JPH06505978A - Anti-aggregating peptides containing aromatic esters or amides - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Field of the invention: Anti-aggregating peptides containing aromatic esters or amides The present invention provides novel peptides that inhibit platelet aggregation, and pharmaceutical compositions containing the peptides. and methods of using the peptide. Especially books in combination with fibrinolytic agents Methods of using the peptides of the invention are described.

Background of the invention A blood clot is the result of a process that starts the clotting cascade. It is high in fibrin Consists of aggregations of platelets entangled in a molecular network. This process usually results in tissue damage initiated as a result of and has the effect of slowing or obstructing blood flow in blood vessels . such as atherosclerotic arterial plaques, inflammation of blood vessels (phlebitis) and sepsis Etiological factors not directly related to tissue damage also initiate thrombus formation. Inappropriate thrombosis The formation of an aneurysm, and the resulting reduction in blood flow, can lead to stroke, pulmonary embolism, and It may also have pathological consequences such as heart disease.

Platelets play an important role in blood clot formation. Current antithrombotic treatments are Rostacycline analogues, cyclooxygenase inhibitors, thromboxane synthesis inhibitors Platelet/endothelial cell arachidonic acid as a toxic agent and thromboxane receptor antagonist Salts - drugs that modify the prostaglandin system; as well as anti-aggregating agents such as heparin use. These drugs inhibit one of two distinct stages of platelet aggregation. Or inhibit both. ADP (adenosine diphosphate), collagen, epine The first phase, which is a response to chemical irritants such as furin or thrombin, Resulting in early activation of platelets. After this, it is initiated by the platelets themselves and the potassium , thromboxane A2 (TXA2) synthesis and further from platelet storage granules. A second phase follows, characterized by the release of ADP, which further activates platelets. sexualize.

Prostacycli, also called prostaglandin I* (PGIT) and stable PCIt analogs inhibit both the primary and secondary phases of platelet aggregation. inhibit.

However, the use of such analogs was associated with undesirable changes in blood pressure. Eye Aiken et al., Prostaglandin system (P rostaglandin) s), 19.629-43 (1980).

Cyclooxygenase inhibitors and thromboxane synthetase inhibitors are TX It acts to block the production of A4. TxA2 antagonists are TxA receptors Blocks the action of TxA by binding to TxA. These therapies reduce blood It only acts on the second stage of plate activation. The use of cyclooxygase inhibitors is recommended for ulcers It was associated with side effects on development and prostacyclin synthesis.

Heparin prevents the activation of fibrinogen by thrombin, thereby , prevents activation of the GPIIb-Tlla receptor by thrombin. this is, It inhibits only the first stage of platelet aggregation and inhibits collagen, ADP and epinephrine production. It has little effect on platelet activation by other means.

Cyclooxygenase inhibitors, prostaglandin analogs and heparin are may inhibit the primary or secondary phase of platelet/fibnogen activation. Therefore, it indirectly inhibits platelet aggregation. Therefore, the first phase of platelet activation or selectively that directly blocks platelet aggregation, whether occurring from the secondary stage. Requires therapeutic products.

Platelet aggregation is primarily mediated through the GPI 1b-11Ia platelet receptor complex. It seems that it will be mediated. Phono Willebrand factor, plasma proteins, and fibrillar Nogen binds and binds GPI 1b-II la receptors on adjacent platelets. It can be cross-linked, thereby allowing platelet aggregation. fib Ronectin, vitronectin and trompospondin are GPIIb-111 This is a protein that has also been shown to bind to a. Fibronectin is found in plasma and Found as a structural protein in the intravesicular matrix. Structural proteins and GPI 1b-111a The bonds between may function to attach platelets to the damaged blood vessel wall.

a peptide fragment of human plasma fibronectin, and facilitates cell attachment, and RGD that enhances phagocytosis (one about Arg-Gly-Asp) A synthetic peptide containing the letter amino acid symbol) sequence is described in U.S. Patent No. 517,686. No. 4,589,881, U.S. Patent No. 4,661,111 and Disclosed in US Pat. No. 4,614,517. WO89105150(P CT US88104403) contains linear and cyclic peptides containing RGD sequences. Chido was also reported. Peptides containing the RGD sequence can inhibit platelet aggregation. It was reported that. Niebelstein et al. Thromb, and Hemostas is), 58.2133 (1987)] -]RGDS-pepti GPIlb-induced aggregation and adhesion of platelets to fibronectin, It was reported that they interact with each other through the I11a complex. U.S. Patent No. 4,68 No. 3,291 describes peptides containing Arg and Lys and fibrino An RGD-sequence that inhibits the binding of A-gen to platelets and inhibits platelet aggregation. is disclosed. The disadvantage of these peptides is their poor stability in plasma. qualitative and their low potency. European Patent No. 0275748 describes GPI A linear protein that binds to the 1b-11Ia receptor and inhibits platelet aggregation. Tetrapeptide-hexapeptide and cyclic hexapeptide-octapeptide is disclosed. The reported cyclic peptide has a diacetate between two cysteinyl residues. Formed via sulfide bridges. Other linear and cyclic peptides are European patented It is disclosed in Application Publication No. A0341915 (the contents of which are cited herein). ). One compound disclosed therein is Ac-Arg-Gl y-A sp-NHCH2CH2Ph.

The present invention provides that the carboxy terminus of the peptide is an aryl alcohol or an aryl amine. to provide a compound which is substituted with an aryl ester or amide to form an aryl ester or amide It is. The compounds of the present invention are capable of binding GP-11blIIa to fibrinogen. It is surprisingly effective. Thus, the advantages of the compounds of the present invention are , their ability to inhibit platelet aggregation and their improved activity.

Recent advances in the treatment of occluded arteries and deep vein thrombosis allow blood flow to be re-established or to improve, fibrinolytic agents are used to dissolve blood clots or emboli.

anistreplase, tissue plasminogen activity factor (tPA), urokinase (UK), pro-urokinase (pUK), and streptokinase (SK), and its mutants and derivatives. Fibrinolytic agents hydrolyze fibrin at specific sites, thereby It is a proteolytic enzyme that disrupts the bulin network. Their in vivo action is Proteolytic activation of plasminogen in the blood to form plasmin and it dissolves fibrin clots. smaller peptides of fibrin lysis has the effect of dissolving thrombi or emboli. However, such treatment A recurring problem with therapy is reocclusion of blood vessels due to secondary thrombus formation.

Fibrinolytic therapy is most commonly used to reestablish flow in clotted blood vessels. used for. However, fibrinolytic therapy does not address the factors responsible for the initiation of blood clots. Do not turn the child over. To this end, anticoagulants such as heparin are used to prevent reocclusion. It can also be prevented. In fact, patients with severe stenosis in their arteries receive high doses of heparin. The risk of rethrombosis after reperfusion is very high even in the presence of. Gold ) et al., Circulation, 73.347-52 (1986) See.

Furthermore, the use of SK and tPA has been shown to increase platelet hyperaggregation (hyperaggregation). (gability). Ohlstein et al., Trombo Sys Research (T hromb.

Res, ), 4.575-85 (1987). High dose (7) tPA1: : This procedure may be accompanied by systemic bleeding and is not recommended to prevent reocclusion. Not possible. Therefore, methods of preventing rethrombosis after fibrinolytic therapy are needed.

U.S. Patent Application No. 917,122 describes a method for inhibiting reocclusion after reperfusion and T for use in a method for lowering the dose of tPA required for primary lysis. xA, antagonists are disclosed. Yasuda et al. [Clinical Lisa (CIin, Res.), 34.2 (1986)] Re-occlusion by fibrin-rich platelet thrombus after embolism is associated with GPIlb-II in dogs. was shown to be inhibited by a murine monoclonal antibody against Ia. Main departure A novel method for vascular reclosure by administering a peptide that directly inhibits platelet aggregation. A method for inhibiting occlusion is described.

Summary of the invention In one embodiment, the present invention provides formula (I): D-E-Asp-Q-Y (I) [In the formula, Q is NH, NCH, or C9 Y is unsubstituted or 1 to 3 C. alkyl, trifluoromethyl, )10gen , OR', SR', (CHz), Ar, C0NR, R2, C0tR2 or R1 Phenyl, naphthyl or Het substituted by R4N; W is NHR' , NR'C(=NH)NHR', NR'C(=NH)R' or (C=NH)N HR’.

U is CO, SOz, NHCOl (CH2), 5OzNH, NH302 or ■ ], Ro, R4Rs N, Ra Rs N CO, R' O, Y N R', A is absent or Asn, Gln, AIa, Pro or Abu; B is Arg, I-IArg, NArg, (Me2)A rg, (Etz)A r g, Ab u.

Ala, Gly, Hi 5SOrn, Lys, Phg or its a-R” substitution derivative D- or L-amino acids selected from conductor, Dtc, Tpr or Pro: E is Gly, Sar, CHICo, 0CHtCO, or NHco; Ro is H, C,, alkyl or Ar (CHJ-:X is R4R4N, R4R8N-Co or is H; R1 and R2 are H, C, alkyl or (CH2)-A r; R 4 is H or C1-6 alkyl: Rs is Rs, Rg CO1Ra OCOSRe OCH (Rs’) CO.

Rs　N　HCH(Rso)Co, R55CH(Reo)Co, RgSO2 or l; 1Raso; R6 and R6゛ are H, C, -6 alkyl, C8-7 cycloa Alkyl, Ar5Ar-C1-4 alkyl, Het, Het-C1-4 alkyl , Ar (C1-12), CH (ArXCH2) or Ar-C3-7 Lukiru; Ar is phenyl or 1-3 01-6 alkyl, trifluoromethyl, halo phenyl substituted by gen, R'O or R'S.

m is (1) If W is NR'-(C=NH)NHR', then 0 to 4° (2) W is (C=NH)NHR' or NR' (C=NH)R', 0 to 5; or Taha (3) If W is NHR', 0~6゜q is (1) W is NR'-(C=NH) If NHR', 1 to 4: (2) W is (C=NH)NHR' or NR' When (C=NH)R', 2 to 5. or (3) When W is NHR', it is 3 to 6] or its drug It is a pharmaceutically acceptable salt.

One feature of the invention is that the aryl group is attached to the Asp residue via nitrogen or oxygen. This compound had high activity in inhibiting platelet aggregation.

Another feature of the present invention is that the α-amino group of Arg is modified to incorporate the amino group into the ring. It is a novel active compound containing

Yet another feature of the invention is that the α-amino group of Arg is removed or Novel active compounds that are substituted with another substituent.

The present invention comprises a compound of formula (I) and a pharmaceutically acceptable carrier. Also a pharmaceutical composition for the inhibition of platelet aggregation and clot formation.

The present invention further provides for administering an effective amount of the compound represented by formula (I) into the body. A method of inhibiting platelet aggregation in a mammal in need thereof.

In another aspect, the invention provides an effective amount of a fibrinolytic agent and a fibrinolytic agent having the formula (I) arteries in mammals after fibrinolytic treatment, which consists of administering compounds into the body or provide a method of inhibiting venous reocclusion. The present invention is effective against seizures, transient It is also a method for treating cerebral ischemic attack or myocardial infarction.

The present invention also provides a fibrinolytic agent and a compound of formula (I) in a pharmaceutical carrier. Fibrinolysis and reperfusion in mammals consisting of arterial or intravenous It is also a pharmaceutical composition that inhibits reocclusion in the vein.

Finally, the invention provides that from the fibrinolytic agent and the compound of formula (1) in the container A kit for use in a method for performing fibrinolytic therapy.

Detailed description of the invention The present invention consists of the sequence Gay-Asp and is directly bonded to the carboxy group of Asp. Describes acyclic peptidomimetic compounds with combined aryl ester or amide moieties. I will post it. Compounds of the invention inhibit platelet aggregation and GPIlb-111a It appears to interact with receptors and other attachment proteins. Asp residue of aryl group Binding to gives high activity in inhibiting platelet aggregation.

The compound of the present invention is a peptide represented by the above formula (I).

Suitably, Y is optionally halogen, Cl-4 alkyl, Cl-4 alkyl substituted by C, C1-, alkylthio, C0NR1Rt* or CO,R3 phenyl or naphthyl, which may be

Preferably QA is NH.

Preferably Y is phenyl or naphthyl.

Preferably, Y is optionally chloro, Cl-4 alkyl, Cl-4 alkylthio or phenyl optionally substituted with carboxy.

Preferably, V is X A B R'N, R4R5NC0 or R, R3N, Here, R' or R4 is H or methyl.

Preferred Nyl, V is ArCON (CH3) or HetCO-N (CHs).

Preferably, V is R, R, N, where R4 is H or methyl, and R .

is R, CO or Rsoco.

Preferably E is Gly, Sar, NHCO or CH2C0. Preferably , E is Gly.

Preferably Het is chenyl or pyridyl.

Therefore, when W is -NR'(C=NH)NHR', m is preferably 2 ~3; W is -(C=NH)NHR' or -NR'(C=NH)R' When W is -NHR', m is preferably 3 to 4; when W is -NHR', m is preferably is 4-5.

It depends on the positional relationship between the substituents (CH,) and -W. Therefore, W is −NR When '(C=NH)NHR', m is preferably 0 to 0 for the para position. 1, 0 to 2 for the meta position, and 1 to 3 for the ortho position. W is -(C= When NH)NHR' or -NR'(C=NH)R', m is preferably , 0 to 2 for the para position, 1 to 3 for the meta position, and 2 to 2 for the ortho position. It is 4. When W is -NHR', m is preferably 0 for the para position. -3, 2-4 for the meta position, and 3-5 for the ortho position. Generally para Preferably.

The meaning of X in the formulas described herein with respect to X-A or X-B is If absent, indicate the amino group of these amino acids. One or more compounds of the present invention If the chiral center of and each unique non-racemic compound to be resolved. Substituents R3, R2, R1, R ,, Ro, R6', Ar, R', Q, nSp and q have any one meaning are independent of their meaning in other cases.

As used herein, C1-4 alkyl refers to methyl, ethyl, n-propylene isobutyl, isopropyl, n-butyl, isobutyl and n-butyl. do. C3-6 alkyl includes these groups and also pentyl and hexyl. sil, and those such as n-pentyl, isopentyl and neopentyl Examples include simple aliphatic isomers of

As used herein, Ar is phenyl, or 1 or 2 Cl-8 Substituted by alkyl, trifluoromethyl, halogen, R'O or R'S phenyl. Regarding the substitution of R'O and R'S for Ar and R′ is H, resulting in hydroxy or mercapto: for C+-sC+-, resulting in C1-6 alkoxy or C16 alkyl thio: or Ar(CHz)□, resulting in 1 or more as desired. or two C1-4 alkyl, Cl-4 alkoxy or halogen Phenoxy, phenyl C1-, alkoxy, phenyl which may be substituted on yields nylthio or phenylCl-3 alkylthio. Methyl or is benzyloxy and benzylthio optionally substituted by methoxy Can be mentioned.

Y is a substituted aromatic or heteroaromatic group. That is, Y is 1 or or 2 C1-6 alkyl, trifluoromethyl, halogen, R'O, R'S .

Substituted by (CH2)-Ar, C0NRIR2, CO2R2 or R2H,N phenyl or naphthyl, which may be Het or heteroaryl contains 1 to 3 heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. a 5- or 6-membered monocyclic aromatic ring, or a 9- or 10-membered bicyclic aromatic ring ring, which is stable and accessible by conventional chemical synthesis. Ru. Heterocycles include pyridyl, furyl, chenyl, indolyl, quinolinyl, Mention may be made of benzofuryl and benzothenyl. available through chemical synthesis up to 3 substitutions on the phenyl, naphthyl or Het ring that are present and stable Any readily available combination of groups is within the scope of this invention.

substituted by 1 or 2 C0-6 alkyl or R'0 groups a 5- or 6-membered monocyclic ring containing 1 or 2 nitrogen atoms or 9 Typical examples include phthalimidyl, piperidinyl, pyrrolyl, isoquino Lyle, 2-pyridonyl, 1,3-dihero 2H-isoindolyl, 2-pyrrolidi Nonyl, imidazolyl and morpholinyl are mentioned.

The present invention provides that either the peptide bond or -CONH- is replaced by an isosteric bond. Contains compounds that are Examples of peptide isosteres include -NHCO-1-CH=CH -1-CHtCH, -1-COCHt-1-COO-1-CHOHCH! -1- CHtN R4-1-C3NH- and -CH,S- are mentioned.

Specific compounds of the invention include: N-[[5-(aminoiminomethyl)amino-1-oxopentyl]-Gly- Asp-phenylamide; N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-ox [sopentyl]-Gay-Asp-phenylamide: N-[(2S)-2-(1, 3-dihydro-2H-isoindolyl)-5-(aminoiminomethyl)-amino -1-oxopentyl]-Gay-Asp-phenylamide; N-[N'-(4,4-diphenylbutyryl)-N-methyl-arginyl]-〇 1y-Asp-phenylamide: N-[(2S)-(1-(2-pyrrolidinonyl))-5-(aminoiminomethyl ) Amino-1-oxopentyl]-Gly-Asp-phenylamide: N'-( phthaloyl)-A r g-G ] y-A s p-phenylamide; Nzoyl-N'MeArg-Gly-Asp-(4-carboxy)phenylamide Do.

N'acetyl-A r g-G]y-As p-phenylamide.

N'benzoyl-Arg-Gly-Asp-phenylamide: N'benzyloxy Cycarbonyl-Arg-Gly-ASp-phenylamide; D-Phg-Arg -Gly-Asp-phenylamide; N'benzyloxycarbonyl-N'methy Lu-〇rn-Gly-Asp-phenylamide: 4-(aminoiminomethyl)aminobenzoyl-8ar-Asp-phenylami Do; N#benzoyl-N'MeArg-Gly-Asp-(2-chloro)phenyla Mid; N″benzoylN’MeArg-Gly-Asp-(2-methoxy)phenyla Mid: N1 benzoyl-N'MeArg-Gly-MeAsp-(2-hydroxy) phenylamide; N1benzoyl-Arg-NHNHCO-Asp-(N-methyl)phenyla MiAsp-phenylamide Preferred compounds of the invention are: N'-(2-methylbenzoyl)-N'-MeArg-Gly-AS+)-( 2-methyl) phenylamide; N'benzoyl-N'MeArg-Gly-Asp-feramide.

N'acetyl-N'MeA r g-G Iy-A s p-phenylamide ;N'acetyl-N'MeArg-Gly-Asp-(2-carboxy)phenylene Ruamide: N'(2-methylthio)benzoyl-N'MeA r g-G I y-A s p-(2-methylthio)phenylamide.

N'benzoyl-N'MeArg-G1 y-Asp-(4-chloro)phenyl Amide.

N1benzoyl-N&MeArg-GlcyAsp-(3-carboxy)pheny Ruamide: and N'(2-thenylcarbonyl)-N'MeA r g-G l y-A s It is p-phenylamide.

Peptide peptides are described herein using nomenclature commonly used in the art. Write down.

Alanine Ala Leucine Leu Arginine Arg Lysine Lys Asparagine Asn Methionine Met Aspartic acid Asp Phenyl Alanine Phe Cystine Cys Proline Pr.

Glutamine Gin Serine Glutamic acid Glu Threonine Thr Glycine Gly Tryptophan Trp histidine His tyrosine Tyr isoleucine Ile Nokuri Val Asparagine or Aspartic Acid Asx Glutamine or Glutamine Minic acid GlxAccording to the conventional expression, the amino terminus is on the left and the carboxy terminus is on the right. It is. Unless otherwise specified, all chiral amino acids (AA) are in the L-absolute configuration. Consider it as a thing.

Abu represents aminobutyric acid and Dtc represents 5,5-dimethylthiazolidine-4-cal. Tpr represents thiazolidine-4-carboxylic acid; represents moarginine, NArg represents noarginine, (Me2) A r g I; LN', represents N''-dimethylarginine, (Eh)Arg is N', N-diethylarginine, D-Phg represents D-phenylglycine, O rn represents ornithine.

t-Bu represents a tertiary butyl group, Boc represents t-butyloxycarbonyl group, Fmoc represents a fluorenylmethoxycarbonyl group, Ph1i represents a nyl group, phth represents a phthaloyl group, and Cbz represents a carbobenzyloxy group. , BrZ represents a 0-bromobenzyloxycarbonyl group, and C]Zlto - represents a chloropennoroxycarbonyl group, Bzl represents a benzyl group, and 4- MBzl represents 4-methylbenzyl group, Ac represents acetyl, AlkliC I-4 represents alkyl, Nph represents 1- or 2-naphthyl, and cHex represents Represents chlorhexyl.

DCC stands for dicyclohexylcarbodiimide and DMAP stands for dimethylaminopyridine. lysine, DIEA stands for diisopropylethylamine, and EDC stands for N-ethylamine. Represents thyl-N'(dimethylaminopropyl)-carbodiimide, HOBt is 1 - represents hydroxybenzotriazole, THF represents tetrahydrofuran, DMF stands for dimethylformamide and PPA stands for 1-propanephosphonic acid cyclic anhydride. DPPA stands for diphenylphosphoryl azide, BOP stands for hexaph Benzotriazol-1-yloxy-tris(dimethylamino)fluorophosphate represents phosphonium, HF represents hydrofluoric acid, and TFA represents trifluoroacetic acid. represent.

As used herein, a binding agent is used to form a peptide bond. Reagents are shown. Typical binders are carbodiimides, activated anhydrides and esters and acyl halides. EDC, DCCSDPPA, PPA, BOP reagents, such as HOBt, N-hydroxysuccinimide and oxalyl chloride. Reagents are typical.

α-R° substitution induction of the amino acids of the present invention, sometimes written as (α-R’)AA The symbol indicates an amino acid that is monosubstituted on the α-amino group by Ro, where R o is C+-S alkyl or Ar(CH2)*. Preferably, Ro is Methyl or benzyl. Ro is preferably methyl. Each (a - M e) A r g and (α-Me)Gly, N”-methylarginine and N'-Methylglycine is herein referred to as MeA, according to conventional and conventional notation. Also written as rg and Sar (sarcosine). All other N-α-substituted amino No acids will have the symbol α- in their expression.

Peptides are described by Merrifield [Journal of American Chemical Society (J, Av, Chem, Soc, ), 85.2149 (1964)] or as known in the art. It is prepared by a common liquid phase method. Journal of Mesoinnal Ke Mistry (J, Med, Chet), 29.984 (1986) and Journal of Mesointernal Chemistry (J, Med, C Het), 30.2291 (1987) by Ali et al. The generally disclosed peptide synthesis method is a general technique description and is not covered herein. It shall be cited in the specification. The combination of solid phase and liquid phase synthesis methods is -, tetra- or penta-peptide fragments are prepared by phase-phase synthesis and then convergent synthesis (co) combined or further modified by liquid phase synthesis. nvergent 5 synthesis). liquid Phase synthesis methods are generally preferred for the smaller peptides of the invention.

The reactive functional groups on the side chains of each synthetic fragment, amino acid or peptide, are Preferably, it is protected as known in the wild. For example, Boa.

The Cbz or Fmoc group is used for the protection of amino groups, especially α-amino groups. It can be done. Boa groups are generally preferred for protection of alpha-amino groups. t-B u.

cHex or benzyl ester retains the carboxy side chain of Asp or Glu. can be used for protection. A benzyl group or a suitably substituted benzyl group is mercapto group or other thiol-containing residues of stene: or serine or thiol-containing residues. Used to protect leonine hydroxyl. Tonle group is the imitation of His. Used for protection of midazolyl group and for protection of guanidino nitrogen of Arg is a tonole group or a nitro group. suitably substituted carbobenzylox The hydroxyl group or benzyl group is the hydroxyl group of TyrSSer or Thr, or It can be used for the ε-amino group of Taharishin. The phthaloyl group also can be used for the protection of the ε-amino group of benzyloxycarbonyl or Preferred substitutions for the benol protecting group include chloro, bromo, nitro or ortho with methyl. and/or para substitution and are used to alter the reactivity of the protecting group.

With the exception of the Boa group, protecting groups are most conveniently removed by mild acid treatment. It is. These protecting groups can be modified by catalytic hydrogenation, as is known in the art. , ammonia in sodium water or HF treatment.

Solution-phase synthesis of peptides combines conventional methods used to form amide bonds. done using. Typically, the H-Q-Y fragment is optionally 1-hydroxy of benzotriazole (HOBt) and dimethylaminopyridine (DMAP) In the presence of a catalyst such as N,N'dicyclohexylcarbodiimide (DCC), The free amino or hydroxyl is attached to a suitable side chain protected Asp via a group. optionally in the presence of a base. , activated ester, anhydride or acid of the free carboxyl of the protected Boc-amino acid Other methods such as halide formation and subsequent reaction of protected amino acids with free amines Also suitable are methods. For example, a protected Boc-amino acid or peptide salt in the presence of a base such as tylmorpholine, DMAP or trialkylamine. In an anhydrous solvent such as methylene chloride or tetrahydrofuran (THE), chloride isobutyl acid to form an "activated anhydride," which is then React with protected amino acids or free amines of the peptide. by these methods The Boc-Asp-Q-Y intermediate formed can be converted to an amino terminal using conventional methods. selectively deprotected at the ends and converted to other peptides or amino acids using similar methods. combine.

When using a solid phase method, for example, if Y has a carboxyl group that can be attached to lysine, When the peptide, or convenient fragment thereof, is It is created by starting and working towards the amino terminus of the peptide. The solid phase synthesis method is In general, U.S. Pat. Lubechka Nmika Acta (Helv, Chew, Acta), 56.14 76 (1973)], benzhydrylamine resin (BH A), methylbenzhydrylamine resin (MBHA), chloromethyl resin (CM R), hydroxymethyl resin (HMR) or 5ASRIN resin. Add a properly protected carboxyl group of a Y residue such as Boc-NH-Y to the resin. It can be started by making a covalent bond. The carboxy terminus of the peptide product If ruboxiamide is to be used, BHA or MBHA support resins are used. It will be done. If the carboxy terminus of the peptide product should be a carboxyl group , CMR or HMR supports are commonly used, which include carboxyamide Or it may be used to produce esters.

After binding the first protected residue to the resin, the amino group is deprotected by treatment with a mild acid. The free carboxyl of the second protected AA is bonded to the amino group of. This process is The intermediates are carried out continuously without isolation until the desired peptide is formed. Then, The completed peptide is unblocked and/or removed from the support resin in either order. Can be separated.

If the Y residue does not have a carboxyl group and solid phase synthesis is desired, the peptide may be attached to CMR or HMR via the β-carboxyl group of Asp. . For example, the synthesis method may include a suitably side-chain protected Athnochloride via a liquid phase synthesis method. By attaching arylamine or aryloxy groups to gic acid residues You can start. Next, the side chain carboxyl groups are selectively deprotected and the chloromethyl binds to fat (CMR). Benzyl esters can be selectively decomposed by hydrogenation. is a preferred side chain protecting group to be protected. Then, the amino group is removed by treatment with acid. Release and perform solid phase peptide synthesis using standard methods.

Since Y does not have a carboxylic acid group and has a substituent that is incompatible with hydrogenation, solid phase synthesis is not possible. If this method is desired, 1-butylene is added to protect the side chain carboxyl of aspartic acid. Using methyl esters or other acid free radicals. In this case, Asuno (lagic acid The mino group is a basic radical such as the fluorenylmethoxycarbonyl group (Fmoc). protected by Solution of aspartic acid to arylamine or phenol After phase combination, selective deprotection of the t-butyl ester is performed by mild acid hydrolysis. Ru. The side chain carboxyl group of Asp is attached to the resin by conventional methods. Next Then, for the next solid-phase peptide synthesis method, fluorenyl methoxychloride was prepared using a grade base. Remove the cycarbonyl group.

The preferred method for cleaving the peptide from the supporting resin is anisole or dimethane. Denture with anhydrous HF in the presence of a suitable cation scavenger such as toquinobenzene. It is to process lipid-supporting peptides. This method simultaneously protects the sulfur. Remove the oalkyl group (remove all protecting groups and separate the peptide from the resin. The peptides hydrolyzed from CMR or HMR in the method are carboxylic acids and B What is separated from the HA resin is obtained as a carboxamide. Peptide is A When bonded through the β-carboxyl of sp, the carboxyl group is preferred. In this case, CMR or HMR is used. If the peptide is attached via the Y residue The choice of resin will depend on whether acid or carboxamide is desired. There will be.

Modification of the terminal amino group of a peptide can be accomplished by, as generally known in the art, It is carried out by alkylation, sulfonylation or acylation. These modifications are carried out on the D residue or synthesized before incorporation into the peptide, and the terminal performed on the peptide after the mino group is released but before the protecting group is removed. It's fine.

Typically, acetylation is performed in the presence of a tertiary amine, an acyl halide, or For free amino groups using the corresponding alkyl or arylic acid anhydride It will be done. Monoalkylation is lithium cyanoborohydride or cyanoborohydride a suitable aliphatic aldoxide or ketone in the presence of a slow reducing agent such as sodium chloride. most conveniently carried out by reductive alkylation of the amino group with . dialkylation by treating the amino group with excess alkyl halide in the presence of a base. It can be done.

A r gs HA r gs (Mez) A r gs (E tg) A r g, A I a s G 1 y s HI S 5Abu, Orn, Lys α-R′-substituted derivatives of amino acids of the present invention, including derivatives of It is prepared by a method. The substituent R' is C+-S alkyl or A as defined above. It may be r(CHz)-. Typical methods for producing these derivatives are described in U.S. patents. No. 4,687.758; Cheung et al., Canadian Journal Le of Chemistry (Can, J, Chet), 55.906. (197 7), F. reidinger et al., Journal of O. - Ganic Chemistry (J, Org, Chem,), 48.77 (198 2); and Shuman et al., Peptide: 7th American Peptide Peptides: Proceedings of the Symposium f the 7 th A11erican Peptide Symposi umj, li Edited by Rich, D., Gross, E., Pi. As Chemical Cumber--(Pierce Cheu+1cal Co,) , Rockford, IL, 617 (1981), which Referenced herein.

Acid addition salts of peptides are prepared by adding the parent compound and excess hydrochloric, hydrobromic, sulfuric, or Preferably from acids such as phosphoric acid, acetic acid, maleic acid, succinic acid or methanesulfonic acid. Prepared by standard methods in suitable solvents. The acetate form is particularly useful. Tolerance Some compounds form internal salts or zwitterions. Cationic salts are Excesses such as hydroxides, carbonates or alkoxides containing thiones by treating the parent compound with an alkaline reagent: or with a suitable organic amine. Manufactured by Li", Na', K+, Ca"1+, Mg" and NH," A cation is an example of a cation present in a pharmaceutically acceptable salt.

The present invention consists of a peptide represented by formula (1) and a pharmaceutically acceptable carrier. A pharmaceutical composition is provided. Peptides produced according to the above and other Peptide, or fibronectin, fibrinogen or phonovirus Pharmaceutical compositions of polypeptide derivatives of brand factors can be prepared as solutions for parenteral administration or It may be formulated as a lyophilized powder. Powders may be mixed with a suitable diluent or may be reconstituted by addition of other pharmaceutically acceptable carriers. Liquid formulations are Generally, they are buffered, isotonic, or aqueous solutions. Examples of suitable diluents include typically isotonic Saline solution, standard 5% dextrose in water or buffered sodium acetate Alternatively, an ammonium acetate solution may be used. Such formulations are especially suitable for parenteral administration. Suitable, but also used for oral administration or with a meter for insufflation methods. It may be contained in a metered dose inhaler or nebulizer. Polyvinylpyrrolidone, gelatin , hydroxycellulose, gum arabic, polyethylene glycol, mannitol It is desirable to add excipients such as sodium chloride or sodium citrate. Yes.

Alternatively, these peptides can be encapsulated or tabletted for oral administration. or may be prepared in an emulsion or syrup. pharmaceutically Acceptable solid or liquid carriers are used to enhance or stabilize the composition. Alternatively, it may be added to make the composition easier to prepare. As a solid carrier, Starch, lactose, calcium sulfate dihydrate, clay, magnesi stearate um or stearic acid, talc, pectin, gum arabic, agar or gelatin Chin is an example. Liquid carriers include syrup, peanut oil, olive oil, physiological Includes saline and water. The carrier may also be used alone or together with wax. Glyceryl monostearate or glyceryl distearate Also included are sustained release materials. The amount of solid carrier will vary, but preferably It would be about 2019 to about 19 per digit. Pharmaceutical preparations are crushed for tablet form , mixed, granulated, and then optionally compressed or gelatin hard capsules. Conventional pharmaceutical techniques including grinding, mixing and then filling for cell forms prepared according to the art. When a liquid carrier is used, the preparation It may be in the form of xyl, emulsion or aqueous or non-aqueous suspension. It takes Liquid formulations may be administered directly orally or filled into soft gelatin capsules. It's fine.

For rectal administration, the peptides of the invention may be formulated with cocoa butter, glycerin, gelatin, etc. or combined with excipients such as polyethylene glycol and formed into suppositories. Good too.

The present invention provides for in vivo administration of a peptide of formula (I) and a pharmaceutically acceptable carrier. A method for inhibiting platelet aggregation and clot formation in mammals, particularly humans, comprising It also has something to offer. Indications for such treatment include acute myocardial infarction (A Ml), deep vein thrombosis, pulmonary embolism, dissecting aneurysm, transient ischemic attack (TIA) generalized disease, including seizures and other infarction-related diseases, as well as unstable angina. Intravascular coagulation (DIC), sepsis, surgical or infectious shock, postoperative or and postpartum injuries, cardiopulmonary bypass surgery, incompatible blood transfusions, placental abruption, thrombotic blood Chronic or acute conditions such as plateletopenic purpura (TTP), snake venom and immune diseases The hyperaggregation of is believed to be responsive to such treatment. Furthermore, the peptide of the present invention methods for preventing metastatic disease, preventing or treating fungal infections, and determining whether bone resorption is the cause. It may be used in methods of prevention or treatment of diseases in children.

The peptide is such that the concentration of drug in plasma is sufficient to inhibit platelet aggregation. The drug is administered to the patient orally or parenterally. Contains the peptide The pharmaceutical composition may be administered at a dose of about 0.2 to about 50 mg/kg in a manner consistent with the patient's condition. administered in amounts. For acute treatment, parenteral administration is preferred. Excessive cohesiveness For persistent conditions, administer the drug in 5% dextrose in water or normal saline. Intravenous infusions of putido are most effective, but intramuscular porous injections are also sufficient.

For chronic but non-critical platelet aggregation, capsules or tablets Oral administration or porous intramuscular injection is preferred. The peptide is approximately 0° Administered 1 to 4 times daily at doses of 4 to about 50 mg/At, about 0.4 to about 200 mg/At. A total daily dose of 9/day is achieved at W97.

The present invention further provides for in vivo administration of a peptide represented by formula (I) and a fibrinolytic agent. A method of inhibiting arterial or venous re-occlusion after fibrinolytic treatment is also provided. It is. Does administration of peptides in fibrinolytic therapy completely prevent reocclusion? , or were found to delay the time to reocclusion.

As used in connection with the present invention, the term fibrinolytic agent refers to the lysis of fibrin clots. Compounds, either natural or synthetic, that cause them directly or indirectly It also means things. Plasminogen activator is a well-known fibrinolytic agent. It's a group. Useful busminogen activators include, for example, Anist. Leplase, urokinase (UK), prourokinase (pUK), streptococcus kinase (SK), tissue plasminogen activator (tPA); chemically modified or one or more amino acids added, deleted or substituted or, for example, by replacing the active site of one plasminogen activator with another protein. Fibrin-binding domain or fibrin-binding portion of lasminogen activator One or more functional domains can be added, deleted or It includes modified mutants or variants thereof. Other mutants include 1 Examples include tPA molecules in which more than one glycosylation site has been modified. plasminogen Preferred activators are those that increase the serum half-life of plasminogen activator. tP in which the primary amino acid sequence has been modified in the growth factor domain to increase It is a mutant of A. tPA growth factor mutants are, for example, Robin European Patent Application Publication No. A0297589 and Brown ( European Patent Application Publication No. A0240334 and UK It is disclosed in Patent No. 8815135.2. Other variants include the European patent No. 0028489, European Patent No. 0155387 and European Patent No. 02978 Hybrid proteins such as those disclosed in No. 82 (these all of which are cited herein). Anistreblase is used in the present invention. is a preferred hybrid protein for use. Fibrinolytic agents are simply derived from natural sources. may be isolated, but are generally produced by conventional methods of genetic engineering.

Useful formulations of tPA, SK, UK and pUK are described, for example, in European patent application publication No. A0211592, German Patent Application No. 3032606, European Patent Application No. AOO92182 and U.S. Pat. No. 4.568.543 ( all of which are cited herein). Typically, the fibrinolytic agent is Sodium acetate or ammonium acetate buffered to pH 35-5.5 or Buffered isotonic aqueous solutions such as sodium adipate or ammonium adipate May be formulated in liquid. Polyvinylpyrrolidone, gelatin, hydroxycellulose gum arabic, polyethylene, glycol, mannitol and sodium chloride Further excipients may be added, such as aluminum. Such compositions can be lyophilized. can be done.

The pharmaceutical composition uses both the peptide and the fibrinolytic agent in the same container. However, formulation in separate containers is preferred. Both drugs in solution When administered in a form, they can be placed in an infusion/injection system for co-administration or in tandem. It can be contained in the storage compartment.

Indications for such treatment include myocardial infarction, deep vein thrombosis, pulmonary embolism, occlusion and other occlusion-related diseases. The peptide may be tPA or other Immediately before, simultaneously with, or immediately after parenteral administration of the fibrinolytic agent. administered later. After reperfusion is established to maximally inhibit post-treatment reocclusion , it has been demonstrated that it is desirable to continue treatment with the peptide for a sufficient period of time. Ru. An effective dose of tPA, SK, UK or pUK is between 5 and 5 u/kg; An effective dose of the peptide is about 0.1-25119/AI9.

Box for convenient administration of simultaneous or separate inhibitors and fibrinolytic agents. For parenteral administration as described above in a single container, such as a carton or other container. and an effective amount of the inhibitor for parenteral administration as described above. 'Individual bottles, bags, vials or bottles each containing PA or other fibrinolytic agents or other containers are prepared. Such kits may include, for example, drugs in separate containers or in the same container, and for reconstitution in lyophilized plugs. It can consist of a container of solution. This variant allows for two chambers in a single container. Contains reconstitution solution and lyophilized plug, which must be mixed before use. can be set. For such a device, the fibrinolytic agent and the peptide are For example, packed separately in two containers or lyophilized together as a powder and may be obtained in a single container.

If both drugs are administered in solution form, they can be used in an infusion/injection system for simultaneous administration. or can be contained in a tandem device. For example, platelet aggregation inhibitors Intravenous form or tube to fibrinolytic agent in second infusion bag. It may be in an infusion bag that is serially connected through the infusion bag. using such a system , patients receive an initial porous injection or infusion of a peptide inhibitor followed by fibrinolytic You can receive antidote infusions.

The pharmacological activity of the peptide was evaluated by the following test: Thrombus formation in v Ivo inhibition can be achieved using the prostaglandins (Pro) of Aiken et al. staglandins), 19.629-43 (1980). Systemic effects and hemodynamics of peptide infusions in anesthetized dogs according to methods This is demonstrated by recording the positive effects.

Inhibition of blood cell and plate aggregation Blood was collected from an adult naive mongrel dog (citrate was added to prevent clotting). salt added). Platelet-rich plasma, by centrifugation at 150×g for 10 minutes at room temperature PRP was prepared. Wash by centrifuging the PRP for 10 minutes at 800Xg platelets were prepared. The obtained cell pellet was immersed in a Ca + 4-free Tyro. washed twice in buffer (pH 6,5), then containing 1.811 MCa← Resuspend at 3X10' cells/ml in tie buffer (pH 7.4). Ta. After 3 minutes of addition of peptide, the effect in all assays of platelet aggregation was determined. Added dynamic drug. The final agonist concentration was 0.1 units/III thrombin and 211M ADP (Sigma). Chronolog Lumi - Ablebometer (Chrono-LogL umi-A ggregom Aggregation was monitored with ether). Using optical transmittance 5 minutes after addition of agonist , percent according to the formula 9% agglomeration = [(90-CR)10(90-10)]X100 The agglomeration was calculated [where CR is the chart measurement, 90 is the baseline, and 10 is the PR P blank measurement value]. Plot % inhibition of aggregation versus peptide concentration IC1o was determined by: Peptides were assayed at 200a+M, A suitable A response curve was established by serial dilution by a factor of 2.

Before the addition of agonist to assess the stability of the peptide towards plasma proteases. The peptides were incubated in PRP for 3 hours (rather than 3 minutes).

Compounds for their ability to inhibit canine platelet aggregation in response to stimulation by ADP Assayed. Examples 1 to 5. Compounds of 12 to 13 and 15 are about 0.1 to 1° IC, in the range of 0 μM. having: Examples 6-9, 11.19-21.26 and Compound 27 had an IC of approximately 2-40 μM. and the compound of Example 10 has 200 It had an IC5° exceeding MM. The compound of Example 5 is a preferred compound.

The following examples do not limit the scope of the invention in any way, and the invention The method for producing and using the compound is explained. Other specific examples of , are readily apparent and available to those skilled in the art.

Example In the examples below, all temperatures are in degrees Celsius. Amino acid analysis is done by Dionex. The test was carried out using Dionex Autoion 100. Pe Analysis for peptide content is based on amino acid analysis. Mass spectrum is fast atoms Performed on a VGZab mass spectrometer using bombardment. thin layer chromadophy For this purpose, EM silica gel thin layer (0,25,) plates were used. ODS is Represents a tadecylsilyl silica gel chromatography support. Eluent composition The abbreviations used to represent n-BuOH: n-butanol, HOAc: acetic acid , H2O: water, EtOAc: ethyl acetate, 1-ProH: isopropanol, P : pyridine and CA: chloroacetic acid. HPLC is an isocratic gradient Base with CRIB recording integrator in either continuous gradient mode or continuous gradient mode By Beckman 344 gradient chromatography system It was conducted. Ultrasphere” and Ultrasphere Ultrasphere’ ODS are each located in Fuller, California. Beckman Instruments, Inc. Silica gel and oil manufactured by N Instruments Inc. It is a tadecylsilyl run chromatography support. Dynamax (D ynamax') and Dynamax' C18 silicage and octadenyl silane chromatography supports, respectively. Rainin Instruments Company (Rainin) Manufactured by Instrments COI). Sefadesokes (S) ephadex) is manufactured by Pharmacia Pharma, Bislitowey, New Chassis. In Chemicals (Pharmacia Fine Che Ilicals) It is a cross-linked poly(dextran) produced by Celite ) is a Mansville Corporation of Denver, Colorado. A filter aid consisting of acid-washed diatomaceous earth silica manufactured by LeCorp, ) It is. The solid phase peptide synthesis method was performed using an automated Beckman 990 It was done using a generator. Where indicated, peptide purity is determined by HPLC chromatography. It is based on the integral of grams. MeArg is US Pat. No. 4,687,758 (1987) by the method disclosed by Alt et al. was created.

G ] y (OMe)-hydrochloride (6,39,5Qmmol) in dry DMF ( 10011) Drop DIEA (19.3 mL 110 mmol) into the medium-cooled suspension to a neutral pH of 1:. Next, HOBt (7, 49, 55 mmol) and and Boc-N’MeArg(Tos) (22.1q, 50+tmol). Ta. The reaction mixture was stirred for several minutes and then treated with EDC (10,559,55 m mol) was added. The reaction mixture was warmed to room temperature and stirring continued overnight. true solvent Removed in vacuo and the residue was partitioned between ethyl acetate and water. IN organic extract HCI (3x), water (3x), 10% Na2COs (3x), water (3x) and and saturated salt solution (1x). Organic extract with anhydrous sodium sulfate Dry, filter, and then concentrate the title compound to a white solid (21.549.8 4%).

b) N'MeArg(Tos)-GIy(OMe) (2) B for 45 min at room temperature oC-N’MeArg(Tos)-Gly(OMe) (1) (26g, 5mmo l) was treated with 50% TFA in methylene chloride. Remove the solvent and dissolve the residue in chloride solution. Traces of TFA were removed by evaporation from the tyrene several times. Residue with ether and used in the next step without further purification.

c) N'(2-methyl)benzoyl-N'MeArg(Tos)-Gly(OM e) (3) N'MeA r g (T o 5) - G 1 y (OMe) (2) (5 mmol) of DIEA (1,92 g, 3.5NZ) was added to make the pH neutral (pH 7.5-8°0). HOBt (745m+9) and then 2-methylbenzoic anhydride (5,5mmol ) (produced from 2-methylbenzoic acid 1.379 and DCC 1.39) added. The reaction mixture was stirred at room temperature overnight. Remove the solvent under vacuum and dissolve the residue in vinegar. Dissolved in ethyl acid (120*J), water (3X40mA'), 10% sodium carbonate (2 x 40 ml), water (2 x 40 ml) and saturated salt solution. . The organic extract was dried over anhydrous sodium sulfate, filtered, and concentrated to the title The compound was obtained as a white fluffy solid (2.29).

d) N"(2-methyl)benzoyl-N"MeArg(Tos)-GI　y　( 4) N'(2-methyl)benzoyl-N'MeArg(Tos)-GI y(O Solution of Me) (3) (2,21,4, l mol) in methanol (10++1) IN NaOH (5++A'.

A solution of 5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour to complete the esterification. Completed water splitting. The solvent was removed and the residue was redissolved in water (50,N) and diluted with ethyl acetate. Back-extracted with chill and acidified with 3N HCl+ to acid p)((2,86). The liquid was extracted with ethyl acetate, the organic layer was washed twice with water, and dried over anhydrous sodium sulfate. filtered and concentrated to give the title compound as a white fluffy solid (1. 539).

e) Boc-Asp(0-cHex)-(2-methyl)phenylamide (5)B THF of oc-Asp(0-cHex)(3,29,10IIIlo1) (50mA') and N-methylmorpholine (1,21*L 1-1 ooao l) Add isobutyl chloroformate (1,43d, 11 mmol) dropwise to the medium-cooled solution. Ta. The reaction mixture was stirred for several minutes and then 2-methylaniline (1,17 ml. 11 mmol) of THF (5,0++4 medium solution) was added.The reaction mixture was Warm to room temperature and stir for 18 hours. After the reaction was completed (TLC monitored), The reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (300+l) and 5% Citric acid aqueous solution (3x), water (3x), 10% NaHCOs aqueous solution (3x), water (3x) and saturated salt solution (1x). Dry organic extract anhydrous Na25O4), filtered and then concentrated to a brownish solid. that Recrystallization from ter-hexane gave the title compound (1.7 g).

f) Asp(0-cHex)-(2-methyl)phenylamide (6) Boc-A sp(0-cHex)-(2-methyl)feramide (5) (1,2g, 3 mmol) in 50% TFA in methylene chloride (10+J) for 45 min at room temperature. treated with liquid. The solvent was removed and the residue was evaporated several times from methylene chloride to remove traces. of TFA was removed. The product is precipitated as its TFA salt by addition of ether. I was honored. The solid was collected and air dried.

Asp(0-cHex)-(2-methyl)phenylamide (6) (3+on+ DIEA (1.2 ml, 6.6 mmol) was added to bring the pH to neutral. Then, N-hydroxybenzotria Add sol (5011Ig, 3, 3111101), then DMF (5 N'(2-methyl)benzoyl-N'MeArg(To5)-G in mA') IV (1,53g, 2,96 w+ol) was added. Carry out the reaction in a cool place. Stir for 1 minute, then N-ethyl-N-(dimethylaminopropyl)carbodi Imide (EDC) (633 mq, 3.3 ma+ol) was added dropwise. reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was concentrated to dryness. oily residue Dissolved in ethyl acetate (10Q++1), water (3X40mA'), 5% citric acid water solution (2x40mz), water (2x4Q++/), 10% NaHCOs aqueous solution (2 X4(b/), successively with water (3X40ml) and saturated salt solution (IX40ml) I washed it. The organic extracts were dried (anhydrous NazSOs), filtered, and then concentrated. The title compound (1,59,71%) was obtained which was supported by the mass spectrum. Ta.

Protected linear peptide (-7) (1,439) was incubated with anisotropy for 1 h at 0°C. The mixture was treated with anhydrous HF (30 ml) in the presence of 3 m/ml. H under vacuum at 0℃ F was removed and the residue was triturated with ether to give an oil. 0.2 residue The crude peptide (1,13 9) was obtained.

Gel filtration (Sephadex' G-15, 1% acetic acid/water) The aliquots were purified by Appropriate fractions are collected and lyophilized to produce semi-purified peptides. (109119) was obtained. Half aliquots of semi-purified peptide (25s+) HPLC (5μ, Altex Ul, tra sphere’) ODS, I Q++++X 25cm, isocratic , A, acetonitrile, B: water-0.1% trifluoroacetic acid, 20% acetonitrile The title compound (20 u ) was obtained. MS (FAB) m/e 568.1 [M+H]”; HPLCk’ 10.93 (5μ Altex Ultra Sphere (Altex Ultra sphere") ODS, gradient, A niacetonitrile, B: water-0.1% tri Fluoroacetic acid, 10% to 50% acetonitrile within 20 minutes, UV at 220 nm detection), k' 6.17 (5μ Altex Ultrasphere (ALtex Ultrasphere+) ODS, Isocratic, 20% acetonitrile Amino acid analysis: Asp (1,00), Gly (0,97).

Example 2 N'benzoyl-N'MeArg-Gly-Asp-phenylamide (15) DMF (8 ++A') Add aniline (733 μl, 8001), HOBt (1,5 99,1,3 molar equivalents) and DTEA (280 μl) were added. reaction mixture The mixture was stirred for several minutes and EDC (1,929,10 mwol) was added. reaction mixture The mixture was allowed to warm to room temperature and stirred overnight.

The reaction mixture was concentrated and the residue was dissolved in ethyl acetate, water, IN hydrochloric acid (2x), water , 5% sodium bicarbonate, water and saturated sodium chloride. Yes The extract was dried (anhydrous NatSO4), filtered, and then concentrated to yield the title compound. (2.459.76%).

b) Asp(○-Bzl)-phenylamide (10) Boc-Asp(0-Bz I)-Phenylamide (9) (2,439,5, l mmol) at room temperature Treated with a 50% TFA solution in methylene chloride (20 ml) for 45 minutes.

The solvent was removed and the residue was evaporated several times from methyl chloride to remove traces of TFA. Ta. The product was precipitated as its TFA salt by addition of ether. solid Collected and air dried to give the title compound (3.369).

c) Boc-N’MeArg(Tos)-Gay (11) B o c-N’M eA r g (To 5) - G] y (OMe) (1) (14, 89, 2 IN NaOH (35 wL) to a solution of 8.8 mmol) in acetone (60 mA) 1.2 equivalents) was added dropwise. Bring the reaction mixture to room temperature to complete the ester hydrolysis. Stir at room temperature for 1 hour. The solvent was removed and the residue was redissolved in water (50++1) and vinegar Any unreacted esters were removed by back extraction with ethyl acetate and acidified with 3N HCl. (pH 2,86). Extract the acidic solution with ethyl acetate and wash the organic layer with water (2x). dried over anhydrous sodium sulfate, filtered, and concentrated to give the title compound in white. Obtained as a colored fluffy solid (10.539.73%).

DM of Asp(0-BZl)-phenylamide (10) (6,1m+1ol) Add DIEA (160 μ/, 1.5 equivalents) to the medium cold solution of F (7,5++1). The pH was made neutral.

Added N-hydroxybenzotriazole (1:21g, 7,9i+mol) and then Boc-N’MeA rg(T o5)-G1y (3,669,7,32 mmol) was added. reaction The mixture was stirred in a cold place for several minutes, and EDC (1,49,7,32 mmol) was added dropwise. reaction The mixture was warmed to room temperature and stirred for 18 hours. The reaction mixture was concentrated to dryness. oily residue The distillate was taken up in ethyl acetate (125,Z), water (3 x 40 ml), IN hydrochloric acid (2 X 4ffi ml>, water (2X40m/), 5% NaHCOs aqueous solution (2X4 0 m/), water (3X40 vol) and saturated salt solution (IX40++/) successively Washed. The organic solvent was dried with anhydrous NazSO, filtered, and then concentrated. The title compound was obtained (4.539.95%).

Boc-N’MeArg(Tos)-Gly-Asp(0-Bz　I)-pheni chloramide (12) (2,459,3,14 mmol) at room temperature for 45 minutes. Treated with 50% TFA in methylene (2Qmj'). Remove solvent and salt residue Traces of TFA were removed by several evaporations from methylene chloride. the residue with ether Trituration together gave the TFA salt of the title compound (2.72g).

N’MeA r g(To 5)-G l y-A sp(0-Bz) )-phenylamide (13) (970ig, 1, 12 mmol) in DMF Add DIEA (293 μt, 15 equivalents) to the solution in (2,Q*/) to adjust the pH. sum (7.0). Then, benzoyl chloride (137 μl, 1.18 mmol ) was added. The reaction mixture was stirred for 2 hours. Remove the solvent under vacuum and remove the residue. Take up in ethyl acetate (75 mA'), water (3x40++1), IN hydrochloric acid (2x4 0ml), water (2X40@l), 5% NaHCO3 aqueous solution (2X40ml), water Washed successively with (3X4Q++A) and saturated salt solution (IX40ml). Yes The organic solvent was dried with anhydrous Na25O, filtered, and then concentrated to give the crude peptide. (0,699) was obtained. Flash chromatography (Nrika, 2) of peptides ．． Purification by 5% methanol/ethyl acetate gave the title compound (2701 9).

The protected linear peptide (14) (184-9) was incubated with anisotropy for 1 h at 0°C. treated with anhydrous HF (Lowl) in the presence of 1 mA. F was removed and the residue was triturated with ether. Dissolve the residue in 0.2M acetic acid The crude peptide was obtained by washing several times with ether and freeze-drying (108 mw). Peptides were purified by flash chromatography (silica ODS, 10-20% acetic acid Tonitrile/water-0.1% TFA gradient) was used to obtain the title peptide. (71 mq). MS (FAB) mle 540 [M+H]”; HPLC k’　4.44　(5μ Altex-Ultrasphere (Altex Ult rasphere") ODS, gradient, A niacetonitrile, B: water - 0.1% Trifluoroacetic acid, 10% to 50% acetonitrile within 15 minutes, U at 220 stops V detection); Amino acid analysis: Asp (1,00), cry (1,08).

N’MeA　r　g(Tos　-G　y-A　sp(0-Bz　I )-phenylamide (13) (87019, lm+aol) in DMF (2, DIEA (2611', 1.5 eq.) was added to the solution in 0 ml') to neutralize the pH. (7,0) was obtained. Next, acetic anhydride (100μ111, 05 mmol) was added Added. The reaction mixture was stirred for 30 minutes. Remove the solvent under vacuum and dissolve the residue in vinegar. Dissolved in ethyl acid (75++7), water (3X4(bl), IN hydrochloric acid (2X4Q+ +1), water (2X40++1'), 5% NaHCOs aqueous solution (2X40+1), Washed successively with water (3x4011) and saturated salt solution (IX40 mA').

The organic solvent was dried (anhydrous Na2sO4), filtered and concentrated to give the title compound ( 0.57v, 79%).

b) N'acetyl N'MeArg-Gly-Asp-phenylamide (17) Protected linear peptide (16) (56519,780 ml aol) was incubated at 0°C. Treated with anhydrous HF (10++1+) in the presence of anisole (1d) for 1 hour.

The HF was removed under vacuum at 0° C. and the residue was triturated with ether. 0 residue ．． The crude peptide (30 0-9) was obtained. Gel filtration of peptides (Sephadex) ) G-15, 1% acetic acid/water). Collect appropriate fractions and lyophilize A semi-purified peptide (241 g) was obtained. Flat chromatography of peptides (silica ODS, 5% acetonitrile/water - 0.1% TFA) Further purification gave the title compound (236Q). MS (FAB) mle 478 ．． 2 [M1H]”; HPLCk’2.71 (5μ Artex Ultrasf Air (Altex Ultrasphere R) 0DS1 gradient, A near acetony Tolyl, B: Water-0.1% trifluoroacetic acid, 10%-50% acetic acid within 15 minutes. tonitrile, UV detection at 220 nm); amino acid analysis.

Asp (1,00), Gly (1,02).

Example 4 N6 acetyl-N’MeA r g-G] V-A s p-(2-carboxy Preparation of c)-phenylamide (18) 1. For Qmmolmml, protect tetrapeptyl according to the following method. Resin Bo c-N’MeA r g (To 5)-G l y-A s p ( 0-cHex)-2-ami7'benzoic acid (0-Bzl-resin) was prepared.

Starting at the carboxyl terminus, successive protected amino acids are added until the desired sequence is obtained. and added. To protect the alpha-amino group, t-butyloxycarbonyl ( Boc) group was used.・Arginine, tono with side chain functional groups protected as follows (Tos); aspartic acid, cyclohexyl ester (0-cHe):).

Methyl chloride 12 The BoC group was removed by treatment with 950% trifluoroacetic acid (TFA). removed. 7% diisopropylethylamine (DIEA) in methylene chloride Neutralization of the amine-TFA salt was performed by treatment with ). 3 equivalents of Bo in DMF c-amino acid and 3 equivalents of 1-hydroxybenzotriazole (HOBt). and 3 equivalents of dicyclohequinolcarbodiimide (DCC) in methylene chloride was used to attach amino acids to the growing peptide. Confirmed by ninhydrin test The completeness of the bond was inspected and the bond was repeated if desired. Positive ninhydrin test failed In case of complete binding, rebinding was achieved using BOP reagent. General The rotocalls are listed below.

1. Wash with methylene chloride 1X1 minute 2. Wash with 50% TFA 1X1 minute 3. Deblock with 50% TFA 1 x 20 minutes 4 Wash with methylene chloride 6 x 1 minute Neutralize with 5.7% DIEA 3X2 minutes 6. Wash with methylene chloride 4x1 minute 7. Wash with dimethylformamide 2X 1 min 8. Boc-AA + in DMF HOBt Do not drain 9, DCC in methylene chloride for 2 hours 10. Wash with dimethylformamide 2X 1 minute 11. Wash with methylene chloride 3X 1 minute Gisin's method [Helvetica, stain force, acta (Helv, Ch. ew, Acta), 56.1476 (1973)], BoC-2- Boc-2-aminobenzoic acid from cesium aminobenzoate and chloromethyl resin Acid-(〇-resin) was prepared. The synthesis was started in step 1 for the addition of Asp.

The N-terminal BoC group was removed with 50% TFA in methylene chloride and the resulting TFA salt After neutralization with 7% DIEA in methylene chloride, the resin-bound peptide was washed with acetic anhydride:pi Acetylate with lysine/methylene chloride (1:1:2.20.1> for 30 minutes. A protected peptide-resin intermediate (1,649) was obtained.

anhydrous liquid HF (1 (bl)) in the presence of anisole (1 w/) for 50 min at 0 °C. The peptide was cleaved from the resin by removing the side chain protecting group. under vacuum The HF was removed and the resin was washed with ethyl ether and air dried. Next, add 1 resin The crude peptide 2 was extracted with % acetic acid/water (2X30 mA') and then lyophilized. I got 001+9. Half HPLC (5 μ Vydac, 2 cm x 25 cm, iso Raftic, 10% acetonitrile/water 1% trifluoroacetic acid, 300n An aliquot (50 μg) was purified by UV detection using 0,019) was obtained. MS (FAB) m/e 522 [M1H]”; HPLC k'　6.7　(5μVydac, step gradient, A: Niacetonitrile, B: Water -0.1% trifluoroacetic acid, within 5 minutes 1% to 5% acetonitrile, within 25 minutes Amino acid analysis: Asp (1,0 0), Gly (1,04).

Example 5 No(2-methylthio)benzoylN'MeArg-Gly-Asp-(2-methylthio)benzoyl Chil Boc-Asp(0-cHex) (630g+9.2umol) and N- Methylmorpholine (242 mg, 1.2 mol/equivalent) in THF (10 mA') Isobutyl chloroformate (311μ!, 1.2 mol/equivalent) was added dropwise to the medium-cooled solution. . The reaction mixture was stirred for several minutes and 2-methylmercaptoaniline (295 votes 9.1 ．． 2 mol/equivalent) solution was added. Warm the reaction mixture to room temperature and stir for 18 hours. did. After the reaction is complete (TLC monitored), the reaction mixture is filtered and the filtrate is Evaporated to dryness. The obtained residue was dissolved in ethyl acetate, and 5% aqueous citric acid solution (3 ×), water (3×), 10% aqueous NaHCOs (3×), water (3×) and saturated Washed successively with aqueous sodium chloride solution (IX). Dry organic extract anhydrous (2CO3), filtered, and concentrated to give the title compound (860-g).

b) Asp(0-cHex)-(2-methylthio)feramide (20)Bo c-Asp(0-cHex)-(2-methylthio)feramide (19) (8 60-9) in 50% TFA solution in methylene chloride (10 ml) for 60 min at room temperature. Processed with. The solvent was removed and the residue was evaporated several times from methylene chloride to remove traces. TFA was removed and then triturated with ether to obtain the TFA salt of the title compound. Ta.

Asp(0-cHex)-(2-methylthio)phenylamide (1,97mmo l) Add DIEA (343 μ/) to the cold solution of (20) in DMF (21+7’) was added to make the pH neutral. Then, N-hydroxy-benzotriazole ( 320119,2,37+n+ol) and then Boa-Gly (3 80m+9.2.17ma+ol) was added. The reaction was stirred in the cold for a few minutes and E DC (416u, 2.17wol) was added dropwise.

The reaction mixture was warmed to room temperature and stirred for 18 hours. The reaction mixture was concentrated to dryness. profit The resulting residue was dissolved in ethyl acetate, diluted with 10% ICO, water (lx), and 5% quench. washed successively with acid (1x), water (2x) and saturated NaCl solution (1x). . The organic extract was dried (anhydrous NazSCL), filtered, and then concentrated to the title A compound was obtained (770 shoulder g).

d) Gly-Asp(0-cHex)-(2-methylthio)phenylamide (2 2) Boc-Gly-Asp(0-cHex)-(2-methylthio)phenyla Mido (21) (770 liters) was dissolved in methylene chloride (5 liters) for 60 minutes at room temperature. % TFA solution. The solvent was removed and the residue was evaporated several times from methylene chloride. Traces of TFA were removed. The residue was triturated with ether to give the title compound. Obtain TFA salt (609,5 rich 9).

Gay-Asp(0-cHex)-(2-methylthio)phenylamide (22) DIE into a cold solution of (594M9.1.17m+ol) in DMF (2ffil) A (204 μl) was added to bring the pH to neutral. N-hydroxybenzotriazo (19 (bv, 1.4 mmol)) and then Boc-N'MeAr g(Tos) (570 mg, 1.29 mmol) was added. Carry out the reaction in a cool place. After stirring for a minute, EDC (24719, 1,29 mmol) was added dropwise. reaction mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was concentrated to dryness. Residual obtained Dissolve the material in ethyl acetate, add 10% KzCOs (2X), water (1X), 5% KzCOs. Washed successively with phosphoric acid (3x), water (2x) and saturated NaCl solution. organic extraction The output was dried with anhydrous Na2S04), filtered, and then concentrated to yield the title compound. (920 inches).

f) N'MeArg(Tos)-Gly-Asp(0-cHex)-(2-methyl ruthio)-phenylamide (24) Boc-N’MeArg(Tos)-Gly-Asp(0-cHex)-(2- Methylthio)-phenylamide (23) (920-9) was treated with salt for 60 min at room temperature. Treated with 50% TFA solution in methylene chloride (5 m/). Remove solvent and remove residue Traces of TFA were removed by several evaporations from methylene chloride, then with ether. Trituration together gave the TFA salt of the title compound (635.519).

g) 2-Methylmercaptobenzoic acid (25) 2-mercaptobenzoic acid (59,3 2,5 mmol) of THF (30 m/) and triethylamine (5,4 mA ) Methyl iodide (2,2 wing I) in THF (2 mA') was added dropwise to the cold solution.

The reaction mixture was stirred in the cold under an inert atmosphere for 18 hours. triethylamine The salts were filtered off and the filtrate was concentrated to dryness. Dissolve the residue in ethyl acetate (125,1) , washed successively with 5% citric acid (2x), water (3x) and saturated NaCl solution. Ta. The organic extract was dried (anhydrous Mg5O4), filtered, and then concentrated to the title The compound was obtained as a solid (4.09).

h) N'(2-methylmercaptobenzoyl)N'-MeArg(Tos)-G ly-Asp(0-cHex)-(2-methylthio)phenylamide (26) N”MeArg(Tos)-Gly-Asp(0-cHex)-(2-methylthi e) DM of phenylamide (24) (635 shoulder 9.0, 760111101) Add DIEA (133 μl, 1.0 eq.) to the cold solution in F (2 ml) to adjust the pH. was made neutral. Add N-hydroxybenzotriazole (37119) and then Then, 2-methylmercaptobenzoic acid (25) (463 u) was added. reaction The mixture was stirred in a cold place for several minutes, and EDC (528-9) was added dropwise. Bring the reaction mixture to room temperature Warmed and stirred for 28 hours, then concentrated to dryness. Dissolve the residue in ethyl acetate , 10% 2CO3 (2X), water (2X), 5% citric acid (2X), water (2X) and saturated NaCl solution (1x). Clean organic solvents and anhydrous NazSO4), and then concentrated to obtain the crude peptide (1,19).

Peptides were purified by chromatography (silica, methylene chloride:methanol 91). The title compound (610-9) was obtained by purification.

i) N'(2-methylthio)benzoyl-N'MeArg-Gly-Asp-( 2-Protected linear peptide (26) (270 mq) was incubated at 0°C for 1 hour. Treated with anhydrous HF (5 mA) in the presence of Nisole (0,5++7). under vacuum , HF was removed at 0 °C and the residue was triturated with ether to obtain the crude peptide (17 9++g) was obtained.

Gel filtration (Sephadex') G-15, 1% acetic acid/water ) was used to purify aliquots (130u). Collect appropriate fractions and lyophilize. A semi-purified peptide (107 mw) was obtained. The peptide was flash chromatographed. Fi (silica ODS, gradient eluent, 10-50% acetonitrile/water - 0,1 % TFA) to obtain a purer peptide (75.4u).

Half-preparative HPLC (5μ Altex Ultrasphere (Altex Ultr) asphere') ODS, I Qmm x 25c (isocratic, 22 % acetonitrile/water - 0.1% trifluoroacetic acid, 22On! 1 for UV detection) Final purification by gave the title compound. MS (FAB) m/e 632 [ M+H]”+HPLCk’ 7.5 (5μ Artex Ultrasphere ( Altex Ultrasphere’) ODS, Gradient, A Niacetonitrile , B: water - 0,1% trifluoroacetic acid, 10% to 50% acetonitol within 40 minutes Ril, UV detection at 220um), k' 5.9 (5μ Artex Ultra Sphere (Altex Ultrasphere R) ODS, 23% acetoni Tolyl/water - 0.1% trifluoroacetic acid, UV detection at 2201 m), amino acid content Analysis: Asp (1,00), Gly 5-iodopenta under argon atmosphere A solution of ethyl nitrate (1,669,65 mmol) in DMF (25 mj') was Liumimidonicarbonic acid t-butyl methyl ester (1,389,5,5 mmol) was added. The resulting mixture was stirred at room temperature for 4 hours under an argon atmosphere. Mixed The mixture was poured into HzO and extracted with Ebo (3X 100 xi). The organic extract was washed with HzO (3x501/) and then dried (Na2 sO4). The solvent was removed under reduced pressure and the residue was subjected to flash chromatography. The title compound was purified by eluting with 25% EtOAc/hexanes. Obtained as a colorless oil (2.09.100%).

b) 5-[[(1,1-dimethylethoxy)carbonyl]amino]pentanoic acid 5 -[N-(t-butoxycarbonyl)-N-(methoxycarbonyl)aminocope Ethyl tanthanate (2,09,6,6viol) (7) MeOH (3C1+1' ) Nakomizo liquid 1:0.95 NNaOH (30wl, 28, 5 l1ffial ) was added and the resulting mixture was stirred at room temperature.

After stirring for 5 hours, the resulting clear solution was concentrated under reduced pressure. Acidify the concentrate with glacial acetic acid The mixture was extracted with CHxClx (3X 100++1). Combined with The extract was dried (Na2SO4) and the solvent was removed in vacuo to give a white solid ( 1. Oy, 71%): Melting point 45-47°C.

c) Boc-Asp(0-Bz　I)-phenylamide aniline (2,81hf 30.9 mmol) and diisopropylethylamine (4°Og, 30.9 Dry DMF (32 mJ) of 關o1) Nakazo liquid was cooled to 0°C under an argon atmosphere. Ta. Add HOBt hydrate (4,3y, 32.0mmol) to this solution, and then Ite, Boc-Asp (0-Bz I) (10.0g, 30, 9 mmol) was added. The resulting mixture was stirred at 0 °C until dissolution occurred and EDC hydrochloride (5,99,3o, Ommol) was added dropwise. Slowly bring the resulting mixture to room temperature and stirred for 2 hours.

The mixture was partitioned between HzO (500H1> and EtoAc (2oo@l).

The aqueous phase was extracted with EtOAc (200 zl) and the combined organic extracts were purified with HzO ( 3X 75zl) and saturated NaCA' aqueous solution (75,1), Dried (Na2S 04).

The solvent was removed in vacuo and the residue was then combined with 2y% EtOΔC/hexane. Shattered. The solid that formed was collected by filtration to give the title compound (7.79 ).

The filtrate was concentrated under reduced pressure to obtain additional material (2.3y, 81% total yield).

d) Asp(Bz　1)-phenylamide trifluoroacetate Argon atmosphere Boc-Asp(Bz　])-phenylamide (5,75 g, 14. 4vaol) (’) 1: 2 to’J full, to acetic acid/CfhClx (30 mA') The Nakamizo solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated under reduced pressure; The residue was treated with Et, 0 (3x3(b/)) and then evaporated to give a white powder. (6.Oy, 1.00%).

e) Boc-Gly-Asp(Bzl)-phenylamide 0 under argon atmosphere Asp(Bzl)-phenylamide trifluoroacetate (5,99 , 14,3 mmol) in dry DMF was diisopropylethyl 7mine (1 , 859,14.3 mmol) and then HOBt hydrate (2.16 g , 16.0111101) was added. Boc-glycine (2,59, 14,3 mmol) and the resulting mixture was stirred at O’C until dissolution occurred. Stirred. EDC hydrochloride (2,749,14,3 ma+ol) was added dropwise. obtained The mixture was slowly warmed to room temperature and stirred for 2 hours. The mixture was heated to HzO (200 m1A') and EtOAc (1001/). The aqueous phase was mixed with EtOAc. c (100@1) and the combined organic extracts were extracted with HzO (3X 50ml) and saturated NaCA' water solution' g & (40 mA') were continuously washed and dried. (Na2SO4). The solvent was removed in vacuo and the residue was flash chromatographed. Purified by eluting with 60% EtOAc/hexanes to give a white A colored solid was obtained (2.4g, 38%).

f) Gly-Asp(Bzl)-phenylamide trifluoroacetate al Bo c-G] y-A sp(Bz])-fe under the Gon atmosphere Nylamide (2,49,5,3ymol) in 1:2 trifluoroacetic acid/CH2C 12(15++/) Namomizo liquid was stirred at room temperature for 1 hour. Concentrate the reaction mixture under reduced pressure and the residue was treated with Et20 (3x3017) and then evaporated to a white powder. (2.5 g, 100%).

Gay-Asp(Bzl)-phenylamide at 0 °C under argon atmosphere. A solution of trifluoroacetate (2,0y, 4.2 mmol) in dry DMF was Add propylethylamine (0,559,4,3o+mol), then add H OBt hydrate (0°599.4.4a++ mol) was added. the resulting mixture 5-[[(1,1-dimethylethquine)carbonyl]amino]pentanoic acid (0 , 92y, 4.2 mmoυ was added and stirring continued for 10 minutes, resulting in dissolution.

EDC hydrochloride (Region 819.4, 211111101) was added dropwise. the resulting mixture The mixture was warmed gently to room temperature and stirred for 2 hours. The mixture was left at room temperature overnight and then stirred for 2 hours. Ite, HzO (100mA') Oyohi EtOAc (75mA') l: Distribution I let it happen. The aqueous phase was extracted with EtOAc (75 m/) and the combined organic extracts were zO (3X30g/) and saturated NaCl water me (30ml>t' continuous fJc Schiff JLC purification L, next iteration, and drying (Na2S 04). Remove solvent in vacuum and the residue was subjected to Furanoyu chromatography with 7% MeOH/CH2Cz Purification by elution with 2 gave the title compound (0.89, 34%). child After trituration with EtOAc, a white solid was formed.

h) N-[5-[[(1,1-dimethylethquin)carbonyl]az-]-1- Oxy~ pentyl]-Gly-Asp-phenylamide N-[5-[[(1,1-dimethyl ethoxy)carbonyl]amino]-1-oxopentyl]-G I y-As p(0-Bzl)-phenylamide (0,72g, 1,3 mmol) Add 10% palladium on activated carbon (150 mp) to anhydrous EtOH (75 ml). Added. The resulting mixture was hydrogenated with 3-psiH2 for 3 hours. Remove the mixture Air and then filtered through a pad of Celite. Strain the filtrate Concentration in vacuo gave a white solid (0.58q, 97%).

N-[5-[[(1,1-dimethylethquino)carbo Nyl]amino]-1-oxopentyl-Gay-Asp-phenylamide (0 ,589, ], , 3 mmol) of 1,4 trifluoroacetic acid/CH2 (J '2 (25+17') Namomizo liquid was stirred at room temperature for 1 hour. Reaction mixture under reduced pressure Concentration gave the title compound, which was used without further purification.

j) N-[[5-(aminoiminomethyl)aminoco-1-oxopentyl]-G lyN-[5-amino-1-oxopentyl]-Gly-Asp-phenylami (0.59.12 mmol) was placed in distilled H,O and diluted with 0.95N NaOH. The pH was adjusted to 10. Hydrogen sulfate O-methylisourea (0,249,1,4mm ol) was added and the pH was readjusted to 10 with 0°95N NaOH. The resulting mixture The mixture was stirred at room temperature overnight. Reaction analysis by thin-layer chromatography The reaction was found to be incomplete, so 0-methylisourea hydrogen sulfate ( 2.4g, 12. (hcaol> was added. 10% NaOH'tl-pH was added. >10 and then the mixture was stirred at room temperature overnight under an argon atmosphere.

The pH was adjusted to pH 6 with 3N HCJ and the mixture was then kept under argon atmosphere for several days. Stored at −25° C. under air. After thawing, collect the white solid formed by filtration. collected. The filtrate was lyophilized, the combined solid was triturated with MeOH, and then Filtered. The filtrate was concentrated in vacuo and the residue was subjected to reverse phase HPLC with 0.1% TFA. The title compound was purified by elution with 25% MeOH/H20 containing (0.21y, 40%) was obtained.

Example 7 N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-ox Preparation of [Sopentyl]-Gly-Asp-phenylamide (29) Argon atmosphere Below, (4R)-4-benzyl-2-oxazolidinone (20, 0.09,0.132 mol) of n-butyllithium (2.0.0.0,132 mol) in THF. A 5M solution (52.8 ml, O, )32 mol) was slowly added. obtained The solution was stirred for 10 minutes at -78°C and then added with pre-chilled phenyl chloride. Solution of cetyl (17,5s+1.0.132mol) in THF (5(bA') (-78°C) was added slowly. The resulting solution was warmed to room temperature and stirred overnight. Ta. The solvent was removed under reduced pressure and the residue was dissolved in Et*O(1,(D’) and H,0(150 Tomi I) was distributed. The organic extract was immersed in HzO (100 W/> and NaCl saturated water. The solution was washed successively with 75 blades and dried (NazSO4).The solvent was removed under reduced pressure. Removed solid residue (10%): suspended in tOAc/hexane (500,/) . Insoluble matter was removed by decantation. Upon cooling, a white crystalline solid emerges from solution. was formed and collected by filtration (15.29.39%). Concentrate the filtrate under reduced pressure. Ta. The residue was combined with the above insoluble material and subjected to flash chromatography for 30 min. Purification eluting with % EtOAc/hexane gave additional material (total yield: 2 3.09.59%). Melting point 69-71°C.

4-(R)-benzyl-3-phenylacetate at -78°C under argon atmosphere Newly prepared thyl-2-oxazolidinone (23.7g, 8Q, 3mmol) Potassium bis(trimethylsilyl)amide was added to the solution in distilled THF (600W/). A solution of (16117.80.3 o1 0.5M solution in toluene) was added dropwise. . The resulting solution was stirred at -78°C for 20 minutes and then precooled. Allyl bromide (10,5,/.

A solution of 121 o1) in THF (40 mA') was added dropwise. the resulting mixture Stir at -78°C for 1 hour, then warm to room temperature and stir overnight. Glacial acetic acid (1 4,017') was added to the reaction mixture, which was then filtered. Reduce pressure of filtrate Concentrate and partition the residue between Et, O(1,OA') and H20 (200-mf) I let it happen. The organic extract was dissolved in H2O (2X 150-1), 5% NaHCO, aqueous solution ( 150W/) and NaCl saturated aqueous solution (150wj’), then and dried (Naz S O4). Remove the solvent in vacuo and flush the residue. chromatography and eluting with 30% EtOAc/hexane. It was purified by Both parts containing the pure (2R,4R) diastereomer were combined. . The remaining material is a mixture of (2R,4R) and (2S,4R) diastereomers. It consisted of This mixture was triturated with cyclohexane to further purify it. (2R,4R) diastereomer was obtained (total yield: 11.79.43%).

Melting point 73-75°C.

c) Methyl (2R)-2-phenyl-4-pentenoate - 20°C, argon atmosphere A solution of methoxymagnesium bromide under [methyl bromide magnesium at -20 °C] Nesium (16.1 ml of a 3.0 M solution in EtzO) was dissolved in methanol (20 ml ) to (2R,4R)-4-benzyl-3-(14+so-2-f) phenyl-venter-4-enyl)-2-oxazolidinone (8,19,24,2m mol) in methanol (200 liters) was slowly added. The obtained solution The liquid was stirred at -20°C for 1 hour, then warmed to 0°C and stirred for 1.5 hours. p Quench the reaction by adding H7 phosphate buffer (200 l) and Stirred at 0°C for 10 minutes. The mixture was heated to CHzClt (700W/> and N H 4Cl/Na Cl aqueous solution (1:1 w/w, 700W/) . The aqueous layer was extracted with CH2Cl2 (3X 200II>) and the combined organic extracts was dried (Na2SOJ. The solvent was removed in vacuo and the residue was dissolved in hexane (2X4 0++/) and then stirred. Decant the solid formed So it was removed and the solvent was removed under reduced pressure to give the title compound (3.79.80%).

d) methyl (2R)-5-iodo-2-phenylpencunate under an argon atmosphere, Methyl (2R)-2-phenyl-4-pentenoate (37 g, 19. Add BH3・THF (1 in THF) to a solution of 5 o1) in anhydrous THF (50@l). , 6.5*L of 0M solution (6.5 o1) was added. The resulting solution was heated at 50°C. Add soaked anhydrous methanol (1,511) to the reaction mixture, which was heated for 1 hour and then cooled to room temperature. dropwise to the reaction mixture, then quickly add anhydrous sodium acetate (16 g, 19.5 mu o1) in anhydrous methanol (1811) and iodine monochloride (CH2C12 13.0 d, 13.0 + mol) of a 1°0M solution in medium was added. the resulting mixture The mixture was stirred at room temperature for 45 min and then treated with sodium thiosulfate in H2O (800 +J) Pour it inside.

The mixture was extracted with EhO (3X 275 m/) and the combined organic extracts were dried. (Na2SO4). The solvent was removed in vacuo and the residue was flash chromatographed. Purified by chromatography eluting with 10% EtOAc/hexanes; The title compound was obtained as a colorless oil (2.49.39%).

e) (2R)-5-[N-(t-butoxycarbonyl)-N-(carbomethoxy )ami/]-2-phenylpentanoic acid methyl ester (2R )-5-iodo-2-phenyl-methyl benchunate (199,5,9 mmol) Potassium t-butoxymethyliminodicarboxylate was added to a solution in DMF (25 mA'). (1,39,6, Ommol) was added. The resulting mixture was heated at room temperature for 30 minutes. Stirred. The mixture was poured into HsO (200 ml) containing sodium thiosulfate. , extracted with EhO (3×100+*/). The combined organic extracts were purified with H2O (3X 2Qg/) and dried (Na2SO4). Remove the solvent under reduced pressure and remove the oil. Obtained.

The aqueous washings were further extracted with CHtCb (3 x 100 ml) and the combined C The Ht CI 2 extract was washed with H2O (3x 25 mA') and dried ( Na2　S　O4). The solvent was removed in vacuo and the residue was flash chromatographed. Purified by eluting with 25% EtOAc/hexane and giving the title The compound was obtained as a colorless oil (13g, 60%).

(2R)-5-[(N-t-butquinecarbonyl)-(N-carbomethoxy)a Minoc-2-phenylpentanoic acid methyl ester (1,39,3,,6 mmol ) in methanol (25,1) with 0.95N NaOH (15,0 mA', 14.2 mmol) was added and the resulting mixture was stirred at 0°C. 2 hours at 0℃ After stirring, the mixture was warmed to room temperature and stirred for an additional 3 hours. The mixture was heated with H2 Pour into O(15QmA'), acidify with acetic acid, CH2Cl2 (3X 100 ml>. The combined organic extracts were dried (NazSO4). true solvent Removal in vacuo and NMR analysis showed the presence of starting material. methane residue (15d) and 0.95N NaOH (15@l, 14, 2 + u +ol) was added. The mixture was stirred at room temperature for 3 hours. The resulting solution was diluted with H2O (200ml), acidified with acetic acid, then CHzCA'z (3X1 25 ml). Dry the combined organic extracts (Na2SO4) and remove the solvent. Removal in vacuo gave the title compound (1.0 g, 96%). [akoo2s(1, CHsOH)aminoco-1-oxopentyl]-glycyl-NI-phenyl- -Asunokurutoamide benzyl ester Gay-As+)(0-Bz　])-phenyl at 0°C under argon atmosphere A solution of amide (1,44 g, 3, l mmol) in dry DMF (5 Add diisopropylethylamine (0.40 g, 3.1 mmol) to Then, HOBt hydrate (0,439,3,2 mmol) was added. to the mixture (2R)-5-[(t-butoxycarbonyl)ami-2-phenylpentanoic acid A solution of (0,909,3,1關01) in DMF (3 mA) was added, and the obtained The mixture was stirred at 0°C for 10 minutes. EDC hydrochloride (0°599.3, lm mol) was added dropwise. The resulting mixture was slowly warmed to room temperature and placed under an argon atmosphere. It was kept under ambient air overnight. The mixture was purified with H2O (1500 mA') and EtOAc. The aqueous layer was extracted with EtOAc (50@1) and combined. The organic extracts were dissolved in H2O (3X 2081) and a saturated aqueous NaCl solution (25 1) and dried (Na2SO4). Remove solvent in vacuum and the residue was subjected to flash chromatography in 5% MeOH/CH2C4. Purification by elution with gave a white solid (1.19.57%).

h) N-[(2R)-2-phenyl-5-[(1,1-dimethylethoxycarboxylate) aminoco-1-oxopentyl]-G]y=Asp-phenylamide N- [(2S)-2-phenyl-5-[(1,1-dimethylethoxycarbonyl)a Minococ-1-oxopentyl]-Gay-As'p-phenylamide (059, 079 mmol) in anhydrous EtOH (100++/) on activated carbon. Add radium (250 inches) and stir the resulting mixture in water at 30 pSiHz for 45 minutes. After addition, the mixture was degassed and filtered through a pad of Celite (C61ite). Ta. The filtrate was concentrated under reduced pressure to obtain the title compound (0.43 g, 100%).

N-[(2R)-2-phenyl-5-[(1,1-di methylethoxycarbonyl)aminoco-1-oxopentyl]-Gly-Asp - Phenylamide (0,439, Q, 8m5ol) of trifluoroacetic acid (1,0 The solution in ml) and CHIClt (5 ml) was stirred at room temperature for 1.5 hours. The reaction mixture was concentrated in vacuo and the residue was treated repeatedly with Et, O and evaporated to white. A powder was obtained (0.439.98%).

N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-ox [sopentyl]-Gly-Asp-phenylamide (0,449, Q, 8an ol) in distilled water (5 ml) and adjusted the pH to >12 with 1,0N NaOH. Ta. Add 0-methylisourea hydrogen sulfate (1,49,7,9 mmol), 1. The pH was adjusted to >12 with ON NaOH. The resulting mixture was stirred at room temperature overnight. Ta. The pH was adjusted to 6 with 3N HCl and the mixture was lyophilized. Me the residue It was triturated with OH (5 tomA') and filtered. Concentrate the filtrate under reduced pressure and separate the residue. Subjected to reverse phase HPLC and eluted with 0.1:25.75 TFA/CH3CN/HIO. It was purified by The isolated material was lyophilized to give the title compound ( 1.6g, 41%). Amino acid analysis.

Asp (1,00), Gly (1,05).

Aniline (1.45 mL 15.9 anol) and diisopropylethylamide A solution of (2°059.15.9 ml 1 ol) in dry DMF (16 mA') was It was cooled to 0° C. under an argon atmosphere. Add HOBt hydrate (2,239 ,16,5關01) and then Boc-Asp(0-cHex)(5, Qg, 15, 9 mmol) was added.

The resulting mixture was stirred at 0 °C until dissolution occurred, then EDC hydrochloride ( 3,059,15,9III1101) was added dropwise. Slowly mix the resulting mixture The mixture was slowly warmed to room temperature and stirred for 2 hours. The mixture was heated to HzO (250 mA') and Partitioned into EtOAc (150ml). The aqueous layer was treated with EtOAc (150 mg ) and the combined organic extracts were saturated with HzO (3 x 50 l) and NaCl saturated It was washed successively with aqueous solution (40 liters) and dried (Na2SO4). vacuum the solvent The residue was triturated with 20% EtOAc/hexane to give a white solid. (3.859.62%).

b) Asp(0-cHex)-phenylamide trifluoroacetate argon atmosphere Boc-Asp(0-cHex)-phenylamide (3,8y, 9.7 A solution of 1=1 trifluoroacetic acid/CHtCb (20 wl) was prepared at room temperature. Stirred for 30 minutes. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in Et20 (3X 3Q si') and evaporated to give a white powder (4.09, -'100%).

c) N-[N”-(1,1-dimethylethquinecarbonyl)-NS-[imino[ [4-(methylphenyl)sulfonyl]amino]methyl]-L-olnitylco glycine benzyl ester Gly-OBzl hydrochloride (70 g, 35 mmo l) in dry DMF (40++/) was diisopropylethylamine (4, 59.35 ml) was added. Add HOBt hydrate (5,29,38 , 5o1) and then N”-(1,1-dimethylethoxycarbonyl )-N'-[imino[[4-(methylphenyl)sulfonyl]amine]methyl] -L-ornithine (15,09,35 mmol) was added. the resulting mixture Stir at 0 °C until dissolution occurs, then add EDC hydrochloride (6,79,35 mmol l) was added dropwise. The resulting mixture was slowly warmed to room temperature and stirred overnight. Applicable The mixture was partitioned between HtO (350ml') and EtOAc (75ml).

The aqueous layer was extracted with EtOAc (2X 75m/) and the combined organic extracts were Washed with O (3X 4 Qml) and dried (Na2SO4). The residue was flash chromatographed in 7% MeOH/ Purification by elution with CH2C/2 gave the title compound (9.09.49 %).

Compound of Example 8(C) (15, Oq, 26o+mo l) in trifluoroacetic acid (2011) and CH2Cl2 (30++1) The solution was stirred at room temperature for 1 hour. The reaction mixture was concentrated under reduced pressure, and the residue was dissolved in Et20 ( 3X 3011) and evaporated to give a white powder (15.69.100% ).

Diisopropylethylamine (0,989,7,6a) under argon atmosphere Example 8(d) compound (1) was added to a solution of Example 8(d) in CH2Cl2 (35ml) , 5g, 2.5+amol) were added. α,i゛-dichloro is added to the resulting mixture. -0-xylene (0.44 g, 2.510101) was added and the reaction mixture was Stirred overnight at room temperature under an argon atmosphere. The mixture was dissolved in H,o (2X 2Qm The organic extract was concentrated under reduced pressure.

The residue was subjected to flagge chromatography to give 5% M e OH/CH2C. Purification by eluting with 0.12 g of 1.2 g to give the title compound as a white solid (0.1 g). 659.46%).

f) N-[(2S)-2-(1,3-dihydro-2H-isoindolyl)-5- [imino[[4-(methylphenyl)sulfonyl]amino]methyl]amino]- 2-oxopentylcoglycine Compound of Example 8(e) (0.55 g, 0.95 a+mol) in anhydrous ethanol Add 10% palladium on activated carbon (20019) to the solution in 15Q ml did. The resulting mixture was hydrogenated at 30 psiH for 4.5 hours. the mixture It was degassed and filtered through Celite filter. The filtrate was concentrated in vacuo to give the title compound. (0.459.98%).

g) N-'[(2S)-2-(1,3-dihydro-2H-isoindolyl)-5 -(aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp(0 -cHex) monophenylamide AS+)(0-cHex)-phenylamide at 0°C under argon atmosphere. Trifluoroacetate (275 recommended 9.0, 681 lnal) in dry DMF (5 Add diisopropylethylamine (88 my, 0.68 anol) to the solution in Added. Add HOBt hydrate (110119, Q, 3anol) to this solution Then, the compound of Example 8(f) (330m+9, Q, 58anol) was added. The resulting mixture was stirred at 0 °C until dissolution occurred and diluted with EDC hydrochloric acid. Salt (130 mgg, 0.68 anol) was added dropwise. Slowly mix the resulting mixture The mixture was warmed to room temperature and stirred overnight. The mixture was diluted with HtO (100 mg) and Et Partitioned into OAc (40wf). EtOAc (2X 40It’) for the aqueous layer The combined organic extracts were washed with HtO (3x 20111) and dried. (Na2SO,). The solvent was removed in vacuo and the residue chromatographed. Purified by eluting with 5% MeOH/CH2Cl 1 to give the title compound as a white solid (260 mv, 50%).

h) ``Mata criminal 2 discharges - 2 views 2 illusions 2 phantom cups above n sky'' 2 to 2 noimino methyl) a Mino-1-oxopentyl]-Gly-Asp-phenyla 1 Compound of Example 8(g) (150 mg, 0.2 anol) in anisole (1,5 -) The solution was transferred to a hydrogen fluoride reaction vessel. Hydrogen fluoride was introduced into this at -78℃. The resulting solution was heated to 0°C. After stirring at 0°C for 1 hour, hydrogen fluoride was added. Removed in vacuo. Ether (100 IA') was added to the residue, then 1 Stir vigorously for an hour. The solid formed was collected by quick filtration and subjected to reverse phase H Purified by PLC eluting with 40% MeOH/H20. frozen After drying, the title compound was obtained as a hygroscopic powder (30 mg, 30%).

Example 9 N'-(4,4-siphaerbutyryl)-N1-methyl-Arg-Gly-As p-glynon benzyl ester hydrochloride (0,919,4,5 mmol) and di- Sopropylethylamine (0,58q, 4.5noool) in dry DMF (7 The solution in s+4') was cooled to 0° C. under an argon atmosphere. In this solution, HOBt hydrate (0,669,4゜9　Ilmol　) and then Boc-Me Arg(Tos) (2,009,4,5 mmol) was added. the resulting mixture was stirred at 0 °C until dissolution occurred and added with EDC hydrochloride (0,869,4,5 mm ol) was added dropwise. The resulting solution was slowly warmed to room temperature and placed under an argon atmosphere. Kept under overnight. The mixture was mixed with 820 (100 m/) and EtOAc (75 m/) ++l) 1m distribution c' seta. The aqueous layer was extracted with EtOAc (75i+/), The combined organic extracts were combined with HsO (3X 20++4') and NaCl saturated aqueous solution. (30 shoulder l) and dried (NazSO4). Remove the solvent in vacuo and the residue was purified by flash chromatography to give a white solid. (2.40y, 89%).

Boc-MeArg(Tos)-Gly-OBz I under argon atmosphere (2,409,4, (lu+ol) of trifluoroacetic acid (101/) and CH The solution in zC4 (30ml) was stirred at room temperature for 90 minutes. The reaction mixture was concentrated under reduced pressure. , the residue was treated with 50% Et20/hexane (3X30ml) and evaporated to white. A colored solid was obtained (2.5 g, 100%).

c) N-[N"-(4,4-diphenylbutyryl-Ns(imino[[4-( methylphenyl)sulfonyl]amino]methyl]N! -Methyl-L-ornityl ]-Glycylbenzyl ester MeArg(Tos)-Gly-OBzl (2,59,4,11Il101) and drying of diisopropylethylamine (0,55 g, 4.1!16101) The solution in DMF (6 shoulders) was cooled to 0° C. under an argon atmosphere. in this solution HOBt hydrate (0,629,4°6 o1) was added, then 4,4-diphthalate Phenylbutanoic acid (1.2 g, 4, l mol) was added. the resulting mixture was stirred at 0 °C until dissolution occurred and EDC hydrochloride (0,809,4,Ilm ol) was added dropwise. The resulting solution was slowly warmed to room temperature and placed under an argon atmosphere. Kept under overnight. The mixture was heated with N20 (300++1>) and EtOAc (7 (1+A'). The aqueous layer was extracted with EtOAc (2X70mA') and The combined organic extracts were washed with HzO (3X 40++1>) and dried (Na 2SO<).

The solvent was removed in vacuo and the residue was flash chromatographed to 5% M Purification by elution with eOH/CHzCz2 gave the title compound as a white solid. (1.4 g, 48%).

d) N'-(4,4-diphenylbutyryl)-N'MeArg(Tos)-GI yN'-(4,4-Sifny-1-lubutyryl)-N'MeArg(Tos) -GI y-OBZI (1,369, l, 9 mmol) in anhydrous -EtO Add 10% palladium on activated carbon (350-g) to a solution in H (100++1> Ta. The resulting mixture was hydrogenated with 3QpsiH2 for 3 hours. Degas the mixture , filtered through a pad of Celite. The filtrate was concentrated in vacuo to give a white solid (1. 009.83%).

e) N’-(4,4-diphenylbutyryl)-N”MeA r g (To 5) -G I y -Asp(0-cHex)-phenylamideAsp(0-cHe x)-feramide trifluoroacetate (0.65 g, 1°6 mmol) and diisopropylethylamine (0,219,1,6 grade o1) in dry DMF The solution in (4 mA') was cooled to 0° C. under an argon atmosphere. HOB to this solution t hydrate (0,22y, 1.7 mmol) and then Example 9(d) A solution of the compound (1,09, l, 5 mmol) in dry DMF (3++1) Addition of liquid 1. f:. The resulting lettuce mixture was stirred at 0°C for 5 hours and then diluted with EDC hydrochloride (0, 31 g, 1.6 dragon o1) was added dropwise. The resulting solution was slowly warmed to room temperature. , and kept under an argon atmosphere overnight.

The mixture was partitioned between HzO (150++1) and EtOAc (50ml) . The aqueous layer was extracted with EtOAc (50 ml) and the combined organic extracts were purified with HzO ( Wash sequentially with 3X 30 ml A’) and NaCl saturated aqueous solution (30,1). , dried (NazSO4). The solvent was removed in vacuo and the residue was flash chromatographed. Purify by chromatography and elution with 5% MeOH/CHzC12. The title compound was obtained as a white solid (0.759.53%).

Anisole (200-g, 0, 22 mmol) of the compound of Example 9(e) 1,517') solution was transferred to a hydrogen fluoride reaction vessel. This was added to the liquid film at -78°C. Hydrogen fluoride (15 ml) was introduced and the resulting solution was warmed to 0°C. 1 o'clock at 0°C After stirring for a while, the hydrogen fluoride was removed in vacuo. Ether (75++A ') was added and then stirred. The solid formed was collected by filtration and subjected to reverse phase HP Subjected to LC and eluted with 60% MeOH10, N20 containing 1% TFA. It was purified by Appropriate fractions were combined and concentrated under reduced pressure. Freeze-dry the concentrate overnight A white powder was obtained (1519.10%).

The crude product was purified by semi-fractional HPLC to obtain the purified compound, which was lyophilized. The title compound was obtained (1519). TLCR20,65(n-BuOH:HO Ac:N201: 1: 1).

Example 10 Preparation of Amino-1-oxopentyl]-Gay-Asp-phenylamide (32) A solution of potassium carbonate (560 votes 9.4, 0 o1) in H,0 (10 shoulders) at 0°C Arg(Tos) was added to. After stirring for several minutes at 0°C, 4-bromobutanoyl chloride (185 Oe + mol) and stirred the resulting mixture for 30 minutes. and extracted with Et and O. The aqueous layer was acidified with cold dilute aqueous HCI and EtOAc. Extracted. The solvent was removed in vacuo to give the title compound (0.4g, 87%).

N'-(4-bromo-1-oxobutyl)-A at 0°C under argon atmosphere DMF of r g (Tos) (2,49,5,Qmmol) (30++1 ) of sodium hydride (1.1 g of a 60% suspension in mineral oil, 27.5 mmo 1) was added and the resulting mixture was warmed to room temperature and stirred overnight. Place the mixture on ice. Poured into H20, acidified with dilute aqueous HCl at 0<0>C and extracted with EtOAc.

The aqueous layer is made of Amberlite' XAD-2 (1309). It was placed on a column and filtered by gravity to obtain the title compound (1,49). Vacuum organic extract An additional 0.59 of material was obtained by concentrating in silica (73% overall yield). Elemental analysis (C+ tH24NsO5S・0.85H20): Theoretical value: C49,59,H6, 29, N 13.60: Actual value C49,97, H6, 22°N13.24° c) N-[(2S) 2(1(2-pyrrolidinonyl))-5'-(aminoimide [methyl)-amino-1-oxopentyl]-Gly-Asp(0-cHex) -Phenyrue Mutualjou 4NHC1/dioxane 5m/l, -Boc-Gay-Asp(0-cHex) -Dissolve phenylamide (496IIg, 1.11+uol) at room temperature for 20 minutes Stir for a while. Dissolve the residue in methylene chloride and evaporate three times to ensure HCI removal. It came true. The residue was dissolved in 5 ml of DMF and diisopropylethylamine (1 77u, 1.37meal) was added. The pH was adjusted to neutral with base. H.O.B. t (193my, 1.43mmol), N-[(2S)-2-(1-(2-pi) loridinonyl))-5-((toluenesulfonyl)amino-iminomethyl)ami Nor-1-oxopentanoic acid (550 mg, 1.39 mmol) and 1-(dimethyl thylaminopropyl)-3-ethylcarbodiimide hydrochloride (266■9.1 , 39++osol) were added continuously. The reaction was stirred at room temperature for 18 hours. Ta. The DMF was removed in vacuo and the residue was dissolved in ethyl acetate/chloroform (approximately 1.1) Dissolved in NaH3O4 (3X) and washed with 5% NaHCOs (3X). , dry Mg5OJ, filtered and then concentrated in vacuo. 60919 (yield 76%). The crude product was purified by flash chromatography (silica gel, 10% methane). (tanol/chloroform). Collect appropriate fractions and evaporate the solvent. (463 hits, yield 53%). NMRδ(CD(Js) 1.1 2.1(m , 14 H), 23 (s, 5H), 2.9 (d, 2H), 3.0-3.7 (m, broad, 3H), 3.85-4.0 (m, 2H), 4.55-5.1 (m, Broad, 3H), 6.25-6.65 (m.

Broad, 2H), 6.9-7.9 (m, 9H), 7.9-8.1 (m, I H); TLCR, 0,35 (15% methanol/chloroform): MS (FA B) m/e 726 [M+H]'.

d) N-[(2S)-2-(1-(2-pyrrolidinonyl))-5-(aminoimide Amino-1-oxopentyl]-Gly-Asp-phenylamide The protected peptide of Example 10(c) (21519,291 nmol) was added at 0°C for 1 hour. During that time, it was treated with 5 ml of anhydrous HF. The HF was removed in vacuo and the residue was dissolved in glacial acetic acid. The crude peptide salt was obtained by lyophilization (159m+9.99%). 50 fables Filter and preparative HPLC (C18 reverse phase, 13% 7-1=tonitrile-Q, 1% TF A/Water-〇, 1% TFA). Remove the solvent and dissolve the peptide in 10% acetic acid. It was dissolved and lyophilized (23 g). HPLCk' 2.58 (C18 reverse phase, Gradient, A: Niacetonitrile-0,1% trifluoroacetic acid, B: Water-0,1% trifluoroacetic acid. Fluoroacetic acid, 10%-50% over 15 minutes, UV detection at 220um), k'　2.34　(C18 reverse phase, 17% acetonitrile/water-0.1% triflu (oroacetic acid, UV detection at 22 onm); TLCR, 0.60 (butanol:acetic acid : water 1: 1: 1), R, 0,54 (butanol: ethyl acetate: acetic acid: water 1: 1: 1: 1); MS (FAB) m/e 490.2 [M+ H]”;7 MiJWI1 analysis: Asp (1,0), Gly (0,85), Ar g(0,21); peptide content 69.87% (peptide was heated at 160"C for 1 hour , 2:1:0. O05%v:v:wHC1':TFA:hydrolyzed with phenol) .

Example 11 N'-(phthaloyl)-A r g-G Iy-A p-phenylamide (33) key A r g (Tos) (3,289,10,Ommol) Phthalic anhydride (1,489,10, Ommol) in a solution in toluene (40 ml) and triethylamine (0,381,30°Qmmol) were added. obtained The mixture was refluxed using a Dean-S tark trap. Heated under flowing water. After refluxing for 2 hours, the reaction mixture was cooled and concentrated under reduced pressure. Residue Methylene chloride was added to the distillate and dissolved by heating and stirring for 30 minutes. the solution H! Washed with O and the solvent removed in vacuo. White flaky solid with Et and O and collected by filtration to give the title compound (4.139.90%).

Elemental analysis (CnHzzN<0sS)・Theoretical value: C55,01,H4,84,N 12.22; Actual value: C55, 55, H5, 19, N 11.77° 4NHC 1/Dioxane 5++A! 1. Knee Boc-Gly-Asp (0-cyclofheki sil)-phenylamide (49619,1,11 mmol) was dissolved at room temperature. Stirred for 0 minutes. The residue was dissolved in methylene chloride and evaporated three times to remove HCA'. I made sure to leave. Dissolve the residue in DMF 5++/diisopropylethyl alcohol. Min (17719.1, 37 mmol) was added. Neutralize the pH by adding base Adjusted to gender. HOBt (19311g, 1.43 mmol), phthaloyl -A r g (To 5) -OH (637119, 1,39 amol) and 1 -(dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (26619 , 1, 39 mmol) were added successively and the reaction was stirred at room temperature for 18 hours. DM F was removed in vacuo and the residue was taken up in ethyl acetate/chloroform (approximately 1.1). , l　N　NaHS　3 times with O4, 3 times with 5% NaHCOs, and dry M g5O<), filtered and then evaporated. 817m (yield 94%) was obtained.

The crude product was purified by flash chromatography (silica gel, 10% methanol/ (chloroform). Collect appropriate aliquots according to tlc and remove solvent. and the residue was dried in vacuo (48019.55%). NMRδ(CDCl 2) 1.11-1.9 (m, 13H), 2.05-2.2 (br s, 3H ), 2.35 (s.

3H), 2.9 (6,5H), 3.0-3.3 (brs, LH), 3.8 5-4.0 (m.

LH), 4.55-5.1 (m, broad, 3H), 6.25-6.65 ( m, broad.

3H), 6.9-7.9 (m, 13H), 8.0 (s, LH): TLCR2 0,37 (15% methanol/chloroform); MS (FAB) m/e 78 8 [M+H]”.

c) N'-(phthaloyl)-Ar g-G 1 y-As p-phenylamide The protected peptide of Example 11(b) (200 mg, 254 nmol) was added to anhydrous HF. 5++/ for 1 hour at 0°C. The HF was removed in vacuo and the residue was dissolved in glacial acetic acid. The crude peptide salt was thawed and lyophilized (135 mg, 87%). 100+9 and gel filtration (Sephadex") G-10,10 % acetic acid). Appropriate fractions were collected and lyophilized to yield the title compound. (25+Ig). HPLCk’　3.49　(C18 reverse phase, gradient, A Niacetonium Tolyl, B: water-0,1% trifluoroacetic acid, 10%-50% over 15 minutes acetonitrile, UV detection with 220nIIl), k' 7.56 (C18 reverse phase , 20% CH3CN/water-0,1% trifluoroacetic acid, UV detection at 220 um ); TLCR10,77 (butanol:acetic acid:water, 1.1:1), 064 (butanol:acetic acid:water, 1.1:1); Nor: Ethyl acetate: Acetic acid: Water, 1: 1: 1: 1); MS (FA B) m/e 552.2 [M1H]"" diamino acid analysis: Asp (0,98 ), Gly (0,98), Arg (0,84); peptide content 67.01% ( 1 hour at 160℃, 2+1+0.005%v:v:wH(J'/TFA /hydrolyzed peptide with phenol).

N'MeA r g (To 5) - G l y (OMe) (2) (5mmo DI EA (2.88 ml, 16, 5 mmol) was added to make the pH neutral (pH 7.5-8.0). Then salt Benzoyl dihydride (0.64++1.5.5 mmol) was added. The reaction mixture Stirred overnight at room temperature, the ε solvent was removed under vacuum and the residue was dissolved in ethyl acetate (60 m/s). water (3X20++1), 10% sodium bicarbonate aqueous solution (2X20m /), water (2X20+1), 5% citric acid aqueous solution (2X20 liters), water (2X 2Q I/) and saturated salt solution. Dry the organic solvent with anhydrous sulfur sodium chloride), filtered and concentrated to give the title compound as a white fluffy solid. (1.94 g).

b) N'benzoyl-N'MeArg(Tos)-GayN'benzoyl-N' MeArg(Tos)-Gly(OMe)(3)(3,59,6°811101 ) (1) A solution of 1N NaOH (8,25 mf) in methanol (15 mA) , 8°12 mmol) was added dropwise. The reaction mixture was stirred for 1 hour at room temperature to The hydrolysis was completed. Remove the solvent and redissolve the residue in water (80@A’). , back-extracted with ethyl acetate and acidified with 3N HCl to acidic pH (1,35). Applicable The acidic solution was extracted with ethyl acetate, the organic layer was washed twice with water, and anhydrous sodium sulfate was added. dried, filtered and concentrated to give the title compound as a white fluffy solid. (3.39).

c) Boc-Asp(0-cHex)-(4-rioo)7 aleamide Boc- Asp(0-cHex) (6, 29, 20 mmol) in THF (100 sO) and N-methylmorpholine (2.42 ml, 22+++nol) in the solution. Isobutyl roroformate (2,86ml 1.22++5ol) was added dropwise. the reaction mixture Stir the mixture for a few minutes, then add 4-chloroaniline (2.8 g, 22+ smol) A solution of 10% in THF (10%) was added. The reaction mixture was warmed to room temperature and 18 Stir for hours. Once the reaction was completed, the reaction mixture was concentrated to dryness. residue was dissolved in ethyl acetate (300,1), water (3x), IN HCI (2x), water (3x), 10% aqueous NaHCO3 (3x), water (3x) and saturated salt solution. Washed continuously. The organic extract was dried over anhydrous NazSO4), filtered and then , and concentrated to give a white solid. The title compound was recrystallized from ethyl acetate-hexane. (2.49).

d) Boc-Asp (0-cHex )-(4-chloro)phenylamide (1,449,34+mol) in methylene chloride The mixture was treated with a 50% TFA solution in water (10 ml) for 45 minutes at room temperature. remove the solvent, The residue was evaporated several times from methylene chloride to remove traces of TFA. Aete The solid was collected and air dried to precipitate the product as its TFA salt. Ta.

e) N'benzoyl N'MeArg(Tos)-Gly-Asp(0-cHex )-(4-chloro)phenylamide Asp(0-cHex)-(4-chloro)phene dissolved in DMF (811/) DI EA (1.5 ml, 8 ．． 6+aa+ol) was added to make the pH neutral. Next, HOBt (300 ++y, 2.2+u+ol), then N1 in DMF (5++/) Benzoyl-N'MeA rg (To 5)-G] V (1,009,2 mmol) was added. The reaction was stirred in the cold for several minutes, then N-ethyl- N.

-(dimethylaminopropyl)carbodiimide (EDC) (49219,2,5 7a+mol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 hours. . The reaction mixture was concentrated to dryness. Dissolve the oily residue in ethyl acetate (100,1) water (3X), IN HCI (2X), water (3X), 10% NaHCO, water soluble Washed successively with liquid (3x), water (3x) and saturated salt solution. The organic extract Dry anhydrous Na25O4), filter and then concentrate to give the title compound (1 ．． 749). Flash chromatography (silica gel, gradient 2-8% methane) Purification by diluent/methylene chloride) gave the title compound (1.1 g).

Protected linear peptide of Example 12(e) in the presence of anisole (2 ml) (900@9) was treated with anhydrous HF (20 ml) at 0° C. for 1 hour. Under vacuum, 0° The HF was removed with C and the residue was triturated with ether to give an oil. residue Dissolved in 0°2M acetic acid, washed several times with ether, and lyophilized to obtain the crude product. (775-9). The peptide was purified by flash column chromatography (silica O D.S.

Purification by gradient 20-26% acetonitrile/water-0.1% TFA) yielded the title compound. Putido was obtained (343W9). MS (FAB-) m/e 574.4 [M+ H]", 572.2 [M-HF-; HPLCk' 9.94 (5μ Artetsu Kusu Ultrasphere (Altex Ultrasphere) OD S , 4.5 ++++ % trifluoroacetic acid, 1-50% A within 20 minutes, UV detection at 220 nm), and' 6.88 (21% acetonitrile/water-01% trifluoroacetic acid, UV detection at 220 stop); TLCR20,74 (n-BuOH:HzO:AcO H: EtOAc 1: 1: 1: 1) and R+ 0.74 (n- BuOH:AcOH:HIO:pyridine 15:5:10:10); Amino acid analysis :Asp (1,00), Gly (1,09).

Example 13 N'benzoyl-N'MeArg-Gly-As+) (3-carboxy)-F Benzyl alcohol (2,749,20 o1) of en-3-aminobenzoic acid (2,749,20 o1) Benzenesulfonic acid (3,489, 22 mmol) was added. The reaction mixture was collected using a Digi-Stark trap. The mixture was heated for 4 hours. The alcohol was distilled off, the residue was dissolved in ethyl acetate, water, 1 Washing sequentially with 0% aqueous NaHCOs (3x), water (3x) and saturated salt solution did. The organic extract was dried over anhydrous Na2S (O4), filtered, and then concentrated. The title compound was obtained (1.5g).

THF of Boc-Asp(0-cHex) (0,9159,2,9ma+ol) (15++f) and N-methylmorpholine (0,35++1.3.2mmo l) Add isobutyl chloroformate (0.42ml 1.3.2mmol) dropwise to the medium shade solution. did. The reaction mixture was stirred for several minutes and then added with benzyl 3-aminobenzoate (0 , 669,2,9 o1) in THF (2 m4) was added. 'Nuclear reaction mixture The mixture was allowed to warm to room temperature and stirred for 18 hours. As soon as the reaction is complete, remove the reaction mixture. The mixture was concentrated to dryness. Dissolve the residue in ethyl acetate, water (3x), 5% citric acid Aqueous solution (3X), -water (3X), 10% NaHCO3 aqueous solution (3X), water (3X ) and saturated salt solution. The organic extract was dried and anhydrous Na25 ( L), filtered and concentrated to give a brownish solid (1°469). Hula chromatography (silica gel, gradient 0.3-0.8% methanol/salt) Purification by methylene chloride) gave the title compound (0.649).

c) Asp(0-cHex)-(3-benzyloxycarbonyl)phenyla MidoAsp(0-cHex)-(3-benzyloxycarbonyl)phenylamide (0°641.1.2 of) with 50% TF in methylene chloride (12@A’) It was treated with solution A for 60 minutes at room temperature. Remove the solvent and extract the residue from methylene chloride. Traces of TFA were removed by evaporation several times. The product as its TFA salt Crystallized from tel-hexane (0.69).

d) N'benzoyl-N'MeArg(Tos)-Gly-Asp(0-cHe x)-(3-benzyloxycarbonyl)phenylamide Asp(0-cHex )-(3-benzyloxycarbonyl)phenylamide (600 mg, 1.11 mmol) of DMF (10++1) DI EA (0,43 votes 1.2°45101) was added to make the pH neutral. Then, HOBt(165 ++v, 1,22 mmol) and then N'vegetate in DMF (5 mA). Nzoyl-N’MeArg(Tos)-Gay (5591+9.1.11mm ol) was added. The reaction was stirred in the cold for a few minutes, then N-ethyl-N'- (dimethylaminopropyl)carbodiimide (EDC) (2341+9.1.2 2 ml 1 ol) was added dropwise. The reaction mixture was warmed to room temperature and stirred for 18 hours. did. The reaction mixture was concentrated to dryness. Dissolve the residue in ethyl acetate (10011) water (3x), 5% citric acid aqueous solution (3x), water (3x), 10% NaHCO , aqueous solution (3x), water (3x) and saturated salt solution. organic extraction The product was dried in anhydrous Na2S04), filtered, and then concentrated to yield the title compound. (0.879). Flash chromatography (silica gel, gradient 3-5 % methanol/methylene chloride) to give the title compound (0.449) .

The protected linear peptide of Example 13(d) in the presence of anisole (1d) 0,449) was treated with anhydrous HF (10@A') at 0°C for 1 hour. Under vacuum, 0℃ The HF was removed and the residue was triturated with ether to an oil. 0.2 residue Dissolved in M acetic acid, washed several times with ether, and lyophilized to obtain the crude product (22 019). The peptide was purified by flash chromatography (silica ODS, gradient position). 〇-15% acetonitrile/water (1% TFA) to obtain the title peptide. (91u). MS (FAB) m/e 584 [M+H]”, 582 [ M-Hl-; HPLCk' 9.73 (5μ Artex Ultrasphere (Altex Ultrasphere”) OD S, 4.5ms+X 25 cm, gradient, A: Niacetonitrile, B: Water-0.1% trifluoroacetic acid, 20 min. UV detection at 1%-50% A, 22Or+U) and to' 13.08 (12% acetonitrile/water - 0,1% trifluoroacetic acid, UV at 220ni detection); TLCR70,58 (n-BuOH:N20:AcOH:EtOAc 1: 1: 1: 1) and R+ 0.56 (n BuOH:Ac OH:H,O:pyridine 15:5:10:10); Amino acid analysis: Asp (1° 00), Gly(1,01).

Example 14 N1benzoyl-N'MeArg-Gly-Asp-(4-carboxy)phae 4-aminobenzoic acid (4,129,30 mmol) in benzyl alcohol (3 Benzenesulfonic acid (5,229,33mmol l) was added.

The reaction mixture was heated using a Dee 2 Starktra knob for 4 hours. The Al Distill the coal and dissolve the residue in ethyl acetate, water, 10% NaHCO3 (2X ), water (3x) and saturated salt solution (IX). Dry organic extract Dry anhydrous Na2S04), filter and concentrate to give the title compound (1. 629).

Boc-Asp(0-cHex) (0,8839,2,8 mmol) in THF (10++6) and N-methylmorpholine (0,34 g/, 3,1 wol) Isobutyl chloroformate (0,40,j', 3.1 wol) was added dropwise to the negative solution.

The reaction mixture was stirred for several minutes, then benzyl 4-aminobenzoate (0,59 A solution of 49.2.5 mmol) in THF (2 mA) was added. the reaction mixture was allowed to warm to room temperature and stirred for 18 hours. As soon as the reaction is complete, the reaction mixture was concentrated to dryness. Dissolve the residue in ethyl acetate, water (3x), 5% citric acid in water solution (3x), water (3x), 10% NaHCOs aqueous solution (3x), water (3x) and and saturated salt solution. The organic extract was dried (anhydrous NazSOn), Filtration and concentration gave the title compound as a yellowish solid (1.18 g).

Boc-Asp-(0-cHex)-(4-benzyloxycarbonyl)tuuni Ruamine (1.1 g, 2.1 mol) was mixed with 50% TFA/methylene chloride (20, 6) for 90 minutes at room temperature. Remove the solvent and extract the residue from methylene chloride several times. Evaporation removed traces of TFA. the product from ether-hexane, Crystallized as its TFA salt (0.97v).

Asp(0-cHex)-(4-benzyloxycarbonyl)phenylamide ( 092 g, 1.71 mmol) of DMF (15 mA') in a medium shade solution. EA (0.66 mr, 3.78 mmol) was added to bring the pH to neutral. H.O. Add Bt (0,2549,1,88l1liol), then DMF ( N'benzoyl-N'MeA rg(Tos)-Gl in 5++4') y (0,8619,1,71 mmol) was added. Stir the reaction in the cold for a few minutes. The mixture was stirred and then EDC (0,369,1,88 dragon o1) was added dropwise. the reaction mixture The mixture was allowed to warm to room temperature and stirred for 18 hours. The reaction mixture was concentrated to dryness. residue was dissolved in ethyl acetate (100+A'), water (3 x χ, -5% citric acid aqueous solution ( 3X), water (3X), 10% aqueous NaHCOs (3X), water (3X) and saturated Washed successively with Japanese salt solution. The organic extract was dried with anhydrous Na25O4) and filtered. and then concentration to give the title compound (1.069). Flash-crunch the product. Purified by chromatography (silica, 3% methanol/methylene chloride) and labeled. The following compound was obtained (0.579).

e) N'benzoyl-N'MeArg-G1y-Asp-(4-carboquine) The protected linear peptide (0,569) of Hue Example 14(d) was treated with anhydrous HF (1 (bl)) for 1 hour at 0°C in the presence of , the HF was removed at 0° C. and the residue was triturated with ether. 0.2M residue Dissolved in acetic acid, washed several times with ether and lyophilized to obtain the crude product (230 u). The peptide was purified by flash chromatography (silica ODS, gradient apparatus 0- Purification with 13% acetonitrile/water-0.1% TFA) gave the title peptide. (1819). MS (FAB) m/e 584.2 [M+H]”, HPL Ck' 10.0 (5μ Fulltex Ultrasphere (Altex Ul) trasphere') ODS, 4.511X25cm, gradient, A. Aceto Nitrile, B: water - 01% trifluoroacetic acid, 1% - 50% A, 2 within 20 minutes UV detection at 20 nm) and '8.55 (13% acetonitrile/water- 0,1% TFA, UV detection at 220 nm), TLCR.

0.71 (n-BuOH:HzO:AcOH:EtOAc 1: 1: 1:1) and R10,58 (n-BuOH:AcOH:HzO:pyrinone 1 5:5:10:10); Amino acid analysis Asp (1,08), GIY ( 1,00).

Example 15 N'(2-thenylcarbonyl)-N'MeA r g-G l y-A s p-phenyla Boc-Asp (0-cHex) (31,54v, l Q Qm mol) of THF (500ni) and N-methylmorpholine (12,09m/mol) , 11 Qmmol) isobutyl chloroformate (14,2717', 110℃mo1. ) was added dropwise. The reaction mixture was stirred for several minutes and then anilined. A solution of ChL (11 Qmmol) was added. The reaction mixture was allowed to cool to room temperature. and stirred for 18 hours. As soon as the reaction is complete, concentrate the reaction mixture to dryness. Ta. The residue was dissolved in ethyl acetate (80011), water (3x), 5% citric acid solution. solution (2x), water (3x), 10% NaHCO3 aqueous solution (2x), water (3x) and and saturated salt solution. Dry the organic extract with anhydrous NazSO<) , filtered and then concentrated to give a white solid. The product was dissolved in ethyl acetate-hexane. The title compound was obtained by recrystallization from (31.429).

b) Asp(0-cHex)-phenylamide Boc-Asp(0-cHex) - Phenylamide (11 g, 28.9 mmol) in 50% TFA/methylene chloride (120 ml) for 90 minutes at room temperature. Remove the solvent and dissolve the residue in methane chloride. Traces of TFA were removed by evaporation several times. to add ether The product was precipitated as its TFA salt (11.29).

Example 14(d)(7) Procedure Nishita Katte, Asp (in DMF (30*r) 0-cHex)-feramide (4,2y, 9 mmol) in DMF (5 m DIEA (3.45ml, 19.8ml1ol), HOBt (1 ,349,9,9mmol) and N'Boc-N'MeAr g(Tos) Reacts with -G l y (11) (3,749,7,5fflIIlO1) The title compound was obtained (5.669).

d) Procedure for N'MeArg-Gay-Asp-7-Ramide Example 14(e) The protected peptide of Example 15(c) was treated with TFA to obtain the title compound. (1.0749).

N’MeA r g-G I y-A s p-phenylamide (27) (0, 163g, 0゜37mmol) of DMF (5++/) DIEA in a medium solution (100/,!J, 0.555au+ol) to neutralize the pH (pH 7,5 ~8.0)-. Then, 2-thenylcarboxylic anhydride (0.37 mmol ) (Prepared from 2-thenylcarboxylic acid (96,419, Q, 75 mmol) ) and DCC (76,419,0,37 mmol) were added. the reaction Stir in the cold for several minutes, warm to room temperature, and stir for 18 hours. Concentrate the reaction mixture It was dried and solidified. The residue was then subjected to gel filtration (Sephadex'). G-15, 1% acetic acid/water). Collect appropriate fractions and lyophilize. A semi-purified product was obtained (45u). Flash chromatography of the peptide (Silica ODS.

Further purification was performed using a gradient device (0-16% acetonitrile/water-0.1% TFA). The title compound was obtained (16.3 mg), MS (FAB) m/e 546.2 [M+H]', 544.4 [M-HF-; HPLCk' 16 (5μ Al ``Altex Ultrasphere'' OD S, 4.5mmX 25c gradient, A Niacetonitrile, B: Water - 0.1% trifluoroacetic acid, 1-50% A within 20 minutes, UV detection at 220°C), and and '9.02 (10% acetonitrile/water - 0.1% trifluoroacetic acid, 220nrt' UV detection). TLCR10,83(n-BuOH:HtO:A cOH:EtOAc 1:1:1:1); and R,0,64(n -BuOH: AcOH: HzO: pyridine 15:5:10:10) diamino Acid analysis: Asp (1,00), Gly (0,98).

GI　y(OBz　1) p-toluenesulfonate (2,939,8,7mmo l) Dry medium temperature suspension IRA in DMF (71,51 mA', 410 o1) was added dropwise to bring the pH to neutral. Next, HOBt (1.17g, 8.7fflI 1101) and N’Boc-Arg(Tos) (3,389,7,89m mol) was added. The reaction mixture was stirred for several minutes and then treated with DDC (1 , 789,8,7 mmol) was added. The reaction mixture was warmed to room temperature and overnight Continued stirring. Remove the solvent under reduced pressure and flash chromatograph the residue. (silica, gradient 2-10% methanol/chloroform) to produce the title compound. (3.05v).

b) Boc-Arg(Tos)-Gay N'Boc in methanol (50ml) -Arg(Tos)-Gly (0,659,1,12 o1) and carbon superactivation Polymerized palladium (100 liters) was reacted for 5 hours in a Parr shaker. Ta. The catalyst was removed by filtration and the filtrate was concentrated to give the title compound.

Following the procedure of Example 14(d), Asp( 0-Bzl)-phenylamide (10) (2,241110101) as DI EA (0,4@l, 2.24a+ol), HOBt (0,39,2,24 mmol), Bop reagent (0,999,2,24 mmol) and Boc- A　r　g(To　5)-G　y　y　(2.24mmol) to react with crude The product was obtained. This was subjected to flash chromatography (silica, 5% methanol). /chloroform) to give the title compound (0.499).

cl)Arg(Tos)-Gly-Asp(0-BzI)-phenylamide Boc-Arg(Tos)-Gay-Asp(0-Bz　])-Feramide (22019,0,287 mmol) in 50% TFA/methylene chloride (10++ A') for 4 hours at room temperature. Remove the solvent and extract the residue from methylene chloride several times. Evaporation removed traces of TFA. The residue was treated with HCl-dioxane, Evaporation from toluene several times and drying under vacuum gave the title compound as its hydrochloride salt. Ta.

DMF of Arg(Tos)-Gly-Asp(0-Bzl)-phenylamide Triethylamine (43 μm) was added to the solution in (20 ml) until neutral pH.

Acetic anhydride (30 μl) was added and the reaction mixture was then stirred for 18 hours. true The solvent was removed under vacuum and the residue was subjected to flash chromatography (15% methanol). The title compound was obtained (0.149).

f) N'acetyl-Arg-Gly-Asp-feramide anisole (1 m The protected peptide (120my) of Example 1fi(e) was treated with anhydrous H The mixture was treated with F (10 ml) at 0°C for 1 hour. Remove the HF under vacuum at 0 °C, then , and the residue was triturated with ether. The residue was dissolved in 0.2M acetic acid and The crude product was obtained (35u) by washing with water several times and freeze-drying. distribute the peptide Column chromatography (Sephadex') G-15 , 4, 1:1 butanol:acetic acid:water). Collect appropriate fractions and freeze Drying gave a semi-purified product (25u). Half number HPLC (5μODS silica , 20% acetonitrile/water - 0, 1% TFA) to obtain the purified standard. The following compound was obtained (1819). MS (FAB) m/e 464.2 [M+H] ”, 462 [M-HF-; HPLCk’ 3.35 (20% acetonitrile/ Water - 0.1% TFA.

UV detection at 220 stop); TLCR + 0.15 (n BuOH:HtO:A c0H4:1:1); Amino acid analysis: Asp (1,00), Gly (0,97 ), ArgN1 benzoyl-A r g-G 1 y-A s p-phenyla Preparation of Mido (39) Arg(Tos)-Gay-Asp(0-Bzl)-Fe Triethyl to a solution of Nilamide (0,313 anal) in DMF (5++/) Amine (54 μl) was added to neutral pH. Add benzoyl chloride (36 μl). and the reaction mixture was stirred for 18 hours. Remove the solvent and flush the residue under vacuum. chromatography (silica, 5-10% methanol/chloroform) Further purification gave the title compound.

b) N”benzoyl-Arg-Gly-Asp-feramide Example 14 (e ), the protected peptide was treated with anhydrous HF and isolated.

Example 18 N1 benzyloxycarbonyl-A r g-G] y-A s p-phenylene (4 Cbz-Arg to G according to the procedure of Example 1(a)) -Asp(0-tBu)-coupled with phenylamide in DMF, Isolation using DIEASEDC and HOBT gave the title compound.

The compound of Example 18(a) was dissolved in dioxane and treated with hydrochloric acid to give the title compound D. -Preparation of Phg-Arg-Gay-Asp-phenylamide (41)Cbz-D - Acid fluoride was obtained by treating phenylglycine with cyanuryl fluoride. Cbz- D-phenylglycyl fluoride and A r g (To 5)-G I y -Asp(0-cHex)-phenylamide was coupled, resulting in the product Treatment of the material with HF gave the desired compound. MS (FAB) m/e 555 [M+ H]”.

Journal of Organic Chemistry (J, Org, Ches , ) 48.77 (1983) by a method similar to that described in N'Cbz -Orn(Phth) was converted to N'Cbz-N'MeOrn(Phth).

N’Cbz-N’Me-Orn (Pht h) and G l y -A sp (0-bonyl-N'MeOrn(Phth )-Gly-Asp(0-Bz　l)-phenylamide was obtained. palladium catalyst The benzyloxycarbonyl group (Cbz) was removed by hydrogenation above, resulting in The amine was benzoylated and the resulting amino group was subjected to the procedure of Example 17(a). It was benzoylated. The obtained peptide was treated with hydranone to obtain the title compound. Obtained. MS (FAB) m/e 498 [M+H]".

Example 21 4-(aminoiminomethyl)aminobenzoyl-3ar-Asp-phenylami 4-guanidinobenzoic acid hydrochloride and 5ar according to the procedure of Example 14(d) -Asp(0-Bzl)-phenylamide was concentrated with EDC to form 4-guanimide. Dinobenzoyl-3ar-Asp(0-Bzl)-phenylamide was obtained. para Treatment of this product with hydrogen in the presence of a catalyst gave the title compound. MS (F AB) m/e 441 [M+H]'.

Except replacing 4-chloroaniline with 2-chloroaniline in step 12(c) prepared the title compound using the procedure of Example 12.

Example 23 N'benzoyl-N'MeArg-Gly-Asp (2-methoxy) 7 ale Preparation of amide (45) The procedure was carried out except for replacing 4-chloroaniline with 2-methoxyaniline in step 12(c). The title compound was prepared using the procedure of Example 12.

Benzyl 3-aminobenzoate in step 13(b) to 2-benzyloxyaniline The title compound was prepared using the procedure of Example 13 with the following changes.

a) Boa-Arg(NOx)-0CH3B　c　　c　-A　r　g(Now) Diazomethane to a solution of (2,09,5,5wol) in MeOH/CHtC4z A solution of 100% in Et,O was added. The reaction mixture was mixed with 5% aqueous NaICO3 solution and H , O and dried. The solvent was removed in vacuo to give the title compound ( 1,69,87%), which was used without further purification.

b) Boc-Arg(Now)-NHNH2Boc-Arg(Not)-0C A solution of Hs (1.6 g, 4.8 mmol) in MeOH with 62% hydrazine hydrate (1,2ar, 24wol) was added. The reaction mixture was stirred at room temperature for 48 hours. Stirred. The resulting mixture was concentrated under reduced pressure and the residue was subjected to flash chromatography. (silica, gradient 5-15% MeOH/CH2C1m) to give a colorless oil. (0,749,46%), which solidified on standing.

c) Boc-Asp(0-Bz I)-(N-methyl)phenylamide Boc- In a solution of Asp(0-Bz　]) (22,0g6.211111o1) in DMF N-methylaniline (0,99e, 9.3 mmol), HOBT hydrate (0,9 2y, 6.8 mmol) and DCC (1,39,6,2 mmol) were added. . The resulting mixture was stirred at room temperature overnight. The reaction mixture was concentrated under reduced pressure. residue was dissolved in CH2Cl2, washed with 0.5N HCl and HIO, and dried (M gSO<). The solvent was removed in vacuo and the residue was subjected to flash chromatography. Further purification gave a viscous oil.

d) O=C-Asp(0-BzI)-(N-methyl)phenylamide at 0°C. Boc-Asp(0-Bz　1)-(N-methyl)phenylamide (0゜9 CH2Cl2 (8a r) medium solution was added. After stirring at 0°C for 45 minutes, the mixture was concentrated under reduced pressure and poured into a tube. Treated azeotropically with luene several times. The residue was treated with 20% phosphorus in toluene (4'2*I). A solution of gen was added. The resulting mixture was heated under reflux for 15 minutes and then brought to room temperature. The mixture was cooled to room temperature and concentrated under reduced pressure. Treat the residue azeotropically with toluene for further purification Used without.

Example 25 A solution of the compound of (c) in CH3CN was added with Boc-Arg(N ow) -NHNH! Dissolution of (0,749,2,2111101) in CHICN The solution was heated and the resulting solution was stirred at room temperature overnight. Remove the solvent in vacuo and remove the remaining The material was subjected to flash chromatography (silica, 10% MeOH/CHZCA'x ) to give the title compound (0.349.23%).

f) A r g-NHNHCO-A p-(N-methyl) phenylamine Example 25 Compound (e) (0,309,0,45 mmol) was added with trifluoride. roacetic acid (0.7ar, 11.2 mmol) in CH2Cl2 (0.7111) Upon addition of the solution, the resulting mixture was stirred at room temperature for 15 minutes and then concentrated under reduced pressure. Ta. Treat the residue azeotropically with toluene, then dissolve the residue in CH,Cl2 did. Add triethylamine (0,1,3ij', l, Q mmol) to this solution. and then benzoyl chloride (0.05ar, 0.44 mmol) Added. The resulting mixture was stirred at room temperature overnight, then concentrated under reduced pressure. residue by flash chromatography (silica, 10% MeOH/CH2C1z) Further purification gave the title compound (0.15g, 50%).

Example 25 (f) compound (150 u, 0, 22 mol) in MeOH (3 0f) in solution in glacial acetic acid (0,3,7') and 10% palladium on activated carbon (19 -9) was added. The resulting mixture was hydrogenated at 45 psi H2 for 3 hours. child At that time, thin layer chromatography showed only the presence of starting material. the mixture Degassed and added 10% palladium on activated carbon (90-g). the reaction mixture The material was hydrogenated (45 psi) overnight. At this time, once again, the thin layer chromatography Therefore, only the presence of starting material was indicated. The mixture was coated with Ce'l'ite. ”) and then concentrated under reduced pressure. The residue was diluted with methanol (3 0ml), glacial acetic acid (03Mt) and 10% palladium on activated carbon (100-v) was added and the mixture was hydrogenated (46 psi) for 7 hours. The mixture was washed with celite ( The crude product was filtered through a pad of Half the number HPLCk' 3.86 (Dynamax (Dynas+ax) C18, 17% acetonitrile/water - 0,1% trifluoroacetic acid, at 260 uM (UV detection) to give the purified title compound, which contains a small amount of glacial acetic acid. Lyophilized from H,O (19++9.10%). MS (FAB) m/e54 1 [M+H]”; TLCR10,87 (n BuOH:HOAc:HzO:Vinegar Ethyl acid 1: 1: 1: 1); RcO, 88 (n-BuOH:HOA c: H, 0: Virigi: / 15: 5: 10: 10); HPLCk' 7.86 (Dynamax (D ynaw + ax) C18, gradient, A Niace Tonitrile, B: water-0,1% trifluoroacetic acid, 10-80% A within 30 minutes , UV detection at 260 nM).

Using the procedure of Example 2 except replacing aniline with phenol in step 2(a). The title compound was prepared.

N-methylaniline (15g, 14.Qmmol) in dry DMF (10+1 ) DIEA (12ar, 5.Qmmol) was added dropwise to the solution to adjust the pH to 8. did. Next, Boc-D-A rg (Tos) (5 g, 11.7 mmo l) and HOBt (2,19,17, Qmmol) were added. the reaction mixture Stir the mixture for a few minutes, then add EDC (2,59,13,1 mmol) for 2 minutes. It dripped. The reaction mixture was stirred at room temperature overnight. Further D to the reaction mixture I EA (1,2ar, 6.Qmmol) and EDC (2,09,10.5 關o1) was added to complete the slow reaction. After one week, remove the solvent under reduced pressure and remove the remaining The material was partitioned between ethyl acetate and 10% ICOs. The organic extract was dissolved in water, 5% Washed successively with enoic acid, water and saline. Organic extract anhydrous sodium sulfate dried, filtered, concentrated and then combined with ether/hexane (1/1) to a powder. Trituration gave the title compound as a white solid (2.49.40%).

b) D-Arg(Tos)-(N-methyl)phenylamide Boc-D-Arg (Tos)-(N-methyl)phenylamide (2,:39.4.41■ol) Treated with 50% TFA in methylene chloride (20,/) for 1 hour at room temperature. remove solvent The residue was evaporated several times from methylene chloride to remove traces of TFA.

The residue was triturated with ether and used for the next step without further purification.

D-A　g(To　5)-(N-methyl)phenylamide in methylene chloride ( Add triethylamine (1,31/, 9.3 mmol) to the medium temperature solution (5 ml). Ta. Then, malonylmethyl chloride (500 μl, 4.7+n+ol) was added. Ta. The reaction mixture was allowed to warm to room temperature and continued to stir overnight. The reaction mixture was placed in water. Pour and wash the organic layer sequentially with 5% citric acid, water, 5% NaHCO1, water and brine. Washed. The organic layer was dried over anhydrous sodium sulfate, filtered, and concentrated. The title compound was obtained as a white solid (179.81%).

1-(D-Arg(Tos)-N-methylanilide)-methylmalonate (0 , 789, 1,5 mmol) in methanol (1,51 A) with 1N NaOH A solution of (2.5 ml, 2.5 Ill+nol) was added dropwise. The reaction solution was brought to room temperature. The mixture was stirred for 18 hours. IMHC1! Adjust the pH of the solution to 6 using Concentrated. The pH was adjusted to 2 using IMHCj' and an oil precipitated. water and Trituration together and drying in vacuo gave the title compound as a white solid (0.60 89).

Asp(0-cHex)-phenylamide A3p(0-cHex)-phenyla Add DIEA (0.2 ml, 1.1 m 1 ol) was added dropwise to adjust the pH to 8. Next, HoBt (0,188g, l, 2+u+ol) and 1-(D-Arg(Tos)-N-methylanilide)- Malonic acid (6) (0.57 g, 1.1+mol) was added. The reaction mixture Stir for a few minutes, then add EDC (0,216q, 1.IIllmOl) dropwise. Ta. The mixture was stirred for 18 hours. Remove the solvent under vacuum and dissolve the residue in ethyl acetate. and 10% KtCOs. The organic extract was mixed with water, 5% citric acid, and water. and saline.

The organic layer was dried over anhydrous sodium sulfate, filtered, and then concentrated to a white solid, This was used in the next step without further purification.

The compound of Example 27(e) was dissolved in anhydrous HF (10 ml) at 0°C for 1 hour. The HF was removed under vacuum at 0°C and the residue was evaporated with ether. ground into an oily substance. The residue was dissolved in 0.2M acetic acid and lyophilized to obtain a crude The obtained product was obtained (0.269). The product was filtered through gel filtration (Sephadex). hadex') G-25,0,2M acetic acid) to obtain the title compound, f= (0,0289). MS (FAB) m/e 540.3 [M+H]”;H PLCk' 10.7 (5μ Altex Ultrasphere (Altex) Ultrasphere'') ODS, gradient, A, acetonitrile, B: water-0 1% trifluoroacetic acid, 10%-50% A within 20 minutes, UV inspection at 220 nm ), k'7.3 (Ultrasphere R ODS.

22% acetonitrile/water 1% trifluoroacetic acid, UV detection at 220 nm );TLCR70,76(n-BuOH:R20:AcOH:EtOAc 1 +1:1:1);R30,84(n-BuOH:AcOH:R20:Pi Rigi: /15:5:10:10), Amino acid analysis: Asp (1,00), Ar g (0,99).

Parenteral dosage unit composition A preparation containing the compound 20m+9 of Example 1 or 2 as a sterile dry powder was prepared below. Prepare as follows: 20 ml of the core compound is dissolved in 15 ml of distilled water. sterile The solution is filtered into 25 ml multi-dose ampoules under conditions and then lyophilized. . 20g+ 5% Dextrose in Water (D5W) for intravenous or intramuscular injection The powder is reconstituted by adding 11. This allows the dose to be determined by Subsequently, a metered amount of this dosage unit is added to D5W for injection. may be diluted by adding to another amount of A separate bottle or bag for a intravenous drip or other injection-infusion system In addition to the dispensing system, 50 mg of the compound of Example 3 was added to 75 mg of lactose and stearic acid. By mixing and grinding with magnesium ester Prepare the cells.

The resulting powder is screened and filled into hard gelatin capsules.

Sucrose 20119, calcium sulfate dihydrate 15019 and Example 3 By mixing and granulating compound 50maro9 with 10% gelatin solution , to prepare tablets for oral administration. Screen wet granules, dry and starch 10 Mix with shoulder 9, talc 5 paddy and stearic acid 3IQ and compress into tablets.

The foregoing description provides instructions for making and using the present invention. However, the main The present invention is not limited to the specific examples described in this specification (and does not apply to the following claims). Including all modifications within the scope.

international search report Continuation of front page (51) Int, C1, 5 identification symbol Internal office reference number A61K 37154 AB N 8314-4CCO7D 209/44 9284-4C23310093 60-4C 333/16 9165-4C 333/20 9165-4C C07K 5102 8318-4H (72) Inventor: Pondinel, William Nidward, American Pencil 1512 I Franklin Lane, Uniin, Luvenia 19087 (72) Inventor Ku, Thomas Wenfu Pennsylvania, USA 1413 Southwind Way, Drescher, State 19025 (72) Inventor: Samanen, James Martin United States Pencil 19460 Jug Hallow Road, Phoenixville, NY (no street address shown) death)

Claims (1)

[Claims] 1. formula: D-E-Asp-Q-Y [In the formula, Q is NH, NCH3 or O; Y is unsubstituted or 1 to 3 C1-6 alkyl, trifluoromethyl, halogen , OR', SR', (CH2)nAr, CONR1R2, CO2R2 or R4 Phenyl, naphthyl or Het substituted by R4N; D is ▲ formula, There are chemical formulas, tables, etc. ▼ or ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ W is NHR ', NR'C(=NH)NHR', NR'C(=NH)R' or (C=NH) NHR′; U is CO, SO2, NHCO, (CH2)n, SO2NH, NHSO2 or O C(=O); V is H, R', R4RsN, R4RsNCO, R'O, Y-NR', x-A-B -NR' or ▲There is a mathematical formula, chemical formula, table, etc. ▼A does not exist, Asn, Gln, Ala, Pro or Abu; B is Arg, HArg, NArg, (M e2) Arg, (Et2) Arg, Abu, Ala, Gly, His, Orn, Lys, Phg or its α-R′ substituted derivative, Dtc, Tpr or Pro D- or -amino acid selected from; E is Gly, Sar, CH2CO, OC H2CO or NHCO; R' is H, C1-6 alkyl or Ar(CH2)p ;X is R4R4N, R4R5N-CO or H; R1 and R2 are H, C1-6 Alkyl or (CH2)pAr; R4 is H or C1-6 alkyl; R5 is R6, R6CO, R6OCO, R6OCH(R6')CO, R6NHCH (R6')CO, R6SCH (R6')CO, R6SO2 or R6SO; R6 and R6' is H, C1-6 alkyl, C3-7 cycloalkyl, Ar, Ar- C1-4 alkyl, Het, Het-C1-4 alkyl, ha-(CH2)pC H(Ar)(CH2)p or Ar-C3-7 cycloalkyl; Ar is phenyl or 1 to 3 C1-6 alkyl, trifluoromethyl, halogen, R'O or phenyl substituted by R'S; ▲Mathematical formulas, chemical formulas, tables, etc. ▼ is a 5- or 6-membered monocyclic or bicyclic ring containing 1 or 2 nitrogen atoms ; m is (1) 0 to 4 when W is NR'-(C=NH)NHR'; (2) W is (C=NH)NHR' or NR'(C=NH)R', 0 to 5; or (3) When W is NHR', 0 to 6; n is 0 to 2; P is 0-3; q is (1) 1 to 4 when W is NR'-(C=NH)NHR'; (2) when W is (C=NH)NHR' or NR'(C=NH)R', 2 to 5; or Taha (3) When W is NHR', it is 3 to 6] or its pharmaceutical compound Pharmaceutically acceptable salts. 2. Y is optionally halogen, C1-4 alkyl, C1-4 alkoxy, C1- Optionally substituted by 4 alkylthio, CONR1R2 or CO2R3 2. A compound according to claim 1, which is phenyl or naphthyl. 3. 3. A compound according to claim 2, wherein Y is phenyl or naphthyl. 4. 2. The compound according to claim 1, wherein D is ▲ has a mathematical formula, a chemical formula, a table, etc. ▼. 5. 2. A compound according to claim 1, wherein Q is NH. 6. 2. A compound according to claim 1, wherein V is R4R5N. 7. 5. A compound according to claim 4, wherein E is Gly. 8. Claims that R4 is H or methyl and R5 is R6CO or R6OCO A compound according to item 6. 9. A claim in which V is ArCO-N(CH3) or HetCO-N(CH3) 4. The compound described in 4. 10. Y is optionally chloro, C1-4 alkyl, C1-4 alkylthio or 9. The compound according to claim 8, which is phenyl optionally substituted with carboxy. 11. N-[[5-(aminoiminomethyl)amino-1-oxopentyl]-G ly-Asp-phenylamide, N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-ox sopentyl]-Gly-Asp-phenylamide, N-[(2S)-2-(1, 3-dihydro-2H-isoindolyl)-5-(aminoiminomethyl)amino- 1-oxopentyl]-Gly-Asp-phenylamide, N-[Nα-(4, 4-diphenylbutyryl)-Nα-methyl-arginyl]-Gly-Asp-F phenylamide, Nα-(phthaloyl)-Arg-Gly-Asp-phenylamide, Nαbenzo yl-NαMeArg-Gly-Asp-(4-carboxy)phenylamide, Nα acetyl-Arg-Gly-Asp-phenylamide, Nα benzoyl-A rg-Gly-Asp-phenylamide, Nαbenzyloxycarbonyl-Ar g-Gly-Asp-phenylamide, D-Phg-Arg-Gly-Asp- Phenylamide, Nα benzyloxycarbonyl-Nα methyl-Orn-Gly -Asp-phenylamide, 4-(aminoiminomethyl)aminobenzoyl-Sar-Asp-phenylami Do, Nαbenzoyl-NαMeArg-Gly-Asp-(2-chloro)phenyla Mido, Nαbenzoyl-NαMeArg-Gly-Asp-(2-methoxy)phenyl Amide, Nαbenzoyl-NαMeArg-Gly-MeAsp-(2-hydroxy)ph phenylamide, Nαbenzoyl-Arg-NHNHCO-Asp-(N-methyl)phenylamide Do, Nα(OCCH2CO)-Arg-(N-methyl)phenylamide, Asp-F phenylamide Nα-(2-methylbenzoyl)-Nα-MeArg-Gly-Asp-(2- methyl) phenylamide, Nαbenzoyl-NαMeArg-Gly-Asp-phenylamide, Nαacetyl Chil-NαMeArg-Gly-Asp-phenylamide, Nα acetyl-Nα MeArg-Gly-Asp-(2-carboxy)phenylamide, Nα(2-methylthio)benzoyl-NαMeArg-Gly-Asp-(2- methylthio) phenylamide, Nαbenzoyl-NαMe∧rg-Gly-Asp-(4-chloro)phenyla Mido, Nαbenzoyl-NαMeArg-Gly-Asp-(3-carboxy)pheny Ruamide, and Nα(2-thienylcarbonyl)-NαMeArg-Gly-Asp-phenyl 2. The compound according to claim 1, which is an amide. 12. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier. . 13. Use of a compound according to claim 1 in the manufacture of a medicament. 14. A method for inhibiting platelet aggregation comprising administering a compound according to claim 1. 15. Stroke or - hyperencephalic ischemic episode consisting of administering a compound according to claim 1. How to treat the disease. 16. A method for treating myocardial infarction comprising administering the compound according to claim 1. 17. A mammal comprising administering a fibrinolytic agent and a compound according to claim 1. A method for inhibiting thrombolysis and reocclusion of arteries or veins. 18. Fibrinolytic agents include anistreplase, streptokinase (SK), and uro Kinase (UK), prourokinase (pUK) or tissue plasminogen activity 17. The agent according to claim 16, which is a sexualizing factor (tPA) or a mutant or derivative thereof. the method of.