EP0851760A1 - Enzyme de conversion d'interleukines et apoptose - Google Patents

Enzyme de conversion d'interleukines et apoptose

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Publication number
EP0851760A1
EP0851760A1 EP96930620A EP96930620A EP0851760A1 EP 0851760 A1 EP0851760 A1 EP 0851760A1 EP 96930620 A EP96930620 A EP 96930620A EP 96930620 A EP96930620 A EP 96930620A EP 0851760 A1 EP0851760 A1 EP 0851760A1
Authority
EP
European Patent Office
Prior art keywords
cephem
acetoxymethyl
carboxamide
dioxide
methoxy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96930620A
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German (de)
English (en)
Other versions
EP0851760A4 (fr
Inventor
Mark Alan Levy
John Gerald Gleason
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SmithKline Beecham Corp
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SmithKline Beecham Corp
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Publication of EP0851760A1 publication Critical patent/EP0851760A1/fr
Publication of EP0851760A4 publication Critical patent/EP0851760A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D501/00—Heterocyclic compounds containing 5-thia-1-azabicyclo [4.2.0] octane ring systems, i.e. compounds containing a ring system of the formula:, e.g. cephalosporins; Such ring systems being further condensed, e.g. 2,3-condensed with an oxygen-, nitrogen- or sulfur-containing hetero ring
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention is to the discovery of a new method to block excessive or inappropriate apoptosis in a mammal.
  • necrosis is usually the result of severe trauma and is a process that involves loss of membrane integrity and uncontrolled release of cellular contents, often giving rise to inflammatory responses.
  • apoptosis is a more physiological process that occurs in a controlled manner and is generally non ⁇ inflammatory in nature. For this reason apoptosis is often referred to as programmed cell death.
  • the name itself (apoptosis: Greek for "dropping off, for example leaves from trees) implies a cell death that is part of a normal physiological process (Kerr et al.. Br. J. Cancer. 26: 239-257 (1972)).
  • Apoptosis appears to be a carefully controlled series of cellular events which ultimately leads to death of the cell. This process for elimination of unwanted cells is active and requires expenditure of cellular energy.
  • the morphological characteristics of apoptosis include cell shrinkage and loss of cell-cell contact, condensation of nuclear chromatin followed by fragmentation, the appearance of membrane ruffling, membrane blebbing and apoptotic bodies. At the end of the process, neighboring cells and macrophages phagocytose the fragments from the apoptotic cell. The process can be very fast, occurring in as little as a few hours (Bright et al., Biosci. Rep.. 14: 67-82 (1994)).
  • the best defined biochemical event of apoptosis involves the orderly destruction of nuclear DNA.
  • Signals for apoptosis promote the activation of specific calcium- and magnesium-dependent endonucleoases that cleave the double stranded DNA at linker regions between nucleosomes. This results in production of DNA fragments that are multiples of 180-200 base pair fragments (Bergamaschi et al., Haematologica. 79: 86-93 (1994); Stewart, JNCI, 86: 1286-1296 (1994)). When examined by agarose gel electrophoresis, these multiple fragments form a ladder pattern that is characteristic for most cells undergoing apoptosis.
  • TNF / nerve growth factor family of receptors which include CD40 and Fas/Apo-1 (Bright et al., supra).
  • genes that appear to be required for induction of apoptosis are Ced-3 and Ced-4. These genes must function in the dying cells and, if either gene is inactivated by mutation, cell death fails to occur (Yuan et al., Devel. Biol.. 138: 33-41 (1990)).
  • genes that have been linked with induction of apoptosis include the proto-oncogene c-myc and the tumor suppresser gene p53 (Bright et al., supra; Symonds et al., Cell. 78: 703-711 (1994)).
  • Ced-9 An example in C. elegans is Ced-9. When it is abnormally activated, cells survive that would normally die and, conversely, when Ced-9 is inactivated cells die that would normally live (Stewart, B.W., supra).
  • bcl-2 A mammalian counterpart is bcl-2, which had been identified as a cancer-causing oncogene.
  • This gene inhibits apoptosis when its product is overexpressed in a variety of mammalian cells, rendering them less sensitive to radiation, cytotoxic drugs and apoptotic signals such as c-myc (Bright et al., supra).
  • Some virus protein have taken advantage of this ability of specific proteins to block apoptosis by producing homologous viral proteins with analogous functions.
  • An example of such a situation is a protein produced by the Epstein Barr virus that is similar to bcl-2, which prevents cell death and thus enhances viral production (Wells et al.. J. Reprod. Fertil.. 101: 385-391 (1994)).
  • Apoptosis is an important part of normal physiology. The two most often sited examples of this are fetal development and immune cell development. In development of the fetal nervous system, over half of the neurons that exist in the early fetus are lost by apoptosis during development to form the mature brain (Bergamaschi et al., Haematologica. 79: 86-93 (1994)). In the production of immune competent T cells (and to a lesser extent evidence exists for B cells), a selection process occurs that eliminates cells that recognize and react against self. This selection process is thought to occur in an apoptotic manner within areas of immune cell maturation (Williams, G. T., J. Pathol.. 173: 1-4 (1994V. Krammer et al.. Curr. Opin. Immunol.. 6: 279-289 (1994)).
  • Dysregulation of apoptosis can play an important role in disease states, and diseases can be caused by both excessive or too little apoptosis occurring.
  • An example of diseases associated with too little apoptosis would be certain cancers.
  • There is a follicular B-cell lymphoma associated with an aberrant expression of functional bcl-2 and an inhibition of apoptosis in that cell (Bergamaschi et al., supra).
  • There are numerous reports that associate deletion or mutation of p53 with the inhibition of apoptosis and the production of cancerous cells Kerr et al., Cancer. 73: 2013-2026 (1994 : Ashwell et al.. Immunol. Today. 15: 147-151, (1994)).
  • one example of excessive or inappropriate apoptosis is the loss of neuronal cells that occurs in Alzheimer disease, possible induced by ⁇ -amyloid peptides (Barr et al.,
  • ICE transfection-induced apoptosis could be antagonized by overexpression of bcl-2, the mammalian oncogene that can prevent programmed cell death (Miura et al., supra). Additional experiments were performed using the crmA gene.
  • This gene of the cowpox virus encodes a serpin protein, a family of proteins that are inhibitors of proteases (Ray et al., Cell, 69: 597-604 (1992)).
  • the protein of crmA has been shown to inhibit processing of pro- interleukin -l ⁇ by ICE. (Gagliardini et al. Science. 263: 826-828 (1994)) showed that microinjection of the crmA gene into dorsal root ganglion neurons prevented cell death induced by nerve growth factor deprivation. This result shows that ICE is involved in neuronal cell apoptosis.
  • Brain interleukin- 1 has been reported to be elevated in Alzheimer disease and Down syndrome (Griffin et al., Proc. Natl. Acad. Sci. U. S. A.. 86: 7611-7615 (1989)).
  • interleukin-l can increase the mRNA and production of ⁇ -amyloid protein, a major component of senile plaques in Alzheimer disease as well as in brains of people with Down syndrome and with aging (Forloni et al., Mol. Brain Res.. 16: 128-134 (1992V Buxbaum et al.. Proc.
  • the present invention is to the novel compounds of Formula (I), their pharmaceutical compositions, and to the novel inhibition of ICE and ICE-like proteins for use in the treatment of apoptosis, and disease states caused by excessive or inappropriate cell death.
  • Another aspect of the present invention is to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the present invention is to a method for the treatment of diseases or disorders associated with excessive IL-l ⁇ convertase activity, in a mammal in need thereof, which method comprises administering to said mammal an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention is to a method of preventing or reducing apoptosis in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • Another aspect of the present invention is to a method of blocking or decreasing the production of IL-l ⁇ and/or TNF, in a mammal, preferably a human, in need of such treatment which method comprises administering to said mammal or human an effective amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof.
  • the compounds of the present invention may contain one or more asymmetric carbon atoms, in particular positions 6 and 7, and may exist in racemic and optically active forms. All of these compounds are included within the scope of the present invention.
  • the compound has a 6R, 7S configuration.
  • Rl is hydrogen, halogen, or an optionally substituted alkoxy
  • R2 is NR a Rb
  • R a is hydrogen, or Ci-4 alkyl
  • Rb is Ci-io alkyl, optionally substituted arylCi-4 alkyl, optionally substituted heteroaryl Ci-4 alkyl, optionally substituted C3-.7 cycloalkyl, or R a and Rb together with the nitrogen to which they are attached form a 5 to 10 membered ring which optionally contains an additional heteroatom selected from oygen, nitrogen or sulfur
  • R3 is hydrogen, -OC(O)R5, or S(O)n R ⁇ , or bromo;
  • R4 is hydrogen, -OC(O)R5, bromo or S(O)n R6, provided that when R3 is hydrogen, R4 is other than hydrogen;
  • R5 is C 1-6 alkyl, C3-7 cycloalkyl, optionally substituted aryl or optionally substituted arylalkyl; R is optionally substituted aryl, or optionally substituted heteroaryl; m is an integer having a value of 1 or 2; n is 0, or an integer having a value of 1 or 2; or a pharmaceutically acceptable salt thereof.
  • Ri is hydrogen, halogen, or an optionally substituted Ci-4 alkoxy.
  • the carbon chain may be optionally substituted, one or more times, independently by hydroxy, halogen, alkoxy, C(O)H, C(O)2Rc, or C(O)CH3 moieties; wherein R c is hydrogen, Ci-6 alkyl, aryl, or arylCi-4alkyl.
  • Rl is methoxy.
  • R2 is NR a Rb;
  • Rb is Ci-10 alkyl, optionally substituted arylCi-4 alkyl, optionally substituted heteroaryl Ci-4 alkyl, optionally substituted C3-7 cycloalkyl, or R a and Rb together with the nitrogen to which they are attached form a 5 to 10 membered ring which optionally contains an additional heteroatom selected from oygen, nitrogen or sulfur; and
  • R a is hydrogen, or Ci-4 alkyl, preferably hydrogen, or methyl.
  • Rb an optionally substituted benzyl.
  • the alkyl group in the arylalkyl moiety may be branched or straight, such as a methylene or a substituted methylene group , i.e., -CH(CH3) - aryl.
  • R a is hydrogen, or Ci-4 alkyl, preferably hydrogen, or methyl.
  • the optionally substituted aryl moiety of the arylalkyl group may be substituted one to three times independently by hydroxy, halogen, alkyl or alkoxy.
  • n is 1 or 2.
  • m is 2.
  • R3 is hydrogen, -OC(O)R5, S(O)n-R6, or bromo; provided that when R3 is hydrogen, then R4 is other than hydrogen.
  • R3 is -OC(O)R5, the R5 group is suitably Ci-6 alkyl, C3-7 cycloalkyl, optionally substituted aryl, or optionally substituted arylalkyl; preferably R5 is Ci- alkyl, more preferably methyl.
  • R6 is suitably an optionally substituted aryl, or an optionally substituted aryl heteroaryl; and n is 0, or an integer having a value of 1 or 2.
  • R ⁇ is heteroaryl, as defined below, it is preferably a triazole, oxadiazole, or tetrazole moiety.
  • R is aryl, as also defined below, it is preferably a phenyl; the n value is preferably 1 or 2.
  • n is preferably 0.
  • the heteroaryl or aryl ring may be optionally substituted one or more times independently by hydroxy, halogen, alkyl or alkoxy, preferably alkyl, more preferably methyl.
  • R4 is hydrogen, bromine, -OC(O)R5, or S(O) n 6 > wherein R5 and R are as defined above in the R3 group.
  • excessive IL-l ⁇ convertase activity is used herein to mean an excessive expression of the protein, or activation of the enzyme.
  • C ⁇ _ alkyl or “alkyl” is used herein to mean both straight and branched chain radicals of 1 to 6 carbon atoms, unless the chain length is otherwise specified, including, but not limited to, methyl, ethyl, n-propyl, w ⁇ -propyl, w-butyl, sec- butyl, wo-butyl, tert-butyl, and the like.
  • heteroaryl (on its own or in any combination, such as “heteroaryloxy”, or
  • heteroaryl alkyl is used herein to mean a 5-10 membered aromatic ring system in which one or more rings contain one or more heteroatoms selected from the group consisting of N, O or S, such as, but not limited, to pyrrole, pyrazole, furan, thiophene, quinoline, isoquinoline, quinazolinyl, pyridine, pyrimidine, oxazole, oxadiazole, tetrazole, thiazole, thiadiazole, triazole, imidazole, benzimidazole, benzothiaphene, benzopyrrole, or benzofuran.
  • N O or S
  • aryl (on its own or in any combination, such as “aryloxy”, or “arylalkyl”) is used herein to mean a phenyl and naphthyl ring.
  • cycloalkyl is used herein to mean cyclic radicals, preferably of 3 to 7 carbons, including but not limited to cyclopropyl, cyclopentyl, cyclohexyl, and the like.
  • halo or halogens, is used herein to include, unless otherwise specified, chloro, fluoro, bromo and iodo.
  • core a (I) is numbered as follows:
  • the present invention is to the inhibition of ICE and ICE-like proteases by compounds of Formula (I).
  • ICE-like proteases are fragment, homologs, analogs and derivatives of the polypeptides Interleukin-l ⁇ converting enzyme (or convertase). These analogs are structurally related to the ICE family. They generally encode a protein (s) which exhibits high homology to the human ICE over the entire sequence. Preferably, the pentapeptide QACRG is conserved.
  • the ICE like proteases which may include many natural allelic variants (such as substitutions, deletion or addition of nucleotides) does not substantially alter the function of the encoded polypeptide.
  • ICE protease retain essentially the same biological function or activity as the ICE protease, although it is recognized that the biological function may be enhanced or reduced activity.
  • the suitable activity is not IL-l ⁇ convertase activity, but the ability to induce apoptosis or involved in programmed cell death in some manner.
  • Suitable ICE like proteases encompasses within this invention are those described in PCT US94/07127 filed 23 June 1994, Attorney Docket No.: 325800-184; and in USSN 08/334,251 , filed 1 November 1994, Attorney Docket No.: 325800-249 whose disclosures are inco ⁇ orated herein by reference in their entirety.
  • blocking or inhibiting, or decreasing the production of IL-l ⁇ and/or TNF refers to: a) a decrease of excessive levels, or a down regulation, of the cytokine in a human to normal or sub-normal levels by inhibition of the in vivo release of the cytokine; or b) a down regulation, at the genomic level, of excessive in vivo levels of the cytokine (IL-l or TNF) in a human to normal or sub-normal levels; or c) a down regulation, by inhibition of the direct synthesis of the cytokine (IL- 1 , or TNF) as a postranslational event; or d) a down regulation, at the translational level, of excessive in vivo levels of the cytokine (IL-l, or TNF) in a human to normal or sub-normal levels.
  • the blocking or inhibiting, or decreasing the production of IL-l ⁇ and/or TNF is a discovery that the compounds of Formula (I) are inhibitors of the cytokines, IL-l and TNF is based upon the effects of the compounds of Formulas (I) on the production of the IL- 1 and TNF in in vitro and in vivo assays which are well known and recognized in the art, some of which are described herein.
  • t-Butyl ester (2-Scheme I) is synthesized by treating commercially available 7- aminocephalosporonic acid (1-Scheme I) with isobutylene and sulfuric acid in dioxane. Following the procedure of Doherty et al. (J. Med. Chem. 1990, 33, 2513-2521, which is inco ⁇ orated herein by reference), 7-alkoxy substituted 3a-Scheme I and 3b-Scheme I are produced as a separable mixture. Deprotection of 3-Scheme I with trifluoroacetic acid/anisole at 0°C gives the free acid 4-Scheme I.
  • Alkoxy derivative 9-Scheme 2 is obtained in one step from 8-Scheme 2 by treatment with NaNO2 and the alcohol in perchloric acid (Alpegiani et al. US 5,254,680, which is inco ⁇ orated herein by reference).
  • the amide 9-Scheme 2 is formed by amine coupling with acid 8-Scheme 2 by procedures described for 5-Scheme I; oxone oxidation of 9- Scheme 2 yields 10-Scheme 2.
  • the following derivatives can be synthesized according to procedures outlined by Alpegiani et al. J. Med. Chem.
  • Example 1(b) To the amide of Example 1(b) (0.27 g, 0.62 mmol) in acetonitrile (5 mL) at 0 °C was added a solution of Oxone (0.57 g, 1.9 mmol) in water (5 mL) and the solution was stirred at room temperature for 15 h. To the mixture was added 20% sodium metabisulfite, followed by water and the mixture was extracted twice with methylene chloride. The organic extract was dried (MgSO4), filtered and concentrated in vacuo to give an oil. The oil was purified by flash chromatography (silica gel, 50-70% ethyl acetate/hexanes) to yield the title compound (0J7 g, 56%).
  • Example 5 N-rS-(-V ⁇ -MethylbenzyH-(6R.7SV3-acetoxymethyl-7-methoxy-3-cephem-4- carboxamide- 1.1 -dioxide Following the procedure of Example 1 , except substituting S-(-)- ⁇ - methylbenzylamine for 3,4-dichlorobenzylamine, the title compound was prepared. MS(ES+) m/e 423 [M+H]+.
  • Example 6 N-Benzyl-(6R.7S -3-acetoxymethyl-7-methoxy-3-cephem-4-carboxamide-lJ -dioxide Following the procedure of Example 1, except substituting benzylamine for 3,4- dichlorobenzylamine, the title compound was prepared. MS(ES + ) m/e 409 [M+H]+.
  • Example 7 N-fR-(+Va-Methylbenzyll-(6R.7SV3-acetoxymethyl-7-methoxy-3-cephem-4- carboxamide- 1.1 -dioxide
  • Example 10 N-(3.4-DichlorobenzylV(6R.7SV3-acetoxymethyl-7-benzyloxycarbonylmethylenoxy-3- cephem-4-carboxamide- 1.1 -dioxide Prepared according to the procedure of Example 1 except substituting benzyl glycolate for methanol and concentrating the methylene chloride solution to a small volume before its addition. MS(ES+) m/e 611 [M+H]+.
  • Example 12 N-(4-Chlorobenzyl " )-(6R.7SV3-acetoxymethyl-7-methoxy-3-cephem-4-carboxamide- 1J -dioxide
  • Example 15 N-(3-Trifluoromethylbenzyl)-(6R.7S)-3-acetoxymethyl-7-methoxy-3-cephem-4- carboxamide- 1.1 -dioxide Following the procedure of Example 1 , except substituting 3- trifluoromethylbenzylamine for 3,4-dichlorobenzylamine, the title compound was prepared. MS(ES+) m/e 477 [M+H]+.
  • Example 20 N-(4-NitrobenzylV(6R.7SV3-acetoxymethyl-7-methoxy-3-cephem-4-carboxamide-lJ- dioxide Following the procedure of Example 1, except substituting 4-nitrobenzylamine for 3, 4-dichlorobenzy lamine, the title compound was prepared. MS(ES + ) m/e 454 [M+H]+.
  • Example 23 N-(3.4-DichlorobenzylV(6R.7RV7-(2'. 2'. 2'-trichloroethoxycarboxamidoV3- acetoxymethyl-3-cephem-4-carboxamide- 1.1 -dioxide a) (6R.7RV7-(2'. 2'. 2'-trichloroethoxycarboxamido)-3-acetoxymethyl-3-cephem-4- carboxylic acid.
  • Example 24 N-Cyclohexanemethylene-(6R.7S)-3-acetoxymethyl-7-methoxy-3-cephem-4- carboxamide- 1.1 -dioxide Following the procedure of Example 1, except substituting cyclohexanemethylamine for 3,4-dichlorobenzylamine, the title compound was prepared. MS(ES+) m/e 415 [M+H]+.
  • Example 25 N-Furfuryl-(6R.7S)-3-acetoxymethyl-7-methoxy-3-cephem-4-carboxamide- 1.1 -dioxide Following the procedure of Example 1, except substituting furfurylamine for 3, 4-dichlorobenzy lamine, the title compound was prepared. MS(ES + ) m/e 399 [M+H]+.
  • BIOLOGICAL ASSESSMENTS Assay I - DNA Ladder:
  • the present invention can utilize a model that measures apoptosis, by measuring the production of DNA ladders visualized on agarose gels.
  • the observation of DNA ladders has been a hallmark of the apoptosis response for many years.
  • the model used in our studies is the production of apoptosis in human monocytic HL-60 cells by the anti-cancer ether lipid l-O-octadecyl-2-O-methyl-.sn-3-phosphocholine (ET-I8-OCH3) and tumor necrosis factor ⁇ (TNF).
  • ET-I8-OCH3 tumor necrosis factor ⁇
  • the general method is to treat HL-60 cells with 6 ⁇ M ET- 18-OCH3 or 10 units of TNF for 24 hours, followed by extraction of small molecular weight DNA and removal of protein and RNA. The DNA is separated on a agarose gel and visualized with ethidium bromide staining. An internal standard is added to the cells just prior to extraction and preparation of DNA. Drugs are provided to cells 10 minutes prior to the apoptotic insult. This method provides a qualitative assessment of the ability of compounds to inhibit apoptosis.
  • HL-60 cells (American Type Cell Culture) are grown and kept at log phase in RPMI
  • RPMI complete 1640 w/L-glutamine and 10 % heat inactivated Fetal Bovine Serum. • On the day of the experiment, the desired number of cells (for example, 5 x 10 ⁇ cells/treatment group) are resuspend in RPMI complete to give a final cell concentration of approximately 0.5 x 10 ⁇ cells/ml. For each treatment group, 10 mis of cell suspension are placed in a culture flask. Cells are incubated for 2 hours at 37°C. Exposures:
  • CHCI3 is prepared, and diluted in RPMI complete to 600 ⁇ M. 100 ⁇ l of 600 ⁇ M ET-I8-OCH3 is added into 10 ml treatment groups yielding a final concentration of 6 ⁇ M. The cell suspensions are then incubated overnight (18 hours). For a typical exposure to TNF, 300 to 3000 units of TNF are added to 10 ml of cell suspension.
  • Cells are lysed by resuspending them into 200 ⁇ L of cold, sterile detergent buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-100) and transferring the approximately 250 ⁇ L volume to sterile, 1.5 mL eppendorf tubes on ice. Tubes are incubated for 30 min. at 4°C, with mild shaking.
  • sterile detergent buffer 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.2% Triton X-100
  • Tubes are spun in a Microfuge for 15 min., the supernatant collected, taking care to avoid cellular debris.
  • a DNA standard for example, Sigma # D 5042, 123 bp ladder
  • Sigma # D 5042, 123 bp ladder is run on each gel.
  • Assay II Inhibition of ICE Source of Enzyme Human ICE was cloned and expressed in E. coli as its inactive precursor (p45) bearing a hexa-His flag on its amino-terminal end. Following harvesting, the cells were lysed, centrifuged, and the pellet containing the p45 solubilized with phosphate buffered 7 M urea at pH 7.5. The flagged p45 was applied to a Ni-nitrilo-acetic acid column, washed, and eluted with 300 mM imidazole. This yielded a highly enriched proenzyme preparation ( 3 90% pure p45).
  • Catalytic autoproteolytic activation to pl0/p20 dimer was achieved by concentrating the p45 on a Centricon ultrafiltration membrane (Amicon) at 10 _C for several hours.
  • the formation of the catalytic subunits (plO and p20) in activated samples was demonstrated by correlating time-dependent generation of ICE activity with pl0/p20 signals in Western blots and by reversed-phase HPLC. Formation of authentic plO and p20 was also confirmed by N-terminal sequence and MALD-mass spectral analyses of samples purified by reversed-phase HPLC.
  • the activated enzyme was stored frozen at -80 _C.
  • ICE was assayed at 25 _C in 96-well plates using the fluorogenic tetrapeptide substrate N-acetyl-L-tyrosyl-L-valyl-L-alanyl-L-aspartyl-7-amido-4-methylcoumarin (Ac-YVAD-AMC).
  • the assays were conducted at pH 7.5 in a buffered system containing 25 mM Hepes, 10% sucrose, 0.1% CHAPS, and 20-50 uM DTT. The concentration of substrate was fixed at 20 uM. Fluorescence of the liberated 7-amino-4- methylcoumarin was continuously monitored at 460 nm following excitation at 360 nm.
  • the compounds of the present invention will generally be administered in a standard pharmaceutical composition obtained by admixture with a pharmaceutical carrier or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutical carrier or diluent selected with regard to the intended route of administration and standard pharmaceutical practice.
  • they may be administered orally in the form of tablets containing such excipients as starch or lactose, or in capsule, ovules or lozenges either alone or in admixture with excipients, or in the form of elixirs or suspensions containing flavouring or colouring agents.
  • They may be injected parenterally, for example, intravenously, intramuscularly or subcutaneously.
  • parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the choice of form for administration as well as effective dosages will vary depending, inter alia, on the condition being treated. The choice of mode of administration and dosage is within the skill of the art.
  • the compounds of the present invention particularly those noted herein or their pharmaceutically acceptable salts which are active when given orally, can be formulated as liquids, for example syrups, suspensions or emulsions, tablets, capsules and lozenges.
  • a liquid formulation will generally consist of a suspension or solution of the compound or pharmaceutically acceptable salt in a suitable liquid carrier(s) for example, ethanol, glycerin, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a suitable liquid carrier(s) for example, ethanol, glycerin, non-aqueous solvent, for example polyethylene glycol, oils, or water with a suspending agent, preservative, flavouring or colouring agent.
  • a composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • suitable pharmaceutical carrier(s) routinely used for preparing solid formulations.
  • examples of such carriers include magnesium stearate, starch, lactose, sucrose and cellulose.
  • a composition in the form of a capsule can be prepared using routine encapsulation procedures.
  • pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule.
  • the composition is in unit dose form such as a tablet or capsule.
  • Typical parenteral compositions consist of a solution or suspension of the compound or pharmaceutically acceptable salt in a sterile aqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrroiidone, lecithin, arachis oil or sesame oil.
  • a sterile aqueous carrier or parenterally acceptable oil for example polyethylene glycol, polyvinyl pyrroiidone, lecithin, arachis oil or sesame oil.
  • the solution can be lyophilized and then reconstituted with a suitable solvent just prior to administration.
  • a typical suppository formulation comprises a compound or a pharmaceutically acceptable salt thereof which is active when administered in this way, with a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • a binding and/or lubricating agent such as polymeric glycols, gelatins or cocoa butter or other low melting vegetable or synthetic waxes or fats.
  • the pharmaceutically acceptable compounds of the invention will normally be administered to a subject in a daily dosage regimen.
  • a daily dosage regimen for a patient this may be, for example, from about .001 to about lOOmg/kg, preferably from about 0.001 to about lOmg/kg animal body weight.
  • a daily dose, for a larger mammal is preferably from about 1 mg to about 1000 mg, preferably between 1 mg and 500 mg or a pharmaceutically acceptable salt thereof, calculated as the free base, the compound being administered 1 to 4 times per day.
  • Unit dosage forms may contain from aobut 25 ⁇ g to about 500mg of the compound.
  • Bone remodeling involves the initial reso ⁇ tion by osteoclasts, followed by bone formation by osteoblasts. Recently, there have been a number of reports of apoptotic events occurring during this process. Apoptotic events have been observed in both the bone forming and bone resorbing cells in vitro and indeed at the sites of these remodeling units in vivo.
  • Apoptosis has been suggested as one of the possible mechanisms of osteoclast disappearance from reversal sites between reso ⁇ tion and formation.
  • TGF- ⁇ 1 induces apoptosis (approx. 30%) in osteoclasts of murine bone marrow cultures grown for 6 days in vitro .
  • the anti-reso ⁇ tive bisphosphonates (clodronate, pamidronate or residronate) promote apoptosis in mouse osteoclasts in vitro and in vivo . (Hughes, et al., supra at S347).
  • M-CSF which has previously been found to be essential for osteoclast formation can suppress apoptosis, suggesting not only that maintenance of osteoclast populations, but also that formation of these multinucleated cells may be determined by apoptosis events.
  • Fuller, et al., J. Bone Min. Res. 8 , S384 (1993); Perkins, et al., J. Bone Min. Res. 8 , S390 (1993) Local injections of IL-l over the calvaria of mice once daily for 3 days induces intense and aggressive remodeling. (Wright, et al., J. Bone Min. Res. 9 , S174 (1994)).
  • one aspect of the present invention is the promotion of apoptosis in osteoclasts as a novel therapy for inhibiting reso ⁇ tion in diseases of excessive bone loss, such as osteoporosis, using compounds of Formula (I) as defined herein.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to enhance bone formation using compounds of Formula (I) as defined herein.
  • Osteoarthritits is a degenerative disease characterized by progressive erosion of articular cartilage.
  • Chondrocytes are the single cell-type found in articular cartilage and perturbations in metabolism of these cells may be involved in the pathogenesis of OA.
  • Damage to cartilage initiates a specific reparative response which involves an increase in the production of proteoglycan and collagen in an attempt to reestablish normal matrix homeostasis.
  • the 3-dimensional collagen network is disrupted and cell death of chondrocytes occurs in OA lesions (Malemud, et al.: Regulation of chondrocytes in osteoarthritis. In: Adolphe, M. ed. Biological Regulation of Chondrocytes.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat osteoarthritis, using compounds of Formula (I) as defined herein.
  • liver degenerative conditions of the liver are linked to hepatocellular apoptosis. These conditions include chemical-, infectious- and immune/inflammatory-induced hepatocellular degeneration.
  • Apoptosis of liver cells has been observed in liver degenerative states induced by a variety of chemical agents, including acetaminophen (Ray, et a l.,(1993) FASEB. J. 7, 453-463), cocaine (Cascales, et al., (1994) Hepatology 20, 992-1001) and ethanol (Baroni, etal., (1994) /. Hepatol. 20, 508-513).
  • Infectious agents and their chemical components that have been shown to induce apoptosis include hepatitis ((Hiramatsu, et al., (1994) Hepatology 19, 1354- 1359; Mita, et al., (1994) Biochem. Biophys. Res. Commun. 204, 468-474)), tumor necrosis factor and endotoxin . (Leist, et al., (1995) J. Immunol. 154, 1307-1316; and Decker, K. (1993) Gastroenterology 28(S4), 20-25).
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat degenerative liver diseases., using compounds of Formula (I) as defined herein.
  • Apoptosis is recognized as a fundamental process within the immune system where cell death shapes the immune system and effects immune functions. Apoptosis also is implicated in viral diseases (e.g AIDS). Recent reports indicate that HIV infection may produce an excess of apoptosis, contributing to the loss of CD4 T cells. Of additional interest is the observation that APO-1/Fas shares sequence homology with HIV-l gpl20.
  • Another aspect of the present invention is the inhibition of apoptosis as a novel therapy to treat viral diseases, using compounds of Formula (I) as defined herein. Additional therapeutic directions and other indications in which inhibition of apoptotic cysteine proteases is of therapeutic utility, along with relevant citations in support of the involvement for apoptosis in each indication, are presented below in Table 1.
  • JL- 1 and TNF inhibiting effects of compounds of the present invention are determined by the following in vitro assays: Interleukin - 1 (IL-l)
  • Human peripheral blood monocytes are isolated and purified from either fresh blood preparations from volunteer donors, or from blood bank buffy coats, according to the procedure of Colotta et al, J Immunol, 132, 936 (1984). These monocytes (lxlo are plated in 24-well plates at a concentration of 1-2 million/ml per well. The cells are allowed to adhere for 2 hours, after which time non-adherent cells are removed by gentle washing. Test compounds are then added to the cells for about lhour before the addition of lipopolysaccharide (50 ng/ml), and the cultures are incubated at 37°C for an additional 24 hours. At the end of this period, culture super-natants are removed and clarified of cells and all debris.
  • Culture supernatants are then immediately assayed for IL-l biological activity, either by the method of Simon et al., J. Immunol. Methods, 84, 85, (1985) (based on ability of JL-1 to stimulate a Interleukin 2 producing cell line (EL-4) to secrete JL-2, in concert with A23187 ionophore) or the method of Lee et al, J. ImmunoTherapy, 6 (1), 1-12 (1990) (ELISA assay).
  • TNF Tumour Necrosis Factor
  • Human peripheral blood monocytes are isolated and purified from either blood bank buffy coats or platelet pheresis residues, according to the procedure of Colotta, R. et al., J Immunol, 132(2), 936 (1984).
  • the monocytes are plated at a density of 1x10 ⁇ cells/ml medium/well in 24-well multi-dishes. The cells are allowed to adhere for 1 hour after which time the supernatant is aspirated and fresh medium (1ml, RPMI- 1640, Whitaker Biomedical Products, Whitaker, CA) containing 1% fetal calf serum plus penicillin and streptomycin (10 units/ml) added.
  • the cells are incubated for 45 minutes in the presence or absence of a test compound at InM-lOmM dose ranges (compounds are solubilized in dimethyl sulfoxide/ethanol, such that the final solvent concentration in the culture medium is 0.5% dimethyl sulf oxide/0.5% ethanol).
  • Bacterial lipopoly-saccharide E. coli 055:B5 [LPS] from

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Abstract

L'invention concerne des composés nouveaux de formule (I), leurs compositions pharmaceutiques et une inhibition nouvelle de protéines de l'enzyme de conversion d'interleukines et de protéines analogues à l'enzyme de conversion d'interleukines destinées à être utilisées pour traiter l'apoptose et les états pathologiques causés par une mort cellulaire excessive ou inappropriée.
EP96930620A 1995-08-31 1996-08-29 Enzyme de conversion d'interleukines et apoptose Withdrawn EP0851760A4 (fr)

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US308395P 1995-08-31 1995-08-31
PCT/US1996/013867 WO1997007805A1 (fr) 1995-08-31 1996-08-29 Enzyme de conversion d'interleukines et apoptose
US3083 2001-12-06

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WO1999046248A1 (fr) 1998-03-09 1999-09-16 Vertex Pharmaceuticals Incorporated DERIVES DE LA 1,2-DIAZEPANE INHIBITEURS DE L'ENZYME DE CONVERSION DE L'INTERLEUKINE-1$g(b)
BR9909660A (pt) 1998-03-19 2000-11-21 Vertex Pharma Inibidores de caspases
US6300341B1 (en) 1998-09-30 2001-10-09 The Procter & Gamble Co. 2-substituted heterocyclic sulfonamides
US6307049B1 (en) 1998-09-30 2001-10-23 The Procter & Gamble Co. Heterocyclic 2-substituted ketoamides
PE20011350A1 (es) 2000-05-19 2002-01-15 Vertex Pharma PROFARMACO DE UN INHIBIDOR DE ENZIMA CONVERTIDORA DE INTERLEUCINA-1ß (ICE)
US7531570B2 (en) 2004-05-27 2009-05-12 Vertex Pharmaceuticals Incorporated Treatment of diseases using ICE inhibitors
US9956260B1 (en) 2011-07-22 2018-05-01 The J. David Gladstone Institutes Treatment of HIV-1 infection and AIDS

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EP0124081A2 (fr) * 1983-05-02 1984-11-07 Merck & Co. Inc. Céphalosporinesulfones comme agents anti-inflammatoires et anti-dégénératifs, compositions pharmaceutiques les contenant et procédés pour les fabriquer
EP0267723A2 (fr) * 1986-11-12 1988-05-18 Merck & Co. Inc. Sulfones de céphalosporine substituées comme agents anti-inflammatoires et anti-dégénératifs
WO1995002603A2 (fr) * 1993-07-14 1995-01-26 Pharmacia S.P.A. Utilisation des derives de cepheme comme agents anti-metastatiques

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EP0124081A2 (fr) * 1983-05-02 1984-11-07 Merck & Co. Inc. Céphalosporinesulfones comme agents anti-inflammatoires et anti-dégénératifs, compositions pharmaceutiques les contenant et procédés pour les fabriquer
EP0267723A2 (fr) * 1986-11-12 1988-05-18 Merck & Co. Inc. Sulfones de céphalosporine substituées comme agents anti-inflammatoires et anti-dégénératifs
WO1995002603A2 (fr) * 1993-07-14 1995-01-26 Pharmacia S.P.A. Utilisation des derives de cepheme comme agents anti-metastatiques

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Title
M. ALPEGIANI ET AL.: "Cephem Sulfones as Inactivators of Human Leukocyte Elastase. 5. 7-alpha-Methoxy- and 7-alpha-Chloro-1,1-dioxocephem 4-Ketones" J. MED. CHEM., vol. 37, no. 23, 1994, pages 4003-4019, XP002077984 *
P. E. FINKE ET AL.: "Inhibition of Human Leukocyte Elastase. 4. Selection of a Substituted Cephalosporin (L-658,758) as a Topical Aerosol" J. MED. CHEM., vol. 35, no. 21, 1992, pages 3731-3744, XP002077983 *
See also references of WO9707805A1 *

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WO1997007805A1 (fr) 1997-03-06
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