WO2005035497A1 - Caspase inhibitor comprising 2-alkyl-4-oxobutanoyl group and pharmaceutical composition thereof - Google Patents

Caspase inhibitor comprising 2-alkyl-4-oxobutanoyl group and pharmaceutical composition thereof Download PDF

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WO2005035497A1
WO2005035497A1 PCT/KR2004/002554 KR2004002554W WO2005035497A1 WO 2005035497 A1 WO2005035497 A1 WO 2005035497A1 KR 2004002554 W KR2004002554 W KR 2004002554W WO 2005035497 A1 WO2005035497 A1 WO 2005035497A1
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sac
scac
compound
amino
acid
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PCT/KR2004/002554
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French (fr)
Inventor
Hye-Kyung Chang
Yeong-Soo Oh
Cheol-Won Park
Yong-Jin Jang
Sung-Sub Kim
Min-Jung Kim
Mi-Jeong Park
Jung-Gyu Park
Sun-Hwa Lee
Hee-Dong Park
Ae-Ri Kim
Ki-Sook Park
Kyeong-Sik Min
Tae-Soo Lee
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Lg Life Sciences Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C235/00—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms
    • C07C235/70—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton
    • C07C235/72—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms
    • C07C235/74—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups and doubly-bound oxygen atoms bound to the same carbon skeleton with the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of a saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C07D217/16—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals substituted by oxygen atoms

Definitions

  • the present invention relates to a novel compound of formula 1, pharmaceuticaly acceptable salt, physiofogicaly hydr ysable ester, hydrate, solvate, and stereoisomer thereof usable as an inhibitor against caspases including caspase-1 [interleukin-l ⁇ converting enzyme, ICE], caspase-3 [apopain/CPP-32] and caspase-8, 9 and a pharmaceutical composition comprising the compound of the formula 1.
  • Caspase is a new kind of cysteine protease in the tetramer form of ⁇ ⁇ discovered 2 2 during the last 10 years. About 14 kinds of caspase have been known until now.
  • Caspase-1 (ICE), one of them, is a kind of cytokines and relates to converting inactive prointerleukin-l ⁇ to active interleukin-l ⁇ .
  • Interleukin-1 consists of interleukin-l ⁇ and interleukin-l ⁇ , both of which are synthesized in monocytes in the form of precursor having 31kDa. Only prointerleukin-l ⁇ is activated by ICE. The positions hydrofyzed by caspase-1 are Asp -Gly and Asp -Ala .
  • Interleukin-l ⁇ has been reported to act as an important mediator in causing inflammation (1, 3).
  • Caspase-1 has been discovered for the first time in 1989, and in two independent study groups, its three dimensional structure was discovered by X-ray crystalographic method.
  • Caspase-3 (CPP-32) is broady studied about its r e or action mechanism, and its three dimensional structure was determined in 1996 (2).
  • Caspase-3 (apopain) activated from procaspase-3 is hydrofyzed at (P4)Asp-X-X-Asp(Pl) motif, and known substrates indude pdy(ADP-ribose) pofymerase, Ul 70,000 Mr smal nudear ribonudeoprotein and catalytic subunit of 460,000 Mr DNA-dependent protein kinase, etc.
  • the X-ray structure of caspase-7 has been reported to be very similar to that of caspase-3 (4).
  • caspase-8 and 9 are present in the upstream of caspase-3, 6, 7, and these caspases are known to relate to apoptosis cascade.
  • the X-ray structure of caspase-8 was determined in 1999 (5), and particularly by administering inhibitors thereof, may be advantageously used for treating diseases related to apoptosis.
  • Caspase inhibitors mean those compounds that inhibit the activity of caspase, and so control such symptoms as inflammation, apoptosis, etc. caused by caspase activity.
  • Diseases or symptoms that may be treated or attenuated by administering the inhibitors indude the fob wing: dementia, cerebral stroke, Brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, liver diseases by hepatitis virus, acute hepatitis, fiJminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, or ischemic cardiac diseases (6).
  • Both of the above inhibitors exhibit activity based on the common mechanism to irreversibly inactivate the enzyme, thereby suppressing eel apoptosis (irreversible, broad-spectrum inhibitor). Comparing irreversible and reversible inhibitors (7), it has been reported that irreversible inhibitor has much more effective inhibitory activity. Both IDN-1965 of IDUN Co. and MX-1013 of Maxim Co. are reported to show activity in eel apoptosis model for hepatic injury (8, 9). These compounds are now in the predinical test stage. The irreversible inhibitor, IDN-6556, whose structure has not been reported, is now in the stage of phase II dinical test as a therapeutic agent for hepatic injury (10, 11).
  • Diabetes Diabetes 2002 Jun;51(6):1938-48, Hyperglycemia-induced apoptosis in mouse myocardium: mitochondria! cytochrome C-mediated caspase-3 activation pathway.
  • Cai L Li W, Wang G, Guo L, Jiang Y, Kang YJ.
  • the present inventors newly designed and synthesized compounds which have a chemical structurecloudamentaly different from inhibitors reported up to date, measured their binding ability to caspases, and enabled them to have high selectivity for similar enzymes. As a result, the inventors have discovered that a compound of the f olo wing formula (1) does meet the expected purpose of the present invention, and completed the present invention.
  • R, R , R , R , X and Ar are defined as described bebw. 1 2 3
  • the present invention provides a novel compound of the above formula 1 having effective inhibitory activity against caspases, and pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof.
  • It is another object of the present invention to provide a pharmaceutical composition comprising the compound of the formula 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as active ingredients.
  • SCAC Simple Cydo Alkyl Chain
  • Aryl group (Ar, bebw) indudes both the aromatic and heteroaromatic groups.
  • the aromatic group means a 5 to 15-membered single or fiised unsaturated cyde.
  • One or more hydrogens may be replaced with a group(s) selected from the fdbwing: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, hab, hydroxy, nitro, thio, alkyl, cycbalky], alkoxy, arybxy, su ⁇ bxy, and guanido group.
  • the heteroaromatic group means the aromatic group containing 1 to 5 hetero atoms selected from a group consisting of oxygen, sufir, and nitrogen.
  • one or more hydrogens may be replaced with a group(s) selected from the fdbwing: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, hab, hydroxy, nitro, thio, alkyl, cycbalky], alkoxy, aryl, arybxy, su ⁇ bxy, and guanido group.
  • the aryl group indudes phenyl, 1 -naphthyl, 2-naphthyl, pyridinyl, pyrimidinyl, quindinyl, benzothienyl, inddyl, pyrazinyl, isoinddyl, isoquindyl, qunazdu yl, quinoxa ⁇ nyl, phthalazinyl, imidazdiny], isoxazdinyl, pyrazdyl, oxazdyl, thiazdyl, inddizinyl, indazdyl, benzothiazdyl, benzimidazdyl, benzofiiranyl, thienyl, pyrrdyl, oxadiazdyl, thiadiazdyl, triazdyl, tetrazdyl, oxazdopyridinyl,
  • the protecting group of ester is a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form.
  • the present invention relates to the compound of the fdbwing formula (1), pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof having inhibitory activity against capsizes.
  • R represents simple alkyl chain (-SAC), cydoalkyl chain (-SCAC), aryl group (-Ar), alkyl chain substituted by aryl (-SAC-Ar), or hydrogen
  • R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of al the natural amino acids
  • the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 1 becomes a stereocenter due to the R group; or the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a su ⁇ bnamide 2 4 4 form (-CONHSO R wherein R
  • R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of the 2 natural amino acids; and the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 2 becomes a stereocenter due to the R group; the compound of formula (1) may have a 2 protecting group in an ester form (-CO R wherein R is -SAC) or a svJfonamide form 2 6 6 (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceuticaly 2 1 1 acceptable salt, when R is a carboxylic acid as side chain residue of an amino acid; or 2 the compound of formula (1) may also exist in the form of pharmaceuticaly acceptable salt when R consists of a base as side chain residue of an amino acid, or R 2 2 fiirther represents H; -(CH ) OR wherein R is -SAC, -SCAC, -Ar, or
  • Ar represents aryl (- Ar)
  • R represents H
  • R represents -SAC, or H
  • X represents -COCH N , -COCH F, -COCH CI, -COCH Br, -COCH I, -COCH 2 2 2 2 2 2 2 2 OAr, -COCH OCOAr or -COCH SR (R is -SAC, -SCAC, -Ar or -SAC-Ar). 2 2 2 15 15 15
  • Particularly preferred compounds are those selected from the fdbwing group:
  • composition according to the present invention inhibit caspases, and thus is usefii for the treatment or prevention of dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, liver diseases by hepatitis virus, acute hepatitis, fifa ⁇ iant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, or ischemic cardiac diseases.
  • composition according to the present invention may be formulated and administered as oral preparation, injection, or patch.
  • the compound of formula 1 according to the present invention can be synthesized as shown in the fdbwing Reaction Schemes 1 and 2.
  • those illustrated in the fdbwing Reaction Schemes represent only the typical processes used in the present invention.
  • the order of units may be modified with no limit, and so the processes are not restricted to those explained bebw.
  • ⁇ -keto phosphonate (111) is synthesized from aromatic methyl ester (II) and methyl dimethyl phosphonate.
  • the resulting compound (111) is then reacted with 2-ketoester (IV) by using the Wittig Reaction to give a 4-oxo-2-alkyl-2-butenoic ester (V).
  • the compound (V) in the form of trans/cis mixture is hydrogenated to give a derivative (VI). If the protecting group P is needed to replace by a new substituent, P is removed and P is introduced again.
  • reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure and separated by cdumn chro- matography (50%-80% ethyl acetate/hexane) to give the title compound (1.68 g, 95%) as yebw sdid.
  • anhydrous tetrahydrofiiran (40 ⁇ ) was added to sodium hydride (60% dispersion in mineral oil, 216 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere.
  • Lithium chbride (415 mg, 2.0 eq) was added thereto and the mixture was fiirther stined at 0 °C for about 10 minutes.
  • Methyl 2-oxobutylate (630 mg, 1.1 eq) dissdved in anhydrous tetrahydrofiiran (10 m ⁇ ) was added threrto, and stined at 0 °C for 30 minutes and at room temperature for 2 hours. The reaction was completed with saturated ammonium chbride sdution, and then extracted twice with ethyl acetate (100 m ⁇ ).
  • reaction was stopped by acetic acid, and the reaction sdution was distiled under reduced pressure to remove methand.
  • the residue was extracted with ethyl acetate (50 M x 2), washed with water and aqueous sodium chbride sdution, dried (anhydrous Na SO ), con- 2 4 centrated under reduced pressure, and separated-purified by column chromatography (ethyl acetate-hexane, 1:5), to give the title compound (4.80 g, 86%) in diastereomeric form.
  • anhydrous tetrahydrofiiran (10 rd ) was added to sodium hydride (60% dispersion in mineral oil, 34 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere.
  • Dim ethyl 2-(l,3-dimethyl-l H - indd-2-yl)-2-oxoethylphosphonate (230 mg, 0.78 mmd) prepared in Preparation 2 dissdved in anhydrous tetrahydrofiiran (5 M ) was added to the mixture. The resulting mixture was stined for about 10 minutes.
  • Lithium chbride (66 mg, 2.0 eq) was added thereto and the mixture was fiirther stined at 0 °C for about 10 minutes.
  • Ethyl - 3-methyl 2-oxobutylate (124 mg, 0.13 M , 1.1 eq) dissdved in anhydrous tetrahydrofiiran (3 M ) was added thererto, and stined at 0 °C for 30 minutes and at room temperature for 2 hours.
  • the reaction was completed by saturated ammonium chbride sdution, and then the reaction was extracted twice with ethyl acetate (30 M ).
  • the sdvent was distiled under reduced pressure, and the residue was extracted with ethyl acetate (300 M x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and 2 4 purified by cdumn chromatography (30% ethyl acetate/hexane), to give a compound (2.35 g, 79%). To this compound and Dess-Martin reagent (5.41g, 3.0 eq) was added dry dichbromethane (50 mil ), and the mixture was stined for one hour at room temperature.
  • the reaction was stopped by isopropyl alcohd (6 ⁇ ).
  • the mixture was filtrated through Celite under reduced pressure to remove sdid, and the filtrate was extracted with ethyl acetate (50 mil x 2), washed with water, saturated aqueous sodium bicarbonate sdution, and aqueous sodium chbride sdution, dried (anhydrous Na SO 2 4 ), concentrated under reduced pressure, and preliminarily purified by column chromatography (36% ethyl acetate-hexane), to give the diastereomeric title compound (2.11 g).
  • reaction was stopped by isopropyl alcohd (0.05 mil ). After the common treatment, the reaction was purified by Prep TLC (25% ethyl acetate-hexane) to give the diastereomeric title compound (14 mg, 69%).
  • Methyl dimethyl phosphonate (4.84 g, 4.17 mil , 3.0 eq ) was dissdved in anhydrous tetrahydrofiiran (about 100 mil ) under nitrogen atmosphere, the mixture was kept at -78°C, and n-buty ⁇ thium (2.5M in hexan, 15.6 mil , 3.0 eq) was added thereto. The mixture was stined at -78 - -60 °C for one hour.
  • the methyl 1-isoquindinyl carboxylate (2.44 g, 13.0 mmd, prepared from 1-isoquindinecarboxvlic acid according to the same procedure as Preparation 1) was dissdved in anhydrous tetrahydrofiiran (20 mil ) and added to the mixture at -60 °C . Th e resulting mixture was stined at -60 °C for 2 hours while it was sbwly warmed to room temperature. Oxalic acid dihydrate (5.41 g, 3.3 eq) was dissdved in methand (12 mil ), and then added to the mixture.
  • reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure and separated by cdumn chromatography (50%-80% ethyl acetate/hexane) to give the title compound (3.59 g, 99%) as yebw sdid.
  • Methyl 2-oxobutylate (2.4 g, 1.1 eq) dissdved in anhydrous tetrahydrofiiran (20 mil ) was added threrto at 0 °C, and the mixture was stined at room temperature for 2.5 hours. The reaction was completed by saturated ammonium chbride sdution, and then extracted twice with ethyl acetate (100 mil ). The filtrate was washed with water, saturated sodium bicarbonate sdution (NaHCO , 50 mil x 2), and aqueous sodium chbride sdution in turn, dried (anhydrous 3 Na SO ), and concentrated under reduced pressure . The residue was separated by 2 4 cdumn chromatography (20% ethyl acetate/hexane) to give the title compound (2.7 g, 68%) as yebw liquid in the form of cis.
  • the above compound (1.0 g, 3.66 mmd) was dissdved in dichbtomethane (30 mil ), and then was added to the above mixture sbwly. The mixture was stined at -78 °C for 15 minutes. DIPEA (3.19 mil , 5.0 eq) was added to the reaction mixture and the mixture was stirred at -78 °C for 10 minutes. The temperature was sbwly raised to room temperature. Ethyl acetate/hexane (100 mil ) was added to the mixture, and organic layer was washed with 0.5N HC1 and saturated sodium chbride sdution, dried (anhydrous Na SO ), and concentrated under reduced 2 4 pressure.
  • the mixture was filtrated through Celite under reduced pressure to remove sdid, and the filtrate was extracted with ethyl acetate (30 mil x 2), washed with water, saturated aqueous sodium bicarbonate sdution and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and preliminarily purified by cdumn 2 4 chromatography (30%-40% ethyl acetate-hexane), to give the diastereomeric title compound (192 mg, 80%).
  • anhydrous tetrahydrofiiran (30 mil ) was added to sodium hydride (60% dispersion in mineral oil, 276 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere.
  • the phosphonate compound prepared in Preparation 30 (1.76 g, 6.27 mmd) was dissdved in anhydrous tetrahydrofiiran (10 mil ), which was added to the mixture.
  • the resulting mixture was stined for about 10 minutes.
  • Lithium chbride (532 mg, 2.0 eq) was added thereto and the resulting mixture was fiirther stirred at 0 °C for about 10 minutes.
  • Caspase-1 and caspase-8 known as cysteine proteases in the form of ⁇ ⁇ were 2 2 expressed, purified, and activated by modifying a method known in Thornberry, N. A. et , Nature, 1992, 356, 768. Thornberry, N. A. Methods in Enzymology, 1994, 244, 615. Walker, N. P. C et al. Cell, 1994, 78, 343, and caspase-9 was also purified by a similar method, and the inhibitory activity against them was tested. Briefly describing, plO and p20 subunits (Thornberry, N. A.
  • the enzyme reaction was carried out at 25 °C in a buffer sdution containing 50mM HEPES (pH 7.50), 10%(w/v) sucrose, 0.1%(w/v) CHAPS, lOOmM NaCl, ImM EDTA, and lOmM DTT in the presence of 50 ⁇ M AcYVAD-AFC for lOnM caspase-1, 50 ⁇ M AcDEVD- AFC for 2.1nM caspase-8, and 150 ⁇ M AcLEHD-AFC for 200nM caspase-9.
  • the inhibitory constants K and K of the inhibitors were determined by measuring the i obs reaction vebcity to the lapse of time with using a fluorescent spectrometer and by obtaining the initial rate constant. K was calculated from the Lineweaver Burk Pbt, and K from the fobwing Equation 1. obs
  • K -h ( ⁇ -A /A )/t obs t oo [527] in which [528] A means deavate rate (%) at time t, and t [529] A means the maximum deavage rate (%). [530] [531] Spectra MAX GeminiXS Fluorescent Spectrometer of Mdeciiar Device Co. was used at the excitation wavelength of 405nm and the emission wavelength of 505nm.
  • Rat 250-300 g, Bio Genomics , Korea
  • pdtethylene tube was inserted into, and fixed to, the jugular vein and carotid artery of the test animal.
  • the canular was extracted out of the r ear of rat neck, which was connected to the end of the canular with the bng polyethylene tube protected by spring.
  • the drug was dissdved in a mixed sdution of PEG400, EtOH, TWEEN80, and phosphate buffer, and administered to the jugular vein of the test animal in the amount of 10 mg/kg.
  • rat was starved for 18 hours from the drug administration.
  • the drug was dissdved in a mixed solution of PEG400, EtOH, TWEEN80, and phosphate buffer, and administered to the jugular vein of the test animal through oral sonde in the amount of 20 mg/kg.
  • Bbod was colected before the drug administration (contrd), and at 1, 5, 15, 30, 60, 90, 120, 180 and 240 minutes after the oral administration for intravenous injection test, and at 15, 30, 60, 90, 120, 180 and 240 minutes after the oral administration for oral administration test.
  • About 200 fd of bbod was colected from the carotid artery by using heparin-treated syringe.
  • the colected bbod was centrifiiged to separate bbod plasma, protein was removed therefrom, and then the resultant was centrifiiged and analyzed by HPLC Calibration curve was determined at the concentration of 0.2-10 ⁇ g I mil , and the drug was analyzed by Shiseido Capcel-Pak C reverse column.
  • HPLC consists of Class-LCI 0A system 18 contrd software, CBM-IOA communication bus module, two of LC- 10 AD pump, SIL- 10AXL autoinjector with sample coder, SPD-10AV ultravidet (UV) detector (Shimadzu, Tokyo, Japan), and GLP-2050+ laser printer (LG Electronics, Seoul, Korea).
  • the compound Ia-2 was analyzed by UV lamp using wavelength of 329 nm. The flow rate was 1 mil /minute. As ehient, acetonitrile and 20 mM ammonium acetate was used at 31% and 69%, respectively. The retention time of drug was about 9 minutes.
  • IDN1965 was analyzed by UV lamp using wavelength of 293 nm.
  • the flow rate was about 1 mil /minute.
  • acetonitrile and 20 mM ammonium acetate was used at 32% and 68%, respectively.
  • the retention time of drug was about 10 minutes.
  • the data were represented by relation graph of plasma-drug concentration against time. P harmacokinetic parameters, half life (t ), maximum concentration (C ), time 1/2 max of maximum concentration (T ), AUC , AUC , systemic deareance (CL), max inf last distributed volume (Vd) were determined using Win-Nonfin Program (Scientific Consultion Co., USA) to non-compartment model (cf.: "p harmacokinetic parameter value after intravenous injection and oral administration in rat")
  • Step 1) Preparation of bbod sample
  • Female Balb/c mice (6 weeks, Charles River Laboratory, Osaka , Japan ) were kept under the conditions of 22 °C , 55% of relative humidity, and light-darkness cyde of 12 hours. Food and water were supplied ad libitum. In pyrogen-free saline were dissdved LPS (fipopdysaccaride) and D-galactosamine in concentrations of 0.4 mg / mil and 280 mg / mil , respectively, and their 1:1 mixture was injected into the mice in the amount of 5 mil / kg .
  • LPS fupopdysaccaride
  • bbod samples were obtained from their hearts.
  • Step 2 Determination of the activity of plasma aminotransf erase
  • the plasma ALT activity was determined for the bbod samples obtained in Step 1 by using ALT assay kit (Asan Pharm. Co., Seoul , Korea ) according to the manufacturer's instruction. As a result, it appeared that the injection of LPS and D- galactosamine steeply increases the ALT activity in plasma, and the test compounds inhibit the increased enzyme activity in dose-dependent manner. Based on these results, ED values of the test compounds were calculated with using Prism software 50 of GraphPad Co. (cf.: Table 3 - "ED vahie for inhibiting the ALT activity").
  • mice Female Balb/c mice (6 weeks, Charles River Laboratory, Osaka , Japan ) were kept under the conditions of 22 °C , 55% of relative humidity, and light-darkness cyde of 12 hours. Food and water were supplied ad libitum. In pyrogen-free saline were dissdved LPS (fipopdysaccaride) and D-galactosamine in concentrations of 0.4 mg / mil and 280 mg / mil , respectively, and their 1 : 1 mixture was injected to the mice in the amount of 5 mil / kg .
  • LPS fupopdysaccaride
  • D-galactosamine in concentrations of 0.4 mg / mil and 280 mg / mil
  • the number of survival mice was determined over 72 hours to estimate the survival rate (cf.: "survival rate (%) in induced acute hepatitis model").
  • compound (Ia-2) according to the present invention showed an increased inhibitory activity by 2 times or more against caspase-8 and Jurkat eel compared with IDN-1965, and by about 3 times against LPS-induced acute hepatitis model (mouse). Also, regarding the pharmacokinetic test, the compound showed highuy improved half-life and deareance parameter values at the intravenous injection. The compound of the present invention showed the most remarkable effects than the IDN-1965 in terms of oral absorption rate and survival rate. In the case of survival test ( iv administration), the contrd groups were al dead, whereas the groups into which compound (Ia-1) of the present invention are administered showed a surprising survival rate of 90%.
  • IDN-1965 did not show any inhibitory effect against ALT activity upon oral administration in LPS-induced acute hepatitis model, whereas the compound (Ia-2) of the present invention showed inhibitory effect against ALT activity in both cases of intraperitoneal and oral administration. Also, compound (Ia-2) of the present invention showed highly improved effects in both aspects of phtsiochemical properties and solubility. Thus, the compound (Ia-2) according to the present invention can be used as injection preparation.
  • the compound (Ia-2) of the present invention showed highly improved effects, compared with the IDN-1965 in both aspects of activity and physico-chemical property.

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Abstract

The present invention relates to a compound of the formula (1), pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or steroisomer thereof having inhibitory activity against caspases. The compound contains unknown new 2-alkyl-4-oxobutanoyl group. Further, the present invention relates to a process for preparing the compound of formula 1 and a method for determining its binding ability for caspases. The compound of formula 1 can be used effectively for the treatment or relief of various diseases caused by caspases.

Description

Description CASPASE INHIBITOR COMPRISING 2-ALKYL-4-OXOBUTANOYL GROUP AND PHARMACEUTICAL COMPOSITION THEREOF
[i] TECHNICAL FIELD
[2]
[3] The present invention relates to a novel compound of formula 1, pharmaceuticaly acceptable salt, physiofogicaly hydr ysable ester, hydrate, solvate, and stereoisomer thereof usable as an inhibitor against caspases including caspase-1 [interleukin-lβ converting enzyme, ICE], caspase-3 [apopain/CPP-32] and caspase-8, 9 and a pharmaceutical composition comprising the compound of the formula 1.
[4] BACKGROUND ART
[5]
[6] Caspase is a new kind of cysteine protease in the tetramer form of α β discovered 2 2 during the last 10 years. About 14 kinds of caspase have been known until now. Caspase-1 (ICE), one of them, is a kind of cytokines and relates to converting inactive prointerleukin-lβ to active interleukin-lβ. Interleukin-1 consists of interleukin-lα and interleukin-lβ, both of which are synthesized in monocytes in the form of precursor having 31kDa. Only prointerleukin-lβ is activated by ICE. The positions hydrofyzed by caspase-1 are Asp -Gly and Asp -Ala . The hydrolysis of the latter position gives interleukin-lβ. Interleukin-lβ has been reported to act as an important mediator in causing inflammation (1, 3). Caspase-1 has been discovered for the first time in 1989, and in two independent study groups, its three dimensional structure was discovered by X-ray crystalographic method.
[7]
[8] Caspase-3 (CPP-32) is broady studied about its r e or action mechanism, and its three dimensional structure was determined in 1996 (2). Caspase-3 (apopain) activated from procaspase-3 is hydrofyzed at (P4)Asp-X-X-Asp(Pl) motif, and known substrates indude pdy(ADP-ribose) pofymerase, Ul 70,000 Mr smal nudear ribonudeoprotein and catalytic subunit of 460,000 Mr DNA-dependent protein kinase, etc. The X-ray structure of caspase-7 has been reported to be very similar to that of caspase-3 (4).
[9] [10] Caspase-8 and 9 are present in the upstream of caspase-3, 6, 7, and these caspases are known to relate to apoptosis cascade. The X-ray structure of caspase-8 was determined in 1999 (5), and particularly by administering inhibitors thereof, may be advantageously used for treating diseases related to apoptosis.
[11]
[12] Caspase inhibitors mean those compounds that inhibit the activity of caspase, and so control such symptoms as inflammation, apoptosis, etc. caused by caspase activity. Diseases or symptoms that may be treated or attenuated by administering the inhibitors indude the fob wing: dementia, cerebral stroke, Brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, liver diseases by hepatitis virus, acute hepatitis, fiJminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, or ischemic cardiac diseases (6).
[13]
[14] Among caspase inhibitors known until now, the most noted irreversible inhibitors are the folowing:
[15]
Figure imgf000003_0001
[16]
[17] Both of the above inhibitors exhibit activity based on the common mechanism to irreversibly inactivate the enzyme, thereby suppressing eel apoptosis (irreversible, broad-spectrum inhibitor). Comparing irreversible and reversible inhibitors (7), it has been reported that irreversible inhibitor has much more effective inhibitory activity. Both IDN-1965 of IDUN Co. and MX-1013 of Maxim Co. are reported to show activity in eel apoptosis model for hepatic injury (8, 9). These compounds are now in the predinical test stage. The irreversible inhibitor, IDN-6556, whose structure has not been reported, is now in the stage of phase II dinical test as a therapeutic agent for hepatic injury (10, 11).
[18]
[19] References:
[20] (1) Inflammation: Basic Principles and Clinical Correlates, 2nd ed., ed by Gain, Goldstein and Snyderman. Raven Press Ltd., New York . 1992, pp211-232; Blood, 1996, 87(6), 2095-2147. [21] (2) Wilson, K. P. et ύ, Nature, 1994, 370. 270; Walker, N. P. C et al. Cell, 1994,78 , 343; Nαtwre Structural Biology, 1996, 3(7), 619. [22] (3) Thornberry, Ν. A. et , Nature, 1992, 356. 768; Nαtwre Biotechnology, 1996,14, 297; Protein Science, 1995, 4, 3; Nαtwre, 1995, 376(July 6), 37; Protein Science, 1995, 4, 2149.
[23] (4) Wei, Y. et , Chemistry and Biology, 2000, 7, 423.
[24] (5) Blanchard H. et , Structure, 1999, 7, 1125; Blanchard H. et , J. ofMol. Biol., 2000, 302, 9. [25] (6) References for caspase-related diseases
[26] Dementia: Arch Νeurd 2003 Mar;60(3):369-76, Caspase gene expression in the brain as a fiinction of the dinical progression of Alzheimer disease. Pompl PΝ, Yemul 5, Xiang Z, Ho L, Haroutunian V, Purohit D, Mohs R, Pasinetti GM [27]
[28] Cerebral stroke: Proc Νatl Acad Sci USA 2002 Νov 12;99(23):15188-93, Caspase activation and neuroprotection in caspase-3-deficient mice after in vivo cerebral ischemia and in vitro oxygen glucose deprivation. Le DA, Wu Y, Huang Z, Matsushita K, Plesnila Ν, Augustinack JC, Hyman BT, Yuan J, Kuida K, Flavel RA, Moskowitz MA.
[29]
[30] Brain impairment due to AIDS: J Νeurosci 2002 May 15;22(10):4015-24, Caspase cascades in human immunodeficiency virus-associated neurodegeneration. Garden GA, Budd SL, Tsai E, Hanson L, Kaul M, D'Emffia DM, Friedander RM, Yuan J, Masliah E, Lipton S A.
[31]
[32] Diabetes: Diabetes 2002 Jun;51(6):1938-48, Hyperglycemia-induced apoptosis in mouse myocardium: mitochondria! cytochrome C-mediated caspase-3 activation pathway. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ.
[33]
[34] Gastric ulcer: J Physic! Pharmac 1998 Dec;49(4):489-500, Rde of basic fibroblast growth factor in the suppression of apoptotic caspase-3 during chronic gastric ulcer healing. Sbmiany BL, Piotrowski J, Sbmiany A.
[35]
[36] Cerebral injury by hepatitis: J Viral Hepat 2003 Mar;10(2): 81-6, Cerebral dysfiinction in chronic hepatitis C infection. Forton DM, Taybr-Robinson SD , Thomas HC
[37]
[38] Fulminant hepatic failure: Gastroenterdogy 2000 Aug; 119(2): 446-60, Tumor necrosis factor alpha in the pathogenesis of human and murine finminant hepatic failure. Streetz K, Leifeld L, Grundmann D, Ramakers J, Eckert K, Spengler U, Brenner D, Manns M, Trautwein C
[39]
[40] Sepsis: Nat Immund 2000 Dec;l(6):496-501, Caspase inhibitors improve survival in sepsis: a critical rde of the lymphocyte. Hotchkiss RS, Chang KC, Swanson PE, Tinsley KW, Hui JJ, Klender P, Xanthoudakis S, Roy S, Black C, Grimm E, Aspiotis R, Han Y, Nichdson DW, Karl IE.
[41]
[42] Organ transplantation rejection: Xenotransplantation 2001 May;8(2): 115-24, In vitro prevention of eel-mediated xeno-graft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney eels. Fujino M, Li XK, Suda T, Hashimoto M, Okabe K, Yaginuma H, Mkoshiba K, Guo L, Okuyama T, Enosawa S, Amemiya H, Amano T, Suzuki S.
[43]
[44] Rheumatic arthritis: Prog Med Chem 2002;39: 1-72, Caspase inhibitors as anti- inflammatory and antiapoptotic agents. Graczyk PP.
[45]
[46] Ischemic cardiac diseases: Am J Physid Heart Circ Physid 2002 Sep; 283(3): H990-5, Hypoxia-induced deavage of caspase-3 and DFF45/ICAD in human failed cardiomyocytes. Todor A, Sharov VG, Tanhehco EJ, Silverman N, Bernabei A, Sabbah HN.
[47]
[48] Anti-inflammation: J Immund 2003 Mar 15; 170(6): 3386-91, A broad-spectrum caspase inhibitor attenuates alergic airway inflammation in murine asthma model. Iwata A, Nishio K, Winn RK, Chi EY, Henderson WR Jr, Harlan JM
[49]
[50] (7) Wu J. et , Methods: A Companion to Methods in Enzymology, 1999, 17, 320.
[51] (8) Hoglen N. C et , J. of Pharmacoloy and Experimental Therapeutics, 2001, 297, 811.
[52] (9) Jaeschke H. et , Toxicology and Applied Pharmacology, 2000, 169, 11. DISCLOSURE OF THE INVENTION
[54]
[55] The present inventors newly designed and synthesized compounds which have a chemical structure fiindamentaly different from inhibitors reported up to date, measured their binding ability to caspases, and enabled them to have high selectivity for similar enzymes. As a result, the inventors have discovered that a compound of the f olo wing formula (1) does meet the expected purpose of the present invention, and completed the present invention.
[56] O R2
R3 0 R R1 [57] (1)
[58]
[59] in which
[60] R, R , R , R , X and Ar are defined as described bebw. 1 2 3
[61]
[62] Therefore, the present invention provides a novel compound of the above formula 1 having effective inhibitory activity against caspases, and pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof. [63] [64] It is another object of the present invention to provide a pharmaceutical composition comprising the compound of the formula 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as active ingredients. [65] BEST MODE FOR CARRYING OUT THE INVENTION [66] [67] Prior to ilustrating the present invention in detail, the fdowing important terms are defined: [68] [69] a) Simple Alkyl Chain (SAC, bebw) means a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form. [70]
[71] b) Simple Cydo Alkyl Chain (SCAC, bebw) means a cydic radical having 3 to 10 carbon atoms.
[72]
[73] c) Aryl group (Ar, bebw) indudes both the aromatic and heteroaromatic groups. The aromatic group means a 5 to 15-membered single or fiised unsaturated cyde. One or more hydrogens may be replaced with a group(s) selected from the fdbwing: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, hab, hydroxy, nitro, thio, alkyl, cycbalky], alkoxy, arybxy, suϊbxy, and guanido group. The heteroaromatic group means the aromatic group containing 1 to 5 hetero atoms selected from a group consisting of oxygen, sufir, and nitrogen. Likewise, one or more hydrogens may be replaced with a group(s) selected from the fdbwing: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, hab, hydroxy, nitro, thio, alkyl, cycbalky], alkoxy, aryl, arybxy, suϊbxy, and guanido group.
[74]
[75] The aryl group indudes phenyl, 1 -naphthyl, 2-naphthyl, pyridinyl, pyrimidinyl, quindinyl, benzothienyl, inddyl, pyrazinyl, isoinddyl, isoquindyl, qunazdu yl, quinoxaϋnyl, phthalazinyl, imidazdiny], isoxazdinyl, pyrazdyl, oxazdyl, thiazdyl, inddizinyl, indazdyl, benzothiazdyl, benzimidazdyl, benzofiiranyl, thienyl, pyrrdyl, oxadiazdyl, thiadiazdyl, triazdyl, tetrazdyl, oxazdopyridinyl, imidazopyridinyl, isothiazdyl cinndinyl, carbazdyl, isochromany], chromanyl, tetrahydroisoquindiny], isoinddinyl, isobenzotetrahydrofiiranyl, isobenzotetrahydro-thienyl, isobenzothienyl, benzoxazdyl, pyridopyridinyl, benzotetrahydrofiirany], benzotetrahydrothienyl, purinyl benzodioxdyl, triazinyl, phenoxazinyl, phenothiazinyl, pteridinyl, benzothiazdyl, imidazopyridinyl, imidazothiazdyl, dihydrobenzisoxazinyl, ben- zisoxazinyl, benzoxazinyl, dihydrobenzisothiopyranyl, benzopyranyl, benzoth- iopyrany], coumarinyl, isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide, tetrahydroquindinyl-N-oxide, dihydroquindiny], dihydroquindinony], dihydroiso- quindinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindcfinonyl, ben- zodioxanyl), benzoxazdinonyl, pyrrdyl-N-oxide, pyrimidinyl-N-oxide, pyrazinyl- N-oxide, quindinyl-N-oxide, inddyl-N-oxide, inddinyl-N-oxide, isoquindyl- N-oxide, qunazdinyl-N-oxide, quinoxatnyl-N-oxide, phthalazinyl-N-oxide, imi- dazdinyl-N-oxide, isoxazdyl-N-oxide, oxazdyl-N-oxide, thiazdyl-N-oxide, in- ddizinyl-N-oxide, indazdyl-N-oxide, benzothiazdyl-N-oxide, benzimidazdyl- N-oxide, pyrrdyl-N-oxide, oxadiazdyl-N-oxide, thiadiazdyl-N-oxide, triazdyl- N-oxide, tetrazdyl-N-oxide, etc.
[76]
[77] d) Simple Alkyl Chain substituted by Aryl (S AC-Ar, bebw) means a straight-chain or branched alkyl which has 1 to 8 carbon atoms and is substituted by the above mentioned aryl group.
[78]
[79] e) Natural amino acid indudes the fdbwing: Glycine, Alanine, Vaϋne, Leucine, Isdeucine, Serine, Threonine, Cysteine, Methionine, Prdine, Aspartic acid, Asparagine, Gktamic acid, Glutamine, Lysine, Arginine, Histidine, Phenylalanine, Tyrosine, and Tryptophan
[80]
[81] f) The protecting group of ester is a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form.
[82]
[83] Further, the present specification indudes the fdbwing abbreviations:
[84] N-chbrosuccinimide: NCS
[85] N-methyknorphdine: NMM
[86] N,N-dimethyl formamide: DMF
[87] Dimethylsulfoxide: DMSO
[88] Triethylamine: TEA
[89] 4-(Dimethylamino)pyridine: DMAP
[90] NN-Diisopropylethylamine: DIPEA
[91 ] O-(7-Azabenzotriazd- 1 -yl)-Ν,Ν,Ν',Ν'-tetramethyluroniumhexafluorophosphate: HATU
[92] l-(3-dimethylaminopropyl)-3-ethylcarbodiimide: EDC
[93] 1-hydroxybenzotriazde hydrate: HOBt
[94] trifluoroacetic acid: TFA
[95] t-butoxy carbonyl: Boc
[96] benzybxycarbonyl: Cbz
[97] methyl: Me
[98] ethyl: Et
[99] equivalent eq
[100]
[101] The present invention relates to the compound of the fdbwing formula (1), pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof having inhibitory activity against capsizes.
[102]
Figure imgf000009_0001
[103] (1)
[104] [105] The substituents R, R , R , R , Ar and X present in the formula 1 listed above are 1 2 3 specificaly defined as fdbws. [106] [107] I) R represents simple alkyl chain (-SAC), cydoalkyl chain (-SCAC), aryl group (-Ar), alkyl chain substituted by aryl (-SAC-Ar), or hydrogen, [108] [109] II) R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of al the natural amino acids; and the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 1 becomes a stereocenter due to the R group; or the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a suϊbnamide 2 4 4 form (-CONHSO R wherein R is -SAC), or may exist in the form of pharma- 2 5 5 ceuticaly acceptable salt, when Rl is a carboxylic acid as side chain residue of an amino acid; or the compound of formula (1) may also exist in the form of pharmaceuticaly acceptable salt when R consists of a base as side chain residue of an amino acid,
[110]
[111] 111) R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of the 2 natural amino acids; and the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 2 becomes a stereocenter due to the R group; the compound of formula (1) may have a 2 protecting group in an ester form (-CO R wherein R is -SAC) or a svJfonamide form 2 6 6 (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceuticaly 2 1 1 acceptable salt, when R is a carboxylic acid as side chain residue of an amino acid; or 2 the compound of formula (1) may also exist in the form of pharmaceuticaly acceptable salt when R consists of a base as side chain residue of an amino acid, or R 2 2 fiirther represents H; -(CH ) OR wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar, and n 2 n 8 8 = 1 or 2; or -(CH ) OQ=O)R wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar, and n = 1 or 2, [112] [113] IV) R represents simple alkyl chain ( -SAC), cycbalkyl chain (-SCAC), aryl 3 group (-Ar), alkyl chain substituted by aryl (-SAC-Ar), or hydrogen, [114]
[115] V) Ar represents aryl (- Ar),
[116] 1 1
[117] VI) Rand adjacent R together may form a cyde, where -R-R - is -(CH ) -, -(CH ) - 2 n 2 n O-(CH ) -, or -(CH ) -NHR -(CH ) - wherein n+m<9 and R is -SAC, -SCAC, -Ar, - 2 m 2 n 9 2 m 9 SAC-Ar, -Q=O)-SAC, -Q=O)-SCAC, -Q=O)-Ar, or -Q=O)-SAC-Ar, [118] [119] VII) X represents -CN; -CHO; -Q=O)OR wherein R is -SAC, -SCAC, -Ar, or - 10 10 SAC-Ar; -C(=O)CH OR wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; -C(=O)CH 2 10 10 2 OQ=O)R wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-CO R wherein 10 10 2 10 R is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-SO R wherein R is -SAC, -SCAC, 10 2 10 10 -Ar, or -SAC-Ar; -CONR R wherein R and R each are -H, -SAC, -SCAC, -Ar, or 11 12 11 12 -SAC-Ar; or -COCH -W wherein W is -N , -F, -CI, -Br, -I, -NR R (R andR each 2 2 13 14 13 14 are -SAC, -SCAC, -Ar, or -SAC-Ar, or together may form cydic group), -SR (R is ■ SAC, -SCAC, -Ar, or -SAC-Ar), or is the fdbwing formula:
[120]
Figure imgf000010_0001
[121]
[122] wherein
[123] Y is H, -OH, -OR (R = -SAC or -SCAC), -C(=O)R (R = -H, -SAC, or - 16 16 17 17 SCAC), -F, -CI, -Br, -I, -CN, -N , -CO H, CF , -CO R (R = -SAC or -SCAC), - 3 2 3 2 18 18 O(=O)NHR (R = -SAC or -SCAC), or -C(=O)NR R (R and R each are -SAC, - 18 18 19 20 19 20 SCAC, -Ar, or -SAC-Ar ), [124] R is H, or -SAC 21
[125]
[126] Preferred compounds among the compound of formula (1) above are those wherein
[127] I) R represents H;
[128] [129] II) R represents -(CH ) COOH, -(CH ) COOR(R=SAC), or -(CH ) CONHSO R 1 2 2 2 2 2 2 2 22 (R = SAC); 22
[130]
[131] III) R represents H, -SAC, -Ar, or -(CH ) O R (R = -SAC, -SCAC, -Ar, or - 2 2 n m SAC-Ar, n = 0, 1 or 2, and m = 0 or 1); [132]
[133] IV) R represents -SAC, or H,
[134]
[135] V) Ar represents aryl (-Ar).
[136] [137] VI) X represents -COCH N , -COCH F, -COCH CI, -COCH Br, -COCH I, -COCH 2 2 2 2 2 2 OAr, -COCH OCOAr or -COCH SR (R is -SAC, -SCAC, -Ar or -SAC-Ar). 2 2 2 15 15
[138]
[139] Particularly preferred compounds are those selected from the fdbwing group:
[140] (1) 3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-ethyl-4-oxobutanoyl] amino }-5-fluoro-4-oxopentanoic acid ( la); [141] (2) (3S)-3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-ethyl-4-oxobutanoyl] amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid ( lb); [142] (3) (3S)-5-[(2,6-Dichbrobenzoyl)oxy]-3-{ [4-(l,3-Dimethyl-l H- indd- 2-yl)-2-ethyl-4-oxobutanoyl] amino }-4-oxopentanoic acid ( Ic); [143] (4) 3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-isopropyl-4-oxobutanoyl] amino }-5-fluo-ro-4-oxopentanoic acid (Id); [144] (5) (3S)-5-[(2,6-Dichbrobenzoyl)oxy] - 3-[(2-methyl-4-oxo-4-phenylbutanoyl)-amino]-4-oxopentanoic acid ( le); [145] (6) (3S)-5-[(2,6-Dichbrobenzoyl)oxy] - 3-[(2-isopropyl-4-oxo-4-phenylbutanoyl)-amino]-4-oxopentanoic acid ( If); [146] (7) (3S)-3- { [(2R)-2-Isopropyl-3-methyl-4-oxo-4-phenylbutanoyl] amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid ( lg); [147] (8) (3S)-3-{[2-Ethyl-4-(l-naphthyl)-4-oxobutanoyl] amino}-4-oxo-5-(2,3,5,6-tetra-fluorophenoxy)pentanoic acid (Di); [148] (9) 3-{[2-Ethyl-4-(l-isoquindinyl)-4-oxobutanoyl] amino }-5-fluoro-4-oxopentanoic acid ( Η); and [149] (10) 5-FTuoro-3-{[2-isopropyl-4-(l-isoquincfinyl)-4-oxobutanoyl] amino }-4-oxo-pentanoic acid(Ij). [150] [151] It is another obj ect of the present invention to provide a pharmaceutical composition comprising the compound of the formula 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as active ingredients. The composition according to the present invention inhibit caspases, and thus is usefii for the treatment or prevention of dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, liver diseases by hepatitis virus, acute hepatitis, fifaώiant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, or ischemic cardiac diseases. It is a fiirther object of the present invention to provide a composition for preventing inflammation and apoptosis, comprising the compound of the formula 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as active ingredient.
[152]
[153] Preferably, the composition according to the present invention may be formulated and administered as oral preparation, injection, or patch.
[154]
[155] The compound of formula 1 according to the present invention can be synthesized as shown in the fdbwing Reaction Schemes 1 and 2. However, those ilustrated in the fdbwing Reaction Schemes represent only the typical processes used in the present invention. The order of units may be modified with no limit, and so the processes are not restricted to those explained bebw.
[156]
[157] First, β-keto phosphonate (111) is synthesized from aromatic methyl ester (II) and methyl dimethyl phosphonate. The resulting compound (111) is then reacted with 2-ketoester (IV) by using the Wittig Reaction to give a 4-oxo-2-alkyl-2-butenoic ester (V). The compound (V) in the form of trans/cis mixture is hydrogenated to give a derivative (VI). If the protecting group P is needed to replace by a new substituent, P is removed and P is introduced again.
[158]
[159] Reaction Scheme 1
[160]
Figure imgf000013_0001
Figure imgf000013_0002
[161]
[162] in which
[163] VI
[164] a)Ar = 2-(l,3-DimethylJnddy;R =Et,P =Me
[165] b)Ar = 2-(l,3-DimethylJnddy;R =Et,P =Bn
[166] c)Ar = 2-(l,3-DimethylJnddy;R = -Pr,P =Et
[167] d)Ar = phenyl R =Me,P =Me
[168] e)Ar = phenyiR = -Pr, P = Me
[169] h) Ar = 1 -naphthyl, R = Et, P = Me
[170] i) Ar = 1-isoquinolinyi R = Et, P = Me
[171] j) Ar = 1-isoquinolinyi R = = *-Pr, P = Et. 2 1
[172]
[173] The compound (VI) is hydrdyzed to give a carboxylic acid derivative (VII). This obtained derivative (VII) is coupled with an aspartic acid derivative (VH1) to give a compound (IX), which is then subjected to Dess-Martin periodinane oxidation reaction, and deprotection reaction to give the desired compound of formula (1).
[174]
[175] Reaction Scheme 2
[176] Dess-Martin
Figure imgf000014_0001
Figure imgf000014_0002
[177]
[178] in which
[179] X, I
[180] a) Ar = 2-(l,3-DimethylJnddy; R = Et, W = F 2
[181] b) Ar = 2-(l,3-DimethylJnddy; R = Et, W = OPh (2,3,5,6-tetrafluoro) 2
[182] c) Ar = 2-(l,3-DimethylJnddy; R = Et, W = OCOPh (2,6-dichbro) 2
[183] d) Ar = 2-(l,3-DimethylJnddy; R = *-Pr, W = F 2
[184] e) Ar = phenyl R = Me, W = OCOPh (2,6-dichbro) 2
[185] f) Ar = phenyl, R = ?-Pr, W = OCOPh (2,6-dichbro) 2
[186] h) Ar = 1 -naphthyl, R = Et, W = OPh (2,3,5,6-tetrafluoro) 2
[187] i) Ar = 1 -isoquinolinyi R = Et, W = F
[188] j) Ar = 1-isoquinolinyi R = = ϊ-Pr, W = F.
[189]
[190] The compound (VII) is di-alkylated, into which R group is intrioduced, and then 3 the compound is reacted according to the same procedure as Reaction Scheme 2 to give a compound (lg). Alkyl ether is deprotected by t chbroacetic acid (cf: Reaction Scheme 3).
[191]
[192] Reaction Scheme 3
[193]
Figure imgf000015_0001
Figure imgf000015_0002
[194] [195] in which [196] X, I [197] g) Ar = phenyi R = *-Pr, R = Me, W = OPh (2,3,5,6-tetrafluoro) 2 3 [198] [199] The fiinctional group W in the compound (I) of Reaction Schemes 2 and 3 may be formed through several steps after the compound (VII) is combined with aspartic acid (β-t-Bu) methylester, or through reacting the compound (VIII) already having desired X with the compound (VII) (Ref.: WO 00/23421). Further, when W is F, the compound may be prepared according to a method known in Tetrahedron Letters, 1994, 55(52), 9693-9696.
[200] [201] Reaction Scheme 4 [202]
Figure imgf000016_0001
[203] [204] in which
Figure imgf000016_0002
[206] a) W = OPh (2,3,5,6-tetrafluoro) [207] b) W = OCOPh (2,6-dichbro) [208] c) W = F. [209] [210] The structures of the typical compounds synthesized according to the present invention are ilustrated bebw, to ilustrate synthetic methods of the compounds through the fobwing examples. However, it should be understood that these examples are intended to ilustrate the present invention, and cannot limit the scope of the present invention in any manner.
[211]
[212]
Figure imgf000016_0003
[213] (2)
Figure imgf000017_0001
[214] (3)
Figure imgf000017_0002
[215] (4)
[216]
Figure imgf000017_0003
[217] (6)
Figure imgf000017_0004
[219] (8)
Figure imgf000017_0005
[220] (9)
Figure imgf000018_0001
[222]
[223] Synthetic Examples and Examples showing the Binding activity
[224] Preparation 1: M ethyl l,3-Dimethyl-l/7-indol-2-carboxylate
[225]
Figure imgf000018_0002
[226] 1,3-Dimethyl- iH-indd-2-carboxylic acid (4.39 g, 23.2 mmd) was dissdved in DMF (90 mH ), and K CO (6.41 g, 2.0 eq) and Mel (9.88g, 3.0 eq) were added thereto. 2 3 The mixture was stured at room temperature for 2 hours and then concentrated under reduced pressure. The residue was extracted with ethyl acetate (50 M x 2), washed with water, saturated sodium bicarbonate sdution, and aqueous sodium chbride sdution in turn, dried (anhydrous Na SO ), concentrated under reduced pressure, and 2 4 purified by cdumn chromatography (10% ethyl acetate-hexane), to give the title compound (4.36 g, 92%) as slightly yebw sdid [227] NMR (500MHz, CDCl ) δ 7.67 (d, 1H), 7.38 (t, 1H), 7.32 (d, 1H), 7.14 (t, 1H), 3 3.92 (s, 3H), 3.89 (s, 3H), 3.80 (s, 3H), 2.66 (s, 3H) [228] [229] Preparation 2: Dim ethyl 2-(l,3-Dimethyl-lff - indol-2-yl)-2-oxoethylphospho-nate
[230]
Figure imgf000018_0003
[231] Methyl dimethyl phosphonate (2.20 g, 1.89 β , 3 eq ) was dissdved in anhydrous tetrahydrofiiran (about 40 M ) under nitrogen atmosphere, the mixture was kept at -78° C, and n-butyfithium (2.5M in hexan, 7.08 M , 3.0 eq) was added thereto. The mixture was stured at -78 ~ -60 °C for one hour. The methyl 1,3-dimethyl-l H - indd-2-carboxylate prepared in Preparation 1 (1.20 g, 5.90 mmd) was dissdved in anhydrous tetrahydrofiiran (10 M ) and was added to the mixture at -60 °C . Th e resulting mixture was stined at -60 °C for 2 hours while it was sbwly warmed to room temperature. Oxalic acid dihydrate (2.45 g, 3.3 eq) was dissdved in methand (6 M ), and then added to the mixture. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure and separated by cdumn chro- matography (50%-80% ethyl acetate/hexane) to give the title compound (1.68 g, 95%) as yebw sdid.
[232]
[233] NMR (500MHz, CDCl ) δ 7.67 (d, 1H), 7.38 (t, 1H), 7.32 (d, 1H), 7.14 (t, 1H), 3 3.94 (s, 3H), 3.82 (s, 3H), 3.80 (s, 3H), 3.68 (d, 2H), 2.67 (s, 3H) [234] [235] Preparation 3: M ethyl 4-(l,3-dimethyl-l y - indol-2-yl)-2-ethyl-4-oxo-2-butenoate
[236]
Figure imgf000019_0001
[237] At 0 °C , anhydrous tetrahydrofiiran (40 τ ) was added to sodium hydride (60% dispersion in mineral oil, 216 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere. Dim ethyl 2-(l,3-Dimethyl-l H - indd-2-yl)-2-oxoethylphosphonate prepared in Preparation 2 (1.45 g, 4.90 mmd) dissdved in anhydrous tetrahydrofiiran (20 mβ ) was added to the mixture. The resulting mixture was stined for about 10 minutes. Lithium chbride (415 mg, 2.0 eq) was added thereto and the mixture was fiirther stined at 0 °C for about 10 minutes. Methyl 2-oxobutylate (630 mg, 1.1 eq) dissdved in anhydrous tetrahydrofiiran (10 mβ ) was added threrto, and stined at 0 °C for 30 minutes and at room temperature for 2 hours. The reaction was completed with saturated ammonium chbride sdution, and then extracted twice with ethyl acetate (100 mβ ). The filtrate was washed with water, saturated sodium bicarbonate sdution (NaHCO , 50 M x 2), and saturated sodium 3 chbride sdution in turn, dried (anhydrous Na SO ), and concentrated under reduced 2 4 pressure . The residue was separated by cdumn chromatography (10%-20% ethyl acetate/hexane) to give the title compound (1.17 g, 83%, cis/trans = 3:1) as yebw liquid. [238]
[239] Trans isomer
[240] NMR (500MHz, CDCl ) δ 7.67 (d, IH), 7.56 (s, IH), 7.39 (t, IH), 7.34 (d, IH), 3 7.14 (t, IH), 3.98 (s, 3H), 3.86 (s, 3H), 2.65 (qt, 2H), 2.55 (s, 3H), 1.14 (t, 3H) [241]
[242] Cis isomer
[243] NMR (500MHz, CDCl ) δ 7.65 (d, IH), 7.37 (t, IH), 7.32 (d, IH), 7.13 (t, IH), 3 6.67 (s, IH), 3.93 (s, 3H), 3.67 (s, 3H), 2.54 (s, 3H), 2.51 (qt, 2H), 1.20 (t, 3H) [244] [245] Preparation 4: Methyl 4-(l,3-dimethyl-l y - indol-2-yl)-2-ethyl-4-oxo-butanoate (VIa)
[246]
Figure imgf000020_0001
[247] The compound prepared in Preparation 3 (1.16 g, 4.06 mmd) was dissdved in ethyl acetate (30 mβ ), and Pd/C (400 mg, 10%, Aldrich) was added thereto. The resulting mixture was reacted by using Parr Hydrogenater under 40 psi of hydrogen atmospheric pressure for 6 hours. The reaction was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography (10% ethyl acetate/hexane) to give the title compound (1.00 g, 94%) as white solid.
[248]
[249] NMR (500MHz, CDCl ) δ 7.67 (d, IH), 7.36 (t, IH), 7.32 (d, IH), 7.18 (t, IH), 3 3.93 (s, 3H), 3.72 (s, 3H), 3.41 (dd, IH), 3.08-3.00 (m, 2H), 2.66 (s, 3H), 1.80-1.64 (m, 2H), 0.99 (t, 3H) [250] [251] Preparation 5: 4-(l^-Dimethyl-l /-indol-2-yl)-2-ethyl-4-oxobutanoic acid
[252]
Figure imgf000020_0002
[253] The compound prepared in Preparation 4 (500 mg, 1.74 mmd) was dissdved in the mixture of distiled tetrahydrofiiran (1 0 ml ) and MeOH (5 mβ ). IN NaOH (5.2 πtβ , 3.0 eq) was added thereto, and the mixture was stined for one day. T he mixture was neutralized by IN HC1 and disuled under reduced pressure to remove most of tetrahydrofiiran. The residue was dissdved in excess ethyl acetate (50 M ), washed with saturated sodium chbride sdution, dried (anhydrous Na SO ), and concentrated 2 4 under reduced pressure to give the title compound (500 mg, quantitative) as slightly yebw powder. This compound was used in the next reaction without any fiirther purification.
[254]
[255] MS: [M+H] 274
[256]
[257] Preparation 6: Benzyl 4-(l,3-Dimethyl-l/y - indol-2-yl)-2-ethyl-4-oxobutanoate (VIb)
[258]
Figure imgf000021_0001
[259] The compound prepared in Preparation 5 (360 mg, 1.32 mmd) was dissdved in DMF (5 M ), K CO (365 mg, 2.0 eq) and benzyl bromide (271 mg, 0.19 m£ , 1.2 eq) 2 3 were added thereto, and the mixture was stined at room temperature for 4 hours. The resulting mixture was extracted with ethyl acetate, and the filtrate was washed with water, saturated sodium bicarbonate, amd saturated sodium chbride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure. The residue was 2 4 separated by column chromatography (5%-10% ethyl acetate/hexan) to give the title compound (350 mg, 73%) as slightly yebw liquid. [260] [261] NMR (400MHz, CDCl ) δ 7.68 (d, IH), 7.38-7.30 (m, 7H), 7.16 (t, IH), 5.17 3 (Abq, 2H), 3.90 (s, 3H), 3.44 (dd, IH), 3.1 l(m, IH), 3.03 (m, IH), 2.66 (s, 3H), 1.84-1.68 (m, 2H), 0.99 (t, 3H) [262] [263] Preparation 7: Chiral Resolution and Deprotection of benzyl 4-(l^-dimethyl-lff - indol-2-yl)-2-ethyl-4-oxobutanoate [264] [265] Each enantiomer was separated by Chiral OD Cdumn (Daicel Chemical Industries, 2.00cm x 25cm, OD00CJ-1C005, 3% i-PrOH in Hexane, 220nm). The protecting group of the separated enantiomer was removed under hydrogen babon to give the title compound (quantitative) as chiral title compound.
[266]
[267] MS: [M+H] 274
[268]
[269] Preparation 8: tert- tγl 3-{[4-(l,3-dimethyl-l y - indol-2-yl)-2-ethyl-4-oxo-butanoyl]amino}-5-fluoro-4-oxopentanoate (Xa)
[270]
Figure imgf000022_0001
[271] The carboxylic acid derivative prepared in Preparation 5 or Preparetion 7 (237 mg, 0.87 mmd) and amino alcohd derivative VHlc [Ref: Tetrahedron Letters, 1994, 35(52), 9693, 200 mg, 1.1 eq] were reacted in DMF sdvent according to the common method using HATU (430 mg, 1.3 eq) and TEA (264 mg, 0.36 τ , 3.0 eq), which was then subjected to the workup. The residue was purified by cdumn chromatography (25%-30%-40% EA Hex) to give a compound (250 mg, 62%). To this compound and Dess-Martin reagent (687 mg, 3.0 eq) was added dry dichbromethane (4 mβ ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (3 mβ ). After the common treatment, the reaction was purified by cdumn chromatography (36% ethyl acetate-hexane) to give the diastereomeric title compound (180 mg, 72%).
[272]
[273] NMR (500MHz, CDCl ) δ 7.69 (d, IH), 7.39 (t, IH), 7.34 (m, IH), 7.16 (t, IH), 3 6.89 & 6.85 (two d, IH), 5.47-5.05 (m, 2H), 4.92-4.84 (two m, IH), 3.96-3.94 (two s, 3H), 3.53-3.41 (m, IH), 3.08-2.97 (m, 2H), 2.86-2.68 (m, 2H), 2.68 & 2.67 (two s, 3H), 1.80 (m, IH), 1.63 (m, IH), 1.46 (s, 9H), 1.02 (m, 3H) [274] [275] Example 1: 3-{[(2Λ or 2S)-4-(l,3-Dimethyl-l y - indol- 2-yl)-2-ethyl-4-oxobutanoyl]amino}-5-fluoro-4-oxopentanoic acid (Ia-1 or Ia- 2) [276]
Figure imgf000023_0001
[277] The compound prepared in Preparation 8 (44 mg) was dissdved in dichbromethane (2 τ ), and TFA (1 τ ) was added thereto at 0 °C . Th e resulting mixture was stined for 2 hours while it was sbwly warmed to room temperature and concentrated under reduced pressure to give the title compound (Ia-1, Ia-2) quantitatively.
[278]
[279] NMR (400MHz, DMSO-d ) δ 8.64 & 8.52 (two br s, IH), 7.73 (d, IH), 7.49 (d, 6 IH), 7.34 (t, IH), 7.11 (t, IH), 5.16 (br, 2H), 4.61 & 4.46 (two m, IH), 3.84 & 3.83 (two s, 3H), 3.25 (m, IH), 2.99 (m, IH), 2.88-2.60 (m, 3H), 2.60 & 2.59 (two s, 3H), 1.54 (m, 2H), 0.90 (m, 3H) [280] MS: M+ H 405
[281] [282] Preparation 9: tert-B ty\ (35)-3-{[(benzyloxy)carbonyl] amino}-4-hydroxy-5-(2,3,5,6-tetrafluorophenoxy)pentanoate (Xllla) [283]
Figure imgf000023_0002
[284] To N-benzybxycarbonyl-β-t-butylaspartic acid (18.5 g, 55.7 mmd) and NMM (6.78 mil , 1.1 eq) was added anhydrous tetrahydrofiiran (180 M ) under nitrogen atmosphere. The reaction solution was kept at -15 °C . Isobutyl chbroformate (7.63 M , 1.05 mmd) was added thereto, and the mixture was stined for about 20 minutes. To the reaction mixture kept at 0 °C was added diazomethane-ether solution (synthesized from 2.0 eq of l-methyl-3-nitro-l-nitroso-guanidine, 60 M ) to prepare diazoketone derivative (-30 minutes). 30% HBr/AcOH (23.3 m£ , 2.0 eq) was added thereto to prepare bromomethylketone derivative (30-60 minutes). The compound was extracted with ethyl acetate, washed twice with saturated aqueous sodium bicarbonate sdution, and once with aqueous sodium chbride sdution, dried (anhydrous Na SO ), and con- 2 4 centrated under reduced pressure, to give bromomethyl ketone derivative (23 g). [285]
[286] The obtained bromomethyl ketone derivative (23 g, 55.7 mmd) and 2,3,5,6-tetrafluorophend (10.2 g, 1.1 eq) were dissdved in dimethylformamide (150 M ), KF (8.14 g, 2.5 eq) was added thereto, and the mixture was stined at room temperature for two hours, and treated by the common method, to give a 2,3,5,6-tetrafluorophenoxy methyl ketone derivative. This compound was dissdved in methand (150 τd ) and reacted by adding NaBH (4.24 g) sbwly (0 °C -room 4 temperature, 2 hours). The reaction was stopped by acetic acid, and the reaction sdution was disuled under reduced pressure to remove methand. The residue was extracted with ethyl acetate (200 M x 2), washed with water and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and 2 4 separated-purified by column chromatography (15% ethyl acetate/hexane), to give the title compound (20.2 g, 74%) in diastereomeric form. [287] [288] Preparation 10: t-Butyl (3S) - 3-amino-4-hydroxy-5-(2,3,5,6-tetrafluorophen-oxy)pentanoate (Villa)
[289]
Figure imgf000024_0001
[290] The compound prepared in Preparation 9 was dissdved in methand (300 M ) and Pd/C (10%, 1.50 g) was added thereto. The benzybxycarbonyl group of the compound was removed (Pd/C) under hydrogen babon for 3 hours to give the title compound (95%).
[291]
[292] H-NMR (400 MHz, DMSO-d ) δ 8.2 (br, 2H), 7.6-7.5(m, IH), 5.9(m, IH), 6 4.3-4.1(m, 3H), 3.6(m, IH), 2.7(m, IH), 1.4(s, 9H) [293] [294] Preparation 11: tert-B tγl (35)-3-{[4-(l,3-dimethyl-lff - indol-2-yl)-2-ethyl-4-oxobutanoyl]amino}-4-oxo-5-(2^ ,6-tetrafluorophenoxy)pen tanoate (Xb) [295]
Figure imgf000025_0001
[296] The carboxylic acid derivative prepared in Preparation 5 (152 mg, 0.557 mmd) and the amino alcohd derivative Villa prepared in Preparation 10 (216 mg, 1.1 eq) were reacted in DMF sdvent (2 M ) according to the common method by using HATU (275 mg, 1.3 eq) and TEA (169 mg, 0.23 τd , 3.0 eq), which was then subjected to the workup. The residue was purified by cdumn chromatography (20%-30%-40% EA/Hex) to give a compound (246 mg, 80%). To this compound and Dess-Martin reagent (502 mg, 3.0 eq) was added dry dichbromethane (5 M ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (1 β ). After the common treatment, the reaction was purified by cdumn chromatography (20%-25% ethyl acetate-hexane) to give the diastereomeric title compound (183 mg, 76%).
[297]
[298] NMR (500MHz, CDCl ) δ 7.66 (d, IH), 7.36 (t, IH), 7.30 (d, IH), 7.13 (t, 3 1H),6.95 & 6.88 (two d, IH), 6.71 & 6.63 (two m, IH), 5.43-5.06 (two set of AB, 2H), 4.93 & 4.84 (m, IH), 3.45 (two set of dd, IH), 3.08-2.94 (m, 2H), 2.85-2.65 (m, 2H), 2.65 &2.64 (two s, 3H), 1.80 (m,lH), 1.61 (m, IH), 1.45 & 1.44 (two s, 9H), 1.01 & 0.99 (two t, 3H).
[299]
[300] Example 2: (3S)-3-{[4-(l,3-Dimethyl-l y - indol- 2-yl)-2-ethyl-4-oxobutanoyl]-amino} - 4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (lb)
[301]
Figure imgf000025_0002
[302] The compound prepared in Preparation 11 (180 mg) was dissdved in dichbromethane (4 τd ), and TFA (2 τd ) was added thereto at 0 °C . Th e resulting mixture was stined for 2 hours while it was sbwly warmed to room temperature and concentrated under reduced pressure to give the title compound (160 mg, 98%).
[303] [304] NMR (500MHz, DMSO-d ) δ 12.50 (br, IH), 8.74-8.59 (two d, IH), 7.73 (d, IH), 6 7.52-7.44 (m, 2H), 7.35 (t, IH), 7.12 (t, IH), 5.38-5.13 (m, 2H), 4.67 & 4.47 (two m, IH), 3.82 & 3.79 (two s, 3H), 3.28 (m, IH), 3.00 (m, IH), 2.87-2.72 (m, 2H), 2.59 (two s, 3H), 2.55 (m,lH), 1.54 (m, 2H), 0.88 (two t, 3H) [305] MS: [M+H] 551
[306] [307] Preparation 12: ( 3S)-3-{[(Benzyloxy)carbonyl] amino}-5-(t-butoxy)-2-hydroxy-5-oxopentyl-2,6-dichlorobenzoate (Xlllb) [308]
Figure imgf000026_0001
[309] To N-benzybxycarbonyl-β-t-butylaspartic acid (5.03 g, 15.6 mmd) and NMM (1.90 M , 17.1 mmd) was added anhydrous tetrahydrofiiran (60 M ) under nitrogen atmosphere. The reaction solution was kept at -15 °C . Isobutyl chbroformate (2.12 M , 16.3 mmd) was added thereto, and the mixture was stined for about 20 minutes. To the reaction mixture kept at 0 °C was added diazomethane-ether solution (synthesized from 2.0 eq of l-methyl-3-nitro-l-nitroso-guanidine, 60 M ) to prepare diazoketone derivative (-30 minutes). 30% HBr/AcOH (6.42 m£ , 2.0 eq) was added thereto to prepare bromomethylketone derivative (30-60 minutes). The compound was extracted with ethyl acetate, washed twice with saturated aqueous sodium bicarbonate sdution and once with aqueous sodium chbride sdution, dried (anhydrous Na SO ), and con- 2 4 centrated under reduced pressure, to give bromomethyl ketone derivative (6.4 g).
[310]
[311] The obtained bromomethyl ketone derivative (4.36 g) and 2,6-dichbrobenzoic acid (2.28 g, 1.1 eq) were dissdved in dimethylformamide (18 rd ), KF (1.58 g, 2.5 eq) was added thereto, and the mixture was stined at room temperature for two hours and treated by the common method to give 2,6-dichbrobenzoxy methyl ketone derivative. This compound was dissdved in methand (20 M ) and reacted by adding NaBH (412 4 mg)-methand sdution (40 M ) (0 °C -room temperature, 2 hours). The reaction was stopped by acetic acid, and the reaction sdution was distiled under reduced pressure to remove methand. The residue was extracted with ethyl acetate (50 M x 2), washed with water and aqueous sodium chbride sdution, dried (anhydrous Na SO ), con- 2 4 centrated under reduced pressure, and separated-purified by column chromatography (ethyl acetate-hexane, 1:5), to give the title compound (4.80 g, 86%) in diastereomeric form.
[312]
[313] H-NMR (400 MHz, CDCl ) δ 7.3-7.2 (m, 8H), 5.9(m, IH), 5.2(m, 4H), 4.7(m, 3 IH), 2.9(m, IH), 2.7(m, IH), 1.4(s, 9H) [314] [315] Preparation 13: (3S)-3-Amino-5-(t-butoxy)-2-hydroxy-5-oxopentyl-2,6-dichlorobenzoate (Vlllb)
[316]
Figure imgf000027_0001
[317] The benzybxycarbonyl group of the compound prepared in Preparation 12 was removed (Pd/C) under hydrogen babon for 40 minutes to give the title compound (100%).
[318]
[319] H-NMR (400 MHz, DMSO-d ) δ 8.2(br, 2H), 7.6-7.5(m, 3H), 6.1(m, IH), 6 4.4-3.9(m, 3H), 3.0-2.6(m, 2H), 1.4(s, 9H) [320] [321] Preparation 14: (3S)-5-(tert -Butoxy )-3-{[4-(l,3-dimethyl-lff - indol- 2-yl)-2-ethyl-4-oxobutanoyl]amino}-2^-dioxopentyl-2,6-dichlorobenzoate (Xc) [322]
Figure imgf000027_0002
[323] The carboxylic acid derivative prepared in Preparation 5 or Preparation 7 (250 mg, 0.87 mmd) and the amino alcohd derivative Vlllb prepared in Preparation 13 (398 mg, 1.1 eq) were reacted in DMF sdvent according to the common method using HATU (430 mg, 1.3 eq) and TEA (264 mg, 0.36 τd , 3.0 eq), which was then subjected to the workup. The residue was purified by cdumn chromatography (25%-30%-40% EA/Hex) to give a compound (530 mg, 96%). To this compound and Dess-Martin reagent (10.6 g, 3.0 eq) was added dry dichbromethane (4 M ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (1 β ). After the common treatment, the reaction was purified by cdumn chromatography (36% ethyl acetate-hexane) to give the diastereomeric title compound (289 mg, 55%).
[324]
[325] NMR (500MHz, CDCl ) δ 7.69 (d, IH), 7.40-7.27 (m, 5H), 7.14 (m, IH), 7.03- 3 6.69 (two d, IH), 5.51-5.18 (two set of ABq, 2H), 5.00 & 4.90 (m, IH), 3.95 &3.93 (two s, 3H), 3.53-3.43 (m, IH), 3.08-2.96 (m, 2H), 2.88-2.70 (m, 2H), 2.68 &2.66 (two s, 3H), 1.84 (m,lH), 1.66 (m, IH), 1.47 & 1.45 (two s, 9H), 1.06 & 1.02 (two t, 3H)
[326]
[327] Example 3: (3S)-5-[(2,6-Dichlorobenzoyl)oxy]-3-{[4-(l,3-dimethyl-l H - indol-2-yl)-2-ethyl-4-oxobutanoyl]amino} - 4-oxopentanoic acid (Ic)
[328]
Figure imgf000028_0001
[329] The compound prepared in Preparation 14 (280 mg) was dissdved in dichbromethane (4 τd ), and TFA (2 τd ) was added thereto at 0 °C . Th e resulting mixture was stined for 2 hours while it was sbwly warmed to room temperature and concentrated under reduced pressure to give the title compound (quantitative).
[330]
[331] NMR (500MHz, DMSO-d ) δ 12.46 (br, IH), 8.75-8.62 (two br, IH), 7.74 (two d, 6 IH), 7.61-7.53 (m, 3H), 7.49 (two d, IH), 7.34 (t, IH), 7.12 (t, IH), 5.43-5.05 (two br, 2H), 4. 76 & 4.55 (two m, IH), 3.85 (two s, 3H), 3.29 (m, IH), 3.04 (m, IH), 2.92-2.73 (m, 2H), 2.61 (two s, 3H), 2.61 (m, IH), 1.58 (m, 2H), 0.92 (two t, 3H)
[332] MS: M+H 575
[333]
[334] Preparation 15: Ethyl 4-(l,3-dimethyl-l y - indol- 2-yl)-2-isopropyl-4-oxo-2-butenoate
[335]
Figure imgf000028_0002
[336] At 0 °C , anhydrous tetrahydrofiiran (10 rd ) was added to sodium hydride (60% dispersion in mineral oil, 34 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere. Dim ethyl 2-(l,3-dimethyl-l H - indd-2-yl)-2-oxoethylphosphonate (230 mg, 0.78 mmd) prepared in Preparation 2 dissdved in anhydrous tetrahydrofiiran (5 M ) was added to the mixture. The resulting mixture was stined for about 10 minutes. Lithium chbride (66 mg, 2.0 eq) was added thereto and the mixture was fiirther stined at 0 °C for about 10 minutes. Ethyl - 3-methyl 2-oxobutylate (124 mg, 0.13 M , 1.1 eq) dissdved in anhydrous tetrahydrofiiran (3 M ) was added thererto, and stined at 0 °C for 30 minutes and at room temperature for 2 hours. The reaction was completed by saturated ammonium chbride sdution, and then the reaction was extracted twice with ethyl acetate (30 M ). The filtrate was washed with water, saturated sodium bicarbonate solution, and aqueous sodium chbride sdution in turn, dried (anhydrous Na SO ), and concentrated 2 4 under reduced pressure . The residue was separated by cdumn chromatography (10%-20% ethyl acetate/hexane) to give the title compound (241 mg, 99%, cis/trans = 235 mg:7 mg) as yebw liquid.
[337]
[338] Trans isomer
[339] NMR (500MHz, CDCl ) δ 7.66 (d, IH), 7.38 (t, IH), 7.33 (d, IH), 7.32 (s, IH), 3 7.13 (t, IH), 4.29 (qt, 2H), 3.99 (s, 3H), 3.28 (septet, IH), 2.56 (s, 3H), 1.34 (t, 3H), 1.22 (d, 6H) [340]
[341] Cis isomer
[342] NMR (500MHz, CDCl ) δ 7.66 (d, IH), 7.37 (t, IH), 7.32 (d, IH), 7.14 (t, IH), 3 6.64 (s, IH), 4.18 (qt, 2H), 3.92 (s, 3H), 2.83 (septet, IH), 2.56 (s, 3H), 1.21 (d, 6H), 1.19 (t, 3H) [343] [344] Preparation 16: Ethyl 4-(l,3-dimethyl-Lff - indol-2-yl)-2-isopropyl-4-oxo-butanoate (Vic) [345]
Figure imgf000029_0001
[346] The compound prepared in Preparation 15 (230 mg, 0.734 mmd) was dissdved in ethyl acetate (10 mβ ), and Pd/C (60 mg, 10%, Aldrich) was added thereto. The re suiting mixture was reacted by using Parr Hydrogenater under 40 psi of hydrogen atmospheric pressure for one day. The reaction was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was separated by column chromatography (10% ethyl acetate/hexane) to give the title compound (151 mg, 65%) as solid.
[347] [348] NMR (500MHz, CDCl ) δ 7.69 (d, IH), 7.37 (t, IH), 7.33 (d, IH), 7.15 (t, IH), 3 4.19 (m, 2H), 3.94 (s, 3H), 3.45 (dd, IH), 3.00-2.95 (m, 2H), 2.67 (s, 3H), 2.09 (septet, IH), 1.28 (t, 3H), 1.03 (d, 3H), 1.02 (d, 3H)
[349] [350] Preparation 17: tert -Butyl- 3-{[4-(l^-dimethyl-lff - indol- 2-yl)-2-isopropyl-4-oxobutanoyl]amino}-5-fluoro-4-oxopentanoate (Xd)
[351]
Figure imgf000030_0001
[352] The title compound was obtained according to the same procedure as Preparation
[353] [354] NMR (500MHz, CDCl ) δ 7.69 (d, IH), 7.37 (t, IH), 7.34 (d, IH), 7.15 (t, IH), 3 6.84 & 6.78 (two d, IH), 5.49-5.05 (m, 2H), 4.90 & 4.81 (two m, IH), 3.94 &3.92 (two s, 3H), 3.54-3.43 (m, IH), 3.06-2.96 (m, 2H), 2.81-2.65 (m, 2H), 2.69 &2.67 (two s, 3H), 2.00 (m,lH), 1.47 & 1.46 (two s, 9H), 1.05 (m, 6H)
[355] [356] Example 4: 3-{[4-(l,3-Dimethyl-l /-indol-2-yl)-2-isopropyl-4-oxobutanoyl] amino}-5-fluoro-4-oxopentanoic acid (Id)
[357]
Figure imgf000030_0002
[358] The compound prepared in Preparation 17 was reacted according to the same procedure as Example 1 to give the title compound.
[359] [360] NMR(500MHz, CDCl ) δ 7.69 (d, IH), 7.39 (d, IH), 7.34 (t, IH), 7.11 (t, IH), 3 4.85-4.67 (two m, IH), 4.70-4.25 (m, 2H), 3.90 (three s, 3H), 3.42 (m, IH), 3.07 (m, IH), 2.90-2.76 (m, 3H), 2.67 (three s, 3H), 1.96 (septet, IH), 1.03 (m, 6H)
[361] MS [M+H] 419
[362]
[363] Preparation 18: Methyl 2-methyl-4-oxo-4-phenyl-2-butenoate
[364]
Figure imgf000031_0001
[365] Methyl benzoate was reacted according to the same procedure as Preparation 2 to prepare a phosphate compound. The obtained compound was reacted according to the same procedure as Preparation 3 to give the title compound, except that methyl pyruvate was used instead of methyl 2-oxobutylate.
[366]
[367] Trans isomer
[368] NMR (400MHz, CDCl ) δ 7.9 (m, 2H), 7.7 (m, IH), 7.5 (m, IH), 7.4 (m, 2H), 3.8 3 (s, 3H), 2.1 (s, 3H) [369]
[370] Cis isomer
[371] NMR (400MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 6.7 (m, IH), 3.6 3 (s, 3H), 2.1 (s, 3H) [372] [373] Preparation 19: Methyl 2-methyl-4-oxo-4-phenylbutanoate (VId)
[374]
Figure imgf000031_0002
[375] The cis/trans mixture prepared in Preparation 18 (220 mg, 1.1 mmd) and Pd/C (50 mg, 5%) were dissdved in ethyl acetate (3 M ), which was stined under hydrogen babon atmospheric pressure for 6 hours. The mixture was filtrated through Celite under reduced pressure to remove sdid, and the filtrate was extracted with ethyl acetate (20 M x 2), and distiled under reduced pressure, to give the title compound (200 mg, 88%) as sdid.
[376] [377] NMR (400 MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 3.7 (s, 3H), 3.4 3 (m, IH), 3.1 (q, IH), 3.0 (m, IH), 1.2 (d, J = 7.2Hz, 3H) [378] [379] Praparation 20: 2-Methyl-4-oxo-4-phenylbutanoic acid
[380]
Figure imgf000032_0001
[381] The compound prepared in Preparation 19 (VId, 1.1 g, 5.38 mmd) was dissdved in the distiled tetrahydrofiiran (12 0 M ), and IN aqueous sodium hydroxide sdution (8.1 mil , 1.5 eq) was added thereto. After about 4 hours, t he mixture was neutralized by IN aqueous HC1 solution and distiled under reduced pressure to remove most of tetrahydrofiiran. The residue was dissdved in excess ethyl acetate (>700 M ), washed with water, dried (anhydrous Na SO ), and concentrated under reduced pressure, to 2 4 give the title compound (1.03 g, 99%) as white powder. This compound was used in the next reaction without any fiirther purification. [382] [383] NMR (400MHz, DMSO-d ) δ 12.1 (br, IH), 7.9 (d, J = 4Hz, 2H), 7.6 (m, IH), 7.5 6 (m, 2H), 3.4(m, IH), 3.0 (m, IH), 2.8 (m, IH), 1.1 (d, J = 8Hz, 3H) [384] [385] Preparation 21: (3S)-5-(t-Butoxy)-3-[(2-methyl-4-oxo-4-phenylbutanoyl)-amino]-2^-dioxopentyl-2 ,6-dichlorobenzoate (Xe) [386]
Figure imgf000032_0002
[387] A mixture of the carboxylic acid derivative prepared in Preparation 20 (1.03 g, 5.38 mmd), amino alcohd derivative (VlUb, 2.68 mg, 1.2 eq), and HATU (2.86 g, 1.4 eq) was coded to 0 °C , triethylamine (3.0 M , 4.0 eq) was added thereto in a sdvent of DMF(30 M ), and the resulting mixture was reacted for 5 hours. The sdvent was distiled under reduced pressure, and the residue was extracted with ethyl acetate (300 M x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and 2 4 purified by cdumn chromatography (30% ethyl acetate/hexane), to give a compound (2.35 g, 79%). To this compound and Dess-Martin reagent (5.41g, 3.0 eq) was added dry dichbromethane (50 mil ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (6 ιβ ). The mixture was filtrated through Celite under reduced pressure to remove sdid, and the filtrate was extracted with ethyl acetate (50 mil x 2), washed with water, saturated aqueous sodium bicarbonate sdution, and aqueous sodium chbride sdution, dried (anhydrous Na SO 2 4 ), concentrated under reduced pressure, and preliminarily purified by column chromatography (36% ethyl acetate-hexane), to give the diastereomeric title compound (2.11 g).
[388]
[389] NMR (400MHz, CDCl ) δ 7.9 (t, J = 8Hz, 2H), 7.6 (m, IH), 7.5 (m, 2H), 7.4-7.2 3 (m, 3H), 6.9 (IH, NH), 5.3-5.1 (m, IH), 4.9 (m, IH), 3.0 (m, 2H), 2.7 (m, IH), 1.4 (s, 9H), 1.3 (m, 3H) [390] [391] Example 5: (3S)-5-[(2,6-Dichlorobenzoyl)oxy] - 3- [(2-methyl-4-oxo-4-phenyl-butanoyl)amino] -4-oxopentanoic acid (le) [392]
Figure imgf000033_0001
[393] The compound prepared in Preparation 21 (190 mg) was dissdved in dichbromethane (2 mil ), and TFA (1 mil ) was added thereto at 0 °C . The mixture was stined for 2 hours while it was sbwly warmed to room temperature, and then concentrated under reduced pressure. This compound was dissdved in distiled tetrahydrofiiran (3 mil ), and IN aqueous sodium carbonate sdution (0.34 mil , 1.0 eq) was added thereto. After about 1 hour, t he mixture was neutralized by IN aqueous HC1 sdution and distiled under reduced pressure to remove most of tetrahydrofiiran. The residue was dissdved in excess ethyl acetate (10 mil ), washed with water, dried (anhydrous Na SO ), and concentrated under reduced pressure, to give the title 2 4 compound (le) quantitatively.
[394] [395] NMR (400MHz, CDCl ) δ 8.1 (t, J = 8Hz, 2H), 7.7 (m, IH), 7.6 (m, 2H), 7.5-7.4 3 (m, 3H), 6.9 (IH, NH), 5.4-5.2 (m, IH), 5.0 (m, IH), 3.2 (m, 2H), 2.8(m, IH), 1.3 (m, 3H) [396] MS [M+Na]+ 516
[397]
[398] Preparation 22: Methyl (E)-2-isopropyl-4-oxo-4-phenyl-2-butenoate
[399]
Figure imgf000034_0001
[400] Methyl benzoate was reacted according to the same procedure as Preparation 2 to prepare a phosphate compound. The obtained compound was reacted according to the same procedure as Preparation 3 to give the title compound, except that methyl 3-methyl-2-oxobutylate was used instead of methyl 2-oxobutylate.
[401]
[402] NMR (400MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 6.7 (s, IH), 3.6 3 (s, 3H), 2.8 (m, IH), 1.2 (d, J = 6.8Hz, 6H) [403]
[404] Preparation 23: Methyl 3-isopropyl-4-oxo-4-phenylbutanoate (Vie)
[405]
Figure imgf000034_0002
[406] The compound prepared in Preparation 22 was reacted according to the same procedure as Preparation 19 to give the title compound. [407] [408] NMR (500MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 3.6 (s, 3H), 3.4 3 (m, IH), 3.0-2.9 (m, IH), 2.9 (m, IH), 2.0 (m, IH), 0.9 (m, 6H) [409] [410] Preparation 24: (3S)-5-(t-Butoxy)-3-[(2-isopropyl-4-oxo-4-phenylbutanoyl)-amino]-2,5-dioxopenty 1-2,6-dichlorobenzoate (Xf) [411]
Figure imgf000035_0001
[412] The compound prepared in Preparation 23 was reacted according to the same procedure as Preparation 20 to give a compound, which was reacted according to the same procedure as Preparation 14 to give the title compound.
[413]
[414] NMR (500MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 7.3 (m, 3H), 6.9 3 (IH, NH), 5.4-4.9 (m, 2H), 4.8 (m, IH), 3.6 (m, IH), 3.1 (m, IH), 2.9 (m, IH), 2.7 (m, IH), 2.6 (m, IH), 2.0 (m, IH), 1.4 (s, 9H), 0.9 (m, 6H) [415] [416] Example 6: (3S)-5-[2,6-Dichlorobenzoyl]oxy] - 3-[(2-isopropyl-4-oxo-4-phenyl-butanoyl)amino]-4-oxopentanoic acid (If; di- astereomeric mixture) [417]
Figure imgf000035_0002
[418] The compound prepared in Preparation 24 was reacted according to the same procedure as Example 5 to give the title compound. [419] [420] NMR (400MHz, CDCl ) δ 7.9 (m, 2H), 7.5 (m, IH), 7.4 (m, 2H), 7.3 (m, 3H), 6.9 3 (IH, NH), 5.4-4.9 (m, 2H), 4.8 (m, IH), 3.6 (m, IH), 3.1 (m, IH), 2.9 (m, IH), 2.7 (m, IH), 2.6 (m, IH), 2.0 (m, IH), 0.9 (m, 6H) [421]
[422] If (single diastereomer)
[423] (2S)-2-Isopropyl-4-oxo-4-phenylbutanoic acid which is chiral carboxylic acid derivative (J. Med. Chem. 1998, 41, 2461-2480) was reacred according to the same procedure as the above to give the chiral compound (If). [424] [425] Preparation 25: (IR, 3Z)-2-Isopr opyl- 4-methoxy-3-methyl-4-phenyl-3-butenoic acid
[426]
Figure imgf000036_0001
[427] The chiral carboxylic acid derivative mentioned in Example 6 (160 mg, 0.726 mmd, J. Med. Chem. 1998, 41, 2461-2480) was dissdved in THF (3 mH ), and NaH (60% dispersion in mineral oil, 73 mg, 2.5 eq) was added thereto at -78 °C . The temperature was raised to -40 °C and then coded to -78 °C again. Mel (0.18 mil , 4.0 eq) was added thereto at the same temperature, and then the resulting mixture was stined for 3 hours while it was sbwly warmed to room temperature. The reaction was stopped by 0.5N aqueous HC1 sdution. The reaction mixture was extracted with ethyl acetate (10 mil ), washed with water, dried (anhydrous Na SO ), concentrated under 2 4 reduced pressure, to give the title compound (130 mg, 71%). [428] [429] Preparation 26: tert -Butyl (35)-3-{[(2Λ, 3Z )-2-isopropyl-4-methoxy-3-methyl-4-phenyl-3-butenoyl]amino}-4-oxo-5-(2,3,5,6-te trafluorophenoxy)pentanoate [430]
Figure imgf000036_0002
[431] The compound prepared in Preparation 25 (35 mg, 0.14 mmd) and amino alcohd derivative Villa (60 mg, 1.2 eq) were reacted in DMF sdvent according to the common method using HATU (80 mg, 1.5 eq) and TEA (57 mg, 0.078 vd , 4.0 eq), which was then subjected to the workup. The residue was purified by cdumn chromatography (20% EA/Hex) to give a compound (20.3 mg, 25%). To this compound and Dess-Martin reagent (44 mg, 3.0 eq) was added dry dichbromethane (1 mil ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (0.05 mil ). After the common treatment, the reaction was purified by Prep TLC (25% ethyl acetate-hexane) to give the diastereomeric title compound (14 mg, 69%).
[432] [433] Preparation 27: t-Butyl (3S)-3-{[(2R)-2-isopropyl-3-methyl-4-oxo-4-phenylbutanoyl]amino}-4-oxo-5-(2,3,5 ,6-tetrafluorophenoxy)pentanoate (lg)
[434]
Figure imgf000037_0001
[435] The compound prepared in Preparation 26 (14 mg, 0.024 mmd) was dissdved in CH CI (1 mil ), and trichbroacetic acid (11.8 mg, 3.0 eq)/CH CI sdution was added 2 2 2 2 thereto at 0 °C . The resulting mixture was stined for 3 hours while it was sbwly warmed to room temperature. The mixture was distiled under reduced pressure, and the residue was extracted with ethyl acetate (10 mil ), washed with water, dried (anhydrous Na SO ), concentrated under reduced pressure, and purified by Prep TLC 2 4 (33% ethyl acetate-hexane), to give the title compound (7 mg, 49%).
[436]
[437] NMR (500MHz, CDCl ) δ 8.0 (m, IH), 7.5 (m, 2H), 7.3 (m, 2H), 6.8 (m, IH), 5.3 3 (m, IH), 5.1 (m, IH), 4.5-4.3 (m, IH), 3.0-2.9 (m, IH), 2.6-2.4 (m, IH), 2.3 (m, 2H), 1.4 (s, 9H), 1.0 (m, 6H), 0.9(m, 3H) [438] MS [M+H]+ 568
[439] [440] Example 7: (3S)-3-{[(2R)-2-Isopropyl-3-methyl-4-oxo-4-phenylbutanoyl] - amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (lg)
[441]
Figure imgf000037_0002
[442] The compound prepared in Preparation 27 was reacted according to the same procedure as Example 5 to give the title compound. [443] [444] NMR (400MHz, CDCl ) δ 7.9 (br, IH), 7.5-7.1 (m, 5H), 6.7 (m, IH), 4.9-4.8 (m, 3 IH), 4.5 (m, IH), 4.2 (m, IH), 4.0 (m, IH), 3.0-2.8 (m, IH), 2.4 (m, IH), 2.1 (m, IH), 1.8 (m, IH), 1.3 (m, 3H), 0.9 (m, 6H).
Figure imgf000038_0001
[446]
[447] Preparation 28: Methyl 2-ethyl-4-(l-naphthyl)-4-oxobutanoate (VHi)
[448]
Figure imgf000038_0002
[449] l-Naphthylcarboxylic acid was reacted according to the same procedure as Preparations 1, 2, 3 or 4 to give the title compound. [450] [451] NMR (500MHz, CDCl ) δ 8.5 (d, J = 8.2Hz, IH), 8.0-7.8 (m, 3H), 7.6-7.4 (m, 3H), 3 3.5 (q, J = 10Hz, IH), 3.9 (s, 3H), 3.0 (m, 2H), 1.8-1.7 (m, 2H), 0.9 (t, J = 14Hz, 3H). [452] [453] Preparation 29: t-Butyl (3S)-3-{[2-ethyl-4-(l-naphthyl)-4-oxobutanoyl] - amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate (Xh) [454]
Figure imgf000038_0003
[455] The compound prepared in Preparation 28 and amino alcohd derivative Villa were reacted according to the same procedure as Preparations 5 and 8 to give the title compound.
[456]
[457] NMR (500MHz, CDCl ) δ 8.5 (d, J = 8.7Hz, IH), 7.9 (m, 2H), 7.8 (m, IH), 7.5 (m, 3 3H), 6.9 (m, IH), 6.7-6.5 (m, IH), 5.3-5.2 (dd, J = 7.2, 17.4Hz, IH), 5.1 (m, IH), 4.9-4.8 (m, IH), 3.6 (m, IH), 3.0 (m, 2H), 3.3 (m, IH), 2.7 (m, IH), 1.8 (m, IH), 1.4 (m, IH), 1.0 (m, 3H) [458] [459] Example 8: (3S)-3-{[2-Ethyl-4-(l-naphthyl)-4-oxobutanoyl] amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (Di) [460]
Figure imgf000039_0001
[461] The compound prepared in Preparation 29 was reacted according to the same procedure as Example 5 to give the title compound. [462] [463] NMR (400MHz, DMSO-d ) δ 8.3 (m, IH), 8.1 (m, IH,) 8.0 (m, IH), 7.9 (m, IH), 6 7.6-7.4 (m, 4H), 5.2 (br, 2H), 4.6-4.5 (m, IH), 3.3 (m, 2H), 2.8 (m, IH), 2.7-2.5 (m, 2H), 1.5 (m, IH), 0.9-0.8 (m, 3H) [464] MS [M+MeOH+Naf 588
[465]
[466] Preparation 30: Dimethyl 2-(l-isoquinolinyl)-2-oxoethyl phosphonate
[467]
Figure imgf000039_0002
[468] Methyl dimethyl phosphonate (4.84 g, 4.17 mil , 3.0 eq ) was dissdved in anhydrous tetrahydrofiiran (about 100 mil ) under nitrogen atmosphere, the mixture was kept at -78°C, and n-butyϊthium (2.5M in hexan, 15.6 mil , 3.0 eq) was added thereto. The mixture was stined at -78 - -60 °C for one hour. The methyl 1-isoquindinyl carboxylate (2.44 g, 13.0 mmd, prepared from 1-isoquindinecarboxvlic acid according to the the same procedure as Preparation 1) was dissdved in anhydrous tetrahydrofiiran (20 mil ) and added to the mixture at -60 °C . Th e resulting mixture was stined at -60 °C for 2 hours while it was sbwly warmed to room temperature. Oxalic acid dihydrate (5.41 g, 3.3 eq) was dissdved in methand (12 mil ), and then added to the mixture. The reaction mixture was filtered through Celite, and the filtrate was concentrated under reduced pressure and separated by cdumn chromatography (50%-80% ethyl acetate/hexane) to give the title compound (3.59 g, 99%) as yebw sdid.
[469]
[470] NMR (400MHz, CDCl ) δ 9.03 (d, IH), 8.61 (d, IH), 7.88 (m, 2H), 7.73 (m, 2H), 3 4.21 (d, J = 24Hz, 2H), 3.77 & 3.75 (two s, 6H) [471] [472] Preparation 31: M ethyl 2-ethyl-4-(l-isoquinolinyl)-4-oxobutanoate (VH)
[473]
Figure imgf000040_0001
[474] At 0 °C , anhydrous tetrahydrofiiran (100 mil ) was added to sodium hydride (60% dispersion in mineral oil, 649 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere. The phosphonate compound prepared in Preparation 30 (4.12 g, 14.75 mmd) was dissdved in anhydrous tetrahydrofiiran (20 mil ) and was added to the mixture. The resulting mixture was stined for about 10 minutes. Lithium chbride (1.25 g, 2.0 eq) was added thereto and the resulting mixture was fiirther stirred at 0 °C for about 10 minutes. Methyl 2-oxobutylate (2.4 g, 1.1 eq) dissdved in anhydrous tetrahydrofiiran (20 mil ) was added threrto at 0 °C, and the mixture was stined at room temperature for 2.5 hours. The reaction was completed by saturated ammonium chbride sdution, and then extracted twice with ethyl acetate (100 mil ). The filtrate was washed with water, saturated sodium bicarbonate sdution (NaHCO , 50 mil x 2), and aqueous sodium chbride sdution in turn, dried (anhydrous 3 Na SO ), and concentrated under reduced pressure . The residue was separated by 2 4 cdumn chromatography (20% ethyl acetate/hexane) to give the title compound (2.7 g, 68%) as yebw liquid in the form of cis.
[475]
[476] The obtained cis isomer (2.7 g, 10.03 mmd) was dissdved in ethyl acetate (50 mil ), and Pd/C (800 mg, 10%, Aldrich) was added thereto. The resulting mixture was reacted by using Pan Hydrogenater under 40 psi of hydrogen atmospheric pressure for 2 hours. The reaction was filtered through Celite, and the filtrate was concentrated under reduced pressure. The residue was separated by cdumn chromatography (20% ethyl acetate/hexane) to give per-reduced (ketone was also reduced) methyl 2-ethyl-4-(l-isoquinolinyl)-4-hydroxybutanoate (1.95 g, 71%) as yebw liquid.
[477]
[478] NMR (400MHz, CDCl ) δ 8.43 (d, IH), 8.28 (d, IH), 7.85 (d, IH), 7.73-7.59 (m, 3 2H), 7.59 (d, IH), 5.40 (m, IH), 3.83 (s, 3H), 3.05 (m, IH), 2.37 (t, IH), 1.69-1.46 (m, 3H), 0.89 (t, 3H) [479] [480] Oxalyl chbride (0.51 mil , 1.6 eq) was dissdved in dichbromethane (30 mil ), DMSO (1.04 mil , 4.0 eq) was added thereto at -78 °C , and the resulting mixture was stined for about 10 minutes. The above compound (1.0 g, 3.66 mmd) was dissdved in dichbtomethane (30 mil ), and then was added to the above mixture sbwly. The mixture was stined at -78 °C for 15 minutes. DIPEA (3.19 mil , 5.0 eq) was added to the reaction mixture and the mixture was stirred at -78 °C for 10 minutes. The temperature was sbwly raised to room temperature. Ethyl acetate/hexane (100 mil ) was added to the mixture, and organic layer was washed with 0.5N HC1 and saturated sodium chbride sdution, dried (anhydrous Na SO ), and concentrated under reduced 2 4 pressure. The residue was separated by column chromatography (25%-30% ethyl acetate/hexan) to give the title compound (900 mg, 91%) as yebw liquid. Each enantiomer was separated by Chiral OD Cdumn (Daicel Chemical Industries, 2.00cm x 25cm, OD00CJ-1C005, 3% i-PrOH in Hexane, 220nm).
[481]
[482] NMR (400MHz, CDCl ) δ 8.89 (d, IH), 8.58 (d, IH), 7.86 (d, IH), 7.82 (d, IH), 3 7.73-7.65 (m, 2H), 3.74 (dd, IH), 3.70 (s, 3H), 3.54 (dd, IH), 3.05 (m, IH), 1.77 (m, 2H), 0.98 (t, 3H) [483] [484] Preparation 32: tert -Butyl 3-{[2-ethyl-4-(l-isoquinolinyl)-4-oxobutanoyl] -amino}-5-fluoro-4-oxopentanoate (Xi)
[485]
Figure imgf000041_0001
[486] The compound prepared in Preparation 31 was hydrdyzed. The mixture carboxylic acid derivative of the obtained each isomer (160 mg, 0.622 mmd), amino alcohd derivative VIHc (130 mg, 1.05 eq), HOBt (92 mg, 1.1 eq), and DMAP (88 mg, 1.1 eq) was dissdved in THF (8 vd ), which was then coded to 0 °C . EDC (132 mg, 1.10 eq) was added thereto and the resulting mixture was stined for one day while it was sbwly warmed to room temperature. The residue was extracted with ethyl acetate (30 mil x 2), washed with water, aqueous sodium bicarbonate sdution, and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and 2 4 purified by cdumn chromatography (30%-40% ethyl acetate/hexane), to give a compound (249 mg, 91%). To this compound and Dess-Martin reagent (450 mg, 2.0 eq) was added dry dichbromethane (6 mil ), and the mixture was stirred for one hour at room temperature. The reaction was stopped by isopropyl alcohd (1 mil ). The mixture was filtrated through Celite under reduced pressure to remove sdid, and the filtrate was extracted with ethyl acetate (30 mil x 2), washed with water, saturated aqueous sodium bicarbonate sdution and aqueous sodium chbride sdution, dried (anhydrous Na SO ), concentrated under reduced pressure, and preliminarily purified by cdumn 2 4 chromatography (30%-40% ethyl acetate-hexane), to give the diastereomeric title compound (192 mg, 80%). [487] [488] NMR (400MHz, CDCl ) δ 8.88 ( two d, IH), 8.59 (m, IH), 7.86 (m, 2H), 3 7.74-7.67 (m, 2H), 6.94 (two d, IH, NH), 5.44-5.07 (m, 2H), 4.90 (m, IH), 3.76 (m, IH), 3.57 (m, IH), 3.02 (m, IH), 2.84-2.77 (m, 2H), 1.81 (m, IH), 1.66 (m, IH), 1.02 (two t, 3H) [489] [490] Example 9: 3-{[2-Ethyl-4-(l-isoquinolinyl)-4-oxobutanoyl] amino}-5-fluoro-4-oxopentanoic acid (Ii-a, Ii-b) [491]
Figure imgf000042_0001
[492] The mixture of each diastereomer prepared in Preparation 32 was reacted according to the same procedure as Example 1 to give the title compound. [493] [494] NMR (500MHz, DMSO-d ) δ 12.20 (br, IH), 8.78-8.26 (m, 3H), 8.12-7.73 (m, 6 4H,) 5.94-4.56 (br m, 2H), 7.9 (m, IH), 4.37-4.32 (two m, IH), 3.12-2.50 (m, 4H), 2.37-2.05 (m, 2H), 1.78-1.29 (m, 2H), 0.88-0.72 (m, 3H) [495] MS [M+H] 389
[496] [497] Preparation 33: Ethyl (Z) - 2-isopropyl-4-(l-isoquinolinyl)-4-oxo-2-butenoate [498]
Figure imgf000043_0001
[499] At 0 °C , anhydrous tetrahydrofiiran (30 mil ) was added to sodium hydride (60% dispersion in mineral oil, 276 mg, 1.1 eq, washed with anhydrous tetrahydrofiiran in advance) under nitrogen atmosphere. The phosphonate compound prepared in Preparation 30 (1.76 g, 6.27 mmd) was dissdved in anhydrous tetrahydrofiiran (10 mil ), which was added to the mixture. The resulting mixture was stined for about 10 minutes. Lithium chbride (532 mg, 2.0 eq) was added thereto and the resulting mixture was fiirther stirred at 0 °C for about 10 minutes. Ethyl 3-methyl-2-oxobutylate (994 mg, 1.1 eq) dissdved in anhydrous tetrahydrofiiran (15 mil ) was added threrto at 0 °C and stined at room temperature for one day. The reaction was completed by saturated ammonium chbride sdution, and then the reaction was extracted twice with ethyl acetate (100 mil ). The filtrate was washed with water, saturated sodium bicarbonate sdution (NaHCO , 50 mil x 2) and aqueous sodium chbride sdution in turn, 3 dried (anhydrous Na SO ), and concentrated under reduced pressure . The residue was 2 4 separated by column chromatography (20% ethyl acetate/hexane) to give the title compound (1.7 g, 91%) as yebw liquid in the form of cis. [500] [501] NMR (500MHz, CDCl ) δ 8.98 (d, IH), 8.61 (d, IH), 7.87 (d, IH), 7.82 (d, IH), 3 7.74-7.66 (m, 2H), 7.38 (s, IH), 4.30 (qt, 2H), 2.84 (m, IH), 1.29 (t, 3H), 1.16 (d, 6H) [502] [503] Preparation 34: Ethyl 2-isopropyl-4-(l-isoquinolinyl)-4-oxobutanoate ( Ij)
[504]
Figure imgf000043_0002
[505] The compound prepared in Preparation 33 (1.45 g, 10.03 mmd) was dissdved in ethyl acetate (50 mϋ ), and Pd/C (100 mg, 10%, Aldrich) was added thereto. The resulting mixture was reacted by using Parr Hydrogenater under 40 psi of hydrogen atmospheric pressure for 4 hours. The reaction was filtered through Celite, and the filtrate was concentrated under reduced pressure to give per-reduced (ketone was also reduced) ethy 2-isopropyl-4-(l-isoquinolinyl)-4-hydroxybutanoate (1.31 g, 71%) as yebw liquid. To this compound (400 mg) and Dess-Martin reagent (900 mg, 3.0 eq) was added dry dichbromethane (10 mil ), and the mixture was stined for one hour at room temperature. The reaction was stopped by isopropyl alcohd (1 mil ). The resultant was filtered through Celite, treated commonly, and then purified by cdumn chromatography (10% ethyl acetate-hexane) to give the enantiomeric title compound (223 mg, 56%).
[506]
[507] NMR (500MHz, CDCl ) δ 8.85 (d, IH), 8.59 (d, IH), 7.86 (d, IH), 7.82 (d, IH), 3 7.73-7.64 (m, 2H), 4.19 (m, 2H), 3.74 (dd, IH), 3.50 (dd, IH), 2.99 (m, IH), 2.16 (m, IH), 1.26 (t, 3H), 1.04 (two d, 6H) [508] [509] Preparation 35: tert -Butyl 5-fluoro-3-{[2-isopropyl-4-(l-isoquinolinyl)-4-oxo-butanoyl]amino}-4-oxopentano ate (Xj)
[510]
Figure imgf000044_0001
[511] The title compound was prepared according to the same procedure as Preparation 32, except that the compound of Preparation 34 was used instead of the compound of Preparation 31. [512] [513] NMR (400MHz, CDCl ) δ 8.86 (m, IH), 8.60 (m, IH), 7.89-7.82 (m, 2H), 3 7.74-7.64 (m, 2H), 6.88 (m, IH), 5.50-5.07 (m, 2H), 4.90 (m, IH), 3.87-3.74 (m, IH), 3.61-3.55 (m, IH), 3.08-2.99 (m, IH), 2.84-2.66 (m, 2H), 2.04 (m, IH), 1.48 & 1.46 (two s, 9H), 1.06 (m, 6H) [514] [515] Example 10: 5-Fluoro-3-{[2-isopropyl-4-(l-isoquinolinyl)-4-oxobutanoyl] ■ amino}-4-oxopentanoic acid (Ij) [516]
Figure imgf000045_0001
[517] The compound prepared in Preparation 35 was reacted according to the same procedure as Example 1 to give the title compound. [518] [519] NMR (500MHz, DMSO-d ) δ 8.78-8.26 (m, 3H), 8.12-7.73 (m, 4H,) 5.94-4.56 (br 6 m, 2H), 7.9 (m, IH), 4.42-4.31 (two m, IH), 3.12-2.50 (m, 4H), 2.36-2.05 (m, 2H), 2.03 (m, IH), 0.88-0.72 (m, 6H) [520] MS [M+ H]+ 403
[521]
[522] Experiment 1: Assay
[523] Caspase-1 and caspase-8 known as cysteine proteases in the form of α β were 2 2 expressed, purified, and activated by modifying a method known in Thornberry, N. A. et , Nature, 1992, 356, 768. Thornberry, N. A. Methods in Enzymology, 1994, 244, 615. Walker, N. P. C et al. Cell, 1994, 78, 343, and caspase-9 was also purified by a similar method, and the inhibitory activity against them was tested. Briefly describing, plO and p20 subunits (Thornberry, N. A. et , Nature, 1992, 356, 768) were expressed in E.coli and purified by nickel cdumn and anionic exchange chromatography to give caspase-1, caspase-8, and caspase-9. The fluorescent substrate Ac YV AD- AFC for thus obtained caspase-1, AcDEVD-AFC for caspase-8, and AcLEHD-AFC for caspase-9 were used for determining specific activity of the synthesized inhibitors. The enzyme reaction was carried out at 25 °C in a buffer sdution containing 50mM HEPES (pH 7.50), 10%(w/v) sucrose, 0.1%(w/v) CHAPS, lOOmM NaCl, ImM EDTA, and lOmM DTT in the presence of 50μM AcYVAD-AFC for lOnM caspase-1, 50μM AcDEVD- AFC for 2.1nM caspase-8, and 150μM AcLEHD-AFC for 200nM caspase-9. The inhibitory constants K and K of the inhibitors were determined by measuring the i obs reaction vebcity to the lapse of time with using a fluorescent spectrometer and by obtaining the initial rate constant. K was calculated from the Lineweaver Burk Pbt, and K from the fobwing Equation 1. obs
[524]
[525] Equation 1
[526] K = -h (\-A /A )/t obs t oo [527] in which [528] A means deavate rate (%) at time t, and t [529] A means the maximum deavage rate (%). [530] [531] Spectra MAX GeminiXS Fluorescent Spectrometer of Mdeciiar Device Co. was used at the excitation wavelength of 405nm and the emission wavelength of 505nm.
[532] [533] The in vivo inhibitory activity of the inhibitors was determined by subjecting Jurkat eel to apoptosis by Fas antibody and by detecting the ccbr change according to the WST-1 method known in Francoeur A.M and AssaUan A. (1996) Bioche ica 3, 19-25 to observe the amount of live Jurkat eels when the eels were treated by the inhibitor. Spectra MAX 340 Spectrometer of Mdecular Device Co. was used at the absorbance wavelength of 440nm.
[534] [535] Table 1
Figure imgf000046_0001
[536] [537] Experiment 2: Pharmacokinetic test in rat
[538] Rat (250-300 g, Bio Genomics , Korea ) was anesthetized under ether, and pdtethylene tube was inserted into, and fixed to, the jugular vein and carotid artery of the test animal. To enable the rat to move freely, the canular was extracted out of the r ear of rat neck, which was connected to the end of the canular with the bng polyethylene tube protected by spring. After rat comes out of the anesthetia in metabolic cage, in the intravenous injection test, the drug was dissdved in a mixed sdution of PEG400, EtOH, TWEEN80, and phosphate buffer, and administered to the jugular vein of the test animal in the amount of 10 mg/kg. For the oral administration, rat was starved for 18 hours from the drug administration. For oral administration test, the drug was dissdved in a mixed solution of PEG400, EtOH, TWEEN80, and phosphate buffer, and administered to the jugular vein of the test animal through oral sonde in the amount of 20 mg/kg. Bbod was colected before the drug administration (contrd), and at 1, 5, 15, 30, 60, 90, 120, 180 and 240 minutes after the oral administration for intravenous injection test, and at 15, 30, 60, 90, 120, 180 and 240 minutes after the oral administration for oral administration test. About 200 fd of bbod was colected from the carotid artery by using heparin-treated syringe. The colected bbod was centrifiiged to separate bbod plasma, protein was removed therefrom, and then the resultant was centrifiiged and analyzed by HPLC Calibration curve was determined at the concentration of 0.2-10 βg I mil , and the drug was analyzed by Shiseido Capcel-Pak C reverse column. HPLC consists of Class-LCI 0A system 18 contrd software, CBM-IOA communication bus module, two of LC- 10 AD pump, SIL- 10AXL autoinjector with sample coder, SPD-10AV ultravidet (UV) detector (Shimadzu, Tokyo, Japan), and GLP-2050+ laser printer (LG Electronics, Seoul, Korea). The compound Ia-2 was analyzed by UV lamp using wavelength of 329 nm. The flow rate was 1 mil /minute. As ehient, acetonitrile and 20 mM ammonium acetate was used at 31% and 69%, respectively. The retention time of drug was about 9 minutes. IDN1965 was analyzed by UV lamp using wavelength of 293 nm. The flow rate was about 1 mil /minute. As ehient, acetonitrile and 20 mM ammonium acetate was used at 32% and 68%, respectively. The retention time of drug was about 10 minutes. The data were represented by relation graph of plasma-drug concentration against time. P harmacokinetic parameters, half life (t ), maximum concentration (C ), time 1/2 max of maximum concentration (T ), AUC , AUC , systemic deareance (CL), max inf last distributed volume (Vd) were determined using Win-Nonfin Program (Scientific Consultion Co., USA) to non-compartment model (cf.: "p harmacokinetic parameter value after intravenous injection and oral administration in rat")
[539] [540] Table 2
Figure imgf000048_0001
[541] [542] Experiment 3: Therapeutic effect for LPS-induced acute hepatitis in mouse
[543] Step 1) Preparation of bbod sample [544] Female Balb/c mice (6 weeks, Charles River Laboratory, Osaka , Japan ) were kept under the conditions of 22 °C , 55% of relative humidity, and light-darkness cyde of 12 hours. Food and water were supplied ad libitum. In pyrogen-free saline were dissdved LPS (fipopdysaccaride) and D-galactosamine in concentrations of 0.4 mg / mil and 280 mg / mil , respectively, and their 1:1 mixture was injected into the mice in the amount of 5 mil / kg . Immediately after the injection of LPS and D-galactosamine, into the mice was intraperitonealy injected a vehide (a mixture of PEG400: ethand: TweenδO = 15: 7.5: 2.5 was diluted by five times with saline) wherein the test compound is dissdved or the vehide abne. After 8 hours from the drug injection, bbod samples were obtained from their hearts.
[545] [546] Step 2: Determination of the activity of plasma aminotransf erase [547] The plasma ALT activity was determined for the bbod samples obtained in Step 1 by using ALT assay kit (Asan Pharm. Co., Seoul , Korea ) according to the manufacturer's instruction. As a result, it appeared that the injection of LPS and D- galactosamine steeply increases the ALT activity in plasma, and the test compounds inhibit the increased enzyme activity in dose-dependent manner. Based on these results, ED values of the test compounds were calculated with using Prism software 50 of GraphPad Co. (cf.: Table 3 - "ED vahie for inhibiting the ALT activity").
[548] [549] Table 3
Figure imgf000049_0001
[550] [551] Experiment 4: Effect for increasing the survival rate in LPS- induced acute hepatitis model
[552] Female Balb/c mice (6 weeks, Charles River Laboratory, Osaka , Japan ) were kept under the conditions of 22 °C , 55% of relative humidity, and light-darkness cyde of 12 hours. Food and water were supplied ad libitum. In pyrogen-free saline were dissdved LPS (fipopdysaccaride) and D-galactosamine in concentrations of 0.4 mg / mil and 280 mg / mil , respectively, and their 1 : 1 mixture was injected to the mice in the amount of 5 mil / kg . Immediately after the injection of LPS and D-galactosamine, into the mice was intraperitonealy injected a vehide (a mixture of PEG400: ethand: TweenδO = 15: 7.5: 2.5 was diluted by five times with saline) wherein the test compound is dissdved or the vehide abne. Thereafter, the number of survival mice was determined over 72 hours to estimate the survival rate (cf.: "survival rate (%) in induced acute hepatitis model").
[553] [554] Table 4
Figure imgf000050_0001
[555] [556] (Advance) [557] Compound (Ia-2) according to the present invention and IDN-1965 known in USP 6,200,969 Bl were tested, and the results are shown in the fobwing table 5.
[558] [559] Table 5
Figure imgf000050_0002
[560] [561] As can be seen from the above Table 5, compound (Ia-2) according to the present invention showed an increased inhibitory activity by 2 times or more against caspase-8 and Jurkat eel compared with IDN-1965, and by about 3 times against LPS-induced acute hepatitis model (mouse). Also, regarding the pharmacokinetic test, the compound showed highuy improved half-life and deareance parameter values at the intravenous injection. The compound of the present invention showed the most remarkable effects than the IDN-1965 in terms of oral absorption rate and survival rate. In the case of survival test ( iv administration), the contrd groups were al dead, whereas the groups into which compound (Ia-1) of the present invention are administered showed a surprising survival rate of 90%. IDN-1965 did not show any inhibitory effect against ALT activity upon oral administration in LPS-induced acute hepatitis model, whereas the compound (Ia-2) of the present invention showed inhibitory effect against ALT activity in both cases of intraperitoneal and oral administration. Also, compound (Ia-2) of the present invention showed highly improved effects in both aspects of phtsiochemical properties and solubility. Thus, the compound (Ia-2) according to the present invention can be used as injection preparation.
[562] As shown in the above Table 5, the compound (Ia-2) of the present invention showed highly improved effects, compared with the IDN-1965 in both aspects of activity and physico-chemical property.

Claims

Claims
[ 1 ] 1. A compound of the fobwing f ormula ( 1 ) :
Figure imgf000052_0001
(1) in which I) R represents simple alkyl chain (-SAC), cydoalkyl chain (-SCAC), aryl group (-Ar), alkyl chain substituted by aryl (-SAC-Ar), or hydrogen, II) R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of al the natural amino acids; and the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 1 becomes a stereocenter due to the R group; or the compound of formula (1) may have a protecting group in ester form (-CO R wherein R is -SAC) or 2 4 4 sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form 2 5 5 of pharmaceuticaly acceptable salt, when R is carboxylic acid as side chain residue of an amino acid; or the compound of formula (1) may also exist in the form of pharmaceuticaly acceptable salt when R consists of a base as side chain residue of an amino acid, III) R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of the 2 natural amino acids; and the compound of formula (1) may exist in a specific di- astereomeric form, or mixtures thereof, when the carbon to which R is attached 2 becomes a stereocenter due to the R group; the compound of formula (1) may 2 have a protecting group in ester form (-CO R wherein R is -SAC) or 2 6 6 sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form 2 1 1 of pharmaceuticaly acceptable salt, when R is carboxylic acid as side chain 2 residue of an amino acid; or the compound of formula (1) may also exist in the form of pharmaceuticaly acceptable salt when R consists of a base as side chain 2 residue of an amino acid, or R further represents H; -(CH ) OR wherein R is -SAC, -SCAC, -Ar, or - 2 2 n 8 8 SAC-Ar, and n = 1 or 2; or -(CH ) OQ=O)R wherein R is -SAC, -SCAC, -Ar,
2 n 8 8 or -SAC-Ar, and n = 1 or 2, IV) R represents simple alkyl chain (-SAC), cydoalkyl chain (-SCAC), aryl 3 group (-Ar), alkyl chain substituted by aryl (-SAC-Ar), or hydrogen, V) Ar represents aryl (-Ar), VI) R and adjacent R together may form a cyde, where -R-R - is -(CH ) -, -(CH 2 n ) -O-(CH ) -, or -(CH ) -NHR -(CH ) - wherein n+m<9 and R is -SAC, - 2 n 2 m 2 n 9 2 m 9 SCAC, -Ar, -SAC-Ar, -Q=O)-SAC, -Q=O)-SCAC, -Q=O)-Ar, or - Q=O)-SAC-Ar, Vπ) X represents ?CN; -CHO; -O(=O)OR , wherein R is -SAC, -SCAC, -Ar, 10 10 or -SAC-Ar; -Q=O)CH OR wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; - 2 10 10 Q=O)CH OQ=O)R wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; 2 10 10 CH=CH-CO R wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-SO R 2 10 10 2 wherein R is -SAC, -SCAC, -Ar, or -SAC-Ar; -CONR R wherein R and 10 10 11 12 11 R each are ?H, -SAC, -SCAC, -Ar, or -SAC-Ar; or ?COCH -W wherein W is - 12 2 N , -F, -CI, -Br, -I, -NR R (R and R each are -SAC, -SCAC, -Ar, or - 2 13 1 13 1 SAC-Ar, or together may form cydic group), -SR (R is -SAC, -SCAC, -Ar, or -SAC-Ar), or is the fobwing formula:
Figure imgf000053_0001
wherein Y is H, -OH, -OR (R = -SAC or ?SCAC), -Q=O)R (R = -H, -SAC, or - 16 16 17 17 SCAC), -F, -CI, -Br, -I, -CN, -N , -CO H, CF , -CO R (R = -SAC or -SCAC), 3 2 3 2 18 18 -C(=O)NHR (R = -SAC or -SCAC), or -C(=O)NR R (R and R each are - 18 18 19 20 19 20 SAC, -SCAC, -Ar, or -SAC-Ar ), R is H, or ?SAC, 21 pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof. [4] 2. The compound according to daim 1 wherein I) R represents H; π) R represents ?(CH ) COOH, -(CH ) COOR (R=SAC), or ?(CH ) CONHSO 1 2 2 2 2 2 2 2 R (R = SAC); 22 22 HI) R represents H, -SAC, -Ar, or -(CH ) O R (R = -SAC, -SCAC, -Ar, or - 2 2 n m SAC-Ar, n = 0, 1 or 2, and m = 0 or 1); IV) R represents -SAC, or H, 3 V) Ar represents aryl (-Ar). VI) X represents -COCH N , -COCH F, -COCH CI, -COCH Br, -COCH I, - 2 2 2 2 2 2 COCH OAr, -COCH OCOAr or -COCH SR (R is -SAC, -SCAC, -Ar or - 2 2 2 15 15 SAC-Ar). [5] 3. The compound according to daim 1 which is selected from the fobwing group: (1) 3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-ethyl-4-oxobutanoyl] amino }-5-fluoro-4-oxopentanoic acid (la); (2) (3S)-3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-ethyl-4-oxobutanoyl] amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (lb); (3) (3S)-5-[(2,6-Dichbrobenzoyl)oxy]-3-{[4-(l,
3-Dimethyl-l H- mdd-2-yl)-2-e yl-4-oxobutanoyl]amino}-4-oxopentanoic acid (Ic);
(4) 3-{ [4-(l,3-Dimethyl-l H-indd-2-yl)-2-isopropyl-4-oxobutanoyl] amino }-5-fluo-ro-4-oxopentanoic acid (Id); (5) (3S)-5-[(2,6-Dichbrobenzoyl)oxy] - 3-[(2-methvl-4-oxo-4-phenylbutanoyl)-amino]-4-oxopentanoic acid (le); (6) (3S)-5-[(2,6-Dichbrobenzoyl)oxy] - 3-[(2-isopropyl-4-oxo-4-phenylbutanoyl)-amino]-4-oxopentanoic acid (If); (7) (3S)-3-{ [(2R)-2-Isopropyl-3-methyl-4-oxo-4-phenylbutanoyl] amino}-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid (lg); (8) (3S)-3-{ [2-Ethyl-4-(l-naphthyl)-4-oxobutanoyl] amino}-4-oxo-5-(2,3,
5,6-tetra-fluorophenoxy)pentanoic acid (Di); (9) 3-{ [2-Ethyl-4-(l-isoquindinyl)-4-oxobutanoyl] amino }-5-fluoro-4-oxopentanoic acid (Η); and (10) 5-Fhιoro-3-{ [2-isopropyl-4-(l-isoquinolinyl)-4-oxobutanoyl] amino }-4-oxo-pentanoic acid(Tj).
[6] 4. A pharmaceutical composition for inhibiting capases comprising the compound of formula 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as defined in daim 1 as active ingredient together with pharmaceuticaly acceptable carrier.
[7] 5. A pharmaceutical composition for preventing inflammation and apoptosis comprising the compound as defined daim 1, pharmaceuticaly acceptable salt, physidogicaly hydroh/sable ester, hydrate, sdvate, or steroisomer thereof, as defined in daim 1 as active ingredient together with pharmaceuticaly acceptable carrier.
[8] 6. A pharmaceutical composition for the treatment or prevention of dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, liver diseases by hepatitis virus, acute hepatitis, finminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, or ischemic cardiac diseases, comprising the compound as defined daim 1, pharmaceuticaly acceptable salt, physidogicaly hydrdysable ester, hydrate, sdvate, or steroisomer thereof, as defined in daim 1 as active ingredient together with pharmaceuticaly acceptable carrier.
[9] 7. The composition according to daim 6 for the treatment of acute hepatitis.
[10] 8. The composition according to daim 7 which is formulated as oral preparation, injection, or patch.
PCT/KR2004/002554 2003-10-10 2004-10-06 Caspase inhibitor comprising 2-alkyl-4-oxobutanoyl group and pharmaceutical composition thereof WO2005035497A1 (en)

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