WO2006033551A1 - Caspase inhibitors containing dicarbonylamino-isoxazoline - Google Patents

Caspase inhibitors containing dicarbonylamino-isoxazoline Download PDF

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Publication number
WO2006033551A1
WO2006033551A1 PCT/KR2005/003136 KR2005003136W WO2006033551A1 WO 2006033551 A1 WO2006033551 A1 WO 2006033551A1 KR 2005003136 W KR2005003136 W KR 2005003136W WO 2006033551 A1 WO2006033551 A1 WO 2006033551A1
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Prior art keywords
sac
carbonyl
scac
amino
dihydro
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PCT/KR2005/003136
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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
Tae-Kyo Park
Kyeong-Sik Min
Tae-Soo Lee
Sun-Hwa Lee
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Lg Life Sciences Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/04—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having one double bond between ring members or between a ring member and a non-ring member
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms

Definitions

  • the present invention relates to a dicarbonylamino-isoxazoline derivative, or phar ⁇ maceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an inhibitor against various caspases including caspase- l[interleukin-l ⁇ -con verting enzyme, ICE], caspase-3[apopain/CPP-32], caspase-8, and caspase-9, and a therapeutic composition for preventing inflammation and apoptosis comprising the same.
  • caspase- l[interleukin-l ⁇ -con verting enzyme, ICE], caspase-3[apopain/CPP-32], caspase-8, and caspase-9 a therapeutic composition for preventing inflammation and apoptosis comprising the same.
  • Caspase is a new kind of cysteine protease in the form of ⁇ ⁇ tetramer discovered during the last 10 years. About 14 kinds thereof have been known until now.
  • Caspase- l(ICE) is a kind of cytokine and participates in converting the inactive prointerleukin-l ⁇ to the 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 3IkDa. Only prointerleukin-l ⁇ is activated by ICE.
  • caspase- 1 The positions hydrolyzed by caspase- 1 are Asp -GIy and Asp -Ala . The hydrolysis of the latter position gives interleukin-l ⁇ . 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 by two independent study groups, the three dimensional structure thereof was determined by X-ray crystallographic method.
  • Caspase-3(CPP-32) is broadly studied for its role or mechanism for action, and its three dimensional structure was determined in 1996(2).
  • Caspase-3(apopain) activated from procaspase-3 hydrolyzes (P 4 )Asp-X-X-Asp(P 1 ) motif, and the known substrates include poly(ADP-ribose) polymerase, Ul 70,000 Mr small nuclear ribonucleoprotein, 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 participate in the apoptosis cascade.
  • the X-ray structure of caspase-8 was determined in 1999(5), and particularly the inhibitors thereof may be advantageously used for treating the 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 the caspase activity.
  • Diseases or symptoms that may be treated or attenuated by administering the inhibitors include the following: dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, hepatitis-induced hepatic diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, ischemic cardiac diseases, and liver cirrhosis(6).
  • Cirrhosis i) J Pharmacol Exp Ther. 2004 Mar;308(3): 1191-6, The caspase inhibitor
  • Idn-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse.
  • Canbay A. Fledstein A., Baskin-Bey E., Bronk FS. Gores GJ.; ii) Hepatology. 2004 Feb;39(2):273-8, Apoptosis: the nexus of liver injury and fibrosis.
  • Canbay A Friedman S, Gores GJ.; iii) Hepatology. 2003 Nov;38(5): 1188-98, Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression.
  • Canbay A, Feldstein AE Higuchi H, Werneburg N, Grambihler A, Bronk SF, Gores GJ.
  • the present inventors newly designed and synthesized some compounds which can be used as an effective inhibitor against caspases and have a distinctive structure and high selectivity for similar enzymes, and determined their binding ability and inhibitory activity for caspases. As a result, the inventors have discovered that a compound of the following formula (1) does meet such requirements, and completed the present invention.
  • A, B, R, R , R and X are defined as described below.
  • the present invention provides the novel dicarbonylamino-isoxazoline derivative of formula (1), or pharmaceutically acceptable salt, physiologically hy- drolysable ester, hydrate, solvate, or stereoisomer thereof having effective inhibitory activity against caspases.
  • a composition, a use, or a method for inhibiting caspases specifically a therapeutic composition, a use, or a method for preventing inflammation and apoptosis, comprising the compound of formula (1), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an active ingredient together with the phar ⁇ maceutically acceptable carrier.
  • a) Simple Alkyl Chain means a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form.
  • Aryl group (Ar, below) includes both the aromatic and heteroaromatic groups.
  • the aromatic group means a 5 to 15-membered single or fused unsaturated cycle.
  • One or more hydrogens may be replaced with a group(s) selected from the following: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl, alkoxy, aryloxy, sulfoxy, and guanido group.
  • the heteroaromatic group means the aromatic group containing 1 to 5 hetero atoms selected from a group consisting of oxygen, sulfur, and nitrogen.
  • one or more hydrogens may be replaced with a group(s) selected from the following: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl, alkoxy, aryl, aryloxy, sulfoxy, and guanido group.
  • a group(s) selected from the following: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl, alkoxy, aryl, aryloxy, sulfoxy, and guanido group.
  • an alkyl group can be added to the nitrogen atom to convert the pyridyl group to a pyridinium group having (+)-charge on the nitrogen atom.
  • the aryl group includes phenyl, biphenyl, 1-naphthyl, 2-naphthyl, pyridinyl, N- alkyl-pyridinium, pyrimidinyl, quinolinyl, benzothienyl, indolyl, pyrazinyl, isoindolyl, isoquinolyl, qunazolinyl, quinoxalinyl, phthalazinyl, imidazolinyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxa- zolopyridinyl, imidazopyridinyl,
  • the protecting group of simple ester is a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form.
  • R represents H, simple alkyl chain ( -SAC), s imple cycloalkyl chain (-SCAC), aryl group (-Ar), or simple alkyl chain substituted by aryl (-SAC-Ar), [102]
  • [103] II) Represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety,
  • R 2 represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety, or R further represents H; -(CH ) OR wherein R is -SAC, -SCAC, -Ar, or - SAC-Ar, and n
  • V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
  • Rand R may form a cycle together with the carbon atom to which they are attached, where -R-R 1 - is -(CH ) -, -(CH ) -0-(CH ) -, or -(CH ) -NR 10 -(CH ) -
  • R 24 is H or -SAC.
  • R represents H
  • V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
  • X represents -COCHN 2 , -COCH 2 F, -COCH 2 Cl, -COCH 2 Br, -COCH 2 I, -COCH 2
  • OAr, -COCH OCOAr, or -COCH SR 17 (R 17 is -SAC, -SCAC, -Ar, or -SAC-Ar).
  • R 17 is -SAC, -SCAC, -Ar, or -SAC-Ar.
  • the more preferred compounds are those selected from the group consisting of the following: [129] (3S)-3- ⁇ [(3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl] amino ⁇ -5-(2,6-dichlorobenzoyl)oxy] -4-oxopentanoic acid( 1 ) ; [130] (3S)-3-( ⁇ [5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl ⁇ amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)
  • the resulting oxime derivative (II) is treated by NCS (N-chlorosuccinimide) in dimethylformamide solution to give a hydroxamoyl chloride derivative (III).
  • hydroxamoyl chloride derivative (III) is reacted with an acrylate derivative (IV) (see the following Reaction Scheme 5) to give an isoxazoline derivative (V), which is then hydrolyzed, if needed, to give a deprotected isoxazoline derivative (VI).
  • the oxime derivative (II), the acrylate derivative (IV), and NaOCl may react together in a reaction vessel (in situ) to directly give the isoxazoline derivative (V).
  • isoxazoline derivative (VII) is deprotected and fused with an amine group to give an isoxazoline derivative (VIII), which may be hydrolyzed to give the deprotected isoxazoline derivative (VI).
  • the functional group Z in the compound (I) of Reaction Scheme 3 may be formed first by synthesizing the compound (XI) already having the desired Z group as depicted in the following Reaction Scheme 4, and by coupling the compound (XI) with the carboxylic acid compound (VI) on the left side (see WO 00/23421). Or, the desired Z group may be introduced later according to the process of Reaction Scheme 4 after the carboxylic acid compound (VI) is combined with an aspartic acid ( ⁇ -t-Bu) methyl ester and hydrolyzed.
  • Z is F
  • the racemic compound may be prepared according to a method known in Tetrahedron Letters, 1994, 35(52), 9693-9696.
  • the compound (IV) can be easily prepared from a known compound (XII) (Synthesis, 1982, p 924) as depicted in the following Reaction Scheme 5, or in case the compound (IV) is methyl (ethyl) 2-i-propylacrylate, the compound (IV) can be prepared from dimethyl(diethyl)malonate (/. Chemical Society Perkin Trans. 1 1997, 1559-1570).
  • the compound of formula (1) according to the present invention has a broad spectrum of inhibitory activity against caspases as demonstrated by the results of the following Experiments, and so has an effect for preventing inflammation and apoptosis.
  • the present invention provides a composition for inhibiting caspases, specifically a therapeutic composition for preventing inflammation and apoptosis, comprising the compound of formula (1), or pharmaceutically acceptable salt, physio ⁇ logically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an active ingredient together with the pharmaceutically acceptable carrier.
  • the composition of the present invention has a therapeutic effect for dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injure by hepatitis, hepatitis-induced hepatic diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, cardiac cell apoptosis due to ischemic cardiac diseases, or liver cirrhosis.
  • Caspase inhibitor particularly the compound of formula (1)
  • an effective amount of the caspase inhibitor, particularly the compound of formula (1), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof is mixed with a pharmaceutically acceptable carrier that may take a wide variety of forms depending on the formulation to be prepared.
  • the caspase inhibitor compound may be formulated as a parenteral injection, or percutaneous or oral preparation, depending on its application purpose. It is especially advantageous to formulate the composition in a unit dosage form for ease of admin ⁇ istration and uniformity of dosage.
  • any usual pharmaceutical carrier may be used.
  • water, glycols, oils, alcohols and the like may be used for such oral liquid preparations as suspensions, syrups, elixirs and solutions; or starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like may be used for such solid preparations as powders, pills, capsules and tablets. Due to their ease of administration, tablets and capsules are the most advantageous dosage unit forms. It is also desirable for tablets and pills to be formulated into enteric-coated preparation.
  • sterile water is usually used as the carrier, though other ingredients such as solubility aids may be used.
  • injections for example, sterilized aqueous or oily suspension for injection, can be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent.
  • Solvents that can be used for preparing injections include water, Ringer's fluid, and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non- stimulative fixing oil including mono- or di-glyceride may be used for this purpose.
  • Fatty acid such as oleic acid may also be used for injections.
  • the carrier may include a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives having no significant skin irritation. Said additives may facilitate the admin ⁇ istration through the skin and/or may assist preparation of a desired composition.
  • These percutaneous preparations are administered via various manners, e.g., as a transdermal patch, a spot-on, or an ointment.
  • the caspase inhibitor specifically the compound of formula (1)
  • the total daily dosage may be ad ⁇ ministered once or over several times.
  • specific administration dosage for an individual patient can be varied with specific compound used, body weight, gender, hygienic condition, or diet of subject patient, time or method of administration, excretion rate, mixing ratio of agent, severity of disease to be treated, etc.
  • reaction mixture was filtered through a cellit to remove the precipitate, and washed with ethylether.
  • the organic layer was washed with water and saturated aqueous sodium bicarbonate solution (300 D x 2), dried (anhydrous Na SO ), and concentrated under reduced pressure to give
  • 2-ethylacrylate prepared in Preparation 3 (1.54g, 1.2eq) were dissolved in anhydrous ether (100 D ) under nitrogen atmosphere, and maintained at -78°C. Triethylamine (2.79 D , 2.02g, 2.0eq) was added thereto. The mixture was stirred overnight while slowly warming it to room temperature. Water (100 D ) was added thereto, and the mixture was extracted with ethyl acetate (100 D x 2), washed with water (100 D ), dried (anhydrous Na SO ), and concentrated under reduced pressure.
  • the glyoxal was dissolved in ethanol (20 D )-water (10 D ), hydroxylamine hydrochloride (415mg, l.leq) and anhydrous sodium carbonate (Na CO , 320mg, 0.55eq) were added thereto at 0 0 C, and the mixture was stirred for 1 hour at room temperature. Saturated aqueous sodium chloride solution (30 D ) was added thereto, and the mixture was extracted twice with ethyl acetate (60 D ), dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the title yellow oxime compound (530mg, 49%).
  • 2-ethylacrylate (374mg, l.leq) were dissolved in anhydrous ether (30 D ) under nitrogen atmosphere, maintained at 0 0 C, and triethylamine (0.74 D , 538mg, 2.0eq) was added thereto. While slowly warming the mixture to room temperature, it was stirred for 2 hours. Water (30 D ) was added thereto, and the mixture was extracted with ethyl acetate (50 D x 2), washed with saturated aqueous sodium bicarbonate solution and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under
  • diazomethane-ether solution (synthesized from 2.0eq of l-methyl-3-nitro-l-nitroso-guanidine, 20 D ) was added thereto to give diazoketone derivative (1 hour), which was then extracted with ethyl acetate, washed with water, saturated aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was purified by column chromatography (10-15% EA/Hex) to give the title diazo derivative (1.50g, 76%, yellow powder).
  • Caspase- 1 and caspase-8 known as cysteine proteases in the form of ⁇ ⁇ were expressed, purified, and activated by modifying a method known in Thornberry, N. A. et al, 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, p 10 and p20 subunits (Thornberry, N. A.
  • the enzyme reaction was carried out at 25 0 C with various concentrations of the inhibitors in a buffer solution containing 2OmM HEPES ⁇ H 7.40), 10%(w/v) sucrose, 0.1%(w/v) CHAPS, 10OmM NaCl, ImM EDTA, and 1OmM DTT in the presence of 25 ⁇ M AcYVAD-AFC for 1OnM caspase- 1, 25 ⁇ M AcDEVD-AFC for 2.InM caspase-8, and 150 ⁇ M AcLEHD-AFC for 20OnM caspase-9.
  • the inhibitory constants K i and K obs of the inhibitors were determined by measuring the reaction velocity with the time lapse using a fluorescent spectrometer and by obtaining the initial rate constant. K was calculated from the Lineweaver Burk Plot, and K from the following Equation 1. obs
  • K -In (I-A /A )/t obs t oo
  • A means cleavage rate (%) at time t
  • A means the maximum cleavage rate (%).
  • Jurkat cell (ATCC TIB- 152) to apoptosis using anti-Fas monoclonal antibody (Upstate Biotech 05-201) and by detecting the color change according to the WST-I method (TAKARA MK400) known in Francoeur A.M. and Assalian A. (1996) Biochemica 3, 19-25 to observe the amount of alive Jurkat cells when the cells were treated by the inhibitor.
  • Spectra MAX 340 Spectrometer of Molecular Device Co. was used at the absorbance wavelength of 440nm.
  • Step 2 Determination of the activity of plasma aminotransferase
  • the plasma ALT activity was determined for the blood samples obtained in Step 1 using ALT assay kit (Asan Pharm. Co., Seoul , Korea ) according to the manufacturer's instruction. The results appeared that the injection of LPS and D-galactosamine sharply increases the ALT activity in plasma, and the test compounds inhibit the increased enzyme activity in a dose-dependent manner. Based on these results, ED values of the test compounds were calculated using Prism software of GraphPad Co.

Abstract

The present invention relates to a dicarbonylamino-isoxazoline derivative as an inhibitor against various caspases and a therapeutic composition for preventing inflammation and apoptosis comprising the same.

Description

Description
CASPASE INHIBITORS CONTAINING DICARBONYLAMINO-
ISOXAZOLINE
[i]
TECHNICAL FIELD
[2]
[3] The present invention relates to a dicarbonylamino-isoxazoline derivative, or phar¬ maceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an inhibitor against various caspases including caspase- l[interleukin-lβ-con verting enzyme, ICE], caspase-3[apopain/CPP-32], caspase-8, and caspase-9, and a therapeutic composition for preventing inflammation and apoptosis comprising the same.
[4]
BACKGROUND ART
[5] Caspase is a new kind of cysteine protease in the form of α β tetramer discovered during the last 10 years. About 14 kinds thereof have been known until now. Caspase- l(ICE), one of them, is a kind of cytokine and participates in converting the inactive prointerleukin-lβ to the 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 3IkDa. Only prointerleukin-lβ is activated by ICE. The positions hydrolyzed by caspase- 1 are Asp -GIy 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 by two independent study groups, the three dimensional structure thereof was determined by X-ray crystallographic method.
[6]
[7] Caspase-3(CPP-32) is broadly studied for its role or mechanism for action, and its three dimensional structure was determined in 1996(2). Caspase-3(apopain) activated from procaspase-3 hydrolyzes (P 4 )Asp-X-X-Asp(P 1 ) motif, and the known substrates include poly(ADP-ribose) polymerase, Ul 70,000 Mr small nuclear ribonucleoprotein, 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).
[8]
[9] Caspase-8 and 9 are present in the upstream of caspase-3,6,7 and these caspases are known to participate in the apoptosis cascade. The X-ray structure of caspase-8 was determined in 1999(5), and particularly the inhibitors thereof may be advantageously used for treating the diseases related to apoptosis.
[10] [H] Caspase inhibitors mean those compounds that inhibit the activity of caspase, and so control such symptoms as inflammation, apoptosis, etc. caused by the caspase activity. Diseases or symptoms that may be treated or attenuated by administering the inhibitors include the following: dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis virus, hepatitis-induced hepatic diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, ischemic cardiac diseases, and liver cirrhosis(6).
[12] [13] Among the caspase inhibitors known until now, the most noted irreversible inhibitors are the following:
[14]
Figure imgf000003_0001
IDN-1965 MX-1013
[15] [16] Both the above inhibitors exhibit their activity based on the common mechanism that they irreversibly inactivate the enzyme to suppress the cell apoptosis (irreversible, broad- spectrum inhibitor). It has been reported that irreversible inhibitor has much more effective inhibitory activity than reversible inhibitor (7). Both IDN-1965 of IDUN Co. and MX-1013 of Maxim Co. are reported to show activity in cell apoptosis model for hepatic injury (8, 9). These compounds are now in the stage of preclinical test. The irreversible inhibitor IDN-6556, the structure of which has been recently reported, is now in the stage of phase II clinical test as a therapeutic agent for hepatic injury (10, 11).
[17]
Figure imgf000003_0002
IDN-6556
[18] [19] References: [20] (1) Inflammation: Basic Principles and Clinical Correlates, 2nd ed., ed by Gallin, Goldstein and Snyderman. Raven Press Ltd., New York . 1992, pp211-232; Blood,
1996, 87(6), 2095-2147. [21] (2) Wilson, K. P. et al, Nature, 1994,370. 270; Walker, N. P. C. et al. Cell, 1994,
78, 343; Nature Structural Biology, 1996, 3(7), 619. [22] (3) Thornberry, N. A. et al, Nature, 1992,356. 768; Nature Biotechnology, 1996, 14
, 297; Protein Science, 1995, 4, 3; Nature, 1995, 376(JuIy 6), 37; Protein Science,
1995, 4, 2149.
[23] (4) Wei, Y. et al, Chemistry and Biology, 2000, 7, 423.
[24] (5) Blanchard H. et al, Structure, 1999, 7, 1125; Blanchard H. et al, /. of MoI. Biol.,
2000, 302, 9.
[25] (6) References for caspase related diseases
[26] Dementia: Arch Neurol 2003 Mar;60(3):369-76, Caspase gene expression in the brain as a function of the clinical progression of Alzheimer disease. Pompl PN, Yemul
S, Xiang Z, Ho L, Haroutunian V, Purohit D, Mohs R, Pasinetti GM. [27] [28] Cerebral stroke: Proc Natl Acad Sci U S A 2002 Nov 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 N, Augustinack JC, Hyman BT, Yuan J, Kuida K, Flavell RA, Moskowitz
MA. [29] [30] Brain impairment due to AIDS: J Neurosci 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'Emilia DM, Friedlander RM, Yuan J,
Masliah E, Lipton SA. [31] [32] Diabetes: Diabetes 2002 Jun;51(6): 1938-48, Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. [33] [34] Gastric ulcer: J Physiol Pharmacol 1998 Dec;49(4):489-500, Role of basic fibroblast growth factor in the suppression of apoptotic caspase-3 during chronic gastric ulcer healing. Slomiany BL, Piotrowski J, Slomiany A. [35] [36] Cerebral injury by hepatitis virus: J Viral Hepat 2003 Mar; 10(2):81-6, Cerebral dysfunction in chronic hepatitis C infection. Forton DM, Taylor-Robinson SD ,
Thomas HC. [38] Fulminant hepatic failure: Gastroenterology 2000 Aug; 119(2):446-60, Tumor necrosis factor alpha in the pathogenesis of human and murine fulminant hepatic failure. Streetz K, Leifeld L, Grundmann D, Ramakers J, Eckert K, Spengler U, Brenner D, Manns M, Trautwein C.
[39]
[40] Sepsis: Nat Immunol 2000 Dec;l(6):496-501, Caspase inhibitors improve survival in sepsis: a critical role 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, Nicholson DW, Karl IE.
[41]
[42] Organ transplantation rejection: Xenotransplantation 2001 May;8(2): 115-24, In vitro prevention of cell-mediated xeno-graft rejection via the Fas/FasL-pathway in CrmA-transducted porcine kidney cells. Fujino M, Li XK, Suda T, Hashimoto M, Okabe K, Yaginuma H, Mikoshiba K, Guo L, Okuyama T, Enosawa S, Amemiya H, Amano T, Suzuki S.
[43]
[44] Rheumatic arthritis: Prog Med Chem 2002;39:l-72, Caspase inhibitors as anti¬ inflammatory and antiapoptotic agents. Graczyk PP.
[45]
[46] Ischemic cardiac diseases: Am J Physiol Heart Circ Physiol 2002
Sep;283(3):H990-5, Hypoxia-induced cleavage 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 Immunol 2003 Mar 15;170(6):3386-91, A broad- spectrum caspase inhibitor attenuates allergic airway inflammation in murine asthma model. Iwata A, Nishio K, Winn RK, Chi EY, Henderson WR Jr, Harlan JM.
[49]
[50] Cirrhosis: i) J Pharmacol Exp Ther. 2004 Mar;308(3): 1191-6, The caspase inhibitor
Idn-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse. Canbay A., Fledstein A., Baskin-Bey E., Bronk FS. Gores GJ.; ii) Hepatology. 2004 Feb;39(2):273-8, Apoptosis: the nexus of liver injury and fibrosis. Canbay A, Friedman S, Gores GJ.; iii) Hepatology. 2003 Nov;38(5): 1188-98, Kupffer cell engulfment of apoptotic bodies stimulates death ligand and cytokine expression. Canbay A, Feldstein AE, Higuchi H, Werneburg N, Grambihler A, Bronk SF, Gores GJ.
[51]
[52] (7) Wu J. et al, Methods: A Companion to Methods in Enzymology, 1999, 17, 320. [53] (8) Hoglen N. C. et al, /. of Pharmacoloy and Experimental Therapeutics, 2001,
297, 811.
[54] (9) Jaeschke H. et al, Toxicology and Applied Pharmacology, 2000, 169, 11.
[55] (10) Hoglen N.C. et al, /. Pharmacol Exp. Ther., 2004, 309(2):634. Charac¬ terization of IDN-6556 (3-[2-(2-tert-butyl-phenylaminooxalyl)-amino]-propionylamino ]-4-oxo-5-(2,3,5,6-tetrafluoro-phenoxy)-pentanoic acid): a liver-targeted caspase inhibitor;
[56] (11) Canbay A et al, /. Pharmacol. Exp. Ther., 2004, 308(3), 1191. The caspase inhibitor IDN-6556 attenuates hepatic injury and fibrosis in the bile duct ligated mouse.
[57]
DISCLOSURE OF THE INVENTION
[58]
[59] The present inventors newly designed and synthesized some compounds which can be used as an effective inhibitor against caspases and have a distinctive structure and high selectivity for similar enzymes, and determined their binding ability and inhibitory activity for caspases. As a result, the inventors have discovered that a compound of the following formula (1) does meet such requirements, and completed the present invention.
[60]
Figure imgf000006_0001
[61] (1)
[62] in which
[63] A, B, R, R , R and X are defined as described below.
[64]
[65] Therefore, the present invention provides the novel dicarbonylamino-isoxazoline derivative of formula (1), or pharmaceutically acceptable salt, physiologically hy- drolysable ester, hydrate, solvate, or stereoisomer thereof having effective inhibitory activity against caspases.
[66]
[67] It is another object of the present invention to provide a composition, a use, or a method for inhibiting caspases, specifically a therapeutic composition, a use, or a method for preventing inflammation and apoptosis, comprising the compound of formula (1), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an active ingredient together with the phar¬ maceutically acceptable carrier. [69] First of all, the important terms in the present invention are defined as follows:
[70]
[71] a) Simple Alkyl Chain (SAC, below) means a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form.
[72]
[73] b) Simple CycloAlkyl Chain (SCAC, below) means a cyclic radical having 3 to 10 carbon atoms.
[74]
[75] c) Aryl group (Ar, below) includes both the aromatic and heteroaromatic groups.
The aromatic group means a 5 to 15-membered single or fused unsaturated cycle. One or more hydrogens may be replaced with a group(s) selected from the following: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl, alkoxy, aryloxy, sulfoxy, and guanido group. The heteroaromatic group means the aromatic group containing 1 to 5 hetero atoms selected from a group consisting of oxygen, sulfur, and nitrogen. Likewise, one or more hydrogens may be replaced with a group(s) selected from the following: acyl, amino, carboalkoxy, carboxy, carboxyamino, cyano, halo, hydroxy, nitro, thiol, alkyl, cycloalkyl, alkoxy, aryl, aryloxy, sulfoxy, and guanido group. Or, for example, in the case of pyridyl, an alkyl group can be added to the nitrogen atom to convert the pyridyl group to a pyridinium group having (+)-charge on the nitrogen atom.
[76]
[77] The aryl group includes phenyl, biphenyl, 1-naphthyl, 2-naphthyl, pyridinyl, N- alkyl-pyridinium, pyrimidinyl, quinolinyl, benzothienyl, indolyl, pyrazinyl, isoindolyl, isoquinolyl, qunazolinyl, quinoxalinyl, phthalazinyl, imidazolinyl, isoxazolyl, pyrazolyl, oxazolyl, thiazolyl, indolizinyl, indazolyl, benzothiazolyl, benzimidazolyl, benzofuranyl, thienyl, pyrrolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxa- zolopyridinyl, imidazopyridinyl, isothiazolyl, cinnolinyl, carbazolyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl, isoindolinyl, isobenzotetrahydrofuranyl, isoben- zotetrahydro-thienyl, isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahy- drofuranyl, benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl, phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl, imidazothiazolyl, dihy- drobenzisoxazinyl, benzisoxazinyl, benzoxazinyl, dihydrobenzisothiopyranyl, ben- zopyranyl, benzothiopyranyl, coumarinyl, isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide, tetrahydroquinolinyl-N-oxide, dihydroquinolinyl, dihydro- quinolinonyl, dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl, isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl-N-oxide, pyrimidinyl- N-oxide, pyrazinyl-N-oxide, quinolinyl-N-oxide, indolyl-N-oxide, indolinyl-N-oxide, isoquinolyl-N-oxide, qunazolinyl-N-oxide, quinoxalinyl-N-oxide, phthalazinyl-
N-oxide, imidazolinyl-N-oxide, isoxazolyl-N-oxide, oxazolyl-N-oxide, thiazolyl-
N-oxide, indolizinyl-N-oxide, indazolyl-N-oxide, benzothiazolyl-N-oxide, benz- imidazolyl-N-oxide, pyrrolyl-N-oxide, oxadiazolyl-N-oxide, thiadiazolyl-N-oxide, triazolyl-N-oxide, tetrazolyl-N-oxide, etc. [78] [79] d) Simple Alkyl Chain substituted by Aryl (SAC-Ar, below) means a straight-chain or branched alkyl which has 1 to 8 carbon atoms and is substituted by the above mentioned aryl group. [80] [81] e) Natural amino acid includes the following: Glycine, Alanine, Valine, Leucine,
Isoleucine, Serine, Threonine, Cysteine, Methionine, Proline, Aspartic acid,
Asparagine, Glutamic acid, Glutamine, Lysine, Arginine, Histidine, Phenylalanine,
Tyrosine, and Tryptophan. [82] [83] f) The protecting group of simple ester is a hydrocarbon having 1 to 8 carbon atoms in either linear or branched isomeric form. [84]
[85] Further, the present specification includes the following abbreviations:
[86] N-chlorosuccinimide: NCS
[87] N-methylmorpholine: NMM
[88] O-(7-azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluroniumhexafluorophosphate:
HATU
[89] N,N-dimethyl formamide: DMF
[90] l-(3-dimethylaminopropyl)-3-ethylcarbodiimide: EDC
[91] 1-hydroxybenzotriazole hydrate: HOBt
[92] trifluoroacetic acid: TFA
[93] t-butoxycarbonyl: Boc
[94] benzyloxycarbonyl: Cbz
[95] methyl: Me
[96] ethyl: Et
[97] equivalent: eq
[98] [99] The substituents included in the above formula (1) are specifically defined as follows. [100] [101] I) R represents H, simple alkyl chain ( -SAC), s imple cycloalkyl chain (-SCAC), aryl group (-Ar), or simple alkyl chain substituted by aryl (-SAC-Ar), [102]
[103] II) Represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety,
[104]
[105] III) R2 represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; the compound of formula (1) may have a protecting group in an ester form (-CO R wherein R is -SAC) or a sulfonamide form (-CONHSO R wherein R is -SAC), or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety, or R further represents H; -(CH ) OR wherein R is -SAC, -SCAC, -Ar, or - SAC-Ar, and n = 1 or 2; or -(CH 2 ) n OC(=O)R8 wherein R8 is -SAC, -SCAC, -Ar, or -
SAC-Ar, and n = 1 or 2, [106] [107] IV) A represents -(NR9) - wherein R9 is H, -SAC, -SCAC, -Ar, or -SAC-Ar, and n=0-l, [108]
[109] V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
[HO] [111] VI) Rand R may form a cycle together with the carbon atom to which they are attached, where -R-R1- is -(CH ) -, -(CH ) -0-(CH ) -, or -(CH ) -NR10-(CH ) -
2 n 2 n 2 m 2 n 2 m wherein n+m<9 and R10 is -SAC, -SCAC, -Ar, -SAC-Ar, -Q=O)-SAC, -Q=O)-SCAC,
-Q=O)-Ar, or -Q=O)-SAC-Ar, [112] [113] VII) X represents -Q=O)CH2OR11 wherein R11 is -SAC, -SCAC, -Ar, or -SAC-Ar; -
Q=O)CH OQ=O)R12 wherein R12 is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-CO R
13 wherein R13 is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-SO2R14 wherein R14 is - SAC, -SCAC, -Ar, or -SAC-Ar; -Q=O)CH=CH ; -COCHN ; or -COCH -W wherein W is -F, -Cl, -Br, -I, -NR15R16 (R15 and R16 each are -SAC, -SCAC, -Ar, or -SAC-Ar, or together may form 3- to 6-membered saturated or unsaturated cyclic group), -SR (R is -SAC, -SCAC, -Ar, or -SAC-Ar), or is the following formula: [114]
Figure imgf000010_0001
[115]
[116] wherein
[117] Y is H, -OH, -OR18 (R18 = -SAC or -SCAC), -C(=0)R19 (R19= -H, -SAC, or -
SCAC), -F, -Cl, -Br, -I, -CN, -N3, -CO2H, -CF3, -CO2R20 (R20 = -SAC or -SCAC), -
C(=0)NHR21 (R21 = -SAC or -SCAC), or -Q=O)NR22R23 (R22 and R23 each are -SAC, -
SCAC, -Ar, or -SAC-Ar ), and [118] R24 is H or -SAC.
[119] [120] The preferred compounds among the compound of formula (1) above are those wherein
[121] I) R represents H;
[122] II) R1 represents -CH COOH, -CH COOR3 (R3 = -SAC), or -CH CONHSO R4 (R4 =
-SAC); [123] III) R2 represents H, -SAC, -Ar, or -(CH ) OR7 (R7 = -SAC, -SCAC, -Ar, or -
SAC-Ar, and n = l or 2);
[124] IV) A represents -(NR9) - (R9 is H, -SAC, -SCAC, -Ar, or -SAC-Ar, and n=O-l),
[125] V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
[126] VI) X represents -COCHN2, -COCH2F, -COCH2Cl, -COCH2Br, -COCH2I, -COCH2
OAr, -COCH OCOAr, or -COCH SR17 (R17 is -SAC, -SCAC, -Ar, or -SAC-Ar). [127] [128] The more preferred compounds are those selected from the group consisting of the following: [129] (3S)-3-{ [(3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl] amino } -5-(2,6-dichlorobenzoyl)oxy] -4-oxopentanoic acid( 1 ) ; [130] (3S)-3-({ [5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(2); [131] 3-({ [5-Ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl}amino)-5-fluoro-4-oxopentanoic acid(3); [132] 3-({ [5-Ethyl-3-(l-isoquinolinylcarbonyl)-4,5-dihydro-5-isoxazolyl]carbonyl} amino)-5-fluoro-4-oxopentanoic acid(4); [133] (3S)-3-[({5-ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl} carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(5); [134] 3-[({5-Ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl}carbonyl) amino] -5-fluoro-4-oxopentanoic acid(6); [135] (35)-3-[({3-[([l,l'-biphenyl]-2-ylamino)caibonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenox y)pentanoic acid(7); [136] 3-[({3-[([l,r-Biphenyl]-2-ylamino)carbonyl]-5-ethyl-4,5-dihydro-5-isoxazolyl} carbony 1) amino] - 5 -fluoro-4-oxopentanoic acid( 8 ) ; [137] 3-{ [(5-Ethyl-3-{ [(2'-methyl[l,r-biphenyl]-2-yl)amino] carbonyl}-4,5-dihydro-5-isoxazolyl)carbonyl] amino }-5-fluoro-4-oxopentanoic acid(9); [138] 3-[({3-[3,4-Dihydro-l(2H)-quinolinylcarbonyl]-5-ethyl-4,5-dihydro-5-isoxazolyl} carbonyl)amino]-5-fluoro-4-oxopentanoic acid(lθ); [139] (3S)-3-[({3-[3,4-dihydro-2(lH)-isoquinolinylcarbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenox y) pentanoic acid(l l); [140] 3-{ [(3-{ [2-(tert-Butyl)anilino]carbonyl}-5-ethyl-4,5-dihydro-5-isoxazolyl) carbonyl] amino } -5-fluoro-4-oxopentanoic acid( 12) ; [141] 3-{ [(3-{ [2-(tert-Butyl)anilino]carbonyl}-5-isopropyl-4,5-dihydro-5-isoxazolyl) carbonyl] amino }-5-fluoro-4-oxopentanoic acid(13); and [142] 3-{ [(3-{ [2,5-Di(tert-butyl)anilino] carbonyl}-5-isopropyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopenta noic acid(14). [143] [144] The processes for preparation of the novel dicarbonylamino-isoxazoline derivative of formula (1) showing an inhibitory activity against caspases are depicted in the following Reaction Schemes 1 to 5. However, those illustrated in the following
Reaction Schemes represent only the typical processes used in the present invention.
The manipulation order may be changed with no limit, and so the processes are not restricted to those explained below. [145]
[146] Reaction Scheme 1
[147]
Figure imgf000012_0001
III
Figure imgf000012_0002
V Vl
B = Ar, A = none
[148] in which [149] P represents simple alkyl chain. [150] VI a) B=Phenyl, R2=Et [151] b) B=l-Naphthyl, R2=Et [152] c) B=l-Isoquinolinyl, R =Et [153] [154] In Reaction Scheme 1 above, a glyoxal derivative is reacted with hydroxylamine-hy- drochloride and sodium carbonate in a solvent mixture of alcohol-water to give an oxime derivative (II) (a mixture of syn and anti oximes). The resulting oxime derivative (II) is treated by NCS (N-chlorosuccinimide) in dimethylformamide solution to give a hydroxamoyl chloride derivative (III). Thus obtained hydroxamoyl chloride derivative (III) is reacted with an acrylate derivative (IV) (see the following Reaction Scheme 5) to give an isoxazoline derivative (V), which is then hydrolyzed, if needed, to give a deprotected isoxazoline derivative (VI). If appropriate, the oxime derivative (II), the acrylate derivative (IV), and NaOCl may react together in a reaction vessel (in situ) to directly give the isoxazoline derivative (V).
[155] [156] Reaction Scheme 2 [157]
Figure imgf000013_0001
VII
Figure imgf000013_0002
VIII Vl
B = Ar, A = -NH-
[158] VI e) B = 1-Naphthyl, A = NH, R2 = Et [159] f) B = [l,l'-Biphenyl]-2-yl, A = NH, R2 = Et [160] g) B = (2'-Methyl[U'-biphenyl]-2-yl), A = NH, R2 = Et [161] h) B-A = 3,4-Dihydro-l(2H)-quinolinyl, R2 = Et [162] i) B-A = 3,4-Dihydro-2(lH)-isoquinolinyl, R2 = Et [163] j) B = 2-t-Butyl-l -phenyl, A = NH, R2 = Et, [164] k) B = 2-t-Butyl-l -phenyl, A = NH, R2 = i-Pr [165] [166] In Reaction Scheme 2, glycine t-butyl ester • HCl synthesized according to a known process (/. of Chemical Society, PT 1, 1997, 3005) is used to give tert-butyl 2-chloro-2-(hydroxyimino)acetate, which is then reacted with the acrylate derivative (IV) to give an isoxazoline derivative (VII). Thus obtained isoxazoline derivative (VII) is deprotected and fused with an amine group to give an isoxazoline derivative (VIII), which may be hydrolyzed to give the deprotected isoxazoline derivative (VI).
[167] [168] Reaction Scheme 3 [169]
Dess-Wartin
Figure imgf000014_0001
Figure imgf000014_0002
( X - COCHiZ) (Z=OR11, OQ=O)R12 , or W)
[170] [171] I a) B = Phenyl, A = None, R = Et, Z = OCO-Ph(2,6-dichloro) [172] b) B = 1-Naphthyl, A = None, R2 = Et, Z = OPh (2,3,5,6-tetrafluσro) [173] c) B = 1-Naphthyl, A = None, R2 = Et, Z = F [174] d) B = 1-Isoquinolinyl, A = None, R2 = Et, Z = F [175] e) B = 1-Naphthyl, A = NH, R2 = Et, Z = OPh (2,3,5,6-tetrafluσro) [176] f) B = 1-Naphthyl, A = NH, R2 = Et, Z = F [177] g) B = [l,l'-Biphenyl]-2-yl, A = NH, R2 = Et, Z = OPh (2,3,5,6-tetrafluoro) [178] h) B = [l,l'-Biphenyl]-2-yl, A = NH, R2 = Et, Z = F [179] i) B = (2'-Methyl[l,l'-biphenyl]-2-yl), A = NH, R2 = Et, Z = F [180] j) B-A = 3,4-Dihydro-l(2H)-quinolinyl, R2 = Et, Z = F [181] k) B-A = 3,4-Dihydro-2(lH)-isoquinolinyl, R2 = Et, Z = OPh (2,3,5,6-tetrafluoro) [182] 1) B = 2-t-Butyl-l -phenyl, A = NH, R2 = Et, Z = F [183] m) B = 2-t-Butyl-l -phenyl, A = NH, R2 = i-Pr, Z = F [184] [185] As depicted in Reaction Scheme 3 above, the carboxylic acid derivative (VI) is coupled with an aspartic acid derivative (XI) (see the following Reaction Scheme 4) to give a compound (IX), which is then subjected to Dess-Martin periodene oxidation reaction, and deprotection reaction, if needed, to give the desired compound of formula (1).
[186] [187] The functional group Z in the compound (I) of Reaction Scheme 3 may be formed first by synthesizing the compound (XI) already having the desired Z group as depicted in the following Reaction Scheme 4, and by coupling the compound (XI) with the carboxylic acid compound (VI) on the left side (see WO 00/23421). Or, the desired Z group may be introduced later according to the process of Reaction Scheme 4 after the carboxylic acid compound (VI) is combined with an aspartic acid (β-t-Bu) methyl ester and hydrolyzed. When Z is F, the racemic compound may be prepared according to a method known in Tetrahedron Letters, 1994, 35(52), 9693-9696.
[188] [189] Reaction Scheme 4 [190]
Figure imgf000015_0001
Figure imgf000015_0002
Xi
[191] [192] XI a) Z = OCOPh (2,6-dichloro) [193] b) Z = OPh (2,3,5,6-tetrafluoro) [194] c) Z = F (racemic) [195] [196] The acrylate derivative (IV) used as a reactant in the above Reaction Schemes 1 and 2 may be prepared via two (2) pathways. That is, the compound (IV) can be easily prepared from a known compound (XII) (Synthesis, 1982, p 924) as depicted in the following Reaction Scheme 5, or in case the compound (IV) is methyl (ethyl) 2-i-propylacrylate, the compound (IV) can be prepared from dimethyl(diethyl)malonate (/. Chemical Society Perkin Trans. 1 1997, 1559-1570).
[197] [198] Reaction Scheme 5 [199]
Figure imgf000016_0001
[200]
[201] IV a) P = Et, R2= Me (Commercially available)
[202] b) P = Et, R2 = Et
[203] c) P = Me, R2 = i-Pr
[204] d) P = Et, R2 = n-Pr
[205] e) P = Et, R2 = n-Bu
[206] f) P = Et, R2 = i-Bu
[207] g) P = Et, R2 = CH Ph
[208] h) P = Et, R2 = CH OPh
[209] i) P1 = Et, R2 = CH2OMe
[210]
[211] The compound of formula (1) according to the present invention has a broad spectrum of inhibitory activity against caspases as demonstrated by the results of the following Experiments, and so has an effect for preventing inflammation and apoptosis. Thus, the present invention provides a composition for inhibiting caspases, specifically a therapeutic composition for preventing inflammation and apoptosis, comprising the compound of formula (1), or pharmaceutically acceptable salt, physio¬ logically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as an active ingredient together with the pharmaceutically acceptable carrier. Specifically, the composition of the present invention has a therapeutic effect for dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injure by hepatitis, hepatitis-induced hepatic diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, cardiac cell apoptosis due to ischemic cardiac diseases, or liver cirrhosis.
[212]
[213] Caspase inhibitor, particularly the compound of formula (1), may be formulated into various pharmaceutical forms for administration purpose. To prepare the phar¬ maceutical composition according to the present invention, an effective amount of the caspase inhibitor, particularly the compound of formula (1), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof, is mixed with a pharmaceutically acceptable carrier that may take a wide variety of forms depending on the formulation to be prepared.
[214]
[215] The caspase inhibitor compound may be formulated as a parenteral injection, or percutaneous or oral preparation, depending on its application purpose. It is especially advantageous to formulate the composition in a unit dosage form for ease of admin¬ istration and uniformity of dosage.
[216]
[217] For the oral preparation, any usual pharmaceutical carrier may be used. For example, water, glycols, oils, alcohols and the like may be used for such oral liquid preparations as suspensions, syrups, elixirs and solutions; or starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like may be used for such solid preparations as powders, pills, capsules and tablets. Due to their ease of administration, tablets and capsules are the most advantageous dosage unit forms. It is also desirable for tablets and pills to be formulated into enteric-coated preparation.
[218]
[219] For the parenteral preparation, sterile water is usually used as the carrier, though other ingredients such as solubility aids may be used. Injections, for example, sterilized aqueous or oily suspension for injection, can be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent. Solvents that can be used for preparing injections include water, Ringer's fluid, and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non- stimulative fixing oil including mono- or di-glyceride may be used for this purpose. Fatty acid such as oleic acid may also be used for injections.
[220]
[221] For the percutaneous administration, the carrier may include a penetration enhancing agent and/or a suitable wetting agent, optionally combined with suitable additives having no significant skin irritation. Said additives may facilitate the admin¬ istration through the skin and/or may assist preparation of a desired composition. These percutaneous preparations are administered via various manners, e.g., as a transdermal patch, a spot-on, or an ointment.
[222]
[223] When the caspase inhibitor, specifically the compound of formula (1), is used for clinical purpose, it is preferable to administer to the subject patient in an amount ranging from 0.1 to 100 D per D of body weight a day. The total daily dosage may be ad¬ ministered once or over several times. However, specific administration dosage for an individual patient can be varied with specific compound used, body weight, gender, hygienic condition, or diet of subject patient, time or method of administration, excretion rate, mixing ratio of agent, severity of disease to be treated, etc.
[224]
[225] The present invention will be more specifically explained by the following examples. However, it should be understood that these examples are intended to illustrate the present invention but not in any manner to limit the scope of the present invention. In the following examples, the compounds of Examples 3 to 14 were prepared according to the same procedure as Example 1 or 2.
[226]
[227] Preparation 1
[228] 2-Oxo-2-phenylacetaldehyde oxime
[229] Phenyl glyoxal hydrate (5.09g, 33.5mmol) was dissolved in ethanol (60 D )-water
(30 D ), hydroxylamine hydrochloride (2.8Og, 1.2eq) and anhydrous sodium carbonate (Na CO , 2.13g, 0.6eq) were added thereto at 00C, and the mixture was stirred for one hour at room temperature. Saturated aqueous sodium chloride solution (100 D ) was added thereto, and the mixture was extracted twice with ethyl acetate (300 D ). The extract was washed with dilute aqueous sodium bicarbonate solution (NaHCO , 100 D x 2), dried (anhydrous Na SO ), and concentrated under reduced pressure to give the title yellow oxime compound in a stoichiometric yield (5.06g).
[230] 1 H-NMR (500MHz, CDCl3) δ 8.35(bs, IH), 8.05(m, 3H), 7.59(t, IH), 7.48(m, 2H)
[231]
[232] Preparation 2
[233] 7V-hydroxy-2-oxo-2-phenylethanimidoyl chloride
[234] The oxime compound prepared in Preparation 1 (33.5 mmol) was dissolved in dimethylformamide (100 D ), and N-chlorosuccinimide (4.7Og, 1.05eq) was added thereto. This solution was stirred for one hour in a water bath of about 400C, and the volatile solvent was removed by distillation under reduced pressure. The residue was dissolved in ethyl acetate-hexane (1:1, 150 D ), washed with water (100 D x 3), dried
(anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the title compound (3.86g, 99%). This compound was used in the next reaction without further purification.
[235] 1 H-NMR (500MHz, CDCl ) δ 8.75(s, IH), 7.99(d, 2H), 7.62(t, IH), 7.47(t, 2H)
[236]
[237] Preparation 3
[238] Ethyl 2-ethylacrylate
[239] To CuCN (26.9g, pre-dried under vacuum) was added about 500 D of anhydrous tetrahydrofuran under nitrogen atmosphere. The mixture was maintained at -780C, and
100 D of methyl magnesium bromide (3.0M in ethylether) was slowly added thereto, with stirring by mechanical stirrer. The thick mixture was stirred for about 30 minutes at -78°C, and ethyl 2-bromomethylacrylate (28.9g, 150 mmol, Synthesis: Villieras, J. and Rambaud, M. Synthesis, 1982, 914) dissolved in about 30 D of anhydrous tetrahydrofuran was slowly added thereto. The reaction mixture was slowly warmed to room temperature over 2 hours. Saturated aqueous ammonium chloride solution (-50 D ) was slowly added thereto to complete the reaction. The reaction mixture was filtered through a cellit to remove the precipitate, and washed with ethylether. The organic layer was washed with water and saturated aqueous sodium bicarbonate solution (300 D x 2), dried (anhydrous Na SO ), and concentrated under reduced pressure to give
2 4 transparent liquid (26.7g, stoichiometric yield). This liquid was identified to be the title compound having about 75% weight purity by 1H-NMR (CDCl , 500MHz) analysis.
[240] l H-NMR (500 MHz, CDCl ) δ 6.12(1H, s), 5.50(1H, s), 4.20(2H, q, J = 7.3 Hz),
2.31(2H, qt), 1.28(3H, t, J = 7.3 Hz), 1.07(3H, t, J = 7.8Hz)
[241]
[242] Preparation 4
[243] Ethyl 3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolecarboxylate
[244] Hydroxamoyl chloride prepared in Preparation 2 (1.84, lO.Ommol) and ethyl
2-ethylacrylate prepared in Preparation 3 (1.54g, 1.2eq) were dissolved in anhydrous ether (100 D ) under nitrogen atmosphere, and maintained at -78°C. Triethylamine (2.79 D , 2.02g, 2.0eq) was added thereto. The mixture was stirred overnight while slowly warming it to room temperature. Water (100 D ) was added thereto, and the mixture was extracted with ethyl acetate (100 D x 2), washed with water (100 D ), dried (anhydrous Na SO ), and concentrated under reduced pressure. The residue was separated by column chromatography (10% ethyl acetate-hexane) to give the title compound (1.05g, 38%), which was then analyzed by H-NMR to be a mixture of about 1:1 di- astereomers.
[245] l H-NMR (500MHz, CDCl ) δ 8.20(d, 2H), 7.59(t, IH), 7.47(t, 2H), 4.28(m, 2H),
3.78(d, IH), 3.30(d, IH), 2.06(m, 2H), 1.32(t, 3H), 1.00(t, 3H)
[246]
[247] Preparation 5
[248] S-Benzoyl-S-ethyl^S-dihydro-S-isoxazolecarboxylic acid
[249] The compound prepared in Preparation 4 (260mg) was dissolved in distilled tetrahydrofuran (10 D ), and IN aqueous sodium hydroxide solution (1.42 D , 1.5eq) was added thereto. After about 3 hours, the solution was neutralized by IN aqueous hy¬ drochloric acid solution, and distilled under reduced pressure to remove most of the tetrahydrofuran. The residue was dissolved in excess ethyl acetate (50 D ), washed with aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the title compound in a stoichiometric yield. This compound was used in the next reaction without further purification.
[250] Mass : M+H 248
[251]
[252] Preparation 6
[253] (3^-3-{[(benzyloxy)carbonyl]amino}-5-(terf-butoxy)-2-hydroxy-5-oxopentyl
2,6-dichlorobenzoate
[254] To N-benzyloxycarbonyl-β-t-butyl-aspartic acid (5.03g, 15.6mmol) and NMM
(1.90 D , π.lmmol) was added anhydrous tetrahydrofuran (60 D ) under nitrogen atmosphere, which was then maintained at -15°C. Isobutylchloroformate (2.12 D , 16.3mmol) was added thereto, and the mixture was stirred for about 20 minutes. While maintaining the reaction temperature at 00C, diazomethane-ether solution (synthesized from 2.0eq of l-methyl-3-nitro-l-nitroso-guanidine, 60 D ) was added thereto to give diazoketone derivative (-30 minutes). 30% HBr/ AcOH (6.42 D , 2.0eq) was added thereto to give bromomethylketone derivative (30-60 minutes), which was then extracted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution (x2) and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the bromomethylketone derivative (6.4g).
[255]
[256] The bromomethylketone derivative (4.36g) and 2,6-dichlorobenzoic acid (2.28g, l.leq) were dissolved in dimethylformamide (18 D ), KF (1.58g, 2.5eq) was added thereto, and the mixture was stirred for 2 hours. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution (x2) and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated again under reduced pressure to give 2,6-dichlorobenzoyloxymethylketone derivative. This compound was dissolved in methanol (20 D ) and reacted by adding NaBH 4 (412mg)-methanol solution (40 D )
(-10°C-r.t., 2 hours). The reaction was stopped by acetic acid, and distillation under reduced pressure was carried out to remove methanol. The residue was extracted with ethyl acetate (50 D x 2), washed with water and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure. The residue was separated-purified by column chromatography (ethyl acetate-hexane, 1:5) to give the title compound (4.8Og, 86%) in a diastereomeric form.
[257] * H-NMR (400MHz, CDCl ) δ 7.3-7.2(m, 8H), 5.9(m, IH), 5.2(m, 4H), 4.7(m,
IH), 2.9(m, IH), 2.7(m, IH), 1.4(s, 9H)
[258]
[259] Preparation 7
[260] (3»S>3-amino-5-(terf-butoxy)-2-hydroxy-5-oxopentyl 2,6-dichlorobenzoate
[261] The compound prepared in Preparation 6 was dissolved in methanol (300 D ), Pd/C (10%, 1.5Og) was added thereto, and benzyloxycarbonyl group was removed (PdJC) for 3 hours in a hydrogen balloon to give the title compound (100%).
[262] * H-NMR (400MHz, DMSO-d ) δ 8.2(br, 2H), 7.6-7.5(m, 3H), 6. l(m, IH),
4.4-3.9(m, 3H), 3.0-2.6(m, 2H), 1.4(s, 9H)
[263]
[264] Preparation 8
[265] (3^)-3-{[(3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-( tert - butoxy)-2,5-dioxopentyl 2,6-dichlorobenzoate
[266] A mixture of the carboxylic acid derivative prepared in Preparation 5 (Via, 80mg,
0.324mmol), the amino alcohol derivative prepared in Preparation 7 (147mg, l.leq), and HATU (160mg, 1.3eq) was cooled to 00C, triethylamine (0.14 D , 3.0eq) in DMF (5 D ) solvent was added thereto, and the resulting mixture was reacted for 5 hours. The solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate (30 D x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under
2 4 reduced pressure. The residue was purified by column chromatography (20-30% ethyl acetate-hexane) to give Compound [IX(a)] (172mg, 87%). To this compound and Dess-Martin reagent (356mg, 3.0eq) was added anhydrous dichloromethane (4 D ), which was then stirred for 1 hour at room temperature. Isopropyl alcohol (I D ) was added thereto to stop the reaction. The solid was removed by filtration through cellite under reduced pressure, and the filtrate was extracted with ethyl acetate (20 D x 2). The extract was washed with water, saturated aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was primarily purified by column chromatography (20-25% ethyl acetate-hexane) to give the title compound (138mg, 82%) in a di- astereomeric form.
[267] * H-NMR (500MHz, CDCl ) δ 8.15(m, 2H), 7.70(m, IH), 7.59(m, IH), 7.45(m,
2H), 7.34-7.27(m, 3H), 5.18-5.02(m, 2H), 4.95(m, IH), 3.72(d, IH), 3.35(two d, IH), 2.97-2.79(m, 2H), 2.17(m,lH), 1.99(m, IH), 1.45 & 1.38(two s, 9H), 1.06 & 1.01(two t, 3H)
[268]
[269] Example 1
[270] (35)-3-{[(3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl] amino}-5-[(2,6-dichlorobenzoyl)oxy]-4-oxopentanoic acid
[271] The compound prepared in Preparation 8 (118mg) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 2 hours, con¬ centrated under reduced pressure, and purified by Prep-TLC (10% methanol- dichloromethane) to give the title compound (91mg, 85%, white powder).
[272] 1 H-NMR (400MHz, DMSO-d ) δ 8.71(bs, IH), 8.03(d, 2H), 7.69(m, IH),
7.61-7.36(m, 5H), 5.16(m, 2H), 4.82(m, IH), 3.60(m, 2H), 3.11-2.70(m, 2H), 2.06-1.91(m,2H), 0.89(m, 3H)
[273]
[274] Preparation 9
[275] 2-(l-Naphthyl)-2-oxo-acetaldehyde oxime
[276] To l'-acetonaphthone (5.11g, 30.0mmol) and CuBr (8.04g, 1.2eq) was added ethyl acetate (100 D ), which was then refluxed over one day. CuBr(0.3eq) was added thereto further, and the mixture was refluxed again for 3 hours until l'-acetonaphthone was removed. The solid was removed by filtration through cellite, and the organic layer was washed 2-3 times with water, washed with aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure to give
2 4
7.7g of crude compound.
[277]
[278] The compound obtained above (2.38g, 9.55mmol) was dissolved in DMSO (20 D ), and stirred for one day at room temperature. The reaction mixture was dissolved in ethyl acetate (100 D ), washed with water (100 D x 3) and aqueous sodium chloride solution, and purified by column chromatography (50% ethyl acetate -hexane) to give glyoxal (1.Og, Purity 50%). The glyoxal was dissolved in ethanol (20 D )-water (10 D ), hydroxylamine hydrochloride (415mg, l.leq) and anhydrous sodium carbonate (Na CO , 320mg, 0.55eq) were added thereto at 00C, and the mixture was stirred for 1 hour at room temperature. Saturated aqueous sodium chloride solution (30 D ) was added thereto, and the mixture was extracted twice with ethyl acetate (60 D ), dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the title yellow oxime compound (530mg, 49%).
[279] 1 H-NMR (500MHz, CDCl3) δ 8.84(d, IH), 8.25(bs, IH), 8.09(s, IH), 8.03(d, IH),
7.91(d, IH), 7.86(d, IH), 7.62-7.51(m, 3H)
[280]
[281] Preparation 10
[282] 7V-hydroxy-2-(l-naphthyl)-2-oxoethanimidoyl chloride
[283] The oxime compound prepared in Preparation 9 (530mg, 2.66mmol) was dissolved in dimethylformamide (10 D ), and N-chlorosuccinimide (373mg, 1.05eq) was added thereto. This solution was stirred for 1 hour in a water bath of about 400C, and the volatile solvent was removed by distillation under reduced pressure. The residue was dissolved in ethyl acetate-hexane (1:1, 50 D ), washed with water (20 D x 2) and saturated aqueous sodium chloride solution (30 D ), dried (anhydrous NaSO ), and con¬ centrated under reduced pressure to give the title compound (621mg) in a stoi- chiometric yield. This compound was used in the next reaction without further pu¬ rification.
[284]
[285] Preparation 11
[286] Ethyl 5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolecarboxylate
[287] The hydroxamoyl chloride prepared in Preparation 10 (2.66mmol) and ethyl
2-ethylacrylate (374mg, l.leq) were dissolved in anhydrous ether (30 D ) under nitrogen atmosphere, maintained at 00C, and triethylamine (0.74 D , 538mg, 2.0eq) was added thereto. While slowly warming the mixture to room temperature, it was stirred for 2 hours. Water (30 D ) was added thereto, and the mixture was extracted with ethyl acetate (50 D x 2), washed with saturated aqueous sodium bicarbonate solution and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under
2 4 reduced pressure. The residue was purified by column chromatography (10% ethyl acetate-hexane) to give the title compound (500mg, 57%).
[288] 1 H-NMR (500MHz, CDCl ) δ 8.42(d, IH), 8.08(d, IH), 8.05(d, IH), 7.91(d, IH),
7.62-7.52(m, 3H), 4.32(m, 2H), 3.87(d, IH), 3.39(d, IH), 2.11(m, 2H), 1.36(t, 3H), 1.04(t, 3H)
[289]
[290] Preparation 12
[291] 5-Ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolecarboxylic add
[292] The compound prepared in Preparation 11 (500mg) was dissolved in distilled tetrahydrofuran (10 D ), and IN aqueous sodium hydroxide solution (1.69 D , 1.5eq) was added thereto. After about 0.5 hour, the solution was neutralized by IN aqueous hy¬ drochloric acid solution, and distilled under reduced pressure to remove most of the tetrahydrofuran. The residue was dissolved in excess ethyl acetate (50 D ), washed with aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under
2 4 reduced pressure. The residue was purified by column chromatography (30% EA/ Hex- 10% to 20% MeOH/CH Cl ) to give the title compound (440mg, 96%) as yellow powder.
[293] Mass : M+H 298
[294]
[295] Preparation 13
[296] tert-Butγl (3S)-3-{[(benzyloxy)carbonyl] - amino}-4-hydroxy-5-(2,3,5,6-tetrafluorophenoxy)pentanoate
[297] To N-benzyloxycarbonyl-β-t-butyl-aspartic acid (18.5g, 55.7mmol) and NMM
(6.78 D , l.leq) was added anhydrous tetrahydrofuran (180 D ) under nitrogen atmosphere, and the mixture was maintained at -15°C. Then, isobutylchloroformate (7.63 D , 1.05eq) was added thereto, and the mixture was stirred for about 20 minutes. While maintaining the reaction temperature at 00C, diazomethane-ether solution (synthesized from 2.0eq of l-methyl-3-nitro-l-nitroso-guanidine, 60 D ) was added thereto to give diazoketone derivative (~3 minutes). 30% HBr/ AcOH (23.3 D , 2.0eq) was added thereto to give bromomethylketone derivative (30-60 minutes), which was then extracted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution (x2) and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure to give the bromomethylketone derivative (23g) in a stoichiometric yield.
[298]
[299] The bromomethylketone derivative (23g, 55.7mmol) and 2,3,5,6-tetrafluorophenol
(10.2g, l.leq) were dissolved in dimethylformamide (150 D ), KF (8.14g, 2.5eq) was added thereto, and the mixture was stirred for 2 hours at room temperature. The mixture was concentrated under reduced pressure, extracted with ethyl acetate, washed with saturated aqueous sodium bicarbonate solution (x2) and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated again under reduced pressure to
2 4 give 2,3,5,6-tetrafluorophenoxymethylketone derivative. This compound was dissolved in methanol (150 D ) and reacted by slowly adding NaBH 4 (4.24g) (0°C-r.t., 2 hours).
The reaction was stopped by acetic acid, and distillation under reduced pressure was carried out to remove methanol. The residue was extracted with ethyl acetate (200 D x 2), washed with water and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4
), and concentrated under reduced pressure. The residue was separated-purified by column chromatography (15% ethyl acetate/hexane) to give the title compound (20.2g,
74%) in a diastereomeric form. [300] Mass : M+H 488
[301]
[302] Preparation 14
[303] terf-Butyl (3^-3-amino-4-hydroxy-5-(2,3,5,6-tetrafluorophenoxy)-pentanoate
(XIb) [304] The compound prepared in Preparation 13 was dissolved in methanol (300 D ), Pd/C
(10%, 1.50g) was added thereto, and benzyloxycarbonyl group was removed (Pd/C) for 3 hours in a hydrogen balloon to give the title compound (95%). [305] 1 H-NMR (400MHz, DMSO-d ) δ 8.2(br, 2H), 7.6-7.5(m, IH), 5.9(m, IH),
4.3-4.1(m, 3H), 3.6(m, IH), 2.7(m, IH), 1.4(s, 9H) [306]
[307] Preparation 15
[308] fert-Butyl (35)-3-({[5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl}-amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoate [309] A mixture of the carboxylic acid derivative prepared in Preparation 12 (140mg, 0.471mmol), the aminoalcohol derivative prepared in Preparation 14 (182mg, l.leq) and HATU (233mg, 1.3eq) was cooled to 00C, triethylamine (0.20 D , 3.0eq) in DMF (5 D ) solvent was added thereto, and the resulting mixture was reacted for 5 hours. The solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate (30 D x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under
2 4 reduced pressure. The residue was purified by column chromatography to give Compound[IX(b)] (248mg, 83%). To this compound and Dess-Martin reagent (482mg, 3.0eq) was added anhydrous dichloromethane (8 D ), which was then stirred for 1 hour at room temperature. Isopropyl alcohol (I D ) was added thereto to stop the reaction. The solid was removed by filtration through cellite under reduced pressure. The residue was extracted with ethyl acetate (30 D x 2), washed with water, saturated aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was primarily purified by column chromatography (20-25% ethyl acetate-hexane) to give the title compound (150mg, 65%) in a diastereomeric form.
[310] 1 H-NMR (500MHz, CDCl ) δ 8.35(t, IH), 7.97(m, 2H), 7.82(d, IH), 7.52-7.42(m,
4H), 6.70(m, IH), 5.08-4.90(m, 3H), 3.72(two d, IH), 3.34(two d, IH), 2.96-2.72(m, 2H), 2.11(m, IH), 1.95(m, IH), 1.34 & 1.28(two s, 9H), 0.97(two t, 3H)
[311]
[312] Example 2
[313] (35)-3-({[5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl}amino)-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid
[314] The compound prepared in Preparation 15 (150mg, 0.238mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and purified by Prep-TLC (10% methanol- dichloromethane) to give the title compound (109mg, 80%, white powder).
[315] * H-NMR (500MHz, DMSO-d ) δ 8.70(two d, IH), 8.20(m, 2H), 8.05(m, IH),
7.98(m, IH), 7.61(m,4H), 5.24(m, 2H), 4.81(two q, IH), 3.67(two d, IH), 3.47(two d, IH), 2.84(m, IH), 2.65(m, IH), 2.00(m, 2H), 0.91(m, 3H)
[316] Mass : M+H 575
[317]
[318] Preparation 16
[319] tert-Butyl 3-({[5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl]carbonyl} amino)-5-fluoro-4-oxopentanoate
[320] A mixture of the carboxylic acid derivative prepared in Preparation 12 (170mg,
0.572mmol), the aminoalcohol derivative (XIc, 135mg, l.leq), and HATU (283mg, 1.3eq) was cooled to 00C, triethylamine (0.24 D , 3.0eq) in DMF (5 D ) solvent was added thereto, and the mixture was reacted for 5 hours. The solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate (30 D x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was purified by column chromatography (30-40% ethyl acetate-hexane) to give Compound [IX(c)] (200mg, 72%). To this compound and Dess-Martin reagent (520mg, 3.0eq) was added anhydrous dichloromethane (6 D ), which was then stirred for 1 hour at room temperature. Isopropyl alcohol (I D ) was added thereto to stop the reaction. The solid was removed by filtration through cellite under reduced pressure. The filtrate was extracted with ethyl acetate (30 D x 2), washed with water, saturated aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure. The residue was purified by column chromatography (30% ethyl acetate-hexane) to give the title compound (186mg, 93%) in a diastereomeric form.
[321] * H-NMR (500MHz, CDCl ) δ 8.41(m, IH), 8.04(m, 2H), 7.90(d, IH),
7.65-7.52(m, 4H), 5.20-4.94(m, 3H), 3.78(m, IH), 3.41(two d, IH), 3.03-2.7(m, 2H), 2.19(m, IH), 2.01(m, IH), 1.42 & 1.35(two s, 9H), 1.04(two t, 3H)
[322]
[323] Example 3
[324] 3-({[5-Ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl] carbonyl}amino)-5-fluoro-4-oxopentanoic add
[325] The compound prepared in Preparation 16 (180mg, 0.371mmol) was reacted according to the same procedure as Example 2 to give the title compound (quantitative yield).
[326] * H-NMR (500MHz, DMSO-d ) δ 8.65(m, IH), 8.21(m, 2H), 8.02(m, 2H), 7.63(m,
3H), 5.25(m, 2H), 4.79(m, IH), 3.68(m, IH), 3.46(m, IH), 2.83(m, IH), 2.63(m, IH), 2.00(m, 2H), 0.90(m, 3H)
[327]
[328] Preparation 17
[329] 2-Diazo-l-(l-isoquinolinyl)-l-ethanone
[330] To 1-isoquinolinecarboxylic acid (1.73g, lO.Ommol) and NMM (1.50 D , 1.4eq) was added anhydrous tetrahydrofuran (10 D ) under nitrogen atmosphere. While maintaining the mixture at 00C, isobutylchloroformate (1.36 D , 1.05eq) was added thereto and the mixture was stirred for about 2 hours. While maintaining the reaction mixture at 00C, diazomethane-ether solution (synthesized from 2.0eq of l-methyl-3-nitro-l-nitroso-guanidine, 20 D ) was added thereto to give diazoketone derivative (1 hour), which was then extracted with ethyl acetate, washed with water, saturated aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was purified by column chromatography (10-15% EA/Hex) to give the title diazo derivative (1.50g, 76%, yellow powder).
[331] * H-NMR (500MHz, CDCl ) δ 9.29(d, IH), 8.5 l(d, IH), 7.88-7.83(m, 2H),
7.76-7.69 (m, 2H), 6.7 l(s, IH)
[332]
[333] Preparation 18
[334] 2-(l-Isoquinolinyl)-2-oxoacetaldehyde oxime
[335] The diazo derivative prepared in Preparation 17 (670mg, 3.40mmol) was dissolved in acetone (5 D ), dimethyldioxirane-acetone solution (0.1M, 30 D ) was added thereto, and the mixture was stirred for 2-3 minutes at room temperature. Water was added thereto, and the mixture was distilled under reduced pressure to remove acetone. The residue was extracted with ethyl acetate, washed with aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure. The
2 4 residue was dissolved in ethanol-water (2:1, 20 D ), hydroxylamine hydrochloride (236mg, l.Oeq) and anhydrous sodium carbonate (Na CO , 180mg, 0.5eq) were added thereto at 00C, and the mixture was stirred for 30 minutes at room temperature. Saturated aqueous sodium chloride solution (30 D ) was added thereto. The mixture was extracted twice with ethyl acetate (60 D ), dried (anhydrous Na 2SO 4 ), and concentrated under reduced pressure. The residue was purified by column chromatography (20-30%
EA/Hex) to give the title oxime derivative (306mg, 45%) having yellow color. [336] 1 H-NMR (500MHz, CDCl ) δ 10.28(bs, IH), 8.69(d, IH), 8.62(s, IH), 8.57(d,
IH), 7.89(d, IH), 7.84(d, IH), 7.77-7.69(m, 2H) [337]
[338] Preparation 19
[339] Ethyl 5-ethyl-3-(l-isoquinolinylcarbonyl)-4,5-dihydro-5-isoxazolecarboxylate
[340] The compound of Preparation 18 was reacted according to the same procedure as
Preparations 10 and 11 to give the title compound. [341] 1 H-NMR (400MHz, CDCl ) δ 8.63(d, IH), 8.26(d, IH), 7.90(d, IH), 7.82(d, IH),
7.74(t, IH), 7.66(t, IH), 4.3 l(m, 2H), 3.93(d, IH), 3.39(d, IH), 2.09(qt, 2H), 1.34(t,
3H), 1.02(t, 3H) [342]
[343] Preparation 20
[344] t-Butyl 3-({[5-ethyl-3-(l-isoquinolinylcarbonyl)-4,5-dihydro-5-isoxazolyl] carbonyl}amino)-5-fluoro-4-oxopentanoate [345] The compound of Preparation 19 was reacted according to the same procedure as
Preparations 12 and 16 to give the title compound. [346] * H-NMR (500MHz, CDCl ) δ 8.61(m, IH), 8.39(m, IH), 7.91(d, IH), 7.84(d, IH),
7.75(t, IH), 7.70-7.56(m, 2H), 5.29-5.00(m, 2H), 4.92(m, IH), 3.86-3.80(two d, IH), 3.47-3.43(two d, IH), 3.04-2.76(m, 2H), 2.23-2.15(m, IH), 2.08-1.98(m, IH), 1.41-1.36(two s, 9H), 1.03(two t, 3H)
[347]
[348] Example 4
[349] 3-({[5-Ethyl-3-(l-isoquinolinylcarbonyl)-4,5-dihydro-5-isoxazolyl]carbonyl} amino)-5-fluoro-4-oxopentanoic acid
[350] The compound prepared in Preparation 20 (108mg, 0.223mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and purified by Prep-TLC (15% methanol- dichloromethane) to give the title compound (68mg, 72%, a little yellow powder).
[351] * H-NMR (500MHz, DMSO-d ) δ 8.60(m, IH), 8.23(m, IH), 8.13-8.06(m, 2H),
7.87(t, IH), 7.76(t, IH), 5.20(bs, 2H), 4.72(m, IH), 3.75-3.69(two d, IH), 3.53-3.46(two d, IH), 2.83-2.65(two bs, 2H), 2.06-1.90(two m, 2H), 0.92-0.84(two t, 3H)
[352] MS ; M+MeOH+H 462
[353]
[354] Preparation 21
[355] tert-Butyl 2-chloro-2-(hydroxyimino)acetate
[356] Glycine t-butyl ester HCl salt (12.Og, 71.6mmmol) synthesized according to a method known in /. of Chemical Society, PT 1, 1997, 3005 was dissolved in water (60 D ), c-HCl solution (5.97 D , l.Oeq) was added thereto once at 00C, and immediately NaNO solution (4.95g in 15 D water, l.Oeq) was added thereto. Again, each l.Oeq of c- HCl and NaNO 2 solutions was added thereto. The mixture was stirred for about 10 minutes at 00C, extracted with ethyl acetate (50 D x 3), washed with aqueous sodium chloride solution, dried (anhydrous Na SO ), and concentrated under reduced pressure
2 4 to give the title compound (6.3g, 49%). [357]
[358] Preparation 22
[359] 3-(tert-Butyl) 5-ethyl 5-ethyl-4,5-dihydro-3,5-isoxazoledicarboxylate
[360] Ethyl 2-ethylacrylate (3.0g, 23.4mmol, l.Oeq) and triethylamine (6.52 D , 2.0eq) were dissolved in anhydrous chloroform (40 D ) under nitrogen atmosphere, and maintained at 00C. The hydroxamoyl chloride prepared in Preparation 21 (4.82g, 1.15eq) was dissolved in chloroform (20 D ), and this solution was slowly added thereto over 1 hour. While slowly warming the mixture to room temperature, it was stirred overnight. Water (50 D ) was added thereto, and the mixture was extracted with ethyl acetate (100 D x 2), washed with water (100 D ), dried (anhydrous NSO ), and con¬ centrated under reduced pressure. The residue was separated by column chro¬ matography (10% ethyl acetate-hexane) to give the title compound (3.4g, 53%).
[361] 1 H-NMR (500MHz, CDCl ) δ 4.26(m, 2H), 3.60(d, IH), 3.06(d, IH), 2.00(m, 2H),
1.54(s, 9H), 1.30(t, 3H), 0.95(t, 3H)
[362]
[363] Preparation 23
[364] Ethyl 5-ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazole carboxylate)
[365] The compound prepared in Preparation 22 (400mg, 1.47mmol) was dissolved in
4.0 N HCl(g)/EtOAc (30 D ), stirred for 2 hours, and concentrated under reduced pressure. To the resulting compound were added 1-naphthylamine (211mg, l.Oeq) and HATU (727mg, 1.3eq). Triethylamine (0.61 D , 3.0eq) dissolved in DMF (5 D ) was added thereto, and the mixture was stirred for one day. The solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate (30 D x 2), washed with water, aqueous sodium bicarbonate solution, and aqueous sodium chloride solution, dried (anhydrous Na 2 SO 4 ), and concentrated under reduced pressure. The residue was purified by column chromatography (10-15% ethyl acetate-hexane) to give the title compound (460mg, 92%). [366] 1 H-NMR (500MHz, CDCl3) δ 8.85(bs, IH), 8.07(d, IH), 7.90(d, IH), 7.87(d, IH),
7.58-7.48(m, 3H), 4.32(m, 2H), 3.78(d, IH), 3.30(d, IH), 2.07(m, 2H), 1.35(t, 3H),
1.03(t, 3H) [367]
[368] Preparation 24
[369] tert-Butγl (35)-3-[({5-ethyl-3-[(l-naphthylamino)carbonyl] -
4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pe ntanoate [370] The compound of Preparation 23 was reacted according to the same procedure as
Preparations 12 and 15 to give the title compound. [371] 1 H-NMR (500MHz, CDCl ) δ 8.82(d, IH), 8.07(m, IH), 7.88(m, 2H), 7.73(m,
2H), 7.54(m, 3H), 6.79(two m, IH), 5.16-4.96(m, 3H), 3.75(dd, IH), 3.33(d, IH),
3.07-2.81(m, 2H), 2.18(m,lH), 2.01(m, IH), 1.43 & 1.41(two s, 9H), 1.03(two t, 3H) [372] Mass M+Na 668
[373]
[374] Example 5
[375] (3^)-3-[({5-ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl} carbonyl)-amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid [376] The compound prepared in Preparation 24 (178mg, 0.276mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and purified by Prep-TLC (7.5% methanol- dichloromethane) to give the title compound (quantitative yield). [377] * H-NMR (500MHz, CD OD) δ 8.13-8.05(m, 2H), 7.96(d, IH), 7.78(m, IH),
7.70-7.61(m, 3H), 7.29-7. l l(m, IH), 3.82-3.46(m, 2H), 3.10-2.92(m, 2H), 2.23(m,
IH), 2.15(m, IH), 1.16(m, 3H) [378]
[379] Example 6
[380] 3-[({5-Ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl} carbonyl)amino]-5-fluoro-4-oxopentanoic acid [381] The compound of Preparation 23 was reacted according to the same procedure as
Preparations 12, 16 and Example 1 to give the title compound. [382] * H-NMR (500MHz, DMSO-d ) δ 10.53(d, IH), 8.61(bs, IH), 7.98-7.83(m, 3H),
7.62-7.52(m, 4H), 5.40-4.45(bs, 2H), 4.81-4.70(m, IH), 3.65-3.58(two set of d, IH),
3.38(m, IH), 2.95-2.68(m, 2H), 2.05-1.90(m, 2H), 0.94-0.80(m, 3H) [383] Mass M+H 444.1
[384]
[385] Preparation 25
[386] Ethyl 3-[([l,l'-biphenyl]-2-ylamino)carbonyl]-5-ethyl-4,5-dihydro-5-isoxazole carboxylate [387] The compound of Preparation 22 was reacted according to the same procedure as
Preparations 23 using 2-aminobiphenyl instead of 1-naphtylamine to give the title compound. [388] * H-NMR (500MHz, CDCl ) δ 8.57(d, IH), 8.42(d, IH), 7.57-7.15(m, 8H), 4.25(m,
2H), 3.68(d, IH), 3.18(d, IH), 1.99(m, 2H), 1.30(t, 3H), 0.94(t, 3H) [389]
[390] Preparation 26
[391] tert-Butγl (3S)-3-[({3-[([l,l'-biphenyl]-2-ylamino)carbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophe noxy)pentanoate [392] The compound of Preparation 25 was reacted according to the same procedure as
Preparations 12 and 15 to give the title compound. [393] 1 H-NMR (400MHz, CDCl ) δ 8.5 l(s, IH), 8.39(m, IH), 7.55-7.22(m, 9H),
6.78(m, IH), 5.08-4.90(m,3H), 3.61(two d, IH), 3.21(two d, IH), 3.03-2.78(m, 2H),
2.08(m, IH), 1.89(m, IH), 1.39(two s, 9H), 0.95(two t, 3H) [394] [395] Example 7 [396] (35)-3-[({3-[([l,l'-biphenyl]-2-ylamino)carbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,64etrafluorophe noxy)pentanoic acid
[397] The compound prepared in Preparation 26 (106mg, 0.158mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and purified by Prep-TLC (7.5% methanol- dichloromethane) to give the title compound (quantitative yield).
[398] * H-NMR (400MHz, DMSO-d ) δ 9.51(m, IH), 8.61(br, IH), 7.74(m, IH), 7.51(m,
IH), 7.42-7.30(m, 8H), 5.18(br, 2H), 4.75(m, IH), 3.42(two d, IH), 3.13(two d, IH), 2.91-2.49(m, 2H), 1.98-1.77(m, 2H), 0.82(two t, 3H)
[399] Mass : M+H 616
[400]
[401] Example 8
[402] 3-[({3-[([l,l'-Biphenyl]-2-ylamino)carbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoic acid
[403] The compound of Preparation 25 was reacted according to the same procedure as
Preparations 12, 16 and Example 1 to give the title compound.
[404] 1 H-NMR (400MHz, DMSO-d ) δ 9.54(m, IH), 8.57-8.5 l(two set of d, IH),
7.75(m, IH), 7.47-7.3 l(m, 8H), 5.22-5.08(m, 2H), 4.76-4.67(m, IH), 3.46-3.12(m, 2H), 2.95-2.53(m, 2H), 1.90-1.81(m, 2H), O.88-O.8O(m, 3H)
[405] Mass M+H 444.1
[406]
[407] Preparation 27
[408] Ethyl 5-ethyl-3-{[(2'-methyl[l,lI-biphenyl]-2-yl)amino] carbonylJ-^S-dihydro-S-isoxazolecarboxylate
[409] The compound of Preparation 22 was reacted according to the same procedure as
Preparation 23 using 2-iodoaniline instead of 1-naphtylamine (yield 25%) and then Suzuki coupling (2-tolylboronic acid, Na CO , PdCl (PPh ) , 110°C, DMF, 81%) to give the title compound.
[410] * H-NMR (400MHz, CDCl ) δ 8.43(m, IH), 8.14(s, IH), 7.40-7.15(m, 7H),
4.25(m, 2H), 3.64(m, IH), 3.14(m, IH), 2.10(s, 3H), 1.94(m, 2H), 1.27(m, 3H), 0.92(m, 3H)
[411]
[412] Preparation 28
[413] fert-Butyl 3-{[(5-ethyl-3-{[(2'-methyl[l,l'-biphenyl]-2-yl)amino] carbonyl}-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopentanoate
[414] The compound of Preparation 27 was reacted according to the same procedure as Preparations 12 and 16 to give the title compound.
[415] 1 H-NMR (400MHz, CDCl ) δ 8.43(m, IH), 8.10(s, IH), 7.42-7.13(m, 8H),
5.13-4.82(m, 3H), 3.59(m, IH), 3.16(m, IH), 3.01-2.70(m, 2H), 2.11(two s, 3H), 2.07(m, IH), 1.87(m, IH), 1.43(two s, 9H), 0.93(two t, 3H)
[416]
[417] Example 9
[418] 3-{[(5-Ethyl-3-{[(2'-methyl[l,l'-biphenyl]-2-yl)amino] carbonyl}-4,5-dihydro-5-isoxazolyl)carbonyl]amino}-5-fluoro-4-oxopentanoic add
[419] The compound prepared in Preparation 28 (61mg, 0.113mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and the resulting white-yellow solid was filtered and washed with hexane, to give the title compound (quantitative yield).
[420] * H-NMR (400MHz, DMSO-d ) δ 8.82(m, IH), 8.54(m, IH), 8.01(m, IH),
7.44-7.14 (m, 7H), 5.21-5.05(m, 2H), 4.70(m, IH), 3.37(m, IH), 3.13(m, IH), 2.81-2.51(m, 2H), 2.05(two s, 3H), 1.92-1.76(m, 2H), 0.80(two t, 3H)
[421] Mass : M+H 484
[422]
[423] Preparation 29
[424] Ethyl 3-[3,4-dihydro-l(2/7)-quinolinylcarbonyl] -
S-ethyl^S-dihydro-S-isoxazolecarboxylate
[425] The compound of Preparation 22 was reacted according to the same procedure as
Preparation 23 using 1,2,3,4-tetrahydroquinoline instead of 1-naphtylamine to give the title compound.
[426] * H-NMR (400MHz, CDCl ) δ 7.26-7.10(m, 4H), 4.28(m, 2H), 4.02-3.78(m, 2H),
3.57(m, IH), 3.13(m, IH), 2.79(m, 2H), 2.07-1.94(m, 4H), 1.31(t, 3H), 0.92(t, 3H)
[427]
[428] Preparation 30
[429] tert-Butγl 3-[({3-[3,4-dihydro-l(2fl)-quinolinylcarbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoate
[430] The compound of Preparation 29 was reacted according to the same procedure as
Preparations 12 and 16 to give the title compound.
[431] * H-NMR (400MHz, CDCl ) δ 7.54(m, IH), 7.27-7.07(m, 4H), 5.16-4.80(m, 3H),
3.97-3.78(m, 2H), 3.61-2.78(m, 4H), 2.14-1.77(m, 4H), 1.43(two s, 9H), 0.97(two t, 3H)
[432]
[433] Example 10
[434] 3-[({3-[3,4-Dihydro-l(2#)-quinolinylcarbonyl] - 5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-5-fluoro-4-oxopentanoic acid
[435] The compound prepared in Preparation 30 (42mg, 0.086mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con¬ centrated under reduced pressure, and purified by trituration with dichloromethane/ hexane to give the title compound (quantitative yield).
[436] * H-NMR (400MHz, DMSO-d ) δ 8.54(m, IH), 7.23-7.1 l(m, 4H), 5.23-5.09(m,
2H), 4.70(m, IH), 3.80-3.75(m, 2H), 3.21(m, IH), 2.78-2.60(m, 4H), 1.94-1.90(m, 4H), 0.94(m, 3H)
[437] Mass : M+H 434
[438]
[439] Preparation 31
[440] Ethyl 3-[3,4-dihydro-2(l/7)-isoquinolinylcarbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolecarboxylate
[441] The compound of Preparation 22 was reacted according to the same procedure as
Preparation 23 using 1,2,3,4-tetrahydroisoquinoline instead of 1-naphtylamine to give the title compound.
[442] * H-NMR (400MHz, CDCl ) δ 7.21-7.09(m, 4H), 5.03(s, IH), 4.80(dd, IH),
4.27(m, 2H), 4.18-3.78(m, 2H), 3.71(dd, IH), 3.31(dd, IH), 2.93(m, 2H), 2.02(m, 2H), 1.32(m, 3H), 0.98(m, 3H)
[443]
[444] Preparation 32
[445] tert-Butyl (35)-3-[({3-[3,4-dihydro-2(L»)-isoquinolinylcarbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophe noxy)pentanoate
[446] The compound of Preparation 31 was reacted according to the same procedure as
Preparations 12 and 15 to give the title compound.
[447] * H-NMR (400MHz, CDCl ) δ 8.13(m, IH), 7.82-7.68(m, 2H), 7.51-7.34(m, 4H),
6.85(m, IH), 6.82-6.58(m, IH), 5.11-4.86(m, 5H), 3.56(two d, IH), 3.12(two d, IH), 2.99-2.75(m, 2H), 2.11(m, IH), 1.91(m, IH), 1.43(two s, 9H), 0.97(two t, 3H)
[448]
[449] Example 11
[450] (3S)-3-[({3-[3,4-dihydro-2(l#)-isoquinolinylcarbonyl] -
5-ethyl-4,5-dihydro-5-isoxazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophe noxy)pentanoic acid
[451] The compound prepared in Preparation 32 (50mg, 0.079mmol) was dissolved in dichloromethane (4 D ), and trifluoroacetic acid (2 D ) was added thereto at 00C. While slowly warming the mixture to room temperature, it was stirred for 1 hour, con- centrated under reduced pressure, and purified by Prep-TLC (7.5% methanol- dichloromethane) to give the title compound (quantitative yield). [452] * H-NMR (500MHz, CD OD) δ 7.20-6.98(m, 5H), 5.17-4.7 l(m, 5H), 3.94-3.85 (m,
2H), 3.52(two d, IH), 3.33(two d, Ih), 3.05-2.78(m, 4H), 2.09(m, IH), 1.97(m, IH),
1.00(two t, 3H) [453] Mass : M+H 580
[454]
[455] Example 12
[456] 3-{[(3-{[2-(terf-Butyl)amϊino] carbonylJ-S-ethyl^jS-dihydro-S-isoxazolyOcarbonyllaminoJ-S-fluoro^-oxopentan oic acid [457] The title compound was obtained according to the same procedure as Preparations
23 (2-tert-butylaniline instead of 1-naphthylamine), 12, and 16, and Example 1. [458] * H-NMR (400MHz, CDCl ) δ 8.57(d, IH), 7,87-7.74(m, IH), 7.45-7.33(m, 2H),
7.22-7.18(m, IH), 5.00-4.70(m, 3H), 3.73-3.70(m, IH), 3.31(d, IH), 3.06-2.82(m, 2H),
2.13(m, IH), 1.99(m, IH), 1.45(s, 9H), 1.01(m, 3H) [459] Mass : M+H 450
[460]
[461] Example 13
[462] 3-{[(3-{[2-(terf-Butyl)-anilino] carbonylJ-S-isopropyl^jS-dihydro-S-isoxazoly^carbonyllaminoJ-S-fluoro^-oxope ntanoic acid [463] The title compound was obtained according to the same procedure as Preparations
22 [methyl 2-i-propylacrylate instead of ethyl 2-ethylacrylate (/. Chemical Society
Perkin Trans. 1 1997, 1559-1570)], 23 (2-tert-butylaniline instead of
1-naphthylamine), 12, and 16, and Example 1. [464] l H-NMR (500MHz, CDCl ) δ 8.50(d, IH), 7.83(m, IH), 7.41(d, IH), 7.17(m, IH),
4.79-4.42(m, 3H), 3.66-3.61(two d, IH), 3.38(d, IH), 3.09-2.73(m, 2H), 2.31(m, IH),
1.43(s, 9H), 1.02(m, 6H) [465] Mass : M+H 464
[466]
[467] Example 14
[468] 3-{[(3-{[2,5-Di(terf-butyl)amϊino] carbonyl}-5-isopropyl-4,5-dihydro-5-isoxazolyl)carbonyl]-amino}-5-fluoro-4-oxop entanoic acid [469] The title compound was obtained according to the same procedure as Preparations
22 [methyl 2-i-propylacrylate instead of ethyl 2-ethylacrylate (/. Chemical Society
Perkin Trans. 1 1997, 1559-1570)], 23 (2,5-di-tert-butylaniline instead of 1-naphthylamine), 12, and 16, and Example 1.
[470] 1 H-NMR (500MHz, CDCl ) δ 8.49(d, IH), 7.89(s, IH), 7.33(d, IH), 7.18(dd, IH),
4.77-4.45(m, 3H), 3.66-3.61(dd, IH), 3.38(d, IH), 3.09-2.75(two m, 2H), 2.31(m, IH), 1.41(s, 9H), 1.31(s, 9H), 1.02(m, 6H)
[471] Mass : M+H 520
[472]
[473] Experiment 1
[474] Determination of the caspase inhibitory effect
[475] Caspase- 1 and caspase-8 known as cysteine proteases in the form of α β were expressed, purified, and activated by modifying a method known in Thornberry, N. A. et al, 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, p 10 and p20 subunits (Thornberry, N. A. et al, Nature, 1992, 356, 768) were expressed in E.coli and purified by nickel column and anionic exchange chromatography to give caspase- 1, caspase-8 and caspase-9. The fluorescent substrates AcYVAD-AFC for 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 0C with various concentrations of the inhibitors in a buffer solution containing 2OmM HEPESφH 7.40), 10%(w/v) sucrose, 0.1%(w/v) CHAPS, 10OmM NaCl, ImM EDTA, and 1OmM DTT in the presence of 25μM AcYVAD-AFC for 1OnM caspase- 1, 25 μM AcDEVD-AFC for 2.InM caspase-8, and 150μM AcLEHD-AFC for 20OnM caspase-9. The inhibitory constants K i and K obs of the inhibitors were determined by measuring the reaction velocity with the time lapse using a fluorescent spectrometer and by obtaining the initial rate constant. K was calculated from the Lineweaver Burk Plot, and K from the following Equation 1. obs
[476]
[477] Equation 1
[478] K = -In (I-A /A )/t obs t oo
[479] in which
[480] A means cleavage rate (%) at time t, and
[481] A means the maximum cleavage rate (%).
[482]
[483] Spectra MAX GeminiXS Fluorescent Spectrometer of Molecular Device Co. was used at the excitation wavelength of 400nm and the emission wavelength of 505nm. [484] [485] The in vitro inhibitory activity of the inhibitors was determined by subjecting
Jurkat cell (ATCC TIB- 152) to apoptosis using anti-Fas monoclonal antibody (Upstate Biotech 05-201) and by detecting the color change according to the WST-I method (TAKARA MK400) known in Francoeur A.M. and Assalian A. (1996) Biochemica 3, 19-25 to observe the amount of alive Jurkat cells when the cells were treated by the inhibitor. Spectra MAX 340 Spectrometer of Molecular Device Co. was used at the absorbance wavelength of 440nm.
[486] [487] Table 1
Figure imgf000036_0001
[488] [489] Experiment 2 [490] Therapeutic effect for LPS-induced acute hepatitis in mouse [491] Step 1) Preparation of blood sample [492] Male 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 cycle of 12 hours. Food and water were supplied ad libitum. In pyrogen-free saline were dissolved LPS (lipopolysaccaride) and D-galactosamine in concentrations of 0.4 D / D and 280 D / D , respectively, and their 1:1 mixture was injected to each mouse in the amount of 5 D / D . Immediately after the injection of LPS and D-galactosamine, vehicle (a mixture of PEG400 : ethanol : TweenδO = 15 : 7.5 : 2.5 was diluted by five times with phosphate buffer) wherein the test compound is dissolved or the vehicle alone was in- traperitoneally injected into the mice. After 8 hours from the drug injection, blood samples were obtained from their hearts.
[493] [494] Step 2: Determination of the activity of plasma aminotransferase [495] The plasma ALT activity was determined for the blood samples obtained in Step 1 using ALT assay kit (Asan Pharm. Co., Seoul , Korea ) according to the manufacturer's instruction. The results appeared that the injection of LPS and D-galactosamine sharply increases the ALT activity in plasma, and the test compounds inhibit the increased enzyme activity in a dose-dependent manner. Based on these results, ED values of the test compounds were calculated using Prism software of GraphPad Co.
[496] [497] Table 2
Figure imgf000037_0001

Claims

Claims
[ 1 ] L A compound of the following formula ( 1 ) :
Figure imgf000038_0001
(D in which
I) R represents H, -SAC, -SCAC, -Ar, or -SAC-Ar,
II) R represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; or the compound of formula (1) may have a protecting group in an ester form of -CO R wherein R is -SAC or a sulfonamide form of -CONHSO R wherein R is -SAC, or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety,
III) R2 represents -SAC, -SCAC, -Ar, -SAC-Ar, or a side chain residue of all 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 becomes a stereocenter due to the R group; the compound of formula (1) may have a protecting group in an ester form of -CO R wherein R is -SAC or a sulfonamide form of -CONHSO R wherein R is -SAC, or may exist in the form of pharmaceutically acceptable salt, when R is a side chain residue of an amino acid containing carboxyl moiety; or the compound of formula (1) may also exist in the form of pharmaceutically acceptable salt when R is a side chain residue of an amino acid containing a base moiety, or R further represents H; -(CH 2 ) n OR wherein R7 is -SAC, -SCAC, -Ar, or -SAC-Ar, and n = 1 or 2; or -(CH ) 0C(=0)R8 wherein R8 is -SAC, -SCAC, -Ar, or -SAC-Ar, and n = 1 or 2,
IV) A represents -(NR9) n - wherein R9 is H, -SAC, -SCAC, -Ar, or -SAC-Ar, and n=0 or 1,
V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
VI) R and R may form a cycle together with the carbon atom to which they are attached, where -R-R1- is -(CH ) -, -(CH ) -0-(CH ) -, or -(CH ) -NR1O-(CH ) -
2 n 2 n 2 m 2 n 2 m wherein n+m<9 and R10 is -SAC, -SCAC, -Ar, -SAC-Ar, -C(=O)-SAC, - C(=O)-SCAC, -C(=O)-Ar, or -C(=O)-SAC-Ar, VII) X represents -C(=O)CH OR11 wherein R11 is -SAC, -SCAC, -Ar, or - SAC-Ar; -C(=O)CH2OC(=O)R12 wherein R12 is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-CO R13 wherein R13 is -SAC, -SCAC, -Ar, or -SAC-Ar; -CH=CH-SO R14 wherein R14 is -SAC, -SCAC, -Ar, or -SAC-Ar; -C(=O)CH=CH2; -COCHN2; or -COCH -W wherein W is -F, -Cl, -Br, -I, -NR15R16 (R15 and R16 each are -
SAC, -SCAC, -Ar, or -SAC-Ar, or together may form 3- to 6-membered s saattuurraatteedd o orr u unnssaattuurraatteedd c cyycclliicc g grroouupp)),, -SR (R is -SAC, -SCAC, -Ar, or - SAC-Ar), or is the following formula:
Figure imgf000039_0001
wherein is H, -OH, -OR18 (R18 = -SAC or -SCAC), -C(=0)R19 (R19 = -H, -SAC, or -
SCAC), -F, -Cl, -Br, -I, -CN, -N3, -CO2H, -CF3, -CO2R20 (R20 = -SAC or -SCAC),
-Q=O)NHR21 (R21 = -SAC or -SCAC), or -Q=O)NR22R23 (R22 and R23 each are -
SAC, -SCAC, -Ar, or -SAC-Ar ), and
R is H or -SAC, or pharmaceutically acceptable salt, physiologically hy- drolysable ester, hydrate, solvate, or stereoisomer thereof.
[4] 2. The compound according to claim 1 wherein
I) R represents H;
II) R1 represents -CH2COOH, -CH2COOR3 (R3 = -SAC), or -CHICONHSO2R4 (R4 = -SAC);
III) R2 represents H, -SAC, -Ar, or -(CH^OR7 (R7 = -SAC, -SCAC, -Ar, or - SAC-Ar, and n = l or 2);
IV) A represents -(NR9) n - (R9 is H, -SAC, -SCAC, -Ar, or -SAC-Ar, and n=0 or
D,
V) B represents H, -SAC, -SCAC, -Ar, or -SAC-Ar, or
VI) X represents -COCHN , -COCH F, -COCH Cl, -COCH Br, -COCH I, - COCH OAr, -COCH OCOAr, or -COCH SR17 (R17 is -SAC, -SCAC, -Ar, or - SAC-Ar), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof.
[5] 3. The compound according to claim 1 which is selected from the group consisting of the following:
(3S)-3-{ [(3-benzoyl-5-ethyl-4,5-dihydro-5-isoxazolyl)carbonyl] amino }-5-(2,6-di chlorobenzoyl)oxy ] -4-oxopentanoic acid( 1 ) ;
(3S)-3-({ [5-ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-4- oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(2); 3-({ [5-Ethyl-3-(l-naphthoyl)-4,5-dihydro-5-isoxazolyl]carbonyl}amino)-5-fluor o-4-oxopentanoic acid(3);
3-({ [5-Ethyl-3-(l-isoquinolinylcarbonyl)-4,5-dihydro-5-isoxazolyl]carbonyl} amino) - 5 -fluoro-4-oxopentanoic acid(4) ;
(3S)-3-[({5-ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl} carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(5);
3-[({5-Ethyl-3-[(l-naphthylamino)carbonyl]-4,5-dihydro-5-isoxazolyl}carbonyl) amino] -5-fluoro-4-oxopentanoic acid(6);
(3S)-3-[({3-[([l,r-biphenyl]-2-ylamino)carbonyl]-5-ethyl-4,5-dihydro-5-isoxazo lyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(7);
3-[({3-[([l,r-Biphenyl]-2-ylamino)carbonyl]-5-ethyl-4,5-dihydro-5-isoxazolyl} carbony 1) amino] - 5 -fluoro-4-oxopentanoic acid( 8 ) ;
3-{ [(5-Ethyl-3-{ [(2'-methyl[l,r-biphenyl]-2-yl)amino]carbonyl}-4,5-dihydro-5-i soxazolyl)carbonyl] amino } -5-fluoro-4-oxopentanoic acid(9) ;
3-[({3-[3,4-Dihydro-l(2H)-quinolinylcarbonyl]-5-ethyl-4,5-dihydro-5-isoxazolyl
} carbonyl)amino]-5-fluoro-4-oxopentanoic acid(lθ);
(3S)-3-[({3-[3,4-dihydro-2(lH)-isoquinolinylcarbonyl]-5-ethyl-4,5-dihydro-5-iso xazolyl}carbonyl)amino]-4-oxo-5-(2,3,5,6-tetrafluorophenoxy)pentanoic acid(l l);
3-{ [(3-{ [2-(ter?-Butyl)anilino]carbonyl}-5-ethyl-4,5-dihydro-5-isoxazolyl) carbonyl] amino } -5-fluoro-4-oxopentanoic acid( 12) ;
3-{ [(3-{ [2-(ter?-Butyl)anilino]carbonyl}-5-isopropyl-4,5-dihydro-5-isoxazolyl) carbonyl] amino }-5-fluoro-4-oxopentanoic acid(13); and
3-{ [(3-{ [2,5-Di(ter?-butyl)anilino]carbonyl}-5-isopropyl-4,5-dihydro-5-isoxazol yl)carbonyl]amino}-5-fluoro-4-oxopentanoic acid(14), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof. [6] 4. A therapeutic composition for preventing inflammation and apoptosis comprising the compound of formula (1), or pharmaceutically acceptable salt, physiologically hydrolysable ester, hydrate, solvate, or stereoisomer thereof as defined in claim 1 as an active ingredient together with the pharmaceutically acceptable carrier. [7] 5. The composition according to claim 4 for the treatment of dementia, cerebral stroke, brain impairment due to AIDS, diabetes, gastric ulcer, cerebral injury by hepatitis, hepatitis-induced hepatic diseases, acute hepatitis, fulminant hepatic failure, sepsis, organ transplantation rejection, rheumatic arthritis, cardiac cell apoptosis due to ischemic cardiac diseases, or liver cirrhosis. [8] 6. The composition according to claim 4 for the treatment of acute hepatitis or liver cirrhosis.
[9] 7. The composition according to claim 4 for the treatment of rheumatic arthritis.
[10] 8. The composition according to any one of claims 4 to 7 which is formulated as an oral preparation, an injection, or a patch.
PCT/KR2005/003136 2004-09-24 2005-09-22 Caspase inhibitors containing dicarbonylamino-isoxazoline WO2006033551A1 (en)

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WO2022123530A1 (en) 2020-12-10 2022-06-16 주식회사 엘지화학 Boronic acid compound

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WO2012130798A1 (en) 2011-03-31 2012-10-04 Bayer Cropscience Ag Herbicidally and fungicidally active 3-phneylisoxazoline-5-carboxamides and 3-phneylisoxazoline-5-thioamides
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