EP2619590A2 - Composés pour le diagnostic de maladies neurodégénératives sur la rétine - Google Patents

Composés pour le diagnostic de maladies neurodégénératives sur la rétine

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Publication number
EP2619590A2
EP2619590A2 EP11804932.9A EP11804932A EP2619590A2 EP 2619590 A2 EP2619590 A2 EP 2619590A2 EP 11804932 A EP11804932 A EP 11804932A EP 2619590 A2 EP2619590 A2 EP 2619590A2
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EP
European Patent Office
Prior art keywords
alkyl
alkenyl
alkynyl
coo
enantiomers
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.)
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Application number
EP11804932.9A
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German (de)
English (en)
Inventor
Boris Schmidt
Daniel Kieser
Alexander BOLÄNDER
Jochen Herms
Roland Hans Heyny-Von Haussen
Jiamin GU
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ludwig Maximilians Universitaet Muenchen LMU
Technische Universitaet Darmstadt
Klinikum Darmstadt GmbH
Original Assignee
Ludwig Maximilians Universitaet Muenchen LMU
Technische Universitaet Darmstadt
Klinikum Darmstadt GmbH
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Application filed by Ludwig Maximilians Universitaet Muenchen LMU, Technische Universitaet Darmstadt, Klinikum Darmstadt GmbH filed Critical Ludwig Maximilians Universitaet Muenchen LMU
Publication of EP2619590A2 publication Critical patent/EP2619590A2/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0039—Coumarin dyes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/04—Indoles; Hydrogenated indoles
    • C07D209/10—Indoles; Hydrogenated indoles with substituted hydrocarbon radicals attached to carbon atoms of the hetero ring
    • C07D209/12—Radicals substituted by oxygen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/26—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders

Definitions

  • the present invention describes compounds having affinity for the A ⁇ protein, for ⁇ -synuclein and / or for tau-PHF aggregates, which are suitable as preferably fluorescent probes for the in vivo diagnosis of neurodegenerative diseases, e.g. Alzheimer's disease and Parkinson's disease.
  • the compounds are characterized by suitable physicochemical properties (such as excitation wavelength, emission wavelength, Stokes shift, extinction) and high affinity and selectivity to the Zieiproteinen.
  • the present invention relates to the use of such compounds for diagnostic purposes and to a method for the diagnosis of neurodegenerative diseases.
  • Alzheimer's or Parkinson's disease brings considerable benefits to the patient in therapy.
  • the reliable diagnosis of Alzheimer's disease is currently only possible by postmortem microscopy.
  • CT Computed Tomography
  • MRI Magnetic Resonance Imaging
  • PET Positron Emission Tomography
  • WO2009155017 discloses radiopharmaceutical compositions which have a high affinity for amyloid plaques and are detected by positron emission tomography.
  • WO2007136996 describes cyanine dyes which are used for the labeling of biomolecules, eg for the in vivo diagnosis of cancer.
  • US20020133019 discloses thioflavine derivatives for ante-mortem in vivo diagnosis of, among others, Alzheimer's disease. Labeled thioflavins bind to amyloid plaques and are detected by gamma imaging, MRI or NMR spectroscopy.
  • CSF cerebrospinal fluid
  • An object of the present invention is to provide suitable diagnostic probes for the detection of neurodegenerative diseases that can be detected by an optical detection method.
  • the present invention solves this problem by the use of certain compounds for the diagnosis of neurodegenerative diseases.
  • these compounds have at least three of the following properties a) -f):
  • TPSA topological polar surface area
  • This stabilization may be by hydrogen bonding or van der Waals interactions of the aryl units with the target protein.
  • Such a twist may be due to 1, 3 allyl stress or by 1, 4 Butadiene voltage of alkylated vinyl aromatic be induced.
  • Particularly preferred compounds or probes are aromatic molecules which have a ⁇ -electron system extended over at least two aromatic rings or aromatic ring systems.
  • compounds of the following classes have three or more of the aforementioned properties, so that the present invention provides the arylaminothiazoles, 2H-ndol-2-ylidene-1-propene-1-yl-indolium cations, benzothiazolylidene-1-propenyl-benzothiazolium cations Benzoxazolylidene-1-propenylbenzoxazolium cations, 4,6-divinylpyrimidines, 3,6-divinylpyridazines, 2,5-divinylpyrazines, [4- (1, 3-benzothiazol-2-yl) phenyl] hydrazones and / or diarylureas, which have an affinity for the A ⁇ protein, ⁇ -synuclein and / or tau-PHF aggregates and are therefore suitable for the diagnosis of neurodegenerative diseases.
  • Compounds of the class of arylaminothiazoles include compounds which preferably have the following general structure:
  • X, Y, Z independently of one another denote carbon or nitrogen and
  • R, R 2, R 3, R 4 independently of one another d-Ce-alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 4 -C 6 -Alkeninyl, C 3 -C 0 cycloalkyl, thioalkyl, alkoxy, CC 6 alkanoyl, C 6 -C 6 - aryl, C 6 -C 6 -heteroaryl, CRCE-haloalkyl, C2-C6 haloalkenyl, C 2 -C 6 haloalkynyl, C 4 -C 6 - Haloalkeninyl, C 3 -C 0 -allococycloalkyl, -H, -OH, -OCH 3) -OC 2 H 5 , -OCF 3 , -OC 2 F 5 , -NH 2 , -N (CH 3 ) 2 , -N (C 2 H 5
  • divinyl aromatics more preferably divinyl nitrogen heteroaromatics, and most preferably divinyl pyrimidines, divinyl pyridines, divinyl pyrazines, divinyl pyridazines and divinyl triazines.
  • Compounds of the class of 4,6-divinylpyrimidines include compounds which preferably have the folloWing general structure:
  • Ar represents one of the following cyclic, heterocyclic, aromatic or heteroaromatic radicals:
  • X means carbon or nitrogen
  • R 1, R 2, R 3 and R 4 are each independently Ci-C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 - alkynyl, C 4 -C 6 -Alkeninyl, C 3 -C 0 - cycloalkyl, thioalkyl, alkoxy, C C6-alkanoyl, C6 -Ci6 aryl, C 6 -C 16 -heteroaryl, dC 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 - haloalkynyl, C 4 - C 6 -haloalkeninyl, C 3 -Cio-halocycloalkyl, -H, -OH, -OCH 3 , - OC 2 H 5 ,
  • Ar represents one of the following cyclic, heterocyclic, aromatic or heteroaromatic radicals:
  • X means carbon or nitrogen
  • R 1, R 2 and R 3 independently of one another CC 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C4-C6 -Alkeninyl, C 3 -C 10 cycloalkyl, Thioaikyl, alkoxy, C 1 -C 6 -alkanoyl, C 6 -C 6 - Aryl, C6-Ci6-heteroaryl, -C 6 haloalkyl, C 2 -C 6 haloalkenyl, C 2 -C 6 haloa kinyl, C 4 -C 6 -Haloalkeninyl, C 3 -C 0 halocycloalkyl, -H,!
  • Compound compounds of the [4- (1,3-benzothiazol-2-yl) phenyl] hydrazones include compounds which preferably have the following general structure:
  • Ar represents one of the following cyclic, heterocyclic, aromatic or heteroaromatic radicals:
  • X means carbon or nitrogen
  • R 1 , R 2 and R 3 independently of one another are C 1 -C 6 -alkyl, C 2 -C 6 -alkenyl, C 2 -C 6 -alkynyl, C 4 -C 6 -alkeninyl, C 3 -C 10 -cycloalkyl, thioalkyl, alkoxy, Ci-Ce-alkanoyl, C 6 -C 16 - aryl, C 6 -C 6 -heteroaryl, CC 6 haloalkyl, C 2 -C 6 -Haioalkenyl, C 2 -C 6 haloalkynyl, C 4 -C 6 -Haloalkeninyl , C 3 -C 10 -halocycloalkyl, -H, -OH, -OCH 3 , -OC 2 H 5 , -OCF 3 , -OC 2 F 5 , -NH 2> -N (CH 3
  • Compounds of the class of 3,6-divinylpyridazines include compounds which preferably have the following general structure:
  • Ar represents one of the following cyclic, heterocyclic, aromatic or heteroaromatic radicals:
  • X means carbon or nitrogen
  • R 1, R 2 and R 3 are independently -C 6 alkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 4 -C 6 -Alkeninyl, C 3 -C 0 cycloalkyl, thioalkyl, alkoxy , C 1 -C 6 -alkanoyl, C 6 -C 6 - Aryl, C 6 -C 6 heteroaryl, Ci-C 6 haloalkyl, C 2 -C 6 -alkenyl -Haloa!, C 2 -C 6 haloalkynyl, C 4 -C 6 -Haloalkeninyl, C 3 -Cio-halocycloalkyl, -H, -OH, -OCH 3 , -OC 2 H 5 , -OCF 3 , -OC 2 F 5 , -NH 2> -N (CH 3 ) 2 , -N (C
  • X, X ⁇ Y, ⁇ ', Z, Z' are independently carbon or nitrogen and R, R 2, R 3, R 4, R 5, R 6 independently of one another 6 -alkyl, C 2 -C 6 alkenyl , C 2 -C 6 -alkynyl, C 4 -C 6 -alkeninyl, C 3 -C 10 -cycloalkyl, thioalkyl, alkoxy, C 1 -C 6 -alkanoyl, C 6 -C 16 -aryl, C 6 -C 6 -heteroaryl , C 1 -C 6 -haloalkyl, C 2 -C 6 -haloalkenyl, C 2 -C 6 -haloalkynyl, C 4 -C 6 -haloalkeninyl, C 3 -C 0 -Ha [ocycloalkyl, -H, -OH, -OCH 3 , -OC 2 H
  • Compounds of the class of 2H-indol-2-ylidene-1-propen-1-yl-indolium cations, benzothiazolylidene-1-propenyl-benzothiazolium cations and benzoxazolylidene-1-propenyl-benzoxazolium cations include compounds which preferably have the following general structure:
  • R is hydrogen, -F, -Cl, -Br, -I, -NO 2 , alkoxy;
  • X represents -Cl, -Br, -I, -OTs, -OMs;
  • Y is O, S, CR R 2 ;
  • R and R 2 are independently -CH 3 or -C 2 H 5 ;
  • Z is O or CH 2 ;
  • n 0, 1, 2 or 3.
  • C r C 6 alkyl means: -CH 3 , -C 2 H 5 , -C 3 H 7 , -CH (CH 3 ) 2, -C 4 H 9, -CH 2 -CH (CH 3 ) 2, - CHiCH ⁇ -C ⁇ Hs , -C (CH 3 ) 3 , -C 5 H 11, -CH (CH 3 ) -C 3 H 7 , -CH 2 -CH (CH 3 ) -C 2 H 5 , -CH (CH 3 ) -CH (CH 3 ) 2 , -CH (CH 3 ) -CH (CH 3 ) 2 , -CH (CH 3 ) -CH (CH 3 ) 2 ,
  • -C ⁇ CH 3 ) 2 -CH (CH 3 ) 2, -C 2 H 4 -C (CH 3 ) 3 , or -CH (CH 3 ) -C (CH 3 ) 3 .
  • Preferred are -CH 3 , -C 2 H 5 , -C 3 H 7 , -CH (CH 3 ) 2 , -C 4 H 9 , -CH 2 -CH (CH 3 ) 2, -CH (CH 3 ) - C 2 H 5, -C ⁇ CH 3 ) 3 and -C 5 Hn.
  • Particularly preferred are -CH 3 , -C 2 H 5, -C 3 H 7 and -CH (CH 3 ) 2 .
  • thioalkyl -S-C 1 -C 6 signifies alkyl wherein C 1 -C 6 -alkyl has the significance given above.
  • the following radicals are preferred: -S-CH 3 , -S-C 2 H 5 , -SC 3 H 7 , -S-CH (CH 3 ) 2 , -SC 4 H 9 , -S-CH 2 -CH (CH 3 ) 2, -S-CH (CH 3 ) -C 2 H 5i -SC (CH 3 ) 3 and -SC 5 Hn.
  • SC 3 H 7, -S-CH (CH 3) 2 and -S-C (CH 3) 3 - particularly preferably -S-CH 3l -SC 2 H 5, are.
  • Ci-C 6 haloalkyl means a Ci-C 6 alkyl group containing at least one halogen atom selected from fluorine, chlorine, bromine, iodine.
  • C 2 -C 6 haloalkenyl means a C 2 -C 6 alkenyl group containing at least one halogen atom selected from fluorine, chlorine, bromine, iodine.
  • C 2 -C 6 Haloalkynyl means a C 2 -C 6 alkynyl group containing at least one halogen atom selected from fluorine, chlorine, bromine, iodine.
  • C4-C6 -Haloalkeninyl means a C4-C6 -Alkeninyl group which contains at least one halogen atom selected from fluorine, chlorine, bromine, iodine and C 3 -C halocycloalkyl 0 represents a C 3 -C -cycloalkyl 0 Group containing at least one halogen atom selected from fluorine, chlorine, bromine, iodine.
  • alkyloxy or "alkoxy” means -O-C 1 -C 6 -alkyl wherein C 1 -C 6 -alkyl has the meaning given above.
  • the following C 1 -C 6 -alkoxy groups are preferred: -O-CH 3 , -O-C 2 H 5 , -O-C 3 H 7 , -O-CH (CH 3 ) 2, -O-C 4 H 9 , -O-CH 2 -CH ⁇ CH 3 ) 2 , -O-CH (CH 3 ) -C 2 H 5 , -O-C (CH 3 ) 3, and -O-C 5 Hn.
  • Especially preferred are -O-CH 3 , -O-C 2 H 5 , -O-C 3 H 7 , -O-CH (CH 3 ) 2 and -O-C (CH 3 ) 3 .
  • CH (CH 3 ) -CH CH-CH 3
  • -CH (CH 3 ) -CH 2 -CH CH-CH 3
  • -CH 2 -CH CH-CH (CH 3 ) 2
  • substituents from the group of phenols methylaniline, dimethylaniline, methyl-2-aminopyridine, dimethyl-2-aminopyridine.
  • Particular preference is given to compounds having three aromatic rings which are connected to one another via vinyl bridges, resulting in extended ⁇ -electron systems.
  • Particular preference is given to compounds of the abovementioned classes with delocalized electrons via at least 15 atoms involved. More preferred are compounds having ⁇ -electron systems extending over at least 20 atoms, and particularly preferred are delocalized electron systems extending over 22 or more atoms.
  • the classification of neurodegenerative diseases is based both on the clinical presentation with typical topographic distribution and cell type of the degenerative process as well as on the deposition of structurally altered proteins such as prion protein, tau, beta-amyloid, alpha-synuclein, TDP-43 and / or Huntington.
  • the fluorescence of the substances according to the invention is either enhanced or significantly shifted when bound to the target proteins. As a result, only the necessary signal-to-noise ratio is possible.
  • diagnosis or diagnosis covers the areas of in vivo, in vitro, ex vivo diagnostics. Generally, the diagnosis or diagnosis is used exclusively or mainly to provide information.This information provides information about:
  • samples derived from the human body are used, e.g. Blood, serum, plasma, seminal fluid, spinal fluid, peritoneal fluid, saliva, sputum, tear fluid, urine, biopsy material or tissue donation. Obtaining such a sample may or may not be part of the diagnostic procedure. In certain embodiments of the present invention, obtaining the sample intended for diagnosis is not a step in the diagnostic method of the invention.
  • the compounds of the invention are detected after binding to the A ⁇ protein, ⁇ -synuclein and / or tau-PHF aggregates in the retina.
  • arylaminothiazoles 2H-indol-2-ylidene-1-propene-1-yl-indolium cations according to the invention, benzothiazolylidene-1-propenylbenzothiazolium cations, benzoxazolylidene-1-propenylbenzoxazolium cations, 4,6-divinylpyrimidines, 3,6-divinylpyridazines, 2,5-divinylpyrazines, [4- (1, 3-benzothiazol-2-yl) phenyl] hydrazones and / or diarylureas are particularly preferably used for the diagnosis of newly rodegenerativen diseases.
  • the invention comprises a method for the diagnosis of neurodegenerative diseases, a) administering at least one compound selected from the group of arylaminothiazoles, 2H-indol-2-ylidene-1-propene-1-yl-indolium cations, benzothiazolylidene-1-propenylbenzothiazolium cations,
  • the invention further encompasses a method for the in vivo detection of A ⁇ protein, ⁇ -synuclein and / or tau-PH F aggregates,
  • Divinylpyrimidines 3,6-divinylpyridazines, 2,5-divinylpyrazines, [4- (1,3-benzothiazol-2-yl) phenyl] hydrazones and diaryl ureas;
  • the compounds mentioned herein preferably act as fluorescent probes. They have a (preferably high) affinity for the Aß protein, a-synuclein and / or tau-PHF aggregates and bind - preferably specifically - to them.
  • the binding of the compounds of the invention to one or more of the above target proteins is optically detectable.
  • the increase in absorbance upon binding to the target protein is preferably characterized by a> 10x increase in signal-to-noise improvement over the free compound and can be determined experimentally, for example, by reducing background noise.
  • Preference is given to compounds which have an extinction coefficient of ⁇ > 10,000 L mor crrf 1 .
  • the determination of the extinction coefficient bz.w the extinction is carried out in certain embodiments at 25 ° C, pH 7, the respective absorption maximum of the compound with DMSO as a solvent.
  • the difference between the excitation maximum and the emission maximum is called the Stokes shift and this value essentially determines how well a compound is suitable for fluorescence studies.
  • the compounds according to the invention are preferably characterized by a Stokes shift> 20 nm. Particular preference is furthermore given to compounds which have a half-life in vivo of> 60 min. For this purpose, in certain embodiments, residence time and excretion rate of correspondingly labeled probes (eg 3 H, C, 8 F) are determined.
  • the affinity of the fluorescent probes is usually determined indirectly by the displacement of fluorescent or radioactive reference ligands.
  • the affinity of the ligands of the invention is characterized by displacement of thioflavin S, thioflavin T or 11 C-PIB with an EC50 ⁇ 300nM. This can e.g. as described in Lockhart et al., March 4, 2005, The Journal of Biological Chemistry, 280, 7677-7684 under Material & Methods, in particular the two paragraphs "Radioligand Competition Assay” and "Fluorescence Competition Assay” ,
  • the diffusivity of a compound through the endothelium of the blood-brain barrier is largely determined by its lipid solubility (lipophilicity) and size.
  • the compounds of the invention have a molecular weight ⁇ 500 g / mol.
  • the log P value and the log D value are model measures of the relationship between lipophilicity (fat solubility) and hydrophilicity (water solubility) of a substance. The expectation is to be able to estimate the distribution coefficients of this substance in other systems with an aqueous and a lipophilic phase with the help of the octanol-water partition coefficient.
  • the log P value is greater than one if a substance is better soluble in fat-like solvents such as / 7-octanol, less than one if it is better soluble in water. Accordingly, log P value is positive for lipophilic and negative for hydrophilic substances. Preference is given to compounds which have a log P value of from 1 to 2.8. In further embodiments, compounds with a log D ⁇ 5 are preferred. The log P value or the log D value are measured by an octanol / water two-phase system and UV / VI S spectroscopy at 25 ° C. and pH 7.
  • TPSA topological polar surface area
  • the compounds according to the invention are further characterized by good photostability (low photobleaching) and by a short-lived singlet excitation against long-lasting triplet excitation.
  • the compounds of the invention have one or more of the following physicochemical properties. Particular preference is given to compounds which have at least three of the following properties a) -f):
  • TPSA topological polar surface area
  • TPSA topological polar surface area
  • TPSA topological polar surface area
  • the compounds according to the invention have at least three of the following properties a) -f), where at least one of the properties a) and d) is selected:
  • TPSA topological polar surface area
  • the compounds according to the invention have at least three of the following properties a) -i):
  • TPSA topological polar surface area
  • the compounds disclosed herein are particularly advantageous for the early diagnosis of new rodegenerative diseases from the group of tauopathies.
  • the group of tauopathies includes newly rodegenerative diseases, the common feature of which is the accumulation of the tau protein, a small molecule phosphoprotein that attaches to and supports the assembly of supporting cytoskeletal proteins (microtubules) in the brain.
  • the compounds referred to herein are used for the early diagnosis of tauopathies, e.g. Alzheimer's disease, corticobasal degeneration, agryophilic grain disease, Pick's disease, FTDP-17 (frontotemporal dementia and parkinsonism of chromosome 17) or progressive supranuclear palsy.
  • tauopathies e.g. Alzheimer's disease, corticobasal degeneration, agryophilic grain disease, Pick's disease, FTDP-17 (frontotemporal dementia and parkinsonism of chromosome 17) or progressive supranuclear palsy.
  • the compounds according to the invention are furthermore advantageous for the early diagnosis of neurodegenerative diseases from the group of synucleinopathies.
  • the group of synucleinopathies includes newly rodegenerative diseases whose common feature is the accumulation of the a-synuclein protein in the brain, e.g. Parkinson's disease.
  • the ⁇ -synuclein protein is a protein of 140 amino acids that normally occurs only in the presynaptic processes of neurons.
  • a detectable optical response is characterized in that a change or appearance of an optical signal can be observed or measured by instruments.
  • the optical response is a change in fluorescence, such as a change in intensity, excitation or emission wavelength, fluorescence lifetime, or fluorescence polarization.
  • the fluorescent probes are administered parenterally.
  • the compounds are administered enterally.
  • the compounds are ora! administered.
  • the compositions containing the compounds of this invention typically contain an effective concentration of the compounds in an aqueous solution or suspension which may further contain buffers, surfactants, thixotropic agents, cosolvents, flavoring agents or the like.
  • the compounds herein are preferably capable of crossing the blood-brain barrier.
  • the compounds used according to the invention can overcome the blood-retinal barrier.
  • the disease-causing protein deposits are also in the retina.
  • the compounds disclosed in the present application are detected after attachment to an A ⁇ protein, ⁇ -synuclein and / or tau-PHF aggregates in the patient's retina.
  • the detection takes place by means of adaptation of the optical fiber optics or the fluorescence microscopy.
  • Detection at the retina is done in one embodiment using adapted retinal scanners.
  • the confocal retinal laser scanner from Heidelberger Engineering can be used (Heidelberg Retina Angiograph 2, HRA2).
  • HRA2 Heidelberg Retina Angiograph 2, HRA2.
  • the detection can also take place via a fundus camera and / or via a laser scanner in combination with fluorescence ⁇ eg ICG) and autofluorescence techniques (eg lipofuscin).
  • fluorescence lifetime microscopy (FLIM) and spectral fluorescence distribution can be used to highly sensitively determine the binding of the probe of the present invention to the target
  • FLIM fluorescence lifetime microscopy
  • spectral fluorescence distribution can be used to highly sensitively determine the binding of the probe of the present invention to the target
  • the tissue-sparing excitation of dyes via multiphoton microscopy further provides a high-penetration method
  • Light sources are both laser and filtered light sources.
  • the detection takes place by means of suitable filter systems or detectors which are known in the prior art. Excitation in the wavelength range of 380-900 nm and emission at 400-1000 nm are preferred. The excitation between 450-500 nm and the emission at 600-650 nm or 600-700 nm is particularly preferred.
  • Alzheimer's dementia is diagnosed due to the assured absence of tau aggregates in the intestinal epithelium.
  • the tau aggregation in the intestinal epithelium correlates inversely with the diagnosis of Alzheimer's dementia: the lack of detection of tau aggregates with the disclosed compounds is a sure sign of Alzheimer's disease.
  • the compounds according to the invention are the following compounds:
  • Example 2 BSc4097: 4,4 ⁇ , 1 ⁇ ) -2,2 ⁇ 2- ( ⁇ ⁇ ) ⁇ -4,6- ⁇ ) 0 ⁇ 3 ( ⁇ -2,1-diyl) bis (N, N-dimethylaniline) Synthesis: 4,6-dimethylpyrimidine-2-thiol (100 mg, 0.64 mmol), 4- (dimethylamino) benzaldehyde (193.5 mg, 1.29 mmol) and Aliquat 336 (25 mg, 0.06 mmol) are dissolved in 5M NaOH solution (10 ml). The solution is heated for 1 h at 1 10 ° C to boiling, then stirred for 3 h at T.
  • BSc4328 4,6-bis ((E) -2- (naphthalen-1-yl) vinyl) pyrimidine
  • BSC4352 4.4, - (1E i 1 'E) -2,2, - (pyrazine-2,5-diyl) bis (ethene-diyl 2 t 1) to (W, W dimethylaniline)
  • BSc4354 2,5-bis (4-ethoxystyryl) pyrazine
  • Nicotinaldehyde (107 mg, 1 mmol) in tetrahydrofuran (30 ml) and NaOH (3 g, 75 mmol) are added to the reaction solution and heated at reflux for 3 hours. The cooled reaction solution is washed twice with water, dried over Na 2 SO 4 and the solvent removed in vacuo. After purification by column chromatography (1: 1 EtOAc / hexane, silica gel) 257 mg, (77.9%) of BSc4337 are obtained.
  • BSc4007 W- (4- (7- (Diethylamirol) -2-oxo-2H-chromen-3-yl) thiazol-2-yl) nicotinohydrazide
  • 6-Substituted 1-methyl-2-PEG-benzo [d] thiazole-3-ium 6-substituted 2-methylbenzo [d] thiazole (5 mmol) is dissolved in a 10 ml flask with 2-ethoxyethyl-4-toluenesulfonates ( 6 mmol). The reaction mixture is heated under argon atmosphere to 120 ° C and stirred for 12 hours, whereby the reaction mixture turns violet. Allow to cool to room temperature. The resulting precipitate is recrystallized from ethyl acetate to give 6-substituted 1-methyl-2-PEG-benzo [d] thiazole-3-ium colorless solid.
  • 6-Substituted 1-methyl-2-PEG-benzo [d] oxazol-3-ium 6-substituted 2-methylbenzo [d] oxazole (5 mmol) is dissolved in a 10 ml flask with 2-ethoxyethyl-4-toluenesulfonates ( 6 mmol). The reaction mixture is heated under argon atmosphere to 120 ° C and stirred for 12 hours, whereby the reaction mixture turns violet. Allow to cool to room temperature. The resulting precipitate is recrystallized from ethyl acetate to give 6-substituted 1-methyl-2-PEG-benzo [d] oxazol-3-ium colorless solid.
  • Betspiel 21 Staining protocol for the compounds of the invention
  • the tissue samples were fixed in 10% buffered Formaiin solution and embedded in paraffin. 4 ⁇ thick sections were made on the microtome and mounted on slides in a water bath. The deparaffination took place via the following steps:
  • the dyes according to the invention were dripped onto the tissue section from 0.01-1% ethanolic or methanolic solution (50-200 ⁇ l) and incubated in a moist, EtOH / MeOH saturated and light-protected chamber for 10 minutes.
  • a moist, EtOH / MeOH saturated and light-protected chamber for 10 minutes.
  • up to 10% DMSO was added and optionally filtered through a syringe filter (0.45pm pore size).
  • the samples were stained with the dyes of the present invention as described in Example 21 and then examined with a Zeiss Axioskop, ABO 100Hg Fluore, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX.
  • a FITC filter or a DAPI filter was used. The results can be seen in FIGS. 1 to 21 and show the binding and visualization of the dyes according to the invention.
  • Radioligand Competition Assay The affinity of the compounds of the invention was checked by a radioligand competition assay. For this, A ⁇ - (1-42) -peptide was incubated in a concentration of 10 mg / ml in PBS with 0.1% BSA together with [125 IJIMPY 0.1 nM and different ligand concentrations for 3 h at 20 ° C and then through a Whatman GF / B Filter filtered.
  • FIG. 1 staining with BSc4258 human brain tissue, patient AD +; amyloid
  • Plaque Plaque
  • FITC filter Zeiss Axioskop
  • ABO 100Hg fluorescent lamp camera: Leica DFC300FX
  • Figure 2 staining with BSc4090 ammone horn, male 89J, AD +, A ⁇ plaque, FITC filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 3 staining with BSc4090 ammone horn, male 89J, AD +, A ⁇ plaque, DAPI Filter, Zeiss Axioskop, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 4 Staining with BSc4097 Ammonium horn, male 89J, AD +, A ⁇ plaque, DAPI filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica
  • Figure 5 Staining with BSc4097 Ammonium horn, male 89J, AD +, A ⁇ plaque
  • Figure 6 Staining with BSc4327 Ammonium horn, male 82J, AD +, A ⁇ in one
  • Figure 8 Staining with BSc4352 Ammonshorn, male 82J, AD +, A ⁇ plaque, DAPI filter, Zeiss Axioskop, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 9 Staining with BSc4352 Ammonium horn, male 82J, AD +, A ⁇ plaque, DAPI Filter, Zeiss Axioskop, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 10 Staining with BSc4353 Ammonium horn, male 82J, AD +, Aß in one
  • Angiopathy FITC filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp,
  • Figure 1 Staining with BSc4353 Ammonium horn, male 89J, AD +, A ⁇ plaque, FITC filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 12 Staining with BSc4354 Ammonium horn, male 82J, AD +, A ⁇ in one
  • Angiopathy FITC filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 13 Staining with BSc4354 Ammonshorn, male 89J, AD +, A ⁇ plaque, FITC filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 14 staining with BSc4342 ammone horn, male 89J, AD +, A ⁇ plaque, DAPI Filter, Zeiss Axioskop, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 5 Staining with BSc4342 Ammonshorn, male 89J, AD +, A ⁇ plaque, DAPI filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • Figure 16 Staining with BSc4337 Ammonshorn, male 89J, AD +, A ⁇ plaque, DAPI filter, Zeiss Axioscope, ABO 100Hg fluorescence ampe, camera: Leica DFC300FX
  • Figure 17 Staining with BSc4337 Ammonshorn, male 89J, AD +, A ⁇ plaque, DAPI filter, Zeiss Axioscope, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX
  • FIG. 18 staining with BSc4007 ammone horn, male 89J, AD, tau fibril,
  • FIG. 19 staining with BSc4138 ammone horn, male 89J, AD, tau fibril,
  • Figure 20 Staining with BSc4138 Ammonium horn, male 89J, AD, A ⁇ plaque, DAPI filter; Zeiss Axioskop, ABO 100Hg fluorescent lamp, camera: Leica DFC300FX)

Abstract

La présente invention concerne des composés présentant une haute affinité pour la protéine Aβ, l'α-synucléine ou pour les agrégats Tau-PHF, lesquels conviennent comme sondes de préférence fluorescentes pour le diagnostic in vivo de maladies neurodégénératives, comme par exemple la démence d'Alzheimer ou la maladie de Parkinson. Les composés se distinguent par des propriétés physicochimiques appropriées (longueur d'onde d'excitation, longueur d'onde d'émission, déplacement de Stokes, extinction) ainsi que par une affinité et une sélectivité élevées vis-à-vis des protéines cibles.
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