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WO2003045375A1 - Nouvelle utilisation de cyclopentabenzofuranes - Google Patents

Nouvelle utilisation de cyclopentabenzofuranes Download PDF

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Publication number
WO2003045375A1
WO2003045375A1 PCT/EP2002/012880 EP0212880W WO03045375A1 WO 2003045375 A1 WO2003045375 A1 WO 2003045375A1 EP 0212880 W EP0212880 W EP 0212880W WO 03045375 A1 WO03045375 A1 WO 03045375A1
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WIPO (PCT)
Prior art keywords
fluorine
chlorine
alkyl
butyl
hydrogen
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PCT/EP2002/012880
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German (de)
English (en)
Inventor
Lutz Assmann
Thomas Fahrig
Irene Gerlach
Martin Hendrix
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Bayer Aktiengesellschaft
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Priority to AU2002356615A priority Critical patent/AU2002356615A1/en
Publication of WO2003045375A1 publication Critical patent/WO2003045375A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/443—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with oxygen as a ring hetero atom

Definitions

  • the invention relates to the use of cyclopentabenzofurans for the manufacture of a medicament for the prophylaxis and / or treatment, in particular for the neuroprotective treatment of Parkinson's disease.
  • NF- ⁇ B nuclear factor- ⁇ B
  • NF- ⁇ Bl p50 and its precursor protein pl05
  • NF- ⁇ B2 p52 and its precursor protein plOO
  • c-Rel c-Rel
  • RelA p65
  • RelB Gosh et al. Ann. Rev. of Immunol. 1998, 16, 225-260
  • NF- ⁇ B is present in unstimulated cells as a cytoplasmic, inactive form by binding to an inhibitory protein (I- ⁇ B).
  • I- ⁇ B After stimulation, I- ⁇ B is rapidly phosphorylated by I- ⁇ B kinases and proteolytically broken down. This releases NF- ⁇ B in its active form and enables its translocation into the cell nucleus. In its capacity as a transcription factor, NF- ⁇ B activates or modulates the expression of specific genes in the cell nucleus (Karin, J. Biol. Chem. 1999, 274, 27339-27342).
  • Parkinson's disease is a chronically progressive, neurodegenerative disease that is characterized by the loss of dopaminergic neurons of the substantia nigra.
  • the main characteristics of early signs and symptoms of Parkinson's disease are resting tremors, slowing of movement, muscle stiffness and unstable posture.
  • WO 00/07579 and WO 00/08007 describe natural or synthetic rocaglamides as inhibitors for NF- ⁇ B-mediated, pathophysiological processes for the treatment of chronic inflammatory diseases such as arteriosclerosis and multiple sclerosis.
  • the present invention therefore relates to the use of compounds of the formula (I) in which
  • R 1 and R 3 each independently represent hydrogen, halogen or alkyl
  • R 2 and R 4 each independently represent halogen, alkyl or optionally substituted alkoxy
  • R 5 represents hydroxy, alkylamino or the radical -NR 12 -CHR 13 -COOR 14 , in which
  • R 12 represents hydrogen or alkyl
  • R stands for one of the residues of a natural or synthetic ⁇ -amino acid, functional groups being optionally protected, or
  • R 12 and R 13 together represent - (CH 2 ) 3 - and - (CH 2 ) 4 -, and
  • R 14 represents alkyl, benzyl or another C-terminal protective group
  • R 6 represents hydrogen or
  • R 5 and R 6 together represent oxygen (oxo), R 7 and R 8 each represent hydrogen,
  • R 9 represents optionally substituted phenyl or hetaryl
  • R 10 represents hydrogen, halogen, alkyl or alkoxy
  • R 11 represents hydrogen, halogen or alkyl
  • the compounds used according to the invention can exist in stereoisomeric forms which either behave like image and mirror image (enantiomers) or which do not behave like image and mirror image (diastereomers).
  • the invention relates to both the enantiomers or diastereomers or their respective mixtures. These mixtures of the enantiomers and diastereomers can be separated into the stereoisomerically uniform constituents in a known manner.
  • the compounds used according to the invention can also be in the form of their salts.
  • salts with organic or inorganic bases or acids may be mentioned here.
  • Physiologically acceptable salts are preferred in the context of the present invention.
  • Physiologically acceptable salts of the compounds used according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulfonic acids.
  • Physiologically acceptable salts of the compounds used according to the invention can be salts of the substances according to the invention with mineral acids, carboxylic acids or sulfonic acids.
  • particular preference is given to Salts with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid,
  • Ethanesulfonic acid toluenesulfonic acid, benzenesulfonic acid, naphthalenedisulfonic acid, Acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, fumaric acid, maleic acid or benzoic acid.
  • Physiologically acceptable salts can also be metal or ammonium salts of the compounds used according to the invention which have a free carboxyl group.
  • metal or ammonium salts which are derived from ammonia, or organic amines, such as ethylamine, di- or.
  • ammonium salts which are derived from ammonia, or organic amines, such as ethylamine, di- or.
  • R and R independently of one another preferably each represent hydrogen, fluorine, chlorine, bromine or CrC 6 alkyl.
  • R 2 and R 4 independently of one another preferably each represent fluorine, chlorine, bromine, dC 6 -alkyl or C 1 -C 4 -alkoxy optionally substituted by fluorine or chlorine.
  • R 5 preferably represents hydroxy, C 1 -C alkylamino or the rest
  • R preferably represents hydrogen
  • R 5 and R 6 also preferably together represent oxygen (oxo).
  • R 7 and R 8 each preferably represent hydrogen.
  • R 9 preferably represents phenyl, methylenedioxyphenyl or 5- or 6-membered hetaryl with 1 or 2 heteroatoms from the series nitrogen, oxygen and sulfur, which can each be optionally substituted up to four times by substituents of the group: halogen, C 1 -C 6 - Alkyl, hydroxy, C1-C4-alkoxy, C ⁇ -C4-alkylthio, Ci-C ⁇ alkylcarbonyl, phenyl, phenoxy, hetaryl,
  • R 10 preferably represents hydrogen, fluorine, chlorine, d-Ce-alkyl or dC 6 -alkoxy.
  • R 11 preferably represents hydrogen, fluorine, chlorine, bromine or d -C 6 alkyl.
  • R 12 preferably represents hydrogen or C 1 -C 4 alkyl.
  • R 13 preferably represents hydrogen, C 1 -C 4 -alkyl optionally substituted by amino or hydroxy, or mercaptomethyl, methylthioethyl, carboxymethyl, carboxyethyl, carbamoylmethyl, carbamoylethyl, guanidino-propyl or optionally substituted by amino, nitro, halogen, hydroxyl or
  • R 12 and R 13 also preferably together represent - (CH 2 ) 3 - or - (CH 2 ) 4
  • R 14 preferably represents Ci-C ⁇ -alkyl, benzyl or another C-terminal protective group.
  • R 1 and R 3 independently of one another particularly preferably each represent hydrogen, fluorine, chlorine, bromine, methyl or ethyl.
  • R 2 and R 4 independently of one another particularly preferably each represent fluorine, chlorine, bromine, C 1 -C 4 -alkyl or methoxy or ethoxy which is optionally substituted by fluorine or chlorine.
  • R particularly preferably represents hydroxyl, C 1 -C -alkylamino or the radical -NR 12 -CHR 13 -COOR 14 .
  • R 6 particularly preferably represents hydrogen.
  • R 5 and R 6 also particularly preferably together represent oxygen (oxo).
  • R 7 and R 8 each particularly preferably represent hydrogen.
  • R 9 particularly preferably represents phenyl, methylenedioxyphenyl or 5- or
  • R 10 particularly preferably represents hydrogen, fluorine, -CC 4 -alkyl or CrC 4 -alkoxy.
  • R 11 particularly preferably represents hydrogen, fluorine, chlorine, bromine or C 1 -C 4 -alkyl.
  • R 12 particularly preferably represents hydrogen or methyl.
  • R 13 particularly preferably represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, 3-aminopropyl, 4-aminobutyl, carboxymethyl, 2-carboxyethyl , Carbamoylmethyl, 2-carbamoylethyl, 3-guanidinopropyl, phenyl, benzyl, 4-hydroxybenzyl, 4-methoxybenzyl, 2-nitrobenzyl, 3-nitrobenzyl, 4-nit ⁇ obenzyl, 2-aminobenzyl, 3-aminobenzyl, 4-aminobenzyl , 3,4-dichlorobenzyl, 4-iodobenzyl, -naphthylmethyl, ß-naphthylmethyl 3-indolylmethyl, 4-imidazolylmethyl, 1,2,3-triazol-l-yl-
  • R 12 and R 13 are also particularly preferably together - (CH 2 ) 3 - or - (CH 2 ) 4 -.
  • R 14 particularly preferably represents C 1 -C 4 -alkyl, benzyl or another C-terminal protective group.
  • R 1 and R 3 very particularly preferably each represent hydrogen, chlorine or
  • R 2 and R 4 very particularly preferably each represent methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, chlorodifluoromethoxy, 2-chloro-1,1,2-trifluoroethoxy, 1,1,2-trifluoroethoxy, 1,1,2,2 -Tetrafluoroethoxy, 2,2,2-
  • R 5 very particularly preferably represents hydroxyl, methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec.-butylamino, tert.-butylamino or the radical -NR 12 -CHR 13 -COOR 14 .
  • R 6 very particularly preferably represents hydrogen.
  • R 5 and R 6 also very particularly preferably stand together for oxygen (oxo).
  • R and R very particularly preferably each represent hydrogen.
  • R 9 very particularly preferably represents phenyl, methylenedioxyphenyl, furyl, thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl or pyridyl, which can in each case optionally be mono- or disubstituted by substituents from the group: fluorine, chlorine, bromine, iodine, methyl, ethyl , n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, trifluoromethyl, difluoromethyl, chlorodifluoromethyl, 2-chloro-l, l, 2-trifluoroethyl, 1,1,2 Trifluoroethyl, 1,1,2,2-tetrafluoroethyl, l, l-difluoro-2,2,2-trichloroethyl, 2,2,2-trifluoroeth
  • R 10 very particularly preferably represents hydrogen, fluorine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, methoxy, ethoxy, n-propoxy, isopropoxy, n -Butoxy, isobutoxy, sec-butoxy or tert-butoxy.
  • R 11 very particularly preferably represents hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl.
  • R 12 very particularly preferably represents hydrogen.
  • R 13 very particularly preferably represents hydrogen, methyl, isopropyl, isobutyl, sec-butyl, hydroxymethyl, 1-hydroxyethyl, mercaptomethyl, 2-methylthioethyl, 3-aminopropyl, 4-aminobutyl, carboxymethyl, 2-carboxyethyl,
  • R 14 very particularly preferably represents methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl or benzyl.
  • R 2 and R 4 each independently represent halogen, alkyl or optionally substituted alkoxy
  • R 5 represents hydroxy, alkylamino or the radical -NR 12 -CHR 13 -COOR 14 , in which
  • R 12 represents hydrogen or alkyl
  • R 13 stands for one of the residues of a natural or synthetic ⁇ -amino acid, functional groups being optionally protected, or
  • R 12 and R 13 together represent - (CH 2 ) 3 - and - (CH 2 ) -, and
  • R 14 represents alkyl, benzyl or another C-terminal protective group
  • R 6 represents hydrogen or
  • R 5 and R 6 together represent oxygen (oxo),
  • R 7 and R 8 each represent hydrogen
  • R 9 represents optionally substituted phenyl or hetaryl
  • R 10 represents hydrogen, alkyl or alkoxy
  • R ⁇ represents hydrogen, halogen or alkyl.
  • R 1 and R 3 each represent hydrogen, fluorine, chlorine, bromine, methyl or ethyl
  • R 2 and R 4 each represent methoxy, ethoxy, trifluoromethoxy, difluoromethoxy, chlorodifluoromethoxy, 2-chloro-1, 1, 2-trifluoroethoxy, 1, 1, 2-trifluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2 , 2,2-trichloro-l, l-difluoromethoxy or 1,1-difluoroethoxy,
  • R 5 represents hydroxy, methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec.-butylamino, tert.-butylamino or the radical -NR 12 -CHR 13 -COOR 14 ,
  • R 6 represents hydrogen, or
  • R 5 and R 6 together represent oxygen (oxo),
  • R and R each represent hydrogen
  • R 9 for optionally single or double by fluorine, chlorine, bromine, iodine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, trifluoromethyl, difluoromethyl, chlorodifluoromethyl, 2- Chloro-l, l, 2-trifluoroethyl, 1,1,2-trifluoroethyl, 1,1,2,2-tetrafluoroethyl, l, l-difluoro-2,2,2-trichloroethyl, 2,2, 2-trifluoroethyl, 1,1,2,3,3,3-hexafluoropyl, 2,2,3,3-tetrafluoropropyl, 2,2,3,3,3-pentafluoropropyl, hydroxy, methoxy, ethoxy, n -Propoxy, isopropoxy, n-butoxy
  • R 11 represents hydrogen, fluorine, chlorine, bromine, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl or tert-butyl,
  • R 12 represents hydrogen
  • R 13 for hydrogen, methyl, iso-propyl, iso-butyl, sec.-butyl, hydroxymethyl, 1-
  • R 14 represents methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, benzyl or another C-terminal protective group.
  • Saturated or unsaturated hydrocarbon radicals such as alkyl or alkenyl can also be used in connection with heteroatoms, e.g. in alkoxy, where possible, be straight-chain or branched.
  • Optionally substituted radicals can be mono- or polysubstituted, whereby in the case of multiple substitutions the substituents can be the same or different.
  • hetaryl generally represents an aromatic, optionally benzo-fused 5- to 7-membered, preferably 5- to 6-membered Heterocycle, which can contain up to 3 heteroatoms from the S, N and / or O series.
  • examples include: indolyl, isoquinolyl, quinolyl, benzorbythiophene, benzo [b] furanyl, pyridyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, imidazolyl, morpholinyl or piperidyl. Furyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, pyridyl and thienyl are preferred.
  • protective groups known from peptide chemistry are listed, for example, in TW Greene, PGM Wuts, Protective Groups in Organic Synthesis, 2 nd Ed., John Wiley & Sons, New York 1991.
  • suitable protective groups are C 1 -C 4 -alkyl and benzyl in particular for hydroxyl or carboxy groups (C-terminal protective groups); Acetyl, trifluoroacetyl, trichloroacetyl, benzoyl, phenylacetyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl, (Cbz), (9-fluorenyl) methoxycarbonyl (Fmoc) and benzyl, in particular for hydroxyl and amino groups (N-terminal protective groups).
  • tert-butyl, benzyl, acetyl, Boc and Cbz are particularly preferred.
  • Parkinson's disease idiopathic
  • Parkinsonism is a chronic, progressive neurological disorder that belongs to a broader classification of neurological disorders, known as Parkinsonism. It is clinically defined by the appearance of at least two of the four cardinal symptoms: bradykinesia, resting tremor, muscle stiffness and postural and movement disorders.
  • the idiopathic form of Parkinson's disease is pathologically characterized by the loss of pigmented nerve cells, particularly in the area of the substantia nigra of the brain. Idiopathic Parkinson's disease accounts for approximately 75% of all Parkinsonism diseases. The remaining 25% of cases are called atypical park sonism and include diseases such as multiple system atrophy,
  • the present invention also includes pharmaceutical preparations which, in addition to inert, non-toxic, pharmaceutically suitable auxiliaries and excipients, contain one or more compounds of the general formula (I) or which consist of one or more compounds of the formula (I), and processes for the preparation of these preparations.
  • the compounds of the formula (I) should be present in these preparations in a concentration of 0.1 to 99.5% by weight, preferably 0.5 to 95% by weight, of the total mixture.
  • the pharmaceutical preparations can also contain other active pharmaceutical ingredients.
  • the pharmaceutical preparations listed above can be prepared in a customary manner by known methods, for example using the excipient or excipients.
  • the new active compounds can be converted in a known manner into the customary formulations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents.
  • the therapeutically active compound should in each case be present in a concentration of about 0.5 to 90% by weight of the total mixture, i.e. in amounts sufficient to achieve the dosage range indicated.
  • the formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, optionally using emulsifiers and / or dispersants, e.g. in the case of the use of water as a diluent, organic solvents can optionally be used as auxiliary solvents.
  • the application is carried out in the usual way, preferably orally, transdermally or parenterally, in particular perlingually or intravenously. It can also be inhaled over
  • Mouth or nose for example using a spray, or topically on the skin.
  • Formulation and the time or interval at which the administration takes place In some cases it may be sufficient to make do with less than the aforementioned minimum quantity, while in other cases the above upper limit must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day.
  • NF- ⁇ B inhibitory activity of the compounds according to the invention can be determined in vitro as follows:
  • the promoter of the human TNF ⁇ gene contains three NF- ⁇ B binding sequences, which are designated as kl, k2 and k3.
  • Lipopolysaccharide or phorbol ester induce NF- ⁇ B release (Lenardo, M.J., et al., Cell 58, 227-229, 1989).
  • Donor blood mononuclear cells are isolated with Vacutainer CPT TM tubes (Becton Dickinson and Company, Franklin Lakes, NJ. 07417-1885) according to the manufacturer's instructions.
  • the Vacutainer CPT tubes contain 1.0 ml of phosphate-buffered saline with 120 USP units of sodium heparin over 3.0 g of a polyester gel that covers 2.0 ml of a Ficoll solution.
  • the monocytes are taken from a zone above the polyester gel and sown for cell culture with a cell density of 250 ⁇ 10 5 cells per well and 96-well microtiter plates.
  • the cells are incubated for 4 to 6 hours in medium RPMI-1640 (Gibco BRL, Life Technologies GmbH, Dieselstrasse 5, 76344 Eggenstein). The culture supernatant is then aspirated off, medium RPMI-1640 is again added and the substances to be tested are usually in concentrations between 0 ⁇ M
  • LPS Bacterial lipopolysaccharide
  • Sigma-Aldrich Chemie GmbH Grünwalder Weg 30, 82039 Deisenhofen, order no .: L 4391, in a concentration of 125 ng / ml to stimulate NF-B- mediated TNF ⁇ gene expression added.
  • L 4391 Bacterial lipopolysaccharide
  • Microtiter plate culture supernatant is taken and the TNF ⁇ content therein is determined quantitatively using commercially available enzyme-linked immunosorbent assays (ELISA).
  • ELISA enzyme-linked immunosorbent assays
  • TNF ⁇ ELISA for concentration determination are e.g. from Sigma-Aldrich
  • the concentration of the TNF ⁇ formed in the culture supernatant of the monocyte culture after LPS stimulation is determined quantitatively with and without inhibitor substance.
  • the results of the TNF ⁇ concentration determination are plotted against each other in an xy-axis diagram.
  • the graph of the y-coordinates (TNF ⁇ concentration in the culture supernatant) and the x-coordinates (concentration of the inhibitor used) enables the inhibition of NF-B-mediated TNF ⁇ synthesis to be read as a function of the concentration of the inhibitor substance.
  • the active substance concentration of the inhibitor added which, for example, inhibits TNF ⁇ synthesis by 50% can be read off from the diagram.
  • This concentration of active substance, which causes 50% inhibition is called the effective inhibitor concentration for 50% inhibition (IC 50 ).
  • Tissue factor is a membrane-bound protein that is the primary initiator of the blood coagulation cascade and a key function in cardiovascular diseases such as unstable angina pectoris, acute implications after plaque rupture, vascular occlusions of various etiology, arteriosclerotic processes and other diseases such as septic shock or cancer.
  • the tissue factor gene is particularly induced in monocytes and endothelial cells by NF- ⁇ B activation.
  • the promoter of the human tissue factor gene contains an NF- ⁇ B binding site which makes a decisive contribution to activating the promoter (P. Oeth et al. Arteriosclerosis, Thrombosis, and Vascular Biology 17, 365-374, 1997).
  • the promoter fragment of the human tissue factor gene which contains the NF- ⁇ B binding sequence, was with oligonucleotide primers of the sequence 5'-TCC CTC GAG ATC TCC CAG AGG CAA ACT GCC AGA T-3 '(5' primer of the position -925) and
  • This expression construct was subjected to the analysis of DNA sequencing and transfected into the monocyte cell line RAW 264.7 (American Type Culture Collection 12301 Parklane Drive, Rockville, Maryland 20852, USA). The transfection, selection and clone analysis were carried out according to standard methods as described (see Transfection of Mammalian Cells in Culture, LG Davis et al. Basic Methods in
  • RAW-A3 Molecular Biology, Elsevier Sei. Publishing Co., New York 1986.
  • RAW-A3 cells are sown in each well in 12-well microtiter plates.
  • the serum concentration in the RPMI medium is gradually reduced from 10% total calf serum to 0.5% and 0.1% serum content in order to reduce the serum-dependent tissue factor promoter activation to a minimum.
  • the NF- ⁇ B inhibitor is added and then serum is added up to a concentration of 15% in the medium for promoter induction.
  • the culture supernatant is aspirated and the cells are used to measure the luciferase activity in accordance with the "Luciferase Assay System" (Technical Bulletin from Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399 USA, products E4030 , E1483, El 501)
  • the cell lysate is incubated with the luciferase assay substrate and measured in a luminometer to measure the emitted light, when plotted in an xy-axis diagram with the respective emitted light (specified in relative light units) as
  • the y coordinate and the inhibitor concentrations as x coordinates result in a graph for f (x), in which the half-maximum y value is to be assigned to an inhibitor concentration x which corresponds to the inhibitory concentration IC 50 in this test.
  • the IC 50 value of the compound according to preparation example 1-1 is 20 ⁇ M and 1-5 2 ⁇ M.
  • the NF- ⁇ B-mediated expression of luciferase by the inhibitors according to the invention with a concentration in the lower micromolar or in the sub-micromolar range indicates the high effectiveness of the inhibition of NF- ⁇ B-mediated gene expression.
  • the NF- ⁇ B-mediated induction of TNF ⁇ synthesis is independent of the different stimuli used by LPS, opsonized zymosan or phorbol myristate acetate (PMA) for the activation of the TNF ⁇ promoter (A. Baldwin, Annual Rev. Immunology 14, 649, 1996).
  • the use of the inhibitors according to the invention should therefore also result in a comparatively strong inhibition of the
  • TNF ⁇ synthesis perform regardless of the type of stimulation of TNF ⁇ synthesis.
  • NF- ⁇ B inhibitors according to the invention inhibit TNF ⁇ synthesis in human monocytes in a comparable manner, independently of the stimulus with IC 50 values in the submicromolar range, as was shown in Example A.
  • the IC50 value according to PMA stimulus is 0.10 ⁇ M for the connection according to preparation example II and 0.09 ⁇ M for connection 1-5.
  • the recruitment of leukocytes from the blood circulation into the extravascular space is essential for inflammatory responses and for repairing tissue damage.
  • the process of leukocyte immigration involves several steps in series. The initial interaction between leukocytes and the endothelium of the blood vessels is determined by TNF and II-1 dependent expression of the
  • Adhesion protein ELAM-1 mediated on the endothelium. It mediates the so-called rolling of the leukocytes along the blood vessel wall.
  • the transcriptional regulation of ELAM-1 expression is dependent both on the nuclear factor- ⁇ B (NF- ⁇ B) activation and binding to the ELAM-1 promoter and on the “AP-1 binding site [MA. Read et., Biol. Chem. Vol. 272, 2753-2761, (1997)].
  • ELAM-1 The influence of compound 1-3 -R on the expression of ELAM-1 was checked in two different test approaches.
  • the adhesion of human neutrophils to TNF- ⁇ stimulated HUVEC cells was measured in a functional approach.
  • the expression of ELAM-1 on the surface of the HUVECs was determined using a fluorescence-labeled ELAM-1-specific monoclonal antibody by means of FACS (cell sorting) analysis.
  • the umbilical cord endothelial cells were grown to confluence in 96-well microtiter plates in EBM + 10% FCS. At the start of the experiment, the medium was exchanged for EBM without FCS and the test substances were then used. After 20 minutes, the HUVECs were stimulated with 10 nM TNF- ⁇ . After 4 hours of incubation, 200 ⁇ l / well of the neutrophil suspension was exchanged. The neutrophils were previously labeled with a 25 ⁇ M BCECP phosphor fluorescent dye solution for 20 minutes. After 30 minutes of incubation, the BCECP neutrophil
  • the solution was drawn off with excess neutrophils and exchanged for 200 ⁇ l / well of 0.5% NaOH solution.
  • the fluorescence of the adhering neutrophils was then measured in the fluorescence photometer.
  • ELAM-1 expression of ELAM-1 on the cell surface could be inhibited by the compound by 50%.
  • the maximum inhibition of cell adhesion was determined to be 50 nM for compound I-3-R.
  • the maximum 50% inhibition of ELAM-1 expression is in good agreement with the simultaneous NF-B and AP-1-dependent regulation of the ELAM-1 promoter.
  • Umbilical cord endothelial cells were cultured as described above and in the presence or absence of 10 ng / ml TNF in the presence or absence incubated by test substances for 4 hours. The cells were dissolved out of the microtiter plates by incubation with 5 mM EDTA in PBS, centrifuged for 5 minutes at 1000 min ⁇ 1 , and in 100 ⁇ l PBS plus 1% bovine serum albumin (BSA, Sigma-Aldrich GmbH, Best No. A 7906) and an anti-ELAM 1 antibody (monoclonal antibody, Becton and Dickinson, Erembodegem, Belgium, order no.
  • BSA bovine serum albumin
  • an anti-ELAM 1 antibody monoclonal antibody, Becton and Dickinson, Erembodegem, Belgium, order no.
  • HUVECs stimulated with TNF for 4 hours showed significantly stronger fluorescence signals after incubation with anti-ELAM-1 antibodies than cells, on the other hand, which were not incubated with TNF.
  • the compound from preparation example 1-3 -R was able to significantly inhibit this induced expression of ELAM-1 in a concentration range between 0.05 ⁇ M and 5 ⁇ M, but the expression of ELAM 1 was not completely inhibited at any of the concentrations examined.
  • reporter gene cell line which contains the interleukin-2 promoter coupled to the luciferase gene.
  • the promoter contains the DNA sequence from
  • the vector used is pGL3; the starting cell line into which the entire construct was stably transfected is SS-1.
  • the culture medium for this cell line was RPMI 1640 (Gibco, Rockille). It also contained: 100 ⁇ g / ml streptomycin, 100 U / ml penicillin, 2 mM L-glutamine, 10% heat-inactivated FBS and 800 ⁇ g / ml G418 sulfate.
  • the reporter gene test cell line was seeded in phenol red-free RPMI with the additions of 1 ⁇ 10 6 cells per well in 96-well plates and with phorbol 12-myristat 13-acetate (PMA; 5 ng / ml) and ionomycin (10 M ; 0.4 ⁇ g / ml) for 24 hours
  • LucLite TM solution Packard, Meriden, CT
  • Luminoskan Luminoskan, Labsystems
  • Cyclopentanbenzofurans were investigated for their inhibitory effect on the interleukin-lß (IL-lß) stimulated release of interleukin-8 (IL-8) from human umbilical cord endothelial cells (HUVEC).
  • IL-1 ⁇ induced IL-8 formation is mediated by an NF- ⁇ B dependent signal transduction pathway.
  • HUVEC P-115 endothelial cells were manufactured by CellSystems (St. Katharinen,
  • the cells were distributed on 96-well microtiter plates with a density of 5000 cells per well. 16 hours before the addition of the test substances and stimulus (IL-1ß), the cell culture medium was replaced by 170 ⁇ l of fresh medium (EBM2 medium, Cell Systems). Then 20 ⁇ l of the test substance was added in the desired concentrations and the IL-8 synthesis and release was induced by adding 10 ⁇ l IL-lß (final concentration 10 ng / ml; Biosource GmbH, Ratingen, Germany). After 6 hours, 150 ⁇ l of the cell culture supernatant were removed and the IL-8 concentration released was determined using an IL-8 ELISA (Biosource GmbH, Ratingen, Germany). The ELISA was operated according to the manufacturer's instructions.
  • the compound from Preparation 1-40 was able to inhibit the release of IL-8 by 50% (IC50) at a concentration of 250 to 300 nM.
  • Example 1 MPP + toxicity in primary rat dopaminergic neurons
  • MPP + l-methyl-4-phenylpyridinium
  • Dopaminergic neurons were obtained from brains of 15-day-old rat embryos. For this purpose, the ventral parts of the midbrain were dissected out, the tissue was enzymatically dissociated and the cell suspension obtained with a density of 80,000
  • a buffered saline solution 25 mM HEPES, 125 mM NaCl, 4.8 mM KC1, 1.2 mM KH 2 PO, 1.3 mM CaCl 2 , 1.2 mM MgCl 2 , 5.6 mM glucose
  • Fig. 1 Primary dopaminergic neurons were treated with MPP + in the presence and absence of various concentrations of the test substance for 24 hours. The functionality of the dopaminergic neurons was then determined by measuring the dopamine uptake. The dopamine uptake of untreated cells (Ko) was set equal to 100% and all other values were calculated in relation. * p ⁇ 0.05,
  • the neuroprotective in vivo effect of the compounds according to the invention was demonstrated in a mouse model for Parkinson's syndrome, the so-called MPTP model.
  • mice were given 3 mg / kg MPTP i.p. on 3 consecutive days. applied (Bezard et al. Neurosci. Lett. 1997, 234, 47-50). The experimental animals then show a reduced number of dopaminergic neurons in the substantia nigra pars compacta.
  • Fig. 2 Mice were administered 4 mg / kg MPTP for three days.
  • the test substance (Subst, 3 mg / kg) was administered i.p. twice a day starting with the first MPTP administration over a period of 10 days. injected.
  • the number of dopaminergic neurons in the substantia nigra was then quantified.
  • the number of tyrosine hydroxylase immunoreactive neurons (TH-LR neurons) in control animals (Ko) became the same

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Abstract

L'invention concerne l'utilisation de cyclopentabenzofuranes pour produire un médicament préventif et/ou thérapeutique, en particulier un médicament permettant de traiter de manière neuroprotectrice la maladie de Parkinson.
PCT/EP2002/012880 2001-11-29 2002-11-18 Nouvelle utilisation de cyclopentabenzofuranes WO2003045375A1 (fr)

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DE10158561.6 2001-11-29
DE10158561A DE10158561A1 (de) 2001-11-29 2001-11-29 Neue Verwendung von Cyclopentabenzofuranen

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054457A1 (fr) * 2003-12-03 2005-06-16 Bayer Cropscience Aktiengesellschaft Procede pour identifier des composes fongicides a partir de pyruvate kinases de champignons
EP1693059A1 (fr) * 2005-02-22 2006-08-23 Deutsches Krebsforschungszentrum Utilisation des dérivés de rocaglamide en tant qu'inhibiteurs NF-À-spécifiques pour le traitement de certaines maladies inflammatoires
JP2007538031A (ja) * 2004-05-18 2007-12-27 バイエル・ヘルスケア・アクチェンゲゼルシャフト 新規なシクロペンタ［ｂ］ベンゾフラン誘導体およびその使用
EP2189158A1 (fr) 2008-11-20 2010-05-26 DKFZ Deutsches Krebsforschungszentrum, Stiftung des Öffentlichen Rechts Combinaison de rocaglamide et de substances induisant l'apoptose pour le traitement du cancer
JP2012509923A (ja) * 2008-11-25 2012-04-26 ユニヴェルシテ・ドゥ・ストラスブール 心臓保護剤及び抗腫瘍剤としてのロカグラオール誘導体
WO2012066002A1 (fr) 2010-11-16 2012-05-24 Universite De Strasbourg Dérivés de flavagline en tant qu'agents neuroprotecteurs
WO2015075165A1 (fr) 2013-11-22 2015-05-28 Deutsches Krebsforschungszentrum Inhibiteurs de translation en chimiothérapie à dose élevée et/ou radiothérapie à dose élevée
CN113149942A (zh) * 2021-02-10 2021-07-23 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) 一种洛克米兰醇酚羟基衍生物、其制备方法和应用
WO2021243468A1 (fr) 2020-06-04 2021-12-09 London Pharmaceuticals And Research Corporation Analogues de cannabidiol rigide utilisés en tant que modulateurs puissants de récepteurs cannabinoïdes et leurs utilisations
CN115073407A (zh) * 2021-03-10 2022-09-20 上海中医药大学 一种具有合成致死性的药用组合物及其应用

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WO2000007579A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft UTILISATION DE DERIVES DE CYCLOPENTABENZOFURANNE POUR LUTTER CONTRE DES MALADIES DEPENDANT DU NF-λB
WO2000008007A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft Derives de cyclopentabenzofuranne et leur utilisation
WO2001072328A2 (fr) * 2000-03-28 2001-10-04 Eli Lilly And Company Methodes de traitement de maladies a l'aide de la proteine c activee

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WO2000008007A2 (fr) * 1998-08-05 2000-02-17 Bayer Aktiengesellschaft Derives de cyclopentabenzofuranne et leur utilisation
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054457A1 (fr) * 2003-12-03 2005-06-16 Bayer Cropscience Aktiengesellschaft Procede pour identifier des composes fongicides a partir de pyruvate kinases de champignons
JP2007538031A (ja) * 2004-05-18 2007-12-27 バイエル・ヘルスケア・アクチェンゲゼルシャフト 新規なシクロペンタ［ｂ］ベンゾフラン誘導体およびその使用
EP1693059A1 (fr) * 2005-02-22 2006-08-23 Deutsches Krebsforschungszentrum Utilisation des dérivés de rocaglamide en tant qu'inhibiteurs NF-À-spécifiques pour le traitement de certaines maladies inflammatoires
EP2189158A1 (fr) 2008-11-20 2010-05-26 DKFZ Deutsches Krebsforschungszentrum, Stiftung des Öffentlichen Rechts Combinaison de rocaglamide et de substances induisant l'apoptose pour le traitement du cancer
JP2012509923A (ja) * 2008-11-25 2012-04-26 ユニヴェルシテ・ドゥ・ストラスブール 心臓保護剤及び抗腫瘍剤としてのロカグラオール誘導体
WO2012066002A1 (fr) 2010-11-16 2012-05-24 Universite De Strasbourg Dérivés de flavagline en tant qu'agents neuroprotecteurs
EP2457907A1 (fr) 2010-11-16 2012-05-30 Université de Strasbourg Dérivés de flavagline en tant qu'agents neuroprotecteurs
WO2015075165A1 (fr) 2013-11-22 2015-05-28 Deutsches Krebsforschungszentrum Inhibiteurs de translation en chimiothérapie à dose élevée et/ou radiothérapie à dose élevée
WO2021243468A1 (fr) 2020-06-04 2021-12-09 London Pharmaceuticals And Research Corporation Analogues de cannabidiol rigide utilisés en tant que modulateurs puissants de récepteurs cannabinoïdes et leurs utilisations
CN113149942A (zh) * 2021-02-10 2021-07-23 贵州省中国科学院天然产物化学重点实验室(贵州医科大学天然产物化学重点实验室) 一种洛克米兰醇酚羟基衍生物、其制备方法和应用
CN115073407A (zh) * 2021-03-10 2022-09-20 上海中医药大学 一种具有合成致死性的药用组合物及其应用

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