WO2017014601A1 - Sonde de fluorescence à base d'indolizino [3,2-c] quinoline - Google Patents

Sonde de fluorescence à base d'indolizino [3,2-c] quinoline Download PDF

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WO2017014601A1
WO2017014601A1 PCT/KR2016/008059 KR2016008059W WO2017014601A1 WO 2017014601 A1 WO2017014601 A1 WO 2017014601A1 KR 2016008059 W KR2016008059 W KR 2016008059W WO 2017014601 A1 WO2017014601 A1 WO 2017014601A1
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compound
quinoline
nmr
mhz
group
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PCT/KR2016/008059
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Korean (ko)
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이지연
김익연
권순범
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서울대학교 산학협력단
연세대학교 산학협력단
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Priority claimed from KR1020160092867A external-priority patent/KR101850607B1/ko
Application filed by 서울대학교 산학협력단, 연세대학교 산학협력단 filed Critical 서울대학교 산학협력단
Publication of WO2017014601A1 publication Critical patent/WO2017014601A1/fr
Priority to US15/877,872 priority Critical patent/US10787448B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention relates to a fluorescent probe composition
  • a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound, and a nucleic acid / protein / cell imaging method using the same.
  • Indolizin and quinoline compounds are representative molecules that are targets in the development of various small molecule drugs. Since these compounds have different pharmacological properties depending on the substituent pattern bound to the aromatic ring, various applications are possible. For example, antifungal agents, antimalarial agents, apoptosis inducing agents, and EGFG kinase inhibitors have been reported.
  • the heterocycle system exhibiting strong fluorescence can be used as a molecular probe to monitor the reaction with the target molecule in biochemical research, and thus the demand for a new fluorophore having inherent photophysical properties. Is increasing. In particular, in monitoring the interaction between the ligand and the target, there is a need for the development of an environment-sensitive fluorophore whose optical properties vary depending on the physicochemical properties of the environment surrounding the molecule.
  • organophosphor development is an essential technology for cell imaging and protein function research, and it is necessary to develop a phosphor that can be applied in an aqueous solution that fluoresces light of a specific wavelength and is not interfered by other components in the cell. .
  • the problem with most of the currently developed organic phosphors is that the solubility in aqueous solutions is poor and the compounds themselves aggregate or cause aggregation of proteins. That is, there is an increasing demand for phosphors that can be used in aqueous solutions and buffer conditions, which can compensate for the disadvantages of existing phosphors, and have good brightness without causing aggregation of proteins.
  • the present inventors made a thorough study to develop a fluorescent probe with more improved properties, and found that some indolinino [3,2-c] quinoline compounds exhibit desirable optical properties.
  • the aim is to provide an aqueous solution system and a biocompatible, useful phosphor platform.
  • the present invention is to devise compounds having excellent environment-sensitivity, fluorescence intensity, photostability, nucleic acid / protein binding, intracellular permeability, and the like, and to provide a method of imaging (imaging) nucleic acids, proteins or cells using them.
  • the present invention provides a fluorescent probe composition
  • a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • R 1, R 2, R 3 , R 4 and R 5 are the same or different and are each independently hydrogen, halogen, C 1- 6 alkyl, C 1- 6 alkoxy, COOR 8, aryl, heteroaryl, , And C 1- 6 is selected from the group consisting of alkynyl,
  • R 6 is selected from the group consisting of C 1-6 alkyl, aryl and heteroaryl,
  • n 0, 1, 2 or 3
  • R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl,
  • R 8 is selected from the group consisting of hydrogen, C 1-6 alkyl and C 1-6 alkynyl,
  • Any one to three carbon atoms of the aryl and heteroaryl are the same or different from each other, and each independently hydrogen, halogen, nitro, , , , , , CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 6 alkyl and C 1- 6 alkoxy.
  • the aryl is characterized in that it is selected from the group consisting of phenyl, naphthyl, anthryl and biaryl.
  • the heteroaryl is selected from the group consisting of pyridyl, pyrimidyl, thiophenyl, pyrrolyl, furanyl and triazolyl.
  • R 1 is hydrogen or COOR 8
  • R 6 is aryl or heteroaryl
  • the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof is characterized in that at least one selected from the group consisting of the following formula (2) to (21).
  • the present invention also provides a nucleic acid, protein or cell detection reagent comprising the composition.
  • the present invention also provides a nucleic acid, protein or cell imaging method using the composition.
  • the present invention also provides an indolinino [3,2-c] quinoline compound represented by the following Chemical Formula 22 or a pharmaceutically acceptable salt thereof.
  • R 1 is hydrogen, COOEt, , And C 1- 6 is selected from the group consisting of alkynyl,
  • R 2 is , , And Is selected from the group consisting of
  • n 0, 1 or 2
  • R 3 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl.
  • the indolinino [3,2-c] quinoline compound used in the fluorescent probe of the present invention is a water-soluble fluorescent compound whose characteristics and application ranges of fluorescence vary greatly depending on the nature and position of the functional group attached to the hetero ring.
  • the shortcomings of the system can be improved and utilized in various fields.
  • the fluorescent probes of the present invention can be applied to nucleic acid and proteins to be applied to functional studies and imaging techniques of various nucleic acids / proteins such as their movements and drug-protein interactions.
  • the fluorescent probe of the present invention has a large difference between the excitation wavelength and the fluorescence wavelength, thereby minimizing self-quenching.
  • the fluorescent probe of the present invention can be controlled to maximize the fluorescence in the organic solvent and fluorescence in the aqueous solution because the wavelength range of the fluorescence is changed according to the substituent and the sensitivity to the surrounding environment is changed (solvatochromic).
  • the fluorescent probe of the present invention is well fluorescence in water or buffer, solubility and can minimize the problem of protein aggregation.
  • the fluorescent probe of the present invention is useful for cellular or tissue imaging technology and intracellular enzyme activity analysis because of its excellent intracellular permeability.
  • FIG. 1 is a diagram showing the chemical formula of five compounds of high fluorescence intensity in an aqueous solution of the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 2 is a diagram showing absorption and emission spectra measured in water of five compounds having high fluorescence intensity in an aqueous solution among the indolinino [3,2-c] quinoline compounds of the present invention.
  • FIG. 3 is a graph of the types of substituents bonded to the indolinino [3,2-c] quinoline compound of the present invention when the R 6 substituent is a phenyl group, and the fluorescence quantum yield (QY) in an aqueous solution. It is a figure which shows a correlation.
  • DW distilled water
  • DMSO dimethyl sulfoxide
  • DMF dimetyl formamide
  • 5A is a diagram showing the results of evaluation of the fluorescence yield in aqueous solution or ethanol of the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 5B is a graph showing quantum yield tendency in an ethanol solvent for eleven compounds having high fluorescence in the indolinino [3,2-c] quinoline compound of the present invention.
  • 6A to 6C show the light stability in the aqueous solution 6a or ethanol 6b with respect to the indolinino [3,2-c] quinoline compound of the present invention, and further, when blue light was irradiated for 2 hours in the aqueous solution ( It is a figure which shows the result of having evaluated the light stability of 6c).
  • 7A to 7D are evaluation of the binding property with DNA of the indolinino [3,2-c] quinoline compound of the present invention, and the compound group (7a) in which the maximum emission wavelength is shifted to a short wavelength while the fluorescence intensity is changed. ), A compound group 7b in which the maximum emission wavelength shifts to a long wavelength while the intensity of fluorescence changes, a compound group in which only the intensity of fluorescence changes, and a compound group 7c in which no change occurs are shown.
  • FIG. 8 is a diagram showing the results of evaluation of the binding properties with HSA protein for the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 9 is a visual confirmation that fluorescence increases upon binding to HSA for the indolinino [3,2-c] quinoline compound of the present invention, and Job's Plot to confirm whether the compound selectively binds 1: 1 with the protein. The figure which shows the result of having performed.
  • FIG. 10 is a diagram visualizing an increase in fluorescence intensity through binding of HSA protein to the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 11 is a diagram showing the results of evaluation of the binding properties with HSA protein for the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 12 is a diagram showing the results confirmed by the FRET phenomenon whether binding to the PDE ⁇ protein for the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 13 is a diagram showing the results of confirming the binding strength (kd) of the indolinino [3,2-c] quinoline compound of the present invention and the intensity of binding force (kd) through fluorescence polarization.
  • FIG. 14 is a diagram showing a result of confirming competitive binding experiment with delta-racin by fluorescence polarization to bind to PDE ⁇ protein to the indolinino [3,2-c] quinoline compound of the present invention.
  • 15 and 16 are diagrams showing the results confirmed by the electrophoresis and fluorescence whether binding to the PDE ⁇ protein for the indolinino [3,2-c] quinoline compound of the present invention.
  • FIG. 17 to 21 show the results of cell permeability evaluation for the indolinino [3,2-c] quinoline compound of the present invention, more specifically, Figures 17 to 19 in the live HeLa cell, Figure 20 is MCF7 And in NIH-3T3 cells, Figure 21 is a diagram showing the results of evaluating cell permeability in Sf21 cells.
  • FIG. 22 is a diagram showing the results of confirming the cell permeability of the indolinino [3,2-c] quinoline compound of the present invention through costaining with DAPI, an existing staining reagent.
  • Figure 23a is a diagram showing the results confirmed by the colocalizaion experiments with the conventional staining reagents DAPI and Mitotracker for the indolizino [3,2-c] quinoline compound of the present invention
  • Figure 23b is fluorescent as the light is applied It is a figure which shows the result of this increasing compound.
  • FIG. 24 is a diagram showing the results obtained by comparing fluorescence stability of the indolinino [3,2-c] quinoline compound of the present invention with a conventional staining reagent, Syto RNAselect or Mitotracker.
  • FIG. 24 is a diagram showing the results obtained by comparing fluorescence stability of the indolinino [3,2-c] quinoline compound of the present invention with a conventional staining reagent, Syto RNAselect or Mitotracker.
  • 25 is a diagram showing the results confirmed by the MTT assay for cytotoxicity to the indolizino [3,2-c] quinoline compound of the present invention.
  • the present invention provides a fluorescent probe composition
  • a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
  • R 1, R 2, R 3 , R 4 and R 5 are the same or different and are each independently hydrogen, halogen, C 1- 6 alkyl, C 1- 6 alkoxy, COOR 8, aryl, heteroaryl, , And C 1- 6 is selected from the group consisting of alkynyl,
  • R 6 is selected from the group consisting of C 1-6 alkyl, aryl and heteroaryl,
  • n 0, 1, 2 or 3
  • R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl,
  • R 8 is selected from the group consisting of hydrogen, C 1-6 alkyl and C 1-6 alkynyl,
  • Any one to three carbon atoms of the aryl and heteroaryl are the same or different from each other, and each independently hydrogen, halogen, nitro, , , , , , CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 6 alkyl and C 1- 6 alkoxy.
  • R 1, R 2, R 3, R 4 and R 5 are the same or different hydrogen, each independently of one another, C 1- 3 alkyl, C 1- 3 alkoxy, COOR 8 , , And it is selected from the group consisting of C 1- 3 alkynyl,
  • R 6 is aryl or heteroaryl
  • n 0, 1 or 2
  • R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 3 alkyl,
  • Any one to three carbon atoms of the aryl and heteroaryl are the same or different from each other, and each independently hydrogen, halogen, , , , , CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 3 alkyl and C 1- 3 alkoxy.
  • halogen may include F, Cl, Br and I.
  • the "aryl” may be selected from the group consisting of phenyl, naphthyl, anthryl and biaryl, wherein the "heteroaryl” is pyridyl, pyrimidyl, thiophenyl, pyrrolyl, furanyl and tria It may be selected from the group consisting of zolyl.
  • R 1 is hydrogen or COOR 8
  • R 6 may be aryl or heteroaryl.
  • C 1- 6 alkyl may be a straight chain or branched the alkyl, specifically methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert- butyl, n-propyl, isopropyl, n It may be selected from the group consisting of hexyl and isohexyl.
  • the "C 1- 6 alkoxy" may be selected from methoxy, ethoxy, propoxy, butoxy, pentoxy group consisting of.
  • the "probe” may be produced in the form of oligonucleotide probes, double stranded DNA probes, RNA probes, and the like, and selection and hybridization conditions of the appropriate probes are based on those known in the art. It can be transformed into In addition, the probe may be an imaging probe, but is not limited thereto.
  • the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof may be, for example, any one or two or more selected from the group consisting of Formulas 2 to 21, but is not limited thereto. no.
  • the present invention also provides an indolinino [3,2-c] quinoline compound represented by the following Chemical Formula 22 or a pharmaceutically acceptable salt thereof.
  • R 1 is hydrogen, COOEt, , And C 1- 6 is selected from the group consisting of alkynyl,
  • R 2 is , , And Is selected from the group consisting of
  • n 0, 1 or 2
  • R 3 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl.
  • the term "pharmaceutically acceptable” is suitable for use in contact with tissue of a subject (eg, a human being) because the benefit / risk ratio is reasonable without excessive toxicity, irritation, allergic reactions or other problems or complications.
  • a compound or composition is within the scope of sound medical judgment.
  • Acid addition salts include inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid and aliphatic mono and dicarboxylates, phenyl-substituted alkanoates, hydroxy alkanoates and alkanes. Obtained from non-toxic organic acids such as dioates, aromatic acids, aliphatic and aromatic sulfonic acids.
  • Such pharmaceutically nontoxic salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, and iodide.
  • the acid addition salts according to the present invention can be dissolved in conventional methods, for example, by dissolving a compound represented by the formula (1) in an excess of an aqueous solution of an acid, which salt is a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. It can be prepared by precipitation using. It may also be prepared by evaporating the solvent or excess acid from the mixture and then drying or by suction filtration of the precipitated salt.
  • Bases can also be used to make pharmaceutically acceptable metal salts.
  • Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving the compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is pharmaceutically suitable to prepare sodium, potassium or calcium salt as the metal salt.
  • the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable negative salt (eg silver nitrate).
  • the compound represented by Formula 1 may be prepared by reacting the compound represented by Formula A with aldehyde in the presence of a catalyst.
  • R 1 , R 2 , R 3 , R 4, and R 5 are as defined above.
  • the catalyst may be any one selected from the group consisting of FeCl 3 , AlCl 3 , BiCl 3 , InCl 3 , p-Toluenesulfonic acid (PTSA) and Pyridinium p-Toluenesulfonic acid (PPTS), in particular, FeCl 3 is preferred because it can be produced in high yield without generation of side reactions.
  • FeCl 3 is preferred because it can be produced in high yield without generation of side reactions.
  • the catalyst may be contained in an amount of 0.1 to 0.3 equivalents, if the content of the catalyst is less than 0.1 equivalent, the catalyst amount is too small, the reactivity is bad, if the content of the catalyst is more than 0.3 equivalent rather than the reactivity is not preferable because not.
  • the reaction can be carried out at 20 °C to 80 °C under a solvent selected from the group consisting of methylene chloride, N, N- dimethylformamide and tetrahydrofuran, in particular at 40 °C to 80 °C under dichloromethane solvent It is preferable to carry out that the reaction yield can be improved.
  • the aldehyde may be selected from C 1- 6 alkyl aldehyde, an aryl aldehyde and heteroaryl aldehydes, without being limited thereto.
  • Specific examples of the aldehyde include 4-bromobenzaldehyde, 4-fluorobenzaldehyde, 3-fluorobenzaldehyde, 4-chlorobenzaldehyde, 3-chlorobenzaldehyde, 4-nitrobenzaldehyde, benzaldehyde, 4-methoxybenzaldehyde, 3-methoxy Oxybenzaldehyde, 4-methylbenzaldehyde, 3,5-dimethoxybenzaldehyde, 3,4-dimethoxybenzaldehyde, 1-naphthylaldehyde, 2-naphthylaldehyde, picolinealdehyde, 5-bromothiophene-2-carbaldehyde , 5-chlorothioph
  • the present invention provides a nucleic acid, protein or cell detection reagent comprising the composition.
  • the material labeled by the fluorescent probe of the present invention is not limited, and examples thereof include antibodies, enzymes, hormones, receptors, antigens, nucleic acids, natural drugs, viral particles, bacterial particles, cells, and the like, preferably nucleic acids, Proteins such as antibodies, enzymes, hormones, receptors, or cells such as blood cells, tissue cells, bacteria.
  • the present invention also provides a nucleic acid, protein or cell imaging method using the composition.
  • the fluorescent probe of the present invention can be applied to imaging by measuring the fluorescence after treating the specimen with the fluorescent probe.
  • fluorescence can be measured by dissolving in polar organic solvents such as dimethyl sulfoxide (DMSO), adding it to a buffer, treating it with a sample and incubating it.
  • polar organic solvents such as dimethyl sulfoxide (DMSO)
  • the probe concentration in the polar organic solvent is not particularly limited, but is generally 2 ⁇ M.
  • the incubation time is not particularly limited and may be appropriately selected depending on the specimen, but generally 5 minutes to 1 hour is preferable.
  • the temperature of the incubation is not particularly limited and a temperature suitable for each sample can be appropriately selected. Generally, the temperature is 0 ° C to 40 ° C.
  • a temperature suitable for the culture for example, cells derived from humans
  • 37 ° C. in the case of tissue for example, cells derived from humans
  • the measurement of fluorescence can also be performed using a commercially available fluorometer, and can be observed using a fluorescence microscope or a confocal laser scanning fluorescence microscope when the position and the property of the enzyme in a cell are investigated.
  • the sample is not particularly limited and may be any that is intended to measure the enzyme activity contained therein, and various cells and tissues may be mentioned as preferable examples.
  • the fluorescence can be measured by incubating as described above after replacing the culture solution of the cell or tissue with the above-described fluorescent probe solution.
  • Flash chromatography was performed on silica gel using hexene, ethyl acetate and dichloromethane as eluent, and all reactions were monitored by thin layer chromatography (0.25 mm silica plate; F-254) and visualized by UV.
  • 1 H and 13 C NMR spectra were recorded on a 400 MHz NMR spectrometer and HRMS was measured with an electrospray ionization (ESI) and Q-TOF mass analyzer.
  • the target compound was obtained by the method of Example 2-1 except that 4-nitrobenzaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 4-methylbenzaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that benzaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that picolinaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 4-methoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 3,5-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 3,4-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
  • the desired compound was obtained by the method of Example 2-1 except that 1-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 2-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except that 5-chlorothiophene-2carbaldehyde was used instead of 4-bromobenzaldehyde.
  • the target compound was obtained by the method of Example 2-1 except for using the 2- (8-methylindolizin-2-yl) aniline compound instead of the 2- (indolizin-2-yl) aniline compound.
  • a 2- (8-methylindolizin-2-yl) aniline compound was used instead of a 2- (indolizin-2-yl) aniline compound and 3,5-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde. Except for the above, the target compound was obtained by the method of Example 2-1.
  • a 2- (8-methylindolizin-2-yl) aniline compound was used in place of the 2- (indolizin-2-yl) aniline compound and 3,4-dimethoxybenzaldehyde was used in place of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • a 2- (8-methylindolizin-2-yl) aniline compound was used instead of a 2- (indolizin-2-yl) aniline compound and 3,5-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde. Except for the above, the target compound was obtained by the method of Example 2-1.
  • the 2- (6-bromoindolizin-2-yl) aniline compound was used instead of the 2- (indolizin-2-yl) aniline compound, and furan-2-carbaldehyde was used instead of 4-bromobenzaldehyde. Except for the above, the target compound was obtained by the method of Example 2-1.
  • the 2- (6-bromoindolizin-2-yl) aniline compound was used instead of the 2- (indolizin-2-yl) aniline compound, and furan-2-carbaldehyde was used instead of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound was used instead of 2- (indolizin-2-yl) aniline compound and 3-fluorobenzaldehyde was used instead of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound was used instead of 2- (indolizin-2-yl) aniline compound and 3-methoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound is used in place of 2- (indolizin-2-yl) aniline compound and 3,4-dimethoxybenzaldehyde is substituted for 4-bromobenzaldehyde. Except for using, the target compound was obtained by the method of Example 2-1.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound was used instead of 2- (indolizin-2-yl) aniline compound, and 2-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound was used instead of 2- (indolizin-2-yl) aniline compound, and 1-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
  • a target compound was obtained by the method of Example 2-1 except for the above.
  • the desired compound was obtained by the method of Example 2-36 (31%, 11 mg) except that 4- (diethylamino) benzaldehyde was used instead of 4- (dimethylamino) benzaldehyde.
  • a 2- (8-methylindolizin-2-yl) aniline compound was used in place of the 2- (indolizin-2-yl) aniline compound, and a thiopin-2-carbaldehyde was used in place of 4-bromobenzaldehyde. Except for the above, the target compound was obtained by the method of Example 2-1.
  • the target compound was obtained by the method of Example 2-1 except that 4-methoxy-3- (morpholinomethyl) benzaldehyde was used instead of 4-bromobenzaldehyde.
  • Ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound is used instead of 2- (indolizin-2-yl) aniline compound and 4-methoxy-3- (instead of 4-bromobenzaldehyde
  • the target compound was obtained by the method of Example 2-1 except that morpholinomethyl) benzaldehyde was used.
  • the target compound was obtained by the method of Example 2-1 except that 1H-indole-7-carbaldehyde was used instead of 4-bromobenzaldehyde.
  • the desired compound was obtained by the method of Example 2-1 except that pyridine-2,6-dikalbaldehyde was used instead of 4-bromobenzaldehyde.
  • the conditions for the compound for use as a biological phosphor include high emission intensity in water, low self-quenching, and stokes shift to minimize homotransfer of energy. Should be large, for the development of medicinal chemistry and fluorophores, solubility should be excellent so as to minimize aggregation with proteins, and in particular, these properties are more necessary conditions for use as protein labeling dyes.
  • the compounds of the present invention generally exhibit a maximum absorption in the range of approximately 380-430 nm, which means that excitation of visible light is possible.
  • maximum emission was observed in the aqueous solution in the range of approximately 480-540 nm.
  • the most noticeable feature is that large Stokes shifts (90-110 nm) are observed in most compounds, which means that there is a clear separation between the excitation and emission wavelengths. That is, the emission spectrum was observed in the range similar to Alexa Fluor 488 or Fluorescein, but the excitation wavelength was quite different from the emission wavelength.
  • the compound of formula (IQ 6) has a high fluorescence having a maximum emission at 514 nm as a structure in which the p -methoxyphenyl group is bonded to the R 6 position. It is believed that the methoxy group at the para position plays an important role in increasing the fluorescence intensity because the electron donor methoxy group exists in the opposite position to the quinoline nitrogen which is deficient in electrons. That is, upon molecular excitation, significant dipole moment changes are expected to occur.
  • the degree of showing the solvent-dependent (ie environmental-sensitive) tendency is different depending on the substituent of the compound, in particular the form of the compound of formula 16 (IQ 55) and quaternary ammonium salt having acetylene
  • the compounds of Formulas 20 and 21 IQ 6-S1-Me and 6-S2-Cl
  • the environment-sensitive optical properties of which the maximum fluorescence wavelength value changed according to the polarity of the solvent were hardly shown.
  • the compound of Formula 19 IQ 5-D
  • IQ 5-D shifted the wavelength indicating the maximum fluorescence to a long wavelength up to 543 nm in an aqueous solution.
  • indolinino [3,2-c] quinoline compound may be adjusted by varying the substituents.
  • environmentally sensitive properties of indolinino [3,2-c] quinoline compounds suggest that they can be used as fluorescent probes useful for monitoring biological processes such as drug-target binding or protein dynamics.
  • an aqueous solution or an ethanol solvent is as follows. The light stability over time was evaluated.
  • the stability of the fluorescence was determined by re-measuring the fluorescence for each cuvette sample from the natural light state to the last 6-7 hours.
  • the indolinino [3,2-c] quinoline compound of the present invention including the IQ 3, 4, 6, and 8 compounds, in which the fluorescence was stably maintained in the aqueous solution for about 6 hours, was
  • X is represented, some of the materials without substituents in the A, B, C, and D rings were selected and irradiated with blue light in an aqueous solution.
  • the DNA oligomer (48bp) used for the experiment contains the tetO sequence, and the nucleotide sequence is as follows.
  • tetO1 5'-CCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTC-3 '
  • tetO2 5'-GAGTGGTAAAATAACTCTATCAATGATAGAGTGTCAACAAAAATTAGG-3 '
  • Each of the oligomers were mixed with the same molar amount by heating at 95 ° C. for 5 minutes, and cooled at room temperature for 2 hours to hybridize, followed by measuring the fluorescence of the indolinino [3,2-c] quinoline compound of the present invention. After addition of the corresponding DNA oligomer to the cuvette, the light was blocked for 1 minute, incubated, and the fluorescence was measured again.
  • the compound of Formula 7 binds to DNA, and the fluorescence is gradually decreased and the wavelength is shifted toward the long wavelength, and the compound of Formula 8 (IQ 18) including Furan (Furan) binds to DNA and is gradually reduced. It was confirmed that the wavelength shifted toward the short wavelength (7d).
  • the properties of the indolinino [3,2-c] quinoline compound of the present invention to bind with the protein was confirmed. Specifically, 13 kinds of compounds having high fluorescence in Tris buffer (pH 7.5) among indolinino [3,2-c] quinoline compounds were treated with BSA (Bovine Serum Albumin) and HSA (Human Serum Albumin) to observe the fluorescence change. It was.
  • BSA Bovine Serum Albumin
  • HSA Human Serum Albumin
  • the compound of formula 9 the compound itself shows an emission near 500 nm when excited at 280 nm
  • HSA itself shows an emission of 340 nm at 280 nm
  • 1 equivalent to HSA The addition of the compound of HSA decreased the intensity of 340 nm emission of the HSA itself, and the emission near 450 nm was also observed, indicating that the fluorescence resonance energy transfer (FRET) phenomenon occurred due to the combination of HSA and the material.
  • FRET fluorescence resonance energy transfer
  • the compound of Formula 9 (IQ 7) is more compound-HSA (third vial), i.e., compound alone compared to compound alone (first vial) or HSA alone (second vial).
  • QY quantum yield
  • the compound of Formula 9 and HSA 1 were obtained through Job's Plot 1: It was confirmed that the binding to 1, and based on the titration (titration), the K D Value was found to be 1.89 ⁇ M (see Fig. 9).
  • Fluorescence Resonance Energy Transition target protein PDE- ⁇ 6 using fluorescence resonance energy transfer (FRET)
  • K-Ras protein has been found to be 20-30% mutated in human cancer cells and has been known to cause cancer. For the past several decades, many studies have been conducted on anti-cancer target proteins. Since affinity binds strongly to picomolar levels and there is no known allosteric site in the protein, no therapeutic agent to directly inhibit K-Ras protein has yet been developed. Recently, studies on the intracellular location and cancer-causing mechanisms of K-Ras protein have been conducted on PDE ⁇ , a target protein that can prevent signal transduction related to cell proliferation of K-Ras protein. In addition, no inhibitors of PDE ⁇ have been reported.
  • the PDE ⁇ protein shows a maximum emission wavelength at 340 nm by tryptophan residue when irradiated with 280 nm light, and the intensity of the maximum emission wavelength of 340 nm decreases when the ligand is bound, or a fluorescence resonance energy transfer, FRET) shows the change of the maximum emission wavelength.
  • the fluorescence of the recombinant PDE ⁇ and / or indolinino [3,2-c] quinoline compound which was separated and purified by E. coli cells Were measured by irradiation with 280 nm light, respectively.
  • the PDE ⁇ protein was set at 0 with 0.5 ⁇ M indolinino [3,2-c] quinoline compound present.
  • the degree of polarization was measured by titration with increasing concentration up to 32 ⁇ M.
  • the polarization value of the indolinino [3,2-c] quinoline compound and the PDE ⁇ protein complex does not change with time, whereas the polarization degree decreases when an excess amount of deltarasine is added.
  • the deltarasin and the IQ compound bind to the same binding site.
  • the half-life of a compound to be measured in the presence of an excess of a competing compound could be measured through a graph in which the degree of polarization decreases with time, whereby a compound having a good affinity (Kd) is obtained. It was found to have a longer half-life.
  • the indolinino [3,2-c] quinoline compounds derived through fluorescence screening in the present invention have a high affinity of 300-500 nM and have fluorescence, and thus, the inhibitor of PDE? In the study, it is expected to be used as a probe compound, and in terms of economy and efficiency, it is also expected to be used as an alternative to the detection method using antibodies.
  • each fraction of the protein (Sol: Soluble, FT: Flow through, W1: Washing 1, WF: Washing final, M: Marker, E1-5: Elution)
  • Experiment was performed by adding compound (IQ 3) (1: 0.5 mM, 2: 0.1 mM), and the protein was isolated and identified as shown in FIG. 15.
  • Example 7-1 binding to the protein was confirmed using a native gel.
  • fluorescence analysis of the compound of formula 13 for 12% native gel using ImageQuant LAS 4000 (Ex 312 nm, Em 585-625 nm) (A) and of the formula 13 for the native gel through Coomassie blue staining Fluorescence analysis (B) of the compound was carried out (1: DMSO added to Lysate, 2: 5 ⁇ M added to Compound 3 in Lysate, 3: 50 ⁇ M added to Compound 3 in Lysate, M: Marker, 4: DMSO added to purified PDE ⁇ ) , 5: 5 ⁇ M compound 3 was added to purified PDE ⁇ , 6: 50 ⁇ M compound 3 was added to purified PDE ⁇ ), and the results are shown in FIG. 16.
  • the indolinino [3,2-c] quinoline compound of the present invention was tested as follows to determine whether the cell permeability is excellent.
  • HeLa cells were seeded in 96 well plates and incubated for 24 hours. The cells were treated with an indolinino [3,2-c] quinoline compound and incubated at 37 ° C., 5% CO 2 conditions for 30 minutes. After removing the medium, the cells were washed with 1 ⁇ DPBS. The washed cells were fixed at 4% formaldehyde at room temperature for 10 minutes and washed with 1 ⁇ DPBS. Cell images were screened with the Operetta HTS imaging system (PerkinElmer).
  • HeLa cells were inoculated with 70-80% of the area of confocal dish (SPL life science) and incubated overnight, followed by removing the existing DMEM medium and mixing with DMEM to prepare the indolinino [3,2-c] quinoline compound (10 uM). Aliquots were incubated at 37 ° C. for 30 minutes ⁇ 1 hour. Then, if necessary, mix the LysoTracker (50nM) in DMEM to remove the existing medium, divide and incubate for 30 minutes at 37 ° C. After incubation, use a confocal laser microscope (Leica TCS SP8 SMD, Leica, Mannheim, Germany) without washing. Confirmed.
  • the DAPI used to stain the nucleus was blue wavelength, whereas the A group compound was found to be dyed at the green wavelength.
  • the compounds sense specific substances or conditions in the cells and thus the light at the long wavelength side. It was confirmed that it can be used as a sensitive probe that turns on.
  • the compounds of Formulas 17 and 18 are markers commonly used at green wavelength and blue wavelength, respectively. Co-localization with Lysotracker was confirmed. As a result, it was confirmed that the fluorescent materials of the present invention can be stained only in selective intracellular organelles without removing the fluorescent material, and also have the advantage of excellent cell permeability and different targets depending on the structure of the compound.
  • the indolinino [3,2-c] quinoline compound of the present invention was superior in cell permeability compared to other commercially available dyes.
  • the compound of formula 11 (IQ 16) was found to be stained almost similarly to DAPI, while the compound of formula 11 was green, while the DAPI was blue, and had many advantages such as less damage to cells. It is expected to be.
  • the compounds of Formula 2 and Formula 12 (IQ 6 and 36) have a similar pattern to RNA select staining RNA, of which Compound 12 is complementary to DAPI known to stain the nucleus. Staining was confirmed.
  • the compound of Formula 8 confirmed that the Pearson's coefficient was 0.8 or more through co-localization experiment with Mitotracker (FIG. 23A). This suggests that the compound of Formula 8 selectively stains mitochondria.
  • the compound of Formula 8 shows an increase in fluorescence with light, which can be confirmed through the IQ 53 having a furan (furan) group (Fig. 23b).
  • Indolizino [3,2-c] quinoline compound of the present invention was confirmed to have superior fluorescence stability compared to Sytoselect or Mitotracker through comparative experiments with other commercially available dyes, and as shown in FIG. Compound (IQ 36) was stained RNA in a similar fashion to the currently commercially available Sytoselect (RNA marker), but was observed to have better stability. In addition, in the case of the compound of Formula 8 (IQ 18), as described above, it was also quantitatively confirmed that the fluorescence increased with light.
  • 96-wells were inoculated with 1 ⁇ 10 4 cells per well and incubated in a 37 ° C. incubator for 24 hours. Thereafter, the indolinino [3,2-c] quinoline compound was treated at various concentrations (0.5, 1, 2, 5 and 10 ⁇ M) and incubated for 24 hours. After incubation, the medium was replaced with 100 ⁇ l, and then 20 ⁇ l of MTT reagent (5 mg / ml in DPBS) was added, followed by incubation for 3 hours. Finally, after dissolving formazin using DMSO, the absorbance at 570 nm was analyzed to examine the effects of drug proliferation. DMSO 0.1% was used as a control.
  • the compounds of the present invention did not show cytotoxicity when treated with MCF7 cells at a concentration of 10 ⁇ M or less for 24 hours, and when treated with A549 cells and Hela cells for 24 hours. There was no cytotoxicity. This confirmed that the compounds of the present invention can be used as a suitable fluorescent material for use in cell imaging.
  • the indolinino [3,2-c] quinoline compound used in the fluorescent probe of the present invention is a water-soluble fluorescent compound whose characteristics and application ranges of fluorescence vary greatly depending on the nature and position of the functional group attached to the hetero ring.
  • the shortcomings of the system can be improved and utilized in various fields.
  • the fluorescent probes of the present invention can be applied to nucleic acid and proteins to be applied to functional studies and imaging techniques of various nucleic acids / proteins such as their movements and drug-protein interactions.
  • the fluorescent probe of the present invention has a large difference between the excitation wavelength and the fluorescence wavelength, thereby minimizing self-quenching.
  • the fluorescent probe of the present invention can be controlled to maximize the fluorescence in the organic solvent and fluorescence in the aqueous solution because the wavelength range of the fluorescence is changed according to the substituent and the sensitivity to the surrounding environment is changed (solvatochromic).
  • the fluorescent probe of the present invention is well fluorescence in water or buffer, solubility and can minimize the problem of protein aggregation.
  • the fluorescent probe of the present invention is useful for cellular or tissue imaging technology and intracellular enzyme activity analysis because of its excellent intracellular permeability.

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Abstract

La présente invention concerne une composition de sonde de fluorescence comprenant un composé à base d'indolizino [3,2-c] quinoléine, et un procédé d'imagerie d'acide nucléique/de protéine/de cellule utilisant cette dernière. Comme le composé de la présente invention démontre une sensibilité environnementale, une intensité de fluorescence, une photostabilité, une liaison à un acide nucléique/une protéine, une perméabilité intracellulaire, etc., excellentes, le composé peut être efficacement utilisé pour diverses études de fonctions de protéine/cellule et des technologies d'imagerie, telles que des cinétiques acide nucléique/protéine, des interactions protéine-médicament, et une imagerie de protéine intracellulaire.
PCT/KR2016/008059 2015-07-23 2016-07-22 Sonde de fluorescence à base d'indolizino [3,2-c] quinoline WO2017014601A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113402538A (zh) * 2021-05-08 2021-09-17 香港理工大学深圳研究院 一种以喹啉鎓离子为骨架的荧光分子及其标记的多肽或蛋白质与制备方法
CN113402538B (zh) * 2021-05-08 2023-12-19 香港理工大学深圳研究院 一种以喹啉鎓离子为骨架的荧光分子及其标记的多肽或蛋白质与制备方法

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