WO1992013552A1 - Anti-aggregatory peptides containing an aromatic ester or amide - Google Patents

Anti-aggregatory peptides containing an aromatic ester or amide Download PDF

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Publication number
WO1992013552A1
WO1992013552A1 PCT/US1992/000999 US9200999W WO9213552A1 WO 1992013552 A1 WO1992013552 A1 WO 1992013552A1 US 9200999 W US9200999 W US 9200999W WO 9213552 A1 WO9213552 A1 WO 9213552A1
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Prior art keywords
asp
gly
phenylamide
mmol
nhr
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PCT/US1992/000999
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French (fr)
Inventor
Fadia El-Fehail Ali
William Edward Bondinell
Thomas Wen-Fu Ku
James Martin Samanen
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Smithkline Beecham Corporation
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Priority to JP4506448A priority Critical patent/JPH06505978A/en
Publication of WO1992013552A1 publication Critical patent/WO1992013552A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0205—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)3-C(=0)-, e.g. statine or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0207—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-(X)4-C(=0), e.g. 'isosters', replacing two amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0815—Tripeptides with the first amino acid being basic
    • C07K5/0817—Tripeptides with the first amino acid being basic the first amino acid being Arg
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • a thrombus is the result of processes which initiate the coagulation cascade. It is composed of an aggregation of platelets enmeshed in a polymeric network of fibrin. This process is normally initiated as a consequence of tissue injury and has the effect of slowing or preventing blood flow in a vessel. Etiological factors which are not Girectly related to tissue injury, such as atherosclerotic plaque, inflammation of the blood vessels (phlebitis) and septicemia, may also initiate thrombus formation. In some instances, the inappropriate formation of a thrombus, and subsequent decrease in blood flow, may have pathological consequences, such as stroke, pulmonary embolism and heart disease.
  • EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation.
  • the cyclic peptides reported are formed via a disulfide bridge between two cysteinyl residues.
  • Other linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 915.
  • One compound disclosed therein is Ac-Arg-Gly-Asp-NHCH 2 CH 2 Ph.
  • the instant invention provides compounds in which the carboxy terminus of the peptide is substituted with an aryl alcohol or aryl amine to form an aryl ester or amide.
  • the compounds of this invention inhibit the binding of GP-IIbllla to fibrinogen and are surprisingly potent.
  • A is absent, Asn, Gin, Ala, Pro or Abu;
  • Ar-C 1-4 alkyl Het, Het-C 1-4 alkyl, Ar-(CH 2 ) p CH(Ar) (CH 2 ) P or Ar-C 3-7 cycloalkyl;
  • Ar is phenyl or phenyl substituted by one to three C 1-6 alkyl, trifluoromethyl, halogen, R'O or R'S; and is a five or six-membered, mono or bicyclic ring containing one or two nitrogen atoms;
  • This invention discloses acyclic peptide-like compounds comprising the sequence Gly-Asp and having an aryl ester or amide moiety attached directly to the carboxy group of Asp.
  • the compounds of this invention inhibit platelet aggregation and are believed to interact with the GPIIb-IIIa receptor and other adhesion proteins.
  • the attachment of an aryl group to the Asp residue confers increased activity for inhibition of platelet aggregation.
  • D is the preferred value of m is
  • W is -NHR', m is preferably 0-3 for para, 2-4 for meta, and 3-5 for an ortho orientation.
  • a para orientation is generally
  • t-Bu refers to the tertiary butyl radical
  • Boc refers to the- t-butyloxycarbonyl radical
  • Fmoc refers to the
  • Coupling reagents as used herein denote reagents which may be used to form peptide bonds.
  • Typical coupling reagents are carbodiimides, activated anhydrides and esters and acyl halides.
  • Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical.
  • ⁇ -R' substituted derivatives of the amino acids of this invention which may be denoted as ( ⁇ -R')AA, indicate amino acids which are mono-substituted on the ⁇ -amino group by R', wherein R* is C 1-6 alkyl or Ar(CH 2 )n.
  • R' is methyl or benzyl.
  • R' is preferably methyl.
  • a combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, tetra-, or penta-peptide fragments may be prepared by solid phase synthesis and either coupled or further modified by solution synthesis.
  • Solution phase synthesis is generally preferred for the smaller peptides of this invention.
  • the reactive functional groups of the sidechains of each synthetic fragment, amino acid or peptide are suitably protected as known in the peptide art.
  • the Boc, Cbz or Fmoc group may be used for protection of an amino group, especially an ⁇ -amino group.
  • the Boc group is generally preferred for protection of the ⁇ -amino group.
  • a t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl of Asp or Glu.
  • a benzyl group or suitably substituted benzyl group is used to protect the mercapto group of cysteine, or other thiol containing residues; or the hydroxyl of serine or threonine.
  • the tosyl group may be used for protection of the imidazolyl group of His, and tosyl or nitro group for protection of the guanidino nitrogen of Arg.
  • a suitably substituted carbobenzyloxy group or benzyl group may be used for the hydroxyl group of Tyr, Ser or Thr, or the ⁇ -amino group of lysine.
  • the phthaloyl group may also be used for the protection of the ⁇ -amino group of lysine. Suitable substitution of the
  • Solid phase methods for instance when Y bears a carboxyl group which may be attached to a resin, the peptide, or a convenient fragment thereof, is built up sequentially starting from the carboxy terminus and working toward the amino terminus of the peptide.
  • Solid phase synthesis is begun by covalently attaching the carboxyl group of the appropriately protected Y residue, such as Boc-NH-Y, to a suitable resin, such as a benzhydrylamine resin (BHA), methylbenzhydrylamine resin (MBHA), chloromethyl resin (CMR), hydroxymethyl resin (HMR) or SASRIN resin, as is generally set forth in U.S. Patent No. 4,244,946 and Gisin, Helv. Chem.
  • a BHA or MBHA support resin is used if the carboxy terminus of the product peptide is to be a carboxamide.
  • a CMR or HMR support is generally used if the carboxy terminus of the product peptide is to be a carboxyl group, although this may also be used to produce a
  • the completed peptide may then be deblocked and/or split from the carrying resin in any order.
  • the peptide may be attached to a CMR or HMR via the ⁇ -carboxyl group of Asp.
  • the synthesis is begun by coupling the aryl amine or aryloxy group, via solution phase synthesis, to an appropriately sidechain-protected aspartic acid residue.
  • the side chain carboxyl group is then selectively deprotected and coupled to a chloromethyl resin (CMR).
  • CMR chloromethyl resin
  • a benzyl ester is a suitable sidechain protecting group which is selectively deprotected by hydrogenation.
  • the amino group is
  • Useful plasminogen activators include, for example, anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue plasminogen activator (tPA) and mutants, or variants, thereof, which retain plasminogen activator activity, such as variants which have been chemically modified or in wliich one or more amino acids have been added, deleted or substituted or in which one or more or functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator with the fibrin binding domain of another plasminogen activator or fibrin binding molecule.
  • a variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use.
  • the fibrinolytic and the peptide may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.
  • both agents When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
  • the platelet aggregation inhibitor may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the fibrinolytic agent in a second infusion bag.
  • a patient can receive an initial bolus-type injection or infusion, of the peptide inhibitor followed by an infusion of the fibrinolytic agent.
  • Boc-Gly-Asp(O-cHex)-(2-methylthio)phenylamide (21) (770 mg) was treated with 50% TFA solution in methylene chloride (5 mL) for 60 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether to yield the TFA salt of the title compound (609.5 mg).
  • Boc-N ⁇ MeArg(Tos)-Gly-Asp(O-cHex)-(2-methylthio)-phenylamide (23) (920 mg) was treated with 50% TFA solution in methylene chloride (5 mL) for 60 min at room temperature The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA, and triturated with ether to yield the TFA salt of the title compound (635.5 mg).
  • the mixture was degassed and filtered through a pad of
  • Boc-Gly-Asp(O-cHex)-phenylamide (496 mg, 1.11 mmol) was dissolved in 5 mL 4N HCl/dioxane and stirred at room temperature
  • Example 1Kb The protected peptide of Example 1Kb (200 mg, 254 nmol) was treated with 5 mL anhydrous HF at 0° for 1 h. The HF was removed in vacuo and the residue was dissolved in glacial acetic acid and lyophilized to yield the crude peptide salt (135 mg, 87%). 100 mg was filtered and purified by gel filtration (Sephadex ® G-10, 10% acetic acid). The appropriate fractions were pooled and lyophilized to yield the title compound (25 mg).
  • Boc-Asp(O-cHex)-(4-chloro)phenylamide (1.44 g, 3.4 mmol) was treated with 50% TFA solution in methylene chloride (10 mL) for 45 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was
  • Boc-Asp (O-cHex) 0.883 g, 2.8 mmol
  • THF 10 mL
  • N-methylmorpholine 0.34 mL, 3.1 mmol
  • isobutylchloroformate 0.40 mL, 3.1 mmol
  • Boc-Asp(O-cHex)-(4-benzyloxycarbonyl)phenylamide (1.1 g, 2.1 mmol) was treated with 50% TFA/methylene chloride (20 mL) for 90 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was
  • Boc-Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide (220 mg, 0.287 mmol) was treated with 50% TFA/methylene chloride (10 mL) at room temperature for 4 H. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was treated with HCl-dioxane and evaporated several times from toulene and dried under vacuum to yield the titled compound as its hydrochloride salt.
  • Boc-Asp(O-Bzl)-(N-methyl)phenylamide (0.91 g, 2.2 mmol) at 0°C was added a solution of trifluoroacetic acid (8 mL) in CH 2 CI 2 (8 mL). After stirring at 0°C for 45 min, the mixture was concentrated under reduced pressure and treated azeotropically with toluene several times. To the residue was added a solution of 20% phosgene in toluene (42 mL). The resulting mixture was heated at reflux for 15 min, then allowed to cool to room temperature and concentrated under reduced pressure. The residue was treated azeotropically with toluene, and used without further purification. e) Boc-Arg(NO 2 )-NHNHCO-Asp(O-Bzl)-(N-methyl)phenylamide
  • Example 25(f) To a solution of the compound of Example 25(f) (150 mg, 0.22 mmol) in MeOH (30 mL) was added glacial acetic acid (0.3 mL) and 10% palladium on activated carbon (19 mg). The resulting mixture was hydrogenated at 46 psi H 2 for 3 h, at which time thin layer chromatographic analysis indicated only the presence of starting material. The mixture was degassed and additional 10% palladium on activated carbon (90 mg) was added. The reaction mixture was hydrogenated (46 psi) overnight at which time thin layer chromatographic analysis again indicated only the presence of starting material. The mixture was filtered through a pad of Celite ® and
  • Boc-D-Arg(Tos)-(N-methyl)phenylamide (2.3 g,4.4 mmol) was treated with 50% TFA in methylene chloride (20 mL) at room temperature for 1 h. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether and used in the next step without further purification.
  • Triethylamine (1.3 mL, 9.3 mmol) was added to a cold solution of D-Arg(Tos)-(N-methyl)phenylamide in methylene chloride (5 mL). This was followed by the addition of methyl malonylchloride (500 ⁇ L, 4.7 mmol). The reaction mixture was allowed to warm to room temperature and stirring was
  • Example 29 20 mg of the compound is dissolved in 15 ml of distilled water. The solution is filtered under sterile conditions into a 25 ml multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 20 ml of 5% dextrose in water (D5W) for intravenous or intramuscular injection. The dosage is thereby determined by the injection volume. Subsequent dilution may be made by addition of a metered volume of this dosage unit to another volume of D5W for injection, or a metered dose may be added to another mechanism for dispensing the drug, as in a bottle or bag for IV drip infusion or other injection-infusion system.
  • D5W dextrose in water
  • a capsule for oral administration is prepared by mixing and milling 50 mg of the compound of Example 3 with 75 mg of lactose and 5 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.
  • Example 30 Oral Dosage Unit Composition A tablet for oral administration is prepared by mixing and granulating 20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 3 with a 10% gelatin solution. The wet granules are screened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearic acid; and compressed into a tablet.

Abstract

This invention relates to compounds of the formula D-E-Asp-Q-Y, wherein: Q is NH, NCH3 or O; Y is phenyl, naphthyl or Het, unsubstituted or substituted by one to three C1-6alkyl, trifluoromethyl, halogen, OR', SR', (CH2)nAr, CONR1R2, CO2R2 or R4R4N; D is (A) or (B); 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 OC(=O); V is H, R', R4R5N, R4R5NCO, R'O, Y-NR', X-A-B-NR' or (C); A is absent, Asn, Gln, Ala, Pro or Abu; B is a D- or L- amino acid chosen from Arg, HArg, NArg, (Me2)Arg, (Et2)Arg, Abu, Ala, Gly, His, Orn, Lys, Phg or an α-R' substituted derivative thereof, Dtc, Tpr or Pro; E is Gly, Sar, CH2CO, OCH2CO or NHCO; R' is H, C1-6alkyl or Ar(CH2)p; X is R4R4N, R4R5N-CO or H; R1 and R2 are H, C1-6alkyl or (CH2)pAr; R4 is H or C1-6alkyl; R5 is R6, R6CO, R6OCO, R6OCH(R6')CO, R6NHCH(R6')CO, R6SCH(R6')CO, R6SO2 or R6SO; R6 and R6' are H, C1-6alkyl, C3-7cycloalkyl, Ar, Ar-C1-4alkyl, Het, Het-C1-4alkyl, Ar-(CH2)pCH(Ar) (CH2)p or Ar-C3-7cycloalkyl; Ar is phenyl or phenyl substituted by one to three C1-6alkyl, trifluoromethyl, halogen, R'O or R'S; and (C) is a five or six-membered, mono or bicyclic ring containing one or two nitrogen atoms; m is (1) 0-4, when W is NR'-(C=NH)NHR'; (2) 0-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or (3) 0-6, when W is NHR'; n is 0-2; p is 0-3; q is: (1) 1-4, when W is NR'-(C=NH)NHR'; (2) 2-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or (3) 3-6, when W is NHR'; or a pharmaceutically acceptable salt thereof, which are effective for inhibiting platelet aggregation, pharmaceutical compositions for effecting such activity, and a method for inhibiting platelet aggregation.

Description

Anti-Aggregatory Peptides Containing
An Aromatic Ester or Amide
Field of the Invention
This invention relates to novel peptides which inhibit platelet aggregation, pharmaceutical compositions containing the peptides and methods of using the peptides. In
particular, a method of using the peptides of this invention in combination with fibrinolytic agents is disclosed.
Background of the Invention
A thrombus is the result of processes which initiate the coagulation cascade. It is composed of an aggregation of platelets enmeshed in a polymeric network of fibrin. This process is normally initiated as a consequence of tissue injury and has the effect of slowing or preventing blood flow in a vessel. Etiological factors which are not Girectly related to tissue injury, such as atherosclerotic plaque, inflammation of the blood vessels (phlebitis) and septicemia, may also initiate thrombus formation. In some instances, the inappropriate formation of a thrombus, and subsequent decrease in blood flow, may have pathological consequences, such as stroke, pulmonary embolism and heart disease.
Platelets play a major role in thrombus formation.
Current antithrombotic therapy employs agents that modify the platelet/endothelial cell arachidonate-prostaglandin system, such as prostacyclin analogues, cyclooxygenase inhibitors, thromboxane synthesis inhibitors and thromboxane receptor antagonists; and anti-coagulants, such as heparin. These agents inhibit one or both of two discernible phases of platelet aggregation. The primary phase, which is a response to chemical stimuli, such as ADP (adenosine diphosphate), collagen, epinephrine or thrombin, causes initial activation of the platelets. This is followed by a secondary phase, which is initiated by the platelets themselves, and is characterized by thromboxane A2 (TXA2) synthesis and the release of additional ADP from platelet storage granules, which further activates platelets.
Prostacyclin, also called prostaglandin I2 (PGI2), and stable PGI2 analogues inhibit both the primary and secondary phases of platelet aggregation However, use of such
analogues has been associated with undesirable changes in blood pressure. See Aiken, et al., Prostaglandins, 19, 629- 43 (1980).
Cyclooxygenase inhibitors and thromboxane synthetase inhibitors act to block the production of TxA2. TxA2
antagonists block the effects of TxA2 by binding the TxA2 receptor. These therapies act only upon the secondary stage of platelet activation. Use of cyclooxygenase inhibitors has been associated with ulcerogenesis and an adverse effect upon prostacyclin synthesis.
Heparin prevents the activation of fibrinogen by
thrombin and thereby prevents the activation of the GPIIb-IIIa receptor by thrombin. This inhibits only the primary phase of platelet aggregation and has little effect upon activation of platelets by other means, such as collagen, ADP and epinephrine.
Cyclooxygenase inhibitors, prostaglandin analogues and heparin all inhibit platelet aggregation indirectly by inhibiting the primary or secondary phase of
platelet/fibrinogen activation. There is therefore a need for selective therapeutic products which block platelet aggregation directly, whether it arises from the primary or secondary phase of platelet activation. Platelet aggregation is believed to be mediated
primarily through the GPIIb-IIIa platelet receptor complex. Von Willebrand factor, a plasma protein, and fibrinogen are able to bind and crosslink GPIIb-IIIa receptors on adjacent platelets and thereby effect aggregation of platelets.
Fibronectin, vitronectin and thrombospondin are proteins which have also been demonstrated to bind to GPIIb-IIIa.
Fibronectin is found in plasma and as a structural protein in the intracellular matpix. Binding between the structural proteins and GPIIb-IIIa may function to cause platelets to adhere to damaged vessel walls.
Peptide fragments of human plasma fibronectin and synthetic peptides containing an RGD (single letter amino acid code for Arg-Gly-Asp) sequence which promote cell attachment and enhance phagocytosis are disclosed in U.S.
Patent 517,686, U.S. Patent 4,589,881, U.S. Patent 4,661,111 and U.S. Patent 4,614,517. Linear and cyclic peptides containing an RGD sequence have also be reported in WO
89/05150 (PCT US88/04403). Peptides which contain an RGD sequence have been reported to inhibit platelet aggregation. Nievelstein et al, (Thromb. and Hemostasis, 58, 2133(1987) have reported that -RGDS- peptides inhibit thrombin induced aggregation and adhesion of platelets to fibronectin, and may interact through the GPIIb-IIIa complex. U.S. Patent
4,683,291 discloses peptides containing Arg and Lys and an -RGD- sequence which inhibit binding of fibrinogen to platelets and inhibit platelet aggregation. A disadvantage of these peptides is their poor stability in plasma and their low potency. EP 0 275 748 discloses linear tetra- to hexapeptides and cyclic hexa- to octapeptides which bind to the GPIIb-IIIa receptor and inhibit platelet aggregation. The cyclic peptides reported are formed via a disulfide bridge between two cysteinyl residues. Other linear and cyclic peptides, the disclosure of which are incorporated herein by reference, are reported in EP-A 0 341 915. One compound disclosed therein is Ac-Arg-Gly-Asp-NHCH2CH2Ph.
The instant invention provides compounds in which the carboxy terminus of the peptide is substituted with an aryl alcohol or aryl amine to form an aryl ester or amide. The compounds of this invention inhibit the binding of GP-IIbllla to fibrinogen and are surprisingly potent. Thus, an
advantage to the compounds of this invention is their ability to inhibit platelet aggregation and their improved activity.
Recent advances for treatment of occluded arteries and deep vein thrombosis employ fibrinolytic agents to lyse thrombi or emboli in order to reestablish or improve blood flow. Fibrinolytic agents, such as anistreplase, tissue plasminogen activator (tPA), urokinase (UK), pro- Urokinase (pUK), and streptokinase (SK), and mutants and derivatives thereof, are proteolytic enzymes which cause fibrin to be hydrolyzed at specific sites and thereby
fragment the fibrin network. Their action in vivo is to proteolytically activate plasminogen in the blood to form plasmin, which causes lysis of the fibrin clot. Lysis of fibrin into smaller peptides has the effect of solubilizing the thrombus or embolus. A recurrent problem with such therapy, however, is the reocclusion of the blood vessel due to formation of a secondary thrombus.
Fibrinolytic therapy is most commonly used for re-establishing flow in a thrombosed blood vessel. However, fibrinolytic therapy does not reverse the factors responsible for the initiation of the thrombus. For this reason,
anticoagulants such as heparin are often used to prevent reocclusion. In fact, patients which have a high degree of stenosis in an artery are at extremely high risk of
rethrombosis after reperfusion, even in the presence of high doses of heparin. See Gold et al., Circ, 73, 347-52 (1986). In addition, use of SK and tPA has been associated with platelet hyperaggregability. See Ohlstein, et al., Thromb . Res., 4, 575-85 (1987). Treatment with higher doses of tPA can be associated with systemic bleeding and is not
recommended for preventing reocclusion. There is, therefore, a need for a method for preventing rethrombosis after
fibrinolytic therapy.
U.S. Patent Application Serial No. 917,122 discloses TxA2 antagonists for use in a method for inhibiting reocclusion following reperfusion and for lowering the dose of tPA required for fibrinolysis. Yasuda et al . (Clin. Res., 34, 2 (1986)) have demonstrated that reocclusion by fibrin rich platelet thrombi, after thrombolysis with tPA, may be inhibited by a murine monoclonal antibody to GPIIb-IIIa in dogs. This invention discloses a new method for inhibiting reocclusion of a blood vessel by administering peptides which directly inhibit platelet aggregation. Summary of the Invention
In one aspect this invention is a compound of the formula (I):
D-E-Asp-Q-Y
(I)
wherein:
Q is NH, NCH3 or O;
Y is phenyl, naphthyl or Het, unsubstituted or
substituted by one to three C1-6alkyl, trifluoromethyl, halogen, OR', SR', (CH2)nAr, CONR1R2, CO2R2 or R4R4N;
Y
Figure imgf000007_0002
or
Figure imgf000007_0001
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 OC (=O);
V is H, R', R4R5N, R4R5NCO, R'O, Y-NR', X-A-B-NR' or
Figure imgf000007_0003
A is absent, Asn, Gin, Ala, Pro or Abu;
B is a D- or L- amino acid chosen from Arg, HArg, NArg, (Me2)Arg, (Et2)Arg, Abu, Ala, Gly, His, Orn, Lys, Phg or an α-R' substituted derivative thereof, Dtc, Tpr or Pro;
E is Gly, Sar, CH2CO, OCH2CO or NHCO;
R' is H, C1-6alkyl or Ar(CH2)p;
X is R4R4N, R4R5N-CO or H;
R1 and R2 are H, C1-6alkyl or (CH2)pAr;
R4 is H or C1-6alkyl;
R5 is R6, R6CO, R6OCO, R6OCH (R6' ) CO, R6NHCH (R6' ) CO,
R6SCH(R6')CO, R6SO2 or R6SO; R6 and R6' are H, C1-6alkyl, C3-7cycloalkyl, Ar,
Ar-C1-4alkyl, Het, Het-C1-4alkyl, Ar-(CH2)pCH(Ar) (CH2)P or Ar-C3-7cycloalkyl;
Ar is phenyl or phenyl substituted by one to three C1-6alkyl, trifluoromethyl, halogen, R'O or R'S; and
Figure imgf000008_0001
is a five or six-membered, mono or bicyclic ring containing one or two nitrogen atoms;
m is: (1) 0-4, when W is NR,-(C=NH)NHR';
(2) 0-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or (3) 0-6, when W is NHR';
n is 0-2;
p is 0-3;
q is: (1) 1-4, when W is NR'-(C=NH)NHR';
(2) 2-5, when -W is (C=NH)NHR' or NR'(C=NH)R'; or (3) 3-6, when W is NHR';
or a pharmaceutically acceptable salt thereof..
One feature of this invention is a compound, wherein an aryl group is attached to the Asp residue via a nitrogen or oxygen, which has increased activity for inhibition of platelet aggregation.
Another feature of this invention is a novel active compound wherein the α-amino group of Arg is modified to incorporate the amino group into a ring.
Yet another feature of this invention is a novel active compound wherein the α-amino group of the Arg is eliminated or replaced by another substituent.
This invention is also a pharmaceutical composition for inhibiting platelet aggregation and clot formation, which comprises a compound of formula (I) and a pharmaceutically acceptable carrier.
This invention is further a method for inhibiting platelet aggregation in a mammal in need thereof, which comprises internally administering an effective amount of an compound of formula (I).
In another aspect, this invention provides a method for inhibiting reocclusion of an artery or vein in a mammal following fibrinolytic therapy, which comprises internally administering an effective amount of a fibrinolytic agent and a compound of formula (I). This invention is also a method for treating stroke, transient ischemia attacks, or
myocardial infarction.
This invention is also a pharmaceutical composition for effecting fibrinolysis and reperfusion, and inhibiting reocclusion in an artery or vein in a mammal, which comprises a fibrinolytic and a compound of formula (I) in a
pharmaceutical carrier.
Finally, this invention is a kit for use in a method for effecting thrombolytic therapy, which comprises, in a
container, a fibrinolytic and a compound of formula (I).
Detailed Description of the invention This invention discloses acyclic peptide-like compounds comprising the sequence Gly-Asp and having an aryl ester or amide moiety attached directly to the carboxy group of Asp. The compounds of this invention inhibit platelet aggregation and are believed to interact with the GPIIb-IIIa receptor and other adhesion proteins. The attachment of an aryl group to the Asp residue confers increased activity for inhibition of platelet aggregation.
The compounds of this invention are peptides of formula (I) as hereinbefore described.
Suitably Y is phenyl or naphthyl, optionally substituted by halogen, C1-4alkyl, C1-4alkoxy, C1-4alkylthio, CONR1R2 or
CO2R3.
Preferably Q is NH;
Suitably Y is phenyl or naphthyl.
Preferably Y is phenyl, optionally substituted by chloro, C1-4alkyl, C1-4alkylthio or carboxy .
Suitably D is
Figure imgf000009_0001
.
Preferably D is .
Figure imgf000009_0002
Suitably V is X-A-B-R'N, R4R5NCO or R4R5N, wherein R' or R4 is H or methyl. Suitably V is ArCO-N(CH3) or HetCO-N(CH3).
Preferably V is R4R5N, wherein R4 is H or methyl and R5 is R6CO or R6OCO.
Suitably E is Gly, Sar, NHCO or CH2CO. Preferably E is Gly.
Suitably Het is thienyl or pyridyl.
When D is
Figure imgf000010_0001
,the preferred value of m is
dependent upon the substitutent W. Accordingly, when W is -NR' (C=NH)NHR', m is preferably 2-3; when W is -(C=NH)NHR' or -NR' (C=NH)R', m is preferably 3-4; and when W is -NHR', m is preferably 4-5. When D is the preferred value of m is
Figure imgf000010_0002
dependent upon the nature of W, and the positional
relationship between U and the substituent (CH2)m-W.
Accordingly, when W is -NR'(C=NH)NHR' , m is preferably 0-1 for para, 0-2 for meta, and 1-3 for an ortho orientation. When W is -(C=NH)NHR' or -NR'(C=NH)R', m is preferably 0-2 for para, 1-3 for meta, or 2-4 for ortho. When W is -NHR', m is preferably 0-3 for para, 2-4 for meta, and 3-5 for an ortho orientation. A para orientation is generally
preferred.
The meaning of X in the formulae herein depicted with regard to X-A or X-B, when A is absent, is intended to denote the amino group of these amino acids. In cases wherein the compounds of this invention may have one or more chiral centers, unless specified, this invention includes each unique nonracemic compound which may be synthesized and resolved by conventional techniques. The meaning of
substituents, R1, R2, R4, R5, R6, R6', Ar, R', Q, n, p and q, at any one occurrence is independent of their meaning at any other occurrence.
C1-4alkyl as applied herein is meant to include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl and t-butyl. C1-6alkyl includes these groups and additionally pentyl and hexyl and the simple aliphatic isomers thereof, such as npentyl, isopentyl and neopentyl.
Ar as applied herein means phenyl or phenyl substituted by one or two C1-6alkyl, trifluoromethyl, halogen, R'O or R'S. With special reference to substitution of R'O and R'S upon Ar, R' may be H, resulting in hydroxy or mercapto;
C1-6alkyl, resulting in C1-6alkoxy or C1-6alkylthio; or
Ar(CH2)n, resulting in phenoxy, phenylC1-3alkoxy, phenylthio or phenylC1-3alkylthio, optionally substituted on the phenyl by one or two C1-4alkyl, C1-4alkoxy or halogen. Benzyloxy and benzylthio, optionally substituted by methyl or methoxy is illustrative.
Y is a substituted aromatic or heteroaromatic group. For instance, Y is phenyl or naphthyl, optionally substituted by one or two C1-6alkyl, trifluoromethyl, halogen, R'O, R'S, (CH2)nAr, CONR1R2, CO2R2 or R4R5N. Het, or heteroaryl, indicates a five or six membered monocyclic aromatic ring, or a nine or ten-membered bicyclic aromatic ring containing one to three heteroatoms chosen from the group of nitrogen, oxygen and sulfur, which are stable and available by
conventional chemical synthesis. Illustrative heterocycles are pyridyl, furyl, thienyl, indolyl, quinolinyl, benzofuryl and benzothienyl. Any accessible combination of up to three substituents on the phenyl, naphthyl or Het ring which is available by chemical synthesis and is stable are within the scope of this invention.
Figure imgf000011_0001
is a five or six-membered mono-, or nine or ten membered bi-cyclic ring containing one or two nitrogen atoms, optionally substituted by one or two C1-6alkyl or R'O groups, such as are available by chemical synthesis and are stable.
Figure imgf000011_0002
is joined to the moiety D via a nitrogen atom.
Representative examples are phthalimidyl, piperidinyl, pyrrolyl, isoquinolyl, 2-pyridonyl, 1,3-dihyro-2H-isoindolyl, 2-pyrrolidinonyl, imidazolyl and morpholinyl.
This invention includes compounds in which any of the peptide linkages, -CONH-, are replaced by an isosteric linkage. Examples of peptide isosteres are -NHCO-, -CH=CH-, -CH2CH2-, -COCH2-, -COO-, -CHOHCH2-, -CH2NR4-, -CSNH- and -CH2S- .
Specific compounds of this invention are:
N-[[5-{aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp- phenylamide;
N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-oxopentyl]-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- phenylamide;
N-[(2S)--(1-(2-pyrrolidinonyl))-5-(aminoiminomethyl)amino-1- oxopentyl]-Gly-Asp-phenylamide;
Nα-(phthaloyl)-Arg-Gly-Asp-phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(4-carboxy)phenylamide;
Nαacetyl-Arg-Gly-Asp-phenylamide;
Nαbenzoyl-Arg-Gly-Asp-phenylamide;
Nαbenzyloxycarbonyl-Arg-Gly-Asp-phenylamide;
D-Phg-Arg-Gly-Asp-phenylamide;
Nαbenzyloxycarbonyl-Nαmethyl-Orn-Gly-Asp-phenylamide;
4-(aminoiminomethyl) aminobenzoyl-Sar-Asp-phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(2-chloro)phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(2-methoxy)phenylamide;
Nαbenzoyl-NαMeArg-Gly-MeAsp-(2-hydroxy)phenylamide;
Nαbenzoyl-Arg-NHNHCO-Asp-(N-methyl)phenylamide; and
Nα(OCCH2CO) -Arg- (N-methyl)phenylamide
Figure imgf000012_0001
Preferred compounds of this invention are :
N**-(2-methylbenzoyl)-Nα-MeArg-Gly-Asp-(2-methyl)phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-phenylamide;
Nαacetyl-NαMeArg-Gly-Asp-phenylamide;
Nαacetyl-NαMeArg-Gly-Asp-(2-carboxy)phenylamide;
Nα(2-methylthio)benzoyl-NαMeArg-Gly-Asp-(2-methylthio)-phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(4-chloro)phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(3-carboxy)phenylamide; and
Nα(2-thienylcarbonyl)-NαMeArg-Gly-Asp-phenylamide. The nomenclature commonly used in the art is used herein to describe the peptides.
3 3
Amino Acid letter Amino Acid letter
code code
Alanine Ala Leucine Leu
Arginine Arg Lysine Lys
Asparagine Asn Methionine Met
Aspartic Acid Asp Phenylalanine Phe
Cysteine Cys Proline Pro
Glutamine Gin Serine Ser
Glutamic Acid Glu Threonine Thr
Glycine Gly Tryptophan Trp
Histidine His Tyrosine Tyr
Isoleucine lie Valine Val
Asparagine or Aspartic : Acid Asx
Glutamine or Glutamic Acid Glx
In accordance with conventional representation, the amino terminus is on the left and the carboxy terminus is on the right. Unless specified otherwise, all chiral amino acids (AA) are assumed to be of the L-absolute configuration. Abu refers to aminobutyric acid, Dtc refers to 5,5-dimethylthiazolidine-4-carboxylic acid, Tpr refers to
thiazolidine-4-carboxylic acid, HArg refers to homoarginine, NArg refers to norarginine, (Me2)Arg refers to N',N"-dimethyl arginine, (Et2)Arg refers to N',N"-diethyl arginine, D-Phg refers to D-phenylglycine and Orn refers to ornithine.
t-Bu refers to the tertiary butyl radical, Boc refers to the- t-butyloxycarbonyl radical, Fmoc refers to the
fluorenylmethoxycarbonyl radical, Ph refers to the phenyl radical, Phth refers to phthaloyl, Cbz refers to the
carbobenzyloxy radical, BrZ refers to the
o-bromobenzyloxycarbonyl radical, ClZ refers to the
o-chlorobenzyloxycarbonyl radical, Bzl refers to the benzyl radical, 4-MBzl refers to the 4-methyl benzyl radical, Ac refers to acetyl, Alk refers to C1-4 alkyl, Nph refers to 1- or 2-naphthyl and cHex refers to cyclohexyl. DCC refers to dicyclohexylcarbodiimide, DMAP refers to dimethylaminopyridine, DIEA refers to diisopropylethyl amine, EDC refers to N-ethyl-N'(dimethylaminopropyl)-carbodiimide, HOBt refers to 1-hydroxybenzotriazole, THF refers to
tetrahydrofuran, DMF refers to dimethyl formamide, PPA refers to 1-propanephosphonic acid cyclic anhydride, DPPA refers to diphenylphosphoryl azide, BOP refers to benzotriazol-1-yloxy- tris(dimethylamino)phosphonium hexafluorophosphate, HF refers to hydrofluoric acid and TFA refers to trifluoroacetic acid.
Coupling reagents as used herein denote reagents which may be used to form peptide bonds. Typical coupling reagents are carbodiimides, activated anhydrides and esters and acyl halides. Reagents such as EDC, DCC, DPPA, PPA, BOP reagent, HOBt, N-hydroxysuccinimide and oxalyl chloride are typical. α-R' substituted derivatives of the amino acids of this invention, which may be denoted as (α-R')AA, indicate amino acids which are mono-substituted on the α-amino group by R', wherein R* is C1-6alkyl or Ar(CH2)n. Suitably, R' is methyl or benzyl. R' is preferably methyl. Nα-methyl arginine and Nα-methyl glycine, which are (α-Me)Arg and (α-Me)Gly
respectively, are also denoted herein as MeArg and Sar
(sarcosine) in accordance with past conventional notation. All other N-α-substituted amino acids will carry the
designation α- in their representation.
The peptides are prepared either by the solid phase technique of Merrifield (J. Am. Chem. Soc, 85, 2149 (1964)) or by common solution methods known to the art. The methods of peptide synthesis generally set forth by Ali et al. in J. Meri. Chem.. 29, 984 (1986) and J. Med . Chem., 30, 2291 (1987) are generally illustrative of the technique and are
incorporated herein by reference. A combination of solid phase and solution synthesis may be used, as in a convergent synthesis in which di-, tri-, tetra-, or penta-peptide fragments may be prepared by solid phase synthesis and either coupled or further modified by solution synthesis. Solution phase synthesis is generally preferred for the smaller peptides of this invention. The reactive functional groups of the sidechains of each synthetic fragment, amino acid or peptide are suitably protected as known in the peptide art. For example, the Boc, Cbz or Fmoc group may be used for protection of an amino group, especially an α-amino group. The Boc group is generally preferred for protection of the α-amino group. A t-Bu, cHex or benzyl ester may be used for the protection of the side chain carboxyl of Asp or Glu. A benzyl group or suitably substituted benzyl group is used to protect the mercapto group of cysteine, or other thiol containing residues; or the hydroxyl of serine or threonine. The tosyl group may be used for protection of the imidazolyl group of His, and tosyl or nitro group for protection of the guanidino nitrogen of Arg. A suitably substituted carbobenzyloxy group or benzyl group may be used for the hydroxyl group of Tyr, Ser or Thr, or the ε-amino group of lysine. The phthaloyl group may also be used for the protection of the ε-amino group of lysine. Suitable substitution of the
benzyloxycarbonyl or benzyl protecting groups is ortho and/or para substitution with chloro, bromo, nitro or methyl, and is used to modify the reactivity of the protective group.
Except for the Boc group, the protective groups are, most conveniently, those which are not removed by mild acid treatment. These protective groups are removed by such methods as catalytic hydrogenation, sodium in liquid ammonia or HF treatment, as known in the art.
Solution synthesis of peptides is accomplished using conventional methods used to form amide bonds. Typically, the. fragment H-Q-Y is coupled via its free amino or hydroxyl group to an appropriately sidechain protected Asp using a suitable carbodiimide coupling agent, such as N, N'
dicyclohexyl carbodiimide (DCC), optionally in the presence of catalysts such as 1-hydroxybenzotriazole (HOBt) and dimethylamino pyridine (DMAP). Other methods, such as the formation of activated esters, anhydrides or acid halides, of the free carboxyl of a protected Boc-amino acid, and
subsequent reaction with the free amine of a protected amino acid, optionally in the presence of a base, are also suitable. For example, a protected Boc-amino acid or peptide is treated in an anhydrous solvent, such as methylene chloride or tetrahydrofuran (THF), in the presence of a base, such as N-methyl morpholine, DMAP or a trialkylamine, with isobutyl chloroformate to form the "activated anhydride", which is subsequently reacted with the free amine of a second protected amino acid or peptide. The intermediate
Boc-Asp-Q-Y formed by these methods may be deprotected selectively, using conventional techniques, at the amino terminus and coupled to other peptides or amino acids using similar techniques.
If solid phase methods are used, for instance when Y bears a carboxyl group which may be attached to a resin, the peptide, or a convenient fragment thereof, is built up sequentially starting from the carboxy terminus and working toward the amino terminus of the peptide. Solid phase synthesis is begun by covalently attaching the carboxyl group of the appropriately protected Y residue, such as Boc-NH-Y, to a suitable resin, such as a benzhydrylamine resin (BHA), methylbenzhydrylamine resin (MBHA), chloromethyl resin (CMR), hydroxymethyl resin (HMR) or SASRIN resin, as is generally set forth in U.S. Patent No. 4,244,946 and Gisin, Helv. Chem. Acta, 56, 1476 (1973). A BHA or MBHA support resin is used if the carboxy terminus of the product peptide is to be a carboxamide. A CMR or HMR support is generally used if the carboxy terminus of the product peptide is to be a carboxyl group, although this may also be used to produce a
carboxamide or ester.
Once the first protected residue has been coupled to the resin, the amino group is deprotected by milu acid treatment, and the free carboxyl of the second protected AA is coupled to this amino group. This process is carried out
sequentially, without isolation of the intermediate, until the desired peptide has been formed. The completed peptide may then be deblocked and/or split from the carrying resin in any order.
If the Y residue does not possess a carboxyl group and solid phase synthesis is desirable, the peptide may be attached to a CMR or HMR via the β-carboxyl group of Asp. For example, the synthesis is begun by coupling the aryl amine or aryloxy group, via solution phase synthesis, to an appropriately sidechain-protected aspartic acid residue. The side chain carboxyl group is then selectively deprotected and coupled to a chloromethyl resin (CMR). A benzyl ester is a suitable sidechain protecting group which is selectively deprotected by hydrogenation. The amino group is
subsequently liberated by treatment with acid and solid phase peptide synthesis is carried out in the usual manner.
If Y possesses no carboxylic acid moeity and has a substituent incompatible with hydrogenation, and solid phase synthesis is desirable, a t-butyl ester or other acid labile group is used for protecting the sidechain carboxyl of the aspartic acid. In this case the amino group of the aspartic acid is protected by a base labile group, such as the fluorenylmethoxycarbonyl moiety (Fmoc). After solution phase coupling of the aspartic acid to an aryl amine or phenol, selective deprotection of the t-butyl ester is accomplished by mild acid hydrolysis. The sidechain carboxyl group of Asp is coupled to the resin by conventional methods. The fluorenylmethoxycarbonyl group is then removed by mild base for subsequent solid phase peptide synthesis.
The preferred method for cleaving a peptide from the support resin is to treat the resin supported peptide with anhydrous HF in the presence of a suitable cation scavenger, such as anisole or dimethoxy benzene. This method
simultaneously removes all protecting groups, except a thioalkyl group protecting sulfur, and splits the peptide from the resin. Peptides hydrolyzed in this way from the CMR or HMR are carboxylic acids, those split from the BHA resins are obtained as carboxamides. If the peptide is linked through the β-carboxyl of Asp, a CMR or HMR is used, since a carboxyl group is desired. If the peptide is coupled through the Y residue, the choice of resin will depend upon whether an acid or carboxamide is desired.
Modification of the terminal amino group of the peptide is accomplished by alkylation, sulfonylation or acylation as is generally known in the art. These modifications may be carried out upon the D residue prior to incorporation into the peptide, or upon the peptide after it has been
synthesized and the terminal amino group liberated, but before the protecting groups have been removed.
Typically, acetylation is carried out upon the free amino group using the acyl halide, or anhydride, of the corresponding alkyl or aryl acid, in the presence of a tertiary amine. Mono-alkylation is carried out most
conveniently by reductive alkylation of the amino group with an appropriate aliphatic aldehyde or ketone in the presence of a mild reducing agent, such as lithium or sodium
cyanoborohydride. Dialkylation may be carried by treating the amino group with an excess of an alkyl halide in the presence of a base.
The α-R" substituted derivatives of the amino acids of this invention, which includes derivatives of Arg, HArg, (Me2)Arg, (Et2)Arg, Ala, Gly, His, Abu, Orn, Lys are prepared by methods common to the chemical art. The R' substituent may be C1-6alkyl or Ar(CH2)n, as hereinbefore defined.
Representative methods for preparing these derivatives are disclosed in U.S. Patent No. 4,687,758; Cheung et al., Can. J. Chem., 55, 906 (1977); Freidinger et al., J. Org. Chem., 48, 77, (1982); and Shuman et al., Peptides: Proceedings of the 7th American Peptide Symposium, Rich, D., Gross, E., Eds, Pierce Chemical Co., Rockford, I11.,617 (1981), which are incorporated herein by reference.
Acid addition salts of the peptides are prepared in a standard manner in a suitable solvent from the parent compound and an excess of an acid, such as hydrochloric, hydrobromic, sulfuric, phosphoric, acetic, maleic, succinic or methanesulfonic. The acetate salt form is especially useful. Certain of the compounds form inner salts or
zwitterions which may be acceptable. Cationic salts are prepared by treating the parent compound with an excess of an alkaline reagent, such as a hydroxide, carbonate or alkoxide, containing the appropriate cation; or with an appropriate organic amine. Cations such as Li+, Na+, K+, Ca++, Mg++ and NH4+ are specific examples of cations present in
pharmaceutically acceptable salts.
This invention provides pharmaceutical composition which comprises a peptide according to formula (I) and a pharmaceutically acceptable carrier. Pharmaceutical
compositions of the peptides prepared as hereinbefore
described and other peptide or polypeptide derivatives of fibronectin, fibrinogen or Von Willebrand's factor, may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulation is especially suitable for parenteral administration, but may also be used for oral administration or contained in a metered dose inhaler or nebulizer for insufflation. It may be desirable to add excipients such as polyvinylpyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate.
Alternately, these peptides may be encapsulated, tableted or prepared in an emulsion or syrup for oral
administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the
composition, or to facilitate preparation of the composition. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. Liquid carriers include syrup, peanut oil, olive oil, saline and water. The carrier may also include a sustained release material such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 g per dosage unit. The pharmaceutical preparations are made following the conventional techniques of pharmacy involving milling, mixing, granulating, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, the
preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly p.o. or filled into a soft gelatin capsule.
For rectal administration, the peptides of this
invention may also be combined with excipients such as cocoa butter, glycerin, gelatin or polyethylene glycols and molded into a suppository.
This invention also provides a method of inhibiting platelet aggregation and clot formation in a mammal,
especially a human, which comprises the internal
administration of a peptide of formula (I) and a
pharmaceutically acceptable carrier. Indications for such therapy include acute myocardial infarction (AMI), deep vein thrombosis, pulmonary embolism, dissecting anurysm, transient ischemia attack (TIA), stroke and other infarct-related disorders, and unstable angina. Chronic or acute states of hyper-aggregability, such as disseminated intravascular coagulation (DIC), septicemia, surgical or infectious shock, post-operative and post-partum trauma, cardiopulmonary bypass surgery, incompatible blood transfusion, abruptio placenta, thrombotic thrombocytopenic purpura (TTP), snake venom and immune diseases, are likely to be responsive to such
treatment. In addition, the peptides of this invention may be used in a method for the prevention of metastatic
conditions, the prevention or treatment of fungal infection, and- the prevention or treatment of diseases in which bone resorption is a factor.
The peptide is administered either orally or
parenterally to the patient, in a manner such that the concentration of drug in the plasma is sufficient to inhibit platelet aggregation. The pharmaceutical composition
containing the peptide is administered at a dose between about .2 to about 50 mg/kg in a manner consistent with the condition of the patient. For acute therapy, parenteral administration is preferred. For persistent states of hyperaggregability, an intravenous infusion of the peptide in 5% dextrose in water or normal saline is most effective, although an intramuscular bolus injection may be sufficient.
For chronic, but noncritical, states of platelet aggregability, oral administration of a capsule or tablet, or a bolus intramuscular injection is suitable. The peptide is administered one to four times daily at a level of about 0.4 to about 50 mg/kg. to achieve a total daily dose of about 0.4 to about 200 mg/kg/day.
This invention further provides a method for inhibiting the reocclusion of an artery or vein following fibrinolytic therapy, which comprises internal administration of a peptide of formula (I) and a fibrinolytic agent. It has been found that administration of a peptide in fibrinolytic therapy either prevents reocclusion completely or prolongs the time to reocclusion.
When used in the context of this invention the term fibrinolytic agent is intended to mean any compound, whether a natural or synthetic product, which directly or indirectly causes the lysis of a fibrin clot. Plasminogen activators are a well known group of fibrinolytic agents. Useful plasminogen activators include, for example, anistreplase, urokinase (UK), pro-urokinase (pUK), streptokinase (SK), tissue plasminogen activator (tPA) and mutants, or variants, thereof, which retain plasminogen activator activity, such as variants which have been chemically modified or in wliich one or more amino acids have been added, deleted or substituted or in which one or more or functional domains have been added, deleted or altered such as by combining the active site of one plasminogen activator with the fibrin binding domain of another plasminogen activator or fibrin binding molecule. Other illustrative variants include tPA molecules in which one or more glycosylation sites have been altered. Preferred among plasminogen activators are variants of tPA in which the primary amino acid sequence has been altered in the growth factor domain so as to increase the serum half-life of the plasminogen activator. tPA Growth factor variants are disclosed, e.g., by Robinson et al., EP-A 0 297 589 and Browne et al., EP-A 0 240 334 and in GB 8815135.2. Other variants include hybrid proteins, such as those disclosed in EP 0 028 489, EP 0 155 387 and EP 0 297 882, all of which are incorporated herein by reference. Anistreplase is a
preferred hybrid protein for use in this invention.
Fibrinolytic agents may be isolated from natural sources, but are commonly produced by traditional methods of genetic engineering.
Useful formulations of tPA, SK, UK and pUK are
disclosed, for example, in EP-A 0 211 592, German Patent Application No. 3032606, EP-A 0 092 182 and U.S. Patent
4,568,543, all of which are incorporated herein by reference. Typically the fibrinolytic agent may be formulated in an aqueous, buffered, isotonic solution, such as sodium or ammonium acetate or adipate buffered at pH 3.5 to 5.5.
Additional excipients such as polyvinyl pyrrolidone, gelatin, hydroxy cellulose, acacia, polyethylene, glycol, mannitol and sodium chlbride may also be added. Such a composition can be lyophilized.
The pharmaceutical composition may be formulated with both the peptide and fibrinolytic in the same container, but formulation in different containers is preferred. When both agents are provided in solution form they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement.
Indications for such therapy include myocardial
infarction, deep vein thrombosis, pulmonary embolism, stroke and other infarct-related disorders. The peptide is
administered just prior to, at the same time as, or just after parenteral administration of tPA or other fibrinolytic agent. It may prove desirable to continue treatment with the peptide for a period of time well after reperfusion has been established to maximally inhibit post-therapy reocclusion. The effective dose of tPA, SK, UK or pUK may be from 0.5 to 5 mg/kg and the effective dose of the peptide may be from about 0.1 to 25 mg/kg.
For convenient administration of the inhibitor and the fibrinolytic agent at the same or different times, a kit is prepared, comprising, in a single container, such as a box, carton or other container, individual bottles, bags, vials or other containers each having an effective amount of the inhibitor for parenteral administration, as described above, and an effective amount of tPA, or other fibrinolytic agent, for parenteral administration, as described above. Such kit can comprise, for example, both pharmaceutical agents in separate containers or the same container, optionally as lyophilized plugs, and containers of solutions for
reconstitution. A variation of this is to include the solution for reconstitution and the lyophilized plug in two chambers of a single container, which can be caused to admix prior to use. With such an arrangement, the fibrinolytic and the peptide may be packaged separately, as in two containers, or lyophilized together as a powder and provided in a single container.
When both agents are provided in solution form, they can be contained in an infusion/injection system for simultaneous administration or in a tandem arrangement. For example, the platelet aggregation inhibitor may be in an i.v. injectable form, or infusion bag linked in series, via tubing, to the fibrinolytic agent in a second infusion bag. Using such a system, a patient can receive an initial bolus-type injection or infusion, of the peptide inhibitor followed by an infusion of the fibrinolytic agent.
The pharmacological activity of the peptides was.
assessed by the following tests:
In Vivo Inhibition of Platelet Aggregation
In vivo inhibition of thrombus formation is demonstrated by recording the systemic and hemodynamic effects of infusion of the peptides into anesthetized dogs according to the methods described in Aiken et al., Prostaglandins, 19, 629-43 (1980). Inhibition of Platelet Aggregation
Blood was collected (citrated to prevent coagulation) from, naive, adult mongrel dogs. Platelet rich plasma, PRP, was prepared by centrifugation at 150 × g for 10 min at room temperature. Washed platelets were prepared by centrifuging PRP at 800 x g for 10 min. The cell pellet thus obtained was washed twice in Tyrode's buffer (pH 6.5) without Ca++ and resuspended in Tyrode's buffer (pH 7.4) containing 1.8 mM Ca++ at 3 × 105 cells/ml. Peptides were added 3 min prior to the agonist in all assays of platelet aggregation. Final agonist concentrations were 0.1 unit/ml thrombin and 2 mM ADP (Sigma). Aggregation was monitored in a Chrono-Log Lumi-Aggregometer. Light transirdttance 5 min after addition of the agonist was used to calculate percent aggregation according to the formula % aggregation = [(90-CR) + (90-10)]
× 100, where CR is the chart reading, 90 is the baseline, and 10 is the PRP blank reading. IC50 were determined by
plotting [% inhibition of aggregation] vs. [concentration of peptide]. Peptides were assayed at 200 mM and diluted sequentially by a factor of 2 to establish a suitable dose response curve.
To assess the stability of the peptide to plasma proteases, the peptides were incubated for 3 h. (rather than 3 min) in the PRP prior to addition of the agonist.
Compounds were assayed for their ability to inhibit the aggregation of dog platelets in response to stimulation by ADP. The compounds of Examples 1-5, 12-13 and 15 had IC50 in the range of about 0.1-1.0 μM; the compounds of Examples 6-9, 11, 19-21, 26 and 27 had IC50 of about 2 to 40 μM; and the compound of Example 10 had an IC50 of greater than 200 μM. The compound of Example 5 is a preferred compound.
The examples which follow are intended to in no way limit the scope of this invention, but are provided to illustrate how to make and use the compounds of this invention. Many other embodiments will be readily apparent and available to those skilled in the art.
EXAMPLES
In the examples which follow all temperatures are in degrees centigrade. Amino acid analysis was performed upon a Dionex autoion 100. Analysis for peptide content is based upon amino acid analysis. Mass spectra were performed upon a VG Zab mass spectrometer using fast atom bombardment. EM silica gel thin layer (0.25 mm) plates were used for thin layer chromatography. ODS refers to an octadecylsilyl silica gel chromatographic support. The abbreviations used to represent the eluent composition are n-BuOH: n-butanol, HOAc: acetic acid, H2O: water, EtOAc: ethyl acetate, i-ProH:
isopropanol, P: pyridine and CA: chloroacetic acid. HPLC was performed upon a Beckman 344 gradient chromatography system with a CRIB recording integrator in either an isocratic or continuous gradient mode. Ultrasphere® and Ultrasphere® ODS are silica gel and octadecylsilane chromatographic supports respectively, manufactured by Beckman Instruments Inc.,
Fullerton, CA. Dynamax® and Dynamax® C18 silica gel and octadecylsilane chromatographic supports respectively, manufactured by Rainin Instruments Co., Woburn, Mass.
Sephadex is a cross-linked poly (dextran) manufactured by
Pharmacia Fine Chemicals, Piscataway, N.J. Celite® is filter aid composed of acid washed diatomaceous silica manufactured by Mansville Corp., Denver, Colorado. Solid phase peptide synthesis was performed using an automated Beckman 990 synthesizer. Where indicated, the purity of the peptide is based upon integration of the HPLC chromatogram. MeArg was prepared by the method disclosed by Ali et al., in U.S.
Patent 4,687,758 (1987). Example 1
Preparation of Nα(2-methyl)benzoyl-NαMeArg-Gly-Asp- (2 - methyl)phenylamide (8)
Figure imgf000026_0001
a) Boc-NαMeArg (Tos ) -Gly(OMe) (1 )
DIEA (19.3 mL, 110 mmol) was added to a cold suspension of Gly(OMe)·hydrochloride (6.3 g, 50 mmol) in dry DMF (100 mL) in a dropwise manner to bring it to a neutral pH. This was followed by addition of HOBt (7.4 g, 55 mmol).and
Boc-NαMeArg(Tos) (22.1 g, 50 mmol). The reaction mixture was stirred for a few minutes, then EDC (10.55 g, 55 mmol) was added at 0°C. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The solvent was removed under vacuum, and the residue was partitioned between ethyl acetate and water. The organic extract was washed successively with 1N HCl (3x), water (3x), 10% Na2CO3 (3x), water (3x) and saturated salt solution (1x). The organic extract was dried with anhydrous sodium sulfate, filtered and concentrated to the titled compound (21.54 g, 84%) as white solid h) NαMeArg (Tos ) -Gly(OMe) (2)
Boc-NαMeArg(Tos)-Gly(OMe) (1) (2.6 g, 5 mmol) was treated with 50% TFA in methylene chloride at room
temperature for 45 min. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether and used in the next step without further purification. c) Nα(2-methyl) benzoyl-NαMeArg (Tos ) -Gly(OMe) (3)
DIEA (1.92 g, 3.5 mL) was added to a cold solution of NαMeArg(Tos)-Gly(OMe) (2) (5 mmol) in DMF (10 mL) to bring the pH to neutrality (pH 7.5 to 8.0). HOBt (745 mg) was added followed by 2-methylbenzoic acid anhydride (5.5 mmol), (prepared from 1.37 g of 2-methylbenzoic acid and 1.3 g of DCC). The reaction mixture was stirred at room temperature overnight. The solvent was removed under vacuum, the residue was dissolved in ethyl acetate (120 mL) and washed
successively with water (3 × 40 mL), 10% sodium carbonate (2 × 40 mL), water (2 × 40 mL) and saturated salt solution. The organic extract was dried over anhydrous sodium sulfate, filtered and concentrated to yield the titled compound as white fluffy solid (2.2 g). d) Nα(2-methyl) benzoyl-NαMeArg (Tos ) -Gly ( 4)
To a solution of Nα(2-methyl)benzoyl-NαMeArg(Tos)-Gly(OMe) (3) (2.2 g, 4.1 mmol) in methanol (10 mL), a solution of 1 N NaOH (5 mL, 5 mmol) was added dropwise. The reaction mixture was stirred at room temperature for an hour to complete ester hydrolysis. The solvent was removed, the residue was redissolved in water (50 mL), back extracted with ethyl acetate, and acidified to acidic pH (2.86) with 3 N HCl. The acidic solution was extracted with ethyl acetate, the organic layer was washed 2x with water, dried with anhydrous sodium sulfate, filtered and concentrated to yield the titled compound as white fluffy solid (1.53 g). e) Boc-Asp(Q-cHex)-(2-methyl)phenylamide (5)
To a cold solution of Boc-Asp (O-cHex) (3.2 g, 10 mmol) in THF (50 mL) and N-methylmorpholine (1.21 mL, 11 mmol), isobutylchloroformate (1.43 mL, 11 mmol) was added dropwise. The reaction mixture was stirred for a few minutes, then a solution of 2-methylaniline (1.17 mL, 11 mmol) in THF (5.0 mL) was added. The reaction mixture was allowed to warm to room temperature, and stirred for 18 h. Upon completion of the reaction (TLC monitored), the reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (300 mL), and washed successively with 5% aqueous citric acid (3x) water (3x), aqueous 10% NaHCO3 (3x water (3x) and saturated salt solution (1x). The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to a brownish solid. It was recrystallized from ether-hexane to yield the titled compound (1.7 g). f) Asp(O-cHex)-(2-methyl)phenylamide (6) Boc-Asp(O-cHex)-(2-methyl)phenylamide (5) (1.2 g, 3 mmol) was treated with 50% TFA solution in methylene chloride (10 mL) for 45 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces, of TFA. The product was precipitated as its TFA salt upon addition of ether. The solid was collected and air dried. g) Nα(2-methylbenzoyl)Nα- MeArg (Tos ) -Gly-Asp(O-cHex)- (2-methyl)phenylamide (7 )
DIEA (1.2 mL, 6.6 mmol) was added to a cold solution of Asp (O-cHex)- (2-methyl) phenylamide (6) (3 mmol) in DMF (5 mL) to bring the pH to neutrality. N-Hydroxybenzotriazole
(501 mg, 3.3 mmol) was then added followed by
Nα(2-methyl)benzoyl-NαMeArg (Tos ) -Gly (1.53 g, 2.96 mmol) in DMF (5 mL). The reaction was stirred in the cold for a few min, then N-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC) (633 mg, 3.3 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The oily residue was dissolved in ethyl acetate (100 mL), and washed successively with water (3 × 40 mL), 5% aqueous citric acid (2 × 40 mL), water (2 × 40 mL) ), aqueous 10% NaHCO3 (2 × 40 mL), water (3 × 40 mL) and saturated salt solution (1 × 40 mL). The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to yield the titled compound
(1.5 g, 71%) supported by mass spectra. h - Nα (2-methyl) benzoyl-NαmethylArg-Gly-Asp- (2-methyl)phenylamide (8)
The protected linear peptide (7) (1.43 g), was treated with anhydrous HF (30 mL) in the presence of anisole (3 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the resiαue was triturated with ether to yield an oil. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude peptide (1.13 g). An aliquot was purified by gel filtration
(Sephadex® G-15, 1% acetic acid/water). The appropriate fractions were pooled and lyophilized to yield a semipurified peptide (109 mg). An aliquot of the semipurified peptide (25 mg) was purified by semiprep HPLC (5 μ, Altex Ultrasphere® ODS, 10 mm × 25 cm, isocratic, A:acetonitrile B:water-0.1% trifluoroacetic acid, 20% acetonitrile, UV detection at 220 nm) to yield purified title compound (20 mg). MS (FAB) m/e 568.1 [M+H]+;. HPLC k' 10.93 (5 μ Altex Ultrasphere®
ODS, gradient, A:acetonitrile B:water-0.1% trifluoroacetic acid, 10%-50% acetonitrile in 20 min, UV detection at 220 nm), k' 6.17 (5 μ Altex Ultrasphere® ODS, isocratic, 20%
acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 220 nm); Amino Acid Analysis: Asp (1.00), Gly (0.97).
Example 2
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-phenylamide (15) a) Boc-Asp(O-Bzl)-phenylamide (9)
To an cold solution of Boc-Asp (O-Bzl) (3.23 g, 10 mmol) in DMF (8 mL), was added aniline (733 μL, 8 mmol), HOBt (1.59 g, 1.3 mol.equiv.) and DIEA (280 μL). The reaction mixture was stirred for a few min, then EDC (1.92 g, 10 mmol) was added. The reaction mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was concentrated, and the residue dissolved in ethyl acetate, washed successively with, water, 1 N hydrochloric acid (2x), water, 5% sodium bicarbonate, water and saturated sodium chloride. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to yield the titled compound
(2.45 g, 76%). b) Asp(O-Bzl)-phenylamide (10)
Boc-Asp(O-Bzl)-phenylamide (9) (2.43 g, 6.1 mmol) was treated with 50% TFA solution in methylene chloride (20 mL) for 45 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was precipitated as its TFA salt upon addition of ether. The solid was collected and air dried to yield the title compound (3.36 g). c) Boc-NαMeArg(Tos)-Gly (11)
A solution of 1 N NaOH (35 mL, 1.2 equiv.) was added in a dropwise manner to a solution of Boc-NαMeArg(Tos)-Gly(OMe)
(1) (14.8 g, 28.8 mmol) in acetone (60 mL) . The reaction mixture was allowed to stir at room temperature for an hour for complete ester hydrolysis. The solvent was removed, and the residue was redissolved in water (50 mL), back extracted with ethyl acetate to remove any unreacted ester, and acidified (pH 2.86) with 3 N HCl. The acidic solution was extracted with ethyl acetate, and the organic layer was washed with water (2x), dried over anhydrous sodium sulfate, filtered and concentrated to yield the titled compound as white fluffy solid (10.53 g, 73%). d) Boc-NαMpΑrg(Tos)-Gly-Αsp(O-Bzl)-phenylamide (12)
DIEA (160 μL, 1.5 equiv.) was added to a cold solution of Asp(O-Bzl)-phenylamide (10) (6.1 mmol) in DMF (7.5 mL) to bring the pH to neutrality. N-Hydroxybenzotriazole (1.21 g, 7.9 mmol) was added followed by Boc-NαMeArg(Tos)-Gly (3.66 g,
7.32 mmol) in DMF (5 mL). The reaction was stirred in the cold for a few min and EDC (1.4 g, 7.32 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The oily residue was taken into ethyl acetate (125 mL), and washed successively with water (3 × 40 mL), 1 N hydrochloric acid (2 × 40 mL), water (2 × 40 mL) , aqueous 5% NaHCO3 (2 × 40 mL), water (3 × 40 mL) and saturated salt solution (1 × 40 mL). The organic solvent was dried (anhydrous Na2SO4), filtered and concentrated to yield the title compound (4.53 g, 95%). e) NαMpΑrg(Tos)-Gly-Αsp(O-Bzl)-phenylamide (13)
Boc-NαMeArg(Tos)-Gly-Asp(O-Bzl)-phenylamide (12)
(2.45 g, 3.14 mmol) was treated with 50% TFA in methylene chloride (20 mL) at room temperature for 45 min. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether to yield the TFA salt of the titled compound (2.72 g) f) Nαbenzoγl-NαMeArp(Tos)-Gly-Asp(O-Bzl)-phenylamide (14)
DIEA (293 μL, 1.5 equiv.) was added to a solution of NαMeArg(Tos)-Gly-Asp(O-Bzl)-phenylamide (13) (970 mg, 1.12 mmol) in DMF (2.0 mL) to a neutral pH (7.0). Benzoyl chloride (137 μL, 1.18 mmol) was then added. The reaction mixture was stirred for 2 h. The solvent was removed under vacuum and the residue was taken into ethyl acetate (75 mL), and washed successively with water (3 × 40 mL), 1 N
hydrochloric acid (2 × 40 mL), water (2 × 40 mL)), aqueous 5% NaHCO3 (2 × 40 mL), water (3 × 40 mL) and saturated salt solution (1 × 40 mL) . The organic solvent was dried
(anhydrous Na2SO4 ), filtered and concentrated to yield the crude peptide (0.69 g). The peptide was purified by flash chromatography (silica, 2.5% methanol/ethyl acetate) to yield the title compound (270 mg). g) Nα-benzoyl-Nα-MeArg-Gly-Asp-phenylamide (15)
The protected linear peptide (14) (184 mg), was treated with anhydrous HF (10 mL) in the presence of anisole (1 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum and the residue was triturated with ether. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude peptide (108 mg). The peptide was purified by flash chromatography (silica ODS, gradient from 10-20% acetonitrile/water-0.1% TFA) to yield the title peptide (71 mg). MS (FAB) m/e 540 [M+H]+; HPLC k' 4.44 (5μ Altex Ultrasphere® ODS, gradient, A: acetonitrile B:water-0.1% trifluoroacetic acid, 10%-50% acetonitrile in 15 min, UV detection at 220 nm); Amino Acid Analysis: Asp
(1.00), Gly (1.08).
Example 3
Preparation of Nαacetyl-NαMeArg-Gly-Asp-phenylamide (17) a) Nαacetyl-NαMeArg(Tos)-Gly-Αsp(O-Bzl)-phenylamide (16)
To a solution of NαMeArg(Tos)-Gly-Asp(O-Bzl)-phenylamide (13) (870 mg, 1 mmol) in DMF (2.0 mL), DIEA (261 μL, 1.5 equiv.) was added to yield a neutral pH (7.0). Acetic anhydride (100 μL, 1.05 mmol) was then added. The reaction mixture was stirred for 30 min. The solvent was removed under vacuum, the residue was dissolved in ethyl acetate (75 mL) and washed successively with water (3 × 40 mL), 1 N hydrochloric acid (2 × 40 mL), water (2 × 40 mL)), aqueous 5% NaHCO3 (2 × 40 mL), water (3 × 40 mL) and saturated salt solution (1 × 40 mL). The organic solvent was dried
(anhydrous Na2SO4), filtered and concentrated to yield the title compound (0.57 g, 79%) h) Nαacetyl-NαMeArg-Gly-Asp-phenylamide (17)
The protected linear peptide (16) (565 mg, 780 mmol), was treated with anhydrous HF (10 mL) in the presence of anisole (1 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether. The residue was taken into 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude peptide (300 mg). The peptide was purified by by gel filtration (Sephadex® G-15, 1% acetic acid/water). The appropriate fractions were pooled and lyophilized to yield a semipurified peptide (241 mg). The peptide was purified further by flash chromatography (silica ODS, 5% acetonitrile/water-0.1% TFA) to yield the title compound (236 mg). MS (FAB) m/e 478.2 [M+H]+; HPLC k' 2.71 (5μ Altex Ultrasphere® ODS, gradient, A:acetonitrile B:water-0.1% trifluoroacetic acid, 10%-50% acetonitrile in 15 min, UV detection at 220 nm) ; Amino Acid Analysis: Asp (1.00), Gly (1.02).
Example 4
Preparation of Nαacetyl-NαMeArg-Gly-Asp-(2-carboxy)-phenylamide (18)
The protected tetrapeptide resin Boc-NαMeArg(Tos)-Gly-Asp(O-cHex)-2-aminobenzoic acid(O-Bzl-resin) was prepared according the following procedure on a 1.0 mmol scale .
Protected amino acids were added sequentially starting from the carboxyl terminus until the desired sequence was obtained. The t-butyloxycarbonyl (Boc) group was used for protection of the alpha-amino group. Side chain functional groups were protected as follows: arginine, tosyl (Tos);
aspartic acid, cyclohexyl ester (O-cHex). Removal of the Boc group was accomplishedby treatment with 50% trifluoroacetic acid (TFA) in methylene chloride. Neutralization of the amine-TFA salt was accomplished by treatment with 7%
diisopropylethylamine (DIEA) in methylene chloride. Amino acids were coupled to the growing peptide using 3 equivalents of Boc-amino acid and 3 equivalents of 1-hydroxybenzotriazole (HOBt) in DMF and 3 equivalents of dicyclohexylcarbodiimide (DCC) in methylene chloride. Completeness of coupling was checked by ninhydrin test and couplings were repeated as necessary. If a positive ninhydrin test indicated incomplete coupling, recoupling was achieved using BOP reagent. The general protocol is given below.
1. Wash with methylene chloride 1 × 1 min
2. Wash with 50% TFA 1 × 1 min 3. Deblock with 50% TFA 1 × 20 min
4. Wash with methylene chloride 6 × 1 min
5. Neutralize with 7% DIEA 3 × 2 min
6. Wash with methylene chloride 4 × 1 min 7. Wash with dimethylformamide 2 × 1 min
8. Boc-AA + HOBt in DMF do not drain
9. DCC in methylene chloride 2 h
10. Wash with dimethylformamide 2 × 1 min 11. Wash with methylene chloride 3 × 1 min
Boc-2-aminobenzoic acid-(O-resin) was prepared from cesium Boc-2-aminobenzoate and a chloromethyl resin according to the procedure of Gisin, Helv. Chem. Acta, 56, 1476 (1973). The synthesis was begun at step 1 for the addition of Asp.
After removal of the N-terminal Boc group with 50% TFA in methylene chloride and neutralizing the resulting TFA salt with 7% DIEA in methylene chloride, the resin-bound peptide was acetylated with acetic anhydride:pyridine:methylene chloride (1:1:2, 20 mL) for 30 min to yield a protected peptide-resin intermediate (1.64 g).
The peptide was cleaved from the resin with removal of the side chain protecting groups by treatment with anhydrous liquid HF (10 mL) in the presence of anisole (1 mL) at 0°C for 50 min.. The HF was removed under vacuum, the resin was washed with ethyl ether and air-dried. The resin was then extracted with 1% acetic acid/water (2 × 30 mL) and
lyophilized to yield 200 mg of a crude peptide. An
aliquot (50 mg) was purified by semipreparative HPLC (5 μ Vydac, 2 cm × 25 cm, isocratic, 10% acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 300 nm) to yield the title compound (20.0 mg). MS (FAB) m/e 522 [M+H]+; HPLC k' 6.7 (5 μ Vydac, step gradient, A: acetonitrile B:water-0.1% trifluoroacetic acid, 1%-5% acetonitrile in 5 min, 5- 10% in 25 min, UV detection at 300 nm); Amino Acid Analysis: Asp (1.00), Gly (1.04).
Example 5
Preparation of Nα(2-methylthio)benzoylNαMeArg-Gly-Asp-(2- methylthio)phenylamide (27) a) Bon-Asp(O-cHex)-(2-methylthio)phenylamide (19) To a cold solution of Boc-Asp(O-cHex) (630 mg, 2 mmol) and N-methylmorpholine (242 mg, 1.2 mole/equiv) in THF (10 mL), was added isobutylchloroformate (311 μL, 1.2 mole/equiv) dropwise. The reaction mixture was stirred for a few
minutes, and a solution of 2-methylmercaptoaniline (295 mg, 1.2 mole/equiv) was added. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. Upon completion of the reaction (TLC monitored), the reaction mixture was filtered and the filtrate was evaporated to dryness. The resulting residue was dissolved in ethyl acetate, and washed successively with 5% aqueous citric acid (3x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and saturated sodium chloride solution (1x). The organic extract was dried (anhydrous K2CO3), filtered and concentrated to yield the title compound (860 mg). b) Asp(O-cHex)-(2-methylthio)phenylamide (20)
Boc-Asp(O-cHex)-(2-methylthio)phenylamide (19) (860 mg) was treated with 50% TFA solution in methylene chloride
(10 mL) for 60 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA, and triturated with ether to yield the TFA salt of the title compound. c) Boc-Gly-Asp(O-cHex)-(2-methylthio)phenylamide (21)
DIEA (343 μL) was added to a cold solution of Asp(O-cHex)-(2-methylthio)phenylamide (1.97 mmol) (20) in DMF (2 mL) to bring the pH to neutrality. N-Hydroxy-benzotriazole
(320 mg, 2.37 mmol) was then added followed by Boc-Gly (380 mg, 2.17 mmol). The reaction was stirred in the cold for a few min and EDC (416 mg, 2.17 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The resulting residue was dissolved in ethyl acetate, and washed successively with 10% K2CO3, water (1x),
5% citric acid (1x) water (2x) and saturated NaCl solution (1x). The organic extract was dried (anhydrous Na2SO4),
mWHJE SHEET filtered and concentrated to yield the title compound (770 mg). d) Gly-Asp(O-cHex)-(2-methylthio)Phenylamide (22)
Boc-Gly-Asp(O-cHex)-(2-methylthio)phenylamide (21) (770 mg) was treated with 50% TFA solution in methylene chloride (5 mL) for 60 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether to yield the TFA salt of the title compound (609.5 mg). e) Boc Nα-MeArg(Tos)-Gly-Asp(O-cHex)-(2-methylthio)- phenylamide (23)
DIEA (204 μL) was added to a cold solution of Gly-Asp(O- cHex)-(2-methylthio)phenylamide (22) (594 mg, 1.17 mmol) in DMF (2 mL), to bring the pH to neutrality.
N-Hydroxybenzotriazole (190 mg, 1.4 mmol) was added followed by Boc-NαMeArg(Tos) (570 mg, 1.29 mmol). The reaction was stirred in the cold for a few min, and EDC (247 mg, 1.29 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The resulting residue was dissolved in ethyl acetate, and washed successively with 10% K2CO3,(2x), water (1x), 5% citric acid (3x), water (2x)) and saturated NaCl solution. The organic extract was dried (anhydrousNa2SO4 ), filtered and
concentrated to yield the title compound (920 mg). f) NαMeArg(Tos)-Gly-Asp(O-cHex)-(2-methylthio)phenylamide
(24)
Boc-NαMeArg(Tos)-Gly-Asp(O-cHex)-(2-methylthio)-phenylamide (23) (920 mg) was treated with 50% TFA solution in methylene chloride (5 mL) for 60 min at room temperature The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA, and triturated with ether to yield the TFA salt of the title compound (635.5 mg). g) 2-methvlmercaptobenzoic acid (25)
To a cold solution of 2-mercaptobenzoic acid (5 g, 32.5 mmol) in THF (30 mL) and triethylamine (5.4 mL), methyl iodide (2.2 mL) in THF (2 mL) was added dropwise. The reaction mixture was stirred in the cold and under an inert atmosphere for 18 h. The triethylamine salt was filtered, and the filtrate was concentrated to dryness. The residue was dissolved in ethyl acetate (125 mL), and washed
successively with 5% citric acid (2x) water (3x)) and saturated NaCl solution. The organic extract was dried
(anhydrous MgSO4), filtered and concentrated to yield the title compound as solid (4.0 g). h) Nα(2-methylmercaptobenzoyl)Nα-MeArg(Tos)-Gly-Asp(O-cHex)- (2-methγlthio)phenylamide (26)
DIEA (133 μL,1.0 equiv.) was added to a cold solution of NαMeArg(Tos)-Gly-Asp(O-cHex)-(2-methylthio)phenylamide (24) (635 mg, 0.76 mmol) in DMF (2 mL), to bring the pH to neutrality. N-Hydroxybenzotriazole (371 mg) was added followed by 2-methymercaptobenzoic acid (25) (463 mg). The reaction was stirred in the cold for a few min and EDC (528 mg) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h and
concentrated to dryness. The residue was dissolved in ethyl acetate, and washed successively with 10% K2CO3 (2x), water (2x), 5% citric acid (2x) water (2x)) and saturated NaCl solution (1x). The organic solvent was dried (anhydrous Na2SO4), filtered and concentrated to yield the crude peptide (1.1 g). The peptide was purified by chromatography (silica, methylene chloride:methanol 9:1) to yield the title compound (610 mg). i) Nα(2-methylthio)benzoyl-NαMeArg-Gly-Asp-(2-methylthio)-phenylamide (27)
The protected linear peptide (26) (270 mg) was treated with anhydrous HF (5 mL) in the presence of anisole (0.5 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether to yield the crude peptide (179 mg). An aliquot (130 mg) was purified by gel filtration (Sephadex® G-15, 1% acetic acid/water). The appropriate fractions were pooled and lyophilized to yield a semipurified peptide (107 mg). The peptide was purified further by flash chromatography (silica ODS, gradient elution, 10-50% acetonitrile/water-0.1% TFA) to yield a more pure peptide (75.4 mg). Final purification by semipreparative HPLC (5 μAltex Ultrasphere® ODS, 10 mm x 25 cm, isocratic, 22% acetonitrile/water-0.1% trifluoroacetic acid, UV
detection at 220 nm) to yield the title compound. MS (FAB) m/e 632 [M+H]+; HPLC k' 7.5 (5μ Altex Ultrasphere® ODS, gradient. A:acetonitrile B:water-0.1% trifluoroacetic acid, 10%-50% acetonitrile in 40 min, UV detection at 220 nm), k' 5.9 (5μ Altex Ultrasphere® ODS, 23% acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 220 nm) ; .Amino Acid Analysis: Asp (1.00), Gly (1.01).
Example 6
Preparation of N-[ [5-(aminoiminomethyl)amino]-1-oxopentyl]- Gly-Asp-phenylamide (28) a) ethyl 5-[N-(t-butoxvcarbonyl)-N-(methoxycarbonyl)amino]-pentanoate
To a solution of ethyl 5-iodopentanoate (1.66 g, 6.5 mmol) in DMF (25 mL) under an argon atmosphere was added potassium imidodicarbonic acid t-butyl methyl ester (1.38 g, 6.5 mmol). The resulting mixture was stirred at room
temperature under an argon atmosphere for 4 h. The mixture was poured into H2O and extracted with Et2O (3 × 100 mL).
The combined organic extracts were washed with H2O (3 × 50 mL) and dried (Na2SO4). The solvent was removed under reduced pressure, and the residue was purified by flash chromatography, eluting with 25% EtOAc/hexanes to provide the title compound as a colorless oil (2.0 g, 100%). b) 5-[ [ (1,1-dimethylethoxy)carbonyl] amino]pentanoic acid To a solution of ethyl 5-[N-(t-butoxycarbonyl)-N- (methoxycarbonyl)amino]pentanoate (2.0 g, 6.6 mmol) in MeOH (30 mL) was added 0.95 N NaOH (30 mL, 28.5 mmol), and the resulting mixture was stirred at room temperature. After stirring for 5 h, the resulting clear solution was
concentrated under reduced pressure. The concentrate was acidified with glacial acetic acid, and the mixture was extracted with CH2CI2 (3 × 100 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed in vacuo to provide a white solid (1.0 g, 71%): m.p. 45-47°C. c) Boc-Asp(O-Bzl)-phenylamide
A solution of aniline (2.84 mL, 30.9 mmol) and
diisopropylethylamine (4.0 g, 30.9 mmol) in dry DMF (32 mL) was cooled to 0°C under an argon atmosphere. To this solution was added HOBt hydrate (4.3 g, 32.0 mmol), followed by Boc-Asp (O-Bzl) (10.0 g, 30.9 mmol). The resulting mixture was allowed to stir at 0°C until dissolution occurred, and EDC hydrochloride (5.9 g, 30.9 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred for 2 h. The mixture was
partitioned between H2O (500 mL) and EtOAc (200 mL). The aqueous phase was extracted with EtOAc (200 mL), and the combined organic extracts were washed successively with H2O (3 × 75 mL) and saturated aqueous NaCl (75 mL) and dried
(Na2SO4). The solvent was removed in vacuo, and the residue was triturated with 20% EtOAc/hexanes. The solid which formed was collected by filtration to provide the title compound (7.7 g). The filtrate was concentrated under reduced pressure to afford additional material (2.3 g, 81% total yield). d) Asp(Bzl)-phenylamide trifluoroacetate
A solution of Boc-Asp(Bzl)-phenylamide (5.75 g, 14.4 mmol) in 1:2 trifluoroacetic acid/CH2Cl2 (30 mL) under an argon atmosphere was allowed to stir at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure, and the residue was treated with Et2O (3 × 30 mL) and evaporated to provide a white powder (6.0 g, 100%). e) Boc-Gly-Asp(Bzl)-phenylamide
To a solution of Asp(Bzl)-phenylamide trifluoroacetate (5.9 g, 14.3 mmol) in dry DMF at 0°C under an argon
atmosphere was added diisopropylethylamine (1.85 g, 14.3 mmol), followed by HOBt hydrate (2.16 g, 16.0 mmol). To the mixture was added Boc-glycine (2.5 g, 14 .3 mmol) , and the resulting mixture was stirred at 0°C until dissolution occurred. EDC hydrochloride (2.74 g, 14.3 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred for 2 h. The mixture was partitioned between H2O (200 mL) and EtOAc (100 mL). The aqueous phase was extracted with EtOAc (100 mL), and the combined organic extracts were washed successively with H2O
(3 × 50 mL) and saturated aqueous NaCl (40 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 60%
EtOAc/hexanes to provide a white solid (2.4 g, 38%). f) Gly-Asp(Bzl)-phenylamide trifluoroacetate
A solution of Boc-Gly-Asp(Bzl)-phenylamide (2.4 g, 5.3 mmol) in 1:2 trifluoroacetic acid/CH2Cl2 (15 mL) under an argon atmosphere was allowed to stir at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was treated with Et2O (3 × 30 mL) and evaporated to provide a white powder (2.5 g, 100%). g) N-[5-[[(1,1-dimethylethoxy)carbonyl]amino]-1-oxopentyl]-Gly-Asp(O-Bzl)-phenylamide
To a solution of Gly-Asp(O-Bzl)-phenylamide
trifluoroacetate (2.0 g, 4.2 mmol) in dry DMF at 0°C under an argon atmosphere was added diisopropylethylamine (0.55 g, 4.3 mmol), followed by HOBt hydrate (0.59 g, 4.4 mmol). To the resulting mixture was added 5-[[(1,1-dimethylethoxy)-carbonyl]amino]pentanoic acid (0.92 g, 4.2 mmol) and stirring was continued for 10 min at which time dissolution occurred. EDC hydrochloride (0.81 g, 4.2 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred for 2 h. The mixture was allowed to stand at room temperature overnight, then partitioned between H2O (100 mL) and EtOAc (75 mL). The aqueous phase was extracted with EtOAc (75 mL), and the combined organic extracts were washed successively with H2O (3 × 30 mL) and saturated aqueous NaCl (30 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 7% MeOH/CH2Cl2 to provide the title compound (0.8 g, 34%), which after trituration with EtOAc formed a white solid. h) N-[5-[[(1,1-dimethylethoxy)carbonyl]amino]-1-oxopentyl]-Gly-Asp-phenylamide
To a solution of N-[5-[[(1,1-dimethylethoxy)carbonyl]-amino]-1-oxopentyl]-Gly-Asp(O-Bzl)-phenylamide (0.72 g, 1.3 mmol) in absolute EtOH (75 mL) was added 10% palladium on activated carbon (150 mg) . The resulting mixture was hydrogenated at 30 psi H2 for 3 h. The mixture was degassed and filtered through a pad of Celite. The filtrate was concentrated in vacuo to provide a white solid (0.58 g, 97%) . i) N-[5-Amino-1-oxopentyl]-Gly-Asp-phenvlamide
A solution of N-[5-[[(1,1-dimethylethoxy)carbonyl]-amino]-1-oxopentyl]-Gly-Asp-phenylamide (0.58 g, 1.3 mmol) in 1 : 4 trifluoroacetic acid/CH2Cl2 (25 mL) under an argon atmosphere was allowed to stir at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure to provide the title compound which was used without further purification. j) N-[[5-(aminoiminomethyl)amino]-1-oxopentyl]-Gly-Asp-pheηylamide
N-[5-Amino-1-oxopentyl]-Gly-Asp-phenylamide (0.5 g, 1.2 mmol) was placed in distilled H2O, and the pH was adjusted to 10 with 0.95 N NaOH. O-Methylisourea hydrogen sulfate (0.24 g, 1.4 mmol) was added, and the pH was again adjusted to 10 with 0.95 N NaOH. The resulting mixture was allowed to stir at room temperature overnight. Analysis of the reaction by thin layer chromatography indicated that the reaction was incomplete, so additional O-methylisourea hydrogen sulfate (2.4 g, 12.0 mmol) was added. The pH was adjusted to >10 with 10% NaOH, and the mixture was allowed to stir under an argon atmosphere at room temperature overnight. The. pH was adjusted to pH 6 with 3 N HCl, and the mixture was stored at -25°C under an argon atmosphere for several days . Upon thawing, a white solid formed which was collected by
filtration. The filtrate was lyophilized, and the combined solids were triturated with MeOH and filtered. The filtrate was concentrated in vacuo, and the residue was purified by reverse phase HPLC, eluting with 25% MeOH/H2O containing 0.1% TFA to provide the title compound (0.21 g, 40%).
Example 7
Preparation of N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1- oxopentyl]-Gly-Asp-phenylamide (29)
Figure imgf000042_0001
a) (4R)-4-benzyl-3-phenylacetyl-2-oxazolidinone
To a solution of (4R)-4-benzyl-2-oxazolidinone (20.0 g, 0.132 mol) in THF at -78°C under an argon atmosphere was added slowly, n-butyl lithium (52.8 mL of 2.5 M solution in THF, 0.132 mol). The resulting solution was stirred at -78°C for 10 min, at which time a pre-cooled (-78°C) solution of phenylacetyl chloride (17.5 mL, 0.132 mol) in THF (50 mL) was slowly added. The resulting solution was allowed to warm. to room temperature and stirred overnight. The solvent was removed in vacuo, and the residue was partitioned between Et2O (1.0 L) and H2O (150 mL). The organic extract was washed successively with H2O (100 mL) and saturated aqueous NaCl (75 mL) and dried (Na2SO4). The solvent was removed under reduced pressure, and the solid residue was suspended in 10% EtOAc/hexanes (500 mL). The insoluble material was removed by decantation. Upon cooling, a white crystalline solid formed from the solution and was collected by
filtration (15.2 g, 39%). The filtrate was concentrated under reduced pressure. The residue was combined with the above insoluble material and purified by flash
chromatography, eluting with 30% EtOAc/hexanes to provide additional material (total yield: 23.0 g, 59%). m.p. 69-71°C. b) (2R,4R)-4-Benzyl-3-(1-oxo-2-phenyl-pent-4-enyl)-2-oxazolidinone
To a solution of 4-(R)-benzyl-3-phenylacetyl-2-oxazolidinone (23.7 g, 80.3 mmol) in freshly distilled THF (600 mL) at -78°C under an argon atmosphere was added dropwise a solution of potassium bis (trimethylsilyl) amide (161 mL of 0.5 M solution in toluene, 80.3 mmol). The resulting solution was stirred at -78°C for 20 min, and a pre-cooled (-78°C) solution of allyl bromide (10.5 mL, 121 mmol) in THF (40 mL) was added dropwise. The resulting mixture was stirred at -78°C for 1 h, then allowed to warm to room temperature and stirred overnight. Glacial acetic acid (14.0 mL) was added to the reaction mixture, which was then filtered. The filtrate was concentrated under reduced pressure, and the residue was partitioned between Et2θ (1.0 L) and H2O (200 mL). The organic extract was washed
successively with H2O (2 × 150 mL), 5% aqueous NaHCO3 (150 mL) and saturated aqueous NaCl (150 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 30%
EtOAc/hexanes. Those fractions containing the pure (2R, 4R) diastereomer were combined. The remaining material consisted of a mixture of the (2R, 4R) and (2S, 4R) diastereomers.
Trituration of this mixture with cyclohexane provided additional pure (2R, 4R) diastereomer (total yield: 11.7g, 43%). m.p. 73-75°C. c) methyl (2R)-2-phenyl-4-pentenoate
To a solution of methoxy magnesium bromide [prepared by the addition of methyl magnesium bromide (16.1 mL of 3.0 M solution in Et2θ, 48.4 mmol) to methanol (200 mL) at -20°C] at -20°C under an argon atmosphere was slowly added a solution of (2R, 4R)-4-benzyl-3-(l-oxo-2-phenyl-pent-4-enyl)- 2-oxazolidinone (8.1 g, 24.2 mmol) in methanol (200 mL) . The resulting solution was stirred at -20°C for 1 h, then allowed to warm to 0°C and stirred for 1.5 h. The reaction was quenched by the addition of a pH 7 phosphate buffer (200 mL) and stirred at 0°C for 10 min. The mixture was partitioned between CH2CI2 (700 mL) and aqueous NH4Cl/NaCl (1:1 w/w, 700 mL). The aqueous phase was extracted with CH2CI2 (3 × 200 mL), and the combined organic extracts were dried (Na2SO4).
The solvent was removed in vacuo, and the residue was treated with hexanes (2 × 40 mL) and stirred. The solid which formed was removed by decantation, and the solvent was removed under reduced pressure to provide the title compound (3.7 g, 80%). d) methyl (2R)-5-iodo-2-phenylpentanoate
To a solution of methyl (2R)-2-phenyl-4-pentenoate (3.7 g, 19.5 mmol) in anhydrous THF (50 mL) at 0°C under an argon atmosphere was added BH3·THF (6.5 mL of 1.0 M solution in THF, 6.5 mmol). The resulting solution was heated at 50°C for 1 h and then allowed to cool to room temperature.
Anhydrous methanol (1.5 mL) was added dropwise to the
reaction mixture followed in rapid succession by a solution of anhydrous sodium acetate (1.6 g, 19.5 mmol) in anhydrous methanol (18 mL) and iodine monochloride (13.0 mL of 1.0 M solution in CH2CI2, 13.0 mmol). The resulting mixture was stirred at room temperature for 45 min, then poured into H2O (800 mL) containing sodium thiosulfate. The mixture was extracted with Et2O (3 × 275 mL), and the combined organic extracts were dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 10% EtOAc/hexanes to provide the title compound as a colorless oil (2.4 g, 39%) . e) (2R)-5-[N-(t-butoxycarbonyl)-N-(carbomethoxy)amino]-2-phenylpentanoic acid methyl ester
To a solution of methyl (2R)-5-iodo-2-phenyl-pentanoate (1.9 g, 5.9 mmol) in DMF (25 mL) under an argon atmosphere was added potassium t-butoxymethyliminodicarboxylate (1.3 g, 6.0 mmol). The resulting mixture was stirred at room
temperature for 30 min. The mixture was poured into H2O (200 mL) containing sodium thiosulfate and extracted with Et2O (3 × 100 mL). The combined organic extracts were washed with H2O (3 × 20 mL) and dried (Na2SO4). Removal of the solvent under reduced pressure provided an oil. The aqueous washes from above were further extracted with CH2CI2 (3 × 100 mL), and the combined CH2CI2 extracts were washed with H2O (3 × 25 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residues were combined and purified by flash
chromatography, eluting with 25% EtOAc/hexanes to provide the title compound as a colorless oil (1.3 g, 60%). f) (2R)-5-[(t-butoxycarbonyl)amino]-2-phenyl-pentanoic acid To a solution of (2R)-5-[(N-t-butoxycarbonyl)-(N-carbomethoxy)amino]-2-phenylpentanoic acid, methyl ester (1.3 g, 3.6 mmol) in methanol (25 mL) was added 0.95 N NaOH (15.0 mL, 14.2 mmol), and the resulting mixture was stirred at 0°C. After stirring at 0°C for 2 h, the mixture was allowed to warm to room temperature and stirred an additional 3 h. The mixture was poured into H2O (150 mL), acidified with acetic acid and extracted with CH2CI2 (3 × 100 mL). The combined organic extracts were dried (Na2SO4). The solvent was removed in vacuo, and NMR analysis indicated the presence of starting material. The residue was dissolved in methanol (15 mL), and 0.95 N NaOH (15 mL, 14.2 mmol) was added. The mixture was allowed to stir at room temperature for 3 h. The resulting solution was poured into H2O (200 mL), acidified with acetic acid and extracted with CH2CI2 (3 × 125 mL). The combined organic extracts were dried (Na2SO4), and the solvent was removed in vacuo to provide the title compound (1.0 g, 96%). [a]D 25 (1, CH3OH) = -35.9°. g) N-[(2R)-2-phenyl-5-[(1,1-dimethylethoxycarbonyl)aminol-1- oxopentyl]-glycyl-N1-phenyl-L-aspartamide benzyl ester
To a solution of Gly-Asp(O-Bzl)-phenylamide (1.44 g, 3.1 mmol) in dry DMF (5 mL) at 0°C under an argon atmosphere was added diisopropylethylamine (0.40 g, 3.1 mmol), followed by HOBt hydrate (0.43 g, 3.2 mmol). To the mixture was added a solution of (2R)-5-[(t-butoxycarbonyl)amino]-2-phenylpentanoic acid (0.90 g, 3.1 mmol) in DMF (3 mL) , and the resulting mixture was stirred at 0°C for 10 min. EDC
hydrochloride (0.59 g, 3.1 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room
temperature, and kept under an argon atmosphere overnight. The mixture was partitioned between H2O (1500 mL) and EtOAc (50 mL). The aqueous phase was extracted with EtOAc (50 mL), and the combined organic extracts were washed successively with H2O (3 × 20 mL) and saturated aqueous NaCl (25 mL) and dried (Na2SO4) . The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 5% MeOH/CH2Cl2 to provide a white solid (1.1 g, 57%). h) N-[(2R)-2-phenyl-5-[(1,1-dimethylethoxycarbonyl)aminol-1-oxopentyll-Gly-Asp-phenylamide
To a solution of N-[(2S)-2-phenyl-5-[(1,1-dimethylethoxycarbonyl)amino]-1-oxopentyl]-Gly-Asp-phenylamide (0.5 g, 0.79 mmol) in absolute EtOH (100 mL) was added 10% palladium on activated carbon (250 mg), and the resulting mixture was hydrogenated at 30 psi H2 for 45 min.
The mixture was degassed and filtered through a pad of
Celite. The filtrate was concentrated under reduced pressure to provide the title compound (0.43 g, 100%). i) N-[(2R)-2-phenyl-5-amino-1-oxopentyl]-Gly-Asp-Phenylamide A solution of N-[(2R)-2-phenyl-5-[(1,1-dimethylethoxycarbonyl)amino]-1-oxopentyl]-Gly-Asp-phenylamide (0.43 g, 0.8 mmol) in trifluoroacetic acid (1.0 mL) and CH2CI2 (5 mL) under an argon atmosphere was allowed to stir at room temperature for 1.5 h. The reaction mixture was concentrated under reduced pressure, and the residue was treated repeatedly with Et2θ and evaporated to provide a white powder (0.43 g, 98%). j) N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp-phenylamide
N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp-phenylamide (0.44 g, 0.8 mmol) was placed in
distilled H2O (5 mL), and the pH was adjusted to >12 with 1.0 N NaOH. O-Methylisourea hydrogen sulfate (1.4 g, 7.9 mmol) was added, and the pH was again adjusted to >12 with 1.0 N NaOH. The resulting mixture was allowed to stir at room temperature overnight. The pH was adjusted to pH 6 with 3 N HCl, and the mixture was lyophilized. The residue was triturated with MeOH (5 mL) and filtered. The filtrate was concentrated under reduced pressure, and the residue was purified by preparative reverse-phase HPLC, eluting with 0.1:25:75 TFA/CH3CN/H2O . The isolated material was
lyophilized to provide the title compound (1.6 g, 41%) .
Amino acid analysis: Asp (1.00), Gly (1.05).
Example 8
Preparation of N- [ (2S) -2- ( 1 , 3-dihydro-2H-isoindolyl) -5-(aminoiminomethyl) amino-1-oxopentyl ]-Gly-Asp-phenylamide (30)
= H ji iΛ HO
HPN"^N s'^**V// NCO2H
H a) Boc-Asp(O-cHex)-phenylamide
A solution of aniline (1.45 mL, 15.9 mmol) and
diisopropylethylamine (2.05 g, 15.9 mmol) in dry DMF (16 mL) was cooled to 0°C under an argon atmosphere. To this solution was added HOBt hydrate (2.23 g, 16.5 mmol), followed by Boc-Asp (O-cHex) (5.0 g, 15.9 mmol). The resulting mixture was allowed to stir at 0°C until dissolution occurred, and EDC hydrochloride (3.05 g, 15.9 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred for 2 h. The mixture was
partitioned between H2O (250 mL) and EtOAc (150 mL). The aqueous phase was extracted with EtOAc (150 mL), and the combined organic extracts were washed successively with H2O (3 × 50 mL) and saturated aqueous NaCl (40 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was triturated with 20% EtOAc/hexanes to provide a white solid (3.85 g, 62%). b) Asp(O-cHex)-phenylamide trifluoroacetate
A solution of Boc-Asp(O-cHex)-phenylamide (3.8 g, 9.7 mmol) in 1:1 trifluoroacetic acid/CH2Cl2 (20 mL) under an argon atmosphere was allowed to stir at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure, and the residue was treated with Et2θ (3 x 30 mL) and evaporated to provide a white powder (4.0 g, 100%) . c) N-[N2-(1,1-dimethylethoxycarbonyl)-N5-Timing[[4- (methylphenyl)sulfonyl]aminolmethyl]-L-ornithyl]-glycine benzyl ester
To a solution of Gly-OBzl hydrochloride (7.0 g, 35 mmol) in dry DMF (40 mL) at 0°C under an argon atmosphere was added diisopropylethylamine (4.5 g, 35 mmol). To this solution was added HOBt hydrate (5.2 g, 38.5 mmol), followed by N2-(1,1-dimethylethoxycarbonyl)-N5-[imino[[4-(methylphenyl)sulfonyl]-amino]methyl]-L-ornithine (15.0 g, 35 mmol). The resulting mixture was allowed to stir at 0°C until dissolution
occurred, and EDC hydrochloride (6.7 g, 35 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred overnight. The mixture was partitioned between H2O (350 mL) and EtOAc (75 mL). The aqueous phase was extracted with EtOAc (2 × 75 mL), and the combined organic extracts were washed with H2O (3 × 40 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 7% MeOH/CH2Cl2 to provide the title compound (9.0 g, 49%) d) N-[N5-[imino[[4-(methylphenyl)sulfonyl]amino]methyl]-L-ornithyl]-glycyl benzyl ester
A solution of the compound of Example 8(c) (15.0 g, 26 mmol) in trifluoroacetic acid (20 mL) and CH2CI2 (30 mL) under an argon atmosphere was allowed to stir at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure, and the residue was treated with Et2θ
(3 × 30 mL) and evaporated to provide a white powder (15.6 g, 100%) . e) N-[(2S)-2-(1,3-dihydro-2H-isoindolyl)-5-[imino[[4-(methylphenyl)sulfonyl]amino]methyl]amino]-2-oxopentyl]-glycine benzyl ester
To a solution of diisopropylethylamine (0.98 g, 7.6 mmol) in CH2CI2 (35 mL) under an argon atmosphere was added the compound of Example 8(d) (1.5 g, 2.5 mmol). To the resulting mixture was added α,α'-dichloro-o-xylene (0.44 g,
2.5 mmol), and the reaction mixture was allowed to stir under an argon atmosphere at room temperature overnight. The mixture was extracted with H2O (2 × 20 mL) , and the organic extract was concentrated under reduced pressure. The residue was purified by flash chromatography, eluting with 5%
MeOH/CH2Cl2 to provide the title compound as a white -solid (0.65 g, 46%). f) N-[(2S)-2-(1,3-dihydro-2H-isoindplyl)-5-[imino[[4- (methylphenyl)sulfonyl]amino]methyl]amino]-2-oxopentyl]-glycine
To a solution of the compound of Example 8(e) (0.55 g, 0.95 mmol) in absolute ethanol (150 mL) was added 10% palladium on activated carbon (200 mg). The resulting mixture was hydrogenated at 30 psi H2 for 4.5 h. The mixture was degassed and filtered through a pad of Celite. The filtrate was concentrated in vacuo to provide the title compound (0.45 g, 98%). g) N-[(2S)-2-(1,3-dihydro-2H-isoindolyl)-5- (aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp(O-cHex)- phenylamide
To a solution of Asp(O-cHex)-phenylamide
trifluoroacetate (275 mg, 0.68 mmol) in dry DMF (5 mL) at 0°C under an argon atmosphere was added diisopropylethylamine (88 mg, 0.68 mmol). To this solution was added HOBt hydrate (110 mg, 0.8 mmol), followed by the compound of Example 8(f) (330 mg, 0.68 mmol). The resulting mixture was allowed to stir at 0°C until dissolution occurred, and EDC hydrochloride (130 mg, 0.68 mmol) was added portionwise. The resulting mixture was allowed to slowly warm to room temperature, and stirred overnight. The mixture was partitioned between H2O (100 mL) and EtOAc (40 mL). The aqueous phase was extracted with EtOAc (2 × 40 mL) , and the combined organic extracts were washed with H2O (3 × 20 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 5% MeOH/CH2Cl2 to provide the title compound as a white solid (260 mg, 50%). h) N-[(2S)-2-(1,3-dihydro-2H-isoindolyl)-5- (aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp-phenylamide
A solution of the compound of Example 8(g) (150 mg, 0.2 mmol) in anisole (1.5 mL) was transferred to a hydrogen fluoride reaction vessel. To this was introduced at -78°C hydrogen fluoride, and the resulting solution was allowed to warm to 0°C. After stirring for 1 h at 0°C, the hydrogen fluoride was removed in vacuo. Ether (100 mL) was added to the residue, which was then stirred vigorously for 1 h. The solid which formed was collected rapidly by filtration and purified by reverse-phase HPLC, eluting with 40% MeOH/H2O. After lyophilization, the title compound was isolated as a hygroscopic powder (30 mg, 30%). Example 9
Preparation of Nα-(4,4-diphenylbutyryl)-Nα-methyl-Arg-Gly-Asp-phenylamide (31)
Figure imgf000051_0001
a) Boc-NαMeArg (Tos) -Gly-OBzl
A solution of glycine benzyl ester hydrochloride (0.91 g, 4.5 mmol) and diisopropylethylamine (0.58 g, 4.5 mmol) in dry DMF (7 mL) was cooled to 0°C under an argon atmosphere. To this solution was added HOBt hydrate (0.66 g, 4.9 mmol), followed by Boc-MeArg(Tos) (2.00 g, 4.5 mmol). The resulting mixture was allowed to stir at 0°C until dissolution
occurred, and EDC hydrochloride (0.86 g, 4.5 mmol) was added portionwise. The resulting solution was allowed to slowly warm to room temperature and kept under an argon atmosphere overnight. The mixture was partitioned between H2O (100 mL) and EtOAc (75 mL). The aqueous phase was extracted with EtOAc (75 mL), and the combined organic extracts were washed successively with H2O (3 × 20 mL) and saturated aqueous NaCl (30 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography to provide a white solid (2.40 g, 89%). b) NαMeArg(Tos)-Gly-OBzl
A solution of Boc-MeArg(Tos)-Gly-OBzl (2.40 g, 4.0 mmol) in trifluoroacetic acid (10 mL) and CH2CI2 (30 mL) under an argon atmosphere was allowed to stir at room temperature for 90 min. The reaction mixtur.e was concentrated under reduced pressure, and the residue was treated with 50% Et2O/hexanes (3 × 30 mL) and evaporated to provide a white solid (2.5 g, 100%). c) N-[N2-(4,4-diphenylbutyryl-N5-[imino[[4-(methylphenyl)- sulfonyl]amino]methyl]-N2-methyl-L-ornithyl]-glycyl benzyl ester
A solution of MeArg (Tos) -Gly-OBzl (2.5 g, 4.1 mmol) and diisopropylethylamine (0.55 g, 4.1 mmol) in dry DMF (6 mL) was cooled to 0°C under an argon atmosphere. To this solution was added HOBt hydrate (0.62 g, 4.6 mmol), followed by 4,4-diphenylbutanoic acid (1.2 g, 4.1 mmol). The
resulting mixture was allowed to stir at 0°C until
dissolution occurred, and EDC hydrochloride (0.80 g, 4.1 mmol) was added portionwise. The resulting solution was allowed to slowly warm to room temperature and kept under an argon atmosphere overnight. The mixture was partitioned between H2O (300 mL) and EtOAc (70 mL). The aqueous phase was extracted with EtOAc (2 × 70 mL), and the combined organic extracts were washed with H2O (3 × 40 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 5%
MeOH/CH2Cl2 to provide the title compound as a white solid (1.4 g, 48%). d) Nα-(4,4-diphenylbutyryl)-NαMeArg(Tos)-Gly
To a solution of Nα-(4,4-diphenylbutyryl)-NαMeArg(Tos)-Gly-OBzl (1.36 g, 1.9 mmol) in absolute EtOH (100 mL) was added 10% palladium on activated carbon (350 mg). The resulting mixture was hydrogenated at 30 psi H2 for 3 h. The mixture was degassed and filtered through a pad of Celite. The filtrate was concentrated in vacuo to provide a white solid (1.00 g, 83%). e) N α- (4 , 4-diphenylbutyryl ) -NαMeArg (Tos) -Gly-Αsp (O-cHex) -phenylamide
A solution of Asp(O-cHex)-phenylamide trifuloroacetate (0.65 g, 1.6 mmol) and diisopropylethylamine (0.21 g, 1.6 mmol) in dry DMF (4 mL) was cooled to 0°C under an argon atmosphere. To this solution was added HOBt hydrate (0.22 g, 1.7 mmol), followed by a solution of the compound of Example 9(d) (1.0 g, 1.6 mmol) in dry DMF (3 mL). The resulting mixture was stirred for 5 min at 0°C, and EDC hydrochloride (0.31 g, 1.6 mmol) was added portionwise. The resulting solution was allowed to slowly warm to room temperature and kept under an argon atmosphere overnight. The mixture was partitioned between H2O (150 mL) and EtOAc (50 mL). The aqueous phase was extracted with EtOAc (50 mL), and the combined organic extracts were washed successively with H2O
(3 × 30 mL) and saturated aqueous NaCl (30 mL) and dried (Na2SO4). The solvent was removed in vacuo, and the residue was purified by flash chromatography, eluting with 5%
MeOH/CH2Cl2 to provide the title compound as a white solid (0.75 g, 53%). f) Nα-(4,4-diphenylbutyryl)-NαMeArg-Gly-Asp-phenylamide
A solution of Example 9(e) (200 mg, 0.22 mmol) in anisole (1.5 mL) was transferred to a hydrogen fluoride reaction vessel. To this was introduced at -78°C liquid hydrogen fluoride (15 mL), and the resulting solution was allowed to warm to 0°C. After stirring for 1 h at 0°C, the hydrogen fluoride was removed in vacuo. Ether (75 mL) was added to the residue, which was then stirred. The solid which formed was collected by filtration and purified by reverse-phase HPLC, eluting with 60% MeOH/H2O containing 0.1% TFA. The appropriate fractions were combined and
concentrated under reduced pressure. The concentrate was lyophilized overnight to provide a white powder (15 mg, 10%) .
The crude product was purified by semiprep HPLC to yield a purified compound that was lyophilized to provide the title compound (15 mg): TLC Rf 0.65 (n-BuOH: HOAc:H2O 1:1:1).
Example 10
Preparation of N-[ (2S) -2- ( 1- (2-Pyrrolidinonyl) ) -5- (aminoiminomethyl ) amino-1-oxopentyl]-Gly-Asp-phenylamide ( 32 )
Figure imgf000054_0001
a) Nα-(4-bromo-1-oxobutyl)-Arg(Tos)
To a solution of potassium carbonate (560 mg, 4.0 mmol) in H2O (10 mL) at 0°C was added Arg(Tos). After stirring for several min at 0°C, 4-bromobutanoyl chloride (185 mg, 1.0 mmol) was added, and the resulting mixture was stirred for 30 min and extracted with Et2O. The aqueous phase was acidified with cold, dilute aqueous HCl and extracted with EtOAc.
Removal of the solvent in vacuo provided the title compound (0.4 g, 87%). b) N-[(2S)-2-(1-(2-pyrrolidinonyl))-5-((toluenesufonyl)amino¬iminomethyl)amino-1-oxopentanoic acid
To a solution of Nα-(4-bromo-1-oxobutyl)-Arg(Tos)
(2.4 g, 5.0 mmol) in DMF (30 mL) at 0°C under an argon atmosphere was added sodium hydride (1.1 g of 60% suspension in mineral oil, 27.5 mmol), and the resulting mixture was allowed to warm to room temperature and stirred overnight. The mixture was poured into ice H2O, acidified at 0°C with dilute aqueous HCl and extracted with EtOAc. The aqueous phase was placed on a column of Amberlite® XAD-2 (130 g) and gravity filtered to provide the title compound (1.4 g). The organic extract was concentrated in vacuo to provide an additional 0.5 g of material (73% total yield).
Anal. Calcd. for C17H24N5O5S·0.85 H20: C 49.59, H 6.29, N 13.60; Found: C 49.97, H 6.22, N 13.24. c) N-[(2S)-2-(1-(2-pyrrolidinonyl))-5-(aminoiminomethyl)-amino-1-oxopentyl]-Gly-Asp(O-cHex)-phenylamide
Boc-Gly-Asp(O-cHex)-phenylamide (496 mg, 1.11 mmol) was dissolved in 5 mL 4N HCl/dioxane and stirred at room
temperature for 20 minutes. The residue was dissolved in methylene chloride and evaporated three times to insure the removal of HCl. The residue was dissolved in 5 mL DMF and diisopropylethylamine (177 mg, 1.37 mmol) was added. The pH was adjusted with base to neutrality. HOBt (193 mg, 1.43 mmol), N-[(2S)-2-(1-(2-pyrrolidinonyl))-5- ((toluenesufonyl)amino-iminomethyl)amino-1-oxopentanoic acid (550mg, 1.39 mmol) and 1-(dimethylamino-propyl)-3-ethylcarbodiimide hydrochloride (266 mg, 1.39 mmol) were added successively. The reaction was allowed to stir at room temperature for 18 h. The DMF was removed in vacuo and the residue dissolved in ethyl acetate/chloroform (approximately 1:1), washed with 1 N NaHSO4 (3X), 5% NaHCO3 (3X), dried (MgSO4), filtered and concentrated in vacuo . 609 mg (76% yield). The crude product was purified by flash
chromatography (silica gel, 10% methanol/chloroform). The appropriate fractions were pooled and the solvent was evaporated (463 mg, 58% yield). NMR δ (CDCl3) 1.1-2.1 (m, 14
H), 2.3 (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, 1H); TLC Rf 0.35 (15%
methanol/chloroform); MS (FAB) m/e 726 [M+H]+. d) N-[(2S)-2-(1-(2-pyrrolidinonyl))-5- (aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp-phenylamide
The protected peptide of Example 10(c) (215 mg, 291 nmol) was treated with 5 mL anhydrous HF at 0°C for 1 h. The HF was removed in vacuo, the residue was dissolved in glacial acetic acid and lyophilized to yield the crude peptide salt (159 mg, 99% ). 50 mg was filtered and purified by
preparative HPLC (C18 reverse phase, 13% acetonitrile-0.1%
TFA/water-0.1% TFA). The solvent was removed and the peptide was dissolved in 10% acetic acid and lyophilized (23 mg). HPLC k' 2.58 (C18 reverse phase, gradient, A:acetonitrile- 0.1% trifluoroacetic acid B:water-0.1% trifluoroacetic acid, 10%-50% over 15 min, UV detection at 220 nm), k' 2.34 (C18 reverse phase, 17% acetonitrile/water-O.1% trifluoroacetic acid, UV detection at 220 nm) ; TLC Rf 0.60 (butanol: acetic acid:water 1:1:1), Rf 0.54 (butanol: ethyl acetate: acetic acid:water 1:1:1:1); MS (FAB) m/e 490.2. [M+ H]+; Amino Acid Analysis: Asp (1.0), Gly (0.85), Arg (0.21); Peptide content 69.87% (peptide was hydrolyzed with 2:1:0.005% v:v:w
HCl/TFA/phenol at 160°C for 1 h).
Example 11 Preparation of Nα-(phthaloyl)-Arg-Gly-Asp-phenylamide (33)
Figure imgf000056_0001
a) Nα-(phthaloyl)Arg(Tos)
To a solution of Arg (Tos) (3.28 g, 10.0 mmol) in toluene (40 mL) were added phthalic anhydride (1.48 g, 10.0 mmol) and triethylamine (0.3 mL, 30.0 mmol). The resulting mixture was heated at reflux using a Dean-Stark trap. After refluxing for 2 h, the reaction mixture was allowed to cool and was concentrated under reduced pressure. Methylene chloride was added to the residue which dissolved upon warming and
stirring for 30 min. The solution was washed with H2O and the solvent was removed in vacuo . The white, flakey solid was triturated with Et2O and collected by filtration to provide the title compound (4.13 g, 90%).
Anal. Calc. for C21H22N4O6S: C 55.01, H 4.84, N 12.22; Found: C 55.55, H 5.19, N 11.77. b) Nα-(phthaloyl)-Arg(Tos)-Gly-Asp(O-cHex)-phenylamide
The Boc-Gly-Asp(O-cyclohexyl)-phenylamide (496 mg, 1.11 mmol) was dissolved in 5 mL 4N HCl/dioxane and stirred at room temperature for 20 minutes. The residue was dissolved in methylene chloride, and evaporated three times to insure the removal of HCl. The residue was dissolved in 5 mL DMF and diisopropylethylamine (177 mg, 1.37 mmol) was added. The pH was adjusted by addition of base to neutrality. HOBt (193 mg, 1.43 mmol), phthaloyl-Arg(Tos)-OH (637 mg, 1.39 mmol), and 1-(-Dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (266 mg, 1.39 mmol) were added successively and the reaction was allowed to stir at room temperature for 18 h. The DMF was removed in vacuo and the residue was taken up in ethyl acetate/chloroform (approximately 1:1), washed three times with 1 N NaHSO4, three times with 5% NaHCO3, dried (MgSO4), filtered, and evaporated. 817 mg (94% yield) was obtained. The crude product was purified by flash
chromatography (silica gel, 10% methanol/chloroform). The appropriate fractions were pooled according to tic, the solvent was removed and the residue was dried in vacuo (480 mg, 55%). NMR δ (CDCl3) 1.11-1.9(m, 13H), 2.05-2.2(br s,
3H), 2.35(s, 3H), 2.9(d, 5H) , 3.0-3.3(br s, 1H), 3.85-4.0 (m, 1H), 4.55-5.1(m, broad, 3H), 6.25-6.65 (m, broad, 3H), 6.9-7.9(m, 13H), 8.0 (s, 1H); TLC Rf 0.37 (15%
methanol/chloroform); MS (FAB) m/e 788 [M+H]+. c) Nα-(phthaloyl)-Arg-Gly-Asp-phenylamide
The protected peptide of Example 1Kb) (200 mg, 254 nmol) was treated with 5 mL anhydrous HF at 0° for 1 h. The HF was removed in vacuo and the residue was dissolved in glacial acetic acid and lyophilized to yield the crude peptide salt (135 mg, 87%). 100 mg was filtered and purified by gel filtration (Sephadex® G-10, 10% acetic acid). The appropriate fractions were pooled and lyophilized to yield the title compound (25 mg). HPLC k' 3.49 (C18 reverse phase, gradient, A: acetonitrile B:water-0.1% trifluoroacetic acid, 10%-50% acetonitrile over 15 minutes, UV detection at 220 nm), k' 7.56 (C18 reverse phase, 20% CH3CN/water-0.1% trifluoroacetic acid, UV detection at 220 nm); TLC Rf 0.77 (butanol:acetic acid:water, 1:1:1), 0.64 (butanol: ethyl acetate:acetic acid: water, 1:1:1:1); MS (FAB) m/e 552.2 [M+H] +; Amino Acid Analysis : Asp (1. 0) , Gly (0 . 98) , Arg (0 . 84) ; Peptide content 67 .01% (peptide was hydrolyzed with 2 : 1 : 0 .005% v:v:w HCl/TFA/phenol at 160°C for 1 h. ) . Example 12
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-(4-chloro)
phenylamide (34) a) Nαbenzoyl-NαMeArg(Tos)-Gly(OMe)
DIEA (2.88 mL, 16.5 mmol) was added to a cold solution of NαMeArg(Tos)-Gly(OMe) (2) (5 mmol) in DMF (5 mL) to bring the pH to neutrality (pH 7.5 to 8.0). Benzoyl chloride (0.64 mL, 5.5 mmol) was then added. The reaction mixture was stirred at room temperature overnight. The solvent was removed under vacuum and the residue was taken into ethyl acetate (60 mL), and washed successively with water (3×20 mL), aqueous 10% sodium bicarbonate (2×20 mL), water (2×20 mL), aqueous 5% citric acid (2×20 mL), water (2×20 mL) and saturated salt solution. The organic solvent was dried
(anhydrous sodium sulfate), filtered and concentrated to yield the titled compound as white fluffy solid (1.94 g). b) Nαbenzoyl-NαMeArg(Tos)-Gly
To a solution of Nαbenzoyl NαMeArg(Tos)-Gly(OMe) (3)
(3.5 g, 6.8 mmol) in methanol (15 mL), a solution of 1 N NaOH (8.25 mL, 8.12 mmol) was added dropwise. The reaction mixture was stirred at room temperature for an hour to complete ester hydrolysis. The solvent was removed, the residue was redissolved in water (80 mL), back extracted with ethyl acetate, and acidified to acidic pH (1.35) with 3 N HCl. The acidic solution was extracted with ethyl acetate, the organic layer was washed 2x with water, dried with anhydrous sodium sulfate, filtered and concentrated to yield the titled compound as white fluffy solid (3.3 g). c) Boc-Asp(O-cHex)-(4-chloro)phenylamide
To a cold solution of Boc-Asp (O-cHex) (6.2 g, 20 mmol) in THF (100 mL) and N-methylmorpholine (2.42 mL, 22 mmol), isobutylchloroformate (2.86 mL, 22 mmol) was added dropwise. The -reaction mixture was stirred for a few minutes, then a solution of 4-chloroaniline (2.8 g, 22 mmol) in THF (10 mL) was added. The reaction mixture was allowed to warm to room temperature, and stirred for 18 h. Upon completion of the reaction, the reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (300 mL), and washed successively with water (3x), 1 N HCl (2x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and saturated salt solution. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to white solid. It was
recrystallized from ethyl acetate-hexane to yield the titled compound (4.2 g). d) Asp(O-cHex)-(4-chloro)phenylamide
Boc-Asp(O-cHex)-(4-chloro)phenylamide (1.44 g, 3.4 mmol) was treated with 50% TFA solution in methylene chloride (10 mL) for 45 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was
precipitated as its TFA salt upon addition of ether. The solid was collected and air dried. e) Nαbenzoyl NαMeArg(Tos)-Gly-Asp(O-cHex)-(4-chlpro)phenylamide
DIEA (1.5 mL, 8.6 mmol) was added to a cold solution of Asp(O-cHex)-(4-chloro)phenylamide (2.28 mmoi), dissolved in
DMF (8 mL), to bring the pH to neutrality. HOBt (300 mg, 2.2 mmol) was then added followed by Nαbenzoyl-NαMeArg(Tos)-Gly (1.00 g, 2 mmol) in DMF (5 mL). The reaction was stirred in the cold for a few min, then N-ethyl-N'-(dimethylaminopropyl)carbodiimide (EDC) (492 mg, 2.57 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The oily residue was dissolved in ethyl acetate (100 mL), and washed successively with water (3x), 1 N HCl (2x), water (3x), aqueous 10% NaHCO3 (2x), water (3x) and saturated salt solution. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to yield the titled compound (1.74 g). It was purified by a flash chromatography (silica, gradient 2-8% methanol/methylene chloride) to yield the titled product (1.1 g) . f) Nαbenzoyl-NαMeArg-Gly-Asp-(4-chloro)phenylamide
The protected linear peptide of Example 12(e) (900 mg) was treated with anhydrous HF (20 mL) in the presence of anisole (2 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether to yield an oil. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude product (775 mg). The peptide was purified by flash column chromatography (silica ODS, gradient from 20-26% acetonitrile/water-0.1% TFA) to yield the titled peptide (343 mg) . MS (FAB) m/e 574.4 [M+H]+, 572.2 [M-H]-; HPLC k' 9.94 (5 μ Altex Ultrasphere®ODS, 4.5 mm × 25 cm, gradient,
A:acetonitrile B:water-0.1% trifluoroacetic acid, 1-50% A in 20 min, UV detection at 220 nm), and k' 6.88 (21%
acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 220 nm); TLC Rf 0.74 (n-BuOH:H2O:AcOH:EtOAc 1:1:1:1), and Rf 0.74 (n-BuOH:AcOH:H2O:pyridine 15:5:10:10); Amino Acid
Analysis: Asp (1.00), Gly (1.09).
Example 13 Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-(3-carboxy)-phenylamide (35) a) benzyl-3-aminobenzoate
Benzenesulfonic acid (3.48 g, 22 mmol) was added to a solution of 3-aminobenzoic acid (2.74 g, 20 mmol) in benzyl alcohol (20.6 mL, 200 mmol). The reaction mixture was heated using Dean-Stark trap for 4 h. The alcohol was distilled and the residue was dissolved in ethyl acetate and washed successively with water, aqueous 10% NaHCO3 (3x), water (3x) and saturated salt solution. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to yield the titled compound (1.5 g). b) Boc-Asp(O-cHex)-(3-benzyloxycarbonyl)phenylamide
To a cold solution of Boc-Asp(O-cHex) (0.915 g, 2.9 mmol) in THF (15 mL) and N-methylmorpholine (0.35 mL, 3.2 mmol), isobutylchloroformate (0.42 mL, 3.2 mmol) was added dropwise. The reaction mixture was stirred for a few minutes, then a solution of benzyl-3-aminobenzoate (0.66 g, 2.9 mmol) in THF (2 mL) was added. The reaction mixture was allowed to warm to room temperature, and stirred for 18 h. Upon completion of the reaction, the reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate, and washed successively with water (3x), 5% aqueous citric acid (3x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and saturated salt solution. The organic extract was dried (anhydrousNa2SO4), filtered and concentrated to brownish solid (1.46 g). It was purified by a flash
chromatography (silica, gradient 0.3-0.8% methanol/methylene chloride) to yield the titled product (0.64 g). c) Asp(O-cHex)-(3-benzyloxycarbonyl)phenylamide
Asp(O-cHex)-(3-benzyloxycarbonyl)phenylamide (0.64 g, 1.2 mmol) was treated with 50% TFA solution in methylene chloride (12 mL) for 60 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was crystallized as its TFA salt from etherhexane (0.6 g). d) Nαbenzoyl-NαMeArg(Tos)-Gly-Asp(O-cHex)-(3-benzyloxycarbonyl)phenylamide
DIEA (0.43 mL, 2.45 mmol) was added to a cold solution of Asp(O-cHex)-(3-benzyloxycarbonyl) phenylamide (600 mg, 1.11 mmol) in DMF (10 mL), to bring the pH to neutrality. HOBt (165 mg, 1.22 mmol) was then added followed by Nαbenzoyl- NαMeArg(Tos)-Gly (559 mg, 1.11 mmol) in DMF (5 mL). The reaction was stirred in the cold for a few min, then N-ethyl- N'-(dimethylaminopropyl)carbodiimide (EDC) (234 mg, 1.22 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (100 mL), and washed successively with water (3x), 5% aqueous citric acid (3x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and
saturated salt solution. The organic extract was dried
(anhydrous Na2SO4), filtered and concentrated to yield the titled compound (0.87 g). It was purified by a flash chromatography (silica, gradient 3-5% methanol/methylene chloride) to yield the titled product (0.44 g). e) Nαbenzoyl-NαMeArg-Gly-Asp-(3-carboxy)phenylamide
The protected linear peptide of Example 13(d) (0.44 g) was treated with anhydrous HF (10 mL) in the presence of anisole (1 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether to an oil. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude product (220 mg). The peptide was purified by flash
chromatography (silica ODS, gradient 10-15%
acetonitrile/water-0.1% TFA) to yield the titled peptide (91 mg). MS (FAB) m/e 584 [M+H] +, 582 [M-H]-; HPLC k' 9.73 (5 μ
Altex Ultrasphere®ODS, 4.5 mm × 25 cm, gradient,
A: acetonitrile B:water-0.1% trifluoroacetic acid, l%-50% A in 20 min, UV detection at 220 nm), and k' 13.08 (12%
acetonitrile/water- 0.1% trifluoroacetic acid, UV detection at 220 nm) ; TLC Rf 0.58 (n-BuOH:H2O:AcOH:EtOAc 1:1:1:1), and Rf 0.56 (n-BuOH:AcOH:H2O:pyridine 15:5:10:10); Amino Acid Analysis: Asp (1.00), Gly (1.01). Example 14
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-(4-carboxy)phenylamide (36) a) benzyl-4-aminobenzoate
Benzenesulfonic acid (5.22 g, 33 mmol) was added to a solution of 4-aminobenzoic acid (4.12 g, 30 mmol) in benzyl alcohol (31 mL, 300 mmol). The reaction mixture was heated using a Dean-Stark trap for 4 h. The alcohol was distilled and the residue was dissolved in ethyl acetate and washed successively with water, aqueous 10% NaHCO3 (2x), water (3x) and saturated salt solution (1x). The organic extract was dried (anhydrousNa2SO4), filtered and concentrated to yield the titled compound (1.62 g). b) Boc-Asp(O-cHex)-(4-benzyloxycarbonyl)phenylamide
To a cold solution of Boc-Asp (O-cHex) (0.883 g, 2.8 mmol) in THF (10 mL) and N-methylmorpholine (0.34 mL, 3.1 mmol), isobutylchloroformate (0.40 mL, 3.1 mmol) was added dropwise. The reaction mixture was stirred for a few
minutes, then a solution of benzyl-4-aminobenzoate (0.594 g, 2.6 mmol) in THF (2 mL) was added. The reaction mixture was allowed to warm to room temperature, and stirred for 18 h. Upon completion of the reaction, the reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate, and washed successively with water (3x), 5% aqueous citric acid (3x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and saturated salt solution. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to yield the titled compound as a yellowish solid (1.18 g).
C) Asp(O-cHex)-(4-benzyloxycarbonyl)phenylamide
Boc-Asp(O-cHex)-(4-benzyloxycarbonyl)phenylamide (1.1 g, 2.1 mmol) was treated with 50% TFA/methylene chloride (20 mL) for 90 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was
crystallized as its TFA salt from ether-hexane (0.97 g). d) Nαbenzoyl-NαMeArg (Tos ) -Gly-Asp (O-cHex) - (4- benzyloxycarbonyl)phenylamide
DIEA (0.66 mL, 3.78 mmol) was added to a cold solution of Asp(O-cHex)-(4-benzyloxycarbonyl)phenylamide (0.92 g, 1.71 mmol) in DMF (15 mL) to bring the pH to neutrality. HOBt (0.254 g, 1.88 mmol) was added followed by Nαbenzoyl- NαMeArg(Tos)-Gly (0.861 g, 1.71 mmol) in DMF (5 mL). The reaction was stirred in the cold for a few min, then EDC (0.36 g, 1.88 mmol) was added portionwise. The reaction mixture was allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (100 mL), and washed successively with water (3x), 5% aqueous citric acid (3x), water (3x), aqueous 10% NaHCO3 (3x), water (3x) and saturated salt solution. The organic extract was dried
(anhydrous Na2SO4), filtered and concentrated to yield the titled compound (1.06 g). The product was purified by flash chromatography (silica, 3% methanol/methylene chloride) to yield the titled compound (0.57 g). e) Nαbenzoyl-NαMeArg-Gly-Asp-(4-carboxy)phenylamide
The protected linear peptide of Example 14(d) (0.56 g), was treated with anhydrous HF (10 mL) in the presence of anisole (1 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude product (230 mg). The peptide was purified by flash chromatography (silica ODS, gradient 10-13% acetonitrile/water-0.1% TFA) to yield the titled peptide (18 mg). MS (FAB) m/e 584.2 [M+H]+; HPLC k' 10.0 (5 μ Altex Ultrasphere® ODS, 4.5 mm x 25 cm, gradient. A:acetonitrile B:water-0.1% trifluoroacetic acid, 1-50% A in 20 min, UV detection at 220 nm), and k' 8.55 (13% acetonitrilewater-0.1% TFA, UV detection at 220 nm). TLC Rf 0.71 (n-BuOH:H2O:AcOH:EtOAc 1:1:1:1); and Rf 0.58
(n-BuOH:AcOH:H2O:pyridine 15:5:10:10); Amino Acid Analysis: sp (1.08), Gly (1.00). Example 15
Preparation of Nα(2-thienylcarbonyl)-NαMeArg-Gly-Asp-phenylamide (37) a) Boc-Asp(O-cHex)-phenylamide
To a cold solution of Boc-Asp(O-cHex) (31.54 g, 100 mmol) in THF (500 mL) and N-methylmorpholine (12.09 mL, 110 mmol), isobutylchloroformate (14.27 mL, 110 mmol) was added dropwise. The reaction mixture was stirred for a few min, then a solution of aniline (10 mL, 110 mmol) was added. The reaction mixture was allowed to warm to room temperature, and stirred for 18 h. Upon completion of the reaction, the reaction mixture was concentrated to dryness. The residue was dissolved in ethyl acetate (800 mL), and washed
successively with water (3x), 5% aqueous citric acid (2x), water (3x), aqueous 10% NaHCO3 (2x), water (3x) and
saturated salt solution. The organic extract was dried (anhydrous Na2SO4), filtered and concentrated to white solid. The product was recrystallized from ethyl acetate-hexane to yield the titled compound (31.42 g). b) Asp(O-cHex)-phenylamide
Boc-Asp(O-cHex)-phenylamide (11 g, 28.2 mmol) was treated with 50% TFA/methylene chloride (120 mL) for 90 min at room temperature. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The product was precipitated as its TFA salt upon addition of ether (11.2 g). c) NαBoc-NαMeArg(Tos)-Gly-Asp(O-cHex)-phenylamide .
Asp(O-cHex)-phenylamide (4.2 g, 9 mmol) in DMF (30 mL) was reacted with DIEA (3.45 mL, 19.8 mmol), HOBt (1.34 g, 9.9 mmol) and NαBoc-NαMeArg(Tos)-Gly (11) (3.74 g, 7.5 mmol) in DMF (5 mL) according to the procedure of Example 14(d) to yield the titled compound (5.66 g). d) NαMeArg-Gly-Asp-phemylamide
The protected peptide of Example 15 (c) was treated with TFA according to the procedure of Example 14 (e) to yield the titled compound (1.074 g). f) Nα(2-thienylcarbonyl)-NαMeArg-Gly-Asp-phenylamide
DIEA (100 μL, 0.555 mmol) was added to a cold solution of NαMeArg-Gly-Asp-phenylamide (27) (0.163 g, 0.37 mmol) in DMF (5 mL), to bring the pH to neutrality (pH 7.5 to 8.0). 2-thienylcarboxylic acid anhydride (0.37 mmol), (prepared from 2-thienylcarboxylic acid (96.4 mg, 0.75 mmol) and DCC (76.4 mg, 0.37 mmol)) was added. The reaction was stirred in the cold for a few min, allowed to warm to room temperature and stirred for 18 h. The reaction mixture was concentrated to dryness. The residue was then purified by gel filtration (Sephadex® G-15, 1% acetic acid/water). The appropriate fractions were pooled and lyophilized to yield a semipurified product (45 mg). The peptide was further purified by flash chromatography (silica ODS, gradient 10-16%
a'cetonitrile/water-0.1% TFA) to yield the titled compound
(16.3 mg). MS (FAB) m/e 546.2 [M+H]+, 544.4 [M-H]-; HPLC k' 16 (5 μ Altex Ultrasphere®ODS, 4.5 mm × 25 cm, gradient,
A:acetonitrile B:water-0.1% trifluoroacetic acid, 1-50% A in 20 min, UV detection at 220 nm), and k' 9.02 (10%
acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 220 nm). TLC Rf 0.83 (n-BuOH:H2O:AcOH:EtOAc 1:1:1:1); and Rf 0.64 (n-BuOH:AcOH:H2O:pyridine 15:5:10:10); Amino Acid
Analysis: Asp (1.00), Gly (0.98). Example 16
Preparation of Nαacetyl-Arg-Gly-Asp-phenylamide (38) a) Boc-Arg(Tos)-Gly(OBzl)
DIEA (71.51 mL, 410 mmol) was added to a cold suspension of Gly(OBzl) p-toulene sulfonic acid salt (2.93 g, 8.7 mmol), in dry DMF in a dropwise manner to bring it to a neutral pH. This was followed by the addition of HOBt (1.17 g, 8.7 mmol) and NαBoc-Arg (Tos) (3.38 g, 7.89 mmol). The reaction mixture was stirred for a few minutes, then DDC (1.78 g,. 8.7 mmol) was added at 0°C. The reaction mixture was allowed to warm to room temperature and stirring was continued overnight. The solvent was removed under vacuum, and the residue was purified by a flash chromatography (silica, gradient 2-10% methanol/chloroform) to yield the titled compound (3.05 g). b) Boc-Arg(Tos)-Gly
NαBoc-Arg(Tos)-Gly (0.65 g, 1.12 mmol) in methanol (50 mL) and activated palladium on carbon (100 mg) were reacted in a Parr shaker for 5 h. The catalyst was removed by filteration and the filterate was concentrated to yield the titled compound. c) Boc-Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide
Asp(O-Bzl)-phenylamide (10) (2.24 mmol) in DMF (100 mL) was reacted with DIEA (0 . 4 mL, 2 .24 mmol) , HOBt (0 .3 g, 2 . 24 mmol), Bop reagent) (0.99 g, 2.24 mmol) and Boc-Arg(Tos)-Gly (2.24 mmol) according to the procedure of Example 14(d) to yield a crude product. This was purified by flash
chromatography (silica, 5% methanol/chloroform) to yield the titled compound (0.49 g). d) Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide
Boc-Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide (220 mg, 0.287 mmol) was treated with 50% TFA/methylene chloride (10 mL) at room temperature for 4 H. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was treated with HCl-dioxane and evaporated several times from toulene and dried under vacuum to yield the titled compound as its hydrochloride salt. e) Nαacetyl-Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide
Triethylamine (43 μL) was added to a solution of
Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide in DMF (20 mL) to a neutral pH. Acetic anhydride (30 μL) was added, and the
MKΓ/ΓUΓE su reaction mixture was stirred for 18 h. The solvent was removed under vacuum and the residue was purified by flash chromatography (15% methanol/chloroform) to yield the titled compound (0.14 g). f) Nαacetyl-Arg-Gly-Asp-Phenylamide
The protected peptide of Example 16(e) (120 mg) was treated with anhydrous HF (10 mL) in the presence of anisole (1 mL) at 0°C for 1 h. The HF was removed at 0°C under vacuum, and the residue was triturated with ether. The residue was dissolved in 0.2 M acetic acid, washed several times with ether and lyophilized to yield a crude product (35 mg). The peptide was purified by partition column
chromatography (Sephadex®G-15, 4:1:1 butanol:acetic
acid:water). The appropriate fractions were pooled and lyophilized to yield a semi-purified product (25 mg). It was further purified by semipreparative HPLC (5 μODS silica, 20% acetonitrile/water-0.1% TFA) provided the purified titled compound (18 mg). MS (FAB) m/e 464.2 [M+H]+, 462 [M-H]-; HPLC k' 3.35 (20% acetonitrile/water-0.1% TFA, UV detection at 220 nm); TLC Rf 0.15 (n-BuOH:H2O:AcOH 4:1:1); Amino Acid
Analysis: Asp (1.00), Gly (0.97), Arg (1.06).
Example 17 Preparation of Nαbenzoyl-Arg-Gly-Asp-phenylamide (39) a) Nαbenzoyl-Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide
Triethylamine (54 μL) was added to a solution of
Arg(Tos)-Gly-Asp(O-Bzl)-phenylamide (0.313 mmol) in DMF (5 mL) to a neutral pH. Benzoyl chloride (36 μL) was added, and the reaction mixture was stirred for 18 h. The solvent was removed under vacuum and the residue was purified by flash chromatography (silica, 5-10% methanol/chloroform) to yield the titled compound. b) Nαbenzoyl-Arg-Gly-Asp-phenylamide
The protected peptide is treated with anhydrous HF and isolated according to the procedure of Example 14 (e).
Example 18
Preparation of Nαbenzyloxycarbonyl-Arg-Gly-Asp-phenylamide (40) a) Nαbenzyloxycarbonyl-Arg-Gly-Asp(O-tBu)-phenylamide
Cbz-Arg was coupled to Gly-Asp(O-tBu)-phenylamide and isolated using DIEA, EDC and HOBT in DMF according to the procedure of Example 1(a) to give the titled compound. b) Nαbenzyloxycarbonyl-Arg-Gly-Asp(O-tBu)-phenylamide
The compound of Example 18 (a) is dissolved in dioxane and treated with hydrochloric acid to afford the titled compound.
Example 19
Preparation of D-Phg-Arg-Gly-Asp-phenylamide (41)
Cbz-D-Phenylglycine was treated with cyanuryl fluoride to afford the acid fluoride. Cbz-D-Phenylglycinyl fluoride and Arg(Tos)-Gly-Asp(O-cHex)-phenylamide were coupled and the resulting product was treated with HF to afford the desired product. MS (FAB) m/e 555 [M+H]+.
Example 20 Preparation of Nαbenzyloxycarbonyl-Ngmethyl-Orn-Gly-Asp- Phenylamide (42)
NαCbz-Orn(Phth) was converted to NαCbz-NαMeOrn(Phth) by analogy with the method described in J. Org. Chem. 48, 77 (1983). NαCbz-NαMe-Orn(Phth) and Gly-Asp(O-Bzl)-phenylamide were coupled with EDC following the procedure of Example
14(d) to afford Nαbenzyloxycarbonyl-NαMeOrn(Phth)-Gly-Asp(O-Bzl)-phenylamide. The benzyloxycarbonyl group (Cbz) was removed by hydrogenation over a palladium catalyst, and the resulting amine was benzoylated, and the resulting amino group was benzoylated according to the procedure of Example 17 (a). Treatment of the resulting peptide with hydrazine provided the titled compound. MS (FAB) m/e 498 [M+H]+.
Example 21
Preparation of 4-(aminoiminomethyl)aminobenzoyl-Sar-Asp- phenylamide (43)
4-Guanidinobenzoic acid hydrochloride and Sar-Asp(O- Bzl)-phenylamide were condensed with EDC following the procedure of Example 14 (d) to yield 4-guanidinobenzoyl-Sar- Asp(O-Bzl)-phenylamide. Treatment of this product with hydrogen in the presence of a palladium catalyst yielded the titled compound. MS (FAB) m/e 441 [M+H]+.
Example 22
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-(2-chloro)-phenylamide (44)
Using the procedure of Example 12, except substituting 2-chloroaniline for 4-chloroaniline in step 12(c), the titled compound is prepared. Example 23
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-(2-methoxy)-phenylamide (45)
Using the procedure of Example 12, except substituting 2-methoxyaniline for 4-chloroaniline in step 12 (c), the titled compound is prepared.
Example 24 Preparation of Nαbenzoyl-NαMeArg-Gly-MeAsp-(2-hydroxy)-phenylamide (46) Using the procedure of Example 13, except substituting 2-benzyloxyaniline for benzyl 3-aminobenzoate in step 13 (b), the titled compound is prepared. Example 25
Preparation of Nαbenzoyl-Arg-NHNHCO-Asp-(N-methyl)-phenylamide (47)
Figure imgf000071_0001
a) Boc-Arg(NO2)-OCH3
To a solution of Boc-Arg(NO2) (2.0 g, 05.5 mmol) in MeOH/CH2Cl2, a solution of diazomethane in Et2O was added. The reaction mixture was washed successively with 5% aqueous Na2CO3 and H2O and dried. Removal of the solvent in vacuo provided the titled compound (1.6 g, 87%) which was used without further purification. b) Boc-Arg(NO2)-NHNH2
To a solution of Boc-Arg(NO2)-OCH3 (1.6 g, 4.8 mmol) in
MeOH, 62% hydrazine hydrate (1.2 mL, 24 mmol) was added. The resulting mixture was stirred at room temperature for 48 h. The- reaction mixture was concentrated under reduced pressure, and the residue was purified by flash chromatography (silica, gradient 5-15% MeOH/CH2Cl2) to provide a colorless oil
(0.74-g, 46%) which solidified upon standing. c) Boc-Asp(O-Bzl)-(N-methyl)phenylamide
To a solution of Boc-Asp(O-Bzl) (2.0 g, 6.2 mmol) in DMF were added N-methylaniline (0.99 g, 9.3 mmol), HOBT hydrate (0.92 g, 6.8 mmol) and DCC (1.3 g, 6.2 mmol). The resulting mixture was allowed to stir at room temperature overnight. The reaction mixture was concentrated under reduced pressure. The residue was dissolved in CH2CI2, washed successively with 0.5 N HCl and H2O and dried (MgSO4). The solvent was removed in vacuo, and the residue was purified by flash
chromatography to provide a gummy oil. d) Q=C-Asp(O-Bzl)-(N-methyl)phenylamide
To Boc-Asp(O-Bzl)-(N-methyl)phenylamide (0.91 g, 2.2 mmol) at 0°C was added a solution of trifluoroacetic acid (8 mL) in CH2CI2 (8 mL). After stirring at 0°C for 45 min, the mixture was concentrated under reduced pressure and treated azeotropically with toluene several times. To the residue was added a solution of 20% phosgene in toluene (42 mL). The resulting mixture was heated at reflux for 15 min, then allowed to cool to room temperature and concentrated under reduced pressure. The residue was treated azeotropically with toluene, and used without further purification. e) Boc-Arg(NO2)-NHNHCO-Asp(O-Bzl)-(N-methyl)phenylamide
To a solution of the compound of Example 25 (c) in CH3CN was added a solution of Boc-Arg(NO2)-NHNH2 (0.74 g, 2.2 mmol) in CH3CN, and the resulting solution was allowed to stir at room temperature overnight. The solvent was removed in vacuo, and the residue was purified by flash chromatography, (silica, 10% MeOH/CH2Cl2) to provide the titled compound
(0.34 g, 23%). f) Arg-NHNHCO-Asp-(N-methyl)phenylamide
To the compound of Example 25(e) (0.30 g, 0.45 mmol) was added a solution of trifluoroacetic acid (0.7 mL, 11.2 mmol) in CH2CI2 (0.7 mL). The resulting mixture was stirred at room temperature for 15 min, then concentrated under reduced pressure. The residue was treated azeotropically with toluene, and the residue was then dissolved in CH2CI2. To this solution was added triethylamine (0.13 mL, 1.0 mmol), followed by benzoyl chloride (0.05 mL, 0.44 mmol). The resulting mixture was stirred at room temperature overnight, and then concentrated under reduced pressure. The residue was purified by flash chromatography (silica, 10%
MeOH/CH2Cl2) to provide the titled compound (0.15 g, 50%). g) Nαbenzoyl-Arg-NHNHCO-Asp-(N-methyl)phenylamide
To a solution of the compound of Example 25(f) (150 mg, 0.22 mmol) in MeOH (30 mL) was added glacial acetic acid (0.3 mL) and 10% palladium on activated carbon (19 mg). The resulting mixture was hydrogenated at 46 psi H2 for 3 h, at which time thin layer chromatographic analysis indicated only the presence of starting material. The mixture was degassed and additional 10% palladium on activated carbon (90 mg) was added. The reaction mixture was hydrogenated (46 psi) overnight at which time thin layer chromatographic analysis again indicated only the presence of starting material. The mixture was filtered through a pad of Celite® and
concentrated under reduced pressure. Methanol (30 mL), glacial acetic acid (0.3 mL) and 10% palladium on activated carbon (100 mg) was added to the residue, and the mixture was hydrogenated (46 psi) for 7 h. The mixture was filtered through a pad of Celite® and concentrated under reduced pressure. The crude product was purified by semiprep HPLC k' 3.86 (Dynamax C18, 17% acetonitrile/water-0.1%
trifluoroacetic acid, UV detection at 260 nM) to yield the purified titled compound, which was lyophilyzed from H2O containing a small amount of glacial acetic acid (19 mg, 10%). MS (FAB) m/e 541 [M+H]+; TLC Rf 0.87 (n-BuOH:HOAc: H2O:EtOAc 1:1:1:1); Rf 0.88 (n-BuOH:HOAc:H2O:pyridine
15:5:10:10); HPLC k' 7.86 (Dynamax C18, gradient,
A:acetonitrile B:water-0.1% trifluroacetic acid 10-80% A in 30 min, UV detection at 260 nM).
Example 26
Preparation of Nαbenzoyl-NαMeArg-Gly-Asp-phenyl ester (48) Using the procedure of Example 2, except substituting phenol for aniline in step 2(a), the titled compound was prepared. Example 27
Figure imgf000074_0001
Figure imgf000074_0002
a) Boc-D-Arg (Tos) - (N-methyl) phenylamide
DIEA (1.2 mL, 6.9 mmol) was added to a solution of N-methylaniline (1.5 g,14.0 mmol) in dry DMF (10mL) in a dropwise manner to bring the pH to 8. This was followed by the addition of Boc-D-Arg(Tos) (5 g,11.7 mmol) and HOBt (2.7 g, 17.9 mmol). The reaction mixture was stirred for a few min, then EDC (2.5 g, 13.1 mmol) was added portionwise over a period of 2 min. The reaction mixture was stirred at room temperature overnight. Additional amount of DIEA (1.2 mL, 6.9 mmol) and EDC (2.0 g, 10.5 mmol) was added to the reaction mixture to drive the slow reaction to completion. After one week the solvent was removed under vacuum, and the residue was partitioned between ethyl acetate and 10% K2CO3. The organic extract was washed successively with water, 5% citric acid, water, and brine. The organic extract was dried with anhydrous sodium sulfate, filtered, concentrated and triturated with ether/hexane (1/1) to give the titled
compound (2.4 g,-40%) as a white solid. b) D-Arg(Tos)-(N-methyl)phenylamide
Boc-D-Arg(Tos)-(N-methyl)phenylamide (2.3 g,4.4 mmol) was treated with 50% TFA in methylene chloride (20 mL) at room temperature for 1 h. The solvent was removed and the residue was evaporated several times from methylene chloride to eliminate traces of TFA. The residue was triturated with ether and used in the next step without further purification.
SUBSTITUTE SHEET c) Nα(CH3O2OCH2CO)-p-Arg(Tos)-(N-methyl)phenylamide
Triethylamine (1.3 mL, 9.3 mmol) was added to a cold solution of D-Arg(Tos)-(N-methyl)phenylamide in methylene chloride (5 mL). This was followed by the addition of methyl malonylchloride (500 μL, 4.7 mmol). The reaction mixture was allowed to warm to room temperature and stirring was
continued overnight. The reaction mixture was poured into water, and the organic layer was washed successively with 5% citric acid, water, 5% NaHCO3, water, and brine. The organic layer was dried with anhydrous sodium sulfate, filtered and concentrated to yield the titled compound (1.7 g, 81%) as a white solid. d) N α (H2OCCH2CO) -D-Arg (Tos ) - (N-methyl ) phenylamide
To a solution of 1-(D-Arg(Tos)-N-methylanilide)-methylmalonate (0.78 g,1.5 mmol) in methanol (1.5 mL), a solution of IN NaOH (2.5 mL,2.5 mmol) was added dropwise. The reaction solution was stirred at room temperature for 18 h. The pH of the solution was adjusted to 6 using 1M HCl and the solution was concentrated. The pH was adjusted to 2 using 1M HCl, and an oil precipitated. Trituration with water and drying in vacuo yielded the titled compound as a white solid (0.608 g) e
Figure imgf000075_0001
DIEA (0.2 mL, 1.1 mmol) was added to a solution of
Asp(O-cHex)-phenylamide in dry DMF (2 mL) in a dropwise manner to bring it to a pH 8. This was followed by the addition of HOBt (0.188g, 1.2mmol) and 1-(D-Arg(Tos)-N-methylanilide)-malonic acid (6) (0.57g, 1.1mmol). The reaction mixture was stirred for a few minutes, then EDC (0.216 g,1.1 mmol) was added portionwise. The reaction mixture was stirred for 18h. The solvent was removed under vacuum, and the residue was partitioned between ethyl acetate and 10% K2CO3 . The organic extract was washed successively with water, 5% citric acid, water, and brine. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to a white solid which was used in the next step without further purification. f)
Figure imgf000076_0001
The compound of Example 27 (e) was treated with anhydrous
HF (10 mL) in the presence of anisole (2 mL) at 0°C for 1 h.
The HF was removed at 0°C under vacuum, and the residue was triturated with ether to an oil. The residue was dissolved in 0.2M acetic acid and lyophilized to yield a crude product (0.26g). The product was purified by gel filtration
(Sephadex® G-25, 0.2M acetic acid) to yield the titled compound (0.028 g) . MS (FAB) m/e 540.3 [M+H]+ ; HPLC k' 10.7 (5μ Altex Ultrasphere® ODS,. gradient. A: acetonitrile B:
water-0.1% trifluoroacetic acid, 10%-50% A in 20 min, UV detection at 220 nm), k' 7.3 (Ultrasphere® ODS, 22% .
acetonitrile/water-0.1% trifluoroacetic acid, UV detection at 220 nm); TLC Rf 0.76 (n-BuOH:H2O:AcOH:EtOAc 1:1:1:1); Rf 0.84 (n-BuOH:AcOH:H2O:pyridine 15:5:10:10); Amino Acid Analysis: Asp (1.00), Arg (0.99).
Example 28
Parenteral Dosage Unit Composition
A preparation which contains 20 mg of the compound of Example 1 or 2 as a sterile dry powder is prepared as
follows: 20 mg of the compound is dissolved in 15 ml of distilled water. The solution is filtered under sterile conditions into a 25 ml multi-dose ampoule and lyophilized. The powder is reconstituted by addition of 20 ml of 5% dextrose in water (D5W) for intravenous or intramuscular injection. The dosage is thereby determined by the injection volume. Subsequent dilution may be made by addition of a metered volume of this dosage unit to another volume of D5W for injection, or a metered dose may be added to another mechanism for dispensing the drug, as in a bottle or bag for IV drip infusion or other injection-infusion system. Example 29
Oral Dosage Unit Composition
A capsule for oral administration is prepared by mixing and milling 50 mg of the compound of Example 3 with 75 mg of lactose and 5 mg of magnesium stearate. The resulting powder is screened and filled into a hard gelatin capsule.
Example 30 Oral Dosage Unit Composition A tablet for oral administration is prepared by mixing and granulating 20 mg of sucrose, 150 mg of calcium sulfate dihydrate and 50 mg of the compound of Example 3 with a 10% gelatin solution. The wet granules are screened, dried, mixed with 10 mg starch, 5 mg talc and 3 mg stearic acid; and compressed into a tablet.
The foregoing is illustrative of the making and using of this invention. This invention, however, is not limited to the precise embodiments described herein, but encompasses all modifications within the scope of the claims which follow.

Claims

What is claimed is:
1. A compound of the formula: D-E-Asp-Q-Y
wherein:
Q is NH, NCH3 or O;
Y is phenyl, naphthyl or Het, unsubstituted or
substituted by one to three C1-6alkyl, trifluoromethyl, halogen, OR', SR', (CH2)nAr, CONR1R2, CO2R2 or R4R4N;
D is
Figure imgf000078_0001
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 OC(=O);
V is H, R', R4R5N, R4R5NCO, R'O, Y-NR', X-A-B-NR' or
Figure imgf000078_0002
A is absent, Asn, Gln, Ala, Pro or Abu;
B is a D- or L- amino acid chosen from Arg, HArg, NArg, (Me2)Arg, (Et2)Arg, Abu, Ala, Gly, His, Orn, Lys, Phg or an α-R' substituted derivative thereof, Dtc, Tpr or Pro;
E is Gly, Sar, CH2CO, OCH2CO or NHCO;
R' is H, C1-6alkyl or Ar(CH2)p;
X is R4R4N, R4R5N-CO or H;
R1 and R2 are H, C1-6alkyl or (CH2)pAr;
R4 is H or Ci-ealkyl;
R5 is R6, R6CO, R6OCO, R6OCH (R6') CO, R6NHCH(R6') CO, R5SCH(R6')CO, R6SO2 or R6SO;
R6 and R6' are H, C1-6alkyl, C3-7cycloalkyl, Ar,
Ar-C1-4alkyl, Het, Het-C1-4alkyl, Ar-(CH2)pCH(Ar) (CH2)p or Ar-C3-7Cycloalkyl;
Ar is phenyl or phenyl substituted by one to three C1-6alkyl, trifluoromethyl, halogen, R'O or R'S; and
Figure imgf000078_0003
is a five or six-membered, mono or bicyclic ring containing one or two nitrogen atoms;
m is: (1) 0-4, when W is NR'- (C=NH)NHR';
(2) 0-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or
(3) 0-6, when W is NHR ' ; n is 0-2 ;
p is 0-3 ;
q is : ( 1) 1-4 , when W is NR ' - (C=NH) NHR ' ;
(2) 2-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or (3) 3-6, when W is NHR';
or a pharmaceutically acceptable salt thereof.
2. A compound according to claim 1 wherein Y is phenyl or naphthyl, optionally substituted by halogen, C1-4alkyl,
C1-4alkoxy, C1-4alkylthio, CONR1R2 or CO2R3.
3. A compound according to claim 2 wherein Y is phenyl or naphthyl.
4. A compound according to claim 1 wherein D is
Figure imgf000079_0001
5. A compound according to claim 1 wherein Q is NH.
6. A compound according to claim 1 wherein V is R4R5N.
7. A compound according to claim 4 wherein E is Gly.
8. A compound according to claim 6 wherein R4 is H or methyl and R5 is R6CO or R6OCO.
9. A compound according to claim 4 wherein V is ArCO-N(CH3) or HetCO-N(CH3).
10. A compound according to claim 8 wherein Y is phenyl, optionally substituted by chloro, C1-4alkyl, C1-4alkylthio or carboxy.
11. A compound according to claim 1 which is:
N-[[5-(aminoiminomethyl)amino-1-oxopentyl]-Gly-Asp- phenylamide;
N-[(2R)-2-phenyl-5-(aminoiminomethyl)amino-1-oxopentyl]-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- phenylamide;
Nα-(phthaloyl)-Arg-Gly-Asp-phenylamide;
Nαbenzoyl- NαMeArg-Gly-Asp-(4-carboxy)phenylamide;
Nαacetyl-Arg-Gly-Asp-phenylamide;
Nαbenzoyl-Arg-Gly-Asp-phenylamide;
Nαbenzyloxycarbonyl-Arg-Gly-Asp-phenylamide;
D-Phg-Arg-Gly-Asp-phenylamide;
Nαbenzyloxycarbonyl- Nαmethyl-Orn-Gly-Asp-phenylamide;
4-(aminoiminomethyl)aminobenzoyl-Sar-Asp-phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(2-chloro)phenylamide;
Nαbenzoyl- NαMeArg-Gly-Asp-(2-methoxy)phenylamide;
Nαbenzoyl-NαMeArg-Gly-MeAsp-(2-hydroxy)phenylamide;
Nαbenzoyl-Arg-NHNHCO-Asp-(N-methyl)phenylamide;
Nα(OCCH2CO)-Arg-(N-methyl)phenylamide;
Figure imgf000080_0001
Nα-(2-methylbenzoyl)- Nα-MeArg-Gly-Asp-(2-methyl)phenylamide;
Nαbenzoyl- NαMeArg-Gly-Asp-phenylamide;
Nαacetyl- NαMeArg-Gly-Asp-phenylamide;
Nαacetyl-NαMeArg-Gly-Asp-(2-carboxy)phenylamide;
Nα(2-methylthio)benzoyl-NαMeArg-Gly-Asp-(2-methylthio)-phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(4-chloro)phenylamide;
Nαbenzoyl-NαMeArg-Gly-Asp-(3-carboxy)phenylamide; and
Nα(2-thienylcarbonyl)-NαMeArg-Gly-Asp-phenylamide.
12. A pharmaceutical composition comprising a compound according to claim 1 and a pharmaceutically acceptable carrier.
13. The use of a compound according to claim 1 in the manufacture of a medicament.
14. A method for effecting inhibition of platelet
aggregation which comprises administering a compound
according to claim 1.
15. A method for treating stroke or transient ischemia attacks which comprises administering a compound according to claim 1.
16. A method for treating myocardial infarction which comprises administering a compound according to claim 1.
17. A method for effecting thrombolysis and inhibiting reocclusion of an artery or vein in a mammal which comprises administering a fibrinolytic agent and a compound according to claim 1.
18. A method according to claim 16 in which the fibrinolytic is anistreplase, streptokinase (SK), urokinase (UK), prourokinase (pUK) or tissue plasminogen activator (tPA) or a mutant or derivative thereof.
ABSTRACT OF THE INVENTION
This invention relates to compounds of the formula:
D-E-Asp-Q-Y
wherein :
Q is NH, NCH3 or O;
Y is phenyl, naphthyl or Het, unsubstituted or
substituted by one to three C1-6alkyl, trifluoromethyl, halogen, OR', SR1, (CH2)nAr, CONR1R2, CO2R2 or R4R4N;
D is
Figure imgf000082_0001
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 OC (=O);
V is H, R', R4R5N, R4R5NCO, R'O, Y-NR', X-A-B-NR' or
Figure imgf000082_0002
A is absent, Asn, Gin, Ala, Pro or Abu;
B is a - D- or L- amino acid chosen from Arg, HArg, NArg, (Me2)Arg, (Et2)Arg, Abu, Ala, Gly, His, Orn, Lys, Phg or an α-R' substituted derivative thereof, Dtc, Tpr or Pro;
E is Gly, Sar, CH2CO, OCH2CO or NHCO;
R' is H, C1-6alkyl or Ar(CH2)p;
X is R4R4N, R4R5N-CO or H;
R1 and R2 are H, C1-6alkyl or (CH2)pAr;
R4 is H or C1-6alkyl;
R5 is R6, R6CO, R6OCO, R6OCH (R6') CO, R6NHCH (R6')CO, R6SCH(R6')CO, R6SO2 or R6SO;
R6 and R6' are H, C1-6alkyl, C3-7cycloalkyl, Ar,
Ar-C1-4alkyl, Het, Het-C1-4alkyl, Ar-(CH2)pCH(Ar) (CH2)p or Ar-C3-7cycloalkyl;
Ar is phenyl or phenyl substituted by one to three C1-6alkyl, trifluoromethyl, halogen, R'O or R'S; and
Figure imgf000082_0003
is a five or six-membered, mono or bicyclic ring containing one or two nitrogen atoms;
m is: (1) 0-4, when W is NR'-(C=NH)NHR';
(2) 0-5, when W is (C=NH)NHR' or NR'(C=NH)R'; or
(3) 0-6, when W is NHR'; n is 0-2;
p is 0-3;
q is: (1) 1-4, when W is NR'- (C=NH)NHR' ;
(2) 2-5, when W is (C=NH)NHR' or NR' (C=NH) R' ; or
(3) 3-6, when W is NHR';
or a pharmaceutically acceptable salt thereof, which are effective for inhibiting platelet aggregation, pharmaceutical compositions for effecting such activity, and a method for inhibiting platelet aggregation.
PCT/US1992/000999 1991-02-05 1992-02-05 Anti-aggregatory peptides containing an aromatic ester or amide WO1992013552A1 (en)

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WO1993024448A1 (en) 1992-06-03 1993-12-09 Fuji Photo Film Co., Ltd. Amino acid derivative and use thereof
FR2693107A1 (en) * 1992-07-01 1994-01-07 Chauvin Laboratoire Means for the prevention of secondary cataract.
EP0584066A1 (en) * 1991-04-11 1994-03-02 Rhone-Poulenc Rorer International (Holdings) Inc. Anti-thrombotic peptide and pseudopeptide derivatives
WO1994022907A1 (en) * 1993-03-26 1994-10-13 Hoechst Aktiengesellschaft New urea derivates, their preparation and their use
EP0677043A1 (en) * 1992-12-29 1995-10-18 Smithkline Beecham Corporation Platelet aggregation inhibiting compounds
US5494922A (en) * 1993-06-28 1996-02-27 Zeneca Limited Allophanic acid derivatives
US5556977A (en) * 1993-03-29 1996-09-17 Zeneca Limited Heterocyclic derivatives
US5563141A (en) * 1993-03-29 1996-10-08 Zeneca Limited Heterocyclic compounds
US5576334A (en) * 1993-06-28 1996-11-19 Zeneca Limited Acylurea derivatives
US5602155A (en) * 1995-01-17 1997-02-11 G. D. Searle & Co. Platelet aggregation inhibitors
US5612373A (en) * 1993-06-28 1997-03-18 Zeneca Limited Certain diacyl hydrazine derivatives
US5639765A (en) * 1995-01-17 1997-06-17 G. D. Searle & Co. Guanidinoalkyl glycine β-amino acids useful for inhibiting bone loss
US5652242A (en) * 1993-03-29 1997-07-29 Zeneca Limited Heterocyclic derivatives
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5681820A (en) * 1995-05-16 1997-10-28 G. D. Searle & Co. Guanidinoalkyl glycine β-amino acids useful for inhibiting tumor metastasis
US5750754A (en) * 1993-03-29 1998-05-12 Zeneca Limited Heterocyclic compounds
US5753659A (en) * 1993-03-29 1998-05-19 Zeneca Limited Heterocyclic compouds
US5763408A (en) * 1992-06-03 1998-06-09 Fuji Photo Film Co., Ltd. Amino acid derivatives and application thereof
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6326403B1 (en) * 1998-07-15 2001-12-04 Merck Patent Gesellschaft Mit Diacylhydrazine derivatives as integrin inhibitors
WO2002066421A1 (en) 2001-02-21 2002-08-29 Provincia Italiana Della Congregazione Dei Figli Dell'immacolata Concezione - Istituto Dermopatico Dell'immacolata Rgd-analog non-peptidic molecules having anti-adhesive, anti-migration and anti-proliferative effects
US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis

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US6521594B1 (en) 1990-04-06 2003-02-18 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5780303A (en) * 1990-04-06 1998-07-14 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US6017877A (en) * 1990-04-06 2000-01-25 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
US5672585A (en) * 1990-04-06 1997-09-30 La Jolla Cancer Research Foundation Method and composition for treating thrombosis
EP0584066A1 (en) * 1991-04-11 1994-03-02 Rhone-Poulenc Rorer International (Holdings) Inc. Anti-thrombotic peptide and pseudopeptide derivatives
EP0584066A4 (en) * 1991-04-11 1994-10-12 Rhone Poulenc Rorer Int Anti-thrombotic peptide and pseudopeptide derivatives.
US5763408A (en) * 1992-06-03 1998-06-09 Fuji Photo Film Co., Ltd. Amino acid derivatives and application thereof
WO1993024448A1 (en) 1992-06-03 1993-12-09 Fuji Photo Film Co., Ltd. Amino acid derivative and use thereof
WO1994001456A1 (en) * 1992-07-01 1994-01-20 Laboratoire Chauvin Oligopeptide collagen derivatives and their use in secondary cataract prevention
FR2693107A1 (en) * 1992-07-01 1994-01-07 Chauvin Laboratoire Means for the prevention of secondary cataract.
EP0677043A1 (en) * 1992-12-29 1995-10-18 Smithkline Beecham Corporation Platelet aggregation inhibiting compounds
EP0677043A4 (en) * 1992-12-29 1995-11-22
WO1994022907A1 (en) * 1993-03-26 1994-10-13 Hoechst Aktiengesellschaft New urea derivates, their preparation and their use
US5703050A (en) * 1993-03-26 1997-12-30 Hoechst Aktiengesellschaft Urea derivatives, their preparation and use
US5753659A (en) * 1993-03-29 1998-05-19 Zeneca Limited Heterocyclic compouds
US5750754A (en) * 1993-03-29 1998-05-12 Zeneca Limited Heterocyclic compounds
US5556977A (en) * 1993-03-29 1996-09-17 Zeneca Limited Heterocyclic derivatives
US5563141A (en) * 1993-03-29 1996-10-08 Zeneca Limited Heterocyclic compounds
US5652242A (en) * 1993-03-29 1997-07-29 Zeneca Limited Heterocyclic derivatives
US5728701A (en) * 1993-03-29 1998-03-17 Zeneca Limited Heterocyclic derivatives
US5576334A (en) * 1993-06-28 1996-11-19 Zeneca Limited Acylurea derivatives
US5760057A (en) * 1993-06-28 1998-06-02 Zeneca Limited Certain (piperidin-4-yl-alkanoyl)carbazoyl!-carboxy-phenoxy derivatives
US5612373A (en) * 1993-06-28 1997-03-18 Zeneca Limited Certain diacyl hydrazine derivatives
US5981531A (en) * 1993-06-28 1999-11-09 Zeneca Limited Acid derivatives
US5494922A (en) * 1993-06-28 1996-02-27 Zeneca Limited Allophanic acid derivatives
US5602155A (en) * 1995-01-17 1997-02-11 G. D. Searle & Co. Platelet aggregation inhibitors
US5639765A (en) * 1995-01-17 1997-06-17 G. D. Searle & Co. Guanidinoalkyl glycine β-amino acids useful for inhibiting bone loss
US5681820A (en) * 1995-05-16 1997-10-28 G. D. Searle & Co. Guanidinoalkyl glycine β-amino acids useful for inhibiting tumor metastasis
US6326403B1 (en) * 1998-07-15 2001-12-04 Merck Patent Gesellschaft Mit Diacylhydrazine derivatives as integrin inhibitors
WO2002066421A1 (en) 2001-02-21 2002-08-29 Provincia Italiana Della Congregazione Dei Figli Dell'immacolata Concezione - Istituto Dermopatico Dell'immacolata Rgd-analog non-peptidic molecules having anti-adhesive, anti-migration and anti-proliferative effects
US6627769B2 (en) 2001-02-21 2003-09-30 Provincia Italiana Della Congregzaione Dei Figli Dell'immacolata Concezione - Istituto Dermopatico Dell'immacolata RGD-analog non-peptidic molecules having anti-adhesive, anti-migration and anti-proliferative effects

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EP0570507A4 (en) 1994-09-21
EP0570507A1 (en) 1993-11-24

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