WO2000055156A1 - Substituted aromatic amidine derivative and medicinal composition comprising the same - Google Patents

Substituted aromatic amidine derivative and medicinal composition comprising the same Download PDF

Info

Publication number
WO2000055156A1
WO2000055156A1 PCT/KR2000/000216 KR0000216W WO0055156A1 WO 2000055156 A1 WO2000055156 A1 WO 2000055156A1 KR 0000216 W KR0000216 W KR 0000216W WO 0055156 A1 WO0055156 A1 WO 0055156A1
Authority
WO
WIPO (PCT)
Prior art keywords
compound
ethyl
oxoethyl
amidino
acid
Prior art date
Application number
PCT/KR2000/000216
Other languages
French (fr)
Inventor
Bon-Am Koo
Woong-Hyun Nam
Chan-Hee Park
Original Assignee
C & C Research Laboratories
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 C & C Research Laboratories filed Critical C & C Research Laboratories
Priority to JP2000605585A priority Critical patent/JP3377989B2/en
Priority to AU33330/00A priority patent/AU3333000A/en
Publication of WO2000055156A1 publication Critical patent/WO2000055156A1/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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0819—Tripeptides with the first amino acid being acidic

Definitions

  • the present invention relates to an aromatic amidine derivative represented by the following formula (1):
  • A represents G-C ⁇ -alkylene which may be substituted by C ⁇ -C -alkyl, halogeno-C)-C 4 -alkyl, Ci-CValkoxy-G-Gj-alkyl or hydroxy-C i -C 4 -alky 1.
  • R represents hydrogen, or represents G-CValky] which may be substituted by G-Ci-alkyl or phenyl,
  • R ⁇ and R independently of one another represent hydrogen, or represent G-C ⁇ -alkyl or G-Cs-alkanoyl each of which may be mono- or poly-substituted by carboxy, halogen. earbamoyl, amino. methylsulfonvlamino. C ⁇ -C 4 -alkylamino. di((VC ⁇ -alkyl)amino, hydroxy. C ⁇ -C 4 -alkoxycarbonyl or G-Cj-alkoxycarbamoyl. which can be preferably used for oral administration and has potent and selective activity on thrombin inhibition, salt or isomer thereof, process for preparing same, and composition for thrombin inhibition comprising the same as an active ingredient.
  • anti-coagulants which prevent fibrins from being formed can be used for the prevention of thrombosis.
  • zymogens inactivated enzymes
  • the last stage of the blood coagulation process is the step wherein fibrin conglobation is formed from fibrinogen catalyzed by thrombin which has been formed from prothrombin by the action of factor Xa. That is, thrombin as an enzyme for blood coagulation acts a decisive role in angiostasis and thrombosis. Therefore, it is anticipated that any substance having the inhibitory activity against thrombin may inhibit the platelet activity and the formation/stabilization of fibrin, and consequently be used as an effective anti-coagulant.
  • the thrombin inhibitors also activate factors y and ⁇ through a positive feed back reaction.
  • thrombin inhibitors have been developed as an effective remedy for thrombosis and anti-coagulant.
  • three peptide derivatives such as PPACK[D-Phe-Pro-Arg-CH Cl](Thromb. Res., 14, 969(1979)), D-Phe-Pro- Arg, Boc-D-Phe-Pro-Arg and D-MePhe-Pro-Arg(J. Med. Chem., 33, 1729 (1990)), DuP-714[Ac-(D)-Phe- Pro-boroArg-OH](J. Biol. Chem., 265, 18289(1990)), Efegatran [D-MePhe- Pro-Arg . H 2 S0 4 ](Thromb.
  • the present inventors have extensively studied to develop an effective thrombin inhibitor which can be administered orally and has a selective inhibitoiy activity against thrombin.
  • the compound of formula (1 ) as defined above, exhibits a superior inhibitoiy activity against thrombin when administered orally, and then completed the present invention.
  • the present invention provides an aromatic amidine derivative of formula ( 1 ) as defined above, which is a thrombin inhibitor appropriate for oral administration due to its improved phamiacokinetic property, and pharmaceutical!) acceptable salt and stereoisomer thereof.
  • the present invention also provides a process for preparing the compound of formula (1), salt and stereoisomer thereof, and a composition for thrombin inhibition comprising said compound as an active ingredient.
  • the present invention relates to a novel aromatic amidine derivative of the following formula (1):
  • A represents C2-Co-alkylene which may be substituted by G-C -alkyl, halogeno-G-C 4 -alkyl, C ⁇ -C -alkoxy-G-C 4 -alkyl or hydroxy-C ⁇ -C -al yl,
  • R represents hydrogen, or represents G-C -alkyl which may be substituted by G-C 4 -alkyl or phenyl,
  • R and R independently of one another represent hydrogen, or represent G-C 4 -alkyl or C ⁇ -C 5 -alkanoyl each of which may be mono- or poly-substituted by carboxy, halogen, carbamoyl, amino, methylsulfonylamino, G-C ⁇ -alkylamino. di(G-C4-alkyl)amino, hydroxy, G-C 4 -alkoxycarbonyl or G-C ⁇ -alkoxycarbamoyl.
  • the preferred compound includes those wherein A represents ethylene, propylene or butylene which may be substituted by methyl, ethyl, fluoromethyl, methoxymethyl or hydroxymethyl.
  • the preferred compound includes those wherein R represents hydrogen, methyl, isopropyl or benzyl.
  • the preferred compound includes those wherein R and R independently of one another represent hydrogen, methyl, ethyl, 3-carboxypropyl, carboxymethyl, 3-amino-3- carboxy-propanoyl, 3-carbamoyl-3-amino-propanoyl, 4-amino-4-carboxy- butyryl, 4-carbamoyl-4-amino-butyryl, 3 -met- hylsulfonylamino-3-carboxy- propanoyl, 3-diethylamino-3-carboxy-propanoyl, 3-hydroxy-3-carboxy- propanoyl or 3-carbamoyl-2-amino-propanoyl.
  • the compound of formula (1) according to the present invention can form a pharmaceutically acceptable salt.
  • Such salt includes non-toxic acid addition salt containing pharmaceutically acceptable anion, for example a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydriodic acid, etc., a salt with organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trofluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., or a salt with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; and metal addition salt for example a salt with alkali metal such as sodium, potassium, etc. Further, there can be mentioned those salts with other acids
  • the compound of formula (1) includes an asymmetric carbon atom at 2-position of pyrrole ring, and may contain additional asymmetiic carbon atoms depending on the substituents A, R , R and R . Therefore, the compound of formula ( 1 ) can exist as a pure stereoisomer such as enantiomer of R or S, diastereomer, etc., or a mixture thereof including racemate. Therefore, the present invention also includes each of these stereoisomers and their mixtures.
  • novel compound of formula ( 1 ) as defined above can be prepared according to the following processes, and thus, it is an object of the present invention to provide the processes for preparing the compound of formula (1).
  • the compound of formula (1 ) can be prepared by a process characterized in that
  • R is the same with R except that R is not hydrogen and X represents active leaving group, preferably hydroxy or halogen, and a compound represented by the following formula (8):
  • the condensation reaction of the compound of formula (6) with the compound of formula (5) is earned out in a suitable solvent in the presence of a condensing agent according to a conventional manner.
  • a suitable solvent any conventional solvents which do not adversely affect the reaction, preferably dichlorom ethane, dichloroethane, etc. can be used.
  • carboimides such as N,N-diethylcarbodiimide, l-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride[WSCI ⁇ HC1], N,N-di-cyclohexyl- carbodiimide, etc. can be mentioned.
  • This reaction may be optionally proceeded using a reaction-aid such as l-hydroxybenzotriazole[HOBT], an acid acceptor such as N.N-diisopropylethylamine[DIPEA], etc.
  • the reaction can be generally earned out under cooling or heating, preferably at temperatures ranging from 0°c to 20 °Q .
  • the starting compound of formula (5) can be prepared by referring to J. Med. Chem, 1996, 39, 4531-4536; J. Org. Chem, 1982, 47, 104-109; J. Org. Chem, 1984, 49, 2286-2288; Tetrahedron: Asymmetry, Vol. 8, No. 2, 327-335, 1997, etc.. and the compound of formula (6) can be prepared by referring to the specification of the earlier Korean patent application No. 97-22566.
  • tert-butoxycarbonyl group may be used as the amino-protecting group, where the protecting group may be removed by conventional processes for removing the carbamate based amino-protecting group, specifically by hydrolyzation in the presence of an acid(organic acid such as formic acid, acetic acid, trifluoroacetic acid, benzenesulfonic acid, etc., or inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.).
  • an acid(organic acid such as formic acid, acetic acid, trifluoroacetic acid, benzenesulfonic acid, etc.
  • inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.
  • the deprotection reaction is usually carried out in an ordinary solvent which does not adversely affect the reaction, preferably in dichloromethane, dichloroethane, chlorofonn. 1,4-dioxane, etc.
  • Reaction temperature is not critical, but generally ranges between 0°c and 30 °Q .
  • the last step is to give the compound of formula ( 1 ) by converting the cyano group in the compound of fo ⁇ nula (3) to amidino group.
  • the compound of formula (3) may be reacted under hydrogen halide. preferably hydrogen chloride gas and then reacted under the introduction of ammonia gas into the reaction solution.
  • This reaction can generally be cairied out in a solvent, for example, C ⁇ -C 4 -alcohols such as ethanol, propanol. etc., aliphatic ethers such as diethylether, etc.. halogenated hydrocarbons such as chlorofonn.
  • aprotic solvents such as benzene, etc., polar solvents such as N,N-dimethylformamide, dimetbylsulfoxide, etc., or mixtures thereof.
  • polar solvents such as N,N-dimethylformamide, dimetbylsulfoxide, etc.
  • G-C 4 -alcohols such as ethanol is used as the solvent.
  • Reaction temperature and time are not critical, but the reaction is generally carried out under cooling or heating for 2 to 72 hours, preferably at 0°c to 30 °C for 12 to 40 hours.
  • the compound of formula (3) is coupled with either one or both of the compounds of formulae (7) and (8) to give the compound of formula (2) which is then changed to the compound of formula (lb) through the conversion of cyano group to amidino group.
  • Coupling reaction is preferably earned out in an inert solvent.
  • the solvent which can be preferably used for this purpose, acetone, 1,4-dioxane, acetonitrile, chloroform, dichloromethane, dichloroethane, tetiahydrofuran, dimethyl- sulfoxide, N,N-dimethylformamide or mixtures thereof can be mentioned.
  • This reaction is also earned out optionally in the presence of an acid acceptor.
  • inorganic base for example, hydroxides, carbonates or bicarbonates of alkali metal or alkaline earth metal such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, magnesium carbonate, sodium bicarbonate, etc.
  • organic base such as triethylamine, trimethylamine, pyridine, N,N-diisopropylethylamme, etc.
  • acid acceptors are triethylamine, potassium carbonate, sodium carbonate or
  • N.N-diisopropylethylamine This reaction is also earned out optionally in the presence of a condensing agent.
  • a condensing agent which can be preferably used for this memepose.
  • carboimides such as N,N-diethylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, etc. can be mentioned.
  • the condensing agent l-hydroxybenzotriazole, etc. as the reaction-aid and N,N-diisopropylethylamine, etc. as the acid acceptor may be used.
  • Reaction temperature and time are not critical in this reaction, but the reaction is generally carried out under cooling to heating for 2 to 24 hours, preferably at 0 °c to 70 °c for 2 to 18 hours.
  • the hydrolysis reaction can be carried out in a solvent selected from a group consisting of water, alcohols and tetrahydrofuran and in the presence of a base such as hydroxides of alkali metal or inorganic acid.
  • a solvent selected from a group consisting of water, alcohols and tetrahydrofuran and in the presence of a base such as hydroxides of alkali metal or inorganic acid.
  • Particularly preferable solvent includes a solvent mixture of water and alcohol
  • base includes potassium hydroxide, sodium hydroxide or lithium hydroxide
  • inorganic acid includes hydrochloric acid.
  • Reaction temperature and time are not critical in this reaction, but the reaction is generally carried out under cooling or heating for 2 to 72 hours, preferably at 0°c to 30°c for 12 to 24 hours.
  • the present invention also provides a pharmaceutical composition for thrombin inhibition comprising an effective amount of the compound of formula (1), as defined above, or a pharmaceutically acceptable salt or a stereoisomer thereof as an active ingredient together with a pharmaceutically acceptable earner.
  • composition according to the present invention exhibits a potent inhibitoiy activity against thrombin, it can be advantageously used for the prevention and treatment of thrombosis.
  • the compound of fo ⁇ nula (1) of the present invention shows a superior effect when it is orally administered, it is expected to be effectively utilized for this purpose.
  • the active compound according to the present invention When used for clinical potpose, it is administered generally in an amount ranging from 0.1 to 50 g, preferably in an amount ranging from 0.5 to 20], ⁇ per k of body weight a day.
  • the specific administration dosage for the patient can be deteimined by an expert depending on the specific compound used, body weight of the subject patient, sex, hygienic condition, diet, time or method of administration, excretion rate, mixing ratio of the agent, severity of the disease to be treated, etc.
  • the compound of the present invention may be administered in the fo ⁇ n of injections or oral preparations.
  • solid preparations for oral administration, capsules, tablets, pills, powders and granules, preferably capsules and tablets can be mentioned. It is also desirable for tablets and pills to be formulated into enteric-coated preparation.
  • the solid preparations may be prepared by mixing the active compound of formula (1) according to the present invention with at least one carrier selected from a group consisting of inactive diluents such as sucrose, lactose, starch, etc., lubricants such as magnesium stearate, disintegrating agent and binding agent.
  • the composition comprising the active compound of formula (1 ) has the feature to exhibit an excellent pharmacological effect when it is applied in the form of an oral preparation.
  • the present inventors carried out phamiacokinetic experiments on animal models of mouse and dog, and as a result have identified that the pharmaceutical composition of the present invention has the characteristics to be sustained during a long time in blood when it is administered orally.
  • the compound (1) of the present invention can be used more effectively as an oral preparation than any other thrombin inhibitors developed earlier.
  • aqueous or oily suspension for injection comprising the active compound of formula (1)
  • suitable dispersing agent, wetting agent, or suspending agent suitable dispersing agent, wetting agent, or suspending agent.
  • Solvents which can be used for preparing injections include aqueous solvents such as water, Ringer's fluid and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non-stimulative fixing oil including mono-, di-glyceride may be used for this purpose. Fatty acid such as oleic acid may also be used for injections.
  • the compound of formula (1) does not exhibit acute toxicities to mammals such as mouse and dog, and simultaneously exhibits the desirable potent inhibitory activity against thrombin.
  • N-(3S)-2-oxoazaperhydroepin-3-yl(t-butoxy)carboxamide(1.0g) was dissolved in THF(20 m ), 1.0M THF solution(5.26 m £) of lithium bis(trimethylsilyl)amide was added dropwise, and then the resulting mixture was stirred for 30 minutes at room temperature.
  • the reaction solution was cooled in ice bath, ethyl bromoacetate (0.64 m (>) was added dropwise thereto, and the mixture was stirred for further 2 hours. Water was added to the reaction solution, which was then extracted with methylene chloride. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure.
  • step b) The compound(l ⁇ mg) prepared in step b) above was dissolved in ethanol (5 m #) saturated with HC1 gas, and the resulting solution was sti ⁇ ed for 18 hours at room temperature.
  • the reaction solution was concentrated under reduced pressure and dried under vacuum.
  • the residue thus obtained was dissolved in ethanol(5 m ) saturated with ammonia gas and sti ⁇ ed for 2 days at room temperature.
  • step a) The compound(180mg) prepared in step a) above was dissolved in acetonitrile(10 m £), diisopropylethylamine(0.097 m £) and ethyl 4-bromo butyrate(0.072 m £) were added thereto, and the resulting mixture was sti ⁇ ed for 3 hours at reflux temperature.
  • the compound(140mg) prepared in step b) above was dissolved in ethanol ( 10 n ⁇ (>) saturated with HC1 gas, and the resulting solution was stirred for 18 hours at room temperature.
  • the reaction solution was concentrated under reduced pressure and dried under vacuum.
  • the residue thus obtained was dissolved in ethanol(10 m
  • the reaction solution was concentrated under reduced pressure to remove the solvent and the residue thus obtained was dissolved in a solvent mixture of water(3
  • To this solution was added 85% potassium hydroxide(27mg) and the resulting mixture was sti ⁇ "ed for 1 day at room temperature.
  • Example 7-b The compound(24mg) prepared in Example 7-b) was reacted according to the same procedure as Example 2-b) to give the title compound(280mg) as a pale yellow foam.
  • Example 7-b The compound(505mg) prepared in Example 7-b) was reacted according to the same procedure as Example 6-a) to give the title compound(669mg) as a white foam.
  • Example 10-b The compound(265mg) prepared in Example 10-b) was reacted according to the same procedure as Example 2-b) to give the title compound(190mg) as a white foam.
  • step b) The compound(3.1g) prepared in step b) above was introduced into a 25 m f) volumetiic flask, and reacted according to the same procedure as Example 1 -a) to give the title compound(3.99g) as a white foam.
  • step d) The compound(200mg) prepai'ed in step d) above was reacted according to the same procedure as Example 1-c) to give the title compound(l ⁇ mg) as a white foam.
  • Example 13-d The compound(630mg) prepared in Example 13-d) was reacted according to the same procedure as Example 2-b) to give a compound(600mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound(200mg) as a white foam.
  • Example 13-d The compound(466mg) prepared in Example 13-d) was reacted according to the same procedure as Example 6-a) to give a compound(546mg) as a white foam.
  • the resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound( 120mg) as a white foam.
  • the compound(52 lmg) prepared in Example 13-d) was introduced into a 250 m £ volumetiic flask and dissolved in THF/DMF(4/1, v/v, 100 m £).
  • Sodium bicarbonate(229mg) and sodium iodide(catalytic amount) were added thereto at room temperature and allylbromide(0.43 m ( was slowly added dropwise.
  • the reaction mixture was stiired and refluxed for 4 hours to complete the reaction. Water was added thereto in order to stop the reaction and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure.
  • step a) The compound(400mg) prepai'ed in step a) above was introduced into a 100 n ⁇ () volumetric flask and dissolved in dry THF(30 n ⁇ ⁇ ).
  • Pd 2 dibenzylideneacetone;dba)3 • CHCi3(37mg), dppb(l,4-bis(diphenylphos- phinobutane))(64mg) and 2-mercaptobenzoic acid( 120mg) were added thereto and the resulting mixture was stirred for 1 hour at room temperature. After the reaction was completed, water was added to stop the reaction, and the reaction solution was extracted three times with ethyl acetate.
  • step b) The compound(282mg) prepared in step b) above was introduced into a 100m() volumetric flask and dissolved in THF/DMF(4/1, v/v, 50 mC), and then sodium bicarbonate( 103mg) was added thereto at room temperature.
  • the reaction solution was stirred for about lOminutes, ethyl iodide(0.2 m £) was slowly added dropwise thereto, and the resulting mixture was sti ⁇ ed and refluxed for about 4 hours.
  • water was added in order to stop the reaction, and the reaction solution was extracted three times with ethyl acetate. The organic extracts were combined and the solvent was evaporated under reduced pressure.
  • step c) The compound( 1.34g) prepared in step c) above was introduced into a 250 n ⁇ p volumetiic flask and dissolved in diy THF(50 m ⁇ ). Then, Pd (dibenzylideneacetone;dba) 3 • CHCl 3 (79mg), dppb( l,4-bis(diphenylphos- phinobutane))(233mg) and 2-mercaptobenzoic acid(465mg) were added thereto and the resulting mixture was sti ⁇ ed for 2 horns at room temperature. After the reaction was completed, water was added in order to stop the reaction, and the reaction solution was extracted three times with ethyl acetate.
  • step d) The compound(322mg) prepared in step d) above was reacted according to the same procedure as Example 6-a) to give a compound(257mg) as a white foam.
  • the resulting compound was reacted according to the same procedme as Example 1-c) to give Compound(a)(140mg) and Compound 17(45mg) in the form of a white foam.
  • Example 1-b The compound(300mg) prepared in Example 13-d) and (3R)-3- [(t-butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(350mg) were reacted according to the same procedure as Example 1-b) to give a compound(400mg) as a white foam.
  • the resulting compound was reacted according to the same procedure as Example 1-c) to give Compound(a)( 120mg) and Compound 19(20mg) in the fo ⁇ n of a white foam.
  • Example 13-d The compound(500mg) prepared in Example 13-d) was reacted according to the same procedure as Example 6-a) to give a compound(600mg) as a white foam.
  • the resulting compound(600mg) was dissolved in methylene chloride(50 m ), ti-iethylamine(0.3 n] (>) and methanesulfonylchloride(1.5 £) were slowly added dropwise thereto, and the mixture was stirred for 1 hour at room temperature. After the reaction was completed, water was added in order to stop the reaction, and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure.
  • Example 13-d The compound(500mg) prepared in Example 13-d) was reacted according to the same proceedme as Example 6-a) to give a compound(620mg) as a white foam.
  • the resulting compound(620mg) was dissolved in methylene chloride(50 m ), triethylamine(0.3 m £) and ethyl iodide( 1.3 m ) were slowly added dropwise thereto, and the whole mixture was stiired for 1 hour at room temperature. After the reaction was completed, water was added to stop the reaction, and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure.
  • Example 13-d The compound(300mg) prepared in Example 13-d) and (4S)-2.2-dimethyl-5- oxo-l,3-dioxolane-4-carboxylic acid(250mg) were reacted according to the same procedure as Example 1-b) to give a compound(280mg) as a white foam.
  • the resulting compound(280mg) was reacted according to the same procedure as Example 2-c) to give the title compound(l ⁇ mg) as a white foam.
  • Example 13-d The compound(300mg) prepared in Example 13-d) and (4R)-4-[(t- butoxy)carbonylamino]-4-[benzyloxycarbonyl]butanoic acid(330mg) were reacted according to the same procedure as Example 1 -b) to give a compound(450mg) as a white foam.
  • the resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound(200mg) as a white foam.
  • Example 13-d The compound(300mg) prepared in Example 13-d) and (2S)-2-[(t- butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(330mg) were reacted according to the same procedure as Example 1-b) to give a compound(350mg) as a white foam.
  • the resulting compound was reacted according to the same procedure as Example 1 -c) to give the title compound(l ⁇ mg) as a white foam.
  • Example 13-d The compound(300mg) prepared in Example 13-d) and (2R)-2-[(t- butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(330mg) were reacted according to the same procedure as Example 1-b) to give a compound(350mg) as a white foam.
  • the resulting compound was reacted according to the same procedure as Example 1-c) to give the title compound(120mg) as a white foam.
  • the compound(450mg) prepared in step a) above was dissolved in a solvent mixture of diy THF and diethylether(3 m £/10 m ⁇ ), lithium borohydride(2.0M in THF/0.83 m ) and lithium triethylborohydride(1.0M in THF/0.17 ni ( were slowly added dropwise thererto, and the whole mixture was sti ⁇ ed for 2 hours at room temperature. Mefhanol(2 m e) was slowly added dropwise thereto under ice-cooling and the mixture was sti ⁇ ed for 10 minutes.
  • reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using methylene chloride:methanol( 10: l, v/v) as an eluent to give the title compound(160mg) as a white foam.
  • step b) The compound(160mg) prepared in step b) above was dissolved in methylene chloride(5 n ⁇ £), (diethylamino)sulfoti-ifluoride(0.096 I]) ) was added thereto under ice-cooling, and the whole mixture was stirred for 24 hours at room temperature.
  • the reaction solution was poured into a cold and saturated aqueous solution of sodium bicarbonate(10 m .g), which was then extracted with methylene chloride.
  • the organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure.
  • the resulting residue was purified by silica gel column chromatography using methylene chloride:methanol(20: l, v/v) as an eluent to give the title compound(50mg) as a brown oil.
  • step c) The compound(50mg) prepared in step c) above was reacted according to the same_ procedure as Example 1-a) to give the title compound(20mg) as a pale yellow foam.
  • Example 25 -e The compound(40mg) prepared in Example 25 -e) was reacted according to the same procedure as Example 2-a) to give a compound(15mg) as a white foam. The resulting compound was further reacted according to the same procedure as Example 6-a) to give a compound(15mg) as a white foam. The resulting compound( 15mg) was further reacted according to the same procedure as Example 2-c) to give the title compound(3mg) as a pale yellow solid.
  • step c) The compound(371mg) prepai'ed in step c) above was reacted according to the same procedure as Example 2-a) to give a compound(190mg) as a pale yellow foam.
  • the resulting compound was reacted according to the same procedure as Example 6-a) to give the title compound(303mg) as a white foam.
  • step d) The compound(303mg) prepared in step d) above was reacted according to the same procedure as Example 2-c) to give the title compound(90mg) as a pale yellow solid.
  • the compounds according to the present invention were dissolved in 50%) methanol in various concentrations. Then, 20 ⁇ i each thereof were introduced into each well of a microplate, to each of which 160 ⁇ £ of the reaction medium containing 125mM NaCl, 50mM Tris-HCl(pH8.0) and 2mM synthetic substiate(N-benzoyl-Phe-Val-Arg-p-nitroanilide, Sigma B-7632) was added. 20 ⁇ of human thrombin solution(5 units/ ⁇ , Sigma T-6759, manufactured by Sigma Co.) containing 0.1%> bovine serum albumin was added to each well to initiate the enzymatic reaction at room temperature.
  • the degree of hydrolysis of substrate was determined by measuring the absorbance at 405nm.
  • the thrombin inhibitory activity was represented by IC50 value which indicates the concentration of the test compound showing half the change of absorbance in the well that did not contain the test compound.
  • the thrombin inliibitory activity of the compound according to the present invention is shown in the following Table 2.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Epidemiology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

The present invention relates to novel aromatic amidine derivatives which have a potent and selective thrombine inhibitory activity and which may be administered orally, process for preparing the same, and composition for thrombine inhibition comprising the same as an active ingredient.

Description

SUBSTITUTED AROMATIC AMIDINE DERIVATIVE AND MEDICINAL COMPOSITION COMPRISING SAME
TECHNICAL FIELD
The present invention relates to an aromatic amidine derivative represented by the following formula (1):
Figure imgf000003_0001
in which
A represents G-Cβ-alkylene which may be substituted by Cι-C -alkyl, halogeno-C)-C4-alkyl, Ci-CValkoxy-G-Gj-alkyl or hydroxy-C i -C4-alky 1.
R represents hydrogen, or represents G-CValky] which may be substituted by G-Ci-alkyl or phenyl,
R^ and R independently of one another represent hydrogen, or represent G-Cα-alkyl or G-Cs-alkanoyl each of which may be mono- or poly-substituted by carboxy, halogen. earbamoyl, amino. methylsulfonvlamino. Cι-C4-alkylamino. di((VCι-alkyl)amino, hydroxy. Cι-C4-alkoxycarbonyl or G-Cj-alkoxycarbamoyl. which can be preferably used for oral administration and has potent and selective activity on thrombin inhibition, salt or isomer thereof, process for preparing same, and composition for thrombin inhibition comprising the same as an active ingredient.
BACKGROUND ART
In thrombosis, platelet aggregation and fibrin conglobation cause occlusion of blood vessel. Therefore, anti-coagulants which prevent fibrins from being formed can be used for the prevention of thrombosis.
Lots of zymogens (inactivated enzymes) to be activated through enzymatic reaction cascade are involved in the blood coagulation system. The last stage of the blood coagulation process is the step wherein fibrin conglobation is formed from fibrinogen catalyzed by thrombin which has been formed from prothrombin by the action of factor Xa. That is, thrombin as an enzyme for blood coagulation acts a decisive role in angiostasis and thrombosis. Therefore, it is anticipated that any substance having the inhibitory activity against thrombin may inhibit the platelet activity and the formation/stabilization of fibrin, and consequently be used as an effective anti-coagulant. The thrombin inhibitors also activate factors y and γ through a positive feed back reaction.
Recently, many thrombin inhibitors have been developed as an effective remedy for thrombosis and anti-coagulant. For example, three peptide derivatives such as PPACK[D-Phe-Pro-Arg-CH Cl](Thromb. Res., 14, 969(1979)), D-Phe-Pro- Arg, Boc-D-Phe-Pro-Arg and D-MePhe-Pro-Arg(J. Med. Chem., 33, 1729 (1990)), DuP-714[Ac-(D)-Phe- Pro-boroArg-OH](J. Biol. Chem., 265, 18289(1990)), Efegatran [D-MePhe- Pro-Arg . H2S04](Thromb. Haemost., 67, 325 (1992)), Inogatran [HOOC- CH2-(R)Cha-Pic-Nag, wherein Cha means cyclohexylamine. Pic means pipecolic acid, and Nag means noragmarine](WO 93/1 1 152. Blood Coag. Fibrinol., 1, 69 ( 1996)) and CVS-1 123[(CH3CH2CH2)2-CHCO-Asp(OCH3)- Pro-Arg](WO 93/ 15756). and piperidineamide derivatives such as Argatroban(US 4258192; Thromb. Haemost, 18, 13 ( 1992)) and NAPAP(J. Biol. Chem., 266. 20085(1991)) can be mentioned. However, these compounds have the problems that they are not satisfactoiy in the aspects of bioavailability in the case of oral administration, selective inhibitoiy activity against thrombin compared with other serine proteases, stability, durability of action and toxicity shown at the therapeutic dose.
DISCLOSURE OF INVENTION
Thus, the present inventors have extensively studied to develop an effective thrombin inhibitor which can be administered orally and has a selective inhibitoiy activity against thrombin. As a result, we identified that the compound of formula (1 ), as defined above, exhibits a superior inhibitoiy activity against thrombin when administered orally, and then completed the present invention.
Therefore, the present invention provides an aromatic amidine derivative of formula ( 1 ) as defined above, which is a thrombin inhibitor appropriate for oral administration due to its improved phamiacokinetic property, and pharmaceutical!) acceptable salt and stereoisomer thereof. The present invention also provides a process for preparing the compound of formula (1), salt and stereoisomer thereof, and a composition for thrombin inhibition comprising said compound as an active ingredient.
DETAILED DESCRIPTION OF INVENTION
The present invention relates to a novel aromatic amidine derivative of the following formula (1):
Figure imgf000006_0001
, pharmaceutically acceptable salt and stereoisomer thereof, in which
A represents C2-Co-alkylene which may be substituted by G-C -alkyl, halogeno-G-C4-alkyl, Cι-C -alkoxy-G-C4-alkyl or hydroxy-C ι -C -al yl,
R represents hydrogen, or represents G-C -alkyl which may be substituted by G-C4-alkyl or phenyl,
R and R independently of one another represent hydrogen, or represent G-C4-alkyl or Cι-C5-alkanoyl each of which may be mono- or poly-substituted by carboxy, halogen, carbamoyl, amino, methylsulfonylamino, G-Cα-alkylamino. di(G-C4-alkyl)amino, hydroxy, G-C4-alkoxycarbonyl or G-Cα-alkoxycarbamoyl.
Among said compound of formula (1), the preferred compound includes those wherein A represents ethylene, propylene or butylene which may be substituted by methyl, ethyl, fluoromethyl, methoxymethyl or hydroxymethyl.
Among the compound of formula (1), the preferred compound includes those wherein R represents hydrogen, methyl, isopropyl or benzyl.
Among the compound of formula (1), the preferred compound includes those wherein R and R independently of one another represent hydrogen, methyl, ethyl, 3-carboxypropyl, carboxymethyl, 3-amino-3- carboxy-propanoyl, 3-carbamoyl-3-amino-propanoyl, 4-amino-4-carboxy- butyryl, 4-carbamoyl-4-amino-butyryl, 3 -met- hylsulfonylamino-3-carboxy- propanoyl, 3-diethylamino-3-carboxy-propanoyl, 3-hydroxy-3-carboxy- propanoyl or 3-carbamoyl-2-amino-propanoyl.
Typical examples of the compound of formula (1) according to the present invention are exemplified in the following and summaiized in Table 1:
2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydroepinyl)acetyl)pyrrolidi n-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Compound 1);
4- { [(3S)- 1 -(2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyrrolidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoic acid(Compound 2);
2- { [(3 S)- 1 -(2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyrrolidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino} acetic acid(Compound 3);
4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyrrolιdiny l)-l -methyl-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoic acid(Compound 4);
4-{[(3S)- l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]ethylamino}butanoic acid(Compound 5);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr rolidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]carbamoyl}-2-aminopiOpano ic acid(Compound 6);
2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxopyπOlidinyl)acetyl)pyrrolidin-2-yl ]ethyl}-l-ethylindole-6-carboxamidine(Compound 7);
4-{[(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethylmdol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxopyπolidin-3-yl]amino}butanoic acid(Compound 8);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxopyιτolidin-3-yl]carbamoyl}-2-aminopropanoic acid(Compound 9);
2-{2-[(2S)- l -((2S)-2-((3S)-3-amino-2-oxopyiTolidinyl)-3-methylbutano yl)pyiTolidin-2-yl]ethyl }- 1 -ethylindole-6-carboxamidine(Compound 10);
4- { [(3 S )- 1 -(( 1 S)-2-((2S )-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethy l)pyιτol idinyl)-l-(isopropyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]amino}butanoic acid(Compound 1 1 );
2-{ [(3S)- 1 -(( 1 S)-2-((2S)-2-(2-(6-amidino- 1 -ethylιndol-2-yl)ethyl)pyrrol idinyl)-2-oxo- 1 -benzylethyl)-2-oxopyπ'olidin-3-yl]amino }acetic acid(Compound 12);
2-{2-[ l -(2-((3S)-3-amino-2-oxopiperidyl)acetyl)-(2S)-pyiTolidin-2-yl]et hyl }- 1 -ethylindole-6-carboxamidine(Compound 13 );
4-{ [(3S)- l -(2-((2S)-2-(2-(6-amidino- l -ethylindol-2-yl)ethyl)pyiTolidiny l)-2-oxoethyl)-2-oxo-3-piperidyl]amino }butanoic acid(Compound 14); 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminopiOpanoic acid(Compound 15);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbamoyl}-(2S)-2-aminopropan oic acid(Compound 16);
2-{2-[ l -(2-((3S)-3-((3S)-3-amino-3-carbamoyl-N-ethylpropanoylamino )-2-oxopiperidy])acetyl)-(2S)-pyrrolidin-2-yl]ethyl }- 1 -ethylindole-6-carboxami dine(Compound 17);
(2R)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr rolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminopropanoic acid(Compound 18);
2-{2-[l -(2-((3S)-3-((3R)-3-amino-3-carbamoylpropanoylamino)-2-oxo piperidyl)acety])-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-caιboxamidine(C ompound 19);
4-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyrrolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminobutanoic acid(Compound 20);
2-{2-[ l-(2-((3S)-3-((4S)-4-amino-4-carbamoylbutanoylamino)-2-oxopi peridyl)acetyl)-(2S)-pyrrolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Co mpound 21);
3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l -ethylιndol-2-yl)ethyl)pyπOlidι nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-[(methylsulfonyl)amin o]propanoic acid(Compound 22);
3-{N-[(3S)- l -(2-((2S)-2-(2-(6-amidino- l -ethylindol-2-yl)ethyl)pyιτolidι nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl }-(2S)-2-(diethylamino)propan oic acid(Compound 23);
3-{N-f(3S)- l -(2-((2S)-2-(2-(6-amιdino- l -ethylindol-2-yl)ethyl)pyπOlιdι nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl }-(2S -2-hydiOxypropanoιc acid(Compound 24);
(2R)-4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr rolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminobutanoic acid(Compound 25);
2-{2-[l -(2-((3S)-3-((2S)-2-amino-3-carbamoylpropanoylamino)-2-oxop iperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Co mpound 26);
2-{2-[l-(2-((3S)-3-((2R)-2-amino-3-carbamoylpropanoylamino)-2-oxo piperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(C ompound 27);
2-{2-[(2S)-l-(2-((3S,6S)-3-amino-6-(fluoiOmethyl)-2-oxopiperidyl)acet yl)pyrrolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Compound 28);
(2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethylmdol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-piperidyl)]cai-bamoyl }-2-a minopropanoic acid(Compound 29); and
(2S)-3-{N-[ l-(2-((2S)-2-(2-(6-amidino-l-ethylmdol-2-yl)ethyl)pyπOlidι nyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl)]caι-bamoyl}-2-aminop ropanoic acid(Compound 30).
Table la
Figure imgf000011_0001
JO
Table lb
Figure imgf000012_0001
Table lc
Figure imgf000013_0001
Among the typical examples of the compound of formula (1), more particularly preferred compounds are mentioned below:
4-{ [(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidmy l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoic acid(Compound 2);
4-{ [(3S)- l -(2-((2S)-2-(2-(6-amidino-l -ethylindol-2-yl)ethyl)pyrrolidiny l)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]ethylamino }butanoic acid(Compound 5);
(2S)-3-{N-[(3S)- 1 -(2-((2S)-2-(2-(6-amidιno- 1 -ethylindol-2-yl)ethy l)pyr rolidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]carbamoyl}-2-aminopropano ic acid(Compound 6);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxopyrrolidin-3-yl]carbamoyl}-2-aminopropanoic acid(Compound 9);
4-{ [(3S)-l-(2-((2S)-2-(2-(6-amidmo-l-ethylindol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxo-3-piperidyl]amino}butanoic acid(Compound 14);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminopropanoic acid(Compound 15);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amιdmo-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbamoyl}-(2S)-2-aminopropan oic acid(Compound 16);
4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminobutanoic acid(Compound 20);
3- (N-[(3S)-l-(2-((2S)-2-(2-(6-amιdmo-l-ethylιndol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-(diethylamino)piOpan oic acid(Compound 23);
3 - { N -[(3 S )- 1 -(2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyιτolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-hydroxypropanoic acid(Compound 24);
(2R)-4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino- l-ethylindol-2-yl)ethyl)pyι- rolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminobutanoic acid(Compound 25);
(2S)-3-{N-[ l -(2-((2S)-2-(2-(6-amιdιno- l -ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-piperidyl)]carbamoyl }-2-a minopropanoic acid(Compound 29); and
(2S)-3-{ N-f l -(2-((2S)-2-(2-(6-amidιno-l -ethylmdol-2-yl)ethyl)pyπOlιdi nyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminop ropanoic acid(Compound 30).
Also, the compound of formula (1) according to the present invention can form a pharmaceutically acceptable salt. Such salt includes non-toxic acid addition salt containing pharmaceutically acceptable anion, for example a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydriodic acid, etc., a salt with organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trofluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, etc., or a salt with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; and metal addition salt for example a salt with alkali metal such as sodium, potassium, etc. Further, there can be mentioned those salts with other acids or bases generally known and used in the technical field to which aromatic amidine derivatives or lactam derivatives pertain. They can be produced by conventional conversion methods.
The compound of formula (1) includes an asymmetric carbon atom at 2-position of pyrrole ring, and may contain additional asymmetiic carbon atoms depending on the substituents A, R , R and R . Therefore, the compound of formula ( 1 ) can exist as a pure stereoisomer such as enantiomer of R or S, diastereomer, etc., or a mixture thereof including racemate. Therefore, the present invention also includes each of these stereoisomers and their mixtures.
The novel compound of formula ( 1 ) as defined above can be prepared according to the following processes, and thus, it is an object of the present invention to provide the processes for preparing the compound of formula (1).
More specifically, the compound of formula (1 ) can be prepared by a process characterized in that
(a) a compound represented by the following formula (6):
Figure imgf000016_0001
is condensed with a compound represented by the following formula (5):
Figure imgf000016_0002
wherein A and R are defined as previously described and P represents an am ino-pro tectmg group, to give a compound represented by the following formula (4):
Figure imgf000017_0001
wherein A, R1 and P are defined as previously described, the resulting compound of formula (4) is deprotected to give a compound represented by the following formula (3):
Figure imgf000017_0002
wherein A and R1 are defined as previously described, and then the cyano group in the resulting compound of formula (3) is converted to amidino group to give a compound represented by the following foπnula
( l a):
Figure imgf000017_0003
wherein A and R are defined as previously described, or
(b) the compound of formula (3) is coupled with either one or both of a compound represented by the following formula (7):
R2'-X (7)
2' 2 2" wherein R is the same with R except that R is not hydrogen and X represents active leaving group, preferably hydroxy or halogen, and a compound represented by the following formula (8):
R3'-X (8)
wherein R is the same with R except that R" is not hydrogen and X is defined as previously described, to give a compound represented by the following formula (2):
Figure imgf000018_0001
1 ' wherein A, R , R~ and R are defined as previously described, m and n independently of one another represent an integer of 0 or 1. and p represents an integer of 2-(m+n). and then the cyano group in the resulting compound of formula (2) is converted to amidino group to give a compound represented by the following formula ( lb);
Figure imgf000019_0001
wherein A, R , R , R , m, n and p are defined as previously described, or
(c) if necessaiy, the compound of formula (lb) is hydrolyzed to give the compound of formula (1 ) having a carboxy group.
The above processes will be explained in more detail hereinafter.
First, in process (a), the condensation reaction of the compound of formula (6) with the compound of formula (5) is earned out in a suitable solvent in the presence of a condensing agent according to a conventional manner. As the solvent, any conventional solvents which do not adversely affect the reaction, preferably dichlorom ethane, dichloroethane, etc. can be used. Also, as the preferable condensing agent, carboimides such as N,N-diethylcarbodiimide, l-(3-dimethylamino propyl)-3-ethylcarbodiimide hydrochloride[WSCI ■ HC1], N,N-di-cyclohexyl- carbodiimide, etc. can be mentioned. This reaction may be optionally proceeded using a reaction-aid such as l-hydroxybenzotriazole[HOBT], an acid acceptor such as N.N-diisopropylethylamine[DIPEA], etc. The reaction can be generally earned out under cooling or heating, preferably at temperatures ranging from 0°c to 20 °Q . The starting compound of formula (5) can be prepared by referring to J. Med. Chem, 1996, 39, 4531-4536; J. Org. Chem, 1982, 47, 104-109; J. Org. Chem, 1984, 49, 2286-2288; Tetrahedron: Asymmetry, Vol. 8, No. 2, 327-335, 1997, etc.. and the compound of formula (6) can be prepared by referring to the specification of the earlier Korean patent application No. 97-22566.
Then, deprotection reaction to remove the amino-protecting group is earned out on the compound of formula (4) thus obtained to give the compound of formula (3). For example, tert-butoxycarbonyl group may be used as the amino-protecting group, where the protecting group may be removed by conventional processes for removing the carbamate based amino-protecting group, specifically by hydrolyzation in the presence of an acid(organic acid such as formic acid, acetic acid, trifluoroacetic acid, benzenesulfonic acid, etc., or inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, etc.). The deprotection reaction is usually carried out in an ordinary solvent which does not adversely affect the reaction, preferably in dichloromethane, dichloroethane, chlorofonn. 1,4-dioxane, etc. Reaction temperature is not critical, but generally ranges between 0°c and 30 °Q .
The last step is to give the compound of formula ( 1 ) by converting the cyano group in the compound of foπnula (3) to amidino group. In this step, the compound of formula (3) may be reacted under hydrogen halide. preferably hydrogen chloride gas and then reacted under the introduction of ammonia gas into the reaction solution. This reaction can generally be cairied out in a solvent, for example, Cι-C4-alcohols such as ethanol, propanol. etc., aliphatic ethers such as diethylether, etc.. halogenated hydrocarbons such as chlorofonn. etc., aprotic solvents such as benzene, etc., polar solvents such as N,N-dimethylformamide, dimetbylsulfoxide, etc., or mixtures thereof. Particularly preferably, G-C4-alcohols such as ethanol is used as the solvent. Reaction temperature and time are not critical, but the reaction is generally carried out under cooling or heating for 2 to 72 hours, preferably at 0°c to 30 °C for 12 to 40 hours.
In process (b), the compound of formula (3) prepared in the second step of the above process (a) is used as the starting material.
That is, the compound of formula (3) is coupled with either one or both of the compounds of formulae (7) and (8) to give the compound of formula (2) which is then changed to the compound of formula (lb) through the conversion of cyano group to amidino group. Coupling reaction is preferably earned out in an inert solvent. As the solvent which can be preferably used for this purpose, acetone, 1,4-dioxane, acetonitrile, chloroform, dichloromethane, dichloroethane, tetiahydrofuran, dimethyl- sulfoxide, N,N-dimethylformamide or mixtures thereof can be mentioned. This reaction is also earned out optionally in the presence of an acid acceptor. As the acid acceptor which can be preferably used for this purpose, inorganic base, for example, hydroxides, carbonates or bicarbonates of alkali metal or alkaline earth metal such as sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, magnesium carbonate, sodium bicarbonate, etc., or organic base such as triethylamine, trimethylamine, pyridine, N,N-diisopropylethylamme, etc. can be mentioned. Particularly preferable acid acceptors are triethylamine, potassium carbonate, sodium carbonate or
N.N-diisopropylethylamine. This reaction is also earned out optionally in the presence of a condensing agent. As the condensing agent which can be preferably used for this puipose. carboimides such as N,N-diethylcarbodiimide, l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, etc. can be mentioned. When the condensing agent is used, l-hydroxybenzotriazole, etc. as the reaction-aid and N,N-diisopropylethylamine, etc. as the acid acceptor may be used. Reaction temperature and time are not critical in this reaction, but the reaction is generally carried out under cooling to heating for 2 to 24 hours, preferably at 0 °c to 70 °c for 2 to 18 hours.
The process from the compound of formula (2) thus obtained to the compound of formula (lb) through the conversion of cyano group to amidino group can be proceeded according to the same manner as explained in process (a).
As stated above for process (c), in case either one or both of the substituents R and R in the compound of formula (lb) obtained in process (b) are carbamoyl or ester group, those groups may be hydrolyzed to give the compound of formula (1) having a carboxy group. The hydrolysis reaction can be carried out in a solvent selected from a group consisting of water, alcohols and tetrahydrofuran and in the presence of a base such as hydroxides of alkali metal or inorganic acid. Particularly preferable solvent includes a solvent mixture of water and alcohol, base includes potassium hydroxide, sodium hydroxide or lithium hydroxide, and inorganic acid includes hydrochloric acid. Reaction temperature and time are not critical in this reaction, but the reaction is generally carried out under cooling or heating for 2 to 72 hours, preferably at 0°c to 30°c for 12 to 24 hours.
The aforementioned processes will be more specifically explained by the following examples. As stated above, the compound of formula (1) prepared according to the aforementioned processes has a superior and selective inhibitory activity against thrombin and therefore, the present invention also provides a pharmaceutical composition for thrombin inhibition comprising an effective amount of the compound of formula (1), as defined above, or a pharmaceutically acceptable salt or a stereoisomer thereof as an active ingredient together with a pharmaceutically acceptable earner.
Since the composition according to the present invention exhibits a potent inhibitoiy activity against thrombin, it can be advantageously used for the prevention and treatment of thrombosis. Particularly, since the compound of foπnula (1) of the present invention shows a superior effect when it is orally administered, it is expected to be effectively utilized for this purpose.
When the active compound according to the present invention is used for clinical puipose, it is administered generally in an amount ranging from 0.1 to 50 g, preferably in an amount ranging from 0.5 to 20],^ per k of body weight a day. However, the specific administration dosage for the patient can be deteimined by an expert depending on the specific compound used, body weight of the subject patient, sex, hygienic condition, diet, time or method of administration, excretion rate, mixing ratio of the agent, severity of the disease to be treated, etc.
The compound of the present invention may be administered in the foπn of injections or oral preparations.
As the solid preparation for oral administration, capsules, tablets, pills, powders and granules, preferably capsules and tablets can be mentioned. It is also desirable for tablets and pills to be formulated into enteric-coated preparation. The solid preparations may be prepared by mixing the active compound of formula (1) according to the present invention with at least one carrier selected from a group consisting of inactive diluents such as sucrose, lactose, starch, etc., lubricants such as magnesium stearate, disintegrating agent and binding agent.
As mentioned above, the composition comprising the active compound of formula (1 ) has the feature to exhibit an excellent pharmacological effect when it is applied in the form of an oral preparation. The present inventors carried out phamiacokinetic experiments on animal models of mouse and dog, and as a result have identified that the pharmaceutical composition of the present invention has the characteristics to be sustained during a long time in blood when it is administered orally.
Therefore, it is anticipated that the compound (1) of the present invention can be used more effectively as an oral preparation than any other thrombin inhibitors developed earlier.
Injections, for example, sterilized aqueous or oily suspension for injection comprising the active compound of formula (1), can also be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent. Solvents which can be used for preparing injections include aqueous solvents such as water, Ringer's fluid and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non-stimulative fixing oil including mono-, di-glyceride may be used for this purpose. Fatty acid such as oleic acid may also be used for injections.
According to the experiments caπied out by the present inventors, it has been identified that the compound of formula (1) does not exhibit acute toxicities to mammals such as mouse and dog, and simultaneously exhibits the desirable potent inhibitory activity against thrombin.
The present invention will be more specifically explained in the following examples and experiments. However, it should be understood that the following examples and experiments are intended to illustrate the present invention but not in any manner to limit the scope of the present invention.
BEST MODE FOR CARRYING OUT THE INVENTION
Example 1
Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydro- epinyl)acetyl)pyrrolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Compo und 1)
a) Synthesis of 2-{(3S)-3-[(t-butoxy)carbonylamino]-2-oxoazaper- hydioepinyl} acetic acid
N-(3S)-2-oxoazaperhydroepin-3-yl(t-butoxy)carboxamide(1.0g) was dissolved in THF(20m ), 1.0M THF solution(5.26m£) of lithium bis(trimethylsilyl)amide was added dropwise, and then the resulting mixture was stirred for 30 minutes at room temperature. The reaction solution was cooled in ice bath, ethyl bromoacetate (0.64m(>) was added dropwise thereto, and the mixture was stirred for further 2 hours. Water was added to the reaction solution, which was then extracted with methylene chloride. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using n-hexane: ethyl acetate=2: l (v/v) as an eluent to give a pale yellow oil. This oil was dissolved in a solvent mixture of water(10m{>) and methanol(20m ). Potassium hydroxide(85%; 756mg) was added and the resulting mixture was stirred for 3 hours at room temperature. The solvent was removed by concentration under reduced pressure, and then the residue was acidified to pH 4 using a 10% citric acid solution and extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and dried under vacuum to give the title compound(1.68g) as a white foam.
Η-NMR(300MHz, CDC ) δ : 5.90(d, 1H, J=6.2Hz), 4.43-4.38(m, 1H), 4.21 (d, 2H, J=1.9Hz), 3.66-3.75(m, 1H), 3.20-3.25(m, 1H), 1.98-2.09(m, 2H), 1.83- 1.76(m, 3H). 1.63-1.58(m, 1H), 1.44(s, 9H)
b) Synthesis of N-{(3S)-l -[2-((2S)-2-(2-(6-cyano- l-ethylindol-2- yl)ethyl)pyιτolidinyl)-2-oxoethyl]-2-oxoazaperhydroepin-3-yl}(t-butoxy)carbo xamide
2-[2-((2S)-Pyτrolidin-2-yl)ethyl]- l -ethylindole-6-carbonitιile(400mg, Korean Patent Appln. No. 97-22566) and the compound(515mg) prepared in step a) above were dissolved in methylene chloride(20m#). DIPEA(0.32ni( , HOBT(238mg) and WSCI • HCl(373mg) were sequentially added thereto and the resulting mixture was stirred for 3 hours at room temperature. To the reaction solution was added water, and the mixture was extracted with methylene chloride. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol=40: l(v/v) as an eluent to give the title compound(590mg) as a white foam.
Η-NMR(300MHz, CDCI3) δ : 7.61-7.53(m, 2H), 7.29-7.26(m, 1 H), 6.36(s, 1H), 5.77-5.75(m, 1H), 4.43-4.41(m, 1H), 4.26-4.05(m, 5H), 3.75-3.72(m, 1H), 3.52-3.49(m, 2H), 3.26-3.21(m, 1H), 2.78-2.75(m, 2H), 2.27-2.24(m, 1H), 2.08-1.97(m, 5H), 1.79-1.74(m. 6H), 1.44(s, 9H), 1 .38-1.34(m, 3H)
c) Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydro- epinyl)acetyl)pyιτolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine
The compound(lθθmg) prepared in step b) above was dissolved in ethanol (5m#) saturated with HC1 gas, and the resulting solution was stiπed for 18 hours at room temperature. The reaction solution was concentrated under reduced pressure and dried under vacuum. The residue thus obtained was dissolved in ethanol(5m ) saturated with ammonia gas and stiπed for 2 days at room temperature. The reaction solution was concentrated under reduced pressure and the residue thus obtained was purified by NH-silica column chromatography using ethyl acetate:methanol=3: l(v/v) as an eluent to give the title compound(15mg) as a pale yellow solid.
ES-MS : 453(M+ 1)'
Η-NMR(300MHz. CDiOD) g : 7.83-7.81 (m. 1H), 7.57-7.52(m, 1 H). 7.35-7.31 (m, 1 H), 6.38(s, 1 H), 4.35-4.19(m. 5H). 4.00-3.93(m, 1H), 3.82-3.78(m. 1H), 3.52-3.46(m. 3H), 2.77-2.74(m. 2H). 2.16-2.12(m, 1H), 1.96-1.91(m, 5H), 1.77-1.51(m, 6H), 1.31-1.27(m, 3H) IR(KBr) cm"1 : 3300, 1640, 1530, 1470
Example 2
Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol-2-yI) ethyl)pyrroHdinyI)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoi c acid (Compound 2)
a) Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydro- epinyl)acetyl)pyιτolidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
The compound(480mg) prepared in Example 1 -b) was dissolved in methylene chloride(10m ), tiifluoroacetic acid(5nl£) was added dropwise under ice-cooling, and the resulting mixture was stirred for 3 hours. The solvent was removed by concentration under reduced pressure, and then the residue was adjusted to pH 9 by the addition of saturated aqueous solution of sodium bicarbonate and extracted with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol=10: l(v/v) as an eluent to give the title compound(320mg) as a pale yellow foam.
Η-NMR(300MHz, CDC1 ) : 7.61 -7.53(m, 2H), 7.29-7 26(m, 1 H), 6.37(s, 1 H), 4.36-3.98(m, 5H), 3.75-3.71(m, 2H), 3.52-3.48(m. 2H), 3.21 -3.17(m. 1 H), 2.78-2.74(m, 2H), 2.26-2.22(m, 1 H), 2.09-1.97(m. 5H), 1 .78- 1 .67(m. 6H), 1.35(3t, H, J=7.2Hz)
b) Synthesis of ethyl 4-{[(3S)-l -(2-((2S)-2-(2-(6-cyano- l-ethylιndol- 2-yl)ethyl)pynOlidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butano ate
The compound(180mg) prepared in step a) above was dissolved in acetonitrile(10m£), diisopropylethylamine(0.097m£) and ethyl 4-bromo butyrate(0.072m£) were added thereto, and the resulting mixture was stiπed for 3 hours at reflux temperature. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using methylene chloride:methanol= 20: l(v/v) as an eluent to give the title compound (l lOmg) as a white foam.
Η-NMR(300MHz, CDC ) δ : 7.63-7.53(m, 2H), 7.32-7.27(m, 1 H), 6.37(s, 1H), 4.29-3.96(m, 8H), 3.74-3.69(m, 2H), 3.46-3.43(m, 1H), 3.26-3.12(m, 3H), 2.89-2.78(m, 2H), 2.54-2.47(m, 2H), 2.30-2.25(m, 1H), 2.09-2.01(m, 5H), 1.80-1.75(m, 6H), 1.62-1.57(m, 2H), 1.35(t, 3H, J=7.2Hz), 1.24(t, 3H, J=7.1Hz)
c) Synthesis of 4-{ [(3S)- l -(2-((2S)-2-(2-(6-amidino- l -ethylmdol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]amino}butanoic acid
The compound(140mg) prepared in step b) above was dissolved in ethanol ( 10nι(>) saturated with HC1 gas, and the resulting solution was stirred for 18 hours at room temperature. The reaction solution was concentrated under reduced pressure and dried under vacuum. The residue thus obtained was dissolved in ethanol(10m|>) saturated with ammonia gas and stirred for 2 days at room temperature. The reaction solution was concentrated under reduced pressure to remove the solvent and the residue thus obtained was dissolved in a solvent mixture of water(3|n( and ethanol(6nι()). To this solution was added 85% potassium hydroxide(27mg) and the resulting mixture was stiπ"ed for 1 day at room temperature. The solvent was removed by concentration under reduced pressure and the residue was purified by NH-silica column chromatography using ethyl acetate:methanol=l : l (v/v) as an eluent to give the title compound(40mg) as a white solid.
ES-MS : 539(M+1)+
Η-NMR(300MHz, CD3OD) : 7.88-7.86(m, 1H), 7.54-7.50(m, 1 H), 7.36-7.33 (m, 1H), 6.33(s, 1H), 4.39-4.36(m, 1H), 4.22-4.18(m, 3H), 3.96-3.88(m, 2H), 3.58-3.50(m, 4H), 3.25-3.21(m, 2H), 2.76-2.73(m, 2H), 2.53-2.48(m, 2H), 2.08-1.66(m, 14H), 1.26(t, 3H, J=7.2Hz)
IR(KBr) cm"' ; 3400, 1640, 1570, 1410
Example 3
Synthesis of 2-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindoI-2- yl)ethyI)pyrrolidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yljamino}acetic acid (Compound 3)
a) Synthesis of ethyl 2-{[(3S)-l -(2-((2S)-2-(2-(6-cyano-l-ethylmdol- 2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}acetate
The compound( 140mg) prepared in Example 2-a) was dissolved in methylene chloride(7m£), niethylamine(0.059m >) and ethyl bromoacetate (0.043 n](>) were added thereto, and the resulting mixture was stirred for 2 hours at room temperature. The reaction solution was stiπed for further 3 hours at reflux temperature. The solvent was removed by concentration under reduced pressure to give a residue which was then purified by silica gel column chromatography using methylene chloride: methanol=20: l (v/v) as an eluent to give the title compound( 130mg) as a pale yellow foam. L Η-NMR(300MHz, CDC ) δ : 7.61-7.53(m, 2H). 7.31-7.23(m, 1 H), 6.37(s, 1H), 4.36-3.96(m, 8H), 3.61-3.38(m, 6H), 2.80-2.74(m, 2H), 2.30-2.24(m, 1H), 1.98-1.85(m, 5H), 1.80-1.73(m, 6H), 1.35(t, 3H, J=7.2Hz), 1.27-1.22(m, 3H)
b) Synthesis of 2-{[(3S)-l-(2-((2S)-2-(2-(6-amidmo- l -ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]amino}acetic acid
The compound(120mg) prepared in step a) above was reacted according to the same procedm-e as Example 2-c) to give the title compound(35mg) as a white solid.
ES-MS : 511(M+1)J
Η-NMR(300MHz, CD3OD) δ : 7.89-7.82(m, 1H). 7.52-7.48(m, 1H), 7.35-7.29 (m, 1H), 6.3 l(s, 1H), 4.38-4.35(m, 1H). 4.23-4.16(m, 3H), 3.95-3.81 (m, 2H), 3.55-3.48(m, 4H), 3.21-3.10(m, 2H), 2.74-2.71(m, 2H), 2.07- 1 .79(m, 6H), 1.63-1.46(m, 6H), 1.26(t, 3H, J=7.2Hz)
IR(KBr) cm"1 : 3350. 1640, 1590, 1530, 1470, 1410
Example 4
Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl) ethyl)pyrroIidinyl)-l-methyl-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amin o}butanoic acid(Compound 4)
a) Synthesis of 2-{(3S)-3-[(t-butoxy)carbonylamino]-2-oxoazaper- hydroepinyl } propanoic acid
N-(3S)-2-oxoazaperhydroepin-3-yl(t-butoxy)carboxamide(lg) and ethyl 2-biOmopropanoate(0.74m(>) were reacted according to the same procedure as Example 1 -a) to give the title compound(869mg) as a white foam.
Η-NMR(300MHz, CDCh) δ : 5.95(brs, IH), 5.20-5.04(m, IH),
4.40-4.28(m, IH), 3.48-3.18(m, 2H), 2.06-1.50(m, 6H), 1.42(s, 9H), 1.34-1.32(m, 3H)
b) Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydro- epinyl) propanoyl)pyιτolidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
2-[2-((2S)-Pyττolidin-2-yl)et yl]-l -ethylindole-6-carbonitrile(700mg) and the compound(837mg) prepared in step a) above were reacted according to the same procedure as Example 1-b) to give N-{(3S)-l-[2- ((2S)-2-(2-(6-cyano-l-ethylindol-2-yl)ethyl)pyπOlιdinyl)-l-methyl-2-oxoethyl] -2-oxoazaperhydiOepin-3-yl}(t-butoxy)carboxamide as a pale yellow oil. This compound was reacted according to the same procedme as Example 2-a) to give the title compound(628mg) as a white foam.
ES-MS : 550(M+1)"
Η-NMR(300MHz, CDCI3) : 7.59-7.53(m, 2H), 7.30-7.28(m, IH), 6.40-6.37 (m. I H), 5.41-5.36(m, IH), 4.40-4.3 l(m, IH), 4.22-4.10(m, 2H), 3.67-3.61(m, IH), 3.54-3.40(m, 2H), 3.30-3.16(m, 2H), 2.78-2.72(m, 2H), 2.38-2.22(m, 2H), 2.05-1.63(m, 1 1H), 1.38-1.32(m, 6H)
c) Synthesis of 4-{[(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethylmdol-2- yl)ethyl)pyιτolidinyl)-l-methyl-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino }butanoic acid
The compound(417mg) prepared in step b) above was reacted according to the same procedure as Example 2-b) to give ethyl 4-{ [(3S)- l -(2-((2S)-2-(2-(6-cyano-l-ethylindol-2-yl)ethyl)pynOlidinyl)-l-methyl-2-oxo ethyl)-2-oxoazaperhydroepin-3-yl]amino }butanoate as a white foam. This compound was reacted according to the same procedure as Example 2-c) to give the title compound(130mg) as a pale yellow solid.
ES-MS : 277(1/2M+1)+
Η-NMR(300MHz, CD3OD) δ : 7-92(s. I H), 7.61-7.58(m, IH), 7.42-7.38(m, IH), 6.42(s, IH), 5.30-5.26(m, I H), 4.30-4.16(m, 4H), 3.60-3.38(m, 6H), 2.83- 2.78(m, 2H), 2.60-2.50(m, 2H), 2.15-1.65(m, 14H), 1.36-1.24(m, 6H)
IR(KBr) cm"1 : 3400, 2950, 1630, 1570. 1530, 1470
Example 5
Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yI) ethyI)pyrroIidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]ethylamino}but anoic acid(Compound 5)
a) Synthesis of ethyl 4-{[(3S)-l-(2-((2S)-2-(2-(6-cyano-l-ethylindol- 2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]ethylamino}bu tanoate
The compound( 160mg) prepared in Example 2-b) was dissolved in DMF(lm ), potassium carbonate(162mg) and ethyl iodide(0.047m<>) were added thereto, and the resulting mixture was stiπed for 18 hours at room temperature. Water was added to the reaction solution and the resulting mixture was extracted with methylene chloride. The organic layer was dried over anhydrous sodium sulfate. filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol=40: l(v/v) as an eluent to give the title compound(I OOmg) as a pale yellow oil.
Η-NMR(300MHz, CDCb) δ : 7.58-7.50(m. 2H), 7.27-7.23(m. IH), 6.34(s, IH), 4.25-4.00(m, 8H), 3.65-3.60(m, 2H), 3.47-3.41(m, 4H), 2.76-2.62(m, 4H), 2.29-2.23(m, 3H), 2.02-1.92(m, 5H), 1.80-1.65(m, 8H), 1.32(t, 3H, J=7.2Hz), 1.22(t, 3H, J=7.1Hz), 0.98(t, 3H, J=7.2Hz)
b) Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]ethylamino}buta noic acid
The compound(145mg) prepared in step a) above was reacted according to the same procedure as Example 2-c) to give the title compound(60mg) as a white solid.
ES-MS : 567(M+1)+
1H-NMR(300MHz, CD3OD) δ : 7-92(s, IH), 7.62-7.58(m, IH), 7.40-7.35(m, IH), 6.40(s, IH), 4.35-4.20(m, 4H), 4.10-4.02(m, IH), 3.80-3.76(m, IH), 3.52-3.42(m, 4H), 2.82-2.68(m, 6H), 2.12-1.98(m. 6H), 1.78-1.62(m, 8H), 1.36(t, 3H, J=7.2Hz), 1.03-0.98(m, 3H)
Example 6
Synthesis of (2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyI- indoI-2-yI)ethyI)pyrrolidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yI]carb amoyI}-2-aminopropanoic acid(Compound 6)
a) Synthesis of benzyl (2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-cyano-l - ethylindol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]car bamoy 1 } -2-[(t-butoxy)carbonylamino]propanoate
The compound( 150mg) prepared in Example 2-a) was dissolved in methylene chloride( 10m( , (3S)-3-[(t-butoxy)carbonylamino]-3-[benzyloxy carbonyl]propanoic acid( 134mg), HOBT(56mg), DIPEA(54mg) and WSCI • HCl(99mg) were sequencially added thereto under ice-cooling, and then the resulting mixture was stirred for 3 hours. Water was added to the reaction solution, which was then extracted with methylene chloride. The organic layer was dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol= 30: l (v/v) as an eluent to give the title compound(242mg) as a white foam.
Η-NMR(300MHz, CDCb) δ : 7.57-7.5 l (m. 2H), 7.30-7.25(m, 6H), 6.90(brs, IH), 6.34(s, IH), 5.75(brs, IH), 5.20-5.10(s, 2H), 4.58-4.5 l(m, 2H), 4.22- 4.00(m, 5H), 3.68-3.59(m. IH), 3.48-3.44(m, 2H), 3.26-3.20(m, IH), 2.94-2.88 (m, IH), 2.76-2.65(m. 3H), 2.32-2.25(m. IH), 2.01-1.89(m, 6H), 1.74-1.68(m, 5H), 1.38(s, 9H), 1.34-1 .31(m, 3H)
b) Synthesis of (2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyl indol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]carbamo yl}-2-aminopropanoic acid
The compound(242mg) prepared in step a) above was reacted according to the same procedure as Example 2-c) to give the title compound(72mg) as a white solid.
ES-MS : 569(M+1 )"
Η-NMR(300MHz. CD3OD) δ : 7.96-7.92(m, IH), 7.64-7.59(m, I H), 7.43-7.38 (m. I H), 6.40(s. IH), 4.76-4.72(m. I H), 4.50-4.42(m, IH), 4.28-4.20(m, 4H), 4.00-3.96(m, IH), 3.72-3.51(m, 4H), 3.42-3.25(m, 2H), 2.82-2.77(m, 2H), 2.46-2.41(m, IH). 2.15-1.56(m, 1 I H), 1.36-1.32(m, 3H)
Example 7 Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxopyrroIidinyl) acetyl)pyrroIidin-2-yl]ethyl}-l-ethyIindole-6-carboxamidine(Compound 7)
a) Synthesis of N-{(3S)-l-[2-((2S)-2-(2-(6-cyano-l-ethylindol-2- yl)ethyl)pyrrolidinyl)-2-oxoethyl]-2-oxopyrrolidin-3-yl }(t-butoxy)carboxamide
2-[2-((2S)-PyπOlidin-2-yl)ethyl]-l-ethylindole-6-carbonitrile(500mg) and 2-{(3S)-3-[(t-butoxy)carbonylamino]-2-oxopyπOlidinyl}acetic acid(483 mg, see: J. Org. Chem. 1982, 47, 104-109) were reacted according to the same procedure as Example 1-b) to give the title compound(600mg) as a white foam.
Η-NMR(300MHz, CDC13) δ : 7.54-7.48(m, 2H), 7.26-7.22(m, IH), 6.35(s, IH), 5.08(brs, IH), 4.22-3.98(m. 6H), 3.48-3.36(m, 4H), 2.77-2.72(m, 2H), 2.62-2.55(m, IH), 2.30-2.24(m, IH), 2.00-1.68(m, 6H), 1.42(s, 9H), 1.32(t, 3H, J=7.1Hz)
b) Synthesis of 2-{2-[(2S)-l-(2-((3S)-3-ammo-2-oxopyπOlidinyl) acetyl) pyπOlidin-2-yl]ethyl }-l-ethylindole-6-carbonitιϊle
The compound(600mg) prepared in step a) above was reacted according to the same procedme as Example 2-a) to give the title compound(420mg) as a white foam.
1H-NMR(300MHz. CDCI3) δ : 7.58-7.52(m. 2H), 7.27-7.24(m, I H), 6.34(s, I H), 4.20-3.90(m, 5H), 3.60-3.38(m. 5H), 2.76-2.72(m, 2H), 2.46-2.25(m, 2H), 2.06- 1.92(m, 4H), 1.80-1.70(m. 2H), 1.32(t, 3H, J=7.2Hz)
c) Synthesis of 2-{2-[(2S)-l -(2-((3S)-3-amino-2-oxopyπOlidinyl) acetyl) pyπOlidin-2-yl]ethyl }-l-ethylindole-6-carboxamidine The compound(150mg) prepared in step b) above was reacted according to the same procedure as Example 1-c) to give the title compound(94mg) as a pale yellow solid.
ES-MS : 425(M+1)+
Η-NMR(300MHz, CD3OD) δ : 7-86(s, IH), 7.61-7.57(m, I H). 7.38-7.35(m, I H), 6.42(s, IH), 4.28-4.01(m, 5H), 3.56-3.34(m, 5H). 2.86-2.80(m, 2H), 2.44- 2.20(m, 2H), 2.10-1.96(m, 4H), 1.85-1.76(m, 2H), 1.36(t, 3H, J=7.2Hz)
Example 8
Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yI) ethyl)pyrroIidinyl)-2-oxoethyl)-2-oxopyrrolidin-3-yI]amino}butanoic acid (Compound 8)
a) Synthesis of ethyl 4-{[(3S)-l -(2-((2S)-2-(2-(6-cyano-l-ethylmdol- 2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]amino}butanoate
The compound(24mg) prepared in Example 7-b) was reacted according to the same procedure as Example 2-b) to give the title compound(280mg) as a pale yellow foam.
Η-NMR(300MHz, CDCI3) : 7.56-7.50(m, 2H), 7.27-7.24(m, IH), 6.36(s, IH), 4.20-4.08(m, 6H), 3.90-3.86(m, IH), 3.72-3.42(m, 7H), 2.96-2.72(m. 4H), 2.40-2.26(m, 2H), 2.05- 1.68(111, 10H), 1.34-1.3 l(m, 3H), 1.23-1.19(m, 3H)
b) Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylιndol-2- yl)ethyl) pyιτolidinyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]amino}butanoic acid
The compound(270mg) prepared in step a) above was reacted according to the same procedure as Example 2-c) to give the title compound(30mg) as a pale yellow solid.
ES-MS : 511(M+ 1 )'
Η-NMR(300MHz, CD3OD) δ : 7-92(s, IH), 7.63-7.59(m, IH), 7.42-7.38(m, IH), 6.45(s, IH), 4.28-3.90(m, 6H), 3.58-3.36(m, 6H), 2.86-2.65(m, 4H), 2.42- 2.36(m, IH), 2.23-1.98(m, 6H), 1.86-1.78(m, 3H), 1.40-1.36(m, 3H)
Example 9
Synthesis of (2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l -ethyl indoI-2-yI)ethyl)pyrroIidinyl)-2-oxoethyl)-2-oxopyrro!idin-3-ylJcarbamoyl} -2-aminopropanoic acid(Compound 9)
a) Synthesis of benzyl (2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-cyano-l- ethylindol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]carbamoyl }-2-[(t-butoxy)carbonylamino]propanoate
The compound(505mg) prepared in Example 7-b) was reacted according to the same procedure as Example 6-a) to give the title compound(669mg) as a white foam.
Η-NMR(300MHz, CDCI3) δ : 7.58-7.52(m, 2H), 7.29-7.25(m, I H), 6.34(s, IH), 4.54-4.50(m, IH), 4.20-4.08(m, 6H), 3.90-3.83(m, IH), 3.72-3.68(m, IH), 3.54-3.40(m, 4H), 2.90-2.82(m. 2H), 2.51-2.44(m, 2H), 2.28-2.21(m. IH), 2.08- 1.82(m, 7H), 1.34-1.3 l(m. 3H), 1.23-1.19(m, 3H)
b) Synthesis of (2S)-3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l -ethyl indol-2-yl)ethyl)pyiTolidinyl)-2-oxoethyl)-2-oxopynOlidin-3-yl]carbamoyl}-2-a minopropanoic acid The compound(369mg) prepared in step a) above was reacted according to the same procedure as Example 2-c) to give the title compound(127mg) as a pale yellow solid.
ES-MS : 540(M+1)'
Η-NMR(300MHz, CD^,OD) δ : 7-88(s, IH), 7.61 -7.58(m, IH), 7.42-7.38(m, IH), 6.45(s, I H), 4.64-4.56(m, IH), 4.28-4.14(m, 4H), 4.06-3.98(m, IH), 3.75- 3.70(m, IH), 3.56-3.40(m, 4H), 2.88-2.80(m, 2H), 2.60-2.40(m, 2H), 2.26-1.80 (m. 8H), 1.35-1.30(m, 3H)
Example 10
Synthesis of 2-{2-I(2S)-l-((2S)-2-((3S)-3-amino-2-oxopyrrolid- inyI)-3-methylbutanoyI)pyrroIidin-2-yI]ethyI}-l-ethyIindoIe-6-carboxamidi ne(Compound 10)
a) Synthesis of N- {(3S)-l -[( lS)-2-((2S)-2-(2-(6-cyano- l -ethylιndol-2- yl)etliyl)pynOlidinyl)-l-(isopropyl)-2-oxoethyl]-2-oxopynOlidin-3-yl}(t-butoxy )carboxamide
2-[2-((2S)-Pyrrolidin-2-yl)ethyl]- 1 -ethylindole-6-carbonιtπle(31 Omg) and (2R)- 2-{(3S)-3-[(t-butoxy)caιbonylamino]-2-oxopyτrolidinyl}-3-methyl butanoic acid(370mg, see: J. Org. Chem 1982, 47, 104-109) were reacted according to the same procedure as Example 1-b) to give the title compound(500mg) as a pale yellow foam.
Η-NMR(300MHz, CDCI3) •' 7.60-7.52(m, 2H), 7.28-7.24(m, I H), 6.36(s, IH), 5.00(brs, IH), 4.53-4.46(m, IH), 4.24-4.10(m. 3H), 3.86-3.68(m. IH) 3.60-3.24(m. 4H). 2.78-2.71(m, 2H). 2.64-2.50(m. I H), 2.38-2.20(nι. 2H), 2.05- 1.88(m, 5H), 1.76-1.68(m, 3H). 1.42(s. 9H), ] .36-1 .30(m. 3H), 0.97-0.92(m. 3H). 0.88-0.80(m, 3H) b) Synthesis of 2-{2-[(2S)-l-((2S)-2-((3S)-3-amino-2-oxoρyrroli- dinyl)-3-methylbutanoyl)pyπ-olidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
The compound(600mg) prepared in step a) above was reacted according to the same procedure as Example 2-a) to give the title compound(315mg) as a white foam.
Η-NMR(300MHz, CDCI3) δ •' 7.61-7.54(m, 2H), 7.27-7.24(m, IH), 6.34(s, I H), 4.51-4.45(m, IH), 4.26-4.16(m, 3H), 3.83-3.68(m. IH), 3.62-3.24(111. 4H), 2.78-2.73(m, 2H), 2.64-2.54(m, IH), 2.38-2.20(m, 2H), 2.03-1.88(m, 5H), 1.70-1.58(m, 3H), 1.34(t, 3H, J-7.2Hz), 0.96-0.92(m, 3H), 0.86-0.80(m, 3H)
c) Synthesis of 2-{2-[(2S)-l-((2S)-2-((3S)-3-amino-2-oxopyιτolιdι- nyl)-3-methylbutanoyl)pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine
The compound(49mg) prepared in step b) above was reacted according to the same procedure as Example 1-c) to give the title compound(40mg) as a pale yellow solid.
ES-MS : 234(1/2M+1 )+, 467(M+ 1 )
Η-NMR(300MHz, CD3OD) δ : 7-84(s, IH), 7.59-7.56(m. IH), 7.41-7.38(m. IH), 6.42(s, IH), 4.56-4.50(m, IH), 4.35-4.20(m. 3H), 3.82-3.54(m. 3H). 3.46- 3.20(m, 2H). 2.87-2.80(m, 2H), 2.42-2.17(m. 3H). 2.05-1.68(m, 6H). 1.39-1.36 (m, 3H), 0.96-0.84(m, 6H)
IR(KBr) cm"1 : 3360, 1680, 1630. 1530, 1470. 1430
Example 11
Synthesis of 4-{[(3S)-l-((lS)-2-((2S)-2-(2-(6-amidino-l-ethylindol- 2-yl)ethyl)pyrroIidinyl)-l-(isopropyl)-2-oxoethyI)-2-oxopyrroIidin-3-yl|ami no} butanoic acid(Compound 11) a) Synthesis of ethyl 4-{[(3S)-l-((lS)-2-((2S)-2-(2-(6-cyano-l-ethyl indol-2-yl)ethyl)pynOlidinyl)- l -(isopropyl)-2-oxoethyl)-2-oxopyiTolidin-3-yl] amino }butanoate
The compound(265mg) prepared in Example 10-b) was reacted according to the same procedure as Example 2-b) to give the title compound(190mg) as a white foam.
Η-NMR(300MHz, CDCI3) δ : 7.57-7.52(m, 2H), 7.27-7.24(m, I H), 6.36(s, IH), 4.52-4.46(m, IH), 4.21-4.06(m, 5H), 3.81 -3.22(m, 7H), 2.78-2.60(m, 3H), 2.38-2.20(m, 5H), 2.02-1.70(m, 7H), 1.35-1.3 l(m, 3H), 1 .24-1.20(m, 3H), 0.93- 0.90(m, 3H), 0.85-0.80(m, 3H)
b) Synthesis of 4-{ [(3S)-l-((lS)-2-((2S)-2-(2-(6-amidino-l-ethylindol -2-yl)ethyl)pyπOlidinyl)-l-(isopiOpyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]amino } butanoic acid
The compound(190mg) prepared in step a) above was reacted according to the same procedure as Example 2-c) to give the title compound(20mg) as a white solid.
ES-MS : 277(1/2M+1 )', 554(M+l)f
Η-NMR(300MHz, CDiOD) δ : 7.94-7.90(m, IH), 7.65-7.62(m, I H), 7.45-7.41 (m, I H), 6.46-6.40(m. IH), 4.53-4.48(m, IH), 4.38-4.18(m, 4H). 3.76-3.18(m, 6H). 2.88-2.52(m, 3H), 2.38-1.68(m, 12H), 1.40-1.36(m, 3H), 0.95-0.84(m, 6H)
Example 12
Synthesis of 2-{[(3S)-l-((l S)-2-((2S)-2-(2-(6-amidino-l-ethylindol -2-yl)ethyl)pyrrolidinyl)-2-oxo-l-benzylethyI)-2-oxopyrroIidin-3-yI]amino} acetic acid(Compound 12) a) Synthesis of 2-{2-[(2S)-l-((2S)-2-((3S)-3-amino-2-oxopyrroli- dinyl)-3-phenylpropanoyl)pyιτolidin-2-yl]emyl}-l-ethylindole-6-carbonitrile
2-[2-((2S)-Pyrrolidin-2-yl)ethyl]-l-ethylindole-6-carbonιtι-ile(500mg) and (2R)-2-{(3S)-3-[(t-butoxy)carbonylamino]-2-oxopyπOlidinyl}-3-phenyl propanoic acid(694 mg, see: J. Org. Chem 1982, 47, 104-109) were reacted according to the same procedure as Example 1-b) to give N-{(3S)-l -[(lS)-2-((2S)-2-(2-(6-cyano-l-ethylindol-2-yl)ethyl)pyrrolidinyl)-2- oxo-l -benzylethyl]-2-oxopynolidin-3-yl}(t-butoxy)carboxamide as a pale yellow oil. This compound was reacted according to the same procedure as Example 2-a) to give the title compound(lθθmg) as a pale yellow foam.
Η-NMR(300MHz, CDCI3) δ : 7.56-7.52(m, 2H), 7.28-7.08(m, 6H), 6.34(s, IH), 5.16-5.08(m, IH), 4.16-4.08(m, 2H), 3.86-3.69(m, 2H), 3.41-3.12(m, 5H), 2.96-2.90(m, IH), 2.72-2.60(m, 2H), 2.40-2.10(m, 2H), 2.01- 1 .50(m, 6H), 1.36- 1.32(m, 3H)
b) Synthesis of ethyl 2-{[(3S)-l-((lS)-2-((2S)-2-(2-(6-cyano-l -ethyl indol-2-yl)ethyl)pyπOlidinyl)-2-oxo-l -benzylethyl)-2-oxopyιτolidin-3-yl]amin o } acetate
The compound(lθθmg) prepared in step a) above was reacted according to the same procedure as Example 3-a) to give the title compound(70mg) as a pale yellow oil.
Η-NMR(300MHz. CDCI3) δ : 7.56-7.53(m, 2H), 7.26-7.12(m. 6H ). 6.32(s, IH), 5.18-5.1 l(m, IH), 4.20-4.06(m, 7H), 3.84-3.66(m. 2H). 3.40-3.16(m. 5H), 2.94-2.86(m. IH), 2.68-2.59(m, 2H), 2.30-2.05(m. 3H). 1 .90- 1 .70(m, 5H). 1.41 (t, 3H, J=7.2Hz). 1.24-1.20(m. 3H) c) Synthesis of 2-{[(3S)-l-((lS)-2-((2S)-2-(2-(6-amidino- l-ethyl indol-2-yl)ethyl)pyrrolidinyl)-2-oxo-l -benzylethyl)-2-oxopyπOlidin-3-yl]amin o } acetic acid
The compound(70mg) prepared in step b) above was reacted according to the same procedure as Example 2-c) to give the title compound(25mg) as a pale yellow solid.
ES-MS : 287(1/2M+1)\ 573(M+1)'
Η-NMR(300MHz, CD3OD) δ : 7.98-7.90(m, IH), 7.68-7.64(m, IH), 7.45-7.42 (m, IH), 7.23-7.02(m, 5H), 6.46-6.42(m, IH), 5.12-5.02(m, I H), 4.40-3.95(m, 3H), 3.70-3.35(m, 4H), 3.20-3.04(m, 3H), 2.98-2.70(m, 4H), 2.35-2.00(m, 3H), 1.82- 1.55(m, 5H), 1.38-1.30(m, 3H)
Example 13
Synthesis of 2-{2-[l-(2-((3S)-3-amino-2-oxopiperidyl)acetyl)-(2S)- pyrrolidin-2-yI]ethyI}-l-ethylindole-6-carboxamidine(Compound 13)
a) Synthesis of (2S)-5-amino-2-[(t-butoxy)carbonylamino]pentanoic acid
(2S)-2-[(t-Butoxy)carbonylamino]-5-[(benzyloxy)carbonylamino]penta noic acid (6.0g) was introduced into a 500n]^ volumetric flask and dissolved in methanol (100m ). Palladium/activated carbon(900mg,
15w/w%) was added thereto and the resulting mixture was stiπed for 14 hours under hydrogen atmosphere. After the reaction was completed, the reaction solution was filtered through cellite and the solvent was evaporated under reduced pressure to give the title compound(3.80g) as a white solid.
Η NMR(300MHz. CDC ) δ : 3.95(brs. I H), 3.02(brs. 2H), 1.95-1.50(m, 2H), 1.45(s, 9H)
b) Synthesis of N-[(3S)-2-oxo-(3-piperidyl)](t-butoxy)carboxamide The compound(3.8g) prepared in step a) above was introduced into a 500n]p volumetric flask and dissolved in
Figure imgf000044_0001
at room temperature. WSCI - HCl(6.3g) and HOBT(4.4g) were added thereto and N.N-diisopropylethyl- amine(11.4m^) was added dropwise while stiπing, and then tetra-n-butylammomum iodide(catalytic amount) was added thereto. The reaction solution was stirred for 14 hours at room temperature to complete the reaction. Then, the reaction solution was diluted with water and extracted three times with ethyl acetate. The organic extracts were combined and the solvent therein was evaporated under reduced pressure to give the title compound(2.83g) as a white foam.
Η NMR(300MHZ, CDC13) δ : 6.70(brs, IH), 5.50(brs, IH), 3.95-3.90(m, I H), 3.47-3.10(m, 2H), 2.48-2.40(m, IH), 1.95- 1.65(m, 2H), 1.65-1.50(m, IH), 1.45(s. 9H)
c) Synthesis of 2-{(3S)-3-[(t-butoxy)carbonylamino]-2-oxopiperidyl} acetic acid
The compound(3.1g) prepared in step b) above was introduced into a 25 mf) volumetiic flask, and reacted according to the same procedure as Example 1 -a) to give the title compound(3.99g) as a white foam.
Η NM R(300MHz. CDCb) δ : 5.47(brs. I H). 4.45-3.65(m. 3H), 2.55-2.25(m, I H), 2.00-1.85(m, 2H), 1.60- 1.56(m. I H). 1.45(s, 9H) d) Synthesis of 2-{2-[l-(2-((3S)-3-amino-2-oxopiperidyl)acetyl)-(2S)- pyrrolidin-2-yl]ethyl}- 1 -ethylindole-6-carbonitrile
2-[2-((2S)-Pyrrolidin-2-yl)ethyl]-l-ethylindole-6-carbonitrile(2.59g) and the compound(2.12g) prepared in step c) above were introduced into a 500nι( volumetiic flask, and reacted according to the same procedure as Example 1-b) to give a compound as a light yellow foam. The resulting compound was reacted according to the same procedure as Example 2-a) to give the title compound (1.14g) as a white foam.
Η NMR(300MHZ, CDC13) δ : 7.50-7.45(m, 2H), 7.20-7.16(m, I H), 6.32(s, IH), 4.25-4.05(m, 5H), 3.90-3.85(m, IH). 3.58-3.30(m, 4H), 3.15-3.10(m, 2H), 2.71 -2.65(m, 2H), 2.35-1.65(m, 10H), 1.30(t. 3H, J = 8.33Hz)
e) Synthesis of 2-{2-[l-(2-((3S)-3-amino-2-oxopiperidyl)acetyl)-(2S)- pynolidin -2-yl]ethyl}-l-ethylindole-6-carboxamidine
The compound(200mg) prepai'ed in step d) above was reacted according to the same procedure as Example 1-c) to give the title compound(lθθmg) as a white foam.
ES-MS : 439(M+1 )'
Η-NMR(300MHz. CDiOD) δ : 7-75(s, IH), 7.45-7.41(m, IH), 7.30-7.26(m, IH), 6.45(s. I H), 4.20-3.80(m, 5H), 3.50-3.35(m, 3H), 3.35-3.20(m, 2H), 2.65-2.60(m. 2H), 2.30-1.50(m, 10H), 1.25- 1.20(m. 3H)
Example 14
Synthesis of 4-{|(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol-2-yI) ethyl)pyrroIidinyI)-2-oxoethyl)-2-oxo-3-piperidyl]amino} butanoic acid
(Compound 14) a) Synthesis of 4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl) pyπ-olidinyl)-2-oxoethyl)-2-oxo-3-piperidyl]amino}butanoic acid
The compound(630mg) prepared in Example 13-d) was reacted according to the same procedure as Example 2-b) to give a compound(600mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound(200mg) as a white foam.
ES-MS : 525(M+1)T
Η-NMR(300MHz, CD3OD) δ : 7.90(s, IH), 7.60-7.55(m, IH), 7.40-7.35(m, IH), 6.48(s, IH), 4.40-4.18(m, 3H), 4.12-4.08(m, 2H), 3.65-3.20(m, 5H), 2.90- 2.85(m, 2H), 2.40-1.80(m, 16H), 1.38(t, 3H, J = 8.33Hz)
Example 15
Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol- 2-yl)ethyI)pyrroIidinyl)-2-oxoethyI)-2-oxo-(3-piperidyI)]carbamoyI}-(2S)-2- aminopropanoic acid(Compound 15)
a) Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-amin opropanoic acid
The compound(466mg) prepared in Example 13-d) was reacted according to the same procedure as Example 6-a) to give a compound(546mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound( 120mg) as a white foam.
ES-MS : 554(M+ 1 ) Η-NMR(300MHz, CD3OD) δ : 7.90(s, IH), 7.65-7.30(m, 2H), 6.48(s, IH), 4.50-4.46(m, IH), 4.38-4.00(m, 5H), 3.65-3.30(m, 4H), 2.75-2.70(m, 2H), 2.35- 1.80(m, 16H), 1.38(t, 3H, J = 8.33Hz)
Example 16
Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol- 2-yl)ethyI)pyrrolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethyIcarbamoyl }-(2S)-2-aminopropanoic acid(Compound 16) and 2-{2-[l-(2-((3S)-3-(( 3S)-3-amino-3-carbamoyI-N-ethylpropanoyIamino)-2-oxopiperidyl)acetyl)- (2S)-pyrroIidin-2-yl]ethyl}-l-ethyIindole-6-carboxamidine(Compound 17)
a) Synthesis of 2-{2-[l-(2-((3S)-3-(diprop-2-enylamino)-2-oxopiperi- dyl)acetyl )-(2S)-pyιτolidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
The compound(52 lmg) prepared in Example 13-d) was introduced into a 250m£ volumetiic flask and dissolved in THF/DMF(4/1, v/v, 100 m£). Sodium bicarbonate(229mg) and sodium iodide(catalytic amount) were added thereto at room temperature and allylbromide(0.43m( was slowly added dropwise. The reaction mixture was stiired and refluxed for 4 hours to complete the reaction. Water was added thereto in order to stop the reaction and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride: methanol( 10: l , v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give the title compound(41 lmg) as a yellow oil.
Η NMR(300MHz, CDCI3) δ •• 7.55-7.50(m. 2H), 7.25-7.2 l(m, I H). 6.48(s. I H ). 6.00-5.70(111. 2H), 5.25-5.00(m. 4H). 4.31 -3.85(m. 5H), 3.60-3.30(m, 6H), 3.30-3.10(m, 3H), 2.80-2.76(m, 2H), 2.30-2.25(m, IH), 2.20-1.69(m, 9H), 1.35 (t, 3H, J = 7.41Hz)
b) Synthesis of 2-{2-[l-(2-((3S)-2-oxo-3-(prop-2-enylamino)piperi- dyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
The compound(400mg) prepai'ed in step a) above was introduced into a 100nι() volumetric flask and dissolved in dry THF(30nιø). Pd2(dibenzylideneacetone;dba)3 • CHCi3(37mg), dppb(l,4-bis(diphenylphos- phinobutane))(64mg) and 2-mercaptobenzoic acid( 120mg) were added thereto and the resulting mixture was stirred for 1 hour at room temperature. After the reaction was completed, water was added to stop the reaction, and the reaction solution was extracted three times with ethyl acetate. The organic extracts were combined and the solvent was evaporated under reduced pressm-e. The residue was purified by silica gel column chromatography using ethyl acetate :ethanol(3: l, v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give the title compound(283mg) as a light brown foam.
Η NMR(300MHZ, CDC1 ) δ : 7.65-7.60(m, 2H), 7.20-7.16(m, IH), 6.45(s, I H). 5.95-5.75(m, IH), 5.70-5.50(m. 2H), 4.30-3.85(m, 5H), 3.60-3.15(m. 6H). 2.75-2.70(m, 2H), 2.30-2.25(m. 2H), 2.25-1.69(m, 10H). 1.35(t, 3H, J=7.40Hz)
c) Synthesis of 2-{2-[l-(2-((3S)-3-(ethylprop-2-enylamino)-2- oxopiperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carbonitι-ile
The compound(282mg) prepared in step b) above was introduced into a 100m() volumetric flask and dissolved in THF/DMF(4/1, v/v, 50 mC), and then sodium bicarbonate( 103mg) was added thereto at room temperature. The reaction solution was stirred for about lOminutes, ethyl iodide(0.2m£) was slowly added dropwise thereto, and the resulting mixture was stiπed and refluxed for about 4 hours. After the reaction was completed, water was added in order to stop the reaction, and the reaction solution was extracted three times with ethyl acetate. The organic extracts were combined and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate:ethanol(4: l, v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give the title compound(299mg) as a yellow oil.
Η NMR(300MHZ, CDC13) δ : 7.55-7.50(m, 2H), 7.25-7.20(m, I H), 6.47(s, IH), 5.95-5.75(m, IH), 5.20-5.00(m, 2H), 4.30-3.80(m, 5H), 3.60-3.42(m, 5H), 3.30-3.15(m, 2H), 2.75-2.71(m, 2H), 2.28-2.25(m, IH), 2.10-1.65(m, 9H), 1.35(t, 3H, J = 8.82Hz), 1.05(t, 3H, J = 8.83Hz)
d) Synthesis of 2-{2-[l-(2-((3S)-3-(ethylamino)-2-oxopiperidyl) acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carbonitrile
The compound( 1.34g) prepared in step c) above was introduced into a 250nιp volumetiic flask and dissolved in diy THF(50m^). Then, Pd (dibenzylideneacetone;dba)3 • CHCl3(79mg), dppb( l,4-bis(diphenylphos- phinobutane))(233mg) and 2-mercaptobenzoic acid(465mg) were added thereto and the resulting mixture was stiπed for 2 horns at room temperature. After the reaction was completed, water was added in order to stop the reaction, and the reaction solution was extracted three times with ethyl acetate. The organic extracts were combined and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using ethyl acetate :ethanol(3: l, v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give the title compound(608mg) as a light brown foam.
Η NMR(300MHZ, CDCI3) δ : 7.60-7.55(m, 2H), 7.28-7.25(m, I H). 6.35(s, IH), 4.25-4.20(m, IH), 4.20-3.90(m, 4H), 3.60-3.00(m, 7H), 2.87-2.60(m, 2H), 2.40-1.65(m, 10H), 1.30(t, 3H, J = 8.32Hz), 1.15(t, 3H, J = 8.33Hz)
e) Synthesis of ethyl 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l -ethyl indol-2-yl)ethyl)pyrrolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbam oyl}-(2S)-2-amino-propanoate(a) and 2-{2-[l-(2-((3R)-3-((3S)-3-amino-3- carbamoyl-N-ethylpiOpanoylamino)-2-oxopiperidyl)acetyl)-(2S)-pyπOlidin-2- yl]ethyl}-l-ethylindole-6-carboxamidine (Compound 17)
The compound(322mg) prepared in step d) above was reacted according to the same procedure as Example 6-a) to give a compound(257mg) as a white foam. The resulting compound was reacted according to the same procedme as Example 1-c) to give Compound(a)(140mg) and Compound 17(45mg) in the form of a white foam.
Compound (a);
Η NMR(300MHz, CD3OD) δ : 7-90(s, IH), 7.60-7.56(m, IH), 7.40-7.37(m. IH), 6.45(s, IH), 4.50-4.00(m, 5H), 3.95-3.20(m. 8H), 3.05-2.55(111. 4H), 2.30-1.80(m, 12H), 1.40-1.36(m, 3H), 1.30- 1.26(m, 3H), 1.25- 1 .21(111. 3H)
Compound 17;
ES-MS : 581(M+ 1)'
Η NMR(300MHZ. CD3OD) δ : 7-90(s. IH), 7.60-7.56(m, IH), 7.40-7.37(m, IH), 6.45(s, IH), 4.50-4.00(m, 3H), 3.95-3.20(m, 8H), 3.00-2.55(m, 4H), 2.30- 1.80(m, 12H), 1.40-1.36(m, 3H), 1.30- 1.26(m, 3H)
f) Synthesis of 3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l -ethylmdol-2- yl)ethyl)pynOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbamoyl}-(2S )-2-aminopropanoic acid(Compound 16)
The compound(a)(140mg) prepared in step e) above was dissolved in a solvent mixture of water(lm( and ethanol(2m£), 85% potassium hydroxide(35mg) was added thereto, and the resulting mixtme was stirred for 1 day at room temperature. The solvent was removed under reduced pressure, and the residue was purified by NH-silica column chromatography using ethyl acetate:methanol=l : l (v/v) as an eluent to give the title compound(lθθmg) as a white solid.
ES-MS : 582(M+1)+
Η NMR(300MHZ, CD3OD) : 8.00-7.95(m, IH), 7.65-7.62(m, IH), 7.43-7.40 (m. IH), 6.50(s, IH). 4.50-3.90(m, 6H), 3.70-3.65(m. IH), 3.55-3.40(m. 6H), 3.10-2.60(m, 4H), 2.30- 1.90(m, 10H), 1.40-1.36(m, 3H), 1.30(t, 3H, J-8.33Hz)
Example 17
Synthesis of (2R)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyI indoI-2-yl)ethyl)pyrroIidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbarnoyl}-2 -aminopropanoic acid(Compound 18) and 2-{2-[l-(2-((3S)-3-((3R)-3- amino-3-carbamoylpropanoylamino)-2-oxopiperidyI)acetyl)-(2S)-pyrroIidi n-2-yl|ethyl}-l-ethylindoie-6-carboxamidine(Com pound 19)
a) Synthesis of ethyl (2R)-3-{ N-[(3S)-l-(2-((2S)-2-(2-(6-amidino- l - ethylindol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl} -2-aminopropanoate(a) and 2- { 2-[ 1 -(2-((3 S )-3 -((3 R)-3-amino-3 -carbamoy 1 propanoylamino)-2-oxopiperidyl)acetyl)-(2S)-pyιτolidin-2-yl]ethyl}-l-ethylind ole-6-carboxamidine(Compound 19)
The compound(300mg) prepared in Example 13-d) and (3R)-3- [(t-butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(350mg) were reacted according to the same procedure as Example 1-b) to give a compound(400mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 1-c) to give Compound(a)( 120mg) and Compound 19(20mg) in the foπn of a white foam.
Compound (a);
Η NMR(300MHz, CD3OD) δ : 7.81(s, IH), 7.50(d, IH, J = 8.29Hz), 7.30(d, IH, J = 8.34Hz), 6.45(s, IH), 4.40-4.35(m, IH), 4.30-4.00(m, 6H). 3.68-3.15 (m, 6H), 2.75-2.71(m, 2H), 2.60-2.57(m. IH). 2.45-2.41(π . I H). 2.20-1.60(m, 10H), 1.35(t, 3H, J = 8.33Hz), 1.30-1.26(m. 3H)
Compound 19;
ES-MS : 553(M+1)+
Η N MR(300MHZ, CD3OD) δ : 7.81(s, IH), 7.50(d, IH. J = 8.29Hz), 7.30(d. IH, J = 8.34Hz), 6.45(s, IH), 4.40-4.35(m, IH). 4.30-4.00(111. 4H ). 3.68-3.15 (m, 6H), 2.75-2.70(m, 2H), 2.60-2.55(m. IH). 2.45-2.35(nι. I H). 2.20-1.60(m, 10H), 1.35(t, 3H, J = 8.33Hz)
b) Synthesis of (2R)-3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino- l - ethylindol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl } -2-aminopropanoic acid (Compound 18) Compound(a)(120mg) prepared in step a) above was reacted according to the same procedure as Example 16-f) to give the title compound(80mg) as a white foam.
ES-MS : 554(M+ 1 )"
Η NMR(300MHZ, CDiOD) δ : 7.95(s, IH), 7.50-7.30(m. 2H), 6.35(s, IH), 4.50-4.45(m, I H), 4.30-4.00(m, 4H), 3.60-3.30(m, 9H), 2.80-2.74(m, 2H), 2.20-1.70(m. 10H), 1.35-1.3 l(m, 3H)
Example 18
Synthesis of 4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol -2-yl)ethyI)pyrroIidinyI)-2-oxoethyl)-2-oxo-(3-piperidyI)]carbamoyI}-(2S)-2 -aminobutanoic acid(Compound 20) and 2-{2-[l~(2-((3S)-3-((4S)-4- amino-4-carbamoylbutanoylamino)-2-oxopiperidyl)acetyl)-(2S)-pyrroIidin- 2-yIJethyI}-l-ethylindole-6-carboxamidine(Compound 21)
a) Synthesis of ethyl 4-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethyl indol-2-yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S) -2-aminobutanoate(a) and 2-{2-[ l-(2-((3S)-3-((4S)-4-ammo-4-carbamoyl butanoylamino)-2-oxopiperidyl)acetyl)-(2S)-pyrrolidin-2-yl]ethyl }- 1 -ethylindol e-6-carboxamidine(Compound 21 )
The compound(300mg) prepared in Example 13-d) and (4S)-4- [(t-butoxy)carbonylamino]-4-[benzyloxycarbonyl]butanoic acid(330mg) were reacted according to lie same procedure as Example 1 -b) to give a compound(450mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 1-c) to give Compound(a)(200mg) and Compound 21 (40mg) in the foπn of a white foam. Compound (a);
Η NMR(300MHZ, CD3OD) : 7.90(s, I H), 7.60-7.57(m, IH), 7.40-7.37(m, IH), 6.47(s, IH), 4.50-4.43(m, IH), 4.35-4.05(m, 5H), 3.60-3.25(m, 5H), 2.85- 2.81(m, 2H), 2.50- 1.75(m, 6H), 1.37(t, 3H, J = 7.12Hz), 1.30- 1.26(m, 3H)
Compound 21 ;
ES-MS : 567(M+1)+
Η NMR(300MHz, CD3OD) : 7.90(s. IH), 7.60-7.56(m, IH), 7.40-7.36(m, IH), 6.47(s, IH), 4.50-4.45(m. IH), 4.35-4.05(m, 3H), 3.60-3.25(m, 5H), 2.85- 2.80(m, 2H), 2.50-1.75(m. 6H), 1.37(t, 3H, J = 7.12Hz)
b) Synthesis of 4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-amin obutanoic acid (Compound 20)
Compound(a)(200mg) prepared in step a) above was reacted according to the same procedure as Example 16-f) to give the title compound(150mg) as a white foam.
ES-MS : 568(M+1)+
Η NM R(300MHZ, CD3OD) δ : 7.95(s. I H), 7.60-7.56(m. IH), 7.50-7.47(m, IH), 6.45(s, IH), 4.50-4.00(m. 4H), 3.60-3.27(m, 5H), 2.70-2.65(m. 2H), 2.47-1.75(m, 16H), 1.37(t, 3H. .1 = 7.23Hz)
Example 19
Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol- 2-yl)ethyl)pyrrolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2- |(methyIsulfonyI)amino)propanoic acid(Compound 22) a) Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-[(me thylsulfonyl)amino]propanoic acid
The compound(500mg) prepared in Example 13-d) was reacted according to the same procedure as Example 6-a) to give a compound(600mg) as a white foam. The resulting compound(600mg) was dissolved in methylene chloride(50m ), ti-iethylamine(0.3n](>) and methanesulfonylchloride(1.5 £) were slowly added dropwise thereto, and the mixture was stirred for 1 hour at room temperature. After the reaction was completed, water was added in order to stop the reaction, and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol(10: l, v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give a compound (700mg) as a white foam. The resulting compound was further reacted according to the same procedure as Example 2-c) to give the title compound(220mg) as a white foam.
1H NMR(300MHz, CD OD) δ : 7.94(s, IH), 7.62(d, IH, J =
30Hz), 7.49(d, IH, J - 28Hz), 6.49(s. I H), 5.42-5.38(m. IH),
4.32-4.20(m, 4H), 4.18-4.00 (m, IH), 3.54-3.50(m, 2H), 3.39-3.20(m. 3H), 3.12-2.60(m, 5H), 2.42-1.34(m, 12H), 1.30- 1.26(m, 3H)
Example 20
Synthesis of 3-{N-|(3S)-1-(2-((2S)-2-(2-(6-amidino-l-ethylindol- 2-yl)ethyl)pyrroIidinyI)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2- (diethylamino)propanoic acid(Compound 23) a) Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-(diet hylamino)propanoic acid
The compound(500mg) prepared in Example 13-d) was reacted according to the same procedme as Example 6-a) to give a compound(620mg) as a white foam. The resulting compound(620mg) was dissolved in methylene chloride(50m ), triethylamine(0.3m£) and ethyl iodide( 1.3m ) were slowly added dropwise thereto, and the whole mixture was stiired for 1 hour at room temperature. After the reaction was completed, water was added to stop the reaction, and the reaction solution was extracted three times with methylene chloride. The organic extracts were combined and the solvent was evaporated under reduced pressure. The residue was pmified by silica gel column chromatography using methylene chloride:methanol(10: l, v/v) as an eluent. The fractions containing the product were combined and the solvent was evaporated to give a compound(250mg) as a white foam. The resulting compound was further reacted according to the same procedure as Example 2-c) to give the title compound(80mg) as a white foam.
Η NMR(300MHz, CD3OD) : 8.03(s, IH), 7.70(d, IH, J = 24.0Hz), 7.50(d, IH, J = 23.0Hz), 6.52(d, IH, J = 31.0Hz), 4.62-4.18(m, 7H), 3.58-3.50(111. 2H), 2.95-2.85(m, 2H), 2.78-2.57(m, 4H), 2.55-1.92(m, 10H), 1 .55- 1.10(m, 9H)
Example 21
Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindoI- 2-yI)ethyl)pyπ olidinyl)-2-oxoethyI)-2-oxo-(3-piperidyI)]carbamoyl}-(2S)-2- hydroxypropanoic acid(Compound 24) a) Synthesis of 3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-hydr oxypropanoic acid
The compound(300mg) prepared in Example 13-d) and (4S)-2.2-dimethyl-5- oxo-l,3-dioxolane-4-carboxylic acid(250mg) were reacted according to the same procedure as Example 1-b) to give a compound(280mg) as a white foam. The resulting compound(280mg) was reacted according to the same procedure as Example 2-c) to give the title compound(lθθmg) as a white foam.
ES-MS : 555(M+1)+
Η NMR(300MHz, CD30D) δ : 7.90(s, IH), 7.60-7.56(m, IH), 7.49_7.46(m, IH), 6.48(s, IH), 4.50-4.45(m, IH), 4.38-4.00(m, 5H), 3.65-3.30(m, 4H), 2.75- 2.70(m, 2H), 2.35-1.80(m, 16H), 1.40(t, 3H, J = 8.33Hz)
Example 22
Synthesis of (2R)-4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethyl indoI-2-yI)ethyI)pyrroIidinyI)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2 -aminobutanoic acid(Compound 25)
a) Synthesis of (2R)-4-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l -ethyl indol-2-yl)ethyl)pyrrolidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2- aminobutanoic acid
The compound(300mg) prepared in Example 13-d) and (4R)-4-[(t- butoxy)carbonylamino]-4-[benzyloxycarbonyl]butanoic acid(330mg) were reacted according to the same procedure as Example 1 -b) to give a compound(450mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 2-c) to give the title compound(200mg) as a white foam.
ES-MS : 568(M+1)+ !H NMR(300MHz, CD3OD) δ : 8.00-7.96(m, IH), 7.70-7.38(m, 2H), 6.50(s, IH), 4.60-4.10(m, 6H), 3.70-3.40(m. 3H), 2.90-2.80(m, 2H), 2.50-2.35(m, IH), 2.45-1.80(m, 15H), 1.45(t, 3H, J = 8.3Hz)
Example 23
Synthesis of 2-{2-[l-(2-((3S)-3-((2S)-2-amino-3-carbamoyI- propa- noylamino)-2-oxopiperidyl)acetyI)-(2S)-pyrrolidin-2-yl]ethyl}-l-ethy!indole -6-carboxamidine(Compound 26)
a) Synthesis of 2-{2-[l-(2-((3S)-3-((2S)-2-amino-3-carbamoylprop- anoylammo)-2-oxopiperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl }- 1 -ethylindole-6 -carboxamidine
The compound(300mg) prepared in Example 13-d) and (2S)-2-[(t- butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(330mg) were reacted according to the same procedure as Example 1-b) to give a compound(350mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 1 -c) to give the title compound(l θθmg) as a white foam.
ES-MS : 553(M+1)'
Η NMR(300MHz, CD3OD) δ : 7.90(s. I H). 7.68-7.65(m, IH), 7.45-7.41(m. IH), 6.50(s, IH), 4.50-4.05(m, 5H). 3.80-3.40(m. 4H), 2.97-2.85(m, 2H), 2.62- 2.55(m, IH), 2.50-2.40(m. IH). 2.40-1.85(m. 12H), 1.35(t, 3H, J = 7.20Hz)
Example 24 Synthesis of 2-{2-[l-(2-((3S)-3-((2R)-2-amino-3-carbamoyl- propanoylamino)-2-oxopiperidyl)acetyl)-(2S)-pyrrolidin-2-yl]ethyl}-l-ethyl indole-6-carboxamidine(Compound 27)
a) Synthesis of 2-{2-[l-(2-((3S)-3-((2R)-2-amino-3-carbamoylpropan- oylamino)-2-oxopiperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6- carboxamidine
The compound(300mg) prepared in Example 13-d) and (2R)-2-[(t- butoxy)carbonylamino]-3-[benzyloxycarbonyl]propanoic acid(330mg) were reacted according to the same procedure as Example 1-b) to give a compound(350mg) as a white foam. The resulting compound was reacted according to the same procedure as Example 1-c) to give the title compound(120mg) as a white foam.
ES-MS : 553(M+1)+
Η NMR(300MHz, CD3OD) ■■ 7.90(s, IH), 7.68-7.59(m, IH), 7.45-7.43(111. IH), 6.50(s, IH), 4.50-4.05(m, 5H), 3.80-3.40(m, 4H), 2.97-2.90(m. 2H), 2.62-2.55(m, IH), 2.50-2.45(m, IH), 2.40-1.85(m. 12H), 1.35(1. 3H. J= 7.20Hz)
Example 25: Synthesis of 2-{2-[(2S)-l-(2-((3S,6S)-3-amino-6- (fluoromethyl)-2-oxopiperidyl)acetyl)pyrrolidin-2-yllethyl}-l -ethylindole-6 -carboxamidine(Compound 28)
a) Synthesis of methyl (2S,5S)-5-[(t-butoxy)carbonylamino]-6-oxo piperidine-2-carboxylate
Dimethyl (2S,5S)-2.5-diaminohexan- 1 ,6-dioate dihydrochloride(500 mg, see: . Org. Chem, 1984, 49. 2286-2288) was dissolved in a solvent mixture of ethanol and water(5nιf/5n)c). silver oxide(l)(464mg) was added thereto, and then the mixture was stirred for 30 minutes at room temperature. The insoluble solid was filtered out through cellite. The filtrate was concentrated under reduced pressure and the residue was dissolved in methylene chloride(20m£). To tl is solution were added under ice-cooling triethylamine(0.38n) ) and t-butyldicarbonate (516mg) and the resulting mixture was stirred for 18 hours at room temperature. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using methylene chloride: methanol(20: l, v/v) as an eluent to give the title compound(350mg) as a pale yellow foam.
Η-NMR(300MHz, CDC13) δ : 6.23(brs, IH), 5.45(brs, IH), 4.14-4.06(m, 2H), 3.78(s, 3H), 2.50-2.43(m, IH), 2.28-2.12(m, 2H), 1.58-1.52(m, IH), 1.44 (s, 9H)
b) Synthesis of N-[(3S,6S)-6-(hydroxymethyl)-2-oxo-(3-piperidyl)] (t-butoxy) carboxamide
The compound(450mg) prepared in step a) above was dissolved in a solvent mixture of diy THF and diethylether(3m£/10m^), lithium borohydride(2.0M in THF/0.83m ) and lithium triethylborohydride(1.0M in THF/0.17ni( were slowly added dropwise thererto, and the whole mixture was stiπed for 2 hours at room temperature. Mefhanol(2me) was slowly added dropwise thereto under ice-cooling and the mixture was stiπed for 10 minutes. The reaction solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using methylene chloride:methanol( 10: l, v/v) as an eluent to give the title compound(160mg) as a white foam.
Η-NMR(300MHz. CDCI3) • 6.92(brs, I H), 5.63-5.60(m. I H). 4.08-4.00(m, IH), 3.65-3.54(m, 3H). 2.60(brs, IH), 2.30-2.20(m, IH), 1.95-1 .65(m, 3H), 1.42(s, 9H)
c) Synthesis of N-[(3S,6S)-6-(fluoromethyl)-2-oxo-(3-piperidyl)] (t-butoxy) carboxamide
The compound(160mg) prepared in step b) above was dissolved in methylene chloride(5nι£), (diethylamino)sulfoti-ifluoride(0.096I]) ) was added thereto under ice-cooling, and the whole mixture was stirred for 24 hours at room temperature. The reaction solution was poured into a cold and saturated aqueous solution of sodium bicarbonate(10m.g), which was then extracted with methylene chloride. The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using methylene chloride:methanol(20: l, v/v) as an eluent to give the title compound(50mg) as a brown oil.
Η-NMR(300MHz, CDCI3) : 6.23(brs, IH), 5.46(brs, I H), 4.48-4.20(ni. 2H), 4.07-4.01(m, IH). 3.76-3.70(m, IH), 2.07-1.95(m. IH), 1.80- 1.62(iii. 3H), 1.42 (s, 9H)
d) Synthesis of 2-{(3S,6S)-3-[(t-butoxy)carbonylamino]-6-(fluoro methyl )-2-oxopiperidyl}acetic acid
The compound(50mg) prepared in step c) above was reacted according to the same_ procedure as Example 1-a) to give the title compound(20mg) as a pale yellow foam.
Η-NMR(300MHz. CDCI3) : 5.30(brs, IH), 4.62-4.40(m. 2H), 4.26-4.21 (m, I H), 4.06-3.92(m .2H). 3.74-3.68(m, IH), 2.43-2.35(m. I H). 2.16- 1 .78(m. 3H). 1.42(s. 9H) e) Synthesis of N-{ l-[2-((2S)-2-(2-(6-cyano-l-ethylindol-2-yl)ethyl) pyrrolidinyl)-2-oxoethyl-(3S,6S)-6-(fluoromethyl)]-2-oxo-(3-piperidyl)}(t-buto xy)carboxamide
2-[2-((2S)-Pyrrolidin-2-yl)ethyl]-l-ethylindole-6-carbonitiile(19mg) and the compound(20mg) prepared in step d) above were reacted according to the same procedure as Example 1-b) to give the title compound(25mg) as a pale yellow oil.
1H-NMR(300MHz, CDCI3) : 7.60-7.52(m, 2H), 7.28-7.24(m, IH), 6.41-6.36 (m, IH), 5.30(brs, IH), 4.70-4.40(m, 2H), 4.31-4.22(m, 2H), 4.16-3.96(m, 4H), 3.84-3.76(m, IH), 3.56-3.45(m, 2H), 2.82-2.74(m, 2H), 2.40-1.90(m, 6H), 1.82- 1.64(m, 4H), 1.43(s, 9H), 1.37-1.32(m, 3H)
f) Synthesis of 2-{2-[(2S)-l-(2-((3S,6S)-3-amino-6-(fluoromethyl) -2-oxo-piperidyl)acetyl)pyπOlidin-2-yl]ethyl } - 1 -ethylindole-6-carboxamidine
The compound(25mg) prepared in step e) above was reacted according to the same procedure as Example 1-c) to give the title compound(3mg) as a pale yellow solid.
ES-MS : 236(1/2M+1 )', 471(M+1)+
Η-NMR(300MHz, CD3OD) •• 7.90(s, IH), 7.64-7.6 l(m, IH), 7.42-7.38(m, IH), 6.46(s, IH), 4.60-4.10(m, 8H), 4.05-3.96(m, IH), 3.54-3.48(m, 2H), 2.89- 2.82(m, 2H), 2.26-1.70(m. 10H), 1.42-1.38(m, 3H)
Example 26
Synthesis of (2S)-3-{N-|l-(2-((2S)-2-(2-(6-amidino-l-ethylindol- 2-yl)ethyl)pyrrolidinyI)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-pipe ridyl))carbamoyl}-2-aminopropanoic acid(Compound 29) a) Synthesis of (2S)-3-{N-[l -(2-((2S)-2-(2-(6-amidino-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-piperidyl )] carbarn oyl}-2-aminopropanoic acid
The compound(40mg) prepared in Example 25 -e) was reacted according to the same procedure as Example 2-a) to give a compound(15mg) as a white foam. The resulting compound was further reacted according to the same procedure as Example 6-a) to give a compound(15mg) as a white foam. The resulting compound( 15mg) was further reacted according to the same procedure as Example 2-c) to give the title compound(3mg) as a pale yellow solid.
ES-MS : 293(1/2M+1)\ 586(M+1)^
Η-NMR(300MHz, CD OD) δ : 7.98(s, IH), 7.67-7.64(m, IH), 7.40-7.36(m, IH), 6.42(s, IH), 4.60-4.15(m, 9H), 4.05-3.98(m, IH), 3.54-3.48(m, 2H), 2.89-2.70 (m, 4H), 2.30-1.68(m, 10H), 1.40-1.35(m, 3H)
Example 27
Synthesis of (2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethyIindol- 2-yl)ethyl)pyrroIidinyl)-2-oxoethyl)-6-(methoxymethyI)-2-oxo-(3-piperidyl) ]carbamoyl}-2-aminopropanoic acid(Compound 30)
a) Synthesis of (t-butoxy)-N-[6-(methoxymethyl)-2-oxo-(3-piperidyl)] carboxamide
(t-Butoxy)-N-[6-(hydiOxymethyl)-2-oxo-(3-pιperidyl)]carboxamιde(47 Omg. see: Tetrahedron: Asymmetry, Vol. 8. No. 2, 327-335, 1997) was dissolved in acetonitrile(2I]i{<), silver oxide(I)(0.5g) and methyl iodide(1.5 nj( were added thereto under ice-cooling, and then the mixture was stiπed for 1 day at room temperamre. The insoluble solid was filtered out and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography using methylene chloride:methanol(20: l, v/v) as an eluent to give the title compound(470mg) as a brown oil.
Η-NMR(300MHz, CDCI3) δ : 6.30(brs, IH), 5.52(brs, IH), 4.06-3.98(m, IH), 3.66-3.60(m, IH), 3.40-3.26(m, 5H), 2.40-2.30(m, IH), 2.02-1.92(m, IH), 1.70- 1.58(m, 2H), 1.40(s, 9H)
b) Synthesis of 2-{3-[(t-butoxy)carbonylamino]-6-(methoxymethyl)- 2 -oxopiperidyl} acetic acid
The compound(470mg) prepared in step a) above was reacted according to the same procedure as Example 1-a) to give the title compound(382mg) as a pale yellow foam.
Η-NMR(300MHz, CD3OD) δ : 4.28-4.18(m, 2H), 3.96-4.10(m, 2H), 3.32(s, 3H), 2.10-1.90(m, 4H), 1.40(s, 9H)
c) Synthesis of N-l-[2-(2S)-2-(2-(6-cyano- l -ethylindol-2-yl)ethyl) pyiTolidinyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl))-(t-butoxy) carboxamide
The compound(382mg) prepared in step b) above and 2-[2-((2S)-pyπOlidin-2-yl)ethyl]-l-ethylindole-6-carbonitι-ile(324mg) were reacted according to the same procedure as Example 1-b) to give the title compound(371mg) as a white foam.
Η-NMR(300MHz, CDCh) δ ' 7.60-7.50(m, 2H), 7.42-7.38(m, I H). 6.36(s. IH), 5.32(brs. IH), 4.40-3.90(m. 5H), 3.74-3.38(m, 6H), 3.30(s. 3H). 2.80- 2.75(m. 2H), 2.38-2.20(m. 2H). 2.15- 1.90(m. 6H). 1.82-1.70(m, 2H), 1.40(s, 9H), 1.38-1.32(m, 3H)
d) Synthesis of benzyl (2S)-3-{N-[l-(2-((2S)-2-(2-(6-cyano-l-ethyl indol-2-yl)ethyl)pyrrolidinyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperid yl)]carbamoyl}-2-[(t-butoxy)carbonylamino]propanoate
The compound(371mg) prepai'ed in step c) above was reacted according to the same procedure as Example 2-a) to give a compound(190mg) as a pale yellow foam. The resulting compound was reacted according to the same procedure as Example 6-a) to give the title compound(303mg) as a white foam.
Η-NMR(300MHz, CDCI3) δ : 7.60-7.48(m, 2H), 7.42-7.36(m, 6H), 6.48-6.38 (m, IH), 6.36-6.33(m, IH), 5.80-5.68(m, IH), 5.20-5.14(m, 2H), 4.57-4.51(m, IH), 4.40-3.85(m, 6H), 3.66-3.40(m, 5H), 3.32(s, 3H), 2.95-2.86(m, IH), 2.80- 2.65(m, 3H), 2.40-2.24(m, 2H), 2.10-1.88(m, 6H), 1.80-1.66(m, 2H), 1.42(s, 9H), 1.36-1.32(m, 3H)
e) Synthesis of (2S)-3-{N-[l-(2-((2S)-2-(2-(6-amιdmo-l-ethylindol-2- yl)ethyl)pyπOlidinyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl)]carb amoyl}-2-amino propanoic acid
The compound(303mg) prepared in step d) above was reacted according to the same procedure as Example 2-c) to give the title compound(90mg) as a pale yellow solid.
ES-MS : 598(M+ 1 )
Η-NMR(300MHz. CD3OD) : 8.05-7.90(m. I H). 7.65-7.56(m, IH), 7.48-7.38 (m. I H), 6.46-6.40(m. IH), 4.48-4.40(m, IH). 4.32-4.20(m, 3H), 4.10-3.80(m, 2H). 3.70-3.48(m, 5H), 3.35(s. 3H), 3.15-3.05(m. 2H), 2.88-2.60(m. 4H), 2.40- 1.80(m. 10H), 1.40-1.35(m. 3H) Experiment 1
Inhibitory activity against thrombin and trypsin
The compounds according to the present invention were dissolved in 50%) methanol in various concentrations. Then, 20 βi each thereof were introduced into each well of a microplate, to each of which 160^£ of the reaction medium containing 125mM NaCl, 50mM Tris-HCl(pH8.0) and 2mM synthetic substiate(N-benzoyl-Phe-Val-Arg-p-nitroanilide, Sigma B-7632) was added. 20^ of human thrombin solution(5 units/^, Sigma T-6759, manufactured by Sigma Co.) containing 0.1%> bovine serum albumin was added to each well to initiate the enzymatic reaction at room temperature. After 20 minutes, the degree of hydrolysis of substrate was determined by measuring the absorbance at 405nm. The thrombin inhibitory activity was represented by IC50 value which indicates the concentration of the test compound showing half the change of absorbance in the well that did not contain the test compound. The thrombin inliibitory activity of the compound according to the present invention is shown in the following Table 2.
Experiment 2
Measurement of the thrombin time(TT) in rat plasma
S.D. male rats (6-7 weeks) weighing 220+ 20g which had fasted overnight were used as experimental animals. Blood taken from the hearts of the experimental animals just before the administration of the test compound and at 30, 60, 120 and 240 minutes after oral administration of the test compound dissolved in 50%) PEG 400 was mixed with 0.108M sodium citrate in the ratio of 9: 1 by volume. The mixtures thus obtained were centrifuged at 15,000rpm for 5 minutes at 4 °C to separate the plasma, which was stored at -20 °c until the TT was measured by means of the method described below.
200 # of Owren's buffer was added to 50^. of the plasma and lOO g of the diluted plasma thus obtained was injected into the vial for coagulometer and then incubated for 2 minutes at 37 "£ ■ To this vial was added 100^£ of thrombin at a concentration of 20U/m , which was pre-warmed to 37°c , to measure the time(TT) by which clotting occurred. The ratio of the TT in rat plasma after to before administration of the test compound was calculated. The TT ratios are shown in the following Table 2.
Table 2
Inhibitoiy activity against thrombin and the ratio of TT in rat plasma after or before administration of the test compound
Figure imgf000068_0001
Experiment 3 Pharmacokinetic test
Test method:
S.D. male rats weighing 220+ 20g which had fasted 24 hours were anesthetized with ether and then subjected to cannulation at their femoral veins and arteries. Compound prepared in Example 2 was dissolved in physiological saline and administered to the rats by intravenous injection and oral administration. Blood taken at prescribed intervals of time was immediately mixed with methanol and the mixtme was centrifuged( 15,000rpm, 5min, 4°c ). Then, the supernatant was taken quantitatively and subjected to a HPLC equipped with a DAD(Diode Array Detector) where the concentration of the test compound in blood was measured at 254nm.
Test results:
The concentration of the compound of Example 2 in blood analyzed after intravenous injection and oral administration are shown in Tables 3 and 4, and the phamiacokinetic parameters are described in Table 5. As can be seen from the experimental results shown in these tables, when the compound of Example 2 was administered via intravenous injection, it was rapidly distributed in the body and slowly disappeared. In rats, the compound of Example 2 exhibited a good result, i.e., an elimination half-life of 14 minutes and a bioavailability of 53.6%o. Table 3
Concentiation in blood of the compound of Example 2 following intiavenous injection in the concentration of 5 g/^g in rats
Figure imgf000070_0001
Table 4
Concentration in blood of the compound of Example 2 following oral administiation in the concentration of 50mg/^ in rats
Figure imgf000071_0001
Table 5
Phaimacokinetic parameters of the compound of Example 2 in rats
Figure imgf000071_0002

Claims

1. A compound represented by the following formula (1):
Figure imgf000072_0001
, pharmaceutically acceptable salt or stereoisomer thereof, in which
A represents C2-C6-alkylene which may be substituted by
Cι-C4-alkyl, halogeno-Cι-C4-alkyl, Cι-C -alkoxy-Cι-C -alkyl or hydroxy-C i -C -alkyl,
R represents hydrogen, or represents C]-C -alkyl which may be substituted by Cι-C4-alkyl or phenyl,
R and R independently of one another represent hydrogen, or represent G-C -alkyl or G-Cs-alkanoyl each of which may be mono- or poly-substituted by carboxy, halogen, carbamoyl, amino, methylsulfonylamino, Cι-C -alkylamino, di(Cι-C -alkyl)amino, hydroxy,
Cι-C -alkoxycarbonyl or G-C4-al oxy carbamoyl.
2. The compound according to claim 1, wherein A represents ethylene, propylene or butylene which may be substituted by methyl, ethyl, fluoromethyl, methoxymethyl or hydroxymethyl.
3. The compound according to claim 1, wherein R represents hydrogen, methyl, isopropyl or benzyl.
4. The compound according to claim 1, wherein R and R independently of one another represent hydrogen, methyl, ethyl, 3 -carboxy propyl, carboxymethyl, 3-amino-3-carboxy-propanoyl, 3-carbamoyl-3-amino- propanoyl, 4-amino-4-carboxybutyιyl, 4-caibamoyl-4-amino-butyryl, 3-methylsulfonylamino-3-carboxy-propanoyl, 3-diethylamino-3-carboxy- propanoyl, 3 -hydroxy-3 -carboxy -propanoyl or 3 -carbarn oyl-2-amino- propanoyl.
5. The compound according to claim 1, which is selected from the group consisting of:
2-{2-[(2S)-l-(2-((3S)-3-amino-2-oxoazaperhydroepinyl)acetyl)pyιτolidi n-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Compound 1);
4- { [(3 S )- 1 -(2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pynolidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoic acid(Compound
2);
2- { [(3S )- l-(2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyιτolidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}acetic acid(Compound 3);
4-{ [(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyrrolidiny l)-l -methyl-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]amino}butanoic acid(Compound 4);
4-{ [(3S)-l -(2-((2S)-2-(2-(6-amidιno-l -ethylmdol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]ethylamino}butanoic acid(Compound 5);
(2S)-3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l -ethylindol-2-yl)ethyl)pyι- iOlidinyl)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]carbamoyl}-2-aminopiOpano ic acid(Compound 6);
2-{ 2-[(2S)-l-(2-((3S)-3-amino-2-oxopyπOlidinyl)acetyl)pyπ-olidin-2-yl ]ethyl}-l-ethylindole-6-carboxamidine(Compound 7);
4-{[(3S)-l-(2-((2S)-2-(2-(6-amidmo-l-ethylindol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxopyrrolidin-3-yl]amino} butanoic acid(Compound 8);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxopyιτolidin-3-yl]carbamoyl}-2-aminopropanoic acid(Compound 9);
2-{2-[(2S)-l-((2S)-2-((3S)-3-amino-2-oxopyrrolidinyl)-3-methylbutano yl)pyιτolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Compound 10);
4- { [(3 S )- 1 -(( 1 S)-2-((2S )-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyιτol idinyl)-l -(isopiOpyl)-2-oxoethyl)-2-oxopyπOlidin-3-yl]amino}butanoic acid(Compound 11);
2- { [(3 S )- 1 -(( 1 S)-2-((2S)-2-(2-(6-amidino- 1 -ethylindol-2-yl)ethyl)pyrrol idinyl)-2-oxo- 1 -benzylethyl)-2-oxopyπOlidin-3-yl]amino}acetic acid(Compound 12);
2-{2-[l-(2-((3S)-3-amino-2-oxoρiperidyl)acetyl)-(2S)-pyπOlidin-2-yl]et hyl}-l-ethylindole-6-caι-boxamidine(Compound 13);
4-{ [(3S)- l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlιdmy l)-2-oxoethyl)-2-oxo-3-piperidyl]amino} butanoic acid(Compound 14);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlιdι nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminopropanoic acid(Compound 15);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyιτolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbamoyl}-(2S)-2-aminopiOpan oic acid(Compound 16);
2-{2-[ l -(2-((3S)-3-((3S)-3-amino-3-carbamoyl-N-ethylpropanoylamino )-2-oxopiperidyl)acetyl)-(2S)-pyrrolidin-2-yl]ethyl }- 1 -ethylindole-6-carboxami dine(Compound 17);
(2R)-3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidιno- l -ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminopiOpanoic acid(Compound 18);
2-{2-[l -(2-((3S)-3-((3R)-3-amino-3-carbamoylpropanoylamino)-2-oxo piperidyl)acetyl)-(2S)-pyrrolidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(C ompound 19);
4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyn-olidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminobutanoic acid(Compound 20);
2-{2-[l -(2-((3S)-3-((4S)-4-amino-4-carbamoylbutanoylamino)-2-oxopi peridyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-carboxamidine(Co mpound 21);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-[(methylsulfonyl)amin o]propanoic acid(Compound 22);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]cai'bamoyl}-(2S)-2-(diethylamino)piOpan oic acid(Compound 23);
3-{N-[(3S)-l -(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pynOlιdι nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-hydiOxypropanoic acid(Compound 24);
(2R)-4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminobutanoic acid(Compound 25);
2-{2-[l -(2-((3S)-3-((2S)-2-amino-3-caιbamoylpiOpanoylamino)-2-oxop iperidy])acetyl)-(2S)-pyπOlidin-2-yl]ethyl}-l-ethylindole-6-caι-boxamidine(Co mpound 26);
2-{2-[ l -(2-((3S)-3-((2R)-2-amino-3-caι-bamoylpropanoylamino)-2-oxo piperidyl)acetyl)-(2S)-pyπOlidin-2-yl]ethyl }-l -ethylindole-6-carboxamidine(C ompound 27);
2-{2-[(2S)-l-(2-((3S,6S)-3-amino-6-(fluoromethyl)-2-oxopiperidyl)acet yl)pyπolidin-2-yl]ethyl}- 1 -ethylindole-6-carboxamidine(Compound 28);
(2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-a minopropanoic acid(Compound 29); and
(2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminop ropanoic acid(Compound 30).
6. The compound according to claim 5, which is selected from the group consisting of:
4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidiny l)-2-oxoethyl)-2-oxoazaperhydroepin-3-yl]amino}butanoic acid(Compound
2);
4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyriOlidiny l)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]ethylamino}butanoic acid(Compound 5);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr rolidinyl)-2-oxoethyl)-2-oxoazaperhydiOepin-3-yl]carbamoyl}-2-aminopiOpano ic acid(Compound 6);
(2S)-3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylmdol-2-yl)ethyl)pyι- iOlidinyl)-2-oxoethyl)-2-oxopyιτolidin-3-yl]carbamoyl}-2-aminopropanoic acid(Compound 9);
4-{[(3S)-l-(2-((2S)-2-(2-(6-amidino-l -ethylindol-2-yl)ethyl)pyiTolidiny l)-2-oxoethyl)-2-oxo-3-piperidyl]amino}butanoic acid(Compound 14);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyιτolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminopiOpanoic acid(Compound 15);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyiTolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]-N-ethylcarbamoyl }-(2S)-2-aminopiOpan oic acid(Compound 16);
4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylιndol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-aminobutanoic acid(Compound 20);
3 - { N-[(3 S )- 1 -(2-((2S )-2-(2-(6-amidino- 1 -ethylindol-2-y 1 )ethyl)pyπolidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-(diethylamino)propan oic acid(Compound 23);
3-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-(2S)-2-hydiOxypropanoic acid(Compound 24);
(2R)-4-{N-[(3S)-l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyr iOlidinyl)-2-oxoethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminobutanoic acid(Compound 25);
(2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπOlidi nyl)-2-oxoethyl)-(6S,3S)-6-(fluoromethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-a minopropanoic acid(Compound 29); and
(2S)-3-{N-[l-(2-((2S)-2-(2-(6-amidino-l-ethylindol-2-yl)ethyl)pyπ-olidi nyl)-2-oxoethyl)-6-(methoxymethyl)-2-oxo-(3-piperidyl)]carbamoyl}-2-aminop ropanoic acid(Compound 30).
7. A pharmaceutical composition for thrombin inhibition comprising an effective amount of the compound of formula (1 ) as defined in claim 1 , or a phaimaceutically acceptable salt or a stereoisomer thereof as an active ingredient together with a pharmaceutically acceptable carrier.
8. The composition according to claim 7, which is useful for prevention and treatment of thrombosis.
9. The composition according to claim 7, which is formulated into the oral preparation.
10. A process for preparing the compound of formula (1) as defined in claim 1, pharmaceutically acceptable salt and stereoisomer thereof characterized in that:
(a) a compound represented by the following formula (6):
Figure imgf000078_0001
is condensed with a compound represented by the following formula (5):
Figure imgf000078_0002
wherein A and R are defined as Claim 1 and P represents an amino-protecting group, to give a compound represented by the following formula (4):
Figure imgf000079_0001
wherein A, R1 and P are defined as previously described, the resulting compound of formula (4) is deprotected to give a compound represented by the following formula (3):
Figure imgf000079_0002
wherein A and R are defined as previously described, and then the cyano group in the resulting compound of formula (3) is converted to amidino group to give a compound represented by the following formula (la):
Figure imgf000079_0003
wherein A and R1 are defined as previously described, or
(b) the compound of formula (3) is coupled with either one or both of a compound represented by the following formula (7):
R '-X (7)
9' 9 9' wherein R is the same with R as defined in Claim 1 except that R is not hydrogen and X represents active leaving group, and a compound represented by the following formula (8):
R3'-X (8)
wherein R is the same with R as defined in Claim 1 except that R is not hydrogen and X is defined as previously described, to give a compound represented by the following formula (2):
Figure imgf000080_0001
1 9' wherein A. R , R and R are defined as previously described, m and n independently of one another represent an integer of 0 or 1. and p represents an integer of 2-(m+n), and then the cyano group in the resulting compound of formula (2) is converted to amidino group to give a compound represented by the following formula ( l b):
Figure imgf000081_0001
wherein A, R , R »2' , R , m, n and p are defined as previously described, or
(c) if necessary, the compound of formula (lb) is hydrolyzed to give the compound of formula (1) having a carboxy group.
PCT/KR2000/000216 1999-03-17 2000-03-15 Substituted aromatic amidine derivative and medicinal composition comprising the same WO2000055156A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP2000605585A JP3377989B2 (en) 1999-03-17 2000-03-15 Substituted aromatic amidine derivative and pharmaceutical composition containing the same
AU33330/00A AU3333000A (en) 1999-03-17 2000-03-15 Substituted aromatic amidine derivative and medicinal composition comprising thesame

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR1019990008970A KR20000060566A (en) 1999-03-17 1999-03-17 Substituted aromatic amidine derivatives and pharmaceutical composition comprising the same
KR1999/8970 1999-03-17

Publications (1)

Publication Number Publication Date
WO2000055156A1 true WO2000055156A1 (en) 2000-09-21

Family

ID=19576801

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2000/000216 WO2000055156A1 (en) 1999-03-17 2000-03-15 Substituted aromatic amidine derivative and medicinal composition comprising the same

Country Status (4)

Country Link
JP (1) JP3377989B2 (en)
KR (2) KR20000060566A (en)
AU (1) AU3333000A (en)
WO (1) WO2000055156A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2253612A1 (en) 2005-04-14 2010-11-24 Novartis AG Organic compounds

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4258192A (en) * 1977-12-16 1981-03-24 Mitsubishi Chemical Industries Limited N2 -Arylsulfonyl-L-argininamides and the pharmaceutically acceptable salts thereof
WO1993011152A1 (en) * 1991-12-04 1993-06-10 Aktiebolaget Astra New peptide derivatives
EP0627929A1 (en) * 1992-02-14 1994-12-14 Corvas International, Inc. Inhibitors of thrombosis

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4258192A (en) * 1977-12-16 1981-03-24 Mitsubishi Chemical Industries Limited N2 -Arylsulfonyl-L-argininamides and the pharmaceutically acceptable salts thereof
WO1993011152A1 (en) * 1991-12-04 1993-06-10 Aktiebolaget Astra New peptide derivatives
EP0627929A1 (en) * 1992-02-14 1994-12-14 Corvas International, Inc. Inhibitors of thrombosis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
D.W. BANNER ET AL.: "Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors", J. OF BIOL. CHEM.,, vol. 266, no. 30, 25 October 1991 (1991-10-25), pages 20085 - 20093, XP002085588 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2253612A1 (en) 2005-04-14 2010-11-24 Novartis AG Organic compounds

Also Published As

Publication number Publication date
JP3377989B2 (en) 2003-02-17
KR20000060566A (en) 2000-10-16
JP2002539210A (en) 2002-11-19
AU3333000A (en) 2000-10-04
KR20010101950A (en) 2001-11-15

Similar Documents

Publication Publication Date Title
EP0012845B1 (en) Tetrahydroisoquinoline compounds, process for preparing same and pharmaceutical compositions containing them
AU714324B2 (en) D-amino acid derived inhibitors of cysteine and serine proteases
CN1989104B (en) Cyclic amide derivatives, and their production and use as antithrombotic agents.
FI119812B (en) Process for the preparation of novel therapeutically useful peptide derivatives
JP4237824B2 (en) Novel amino acid derivatives and their use as thrombin inhibitors
KR960001205B1 (en) Novel thiazolidine derivatives and the preparation
SA96160637B1 (en) aroy1- piperidine derivatives
HUT70431A (en) New peptide derivatives
HU223752B1 (en) Pharmaceutical composition for improving gastrointestinal blood circulation
CZ346398A3 (en) D-proline derivatives
AU709070B2 (en) Novel compounds with platelet aggregation inhibitor activity
CA2126026A1 (en) 2-piperazinone compounds and their use
US6201006B1 (en) Aromatic amidine derivatives useful as selective thrombin inhibitors
WO2000000470A1 (en) Amino acid derivatives and drugs containing the same as the active ingredient
HU222353B1 (en) Dipeptide p-amidinobenzylamides with n-terminal sulfonyl or aminosulfonyl radicals and their use
US8071594B2 (en) Substituted diketopiperazines and their use as oxytocin antagonists
EP1444201B1 (en) 2-(3-sulfonylamino-2-oxopyrrolidin-1-yl)propanamides as factor xa inhibitors
WO2000055156A1 (en) Substituted aromatic amidine derivative and medicinal composition comprising the same
WO1993009136A1 (en) Phosphonopeptides with collagenase inhibiting activity
EP0832879A1 (en) Process for preparing intermediates for thrombin inhibitors
KR0163671B1 (en) N-ARYLSULFONYL-Ñß-PROPARGYLGYLCINEAMIDE DERIVATIVES
SK14092001A3 (en) Prodrugs of thrombin inhibitors
KR100405650B1 (en) Orally available peptidic thrombin inhibitors
FR2765220A1 (en) New phenyl sulphonyl amino pentyl derivatives
KR19990043605A (en) Aromatic Amidine Derivatives Useful as Selective Thrombin Inhibitors

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW 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)
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: 1020017009771

Country of ref document: KR

Ref document number: 1020017009767

Country of ref document: KR

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2000 605585

Kind code of ref document: A

Format of ref document f/p: F

WWR Wipo information: refused in national office

Ref document number: 1020017009767

Country of ref document: KR

WWW Wipo information: withdrawn in national office

Ref document number: 1020017009767

Country of ref document: KR

WWP Wipo information: published in national office

Ref document number: 1020017009771

Country of ref document: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
WWW Wipo information: withdrawn in national office

Ref document number: 1020017009771

Country of ref document: KR