WO1994029335A1 - New peptides derivatives - Google Patents

New peptides derivatives Download PDF

Info

Publication number
WO1994029335A1
WO1994029335A1 PCT/SE1994/000534 SE9400534W WO9429335A1 WO 1994029335 A1 WO1994029335 A1 WO 1994029335A1 SE 9400534 W SE9400534 W SE 9400534W WO 9429335 A1 WO9429335 A1 WO 9429335A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
phe
cha
carbon atoms
compound according
Prior art date
Application number
PCT/SE1994/000534
Other languages
French (fr)
Inventor
Karl Thomas Antonsson
Ruth Bylund
Original Assignee
Astra Aktiebolag
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Astra Aktiebolag filed Critical Astra Aktiebolag
Priority to AU69408/94A priority Critical patent/AU685465B2/en
Priority to EP94917871A priority patent/EP0701567A1/en
Priority to JP7501664A priority patent/JPH08511017A/en
Publication of WO1994029335A1 publication Critical patent/WO1994029335A1/en

Links

Classifications

    • 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
    • C07K5/06—Dipeptides
    • C07K5/06139—Dipeptides with the first amino acid being heterocyclic
    • C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C07K5/06—Dipeptides
    • C07K5/06008—Dipeptides with the first amino acid being neutral
    • C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • this invention relates to protease inhibition and treatment of inflammatory diseaes. More specifically this invention relates to new competitive inhibitors of trypsin-like serine proteases such as kininogenases, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds for treatment of inflammatory disorders, e.g. asthma, rhinitis, urticaria, inflammatory bowel diseaes, and arthritis.
  • trypsin-like serine proteases such as kininogenases
  • pharmaceutical compositions containing the compounds as active ingredients e.g. asthma, rhinitis, urticaria, inflammatory bowel diseaes, and arthritis.
  • Kininogenases are serine proteases .that act on kininogens to produce kinins (bradykinin, kallidin, and Met-Lys- bradykinin) . Plasma kallikrein, tissue kallikrein, and mast cell tryptase represent important kininogenases.
  • Kinins are generally involved in inflammation.
  • the active inflammation process is associated with increased permeability of the blood vessels resulting in extravasation of plasma into the tissue.
  • the ensuing plasma exudate contains all the protein systems of circulating blood.
  • the plasma-derived kininogens inevitably will be interacting with different kallikreins, forming kinins continually as long as the active plasma exudation process is ongoing.
  • Plasma exudation occurs independent of the mechanisms that are involved in the inflammation, whether it is allergy, infection or other factors (Persson et al., Editorial, Thorax, 1992, 47:993-1000).
  • Plasma exudation is thus a feature of many diseases including asthma, rhinitis, common cold, and inflammatory bowel diseases. Particulary in allergy mast cell tryptase will be released (Salomonsson et al.. Am. Rev. Respir. Dis., 1992, 146:1535-1542) to contribute to kinin formation and other pathogenic events in asthma, rhinitis, and intestinal diseases.
  • the kinins are biologically highly active substances with smooth muscle effects, sectretory effects, neurogenic effects, and actions that may perpetuate inflammatory processes including activation of phospholipase A 2 and increasing vascular permeability. The latter action potentially induces a vicious circle with kinins providing for the generation of more kinins etc.
  • Tissue kallikrein cleaves primarily low molecular weight kininogen to produce kallidin and plasma kallikrein preferably releases bradykinin from high molecular weight kininogen.
  • arginine chloro ethyl ketones were reported as plasma kallikrein inhibitors by Kettner and Shaw in Biochemistry 1978, 17:4778-4784 and Meth. Enzy . 1981, 80:826-842.
  • esters and amides were reported by Fareed et al. in Ann. N.Y. Acad. Sci. 1981, 370:765-784 to be plasma kallikrein inhibitors.
  • protease enzym inhibitors based on analogues of peptidase substrates, including kallikrein, are described.
  • Inhibitors of trypsin like serine proteases such as thrombin and kallikrein, based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof have been reported in EP-A2-0,293,881.
  • An objective of the present invention is to provide novel and potent kallikrein inhibitors with competitive inhibitory activity towards the enzyme i.e. causing reversible inhibition.
  • a further objective is to obtain inhibitors which can be given orally, dermally, rectally, or via the inhalation route.
  • n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4;
  • a 1 represents a structural fragment of Formulae Ila, lib, He, lid or He; no ⁇ d wherein:
  • p is an integer 0,1 or 2;
  • n is an integer 1, 2, 3, or 4, preferably 2;
  • q is an integer 0,1 or 2, preferably 1;
  • R 1 represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R 11 00C-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R 11 is H or an alkyl group having 1 to 4 carbon atoms, or
  • R 1 represents R 12 00C-l,4-phenyl-CH 2 -, wherein R 12 is H or an alkyl group having 1 to 4 carbon atoms, or
  • R 1 represents R 13 -NH-C0-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and wherein R 13 is H or an alkyl group having 1 to 4 carbon atoms or -CH 2 COOR 12 , wherein R 12 is as defined above, or R 1 represents R 14 S0 2 -, Ph(4-COOR 12 )-S0 2 -, Ph(3-COOR - 1122 )) -S0 2 -, or
  • R 1 represents CO-R 15 , wherein R 15 is an alkyl group having 1-4 carbon atoms, or
  • R 1 represents CO-OR 15 , wherein R 15 is as defined above, or
  • R 1 represent CO-(CH 2 ) p -COOR 12 , wherein R 12 and p are as defined above, or
  • R 1 represents -CH 2 PO(OR 16 ) 2 , wherein R 16 is, individually at each occurrence, H, methyl or ethyl;
  • R 2 represents H or an alkyl group having 1 to 4 carbon atoms or R 21 OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R 22 -(CH 2 ) p -, wherein p is as defined above and R 22 is methyl, phenyl, OH, COOR 21 , and R 21 is H or an alkyl group having 1 to 4 carbon atoms;
  • R 3 represents an alkyl group having 1-4 carbon atoms, or
  • R 3 represents a cyclohexyl- or cyclopentyl group
  • R 3 represents a phenyl group which may or may not be substituted with an alkyl group having 1 to 4 carbon atoms, or with a group OR 21 , wherein R 21 is as defined above or
  • a ⁇ 2 2 represents a structural fragment
  • R 3 and p are as defined above;
  • alkyl group may be straight or branched unless specified otherwise.
  • Alkyl groups having 1 to 4 carbon atoms are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. When unsaturation is referred to, a carbon- carbon double bond is intended. Abbreviations are listed at the end of this specification.
  • compounds of the general Formula I are potent inhibitors of trypsin-like serine proteases and especially plasma and/or tissue kallikrein:
  • Compounds of Formula I having S-configuration on the A 2 amino acid are preferred ones, of those compounds also having R-configuration on the A 1 amino acid are particularly preferred ones.
  • Preferred compounds of the invention include:
  • Particularly preferred compounds are ;
  • the invention also provides compositions and methods for the treatment of physiological disorders and especially inflammatory diseases such as asthma, rhinitis, pancreatitis, uticaria, inflammatory bowel diseaes, and arthritis.
  • An effective amount of Formula I with or without a physiologically acceptable carrier or diluent can be used solely or in combination with other therapeutic agents.
  • the compositions may be administered via oral, dermal, nasal, tracheal, bronchial, parenteral, or rectal routes at varying doses.
  • the compounds inhibit the activity of kallikreins assessed with chromogenic substrates according to known procedures.
  • the anti-inflammatory actions of the present compounds can for example be studied by their inhibition of allergen- induced exudative inflammatory processes in airway mucosa or gut mucosa.
  • K L determinations were made with a chromogenic substrate method, and performed on a Cobas Bio centrifugal analyzer manufactured by Roche (Basel, Switzerland). Residual enzyme activity after incubation of human plasma kallikrein with various concentrations of test compound was determined at three different substrate concentrations, and measured as change in optical absorbance at 405 nm and 37°C.
  • Human plasma kallikrein (E.C.3.4.21.34, Chromogenix AB, M ⁇ lndal, Sweden), 250 ⁇ l of 0.4 nkat/ml in buffer (0.05 mol/1 Tris-HCl, pH 7.4, 1 0.15 adjusted with NaCl) with bovine albumin 5 g/1 (cat no 810033, ICI Biochemicals Ltd, High Wycombe, Bucks, GB) , was incubated for 300 s with 80 ⁇ l of test compound solution in 0.15 mol/1 NaCl containing albumin 10 g/1. An additional 10 ⁇ l of water was supplied in this step. Then 40 ⁇ l of kallikrein substrate (S-2302, Chromogenix AB, 1.25, 2.0 or 4.0 mmol/1 in water) was added together with another 20 ⁇ l of water, and the absorbance change monitored.
  • buffer 0.05 mol/1 Tris-HCl, pH 7.4, 1 0.15 adjusted with NaCl
  • bovine albumin 5 g/1 catalog no 810033
  • the compounds of the Formula I will normally be administered by the oral, rectal, dermal, nasal or parenteral route in the form of pharmaceutical preparations comprising the active ingredient either as a free base or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate and trifluoroacetate and the like in a pharmaceutically acceptable dosage form.
  • a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate and trifluoroacetate and the like in a pharmaceutically acceptable dosage form.
  • the dosage form may be a solid, semisolid or liquid preparation prepared by per se known techniques.
  • the active substance will constitute between 0.1 and 99 % by weight of the preparation, more specifically between 0.1 and 50 % by weight for preparations intended for parenteral administration and between 0.2 and 75 % by weight for preparations suitable for oral administration.
  • Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 g/kg body weight at parenteral administration.
  • a further objective of the invention is the mode of preparation of the compounds.
  • the compounds of Formula I may be prepared by coupling of an N-terminally protected dipeptide (W 1 -A 1 -A 2 -OH) or amino acid (W 1 -A 2 -OH) , when a N- terminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound
  • a 1 , A 2 and n are as defined with Formula I and X is an unprotected or protected guanidino group or a protected amino group, or a group transferable into an amino group, where the amino group is subsequently transferred into an unprotected or protected guanidino group, followed by removal of the protecting group(s) or deprotecting of the N-terminal nitrogen followed by alkylation of the N-terminal nitrogen and deprotection by known methods.
  • the coupling is accordingly done by one of the following methods:
  • a 2 , ⁇ , H ⁇ and X are as defined above followed by deprotection of the W 1 -group and coupling with the N-terminaJ amino acid, in a protected form, leading to the protected / 0534
  • the 1 H NMR and 13 C NMR measurements were performed on BRUKER AC-P 300, BRUKER 200 and BRUKER AM 500 spectrometers, the former operating at a frequency of 500.14 MHz and a 13 C freguency of 125.76 MHz and the latter at H and 13 C freguency of 300.13 MHz and 75.46 MHz respectively.
  • the samples were 10-50 mg dissolved in 0.6 ml of either of the following solvents; CDC1 3 (isotopic purity > 99.8%, Dr. Glaser AG Basel), CD 3 0D (isotopic purity > 99.95%, Dr. Glaser AG Basel) or D 2 0 (isotopic purity > 99.98%, Dr. Glaser AG Basel) .
  • the H and 13 C chemical shift values in CDC1 3 and CD 3 OD are relative to tetra ethylsilane as an external standard.
  • the H chemical shifts in D 2 0 are relative to the sodium salt of 3- (trimethylsilyl)-d 4 -propanoic acid and the 13 C chemical shifts in D 2 0 are referenced relative to 1,4-dioxane (67.3 ppm), both as external standard. Calibrating with an external standard may in some cases cause minor shift differences compared to an internal standard, however, the difference in 1 H chemical shift is less than 0.02 pp_ and in 13 C less than 0.1 ppm.
  • Thin-Layer C hromatography was carried out on commercial Merck S ilicagel 60 F 254 coated glass or aluminium plates. Visualization was by a combination of UV-light, followed by spraying with a solution prepared by mixing 372 ml of Et0H(95%), 13.8 ml of concentrated H 2 S0 4 , 4.2 ml of concentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde or phosphomolybdic acid reagent (5-10 w.t % in Et0H(95%)) and heating.
  • Freeze-drying was done on a Leybold-Heraeus, model Lyovac G T 2 , apparatus.
  • Boc-(R)Cha- O H (1 eq.), HOSu (1.1 eq) and D CC or C ME- C DI ( 1.1 eq) were dissolved in acetonitrile (about 2.5 ml/mmol acid) and stirred at room temperature over night. The precipitate formed during the reaction was filtered off, the solvent evaporated and the product dried in vacuo. (When CME-CDI was used in the reaction the residue, after evaporation of the CH 3 CN, was dissolved in EtOAc and the organic phase washed with water and dried. Evaporation of the solvent gave the title compound) .
  • Boc-NH-(CH 2 ) 3 -NH 2 x HCl (prepared according to Mattingly P.G., Synthesis, 367 (1990)) (3.9 kg, 18.5 mol) in iso-propanol (24 kg) at 60-70° C was added in portions over a 30 minutes period KHC0 3 (4.2 kg, 42 mol). A slow evolution of C0 2 (g) occurs. The mixture was stirred for another 30 minutes followed by addition in portions over a 30 minutes period N-bensyloxycarbonyl-O-methyl isourea (3.74 kg, 18.0 mol). The reaction mixture was stirred at 65-70° C for 16 h, cooled to 20° C and filtered.
  • the precipitate was washed with iso-propanol (10 + 5 L) .
  • the combined filtrates was concentrated at reduced pressure keeping the heating mantle not warmer than 65-70° C.
  • EtOAc 90 L was added.
  • the reaction mixture was cooled to 20-25° C, washed with water (10 and 5 L) and brine (5 L) , and dried with Na 2 S0 4 (2 kg) .
  • After stirring the rection mixture was filtered and the filter cake was washed with EtOAc (11 and 7 L) .
  • the combined filtates were concentrated at reduced pressure keeping the heating mantle not warmer than 40-50° C.
  • Boc-(R)Cha-0H was dissolved in acetonitrile (200 mL) , N-hydroxisuccinimide (9.9 g, 81 mmol) was added. Dicyclohexylcarbodiimide (17.8 g, 81 mmol) was then added slowly and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered off and the Boc- (R)Cha-OSu containing solution was evaporated. Phe-OH (48.7 g, 195 mmol), sodiumhydroxide (10.3 g, 258 mmol), water (270 mL) and finally dimethylformamide (70 mL) were added to a reaction vessel while stirring.
  • Boc-(R)Cha-OSu was dissolved in dimethylformamide (200 mL) and added slowly to the reaction vessel while maintaining the reaction temperature below 5 ⁇ C. After 3 h the solution was evaporated, the residue dissolved in water (1000 mL) and extracted with ethylacetate (2 x 300 mL) . The aqueous phase was acidified with potassium hydrogensulfate (1M) to pH 3 and extracted with ethylacetate
  • H 2 N-(R)Cha-Phe-Nag(Z) 300 mg, 0.55 mmol was dissolved in ethanol (50 mL) and trifluoroaceticacid (56 ⁇ L, 0.73 mmol) was added. The mixture was sonicated and palladium on charcoal (5%, 50 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 19 h. The suspension was filtered through celite and after the solvent was evaporated the title compound (0.13 g, 0.31 mmol) was isolated in 56% yield.
  • H 2 N-(R)Cha-Phe-Nag(Z) 500 mg, 0.91 mmol
  • p-toluenesulphonic acid 173 mg, 0.91 mmol
  • methanol 7.5 L
  • Acetaldehyde 51 ⁇ L, 0.91 mmol
  • sodium cyanoborohydride 86 mg, 1.36 mmol
  • H 2 N-(R)Cha-Phe-Nag(Z) 500 mg, 0.91 mmol was dispersed in acetonitrile (5 mL) .
  • Methyloxalylchloride 104 ⁇ L, 1.14 mmol was added to the slurry. After 60 minutes the starting material was consumed, confirmed by HPLC, and the clear solution was evaporated.
  • the crude methylester was hydrolyzed by dissolving the residue in tetrahydrofuran (4 mL) and adding LiOH (115 mg, 2.73 mmol) dissolved in water (2 mL) . After 90 min.
  • HOOC-CO-(R)Cha-Phe-Nag(Z) (210 mg, 0.34 mmol) was dispersed in tetrahydrofuran (25 mL) and acitic acid (20 mL) was added. Palladium on charcoal (5%, 30 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 25 h. The suspension was filtered through celite and the filter cake was washed with tetrahydrofuran and after the solvent was evaporated the crude product (257 mg) was collected. After azeotropic evaporation with three portions of toluene (tot; 50 mL) and overnight drying under vacuum the product (140 mg, 0.29 mmol) was isolated in 85% yield.
  • compositions A The compounds according to the invention can be formulated in solid dosage forms for oral administration or for topical administration to the intestines.
  • the active constituent is mixed with lactose and micro- crystalline cellulose and magnesium stearate is admixed and tablets are compressed from the mixture.
  • the active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of hydroxypropylmethylcellulose, polyethyleneglycol, talc and titandioxide in water.
  • the active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling microcrystalline cellulose and magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of Eudragit L, dibutylphtalate and talc in isopropanol/aceton.
  • the active constituent is mixed with lactose and micro ⁇ crystalline cellulose and granulated with hydroxypropyl cellulose in water.
  • the granulation is extruded, spheronized and dried.
  • the granules are first coated with ethylcellulose dispersion with acetyltributylcitrate and then with Eudragit L30D dispersion with triethylcitrate and talc.
  • the granules are filled in gelatin capsules each containing 10 mg of active constituent.
  • the active constituent is mixed with lactose and micro ⁇ crystalline cellulose and granulated with hydroxypropyl cellulose in water.
  • the granulation is extruded, spheronized and dried.
  • the granules are coated with a dispersion of
  • Eudragit NE30D, Eudragit SlOO and talc in water The granules are filled in gelatin capsules each containing 100 mg of active constituent.
  • the compounds according to the invention can be formulated in pressurized aerosols or in dry powder inhalers for oral or nasal inhalation.
  • the kininogenase inhibitor is micronized to a particle size suitable for inhalation therapy (mass median diameter ⁇ 4 ⁇ m) .
  • the micronized substance is suspended in a liquid propellant mixture and filled into a container which is sealed with a metering valve.
  • the kininogenase inhibitor can be dissolved in the liquid propellant mixture with the aid of ethanol.
  • the propellants used may by chlorofluorocarbons (CFCs) or hydrofluoroalkanes (HFAs) of different formulae.
  • CFCs chlorofluorocarbons
  • HFAs hydrofluoroalkanes
  • the most frequent used CFCs are trichloromonofluoromethane (propellant 11) and dichlorodifluoromethane (propellant 12) and dichlorotetrafluoroethane (propellant 114).
  • the most frequent used HFAs are tetrafluoromethane (propellant 134a) and heptafluoropropane (propellant 227).
  • surfactant such as sorbitan trioleate, lecithin, oleic acid or other suitable substance may be used to improve the physical stability.
  • Etanol may be used as surfactant or as a medium to increase the solubility of active substance in the propellant mixture.
  • micronized kininogenase inhibitor may be used alone or mixed with a carrier substance such as lactose, mannitol or glucose.
  • a carrier substance such as lactose, mannitol or glucose.
  • Another possibility is to process the micronized powder into spheres which break up during the dosing procedure.
  • This powder or spheronized powder is filled into the drug reservoir in a singledose or multidose inhaler, e.g. the latter being Turbuhaler®.
  • a dosing unit meters the desired dose which is inhaled by the patient.
  • the kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter ⁇ 4 ⁇ m) .
  • 100 mg of the micronized powder is filled into a powder multidose inhaler (Turbuhaler®) .
  • the inhaler is equipped with a dosing unit which delivers a dose of 1 mg.
  • the kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter ⁇ 4 ⁇ m) .
  • 150 mg of the micronized powder is filled into a powder multidose inhaler (Turbuhaler®) .
  • the inhaler is equipped with a dosing unit which delivers a dose of 0.5 mg.
  • Boc tertiary butoxy carbonyl
  • LiOH Lithium hydroxide
  • NMM N-methyl morpholine
  • Ph phenyl
  • n, s, i and t have their usual meanings: normal, iso, sec and tertiary.
  • the stereochemistry for the amino acids is by default (S) if not otherwise stated.

Abstract

A compound of the general formula (I): A1 - A2 -NH-(CH¿2?)n - NH-C(NH)-NH2, wherein n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4; A?1¿ represents a structural fragment of formulae (IIa), (IIb), (IIc), (IId) or (IIe); A2 represents a structural fragment (a), as well as processes for the preparation thereof, the use and the pharmaceutical formulations.

Description

New peptide derivatives
DESCRIPTION
In its broad sense this invention relates to protease inhibition and treatment of inflammatory diseaes. More specifically this invention relates to new competitive inhibitors of trypsin-like serine proteases such as kininogenases, their synthesis, pharmaceutical compositions containing the compounds as active ingredients, and the use of the compounds for treatment of inflammatory disorders, e.g. asthma, rhinitis, urticaria, inflammatory bowel diseaes, and arthritis.
BACKGROUND
Kininogenases are serine proteases .that act on kininogens to produce kinins (bradykinin, kallidin, and Met-Lys- bradykinin) . Plasma kallikrein, tissue kallikrein, and mast cell tryptase represent important kininogenases.
Kinins (bradykinin, kallidin) are generally involved in inflammation. For example, the active inflammation process is associated with increased permeability of the blood vessels resulting in extravasation of plasma into the tissue. The ensuing plasma exudate contains all the protein systems of circulating blood. The plasma-derived kininogens inevitably will be interacting with different kallikreins, forming kinins continually as long as the active plasma exudation process is ongoing. Plasma exudation occurs independent of the mechanisms that are involved in the inflammation, whether it is allergy, infection or other factors (Persson et al., Editorial, Thorax, 1992, 47:993-1000). Plasma exudation is thus a feature of many diseases including asthma, rhinitis, common cold, and inflammatory bowel diseases. Particulary in allergy mast cell tryptase will be released (Salomonsson et al.. Am. Rev. Respir. Dis., 1992, 146:1535-1542) to contribute to kinin formation and other pathogenic events in asthma, rhinitis, and intestinal diseases.
The kinins are biologically highly active substances with smooth muscle effects, sectretory effects, neurogenic effects, and actions that may perpetuate inflammatory processes including activation of phospholipase A2 and increasing vascular permeability. The latter action potentially induces a vicious circle with kinins providing for the generation of more kinins etc.
Tissue kallikrein cleaves primarily low molecular weight kininogen to produce kallidin and plasma kallikrein preferably releases bradykinin from high molecular weight kininogen.
PRIOR ART
Inhibitors of kallikrein based on the amino acid sequence around the cleavage site (-Ser-Pro-Phe-Arg — Ser-Ser-Arg-) have been reported earlier.
The arginine chloro ethyl ketones were reported as plasma kallikrein inhibitors by Kettner and Shaw in Biochemistry 1978, 17:4778-4784 and Meth. Enzy . 1981, 80:826-842.
Likewise, esters and amides were reported by Fareed et al. in Ann. N.Y. Acad. Sci. 1981, 370:765-784 to be plasma kallikrein inhibitors.
In EP-A2-0,195,212 protease enzym inhibitors, based on analogues of peptidase substrates, including kallikrein, are described.
Inhibitors of trypsin like serine proteases, such as thrombin and kallikrein, based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof have been reported in EP-A2-0,293,881.
In WO 92/04371 a series of kallikrein inhibitors with carbonyl-activating or binding groups are described.
DISCLOSURE OF THE INVENTION
An objective of the present invention is to provide novel and potent kallikrein inhibitors with competitive inhibitory activity towards the enzyme i.e. causing reversible inhibition. A further objective is to obtain inhibitors which can be given orally, dermally, rectally, or via the inhalation route.
According to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including stereoisomers, are potent inhibitors of serine proteases and especially kallikreins:
A1 - A2 -NH-(CH2)n- NH-C(NH)-NH2
Formula I
wherein:
n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4;
A1 represents a structural fragment of Formulae Ila, lib, He, lid or He;
Figure imgf000006_0001
no πd wherein:
p is an integer 0,1 or 2;
m is an integer 1, 2, 3, or 4, preferably 2;
q is an integer 0,1 or 2, preferably 1;
R1 represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R1100C-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R11 is H or an alkyl group having 1 to 4 carbon atoms, or
R1 represents R1200C-l,4-phenyl-CH2-, wherein R12 is H or an alkyl group having 1 to 4 carbon atoms, or
R1 represents R13-NH-C0-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and wherein R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COOR12, wherein R12 is as defined above, or R1 represents R14S02-, Ph(4-COOR12)-S02-, Ph(3-COOR -1122)) -S02-, or
Ph(2-C00R12)-S02-, wherein R12 is as defined above and R14 is an alkylgroup having 1-4 carbon atoms, or
R1 represents CO-R15, wherein R15 is an alkyl group having 1-4 carbon atoms, or
R1 represents CO-OR15, wherein R15 is as defined above, or
R1 represent CO-(CH2)p-COOR12, wherein R12 and p are as defined above, or
R1 represents -CH2PO(OR16)2, wherein R16 is, individually at each occurrence, H, methyl or ethyl;
R2 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p is as defined above and R22 is methyl, phenyl, OH, COOR21, and R21 is H or an alkyl group having 1 to 4 carbon atoms;
R3 represents an alkyl group having 1-4 carbon atoms, or
R3 represents a cyclohexyl- or cyclopentyl group, or
R3 represents a phenyl group which may or may not be substituted with an alkyl group having 1 to 4 carbon atoms, or with a group OR21, wherein R21 is as defined above or
R3 represents a 1-napthyl, 2-naphtyl, 4-pyridyl, 3- pyrrolidyl, or a 3-indolyl group which may or may not be substituted with OR21 wherin R21 is as defined above and with p = 1; or
R3 represent a cis- or trans-decalin group with p = 1; or R3 represents Si(Me)3 or CH(R31)2, wherein R31 is a cyclohexyl- or phenyl group;
A ι 22 represents a structural fragment
- NH - CH - C I
Figure imgf000008_0001
wherein R3 and p are as defined above;
An alkyl group may be straight or branched unless specified otherwise. Alkyl groups having 1 to 4 carbon atoms are methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, s-butyl and t-butyl. When unsaturation is referred to, a carbon- carbon double bond is intended. Abbreviations are listed at the end of this specification.
According to the invention it has been found that compounds of the general Formula I, either as such or in the form of physiologically acceptable salts, and including stereoisomers, are potent inhibitors of trypsin-like serine proteases and especially plasma and/or tissue kallikrein: Compounds of Formula I having S-configuration on the A2 amino acid are preferred ones, of those compounds also having R-configuration on the A1 amino acid are particularly preferred ones.
Preferred compounds of the invention include:
H-(R)Cha-Phe-Agm H00C-CH2-(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag HOOC-CH2-(R)Cha-Phe-Nag
CH3-CO-(R)-Cha-Phe-Nag
CH3-CH2-(R)-Cha-Phe-Nag
HOOC-CO-(R)-Cha-Phe-Nag HOOC-CH2-(R)Phe-Phe-Agm
HOOC-CH2-(R)Phe-Cha-Agm
HOOC-CH2-(R)Cha-Cha-Agm
HOOC-CH2-(R)Phe-Phe-Nag
HOOC-CH2-(R)Phe-Cha-Nag HOOC-CH2-(R)Cha-Cha-Nag
HOOC-CH2-(R)Cha-αNal-Agm
HOOC-CH2-(R)Cha-0Nal-Agm
H-(R)Phe-Cha-Agm
H- (R) Phe-Cha-Nag H- (R) Phe-Phe-Agm
H- (R) Phe-Phe-Nag
CH3- (R) Phe-Phe-Agm
CH3- (R) Cha-Phe-Agm
CH3- (R) Phe-Cha-Agm HOOC-CH2- (R) Pro-Phe-Agm
HOOC-CH2- (R) Pro-Phe-Nag
H- (R) Pro-Phe-Agm
H- (R) Pro-Phe-Nag
CH3- (R) Pro-Phe-Agm CH3- (R) Pro-Phe-Nag
Particularly preferred compounds are ;
H- (R) Cha-Phe-Agm HOOC-CH2- (R) Cha-Phe-Agm
H- (R) Cha-Phe-Nag
HOOC-CH2- (R) Cha-Phe-Nag
CH3-CO- (R) -Cha-Phe-Nag
CH3-CH2- (R) -Cha-Phe-Nag HOOC-CO- (R) -Cha-Phe-Nag The best mode according to the invention known at present is to use the compound according to Example 4 namely.
HOOC-CH2-(R)Cha-Phe-Nag
Medical and pharmaceutical use
The invention also provides compositions and methods for the treatment of physiological disorders and especially inflammatory diseases such as asthma, rhinitis, pancreatitis, uticaria, inflammatory bowel diseaes, and arthritis. An effective amount of Formula I with or without a physiologically acceptable carrier or diluent can be used solely or in combination with other therapeutic agents. Depending upon the disorder and patient to be treated the compositions may be administered via oral, dermal, nasal, tracheal, bronchial, parenteral, or rectal routes at varying doses.
The compounds inhibit the activity of kallikreins assessed with chromogenic substrates according to known procedures. The anti-inflammatory actions of the present compounds can for example be studied by their inhibition of allergen- induced exudative inflammatory processes in airway mucosa or gut mucosa.
Determinaton of the inhibition constant KL for plasma kallikrein.
KL determinations were made with a chromogenic substrate method, and performed on a Cobas Bio centrifugal analyzer manufactured by Roche (Basel, Switzerland). Residual enzyme activity after incubation of human plasma kallikrein with various concentrations of test compound was determined at three different substrate concentrations, and measured as change in optical absorbance at 405 nm and 37°C. Human plasma kallikrein (E.C.3.4.21.34, Chromogenix AB, Mδlndal, Sweden), 250 μl of 0.4 nkat/ml in buffer (0.05 mol/1 Tris-HCl, pH 7.4, 1 0.15 adjusted with NaCl) with bovine albumin 5 g/1 (cat no 810033, ICI Biochemicals Ltd, High Wycombe, Bucks, GB) , was incubated for 300 s with 80 μl of test compound solution in 0.15 mol/1 NaCl containing albumin 10 g/1. An additional 10 μl of water was supplied in this step. Then 40 μl of kallikrein substrate (S-2302, Chromogenix AB, 1.25, 2.0 or 4.0 mmol/1 in water) was added together with another 20 μl of water, and the absorbance change monitored.
KL was evaluated from Dixon plots, i.e. diagrams of inhibitor concentration versus 1/ (ΔA/min) , where the data for the different substrate concentrations form straight lines which intercept at x= -KA.
Pharmaceutical preparations
The compounds of the Formula I will normally be administered by the oral, rectal, dermal, nasal or parenteral route in the form of pharmaceutical preparations comprising the active ingredient either as a free base or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, e.g. the hydrochloride, hydrobromide, lactate, acetate, citrate and trifluoroacetate and the like in a pharmaceutically acceptable dosage form.
The dosage form may be a solid, semisolid or liquid preparation prepared by per se known techniques. Usually the active substance will constitute between 0.1 and 99 % by weight of the preparation, more specifically between 0.1 and 50 % by weight for preparations intended for parenteral administration and between 0.2 and 75 % by weight for preparations suitable for oral administration.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 g/kg body weight at parenteral administration.
Preparation
A further objective of the invention is the mode of preparation of the compounds. The compounds of Formula I may be prepared by coupling of an N-terminally protected dipeptide (W1-A1-A2-OH) or amino acid (W1-A2-OH) , when a N- terminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound
H2N-(CH2)n-X
wherein A1, A2 and n are as defined with Formula I and X is an unprotected or protected guanidino group or a protected amino group, or a group transferable into an amino group, where the amino group is subsequently transferred into an unprotected or protected guanidino group, followed by removal of the protecting group(s) or deprotecting of the N-terminal nitrogen followed by alkylation of the N-terminal nitrogen and deprotection by known methods.
The coupling is accordingly done by one of the following methods:
Method I
Coupling of an N-terminally protected dipeptide, prepared by standard peptide coupling, with either a protected- or unprotected amino guanidine or a straight chain alkyla ine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling,shown in the formula λ- A1-A2-OH i H2N-(CH2)n-X Wα- A1'A -NH CH2)n-X wherein A1, A2 and n are as defined in Formula I , Wx is a N- te inal amino protecting group such as tert-butyloxy carbonyl and benzyloxy carbonyl and X is -NH-C(NH)-NH2, -NH-C(NH)-NH- W2, -N(W2)-C(NH)-NH- 2 -NH-C(NW2)-NH-W2 or -NH-W2, Where W2 is an amine protecting group such as tert-butyloxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide. The final compounds can be made in any of the following ways, depending on the nature of the X- group used: Removal of the protecting group(s) (when X= -NH-C(NH)-NH2, -N(W2)-C(NH)-NH- 2, -NH-C(N 2)-NH- 2 or -NH-C(NH)-NH-W2), or a selective deprotection of the V^- group (e.g when X= -NH-C(NH)-NH-W2,-N(W2)-C(NH)-NH- 2, -NH- C(NW2)-NH-W2, W2 in this case must be orthogonal to Wj) followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function (X= NH- 2 , W2 in this case must be orthogonal to W2 or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the V^-group.
Method II
Coupling of an N-terminally protected amino acid, prepared by standard methods, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula
Wj-A2-OR i H2U-(CH2)n-X
Figure imgf000013_0001
wherein A2, π, Hχ and X are as defined above followed by deprotection of the W1-group and coupling with the N-terminaJ amino acid, in a protected form, leading to the protected / 0534
12
peptide described in Method I. The synthesis to the final compounds is then continued according to Method I.
DETAILED DESCRIPTION OF THE INVENTION
The following description is illustrative of aspects of the invention.
EXPERIMENTAL PART
General Experimental Procedures.
The 1H NMR and 13C NMR measurements were performed on BRUKER AC-P 300, BRUKER 200 and BRUKER AM 500 spectrometers, the former operating at a frequency of 500.14 MHz and a 13C freguency of 125.76 MHz and the latter at H and 13C freguency of 300.13 MHz and 75.46 MHz respectively.
The samples were 10-50 mg dissolved in 0.6 ml of either of the following solvents; CDC13 (isotopic purity > 99.8%, Dr. Glaser AG Basel), CD30D (isotopic purity > 99.95%, Dr. Glaser AG Basel) or D20 (isotopic purity > 99.98%, Dr. Glaser AG Basel) .
The H and 13C chemical shift values in CDC13 and CD3OD are relative to tetra ethylsilane as an external standard. The H chemical shifts in D20 are relative to the sodium salt of 3- (trimethylsilyl)-d4-propanoic acid and the 13C chemical shifts in D20 are referenced relative to 1,4-dioxane (67.3 ppm), both as external standard. Calibrating with an external standard may in some cases cause minor shift differences compared to an internal standard, however, the difference in 1H chemical shift is less than 0.02 pp_ and in 13C less than 0.1 ppm. Thin-Layer Chromatography was carried out on commercial Merck Silicagel 60F254 coated glass or aluminium plates. Visualization was by a combination of UV-light, followed by spraying with a solution prepared by mixing 372 ml of Et0H(95%), 13.8 ml of concentrated H2S04, 4.2 ml of concentrated acetic acid and 10.2 ml of p-methoxy benzaldehyde or phosphomolybdic acid reagent (5-10 w.t % in Et0H(95%)) and heating.
Flash chromatography was carried out on Merck Silicagel 60 (40-63 mm, 230-400 mesh) under pressure of N2.
Freeze-drying was done on a Leybold-Heraeus, model Lyovac GT 2 , apparatus.
Protection Procedures
Boc-(R)Cha-OH
To a solution of H-(R)Cha-OH, 21.55 g (125.8 mmol), in 130 ml 1 M NaOH and 65 ml THF was added 30 g (137.5 mmol) of (Boc)2 0 and the mixture was stirred for 4.5 h at room temperature. The THF was evaporated and an additional 150 ml of water was added. The alkaline aqueous phase was washed twice with EtOAc, then acidified with 2 M KHS04 and extracted with 3 x 150 ml of EtOAc. The combined organic phase was washed with water, brine and dried (Na2S04) . Evaporation of the solvent afforded 30.9 g (90.5 %) of the title compound as a white solid.
Preparation of stating Materials
Boc- (R) Cha-OSU
Boc-(R)Cha-OH (1 eq.), HOSu (1.1 eq) and DCC or CME-CDI (1.1 eq) were dissolved in acetonitrile (about 2.5 ml/mmol acid) and stirred at room temperature over night. The precipitate formed during the reaction was filtered off, the solvent evaporated and the product dried in vacuo. (When CME-CDI was used in the reaction the residue, after evaporation of the CH3CN, was dissolved in EtOAc and the organic phase washed with water and dried. Evaporation of the solvent gave the title compound) .
XH-NMR (500 MHz, CDCl3, 2 rotamers ca: 1:1 ratio) S 0.85-1.1 (m, 2H), 1.1-1.48 (m, 4H), 1.5-1.98 (m, 16H; thereof 1.55 (bs, 9H)), 2.82 (bs, 4H), 4.72 (bs, 1H, major rota er) , 4.85 (bs, 1H, minor).
Boc-(R)Cha-Phe-OH
To a stirred mixture of 6.61 g (40 mmol) H-Phe-OH and 1.4 g of NaOH (35 mmol) in 60 ml DMF/H20 (1/1) at + 5 °C was added 3.68 g (10 mmol) Boc-(R)Cha-OSu and the mixture was allowed to reach room temperature. After 3 hours the solvent was evaporated and the residue was dissolved in 150 ml of water. The basic water phase was washed with 2 x 50 ml EtOAc, acidified with 1 M KHS04 and extracted with 2 x 100 L EtOAc. The combined organic phase was washed with 2 x 50 mL water and dried (MgS04) . Filtration and evaporation of the solvent gave 2.86 g (68%) of the title compound.
Figure imgf000016_0001
To a stirred solution of 2.81 g Boc-(R)Cha-Phe-OH (6.71 mmol) and 850 mg HOSu (7.38 mmol) in 30 mL of CH3CN was added 3.13 g CME-CDI (7.38 mmol) and the reaction was left at room temperature for 15 hours. The precipitate formed during the reaction was filtered off, the solvent evaporated and the reasidue was dissolved in 150 mL EtOAc. The organic phase was washed with 1 x 20 mL water, 1 x 20 mL Na2C03(aq), 2 x 20 mL water, 1 x 20 L brine and dried (MgS04) . Filtration followed by evaporation of the solvent gave 2.44 g (70%) of the title compound which was used without further purification. Boc-Nag ( Z )
(i) N-Bensyloxycarbonyl-O-methyl isourea
To a stirred solution of concentrated aqueous NaOH (2.8 L, 50% w/w, 19.1 M, 53 mol) and water (32 L) at 18° C was added in two portions 0-methylisourea hemisulphate (1.7 kg, 94%, 13.0 mol) and O-methylisourea hydrogensulphate (1.57 kg, 99%, 9.0 mol). The reaction mixture was cooled to 3-5° C. Benzyl chloroformiate (3.88 kg, 92%, 20.9 mol) was added over a 20 minutes period under cooling and vigorous stirring. The reaction temperature went from 3 to 8° C during the addition of Z-Cl. The addition funnel was rinsed with 5 litres of water which was added to the reactor. The reaction mixture was stirred at 0-3° C for 18 h, filtered and the crystals was washed with cooled (3° C) water (10 L) . Vacuum drying 25° C, 10-20 mbar) for 48 h gave 3.87 kg (89%) of the title compound as a white crystalline powder.
(ii) Boc-Nag(Z)
To a stirred solution Boc-NH-(CH2)3-NH2 x HCl (prepared according to Mattingly P.G., Synthesis, 367 (1990)) (3.9 kg, 18.5 mol) in iso-propanol (24 kg) at 60-70° C was added in portions over a 30 minutes period KHC03 (4.2 kg, 42 mol). A slow evolution of C02 (g) occurs. The mixture was stirred for another 30 minutes followed by addition in portions over a 30 minutes period N-bensyloxycarbonyl-O-methyl isourea (3.74 kg, 18.0 mol). The reaction mixture was stirred at 65-70° C for 16 h, cooled to 20° C and filtered. The precipitate was washed with iso-propanol (10 + 5 L) . The combined filtrates was concentrated at reduced pressure keeping the heating mantle not warmer than 65-70° C. When approximately 45 litres was distilled off EtOAc (90 L) was added. The reaction mixture was cooled to 20-25° C, washed with water (10 and 5 L) and brine (5 L) , and dried with Na2S04 (2 kg) . After stirring the rection mixture was filtered and the filter cake was washed with EtOAc (11 and 7 L) . The combined filtates were concentrated at reduced pressure keeping the heating mantle not warmer than 40-50° C. When approximately 90 litres of EtOAc was distilled off, toluene (25 L) was added and the evaporation continued. After collection of approximately another 18 litres of destillate, toulene (20 L) was added under vigorous stirring and the resulting mixture was cooled to -1 to 0° C and gently stirred over night (17 h) . The crystal slurry was filtered and the product was washed with cooled toluene (10 and 5 L) . Vacuum drying (10-20 mbar, 40° C) for 24 h gave 4.83 kg (13.8 mol, 76%) of Boc-Nag(Z).
1H-NMR (300 MHz, CDCl3) : δ 1.41 (s, 9H) , 1.6-1.7 (m, 2H) , 3.0-
3.3 (m, 4H) , 4.8-5.0 (bs, 1H) , 5.10 (s, 2H) , 7.2-7.4 (m, 5H) .
Boc-Agm(Z)
(i) Boc-Agm
To a slurry of 14.95 g (65.5 mmol, 1 eq.) of agmatine sulphate (Aldrich) , 13.7 ml of Et3N (98.25 mmol, 1.5 eq.), 165 ml of H20 and 165 ml of THF was added 21.5 g (98.25 mmol, 1.5 eq.) of (Boc)20 during 5 minutes at room temperature. The mixture was stirred vigorously over night, evaporated to dryness and the residue was washed with 2x100 ml of Et20 to give Boc-Agm as a white powder which was used without further purification in the next step.
(ii) Boc-Agm(Z)
To a cold (+5°C) slurry of the crude Boc-Agm from the previous step (ca: 65.5 mmol) in 180 ml of 4N NaOH and 165 ml of THF was added 24 ml (169 mmol, 2.5 eq) of benzyl chloroformate during 10 minutes. After stirring at room temperature for 4 h methanol (150 ml) was added and the stirring was continued for an additional 20 h at room temperature. The organic solvent was evaporated and 200 ml of H20 was added to the residue. The basic water phase was extracted with 1x300 ml and 2x200 ml of EtOAc. The combined organic phases was washed with H20 (2x100ml) , brine (1x100 ml) and dried (MgS04) . Evaporation of the solvent and flash chromathography (CH2Cl2/MeOH, a stepwise gradient of 97/3, 95/5 and 9/1 was used) gave 14.63 g (58%) of pure Boc-Agm(Z) as a white powder.
1H-NMR (CDC13# 500 MHz): δ 1.35-1.40 (m, 2H) , 1.45 (s, 9H) ,
1.5-1.6 (m, 2H), 3.0-3.2 (m, 4H) , 4.65 (bs, 1H) , 5.1 (s, 2H) , 7.25-7.40 (m, 5H) .
13C-NMR (CDC13, 75.5 MHz): δ 25.44, 27.36, 28.21, 65.83, 79.15, 127.47, 127.66, 128.14, 137.29, 156.47, 161.48, 163.30.
H-(R)Cha-Phe-Nag(Z)
(i) Boc-(R)Cha-Phe-OH
Boc-(R)Cha-0H was dissolved in acetonitrile (200 mL) , N-hydroxisuccinimide (9.9 g, 81 mmol) was added. Dicyclohexylcarbodiimide (17.8 g, 81 mmol) was then added slowly and the reaction mixture was stirred overnight at room temperature. The precipitate was filtered off and the Boc- (R)Cha-OSu containing solution was evaporated. Phe-OH (48.7 g, 195 mmol), sodiumhydroxide (10.3 g, 258 mmol), water (270 mL) and finally dimethylformamide (70 mL) were added to a reaction vessel while stirring. Boc-(R)Cha-OSu was dissolved in dimethylformamide (200 mL) and added slowly to the reaction vessel while maintaining the reaction temperature below 5βC. After 3 h the solution was evaporated, the residue dissolved in water (1000 mL) and extracted with ethylacetate (2 x 300 mL) . The aqueous phase was acidified with potassium hydrogensulfate (1M) to pH 3 and extracted with ethylacetate
Figure imgf000020_0001
Figure imgf000020_0002
The collected organic phase was evaporated and H2N-(R)Cha- Phe-Nag(Z) (3 g, 5.5 mmol), was isolated after purification by chromatography on silicagel (230-400 mesh) eluting with CH2Cl2:MeOH:NH4OH (100:4:1 to 100:15:1) in 36% yield. 1H NMR (200 MHz, CDCl3) S (ppm) 7.82 (d, 1H) , 7.4-7.1 (m, 10H) , 5.09 (s, 2H) , 4.42 (q, 1H) , 3.4-2.9 (m, 7H) , 1.8-0.7 (m, 15H) .
Working Examples
Example 1
H-(R)Cha-Phe-Agm x 2 TFA
(i) Boc-(R)Cha-Phe-Agm(Z)
A solution of 729 mg (2 mmol) Boc-Agm(Z) in 15 mL TFA/CH2C12 (1:4) was stirred at room temperature for about 2 h. The solvent was evaporated and the product was dissolved together with 1.03 g (2 mmol) of Boc-(R)Cha-Phe-OSu in 10 mL DMF , the pH was adjusted with NMM to about 9 and the mixture was stirred at room temperature for 5 days. The solvent was evaporated in vacuo and the residue was dissolved in 200 mL EtOAc. The organic phase was washed with 2 x 10 mL of water, 1 M KHS04, 1 M NaOH, water and dried (MgS04) . Evaporation of the solvent followed by flash chromatography (70 g Si02) using a stepwise gradient of 100 mL CH Cl2/MeOH (95/5) followed by 250 mL CH2Cl2/MeOH (9/1) gave 1.09 g (96 %) of the title compound.
1H-NMR (500 MHz, CDCl3, mixture of two rotamers) : major rotamer: δ 0.7-0.9 (m, 2H) , 1.0-1.8 (m, 25H; thereof 1.39 (s, 9H)), 2.9-3.25 (m, 6 H) , 4.02 (m, 1H) , 4.71 (q, 1 H) , 5.05 (S, 2 H) , 7.1-7.4 (m, 10H) . 20
13C-NMR (125 MHz, D20) : carbonyl and guanidine carbons: δ 161.7, 163.6, 172.0, 172.7 and 174.9.
(ii) H-(R)Cha-Phe-Agm x 2 TFA
A solution of 100 mg (0.15 mmol) Boc-(R)Cha-Phe-Agm(Z) in 10 mL CH2C12/TFA (4/1) was stirred at room temperature for 2 h 45 min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H20 (8/1) and hydrogenated over 40 mg 5 % Pd/C at athmospheric pressure for 3 h. The catalyst was filtered off the solvent evaporated and the residue was dissolved in water and freeze dried to give 93 mg (94 %) of the title compound as a white powder.
2H-NMR (500 MHz, D20, mixture of two rotamers) : major rotamer: ό" 0.65-1.75 (m, 17H), 2.86-3.23 (m, 6H) , 3.96 (t, 1H) , 4.59 (dd, 1H) , 7.15-7.4 (m, 5H) .
13C-NMR (125 MHz, D20) : guanidine δ 157.3; carbonyl carbons: δ 171.0 and 173.1.
Example 2
HOOC-CH2-(R)Cha-Phe-Agm x 2 TFA
(i) H-(R)Cha-Phe-Agm(Z)
A solution of 0.99 g (1.49 mmol) Boc-(R)Cha-Phe-Agm(Z) in 30 mL CH2C12/TFA (4/1) was stirred at room temperature for 3 h after which the solvent was evaporated and the residue dissolved in 100 mL CH2C1 . The organic phase was washed with 1 x 30 mL 5 M NaOH, 2 x 30 mL water and dried (MgS04) . Filtration and evaporation of the solvent gave 825 mg (98 %) of the title compound as a white powder. XH-NMR (300 MHz, CDC13) : δ 0.75-1.0 (m, 2H) , 1.05-1.75 (m, 15H) , 2.93-3.34 (m, 7H) , 4.56 (q, 1H) , 7.13-7.39 (m, 10H) .
13C-NMR (75 MHz, CDCL3) : carbonyl and guanidine carbons: δ 161.8, 163.8, 171.8 and 176.5.
(ii) BnOOC-CH2-(R)Cha-Phe-Agm(Z)
A mixture of 282 mg (0.5 mmol) H-(R)Cha-Phe-Agm(Z) , 173 mg (1.25 mmol) K2C03 and 137.5 mg (0.6 mmol) BnOOC-CH2-Br in 16 mL CH3CN/DMF (15/1) was heated to 50 °C for 4 h and 15 minutes after which the solvent evaporated and the residue dissolved in 70 mL EtOAc. The organic phase was washed with 4 x 10 mL water, 10 mL Brine and dried (MgS04) . Evaporation of the solvent followed by flash chromatography (37 g Si02) using CH2Cl2/MeOH(NH3-saturated) (95/5) as eluent afforded 230 mg (64 %) of the desired compound.
^-N (300 MHz, CDC13) : δ 0.7-0.95 (m, 2H) , 1.05-1.75 (m, 15H) , 2.84-3.25 (m, 8H) , 4.56-4.68 (m, 1H) , 4.95 (s, 2H) , 5.12 (s, 2H) , 7.1-7.45 (m, 15H) .
13C-NMR (75 MHz, CDCL3) : carbonyl and guanidine carbons: δ 161.7, 163,6, 171.56, 171.61 and 175.1.
(iii) HOOC-CH2-(R)Cha-Phe-Agm x 2 TFA
To a solution of 230 mg (0.323 mmol) BnOOC-CH2-(R)Cha-Phe- Agm(Z) in 18 mL EtOH/H20 (5/1) was added a small amount (15 drops) of TFA and the mixture was hydrogenated over 70 mg 5 % Pd/C at athmospheric pressure for 6 h. The catalyst was filtered off, the solvent was evaporated and the residue was dissolved in water and freeze dried to afford 223 mg (96%) off the title compound as a white powder. 1H-NMR (300 MHz, CD30D) : δ 0.65-1.85 ( , 17H) , 2.8-3.0 (m, IH) , 3.0-3.3 (m, 5H) , 3.78 (bs, 2H) , 4.0 (bs, IH) , 4.62 (m, IH) , 7.1-7.4 (m, 5H) .
13C-NMR (75 MHz, CD3OD) : guanidine: δ 158.6; carbonyl carbons: δ 168.9, 169.6 and 173.4.
Example 3
H-(R)Cha-Phe-Nag X 2 TFA
(i) Boc-(R)Cha-Phe-Nag(Z)
Prepared in the same way as described for Boc-(R)Cha-Phe- Agm(Z) in Example 1 (i) from Boc-(R)Cha-Phe-OSu (2 mmol) and Boc-Nag(Z) (2 mmol) .Yield = 1.02 g (78 %) .
(ii) H-(R)Cha-Phe-Nag x 2 TFA
A solution of 100 mg (0.15 mmol) Boc-(R)Cha-Phe-Nag(Z) in 10 mL CH2C12/TFA (4/1) was stirred at room temperature for 3 h 35 min after which the solvent was evaporated. The residue was dissolved in 9 ml EtOH/H20 (8/1) and hydrogenated over 40 mg 5 % Pd/C at athmospheric pressure for 3 h. The catalyst was filtered off the solvent evaporated and the residue was dissolved in water and freeze dried to give 97 mg (98 %) of the title compound as a white powder.
1H-NMR (500 MHz, D20, mixture of two rotamers): major rotamer: δ 0.75-1.85 (m, 15H), 2.9-3.45 (m, 6H) , 4.05 (t, IH) , 4.6-4.8 (m, IH; partially hidden by the H-O-D signal), 7.3-7.6 (m, 5H) .
13C-NMR (75 MHz, D20) : guanidine δ 157.6; carbonyl carbons:171.3 and 173.5. Example 4
H00C-CH2-(R)Cha-Phβ-Nag X 2 TFA
(i) H-(R)Cha-Phe-Nag(Z)
Prepared in the same way as described for H-(R)Cha-Phe-Agm(Z) in Example 2 (i) from Boc-(R)Cha-Phe-Nag(Z) . Yield 90 %.
13C-NMR ( 75 MHz, CDC13) : δ 26.0, 26.2, 26.4, 29.5, 32.2,
34.0, 34.2, 36.7, 37.7, 38.3, 42.6, 52.7, 55.1, 66.3, 126.9, 127.7, 127.9, 128.3, 128.6, 129.1, 136.7, 137.5, 161.8, 163.7, 171.5 and 176.6.
(ii) BnOOC-CH2-(R)Cha-Phe-Nag(Z)
A mixture of 275 mg (0.5 mmol) H-(R)Cha-Phe-Nag(Z) , 173 mg (1.25 mmol) K2C03 and 137.5 mg (0.6 mmol) BnOOC-CH2-Br in 15 mL CH3CN was heated to 50 °C for 3 h and 50 minutes after which the solvent evaporated and the residue dissolved in 70 mL EtOAc. The organic phase was washed with 4 x 10 mL water, 10 mL Brine and dried (MgS04) . Evaporation of the solvent followed by flash chromatography (37 g Si02) using CH2Cl2/MeOH(NH3-saturated) (95/5) as eluent afforded 209 mg (60 %) of the desired compound.
1H-NMR (300 MHz, CDCl3) : δ 0.72-0.93 (m, 2H) , 1.0-1.72 (m, 13H) , 2.83-3.25 (m, 9H) , 4.54 (q, IH) , 5.09 (s, 2H) , 5.11 (s,
2H) , 7.05-7.4 (m, 15H) , 7.59 (d, lh; NH) .
13C-NMR (75 MHz, CDC13) : carbonyl and guanidine carbons: δ 161.8, 163.6, 171.3, 171.6 and 175.2.
(iii) HOOC-CH2-(R)Cha-Phe-Nag x 2 TFA To a solution of 209 mg (0.3 mmol) Bn00C-CH2-(R)Cha-Phe- Nag(Z) in 18 mL EtOH/H20 (5/1) was added a small amount (15 drops) of TFA and the mixture was hydrogenated over 70 mg 5 % Pd/C at athmospheric pressure for 4 h. The catalyst was filtered off, the solvent was evaporated and the residue was dissolved in water and freeze dried to afford 190 mg (90%) off the title compound as a white powder.
^-NM (300 MHZ, CD3OD) : δ 0.6-1.38 (m, 6H), 1.4-1.9 (m, 9H) , 2.9-3.4 (m, 6H) , 3.9 (bs, 2H) , 4.1 (bs, IH) , 4.7 (m, Im¬ partially hidden by the H-O-D signal), 7.1-7.45 (m, 5H) .
13C-NMR (75 MHz, CD30D) : guanidine: δ 157.5; carbonyl carbons: δ 169.3, 169.5 and 173.2.
Example 5
H-(R)Cha-Phe-Nag
H2N-(R)Cha-Phe-Nag(Z) (300 mg, 0.55 mmol) was dissolved in ethanol (50 mL) and trifluoroaceticacid (56 μL, 0.73 mmol) was added. The mixture was sonicated and palladium on charcoal (5%, 50 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 19 h. The suspension was filtered through celite and after the solvent was evaporated the title compound (0.13 g, 0.31 mmol) was isolated in 56% yield.
'H NMR (200 MHz, d-HCl+d2-H20) δ (ppm) 7.40-7.00 (m, 5H) , 4.47 (t, IH), 3.86 (t, IH), 3.25-2.65 (m, 6H) , 1.75-0.50 (m, 15H) . TSP-MS found (m/z) -417 (calc . for MH+ (C22H37N602) 417 ) .
Example 6
CH3-CO-(R)Cha-Phe-Nag
(i) CH3-C0-(R)Cha-Phe-Nag(Z) H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol) was dissolved in acetonitrile (7.5 mL) . Acetylchloride (107 mg, 1.36 mmol) dissolved in acetonitrile (1 mL) was then transferred to the reaction vessel. After 30 min. the acylated peptide precipitated as an HCl-salt. Diethylether (5 mL) was added 20 minutes later. The precipitate was filtered off and dried under vacuum at 35°C overnight and the dry product, CH3-CO- (R)Cha-Phe-Nag(Z)xHCl (407 mg, 0.69 mmol) was isolated in 76% yield. IH NMR (200 MHz, CDC13) δ (ppm) 7.4-7.1 (m, 10H) , 5.1 (q,
2H) , 4.7 (m, IH), 4.0 (m, IH) , 3.5-2.9 (m, 6H) , 2.0-0.6 (m, 18H) . TSP-MS found (m/z)=593 (calc. for MH+(C32H45N605)593) .
(ii) CH3-CO-(R)Cha-Phe-Nag
CH3-CO-(R)Cha-Phe-Nag(Z) (400 mg, 0.68 mmol) was dissolved in ethanol (60 mL) and palladium on charcoal (5%, 80 mg) was added. The mixture was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 20 h. the suspension was filtered through celite and after the solvent was evaporated a crude mixture (300 mg) was collected. The crude product (150 mg) was purified by reveresed phase chromatography (C8-gel) eluting with MeCN:NH40Ac (0.1M) (40:60) and the product (100 mg, 0.22 mmol) in 64% yield. 1H NMR (200 MHz, d4-CH3OH) δ (ppm) 7.25-6.85 (m, 5H) , 4.44 (dd, IH) , 4-02 (t, IH), 3.30-2.90 (m, 5H) , 2.72 (dd, IH) , 2.0-0.5 (m, 18H) . TSP-MS found (m/z)459 (calc. for MH+(C24H39N603)459).
Example 7
CH3CH2- (R) Cha-Phe-Nag
(i) CH3CH2- (R) Cha-Phe-Nag (Z)
H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol), p-toluenesulphonic acid (173 mg, 0.91 mmol) and methanol (7.5 L) were added to a reaction vessel which was cooled with ice. Acetaldehyde (51 μL, 0.91 mmol) was added and finally after another 30 min. , sodium cyanoborohydride (86 mg, 1.36 mmol) was added. The mixture was stirred at room temperature for four days and then evaporated. The crude product was purified by chromatography on silicagel (230-400 mesh) eluting with MeCl2: MeOH: NH4OH (90:10:1) yielding CH3CH2-(R)Cha-Phe-Nag(Z) (150 mg, 0.26 mmol) in 29% yield.
1H NMR (200 MHz, d4-MeOH) δ (ppm) 7.39-7.28 (m, 10H) , 5.11 (s, 2H) , 4.63 (t, IH, 3.2-2.9 (m) , 2.42 (m, 2H) , 1.8-0.8 (m, 18H) .
(ii) CH3CH2-(R)Cha-Phe-Nag(Z) 150 mg, 0.26 mmol) was dissolved in EtOH 840 mL) and acetic acid (1 mL) . Palladium on charcoal (5%, 51 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatur for 2 days. The mixture was filtered and the filter cake was washed with MeOH/AcOH (2:1, 40 mL) . The title compound (32 mg, 0.072 mmol) was isolated in 28% yield by chromatography on silicagen (230-400 mesh) eluting with heptane: EtOAc: TEA (30:70:1).
1H NMR (200 MHZ, d4-MeOH) δ (ppm) 7.50-7.10 (m, 5H) , 4.62 (q, IH) , 3.76-3.67 (m, IH) , 3.65-3.56 (m, IH) , 3.51 (t, IH) , 3.35-2.95 (m, 6H) , 2.70 (q, 2H) , 2.0-0.5 (m, 18H) .
Example 8
HOOC-co-(R)Cha-Phe-Nag
(i) HOOC-CO-(R)Cha-Phe-Nag(Z)
H2N-(R)Cha-Phe-Nag(Z) (500 mg, 0.91 mmol) was dispersed in acetonitrile (5 mL) . Methyloxalylchloride (104 μL, 1.14 mmol) was added to the slurry. After 60 minutes the starting material was consumed, confirmed by HPLC, and the clear solution was evaporated. The crude methylester was hydrolyzed by dissolving the residue in tetrahydrofuran (4 mL) and adding LiOH (115 mg, 2.73 mmol) dissolved in water (2 mL) . After 90 min. more LiOH (70 mg, 1.7 mmol) was added and 30 minutes later water (10 mL) was added and the insoluble material were dissolved. After evaporation the dry uncolored powder was slurried in water (10 mL) containing ammonium chloride (150 mg) . The mixture was stirred for 30 min. and then the precipitate was filtered and washed with two portions of water. ~H NMR (200 MHz, d6-DMS0) δ (ppm) 8.7 (d, IH) , 8.2-7.6 (m) ,
7.31-7.22 (m, 10H), 4.94 (s, 2H) , 4.32 (m, IH) , 4.00 (m, IH) , 3.3-2.6 (m, 6H) , 1.7-0.6 (m, 15H) . TSP-MS found (m/z) 623 (calc. for MH+(C32H43N607)623) .
(ii) HOOC-CO-(R)Cha-Phe-Nag
HOOC-CO-(R)Cha-Phe-Nag(Z) (210 mg, 0.34 mmol) was dispersed in tetrahydrofuran (25 mL) and acitic acid (20 mL) was added. Palladium on charcoal (5%, 30 mg) was charged before it was hydrogenated at 45 psi hydrogen pressure in a Parr shaking apparatus for 25 h. The suspension was filtered through celite and the filter cake was washed with tetrahydrofuran and after the solvent was evaporated the crude product (257 mg) was collected. After azeotropic evaporation with three portions of toluene (tot; 50 mL) and overnight drying under vacuum the product (140 mg, 0.29 mmol) was isolated in 85% yield.
*H NMR (200 MHz, D4-MeOH) δ (ppm) 7.19 (m, 5H) , 4.54 (dd, IH) , 4.00 (t, IH) , 4.00 (t, IH), 3.50-2.90 (m, 5H) , 2.70 (t, IH) , 1.90-0.60 (m, 15H) . TSP-MS found (m/z) 489 (calc . for
MH+ (C24H36N6°5) 489) '
Pharmaceutical preparations A. The compounds according to the invention can be formulated in solid dosage forms for oral administration or for topical administration to the intestines. Example Al Plain tablet
Kininogenase inhibitor 10 mg/tablet Lactose anhydrous 250 mg/tablet Microcrystalline cellulose 60 mg/tablet Magnesium stearate 6 mg/tablet
The active constituent is mixed with lactose and micro- crystalline cellulose and magnesium stearate is admixed and tablets are compressed from the mixture.
Example A2 Coated tablet
Kininogenase inhibitor 100 mg/tablet
Lactose 300 mg/tablet
Polyvinylpyrrolidone 40 mg/tablet
Magnesium stearate 8 mg/tablet
Hydroxypropylmethylcellulose 8 mg/tablet
Polyethyleneglycol 1 mg/tablet
Talc 1 mg/tablet
Titandioxid 1 mg/tablet
The active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of hydroxypropylmethylcellulose, polyethyleneglycol, talc and titandioxide in water.
Example A3 Gastro-resistant tablet
Kininogenase inhibitor 10 mg/tablet Lactose 200 mg/tablet Polyvinylpyrrolidone 40 mg/tablet Microcrystalline cellulose 50 mg/tablet
Magnesium stearate 8 mg/tablet
Eudragit L 10 mg/tablet
Dibutylphtalate 1 mg/tablet Talc 2 mg/tablet
The active constituent is mixed with lactose and granulated with polyvinylpyrrolidone in water. After drying and milling microcrystalline cellulose and magnesium stearate is admixed and tablets are compressed. The tablets are coated with a solution of Eudragit L, dibutylphtalate and talc in isopropanol/aceton.
Example A4 Gastro-resistant extended release granules for the small intestine
Kininogenase inhibitor 100 mg/g
Lactose 448 mg/g Microcrystalline cellulose 200 mg/g
Hydroxypropyl cellulose 50 mg/g
Ethylcellulose 20 mg/g
Acetyltributylcitrate 2 mg/g
Eudragit L30D 50 mg/g Triethylcitrate 5 mg/g
Talc 25 mg/g
The active constituent is mixed with lactose and micro¬ crystalline cellulose and granulated with hydroxypropyl cellulose in water. The granulation is extruded, spheronized and dried. The granules are first coated with ethylcellulose dispersion with acetyltributylcitrate and then with Eudragit L30D dispersion with triethylcitrate and talc. The granules are filled in gelatin capsules each containing 10 mg of active constituent. Example *A5
Gastro-resistant extended release granules for the colon
Kininogenase inhibitor 200 mg/g Lactose 400 mg/g Microcrystalline cellulose 200 mg/g
Hydroxypropyl cellulose 50 mg/g
Eudragit NE30D 50 mg/g
Eudragit SlOO 50 mg/g Talc 50 mg/g
The active constituent is mixed with lactose and micro¬ crystalline cellulose and granulated with hydroxypropyl cellulose in water. The granulation is extruded, spheronized and dried. The granules are coated with a dispersion of
Eudragit NE30D, Eudragit SlOO and talc in water. The granules are filled in gelatin capsules each containing 100 mg of active constituent.
B. The compounds according to the invention can be formulated in pressurized aerosols or in dry powder inhalers for oral or nasal inhalation. The kininogenase inhibitor is micronized to a particle size suitable for inhalation therapy (mass median diameter < 4μm) .
For pressurized aerosols the micronized substance is suspended in a liquid propellant mixture and filled into a container which is sealed with a metering valve. Alternatively, the kininogenase inhibitor can be dissolved in the liquid propellant mixture with the aid of ethanol.
The propellants used may by chlorofluorocarbons (CFCs) or hydrofluoroalkanes (HFAs) of different formulae. The most frequent used CFCs are trichloromonofluoromethane (propellant 11) and dichlorodifluoromethane (propellant 12) and dichlorotetrafluoroethane (propellant 114). The most frequent used HFAs are tetrafluoromethane (propellant 134a) and heptafluoropropane (propellant 227).
Low concentrations of surfactant such as sorbitan trioleate, lecithin, oleic acid or other suitable substance may be used to improve the physical stability. Etanol may be used as surfactant or as a medium to increase the solubility of active substance in the propellant mixture.
Example Bl per cent (w/w)
Kininogenase inhibitor 0.5
Trichloromonofluoromethane 15
(propellant 11)
Dichlorodifluoromethane 84 (propellant 12)
Sorbitan trioleate 0.5
Example B2 per cent (w/w)
Kininogenase inhibitor 0.5 Trichloromonofluoromethane 25
(propellant 11)
Dichlorodifluoromethane 74.48
(propellant 12)
Oleic acid 0.02
Example B3 per cent (w/w)
Kininogenase inhibitor 0.2
Trichloromonofluoromethane 15
(propellant 11)
Dichlorodifluorometane 64.78
Ethanol 20
Oleic acid 0.02
Example B4 per cent (w/w)
Kininogenase inhibitor 0.4
Tetrafluoroethane 59.58
(propellant 134a) Heptafluoropropane 20
(propellant 227)
Ethanol 20
Oleic acid 0.02
Example B5 per cent (w/w)
Kininogenase inhibitor 1.0
Heptafluoropropane 93.5
(propellant 227)
Ethanol 5
Sorbitan trioleate 0.5
In a dry powder inhaler the micronized kininogenase inhibitor may be used alone or mixed with a carrier substance such as lactose, mannitol or glucose. Another possibility is to process the micronized powder into spheres which break up during the dosing procedure. This powder or spheronized powder is filled into the drug reservoir in a singledose or multidose inhaler, e.g. the latter being Turbuhaler®. A dosing unit meters the desired dose which is inhaled by the patient.
Example B6
The kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter < 4μm) . 100 mg of the micronized powder is filled into a powder multidose inhaler (Turbuhaler®) . The inhaler is equipped with a dosing unit which delivers a dose of 1 mg.
Example B7
The kininogenase inhibitor is micronized in a jet mill to a particle size suitable for inhalation (mass diameter < 4μm) . 150 mg of the micronized powder is filled into a powder multidose inhaler (Turbuhaler®) . The inhaler is equipped with a dosing unit which delivers a dose of 0.5 mg. ABBREVIATIONS
Ac = Acetyl
Agm = Agmatine
Agm(Z) - ω-N-benzyloxycarbonyl agmatine
Boc = tertiary butoxy carbonyl
Brine = saturated water/NaCl solution
Bn - benzyl
Cha - (S)-/3-cyclohexyl alanine
CME-CDI - l-Cyclohexyl-3-(2-morpholino- ethyl)carbodiimide metho-p- toluenesulfonate
DCC = dicyclohexyl carbodiimide
DMF = dimethyl formamide
Et - ethyl
EtOAc = ethyl acetate
HOSu - N-hydroxysuccinimide
HPLC = High Performance Liquid Chromatography
LiOH = Lithium hydroxide
Me - methyl
Nag - noragmatine
Nag(Z) = ω-N-benzyloxycarbonyl-noragmatine
Nal (S)-naphthylalanine
NMM = N-methyl morpholine
Ph = phenyl
Phe - (S)-phenylalanine
Pro = (S)-proline
Ser = (S)-serine
TFA = trifluoracetic acid
THF = tetrahydrofuran
Z - benzyloxycarbonyl
Prefixes n, s, i and t have their usual meanings: normal, iso, sec and tertiary. The stereochemistry for the amino acids is by default (S) if not otherwise stated.

Claims

1. A compound of the general formula
A1-A2-NH-(CH2)n-NH-C(NH)-NH2 Formula I
wherein:
n is an integer 2, 3, 4, 5, or 6; preferably 3 or 4;
A1 represents a structural fragment of Formulae Ila, lib. He, Hd or He;
Figure imgf000036_0001
Figure imgf000036_0002
wherein:
p is an integer 0,1 or 2;
m is an integer 1, 2, 3, or 4, preferably 2;
q is an integer 0-2, preferably 1; R1 represents H, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group having 2-3 carbon atoms or R11OOC-alkyl-, where the alkyl group has 1 to 4 carbon atoms and R11 is H or an alkyl group having 1 to 4 carbon atoms, or
R1 represents R1 OOC-l,4-phenyl-CH2-, wherein R12 is H or an alkyl group having 1 to 4 carbon atoms, or
R1 represents R13-NH-CO-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted alpha to the carbonyl with an alkyl group having 1 to 4 carbon atoms and where R13 is H or an alkyl group having 1 to 4 carbon atoms or -CH2COOR12, wherein R12 is as defined above, or
R1 represents R14S02-, Ph(4-COOR12)-S02-, Ph(3-COOR12)-S02-, or Ph(2-COOR12)-S02-, wherein R12 is as defined above and R14 is an alkyl group having 1-4 carbon atoms, or
R1 represents CO-R15, wherein R1S is an alkyl group having 1-4 carbon atoms, or
R1 represents CO-OR15' wherein R15 is as defined above, or
R1 represent CO-(CH2)p-COOR12' wherein R12 and p are as defined above, or
R1 represents -CH2PO(OR16)2, wherein R16 is, individually at each occurrence, H, methyl or ethyl;
R2 represents H or an alkyl group having 1 to 4 carbon atoms or R21OOC-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and is possibly substituted in the position which is alpha to the carbonyl group, and the alpha substituent is a group R22-(CH2)p-, wherein p is as defined above and R22 is methyl, phenyl, OH, COOR21, and R21 is H or an alkyl group having 1 to 4 carbon atoms; R3 represents an alkyl group having 1-4 carbon atoms, or
R3 represents a cyclohexyl- or cyclopentyl group, or
R3 represents a phenyl group which may or may not be substituted with an alkyl group having 1 to 4 carbon atoms, or with a group OR21, or
R3 represents a 1-napthyl, 2-naphtyl, 4-pyridyl, 3- pyrrolidyl, or a 3-indolyl group which may or may not be substituted with OR21 and with p = 1; or
R3 represent a cis- or trans-decalin group with p = 1; or
R3 represents Si(Me)3 or CH(R31)2, wherein R31 is a cyclohexyl- or phenyl group;
A2 represents a structural fragment
° a
- NH - CH - C - I
(CH2)p R3 wherein R3 and p are as defined above.
2. A compound according to claim 1 wherein A1 represents Ha or lib.
3. A compound according to claim 1 wherein A1 represents Ha.
4. A compound according to claim 1 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.
5. A compound according to claim 2 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.
6. A compound according to claim 3 wherein R3 is cyclohexyl, cyclopentyl, phenyl, substituted phenyl or other aryl systems and p is 1.
7. A compound according to one or more of the preceding claims wherein R1 represents Rn00C-alkyl-, wherein the alkyl group has 1 to 4 carbon atoms and R11 is H.
8. A compound according to one or more of the preceding claims wherein R3 is cyclohexyl or substituted phenyl.
9. A compound according to one or more of the preceding claims wherein n is 3 or 4.
10. A compound according to one or more of the preceding claims wherein n is 4.
11. A compound according to one or more of the preceding claims wherein p is 1.
12. A compound according to one or more of the preceding claims wherein q is 1.
13. A compound according to one or more of the preceding claims having R-configuration on the amino acid fragment in the A1 position.
14. A compound according to one or more of the preceding claims having S-konfiguration on the amino acid fragment in the A2 position.
15. A compound selected from
H-(R)Cha-Phe-Agm HOOC-CH2-(R)Cha-Phe-Agm H-(R)Cha-Phe-Nag HOOC-CH2-(R)Cha-Phe-Nag CH3-CO-(R)Cha-Phe-Nag CH3-CH2-(R)Cha-Phe-Nag HOOC-CO-(R)Cha-Phe-Nag
either as such or in the form of a physiologically acceptable salt and including stereoisomers.
16. The compound HOOC-CH2-(R)Cha-Phe-Nag, either as such or in the form of a physiologically acceptable salt and including stereoisomers.
17. A process for preparing a compound according to claim 1, which process comprises coupling of a N-terminally protected dipeptide ( 1-A1-A2-0H) or amino acid
Figure imgf000040_0001
when a N- terminally protected amino acid is used a second amino acid is added afterwards using standard methods, to a compound
H2N-(CH2)n-X wherein A1, A2 and n are as defined in Formula I, t^ is an amino protecting group and X is an unprotected or protected guanidino group or a protected amino group, or a group transferable into an amino group, where the amino group is subsequently transferred into an unprotected or protected guanidino group, followed by removal of the protecting group(s) or deprotecting of the N-terminal nitrogen followed by alkylation of the N-terminal nitrogen and deprotection by known methods.
and if desired forming a physiologically acceptable salt, and in those cases where the reaction results in a mixture of stereoisomers, these are optionally separated by standard chromatographic or re-crystallisation techniques, and if desired a single stereoisomer is isolated.
18. A process according to claim 17 which process comprises
a) Method I
Coupling of an N-terminally protected dipeptide, prepared by standard peptide coupling, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling,shown in the formula
»!- A1-A -OH
I H2N-(CH2)n-X W1- A1-A2-NH(CH2)n-X
wherein A1, A2 and n are as defined in Formula I , W1 is a N- r teminal amino protecting group such as tert-butyloxy carbonyl and benzyloxy carbonyl and X is -NH-C(NH)-NH2, -NH-C(NH)-NH- W2, -N( 2)-C(NH)-NH- 2, -NH-C(NW2)-NH-W2 or -NH- 2, where W2 is an amine protecting group such as tert-butyloxy carbonyl or benzyloxy carbonyl, or X is a masked amino group such as azide, giving the protected peptide, and further depending on the nature of the X- group used: Removal of the protecting group(s) (when X= -NH-C(NH)-NH2, -N(W2)-C(NH)-NH-W2, -NH- C(NW2)-NH-W2 or -NH-C(NH)-NH- 2), or a selective deprotection of the χ- group (e.g when X= -NH-C(NH)-NH-W2,-N(W2)-C(NH)-NH- 2, -NH-C(NW2)-NH-W2, W2 in this case must be orthogonal to j) followed by alkylation of the N-terminal nitrogen and deprotection or a selective deprotection/ unmasking of the terminal alkylamino function (X= NH-W2, 2 in this case must be orthogonal to Wχ or X= a masked aminogroup, such as azide) followed by a guanidation reaction, using standard methods, of the free amine and deprotection of the W^-group, or
b) Method II
Coupling of a N-terminally protected amino acid, prepared by standard methods, with either a protected- or unprotected amino guanidine or a straight chain alkylamine carrying a protected or masked amino group at the terminal end of the alkyl chain, using standard peptide coupling, shown in the formula
W1-A2-OH
I H2N-(CH2)n-X
Figure imgf000042_0001
wherein A2, n, λ and X are as defined above followed by deprotection of the 1-group and coupling with the N-terminal amino acid, in a protected form, leading to the protected peptide described in Method I, whereafter the synthesis to the final compounds is continued according to Method I.
19. Use of a compound of the formula I
A1-A2-NH-(CH2)n-NH-C(NH)-NH2
according to claim l.
either as such or in the form of a salt, and as such or having the guanidino group either mono protected at the δ- nitrogen or diprotected at the ^-nitrogens or the y, δ- nitrogens, as a starting material in synthesis of a serine protease inhibitor, and in particular in synthesis of a kininogenase inhibitor.
20. Use according to claim 19, wherein the serine protease inhibitor is a peptidic compound.
21. A compound according to claim 1 for use in therapy.
22. A compound according to claim 21 for use as an antiinflammatory agent.
23. A pharmaceutical preparation comprising an effective amount of a compound according to claims 1 in conjunction with one or more pharmaceutical carriers.
24. A pharmaceutical preparation according to claim 23 for use as an antiinflammatory agent.
25. Use of compound according to claim 1 as an active ingredient for manufacture of a pharmaceutical preparation for inhibition of serine proteases and in particular kininogenases in a human or animal organism.
26. A method for obtaining inhibition of serine proteases and in particular kininogenases in a human or animal organism in need of such inhibition, comprising administering to said organism an inhibitory effective amount of a compound according to claim 1.
27. A compound, a process, a pharmaceutical preparation, a use and a method as claimed in any of claims 1-26 and substantially as described.
PCT/SE1994/000534 1993-06-03 1994-06-02 New peptides derivatives WO1994029335A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU69408/94A AU685465B2 (en) 1993-06-03 1994-06-02 New peptides derivatives
EP94917871A EP0701567A1 (en) 1993-06-03 1994-06-02 New peptides derivatives
JP7501664A JPH08511017A (en) 1993-06-03 1994-06-02 New peptide derivatives

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE19939301911A SE9301911D0 (en) 1993-06-03 1993-06-03 NEW PEPTIDE DERIVATIVES
SE9301911-5 1993-06-03

Publications (1)

Publication Number Publication Date
WO1994029335A1 true WO1994029335A1 (en) 1994-12-22

Family

ID=20390161

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE1994/000534 WO1994029335A1 (en) 1993-06-03 1994-06-02 New peptides derivatives

Country Status (5)

Country Link
EP (1) EP0701567A1 (en)
JP (1) JPH08511017A (en)
AU (1) AU685465B2 (en)
SE (1) SE9301911D0 (en)
WO (1) WO1994029335A1 (en)

Cited By (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5578574A (en) * 1994-03-04 1996-11-26 Eli Lilly And Company Antithrombotic agents
US5599793A (en) * 1994-03-04 1997-02-04 Eli Lilly And Company Antithromobotic agents
US5602101A (en) * 1994-03-04 1997-02-11 Eli Lilly And Company Antithrombotic agents
US5705487A (en) * 1994-03-04 1998-01-06 Eli Lilly And Company Antithrombotic agents
US5710130A (en) * 1995-02-27 1998-01-20 Eli Lilly And Company Antithrombotic agents
US5726159A (en) * 1994-03-04 1998-03-10 Eli Lilly And Company Antithrombotic agents
EP0841933A1 (en) * 1995-07-25 1998-05-20 The Trustees Of The University Of Pennsylvania Bowman-birk inhibitor compositions for treatment of imflammatory disease
US5885967A (en) * 1994-03-04 1999-03-23 Eli Lilly And Company Antithrombotic agents
US5914319A (en) * 1995-02-27 1999-06-22 Eli Lilly And Company Antithrombotic agents
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6692753B2 (en) 1997-05-07 2004-02-17 Trustees Of Tufts College Potentiation of the immune response
US6825169B1 (en) 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
KR100974633B1 (en) * 2002-03-08 2010-08-06 밴티아 리미티드 Selective dipeptide inhibitors of kallikrein
US7879863B2 (en) 2004-03-31 2011-02-01 Ajinomoto Co., Inc. Aniline derivatives
WO2011051671A1 (en) 2009-10-28 2011-05-05 Vantia Limited Aminopyridine derivatives as kallikrein inhibitors
WO2011051672A1 (en) 2009-10-28 2011-05-05 Vantia Limited Azaindole derivatives
WO2011051673A1 (en) 2009-10-28 2011-05-05 Vantia Limited Aminothiazole derivatives useful as klk1 inhibitors
WO2013005045A1 (en) 2011-07-07 2013-01-10 Kalvista Pharmaceuticals Limited Benzylamine derivatives as inhibitors of plasma kallikrein
WO2014006414A1 (en) 2012-07-06 2014-01-09 Kalvista Pharmaceuticals Limited Polymorphs of|n-[(r)-1 -[(s)-1-(4-aminomethyl-benzylcarbamoyl)-2-phenyl-ethylcarbamoy l]-2-(4-e|thoxy-phenyl)-ethyl]-benzamide hydrochloride
WO2014108685A1 (en) * 2013-01-09 2014-07-17 Kalvista Pharmaceuticals Limited Pharmaceutical compositions
WO2016083816A1 (en) 2014-11-27 2016-06-02 Kalvista Pharmaceuticals Limited N-((heteroarylmethyl)-heteroaryl-carboxamide derivatives as plasma kallikrein inhibitors
US9533987B2 (en) 2013-05-23 2017-01-03 Kalvista Pharmaceuticals Limited Heterocyclic derivates
US9670157B2 (en) 2013-01-08 2017-06-06 Kalvista Pharmaceuticals Limited Benzylamine derivatives
US9738641B2 (en) 2013-08-14 2017-08-22 Kalvista Pharmaceuticals Limited Bicyclic inhibitors
US9834513B2 (en) 2013-01-08 2017-12-05 Kalvista Pharmceuticals Limited Benzylamine derivatives
US10221161B2 (en) 2013-08-14 2019-03-05 Kalvista Pharmaceuticals Limited Inhibitors of plasma kallikrein
US10364238B2 (en) 2014-11-27 2019-07-30 Kalvista Pharmaceuticals Limited N-((het) arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
US10752607B2 (en) 2016-06-01 2020-08-25 Kalvista Pharmaceuticals Limited Polymorphs of N-[(6-cyano-2-fluoro)-3-methoxyphenyl)methyl]-3-(methoxymethyl)-1-({4-[(2-oxopyridin-1-yl)methyl]phenyl}methyl)pyrazole-4-carboxamide as kallikrein inhibitors
US10781181B2 (en) 2014-11-27 2020-09-22 Kalvista Pharmaceuticals Limited N-((het) arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
WO2020249977A1 (en) 2019-06-14 2020-12-17 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
WO2020249979A1 (en) 2019-06-14 2020-12-17 Kalvista Pharmaceuticals Limited Treatments of angioedema
EP3760622A1 (en) 2016-05-31 2021-01-06 Kalvista Pharmaceuticals Limited Pyrazole derivatives as plasma kallikrein inhibitors
WO2021028649A1 (en) 2019-08-09 2021-02-18 Kalvista Pharmaceuticals Limited Plasma kallikrein inhibitors
WO2021116679A1 (en) 2019-12-09 2021-06-17 Kalvista Pharmaceuticals Limited Treatments of diabetic macular edema and impaired visual acuity
GB2591730A (en) 2019-12-09 2021-08-11 Kalvista Pharmaceuticals Ltd New polymorphs
US11230537B2 (en) 2016-06-01 2022-01-25 Kalvista Pharmaceuticals Limited Polymorphs of n-[(3-fluoro-4-methoxypyridin-2-yl)methyl]-3-(methoxymethyl)-1-({4-[2-oxopyridin-1-yl)methyl]phenyl} methyl)pyrazole-4-carboxamide as iallikrein inhibitors
US11234939B2 (en) 2017-11-29 2022-02-01 Kalvista Pharmaceuticals Limited Dosage forms comprising a plasma kallikrein inhibitor
US11242333B2 (en) 2013-08-14 2022-02-08 Kalvista Pharmaceuticals Limited Inhibitors of plasma kallikrein
WO2022079446A1 (en) 2020-10-15 2022-04-21 Kalvista Pharmaceuticals Limited Treatments of angioedema
WO2022084693A1 (en) 2020-10-23 2022-04-28 Kalvista Pharmaceuticals Limited Treatments of angioedema
WO2022172006A1 (en) 2021-02-09 2022-08-18 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
WO2023002219A1 (en) 2021-07-23 2023-01-26 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
US11584735B2 (en) 2017-11-29 2023-02-21 Kalvista Pharmaceuticals Limited Solid forms of a plasma kallikrein inhibitor and salts thereof
US11584714B2 (en) 2018-05-29 2023-02-21 Omeros Corporation MASP-2 inhibitors and methods of use
WO2023209381A1 (en) 2022-04-27 2023-11-02 Kalvista Pharmaceuticals Limited Formulations of a plasma kallikrein inhibitor
US11807641B2 (en) 2019-12-04 2023-11-07 Omeros Corporation MASP-2 inhibitors and methods of use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2085444A (en) * 1979-06-12 1982-04-28 Richter Gedeon Vegyeszet Novel Anticoagulant agmatine derivatives and process for the preparation thereof
WO1992004371A1 (en) * 1990-09-07 1992-03-19 Ferring Peptide Research Partnership Kb Kininogenase inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2085444A (en) * 1979-06-12 1982-04-28 Richter Gedeon Vegyeszet Novel Anticoagulant agmatine derivatives and process for the preparation thereof
WO1992004371A1 (en) * 1990-09-07 1992-03-19 Ferring Peptide Research Partnership Kb Kininogenase inhibitors

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Brazilian J. Med. Biol. Res., Volume 20, 1987, L.A.F. FERREIRA et al., "Kallikrein Isolated from Commersial Crystalline Pepsin Preparations", page 511 - page 520. *

Cited By (80)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6825169B1 (en) 1991-10-22 2004-11-30 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US7230074B2 (en) 1991-10-22 2007-06-12 Trustees Of Tufts College Inhibitors of dipeptidyl-aminopeptidase type IV
US5578574A (en) * 1994-03-04 1996-11-26 Eli Lilly And Company Antithrombotic agents
US5602101A (en) * 1994-03-04 1997-02-11 Eli Lilly And Company Antithrombotic agents
US5599793A (en) * 1994-03-04 1997-02-04 Eli Lilly And Company Antithromobotic agents
US5726159A (en) * 1994-03-04 1998-03-10 Eli Lilly And Company Antithrombotic agents
US5705487A (en) * 1994-03-04 1998-01-06 Eli Lilly And Company Antithrombotic agents
US6124277A (en) * 1994-03-04 2000-09-26 Eli Lilly And Company Antithrombotic agents
US5885967A (en) * 1994-03-04 1999-03-23 Eli Lilly And Company Antithrombotic agents
US6090787A (en) * 1994-03-04 2000-07-18 Eli Lilly And Company Antithrombotic agents
US5914319A (en) * 1995-02-27 1999-06-22 Eli Lilly And Company Antithrombotic agents
US5710130A (en) * 1995-02-27 1998-01-20 Eli Lilly And Company Antithrombotic agents
EP0841933A4 (en) * 1995-07-25 1998-08-05 Univ Pennsylvania Bowman-birk inhibitor compositions for treatment of imflammatory disease
EP0841933A1 (en) * 1995-07-25 1998-05-20 The Trustees Of The University Of Pennsylvania Bowman-birk inhibitor compositions for treatment of imflammatory disease
US5965532A (en) * 1996-06-28 1999-10-12 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6875737B1 (en) 1996-06-28 2005-04-05 Trustees Of Tufts College Multivalent compounds for crosslinking receptors and uses thereof
US6100234A (en) * 1997-05-07 2000-08-08 Tufts University Treatment of HIV
US6503882B2 (en) 1997-05-07 2003-01-07 Trustees Of Tufts College Treatment of HIV
US6692753B2 (en) 1997-05-07 2004-02-17 Trustees Of Tufts College Potentiation of the immune response
US6258597B1 (en) 1997-09-29 2001-07-10 Point Therapeutics, Inc. Stimulation of hematopoietic cells in vitro
US6703238B2 (en) 1997-09-29 2004-03-09 Point Therapeutics, Inc. Methods for expanding antigen-specific T cells
US6770628B2 (en) 1998-05-04 2004-08-03 Point Therapeutics, Inc. Hematopoietic stimulation
US6300314B1 (en) 1998-05-04 2001-10-09 Point Therapeutics, Inc. Hematopoietic stimulation
US7067489B2 (en) 1998-05-04 2006-06-27 Point Therapeutics, Inc. Hematopoietic stimulation
US6355614B1 (en) 1998-06-05 2002-03-12 Point Therapeutics Cyclic boroproline compounds
US6890904B1 (en) 1999-05-25 2005-05-10 Point Therapeutics, Inc. Anti-tumor agents
US6949514B2 (en) 1999-05-25 2005-09-27 Point Therapeutics, Inc. Anti-tumor agents
KR100974633B1 (en) * 2002-03-08 2010-08-06 밴티아 리미티드 Selective dipeptide inhibitors of kallikrein
US7879863B2 (en) 2004-03-31 2011-02-01 Ajinomoto Co., Inc. Aniline derivatives
WO2011051671A1 (en) 2009-10-28 2011-05-05 Vantia Limited Aminopyridine derivatives as kallikrein inhibitors
WO2011051672A1 (en) 2009-10-28 2011-05-05 Vantia Limited Azaindole derivatives
WO2011051673A1 (en) 2009-10-28 2011-05-05 Vantia Limited Aminothiazole derivatives useful as klk1 inhibitors
WO2013005045A1 (en) 2011-07-07 2013-01-10 Kalvista Pharmaceuticals Limited Benzylamine derivatives as inhibitors of plasma kallikrein
US9051249B2 (en) 2011-07-07 2015-06-09 Kalvista Pharmaceuticals Limited Benzylamine derivatives as inhibitors of plasma kallikrein
US9234000B2 (en) 2011-07-07 2016-01-12 Kalvista Pharmaceuticals Limited Benzylamine derivatives as inhibitors of plasma kallikrein
WO2014006414A1 (en) 2012-07-06 2014-01-09 Kalvista Pharmaceuticals Limited Polymorphs of|n-[(r)-1 -[(s)-1-(4-aminomethyl-benzylcarbamoyl)-2-phenyl-ethylcarbamoy l]-2-(4-e|thoxy-phenyl)-ethyl]-benzamide hydrochloride
US9512065B2 (en) 2012-07-06 2016-12-06 Kalvista Pharmaceuticals Limited Polymorphs of N-[(R)-1-[(S)-1-(4-aminomethyl-benzylcarbamoyl)-2-phenyl-ethylcarbamoyl]-2-(4-ethoxy-phenyl)-ethyl]-benzamide hydrochloride
US9834513B2 (en) 2013-01-08 2017-12-05 Kalvista Pharmceuticals Limited Benzylamine derivatives
US9670157B2 (en) 2013-01-08 2017-06-06 Kalvista Pharmaceuticals Limited Benzylamine derivatives
WO2014108685A1 (en) * 2013-01-09 2014-07-17 Kalvista Pharmaceuticals Limited Pharmaceutical compositions
US10478409B2 (en) 2013-01-09 2019-11-19 Kalvista Pharmaceuticals Limited Pharmaceutical compositions
US9849100B2 (en) 2013-01-09 2017-12-26 Kalvista Pharmaceuticals Limited Pharmaceutical compositions suitable for parenteral administration comprising aqueous suspensions of benzylamine derivatives having low solubilities
US9533987B2 (en) 2013-05-23 2017-01-03 Kalvista Pharmaceuticals Limited Heterocyclic derivates
EP3305778A1 (en) 2013-05-23 2018-04-11 Kalvista Pharmaceuticals Limited Inhibitors of plasma kallikrein
US11242333B2 (en) 2013-08-14 2022-02-08 Kalvista Pharmaceuticals Limited Inhibitors of plasma kallikrein
US10221161B2 (en) 2013-08-14 2019-03-05 Kalvista Pharmaceuticals Limited Inhibitors of plasma kallikrein
US9738641B2 (en) 2013-08-14 2017-08-22 Kalvista Pharmaceuticals Limited Bicyclic inhibitors
EP4039681A1 (en) 2014-11-27 2022-08-10 Kalvista Pharmaceuticals Limited N-((het)arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
EP3567037A1 (en) 2014-11-27 2019-11-13 Kalvista Pharmaceuticals Limited N-((het)arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
WO2016083816A1 (en) 2014-11-27 2016-06-02 Kalvista Pharmaceuticals Limited N-((heteroarylmethyl)-heteroaryl-carboxamide derivatives as plasma kallikrein inhibitors
US10611758B2 (en) 2014-11-27 2020-04-07 Kalvista Pharmaceuticals Limited N-((het)arylmethyl)-heteroaryl-carboxamides compounds as kallikrein inhibitors
US11084809B2 (en) 2014-11-27 2021-08-10 Kalvista Pharmaceuticals Limited N-((HET)arylmethyl)-heteroaryl-carboxamides compounds as kallikrein inhibitors
US10781181B2 (en) 2014-11-27 2020-09-22 Kalvista Pharmaceuticals Limited N-((het) arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
EP3556752A1 (en) 2014-11-27 2019-10-23 Kalvista Pharmaceuticals Limited N-((het)arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
US11198691B2 (en) 2014-11-27 2021-12-14 Kalvista Pharmaceuticals Limited N-((het)arylmethyl)-heteroaryl-carboxamides compounds as kallikrein inhibitors
US10364238B2 (en) 2014-11-27 2019-07-30 Kalvista Pharmaceuticals Limited N-((het) arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
US11001578B2 (en) 2014-11-27 2021-05-11 Kalvista Pharmaceuticals Limited N-((HET)arylmethyl)-heteroaryl-carboxamides compounds as plasma kallikrein inhibitors
EP3760622A1 (en) 2016-05-31 2021-01-06 Kalvista Pharmaceuticals Limited Pyrazole derivatives as plasma kallikrein inhibitors
US11180484B2 (en) 2016-05-31 2021-11-23 Kalvista Pharmaceuticals Limited Pyrazole derivatives as plasma kallikrein inhibitors
US11230537B2 (en) 2016-06-01 2022-01-25 Kalvista Pharmaceuticals Limited Polymorphs of n-[(3-fluoro-4-methoxypyridin-2-yl)methyl]-3-(methoxymethyl)-1-({4-[2-oxopyridin-1-yl)methyl]phenyl} methyl)pyrazole-4-carboxamide as iallikrein inhibitors
US11739068B2 (en) 2016-06-01 2023-08-29 Kalvista Pharmaceuticals Limited Polymorphs of N-[(3-fluoro-4-methoxypyridin-2-yl)methyl]-3-(methoxymethyl)-1-({4-[(2-oxopyridin-1-yl)methyl]phenyl}methyl)pyrazole-4-carboxamide and salts thereof
EP4151630A1 (en) 2016-06-01 2023-03-22 Kalvista Pharmaceuticals Limited Polymorphs of n-[(3-fluoro-4-methoxypyridin-2-yl)methyl]-3-(methoxymethyl)-1-({4-[2-oxopyridin-1-yl)methyl]phenyl}methyl)pyrazole-4-carboxamide as kallikrein inhibitors
US10752607B2 (en) 2016-06-01 2020-08-25 Kalvista Pharmaceuticals Limited Polymorphs of N-[(6-cyano-2-fluoro)-3-methoxyphenyl)methyl]-3-(methoxymethyl)-1-({4-[(2-oxopyridin-1-yl)methyl]phenyl}methyl)pyrazole-4-carboxamide as kallikrein inhibitors
US11584735B2 (en) 2017-11-29 2023-02-21 Kalvista Pharmaceuticals Limited Solid forms of a plasma kallikrein inhibitor and salts thereof
US11234939B2 (en) 2017-11-29 2022-02-01 Kalvista Pharmaceuticals Limited Dosage forms comprising a plasma kallikrein inhibitor
US11584714B2 (en) 2018-05-29 2023-02-21 Omeros Corporation MASP-2 inhibitors and methods of use
WO2020249979A1 (en) 2019-06-14 2020-12-17 Kalvista Pharmaceuticals Limited Treatments of angioedema
WO2020249977A1 (en) 2019-06-14 2020-12-17 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
EP4282474A2 (en) 2019-06-14 2023-11-29 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
US11613527B2 (en) 2019-08-09 2023-03-28 Kalvista Pharmaceuticals Limited Enzyme inhibitors
WO2021028649A1 (en) 2019-08-09 2021-02-18 Kalvista Pharmaceuticals Limited Plasma kallikrein inhibitors
WO2021028645A1 (en) 2019-08-09 2021-02-18 Kalvista Pharmaceuticals Limited Plasma kallikrein inhibitors
US11807641B2 (en) 2019-12-04 2023-11-07 Omeros Corporation MASP-2 inhibitors and methods of use
GB2591730A (en) 2019-12-09 2021-08-11 Kalvista Pharmaceuticals Ltd New polymorphs
WO2021116679A1 (en) 2019-12-09 2021-06-17 Kalvista Pharmaceuticals Limited Treatments of diabetic macular edema and impaired visual acuity
WO2022079446A1 (en) 2020-10-15 2022-04-21 Kalvista Pharmaceuticals Limited Treatments of angioedema
WO2022084693A1 (en) 2020-10-23 2022-04-28 Kalvista Pharmaceuticals Limited Treatments of angioedema
WO2022172006A1 (en) 2021-02-09 2022-08-18 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
WO2023002219A1 (en) 2021-07-23 2023-01-26 Kalvista Pharmaceuticals Limited Treatments of hereditary angioedema
WO2023209381A1 (en) 2022-04-27 2023-11-02 Kalvista Pharmaceuticals Limited Formulations of a plasma kallikrein inhibitor

Also Published As

Publication number Publication date
AU6940894A (en) 1995-01-03
AU685465B2 (en) 1998-01-22
EP0701567A1 (en) 1996-03-20
SE9301911D0 (en) 1993-06-03
JPH08511017A (en) 1996-11-19

Similar Documents

Publication Publication Date Title
AU685465B2 (en) New peptides derivatives
US4189604A (en) Bestatin
EP0479489B1 (en) Tripeptide antithrombotic agents
AU772024B2 (en) Inhibitors of urokinase and blood vessel formation
IE60128B1 (en) Hydroxylamine derivatives,their preparation and use as medicaments
US6683055B1 (en) Low molecular weight inhibitors of complement proteases
CA2082748A1 (en) Antithrombotic agents
US6831196B2 (en) Urokinase inhibitors
JPH05504775A (en) Inhibitors and substrates of thrombin
JPS62126158A (en) Aminocarbonylrennin inhibitor
JPS61189298A (en) Novel oligopeptidylargininol derivative
EP0124317A2 (en) Proline derivatives
JPH07509731A (en) Piperazide, a substituted phenylalanine derivative, as a thrombin inhibitor
CZ133998A3 (en) Thrommbin inhibitors, process of their preparation and pharmaceutical composition containing thereof
BG60739B2 (en) Tripeptides affecting the central nervous system, and method for their preparation
CA2170896A1 (en) Kininogen inhibitors
US4240975A (en) Bestatin
CA2224721A1 (en) Calpain inhibitors for the treatment of neurodegenerative diseases
CA2370267A1 (en) Prodrugs of thrombin inhibitors
JP2005526762A (en) Inhibitors of urokinase, their production and use
US20050119190A1 (en) Inhibitors of the blood-clotting factor xa, production thereof and use of the same
US6417161B1 (en) Amino acid amidinohydrazones, alkoxyguanidines and aminoguanidines as protease inhibitors
US5922697A (en) Compounds, compositions and methods for inhibiting the binding of proteins containing an SH2 domain to cognate phosphorylated proteins
AU721261B2 (en) Peptide inhibitors of hematopoietic cell proliferation
CA2163011A1 (en) New peptides derivatives

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR BY CA CH CN CZ DE DK ES FI GB GE HU JP KG KP KR KZ LK LU LV MD MG MN MW NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
ENP Entry into the national phase

Ref document number: 1995 302733

Country of ref document: US

Date of ref document: 19950221

Kind code of ref document: A

121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1994917871

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2163011

Country of ref document: CA

WWP Wipo information: published in national office

Ref document number: 1994917871

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWR Wipo information: refused in national office

Ref document number: 1994917871

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1994917871

Country of ref document: EP