational vitro system The compounds of this invention to be suppressed the cytoadherence activity to test, and this test is the competitive trials under Fiberonectin and test compound all exist.At first, the droplet plate is coated with Zeta protein, with the concentration that increases gradually peptide and the known cell of being tested that contains the Zeta protein acceptor added together then.With the cell count that this droplet plate is cultivated, cleaned and dyeing is adsorbed with calculating.This test can directly record the active and adhesion conditioning agent activity of anti-cell adhesion of The compounds of this invention.
Clone U937 buy from American Type Culture Collection (Rockville, MD).Cell cultures is to contain RPMI substratum (the J.R.Scientific Company of 10% foetal calf serum, Woodland Hills, CA) carry out in, Zeta protein is by purifying out in the human plasma according to the described method of people such as Engvall (Int.J.Cancer, 20:1-4 (1977)).
(the 96-well Falcon) coats and spends the night with the droplet plate with the phosphate buffered salt solution (PBS) of 0.1ml 5 μ g/ml Zeta proteins in 4 ℃.In contrast, Xiang Jingzhong adds 5 μ g/ml bovine serum albumins (BSA).Remove on the plate not conjugated protein with the PBS cleaning.In order to block the unreacted position, coat 1 hour titer plate in 37 ℃ of PBS solution with 100 μ l 2.5mg/ml BSA, collect the U937 cell and clean twice, calculate cell count and in Eagle substratum (DMEM) that Dulbecco modifies and BAS (2.5mg/ml), transfer to 2.5 * 10 with Hanks balanced salt solution (HBSS) 6Cell/ml carries out cell in conjunction with test.Test compound is dissolved among the DMEM-BSA and PH is transferred to 7.4 with 7.5% sodium bicarbonate.With 500,250,125,62.5,31.3,15,6,7.8,3.9,1.95 and the concentration of ultimate density 0.98 μ g/ml.This compound (100 μ l) is added in the titer plate well of FN-coating, testing used concentration is to regulate according to the effectiveness of peptide.U937 cell (100 μ l) is added in each well and the droplet plate was cultivated 1 hour in 37 ℃.After the cultivation, this plate cleans once with PBS and solidifies the cell of absorption with the PBS solution of 3% Paraformaldehyde 96, with the 3.7% formaldehyde solution dyeing of 0.5% toluidine blue, under room temperature cell dyeing is spent the night.With the optical density (OD) of toluidine blue staining cell under the vertical road spectrophotometric determination 590nm with the calculations incorporated number (VMAX Kinetic MicroplateReader, Molecular Devices, Menlo Park, CA).This method is to the improvement row method of disclosed method (seeing people such as Cardarelli, PNAS-USA 83:2647-2651 (1986)) formerly.Jurkat-CS-1 adhesion test (Jurkat-24 test)
According to currently known methods (Pierschbacher, PNAS-USA 80:1224 (1983)) with the crosslinked 3-of two different functional groups (2-pyridyl disulfide group) propionic acid N-hydroxyl succinic diamide ester (SPDP) (Sigma, St.Louis MO) is fixed in CS-1 deutero-peptide CLHPGEILDVPST (SEQ.ID.NO.:26) and CLHGPIELVSDPT (SEQ.ID.NO.:27) on the droplet plate.Say simply, with 20 μ g/ml human serum albumin (HSA) the droplet plate was coated 20 hours, handled 1 hour with 10 μ g/ml SPDP then in room temperature.After removing the not binding reagents of putting, add and contain the halfcystine of peptide and make it in 4 ℃ of crosslinked spending the night.Second day, except personnel selection T-leukon Jurkat, as carrying out the test of this cytoadherence described in " U937 cell Zeta protein adhesion test ".Jurkat-endotheliocyte adhesion test
Following test is in representational vitro system The compounds of this invention to be suppressed the cytoadherence activity to test.This test is used to be determined at the adhesion effect between following T-clone Jurkat of test compound existence and endothelial cell monolayer, test compound is added with the concentration that increases gradually with the T cell, and it is added on the endothelial cell monolayer.The also percentage ratio of calculations incorporated cell is cultivated, cleaned to plate.Active and the adhesion conditioning agent activity of cytoadherence inhibitor of The compounds of this invention can be directly measured in this test.
S-generation Human umbilical vein endothelial cells derive from Clonetics (San Diego, CA).(Clonetics, San Diego cultivate in flask CA) in that the EGM-UV substratum that has replenished 10% foetal calf serum is housed with cell, this flask in advance with 0.5% pigskin gelatin coat (Sigma, St.Louis, MO), took a tonic or nourishing food to build up one's health in the every 2-3 of this cell days once, in the time of 4-6 days, be fused to.Detect cell to the antigenic effect of Factor IX, the result shows that in the 12nd generation, cell is positive to this antigen, and the endotheliocyte that surpassed for 12 generations does not use.
T-clone Jurkat derives from American Type Culture Collection and it is cultivated in the RPMI that contains 10% foetal calf serum.This cell cleans twice and is suspended among Dulbecco ' s Minimal Eagle ' the s Media (DMEM) that contains 2.5mg/ml human serum albumin (HSA) again with HBSS.Jurkat cell (1 * 10 6Individual cell/ml) (Sigma, St.Louis, the HSSS solution-dyed that contains 5% foetal calf serum MO), cell dyeed 15 minutes in the darkroom under room temperature, clean twice, and resuspending were in DMEA-HSA solution with 10 μ g/ml diacetic acid fluoresceins.
To knit monolayer endothelial cell 0.1ng/ml (50U/ml) recombinant chou IL-1 (Amgen of the fusion of growing in the culture plate in the 96-well group down in 37 ℃, Thousand Oaks, CA) excite 4 hours, after the cultivation, monolayer cell cleans twice with HBSS and adds 0.1ml DMEM-HSA solution.With Jurkat cell (5 * 10 5Individual cell) combines with the peptide of suitable concentration and 0.1ml Jurkat cell-peptide mixt is added in the monolayer endothelial cell.Generally, the experimental concentration of peptide is 250,50,10 and 2 μ M.For several virtuous peptides, test records the IC of this peptide 50Be 50,10,2 and 0.4 μ M.The droplet plate was put in 5 minutes on ice, makes the Jurkat cell settlement and this plate was cultivated 20 minutes in 37 ℃.After the cultivation, monolayer cell cleans twice with the PBS that contains 1mM calcium chloride and 1mM magnesium chloride, and (IL) reading is measured the fluorescence in each hole to this plate with absolute flat fluorescent for Baxter, Mundelein with Pandex fluorescence concentration analyzer.Percentage degree of adhesion under peptide do not existed is decided to be 100%, calculates the percentage degree of adhesion of peptide under existing.Monolayer is fixed and with microscopy assessment adhesion degree with 3% Paraformaldehyde 96.
Jurkat replaces the U937 extracellular except personnel selection T-leukon, and the test of this cytoadherence is carried out as described in above-mentioned " U937-FN test ".U937 cytoadherence test-results to Zeta protein
Render a service is to represent with μ M office herein, in this test IC 50Be lower than 500 μ M and think that activity is arranged.This being should be understood to need reach 50% compound that suppresses adhesion with higher mole number still has activity, but generally its use is little, because when using in giving human body, needs higher dosage.The compound that activity is lower than 10 μ M is a preferred compound, and what be lower than 100 μ M is not preferred, is lower than 500 μ M and is more preferred and be least preferred greater than 500 μ M.
Therefore, the invention provides the compound that in regulating cytoadherence, has remarkable effectiveness, include but are not limited to the cytoadherence that suppresses with Zeta protein.The data that provided show that also the compound that does not contain the RGD sequence is to FN adhesion effective inhibitors.Jurkat-endotheliocyte adhesion test-results
This test-results is for suppressing the adhesion test result between Jurkat cell and IL-1 activated endotheliocyte.In this test, set IC 50Be lower than 250 μ M for activity (A) is arranged, and IC 50＞250 μ M are non-activity.The above is not IC 50In fact the compound of＞250 μ M does not have activity, but IC 50Lower compound, they do not have enough effectiveness when the people is used.
Therefore, the invention provides the compound that in regulating cytoadherence, has remarkable effectiveness, include but are not limited to the adhesion that suppresses T-cell and endotheliocyte.The specific target acceptor of the special receptor that this relates on and this test compound include but not limited to, the α on the white corpuscle 4β 1, α 4β 7With the VCAM-1 on the endotheliocyte.The data that provided show that the compound that does not contain the RGD sequence is effective cell-cell adhesion inhibiting compounds.
Under tabulate and provided each compound of the present invention gained result in above-mentioned three tests in 3.
The position of selected compounds in sequence table is as follows in the table 3:
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).
Table 3CMPD sequence IC 50(μ M)
FN-U937 Jrkt-EC Jrkt-α 41 C ★(NIC Lys) GDSpC ★＞500 nda nd 2 C ★R (Sar) DSPC ★＞500 nd nd 3 C ★RGNSPC ★＞500 nd nd 4 RC ★NPC ★17 116 21 5 FmocKC ★DPC ★＞500＞200 nd 6 KC ★DPC ★712＞10 nd, 7 FmocAC ★D (thiop) C ★585 5 nd, 8 FmocFC ★D (thiop) C ★＞500 6 nd 9 FmocHC ★D (thiop) C ★＞500 190 nd10 FmocLC ★D (thiop) C ★＞500 8 nd11 FmocRC ★A (thiop) C ★25 15 nd12 FmocRC ★L (thiop) C ★＞500 34 nd13 FmocRC ★F (thiop) C ★15 18 nd14 AC ★D (thiop) C ★124 17 315 RC ★A (thiop) C ★25 37 416 LC ★D (thiop) C ★＞500 15 1.317 FC ★D (thiop) C ★388 46 0.8518 RC ★F (thiop) C ★37 65 919 HC ★D (thiop) C ★637 25 4.720 RC ★L (thiop) C ★69 55 421 FmocGC ★D (thiop) C ★320 1 ndCMPD sequence IC 50(μ M)
FN-U937 Jrkt-EC Jrkt-α 422 FmocpC ★D (thiop) C ★129＜2 2423 AdaC ★D (thiop) C ★＞500 2 0.10424 F7mocAC ★A (thiop) C ★＞500 5 0.57225 (1-FCA) AC ★D (thiop) C ★460＜.4 0.926 (1-FCA) C ★D (thiop) C ★＞500 2 0.927 (1-FCA) RC ★[(p-Cl) Phe] be C (thiop) ★＞500 5 nd28 GC ★D (thiop) C ★57 49 2.829 C ★GKGESPC＞500 nd nd30 (1-FCA) are C (sar) ★D (thiop) C ★278＜0.4 43l (thiop) C ★D (thiop) C ★51 25 0.25532 PC ★D (thiop) C ★433 9 0.72933 (Sar) C ★D (thiop) C ★119＞50 3.434 FmocRC ★(thiop) C ★＞500＞50 nd35 (1-FCA) are C (thiop) ★D (thiop) C ★27 1 nd36 FmocK (AdaC) ★D (thiop) C ★＞500 13 nd37 FmocRC ★(β D) be C (thiop) ★＞500 4 0.30638 FmocK (AnB) C ★D (thiop) C ★＞500 1 nd39 (N-MeR) C ★D (thiop) C ★2 24 240 C ★RGA[(p-Cl) Phe] (thiop) C ★＞500 50 nd41 AdaGGC ★RGA[(p-Cl) Phe] (thiop) C ★L＞50 140 nd42 C ★AGD[(p-Cl) Phe] (thiop) C ★＞500＞50 nd43 AdaGGC ★AGD[(p-Cl) Phe] (thiop) C ★＞500＞50 ndCMPD sequence IC 50(μ M) FN-U937 Jrkt-EC Jrkt-α 4 44 FmocRC ★(AnB) (thiop) C ★21 4 nd, 45 FmocRC ★(AiB) (thiop) C ★＞500＞50 nd 46 FmocRC ★V (thiop) C ★31 26 nd, 47 FmocRC ★[(β-CN) A] be C (thiop) ★＞500＞250 nd 48 (GAC) C ★D (thiop) C ★460＞50 nd 49 (DTC) C ★D (thiop) C ★＞500 2 nd 50 C ★GRGA[(p-Cl) Phe] (thiop) C ★＞500＞50 nd 51 FmocC ★D (thiop) C ★＞500 8 0.315 52 (TCA) C ★D (thiop) C ★656 18 0.097 53 (1-FCA) GC ★D (thiop) C ★603 0.6 nd 54 (5-FINC) C ★D (thiop) C ★＞500 23 nd 55 (CBO) C ★D (thiop) C ★＞500 1 nd 56 FmocKPC ★D (thiop) C ★＞500 2 nd 57 RC ★(AnB) (thiop) C ★67 8 nd, 58 DC ★R (thiop) C ★594＞50 nd, 59 KPC ★D (thiop) C ★603＞50 nd 60 (1-FCA) KC ★E (thiop) C ★＞25 2 nd 61 RC ★(O-Cys) (thiop) C ★＞50 32 nd 62 (1-FCA) C ★(thiop) DC ★＞50＞50 nd 63 FmocRC ★N (thiop) C ★20 15 nd, 64 RC ★((thiop) C of β-Ala) ★119＞50 nd, 65 R ★AD (thiop) D ★＞50＞250＞250
IC 50(μM)
Sequence C MPD FN-U937 Jrkt-EC Jrkt-α 466 (Anb) ★RAD (thiop) D ★21＞250 5167 (Anb) ★RVD (thiop) D ★12＞250 5668 (1-FCA) K ★AD (thiop) D ★＞50＞500 10669 G ★RAD (thiop) D ★＞250＞25070 0.088 (1-FCA) K ★(dA) D (thiop) D ★＞50＞250 nd71 (1-FCA) Orn ★AD (thiop) D ★＞50＞250 nd72 (1-FCA) (dK) ★AD (thiop) (dD) ★＞50＞250 nd73 (1-FCA) (dK ★) (dA) (dD) (dP) (dD ★)＞50＞250＞25074 (1-FCA) (dK ★) AD (thiop) D ★＞50＞250 nd75 G ★RvD (thiop) D ★0.198＞250＞10076 (Anb) ★R (D-Nal) D (thiop) D ★56＞250 nd77 (Anb) ★RFD (thiop) D ★15＞250 nd78 (1-FCA) K ★ADPD ★＞50＞250 nd79 (1-FCA) K ★D (thiop) D ★500＞250 nd80 G ★RAL (thiop) D ★14＞250＞10081 (Aib) ★RAD (thiop) D ★73＞250 nd82 G ★RFD (thiop) D ★0.445＞250＞10083 RGC ★D (thiop) C ★170 111 884 G ★R (3-Br-Tyr) D (thiop) D ★4＞250 (d) b nd85 G ★R (3-Br-Tyr) is C (thiop) ★＞50＞250 nd86 G ★R[(3-pyridyl) A] D (thiop) D ★12 223 nd87 (AMBA) ★RAD (thiop) D ★268＞250 nd
IC 50(μ M) CMPD sequence FN-U937 Jrkt-EC Jrkt-α 488, (1-FCA) C*D, (TIC) C*＞50 11 nd89 G*MD, (thiop) D*＞50＞250 nd90 [, (N-Me) R] C*L, (thiop) C* 6 11 nd91 RC*Y, (thiop) C* 20 250 nd92, (1-FCA) C*D, (thiopP) C*-NH2＞100＞100* 64a does not do b＞250, (d) monolayer endothelial cell breaks away under 250 μ M or bigger concentration.
For described the present invention, the various variations that raw material in the foregoing invention and used preparation and using method are made are conspicuous to those skilled in the art, and therefore, these variations should be believed to comprise in the scope of the invention that limits in described claim.
Sequence table (1) physical data
(ⅰ) applicant: Chang, Shiu-Lan N.
Carderelli，Pina?M.
Lobl，Thomas?J.
(ⅱ) denomination of invention: peptide inhibitors of cell adhesion
(ⅲ) sequence number: 28
(ⅳ) contact address:
(A) address: Birch, Stewart, Kolasch ﹠amp; Bireh,
(B) street: P.O.Box 747
(C) city: Falls Church
(D) state: Virginia
(E) country: USA
(F)ZIP：22040-3487
(ⅴ) computer-reader form:
(A) medium type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: Patent In Release#1.0, Version#1.25
(ⅵ) present request for data:
(A) application number: US 08/001,773
(B) applying date: 08-JAN-1993
(C) classification:
(ⅷ) proxy/agency:
(A) name: Murphy Jr., Gerald M.
(B) registration number: 28,977
(C) pair is rolled up/number of putting on record: 485-103P
(ⅸ) telephone number
(A) phone: 703-241-1300
(B) fax: 703-241-2848
(C) fax: 248345 (2) SEQ ID N0:1:
(ⅰ) sequence signature:
(A) length: 10 amino acid
(B) type: amino acid
(C) chain: strand
(D) topology: straight chain
(ⅱ) molecule type: peptide
(ⅲ) hypothesis: do not have
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: peptide
(B) position: 1..10
(D) out of Memory :/mark=peptide
Cell recognition position ＂ in/note=＂ Zeta protein
(ⅹ ⅰ) sequence description: SEQ ID NO:1:
Gly?Pro?Glu?Ile?Leu?Asp?Val?Pro?Ser?Thr
1 5 10(2)SEQ?ID?NO：2：
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(C) chain: strand
(D) topology: straight chain
(ⅱ) molecule type: peptide
(ⅲ) hypothesis: do not have
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: peptide
(B) position: 1..5
(D) out of Memory :/mark=peptide
/ note=＂ α-4-β-1 integrin identification polypeptide ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:2:
Glu?Ile?Leu?Asp?Val
1 5(2)SEQ?ID?NO：3：
(ⅰ) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(C) chain: strand
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: peptide
(B) position: 1..6
(D) out of Memory :/mark=peptide
Retardance peptide ＂ in the irritated rechallenge of/note=＂ contact
(ⅹ ⅰ) sequence description: SEQ ID NO:3:
Gly?Arg?Gly?Asp?Ser?Pro
1 5(2)SEQ?ID?NO：4：
(ⅰ) sequence signature:
(A) length: 4 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: decorating site
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ with 1-FCA, Ada, GAc, TDC, Fmoc,
5-FINC, CBO or the sarkosine ＂ that derives
(ⅸ) feature:
(A) name/keyword: decorating site
(B) position: 3
(D) out of Memory :/label=modification _ aa
/ note=＂ Thioproline ＂
(ⅸ) feature:
(A) name/keyword: decorating site
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ C-end can be by amidation, as changing in table 1 and 3
Compound 92, or as spreading out with AMP in the claim 8
The terminal ＂ of the C-that gives birth to
(ⅹ ⅰ) sequence description: SEQ ID NO:4:
Cys?Asp?Xaa?Cys
1(2)SEQ?ID?NO：5：
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: peptide
(B) position: 1..7
(D) out of Memory :/mark=peptide
Compound 3 ＂ in/note=＂ table 1 and 3
(ⅹ ⅰ) sequence description: SEQ ID NO:5:
Cys?Arg?Gly?Asn?Ser?Pro?Cys
1 5(2)SEQ?ID?NO：6：
(ⅰ) sequence signature:
(A) length: 8 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: peptide
(B) position: 1..8
(D) out of Memory :/mark=peptide
Compound 29 ＂ in/note=＂ table 1 and 3
(ⅹ ⅰ) sequence description: SEQ ID NO:6:
Cys?Gly?Lys?Gly?Glu?Ser?Pro?Cys
1 5(2)SEQ?ID?NO：7：
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: decorating site
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ fenclonine ＂
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 6
(D) out of Memory :/label=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:7:
Cys?Ala?Gly?Asp?Phe?Xaa?Cys
1 5(2)SEQ?ID?NO：8：
(ⅰ) sequence signature:
(A) length: 9 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 7
(D) out of Memory :/mark=modification _ aa
/ note=＂ fenclonine ＂
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 8
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:8:
Cys?gly?Arg?Ala?Gly?Ala?Phe?Xaa?Gys
1 5(2)SEQ?ID?NO：9：
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ can use Anb deutero-＂
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:9:
Arg?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：10：
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ can use Anb deutero-＂
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:10:
Arg?Val?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：11：
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:11:
Lys?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：12：
(ⅰ) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ IDNO:12:
Gly?Arg?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：13：
(ⅰ) sequence signature:
(A) length: 7 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 3
(D) out of Memory :/mark=modification _ aa
/ note=＂ sarkosine ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:13:
Cys?Arg?Xaa?Asp?Ser?Pro?Cys
1 5(2)SEQ?ID?NO：14：
(ⅰ) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ ornithine ＂
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:14:
Xaa?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：15：
(ⅰ) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ⅱ) molecule type: peptide
(ⅴ) clip types: include
(ⅸ) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(ⅹ ⅰ) sequence description: SEQ ID NO:15:
Gly?Arg?Val?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：16：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ Anb deutero-＂
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:16:
Arg?phe?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：17：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ 1-FCA deutero-＂
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ can be a Thioproline, sees compound 79 ＂
(xi) sequence description: SEQ ID NO:17:
Lys?Ala?Asp?Pro?Asp
1 5(2)SEQ?ID?NO：18：
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:18:
Gly?Arg?Ala?Leu?Xaa?Asp
1 5(2)SEQ?ID?NO：19：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ Aib deutero-＂
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:19:
Arg?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：20：
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:20:
Gly?Arg?Phe?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：21：
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 3
(D) out of Memory :/mark=modification _ aa
/ note=＂ 3-Br halfcystine ＂
(ix) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:21:
Gly?Arg?Tyr?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：22：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 3
(D) out of Memory :/mark=modification _ aa
/ note=" 3-Br halfcystine "
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:22:
Gly?Arg?Tyr?Xaa?Cys
1 5(2)SEQ?ID?NO：23：
(i) sequence signature:
(A) length: 6 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 3
(D) out of Memory :/mark=modification _ aa
/ note=" 3-pyridyl L-Ala "
(ix) feature:
(A) name/keyword: modify the position
(B) position: 5
(D) out of Memory :/mark=modification _ aa
/ note=" Thioproline "
(xi) sequence description: SEQ ID NO:23:
Gly?Arg?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：24：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=" using the AMBA deutero-"
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=" Thioproline "
(xi) sequence description: SEQ ID NO:24:
Arg?Ala?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：25：
(i) sequence signature:
(A) length: 5 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
/ note=＂ Thioproline ＂
(xi) sequence description: SEQ ID NO:25:
Gly?Met?Asp?Xaa?Asp
1 5(2)SEQ?ID?NO：26：
(i) sequence signature:
(A) length: 13 amino acid
(B) type: amino acid
(D) topology: straight chain
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: peptide
(B) position: 1 ... 13
(D) out of Memory :/mark=peptide
/ note=＂ CS-1 deutero-peptide ＂
(xi) sequence description: SEQ ID NO:26:
Cys?Leu?His?Pro?Gly?Glu?Ile?Leu?Asp?Val?Pro
1 5 10
Ser?Thr(2)SEQ?ID?NO：27：
(i) sequence signature:
(A) length: 13 amino acid
(B) type: amino acid
(D) topology: straight chain
(ii) molecule type: peptide
(v) clip types: include
(ix) feature:
(A) name/keyword: peptide
(B) position: 1 ... 13
(D) out of Memory :/mark=peptide
/ note=＂ CS-1 deutero-peptide ＂
(xi) sequence description: SEQ ID NO:27:
Cys?Leu?His?Gly?Pro?Ile?Glu?Leu?Val?Ser?Asp
1 5 10
Pro?Thr(2)SEQ?ID?NO：28：
(i) sequence signature:
(A) length: 4 amino acid
(B) type: amino acid
(D) topology: ring-type
(ii) molecule type: peptide
(v) clip types: include (ix) feature:
(A) name/keyword: modify the position
(B) position: 1
(D) out of Memory :/mark=modification _ aa
/ note=＂ is with 1-FCA or acetylize deutero-＂ (ix) feature:
(A) name/keyword: modify the position
(B) position: 4
(D) out of Memory :/mark=modification _ aa
＂ (xi) sequence description of/note=＂ C-terminal amideization: SEQ ID NO:28:
Cys?Asp?Val?Cys
1

Claims (24)

1. following formula: compound or its pharmaceutically acceptable salt: Wherein
L 1And L 2Be selected from Lys, Pen, Mpr, AnB, AnC, β-Ala, Lys, Orn, Dpr, Asp, Glu respectively or together, have and be suitable for forming L 1And L 2Between amino-acid residue, amino acid analogue and the amino acid analog thing of functional group of ring bridge;
Z is for becoming cyclic group or L 1And L 2Between key;
1 can exist or not exist, when existing, it is selected from Leu, Tyr, Phe, Ile, Pro, Pro analogue, Gly, Ala, Val, falls Leu, falls Val, Trp, D-Nal, Sar, (Ada)-Ala, AnC, AnB, Lys, omega-amino--lower alkanols alkanoic acid, Gly, Ala, Gly-Gly, Ala-Ala, AnC-AnC, AnB-AnB, β-Ala, β-Ala-β-Ala, l-Nal, TTC, TcA, DTC and MTC;
2 can exist or not exist, and when existing, it is selected from Arg, Arg analogue, Lys, Lys analogue, His, Ala, Gly, Sar, Leu, AnB, Phe analogue, Pro, Pro analogue, TCA, TTC, DTC and Dpr;
3 are selected from Gly, Sar, Ala, Ala analogue, d-Ala, Ile, Val, d-Val, d-Nal, Phe, Lys, Arg, Asp, β-Asp, Asn, AiB, AnB, β-Ala, Glu, Met, Leu, Tyr, 3 Br-Tyr, sulfo group third amino, 3,5-two bromos-Tyr and 3,5-two iodos-Tyr;
4 be selected from Asp, d-Asp, Glu, Asn, Gln, Asp and Glu Fm ester, Asp and Glu other replacement alkyl, aryl and alkaryl ester, Asn and Gln alkyl, aryl and alkaryl acid amides, Gly, Ala, β-Ala, Leu, Val, AnB, Phe, neighbour,, right-halo-Phe, to nitro-Phe, Arg, Cys, TIC, Pro and thiop;
5 can exist or not exist, when existing, its be selected from Ser, Thr, Trp, Ala, Val, Phe, neighbour,, right-halo-Phe, to nitro-Phe, to fluoro-Phe, to chloro-Phe, 3,5-two bromos-Tyr, to methoxyl group-Tyr,
Figure A9411599500031
Figure A9411599500032
Wherein m is 2,3 or 4
6 can exist or not exist, when existing, it is selected from Pro, d-Pro, thiop, 1,3-dithia azatropylidene, (1,3-dithiazepine), 1,4-dithia azatropylidene, 1,5-dithia azatropylidene, Pro analogue, 1,1-ACC, Dhp, Hyp, high Pro, Phe, DTC, TTC, TC, MTC, TCA, adjacent halo-Phe, halo-Phe, to halo-Phe, to nitro-Phe, nipecotic acid (isonipecotic acid), N-methylalanine and TLC;
X 1Can exist or not exist, when existing, it is selected from sequence, Ala, AnC, AnB, CBO, omega-amino-lower alkanols alkanoic acid and AMBA, low alkyl group, aryl carboxylic acid, alkaryl carboxylic acid and the SAR of 1-4 D-or L-amino acid and amino acid analogue;
Y 1Can exist or not exist, when existing, it is selected from sequence, Ala, AnC, AnB, CBO, omega-amino-lower alkanols alkanoic acid, AMBA, aminomethyl-pyridine, rudimentary alkanamine, arylamines, alkarylamine and the SAR of 1-4 D-or L-amino acid and amino acid analogue;
X 2At random be N α-substituent R ' or R ' CO-, formic acid, acetate, heterocyclic carboxylic acid, aryl carboxylic acid, heteroaryl carboxylic acid, paraffinic acid, alkenoic acid, the chain acetylenic acid, other mixing functions base straight-chain carboxylic acid of sulfur-bearing and nitrogen, 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, other polyaromatic and heteroaryl carboxylic acid and acetate, QC, CPA, BOC, 5-FINC and CBO;
Y 2Being C-terminal substituting group arbitrarily, it is selected from-OR ', NHR ', NR ' NH 2,-NHNR ' ,-NRS ,-NHNH 2The amino acid that ,-SR ', aminomethyl-pyridine and α-hydroxy-acid group are replaced by tetrazolium;
And each R ' suitable substituting group on the medicine of respectively doing for oneself wherein preferably is selected from the C that hydrogen, straight chain and side chain, end replace and replace 1-C 8Low alkyl group, C 2-C 8Alkenyl, C 2-C 8Alkynyl group, C 6-C 14Aryl, C 7-C 14Alkaryl, C 7-C 14Cycloalkaryl and C 3-C 14Cycloalkyl, for-NR ' 2, the nitrogen-atoms that it can be coupled is formed into cyclic group together, promptly contains aerobic, nitrogen or the sulphur 5-8 unit heterocycle as other ring hetero atom arbitrarily,
Condition is
(1) when 3 being Gly and 2 during for Arg or Arg analogue, 4 be not Asp, Asp analogue, Glu or Glu analogue and
(2) when 3 being Gly and 4 during for Asp, Asp analogue, Glu or Glu analogue, 2 is not Ary or Ary analogue.
2. the compound of claim 1, wherein L 1And L 2Be respectively Cys, but and ring is to be realized by the key of disulfide linkage, lactan, L-lanthionine sample-sulfide or other Cheng Huanqiao.
3. the compound of claim 1, wherein said compound has following array structure:
Figure A9411599500051
NO.:4),
Figure A9411599500061
NO.:4),
Figure A9411599500062
NO.:4),
4. the compound of claim 1, wherein said compound has following array structure: NO.:28),
Figure A9411599500072
NO.:28),
Figure A9411599500073
NO.:4),
Figure A9411599500074
5. the compound of claim 1, wherein Z for be selected from by diketone-, one group of two functional group linking group of diamino-form with the linking group of different two functional groups.
6. following formula: compound
Figure A9411599500081
X wherein 1, X 2, 1,2, L 1, 4,5,6, L 2, Y 1And Y 2Define as claim 1.
7. following formula: compound
Figure A9411599500082
X wherein 2For protecting group 6 is Pro or Pro derivative.
8. according to the compound of claim 7, it is a following formula: compound
Figure A9411599500083
NO.:4) or
Figure A9411599500084
ID.NO.:4)
9. pharmaceutical composition, it contains the compound and the pharmaceutically acceptable carrier of claim 1.
10. pharmaceutical composition, it contains the compound and the pharmaceutically acceptable carrier of claim 6.
11. a pharmaceutical composition, it contains the compound and the pharmaceutically acceptable carrier of claim 7.
12. one kind is prevented or treat the method because of the undesired disease that causes of the cytoadherence of pair cell epimatrix, it comprises to the patient uses scope at the composition of each dosage in the claim 9 of 0.1-100mg/kg.
13. one kind is prevented or treat the method because of the undesired disease that causes of the cytoadherence of pair cell epimatrix, it comprises the composition of using the claim 10 of significant quantity to the patient, to recover the cytoadherence of normal pair cell epimatrix.
14. one kind is prevented or treat the method because of the undesired disease that causes of adhesion between pair cell, it comprises the composition of using the claim 11 of significant quantity to the patient, to recover normal iuntercellular adhesion.
15. one kind suppress white corpuscle and endotheliocyte or with the method for extracellular matrix adhesion, it comprises the composition of the claim 9 of using significant quantity, with suppress described white corpuscle and endotheliocyte and with the adhesion of extracellular matrix.
16. one kind can with the compound specificity bonded antibody of claim 1.
17. a diagnosis is because of existing the method for the disease that can cause with endotheliocyte bonded antibody, it comprises:
I) on a kind of substrate, fix the described compound of at least a claim 1 to form a kind of derived substrates;
Ii) obtain patient's blood or serum sample;
Iii) the described derived substrates of (i) gained is combined with (ii) described blood of gained or serum sample, at this moment, what wherein exist can will combine with described derived substrates with described endothelial cell specific bonded antibody; With
Iv) to detecting with described derived substrates bonded antibody.
18. a diagnosis is because of existing the method for the disease that can cause with extracellular matrix bonded antibody, it comprises:
I) on a kind of substrate, fix the described compound of at least a claim 1 to form a kind of derived substrates;
Ii) obtain patient's blood or serum sample;
Iii) the described derived substrates of (i) gained is combined with (ii) described blood of gained or serum sample, at this moment, what wherein exist can will combine with described derived substrates with described endothelial cell specific bonded antibody; With
Iv) to detecting with described derived substrates bonded antibody.
19. a method of diagnosing the disease that excessively causes because of the cytoadherence with endotheliocyte, it comprises:
I) obtain patient's endothelial tissue sample;
Ii) will contact with the described compound of claim 1 by described endothelial tissue gained cell; With
Iii) measure by the amount of the described compound of described endothelial cell specific bonded.
20. a method of producing the biocompatible surface of prosthese, it comprises with the described compound of claim 1 and coats described prosthetic surface.
21. according to the method for claim 13, wherein said disease is selected from rheumatic arthritis, asthma, transformation reactions, the adult breathes the embarrassed syndromes of stating, cardiovascular disorder, thrombosis or deleterious platelet aggregation, inaccessible again after the thrombolysis, the homotransplantation rejection, graft is to host's rejection, organ transplantation, septic shock, heavily perfusion property damage (reperfusion injury), psoriasis, eczema, contact dermatitis and other dermatitis, osteoporosis, osteoarthritis, atherosclerosis, tumor disease comprises tumour or growth of cancers metastatic tumor, illness in eye such as retinal detachment, I shape diabetes, multiple sclerosis, systemic lupus erythematosus (SLE), inflammation and immune inflammation comprise ophthalmia and enteritis, ulcerative colitis, limitation joint enteritis and other autoimmune disorder.
22. a contraceptive device that suppresses to be fertilized, to suppress spermioteleosis or suppress sperm function, it comprises to the host uses the compound of being fertilized, suppressing spermioteleosis or suppress the claim 1 of sperm function contraception significant quantity suppressing.
23. one kind is used for purifying claim 1 compound is had the macromolecular matrix of avidity, it comprises the compound of claim 1 and insoluble carrier covalent bonds.
24. a purifying is with high-affinity and the macromolecular method of claim 1 compound specificity bonded, it comprises:
I) with a kind of insoluble matrix of claim 1 compound deriving;
Ii) with the macromolecular example reaction that contains described claim 1 compound tool high affinity, this moment described macromole and described claim 1 compound formation one mixture, described mixture combines with described matrix;
Iii) clean decontamination with the solution of unlikely cracking mixture in conjunction with the matrix of the above mixture;
Iv) pass through the described carrier of solution washing with the mixture of described macromole of cleavable and claim 1 compound formation, wash-out goes out described substrate; With
V) reclaim the macromole that the compound of claim 1 is had high affinity.
CN94115995A 1993-01-08 1994-07-11 Peptide inhibitors of cell adhesion Pending CN1114961A (en)

Priority Applications (6)

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SG1996001553A SG52262A1 (en) 1993-01-08 1994-01-07 Peptide inhibitors of cell adhesion
EP94906984A EP0677060A1 (en) 1993-01-08 1994-01-07 Peptide inhibitors of cell adhesion
PCT/IB1994/000026 WO1994015958A2 (en) 1993-01-08 1994-01-07 Peptide inhibitors of cell adhesion
CA002153228A CA2153228A1 (en) 1993-01-08 1994-01-07 Peptide inhibitors of cell adhesion
JP6516421A JPH08505628A (en) 1993-01-08 1994-01-07 Peptide type cell adhesion inhibitor
CN94115995A CN1114961A (en) 1993-01-08 1994-07-11 Peptide inhibitors of cell adhesion

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