Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
  • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
  • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
• • 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
  • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
  • A61K49/0052—Small organic molecules
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
  • C07D491/18—Bridged systems
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
  • C07D491/22—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains four or more hetero rings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
• • 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
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/531—Production of immunochemical test materials
  • G01N33/532—Production of labelled immunochemicals
  • G01N33/533—Production of labelled immunochemicals with fluorescent label

Definitions

• the invention concerns application of compounds based on coumarin derivatives of Troger's base with cyanin substitution. These compounds can be used in the area of selective intracellular localisation, namely as lysosomal probes.
• Lysosomes are no longer considered simple waste organelles, they are going to be studied as developed complex systems which have effect on many cellular processes like apoptosis. Lysosomal disorders can result in damage or loss of neurons and this can result in mental retardation, progressive degeneration and premature death.
• Lysosomal dis- regulation has been also observed with many serious diseases like Alzheimer, Parkinson and Huntington diseases, amyotrophic lateral sclerosis, cancer and cardiovascular, inflammatory and metabolic diseases (Appelqvist H, Waster P, Kagedal K, Ollinger K. : The lysosome: from waste bag to potential therapeutic target J. Mol. Cell. Biol. 2013, 5, 214-226; Degtyarev M, De Maziere A, Klumperman J, Lin K: Autophagy, an achilles' heel AKTing against cancer? Autophagy 2009, 5, 415-418.; Ciechanover, A.
• lysosomes are hard to observe using interference contrast. Therefore, there is effort to find vital dyes for specific colouring of lysosomes in living cells.
• Vast majority of current lysosomal probes is based on organic compounds of alkaline nature that contain a fluorescence unit. These substances are free of charge in neutral to weak alkaline environment which characterises blood and cytosol. But in acid, lysosomal environment, these substances are protonated and thus their lysosomal accumulation occurs.
• Troger's bases Alkaline compounds, called Troger's bases, are one among possible structural motives practical for preparation of lysosomal probes.
• fluorescence lysosomal probes based on Troger's bases have been described, like conjugates with dimethylaminomethyl (Wua Z, Tang M, Tian T, Wua J, Denga Y, Dong X, Tanc Z, Wenga X, Liua, Z, Wanga C, Zhou X.
• lysosomal probes are, above all: a high magnitude of optical coefficient, position of absorption maxima in visible field, high fluorescence and high selectivity for lysosomes.
• Applicability of Troger's bases for fluorescence labelling of lysosomes depends on combination of Troger's base with a suitable fluorescence unit.
• Troger's bases with cyanin substitution show many properties required for building of lysosomal fluorescence probes. (Wua Z, Tang M, Tian T, Wua J, Denga Y, Dong X, Tanc Z, Wenga X, Liua, Z, Wanga C, Zhou X.
• the subject hereof are coumarin derivatives of Troger's bases with cyanin substitution and their application as lysosomal fluorescence probes.
• the subject of the invention are coumarin derivatives of Troger's base with cyanin substitution of general formula I to ⁇ and their application as fluorescence lysosomal probes. These compounds show highly selective lysosomal localisation and they also show high absorption and emission and their absorption maxima are in the visible part of the spectrum.
• the subject of the invention are coumarin derivatives of Troger's base :
• Y is H or alkyl CI to C4;
• R is a cyanin system of general formula I to III; in case that Rl is R then R2, R3 and R4 are H; in case that R2 is R then Rl, R3 and R4 are H; in case that R3 is R then Rl, R2 and R4 are H; in case that R4 is R then Rl, R2 and R3 are H;
• A is alkyl CI to C12 or glycol chains with 1 to 8 glycol (-OCH2CH2-) repeating units terminated with O-alkyl substituent CI to C12 or -OH group or alkyl CI to C8 of sulphonic acid or their corresponding lithium or sodium, or potassium or cesium or rubidium salts or allyl or propargyl or benzyl;
• X is selected from the group containing: acetate, bromide, dihydrogenphosphate, fluoride, phosphate, hexafluorophosphate, hydrogensulphate, chloride, perchlorate, iodide, mesylate, monohydrogenphosphate, formate, nitrate, nonafluorbutylsulphonate, sulphate, tetrafluorob orate, tosylate, triflate, trifluoracetate, trichloracetate, carbonate; n is 1 or 2; and salts of coumarin derivatives of Troger's bases with cyanin substitution with inorganic or organic acids selected from the group containing acetylacetates, adipates, ascorbates, benzoates, besylates, borates, butanoates, citrates, deoxycholates, dihydrogenphosphates, phenylacetates, phosphates, fumarates, gallate, glutarates, hippurates,
• substance 2 with general formula II
• substance 3 with general formula III
• Fig. 1 Structural formula of coumarin derivative of I Troger's base with cyanin substitution
• Fig. 4 Localisation of substances 1 -5 in lysosomes of cells derived from cell lines of human breast carcinoma 4T1. Panels of microscopic photos:
• substance 1 in lysosomes of cells of human breast carcinoma 4T1 green fluorescence emission caused by presence of dimethinium chain of Troger's base
• Fig. 5 Composition of Dulbecco's Modified Eagle's medium containing glucose.
• Fig. 6 General structural formula of coumarin derivative of I, II or III Troger's base with cyanin substitution
• Celles derived from cellular line 4T1 were cultivated in DMEM medium (Dulbecco's Modified Eagle's medium with high content of glucose - for composition see Fig. 5, Sigma USA) completed with 10% fetal bovinne serum (FBS, Thermo Scientific, USA).
• the cells were kept in the logarithmic stage of growth and cultivated under the standard conditions (37°C, 5% C02, 95% moisture).
• the cells were inoculated into plate culture with glass bottom (diameter 35 mm) for microscopic experiments. The cells were incubated under the standard conditions for 16 hours.
• the prepared 4T1 cells were washed twice with pre-heated (37°C) phosphate buffer (PBS; pH 7.4) and incubated in the complete cultivation medium with substances 1 or 2 (for 15 minutes) or with substances 3, 4 or 5 (for 20 minutes). Substances 1 to 5 were used in the form of stock solution with concentration of 20 nM.

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• 2017
  • 2017-09-26 CZ CZ2017-588A patent/CZ2017588A3/cs unknown
• 2018
  • 2018-09-25 WO PCT/IB2018/057412 patent/WO2019064178A1/en not_active Ceased

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