Classifications

• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
  • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
  • C09B23/02—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups
  • C09B23/06—Methine or polymethine dyes, e.g. cyanine dyes the polymethine chain containing an odd number of >CH- or >C[alkyl]- groups three >CH- groups, e.g. carbocyanines
• • 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
  • C07D401/06—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 linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D421/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms
  • C07D421/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings
  • C07D421/06—Heterocyclic compounds containing two or more hetero rings, at least one ring having selenium, tellurium, or halogen atoms as ring hetero atoms containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
• • 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 materials, e.g. electroluminescent or chemiluminescent
  • C09K11/06—Luminescent materials, e.g. electroluminescent or chemiluminescent containing organic luminescent materials
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • 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
  • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
  • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
• • 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
  • C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
• • 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
  • C09K2211/104—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with other heteroatoms

Definitions

• Fluorescent dye preparation method and application thereof
• the invention relates to a novel fluorescent dye, a preparation method and application thereof in the field of fine chemical industry, in particular to a singly charged nitrogen-containing cyanine fluorescent dye, a preparation method thereof, and the use of the fluorescent dye, the conjugate thereof or the same
• a singly charged nitrogen-containing cyanine fluorescent dye a preparation method thereof
• the use of the fluorescent dye, the conjugate thereof or the same The use of the composition in biological dyeing. Background technique
• Fluorescent dyes are widely used as functional pigments in various fields of science and technology. Especially in the fields of life science, clinical medical diagnosis, and immunoassay, research has attracted worldwide attention.
• phenanthridines EB, PI
• AO acridines
• AO imidazoles
• Hoechst DAPI
• Cy cyanine family
• fluorescent dyes such as SYTO
• SYTO fluorescent dyes
• TOPRO TOTO family dyes
• EB ethidium bromide
• PI propidium iodide
• cyanine fluorescent dyes have wide wavelength range, large molar extinction coefficient and moderate fluorescence quantum yield. They are used as biomolecular fluorescent probes, CD and VCD recording materials, photosensitive materials, photosensitizers, and optoelectronics. Conversion materials and the like have been widely used.
• Quinoline-like asymmetric cyanine Light dyes have a high affinity with nucleic acids, and the specificity of non-binding with other biomacromolecules makes them stand out in applications such as genomics technology, nucleic acid quantitative detection, and blood cell analysis. The manner in which such compounds bind to nucleic acids includes electrostatic attraction, base pair insertion, and groove bonding.
• TOTO thiazole orange dimer
• YOYO oxazole yellow dimer
• TOPRPO thiazole orange dimer
• YOYO oxazole yellow dimer
• the present invention is directed to the deficiencies of the prior art and, based on the improvement thereof, provides a novel compound having a simple structure, high sensitivity, long wavelength, and good cell membrane permeability, which has The following structural formula 1 :
• X is C(CH 3 ) 2 , 0, S or Se; m is an integer from 1 to 18;
• RPR 2 is independently selected from H, d. 18 alkyl, OR 7 , -C ⁇ fluorenyl-OR 7 or halogen.
• R 3 is pyrrolyl, imidazolyl, piperidinyl, piperazinyl, morpholinyl, fluorenyl, 1 ⁇ 11 5 or 1 ⁇ 1 6 ;) 2;
• R 4 is ⁇ 18 fluorenyl, benzyl or (CH 2 ) m R 3 , which is optionally substituted by a substituent selected from the group consisting of: H, C 1-18 alkyl, CN, COOH, NH 2 , N0 2 , OH, SH, C 1-6 fluorenyloxy, C 1-6 fluorenylamino, C acylamino, halogen, or d 6 halogenated fluorenyl;
• the base or benzyl group is optionally substituted by a substituent selected from the group consisting of: H, C 1-18 fluorenyl, CN, COOH, NH 2 , N0 2 , OH, SH, d 6 methoxy, d 6 fluorenylamino, d. 6 acylamino, halogen, or. ⁇ haloalkyl;
• Re is the C2-18 yard base
• R 7 is 11 or d_ 18 thiol
• the reaction temperature is 10-180 ° C
• the reaction time is 4-48 hours
• the reaction solvent is selected from the group consisting of: dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene or a mixture thereof.
• the polar organic solvent, the molar ratio of the compound of the formula II to the R 4 Z compound is 1:1 - 1:10;
• the reaction temperature is 50-200 ° C, the reaction time is 15 minutes to 4 hours, the reaction solvent or the reaction solvent is acetic anhydride, acetic acid or a mixture thereof, the first quaternary ammonium intermediate is ruthenium and osmium, ⁇ '-diphenyl
• the molar ratio of formazan is 1:1 - 1:4;
• the reaction temperature is 10-180 ° C
• the reaction time is 4-48 hours
• the reaction solvent is selected from the group consisting of: dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene or a mixture thereof.
• the polar organic solvent, the molar ratio of the compound of the formula V to the compound ( ⁇ 3 ⁇ 4) hail ⁇ is 1:1 to 1:10;
• the reaction temperature is 5-130 Torr
• the reaction time is 10 minutes to 6 hours
• the reaction solvent or reaction solvent is selected from the group consisting of: polarity of dichloromethane, chloroform, methanol, ethanol, ethylene glycol monomethyl ether, or a mixture thereof.
• the organic solvent, the catalyst is a mixture of acetic anhydride and an organic base, and the molar ratio of VI to the compound of formula IV is 1.5:1 to 1:1.5;
• the reaction temperature is 60-140 ° C
• the reaction time is 10 minutes to 2 hours
• the reaction solvent is a polar organic solvent selected from DMF, DMSO, or a mixture thereof
• the sodium salt or potassium salt containing Y and the compound of the formula W The molar ratio of feed is 1: 1 to 10: 1;
• Example 1 is a compound A of Example 2 and a commercial dye ethidium bromide (EB) in a buffer of pH 7.36 and a concentration of 10 mM in tris(hydroxymethyl)carbamidine hydrochloride, before and after binding to calf thymus DNA.
• the relative fluorescence intensity is compared to the map.
• the abscissa is the wavelength (nm) and the ordinate is the relative fluorescence intensity.
• the instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453 ; fluorescence spectrophotometer, model: FP-6500.
• the concentration of Compound A and ethidium bromide (EB) was 1 ⁇ , and the concentration of calf thymus DNA was 100 ⁇ .
• Example 2 is a compound of Example 3 and a commercial dye ethidium bromide ( ⁇ ) in a pH 7.4, concentration of 10 mM tris(;hydroxymethyl)carbamidine hydrochloride buffer before and after binding to calf thymus DNA
• the relative fluorescence intensity is compared to the map.
• the abscissa is the wavelength (nm) and the ordinate is the relative fluorescence intensity.
• the instrument used was a UV-visible spectrophotometer, model: Hp8453; Fluorescence spectrophotometer, Model: FP-6500.
• the concentrations of Compound B and ethidium bromide (EB) were 1 ⁇ , and the concentration of calf thymus DNA was 100 ⁇ .
• Figure 3 is a compound C of Example 4 and a commercial dye ethidium bromide ( ⁇ ) before and after binding to calf thymus DNA in a buffer of tris(;hydroxymethyl)carbamidine hydrochloride at pH 7.24 and a concentration of 10 mM.
• the relative fluorescence intensity is compared to the map.
• the abscissa is the wavelength (nm) and the ordinate is the relative fluorescence intensity.
• the instrument used was a UV-visible spectrophotometer, model: Hp8453; Fluorescence spectrophotometer, Model: FP-6500.
• the concentrations of Compound A and ethidium bromide (EB) were 1 ⁇ , and the concentration of calf thymus DNA was 100 ⁇ .
• Figure 4 is a comparison of fluorescence quantum yields before and after binding to calf thymus DNA in a buffer of tris(hydroxymethyl)carbamidine hydrochloride at pH 7.4 and a concentration of 10 mM in compound ⁇ and a known compound, compound ⁇ 2 . .
• the ordinate is the fluorescence quantum yield.
• the instrument used was an ultraviolet-visible spectrophotometer, model: Hp8453; fluorescence spectrophotometer, model: FP-6500.
• the concentration of Compound A and Compound ⁇ 2 was 1 ⁇
• the concentration of calf thymus DNA was 100 ⁇ .
• Figure 5 is a comparison of the compound hydrazine and the known compound Mi in tris(hydroxymethyl)aminomethane hydrochloride buffer at pH 7.0 and 10 mM, respectively, after binding to bovine serum albumin and calf thymus DNA and before unbinding.
• the instrument used was a fluorescence spectrophotometer, model: FP-6500.
• the concentrations of Compound A and Compound Mi were both 1 ⁇ , and the concentrations of bovine serum albumin and calf thymus DNA were both 40 g/ml.
• Figure 6A is a white field photomicrograph of Compound A staining live cells MCF-7 (human breast cancer cells), 6B It is a fluorescence micrograph of Compound A staining live cells MCF-7. The concentration of Compound A was 2 ⁇ .
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation light channel: Cy5 (633 nm) 0
• Figure 7A is a white field photomicrograph of staining of living cell MCF-7 (human breast cancer cells) by compound D of Example 5, and 7B is fluorescence of compound D staining of living cells MCF-7 micrograph. The concentration of Compound D was 1.5 ⁇ .
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2.
• Fig. 8A is a white field micrograph of the staining of the living cell MCF-7 (human breast cancer cell) by the compound E of Example 6, and 8B is a fluorescence micrograph of the staining of the living cell MCF-7 by the compound E.
• the concentration of Compound A was 3 ⁇ .
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel - Cy5 (633 nm).
• Fig. 9A is a white field micrograph of a known compound staining for living cells MCF-7 (human breast cancer cells;), and 9B is a fluorescence micrograph of compound live cell MCF-7 staining.
• concentration of the compound Mi was 2 ⁇ .
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel - Cy5 (633 nm). detailed description
• alkyl as used herein includes both straight chain alkyl groups and branched chain fluorenyl groups.
• fluorenyl group such as "propyl”
• branched alkyl group such as "isopropyl”
• d -6 alkyl includes d _ 4 alkyl, d 3 alkyl, methyl, ethyl, n-propyl, isopropyl and t-butyl. Similar rules apply to the other groups used in this specification.
• halogen as used herein includes fluoro, chloro, bromo and iodo.
• benzyl refers to a -C3 ⁇ 4-Ph group.
• a benzyl group is modified with “optionally substituted”, it means that the benzyl group may exist in an unsubstituted form or may be substituted at any suitable position by a suitable substituent.
• Suitable substituents include, but are not limited to, H, Cw 8 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, d 6 methoxy, d 6 alkylamino, d -6 amide, halogen, or C ⁇ halogenated fluorenyl and the like, as long as the finally formed compound has the desired properties of the present invention.
• the benzyl group is optionally substituted by COOH, NH 2 , OH, C 1 -6 methoxy, or halogen.
• Y- is used herein to mean an anion, which can be any suitable anion, including but not limited to inorganic anions or organic anions such as halides, C10 4 -, PF 6 ", BF 4 -, CH 3 COO - or OTs".
• X is preferably C(CH 3 ) 2 , 0 or S; more preferably X is C (CH 3 ) 2 or S; most preferably 8.
• Preferably and independently selected from the group consisting of 13 ⁇ 4 or -18 alkyl; more preferably and each independently selected from H or -12 fluorenyl; more preferably and each independently selected from H or d- 6 alkyl; most preferably both are H .
• the benzyl group is optionally substituted by COOH, NH 2 , OH, d- 6 methoxy, or halogen.
• R 5 is preferably a saturated or unsaturated, ⁇ _ 18 alkyl with a straight-chain or branched, or an amino D_ 18 embankment group; more preferably R 5 is a saturated or unsaturated, D_ straight or branched 12 alkyl with; most Preferred are saturated or unsaturated, linear or branched C ⁇ alkyl groups.
• Re is a C 2 -6 fluorenyl group.
• 7 is H or d- 6 fluorenyl.
• Y_ is a halogen ion, C10 4 _, PF 6 " , BF 4 -, CH 3 COO_ or OTs -.
• the present invention provides a process for the preparation of the above compound, the method comprising: separately preparing a first and a second quaternary ammonium salt intermediate, and then reacting one of the first or second quaternary ammonium salt intermediates with The hydrazine, hydrazine-diphenylformamidine is reacted, and then reacted with another intermediate under the action of an organic base such as an amine and acetic anhydride to give the compound.
• an organic base such as an amine and acetic anhydride
• the first is to prepare a first quaternary ammonium salt intermediate, that is, a 4-methylquinoline reaction starting material of the formula ( ⁇ ) is reacted with a compound R 4 Z to obtain a first quaternary ammonium salt intermediate ( ⁇ ), Z.
• Z_ is the halogen anion produced by the reaction or —:
• the reaction temperature is 10-180 ° C
• the reaction time is 4-48 hours
• the reaction solvent is selected from the group consisting of: dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzene or a mixture thereof.
• the molar ratio of the compound of the formula II to the compound R 4 Z is 1: 1- 1 : 10.
• the reaction temperature is 40-140 ° C
• the reaction time is 6-36 hours
• the reaction solvent is a pole selected from the group consisting of: chloroform, acetonitrile, toluene, xylene, o-dichlorobenzene or a mixture thereof.
• the organic solvent, the molar ratio of the compound of the formula II to the compound R 4 Z is 1: 1- 1:6.
• the reaction temperature is 50-12 (TC, the reaction time is 8-24 hours, and the reaction solvent is a polar organic solvent such as acetonitrile, toluene, o-dichlorobenzene or a mixture thereof, the compound of the formula II
• the molar ratio to the compound R 4 Z is 1: 1- 1:3.
• the reaction temperature is 60-110 ° C
• the reaction time is 8-14 hours
• the reaction solvent is a polar organic solvent such as toluene, o-dichlorobenzene or a mixture thereof
• the compound ⁇ compound and the compound R The molar ratio of 4 Z is 1: 1- 1: 1.5.
• the reaction temperature is 50-200 ° C
• the reaction time is 15 minutes to 4 hours
• the reaction solvent or the reaction solvent is acetic anhydride, acetic acid or a mixture thereof
• the first quaternary ammonium intermediate is ruthenium and osmium, ⁇ '-diphenyl
• the molar ratio of formazan is 1: 1 : 1 : 4.
• the reaction temperature is 70-170 ° C
• the reaction time is 20 minutes to 3 hours
• the reaction solvent or the reaction solvent is acetic anhydride, acetic acid or a mixture thereof
• the first quaternary ammonium salt intermediates ⁇ and ⁇ The molar ratio of ⁇ '-diphenylformamidine is 1: 1- 1:3.
• the reaction temperature is from 90 to 160 ° C
• the reaction time is from 30 minutes to 2 hours
• the reaction solvent or the reaction solvent is acetic anhydride, acetic acid or a mixture thereof
• the first quaternary ammonium salt intermediate III The molar ratio to hydrazine, ⁇ '-diphenylformamidine is 1: 1.2-1:3.
• the reaction temperature is from 120 to 160 ° C
• the reaction time is from 30 minutes to 1.5 hours
• the reaction solvent or the reaction solvent is acetic anhydride
• the first quaternary ammonium salt intermediate III and hydrazine ⁇ '-
• the molar ratio of diphenylformamidine is 1: 1.2-1:2;
• a compound having the formula V preferably a substituent-containing 2-methylbenzothiazole, a substituent-containing 2-methylbenzoxazole, a substituent-containing 2-, is prepared by a method similar to the preparation of the compound of the formula III.
• the reaction temperature is 10-180 ° C
• the reaction time is 4-48 hours
• the reaction solvent is selected from the group consisting of: dichloromethane, chloroform, ethanol, acetonitrile, ethyl acetate, toluene, xylene, o-dichlorobenzen
• the reaction temperature is 60-140 ° C
• the reaction time is 6-36 hours
• the reaction solvent is a polarity selected from the group consisting of: chloroform, acetonitrile, toluene, xylene, o-dichlorobenzene or a mixture thereof.
• the organic solvent, the molar ratio of the compound of the formula V to the compound R 3 (CH 2 ) m Z is 1: 1- 1:6.
• the reaction temperature is 80-120 Torr
• the reaction time is 10-24 hours
• the reaction solvent is a polar organic solvent selected from the group consisting of: acetonitrile, toluene, o-dichlorobenzene or a mixture thereof, and the formula V
• the molar ratio of the compound to the compound R 3 (CH 2 ) m Z is from 1:1 to 1:3.
• the reaction temperature is 90-120 ° C
• the reaction time is 12-18 hours
• the reaction solvent is a polar organic solvent such as toluene, o-dichlorobenzene or a mixture thereof
• the molar ratio of R 3 (CH 2 ) m Z to the feed is 1: 1- 1:2.
• the compound of formula (IV) is then reacted with a second quaternary ammonium salt intermediate VI under the action of acetic anhydride and an organic base (e.g., an amine) to provide a ⁇ singly charged nitrogen-containing cyanine compound-
• a second quaternary ammonium salt intermediate VI under the action of acetic anhydride and an organic base (e.g., an amine) to provide a ⁇ singly charged nitrogen-containing cyanine compound-
• the reaction temperature is 5-130 Torr
• the reaction time is 10 minutes to 6 hours
• the reaction solvent or the reaction solvent is a polar organic selected from the group consisting of dichloromethane, chloroform, methanol, ethanol, ethylene glycol monomethyl ether or a mixture thereof.
• Solvent, the catalyst is a mixture of acetic anhydride and an organic base.
• the organic base is preferably selected from the group consisting of: diethylamine, n-propylamine, triethylamine, pyridine, piperidine, or a mixture thereof.
• the molar ratio of the second quaternary ammonium salt intermediate VI to the compound of formula IV is 1.5: 1- 1 : 1.5.
• the reaction temperature is 20-130 ° C
• the reaction time is 30 minutes to 5 hours
• the reaction-free solvent or the reaction solvent is selected from the group consisting of: methanol, ethanol, ethylene glycol monomethyl ether, or a mixture thereof
• the polar organic solvent, the organic base is selected from the group consisting of triethylamine, pyridine, piperidine, or a mixture thereof
• the molar ratio of the second quaternary ammonium salt intermediate VI to the compound of the formula IV is 1.2:1 to 1:1.5.
• the reaction temperature is 30-120 ° C
• the reaction time is 1-4 hours
• the reaction solvent or the reaction solvent is selected from the group consisting of methanol, ethylene glycol monomethyl ether, or a mixture thereof.
• the organic base is selected from the group consisting of triethylamine, pyridine or a mixture thereof, and the molar ratio of the second quaternary ammonium salt intermediate VI to the compound of the formula IV is 1.2: 1- 1: 1.2.
• the reaction temperature is 30-100 ° C
• the reaction time is 1.5-3 hours
• the reaction solvent or the reaction solvent is a polar organic solvent such as methanol, ethylene glycol monomethyl ether or a mixture thereof.
• the organic base in the catalyst is triethylamine, pyridine or a mixture thereof, and the molar ratio of the second quaternary ammonium intermediate VI to the compound of the formula IV is 1:1.
• the compound ring is subjected to negative ion exchange with a sodium or potassium salt containing C10 4 _, PF 6 “, BF 4 “ or CH 3 COO_ according to requirements.
• the reaction temperature is 60-140
• the reaction time is 10 minutes to 2 hours
• the reaction solvent is a polar organic solvent selected from the group consisting of DMF, DMSO, or a mixture thereof, and contains C10 4 _, PF 6 _ , BF 4 _ or CH 3
• the molar ratio of the sodium or potassium salt of COO_ to the compound of the formula ⁇ is 1: 1- 10:1.
• the reaction temperature is 70-130 Torr
• the reaction time is 15 minutes to 1.5 hours
• the reaction solvent is a polar organic solvent such as DMF, DMSO or a mixture thereof.
• the molar ratio of the sodium or potassium salt containing C, PF 6 _, BF 4 " or CH 3 COO_ to the compound of formula VII is from 1:1 to 7:1.
• the reaction temperature is 80-120 ° C
• the reaction time is 20 minutes to 1 hour
• the reaction solvent is DMF, containing C10 4 -, PF 6 ", BF 4 " or CH 3 COO -
• the molar ratio of the sodium or potassium salt to the compound of formula VII is from 1:1 to 4:1.
• the reaction temperature is 90-110 ° C
• the reaction time is 30 minutes
• the reaction solvent is DMF
• the molar ratio of the potassium salt to the hydrazine compound is 1: 1- 2:1.
• the product of the compound of the formula I synthesized by the above method of the present invention can be confirmed by nuclear magnetic resonance spectrum or mass spectrometry. Carbon spectrum and melting point tests can also be used to aid in confirming the structure.
• the present invention also provides a conjugate of the above compound and a composition comprising the above compound or a conjugate thereof.
• the invention also provides a use of the above compounds, conjugates thereof or combinations thereof for biological staining.
• the beneficial effect is that a nitrogen-containing substituent is introduced into the molecule of the new compound, so that the fluorescence quantum yield of the dye and the nucleic acid is increased, and the detection sensitivity is improved.
• the nitrogen-containing substituent introduced by the new compound molecule is non-quaternized (non-positively charged), so it has good cell membrane permeability and an increased application range.
• the introduction of a nitrogen-containing substituent into the molecule of the new compound appropriately increases the polarity of the molecule, reduces the binding force to the hydrophobic region of the molecule such as membrane lipids, proteins, and the like, and exhibits specific binding to the nucleic acid.
• the new dye compound introduces a quinoline heterocycle at one end.
• the maximum wavelength of ultraviolet absorption can be increased by about 80 nm, and the fluorescence emission wavelength can reach 650 nm, avoiding the fluorescence of the organism itself. Background interference.
• the new compound can be used as a light source by using a low-cost, small-sized, and stable red semiconductor laser, which greatly reduces the cost of use.
• the new compound product is small in toxicity, easy to obtain raw materials, and simple in structure.
• the target molecule can be synthesized by 4 to 5 ⁇ reaction, and it is easy to industrialize.
• the compounds of the invention can be used directly in the staining of biological samples in the form of the salts described herein.
• derivatives of the compounds of the invention may also be used in the staining of biological samples, including but not limited to conjugates.
• conjugate refers to a compound formed by the attachment of a fluorescent dye of the invention to other molecules by covalent bonds.
• Molecules that can be conjugated to a fluorescent dye of the invention can be molecules that specifically bind to a cell or cellular component, including but not limited to antibodies, antigens, receptors, ligands, enzymes, substrates, coenzymes, and the like.
• the test sample is incubated with the fluorescent conjugate for a period of time such that the fluorescent conjugate specifically binds to certain cells or cellular components in the test sample, and the binding of the fluorescent conjugate to the cell or cellular component can also be It is called dyeing.
• the staining step can be performed multiple times in sequence, or with multiple conjugates A variety of dyeings are performed.
• the sample is analyzed in a fluorescence activated cell sorter in which the excitation light source excites the fluorescent dye of the present invention in the conjugate, and the measuring device measures the emitted light generated by the excited fluorescent dye.
• compositions comprising a compound of formula I or a conjugate thereof for use in the staining of biological samples.
• compositions of the present invention may comprise, in addition to a compound of formula I or a conjugate thereof, other components required for dyeing biological samples, such as solvents, osmotic pressure adjusting agents, pH adjusting agents, surfactants and the like. These components are known in the industry.
• composition of the present invention may be present in the form of an aqueous solution or may be present in other suitable forms which are formulated as solutions in water prior to use.
• the present invention provides a method of using a compound of Formula I above, or a conjugate thereof, or a composition comprising a compound of Formula I, to stain a biological sample, the method comprising or a compound of Formula I above, or a conjugate thereof, or A step comprising contacting a composition of a compound of formula I with a biological sample.
• contacting as used herein may include contacting in a solution or a solid phase.
• the compounds M 2 In order to demonstrate the optimized improvement of the dye properties after the introduction of the nitrogen-containing groups in the structure of the present invention, the compounds M 2 , the known compounds M 1 and the commercial products are used in Examples 7, 8, 9, 10, 11 and Comparative Example 15.
• the dye ethidium bromide (EBM ⁇ is used as a reference material for comparison.
• the structures of the compounds M 1 and M 2 are as follows.
• reaction solution was poured into diethyl ether to precipitate dark purple granules, which were filtered and dried.
• the dye was separated on a silica gel column and eluted with a gradient of eluent methylene chloride and methanol to yield a blue component with a yield of 60%.
• aqueous solution of a certain concentration of calf thymus DNA was prepared, and the absorbance at 260 nm was measured by an ultraviolet absorption spectrophotometer, and the concentration was determined to be 1.5 mM.
• a solution of Compound A in DMSO (dimethyl sulfoxide) at a concentration of 1 mM and an aqueous solution of ethidium bromide (EB) in a cuvette were added to the calf thymus DNA at a concentration of 1.5 mM.
• Solution 20 (L, finally added pH 7.36, lOmM tris(hydroxymethyl)aminomethane hydrochloride buffer diluted to 3mL, measure its fluorescence intensity.
• the instrument used is an ultraviolet-visible spectrophotometer, model: Hp8453 ; fluorescence spectrophotometer, model: FP-6500.
• the buffer was diluted to 3 mL, placed in a cuvette, and the fluorescence intensity was measured.
• An aqueous solution of a certain concentration of calf thymus DNA was prepared, and the absorbance at 260 nm was measured by an ultraviolet absorption spectrophotometer, and the concentration was determined to be 1.5 mM.
• a solution of Compound B in DMSO (dimethyl sulfoxide) at a concentration of 1 mM and an aqueous solution of ethidium bromide (EB) 3 L were separately placed in a cuvette, and then a small concentration of 1.5 mM was added thereto.
• the bovine thymus DNA solution was 200 ⁇ , and finally diluted with ⁇ 7.4, 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer to 3 mL, and the fluorescence intensity was measured.
• aqueous solution of a certain concentration of calf thymus DNA was prepared, and the absorbance at 260 nm was measured by an ultraviolet absorption spectrophotometer, and the concentration was determined to be 1.5 mM.
• Another 1 mM solution of Compound C in DMSO (dimethyl sulfoxide) and 3 ⁇ g of ethidium bromide (EB) in a cuvette were added to the calf thymus DNA solution at a concentration of 1.5 mM. 200 ⁇ , and finally ⁇ 7.24, 10 mM tris(hydroxymethyl)aminomethane hydrochloride buffer was diluted to 3 mL, and the fluorescence intensity was measured.
• Fluorescence quantum yield of compound A and known compound M l M 2 before and after binding to calf thymus DNA A certain amount of compound A and compound Mi, M 2 in DMSO were added to a pH of 7.4, 10 mM.
• the buffer solution of (hydroxymethyl)carbamidine hydrochloride is adjusted to have a maximum absorption value of ⁇ 0.1 by an ultraviolet-visible spectrophotometer.
• the excitation wavelength was selected to determine the fluorescence intensity. The measurement was performed three times in parallel, and the fluorescence quantum yield was calculated and averaged.
• 6 is a white field photomicrograph of the staining of the living cell MCF-7 by the compound
• 6B is a fluorescence micrograph of the staining of the living cell MCF-7 by the compound A. Nuclear specific staining of MCF-7 cells by Compound A was observed as shown.
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel: Cy5 (633 nm).
• Example 13
• Confocal laser scanning microscopy was used to observe the staining of living cells MCF-7 by compound D: 12 L of PBS buffer with compound D and 1.5 ⁇ was added to the wells of MCF-7 cells in a six-well plate at 37 °C. Incubate 30 mm in a 5% C0 2 cell culture incubator. Then, PBS was shaken and rinsed for 5 min ⁇ 3, and then cell culture medium was added, and the morphology of the cells was observed by confocal laser scanning microscopy (TCS-SP2, Germany). Representative regions were selected, stimulated on a Cy5 (633 nm) channel, and observed three times with an oil microscope (lOOOO x).
• Figure 7A is a white field photomicrograph of Compound D staining live cell MCF-7
• 7B is a fluorescence micrograph of Compound D staining live cell MCF-7. Nuclear specific staining of MCF-7 cells by Compound D was observed as shown.
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel: Cy5 (633 nm;).
• Example 14
• 8 is a white field micrograph of the staining of the living cell MCF-7 by the compound
• 8 ⁇ is a fluorescence micrograph of the compound ⁇ staining of the living cell MCF-7.
• nuclear specific staining of MCF-7 cells was observed.
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel: Cy5 (633 nm). Comparative Example 15
• Confocal laser scanning microscopy was used to observe the staining of living cells MCF-7 by the compound Mi: 12 ⁇ g of PBS buffer with compound at a concentration of 2 ⁇ M was applied to a well-cultured MCF-7 cell in a six-well plate at 37 °C. Incubate 30 mm in a 5% C0 2 cell culture incubator. Then, PBS was vortexed and rinsed for 5 min ⁇ 3, and then cell culture medium was added, and the cell morphology was observed by confocal laser scanning microscopy (TCS-SP2, Germany). A representative region was selected, excited by Cy5 (633 nm) channel, and observed three times with an oil microscope ( ⁇ ⁇ ).
• Figure 9 is a white field photomicrograph of compound live cell MCF-7 staining, and 9 ⁇ is a fluorescence micrograph of compound live cell MCF-7 staining. As shown in the figure, it can be observed that the known compounds stain the nuclear and cytoplasm of MCF-7 cells to a similar extent and are non-specific.
• the instrument used was a confocal laser scanning microscope, model: TCS-SP2. Excitation channel: Cy5 (633 nm).
• fluorescent dye is a use of the novel compounds of the present invention, it is not believed that the compounds of the present invention are used only for fluorescent dyes, and the same effect of the compounds of the present invention as fluorescent dyes is known to those of ordinary skill in the art to which the present invention pertains. Under the consideration of the mechanism, a number of simple inferences can also be made, and other application uses of the compounds of the present invention are considered to be within the scope of the present invention.

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