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.

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