WO2017014601A1 - Indolizino [3,2-c] quinoline-based fluorescent probe - Google Patents

Indolizino [3,2-c] quinoline-based fluorescent probe 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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French (fr)
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/en
Application filed by 서울대학교 산학협력단, 연세대학교 산학협력단 filed Critical 서울대학교 산학협력단
Publication of WO2017014601A1 publication Critical patent/WO2017014601A1/en
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

The present invention relates to a fluorescent probe composition comprising an indolizino [3,2-c] quinoline-based compound, and a nucleic acid/protein/cell imaging method using the same. Since the compound of the present invention demonstrates excellent environmental sensitivity, fluorescence intensity, photostability, nucleic acid/protein binding, intracellular permeability, etc., the compound may be effectively used for various studies of protein/cell functions and imaging technologies, such as nucleic acid/protein kinetics, drug-protein interactions, and intracellular protein imaging.

Description

인돌리지노[3,2-ｃ]퀴놀린계 형광 프로브Indolizino [3,2-c] quinoline-based fluorescent probe
본 발명은 인돌리지노[3,2-c]퀴놀린 화합물을 포함하는 형광 프로브 조성물, 및 이를 이용한 핵산/단백질/세포 이미징 방법에 관한 것이다.The present invention relates to a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound, and a nucleic acid / protein / cell imaging method using the same.
다양한 소분자 약물 개발시 타겟이 되는 분자로서 인돌리진과 퀴놀린계 화합물이 대표적이다. 이들 화합물은 방향고리에 결합된 치환기 패턴에 따라 약리적인 특성이 달라지기 때문에 다양한 응용이 가능한데, 예를 들면, 항곰팡이제, 항말라리아제, 아폽토시스 유도제, EGFG 키나아제 억제제 등이 보고된 바 있다.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.
새로운 화학적 스캐폴드를 고안하기 위한 노력으로서, 상기 인돌리진 화합물과 퀴놀린 화합물을 결합한 혼성구조가 주목받고 있다. 특히, 3-아실인돌리진과 2-아릴퀴놀린은 항암 활성이 알려져 있기 때문에, 3-아실인돌리진과 2-아릴퀴놀린의 혼성구조로서의 신규 헤테로방향족 화합물은 항암 활성 이외에도 형광 센서로서의 가능성이 제기되고 있다.In an effort to devise a new chemical scaffold, a hybrid structure combining the indolizin compound and the quinoline compound has attracted attention. In particular, since 3-acyl indolizine and 2-arylquinoline are known for their anticancer activity, a novel heteroaromatic compound as a hybrid structure of 3-acyl indolizine and 2-arylquinoline has been proposed as a fluorescent sensor in addition to anticancer activity. .
한편, 강한 형광을 나타내는 헤테로사이클 시스템은 생화학적 연구에 있어서 타겟 분자와의 반응을 모니터링할 수 있는 분자 프로브로 이용될 수 있기 때문에, 고유의 광물리적 특성을 갖는 새로운 형광단 (fluorophore)에 대한 수요가 증가하고 있다. 특히, 리간드와 타겟의 상호작용을 모니터링하는 데 있어서, 분자를 둘러싸는 환경의 물리화학적 성질에 따라 광학특성이 달라지는 환경-민감성 형광단에 대한 개발이 요구되고 있다.On the other hand, 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.
또한, 유기형광체 개발은 세포 이미징 및 단백질 기능연구에 필수적인 기술이며, 특정 파장의 빛에 형광을 발하면서 세포 내 다른 구성요소들에 의해 간섭을 받지 않는, 수용액에서 적용 가능한 형광체에 대한 개발이 필요하다. 현재 개발된 대부분의 유기형광체의 문제점은 수용액에서 용해도가 좋지 않아 화합물 자체가 응집하거나 단백질의 응집 (aggregation)을 초래한다는 점이다. 즉, 기존 형광체의 단점을 보완할 수 있는, 수용액 및 버퍼 조건에서도 사용 가능하고 단백질의 응집을 일으키지 않으면서 밝기가 좋은 형광체에 대한 수요가 증가하고 있다. 예를 들면, 종래 많이 사용되는 Alexa 488이나 fluorescein과 같은 형광물질의 경우에는, 여기 (excitation) 파장과 형광을 나타내는 파장이 너무 근접하여 자체적으로 소광 (quenching)되는 단점이 있으며, RNAselect나 Lysotracker 또한, 세포 염색시 광안정성이 낮은 단점이 있는 바, 이를 극복할 수 있는 새로운 형광체에 대한 개발이 필요한 실정이다 (한국등록특허 제10-1019390호 참조).In addition, 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. For example, in the case of fluorescent materials such as Alexa 488 or fluorescein, which are widely used, excitation wavelengths and fluorescence wavelengths are too close to quench themselves, and RNAselect or Lysotracker may also be used. When the cell staining has a disadvantage of low light stability, it is necessary to develop a new phosphor that can overcome this situation (see Korean Patent No. 10-1019390).
이에 본 발명자들은 상기와 같은 문제를 해결하고자, 보다 향상된 특성을 갖는 형광 프로브를 개발하기 위해 예의 연구한 결과, 몇몇 인돌리지노[3,2-c]퀴놀린계 화합물이 바람직한 광학특성을 나타냄을 발견하고, 수용액시스템과 바이오 친화적인, 유용한 형광체 플랫폼을 제공하는 것을 그 목적으로 한다.In order to solve the above problems, 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.
따라서, 본 발명은 환경-민감성, 형광강도, 광안정성, 핵산/단백질 결합, 세포내 투과성 등이 우수한 화합물들을 고안, 이들을 이용하여 핵산, 단백질 또는 세포의 영상화(이미징) 방법 등을 제공하고자 한다.Accordingly, 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.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당업자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.
본 발명은 하기 화학식 1로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는, 형광 프로브 조성물을 제공한다.The present invention provides a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
Figure PCTKR2016008059-appb-I000001
Figure PCTKR2016008059-appb-I000001
상기 화학식 1에서,In Chemical Formula 1,
R1, R2, R3, R4 및 R5는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, C1- 6알킬, C1- 6알콕시, COOR8, 아릴, 헤테로아릴,
Figure PCTKR2016008059-appb-I000002
,
Figure PCTKR2016008059-appb-I000003
및 C1- 6알키닐로 이루어지는 군으로부터 선택되며, 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,
Figure PCTKR2016008059-appb-I000002
,
Figure PCTKR2016008059-appb-I000003
And C 1- 6 is selected from the group consisting of alkynyl,
R6는 C1-6알킬, 아릴 및 헤테로아릴로 이루어지는 군으로부터 선택되고,R 6 is selected from the group consisting of C 1-6 alkyl, aryl and heteroaryl,
n은 0, 1, 2 또는 3이고, n is 0, 1, 2 or 3,
R7은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택되고,R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl,
R8은 수소, C1-6알킬 및 C1-6알키닐로 이루어지는 군으로부터 선택되며,R 8 is selected from the group consisting of hydrogen, C 1-6 alkyl and C 1-6 alkynyl,
상기 아릴 및 헤테로아릴의 임의의 탄소 원자 1 내지 3개는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, 니트로,
Figure PCTKR2016008059-appb-I000004
,
Figure PCTKR2016008059-appb-I000005
,
Figure PCTKR2016008059-appb-I000006
,
Figure PCTKR2016008059-appb-I000007
,
Figure PCTKR2016008059-appb-I000008
, CF3, COO-, C1- 6알킬 및 C1- 6알콕시로 이루어지는 군으로부터 선택되는 치환기와 연결된다.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,
Figure PCTKR2016008059-appb-I000004
,
Figure PCTKR2016008059-appb-I000005
,
Figure PCTKR2016008059-appb-I000006
,
Figure PCTKR2016008059-appb-I000007
,
Figure PCTKR2016008059-appb-I000008
, CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 6 alkyl and C 1- 6 alkoxy.
본 발명의 일 구현예에 있어서, 상기 아릴은 페닐, 나프틸, 안트릴 및 바이아릴로 이루어지는 군으로부터 선택되는 것을 특징으로 한다.In one embodiment of the present invention, the aryl is characterized in that it is selected from the group consisting of phenyl, naphthyl, anthryl and biaryl.
본 발명의 다른 구현예에 있어서, 상기 헤테로아릴은 피리딜, 피리미딜, 티오페닐, 피롤릴, 퓨라닐 및 트리아졸릴로 이루어지는 군으로부터 선택되는 것을 특징으로 한다.In another embodiment of the present invention, the heteroaryl is selected from the group consisting of pyridyl, pyrimidyl, thiophenyl, pyrrolyl, furanyl and triazolyl.
본 발명의 또 다른 구현예에 있어서, 상기 R1은 수소 또는 COOR8이고, R6는 아릴 또는 헤테로아릴인 것을 특징으로 한다.In another embodiment of the present invention, R 1 is hydrogen or COOR 8 , and R 6 is aryl or heteroaryl.
본 발명의 또 다른 구현예에 있어서, 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염은 하기 화학식 2 내지 화학식 21로 이루어진 군에서 선택되는 하나 이상인 것을 특징으로 한다.In another embodiment of the present invention, 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).
Figure PCTKR2016008059-appb-I000009
Figure PCTKR2016008059-appb-I000009
Figure PCTKR2016008059-appb-I000010
Figure PCTKR2016008059-appb-I000010
또한, 본 발명은 상기 조성물을 포함하는, 핵산, 단백질 또는 세포 검출 시약을 제공한다.The present invention also provides a nucleic acid, protein or cell detection reagent comprising the composition.
또한, 본 발명은 상기 조성물을 이용한, 핵산, 단백질 또는 세포 이미징 방법을 제공한다.The present invention also provides a nucleic acid, protein or cell imaging method using the composition.
또한, 본 발명은 하기 화학식 22로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염을 제공한다.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.
[화학식 22][Formula 22]
Figure PCTKR2016008059-appb-I000011
Figure PCTKR2016008059-appb-I000011
상기 화학식 22에서,In Chemical Formula 22,
R1은 수소, COOEt,
Figure PCTKR2016008059-appb-I000012
,
Figure PCTKR2016008059-appb-I000013
및 C1- 6알키닐로 이루어지는 군으로부터 선택되며,R 1 is hydrogen, COOEt,
Figure PCTKR2016008059-appb-I000012
,
Figure PCTKR2016008059-appb-I000013
And C 1- 6 is selected from the group consisting of alkynyl,
R2는
Figure PCTKR2016008059-appb-I000014
,
Figure PCTKR2016008059-appb-I000015
,
Figure PCTKR2016008059-appb-I000016
및
Figure PCTKR2016008059-appb-I000017
로 이루어지는 군으로부터 선택되고,R 2 is
Figure PCTKR2016008059-appb-I000014
,
Figure PCTKR2016008059-appb-I000015
,
Figure PCTKR2016008059-appb-I000016
And
Figure PCTKR2016008059-appb-I000017
Is selected from the group consisting of
n은 0, 1 또는 2 이고, n is 0, 1 or 2,
R3은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택된다.R 3 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl.
(다만, R1이 수소 또는 COOEt이고, n이 0일 때, R2가
Figure PCTKR2016008059-appb-I000018
인 경우는 제외한다.)(However, when R 1 is hydrogen or COOEt, n is 0, R 2
Figure PCTKR2016008059-appb-I000018
Is excluded.)
본 발명의 형광 프로브에 이용되는 인돌리지노[3,2-c]퀴놀린 화합물은 헤테로 고리에 붙은 관능기의 성질과 위치에 따라 형광의 특성과 활용 범위가 크게 달라지는 수용성 형광 화합물로서, 기존의 유기형광체의 단점이 개선되어 다양한 분야에서 활용될 수 있다.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.
또한, 본 발명의 형광 프로브를 핵산, 단백질에 결합시켜 이들의 움직임, 약물-단백질 상호작용 등 다양한 핵산/단백질의 기능 연구 및 이미징 기술에 응용할 수 있다.In addition, 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.
또한, 본 발명의 형광 프로브는 여기 파장과 형광 파장의 차이가 커서 (Stoke shift) 자기 소광(self-quenching)을 최소화할 수 있다.In addition, the fluorescent probe of the present invention has a large difference between the excitation wavelength and the fluorescence wavelength, thereby minimizing self-quenching.
또한, 본 발명의 형광 프로브는 치환기에 따라 형광의 파장대가 달라지고 주변환경에 대한 민감도가 달라지므로, 유기용매에서 형광을 극대화시키고 수용액에서 형광이 잘 나타나게 조절할 수 있다(solvatochromic).In addition, 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).
또한, 본 발명의 형광 프로브는 물이나 버퍼에서도 형광이 잘 나타나며 용해도가 좋고 단백질을 응집시키는 문제를 최소화할 수 있다.In addition, the fluorescent probe of the present invention is well fluorescence in water or buffer, solubility and can minimize the problem of protein aggregation.
또한, 본 발명의 형광 프로브는 세포 내 투과성이 우수하기 때문에 세포 또는 조직내 이미징 기술, 세포내 효소 활성 분석에 유용하다.In addition, 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.
도 1은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물 중 수용액에서 형광강도가 높은 화합물 5종의 화학식을 나타내는 도이다.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.
도 2는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물 중 수용액에서 형광강도가 높은 화합물 5종에 대하여 수중에서 측정된 흡수 및 방출 스펙트럼을 나타내는 도이다.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.
도 3은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물의 R6 치환기가 페닐기일 경우, 여기에 결합하는 치환기의 종류와 수용액에서의 형광 양자수율(fluorescence Quantum Yield; QY)과의 상관 관계를 나타내는 도이다.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.
도 4는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물 중 수용액에서 형광강도가 높은 화합물 5종에 대하여 다양한 용매 중 (DW(distilled water), DMSO(dimethyl sulfoxide), DMF(dimetyl formamide), EtOH(ethanol), EtOH:CHCl3=1:5, MC(methylene chloride))에서의 스펙트럼 분석을 실시한 결과를 나타내는 도이다.FIG. 4 shows five compounds having high fluorescence intensity in an aqueous solution of the indolinino [3,2-c] quinoline compound of the present invention (DW (distilled water), DMSO (dimethyl sulfoxide), DMF (dimetyl formamide) in various solvents. ), EtOH (ethanol), EtOH: CHCl 3 = 1: 5, MC (methylene chloride) is a diagram showing the results of the results of the analysis.
도 5a는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 수용액 또는 에탄올 중에서의 형광수율을 평가한 결과를 나타내는 도이다.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.
도 5b는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물 중 형광광도가 높은 화합물 11종에 대하여 에탄올 용매 중에서의 양자수율 경향성을 나타내는 도이다.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. FIG.
도 5c는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 다양한 용매 중에서의 스펙트럼 분석을 실시한 결과를 나타내는 도이다.It is a figure which shows the result of the spectrum analysis in various solvents about the indolinino [3, 2-c] quinoline compound of this invention.
도 6a 내지 6c는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 수용액(6a) 또는 에탄올(6b) 중에서의 광안정성을 평가하고, 나아가, 수용액 중에서 청색광을 2 시간 조사한 경우(6c)의 광안정성을 평가한 결과를 나타내는 도이다.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).
도 7a 내지 7d는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 DNA와의 결합특성을 평가하고, 이를, 형광의 세기가 변하면서 최대 방출 파장이 단파장으로 이동하는 화합물 그룹(7a), 형광의 세기가 변하면서 최대 방출 파장이 장파장으로 이동하는 화합물 그룹(7b), 형광의 세기만 변하는 화합물 그룹 및 아무 변화가 일어나지 않는 화합물 그룹(7c)으로 구분하여 나타내는 도이다.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.
도 8은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 HSA 단백질과의 결합특성을 평가한 결과를 나타내는 도이다.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.
도 9는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 HSA와 결합 시 형광이 증가함을 시각적으로 확인하고, 화합물이 단백질과 선택적으로 1:1 결합하는지를 확인하기 위하여 Job's Plot을 실시한 결과를 나타내는 도이다.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.
도 10은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 HSA 단백질과의 결합을 통한 형광 세기 증가를 시각화하여 나타내는 도이다.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.
도 11은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 HSA 단백질과의 결합특성을 평가한 결과를 나타내는 도이다.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.
도 12는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 PDEδ 단백질과의 결합하는지를 FRET 현상을 통하여 확인한 결과를 나타내는 도이다.12 is a diagram showing the results confirmed by the FRET phenomenon whether binding to the PDEδ protein for the indolinino [3,2-c] quinoline compound of the present invention.
도 13은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 PDEδ 단백질과의 결합하는지와 더불어 결합력의 세기 (kd)를 형광편광을 통하여 확인한 결과를 나타내는 도이다.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.
도 14는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 PDEδ 단백질과의 결합하는지를 델타라신과의 경쟁적 결합 실험을 형광편광을 통하여 확인한 결과를 나타내는 도이다.FIG. 14 is a diagram showing a result of confirming competitive binding experiment with delta-racin by fluorescence polarization to bind to PDEδ protein to the indolinino [3,2-c] quinoline compound of the present invention. FIG.
도 15 및 도 16은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 PDEδ 단백질과의 결합하는지를 전기영동 및 형광을 통하여 확인한 결과를 나타내는 도이다.15 and 16 are diagrams showing the results confirmed by the electrophoresis and fluorescence whether binding to the PDEδ protein for the indolinino [3,2-c] quinoline compound of the present invention.
도 17 내지 도 21은 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 세포 투과성을 평가한 결과를 나타내는 것으로, 보다 자세하게는 도 17 내지 19는 live HeLa cell에서, 도 20은 MCF7 및 NIH-3T3 cell에서, 도 21은 Sf21 cell에서의 세포 투과성을 평가한 결과를 나타내는 도이다.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.
도 22는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 세포 투과성을 기존 염색 시약인 DAPI와의 costaining을 통하여 확인한 결과를 나타내는 도이다.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.
도 23a는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 세포 투과성을 기존 염색 시약인 DAPI 및 Mitotracker와의 colocalizaion 실험을 통하여 확인한 결과를 나타내는 도이며, 도 23b는 빛이 가해질수록 형광이 증가하는 화합물의 결과를 나타내는 도이다.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.
도 24는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 형광 안정성을 기존 염색 시약인 Syto RNAselect 또는 Mitotracker와의 비교를 통하여 확인한 결과를 나타내는 도이다.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.
도 25는 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물에 대하여 세포 독성을 MTT 분석에 의하여 확인한 결과를 나타내는 도이다.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.
본 발명은 하기 화학식 1로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는, 형광 프로브 조성물을 제공한다.The present invention provides a fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
[화학식 1][Formula 1]
Figure PCTKR2016008059-appb-I000019
Figure PCTKR2016008059-appb-I000019
상기 화학식 1에서,In Chemical Formula 1,
R1, R2, R3, R4 및 R5는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, C1- 6알킬, C1- 6알콕시, COOR8, 아릴, 헤테로아릴,
Figure PCTKR2016008059-appb-I000020
,
Figure PCTKR2016008059-appb-I000021
및 C1- 6알키닐로 이루어지는 군으로부터 선택되며, 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,
Figure PCTKR2016008059-appb-I000020
,
Figure PCTKR2016008059-appb-I000021
And C 1- 6 is selected from the group consisting of alkynyl,
R6는 C1-6알킬, 아릴 및 헤테로아릴로 이루어지는 군으로부터 선택되고,R 6 is selected from the group consisting of C 1-6 alkyl, aryl and heteroaryl,
n은 0, 1, 2 또는 3이고, n is 0, 1, 2 or 3,
R7은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택되고,R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl,
R8은 수소, C1-6알킬 및 C1-6알키닐로 이루어지는 군으로부터 선택되며,R 8 is selected from the group consisting of hydrogen, C 1-6 alkyl and C 1-6 alkynyl,
상기 아릴 및 헤테로아릴의 임의의 탄소 원자 1 내지 3개는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, 니트로,
Figure PCTKR2016008059-appb-I000022
,
Figure PCTKR2016008059-appb-I000023
,
Figure PCTKR2016008059-appb-I000024
,
Figure PCTKR2016008059-appb-I000025
,
Figure PCTKR2016008059-appb-I000026
, CF3, COO-, C1- 6알킬 및 C1- 6알콕시로 이루어지는 군으로부터 선택되는 치환기와 연결된다.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,
Figure PCTKR2016008059-appb-I000022
,
Figure PCTKR2016008059-appb-I000023
,
Figure PCTKR2016008059-appb-I000024
,
Figure PCTKR2016008059-appb-I000025
,
Figure PCTKR2016008059-appb-I000026
, CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 6 alkyl and C 1- 6 alkoxy.
상기 화학식 1에서, 보다 바람직하게는, R1, R2, R3, R4 및 R5는 서로 동일하거나 상이하고, 각각 독립적으로 수소, C1- 3알킬, C1- 3알콕시, COOR8,
Figure PCTKR2016008059-appb-I000027
,
Figure PCTKR2016008059-appb-I000028
및 C1- 3알키닐로 이루어지는 군으로부터 선택되며,In Formula 1, and more preferably, 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 ,
Figure PCTKR2016008059-appb-I000027
,
Figure PCTKR2016008059-appb-I000028
And it is selected from the group consisting of C 1- 3 alkynyl,
R6는 아릴 또는 헤테로아릴이고,R 6 is aryl or heteroaryl,
n은 0, 1 또는 2이고, n is 0, 1 or 2,
R7은 수소, 하이드록시, 할로겐 및 C1- 3알킬로 이루어지는 군으로부터 선택되고,R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 3 alkyl,
상기 아릴 및 헤테로아릴의 임의의 탄소 원자 1 내지 3개는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐,
Figure PCTKR2016008059-appb-I000029
,
Figure PCTKR2016008059-appb-I000030
,
Figure PCTKR2016008059-appb-I000031
,
Figure PCTKR2016008059-appb-I000032
,
Figure PCTKR2016008059-appb-I000033
, CF3, COO-, C1- 3알킬 및 C1- 3알콕시로 이루어지는 군으로부터 선택되는 치환기와 연결된다.Any one to three carbon atoms of the aryl and heteroaryl are the same or different from each other, and each independently hydrogen, halogen,
Figure PCTKR2016008059-appb-I000029
,
Figure PCTKR2016008059-appb-I000030
,
Figure PCTKR2016008059-appb-I000031
,
Figure PCTKR2016008059-appb-I000032
,
Figure PCTKR2016008059-appb-I000033
, CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 3 alkyl and C 1- 3 alkoxy.
본 발명에 있어서, 상기 “할로겐”은 F, Cl, Br 및 I를 포함할 수 있다.In the present invention, the "halogen" may include F, Cl, Br and I.
본 발명에 있어서, 상기 “아릴”은 페닐, 나프틸, 안트릴 및 바이아릴로 이루어지는 군으로부터 선택될 수 있고, 상기 “헤테로아릴”은 피리딜, 피리미딜, 티오페닐, 피롤릴, 퓨라닐 및 트리아졸릴로 이루어지는 군으로부터 선택될 수 있다.In the present invention, 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.
본 발명에 있어서, 상기 R1은 수소 또는 COOR8이고, R6는 아릴 또는 헤테로아릴일 수 있다.In the present invention, R 1 is hydrogen or COOR 8 , R 6 may be aryl or heteroaryl.
또한, 상기 “C1- 6알킬”은 직쇄상 또는 분쇄상 알킬일 수 있으며, 구체적으로 메틸, 에틸, 노말프로필, 이소프로필, 노말부틸, 이소부틸, tert-부틸, 노말프로필, 이소프로필, 노말헥실 및 이소헥실로 이루어지는 군으로부터 선택될 수 있다.In addition, the "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.
본 발명에 있어서, 상기 “C1- 6알콕시”는 메톡시, 에톡시, 프로폭시, 부톡시, 펜톡시로 이루어지는 군으로부터 선택될 수 있다.In the present invention, the "C 1- 6 alkoxy" may be selected from methoxy, ethoxy, propoxy, butoxy, pentoxy group consisting of.
본 발명에 있어서, 상기 "프로브"는 올리고 뉴클레오티드 프로브, 이중쇄 DNA (double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있으며, 적당한 프로브의 선택 및 혼성화 조건은 당업계에 공지된 것을 기초로 변형할 수 있다. 또한, 상기 프로브는 이미징 프로브일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, 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.
본 발명에 있어서, 상기 화학식 1로 표시되는 화합물 또는 이의 약학적으로 허용 가능한 염은 예를 들어, 하기 화학식 2 내지 화학식 21로 이루어지는 군으로부터 선택되는 어느 하나 또는 2종 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, 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.
Figure PCTKR2016008059-appb-I000034
Figure PCTKR2016008059-appb-I000034
Figure PCTKR2016008059-appb-I000035
Figure PCTKR2016008059-appb-I000035
또한, 본 발명은 하기 화학식 22로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염을 제공한다.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.
[화학식 22][Formula 22]
Figure PCTKR2016008059-appb-I000036
Figure PCTKR2016008059-appb-I000036
상기 화학식 22에서,In Chemical Formula 22,
R1은 수소, COOEt,
Figure PCTKR2016008059-appb-I000037
,
Figure PCTKR2016008059-appb-I000038
및 C1- 6알키닐로 이루어지는 군으로부터 선택되며,R 1 is hydrogen, COOEt,
Figure PCTKR2016008059-appb-I000037
,
Figure PCTKR2016008059-appb-I000038
And C 1- 6 is selected from the group consisting of alkynyl,
R2는
Figure PCTKR2016008059-appb-I000039
,
Figure PCTKR2016008059-appb-I000040
,
Figure PCTKR2016008059-appb-I000041
및
Figure PCTKR2016008059-appb-I000042
로 이루어지는 군으로부터 선택되고,R 2 is
Figure PCTKR2016008059-appb-I000039
,
Figure PCTKR2016008059-appb-I000040
,
Figure PCTKR2016008059-appb-I000041
And
Figure PCTKR2016008059-appb-I000042
Is selected from the group consisting of
n은 0, 1 또는 2 이고, n is 0, 1 or 2,
R3은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택된다.R 3 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl.
(다만, R1이 수소 또는 COOEt이고, n이 0일 때, R2가
Figure PCTKR2016008059-appb-I000043
인 경우는 제외한다.)(However, when R 1 is hydrogen or COOEt, n is 0, R 2
Figure PCTKR2016008059-appb-I000043
Is excluded.)
본 발명에서 사용되는 "약학적으로 허용 가능한"이라는 용어는 과도한 독성, 자극, 알러지 반응 또는 기타 문제점 또는 합병증 없이 이득/위험 비가 합리적이어서 대상체 (예: 인간)의 조직과 접촉하여 사용하기에 적합하며 건전한 의학적 판단의 범주 이내인 화합물 또는 조성물을 의미한다.As used herein, 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.
본 발명에서 사용되는 용어 "염"은 약학적으로 허용 가능한 유리산 (free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸디오에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.The term "salt" as used herein is useful with acid addition salts formed with pharmaceutically acceptable free acids. 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. Id, fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suverate , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitro benzoate, hydroxybenzoate, meth Oxybenzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfonate, chlorobenzenesul Nate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1- Sulfonates, naphthalene-2-sulfonates or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면, 화학식 1로 표시되는 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한 이 혼합물에서 용매나 과량의 산을 증발시킨 후 건조시키거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.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.
또한, 염기를 사용하여 약학적으로 허용 가능한 금속염을 만들 수도 있다. 알칼리 금속 또는 알칼리 토금속 염은 예를 들면, 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약상 적합하다. 이에 대응하는 은염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염 (예, 질산은)과 반응시켜 얻는다.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).
본 발명에 있어서, 상기 화학식 1로 표시되는 화합물은 하기 화학식 A로 표시되는 화합물을 촉매 존재하에서 알데히드와 반응시킴으로써 제조될 수 있다.In the present invention, 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.
[화학식 A]          [Formula A]
Figure PCTKR2016008059-appb-I000044
Figure PCTKR2016008059-appb-I000044
상기 화학식 A에서, R1, R2, R3, R4 및 R5는 앞에서 정의한 바와 같다.In Formula A, R 1 , R 2 , R 3 , R 4, and R 5 are as defined above.
본 발명에 있어서, 상기 촉매는 FeCl3, AlCl3, BiCl3, InCl3, PTSA (p-Toluenesulfonic acid) 및 PPTS (Pyridinium p-Toluenesulfonic acid)로 이루어지는 군으로부터 선택되는 어느 하나일 수 있는데, 특히, FeCl3인 것이 부반응물의 생성이 없으면서 높은 수율로 제조할 수 있어 바람직하다.In the present invention, 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.
또한, 상기 촉매는 0.1 내지 0.3 당량으로 함유되는 것일 수 있는데, 촉매의 함량이 0.1 당량 미만이면 촉매량이 너무 적어 반응성이 나쁘며, 촉매의 함량이 0.3 당량을 초과하는 경우에는 오히려 반응성이 저하되므로 바람직하지 않다.In addition, 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.
한편, 상기 반응은 메틸렌클로라이드, N,N-디메틸포름아미드 및 테트라하이드로퓨란으로 이루어지는 군으로부터 선택되는 용매 하의 20 ℃ 내지 80 ℃에서 수행될 수 있으며, 특히, 디클로로메탄 용매 하의 40 ℃ 내지 80 ℃에서 수행되는 것이 반응 수율을 향상시킬 수 있어 바람직하다.On the other hand, the reaction can be carried out at 20 ℃ to 80 ℃ under a solvent selected from the group consisting of methylene chloride, N, N- dimethylformamide and tetrahydrofuran, in particular at 40 ℃ to 80 ℃ under dichloromethane solvent It is preferable to carry out that the reaction yield can be improved.
본 발명에 있어서, 상기 알데히드는 C1- 6알킬알데히드, 아릴알데히드 및 헤테로아릴알데히드 중에서 선택될 수 있으나, 이에 제한되는 것은 아니다. 상기 알데히드의 구체적인 예로, 4-브로모벤즈알데히드, 4-플루오로벤즈알데히드, 3-플루오로벤즈알데히드, 4-클로로벤즈알데히드, 3-클로로벤즈알데히드, 4-니트로벤즈알데히드, 벤즈알데히드, 4-메톡시벤즈알데히드, 3-메톡시벤즈알데히드, 4-메틸벤즈알데히드, 3,5-디메톡시벤즈알데히드, 3,4-디메톡시벤즈알데히드, 1-나프틸알데히드, 2-나프틸알데히드, 피콜린알데히드, 5-브로모티오펜-2-카발데히드, 5-클로로티오펜-2-카발데히드, 퓨란-2-카발데히드 또는 1H-피롤-2-카발데히드를 들 수 있으나 이에 제한되는 것은 아니다.In the present invention, 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-chlorothiophene-2-carbaldehyde, furan-2-carbaldehyde or 1H-pyrrole-2-carbaldehyde.
한편, 본 발명은 상기 조성물을 포함하는, 핵산, 단백질 또는 세포 검출 시약을 제공한다.On the other hand, the present invention provides a nucleic acid, protein or cell detection reagent comprising the composition.
본 발명의 형광 프로브에 의해 표지되는 물질로는 제한이 없으며, 예를 들면 항체, 효소, 호르몬, 리셉터, 항원, 핵산, 천연 약물, 바이러스 입자, 박테리아 입자, 세포 등이 있고, 바람직하게는 핵산, 항체, 효소, 호르몬, 리셉터와 같은 단백질이거나, 혈액세포, 조직세포, 박테리아와 같은 세포이다.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.
또한, 본 발명은 상기 조성물을 이용한, 핵산, 단백질 또는 세포 이미징 방법을 제공한다.The present invention also provides a nucleic acid, protein or cell imaging method using the composition.
본 발명의 형광 프로브는 검체에 형광 프로브를 처리한 후 형광을 측정함으로써 이미징에 응용할 수 있다. 예를 들면, 디메틸설폭사이드 (DMSO)와 같은 극성 유기용매에 녹인 것을 완충액에 더하여 이것을 검체에 처리하고 인큐베이팅하여, 형광을 측정할 수 있다. 극성 유기용매 중의 프로브 농도는 특별히 제한되지 않지만, 일반적으로 2 μM이다.The fluorescent probe of the present invention can be applied to imaging by measuring the fluorescence after treating the specimen with the fluorescent probe. For example, 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. The probe concentration in the polar organic solvent is not particularly limited, but is generally 2 μM.
또한, 인큐베이션 시간은 특별히 제한되지 않으며 검체에 따라 적절히 선택 할 수 있지만, 일반적으로 5분 내지 1시간 정도가 바람직하다. 인큐베이션의 온도는 특별히 제한되지 않으며 각 검체에 적합한 온도를 적절히 선택할 수 있지만, 일반적으로 0 ℃ 내지 40 ℃이며, 검체가 세포 또는 조직인 경우에는, 그 배양에 적합한 온도 (예를 들면, 인간 유래의 세포 또는 조직이면 37 ℃)인 것이 바람직하다. 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. When the sample is a cell or tissue, a temperature suitable for the culture (for example, cells derived from humans) Or 37 ° C. in the case of tissue.
또한, 형광의 측정은 시판되는 형광계를 이용해 수행할 수도 있고, 세포 내 효소의 위치나 성질을 조사한 경우에는, 형광 현미경이나 공초점 레이저 주사 형광 현미경을 이용해 관찰할 수 있다. 또한, 검체로서는, 특별히 제한되지 않으며 그 중에 포함되는 효소 활성을 측정하려고 하는 어느 것 일 수 있고, 바람직한 예로서 각종 세포나 조직을 들 수 있다. 검체가 세포 또는 조직이 경우에는, 세포 또는 조직의 배양액을, 상기한 형광 프로브 용액으로 치환한 후 상기와 같이 인큐베이트를 실시하여 형광을 측정할 수 있다.In addition, 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. In addition, 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. When the sample is a cell or tissue, 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.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
[실시예]EXAMPLE
실시예 1: 실험방법Example 1 Experimental Method
1-1. 크로마토그래피1-1. Chromatography
플래쉬 크로마토그래피는 용리액으로서 헥센, 에틸아세테이트 및 디클로로메탄을 이용하여 실리카겔로 수행하였으며, 모든 반응액은 박막 크로마토그래피 (0.25 mm 실리카 플레이트; F-254)로 모니터링하고 UV로 시각화하였다. 1H and 13C NMR 스펙트럼은 400 MHz NMR 스펙트로미터로 기록하고, HRMS는 ESI (electrospray ionization) 및 Q-TOF 매스 분석기로 측정하였다.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.
1-2. 광학특성 분석1-2. Optical Characteristic Analysis
형광방출 스펙트럼은 JASCO FP-6500 분광형광계 (spectrofluorometer)를 사용하여 20 ℃에서 얻었으며, DMSO에 녹인 인돌리지노[3,2-c]퀴놀린 화합물의 10 mM 원액 (stock solution)을 용매에 희석하였다. 형광양자수율 (fluorescence quantum yield)은 스탠다드 (Φ= 0.86 in water)로서 로다민 6G를 이용하여 종래 알려진 방법으로 결정하였으며, 형광측정에 사용된 슬릿 폭은 excitation 3 nm, emission 5 nm로 하였다. 흡수 스펙트럼은 실온에서 Perkin Elmer Lamda 20 UV/VIS 스펙트로미터로 기록하였다.Fluorescence emission spectra were obtained at 20 ° C. using a JASCO FP-6500 spectrofluorometer and a 10 mM stock solution of the indolinino [3,2- c ] quinoline compound dissolved in DMSO was diluted in a solvent. . Fluorescence quantum yield was determined by a conventionally known method using rhodamine 6G as a standard (Φ = 0.86 in water), and the slit width used for fluorescence measurement was excitation 3 nm and emission 5 nm. Absorption spectra were recorded on a Perkin Elmer Lamda 20 UV / VIS spectrometer at room temperature.
실시예 2: 화합물 합성Example 2: Compound Synthesis
2-1. (IQ 1) 6-(4-Bromophenyl)indolizino[3,2-c]quinoline 합성2-1. (IQ 1) 6- (4-Bromophenyl) indolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 0.14 mmol, 4-브로모벤즈알데히드 0.17 mmol (1.2 당량) 및 FeCl3 0.028 mmol (0.2 당량)을 디클로로메탄 4 ml에 용해시켜 60 ℃에서 16 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 물 3 ml로 세척한 뒤, 물층을 디클로로메탄 3 ml로 한번 더 추출하였다. 유기층을 모아 마그네슘설페이트로 건조시키고, 감압 농축하였다. 농축된 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=30:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.0.14 mmol of 2- (indolizin-2-yl) aniline compound, 0.17 mmol (4-equivalent to 4-bromobenzaldehyde) and 0.028 mmol (0.2 equiv) of FeCl 3 were dissolved in 4 ml of dichloromethane and reacted at 60 ° C. for 16 hours. I was. After the reaction was completed, the reaction mixture was washed with 3 ml of water, and then the water layer was extracted once more with 3 ml of dichloromethane. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The concentrated reaction mixture was purified by silica gel column chromatography using a hexane: ethyl acetate: dichloromethane = 30: 1: 2 solvent to obtain the desired compound.
Yellow solid, mp 205.5-205.8 ℃ (43.4 mg, 83%); IR (ATR) ν = 2921, 2852, 1630, 1480, 1354 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 6.8 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.73-7.68 (m, 1H), 7.69-7.62 (m, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.31 (s, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.48 (d, J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 147.4, 143.1, 139.1, 138.9, 132.6, 131.9, 130.6, 129.7, 127.8, 126.8, 126.0, 123.7, 123.6, 122.5, 121.0, 119.5, 110.1, 92.4; HRMS (ESI) calcd for C21H14BrN2 373.0335 ([M+H]+), found 373.0336.Yellow solid, mp 205.5-205.8 ° C (43.4 mg, 83%); IR (ATR) ν = 2921, 2852, 1630, 1480, 1354 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.39 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 6.8 Hz, 1H), 7.76 (d , J = 8.0 Hz, 2H), 7.73-7.68 (m, 1H), 7.69-7.62 (m, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.31 (s, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.48 (d, J = 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 147.4, 143.1, 139.1, 138.9, 132.6, 131.9, 130.6, 129.7, 127.8, 126.8, 126.0, 123.7, 123.6, 122.5, 121.0, 119.5, 110.1, 92.4; HRMS (ESI) calcd for C 21 H 14 BrN 2 373.0335 ([M + H] + ), found 373.0336.
2-2. (IQ 2) 6-(4-Nitrophenyl)indolizino[3,2-c]quinoline 합성2-2. (IQ 2) 6- (4-Nitrophenyl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 4-니트로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 4-nitrobenzaldehyde was used instead of 4-bromobenzaldehyde.
Orange solid, mp 246.3-246.5 ℃ (26.1 mg, 55%); IR (ATR) ν = 2921, 2850, 1632, 1596, 1505, 1434, 1343 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.47 (d, J = 8.5 Hz, 2H), 8.39 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 6.8 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.69-7.62 (m, 2H), 7.31 (s, 1H), 7.08 (dd, J = 7.2, 8.4 Hz, 1H), 6.48 (t, J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 148.4, 146.4, 145.9, 143.0, 139.2, 132.0, 130.2, 129.7, 128.0, 126.5, 126.4, 124.6, 123.8, 123.7, 122.6, 120.6, 119.7, 110.4, 92.7; HRMS (ESI) calcd for C21H14N3O2 340.1081 ([M+H]+), found 340.1080.Orange solid, mp 246.3-246.5 ° C (26.1 mg, 55%); IR (ATR) ν = 2921, 2850, 1632, 1596, 1505, 1434, 1343 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.47 (d, J = 8.5 Hz, 2H), 8.39 (d, J = 7.6 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.89 (d , J = 8.5 Hz, 2H), 7.77 (d, J = 6.8 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.69-7.62 (m, 2H), 7.31 (s, 1H), 7.08 (dd, J = 7.2, 8.4 Hz, 1H), 6.48 (t, J = 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 148.4, 146.4, 145.9, 143.0, 139.2, 132.0, 130.2, 129.7, 128.0, 126.5, 126.4, 124.6, 123.8, 123.7, 122.6, 120.6, 119.7, 110.4, 92.7; HRMS (ESI) calcd for C 21 H 14 N 3 O 2 340.1081 ([M + H] + ), found 340.1080.
2-3. (IQ 3) 6-(p-Tolyl)indolizino[3,2-c]quinoline 합성2-3. (IQ 3) 6- (p-Tolyl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 4-메틸벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 4-methylbenzaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 85.3-86.0 ℃ (22.9 mg, 53%); IR (ATR) ν = 2980, 1631, 1492, 1435, 1372 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.37 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 7.6 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.27 (s, 1H), 7.02 (t, J = 8.8 Hz, 1H), 6.40 (t, J = 6.8 Hz, 1H), 2.51 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 148.9, 143.2, 139.1, 138.9, 137.0, 131.6, 130.0, 129.7, 128.7, 127.5, 127.0, 125.7, 123.6, 123.4, 122.5, 121.3, 119.3, 109.7, 92.2, 21.7; HRMS (ESI) calcd for C22H17N2 309.1386 ([M+H]+), found 309.1388.Yellow solid, mp 85.3-86.0 ° C (22.9 mg, 53%); IR (ATR) ν = 2980, 1631, 1492, 1435, 1372 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.37 (d, J = 8.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.68 (t , J = 7.6 Hz, 1H), 7.62 (d, J = 8.4 Hz, 2H), 7.56 (d, J = 7.2 Hz, 2H), 7.41 (d, J = 7.6 Hz, 2H), 7.27 (s, 1H) ), 7.02 (t, J = 8.8 Hz, 1H), 6.40 (t, J = 6.8 Hz, 1H), 2.51 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 148.9, 143.2, 139.1, 138.9, 137.0, 131.6, 130.0, 129.7, 128.7, 127.5, 127.0, 125.7, 123.6, 123.4, 122.5, 121.3, 119.3, 109.7, 92.2, 21.7 ; HRMS (ESI) calcd for C 22 H 17 N 2 309.1386 ([M + H] + ), found 309.1388.
2-4. (IQ 4) 6-Phenylindolizino[3,2-c]quinoline 합성2-4. (IQ 4) 6-Phenylindolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that benzaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 141.9-142.8 ℃ (25.5 mg, 62%); IR (ATR) ν = 3046, 2941, 1631, 1487, 1353 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.70 (d, J = 6.8 Hz, 1H), 7.66 (dd, J = 1.2, 8.0 Hz, 2H), 7.45-7.53 (m, 5H), 7.26 (s, 1H), 7.01 (dd, J = 7.2, 8.8 Hz, 1H), 6.38 (t, J = 6.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 148.8, 143.2, 140.0, 139.0, 131.7, 129.7, 129.4, 129.3, 128.8, 127.6, 126.9, 125.8, 123.6, 123.5, 122.5, 121.2, 119.3, 109.8, 92.2; HRMS (ESI) calcd for C21H15N2 295.1230 ([M+H]+), found 295.1225.Yellow solid, mp 141.9-142.8 ° C (25.5 mg, 62%); IR (ATR) ν = 3046, 2941, 1631, 1487, 1353 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.36 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.78 (d, J = 7.2 Hz, 1H), 7.70 (d , J = 6.8 Hz, 1H), 7.66 (dd, J = 1.2, 8.0 Hz, 2H), 7.45-7.53 (m, 5H), 7.26 (s, 1H), 7.01 (dd, J = 7.2, 8.8 Hz, 1H), 6.38 (t, J = 6.4 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 148.8, 143.2, 140.0, 139.0, 131.7, 129.7, 129.4, 129.3, 128.8, 127.6, 126.9, 125.8, 123.6, 123.5, 122.5, 121.2, 119.3, 109.8, 92.2; HRMS (ESI) calcd for C 21 H 15 N 2 295.1230 ([M + H] + ), found 295.1225.
2-5. (IQ 5) 6-(Pyridin-2-yl)indolizino[3,2-c]quinoline 합성2-5. (IQ 5) 6- (Pyridin-2-yl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 피콜린알데히드 (picolinaldehyde)를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that picolinaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 177.9-178.3 ℃ (11.6 mg, 28%); IR (ATR) ν = 3006, 2922, 2852, 1629, 1560, 1434, 1355, 744 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.84 (d, J = 4.4 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.12 (d, J = 7.2 Hz, 1H), 8.07 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.67-7.60 (m, 2H), 7.52 (t, J = 6.0 Hz, 1H), 7.31 (s, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 158.5, 149.2, 147.0, 143.0, 139.2, 137.9, 132.4, 129.7, 128.1, 127.6, 126.2, 125.3, 124.2, 123.72, 123.66, 122.9, 121.1, 119.2, 109.7, 92.4; HRMS (ESI) calcd for C20H14N3 296.1182 ([M+H]+), found 296.1182.Yellow solid, mp 177.9-178.3 ° C (11.6 mg, 28%); IR (ATR) ν = 3006, 2922, 2852, 1629, 1560, 1434, 1355, 744 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.84 (d, J = 4.4 Hz, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.12 (d , J = 7.2 Hz, 1H), 8.07 (d, J = 7.2 Hz, 1H), 8.02 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.67-7.60 (m , 2H), 7.52 (t, J = 6.0 Hz, 1H), 7.31 (s, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 158.5, 149.2, 147.0, 143.0, 139.2, 137.9, 132.4, 129.7, 128.1, 127.6, 126.2, 125.3, 124.2, 123.72, 123.66, 122.9, 121.1, 119.2, 109.7, 92.4 ; HRMS (ESI) calcd for C 20 H 14 N 3 296.1182 ([M + H] + ), found 296.1182.
2-6. (IQ 6) 6-(4-Methoxyphenyl)indolizino[3,2-c]quinoline 합성2-6. (IQ 6) 6- (4-Methoxyphenyl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 4-메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 4-methoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 164.3-164.7 ℃ (29.5 mg, 65%); IR (ATR) ν = 3055, 2998, 1606, 1494, 1440, 1374, 1355, 1024 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.69 (t, J = 7.2 Hz, 1H), 7.66-7.56 (m, 4H), 7.28 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 7.04 (t, J = 7.2 Hz, 1H), 6.43 (t, J = 6.4 Hz, 1H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.4, 148.5, 143.2, 138.9, 132.3, 131.6, 130.1, 129.6, 127.5, 126.9, 125.6, 123.6, 123.4, 122.4, 121.4, 119.3, 114.8, 109.7, 92.1, 55.6; HRMS (ESI) calcd for C22H17N2O 325.1335 ([M+H]+), found 325.1338.Yellow solid, mp 164.3-164.7 ° C (29.5 mg, 65%); IR (ATR) ν = 3055, 2998, 1606, 1494, 1440, 1374, 1355, 1024 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.38 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.94 (d, J = 7.2 Hz, 1H), 7.69 (t , J = 7.2 Hz, 1H), 7.66-7.56 (m, 4H), 7.28 (s, 1H), 7.14 (d, J = 8.4 Hz, 2H), 7.04 (t, J = 7.2 Hz, 1H), 6.43 (t, J = 6.4 Hz, 1H), 3.94 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.4, 148.5, 143.2, 138.9, 132.3, 131.6, 130.1, 129.6, 127.5, 126.9, 125.6, 123.6, 123.4, 122.4, 121.4, 119.3, 114.8, 109.7, 92.1, 55.6 ; HRMS (ESI) calcd for C 22 H 17 N 2 O 325.1335 ([M + H] + ), found 325.1338.
2-7. (IQ 7) 6-(3,5-Dimethoxyphenyl)indolizino[3,2-c]quinoline 합성2-7. (IQ 7) 6- (3,5-Dimethoxyphenyl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 3,5-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 3,5-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 139.7-140.4 ℃ (35.2 mg, 71%); IR (ATR) ν = 3062, 2838, 1592, 1452, 1353, 1149 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.70 (t, J = 7.2 Hz, 1H), 7.67-7.60 (m, 2H), 7.30 (s, 1H), 7.06 (dd, J = 6.4, 8.8 Hz, 1H), 6.79 (d, J = 2.0 Hz, 2H), 6.69-6.64 (m, 1H), 6.47 (t, J = 6.8 Hz, 1H), 3.85 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 161.7, 148.5, 143.0, 141.7, 139.0, 131.6, 129.7, 127.6, 127.1, 125.8, 123.6, 123.6, 122.6, 120.9, 119.2, 110.0, 106.3, 101.9, 92.1, 55.7; HRMS (ESI) calcd for C23H19N2O2 355.1441 ([M+H]+), found 355.1431.Yellow solid, mp 139.7-140.4 ° C (35.2 mg, 71%); IR (ATR) ν = 3062, 2838, 1592, 1452, 1353, 1149 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.39 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.88 (d, J = 7.2 Hz, 1H), 7.70 (t , J = 7.2 Hz, 1H), 7.67-7.60 (m, 2H), 7.30 (s, 1H), 7.06 (dd, J = 6.4, 8.8 Hz, 1H), 6.79 (d, J = 2.0 Hz, 2H) , 6.69-6.64 (m, 1 H), 6.47 (t, J = 6.8 Hz, 1 H), 3.85 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 161.7, 148.5, 143.0, 141.7, 139.0, 131.6, 129.7, 127.6, 127.1, 125.8, 123.6, 123.6, 122.6, 120.9, 119.2, 110.0, 106.3, 101.9, 92.1, 55.7 ; HRMS (ESI) calcd for C 23 H 19 N 2 O 2 355.1441 ([M + H] + ), found 355.1431.
2-8. (IQ 8) 6-(3,4-Dimethoxyphenyl)indolizino[3,2-c]quinoline 합성2-8. (IQ 8) 6- (3,4-Dimethoxyphenyl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 3,4-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 3,4-dimethoxybenzaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 91.3-92.0 ℃ (33.7 mg, 68%); IR (ATR) ν = 3053, 2933, 1602, 1493, 1450, 1352, 1135, 1022 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.38 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.66-7.57 (m, 2H), 7.28 (s, 1H), 7.22 (dd, J = 1.6, 8.4 Hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.05 (t, J = 6.8 Hz, 1H), 6.44 (t, J = 6.8 Hz, 1H), 4.01 (s, 3H), 3.91 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.8, 149.7, 148.5, 143.1, 139.0, 132.4, 131.7, 129.6, 127.6, 127.1, 125.7, 123.6, 123.5, 122.5, 121.30, 121.23, 119.3, 111.9, 111.7, 109.8, 92.2, 56.24, 56.17; HRMS (ESI) calcd for C23H19N2O2 355.1441 ([M+H]+), found 355.1448.Yellow solid, mp 91.3-92.0 ° C (33.7 mg, 68%); IR (ATR) ν = 3053, 2933, 1602, 1493, 1450, 1352, 1135, 1022 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.38 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.90 (d, J = 7.2 Hz, 1H), 7.69 (t , J = 7.6 Hz, 1H), 7.66-7.57 (m, 2H), 7.28 (s, 1H), 7.22 (dd, J = 1.6, 8.4 Hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.05 (t, J = 6.8 Hz, 1H), 6.44 (t, J = 6.8 Hz, 1H), 4.01 (s, 3H), 3.91 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 149.8, 149.7, 148.5, 143.1, 139.0, 132.4, 131.7, 129.6, 127.6, 127.1, 125.7, 123.6, 123.5, 122.5, 121.30, 121.23, 119.3, 111.9, 111.7, 109.8 , 92.2, 56.24, 56.17; HRMS (ESI) calcd for C 23 H 19 N 2 O 2 355.1441 ([M + H] + ), found 355.1448.
2-9. (IQ 9) 6-(Naphthalen-1-yl)indolizino[3,2-c]quinoline 합성2-9. (IQ 9) 6- (Naphthalen-1-yl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 1-나프탈데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The desired compound was obtained by the method of Example 2-1 except that 1-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 205.6-206.3 ℃ (40.0 mg, 83 %); IR (ATR) ν = 3051, 2929, 1631, 1493, 760 cm1; 1H NMR (400 MHz, CDCl3) δ 8.45 (d, J = 7.6 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.76- 7.65 (m, 4H), 7.63(d, J = 9.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.31-7.22 (m, 1H), 7.17 (d, J = 7.2 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.14 (t, J = 6.8 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 147.4, 143.4, 139.0, 137.0, 134.0, 131.6, 131.4, 129.8, 129.6, 128.6, 127.7, 127.3, 126.9, 126.6, 126.5, 126.1, 125.9, 125.3, 123.7, 123.5, 122.7, 122.3, 119.1, 110.0, 92.1; HRMS (ESI) calcd for C25H17N2 345.1386 ([M+H]+), found 345.1383. Yellow solid, mp 205.6-206.3 ° C (40.0 mg, 83%); IR (ATR) ν = 3051, 2929, 1631, 1493, 760 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.45 (d, J = 7.6 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.98 (d , J = 8.4 Hz, 1H), 7.76-7.65 (m, 4H), 7.63 (d, J = 9.0 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 8.4 Hz , 1H), 7.33 (s, 1H), 7.31-7.22 (m, 1H), 7.17 (d, J = 7.2 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.14 (t, J = 6.8 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 147.4, 143.4, 139.0, 137.0, 134.0, 131.6, 131.4, 129.8, 129.6, 128.6, 127.7, 127.3, 126.9, 126.6, 126.5, 126.1, 125.9, 125.3, 123.7, 123.5 , 122.7, 122.3, 119.1, 110.0, 92.1; HRMS (ESI) calcd for C 25 H 17 N 2 345.1386 ([M + H] + ), found 345.1383.
2-10. (IQ 10) 6-(Naphthalen-2-yl)indolizino[3,2-c]quinoline 합성2-10. (IQ 10) 6- (Naphthalen-2-yl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 2-나프탈데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 2-naphthalaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 205.6-206.2 ℃ (22.7 mg, 47%); IR (ATR) ν = 3047, 2920, 1632, 1496, 1352, 745 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.41 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.08 (d, J = 8.4 Hz, 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.71 (t, J = 7.2 Hz, 1H), 7.67-7.59 (m, 3H), 7.59-7.54 (m, 1H), 7.31(s, 1H), 7.01 (t, J = 7.2 Hz, 1H), 6.32 (t, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 143.3, 139.0, 137.4, 133.8, 133.6, 131.8, 129.7, 129.2, 128.7, 128.3, 128.1, 127.7, 127.0, 126.9, 126.8, 126.3, 125.9, 123.7, 123.5, 122.6, 121.4, 119.3, 109.9, 92.3; HRMS (ESI) calcd for C25H17N2 345.1386 ([M+H]+), found 345.1387.Yellow solid, mp 205.6-206.2 ° C (22.7 mg, 47%); IR (ATR) ν = 3047, 2920, 1632, 1496, 1352, 745 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.41 (d, J = 7.6 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.21 (s, 1H), 8.08 (d, J = 8.4 Hz , 1H), 7.99 (d, J = 7.6 Hz, 1H), 7.95 (d, J = 7.2 Hz, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H ), 7.71 (t, J = 7.2 Hz, 1H), 7.67-7.59 (m, 3H), 7.59-7.54 (m, 1H), 7.31 (s, 1H), 7.01 (t, J = 7.2 Hz, 1H) , 6.32 (t, J = 7.2 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 143.3, 139.0, 137.4, 133.8, 133.6, 131.8, 129.7, 129.2, 128.7, 128.3, 128.1, 127.7, 127.0, 126.9, 126.8, 126.3, 125.9, 123.7, 123.5, 122.6 , 121.4, 119.3, 109.9, 92.3; HRMS (ESI) calcd for C 25 H 17 N 2 345.1386 ([M + H] + ), found 345.1387.
2-11. (IQ 11) 6-(5-Chlorothiophen-2-yl)indolizino[3,2-c]quinoline 합성2-11. (IQ 11) 6- (5-Chlorothiophen-2-yl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 5-클로로티오펜-2카발데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 5-chlorothiophene-2carbaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, mp 161.4-161.7 ℃ (36.6 mg, 78%); IR (ATR) ν = 3041, 2937, 1632, 1561, 1493, 1352, 751 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.0 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 7.69 (td, J = 1.2, 6.8 Hz, 1H), 7.66-7.59 (m 2H), 7.26 (s, 1H), 7.19 (d, J = 4.0 Hz, 1H), 7.11-7.04 (m, 2H), 6.57 (td, J = 1.2, 6.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 142.9, 140.5, 139.8, 139.2, 132.7, 132.1, 129.7, 127.8, 127.4, 126.82, 126.75, 126.3, 123.8, 123.6, 122.6, 121.2, 119.5, 110.2, 92.5; HRMS (ESI) calcd for C19H12ClN2S 335.0404 ([M+H]+), found 335.0402.Yellow solid, mp 161.4-161.7 ° C (36.6 mg, 78%); IR (ATR) ν = 3041, 2937, 1632, 1561, 1493, 1352, 751 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.34 (d, J = 8.0 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 7.69 (td, J = 1.2, 6.8 Hz, 1H), 7.66 -7.59 (m 2H), 7.26 (s, 1 H), 7.19 (d, J = 4.0 Hz, 1 H), 7.11-7.04 (m, 2H), 6.57 (td, J = 1.2, 6.4 Hz, 1 H); 13 C NMR (100 MHz, CDCl 3 ) δ 142.9, 140.5, 139.8, 139.2, 132.7, 132.1, 129.7, 127.8, 127.4, 126.82, 126.75, 126.3, 123.8, 123.6, 122.6, 121.2, 119.5, 110.2, 92.5; HRMS (ESI) calcd for C 19 H 12 ClN 2 S 335.0404 ([M + H] + ), found 335.0402.
2-12. (IQ 12) 6-(4-2-12. (IQ 12) 6- (4- BromophenylBromophenyl )-11-) -11- methylindolizino[3,2-c]quinolinemethylindolizino [3,2-c] quinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 226.5-226.8 ℃ (45.0 mg, 83%); IR (ATR) ν = 3042, 2919, 1588, 1498, 1356 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.42 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.80-7.72 (m, 3H), 7.70 (t, J = 6.8 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.29 (s, 1H), 6.89 (d, J = 6.0 Hz, 1H), 6.43 (t, J = 6.8 Hz, 1H), 2.61 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 147.4, 143.1, 140.1, 138.9, 132.5, 131.6, 130.7, 129.6, 128.3, 127.6, 125.9, 124.6, 123.58, 123.56, 122.6, 122.4, 110.2, 90.8, 18.8; HRMS (ESI) calcd for C22H16BrN2 387.0491 ([M+H]+), found 387.0490.Yellow solid, mp 226.5-226.8 ° C (45.0 mg, 83%); IR (ATR) ν = 3042, 2919, 1588, 1498, 1356 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.42 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 7.80-7.72 (m, 3H), 7.70 (t, J = 6.8 Hz, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.57 (d, J = 8.0 Hz, 2H), 7.29 (s, 1H), 6.89 (d, J = 6.0 Hz, 1H), 6.43 (t, J = 6.8 Hz, 1H), 2.61 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 147.4, 143.1, 140.1, 138.9, 132.5, 131.6, 130.7, 129.6, 128.3, 127.6, 125.9, 124.6, 123.58, 123.56, 122.6, 122.4, 110.2, 90.8, 18.8; HRMS (ESI) calcd for C 22 H 16 BrN 2 387.0491 ([M + H] + ), found 387.0490.
2-13. (IQ 13) 11-Methyl-6-2-13. (IQ 13) 11-Methyl-6- (4-nitrophenyl)indolizino(4-nitrophenyl) indolizino [3,2-c][3,2-c] quinolinequinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-니트로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (8-methylindolizin-2-yl) aniline compound instead of 2- (indolizin-2-yl) aniline compound and 4-nitrobenzaldehyde instead of 4-bromobenzaldehyde The target compound was obtained by the method of Example 2-1.
Yellow solid, mp 259.7-260.4 ℃ (35.1 mg, 71%); IR (ATR) ν = 3026, 2920, 1631, 1595, 1441, 1341, 1381, 1102 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 6.8 Hz, 2H), 8.43 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.90 (d, J = 6.8 Hz, 2H), 7.75-7.61 (m, 3H), 7.31 (s, 1H), 6.91 (d, J = 6.4 Hz, 1H), 6.44 (t, J = 6.8 Hz, 1H), 2.62 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 148.4, 146.4, 146.1, 143.0, 140.3, 131.8, 130.3, 129.7, 128.7, 127.9, 126.4, 124.5, 124.3, 123.7, 122.69, 122.67, 121.2, 110.6, 91.2, 18.8; HRMS (ESI) calcd for C22H16N3O2 354.1237 ([M+H]+), found 354.1235.Yellow solid, mp 259.7-260.4 ° C (35.1 mg, 71%); IR (ATR) ν = 3026, 2920, 1631, 1595, 1441, 1341, 1381, 1102 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.48 (d, J = 6.8 Hz, 2H), 8.43 (d, J = 7.6 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.90 (d , J = 6.8 Hz, 2H), 7.75-7.61 (m, 3H), 7.31 (s, 1H), 6.91 (d, J = 6.4 Hz, 1H), 6.44 (t, J = 6.8 Hz, 1H), 2.62 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 148.4, 146.4, 146.1, 143.0, 140.3, 131.8, 130.3, 129.7, 128.7, 127.9, 126.4, 124.5, 124.3, 123.7, 122.69, 122.67, 121.2, 110.6, 91.2, 18.8 ; HRMS (ESI) calcd for C 22 H 16 N 3 O 2 354.1237 ([M + H] + ), found 354.1235.
2-14. 6-(3-Fluorophenyl)-11-methylindolizino[3,2-c]quinoline 합성2-14. 6- (3-Fluorophenyl) -11-methylindolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3-플루오로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (8-methylindolizin-2-yl) aniline compound instead of 2- (indolizin-2-yl) aniline compound and 3-fluorobenzaldehyde instead of 4-bromobenzaldehyde Obtained the desired compound by the method of Example 2-1.
Yellow solid, mp 162.5-163.1 ℃ (35.7 mg, 79%); IR (ATR) ν = 3013, 2918, 1626, 1491, 1435, 1349 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.68 (t, J = 8.4 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 6.87-6.81 (m, 1H), 6.36 (t, J = 7.2 Hz, 1H), 2.59 (s, 3H), 2.51 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.1, 143.2, 140.0, 139.0, 137.1, 131.4, 130.0, 129.7, 128.7, 128.1, 127.4, 125.6, 124.9, 123.5, 122.6, 122.2, 121.8, 109.8, 90.6, 21.7, 18.8; HRMS (ESI) calcd for C23H19N2 323.1543 ([M+H]+), found 323.1542.Yellow solid, mp 162.5-163.1 ° C (35.7 mg, 79%); IR (ATR) ν = 3013, 2918, 1626, 1491, 1435, 1349 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.40 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.68 (t , J = 8.4 Hz, 1H), 7.62 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.40 (d, J = 8.0 Hz, 2H), 6.87-6.81 (m , 1H), 6.36 (t, J = 7.2 Hz, 1H), 2.59 (s, 3H), 2.51 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 149.1, 143.2, 140.0, 139.0, 137.1, 131.4, 130.0, 129.7, 128.7, 128.1, 127.4, 125.6, 124.9, 123.5, 122.6, 122.2, 121.8, 109.8, 90.6, 21.7 , 18.8; HRMS (ESI) calcd for C 23 H 19 N 2 323.1543 ([M + H] + ), found 323.1542.
2-15. (IQ 15) 11-Methyl-6-(p-tolyl)indolizino[3,2-c]quinoline 합성2-15. (IQ 15) 11-Methyl-6- (p-tolyl) indolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-메틸벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (8-methylindolizin-2-yl) aniline compound instead of 2- (indolizin-2-yl) aniline compound and 4-methylbenzaldehyde instead of 4-bromobenzaldehyde The target compound was obtained by the method of Example 2-1.
Yellow solid, mp 176.1-176.5 ℃ (36.6 mg, 80%); IR (ATR) ν = 3042, 2903, 1561, 1496, 1364, 1110 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.39 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.73-7.66 (m, 2H), 7.62 (t, J = 7.2 Hz, 1H), 7.60-7.53 (m, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.28 (dd, J = 1.6, 8.4 Hz, 1H), 7.25 (s, 1H), 6.85 (d, J = 6.8 Hz, 1H), 6.38 (t, J = 7.2 Hz, 1H), 2.58 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.3 (d, JC,F = 247.0 Hz), 147.2, 142.5 (d, JC,F = 287.0 Hz), 142.0 (d, JC,F = 8.0 Hz), 131.6, 131.02 (d, JC,F = 8.0 Hz), 129.6, 128.3, 127.6, 126.0, 124.7, 124.6, 124.5, 123.6, 122.6, 122.5, 121.4, 116.3 (d, JC,F = 6.0 Hz), 116.1 (d, JC,F = 7.0 Hz), 110.2, 90.8, 18.8 ; HRMS (ESI) calcd for C22H16FN2 327.1292 ([M+H]+), found 327.1292.Yellow solid, mp 176.1-176.5 ° C (36.6 mg, 80%); IR (ATR) ν = 3042, 2903, 1561, 1496, 1364, 1110 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.39 (d, J = 8.0 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.73-7.66 (m, 2H), 7.62 (t, J = 7.2 Hz, 1H), 7.60-7.53 (m, 1H), 7.43 (d, J = 8.8 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.28 (dd, J = 1.6, 8.4 Hz, 1H), 7.25 (s, 1H), 6.85 (d, J = 6.8 Hz, 1H), 6.38 (t, J = 7.2 Hz, 1H), 2.58 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 163.3 (d, JC, F = 247.0 Hz), 147.2, 142.5 (d, JC, F = 287.0 Hz), 142.0 (d, JC, F = 8.0 Hz), 131.6 , 131.02 (d, JC, F = 8.0 Hz), 129.6, 128.3, 127.6, 126.0, 124.7, 124.6, 124.5, 123.6, 122.6, 122.5, 121.4, 116.3 (d, JC, F = 6.0 Hz), 116.1 (d , JC, F = 7.0 Hz), 110.2, 90.8, 18.8; HRMS (ESI) calcd for C 22 H 16 FN 2 327.1292 ([M + H] + ), found 327.1292.
2-16. (IQ 16) 6-(3,5-2-16. (IQ 16) 6- (3,5- DimethoxyphenylDimethoxyphenyl )-11-) -11- methylindolizino[3,2-c]methylindolizino [3,2-c] quinoline 합성quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3,5-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 185.8-186.4 ℃ (39.7 mg, 77%); IR (ATR) ν = 2933, 2836, 1596, 1495, 1453, 1365, 1193, 1149 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.69 (t, J = 6.8 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H),7.62 (s, 1H), 6.86 (d, J = 5.2 Hz, 1H), 6.78 (s, 2H), 6.66 (s, 1H), 6.41 (t, J = 7.2 Hz, 1H), 3.83 (s, 6H), 2.58 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 161.6, 148.6, 143.0, 141.7, 140.0, 131.4, 129.7, 128.0, 127.5, 125.8, 125.0, 123.6, 122.6, 122.4, 121.4, 110.1, 106.4, 101.9, 90.6, 55.7, 18.8; HRMS (ESI) calcd for C24H21N2O2 369.1598 ([M+H]+), found 369.1595.Yellow solid, mp 185.8-186.4 ° C (39.7 mg, 77%); IR (ATR) ν = 2933, 2836, 1596, 1495, 1453, 1365, 1193, 1149 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.40 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 6.8 Hz, 1H), 7.69 (t , J = 6.8 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.62 (s, 1H), 6.86 (d, J = 5.2 Hz, 1H), 6.78 (s, 2H), 6.66 (s , 1H), 6.41 (t, J = 7.2 Hz, 1H), 3.83 (s, 6H), 2.58 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 161.6, 148.6, 143.0, 141.7, 140.0, 131.4, 129.7, 128.0, 127.5, 125.8, 125.0, 123.6, 122.6, 122.4, 121.4, 110.1, 106.4, 101.9, 90.6, 55.7 , 18.8; HRMS (ESI) calcd for C 24 H 21 N 2 O 2 369.1598 ([M + H] + ), found 369.1595.
2-17. (IQ 17) 6-(3,4-2-17. (IQ 17) 6- (3,4- DimethoxyphenylDimethoxyphenyl )-11-) -11- methylindolizino[3,2-c]methylindolizino [3,2-c] quinoline 합성quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3,4-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 162.8-163.3 ℃ (35.1 mg, 68%); IR (ATR) ν = 2919, 2845, 1602, 1492, 1411, 1365, 1138 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.40 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.69 (t, J = 7.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.86 (d, J = 6.4 Hz, 1H), 6.39 (d, J = 7.2 Hz, 1H), 4.01 (s, 3H), 3.90 (s, 3H), 2.59 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 149.8, 149.6, 148.6, 143.1, 140.0, 132.4, 131.4, 129.6, 128.1, 127.5, 125.7, 124.9, 123.5, 122.6, 122.3, 121.8, 121.3, 111.9, 111.8, 109.9, 90.7, 56.2, 56.1, 18.8; HRMS (ESI) calcd for C24H21N2O2 369.1598 ([M+H]+), found 369.1595.Yellow solid, mp 162.8-163.3 ° C (35.1 mg, 68%); IR (ATR) ν = 2919, 2845, 1602, 1492, 1411, 1365, 1138 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.40 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.2 Hz, 1H), 7.69 (t , J = 7.2 Hz, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.27 (s, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.09 (d , J = 8.0 Hz, 1H), 6.86 (d, J = 6.4 Hz, 1H), 6.39 (d, J = 7.2 Hz, 1H), 4.01 (s, 3H), 3.90 (s, 3H), 2.59 (s , 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 149.8, 149.6, 148.6, 143.1, 140.0, 132.4, 131.4, 129.6, 128.1, 127.5, 125.7, 124.9, 123.5, 122.6, 122.3, 121.8, 121.3, 111.9, 111.8, 109.9 , 90.7, 56.2, 56.1, 18.8; HRMS (ESI) calcd for C 24 H 21 N 2 O 2 369.1598 ([M + H] + ), found 369.1595.
2-18. (IQ 18) 6-(Furan-2-yl)-11-methylindolizino[3,2-c]quinoline 합성2-18. (IQ 18) 6- (Furan-2-yl) -11-methylindolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 퓨란-2-카발데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (8-methylindolizin-2-yl) aniline compound in place of 2- (indolizin-2-yl) aniline compound and furan-2-carbaldehyde in place of 4-bromobenzaldehyde Then, the target compound was obtained by the method of Example 2-1.
Yellow solid, mp 112.4-113.2 ℃ (33.0 mg, 79%); IR (ATR) ν = 3096, 2922, 1611, 1481, 1438, 1365, 1013 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 6.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 6.8 Hz, 1H), 7.72 (s, 1H), 7.68 (t, J = 6.8 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.24-7.17 (m, 1H), 7.03 (d, J = 3.2 Hz, 1H), 6.87 (s, 1H), 6.72 (s, 1H), 6.54-6.46 (m, 1H), 2.56 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 151.7, 143.2, 143.1, 140.1, 138.5, 131.7, 129.8, 128.0, 127.6, 126.2, 125.0, 123.6, 122.9, 122.5, 122.0, 112.3, 111.5, 110.2, 90.8, 18.8; HRMS (ESI) calcd for C20H15N2O 299.1179 ([M+H]+), found 299.1178.Yellow solid, mp 112.4-113.2 ° C (33.0 mg, 79%); IR (ATR) ν = 3096, 2922, 1611, 1481, 1438, 1365, 1013 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.35 (d, J = 6.4 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 6.8 Hz, 1H), 7.72 (s , 1H), 7.68 (t, J = 6.8 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.24-7.17 (m, 1H), 7.03 (d, J = 3.2 Hz, 1H), 6.87 (s, 1 H), 6.72 (s, 1 H), 6.54-6.46 (m, 1 H), 2.56 (s, 3 H); 13 C NMR (100 MHz, CDCl 3 ) δ 151.7, 143.2, 143.1, 140.1, 138.5, 131.7, 129.8, 128.0, 127.6, 126.2, 125.0, 123.6, 122.9, 122.5, 122.0, 112.3, 111.5, 110.2, 90.8, 18.8 ; HRMS (ESI) calcd for C 20 H 15 N 2 O 299.1179 ([M + H] + ), found 299.1178.
2-19. (IQ 19) Methyl 6-2-19. (IQ 19) Methyl 6- (3,5-dimethoxyphenyl)indolizino[3,2-c]quinoline(3,5-dimethoxyphenyl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3,5-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 205.9-206.3 ℃ (53.7 mg, 93%); IR (ATR) ν = 2936, 2839, 1690, 1598, 1492, 1352, 1134 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 8.0 Hz, 1H), 8.46 (d, J = 9.2 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.74 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 6.74 (s, 2H), 6.70-6.60 (m, 2H), 4.10 (s, 3H), 3.84 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 165.9, 161.9, 147.9, 144.5, 141.6, 141.4, 131.0, 129.8, 128.5, 127.7, 127.64, 127.57, 126.0, 122.4, 121.9, 120.5, 112.3, 106.2, 102.0, 99.4, 55.8, 51.6; HRMS (ESI) calcd for C25H21N2O4 413.1496 ([M+H]+), found 413.1506.Yellow solid, mp 205.9-206.3 ° C (53.7 mg, 93%); IR (ATR) ν = 2936, 2839, 1690, 1598, 1492, 1352, 1134 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.68 (d, J = 8.0 Hz, 1H), 8.46 (d, J = 9.2 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.96 (d , J = 7.2 Hz, 1H), 7.74 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 8.0 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 6.74 (s, 2H ), 6.70-6.60 (m, 2H), 4.10 (s, 3H), 3.84 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.9, 161.9, 147.9, 144.5, 141.6, 141.4, 131.0, 129.8, 128.5, 127.7, 127.64, 127.57, 126.0, 122.4, 121.9, 120.5, 112.3, 106.2, 102.0, 99.4 , 55.8, 51.6; HRMS (ESI) calcd for C 25 H 21 N 2 O 4 413.1496 ([M + H] + ), found 413.1506.
2-20. (IQ 20) Methyl 6-2-20. (IQ 20) Methyl 6- (naphthalen-1-yl)indolizino[3,2-c]quinoline(naphthalen-1-yl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 1-나프탈데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (8-methylindolizin-2-yl) aniline compound in place of 2- (indolizin-2-yl) aniline compound and 1-naphthalaldehyde in place of 4-bromobenzaldehyde Obtained the desired compound by the method of Example 2-1.
Yellow solid, mp 245.8-246.2 ℃ (55.8 mg, 99%); IR (ATR) ν = 3048, 2985, 1697, 1490, 1437, 1350, 1133 cm1; 1H NMR (400 MHz, CDCl3) δ 9.76 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 9.2 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.59 (dd, J = 2.8, 6.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.2 Hz, 1H), 7.74-7.66 (m, 3H), 7.49 (t, J = 6.8 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 6.8, 7.2 Hz, 1H), 7.22 (dd, J = 6.8, 9.2 Hz, 1H), 6.32(t, J = 6.8 Hz, 1H), 4.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.0, 146.8, 145.0, 141.5, 136.7, 134.0, 131.4, 130.8, 129.9, 129.8, 128.6, 128.5, 127.7, 127.5, 127.4, 126.94, 126.87, 126.8, 126.2, 126.1, 124.9, 123.2, 122.5, 120.4, 112.4, 99.3, 51.6; HRMS (ESI) calcd for C27H19N2O2 403.1441 ([M+H]+), found 403.1437.Yellow solid, mp 245.8-246.2 ° C. (55.8 mg, 99%); IR (ATR) ν = 3048, 2985, 1697, 1490, 1437, 1350, 1133 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.76 (d, J = 8.0 Hz, 1H), 8.43 (d, J = 9.2 Hz, 1H), 8.30 (d, J = 8.0 Hz, 1H), 7.59 (dd , J = 2.8, 6.8 Hz, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.2 Hz, 1H), 7.74-7.66 (m, 3H), 7.49 (t, J = 6.8 Hz, 1H), 7.34 (d, J = 7.2 Hz, 1H), 7.31 (d, J = 8.4 Hz, 1H), 7.26 (dd, J = 6.8, 7.2 Hz, 1H), 7.22 (dd, J = 6.8, 9.2 Hz, 1H), 6.32 (t, J = 6.8 Hz, 1H), 4.11 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 166.0, 146.8, 145.0, 141.5, 136.7, 134.0, 131.4, 130.8, 129.9, 129.8, 128.6, 128.5, 127.7, 127.5, 127.4, 126.94, 126.87, 126.8, 126.2, 126.1 , 124.9, 123.2, 122.5, 120.4, 112.4, 99.3, 51.6; HRMS (ESI) calcd for C 27 H 19 N 2 O 2 403.1441 ([M + H] + ), found 403.1437.
2-21. (IQ 21) Methyl 6-2-21. (IQ 21) Methyl 6- (1H-pyrrol-2-yl)indolizino[3,2-c]quinoline(1H-pyrrol-2-yl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 메틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 1H-피롤-2-카발데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Use methyl 2- (2-aminophenyl) indolizin-1-carboxylate compound instead of 2- (indolizin-2-yl) aniline compound and 1H-pyrrole-2-carbaldehyde instead of 4-bromobenzaldehyde A target compound was obtained by the method of Example 2-1 except that was used.
Yellow solid, mp 230.8-231.4 ℃ (42.1 mg, 88 %); IR (ATR) ν = 3323, 3108, 2944, 1690, 1588, 1494, 1439, 1350, 1224 cm1; 1H NMR (400 MHz, CDCl3) δ 10.06 (s, 1H), 9.46 (d, J = 8.0 Hz, 1H), 8.65-8.53 (m, 1H), 8.35 (d, J = 9.2 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.54-7.40 (m, 2H), 7.32 (t, J = 6.8 Hz, 1H), 7.08 (s, 1H), 6.70 (t, J = 6.8 Hz, 1H), 6.60 (s, 1H), 6.41 (s, 1H), 4.09 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.9, 144.3, 141.5, 140.4, 131.3, 128.7, 128.5, 128.3, 127.8, 127.6, 127.5, 125.4, 121.9, 121.8, 120.5, 120.3, 111.8, 110.9, 110.0, 99.2, 51.6; HRMS (ESI) calcd for C21H16N3O2 342.1237 ([M+H]+), found 342.1229. Yellow solid, mp 230.8-231.4 ° C (42.1 mg, 88%); IR (ATR) ν = 3323, 3108, 2944, 1690, 1588, 1494, 1439, 1350, 1224 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 10.06 (s, 1H), 9.46 (d, J = 8.0 Hz, 1H), 8.65-8.53 (m, 1H), 8.35 (d, J = 9.2 Hz, 1H) , 7.98 (d, J = 8.0 Hz, 1H), 7.54-7.40 (m, 2H), 7.32 (t, J = 6.8 Hz, 1H), 7.08 (s, 1H), 6.70 (t, J = 6.8 Hz, 1H), 6.60 (s, 1H), 6.41 (s, 1H), 4.09 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.9, 144.3, 141.5, 140.4, 131.3, 128.7, 128.5, 128.3, 127.8, 127.6, 127.5, 125.4, 121.9, 121.8, 120.5, 120.3, 111.8, 110.9, 110.0, 99.2 , 51.6; HRMS (ESI) calcd for C 21 H 16 N 3 O 2 342.1237 ([M + H] + ), found 342.1229.
2-22. (IQ 22) 9-2-22. (IQ 22) 9- BromoBromo -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(6-브로모인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 퓨란-2-카발데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 229.5-230.0 ℃ (50.2 mg, 89%); IR (ATR) ν = 3057, 3003, 1606, 1489, 1435, 1381, 1242, 1173, 669 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.64-7.57 (m, 3H), 7.51 (d, J = 9.2 Hz, 1H), 7.28 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 9.6 Hz, 1H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.8, 148.5, 143.2, 136.7, 131.7, 131.5, 130.1, 129.7, 127.8, 127.0, 126.5, 126.0, 123.6, 122.3, 121.4, 119.9, 114.9, 104.2, 93.4, 55.7; HRMS (ESI) calcd for C22H16BrN2O 403.0441 ([M+H]+), found 403.0437.Yellow solid, mp 229.5-230.0 ° C (50.2 mg, 89%); IR (ATR) ν = 3057, 3003, 1606, 1489, 1435, 1381, 1242, 1173, 669 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.33 (d, J = 7.6 Hz, 1H), 8.24 (d, J = 8.4 Hz, 1H), 8.05 (s, 1H), 7.69 (t, J = 7.6 Hz , 1H), 7.64-7.57 (m, 3H), 7.51 (d, J = 9.2 Hz, 1H), 7.28 (s, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 9.6 Hz, 1 H), 3.94 (s, 3 H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.8, 148.5, 143.2, 136.7, 131.7, 131.5, 130.1, 129.7, 127.8, 127.0, 126.5, 126.0, 123.6, 122.3, 121.4, 119.9, 114.9, 104.2, 93.4, 55.7 ; HRMS (ESI) calcd for C 22 H 16 BrN 2 O 403.0441 ([M + H] + ), found 403.0437.
2-23. (IQ 23) 9-Bromo-6-(p-tolyl)indolizino[3,2-c]quinoline 합성2-23. (IQ 23) 9-Bromo-6- (p-tolyl) indolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(6-브로모인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-메틸벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (6-bromoindolizin-2-yl) aniline compound instead of 2- (indolizin-2-yl) aniline compound and 4-methylbenzaldehyde instead of 4-bromobenzaldehyde Obtained the desired compound by the method of Example 2-1.
Yellow solid, mp 230.8-231.2 ℃ (23.9 mg, 44%); IR (ATR) ν = 2921, 2852, 1610, 1487, 1435, 667 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.33 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.69 (t, J = 7.2 Hz, 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 9.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H), 7.27 (s, 1H), 7.05 (d, J = 9.6 Hz, 1H), 2.52 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 148.9, 143.2, 139.6, 136.7, 136.2, 131.6, 130.1, 129.8, 128.5, 127.8, 127.0, 126.5, 126.0, 123.6, 122.3, 121.3, 119.8, 104.2, 93.4, 21.7; HRMS (ESI) calcd for C22H16BrN2 387.0491 ([M+H]+), found 387.0490.Yellow solid, mp 230.8-231.2 ° C (23.9 mg, 44%); IR (ATR) ν = 2921, 2852, 1610, 1487, 1435, 667 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.33 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.96 (s, 1H), 7.69 (t, J = 7.2 Hz , 1H), 7.61 (t, J = 7.2 Hz, 1H), 7.55 (d, J = 7.6 Hz, 2H), 7.50 (d, J = 9.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 2H ), 7.27 (s, 1 H), 7.05 (d, J = 9.6 Hz, 1 H), 2.52 (s, 3 H); 13 C NMR (100 MHz, CDCl 3 ) δ 148.9, 143.2, 139.6, 136.7, 136.2, 131.6, 130.1, 129.8, 128.5, 127.8, 127.0, 126.5, 126.0, 123.6, 122.3, 121.3, 119.8, 104.2, 93.4, 21.7 ; HRMS (ESI) calcd for C 22 H 16 BrN 2 387.0491 ([M + H] + ), found 387.0490.
2-24. (IQ 24) 9-2-24. (IQ 24) 9- BromoBromo -6--6- (3,4-dimethoxyphenyl)indolizino[3,2-c]quinoline(3,4-dimethoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(6-브로모인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-브로모벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using 2- (6-bromoindolizin-2-yl) aniline compound instead of 2- (indolizin-2-yl) aniline compound and using 4-bromobenzaldehyde instead of 4-bromobenzaldehyde Then, the target compound was obtained by the method of Example 2-1.
Green solid, mp 188.5-189.0℃ (45.5 mg, 75%); IR (ATR) ν = 3103, 3000, 1600, 1490, [0292] 1460, 1372, 1132, 667 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.70 (t, J = 7.2 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.54 (d, J = 9.6 Hz, 1H), 7.31 (s, 1H), 7.26-7.19 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 4.02 (s, 3H), 3.92 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 150.3, 149.9, 148.3, 143.0, 136.9, 131.8, 131.4, 129.5, 128.0, 127.1, 126.7, 126.1, 123.6, 122.3, 121.4, 121.2, 119.9, 111.9, 111.8, 104.3, 93.5, 56.4, 56.3; HRMS (ESI) calcd for C23H18BrN2O2 433.0546 ([M+H]+), found 433.0543.Green solid, mp 188.5-189.0 ° C. (45.5 mg, 75%); IR (ATR) ν = 3103, 3000, 1600, 1490, 1460, 1372, 1132, 667 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.34 (d, J = 8.0 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.10 (s, 1H), 7.70 (t, J = 7.2 Hz , 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.54 (d, J = 9.6 Hz, 1H), 7.31 (s, 1H), 7.26-7.19 (m, 2H), 7.12 (d, J = 8.0 Hz, 1H), 7.09 (d, J = 9.6 Hz, 1H), 4.02 (s, 3H), 3.92 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 150.3, 149.9, 148.3, 143.0, 136.9, 131.8, 131.4, 129.5, 128.0, 127.1, 126.7, 126.1, 123.6, 122.3, 121.4, 121.2, 119.9, 111.9, 111.8, 104.3 , 93.5, 56.4, 56.3; HRMS (ESI) calcd for C 23 H 18 BrN 2 O 2 433.0546 ([M + H] + ), found 433.0543.
2-25. (IQ 25) 9-Bromo-6-(furan-2-yl)indolizino[3,2-c]quinoline 합성2-25. (IQ 25) 9-Bromo-6- (furan-2-yl) indolizino [3,2-c] quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(6-브로모인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 퓨란-2-카발데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 193.3-193.6 ℃ (47.3 mg, 93%); IR (ATR) ν = 3065, 3013, 1599, 1480, 1313, 668 cm-1; 1H NMR (400 MHz, CDCl3) δ 8.30 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.09 (s, 1H), 7.78 (s, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.26 (s, 1H), 7.15-7.06 (m, 2H), 6.78 (s, 1H); 13C NMR (100 MHz, CDCl3) δ 143.4, 142.9, 138.2, 137.0, 132.1, 129.8, 128.0, 127.6, 126.9, 126.6, 123.6, 122.6, 121.4, 119.8, 112.7, 112.2, 104.8, 93.6; HRMS (ESI) calcd for C19H12BrN2O 363.0128 ([M+H]+), found 363.0126.Yellow solid, mp 193.3-193.6 ° C (47.3 mg, 93%); IR (ATR) ν = 3065, 3013, 1599, 1480, 1313, 668 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 8.30 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.09 (s, 1H), 7.78 (s, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.52 (d, J = 9.2 Hz, 1H), 7.26 (s, 1H), 7.15-7.06 (m, 2H) , 6.78 (s, 1 H); 13 C NMR (100 MHz, CDCl 3 ) δ 143.4, 142.9, 138.2, 137.0, 132.1, 129.8, 128.0, 127.6, 126.9, 126.6, 123.6, 122.6, 121.4, 119.8, 112.7, 112.2, 104.8, 93.6; HRMS (ESI) calcd for C 19 H 12 BrN 2 O 363.0128 ([M + H] + ), found 363.0126.
2-26. (IQ 26) Ethyl 6-2-26. (IQ 26) Ethyl 6- (4-nitrophenyl)indolizino(4-nitrophenyl) indolizino [3,2-c][3,2-c] quinolinequinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-니트로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound instead of 2- (indolizin-2-yl) aniline compound and using 4-nitrobenzaldehyde instead of 4-bromobenzaldehyde Then, the target compound was obtained by the method of Example 2-1.
Yellow solid, mp 245.8-246.1 ℃ (43.2 mg, 75%); IR (ATR) ν = 3112, 2985, 1689, 1597, 1560, 1436, 1349, 1215, 1137 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 9.6 Hz, 1H), 8.48 (d, J = 8.8 Hz, 2H), 8.21 (d, J = 8.0 Hz, 1H), 7.90-7.82 (m, 3H), 7.75 (t, J = 7.2 Hz, 1H), 7.68 (t, J = 7.2 Hz, 1H), 7.37 (dd, J = 6.8, 8.8 Hz, 1H), 6.67 (t, J = 6.4 Hz, 1H), 4.60 (q, J = 7.2 Hz, 2H), 1.57 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.3, 148.5, 146.1, 145.4, 144.5, 141.6, 131.3, 130.2, 129.8, 128.8, 127.8, 127.7, 126.8, 126.5, 124.7, 122.4, 121.4, 121.0, 112.5, 100.1, 60.8, 14.8; HRMS (ESI) calcd for C24H18N3O4 412.1292 ([M+H]+), found 412.1286.Yellow solid, mp 245.8-246.1 ° C (43.2 mg, 75%); IR (ATR) ν = 3112, 2985, 1689, 1597, 1560, 1436, 1349, 1215, 1137 cm -1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.68 (d, J = 8.4 Hz, 1H), 8.51 (d, J = 9.6 Hz, 1H), 8.48 (d, J = 8.8 Hz, 2H), 8.21 (d , J = 8.0 Hz, 1H), 7.90-7.82 (m, 3H), 7.75 (t, J = 7.2 Hz, 1H), 7.68 (t, J = 7.2 Hz, 1H), 7.37 (dd, J = 6.8, 8.8 Hz, 1H), 6.67 (t, J = 6.4 Hz, 1H), 4.60 (q, J = 7.2 Hz, 2H), 1.57 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.3, 148.5, 146.1, 145.4, 144.5, 141.6, 131.3, 130.2, 129.8, 128.8, 127.8, 127.7, 126.8, 126.5, 124.7, 122.4, 121.4, 121.0, 112.5, 100.1 , 60.8, 14.8; HRMS (ESI) calcd for C 24 H 18 N 3 O 4 412.1292 ([M + H] + ), found 412.1286.
2-27. (IQ 27) Ethyl 6-2-27. (IQ 27) Ethyl 6- (3-chlorophenyl)indolizino[3,2-c]quinoline(3-chlorophenyl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3-클로로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Except for using ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound instead of 2- (indolizin-2-yl) aniline compound and 3-chlorobenzaldehyde instead of 4-bromobenzaldehyde Then, the target compound was obtained by the method of Example 2-1.
Yellow solid, mp 214.3-214.5 ℃ (53.3 mg, 95%); IR (ATR) ν = 3111, 2961, 1679, 1631, 1489, 1435, 1386, 1218, 1174, 1026, 741 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 6.4 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.72-7.62 (m, 2H), 7.61-7.51 (m, 2H), 7.51-7.45 (m, 1H), 7.34 (t, J = 7.2 Hz, 1H), 6.65 (t, J = 6.8 Hz, 1H), 4.59 (q, J = 7.2 Hz, 2H), 1.56 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.4, 146.4, 144.5, 141.6, 141.5, 135.7, 131.2, 130.9, 129.8, 129.7, 129.0, 128.6, 127.8, 127.5, 127.2, 126.9, 126.1, 122.4, 121.7, 120.8, 112.3, 99.9, 60.7, 14.8; HRMS (ESI) calcd for C24H18ClN2O2 401.1051 ([M+H]+), found 401.1048.Yellow solid, mp 214.3-214.5 ° C (53.3 mg, 95%); IR (ATR) ν = 3111, 2961, 1679, 1631, 1489, 1435, 1386, 1218, 1174, 1026, 741 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.23 (d, J = 8.0 Hz, 1H), 7.92 (d , J = 6.4 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.72-7.62 (m, 2H), 7.61-7.51 (m, 2H), 7.51-7.45 (m, 1H), 7.34 ( t, J = 7.2 Hz, 1H), 6.65 (t, J = 6.8 Hz, 1H), 4.59 (q, J = 7.2 Hz, 2H), 1.56 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.4, 146.4, 144.5, 141.6, 141.5, 135.7, 131.2, 130.9, 129.8, 129.7, 129.0, 128.6, 127.8, 127.5, 127.2, 126.9, 126.1, 122.4, 121.7, 120.8 , 112.3, 99.9, 60.7, 14.8; HRMS (ESI) calcd for C 24 H 18 ClN 2 O 2 401.1051 ([M + H] + ), found 401.1048.
2-28. (IQ 28) Ethyl 6-2-28. (IQ 28) Ethyl 6- (3-fluorophenyl)indolizino(3-fluorophenyl) indolizino [3,2-c][3,2-c] quinolinequinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3-플루오로벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 202.1-202.4 ℃ (53.8 mg, 100 %); IR (ATR) ν = 3055, 3033, 2980, 1676, 1613, 1490, 1434, 1372, 1135 cm1; 1H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 6.4 Hz, 1H), 7.75 (t, J = 6.8 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.63-7.54 (m, 1H), 7.44-7.25 (m, 4H), 6.68 -6.54 (m, 1H), 4.59 (q, J = 7.2 Hz, 2H), 1.56 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.5, 163.4 (d, JC,F = 247.9 Hz), 146.6, 143.0 (d, JC,F = 295.8 Hz), 141.8 (d, JC,F = 7.5 Hz), 131.4 (d, JC,F = 33.2 Hz), 131.2, 129.8, 128.6, 127.6 (d, JC,F = 24.0 Hz), 127.2, 126.1, 124.6, 124.5, 122.4, 121.7, 120.7, 116.6 (d, JC,F = 20.9 Hz), 116.2, 116.0, 112.3, 99.8, 60.7, 14.8; HRMS (ESI) calcd for C24H18FN2O2 385.1347 ([M+H]+), found 385.1345. Yellow solid, mp 202.1-202.4 ° C (53.8 mg, 100%); IR (ATR) ν = 3055, 3033, 2980, 1676, 1613, 1490, 1434, 1372, 1135 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 8.8 Hz, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.89 (d , J = 6.4 Hz, 1H), 7.75 (t, J = 6.8 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.63-7.54 (m, 1H), 7.44-7.25 (m, 4H) , 6.68 -6.54 (m, 1 H), 4.59 (q, J = 7.2 Hz, 2H), 1.56 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.5, 163.4 (d, JC, F = 247.9 Hz), 146.6, 143.0 (d, JC, F = 295.8 Hz), 141.8 (d, JC, F = 7.5 Hz) , 131.4 (d, JC, F = 33.2 Hz), 131.2, 129.8, 128.6, 127.6 (d, JC, F = 24.0 Hz), 127.2, 126.1, 124.6, 124.5, 122.4, 121.7, 120.7, 116.6 (d, JC , F = 20.9 Hz), 116.2, 116.0, 112.3, 99.8, 60.7, 14.8; HRMS (ESI) calcd for C 24 H 18 FN 2 O 2 385.1347 ([M + H] + ), found 385.1345.
2-29. (IQ 29) Ethyl 6-2-29. (IQ 29) Ethyl 6- (3-methoxyphenyl)indolizino[3,2-c]quinoline(3-methoxyphenyl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3-메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 173.4-173.8 ℃ (51.6 mg, 93%); IR (ATR) ν = 3031, 2957, 1675, 1588, 1488, 1434, 1350, 1109 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.69 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.92 (d, J = 7.2 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 6.8 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.20-7.08 (m, 3H), 6.62 (t, J = 6.8 Hz, 1H), 4.60 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 1.56 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.6, 160.6, 148.0, 144.6, 141.6, 140.9, 131.0, 130.9, 129.8, 128.5, 127.7, 127.54, 127.46, 125.9, 122.4, 122.0, 120.8, 120.6, 115.8, 113.5, 112.1, 99.7, 60.6, 55.6, 14.8; HRMS (ESI) calcd for C25H21N2O3 397.1547 ([M+H]+), found 397.1542.Yellow solid, mp 173.4-173.8 ° C (51.6 mg, 93%); IR (ATR) ν = 3031, 2957, 1675, 1588, 1488, 1434, 1350, 1109 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.69 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 7.92 (d , J = 7.2 Hz, 1H), 7.75 (t, J = 7.2 Hz, 1H), 7.66 (t, J = 6.8 Hz, 1H), 7.52 (t, J = 8.0 Hz, 1H), 7.33 (t, J = 6.8 Hz, 1H), 7.20-7.08 (m, 3H), 6.62 (t, J = 6.8 Hz, 1H), 4.60 (q, J = 7.2 Hz, 2H), 3.87 (s, 3H), 1.56 (t , J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.6, 160.6, 148.0, 144.6, 141.6, 140.9, 131.0, 130.9, 129.8, 128.5, 127.7, 127.54, 127.46, 125.9, 122.4, 122.0, 120.8, 120.6, 115.8, 113.5 , 112.1, 99.7, 60.6, 55.6, 14.8; HRMS (ESI) calcd for C 25 H 21 N 2 0 3 397.1547 ([M + H] + ), found 397.1542.
2-30. (IQ 30) Ethyl 6-2-30. (IQ 30) Ethyl 6- (3,4-dimethoxyphenyl)indolizino[3,2-c]quinoline(3,4-dimethoxyphenyl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 3,4-디메톡시벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 195.5-195.8 ℃ (58.5 mg, 98 %); IR (ATR) ν = 3074, 2969, 1681, 1600, 1497, 1435, 1350, 1132, 1024 cm1; 1H NMR (400 MHz, CDCl3) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.98 (d, J = 7.2 Hz, 1H), 7.73 (t, J = 6.8 Hz, 1H), 7.65 (t, J = 6.8 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.20-7.12 (m, 2H), 7.09 (d, J = 8.0 Hz, 1H), 6.64 (t, J = 7.2 Hz, 1H), 4.59 (q, J = 7.2 Hz, 2H), 4.01 (s, 3H), 3.91 (s, 3H), 1.56 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.5, 150.0, 149.9, 147.9, 144.6, 141.5, 132.1, 131.0, 129.6, 128.4, 127.7, 127.5, 127.4, 125.7, 122.3, 122.13, 122.14, 120.5, 112.1, 112.0, 111.5, 99.6, 60.6, 56.2, 56.2, 14.7; HRMS (ESI) calcd for C26H22N2O4 427.1652 ([M+H]+), found 427.1660. Yellow solid, mp 195.5-195.8 ° C (58.5 mg, 98%); IR (ATR) ν = 3074, 2969, 1681, 1600, 1497, 1435, 1350, 1132, 1024 cm 1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.68 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.98 (d , J = 7.2 Hz, 1H), 7.73 (t, J = 6.8 Hz, 1H), 7.65 (t, J = 6.8 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.20-7.12 (m , 2H), 7.09 (d, J = 8.0 Hz, 1H), 6.64 (t, J = 7.2 Hz, 1H), 4.59 (q, J = 7.2 Hz, 2H), 4.01 (s, 3H), 3.91 (s , 3H), 1.56 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.5, 150.0, 149.9, 147.9, 144.6, 141.5, 132.1, 131.0, 129.6, 128.4, 127.7, 127.5, 127.4, 125.7, 122.3, 122.13, 122.14, 120.5, 112.1, 112.0 , 111.5, 99.6, 60.6, 56.2, 56.2, 14.7; HRMS (ESI) calcd for C 26 H 22 N 2 O 4 427.1652 ([M + H] + ), found 427.1660.
2-31. (IQ 31) Ethyl 6-2-31. (IQ 31) Ethyl 6- (naphthalen-2-yl)indolizino[3,2-c]quinoline(naphthalen-2-yl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 2-나프탈데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, mp 210.3-210.7 ℃ (58.3 mg, 100%); IR (ATR) ν = 3125, 2973, 1679, 1599, 1492, 1434, 1377, 1215, 1105, 1030 cm-1; 1H NMR (400 MHz, CDCl3) δ 9.71 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.18 (s, 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.96-7.90 (m, 2H), 7.75 (t, J = 7.2 Hz, 1H), 7.70-7.64 (m, 2H), 7.64-7.54 (m, 2H), 7.29 (dd, J = 6.8, 9.2 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H), 4.61 (q, J = 7.2 Hz, 2H), 1.57 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.6, 148.0, 144.7, 141.6, 137.0, 133.8, 133.6, 131.1, 129.8, 129.5, 128.7, 128.5, 128.2, 128.1, 127.8, 127.46, 127.44, 127.1, 127.0, 126.0, 125.9, 122.4, 122.2, 120.6, 112.1, 99.7, 60.6, 14.8; HRMS (ESI) calcd for C28H21N2O2 417.1598 ([M+H]+), found 417.1598.Yellow solid, mp 210.3-210.7 ° C (58.3 mg, 100%); IR (ATR) ν = 3125, 2973, 1679, 1599, 1492, 1434, 1377, 1215, 1105, 1030 cm −1 ; 1 H NMR (400 MHz, CDCl 3 ) δ 9.71 (d, J = 8.4 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.18 (s , 1H), 8.07 (d, J = 8.4 Hz, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.96-7.90 (m, 2H), 7.75 (t, J = 7.2 Hz, 1H), 7.70 -7.64 (m, 2H), 7.64-7.54 (m, 2H), 7.29 (dd, J = 6.8, 9.2 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H), 4.61 (q, J = 7.2 Hz, 2H), 1.57 (t, J = 7.2 Hz, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.6, 148.0, 144.7, 141.6, 137.0, 133.8, 133.6, 131.1, 129.8, 129.5, 128.7, 128.5, 128.2, 128.1, 127.8, 127.46, 127.44, 127.1, 127.0, 126.0 , 125.9, 122.4, 122.2, 120.6, 112.1, 99.7, 60.6, 14.8; HRMS (ESI) calcd for C 28 H 21 N 2 O 2 417.1598 ([M + H] + ), found 417.1598.
2-32. (IQ 32) Ethyl 6-2-32. (IQ 32) Ethyl 6- (naphthalen-1-yl)indolizino[3,2-c]quinoline(naphthalen-1-yl) indolizino [3,2-c] quinoline -12-carboxylate-12-carboxylate
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 1-나프탈데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
2-33. (IQ 33) 11-Methyl-6-2-33. (IQ 33) 11-Methyl-6- (naphthalen-1-yl)indolizino[3,2-c]quinoline(naphthalen-1-yl) indolizino [3,2-c] quinoline 합성 synthesis
2-(8-메틸인돌리진-2-일)아닐린 (16 mg, 0.072 mmol) 및 1-나프탈데히드 (0.09 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.007 mmol, 0.1 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=20:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (74%, 19 mg).2- (8-methylindolizin-2-yl) aniline (16 mg, 0.072 mmol) and 1-naphthalaldehyde (0.09 mmol, 1.2 equiv) are dissolved in 1 ml of dry CH 2 Cl 2 and FeCl 3 (0.007 mmol, 0.1 equivalent) and the mixture was reacted at 60 ° C for 19 hours. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 20: 1: 2 to obtain a target compound (74%, 19 mg). .
Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.50 (d, J = 8.0 Hz, 1H), 8.30 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.78-7.66 (m, 4H), 7.50 (t, J = 7.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.78 (d, J = 6.8 Hz, 1H), 6.12 (t, J = 6.8 Hz, 1H), 2.59 (s, 3H).Yellow solid; 1 H NMR (400 MHz, CDCl 3 ) δ 8.50 (d, J = 8.0 Hz, 1H), 8.30 (d, J = 7.6 Hz, 1H), 8.09 (d, J = 8.8 Hz, 1H), 7.99 (d , J = 8.0 Hz, 1H), 7.78-7.66 (m, 4H), 7.50 (t, J = 7.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33 (s, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.09 (d, J = 7.2 Hz, 1H), 6.78 (d, J = 6.8 Hz, 1H), 6.12 (t, J = 6.8 Hz, 1H), 2.59 (s , 3H).
2-34. (IQ 34) 6-2-34. (IQ 34) 6- (Naphthalen-1-yl)pyrido[2',1':2,3]imidazo[4,5-c](Naphthalen-1-yl) pyrido [2 ', 1': 2,3] imidazo [4,5-c] quinoline 합성quinoline synthesis
2-(이미다조[1,2-a]피리딘-2-일)아닐린 (11 mg, 0.05 mmol) 및 1-나프탈데히드 (0.06 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.005 mmol, 0.1 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=5:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (92%, 15.9 mg).2- (imidazo [1,2-a] pyridin-2-yl) aniline (11 mg, 0.05 mmol) and 1-naphthalaldehyde (0.06 mmol, 1.2 equiv) were dissolved in 1 ml of dry CH 2 Cl 2 and , FeCl 3 (0.005 mmol, 0.1 equiv) was mixed and reacted at 60 ° C. for 19 hours. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane, and purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 5: 1: 2 solvent to obtain the target compound (92%, 15.9 mg). .
Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.88 (d, J = 7.6 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H),7.89-7.69 (m, 4H), 7.54 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 7.2 Hz, 1H), 7.50-7.40 (m, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.29-7.22 (m, 1H), 6.53 (t, J = 6.8 Hz, 1H).Yellow solid; 1 H NMR (400 MHz, CDCl 3 ) δ 8.88 (d, J = 7.6 Hz, 1H), 8.36 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.02 (d , J = 8.4 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.89-7.69 (m, 4H), 7.54 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 7.2 Hz , 1H), 7.50-7.40 (m, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.29-7.22 (m, 1H), 6.53 (t, J = 6.8 Hz, 1H).
2-35. (IQ 35) 9-methyl-6-2-35. (IQ 35) 9-methyl-6- (naphthalen-1-yl)indolizino[3,2-c]quinoline(naphthalen-1-yl) indolizino [3,2-c] quinoline 합성 synthesis
2-(6-메틸인돌리진-2-일)아닐린 (16 mg, 0.072 mmol) 및 1-나프탈데히드 (0.20 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.017 mmol, 0.1 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=30:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (34%, 20.4 mg).2- (6-methylindolizin-2-yl) aniline (16 mg, 0.072 mmol) and 1-naphthalaldehyde (0.20 mmol, 1.2 equiv) are dissolved in 1 ml of dry CH 2 Cl 2 and FeCl 3 (0.017 mmol, 0.1 equivalent) and the mixture was reacted at 60 ° C for 19 hours. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 30: 1: 2 to obtain the target compound (34%, 20.4 mg). .
Yellow solid; 1H NMR (400 MHz, CDCl3) δ 8.44 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.14-8.06 (m, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.77-7.63 (m, 4H), 7.59-7.46 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H), 7.34-7.36 (m, 2H), 6.90 (s, 1H), 6.83 (d, J = 9.2 Hz, 1H), 1.78 (s, 3H)Yellow solid; 1 H NMR (400 MHz, CDCl 3 ) δ 8.44 (d, J = 8.0 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 8.14-8.06 (m, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.77-7.63 (m, 4H), 7.59-7.46 (m, 2H), 7.42 (d, J = 8.4 Hz, 1H), 7.34-7.36 (m, 2H), 6.90 (s, 1H ), 6.83 (d, J = 9.2 Hz, 1H), 1.78 (s, 3H)
2-36. (IQ 36) 4-(2-36. (IQ 36) 4- ( Indolizino[3,2-c]quinolinIndolizino [3,2-c] quinolin -6--6- ylyl )-)- N,NN, N -- dimethylanilinedimethylaniline 합성 synthesis
2-(인돌리진-2-일)아닐린 (20 mg, 0.096 mmol) 및 4-(다이메틸아미노)벤즈알데히드 (0.106 mmol, 1.1 당량)를 dry CH2Cl2 0.5 ml에 용해시키고, 2% TFA in CH2Cl2 0.5 ml를 드롭 방식 (dropwise)으로 첨가한 후 상온에서 19시간 동안 반응시켰다. 반응 종료 후, 포화 NaHCO3로 work up 한 뒤, 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=10:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (31%, 10 mg).2- (indolizin-2-yl) aniline (20 mg, 0.096 mmol) and 4- (dimethylamino) benzaldehyde (0.106 mmol, 1.1 equiv) are dissolved in 0.5 ml of dry CH 2 Cl 2 , 2% TFA in 0.5 ml of CH 2 Cl 2 was added dropwise and reacted at room temperature for 19 hours. After completion of the reaction, the mixture was worked up with saturated NaHCO 3 and the reaction mixture was purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 10: 1: 2 to obtain the target compound (31%, 10 mg).
Yellow solid, mp: 173.8-175.0 °C; 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 7.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 7.66 (dd, J = 1.2, 8.4 Hz, 1H), 7.63-7.57 (m, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.25 (s, 1H), 7.02 (dd, J = 6.4, 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.46-6.39 (m, J = 6.8 Hz, 1H), 3.07 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 151.2, 149.3, 143.2, 138.9, 131.7, 129.8, 129.4, 127.5, 127.3, 125.4, 123.6, 123.3, 122.3, 121.6, 119.2, 112.8, 109.6, 92.1, 40.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C23H20N3 338.1652, found 338.1652.Yellow solid, mp: 173.8-175.0 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.35 (d, J = 7.2 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 8.13 (d, J = 7.2 Hz, 1H), 7.66 (dd , J = 1.2, 8.4 Hz, 1H), 7.63-7.57 (m, 2H), 7.55 (d, J = 8.8 Hz, 2H), 7.25 (s, 1H), 7.02 (dd, J = 6.4, 8.4 Hz, 1H), 6.91 (d, J = 8.4 Hz, 2H), 6.46-6.39 (m, J = 6.8 Hz, 1H), 3.07 (s, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 151.2, 149.3, 143.2, 138.9, 131.7, 129.8, 129.4, 127.5, 127.3, 125.4, 123.6, 123.3, 122.3, 121.6, 119.2, 112.8, 109.6, 92.1, 40.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 23 H 20 N 3 338.1652, found 338.1652.
2-37. (IQ 37) 2-37. (IQ 37) N,NN, N -Diethyl-4-(-Diethyl-4- ( indolizino[3,2-c]quinolinindolizino [3,2-c] quinolin -6--6- ylyl )aniline 합성aniline synthesis
4-(다이메틸아미노)벤즈알데히드 대신에 4-(다이에틸아미노)벤즈알데히드를 사용한 것을 제외하고는 실시예 2-36의 방법으로 목적하는 화합물을 얻었다 (31%, 11 mg).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.
Yellow solid, mp: 77.2-78.6 °C; 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 7.6 Hz, 1H), 8.26 (t, J = 6.0 Hz, 2H), 7.72-7.56 (m, 3H), 7.53 (t, J = 8.4 Hz, 2H), 7.26 (s, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 6.45 (t, J = 6.8 Hz, 1H), 3.47 (q, J = 7.2 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 149.5, 148.5, 143.5, 138.7, 131.6, 130.0, 129.6, 127.4, 127.3, 126.4, 125.2, 123.5, 122.3, 121.6, 119.1, 112.2, 109.4, 92.0, 44.6, 12.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C25H24N3 366.1965, found 366.1966.Yellow solid, mp: 77.2-78.6 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.35 (d, J = 7.6 Hz, 1H), 8.26 (t, J = 6.0 Hz, 2H), 7.72-7.56 (m, 3H), 7.53 (t, J = 8.4 Hz, 2H), 7.26 (s, 1H), 7.04 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 2H), 6.45 (t, J = 6.8 Hz, 1H), 3.47 (q, J = 7.2 Hz, 4H), 1.24 (t, J = 7.2 Hz, 6H); 13 C NMR (100 MHz, CDCl 3 ) δ 149.5, 148.5, 143.5, 138.7, 131.6, 130.0, 129.6, 127.4, 127.3, 126.4, 125.2, 123.5, 122.3, 121.6, 119.1, 112.2, 109.4, 92.0, 44.6, 12.7 ; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 25 H 24 N 3 366.1965, found 366.1966.
2-38. (IQ 38) 6-(4-2-38. (IQ 38) 6- (4- MethoxyphenylMethoxyphenyl )-12-) -12- nitroindolizino[3,2-c]quinolinenitroindolizino [3,2-c] quinoline 합성 synthesis
6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀린 (9 mg, 0.028 mmol)을 CH3CN 1 ml에 용해시키고 NBS (0.028 mmol, 1 당량), AgNO3 (0.028 mmol, 1 당량)와 섞은 후 120 °C에서 1시간 동안 반응시켰다. 반응 종료 후, 용매를 감압증류하고 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=10:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (42%, 4.3mg).6- (4-methoxyphenyl) indolizino [3,2-c] quinoline (9 mg, 0.028 mmol) was dissolved in 1 ml of CH 3 CN, NBS (0.028 mmol, 1 equiv), AgNO 3 (0.028 mmol) , 1 equivalent) and reacted at 120 ° C for 1 hour. After the completion of the reaction, the solvent was distilled under reduced pressure and the reaction mixture was purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 10: 1: 2 to obtain the target compound (42%, 4.3 mg).
Orange solid, mp: 265.4-266.9 °C; 1H NMR (400 MHz, CDCl3) δ 9.51 (d, J = 8.4 Hz, 1H), 8.80 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 6.8 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.65 (dd, J = 7.2, 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 6.87 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.9, 147.7, 145.6, 138.0, 131.7, 131.2, 130.10, 130.06, 129.7, 128.0, 127.6, 126.6, 125.5, 120.9, 120.3, 120.0, 115.3, 114.6, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C22H16N3O3 370.1186, found 370.1188.Orange solid, mp: 265.4-266.9 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.51 (d, J = 8.4 Hz, 1H), 8.80 (d, J = 8.8 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 8.18 (d , J = 6.8 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.65 (dd, J = 7.2, 8.4 Hz, 1H), 7.57 (d , J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 6.87 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.9, 147.7, 145.6, 138.0, 131.7, 131.2, 130.10, 130.06, 129.7, 128.0, 127.6, 126.6, 125.5, 120.9, 120.3, 120.0, 115.3, 114.6, 55.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 22 H 16 N 3 O 3 370.1186, found 370.1188.
2-39. (IQ 39) 6-2-39. (IQ 39) 6- (4-Methoxyphenyl)indolizino[3,2-c]quinoline(4-Methoxyphenyl) indolizino [3,2-c] quinoline -12-carbonitrile 합성-12-carbonitrile synthesis
12-브로모-6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀린 (20 mg, 0.05 mmol)을 DMF 0.5 ml에 용해시키고 CuCN (0.40 mmol, 8 당량) 및 NaI (0.1 mmol, 2 당량)와 섞은 후 150 °C에서 1시간 동안 반응시켰다. 반응 종료 후, 용매를 에틸아세테이트 및 H2O로 work up하고 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=20:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (24%, 4.1 mg).12-Bromo-6- (4-methoxyphenyl) indolizino [3,2-c] quinoline (20 mg, 0.05 mmol) was dissolved in 0.5 ml of DMF and CuCN (0.40 mmol, 8 equiv) and NaI ( 0.1 mmol, 2 equivalents) and then reacted at 150 ° C for 1 hour. After completion of the reaction, the solvent was worked up with ethyl acetate and H 2 O and the reaction mixture was purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 20: 1: 2 solvent to obtain the desired compound ( 24%, 4.1 mg).
Yellow solid, mp: 268.0-268.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.98 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.92 (d, J = 8.8 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.74 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.8, 148.2, 144.0, 143.1, 131.3, 131.1, 130.1, 129.9, 129.2, 128.0, 126.9, 123.6, 121.4, 121.3, 118.0, 117.1, 115.1, 112.9, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C23H16N3O 350.1288, found 350.1286.Yellow solid, mp: 268.0-268.8 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.98 (d, J = 8.4 Hz, 1H), 8.27 (d, J = 8.0 Hz, 1H), 8.08 (d, J = 7.2 Hz, 1H), 7.92 (d , J = 8.8 Hz, 1H), 7.79 (t, J = 7.2 Hz, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.42 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 8.4 Hz, 2H), 6.74 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.8, 148.2, 144.0, 143.1, 131.3, 131.1, 130.1, 129.9, 129.2, 128.0, 126.9, 123.6, 121.4, 121.3, 118.0, 117.1, 115.1, 112.9, 55.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 23 H 16 N 3 O 350.1288, found 350.1286.
2-40. (IQ 40) 12-2-40. (IQ 40) 12- ChloroChloro -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀린 (20 mg, 0.062 mmol)을 dry CH2Cl2 1 ml에 용해시키고, NCS (0.062 mmol, 1 당량)와 섞은 후 상온에서 2 시간 동안 반응시켰다. 반응 종료 후, 용매를 감압증류하고 헥산:에틸아세테이트:디클로로메탄=10:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (71%, 15.7 mg).6- (4-methoxyphenyl) indolizino [3,2-c] quinoline (20 mg, 0.062 mmol) was dissolved in 1 ml of dry CH 2 Cl 2 , mixed with NCS (0.062 mmol, 1 equiv) The reaction was carried out at room temperature for 2 hours. After completion of the reaction, the solvent was distilled under reduced pressure and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 10: 1: 2 to give the target compound (71%, 15.7 mg).
Yellow solid, mp: 215.1-216.4 °C; 1H NMR (400 MHz, CDCl3) δ 9.17 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.77-7.62 (m, 3H), 7.58(d, J = 8.4 Hz, 2H), 7.18-7.08 (m, 1H), 7.13 (d, , J = 8.4 Hz, 2H), 6.49 (t, J = 6.4 Hz, 1H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.6, 148.3, 143.6, 134.7, 132.0, 130.1, 129.5, 127.9, 126.7, 125.9, 125.8, 124.0, 123.9, 121.9, 119.8, 116.7, 115.0, 110.6, 96.8, 55.6; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C22H16ClN2O 359.0946, found 359.0945.Yellow solid, mp: 215.1-216.4 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.17 (d, J = 8.0 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.77-7.62 (m, 3H), 7.58 (d, J = 8.4 Hz, 2H), 7.18-7.08 (m, 1H), 7.13 (d,, J = 8.4 Hz, 2H), 6.49 (t, J = 6.4 Hz, 1H ), 3.94 (s, 3 H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.6, 148.3, 143.6, 134.7, 132.0, 130.1, 129.5, 127.9, 126.7, 125.9, 125.8, 124.0, 123.9, 121.9, 119.8, 116.7, 115.0, 110.6, 96.8, 55.6 ; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 22 H 16 ClN 2 O 359.0946, found 359.0945.
2-42. (IQ 42) 12-2-42. (IQ 42) 12- ChloroChloro -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 (30 mg, 0.144 mmol) 및 2-싸이오펜카복살데히드 (2-thiophenecarboxaldehyde)(0.173 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.043 mmol, 0.3 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=10:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (50%, 21.6 mg).2- (indolizin-2-yl) aniline compound (30 mg, 0.144 mmol) and 2-thiophenecarboxaldehyde (0.173 mmol, 1.2 equiv) were dissolved in 1 ml of dry CH 2 Cl 2 , FeCl 3 (0.043 mmol, 0.3 equiv) was mixed and reacted at 60 ° C. for 19 hours. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 10: 1: 2 to obtain the target compound (50%, 21.6 mg). .
Yellow solid, mp: 171.1-178.9 °C; 1H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.69 (t, J = 8.0 Hz, 1H), 7.65-7.56 (m, 3H), 7.40 (d, J = 3.2 Hz, 1H), 7.27 (dd, J = 4.0, 4.8 Hz, 1H), 7.25 (s, 1H), 7.04 (dd, J = 6.4, 8.8 Hz, 1H), 6.48 (t, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 143.1 141.8 140.8, 139.1, 131.8, 129.7, 128.0, 127.83, 127.77, 127.7, 126.9, 126.1, 123.7, 123.6, 122.6, 121.7, 119.3, 110.0, 92.3; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C19H13N2S 301.0794, found 301.0793.Yellow solid, mp: 171.1-178.9 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.35 (d, J = 7.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.03 (d, J = 7.2 Hz, 1H), 7.69 (t , J = 8.0 Hz, 1H), 7.65-7.56 (m, 3H), 7.40 (d, J = 3.2 Hz, 1H), 7.27 (dd, J = 4.0, 4.8 Hz, 1H), 7.25 (s, 1H) , 7.04 (dd, J = 6.4, 8.8 Hz, 1H), 6.48 (t, J = 7.2 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 143.1 141.8 140.8, 139.1, 131.8, 129.7, 128.0, 127.83, 127.77, 127.7, 126.9, 126.1, 123.7, 123.6, 122.6, 121.7, 119.3, 110.0, 92.3; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 19 H 13 N 2 S 301.0794, found 301.0793.
2-43. (IQ 43) Methyl 6-2-43. (IQ 43) Methyl 6- (pyridin-2-yl)indolizino(pyridin-2-yl) indolizino [3,2-c][3,2-c] quinolinequinoline -12-carboxylate 합성-12-carboxylate synthesis
메틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 (30 mg, 0.113 mmol) 및 2-피리딘카복살데히드 (2-pyridinecarboxaldehyde)(0.135 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.043 mmol, 0.3 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=1:1:1 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (54%, 21.4 mg).Methyl 2- (2-aminophenyl) indolizin-1-carboxylate (30 mg, 0.113 mmol) and 2-pyridinecarboxaldehyde (0.135 mmol, 1.2 equiv) in 1 ml dry CH 2 Cl 2 It was dissolved in, FeCl 3 (0.043 mmol, 0.3 equiv) was mixed and reacted for 19 hours at 60 ° C. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane and purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 1: 1: 1 solvent to obtain the target compound (54%, 21.4 mg). .
Yellow solid, mp: 206.7-207.2 °C; 1H NMR (400 MHz, CDCl3) δ 9.71 (d, J = 8.4 Hz, 1H), 8.81 (d, J = 4.8 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 4.0 Hz, 2H), 8.01 (d, J = 7.2 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 6.8 Hz, 1H), 7.54 (q, J = 4.4 Hz, 1H), 7.37 (m, 1H), 6.67 (t, J = 6.8 Hz, 1H), 4.12 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 166.0, 158.1, 149.5, 146.5, 144.6, 141.7, 138.1, 131.7, 129.9, 128.5, 128.2, 127.7, 127.6, 126.4, 125.2, 124.4, 122.8, 122.1, 120.6, 111.9, 51.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C22H16N3O2 354.1237, found 354.1236.Yellow solid, mp: 206.7-207.2 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.71 (d, J = 8.4 Hz, 1H), 8.81 (d, J = 4.8 Hz, 1H), 8.49 (d, J = 9.2 Hz, 1H), 8.26 (d , J = 8.0 Hz, 1H), 8.05 (d, J = 4.0 Hz, 2H), 8.01 (d, J = 7.2 Hz, 1H), 7.76 (t, J = 7.2 Hz, 1H), 7.70 (t, J = 6.8 Hz, 1H), 7.54 (q, J = 4.4 Hz, 1H), 7.37 (m, 1H), 6.67 (t, J = 6.8 Hz, 1H), 4.12 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 166.0, 158.1, 149.5, 146.5, 144.6, 141.7, 138.1, 131.7, 129.9, 128.5, 128.2, 127.7, 127.6, 126.4, 125.2, 124.4, 122.8, 122.1, 120.6, 111.9 , 51.9; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 22 H 16 N 3 O 2 354.1237, found 354.1236.
2-44. (IQ 44) Methyl 6-2-44. (IQ 44) Methyl 6- (thiophen-2-yl)indolizino[3,2-c]quinoline(thiophen-2-yl) indolizino [3,2-c] quinoline -12-carboxylate 합성-12-carboxylate synthesis
메틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 (30 mg, 0.113 mmol) 및 2-싸이오펜카복살데히드 (2-thiophenecarboxaldehyde)(0.135 mmol, 1.2 당량)를 dry CH2Cl2 1 ml에 용해시키고, FeCl3 (0.043 mmol, 0.3 당량)를 섞은 후 60 °C에서 19시간 동안 반응시켰다. 반응종료 후 H2O 및 디클로로메탄으로 work up하고 헥산:에틸아세테이트:디클로로메탄=10:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (80%, 32.3 mg).Methyl 2- (2-aminophenyl) indolizin-1-carboxylate (30 mg, 0.113 mmol) and 2-thiophenecarboxaldehyde (0.135 mmol, 1.2 equiv) were dried with CH 2 Cl 2 1 Dissolved in ml, FeCl 3 (0.043 mmol, 0.3 equiv) was mixed and reacted for 19 hours at 60 ° C. After completion of the reaction, the mixture was worked up with H 2 O and dichloromethane and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 10: 1: 2 to obtain the target compound (80%, 32.3 mg). .
Yellow solid, mp: 196.6-197.0 °C; 1H NMR (400 MHz, CDCl3) δ 9.66 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 9.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 7.2 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 4.4 Hz, 1H), 7.40-7.31 (m, 2H), 7.30-7.26 (m, 1H), 6.69 (t, J = 6.8 Hz, 1H), 4.11 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 165.9, 144.6, 141.7, 141.3, 140.3, 131.2, 129.9, 128.6, 128.23, 128.17, 128.1, 127.7, 127.6, 127.4, 126.4, 122.7, 122.5, 120.6, 112.3, 51.6; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C21H15N2O2S 359.0849, found 359.0851.Yellow solid, mp: 196.6-197.0 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.66 (d, J = 8.4 Hz, 1H), 8.46 (d, J = 9.6 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.07 (d , J = 7.2 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.67 (t, J = 8.0 Hz, 1H), 7.62 (d, J = 4.4 Hz, 1H), 7.40-7.31 (m , 2H), 7.30-7.26 (m, 1 H), 6.69 (t, J = 6.8 Hz, 1 H), 4.11 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 165.9, 144.6, 141.7, 141.3, 140.3, 131.2, 129.9, 128.6, 128.23, 128.17, 128.1, 127.7, 127.6, 127.4, 126.4, 122.7, 122.5, 120.6, 112.3, 51.6 ; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 21 H 15 N 2 O 2 S 359.0849, found 359.0851.
2-45. (IQ 45) 12-(4-2-45. (IQ 45) 12- (4- FluorophenylFluorophenyl )-6-) -6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
12-브로모-6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀린 (20 mg, 0.05 mmol), 아릴보론산 (0.1 mmol, 2 당량), Pd(PPh3)4 (0.0025 mmol, 0.05 당량) 및 K2CO3 (0.15 mmol, 3 당량)을 톨루엔:메탄올:H2O=2:2:1 용매 2.5 ml에 용해시킨 후 100 °C에서 6시간 동안 반응시켰다. 반응 종료 후, 용매를 제거하고 에탄올 및 H2O로 work up한 후, 헥산:에틸아세테이트:디클로로메탄=20:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다. 12-bromo-6- (4-methoxyphenyl) indolizino [3,2-c] quinoline (20 mg, 0.05 mmol), arylboronic acid (0.1 mmol, 2 equiv), Pd (PPh 3 ) 4 (0.0025 mmol, 0.05 equiv) and K 2 CO 3 (0.15 mmol, 3 equiv) were dissolved in 2.5 ml of toluene: methanol: H 2 O = 2: 2: 1 solvent and reacted at 100 ° C. for 6 hours. After the completion of the reaction, the solvent was removed, and the mixture was worked up with ethanol and H 2 O, and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 20: 1: 2 to obtain a target compound.
Yellow solid, mp: 202.3-203.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 7.2 Hz, 1H), 7.68-7.61 (m, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.61-7.53 (m, 2H), 7.41 (d, J = 9.2 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 7.01 (t, J = 6.8 Hz, 1H), 6.46 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 163.8, 161.3, 160.5, 148.6, 133.1, 133.0, 131.0, 130.2, 129.9, 128.3, 127.4, 126.8, 125.3, 124.0, 123.7, 122.7, 120.8, 117.7, 116.2, 116.0, 114.9, 110.3, 108.5, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C28H20FN2O 419.1554, found 419.1555.Yellow solid, mp: 202.3-203.2 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.24 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 2H), 7.95 (d, J = 7.2 Hz, 1H), 7.68-7.61 (m, 1H), 7.64 (d, J = 8.8 Hz, 2H), 7.61-7.53 (m, 2H), 7.41 (d, J = 9.2 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H) , 7.28 (d, J = 8.8 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 7.01 (t, J = 6.8 Hz, 1H), 6.46 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 163.8, 161.3, 160.5, 148.6, 133.1, 133.0, 131.0, 130.2, 129.9, 128.3, 127.4, 126.8, 125.3, 124.0, 123.7, 122.7, 120.8, 117.7, 116.2, 116.0 , 114.9, 110.3, 108.5, 55.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 28 H 20 FN 2 O 419.1554, found 419.1555.
2-46. (IQ 46) 12-(4-2-46. (IQ 46) 12- (4- ChlorophenylChlororophenyl )-6-) -6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline
Yellow solid, mp: 211.6-212.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.24 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 6.8 Hz, 1H), 7.69-7.59 (m, 3H), 7.56 (s, 4H), 7.43 (d, J = 8.8 Hz, 1H),7.37 (t, J = 7.6Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H), 7.02 (t, J = 6.8 Hz, 1H), 6.47 (t, J = 6.4 Hz, 1H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.5, 148.6, 143.8, 137.0, 133.7, 133.6, 132.8, 130.2, 129.9, 129.3, 128.1, 127.5, 126.8, 125.4, 124.0, 123.8, 122.6, 120.9, 117.6, 114.9, 110.4, 108.2, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C28H20ClN2O 435.1259, found 435.1259.Yellow solid, mp: 211.6-212.3 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.24 (d, J = 8.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 7.95 (d, J = 6.8 Hz, 1H), 7.69-7.59 (m, 3H), 7.56 (s, 4H), 7.43 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.6 Hz, 2H), 7.02 (t, J = 6.8 Hz, 1H), 6.47 (t, J = 6.4 Hz, 1H), 3.95 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.5, 148.6, 143.8, 137.0, 133.7, 133.6, 132.8, 130.2, 129.9, 129.3, 128.1, 127.5, 126.8, 125.4, 124.0, 123.8, 122.6, 120.9, 117.6, 114.9 , 110.4, 108.2, 55.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 28 H 20 ClN 2 O 435.1259, found 435.1259.
2-47. (IQ 47) 6-(4-2-47. (IQ 47) 6- (4- MethoxyphenylMethoxyphenyl )-12-) -12- (4-(trifluoromethyl)(4- (trifluoromethyl) phenyl)indolizino[3,2-c]quinolinephenyl) indolizino [3,2-c] quinoline
Yellow solid, mp: 233.3-233.5 °C; 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.68-7.59 (m, 3H), 7.45 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.04 (t, J = 6.8 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.6, 148.7, 143.9, 139.4, 137.0, 132.2, 131.8, 130.1, 130.0, 128.0, 127.6, 126.9, 126.0 (q, JC,F = 3.9 Hz), 125.5, 124.2, 123.9, 122.5, 121.1, 117.4, 115.0, 110.6, 108.0, 55.7; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C29H20F3N2O 469.1522, found 469.1520. Yellow solid, mp: 233.3-233.5 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.25 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.0 Hz, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.85 (d , J = 8.0 Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H), 7.68-7.59 (m, 3H), 7.45 (d, J = 8.8 Hz, 1H), 7.37 (t, J = 7.2 Hz , 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.04 (t, J = 6.8 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H), 3.95 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.6, 148.7, 143.9, 139.4, 137.0, 132.2, 131.8, 130.1, 130.0, 128.0, 127.6, 126.9, 126.0 (q, JC, F = 3.9 Hz), 125.5, 124.2 , 123.9, 122.5, 121.1, 117.4, 115.0, 110.6, 108.0, 55.7; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 29 H 20 F 3 N 2 O 469.1522, found 469.1520.
2-48. (IQ 48) 1-(4-(6-2-48. (IQ 48) 1- (4- (6- (4-Methoxyphenyl)indolizino[3,2-c]quinolin(4-Methoxyphenyl) indolizino [3,2-c] quinolin -12-yl)phenyl)ethanone-12-yl) phenyl) ethanone
Yellow solid, mp: 256.7-258.3 °C; 1H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 8.4 Hz, 1H), 7.97 (d, J = 7.2 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.63-7.60 (m, 1H), 7.48 (d, J = 8.0 Hz, 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.05 (dd, J = 6.8, 8.4 Hz, 1H), 6.49 (t, J = 6.8 Hz, 1H), 3.96 (s, 3H), 2.74 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 198.0, 160.5, 148.6, 143.9, 140.7, 136.9, 136.16, 131.6, 130.1, 130.0, 129.1, 128.0, 127.6, 126.9, 125.4, 124.2, 124.0, 122.5, 121.1, 117.5, 115.0, 110.6, 108.4, 55.7, 26.9; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C30H23N2O2 443.1754, found 443.1753.Yellow solid, mp: 256.7-258.3 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.25 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 8.4 Hz, 1H), 7.97 (d , J = 7.2 Hz, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.64 (d, J = 8.0 Hz, 2H), 7.63-7.60 (m, 1H), 7.48 (d, J = 8.0 Hz , 1H), 7.35 (t, J = 7.2 Hz, 1H), 7.16 (d, J = 8.4 Hz, 2H), 7.05 (dd, J = 6.8, 8.4 Hz, 1H), 6.49 (t, J = 6.8 Hz , 1H), 3.96 (s, 3H), 2.74 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 198.0, 160.5, 148.6, 143.9, 140.7, 136.9, 136.16, 131.6, 130.1, 130.0, 129.1, 128.0, 127.6, 126.9, 125.4, 124.2, 124.0, 122.5, 121.1, 117.5 , 115.0, 110.6, 108.4, 55.7, 26.9; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 30 H 23 N 2 O 2 443.1754, found 443.1753.
2-49. (IQ 49) 12-2-49. (IQ 49) 12- BromoBromo -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀론 (50 mg, 0.154 mmol)을 CH3CN 5 ml에 용해시키고, NBS (0.154 mmol, 1 당량)와 섞은 후, 0 °C에서 1시간 동안 반응시켰다. 반응 종료 후, 용매를 감압증류하고 헥산:에틸아세테이트:디클로로메탄=5:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (96%, 59.8 mg).6- (4-methoxyphenyl) indolizino [3,2-c] quinolone (50 mg, 0.154 mmol) was dissolved in 5 ml of CH 3 CN, mixed with NBS (0.154 mmol, 1 equiv), and 0 The reaction was carried out at 1 ° C for 1 hour. After completion of the reaction, the solvent was distilled under reduced pressure and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 5: 1: 2 to obtain the target compound (96%, 59.8 mg).
Yellow solid, mp: 215.4-216.9 °C; 1H NMR (400 MHz, CDCl3) δ 9.35 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.79-7.62 (m, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.20-7.07 (m, 3H), 6.49 (t, J = 6.8 Hz, 1H), 3.93 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.6, 148.2, 143.8, 136.0, 132.1, 130.1, 129.7, 127.9, 127.1, 126.9, 125.5, 124.4, 123.4, 122.1, 120.8, 117.7, 115.0, 110.7, 81.7, 55.6; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C22H16BrN2O 403.0411, found 403.0414.Yellow solid, mp: 215.4-216.9 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.35 (d, J = 8.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.79-7.62 (m, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.20-7.07 (m, 3H), 6.49 (t, J = 6.8 Hz, 1H), 3.93 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.6, 148.2, 143.8, 136.0, 132.1, 130.1, 129.7, 127.9, 127.1, 126.9, 125.5, 124.4, 123.4, 122.1, 120.8, 117.7, 115.0, 110.7, 81.7, 55.6 ; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 22 H 16 BrN 2 O 403.0411, found 403.0414.
2-50. (IQ 50) 12-2-50. (IQ 50) 12- IodoIodo -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
6-(4-메톡시페닐)인돌리지노[3,2-c]퀴놀론 (12 mg, 0.036 mmol)을 CH3CN 1 ml에 용해시키고, NIS (0.036 mmol, 1 당량)와 섞은 후, 0 °C에서 1시간 동안 반응시켰다. 반응 종료 후, 용매를 감압증류하고 헥산:에틸아세테이트:디클로로메탄=5:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다 (81%, 13.1 mg).6- (4-methoxyphenyl) indolizino [3,2-c] quinolone (12 mg, 0.036 mmol) is dissolved in 1 ml of CH 3 CN, mixed with NIS (0.036 mmol, 1 equiv) and then 0 The reaction was carried out at 1 ° C for 1 hour. After completion of the reaction, the solvent was distilled under reduced pressure and purified by silica gel column chromatography using a solvent of hexane: ethyl acetate: dichloromethane = 5: 1: 2 to obtain the target compound (81%, 13.1 mg).
Yellow solid, mp: 213.7-214.9 °C; 1H NMR (400 MHz, CDCl3) δ 9.62 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.79-7.65 (m, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.50 (t, J = 6.8 Hz, 1H), 3.94 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 160.4, 147.8, 143.6, 138.8, 131.8, 129.9, 129.6, 129.1, 127.8, 127.0, 125.0, 122.1, 119.6, 117.5, 114.8, 110.7, 105.2, 103.3, 55.5; HRMS (ESI-QTOF) m/z [M+H]+ calcd for C22H16IN2O 451.0302, found 451.0302.Yellow solid, mp: 213.7-214.9 ° C; 1 H NMR (400 MHz, CDCl 3 ) δ 9.62 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 7.2 Hz, 1H), 7.79-7.65 (m, 3H), 7.57 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 7.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 6.50 (t, J = 6.8 Hz , 1H), 3.94 (s, 3H); 13 C NMR (100 MHz, CDCl 3 ) δ 160.4, 147.8, 143.6, 138.8, 131.8, 129.9, 129.6, 129.1, 127.8, 127.0, 125.0, 122.1, 119.6, 117.5, 114.8, 110.7, 105.2, 103.3, 55.5; HRMS (ESI-QTOF) m / z [M + H] + calcd for C 22 H 16 IN 2 O 451.0302, found 451.0302.
2-51. (IQ 51) ethyl 6-2-51. (IQ 51) ethyl 6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline -- 12- carboxylate12-carboxylate 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The procedure was carried out except that ethyl 2- (2-aminophenyl) indolizin-1-carboxylate compound was used instead of 2- (indolizin-2-yl) aniline compound and benzaldehyde was used instead of 4-bromobenzaldehyde. The target compound was obtained by the method of Example 2-1.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 9.65 (d, J = 7.0 Hz, 1H), 8.49 (d, J = 9.3 Hz, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 7.1 Hz, 2H), 7.72 (td, J = 6.9, 1.5 Hz, 1H), 7.64 (td, J = 8.3, 1.5 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.33 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.62 (t, J = 7.1 Hz, 1H), 4.56 (q, J = 7.1 Hz, 2H), 3.93 (s, 1H), 1.55 (t, J = 7.1 Hz, 3H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 9.65 (d, J = 7.0 Hz, 1H), 8.49 (d, J = 9.3 Hz, 1H), 8.23 (d, J = 7.7 Hz, 1H), 8.03 (d, J = 7.1 Hz, 2H), 7.72 (td, J = 6.9, 1.5 Hz, 1H), 7.64 (td, J = 8.3, 1.5 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H ), 7.33 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.62 (t, J = 7.1 Hz, 1H), 4.56 (q, J = 7.1 Hz, 2H), 3.93 (s, 1 H), 1.55 (t, J = 7.1 Hz, 3 H).
2-52. (IQ 52) 11-methyl-6-2-52. (IQ 52) 11-methyl-6- (thiophen-2-yl)indolizino[3,2-c]quinoline(thiophen-2-yl) indolizino [3,2-c] quinoline 합성 synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 사이오핀-2-칼브알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 8.35 (dd, J = 7.9, 1.4 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.9 (d, J = 7.1 Hz, 1H), 7.68-7.55 (m, 3H), 7.36 (dd, J = 3.5, 1.1 Hz, 1H), 7.23-7.21 (m, 2H), 6.83 (d, J = 6.6 1H), 6.40 (t, J = 6.9 Hz, 1H), 2.54 (s, 1H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.35 (dd, J = 7.9, 1.4 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.9 (d, J = 7.1 Hz, 1H ), 7.68-7.55 (m, 3H), 7.36 (dd, J = 3.5, 1.1 Hz, 1H), 7.23-7.21 (m, 2H), 6.83 (d, J = 6.6 1H), 6.40 (t, J = 6.9 Hz, 1H), 2.54 (s, 1H).
2-53. (IQ 53) 6-(5-2-53. (IQ 53) 6- (5- chlorofuranchlorofuran -2--2- ylyl )-11-) -11- methylindolizino[3,2-c]methylindolizino [3,2-c] quinoline 합성quinoline synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 2-(8-메틸인돌리진-2-일)아닐린 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 5-클로로퓨란-2-칼브알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.Use 2- (8-methylindolizin-2-yl) aniline compound in place of 2- (indolizin-2-yl) aniline compound and 5-chlorofuran-2-carbaldehyde in place of 4-bromobenzaldehyde Except for using, the target compound was obtained by the method of Example 2-1.
Orange solid, 1H NMR (300 MHz, CDCl3) δ 8.35 (dd, J = 7.7, 1.1 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.1 Hz, 1H), 7.70-7.58 (m, 2H), 7.22 (s, 1H), 7.02 (d, J = 3.3 Hz, 1H), 6.90 (d, J = 6.6 Hz, 1H), 6.57 (t, J = 6.9 Hz, 1H), 6.49 (d, J = 3.3 Hz, 1H), 2.57 (s, 1H).Orange solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.35 (dd, J = 7.7, 1.1 Hz, 1H), 8.22 (d, J = 7.9 Hz, 1H), 8.03 (d, J = 7.1 Hz, 1H ), 7.70-7.58 (m, 2H), 7.22 (s, 1H), 7.02 (d, J = 3.3 Hz, 1H), 6.90 (d, J = 6.6 Hz, 1H), 6.57 (t, J = 6.9 Hz , 1H), 6.49 (d, J = 3.3 Hz, 1H), 2.57 (s, 1H).
2-54. (IQ 54) 6-(4-2-54. (IQ 54) 6- (4- methoxyphenylmethoxyphenyl )-12-((trimethylsilyl)ethynyl)indolizino [3,2-c]quinoline 합성) -12-((trimethylsilyl) ethynyl) indolizino [3,2-c] quinoline synthesis
IQ 50 화합물 0.11 mmol, 트리메틸((트리부틸스테닐)에티닐)실란 0.13 mmol (1.2 당량) 및 Pd(PPh3)4, CuI 0.01 mmol (0.1 당량)을 테트라하이드로퓨란 3 ml에 용해시켜 80 ℃에서 2 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 물 3 ml로 세척한 뒤, 물층을 디클로로메탄 3 ml로 한 번 더 추출하였다. 유기층을 모아 마그네슘설페이트로 건조시키고, 감압 농축하였다. 농축된 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=3:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.0.11 mmol of the IQ 50 compound, 0.13 mmol (1.2 equiv) of trimethyl ((tributylstenyl) ethynyl) silane and 0.01 mmol (0.1 equiv) of Pd (PPh 3 ) 4 , CuI were dissolved in 3 ml of tetrahydrofuran at 80 ° C. Reaction was carried out for 2 hours. After the reaction was completed, the reaction mixture was washed with 3 ml of water, and then the water layer was extracted once more with 3 ml of dichloromethane. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The concentrated reaction mixture was purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 3: 1: 2 solvent to obtain the desired compound.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 9.32 (dd, J = 8.1, 1.1 Hz, 1H), 8.23 (dd, J = 8.4, 1.3 Hz, 1H), 7.91 (dd, J = 8.8, 0.9 Hz, 1H), 7.86 (dt, J = 7.3, 1.1 Hz, 1H), 7.74-7.60 (m, 3H), 7.56 (d, J = 8.8 Hz, 2H), 7.19 (dd, J = 9.0, 6.6 Hz, 1H), 7.11 (d, J = 8.6 1H), 6.52 (td, J = 7.1, 1.5 Hz, 1H), 3.92 (s, 3H), 0.39 (s, 9H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 9.32 (dd, J = 8.1, 1.1 Hz, 1H), 8.23 (dd, J = 8.4, 1.3 Hz, 1H), 7.91 (dd, J = 8.8, 0.9 Hz, 1H), 7.86 (dt, J = 7.3, 1.1 Hz, 1H), 7.74-7.60 (m, 3H), 7.56 (d, J = 8.8 Hz, 2H), 7.19 (dd, J = 9.0, 6.6 Hz, 1H), 7.11 (d, J = 8.6 1H), 6.52 (td, J = 7.1, 1.5 Hz, 1H), 3.92 (s, 3H), 0.39 (s, 9H).
2-55. (IQ 55) 12-2-55. (IQ 55) 12- ethynylethynyl -6--6- (4-methoxyphenyl)indolizino[3,2-c]quinoline(4-methoxyphenyl) indolizino [3,2-c] quinoline 합성 synthesis
IQ 54 화합물 0.12 mmol 및 K2CO3 0.24 mmol (2 당량)을 메탄올 1 ml에 용해시켜 상온에서 3 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 물 3 ml로 세척한 뒤, 물층을 디클로로메탄 3 ml로 한 번 더 추출하였다. 유기층을 모아 마그네슘설페이트로 건조시키고, 감압 농축하였다. 농축된 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=3:1:2 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.0.12 mmol of IQ 54 compound and 0.24 mmol (2 equivalents) of K 2 CO 3 were dissolved in 1 ml of methanol and reacted at room temperature for 3 hours. After the reaction was completed, the reaction mixture was washed with 3 ml of water, and then the water layer was extracted once more with 3 ml of dichloromethane. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The concentrated reaction mixture was purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 3: 1: 2 solvent to obtain the desired compound.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 9.29 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.74-7.61 (m, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.22-7.17 (m, 1H), 7.12 (d, J = 8.6 Hz, 2H), 6.54 (t, J = 6.9 Hz, 1H), 3.92 (s, 3H), 3.73 (s, 1H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 9.29 (d, J = 8.0 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.93 (d, J = 7.3 Hz, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.74-7.61 (m, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.22-7.17 (m, 1H), 7.12 (d, J = 8.6 Hz , 2H), 6.54 (t, J = 6.9 Hz, 1H), 3.92 (s, 3H), 3.73 (s, 1H).
2-56. (IQ 56) 4-(5-(2-56. (IQ 56) 4- (5- ( indolizino[3,2-c]quinolinindolizino [3,2-c] quinolin -6--6- ylyl )-2-methoxybenzyl)morpholine 합성) -2-methoxybenzyl) morpholine synthesis
4-브로모벤즈알데히드 대신에 4-메톡시-3-(몰폴리노메틸)벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 8.36 (d, J = 7.7 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 7.70-7.55 (m, 5H), 7.27 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (dd, J = 9.1, 6.5 Hz, 1H), 6.37 (t, J = 6.9 Hz, 1H), 3.94 (s, 3H), 3.65-3.62 (m, 6H), 2.53-2.50 (m, 4H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.36 (d, J = 7.7 Hz, 1H), 8.22 (d, J = 8.3 Hz, 1H), 7.92 (d, J = 7.3 Hz, 1H), 7.70-7.55 (m, 5H), 7.27 (s, 1H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (dd, J = 9.1, 6.5 Hz, 1H), 6.37 (t, J = 6.9 Hz , 1H), 3.94 (s, 3H), 3.65-3.62 (m, 6H), 2.53-2.50 (m, 4H).
2-57. (IQ 57) ethyl 6-(4-2-57. (IQ 57) ethyl 6- (4- methoxymethoxy -3-(-3- ( morpholinomethylmorpholinomethyl )phenyl) indolizino[3,2-c]quinoline-12-carboxylate 합성) phenyl) indolizino [3,2-c] quinoline-12-carboxylate synthesis
2-(인돌리진-2-일)아닐린 화합물 대신에 에틸 2-(2-아미노페닐)인돌리진-1-카복실레이트 화합물을 사용하고, 4-브로모벤즈알데히드 대신에 4-메톡시-3-(몰폴리노메틸)벤즈알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.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.
Yellow solid, 1H NMR (500 MHz, CDCl3) δ 9.65 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 9.3 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 1.8 Hz, 1H), 7.53 (dd, J = 8.3, 2.0 Hz, 1H), 7.31 (t, J = 9.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 6.8 Hz, 1H), 4.58 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.70-3.55 (m, 6H), 2.50 (s, 4H), 1.54 (t, J = 7.1 Hz, 3H).Yellow solid, 1 H NMR (500 MHz, CDCl 3 ) δ 9.65 (d, J = 8.4 Hz, 1H), 8.47 (d, J = 9.3 Hz, 1H), 8.22 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 7.2 Hz, 1H), 7.71 (t, J = 7.5 Hz, 1H), 7.63 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 1.8 Hz, 1H), 7.53 ( dd, J = 8.3, 2.0 Hz, 1H), 7.31 (t, J = 9.0 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.56 (d, J = 6.8 Hz, 1H), 4.58 ( q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 3.70-3.55 (m, 6H), 2.50 (s, 4H), 1.54 (t, J = 7.1 Hz, 3H).
2-58. (IQ 58) 6-(1H-indol-7-yl)indolizino[3,2-c]quinoline 합성2-58. (IQ 58) 6- (1H-indol-7-yl) indolizino [3,2-c] quinoline synthesis
4-브로모벤즈알데히드 대신에 1H-인돌-7-칼브알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The target compound was obtained by the method of Example 2-1 except that 1H-indole-7-carbaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, 1H NMR (300 MHz, CD3OD) δ 8.52 (dd, J = 7.0, 2.6 Hz, 1H), 8.13 (dd, J = 7.8, 2.8 Hz, 1H), 7.87 (dd, J = 7.1, 2.0 Hz, 1H), 7.75-7.66 (m, 3H), 7.46 (s, 1H), 7.41 (d, J = 6.2 Hz, 1H), 7.37-7.30 (m, 2H), 7.19 (d, J = 3.1 Hz, 1H), 7.08 (dd, J = 9.5, 7.0 Hz, 1H), 6.63 (d, J = 3.1 Hz, 1H), 6.31 (t, J = 7.0 Hz, 1H).Yellow solid, 1 H NMR (300 MHz, CD 3 OD) δ 8.52 (dd, J = 7.0, 2.6 Hz, 1H), 8.13 (dd, J = 7.8, 2.8 Hz, 1H), 7.87 (dd, J = 7.1 , 2.0 Hz, 1H), 7.75-7.66 (m, 3H), 7.46 (s, 1H), 7.41 (d, J = 6.2 Hz, 1H), 7.37-7.30 (m, 2H), 7.19 (d, J = 3.1 Hz, 1H), 7.08 (dd, J = 9.5, 7.0 Hz, 1H), 6.63 (d, J = 3.1 Hz, 1H), 6.31 (t, J = 7.0 Hz, 1H).
2-59. (IQ 6-T) 12-(1-butyl-1H-1,2,3-2-59. (IQ 6-T) 12- (1-butyl-1H-1,2,3- triazoltriazol -4--4- ylyl )-6-(4-) -6- (4- methoxyphenylmethoxyphenyl ) indolizino[3,2-c]quinoline 합성) indolizino [3,2-c] quinoline synthesis
IQ 55 화합물 0.13 mmol, 뷰틸아지드 0.13 mmol (1 당량) 및 소듐아스코베이트 0.06 mmol (0.44 당량), CuSO4.5H2O 0.03 mmol (0.22 당량)을 테트라하이드로퓨란/t-뷰탄올/물 3 ml에 용해시켜 상온에서 12시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 물 3 ml로 세척한 뒤, 물층을 디클로로메탄 3 ml로 한 번 더 추출하였다. 유기층을 모아 마그네슘설페이트로 건조시키고, 감압 농축하였다. 농축된 반응 혼합물을 헥산:에틸아세테이트:디클로로메탄=1:1:1 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.IQ 55 Compound 0.13 mmol, view tilah azide 0.13 mmol (1 eq.) And sodium ascorbyl bait 0.06 mmol (0.44 equiv.), CuSO 4 .5H 2 O 0.03 mmol (0.22 equivalents) in tetrahydrofuran / t- butanol / water 3 It was dissolved in ml and reacted at room temperature for 12 hours. After the reaction was completed, the reaction mixture was washed with 3 ml of water, and then the water layer was extracted once more with 3 ml of dichloromethane. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The concentrated reaction mixture was purified by silica gel column chromatography using hexane: ethyl acetate: dichloromethane = 1: 1: 1 solvent to obtain the desired compound.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 8.29 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 7.1 Hz, 1H), 7.84 (s, 1H), 7.68-7.62 (m, 4H), 7.43 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.54 (t, J = 7.0 Hz, 1H), 4.57 (t, J = 7.2 Hz, 2H), 3.93 (s, 3H), 2.06 (quint, J = 7.3 Hz, 2H), 1.56-1.44 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.29 (d, J = 8.2 Hz, 1H), 7.97 (d, J = 7.1 Hz, 1H), 7.84 (s, 1H), 7.68-7.62 (m , 4H), 7.43 (t, J = 7.8 Hz, 1H), 7.15 (d, J = 8.6 Hz, 2H), 6.54 (t, J = 7.0 Hz, 1H), 4.57 (t, J = 7.2 Hz, 2H ), 3.93 (s, 3H), 2.06 (quint, J = 7.3 Hz, 2H), 1.56-1.44 (m, 2H), 1.04 (t, J = 7.4 Hz, 3H).
2-60. (IQ 6-T-B) N-(2-(2-(2-(2-(4-(6-(4-2-60. (IQ 6-T-B) N- (2- (2- (2- (2- (2- (4- (6- (4- methoxyphenylmethoxyphenyl )) indolizinoindolizino [3,2 -c]quinolin-12-yl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)-6-((3aS,6aR)-5-oxido-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)hexanamide 합성[3,2-c] quinolin-12-yl) -1H-1,2,3-triazol-1-yl) ethoxy) ethoxy) ethoxy) ethyl) -6-((3aS, 6aR) -5-oxido- 2-oxohexahydro-1H-thieno [3,4-d] imidazol-4-yl) hexanamide synthesis
IQ 55 화합물 0.13 mmol, 바이오틴아지드 0.13 mmol (1 당량) 및 소듐아스코베이트 0.06 mmol (0.44 당량), CuSO4.5H2O 0.03 mmol (0.22 당량)을 테트라하이드로퓨란/t-뷰탄올/물 3 ml에 용해시켜 상온에서 24 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 물 3 ml로 세척한 뒤, 물층을 디클로로메탄 3 ml로 한 번 더 추출하였다. 유기층을 모아 마그네슘설페이트로 건조시키고, 감압 농축하였다. 농축된 반응 혼합물을 클로로포름:메탄올=10:1 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.IQ 55 0.13 mmol compound, biotin azide 0.13 mmol (1 eq.) And sodium ascorbyl bait 0.06 mmol (0.44 equiv.), CuSO 4 .5H 2 O 0.03 mmol (0.22 equivalents) in tetrahydrofuran / t- butanol / water 3 It was dissolved in ml and reacted at room temperature for 24 hours. After the reaction was completed, the reaction mixture was washed with 3 ml of water, and then the water layer was extracted once more with 3 ml of dichloromethane. The organic layers were combined, dried over magnesium sulfate, and concentrated under reduced pressure. The concentrated reaction mixture was purified by silica gel column chromatography using chloroform: methanol = 10: 1 solvent to obtain the desired compound.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 8.40 (d, J = 8.4 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.84-7.75 (m, 5H), 7.56 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 8.6 Hz, 1H), 6.84 (t, J = 6.4 Hz, 1H), 4.78 (t, J = 4.7 Hz, 2H), 4.55-4.48 (m, 2H), 4.03 (t, J = 4.8 Hz, 2H), 3.95 (s, 3H), 3.71-3.69 (m, 2H), 3.60-3.58 (m, 2H), 3.45 (t, J = 4.3 Hz, 2H), 3.40 (t, J = 2.9 Hz, 3H), 3.32 (t, J = 4.2 Hz, 2H), 3.23-3.18 (m, 4H), 3.08-3.05 (m, 1H), 2.98-2.91 (m, 2H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.40 (d, J = 8.4 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.27 (s, 1H), 8.16 (d, J = 7.2 Hz, 1H), 7.84-7.75 (m, 5H), 7.56 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 8.6 Hz, 1H), 6.84 (t, J = 6.4 Hz, 1H ), 4.78 (t, J = 4.7 Hz, 2H), 4.55-4.48 (m, 2H), 4.03 (t, J = 4.8 Hz, 2H), 3.95 (s, 3H), 3.71-3.69 (m, 2H) , 3.60-3.58 (m, 2H), 3.45 (t, J = 4.3 Hz, 2H), 3.40 (t, J = 2.9 Hz, 3H), 3.32 (t, J = 4.2 Hz, 2H), 3.23-3.18 ( m, 4H), 3.08-3.05 (m, 1H), 2.98-2.91 (m, 2H).
2-61. (IQ 5-D) 2,6-bis(indolizino[3,2-c]quinolin-6-yl)pyridine 합성2-61. (IQ 5-D) 2,6-bis (indolizino [3,2-c] quinolin-6-yl) pyridine synthesis
4-브로모벤즈알데히드 대신에 피리딘-2,6-디칼브알데히드를 사용한 것을 제외하고는 실시예 2-1의 방법으로 목적하는 화합물을 얻었다.The desired compound was obtained by the method of Example 2-1 except that pyridine-2,6-dikalbaldehyde was used instead of 4-bromobenzaldehyde.
Yellow solid, 1H NMR (300 MHz, CDCl3) δ 8.49 (d, J = 7.3 Hz, 2H), 8.38 (d, J = 7.3 Hz, 2H), 8.33-8.26 (m, 6H), 7.76-7.62 (m, 5H), 7.51 (d, J = 9.0 Hz, 2H), 6.92 (t, J = 9.7 Hz, 2H), 6.31 (t, J = 6.9 Hz, 2H).Yellow solid, 1 H NMR (300 MHz, CDCl 3 ) δ 8.49 (d, J = 7.3 Hz, 2H), 8.38 (d, J = 7.3 Hz, 2H), 8.33-8.26 (m, 6H), 7.76-7.62 (m, 5H), 7.51 (d, J = 9.0 Hz, 2H), 6.92 (t, J = 9.7 Hz, 2H), 6.31 (t, J = 6.9 Hz, 2H).
2-62. (IQ6-S1-Me) 6-(4-2-62. (IQ6-S1-Me) 6- (4- methoxyphenylmethoxyphenyl )-5-) -5- methylindolizino[3,2-c]methylindolizino [3,2-c] quinolin-5-ium methyl sulfate 합성Quinolin-5-ium methyl sulfate synthesis
IQ 6 화합물 0.06 mmol 을 클로로포름 2 mL 에 용해시킨 후 다이메틸설페이트 0.06 mmol (1 당량) 첨가하고 60 ℃에서 36 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 감압 농축하여 목적하는 화합물을 얻었다.0.06 mmol of IQ 6 compound was dissolved in 2 mL of chloroform, and then 0.06 mmol (1 equivalent) of dimethyl sulfate was added and reacted at 60 ° C. for 36 hours. After the reaction was completed, the reaction mixture was concentrated under reduced pressure to obtain the desired compound.
1H NMR (300 MHz, D2O) δ 8.14 (dd, J = 8.1, 1.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.68 (td, J = 6.9, 1.5 Hz, 1H), 7.59 (t, J = 6.9 Hz, 1H), 7.49 (d, J = 9.3 Hz, 1H), 7.41-7.34 (m, 4H), 7.33-7.29 (m, 1H), 7.06 (s, 1H), 6.97 (d, J = 7.2 Hz, 1H), 6.57 (dd, J = 7.2, 1.2 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H). 1 H NMR (300 MHz, D 2 O) δ 8.14 (dd, J = 8.1, 1.2 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.68 (td, J = 6.9, 1.5 Hz, 1H ), 7.59 (t, J = 6.9 Hz, 1H), 7.49 (d, J = 9.3 Hz, 1H), 7.41-7.34 (m, 4H), 7.33-7.29 (m, 1H), 7.06 (s, 1H) , 6.97 (d, J = 7.2 Hz, 1H), 6.57 (dd, J = 7.2, 1.2 Hz, 1H), 3.96 (s, 3H), 3.84 (s, 3H).
2-63. (IQ6-S2-2-63. (IQ6-S2- Cl) 5Cl) 5 -(2--(2- chloroethylchloroethyl )-6-) -6- (4-methoxyphenyl)indolizino[3,2-c]quinolin(4-methoxyphenyl) indolizino [3,2-c] quinolin -5-ium trifluoromethanesulfonate 합성-5-ium trifluoromethanesulfonate synthesis
IQ 6 화합물 0.06 mmol을 아세토나이트릴 2 mL 에 용해시킨 후 2-클로로에틸 트리플루오로메탄설포네이트 0.3 mmol (5 당량) 첨가하고 80 ℃에서 36 시간 동안 반응시켰다. 반응 종료 후, 반응 혼합물을 감압 농축하고 농축된 반응 혼합물을 메탄올:다이클로로메테인=1:20 용매를 이용하여 실리카겔 컬럼 크로마토그래피법으로 정제하여 목적하는 화합물을 얻었다.0.06 mmol of IQ 6 compound was dissolved in 2 mL of acetonitrile, and then 0.3 mmol (5 equivalents) of 2-chloroethyl trifluoromethanesulfonate was added and reacted at 80 ° C. for 36 hours. After the completion of the reaction, the reaction mixture was concentrated under reduced pressure, and the concentrated reaction mixture was purified by silica gel column chromatography using methanol: dichloromethane = 1: 20 solvent to obtain the desired compound.
1H NMR (300 MHz, CDCl3) δ 8.54 (dd, J = 8.3, 1.5 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.96 (td, J = 7.2, 1.5 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.62 (d, J = 8.7 Hz, 2H), 7.58-7.55 (m, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 7.5 Hz, 1H), 6.81 (td, J = 7.2, 1.2 Hz, 1H), 5.09 (t, J = 6.3 Hz, 2H), 4.02 (s, 3H), 3.97 (t, J = 6.3 Hz, 2H). 1 H NMR (300 MHz, CDCl 3 ) δ 8.54 (dd, J = 8.3, 1.5 Hz, 1H), 8.27 (d, J = 8.4 Hz, 1H), 7.96 (td, J = 7.2, 1.5 Hz, 1H) , 7.90 (d, J = 9.0 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.62 (d, J = 8.7 Hz, 2H), 7.58-7.55 (m, 2H), 7.35 (d, J = 8.7 Hz, 2H), 7.16 (d, J = 7.5 Hz, 1H), 6.81 (td, J = 7.2, 1.2 Hz, 1H), 5.09 (t, J = 6.3 Hz, 2H), 4.02 (s, 3H), 3.97 (t, J = 6.3 Hz, 2H).
실시예 3: 형광 프로브의 후보 화합물 선정Example 3: Selection of candidate compounds for fluorescent probes
생물학적 형광체로 이용하기 위한 화합물의 조건으로는, 수중에서 방출강도가 높아야 하고, 자기소광 (self-quenching)이 적어야 하며, 에너지의 호모트랜스퍼 (homotransfer)를 최소화하기 위한 스토크스 시프트 (stokes shift)이 커야한다. 또한, 의약화학 및 형광단 개발을 위하여는, 단백질과의 응집을 최소화할 수 있도록 용해도가 우수해야 하고, 특히 이러한 특성은 단백질 표지 염료로 이용하기 위하여 더욱 필요한 조건이다.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 In addition, 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.
이러한 형광체로서의 조건을 갖는 화합물을 스크리닝하기 위하여, 본 발명의 화학식 1의 화합물들에 대하여, 최대 흡수파장 (λmax ex), 최대 방출파장 (λmax em), 상대적인 형광강도 (Emission Intensitynorm) 등을 측정하였으며, 그 결과를 하기 표 1에 나타내었다.In order to screen compounds having such conditions as phosphors, the maximum absorption wavelength (λ max ex ) and maximum emission wavelength (λ max ) for the compounds of Formula 1 of the present invention. em ), and the relative fluorescence intensity (Emission Intensity norm ) was measured, and the results are shown in Table 1 below.
[표 1]TABLE 1
Figure PCTKR2016008059-appb-I000045
Figure PCTKR2016008059-appb-I000045
a Excited at the longest absorption maxima; b Fluorescence spectra recorded in H2O at 2 μM; c Normalized fluorescence intensity.a Excited at the longest absorption maxima; b Fluorescence spectra recorded in H 2 O at 2 μM; c Normalized fluorescence intensity.
상기 표 1에서 확인할 수 있듯이, 본 발명의 화합물들은 일반적으로 대략 380-430 nm 범위내에서 최대 흡수를 보이는데, 이는 가시광선의 여기 (excitation)가 가능함을 의미한다. 또한, 수용액 상태에서 대략 480-540 nm 범위 내에서 최대 방출을 보였다.As can be seen in Table 1, 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. In addition, maximum emission was observed in the aqueous solution in the range of approximately 480-540 nm.
더욱이, 가장 눈에 띄는 특징은, 대부분의 화합물에서 큰 스토크스 시프트 (90-110 nm)가 관찰된다는 점인데, 이는 여기 파장과 방출 파장 사이에 분명한 분리가 있음을 의미하는 것이다. 즉, 방출 스펙트럼은 Alexa Fluor 488 또는 Fluorescein와 유사한 범위에서 관찰되었으나, 여기 파장은 방출 파장과 꽤 차이가 있었다. Moreover, 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.
이러한 결과는, 본 발명의 화학식 1의 화합물들은 형광체로 이용하기에 적합하고 특히 멀티-형광 기술을 이용한 세포 이미징에 응용될 수 있음을 시사하고 있다.These results suggest that the compounds of formula 1 of the present invention are suitable for use as phosphors and can be particularly applicable to cell imaging using multi-fluorescence techniques.
실시예 4: 광학특성 분석Example 4 Optical Characteristic Analysis
4-1. 치환기 의존적 특성4-1. Substituent dependent properties
상기 실시예 3의 표 1에 기재된 화합물들 중에서, 방출강도가 가장 높은 화합물 IQ 6, 9, 21, 28 및 30을 선정하였다. 이들 화합물들은 각각 도 1에 화학식 2, 3, 4, 5, 6으로 나타내었으며, 이들 화합물의 수용액에서 측정된 흡수 및 방출 스펙트럼을 도 2에 각각 나타내었다.Of the compounds listed in Table 1 of Example 3, compounds IQ 6, 9, 21, 28, and 30 having the highest release strengths were selected. Each of these compounds is represented by Formulas 2, 3, 4, 5, and 6 in FIG. 1, and absorption and emission spectra measured in aqueous solutions of these compounds are shown in FIG. 2, respectively.
그 결과, 도 1 및 2에서 확인할 수 있듯이, 화학식 2의 화합물 (IQ 6)은 R6 위치에 p-메톡시페닐기가 결합된 구조로서 514 nm에서 최대 방출을 갖는 높은 형광을 보이고 있다. 이는, 전자가 부족한 퀴놀린 질소와 반대 위치에 전자 주게 메톡시기가 존재하기 때문으로, para 위치에서의 메톡시기가 형광강도를 높이는데 중요한 역할을 하는 것으로 여겨진다. 즉, 분자 여기 시에, 중요한 쌍극자 모멘트 (dipole moment) 변화가 일어날 것으로 예상된다.As a result, as can be seen in Figures 1 and 2, 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.
대체로 이러한 전자적 효과는 다른 화합물의 결과와도 일치하였는데, 예를 들면 방향고리의 para 위치에 메톡시기가 아닌 브롬 또는 질소기가 결합되어 있는 화합물들 (IQ 2, 13 및 26)은 형광을 유도하지 않았다. 즉, 페닐기에 있는 R 위치의 치환기가 무엇인지에 따라 전자적 영향에 의해 형광 양자수율 (fluorescence Quantum Yield; QY)이 달라지는데, 이러한 상관관계를 도 3에 나타내었다.In general, this electronic effect was consistent with the results of other compounds, for example compounds with bromine or nitrogen groups ( IQ 2, 13 and 26) bound to the para position of the aromatic ring but not methoxy groups ( IQ 2, 13 and 26) did not induce fluorescence. . That is, the fluorescence quantum yield (QY) varies depending on the electronic effect depending on what is the substituent of the R position in the phenyl group, which is shown in FIG.
또한, 화학식 3의 화합물 (IQ 9)은 R6 위치에 있는 나프탈렌 치환기가 강한 형광을 야기시켰다.In addition, the compound of formula 3 (IQ 9) caused strong fluorescence of the naphthalene substituent at the R 6 position.
형광강도가 높은 화합물인 화학식 2 내지 6의 화합물 (IQ 6, 9, 21, 28 및 30)에 대하여, 스토크스 시프트 (stokes shifts)와 형광 양자수율 (Φ)을 측정한 결과를 하기 표 2에 나타내었다.For the compounds of Formulas 2 to 6 ( IQ 6, 9, 21, 28 and 30), which are compounds having high fluorescence intensity, Stokes shifts and fluorescence quantum yield (Φ) were measured. Indicated.
[표 2]TABLE 2
Figure PCTKR2016008059-appb-I000046
Figure PCTKR2016008059-appb-I000046
4-2. 용매 의존적 특성4-2. Solvent dependent properties
형광 프로브에 응용할 수 있는 후보물질로서, 형광강도가 높은 상기 5 종류의 인돌리지노[3,2-c]퀴놀린 화합물 (IQ 6, 9, 21, 28 및 30)에 대하여, 다양한 용매 중에서의 스펙트럼 분석을 실시하고 그 결과를 표 3 및 도 4에 나타내었다.As candidates applicable to fluorescent probes, spectra in various solvents for the above five kinds of indolinino [3,2-c] quinoline compounds ( IQ 6, 9, 21, 28 and 30) having high fluorescence intensity The analysis was performed and the results are shown in Table 3 and FIG. 4.
[표 3]TABLE 3
Figure PCTKR2016008059-appb-I000047
Figure PCTKR2016008059-appb-I000047
상기 표 3 및 도 4에 나타낸 바와 같이, 용매의 극성에 따라 이들 물질의 방출 스펙트럼이 달라짐을 알 수 있었다. 하기 화학식 2, 3, 5 및 6의 화합물 (IQ 6, 9, 28 및 30)은 수용액에서 최대 방출 (peak emission)의 장파장 쪽 옮김 (bathochromic shift)을 유도하여 용매화 발색효과 (solvatochromic effect)를 나타내었다.As shown in Table 3 and Figure 4, it can be seen that the emission spectrum of these materials vary depending on the polarity of the solvent. Compounds of Formulas 2, 3, 5, and 6 ( IQ 6, 9, 28, and 30) induce a solvatochromic effect by inducing a bathochromic shift of peak emission in an aqueous solution. Indicated.
특히, 화학식 2 및 3의 화합물 (IQ 6 및 9)의 경우, 최대 방출은 에탄올 용매 (EtOH)에서 각각 λmax= 511 nm, 516 nm이고, 수용매 (H2O)에서 각각 λmax= 465 nm, 465.5 nm로서, 에탄올에 비하여 수용매에서 현저한 적색 편이 (red-shift)를 보였으며, 화학식 2 및 3의 화합물의 양자 수율은 스탠다드로서 Coumarin 153을 이용하여 에탄올에서 측정하였을 때 각각 0.816 및 0.402로, 상당히 증가하였다. 즉, 이들 화합물은 여기 시에 높은 쌍극자 모먼트 변화를 갖는 것으로 예상된다. In particular, for compounds of Formulas 2 and 3 (IQ 6 and 9), the maximum emission is λ max = 511 nm, 516 nm in ethanol solvent (EtOH), respectively, and λ max = 465 in aqueous solvent (H 2 O), respectively. nm, 465.5 nm, which showed a significant red-shift in the solvent compared to ethanol, and the quantum yields of the compounds of Formulas 2 and 3 were 0.816 and 0.402, respectively, measured in ethanol using Coumarin 153 as a standard. , Significantly increased. That is, these compounds are expected to have high dipole moment changes upon excitation.
이에 반하여, 하기 화학식 4의 화합물 (IQ 21)의 경우, 수용매 (H2O)에서 λmax= 433 nm로서 청색 편이 (blue-shift)의 최대 방출을 보임으로써, 화합물 화학식 2 및 3의 화합물과는 반대의 경향을 보였다.In contrast, for the compound of formula 4 (IQ 21), compounds of formulas 2 and 3 exhibited the maximum emission of blue-shift as λ max = 433 nm in the aqueous solvent (H 2 O). The trend was opposite.
Figure PCTKR2016008059-appb-I000048
Figure PCTKR2016008059-appb-I000048
이에, 수용매 및 에탄올 용매의 양자수율을 비교하기 위하여 본 발명의 화학식 1의 화합물들 (Entry# 1-50)에 대하여, 수용매 또는 에탄올 용매 중에서의 스펙트럼 분석을 실시하고 그 결과를 표 4 및 도 5a/b에 나타내었다.Thus, in order to compare the quantum yields of the solvent and the ethanol solvent, the compounds of the formula 1 (Entry # 1-50) of the present invention were subjected to spectral analysis in the solvent or ethanol solvent and the results are shown in Table 4 and It is shown in Figure 5a / b.
[표 4]TABLE 4
Figure PCTKR2016008059-appb-I000049
Figure PCTKR2016008059-appb-I000049
상기 표 4 및 도 5a/b에 나타낸 바와 같이, 수용액보다 에탄올에서 형광수율이 크게 증가함을 알 수 있었으며(표 4), 총 41 종의 화합물 중 형광수율이 0.8 이상인 화합물이 15 종이었다(도 5a). 또한, 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물을 하기 화학식 X과 같이 나타낼 때, E 고리를 같은 구조로 고정하고 A 고리나 B 고리에 치환된 그룹에 따른 에탄올에서의 양자 수율 경향성을 살펴보면 수소 (proton) 또는 메틸 (methyl) 그룹이 치환되어 있을 경우 양자 수율은 비슷한 경향을 나타냈지만 브로모 (bromo) 그룹이나 에스터 (ester) 그룹이 치환되어 있을 경우는 양자 수율이 감소함을 알 수 있었다(도 5b). As shown in Table 4 and FIG. 5A / B, it was found that the fluorescence yield was significantly increased in ethanol than in aqueous solution (Table 4). Among the 41 compounds, there were 15 compounds having a fluorescence yield of 0.8 or more (FIG. 4). 5a). In addition, when the indolinino [3,2-c] quinoline compound of the present invention is represented by the following Chemical Formula X, the quantum yield in ethanol according to the group in which the E ring is fixed in the same structure and substituted in the A ring or the B ring The tendency showed that the quantum yields were similar when hydrogen or methyl groups were substituted, but the quantum yield was decreased when bromo or ester groups were substituted. It could be seen (Fig. 5b).
[화학식 X][Formula X]
Figure PCTKR2016008059-appb-I000050
Figure PCTKR2016008059-appb-I000050
나아가, 본 발명의 화학식 1의 화합물들 (Entry# 51-57 외)에 대하여, 다양한 용매 중에서의 스펙트럼 분석을 실시하고 그 결과를 표 5 및 도 5c에 나타내었다. Furthermore, for the compounds of formula 1 (Entry # 51-57 et al.) Of the present invention, spectral analysis in various solvents was performed and the results are shown in Table 5 and FIG. 5C.
[표 5]TABLE 5
Figure PCTKR2016008059-appb-I000051
Figure PCTKR2016008059-appb-I000051
상기 표 5 및 도 5c에 나타낸 바와 같이, 화합물의 치환기에 따라 용매의존적 (즉 환경-민감성) 경향을 보이는 정도가 다르며, 특히 아세틸렌을 가지는 하기 화학식 16의 화합물 (IQ 55) 및 4차 암모늄염의 형태인 하기 화학식 20 및 21의 화합물 (IQ 6-S1-Me 및 6-S2-Cl)의 경우는 용매의 극성에 따라 최대 형광 파장 값이 변하는 환경-민감적 광학적 성질이 거의 나타나지 않음을 확인하였다. 또한, 하기 화학식 19의 화합물 (IQ 5-D)은 최대형광을 나타내는 파장이 수용액에서 543 nm 까지 장파장으로 이동하였음을 확인하였다.As shown in Table 5 and Figure 5c, 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 In the case of the compounds of Formulas 20 and 21 (IQ 6-S1-Me and 6-S2-Cl), it was confirmed that the environment-sensitive optical properties of which the maximum fluorescence wavelength value changed according to the polarity of the solvent were hardly shown. In addition, it was confirmed that the compound of Formula 19 (IQ 5-D) shifted the wavelength indicating the maximum fluorescence to a long wavelength up to 543 nm in an aqueous solution.
Figure PCTKR2016008059-appb-I000052
Figure PCTKR2016008059-appb-I000052
따라서, 인돌리지노[3,2-c]퀴놀린 화합물의 이러한 용매-의존적인 광학특성은, 치환기를 다양하게 함으로써 조절될 수 있을 것이다. 또한, 인돌리지노[3,2-c]퀴놀린 화합물의 환경 민감적 특성은 약물-표적 결합 또는 단백질 다이나믹스와 같은 생물학적 공정을 모니터링하는데 유용한 형광 프로브로 이용될 수 있음을 시사하고 있다.Thus, such solvent-dependent optical properties of the indolinino [3,2-c] quinoline compound may be adjusted by varying the substituents. In addition, the 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.
4-3. 수용액중에서의 광안정성4-3. Light stability in aqueous solution
형광 프로브에 응용할 수 있는 후보물질로서, 인돌리지노[3,2-c]퀴놀린 화합물 중 수용액에서 형광의 세기가 높은 물질을 위주로 형광이 얼마나 지속되는지 확인하기 위하여, 하기와 같이 수용액 또는 에탄올 용매에서의 시간에 따른 광안정성을 평가하였다. As candidates for application to fluorescent probes, in order to determine how long fluorescence lasts mainly in materials having high fluorescence intensity in an aqueous solution of an indolinino [3,2-c] quinoline compound, an aqueous solution or an ethanol solvent is as follows. The light stability over time was evaluated.
우선 큐벳을 이용하여 해당 물질의 형광을 측정한 후, 큐벳 샘플을 자연광 상태에서 최종 6~7시간까지 시간별로 형광을 재측정하여 형광의 안정성을 확인하였다.First, after measuring the fluorescence of the material by using a cuvette, 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.
그 결과, 수용액의 경우, 도 6a에 나타낸 바와 같이, IQ 3, 4, 6 및 8 화합물이 수용액에서 6시간까지 형광이 거의 변하지 않고 안정하게 유지됨을 확인하였으며, 에탄올 용매의 경우, 도 6b에 나타낸 바와 같이, 인돌리지노[3,2-c]퀴놀린 화합물의 대부분이 형광의 세기가 높아, 보다 많은 물질을 스크리닝 할 수 있었으며, 수용액에서는 형광이 유지되지 않았던 전자 끌개기 (Electron withdrawing group, EWG)가 존재하는 물질들도 형광이 안정하게 유지 되었고, 형광의 세기도 6시간 동안 거의 변하지 않고 유지됨을 확인하였다.As a result, in the case of the aqueous solution, as shown in Figure 6a, IQ 3, 4, 6 and 8 compound was confirmed that the fluorescence remained stable little change until 6 hours in the aqueous solution, in the case of ethanol solvent, shown in Figure 6b As can be seen, most of the indolinino [3,2-c] quinoline compounds have high fluorescence intensities, allowing more material to be screened, and electron withdrawing groups (EWG), which did not retain fluorescence in aqueous solutions. Fluorescence remained stable even in the presence of materials, and the intensity of fluorescence was confirmed to remain almost unchanged for 6 hours.
다음으로, 상기 실시예로부터, 수용액에서 6 시간 가량 형광이 안정하게 유지된 IQ 3, 4, 6 및 8 화합물을 포함하여, 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물을 하기 화학식 X과 같이 나타낼 때, A, B, C, D 고리에 치환기가 없는 물질 몇 가지를 선정하여 수용액상에서 청색광 (Blue light)을 조사 (irradiation)한 후 형광이 유지되는지 안정성을 실험을 수행하였다.Next, from the above examples, 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 When 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.
[화학식 X][Formula X]
Figure PCTKR2016008059-appb-I000053
Figure PCTKR2016008059-appb-I000053
그 결과, 도 6c에 나타낸 바와 같이 IQ 3, 4, 5 및 6번 물질이 청색광을 2 시간 조사해 준 조건에서도 형광의 세기가 여전히 안정하게 유지되어 형광의 안정성이 우수함을 알 수 있었다. As a result, as shown in FIG. 6C, the fluorescence intensity remained stable even under conditions in which materials IQ 3, 4, 5, and 6 were irradiated with blue light for 2 hours, indicating that the fluorescence stability was excellent.
실시예 5: DNA와의 결합특성 분석Example 5: Analysis of binding properties with DNA
형광분광 광도계를 이용하여, 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물이 DNA와 결합하는 성질을 확인하였다. 우선, 실험에 이용한 DNA 올리고머 (48bp)는 tetO 서열을 포함하는 것으로, 염기서열은 하기와 같다.Using a fluorescence spectrophotometer, the properties of the indolinino [3,2-c] quinoline compound of the present invention to bind with DNA were confirmed. First, the DNA oligomer (48bp) used for the experiment contains the tetO sequence, and the nucleotide sequence is as follows.
tetO1: 5'-CCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTC-3'tetO1: 5'-CCTAATTTTTGTTGACACTCTATCATTGATAGAGTTATTTTACCACTC-3 '
tetO2: 5'-GAGTGGTAAAATAACTCTATCAATGATAGAGTGTCAACAAAAATTAGG-3'tetO2: 5'-GAGTGGTAAAATAACTCTATCAATGATAGAGTGTCAACAAAAATTAGG-3 '
상기 각 올리고머를 같은 몰량으로 95 ℃에서 5분 동안 열을 주어 혼합하고, 2 시간동안 상온에서 냉각하여 혼성화시킨 후, 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물의 형광을 측정하고, 큐벳에 해당 당량의 DNA 올리고머를 첨가한 후에 1 분동안 빛을 차단하여 인큐베이션하여 다시 형광을 재측정하였다.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.
그 결과, 하기 표 6 및 도 7에 나타낸 바와 같이, DNA 올리고머를 첨가하면 형광의 세기가 변하면서 최대 방출 파장이 단파장으로 이동하는 화합물 그룹(7a), 형광의 세기가 변하면서 최대 방출 파장이 장파장으로 이동하는 화합물 그룹(7b), 형광의 세기만 변하는 화합물 그룹 및 아무 변화가 일어나지 않는 화합물 그룹(7c)이 있음을 확인 하였다.As a result, as shown in Table 6 and FIG. 7, when the DNA oligomer is added, the compound group 7a in which the maximum emission wavelength shifts to a short wavelength while the intensity of fluorescence changes, and the maximum emission wavelength is long wavelength while the intensity of fluorescence changes It was confirmed that there is a compound group (7b) to move to, a compound group that changes only the intensity of fluorescence and a compound group (7c) does not change anything.
[표 6]TABLE 6
Figure PCTKR2016008059-appb-I000054
Figure PCTKR2016008059-appb-I000054
특히, 하기 화학식 7의 화합물은 DNA와 결합하여 형광이 점점 감소하고 장파장쪽으로 파장이 이동하였으며, 퓨란 (Furan)을 포함하는 하기 화학식 8의 화합물 (IQ 18)은 DNA와 결합하여 형광이 점점 감소하고 단파장쪽으로 파장이 이동하는 것을 확인하였다(7d). In particular, 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).
Figure PCTKR2016008059-appb-I000055
Figure PCTKR2016008059-appb-I000055
실시예 6: 단백질 결합특성 분석Example 6: Protein Binding Characteristic Analysis
형광분광 광도계를 이용하여, 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물이 단백질과 결합하는 성질을 확인하였다. 구체적으로, 인돌리지노[3,2-c]퀴놀린 화합물 중에서 Tris buffer (pH 7.5)에서 형광이 높은 화합물 13 종을 BSA (Bovine Serum Albumin)와 HSA(Human Serum Albumin)에 처리하여 형광변화를 관찰하였다.Using the fluorescence spectrophotometer, 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.
그 결과, 도 8에 나타낸 바와 같이, 하기 화학식 9의 화합물은, 화합물 자체가 280 nm에서 여기 시 500 nm 근처에서 방출을 보이고, HSA 자체는 280 nm에서 340 nm의 방출을 보이는데, HSA에 1당량의 화합물을 첨가하면 HSA 자체의 340 nm 방출의 세기가 감소하고 450 nm 근처에서의 방출도 관찰되어 HSA와 물질이 결합하여 형광공명에너지전이 (fluorescence resonance energy transfer, FRET) 현상이 일어남을 알 수 있었으며(도 8 위), 화합물이 갖는 399 nm, 421 nm의 파장에서 여기 (excitation)를 주었을 때, HSA가 1당량 첨가되면 물질이 갖는 형광보다 HSA와의 결합으로 인해 형광의 세기가 더 증가하고 파장이 단파장쪽으로 이동되며 피크의 모양도 바뀜을 알 수 있었다(도 8 아래). As a result, as shown in Figure 8, 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. (Fig. 8) When the excitation is given at wavelengths of 399 nm and 421 nm of the compound, when 1 equivalent of HSA is added, the intensity of fluorescence increases and the wavelength is increased due to the binding with HSA than that of the material. Moving toward the shorter wavelength, the shape of the peak was also changed (below Fig. 8).
Figure PCTKR2016008059-appb-I000056
Figure PCTKR2016008059-appb-I000056
또한, 도 9에 나타낸 바와 같이, 상기 화학식 9의 화합물 (IQ 7)은, 화합물 단독 (첫번째 바이얼) 또는 HSA 단독 (두번째 바이얼)에 비하여 화합물+HSA (세번째 바이얼)에서, 즉 화합물 단독보다 HSA와의 결합 시, 형광의 세기가 더 증가하였음을 시각적으로도 확인할 수 있었다. 나아가, 이의 양자수율 (QY)을 구해본 결과, 화합물 자체 보다 HSA와 결합하였을 때 QY가 0.43까지 증가함을 확인하였으며(도 9 위), Job's Plot을 통하여 상기 화학식 9의 화합물과 HSA가 1:1로 결합을 한다는 것을 확인하고, 이를 토대로 적정 (titration)을 진행한 결과, KD Value는 1.89 μM임을 알 수 있었다(도 9 아래).In addition, as shown in FIG. 9, 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). When combined with HSA, it was also visually confirmed that the intensity of fluorescence increased. Furthermore, as a result of obtaining its quantum yield (QY), it was confirmed that when combined with HSA rather than the compound itself, the QY increased to 0.43 (see FIG. 9), and 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 μM (see Fig. 9).
이에, 상기 결과를 바탕으로, 인돌리지노[3,2-c]퀴놀린 화합물과 HSA와의 결합을 통한 형광 세기 증가를 플레이트 웰 (plate well)을 이용하여 시각화 (visualization)하여 도 10에 나타냈으며, 고속 대량 처리 (High throughput) 방식으로 HSA에 결합하는 화합물을 스크린하는 방법을 구축하였다. Accordingly, based on the results, the fluorescence intensity increase through the binding of the indolinino [3,2-c] quinoline compound and HSA was visualized using a plate well, and is shown in FIG. 10. A method of screening a compound that binds to HSA in a high throughput manner was constructed.
나아가, 9 종의 인돌리지노[3,2-c]퀴놀린 화합물 (IQ 3, 4, 7, 8, 9, 10, 16, 17 및 40)이 HSA와 복합체 (complex)를 형성하여 형광의 세기가 증가함을 확인하고, 쿠마린 (Coumarin) 153 (Φ = 0.12 in water)을 기준 물질로 사용하여 형광 단백질의 양자수율 (QY)을 구해본 결과, 도 11에 나타낸 바와 같이, 하기 화학식 10의 화합물 (IQ 17)은 자체의 QY보다 HSA와 결합하였을 때 QY가 0.4까지 증가함을 확인하였다.Furthermore, nine indolinino [3,2-c] quinoline compounds ( IQ 3, 4, 7, 8, 9, 10, 16, 17 and 40) form complexes with HSA to intensify fluorescence As shown in FIG. 11, as shown in FIG. 11, quantum yield (QY) of fluorescent protein was determined using Comarin 153 (Φ = 0.12 in water) as a reference material. (IQ 17) confirmed that the QY increased to 0.4 when combined with HSA rather than its own QY.
Figure PCTKR2016008059-appb-I000057
Figure PCTKR2016008059-appb-I000057
6-1. 6-1. 형광공명에너지전이Fluorescence Resonance Energy Transition (fluorescence resonance energy transfer, FRET)를 이용한 타겟 단백질 (PDE-δ6)의 확인 target protein (PDE-δ6) using fluorescence resonance energy transfer (FRET)
K-Ras 단백질은 인간의 암세포 내에서 20-30 % 돌연변이 된 형태가 발견되며 암을 유발하는 것으로 알려져 지난 수 십 년 동안 항암 목표 단백질로 많은 연구가 진행되고 있으나, K-Ras 단백질과 GTP사이의 친화도 (affinity)가 picomolar 수준으로 강한 결합을 하며 단백질 내 알려진 알로스테릭 자리 (allosteric site)가 존재하지 않기 때문에 K-Ras 단백질을 직접 저해하는 치료제는 아직 개발되지 못하였다. 최근, K-Ras 단백질의 세포 내 위치 및 암 유발 기전에 대한 연구에서 K-Ras 단백질의 세포 증식에 관련된 신호 전달을 막을 수 있는 타겟 단백질인 PDEδ에 관한 연구가 진행되었으나, 현재까지 델타라신 (deltarasin) 외에 PDEδ의 저해제는 보고된 바가 없다. 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 PDEδ, a target protein that can prevent signal transduction related to cell proliferation of K-Ras protein. In addition, no inhibitors of PDEδ have been reported.
상기 PDEδ 단백질은 280 nm 빛을 조사하였을 때 트립토판 잔기에 의해 340 nm에서 최대 방출 파장을 보이며, 리간드가 결합되었을 때 340 nm의 최대 방출 파장의 세기가 감소하거나 형광공명에너지전이 (fluorescence resonance energy transfer, FRET) 현상에 의해 최대 방출 파장의 변화를 보인다. The PDEδ 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.
이에, 본 형광물질이 세포 내 특정 단백질에 선택적으로 결합할 수 있는지 확인하기 위하여, E.Coli cell에서 과발현시켜 분리 정제한 재조합 PDEδ 및/또는 인돌리지노[3,2-c]퀴놀린 화합물의 형광을 각각 280 nm 빛을 조사하여 측정하였다.Therefore, in order to confirm whether the present fluorescent substance can selectively bind to a specific protein in the cell, the fluorescence of the recombinant PDEδ 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.
그 결과, 도 12에 나타낸 바와 같이, 버퍼 내에서 PDEδ 단백질 (빨간색 점선)은 340 nm에서 최대 방출 파장을 보이며, 인돌리지노[3,2-c]퀴놀린 화합물 (파란색 점선)은 약 500 nm에서 최대 방출 파장을 보였다. 또한, PDEδ와 인돌리지노[3,2-c]퀴놀린 화합물을 섞은 후 형광을 측정한 경우 (녹색 실선), PDEδ 단백질의 340 nm에서의 최대 방출 파장은 줄어들며, 인돌리지노[3,2-c]퀴놀린 화합물의 최대 방출 파장은 475 nm로 이동되고, 자체 형광 세기도 증가하는 것을 확인할 수 있었다. As a result, as shown in FIG. 12, in the buffer, PDEδ protein (red dotted line) shows the maximum emission wavelength at 340 nm, and indolinino [3,2-c] quinoline compound (blue dotted line) at about 500 nm The maximum emission wavelength was shown. In addition, when fluorescence was measured after mixing PDEδ with an indolinino [3,2-c] quinoline compound (solid green line), the maximum emission wavelength at 340 nm of the PDEδ protein was reduced and indolinino [3,2- The maximum emission wavelength of the c] quinoline compound was shifted to 475 nm, it was confirmed that the self-fluorescence intensity also increased.
상기의 FRET 현상의 관찰을 통하여 일차적으로 인돌리지노[3,2-c]퀴놀린 화합물이 PDEδ에 결합하는 것을 확인하였으며, IQ 2 화합물과 같이 형광의 세기가 낮거나 IQ 13 화합물과 같이 형광의 변화가 없는 화합물을 제외한 나머지 화합물들에 대하여 결합 확인 실험을 추가적으로 실시 하였다.Through observation of the FRET phenomenon, it was confirmed that the indolinino [3,2-c] quinoline compound binds to PDEδ, and the fluorescence intensity is low like the IQ 2 compound or the fluorescence change like the IQ 13 compound. The binding confirmation experiment was additionally performed on the remaining compounds except those without compounds.
6-2. 형광편광 (fluorescence polarization)을 이용한 적정6-2. Titration using fluorescence polarization
인돌리지노[3,2-c]퀴놀린 화합물이 세포 내 특정 단백질에 결합할 수 있는지 확인하기 위하여, 0.5 μM 인돌리지노[3,2-c]퀴놀린 화합물이 존재하는 상태에서 PDEδ 단백질을 0에서 32 μM까지 농도를 증가시키며 적정 (titration)하여 편광 정도를 측정하였다.To determine if an indolinino [3,2-c] quinoline compound can bind to a specific protein in a cell, the PDEδ protein was set at 0 with 0.5 μM indolinino [3,2-c] quinoline compound present. The degree of polarization was measured by titration with increasing concentration up to 32 μM.
그 결과, 하기 표 7 및 도 13에 나타낸 바와 같이, 편광 정도가 PDEδ 농도-의존적으로 증가하며 쌍곡선 (hyperbolar) 형태를 이루는 것을 관찰할 수 있었으며, 이를 통하여 인돌리지노[3,2-c]퀴놀린 화합물을 스크리닝하여 300-500 nM 수준의 높은 친화도 (affinity)를 갖는 화합물을 확인하고, 추가적인 실험을 실시하기 위하여 그 중 8 종의 화합물 (IQ 1, 3, 4, 6, 8, 9, 10 및 42)을 선정하였다.As a result, as shown in Table 7 and FIG. 13, it was observed that the degree of polarization increases PDEδ concentration-dependently and forms a hyperbolar shape, through which indolinino [3,2-c] quinoline The compounds were screened to identify compounds with high affinity levels of 300-500 nM, and eight of them ( IQ 1, 3, 4, 6, 8, 9, 10) for further experiments. And 42).
[표 7]TABLE 7
Figure PCTKR2016008059-appb-I000058
Figure PCTKR2016008059-appb-I000058
6-3. 경쟁적 결합 (competition binding)을 이용한 6-3. Using competitive binding PDEPDE -- δ6δ6 단백질 결합 확인 Protein binding confirmation
인돌리지노[3,2-c]퀴놀린 화합물과 PDEδ 단백질이 복합체 (complex)를 형성한 후 형광을 갖지 않는 델타라신을 과량 첨가하였을 때, 델타라신과 IQ 화합물이 같은 결합 자리 (binding site)를 공유하면 과량의 델타라신에 의해 IQ 화합물은 free form이 되어 탈편광 (depolarization) 될 것이므로, 상기 실시예 6-2의 형광편광 스크리닝을 통해 도출한 8 종의 화합물 (IQ 1, 3, 4, 6, 8, 9, 10 및 42)에 대해 PDEδ 저해제로 알려진 델타라신 (deltarasin)과 경쟁적 결합 (Competition binding) 실험을 실시하였다. When an indolinino [3,2-c] quinoline compound and a PDEδ protein form a complex and an excess amount of non-fluorescent delta-racin is added, the delta-racin and IQ compounds are bound to the same binding site. Since the IQ compound will be free form and depolarized by the excess deltarasin, the eight compounds ( IQ 1, 3, 4, 6) derived through the fluorescence screening of Example 6-2. , 8, 9, 10, and 42) were subjected to competitive binding experiments with deltarasin known as PDEδ inhibitors.
그 결과, 도 14에 나타낸 바와 같이, 시간에 따라 인돌리지노[3,2-c]퀴놀린 화합물과 PDEδ 단백질 복합체에 의한 편광 수치는 변하지 않는데 반해, 델타라신을 과량 첨가한 경우 편광 정도가 감소하는 것을 확인하였는 바, 델타라신과 IQ 화합물이 같은 결합 부위 (binding site)에 결합하고 있음을 알 수 있었다. 또한, 시간에 따라 편광 정도가 감소하는 그래프를 통해 과량의 경쟁 화합물이 존재할 때 측정하고자 하는 화합물의 반감기 (half-life)를 측정할 수 있었으며, 이를 통하여, 좋은 친화도 (Kd)를 갖는 화합물이 더 긴 반감기를 갖는 것을 확인할 수 있었다. As a result, as shown in FIG. 14, the polarization value of the indolinino [3,2-c] quinoline compound and the PDEδ protein complex does not change with time, whereas the polarization degree decreases when an excess amount of deltarasine is added. As a result, it was found that the deltarasin and the IQ compound bind to the same binding site. In addition, 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.
따라서, 본 발명에서 형광 스크리닝을 통해 도출한 인돌리지노[3,2-c]퀴놀린 화합물들은 Kd값이 300-500 nM 수준의 높은 친화도를 가지며 형광을 가지고 있다는 장점이 있으므로, PDEδ의 저해제에 관한 연구 시, 프로브 화합물로 사용할 수 있을 것으로 기대되며, 경제성 및 효율성 면에서 또한, 항체를 이용한 검출법에 대한 대체 방안으로서의 활용이 기대된다.Therefore, 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.
실시예 7: 형광 단백질로서의 성질을 실제 응용한 예Example 7 Example of Real Application of Properties as Fluorescent Protein
7-1. 타겟 특이성 (Target specificity) 확인7-1. Target specificity
IQ 화합물과 PDEδ 단백질의 결합을 확인하기 위하여, 단백질 각 분획 (Sol: Soluble, FT: Flow through, W1: Washing 1, WF: Washing final, M: Marker, E1-5: Elution)에 하기 화학식 13의 화합물 (IQ 3)(1: 0.5 mM, 2: 0.1 mM)을 첨가하여 실험하였으며, 도 15에 나타낸 바와 같이, 상기 단백질을 분리 동정하였다. In order to confirm the binding of the IQ compound and the PDEδ protein, 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.
Figure PCTKR2016008059-appb-I000059
Figure PCTKR2016008059-appb-I000059
7-2. PDEδ와 화합물 3의 상호작용 확인7-2. Confirmation of PDEδ and Compound 3 Interaction
상기 실시예 7-1의 결과를 바탕으로, native gel을 이용하여 단백질과의 결합을 확인하였다. 구체적으로, ImageQuant LAS 4000을 이용하여 12% native gel에 대한 상기 화학식 13의 화합물의 형광 분석 (Ex 312 nm, Em 585-625 nm) (A) 및 Coomassie blue 염색을 통한 native gel에 대한 화학식 13의 화합물의 형광 분석 (B)을 실시하였으며(1: Lysate에 DMSO 첨가, 2: Lysate에 화합물 3 5 μM 첨가, 3: Lysate에 화합물 3 50 μM 첨가, M: Marker, 4: 정제한 PDEδ에 DMSO 첨가, 5: 정제한 PDEδ에 화합물 3 5 μM 첨가, 6: 정제한 PDEδ에 화합물 3 50 μM 첨가), 그 결과는 도 16에 나타내었다. Based on the results of Example 7-1, binding to the protein was confirmed using a native gel. Specifically, the 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 μM added to Compound 3 in Lysate, 3: 50 μM added to Compound 3 in Lysate, M: Marker, 4: DMSO added to purified PDEδ) , 5: 5 μM compound 3 was added to purified PDEδ, 6: 50 μM compound 3 was added to purified PDEδ), and the results are shown in FIG. 16.
이러한 결과는, 기존의 브래드퍼드 (Bradford) 시약과 같이 단백질을 검출하기 위한 별도의 시약을 사용하지 않고도 형광단백질의 분리 정제가 가능함을 시사하고 있다.These results suggest that it is possible to separate and purify the fluorescent protein without using a separate reagent for detecting a protein, such as a conventional Bradford reagent.
실시예 8: 세포 투과성 분석Example 8: Cell Permeability Assay
본 발명의 인돌리지노[3,2-c]퀴놀린 화합물이 세포 투과성이 우수한지 확인하기 위하여 하기와 같이 실험하였다.The indolinino [3,2-c] quinoline compound of the present invention was tested as follows to determine whether the cell permeability is excellent.
8-1. 형광 이미징8-1. Fluorescence imaging
HeLa 세포를 96 웰 플레이트에 접종하고 24 시간 동안 인큐베이션하였다. 상기 세포에 인돌리지노[3,2-c]퀴놀린 화합물을 처리하고, 37℃, 5% CO2 조건에서 30분 동안 인큐베이션하였다. 배지를 제거한 후, 세포를 1x DPBS로 세척하였다. 세척한 세포를 4 % 포름알데히드로 상온에서 10 분동안 고정하고, 1x DPBS로 세척하였다. 세포 이미지는 Operetta HTS 이미징 시스템 (PerkinElmer)으로 스크린하였다.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).
8-2. live cell 공초점 이미징 (confocal imaging)8-2. live cell confocal imaging
HeLa 세포를 공초점 디쉬 (SPL life science) 면적의 70-80 % 접종하고 밤새 배양시킨 후, 기존 DMEM 배지를 제거하고 DMEM에 섞어서 준비한 인돌리지노[3,2-c]퀴놀린 화합물 (10uM)을 분주하여 30분-1시간 동안 37℃에서 인큐베이션하였다. 이후, 필요한 경우 DMEM에 LysoTracker (50nM)를 섞어서 기존 배지를 제거한 후 분주하여 30분 동안 37℃ 에서 인큐베이션시키고, 인큐베이션이 끝나면 세척 없이 공초점 레이저 현미경 (Leica TCS SP8 SMD, Leica, Mannheim, Germany)으로 확인하였다. 단, lysotracker와 동시염색 (costaining)한 경우 DPBS로 2회 세척한 뒤 확인하였다. 인돌리지노[3,2-c]퀴놀린 화합물은 blue channel (ex:405nm, em:409>nm) 및 green channel (ex:488 nm, em:493>nm)에서 확인하였으며, LysoTracker는 red channel (ex:561 nm, em:566>nm)에서 확인하였다.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. However, co-staining with lysotracker (costaining) was confirmed after washing twice with DPBS. Indolizino [3,2-c] quinoline compounds were identified in the blue channel (ex: 405nm, em: 409> nm) and green channel (ex: 488 nm, em: 493> nm), and LysoTracker showed red channel ( ex: 561 nm, em: 566> nm).
8-3. HeLa cell8-3. HeLa cell
그 결과, 도 17에 나타낸 바와 같이, IQ 화합물이 세포막을 쉽게 투과하여 세포를 염색할 수 있다는 것을 확인하였으며, 이는 10 μM에서 5분 정도로 DNA 마커로 시판되고 있는 DAPI와 비슷한 수준으로 볼 수 있다.As a result, as shown in Figure 17, it was confirmed that the IQ compound can easily penetrate the cell membrane and stain the cells, which can be seen at a level similar to DAPI commercially available as a DNA marker at 10 μM for 5 minutes.
또한, 도 18에 나타낸 바와 같이, IQ 화합물들을 형광을 감지하는 세포 내 소기관과 분자 종류에 따라, A) 핵 (nucleus) 부분에 집중되어 염색되는 화합물 (IQ 7, 8, 16 및 17), B) 세포질 (cytoplasm)과 핵소체 (nucleolus) 부분에 집중적으로 분포 되는 화합물 (IQ 6 및 36), C) 미토콘드리아에 분포되는 화합물 (IQ 18) 및 D) 리소좀 (lysosome) 부분에 집중적으로 분포되는 화합물 (IQ 56 및 57) 등으로 분류할 수 있었으며, 또한 B 그룹 화합물 (IQ 6 및 36)은 세포 내 RNA를 인식하여 형광을 내고, A 그룹 화합물 (IQ 16 및 17)은 DNA를 인식하는 것으로 확인되었다. 이때, 핵을 염색 시킬때 사용하는 DAPI가 blue 파장인데 비해, 상기 A 그룹 화합물은 green 파장에서 염색될 수 있음을 확인하였는 바, 이로써 상기 화합물들이 세포 내의 특정 물질이나 조건을 감지하여 장파장 쪽의 빛에서도 작용 (turn-on)하는 민감한 (sensitive) 프로브로 사용될 수 있음을 확인하였다. In addition, as shown in FIG. 18, A) compounds ( IQ 7, 8, 16, and 17), which are concentrated and stained in the nucleus part according to intracellular organelles and molecular types that detect fluorescence, B ) Compounds intensively distributed in the cytoplasm and nucleolus regions (IQ 6 and 36), C) Compounds distributed in the mitochondria (IQ 18) and D) Compounds intensively distributed in the lysosome region ( IQ 56 and 57), and the group B compounds (IQ 6 and 36) were recognized to fluoresce by intracellular RNA, and the group A compounds (IQ 16 and 17) were identified as DNA. . At this time, the DAPI used to stain the nucleus was blue wavelength, whereas the A group compound was found to be dyed at the green wavelength. Thus, 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.
특히, 도 19에 나타낸 바와 같이, 실시예 8-2의 live 세포 공초점 이미징 (confocal imaging)에서, 하기 화학식 17 및 18의 화합물 (IQ 56 및 57)은 green 파장과 blue 파장에서 각기 상용되는 마커인 Lysotracker와 co-localization됨을 확인하였다. 이로써 본 발명의 형광물질들은 형광물질을 제거하지 않고도 선택적인 세포 내 소기관에만 염색될 수 있다는 장점과 더불어 세포투과성이 우수하고 화합물의 구조에 따라 다른 타겟을 인식한다는 장점이 있음을 확인하였다. In particular, as shown in FIG. 19, in live cell confocal imaging of Example 8-2, the compounds of Formulas 17 and 18 (IQ 56 and 57) 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.
Figure PCTKR2016008059-appb-I000060
Figure PCTKR2016008059-appb-I000060
8-4. MCF7 cell 및 NIH-3T3 cell8-4. MCF7 cell and NIH-3T3 cell
상기와 같은 양상은, 실시예 8-2의 live 세포 공초점 이미징 (confocal imaging)을 통해, HeLa 세포뿐 아니라 유방암 관련 MCF7 세포 (도 20 위) 및 정상 세포인 NIH3T3 세포 (도 20 아래)에서도 같은 분포 양상을 보임을 확인하였다(도 20).This aspect is similar to the HeLa cells as well as breast cancer-related MCF7 cells (top 20) and normal cells NIH3T3 cells (bottom 20) through live cell confocal imaging of Example 8-2. It was confirmed that the distribution pattern (Fig. 20).
8-5. Sf21 cell8-5. Sf21 cell
또한, 인돌리지노[3,2-c]퀴놀린 화합물을 Sf21 세포에 처리한 결과, 도 21에 나타낸 바와 같이, 하기 화학식 10, 11 및 8의 화합물이 세포 내로 투과하여 치환체에 따라 세포 전체가 균일하게 염색되거나 세포질만 염색됨을 확인하였다.In addition, as a result of treating the Sf21 cells with the indolinino [3,2-c] quinoline compound, as shown in FIG. 21, the compounds of the following formulas 10, 11, and 8 penetrated into the cells, and the whole cells were uniform according to the substituents. It was confirmed that the staining or cytoplasmic staining only.
Figure PCTKR2016008059-appb-I000061
Figure PCTKR2016008059-appb-I000061
실시예 9: 기존 개발 시약들과의 세포 투과성 비교Example 9: Cell Permeability Comparison with Existing Development Reagents
기존의 핵을 염색시키는 DAPI와의 동시염색 (costaining)을 통하여 본 발명의 인돌리지노[3,2-c]퀴놀린 화합물이 다른 시판되고 있는 염색약과 비교하여 세포 투과성이 우수함을 확인하였고, 도 22에 나타낸 바와 같이, 특히 화학식 11의 화합물 (IQ 16)은 거의 DAPI와 유사하게 염색되는 것을 확인하였으며, DAPI가 청색인데 반해 화학식 11의 화합물은 녹색으로, 세포에 손상을 덜 줄 수 있는 등 많은 장점이 있을 것으로 기대된다. 또한, 화학식 2 및 하기 화학식 12의 화합물 (IQ 6 및 36)은 RNA를 염색시키는 RNA select와 흡사한 양상을 가짐을 확인할 수 있었으며, 이중 화학식 12의 화합물은 핵을 염색시키는 것으로 알려진 DAPI와 상보적으로 염색시킴을 확인하였다. Through co-staining with DAPI, which stains existing nuclei, it was confirmed that the indolinino [3,2-c] quinoline compound of the present invention was superior in cell permeability compared to other commercially available dyes. As shown, in particular, 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. In addition, it was confirmed that 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.
Figure PCTKR2016008059-appb-I000062
Figure PCTKR2016008059-appb-I000062
또한, 도 23a 및 23b에 나타낸 바와 같이, 화학식 8의 화합물 (IQ 18)은 미토트래커 (Mitotracker)와의 co-localization 실험을 통해 피어슨 상관계수 (Pearson’s coefficient)가 0.8 이상임을 확인하였으며(도 23a), 이를 통해 화학식 8의 화합물이 미토콘드리아를 선택적으로 염색시킴을 알 수 있었다. 또한, 화학식 8의 화합물은 빛을 받을수록 형광이 증가하는 양상을 보이며, 이는 furan (퓨란)기를 가지는 IQ 53을 통해서도 비슷한 경향을 확인 할 수 있었다(도 23b).In addition, as shown in FIGS. 23A and 23B, the compound of Formula 8 (IQ 18) 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. In addition, 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).
실시예 10: 기존 개발 시약들과의 형광 안정성 비교Example 10: Fluorescence Stability Comparison with Existing Development Reagents
본 발명의 인돌리지노[3,2-c]퀴놀린 화합물이 다른 시판되고 있는 염색약과 비교 실험을 통하여 Sytoselect 또는 Mitotracker에 비해 형광 안정성이 우수함을 확인하였고, 도 24에 나타낸 바와 같이, 특히 상기 화학식 12의 화합물 (IQ 36)은 현재 상용화된 Sytoselect (RNA marker)와 비슷한 양상으로 RNA를 염색시키되, 안정성은 더 우수한 것으로 관찰되었다. 또한, 화학식 8의 화합물 (IQ 18)의 경우 위에서 기술 한 바와 같이 빛을 받을수록 형광이 증가하는 양상을 정량적으로도 확인하였다.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.
실시예 11: 세포 독성 확인Example 11: Confirmation of Cytotoxicity
형광 프로브에 응용할 수 있는 후보물질로서, 상기 인돌리지노[3,2-c]퀴놀린 화합물에 대하여, 세포독성 효과를 확인하기 위하여, 하기와 같이 실험하였다.As candidates applicable to fluorescent probes, the following experiments were carried out on the indolinino [3,2-c] quinoline compounds to confirm cytotoxic effects.
96-well에 웰당 1x104 세포를 접종한 후 37 ℃ 인큐베이터에서 24 시간동안 배양하였다. 그 후 다양한 농도 (0.5, 1, 2, 5 및 10 μM)로 인돌리지노[3,2-c]퀴놀린 화합물을 처리하여 24 시간 배양하였다. 배양 후, 배지를 100 μl로 교체 후, MTT시약 (5 mg/ml in DPBS) 20 μl를 첨가한 뒤 3 시간동안 배양하였다. 마지막으로 DMSO를 이용하여 포마진 (formazin)을 녹인 후 570 nm에서 흡광도를 분석하여 약물의 세포증식 억제 효과를 살펴보았다. 대조군으로는 DMSO 0.1 %를 이용하였다. 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 μM) and incubated for 24 hours. After incubation, the medium was replaced with 100 μl, and then 20 μl 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.
그 결과, 도 25에 나타낸 바와 같이, 본 발명의 화합물들은 MCF7 세포l에 10 μM 이하 농도에서 24 시간동안 처리하였을 때도 세포독성이 나타나지 않았으며, A549 세포 및 Hela 세포에 24 시간동안 처리하였을 때도 마찬가지로 세포독성이 나타나지 않았다. 이를 통해 본 발명의 화합물들이 세포 이미징에 사용하기 적합한 형광물질로 사용될 수 있음을 확인하였다.As a result, as shown in FIG. 25, the compounds of the present invention did not show cytotoxicity when treated with MCF7 cells at a concentration of 10 μM 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.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해되어야 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.
본 발명의 형광 프로브에 이용되는 인돌리지노[3,2-c]퀴놀린 화합물은 헤테로 고리에 붙은 관능기의 성질과 위치에 따라 형광의 특성과 활용 범위가 크게 달라지는 수용성 형광 화합물로서, 기존의 유기형광체의 단점이 개선되어 다양한 분야에서 활용될 수 있다.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.
또한, 본 발명의 형광 프로브를 핵산, 단백질에 결합시켜 이들의 움직임, 약물-단백질 상호작용 등 다양한 핵산/단백질의 기능 연구 및 이미징 기술에 응용할 수 있다.In addition, 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.
또한, 본 발명의 형광 프로브는 여기 파장과 형광 파장의 차이가 커서 (Stoke shift) 자기 소광(self-quenching)을 최소화할 수 있다.In addition, the fluorescent probe of the present invention has a large difference between the excitation wavelength and the fluorescence wavelength, thereby minimizing self-quenching.
또한, 본 발명의 형광 프로브는 치환기에 따라 형광의 파장대가 달라지고 주변환경에 대한 민감도가 달라지므로, 유기용매에서 형광을 극대화시키고 수용액에서 형광이 잘 나타나게 조절할 수 있다(solvatochromic).In addition, 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).
또한, 본 발명의 형광 프로브는 물이나 버퍼에서도 형광이 잘 나타나며 용해도가 좋고 단백질을 응집시키는 문제를 최소화할 수 있다.In addition, the fluorescent probe of the present invention is well fluorescence in water or buffer, solubility and can minimize the problem of protein aggregation.
또한, 본 발명의 형광 프로브는 세포 내 투과성이 우수하기 때문에 세포 또는 조직내 이미징 기술, 세포내 효소 활성 분석에 유용하다.In addition, 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.

Claims (10)

  1. 하기 화학식 1로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염을 포함하는, 형광 프로브 조성물.A fluorescent probe composition comprising an indolinino [3,2-c] quinoline compound represented by Formula 1 or a pharmaceutically acceptable salt thereof.
    [화학식 1][Formula 1]
    Figure PCTKR2016008059-appb-I000063
    Figure PCTKR2016008059-appb-I000063
    상기 화학식 1에서,In Chemical Formula 1,
    R1, R2, R3, R4 및 R5는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, C1- 6알킬, C1- 6알콕시, COOR8, 아릴, 헤테로아릴,
    Figure PCTKR2016008059-appb-I000064
    ,
    Figure PCTKR2016008059-appb-I000065
    및 C1- 6알키닐로 이루어지는 군으로부터 선택되며,     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,
    Figure PCTKR2016008059-appb-I000064
    ,
    Figure PCTKR2016008059-appb-I000065
    And C 1- 6 is selected from the group consisting of alkynyl,
    R6는 C1-6알킬, 아릴 및 헤테로아릴로 이루어지는 군으로부터 선택되고,R 6 is selected from the group consisting of C 1-6 alkyl, aryl and heteroaryl,
    n은 0, 1, 2 또는 3이고, n is 0, 1, 2 or 3,
    R7은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택되고,R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl,
    R8은 수소, C1-6알킬 및 C1-6알키닐로 이루어지는 군으로부터 선택되며,R 8 is selected from the group consisting of hydrogen, C 1-6 alkyl and C 1-6 alkynyl,
    상기 아릴 및 헤테로아릴의 임의의 탄소 원자 1 내지 3개는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐, 니트로,
    Figure PCTKR2016008059-appb-I000066
    ,
    Figure PCTKR2016008059-appb-I000067
    ,
    Figure PCTKR2016008059-appb-I000068
    ,
    Figure PCTKR2016008059-appb-I000069
    ,
    Figure PCTKR2016008059-appb-I000070
    , CF3, COO-, C1- 6알킬 및 C1- 6알콕시로 이루어지는 군으로부터 선택되는 치환기와 연결된다.    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,
    Figure PCTKR2016008059-appb-I000066
    ,
    Figure PCTKR2016008059-appb-I000067
    ,
    Figure PCTKR2016008059-appb-I000068
    ,
    Figure PCTKR2016008059-appb-I000069
    ,
    Figure PCTKR2016008059-appb-I000070
    , CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 6 alkyl and C 1- 6 alkoxy.
  2. 제1항에 있어서,The method of claim 1,
    상기 R1, R2, R3, R4 및 R5는 서로 동일하거나 상이하고, 각각 독립적으로 수소, C1- 3알킬, C1- 3알콕시, COOR8,
    Figure PCTKR2016008059-appb-I000071
    ,
    Figure PCTKR2016008059-appb-I000072
    및 C1- 3알키닐로 이루어지는 군으로부터 선택되며,    Wherein R 1, R 2, R 3 , R 4 and R 5 are the same or different and are each independently hydrogen, C 1- 3 alkyl, C 1- 3 alkoxy each other, COOR 8,
    Figure PCTKR2016008059-appb-I000071
    ,
    Figure PCTKR2016008059-appb-I000072
    And it is selected from the group consisting of C 1- 3 alkynyl,
    R6는 아릴 또는 헤테로아릴이고,R 6 is aryl or heteroaryl,
    n은 0, 1 또는 2이고, n is 0, 1 or 2,
    R7은 수소, 하이드록시, 할로겐 및 C1- 3알킬로 이루어지는 군으로부터 선택되고,R 7 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 3 alkyl,
    상기 아릴 및 헤테로아릴의 임의의 탄소 원자 1 내지 3개는 서로 동일하거나 상이하고, 각각 독립적으로 수소, 할로겐,
    Figure PCTKR2016008059-appb-I000073
    ,
    Figure PCTKR2016008059-appb-I000074
    ,
    Figure PCTKR2016008059-appb-I000075
    ,
    Figure PCTKR2016008059-appb-I000076
    ,
    Figure PCTKR2016008059-appb-I000077
    , CF3, COO-, C1- 3알킬 및 C1- 3알콕시로 이루어지는 군으로부터 선택되는 치환기와 연결된다.    Any one to three carbon atoms of the aryl and heteroaryl are the same or different from each other, and each independently hydrogen, halogen,
    Figure PCTKR2016008059-appb-I000073
    ,
    Figure PCTKR2016008059-appb-I000074
    ,
    Figure PCTKR2016008059-appb-I000075
    ,
    Figure PCTKR2016008059-appb-I000076
    ,
    Figure PCTKR2016008059-appb-I000077
    , CF 3, COO-, is connected with a substituent selected from the group consisting of C 1- 3 alkyl and C 1- 3 alkoxy.
  3. 제1항 또는 2항에 있어서, The method according to claim 1 or 2,
    상기 아릴은 페닐, 나프틸, 안트릴 및 바이아릴로 이루어지는 군으로부터 선택되는 것을 특징으로 하는, 조성물.Wherein said aryl is selected from the group consisting of phenyl, naphthyl, anthryl and biaryl.
  4. 제1항 또는 2항에 있어서, The method according to claim 1 or 2,
    상기 헤테로아릴은 피리딜, 피리미딜, 티오페닐, 피롤릴, 퓨라닐 및 트리아졸릴로 이루어지는 군으로부터 선택되는 것을 특징으로 하는, 조성물.Wherein said heteroaryl is selected from the group consisting of pyridyl, pyrimidyl, thiophenyl, pyrrolyl, furanyl and triazolyl.
  5. 제1항에 있어서, The method of claim 1,
    상기 R1은 수소 또는 COOR8이고, R6는 아릴 또는 헤테로아릴인 것을 특징으로 하는, 조성물.Wherein R 1 is hydrogen or COOR 8 , and R 6 is aryl or heteroaryl.
  6. 제1항에 있어서, The method of claim 1,
    상기 화학식 1로 표시되는 화합물은 하기 화학식 2 내지 화학식 19로 이루어진 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는, 조성물. Compound represented by the formula (1) is characterized in that any one or more selected from the group consisting of the formula (2) to formula (19).
    Figure PCTKR2016008059-appb-I000078
    Figure PCTKR2016008059-appb-I000078
    Figure PCTKR2016008059-appb-I000079
    Figure PCTKR2016008059-appb-I000079
  7. 제1항에 있어서, The method of claim 1,
    상기 화학식 1로 표시되는 화합물의 약학적으로 허용 가능한 염은 하기 화학식 20 또는 화학식 21 중에서 선택되는 어느 하나 이상인 것을 특징으로 하는, 조성물. The pharmaceutically acceptable salt of the compound represented by Formula 1 is any one or more selected from the following formula 20 or formula 21, a composition.
    Figure PCTKR2016008059-appb-I000080
    Figure PCTKR2016008059-appb-I000080
  8. 제1항 내지 제7항 중 어느 한 항의 조성물을 포함하는, 핵산, 단백질 또는 세포 검출 시약.A nucleic acid, protein or cell detection reagent comprising the composition of any one of claims 1 to 7.
  9. 제1항 내지 제7항 중 어느 한 항의 조성물을 이용한, 핵산, 단백질 또는 세포 이미징 방법.A nucleic acid, protein or cell imaging method using the composition of any one of claims 1 to 7.
  10. 하기 화학식 22로 표시되는 인돌리지노[3,2-c]퀴놀린 화합물 또는 이의 약학적으로 허용 가능한 염.Indolizino [3,2-c] quinoline compound represented by the following formula (22) or a pharmaceutically acceptable salt thereof.
    [화학식 22][Formula 22]
    Figure PCTKR2016008059-appb-I000081
    Figure PCTKR2016008059-appb-I000081
    상기 화학식 22에서,In Chemical Formula 22,
    R1은 수소, COOEt,
    Figure PCTKR2016008059-appb-I000082
    ,
    Figure PCTKR2016008059-appb-I000083
    및 C1- 6알키닐로 이루어지는 군으로부터 선택되며,    R 1 is hydrogen, COOEt,
    Figure PCTKR2016008059-appb-I000082
    ,
    Figure PCTKR2016008059-appb-I000083
    And C 1- 6 is selected from the group consisting of alkynyl,
    R2는
    Figure PCTKR2016008059-appb-I000084
    ,
    Figure PCTKR2016008059-appb-I000085
    ,
    Figure PCTKR2016008059-appb-I000086
    및
    Figure PCTKR2016008059-appb-I000087
    로 이루어지는 군으로부터 선택되고,    R 2 is
    Figure PCTKR2016008059-appb-I000084
    ,
    Figure PCTKR2016008059-appb-I000085
    ,
    Figure PCTKR2016008059-appb-I000086
    And
    Figure PCTKR2016008059-appb-I000087
    Is selected from the group consisting of
    n은 0, 1 또는 2 이고, n is 0, 1 or 2,
    R3은 수소, 하이드록시, 할로겐 및 C1- 6알킬로 이루어지는 군으로부터 선택된다.R 3 is selected from hydrogen, hydroxy, halogen and the group consisting of C 1- 6 alkyl.
    (다만, R1이 수소 또는 COOEt이고, n이 0일 때, R2가
    Figure PCTKR2016008059-appb-I000088
    인 경우는 제외한다.)    (However, when R 1 is hydrogen or COOEt, n is 0, R 2
    Figure PCTKR2016008059-appb-I000088
    Is excluded.)
PCT/KR2016/008059 2015-07-23 2016-07-22 Indolizino [3,2-c] quinoline-based fluorescent probe WO2017014601A1 (en)

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