CN113735757B - Micro-environment sensitive fluorescent organic small molecular compound and synthetic method and application thereof - Google Patents
Micro-environment sensitive fluorescent organic small molecular compound and synthetic method and application thereof Download PDFInfo
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- CN113735757B CN113735757B CN202111165144.9A CN202111165144A CN113735757B CN 113735757 B CN113735757 B CN 113735757B CN 202111165144 A CN202111165144 A CN 202111165144A CN 113735757 B CN113735757 B CN 113735757B
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- 150000001875 compounds Chemical class 0.000 title claims abstract description 197
- 238000010189 synthetic method Methods 0.000 title description 2
- KAESVJOAVNADME-UHFFFAOYSA-N 1H-pyrrole Natural products C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 claims abstract description 40
- OXHNLMTVIGZXSG-UHFFFAOYSA-N 1-Methylpyrrole Chemical group CN1C=CC=C1 OXHNLMTVIGZXSG-UHFFFAOYSA-N 0.000 claims abstract description 26
- XKJCHHZQLQNZHY-UHFFFAOYSA-N phthalimide Chemical compound C1=CC=C2C(=O)NC(=O)C2=C1 XKJCHHZQLQNZHY-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims description 113
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 100
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 89
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 88
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 73
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 64
- 238000003756 stirring Methods 0.000 claims description 62
- 239000002904 solvent Substances 0.000 claims description 55
- 238000004440 column chromatography Methods 0.000 claims description 54
- 238000010992 reflux Methods 0.000 claims description 50
- 239000000243 solution Substances 0.000 claims description 50
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 50
- 238000010438 heat treatment Methods 0.000 claims description 46
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 44
- 239000011261 inert gas Substances 0.000 claims description 41
- 238000000034 method Methods 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 36
- 238000002360 preparation method Methods 0.000 claims description 31
- 239000012074 organic phase Substances 0.000 claims description 30
- 239000003208 petroleum Substances 0.000 claims description 23
- 238000002156 mixing Methods 0.000 claims description 22
- 239000004327 boric acid Substances 0.000 claims description 21
- 239000002244 precipitate Substances 0.000 claims description 20
- 239000000047 product Substances 0.000 claims description 18
- LTHNHFOGQMKPOV-UHFFFAOYSA-N 2-ethylhexan-1-amine Chemical compound CCCCC(CC)CN LTHNHFOGQMKPOV-UHFFFAOYSA-N 0.000 claims description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 16
- 230000005587 bubbling Effects 0.000 claims description 16
- FXTKWBZFNQHAAO-UHFFFAOYSA-N 3-iodophenol Chemical compound OC1=CC=CC(I)=C1 FXTKWBZFNQHAAO-UHFFFAOYSA-N 0.000 claims description 15
- YNHIGQDRGKUECZ-UHFFFAOYSA-N dichloropalladium;triphenylphosphanium Chemical compound Cl[Pd]Cl.C1=CC=CC=C1[PH+](C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1[PH+](C=1C=CC=CC=1)C1=CC=CC=C1 YNHIGQDRGKUECZ-UHFFFAOYSA-N 0.000 claims description 15
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 14
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 claims description 14
- HLUXBGATERNGEH-UHFFFAOYSA-N n'-(5-methoxy-3,4-dihydro-2h-chromen-3-yl)-n-(4-nitro-2,1,3-benzoxadiazol-7-yl)-n'-propylbutane-1,4-diamine Chemical compound C1OC2=CC=CC(OC)=C2CC1N(CCC)CCCCNC1=CC=C([N+]([O-])=O)C2=NON=C12 HLUXBGATERNGEH-UHFFFAOYSA-N 0.000 claims description 14
- 239000003480 eluent Substances 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 11
- -1 small molecule compound Chemical class 0.000 claims description 11
- 108010071390 Serum Albumin Proteins 0.000 claims description 10
- 102000007562 Serum Albumin Human genes 0.000 claims description 10
- 238000001816 cooling Methods 0.000 claims description 10
- IAQBURSQTCOSBP-UHFFFAOYSA-N 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1h-indole Chemical compound O1C(C)(C)C(C)(C)OB1C1=CC2=CC=CC=C2N1 IAQBURSQTCOSBP-UHFFFAOYSA-N 0.000 claims description 9
- DTUOTSLAFJCQHN-UHFFFAOYSA-N 4-bromo-1,8-naphthalic anhydride Chemical compound O=C1OC(=O)C2=CC=CC3=C2C1=CC=C3Br DTUOTSLAFJCQHN-UHFFFAOYSA-N 0.000 claims description 8
- 229960000583 acetic acid Drugs 0.000 claims description 8
- 238000001035 drying Methods 0.000 claims description 8
- 239000012065 filter cake Substances 0.000 claims description 8
- 239000012362 glacial acetic acid Substances 0.000 claims description 8
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- LTMRRSWNXVJMBA-UHFFFAOYSA-L 2,2-diethylpropanedioate Chemical compound CCC(CC)(C([O-])=O)C([O-])=O LTMRRSWNXVJMBA-UHFFFAOYSA-L 0.000 claims description 7
- HXTWKHXDFATMSP-UHFFFAOYSA-N 4-bromo-2-hydroxybenzaldehyde Chemical compound OC1=CC(Br)=CC=C1C=O HXTWKHXDFATMSP-UHFFFAOYSA-N 0.000 claims description 7
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims description 7
- 229940098779 methanesulfonic acid Drugs 0.000 claims description 7
- PIBWKRNGBLPSSY-UHFFFAOYSA-L palladium(II) chloride Chemical compound Cl[Pd]Cl PIBWKRNGBLPSSY-UHFFFAOYSA-L 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 5
- 239000005457 ice water Substances 0.000 claims description 4
- SRCZQMGIVIYBBJ-UHFFFAOYSA-N ethoxyethane;ethyl acetate Chemical compound CCOCC.CCOC(C)=O SRCZQMGIVIYBBJ-UHFFFAOYSA-N 0.000 claims description 3
- 239000011259 mixed solution Substances 0.000 claims description 3
- 230000001376 precipitating effect Effects 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 23
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical group C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 abstract description 10
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 abstract description 9
- XJHABGPPCLHLLV-UHFFFAOYSA-N benzo[de]isoquinoline-1,3-dione Chemical compound C1=CC(C(=O)NC2=O)=C3C2=CC=CC3=C1 XJHABGPPCLHLLV-UHFFFAOYSA-N 0.000 abstract description 7
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 abstract description 7
- 238000001308 synthesis method Methods 0.000 abstract description 7
- 238000002189 fluorescence spectrum Methods 0.000 abstract description 6
- BLRHMMGNCXNXJL-UHFFFAOYSA-N 1-methylindole Chemical group C1=CC=C2N(C)C=CC2=C1 BLRHMMGNCXNXJL-UHFFFAOYSA-N 0.000 abstract description 5
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Chemical group CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 abstract description 5
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Chemical group C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 abstract description 5
- 239000002243 precursor Substances 0.000 abstract description 5
- CREMABGTGYGIQB-UHFFFAOYSA-N carbon carbon Chemical compound C.C CREMABGTGYGIQB-UHFFFAOYSA-N 0.000 abstract description 4
- 239000011203 carbon fibre reinforced carbon Substances 0.000 abstract description 4
- 229960000956 coumarin Drugs 0.000 abstract description 4
- 235000001671 coumarin Nutrition 0.000 abstract description 4
- 125000006575 electron-withdrawing group Chemical group 0.000 abstract description 3
- 238000006467 substitution reaction Methods 0.000 abstract description 3
- 125000000168 pyrrolyl group Chemical group 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 60
- 238000002474 experimental method Methods 0.000 description 45
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 40
- 230000015572 biosynthetic process Effects 0.000 description 20
- 238000003786 synthesis reaction Methods 0.000 description 20
- 239000000463 material Substances 0.000 description 18
- 239000007850 fluorescent dye Substances 0.000 description 16
- 239000000203 mixture Substances 0.000 description 16
- 238000012512 characterization method Methods 0.000 description 15
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 14
- 239000001257 hydrogen Substances 0.000 description 14
- 229910052739 hydrogen Inorganic materials 0.000 description 14
- 238000001228 spectrum Methods 0.000 description 14
- JZUHIOJYCPIVLQ-UHFFFAOYSA-N 2-methylpentane-1,5-diamine Chemical compound NCC(C)CCCN JZUHIOJYCPIVLQ-UHFFFAOYSA-N 0.000 description 13
- 238000005259 measurement Methods 0.000 description 13
- 230000005284 excitation Effects 0.000 description 12
- 238000012360 testing method Methods 0.000 description 12
- 102100026009 NF-kappa-B inhibitor zeta Human genes 0.000 description 10
- 101710115530 NF-kappa-B inhibitor zeta Proteins 0.000 description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 239000011159 matrix material Substances 0.000 description 6
- QFMVYQPITOGLQP-UHFFFAOYSA-N oxolane dihydrochloride Chemical compound Cl.O1CCCC1.Cl QFMVYQPITOGLQP-UHFFFAOYSA-N 0.000 description 6
- 229910052763 palladium Inorganic materials 0.000 description 6
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Substances [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 108091006905 Human Serum Albumin Proteins 0.000 description 5
- 102000008100 Human Serum Albumin Human genes 0.000 description 5
- 241000283690 Bos taurus Species 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- 238000012546 transfer Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000012984 biological imaging Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- NZSJRHOQFDOJMI-UHFFFAOYSA-N CN1C=CC=C1.C(=O)N(C)C Chemical compound CN1C=CC=C1.C(=O)N(C)C NZSJRHOQFDOJMI-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 238000005481 NMR spectroscopy Methods 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical class [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002284 excitation--emission spectrum Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003233 pyrroles Chemical class 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- 229910015900 BF3 Inorganic materials 0.000 description 1
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- WTEOIRVLGSZEPR-UHFFFAOYSA-N boron trifluoride Chemical compound FB(F)F WTEOIRVLGSZEPR-UHFFFAOYSA-N 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 150000004775 coumarins Chemical class 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 238000012938 design process Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 125000005647 linker group Chemical group 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- IJGRMHOSHXDMSA-UHFFFAOYSA-N nitrogen Substances N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000006862 quantum yield reaction Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
- C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
- C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
- C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
- C07D209/44—Iso-indoles; Hydrogenated iso-indoles
- C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/10—Spiro-condensed systems
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- 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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
Abstract
The invention provides a microenvironment sensitive fluorescent organic small molecular compound and a synthesis method and application thereof, wherein naphthalimide, phthalimide, coumarin and rhodamine are selected as fluorophore precursors with electron withdrawing groups, and electron donating groups such as pyrrole, N-methylpyrrole, indole and N-methylindole are selected, and because substitution reaction is easy to occur at the 2/5 position of the fluorophore precursors, a D-A structure connected with carbon-carbon single bonds can be formed between the fluorophore precursors, the molecule is easy to rotate, so that the novel molecule formed by the naphthalimide, the phthalimide, the coumarin and the rhodamine has viscosity sensitivity, and meanwhile, as ICT molecules, charge separation with different degrees is generated in the molecule under the microenvironment of different polarities, the maximum peak value of the fluorescence emission spectrum of the compound has blue shift or red shift, and the fluorescence intensity is changed, so that the novel molecule has polarity sensitivity.
Description
Technical Field
The invention belongs to the field of microenvironment sensitive fluorescent probes, and particularly relates to a series of novel environment sensitive fluorescent organic small molecular compounds, and a synthesis method and application thereof.
Background
Fluorescent probes are fluorescent active substances that have characteristic fluorescence emission in the ultraviolet-visible-near infrared region and whose fluorescence properties (including fluorescence wavelength, intensity, lifetime, polarization, etc.) change sensitively with the properties of the surrounding environment, such as polarity, viscosity, interacting molecules, etc. Fluorescent probes have been widely used in industrial, agricultural and biochemical research fields due to their sensitive response to specific environmental changes, including physical and chemical changes, and to the specific identification and labeling of specific analytes.
The fluorescent probes commonly used at present are polymer fluorescent probes, organic small molecule fluorescent probes, quantum dots and the like. Among them, the organic small molecular probe has the advantages of strong designability of molecular structure, simple synthesis, good repeatability and the like, and is paid attention to. Organic small molecule fluorescent probes are typically composed of a recognition group, a fluorophore, and a linking moiety. The fluorophore plays a leading role in the whole fluorescent system, and the common organic micromolecular fluorophores at present mainly comprise coumarin, 1, 8-naphthalimide, rhodamine and boron fluoride fluorescent dyes. By modifying the precursors of these fluorophores, probes with specific detection can be obtained.
In addition to chemically reactive fluorescent probes based on chemical interactions, some fluorescent probes are identified or labeled by sensing a physical change in the surrounding environment, by physically affecting their fluorescence, such as an environmentally sensitive polar fluorescent probe and an environmentally sensitive viscosity fluorescent probe. These probe molecules all have the structural characteristics of ICT (intramolecular charge transfer, ICT). Because only a small number of ICT fluorescent dye molecules have their fluorescent properties affected by the environmental viscosity, the number and types of reports for the environmental sensitive viscosity fluorescent probes are small relative to the environmental sensitive polar fluorescent probes.
The most of the reported environment-sensitive viscosity fluorescent probes are D (donor) -A (receptor) or D-pi-A molecular structures composed of nitrogen heterocycle/N, N-dialkylamine-fluorophor. The change in viscosity can affect the rotation of intramolecular bonds, thereby affecting the non-radiative transition process and fluorescence quantum yield of such molecules. However, the bond between D (knob) and A (receptor) of these molecules is a carbon-nitrogen bond, and the conjugated molecules have the property of having partial double bonds, thus greatly reducing the viscosity sensitivity.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a micro-environment sensitive fluorescent organic small molecular compound, a synthesis method and application thereof, wherein the fluorescent organic small molecule has polarity sensitivity and better viscosity sensitivity.
The invention is realized by the following technical scheme:
the micro-environment sensitive fluorescent organic small molecule compound is one of the following compounds:
the preparation method of the microenvironment sensitive fluorescent organic small molecular compound is one of the following methods:
the method comprises the following steps:
stirring and mixing 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol, and heating and refluxing; after the reaction is finished, cooling to room temperature, adding water to generate yellow precipitate, filtering and collecting the precipitate, washing a filter cake, and drying to obtain a compound NAP;
mixing a compound NAP, N-dimethylformamide and N-methylpyrrole or pyrrole, deoxidizing by a bubbling method, stirring under 400nm light in an inert gas atmosphere, removing a solvent under reduced pressure after the reaction is finished, and separating by column chromatography to obtain a product;
or mixing compound NAP, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
The second method is as follows:
mixing phthalimide, 2-ethylhexyl amine and glacial acetic acid under stirring, and heating for reflux; after the reaction is finished, cooling to room temperature, adding water to generate white precipitate, filtering and collecting the precipitate, washing a filter cake, and drying to obtain a compound PMD;
mixing a compound PMD, N-dimethylformamide and N-methylpyrrole or pyrrole, deoxidizing by a bubbling method, and stirring under 400nm light in an inert gas atmosphere; after the reaction is finished, the solvent is removed under reduced pressure, and the product is obtained through column chromatography separation;
or mixing PMD, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
and a third method:
mixing 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid, stirring, and heating for reflux; after the reaction is finished, cooling to room temperature, adjusting the pH value of the solution to 9, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound RHO;
Sequentially adding a compound RHO, N-methylpyrrole and N, N-dimethylformamide into a flask, deoxidizing by a bubbling method, and stirring under 400nm light in an inert gas atmosphere; after the reaction is finished, the solvent is removed under reduced pressure, and the product is obtained through column chromatography separation;
or mixing the compounds RHO, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
the method four:
dropwise adding 3-iodophenol into concentrated sulfuric acid at 0 ℃, stirring, then dropwise adding ethyl acetoacetate, continuously stirring until the reaction is finished, dropwise adding the obtained mixed solution into ice water, precipitating white precipitate, and filtering to obtain a compound COU1;
mixing a compound COU1, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water, stirring under an inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
And a fifth method:
mixing 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water, stirring under inert gas atmosphere, and heating and refluxing; after the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound COU2;
mixing ethanol, compound COU2, diethyl malonate and piperidine under stirring, heating and refluxing to react, removing solvent under reduced pressure, and separating by column chromatography to obtain the final product.
Preferably, the column chromatography is carried out using a petroleum ether-ethyl acetate system as eluent.
Preferably, in the first method, the molar ratio of 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol is 1 (5-10): (800-1000), and the heating reflux temperature is 78-83 ℃; the mole ratio of the compound NAP, N-methylpyrrole or pyrrole and N, N-dimethylformamide is 1 (5-15): 10-25); the illumination reaction temperature at 400nm is 20-25 ℃; the mol ratio of the compound NAP, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000, and the heating reflux temperature is 73-77 ℃;
Preferably, in the second method, the molar ratio of the phthalimide to the 2-ethylhexyl amine to the glacial acetic acid is 1 (5-10) (1000-1300), and the heating reflux temperature is 117-122 ℃; the mole ratio of the compound PMD to the N-methylpyrrole or pyrrole to the N, N-dimethylformamide is 1 (5-15) (10-25), and the 400nm light reaction temperature is 20-25 ℃; the mole ratio of the compound PMD, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3) (1-6) (1/20-1/10) (250-500) (500-1000), and the heating reflux temperature is 73-77 ℃.
Preferably, in the method III, the molar ratio of the 4-diethylamino keto acid, the 3-iodophenol and the methanesulfonic acid is 1 (1-3) (400-800); the heating reflux reaction temperature is 150-155 ℃; the mol ratio of the compound RHO, the N-methylpyrrole and the N, N-dimethylformamide is 1 (5-15): 10-25, and the reaction temperature is 20-25 ℃; the mol ratio of the compound RHO, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3) (1-6) (1/20-1/10) (250-500) (500-1000), and the heating reflux reaction temperature is 73-77 ℃.
Preferably, in the fourth method, the molar ratio of 3-iodophenol, concentrated sulfuric acid and ethyl acetoacetate is 1 (15-35): 1-5, and the reaction temperature is 20-25 ℃; the mol ratio of the compound COU1 to the 1-methyl pyrrole-2-boric acid pinacol ester to the potassium carbonate to the bis-triphenylphosphine palladium dichloride to the tetrahydrofuran to the water is 1 (1-3): (1-6): (1/20-1/10): (250-500): (500-1000), and the reaction temperature is 73-77 ℃.
Preferably, in the fifth method, the molar ratio of 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000, and the heating reflux reaction temperature is 73-77 ℃; the mol ratio of the compound COU2 to the ethanol to the diethyl malonate to the piperidine is 1 (100-200) (1-5) (0.2-0.7), and the heating reflux reaction temperature is 78-83 ℃.
The application of the microenvironment sensitive fluorescent organic small molecular compound in preparing a reagent for detecting serum albumin.
The microenvironment sensitive fluorescent organic small molecular compound is used as a coloring agent in cell washing-free biological imaging.
Compared with the prior art, the invention has the following beneficial technical effects:
The invention designs the molecule of the micro-environment sensitive fluorescent organic small molecule compound: the method selects the fluorophore matrix with the electron withdrawing groups of naphthalimide, phthalimide, coumarin and rhodamine, and selects the electron donating groups of pyrrole, N-methylpyrrole, indole and N-methylindole, and because the 2/5 position of the fluorophore matrix is easy to generate substitution reaction, a D-A structure connected with carbon-carbon single bonds can be formed between the fluorophore matrix, the fluorophore matrix has the characteristic of easy rotation, the fluorophore matrix has viscosity sensitivity, and the D-A structure connected with carbon-carbon single bonds avoids the problem that the viscosity sensitivity is reduced due to the fact that the D-A structure connected with carbon-nitrogen single bonds is conjugated and then has double bond properties. Meanwhile, the novel molecules formed by the two molecules are used as ICT molecules to generate charge separation with different degrees in the micro-environments with different polarities, the maximum peak value of the fluorescence emission spectrum of the compound is blue-shifted or red-shifted, the fluorescence intensity is changed, and the compound has polarity sensitivity. In a large polar solvent (such as water), the compound has almost no fluorescence due to intramolecular twisted charge transfer. However, if serum albumin is added to the aqueous solution, the compound can bind to the hydrophobic site of serum albumin, causing it to fluoresce, and the fluorescence intensity correlates with serum albumin concentration, thereby achieving sensitive detection of serum albumin. Meanwhile, the fluorescence of the compound in the cell culture solution is weaker, but when the compound enters the cell and reaches a nonpolar region in the cell or is combined with protein in the cell, the fluorescence of the compound can be obviously enhanced, so that the fluorescence inside and outside the cell is greatly different, and the compound can also be used for washing-free imaging of living cells. Therefore, pyrrole and derivatives thereof are introduced into the design of fluorescent molecules, a series of fluorescent small organic molecules with microenvironment sensitivity are synthesized, and a new design idea is provided for synthesizing fluorescent molecules with viscosity and polarity sensitivity at the same time.
The synthesis method of the invention has simple operation, and the synthesized compound has better viscosity, polarity sensitivity and AIE (Aggregation-Induced Emission) characteristic.
Drawings
FIGS. 1-13 are nuclear magnetic resonance hydrogen spectra of the compound MNAP, HNAP, INAP, MINAP, MPMD, HPMD, IPMD, MIPMD, MRHO, IRHO, MIRHO, MCOUA and MCOUB, respectively, prepared according to the present invention;
FIGS. 14 to 25 are respectively excitation emission spectra of a compound MNAP, HNAP, INAP, MINAP, MPMD, HPMD, IPMD, MIPMD, MRHO, IRHO, MCOUA, MCOUB produced by the present invention;
FIGS. 26 and 27 are graphs showing fluorescence spectra of IRHO and IPMD, respectively, of the compounds prepared in accordance with the present invention in solutions of different viscosities; FIGS. 28 to 29 are respectively fluorescence spectra of the compounds MCOUA and MCOUB prepared according to the present invention in solutions of different polarities; FIGS. 30 and 31 are respectively graphs showing aggregation-induced emission fluorescence spectra of MNAP and MPMD, which are compounds prepared by the present invention;
FIG. 32 is a graph showing fluorescence spectra of MRHO compounds prepared in accordance with the present invention in different concentrations of bovine/human serum albumin;
FIG. 33 is a photograph of a laser confocal microscope of unwashed/washed cells stained with the compound MNAP prepared in the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
The invention discloses a series of novel microenvironment sensitive fluorescent small organic molecules, which comprise naphthalimide derivatives MNAP, HNAP, INAP and MINAP, phthalimide derivatives MPMD, HPMD, IPMD and MIPMD, rhodamine derivatives MRHO, IRHO and MIRHO, and coumarin derivatives MCOUA and MCOUB.
Wherein, the structural formula of MNAP is:the structural formula of HNAP is: />The INAP has the structural formula: />The MINAP has the structural formula: />The structural formula of MPMD is: />The structural formula of HPMD is: />The structural formula of the IPMD is as follows: />The MIPMD has the structural formula: />The structural formula of MRHO is: />IRHO has the structural formula: />The structural formula of MIRHO is as follows:the structural formula of MCOUA is: / >The structural formula of MCOUB is: />
The invention also discloses a synthesis method of the naphthalimide derivative, the phthalimide derivative, the rhodamine derivative and the coumarin derivative, which comprises the following steps:
1) Preparation of Compound NAP
Adding 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol into a flask, stirring, and heating and refluxing. After the reaction is finished, cooling to room temperature, stopping stirring, adding water to generate yellow precipitate, filtering and collecting the precipitate, washing a filter cake with ethanol, and drying the obtained product to obtain a compound NAP;
wherein, the structural formula of the compound NAP is as follows:
2) Preparation of the Compound MNAP
Adding a compound NAP, N-methylpyrrole and N, N-dimethylformamide into a flask in sequence, stirring under 400nm light in an inert gas atmosphere after deoxidizing by a bubbling method, stopping stirring after the reaction is finished, removing a solvent under reduced pressure, and separating by column chromatography to obtain a compound MNAP;
3) Preparation of Compound HNAP
The NAP, pyrrole and N, N-dimethylformamide are added into a flask in sequence, and after deoxidization by a bubbling method, the mixture is stirred under the light of 400nm in an inert gas atmosphere. Stopping stirring after the reaction is finished, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound HNAP;
4) Preparation of Compound INAP
Sequentially adding the compound NAP, indole-2-boric acid pinacol ester, potassium carbonate, ditriphenylphospholoride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating for reflux. After the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound INAP;
5) Preparation of Compound MINAP
The compound NAP, 1-methylindole-2-boric acid, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water are sequentially added into a flask, and the mixture is stirred and heated for reflux under the inert gas atmosphere. After the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound MINAP;
6) Preparation of Compound PMD
Phthalimide, 2-ethylhexyl amine and glacial acetic acid are added into a flask, stirred and heated for reflux. After the reaction is finished, cooling to room temperature, stopping stirring, adding water to generate white precipitate, filtering and collecting the precipitate, washing a filter cake with water, and drying the obtained product to obtain a compound PMD;
wherein, the structural formula of the compound PMD is as follows:
7) Preparation of Compound MPMD
The PMD, N-methylpyrrole and N, N-dimethylformamide are sequentially added into a flask, and after deoxidization by a bubbling method, the mixture is stirred under the illumination of 400nm in an inert gas atmosphere. Stopping stirring after the reaction is finished, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound MPMD;
8) Preparation of Compound HPMD
The PMD, pyrrole and N, N-dimethylformamide are added into a flask in sequence, and after deoxidization by a bubbling method, the mixture is stirred under the illumination of 400nm in an inert gas atmosphere. Stopping stirring after the reaction is finished, removing the solvent under reduced pressure, and separating by column chromatography to obtain the compound HPMD;
9) Preparation of Compound IPMD
Sequentially adding the compound PMD, indole-2-boric acid pinacol ester, potassium carbonate, ditriphenylphospholoride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating for reflux. After the reaction is finished, extracting the solution by using methylene dichloride, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound IPMD;
10 Preparation of the compound MIPMD
Sequentially adding the compound PMD, 1-methylindole-2-boric acid, potassium carbonate, ditriphenylphosphole palladium dichloride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating for reflux. After the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a compound MIPMD;
11 Preparation of the compound RHO
Adding 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid into a flask, stirring, and heating under reflux. After the reaction was completed, the reaction mixture was cooled to room temperature, stirring was stopped, and the pH of the solution was adjusted to 9 with a saturated sodium carbonate solution. Extracting the solution with dichloromethane, collecting the organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound RHO;
wherein, the structural formula of the compound RHO is as follows:
12 Preparation of Compound MRHO
Sequentially adding the RHO, the N-methylpyrrole and the N, N-dimethylformamide into a flask, deoxidizing by a bubbling method, and stirring under 400nm light in an inert gas atmosphere. Stopping stirring after the reaction is finished, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound MRHO;
13 Preparation of Compound IRHO
Sequentially adding the compounds RHO, indole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating for reflux. After the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound IRHO;
14 Preparation of the compound MIRHO
Sequentially adding the compounds RHO, 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating for reflux. After the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound MIRHO;
15 Preparation of Compound COU1
Slowly dripping 3-iodophenol into 80% concentrated sulfuric acid at 0 ℃ and stirring for 30 minutes under the condition, then dripping ethyl acetoacetate into the solution, continuously stirring the solution to enable the solution to slowly return to room temperature, continuously stirring the solution at the room temperature until the reaction is finished, dripping the mixed solution into ice water, precipitating white precipitate, and filtering to obtain a compound COU1;
wherein, the structural formula of the compound COU1 is as follows:
16 Preparation of Compound MCOUA
Sequentially adding the compound COU1, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water into a flask, stirring under the inert gas atmosphere, and heating and refluxing. After the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound MCOUA;
17 Preparation of Compound COU2
Sequentially adding 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water into a flask, stirring under inert gas atmosphere, and heating and refluxing. After the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound COU2;
Wherein, the structural formula of the compound COU2 is as follows:
18 Preparation of Compound MCOUB
Sequentially adding ethanol, a compound COU2, diethyl malonate and piperidine into a flask, stirring, heating and refluxing until the reaction is finished, stopping stirring, removing a solvent under reduced pressure, and performing column chromatography separation to obtain a compound MCOUB;
the reactant dosage ratio of each step in the method is as follows:
in the step 1), the molar ratio of 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol is 1 (5-10) (800-1000); in the step 1), the specific reaction temperature is 78-83 ℃;
in the step 2), the mole ratio of NAP, N-methylpyrrole and N, N-dimethylformamide is 1 (5-15): 10-25); in the step 2), the specific reaction temperature is 20-25 ℃;
in the step 3), the mole ratio of NAP, pyrrole and N, N-dimethylformamide is 1 (5-15): 10-25; in the step 3), the specific reaction temperature is 20-25 ℃;
in the step 4), the mole ratio of NAP, indole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 4), the specific reaction temperature is 73-77 ℃;
in the step 5), the molar ratio of NAP, 1-methylindole-2-boric acid, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 5), the specific reaction temperature is 73-77 ℃;
In the step 6), the molar ratio of the phthalimide to the 2-ethylhexyl amine to the glacial acetic acid is 1 (5-10) (1000-1300); in the step 6), the specific reaction temperature is 117-122 ℃;
in the step 7), the mole ratio of PMD, N-methylpyrrole and N, N-dimethylformamide is 1 (5-15): 10-25); in the step 7), the specific reaction temperature is 20-25 ℃;
in the step 8), the mole ratio of PMD, pyrrole and N, N-dimethylformamide is 1 (5-15): 10-25; in the step 8), the specific reaction temperature is 20-25 ℃;
in the step 9), the mole ratio of PMD, indole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 9), the specific reaction temperature is 73-77 ℃;
in the step 10), the mole ratio of PMD, 1-methylindole-2-boric acid, potassium carbonate, ditolylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 10), the specific reaction temperature is 73-77 ℃;
in the step 11), the molar ratio of the 4-diethylamino keto acid, the 3-iodophenol and the methanesulfonic acid is 1 (1-3) (400-800); in the step 11), the specific reaction temperature is 150-155 ℃;
In the step 12), the molar ratio of RHO, N-methylpyrrole and N, N-dimethylformamide is 1 (5-15): 10-25); in the step 12), the specific reaction temperature is 20-25 ℃;
in the step 13), the mol ratio of RHO, indole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 13), the specific reaction temperature is 73-77 ℃;
in the step 14), the molar ratio of RHO, 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 14), the specific reaction temperature is 73-77 ℃;
in the step 15), the molar ratio of the 3-iodophenol to the concentrated sulfuric acid to the acetoacetic ester is 1 (15-35) to 1-5; in the step 15), the specific temperature of the room temperature is 20-25 ℃;
in the step 16), the molar ratio of COU1 to 1-methyl pyrrole-2-pinacol borate to potassium carbonate to bis-triphenylphosphine palladium dichloride to tetrahydrofuran to water is 1 to 1 (1-3), 1 to 6, 1/20 to 1/10, 250 to 500 and 500 to 1000; in the step 16), the specific reaction temperature is 73-77 ℃;
in the step 17), the molar ratio of the 4-bromo-2-hydroxybenzaldehyde, the 1-methylpyrrole-2-boric acid pinacol ester, the potassium carbonate, the bis-triphenylphosphine palladium dichloride, the tetrahydrofuran and the water is 1 (1-3): 1-6): 1/20-1/10): 250-500): 500-1000; in the step 17), the specific reaction temperature is 73-77 ℃;
In the step 18), the molar ratio of the compound COU2 to the ethanol to the diethyl malonate to the piperidine is 1 (100-200): 1-5): 0.2-0.7. In step 18), the specific reaction temperature is 78-83 ℃.
The method adopts a column chromatography separation method as a purification operation which is practically used, and the column chromatography separation adopts a petroleum ether-ethyl acetate system as eluent; in the steps 2), 3), 7), 8) and 17), the volume ratio of petroleum ether to ethyl acetate is 10:1; in the steps 4) and 5), the volume ratio of petroleum ether to ethyl acetate is 15:1; in the steps 9) and 10), the volume ratio of petroleum ether to ethyl acetate is 20:1; in the steps 11), 12) and 18), the volume ratio of petroleum ether to ethyl acetate is 5:1; in the step 13), the volume ratio of petroleum ether to ethyl acetate is 3:1; in steps 14) and 16), the volume ratio of petroleum ether to ethyl acetate was 4:1.
The invention is further illustrated by the following examples:
EXAMPLE 1 Synthesis of Compound NAP
1g of 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol are added into a flask, stirred, heated, condensed and refluxed for 2 hours. After the reaction is finished, cooling to room temperature, stopping stirring, adding water to generate yellow precipitate, filtering and collecting the precipitate, washing a filter cake with ethanol, and drying the obtained product to obtain the compound NAP. Three experiments were performed, and the proportions of the different materials and the temperature conditions are shown in Table 1 below.
Table 1 example 1 three sets of experimental conditions
| 4-bromo-1, 8-naphthalic anhydride | 2-ethylhexyl amine | Ethanol | Temperature (temperature) |
| 1g | 0.5mL | 30mL | 78℃ |
| 1g | 0.75mL | 40mL | 80℃ |
| 1g | 1mL | 50mL | 83℃ |
The reaction equation is as follows:
EXAMPLE 2 Synthesis of Compound MNAP
The compound NAP0.232g, N-methylpyrrole, N-dimethylformamide obtained in the first experiment of example 1 was sequentially added into a flask, and after deoxidization by bubbling, the mixture was stirred under 400nm light for 10 hours under an inert gas atmosphere. After the reaction was completed, stirring was stopped, and the solvent was removed under reduced pressure using petroleum ether: the ethyl acetate (10:1) system is used for eluting liquid column chromatography to obtain the compound MNAP. Three experiments were performed and the different material ratios are shown in Table 2 below. The MNAP characterization structure obtained in the first set of experiments was: HRMS calculated for [ C13H13NO2Na ] +: 411.2048; actual measurement value: 411.2045, nuclear magnetic resonance hydrogen spectrum is shown in figure 1.
Table 2 example 2 three sets of experimental conditions
| NAP | N-methylpyrrole | N, N-dimethylformamide |
| 0.232g | 3mL | 6mL |
| 0.232g | 6mL | 15mL |
| 0.232g | 9mL | 10mL |
The reaction equation is as follows:
EXAMPLE 3 Synthesis of Compound MINAP
The compound NAP 0.194g obtained in the first experiment of example 1, 1-methylindole-2-boric acid, potassium carbonate, ditolylphosphine palladium dichloride, tetrahydrofuran and water were sequentially added into a flask, stirred under an inert gas atmosphere, heated, condensed and refluxed for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (15:1) system was separated by column chromatography on an eluent to give compound MINAP. Three experiments were performed and the different material ratios are shown in Table 3 below. The MINAP characterization structure obtained in the first set of experiments was: ([ C) 29 H 30 N 2 O 2 Na] + HRMS calculated values of (2): 461.2199; actual measurement value: 461.2201; the nuclear magnetic hydrogen spectrum is shown in figure 4.
Table 3 example 3 three sets of experimental conditions
| NAP | 1-methylindole-2-boronic acid | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.194g | 0.105g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.194g | 0.3g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.194g | 0.2g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
EXAMPLE 4 Synthesis of Compound PMD
Phthalimide 1g, 2-ethylhexyl amine and glacial acetic acid are added into a flask, stirred, heated to 120 ℃ and condensed and refluxed for 2 hours. After the reaction is finished, cooling to room temperature, stopping stirring, adding water to generate white precipitate, filtering and collecting the precipitate, washing a filter cake with water, and drying the obtained product to obtain the compound PMD. Three experiments were performed and the different material ratios are shown in Table 4 below.
Table 4 example 4 three sets of experimental conditions
| Phthalimide | 2-ethylhexyl amine | Glacial acetic acid | Temperature (temperature) |
| 1g | 0.5mL | 30mL | 120℃ |
| 1g | 0.75mL | 40mL | 122℃ |
| 1g | 1mL | 50mL | 117℃ |
The reaction equation is as follows:
EXAMPLE 5 Synthesis of Compound HPMD
The compound PMD 0.2g obtained in the first set of experiments of example 4, pyrrole, N-dimethylformamide were added sequentially to the flask, deoxygenated by bubbling, and stirred under 400nm light for 10h under inert gas atmosphere. After the reaction is finished, stirring is stopped, the solvent is removed under reduced pressure, and the compound HPMD is prepared by column chromatography separation. Three experiments were performed and the different material ratios are shown in Table 5 below. HPMD characterization structure obtained in the first group of experiments is: [ C 20 H 25 N 2 O 2 ] + HRMS calculated values of (2): 325.1911; actual measurement value: 325.1909; the nuclear magnetic hydrogen spectrum is shown in figure 6.
Table 5 example 5 three sets of experimental conditions
| PMD | Pyrrole compounds | N, N-dimethylformamide |
| 0.2g | 5mL | 6mL |
| 0.2g | 3mL | 15mL |
| 0.2g | 9mL | 12mL |
The reaction equation is as follows:
EXAMPLE 6 Synthesis of Compound IPMD
The compound obtained in the first group of experiments of example 4 PMD 0.169g, indole-2-boronic acid pinacol ester, potassium carbonate, ditriphenylphospholate palladium dichloride, tetrahydrofuran and water were sequentially added into a flask, stirred under an inert gas atmosphere, and heated under reflux for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (20:1) system was separated by column chromatography on an eluent to give the compound IPMD. Three experiments were performed and the different material ratios are shown in Table 6 below. The IPMD characterization structure obtained in the first set of experiments was: [ C 24 H 27 N 2 O 2 ] + HRMS calculated values of (2): 375.2067; actual measurement value: 375.2066; the nuclear magnetic hydrogen spectrum is shown in figure 7.
TABLE 6 example 6 three sets of experimental conditions
The reaction equation is as follows:
EXAMPLE 7 Synthesis of Compound RHO
Adding 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid into a flask, stirring, heating, condensing and refluxing. After the reaction was completed, the reaction mixture was cooled to room temperature, stirring was stopped, and the pH of the solution was adjusted to 9 with a saturated sodium carbonate solution. The solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (5:1) system is used for eluting liquid column chromatography to obtain the compound RHO. Three experiments were performed and the different material ratios are shown in Table 7 below.
Table 7 example 7 three sets of experimental conditions
| 4-diethylaminoketo acid | 3-iodophenol | Methanesulfonic acid | Temperature (temperature) |
| 0.64g | 0.3mL | 20mL | 150℃ |
| 0.64g | 0.6mL | 40mL | 152℃ |
| 0.64g | 0.8mL | 30mL | 155℃ |
The reaction equation is as follows:
EXAMPLE 8 Synthesis of Compound MRHO
The compound RHO 0.3g obtained in the first experiment of example 7, N-methylpyrrole and N, N-dimethylformamide were sequentially added into a flask, and stirred under 400nm light for 10 hours under an inert gas atmosphere after deoxidization by a bubbling method. After the reaction was completed, stirring was stopped, and the solvent was removed under reduced pressure using petroleum ether: the ethyl acetate (5:1) system is used for eluting liquid column chromatography to obtain the compound MRHO. Three experiments were performed and the different material ratios are shown in Table 8 below. The MRHO characterization structure obtained in the first set of experiments is: [ C 29 H 27 N 2 O 3 ] + HRMS calculated values of (2): 451.1977; actual measurement value: 451.2017; the nuclear magnetic hydrogen spectrum is shown in figure 9.
Table 8 example 8 three sets of experimental conditions
The reaction equation is as follows:
EXAMPLE 9 Synthesis of Compound COU2
0.3g of 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water are sequentially added into a flask, and the mixture is stirred, heated, condensed and refluxed for 12 hours under the inert gas atmosphere. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (10:1) system is used for eluting liquid column chromatography to obtain the compound COU2. Three experiments were performed and the different material ratios are shown in Table 9 below.
Table 9 example 9 three sets of experimental conditions
The reaction equation is as follows:
EXAMPLE 10 Synthesis of Compound MCOUB
Ethanol, 0.2g of COU2, diethyl malonate and piperidine which are the compounds obtained in the first experiment of example 9 are sequentially added into a flask, stirred, heated, condensed and refluxed for 12 hours. After the reaction is finished, stirring is stopped, the solvent is removed under reduced pressure, and petroleum ether is used for: the ethyl acetate (5:1) system was separated by column chromatography on an eluent to give compound MCOUB. Three experiments were performed and the different material ratios are shown in Table 10 below. The MCOUB characterization structure obtained in the first set of experiments is: [ C 17 H 16 NO 4 ] + HRMS calculated values of (2): 298.1035; actual measurement value: 298.1087; the nuclear magnetic hydrogen spectrum is shown in figure 13.
Table 10 example 10 three sets of experimental conditions
The reaction equation is as follows:
EXAMPLE 11 Synthesis of Compound HNAP
Experiment of example 1 first setThe obtained compound NAP 0.232g, pyrrole, N-dimethylformamide were added into a flask in this order, and after deaeration by bubbling, the mixture was stirred under 400nm light for 10 hours under an inert gas atmosphere. After the reaction was completed, stirring was stopped, and the solvent was removed under reduced pressure using petroleum ether: the ethyl acetate (10:1) system is used for eluting liquid column chromatography to obtain the compound HNAP. Three experiments were performed and the different material ratios are shown in Table 11 below. The HNAP characterization structure obtained in the first set of experiments is: [ C 24 H 27 N 2 O 2 ] + HRMS calculated values of (2): 375.2067; actual measurement value: 375.2065; the nuclear magnetic hydrogen spectrum is shown in figure 2.
Table 11 example 11 three sets of experimental conditions
| NAP | Pyrrole compounds | N, N-dimethylformamide |
| 0.232g | 5mL | 6mL |
| 0.232g | 3mL | 15mL |
| 0.232g | 9mL | 12mL |
The reaction equation is as follows:
EXAMPLE 12 Synthesis of Compound INAP
The compound NAP 0.194g obtained in the first group of experiments of example 1, indole-2-boronic acid pinacol ester, potassium carbonate, ditriphenylphosphold palladium dichloride, tetrahydrofuran and water were sequentially added into a flask, stirred under an inert gas atmosphere, and heated under reflux for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (15:1) system was separated by column chromatography on an eluent to give compound INAP. Three experiments were performed and the different material ratios were as shown in Table 12 below. The INAP characterization structure obtained in the first set of experiments was: [ C 28 H 29 N 2 O 2 ] + HRMS calculated values of (2): 425.2184; actual measurement value: 425.2208; the nuclear magnetic hydrogen spectrum is shown in figure 3.
Table 12 example 12 three sets of experimental conditions
| NAP | Indole-2-boronic acid pinacol ester | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.194g | 0.2g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.194g | 0.12g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.194g | 0.3g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
EXAMPLE 13 Synthesis of Compound MPMD
0.2g of PMD, 3mL of N-methylpyrrole and 6mL of N, N-dimethylformamide, which are obtained in the first group of experiments in example 4, are sequentially added into a flask, and after deoxygenation by a bubbling method, the mixture is stirred for 10 hours under the illumination of 400nm in an inert gas atmosphere. After the reaction was completed, stirring was stopped, and the solvent was removed under reduced pressure using petroleum ether: the ethyl acetate (10:1) system was separated by column chromatography on an eluent to give compound MPMD. Three groups of experiments were performed, and the mixture ratio conditions of different materials are as follows Table 13. The MPMD characterization structure obtained in the first set of experiments was: [ C 13 H 13 NO 2 Na] + HRMS calculated values of (2): 361.1892; actual measurement value: 361.1894. the nuclear magnetic hydrogen spectrum is shown in figure 5.
Table 13 example 13 three sets of experimental conditions
| PMD | N-methylpyrrole | N, N-dimethylformamide |
| 0.2g | 3mL | 6mL |
| 0.2g | 6mL | 15mL |
| 0.2g | 9mL | 10mL |
The reaction equation is as follows:
EXAMPLE 14 Synthesis of Compound MIPMD
The compound NAP 0.169g obtained in the first experiment of example 1, 1-methylindole-2-boric acid, potassium carbonate, ditolylphosphine palladium dichloride, tetrahydrofuran and water were sequentially added to a flask under an inert gasStirring, heating, condensing and refluxing for 12h under the body atmosphere. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (20:1) system was separated by column chromatography on an eluent to give the compound MIPMD. Three experiments were performed and the different material ratios were as shown in Table 14 below. The first set of experiments resulted in a MIPMD characterization structure of: [ C 25 H 29 N 2 O 2 ] + HRMS calculated values of (2): 389.2224; actual measurement value: 389.2225. the nuclear magnetic hydrogen spectrum is shown in figure 8.
Table 14 example 14 three sets of experimental conditions
| PMD | 1-methylindole-2-boronic acid | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.169g | 0.105g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.169g | 0.3g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.169g | 0.2g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
EXAMPLE 15 Synthesis of Compound IRHO
The compound RHO 0.25g obtained in the first group of experiments of example 7, indole-2-boronic acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water were added into a flask in sequence, stirred under inert gas atmosphere, and heated and refluxed for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (3:1) system was separated by column chromatography on an eluent to give the compound IRHO. Three experiments were performed and the different material ratios are shown in Table 15 below. The IRHO characterization structure obtained in the first set of experiments is: [ C 32 H 27 N 2 O 3 ] + HRMS calculated values of (2): 487.2016; actual measurement value: 487.2014. the nuclear magnetic hydrogen spectrum is shown in figure 10.
Table 15 example 15 three sets of experimental conditions
| RHO | Indole-2-boronic acid pinacol ester | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.25g | 0.2g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.25g | 0.12g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.25g | 0.3g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
EXAMPLE 16 Synthesis of Compound MIRHO
The compound RHO 0.25g obtained in the first group of experiments of example 7, 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water were sequentially added into a flask, stirred under an inert gas atmosphere, heated, condensed and refluxed for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (4:1) system is used for eluting liquid column chromatography to obtain the compound MIRHO. Three experiments were performed and the different material ratios were as shown in Table 16 below. The MIRHO characterization structure obtained in the first group of experiments is as follows: [ C 33 H 29 N 2 O 3 ] + HRMS calculated values of (2): 501.2173; actual measurement value: 501.2167. the nuclear magnetic hydrogen spectrum is shown in figure 11.
Table 16 example 16 three sets of experimental conditions
| RHO | 1-AIndole-2-boronic acid | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.25g | 0.105g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.25g | 0.3g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.25g | 0.2g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
EXAMPLE 17 Synthesis of Compound MCOU1
1.06mL of 3-iodophenol is slowly added into 80% concentrated sulfuric acid at the temperature of 0 ℃ in a dropwise manner, the mixture is stirred for 30min under the condition, then ethyl acetoacetate is added into the mixture in a dropwise manner, the mixture is continuously stirred to enable the mixture to slowly return to the room temperature, the mixture is continuously stirred at the room temperature for 36h, and the reaction is finished. The mixture was dropped into 100mL of ice water to precipitate a white precipitate, which was filtered to obtain compound COU1. The mixture ratio conditions of different materials are shown in the following table 17.
Table 17 example 17 three sets of experimental conditions
| 3-iodophenol | Concentrated sulfuric acid | Acetoacetic acid ethyl ester |
| 1.06mL | 20mL | 1.39mL |
| 1.06mL | 45mL | 4mL |
| 1.06mL | 30mL | 7mL |
The reaction equation is as follows:
EXAMPLE 18 Synthesis of Compound MCOUA
COU 1.26 g, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water obtained in the first group of experiments of example 17 are sequentially added into a flask, stirred under inert gas atmosphere, heated, condensed and refluxed for 12 hours. After the reaction was completed, the solution was extracted with dichloromethane, the organic phase was collected, the solvent was removed under reduced pressure, and petroleum ether was used: the ethyl acetate (4:1) system was separated by column chromatography on an eluent to give compound MCOUA. Three experiments were performed and the different material ratios were as shown in Table 18 below. The MCOUA characterization structure obtained in the first set of experiments is: [ C 15 H 14 NO 2 ] + HRMS calculated values of (2): 240.0980; actual measurement value: 240.1023. the nuclear magnetic hydrogen spectrum is shown in figure 12.
Table 18 example 18 three sets of experimental conditions
| COU1 | 1-methylpyrrole-2-boronic acid pinacol ester | Potassium carbonate | Bis-triphenylphosphine palladium dichloride | Tetrahydrofuran (THF) | Water and its preparation method | Temperature (temperature) |
| 0.26g | 0.21g | 0.138g | 35mg | 10mL | 10mL | 73℃ |
| 0.26g | 0.6g | 0.4g | 17mg | 15mL | 15mL | 77℃ |
| 0.26g | 0.4g | 0.7g | 25mg | 25mL | 25mL | 75℃ |
The reaction equation is as follows:
in order to verify the effect of the present invention, a great deal of laboratory research experiments were performed on the novel micro-environment-sensitive organic fluorescent small molecules prepared according to the above synthesis method, and various experimental conditions are as follows:
1. basic fluorescence behavior characterization
The prepared novel microenvironment sensitive organic fluorescent micromolecules are subjected to excitation emission spectrum characterization by using an Edinburgh instrument FLS 920 single photon counting fluorescence spectrometer, and the results are shown in figures 14-25. As can be seen from fig. 14, the maximum excitation wavelength of the MNAP compound is 400nm and the maximum emission wavelength is 530nm; as can be seen from fig. 15, the maximum excitation wavelength of the compound HNAP was 410nm, and the maximum emission wavelength was 528nm; as can be seen from fig. 16, the maximum excitation wavelength of the compound INAP is 410nm and the maximum emission wavelength is 552nm; as can be seen from fig. 17, the maximum excitation wavelength of compound milpa is 411nm and the maximum emission wavelength is 583nm; as can be seen from fig. 18, the maximum excitation wavelength of compound MPMD is 375nm and the maximum emission wavelength is 525nm; as can be seen from fig. 19, the maximum excitation wavelength of the compound HPMD is 375nm and the maximum emission wavelength is 510nm; as can be seen from fig. 20, the maximum excitation wavelength of the compound IPMD is 380nm and the maximum emission wavelength is 530nm; as can be seen from fig. 21, the compound MIPMD has a maximum excitation wavelength of 380nm and a maximum emission wavelength of 580nm; as can be seen from fig. 22, the compound MRHO has a maximum excitation wavelength of 573nm and a maximum emission wavelength of 637nm; as can be seen from fig. 23, the compound IRHO has a maximum excitation wavelength of 570nm and a maximum emission wavelength of 691nm; as can be seen from fig. 24, the maximum excitation wavelength of the compound MCOUA is 355nm, and the maximum emission wavelength is 451nm; as can be seen from fig. 25, the maximum excitation wavelength of the compound MCOUB was 393nm and the maximum emission wavelength was 514nm.
2. Viscosity sensitivity test
Solvents with different viscosities were formulated using different volume ratios of ethanol to glycerol, and viscosity sensitivity tests were performed on the compounds. As shown in fig. 26 and 27, the fluorescence of the compound IRHO (fig. 26) and IPMD (fig. 27) solutions increased with increasing solvent viscosity. The test results indicate that IRHO and IPMD have viscosity sensitivity.
3. Polarity sensitivity test
The solvents with different polarities are prepared by using chloroform and methanol with different volume ratios, and the polarity sensitivity test is carried out on the compound. As shown in fig. 28 and 29, the fluorescence of the compound MCOUA (fig. 28) and MCOUB (fig. 29) solutions was continuously decreased with increasing polarity of the solvent. The test results show that MCOUA and MCOUB have polarity sensitivity.
4. AIE test
Solvents with different water content were formulated using different volume ratios of methanol to water and AIE testing was performed on the compounds. As shown in fig. 30 and 31, the fluorescence of the solutions of the compounds MNAP (fig. 30) and MPMD (fig. 31) increased continuously with increasing water content. The test results show that MNAP and MPMD have AIE properties.
5. Detection test of Compound MRHO on bovine/human serum Albumin
Serum albumin with different qualities is weighed and dissolved in PBS solution to prepare solutions with different serum albumin concentrations, and the detection test of the compound MRHO on bovine/human serum albumin is carried out. As shown in fig. 32, as the content of bovine serum albumin or human serum albumin in PBS solution increases, hydrophobic cavity sites available for MRHO in serum albumin increases, more MRHO is immobilized in the hydrophobic cavity sites, intramolecular tigt (twisted intramolecular charge transfer, intramolecular torsional charge transfer) is inhibited, and the fluorescence intensity increases. The test results show that MRHO can detect bovine/human serum albumin.
6. Cell wash-free imaging experiment of MNAP compound
The wash-free biological imaging can simplify imaging operation and reduce interference and damage to biological samples. FIG. 33 is a photograph of an unwashed/washed laser confocal microscope of MNAP-stained A549 cells. The MNAP compound has weaker fluorescence in the cell culture fluid, but when the MNAP compound enters the cells and reaches a nonpolar region in the cells or is combined with proteins in the cells, the fluorescence is obviously enhanced, so that the fluorescence inside and outside the cells are greatly different. The result of the picture taken by a laser confocal microscope after the A549 cells dyed by MNAP are unwashed/washed shows that the MNAP can carry out living cell unwashed biological imaging.
For micro-environment sensitive fluorescent small organic molecules, the most important part is the molecular design. In the molecular design process, two important considerations are required: (1) constructing a molecular fragment of a D-A system; (2) bonding means between D and A. Both of which have an important influence on the sensitivity of the fluorescent molecules. Naphthalimide, phthalimide, coumarin and rhodamine are common fluorophore precursors with electron withdrawing groups, and are widely focused due to the advantages of strong designability of molecular structures, simple synthesis, good repeatability and the like; the electron donating groups such as pyrrole, N-methylpyrrole, indole and N-methylindole can easily generate substitution reaction at the 2/5 position, so that a D-A structure connected with a carbon-carbon single bond can be formed between the pyrrole, N-methylpyrrole, indole and N-methylindole and a fluorophore matrix, the molecule is easy to rotate, the viscosity sensitivity is realized, and meanwhile, the novel molecule formed by the pyrrole, N-methylpyrrole, indole and N-methylindole is also polarity sensitivity as an ICT molecule. Therefore, in summary, pyrrole and derivatives thereof are introduced into the design of fluorescent molecules, a series of fluorescent small organic molecules with microenvironment sensitivity are synthesized, and a new design idea is provided for synthesizing fluorescent molecules with viscosity and polarity sensitivity at the same time. The synthesis method of the invention has simple operation, and the synthesized compound has better viscosity, polarity sensitivity and AIE characteristics, and can realize the detection of serum albumin and the washing-free imaging of living cells.
The above is only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited by this, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the claims of the present invention.
Claims (9)
1. The micro-environment sensitive fluorescent organic small molecule compound is characterized by being one of the following compounds:
。
2. the method for preparing the microenvironment-sensitive fluorescent small organic molecule compound according to claim 1, which is characterized by comprising one of the following steps:
the method comprises the following steps:
stirring and mixing 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol, and heating and refluxing; after the reaction is finished, cooling to room temperature, adding water to generate yellow precipitate, filtering and collecting the precipitate, washing a filter cake, and drying to obtain a compound NAP;
mixing a compound NAP, N-dimethylformamide and N-methylpyrrole or pyrrole, deoxidizing by a bubbling method, stirring under 400nm light in an inert gas atmosphere, removing a solvent under reduced pressure after the reaction is finished, and separating by column chromatography to obtain a product;
or mixing compound NAP, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
The second method is as follows:
mixing phthalimide, 2-ethylhexyl amine and glacial acetic acid under stirring, and heating for reflux; after the reaction is finished, cooling to room temperature, adding water to generate white precipitate, filtering and collecting the precipitate, washing a filter cake, and drying to obtain a compound PMD;
mixing a compound PMD, N-dimethylformamide and N-methylpyrrole or pyrrole, deoxidizing by a bubbling method, and stirring under 400nm light in an inert gas atmosphere; after the reaction is finished, the solvent is removed under reduced pressure, and the product is obtained through column chromatography separation;
or mixing PMD, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
and a third method:
mixing 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid, stirring, and heating for reflux; after the reaction is finished, cooling to room temperature, adjusting the pH value of the solution to 9, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound RHO;
Sequentially adding a compound RHO, N-methylpyrrole and N, N-dimethylformamide into a flask, deoxidizing by a bubbling method, and stirring under 400nm light in an inert gas atmosphere; after the reaction is finished, the solvent is removed under reduced pressure, and the product is obtained through column chromatography separation;
or mixing the compounds RHO, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran, water and indole-2-boronic acid pinacol ester or 1-methylindole-2-boric acid, stirring under inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
the method four:
dropwise adding 3-iodophenol into concentrated sulfuric acid at 0 ℃, stirring, then dropwise adding ethyl acetoacetate, continuously stirring until the reaction is finished, dropwise adding the obtained mixed solution into ice water, precipitating white precipitate, and filtering to obtain a compound COU1;
mixing a compound COU1, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis (triphenylphosphine) palladium dichloride, tetrahydrofuran and water, stirring under an inert gas atmosphere, and heating for reflux; after the reaction is finished, extracting the solution by using dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by using column chromatography to obtain a product;
And a fifth method:
mixing 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-boric acid pinacol ester, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water, stirring under inert gas atmosphere, and heating and refluxing; after the reaction is finished, extracting the solution with dichloromethane, collecting an organic phase, removing the solvent under reduced pressure, and separating by column chromatography to obtain a compound COU2;
mixing ethanol, compound COU2, diethyl malonate and piperidine under stirring, heating and refluxing to react, removing solvent under reduced pressure, and separating by column chromatography to obtain the final product.
3. The method for preparing a micro-environment sensitive fluorescent small organic molecule compound according to claim 2, wherein the column chromatography separation is performed by using a petroleum ether-ethyl acetate system as an eluent.
4. The preparation method of the micro-environment sensitive fluorescent small organic molecular compound according to claim 2, wherein in the first method, the molar ratio of 4-bromo-1, 8-naphthalic anhydride, 2-ethylhexyl amine and ethanol is 1 (5-10): (800-1000), and the heating reflux temperature is 78-83 ℃; the mole ratio of the compound NAP, N-methylpyrrole or pyrrole and N, N-dimethylformamide is 1 (5-15): 10-25); the illumination reaction temperature at 400nm is 20-25 ℃; the mole ratio of the compound NAP, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, ditolylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3) (1-6) (1/20-1/10) (250-500) (500-1000), and the heating reflux temperature is 73-77 ℃.
5. The preparation method of the micro-environment sensitive fluorescent small organic molecular compound according to claim 2, wherein in the second method, the molar ratio of phthalimide, 2-ethylhexyl amine and glacial acetic acid is 1 (5-10): (1000-1300), and the heating reflux temperature is 117-122 ℃; the mole ratio of the compound PMD to the N-methylpyrrole or pyrrole to the N, N-dimethylformamide is 1 (5-15) (10-25), and the 400nm light reaction temperature is 20-25 ℃; the mole ratio of the compound PMD, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3) (1-6) (1/20-1/10) (250-500) (500-1000), and the heating reflux temperature is 73-77 ℃.
6. The preparation method of the micro-environment sensitive fluorescent small organic molecule compound according to claim 2, wherein in the third method, the molar ratio of 4-diethylamino keto acid, 3-iodophenol and methanesulfonic acid is 1 (1-3): 400-800; the heating reflux reaction temperature is 150-155 ℃; the mol ratio of the compound RHO, the N-methylpyrrole and the N, N-dimethylformamide is 1 (5-15): 10-25, and the reaction temperature is 20-25 ℃; the mol ratio of the compound RHO, indole-2-boric acid pinacol ester or 1-methylindole-2-boric acid, potassium carbonate, bis-triphenylphosphine palladium dichloride, tetrahydrofuran and water is 1 (1-3) (1-6) (1/20-1/10) (250-500) (500-1000), and the heating reflux reaction temperature is 73-77 ℃.
7. The preparation method of the micro-environment sensitive fluorescent small organic molecular compound according to claim 2, wherein in the fourth method, the molar ratio of 3-iodophenol, concentrated sulfuric acid and ethyl acetoacetate is 1 (15-35): (1-5), and the reaction temperature is 20-25 ℃; the mol ratio of the compound COU1 to the 1-methyl pyrrole-2-boric acid pinacol ester to the potassium carbonate to the bis-triphenylphosphine palladium dichloride to the tetrahydrofuran to the water is 1 (1-3): (1-6): (1/20-1/10): (250-500): (500-1000), and the reaction temperature is 73-77 ℃.
8. The preparation method of the micro-environment sensitive fluorescent organic micromolecule compound according to claim 2, which is characterized in that in the fifth method, the mole ratio of 4-bromo-2-hydroxybenzaldehyde, 1-methylpyrrole-2-pinacol borate, potassium carbonate, ditriphenylphospholorium dichloride, tetrahydrofuran and water is 1 (1-3): (1-6): (1/20-1/10): (250-500): (500-1000), and the heating reflux reaction temperature is 73-77 ℃; the mol ratio of the compound COU2 to the ethanol to the diethyl malonate to the piperidine is 1 (100-200) (1-5) (0.2-0.7), and the heating reflux reaction temperature is 78-83 ℃.
9. The use of the micro-environment-sensitive fluorescent small organic molecule compound of claim 1 in the preparation of a reagent for detecting serum albumin.
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