[go: up one dir, main page]

WO2022100716A1 - Composé de cyanine, colorant contenant le composé de cyanine et utilisation du composé de cyanine - Google Patents

Composé de cyanine, colorant contenant le composé de cyanine et utilisation du composé de cyanine Download PDF

Info

Publication number
WO2022100716A1
WO2022100716A1 PCT/CN2021/130477 CN2021130477W WO2022100716A1 WO 2022100716 A1 WO2022100716 A1 WO 2022100716A1 CN 2021130477 W CN2021130477 W CN 2021130477W WO 2022100716 A1 WO2022100716 A1 WO 2022100716A1
Authority
WO
WIPO (PCT)
Prior art keywords
group
compound
chemical formula
alkyl
cyanine compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/130477
Other languages
English (en)
Chinese (zh)
Inventor
樊江莉
陈庚文
夏天平
叶燚
张子千
姚起超
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Dalian University of Technology
Shenzhen Mindray Bio Medical Electronics Co Ltd
Original Assignee
Dalian University of Technology
Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Dalian University of Technology, Shenzhen Mindray Bio Medical Electronics Co Ltd filed Critical Dalian University of Technology
Priority to CN202180075741.4A priority Critical patent/CN116490500A/zh
Publication of WO2022100716A1 publication Critical patent/WO2022100716A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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

Definitions

  • the present invention relates to the technical field of nucleic acid quantitative detection and biological dyeing, in particular to a cyanine compound, a dye containing the cyanine compound, and the application of the cyanine compound in the quantitative detection of nucleic acid and/or biological dyeing.
  • DNA (DeoxyriboNucleic Acid, deoxyribonucleic acid) is a class of biological macromolecules with genetic information. Normal cells of the organism have relatively stable DNA diploid content, and abnormal changes will occur only when cancer or precancerous lesions with malignant potential occur. Therefore, the specific identification and precise measurement of DNA, especially in living cells, is of great significance in the early diagnosis of cancer.
  • cyanine fluorescent dyes have the advantages of wide wavelength range, large molar extinction coefficient and moderate fluorescence quantum yield. Conversion materials, etc. have been widely used.
  • cyanine dyes Some varieties of cyanine dyes have been commercialized, but most of these commercialized dyes have large molecules and complex structures. identification and detection.
  • excitation light light emitted by a laser, or light absorbed by the dye
  • the excitation light of most commercial cyanine dyes has a relatively large wavelength, such as orange light, which has a problem of poor recognition of tiny objects.
  • a cyanine compound, a dye containing the cyanine compound and the application of the cyanine compound are required to at least partially solve the above problems.
  • a first aspect of the present invention provides a cyanine compound, which has the structure shown in the general formula I,
  • X is selected from the group consisting of C(CH 3 ) 2 , O, S and Se;
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen;
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane the group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl;
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl;
  • Y - is a negative ion.
  • the cyanine compound according to the present invention has good permeability of living cells, can enter cells to stain nucleic acid without destroying the cell membrane, has low toxicity and low carcinogenicity; and the excitation light of the cyanine compound of the present invention is The blue-green light with a smaller wavelength can identify tiny particles and improve the detection ability of small particles; the cyanine compound of the present invention can use ordinary green or blue semiconductor lasers as light sources, which greatly reduces the cost of use; in addition, the present invention
  • the structure of the cyanine compound is simple, the raw materials for its preparation are readily available, the synthesis yield is high, and it is easy to realize industrialization.
  • the X is selected from the group consisting of C(CH 3 ) 2 and S.
  • R 1 and the R 2 are each independently selected from the group consisting of H, C 1 -C 12 alkyl, phenyl, OR 6 and halogen.
  • each of said R 1 and said R 2 is independently selected from the group consisting of H, C 1 -C 6 alkyl, phenyl, OR 6 and halogen.
  • R 1 is selected from the group consisting of H, C 1 -C 6 alkyl, phenyl and halogen;
  • R 2 is H.
  • R 1 is selected from the group consisting of H, methyl, phenyl and Cl.
  • R 3 and the R 4 are each independently selected from C 1 -C 12 alkyl, C 1 -C 12 carboxyl, C 1 -C 12 hydroxyl, C 1 -C 12 NR 5 R 6 , benzyl The group consisting of substituted benzyl and substituted benzyl, wherein the substituent of the substituted benzyl is selected from C 1 -C 12 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy , the group consisting of C 1 -C 6 alkylamino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl.
  • R 3 and the R 4 are each independently selected from C 1 -C 6 alkyl, C 1 -C 6 carboxyl, C 1 -C 6 hydroxyl, C 1 -C 6 NR 5 R 6 , benzyl the group consisting of substituted benzyl and substituted benzyl, wherein the substituent of said substituted benzyl is selected from C 1 -C 6 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy , the group consisting of C 1 -C 6 alkylamino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl.
  • R 3 is selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 hydroxyl, C 1 -C 6 carboxyl, C 1 -C 6 NR 5 R 6 and benzyl;
  • R 4 is selected from the group consisting of C 1 -C 6 alkyl, C 1 -C 6 hydroxyl, C 1 -C 6 carboxyl, and benzyl.
  • R 5 and the R 6 are each independently selected from the group consisting of H and C 1 -C 12 alkyl.
  • R 5 and the R 6 are each independently selected from the group consisting of H and C 1 -C 6 alkyl.
  • R 5 and the R 6 are each independently C 1 -C 6 alkyl.
  • R 5 and the R 6 are ethyl.
  • R 3 is selected from the group consisting of methyl, ethyl, benzyl, 4-(diethylamino)butyl, hydroxypropyl and hexylcarboxy.
  • R 4 is selected from the group consisting of methyl, benzyl, carboxypentyl and hydroxypropyl.
  • the Y - is selected from the group consisting of halogen anion, ClO 4 - , PF 6 - , BF 4 - , CH 3 COO - or OTs - .
  • the cyanine compound comprises chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X, chemical formula XI, chemical formula XII, chemical formula XIII, chemical formula XIV , a structure shown in one of chemical formula XV, chemical formula XVI, chemical formula XVII and chemical formula XVIII,
  • a second aspect of the present invention provides a dye comprising the cyanine compound described in the first aspect.
  • the dyes according to the present invention including cyanine compounds, have good permeability to living cells, can enter cells to stain nucleic acids without destroying cell membranes, have low toxicity and low carcinogenicity; and, the cyanine compounds of the present invention have Both the excitation light and the emission light are blue-green light with a small wavelength, which can identify tiny particles and improve the detection ability of small particles; the cyanine compound of the present invention can use a common green semiconductor laser as a light source, which greatly reduces the use cost;
  • the cyanine compound of the present invention has a simple structure, the raw materials for preparing the cyanine compound are readily available, the synthesis yield is high, and it is easy to realize industrialization.
  • the third aspect of the present invention relates to the application of the cyanine compound described in the first aspect above in the quantitative detection of nucleic acid and/or biological staining; or
  • Fig. 1 shows the absorption spectrum of the compound B after DNA staining according to Example 2 of the present invention
  • Fig. 2 shows the fluorescence spectrum after the compound B according to Example 2 of the present invention dyes DNA
  • Figure 3 shows the absorption spectrum of compound B after RNA staining according to Example 2 of the present invention
  • Fig. 4 shows the fluorescence spectrum after the compound B according to Example 2 of the present invention stains RNA
  • Fig. 5 shows the variation curve of the fluorescence intensity after the compound B of Example 2 of the present invention stains DNA and RNA with nucleic acid concentration
  • Figure 6 is a bright-field photomicrograph of live cells stained with Compound B according to Example 2 of the present invention.
  • Fig. 7 is the fluorescence micrograph after the compound B of Example 2 of the present invention stains the living cell
  • Figure 8 is an overlay of the brightfield photomicrograph in Figure 6 and the fluorescence photomicrograph in Figure 7;
  • FIG. 9 shows the fluorescence spectrum after the compound C according to Example 3 of the present invention stains DNA
  • Figure 10 shows the fluorescence spectrum after the compound C according to Example 3 of the present invention stains RNA
  • Fig. 11 shows the variation curve of the fluorescence intensity after the compound C stained with DNA and RNA according to the embodiment 3 of the present invention as a function of nucleic acid concentration
  • Figure 12 is a brightfield micrograph of live cells stained with Compound C according to Example 3 of the present invention.
  • Figure 13 is a fluorescence micrograph of live cells stained with Compound C according to Example 3 of the present invention.
  • Figure 14 shows the fluorescence spectrum of the compound M after DNA staining according to Example 13 of the present invention.
  • Figure 15 shows the fluorescence spectrum of compound M after RNA staining according to Example 13 of the present invention.
  • Fig. 16 shows the variation curve of the fluorescence intensity after the compound M of Example 13 of the present invention stains DNA and RNA as a function of nucleic acid concentration
  • Figure 17 is a brightfield micrograph of live cells stained with Compound M according to Example 13 of the present invention.
  • Figure 18 is a fluorescence micrograph of live cells stained with Compound M according to Example 13 of the present invention.
  • FIG. 19 is a schematic diagram showing the comparison of nucleic acid fluorescence response performance provided in Example 21 of the present invention.
  • alkyl may be understood in its broadest sense to mean any linear, branched or cyclic alkyl substituent.
  • C 1-18 alkyl as used herein generally refers to a saturated hydrocarbon group having 1 to 18 carbon atoms in configuration, C 1-18 alkyl includes, but is not limited to, C 1-12 alkyl, C 1 -6 alkyl, etc.
  • alkyl generally refers to an unsubstituted alkyl.
  • alkyl includes the substituents methyl (Me), ethyl (Et), n-propyl (nPr), isopropyl (iPr), cyclopropyl, n-butyl (nBu), isobutyl (iBu), sec-butyl (sBu), tert-butyl (tBu), cyclobutyl, 2-methylbutyl, n-pentyl, sec-pentyl, tert-pentyl, 2-pentyl, neopentyl, Cyclopentyl, n-hexyl, sec-hexyl, tert-hexyl, 2-hexyl, 3-hexyl, neohexyl, cyclohexyl, 1-methylcyclopentyl, 2-methylpentyl, n-heptyl, 2-heptyl , 3-heptyl, 4-heptyl, cycloheptyl, cyclo
  • carboxy includes any linear, branched or cyclic carboxy substituent.
  • C 1-18 carboxyl as used herein generally refers to a carboxyl substituted group having 1 to 18 carbon atoms in configuration, and C 1-18 carboxyl includes, but is not limited to, C 1-12 carboxyl, C 1-6 carboxyl, etc. .
  • carboxyl refers to a group having a carboxyl group attached to an alkyl group as previously defined.
  • carboxyl exemplarily includes methylcarboxy, ethylcarboxy (carboxymethyl), propylcarboxy (carboxyethyl), butylcarboxy (carboxypropyl), pentylcarboxy (carboxybutyl), hexylcarboxy (carboxypentyl), heptylcarboxy Carboxyl (carboxyhexyl) and octylcarboxy (carboxyheptyl), and their isomers, etc.
  • hydroxy includes any linear, branched or cyclic hydroxy substituent.
  • C 1-18 hydroxy as used herein generally refers to a hydroxy-substituted group having 1 to 18 carbon atoms in configuration, C 1-18 hydroxy including but not limited to C 1-12 hydroxy, C 1-6 hydroxy, etc. .
  • the term hydroxy refers to a group having a hydroxy group attached to an alkyl group as previously defined.
  • the term hydroxy exemplarily includes hydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxybutyl, hydroxypentyl, hydroxyhexyl, hydroxyheptyl, and hydroxyoctyl, isomers thereof, and the like.
  • alkoxy includes any linear, branched or cyclic alkoxy substituent.
  • alkoxy refers to an alkoxy group attached to an alkyl group as previously defined.
  • the term alkoxy exemplarily includes methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert-butoxy and 2-methyl Butoxy, etc.
  • halogen and “halo” may be understood in the broadest sense to mean preferably fluorine, chlorine, bromine or iodine.
  • Blue-green wavelengths are approximately in the range of 420-560 nm.
  • a first aspect of the present invention provides a cyanine compound, which has the structure shown in general formula I,
  • X is selected from the group consisting of C( CH3 ) 2 , O, S and Se.
  • X is selected from the group consisting of C( CH3 ) 2 and S.
  • R 1 and R 2 are each independently selected from the group consisting of H, C 1 -C 18 alkyl, phenyl, OR 6 and halogen.
  • R 1 and R 2 are each independently selected from the group consisting of H, phenyl, C 1 -C 18 alkyl and halogen.
  • R 1 is selected from the group consisting of H, phenyl, C 1 -C 18 alkyl and halogen.
  • R 1 is selected from the group consisting of H, phenyl, C 1 -C 18 alkyl and Cl.
  • R 2 is selected from the group consisting of H, phenyl and halogen.
  • R 2 is H.
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 , benzyl and substituted benzyl group, wherein the substituent of the substituted benzyl group is selected from C 1 -C 18 alkyl, CN, COOH, NH 2 , NO 2 , OH, SH, C 1 -C 6 alkoxy, C 1 -C 6 alkane The group consisting of amino, C 1 -C 6 amido, halogen and C 1 -C 6 haloalkyl.
  • R 3 and R 4 are each independently selected from the group consisting of C 1 -C 18 alkyl, C 1 -C 18 carboxy, C 1 -C 18 hydroxy, C 1 -C 18 NR 5 R 6 and benzyl .
  • R 3 is preferably a group consisting of C 1 -C 18 alkyl, C 1 -C 18 hydroxyl, C 1 -C 18 carboxyl, C 1 -C 18 NR 5 R 6 and benzyl
  • R 4 is preferably C 1 - the group consisting of C 18 alkyl, C 1 -C 18 hydroxy, C 1 -C 18 carboxy and benzyl.
  • R 3 and R 4 are selected with larger polar groups, which can appropriately increase the molecular polarity, reduce the binding force to water-transporting substances such as membrane lipids and proteins in cells, and improve the specific binding to nucleic acids.
  • R 5 and R 6 are each independently selected from the group consisting of H and C 1 -C 18 alkyl. Preferably, R 5 and R 6 are each independently C 1 -C 18 alkyl.
  • Y - is a negative ion.
  • the above-mentioned C 1 -C 18 alkyl group may further be a C 1 -C 12 alkyl group.
  • the C 1 -C 12 alkyl group may further be a C 1 -C 6 alkyl group.
  • the C1 - C6 alkyl group may be the group consisting of methyl, ethyl, propyl and butyl.
  • R 5 and R 6 are each independently ethyl.
  • R3 and R4 may each independently be methyl, and R3 may also be ethyl.
  • the above-mentioned C 1 -C 6 alkyl group may be a straight-chain alkyl group or a branched-chain alkyl group that is an isomer of the straight-chain alkyl group.
  • the above-mentioned C 1 -C 18 carboxyl group may further be a C 1 -C 12 carboxyl group.
  • the C 1 -C 12 carboxyl group may further be a C 1 -C 6 carboxyl group.
  • the C 1 -C 6 carboxyl group may be the group consisting of methylcarboxy, ethylcarboxy, propylcarboxy, butylcarboxy, pentylcarboxy and hexylcarboxy.
  • R 3 and R 4 may each independently be hexylcarboxy (carboxypentyl).
  • the above-mentioned C 1 -C 6 carboxyl group may be a straight-chain carboxyl group, or may be a branched-chain carboxyl group that is an isomer of a straight-chain carboxyl group.
  • the above-mentioned C 1 -C 18 hydroxyl group may further be a C 1 -C 12 hydroxyl group.
  • the C 1 -C 12 hydroxyl group may further be a C 1 -C 6 hydroxyl group.
  • the C 1 -C 6 hydroxyl group may be the group consisting of hydroxymethyl, hydroxyethyl, hydroxypropyl and hydroxybutyl.
  • R4 may be hydroxypropyl.
  • the above-mentioned C 1 -C 6 hydroxyl groups may be straight-chain hydroxyl groups or branched-chain hydroxyl groups that are isomers of straight-chain hydroxyl groups.
  • C 1 -C 18 NR 5 R 6 may further be C 1 -C 12 NR 5 R 6 .
  • C 1 -C 12 NR 5 R 6 may further be C 1 -C 6 NR 5 R 6 .
  • C 1 -C 6 NR 5 R 6 may be composed of -CH 2 NR 5 R 6 , -(CH 2 ) 2 NR 5 R 6 , -(CH 2 ) 3 NR 5 R 6 , -(CH 2 ) 4 NR 5 R 6 and the group consisting of -(CH 2 ) 5 NR 5 R 6 .
  • R 3 and R 4 are each independently -(CH 2 ) 4 NR 5 R 6 .
  • R3 may be 4-(diethylamino)butyl.
  • the cyanine compound according to the present invention has good permeability of living cells, can enter cells to stain nucleic acid without destroying the cell membrane, and has low toxicity and low carcinogenicity.
  • the excitation light of the cyanine compound of the present invention is blue-green light with a small wavelength, which can identify fine particles and improve the detection capability of small particles.
  • the cyanine compound of the present invention can use a common green semiconductor laser as a light source, which greatly reduces the use cost.
  • the cyanine compound of the present invention according to the present invention has a simple structure, readily available raw materials for its preparation, high synthesis yield, and is easy to realize industrialization.
  • the cyanine compound of the present invention can be a compound comprising chemical formula I, chemical formula II, chemical formula III, chemical formula IV, chemical formula V, chemical formula VI, chemical formula VII, chemical formula VIII, chemical formula IX, chemical formula X, chemical formula XI, chemical formula XII, chemical formula The structure shown in one of XIII, chemical formula XIV, chemical formula XV, chemical formula XVI, chemical formula XVII and chemical formula XVIII,
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is benzyl
  • R 4 is benzyl
  • the first step prepares 2-methylthiobenzothiazole (right side of reaction 1) according to following reaction formula I,
  • the resulting mixture was cooled to room temperature, after which it was poured into a large amount of water. Extract 3 times with an appropriate amount of ethyl acetate, and combine the extracted organic phases. The organic phase was washed twice with distilled water, and then dried over anhydrous magnesium sulfate overnight.
  • the reacted mixture was subjected to suction filtration, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.
  • the 3rd step prepares 3-benzyl-2-thione benzothiazole (the right side of reaction formula III) according to following reaction formula III,
  • the reacted mixture was suction filtered, and then the filter cake was washed three times with 50 mL of toluene to obtain a crude product.
  • the 4th step prepares 3-benzyl-2-ethylsulfanyl benzothiazole (reaction formula IV right side) according to following reaction formula IV,
  • the 5th step prepares compound A (reaction formula V right side) according to following reaction formula V,
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is methyl
  • R 4 is methyl
  • the crude yield of the third step reaction is about 30%.
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is benzyl
  • R 4 is methyl
  • the reaction mixture was cooled to room temperature, after which the mixture was precipitated and filtered, and the filter cake was washed with dichloromethane.
  • the filtrate was dried to obtain a brownish-yellow solid powder, which was 3-benzyl-2-thionebenzothiazole, and the crude yield was about 25%.
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is 4-(diethylamino)butyl
  • R 4 is methyl
  • the microwave tube was heated to 60°C by microwave and the reaction was maintained for 2 hours. After it was cooled down, a large amount of solid was precipitated.
  • the reaction solution was filtered, the obtained filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The filtrate was dried in vacuo to give 2.0 g of a brown solid as 1-methyl-4-iodopyridine quaternary ammonium salt.
  • the reaction vial was microwaved to 60°C for 2 hours. Then, the reaction solution is cooled and filtered, and after the filtrate is concentrated, it is separated by a silica gel column, and a mixture of dichloromethane and methanol is used as an eluent in the separation process.
  • the isolated product was collected to obtain 400 mg of yellow solid powder, namely 3-(4-bromobutyl)-2-((1-methylpyridine-4(1H)-methylene)methyl)benzothiazole, which was The yield is about 32%.
  • the fourth step weigh 0.88mmol (400mg, 1eq) of the 3-(4-bromobutyl)-2-((1-methylpyridine-4(1H)-methylene)methan obtained in the third step base) benzothiazole, 4.4 mmol (321 mg, 5 eq) of diethylamine were weighed and both were added to a 20 mL capacity microwave tube.
  • the above microwave tube was microwave heated to 60°C and maintained for 2 hours. After that, the reaction solution was cooled and filtered, the filtrate was concentrated, and then separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent in the separation process.
  • the isolated product was collected to obtain 150 mg of yellow solid powder, namely compound D, whose name was 3-(4-(diethylamino)butyl)-2-((1-methylpyridine-4(1H)-methylene) ) methyl) benzothiazole quaternary ammonium salt.
  • the yield of compound D was about 38%.
  • X is S
  • R 1 is Cl
  • R 2 is H
  • R 3 is methyl
  • R 4 is methyl
  • reaction solution was evaporated to dryness and separated through a neutral silica gel column.
  • a mixed solvent of dichloromethane and methanol was used as the eluent.
  • the separated yellow components were collected and evaporated to dryness to finally obtain a yellow solid powder, namely Compound E, with a crude yield of about 20%.
  • X is C(CH 3 ) 2
  • R 1 is H
  • R 2 is H
  • R 3 is ethyl
  • R 4 is methyl
  • the microwave tube was microwaved to 140°C, and the reaction was carried out for 3 hours. After the reaction was completed, the temperature of the microwave tube was cooled, and a large amount of solid was precipitated. The reaction solution was filtered, the filter cake was slurried with 20 mL of ethyl acetate at room temperature, and filtered again. The finally obtained solid was dried in vacuo to finally obtain 7.34 g of a red solid, which was 1-ethyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt.
  • the third step weigh 3.17mmol (1g, 1eq) of the 1-ethyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt obtained in the first step, and dissolve it in the reaction flask in 10 mL of acetonitrile.
  • the reaction solution was extracted with dichloromethane, and the organic phase obtained by extraction was washed with water, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated. Then, the concentrated product was separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent during the separation process, and 500 mg of a yellow oily crude product was collected. The crude product was treated with Pre-HPLC to obtain 100 mg of the final product as a yellow oil, which was compound F.
  • X is C(CH 3 ) 2
  • R 1 is H
  • R 2 is H
  • R 3 is benzyl
  • R 4 is methyl
  • the 3rd step in the reactor, add 10 milliliters of methylene chloride, take by weighing the 1-benzyl-2,3,3-trimethyl-3-hydroindole quaternary ammonium salt 3mmol ( 1 g, 1 eq), which was dissolved in dichloromethane. Then add 1 mL of methanol to the reactor
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is methyl
  • R 4 is hydroxypropyl
  • reaction solution was evaporated to dryness and separated through a neutral silica gel column, and a mixed solvent of dichloromethane and methanol was used as the eluent in the separation process.
  • the separated yellow components were collected, evaporated to dryness, and finally a yellow solid powder was obtained, which was compound H, and the crude yield was about 20%.
  • X is C(CH 3 ) 2
  • R 1 is H
  • R 2 is H
  • R 3 is hexylcarboxy
  • R 4 is methyl
  • the reaction solution was extracted with dichloromethane, and the organic phase obtained by extraction was washed with water, washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate and concentrated. Then, the concentrated product was separated through a silica gel column, and a mixture of dichloromethane and methanol was used as an eluent during the separation process, and 500 mg of a yellow oily crude product was collected. The crude product was treated with Pre-HPLC to obtain 100 mg of the final product as a yellow oil, which was compound I.
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is carboxypentyl
  • R 4 is methyl
  • X is S
  • R 1 is H
  • R 2 is H
  • R 3 is hydroxypropyl
  • R 4 is methyl
  • the first step 4.88 mmol of 4-iodopyridine and 9.76 mmol of methyl iodide were added to a round-bottomed flask with a capacity of 25 mL and contained 5 mL of tetrahydrofuran, and the reaction was refluxed at a temperature of 75° C. for 3 h.
  • the mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brown-yellow solid powder, which was 4-iodo-1-methylpyridine quaternary ammonium salt, and the crude yield was about 93%.
  • the second step 670.19 mmol of 2-methylbenzothiazole was added to a double-necked round-bottomed flask with a capacity of 25 mL, and 804.23 mmol of 3-bromo-1-propanol was slowly added dropwise to it. The reaction was continued for about 8 h, and the reactor was cooled to room temperature after completion. The resulting mixture was added to diethyl ether for standing precipitation and filtered, after which the filter cake was washed with a large amount of diethyl ether. A green solid was obtained after drying, which was 3-(3-hydroxypropyl)-2-methylbenzothiazole in about 54% crude yield.
  • the third step 4mL anhydrous methanol is charged into the double-necked round-bottomed flask of 25mL capacity, then 480.08mmol of the obtained 3-(3-hydroxypropyl)- 2-methylbenzothiazole, then add 576.10 mmol of NaHCO 3 (dissolved in 2 mL of water), stir at room temperature for 0.5 h, and then add 576.10 mmol of 4-iodo-1-methyl obtained in the first step above
  • the pyridine was added to a double-necked round-bottomed flask, and the reaction was carried out overnight at 110° C. under reflux conditions.
  • reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered.
  • the crude product obtained by filtration was separated through a neutral silica gel column (the eluent was a mixed solvent of dichloromethane and methanol), and the separated yellow components were collected and evaporated to dryness. Finally, a yellow solid powder is obtained, which is compound K, and its crude yield is about 1.12%.
  • X is S
  • R 1 is methyl
  • R 2 is H
  • R 3 is methyl
  • R 4 is methyl
  • the first step put 5mL of tetrahydrofuran into a 25mL capacity round-bottomed flask, then add 4.88mmol of 4-iodopyridine and 9.76mmol of iodomethane, and react at 75°C and reflux for 3h.
  • the mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brownish-yellow solid powder, which was 4-iodo-1-picoline quaternary ammonium salt, and the crude yield was about 93%.
  • Step 2 Add 612.60 mmol of 2,6-dimethylbenzothiazole to a double-necked round-bottomed flask with a capacity of 10 mL, slowly add 1.23 mmol of methyl iodide dropwise while stirring, and then at a temperature of 70 °C Reflux reaction for 1.5h. After the reaction was completed, the reactor was cooled to room temperature, and ethyl acetate was added to the mixture obtained by the reaction for precipitation and filtration, and then the filter cake was washed with a large amount of ethyl acetate. The filtrate was dried to obtain a white solid, which was 2,3,6-trimethylbenzothiazole, in a crude yield of about 98%.
  • the third step put 4mL of anhydrous methanol into a double-necked round-bottomed flask with a capacity of 25mL, and then add 560.94mmol of 2,3,6-trimethylbenzothiazole obtained in the second step above and 1.12mmol of NaHCO. 3 (dissolved in 1 mL of water), stirred at room temperature for 0.5 h. After dissolving 673.13 mmol of 4-iodo-1-methylpyridine obtained in the first step above in 5 mL of anhydrous methanol, it was added dropwise into a round-bottomed flask, and the reaction was carried out at 110° C. and reflux for 8 h.
  • the obtained reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered.
  • the crude product obtained by filtration is separated by a neutral silica gel column (the mixed solvent of dichloromethane and methanol is used as the eluent), the yellow component obtained by the separation is collected and evaporated to dryness, and finally a yellow solid powder is obtained, which is compound L.
  • the yield is about 4.2%.
  • X is S
  • R 1 is phenyl
  • R 2 is H
  • R 3 is methyl
  • R 4 is methyl
  • the first step put 5mL of tetrahydrofuran into a 25mL capacity round-bottomed flask, then add 4.88mmol of 4-iodopyridine and 9.76mmol of iodomethane, and react at 75°C and reflux for 3h.
  • the mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a brownish-yellow solid powder, which was 4-iodo-1-picoline quaternary ammonium salt, and the crude yield was about 93%.
  • the second step 501.83 mmol of 2-methylnaphthalenethiazole was added to a double-necked round-bottomed flask with a capacity of 25 mL, then 4 mL of chloroform was added, and 1.00 mmol of methyl iodide was slowly added dropwise while stirring.
  • the reaction was carried out under reflux for about 8h.
  • the reactor was cooled to room temperature, and diethyl ether was added to the obtained mixture for precipitation and filtration, and the filter cake was washed with a large amount of diethyl ether.
  • the filtrate was dried to obtain a yellow solid, which was 2,3-dimethylnaphthalenethiazole, and the crude yield was about 56.35%.
  • the 3rd step 4mL anhydrous methanol was charged into the double-necked round bottom flask of 25mL, then 466.62mmol of the 2,3-dimethylnaphthalene thiazole obtained in the second step was added, and 559.95mmol of NaHCO (dissolved ) was added. in 2 mL of water) and stirred at room temperature for 0.5 h. Then, 559.95 mmol of 4-iodo-1-methylpyridine obtained in the first step was added into the reactor, and the reaction was carried out under reflux at a temperature of 110° C. overnight.
  • the resulting reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered.
  • the crude product obtained by filtration is separated by a neutral silica gel column (mixed solvent of dichloromethane and methanol is used as eluent), the separated yellow component is collected, evaporated to dryness, and finally a yellow solid powder is obtained, which is compound M,
  • the crude yield was about 1.40%.
  • the specific preparation method of compound N is as follows: the first step: put 50 mL in a 25 mL round-bottomed flask, then add 50 mmol of sodium hydride and 46 mmol of 2-mercapto, 6-nonane benzothiazole, and reflux at 75 ° C. Condition reaction for 3h. The mixture obtained by the reaction was thoroughly washed with ethyl acetate, and dried to obtain a pale yellow solid powder, which was 2-mercaptomethyl and 6-nonanebenzothiazole, and the crude yield was about 93%.
  • Step 2 Add 45 mmol of 2-mercaptomethyl and 6-nonane benzothiazole into a double-necked round-bottomed flask with a capacity of 25 mL, then add 4 mL of chloroform, and slowly add 1.00 mmol of iodine dropwise while stirring Methane, and then react at 80°C and reflux for about 8h. After the reaction was completed, the reactor was cooled to room temperature, and diethyl ether was added to the obtained mixture for precipitation and filtration, and the filter cake was washed with a large amount of diethyl ether. The filtrate was dried to obtain a yellow solid, which was 1-methyl, 2-mercaptomethyl, 6-nonanebenzothiazole, and the crude yield was about 56.35%.
  • the third step put 4mL of anhydrous methanol into a 25mL double-necked round-bottomed flask, then add 40mmol of 1-methyl, 2-mercaptomethyl, 6-nonane benzothiazole obtained in the second step above, and then Add 559.95 mmol DIEA (dissolved in 2 mL of water), and stir at 80 °C for 0.5 h. Then, 559.95 mmol of 4-iodo-1-methylpyridine obtained in the first step was added into the reactor, and the reaction was carried out under reflux at 80° C. overnight. The resulting reaction solution was washed with water, extracted with dichloromethane, dried and concentrated, and finally crystallized by adding ethyl acetate and filtered.
  • the crude product obtained by filtration is separated by a neutral silica gel column (the mixed solvent of dichloromethane and methanol is used as the eluent), the separated yellow components are collected, evaporated to dryness, and finally a yellow solid powder is obtained, which is compound M,
  • the crude yield was about 5.8%.
  • test result conforms to the structure of chemical formula XVIII.
  • Calf thymus DNA and RNA were stained with the compounds synthesized in Examples 1-14, and absorption (excitation) and fluorescence (emission) spectra were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively. The result is that the excitation light wavelengths of the compounds synthesized in Examples 1-14 are in the blue-green range.
  • Live HeLa cells were stained with the compounds synthesized in Examples 1-14 and observed using a confocal laser scanning microscope. The results show that the compounds synthesized in Examples 1-14 can stain HeLa cells without destroying the cell membrane, and the images are clear.
  • the configuration concentrations are 10 ⁇ g/mL, 20 ⁇ g/mL, 30 ⁇ g/mL, 40 ⁇ g/mL, 50 ⁇ g/mL, 60 ⁇ g/mL, 70 ⁇ g/mL, 80 ⁇ g/mL, 90 ⁇ g/mL, 100 ⁇ g/mL, 110 ⁇ g/mL, 120 ⁇ g/mL mL, 130 ⁇ g/mL, 140 ⁇ g/mL, 150 ⁇ g/mL, 200 ⁇ g/mL of calf thymus DNA in water.
  • a certain amount of compound B was dissolved in DMSO (dimethyl sulfoxide), and a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH of 7.4 and a concentration of 10 mmol/L was added thereto to prepare a certain amount of compound B. concentration of Compound B in buffer solution.
  • a certain amount of compound B buffer solution was mixed with a certain amount of the above-mentioned aqueous solutions of calf thymus DNA with different concentrations, placed in a cuvette and left at 37°C for 3 min, and then the absorption spectrum was measured. And a certain amount of compound B buffer solution and a certain amount of water were used as a comparative test.
  • the instrument used was an ultraviolet-visible spectrophotometer, model Hp8453.
  • the absorption spectrum of compound B as a function of DNA concentration is shown in Figure 1.
  • the nucleic acid concentrations represented by the different curves from top to bottom along the ordinate increase sequentially.
  • the highest curve represents 0 ⁇ g/mL, that is, no DNA;
  • the lowest curve represents the nucleic acid concentration of 200 ⁇ g/mL.
  • compound B has the strongest absorption of light with a wavelength of about 440 nm, and the light near this wavelength is blue-green light.
  • the fluorescence spectrum of compound B as a function of DNA concentration is shown in Figure 2.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn.
  • the curve at the highest position represents a DNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position represents 0 ⁇ g/mL, that is, no DNA.
  • the fluorescence emitted by the excited compound B has the highest intensity near 485 nm, indicating that the compound B can successfully emit fluorescence and can be used as a fluorescent dye.
  • the staining experiment of compound B on calf thymus RNA is similar to the above-mentioned DNA staining experiment process, and will not be repeated here.
  • the absorption spectrum of compound B with the change of calf thymus RNA concentration is shown in FIG. 3
  • the fluorescence spectrum of compound B with the change of calf thymus RNA concentration is shown in FIG. 4 .
  • Fig. 3 taking 440 nm as an example, the nucleic acid concentrations represented by different curves from top to bottom along the ordinate increase sequentially. Among them, the highest curve represents 0 ⁇ g/mL, that is, no RNA; the lowest curve represents the RNA concentration of 200 ⁇ g/mL. It can be seen from the figure that compound B has the strongest absorption of light with a wavelength of about 440 nm, and the light near this wavelength is blue-green light.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease sequentially.
  • the curve at the highest position represents the RNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position represents 0 ⁇ g/mL, that is, no RNA. It can be seen from the figure that the fluorescence emitted by the excited compound B has the highest intensity near 485 nm, indicating that the compound B can successfully emit fluorescence and can be used as a fluorescent dye.
  • compound B has good absorption of blue-green laser with short wavelength, and the short wavelength is conducive to the identification of small particle objects.
  • Common semiconductor green laser or blue laser can be used as the light source, and the cost is low.
  • Figure 5 shows a graph of the fluorescence intensity of compound B stained for DNA and RNA as a function of nucleic acid concentration. In the figure, it is more clearly shown that as the nucleic acid concentration increases, the fluorescence intensity of compound B is higher. In the absence of nucleic acid, compound B is excited and hardly emits light, which is beneficial to reduce the background interference of background fluorescence and can improve the sensitivity.
  • the staining test of compound B on live HeLa cells was a confocal laser scanning microscope, model FV1000IX81, Japan.
  • Compound B was added to PBS buffer to prepare a compound B buffer solution with a concentration of 1 mmol/L.
  • HeLa cells were cultured in a six-well plate, and 10 ⁇ L of the above compound B buffer solution was added thereto. It was then incubated for 30 min in a cell culture incubator at 37°C and 5% CO 2 .
  • the cells were washed three times with PBS, and then the cell culture medium was added, and the cell morphology was observed using a confocal laser scanning microscope. The observation results are shown in the bright field micrograph of the representative area shown in Fig. 6, from which the living cells with complete morphology can be clearly seen.
  • FIG. 8 is an overlay of the brightfield micrograph of FIG. 6 and the fluorescence micrograph of FIG. 7 . In the figure, it can be seen more clearly that the nuclei are stained and the cell structure is intact.
  • Compound B can effectively penetrate the living cell membrane to stain nucleic acids without destroying the cell structure, so that it can be stained and observed in the state of cell survival, which is more conducive to the morphology and type of cells. to identify.
  • Figure 9 shows the fluorescence spectrum of compound C as a function of calf thymus DNA concentration.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn.
  • the curve at the highest position represents a DNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position (almost overlapping the horizontal axis) represents 0 ⁇ g/mL, that is, no DNA.
  • the fluorescence emitted by the excited compound C has the highest intensity near 485 nm, indicating that the compound C can successfully emit fluorescence and can be used as a fluorescent dye.
  • Figure 10 shows the fluorescence spectrum of Compound C as a function of calf thymus RNA concentration.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn.
  • the curve at the highest position represents the RNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position represents 0 ⁇ g/mL, that is, no RNA. It can be seen from the figure that the fluorescence emitted by the excited compound C has the highest intensity near 485 nm, indicating that the compound C can successfully emit fluorescence and can be used as a fluorescent dye.
  • Figure 11 shows a graph of the fluorescence intensity of compound C after staining for DNA and RNA as a function of nucleic acid concentration. In the figure, it is more clearly shown that as the nucleic acid concentration increases, the fluorescence intensity of compound C is higher.
  • Figure 12 shows a bright-field micrograph of a representative area of HeLa live cells stained by compound C, from which the morphologically intact live cells can be clearly seen, and the cell structure is intact and not damaged.
  • Figure 13 shows fluorescence micrographs of representative regions of compound C staining of live HeLa cells. From the figure, it can be clearly seen that each cell can emit fluorescence, which proves that HeLa has been successfully stained with high identification.
  • Figure 14 shows the fluorescence spectrum of Compound M as a function of calf thymus DNA concentration.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease in turn.
  • the curve at the highest position represents a DNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position (almost overlapping the horizontal axis) represents 0 ⁇ g/mL, that is, no DNA. It can be seen from the figure that the fluorescence emitted by the excited compound M has the highest intensity near 520 nm, indicating that the compound M can successfully emit fluorescence and can be used as a fluorescent dye.
  • Figure 15 shows the fluorescence spectrum of Compound M as a function of calf thymus RNA concentration.
  • the nucleic acid concentrations represented by different curves from top to bottom along the ordinate decrease sequentially.
  • the curve at the highest position represents the RNA concentration of 200 ⁇ g/mL;
  • the curve at the lowest position represents 0 ⁇ g/mL, that is, no RNA. It can be seen from the figure that the fluorescence emitted by the excited compound M has the highest intensity near 520 nm, indicating that the compound M can successfully emit fluorescence and can be used as a fluorescent dye.
  • Figure 16 shows a graph of the fluorescence intensity of compound M after staining for DNA and RNA as a function of nucleic acid concentration. In the figure, it is more clearly shown that as the nucleic acid concentration increases, the fluorescence intensity of compound M is higher.
  • Figure 17 shows a bright-field micrograph of a representative area of HeLa living cells stained by compound M, from which the living cells with intact morphology can be clearly seen, and the cell structure is intact and not damaged.
  • Figure 18 shows fluorescence photomicrographs of representative regions of Compound M staining of live HeLa cells. From the figure, it can be clearly seen that each cell can emit fluorescence, which proves that HeLa has been successfully stained with high identification.
  • R1 being a phenyl group In order to study the effect of R1 being a phenyl group on the fluorescence response of nucleic acid, 0, 10, 20, 30 and 40 ⁇ g/ml DNA were added to compound B and compound N at the same concentration, and the fluorescence intensity of the added system was detected. The results are shown in Figure 19, the fluorescence intensity of compound N was significantly higher than that of compound B, which indicated that R1 being a phenyl group significantly enhanced the responsiveness of the compound to nucleic acid, making it more suitable for nucleic acid biological staining. Other compounds in which R1 is phenyl also showed similar results compared to the compounds in which R1 was hydrogen, and had higher nucleic acid fluorescence response performance.
  • the compounds of Example 1 to Example 14 of the present invention can penetrate the living cell membrane without destroying the original cell structure, and can effectively stain nucleic acids; and the wavelength range of the absorbed light is located in the blue-green light region , can use blue or green laser, low cost.
  • Example 2 Compound B blue-green light can no Example 3 Compound C blue-green light can no Example 4 Compound D blue-green light can no Example 5 Compound E blue-green light can no Example 6 Compound F blue-green light can no Example 7 Compound G blue-green light can no Example 8 Compound H blue-green light can no Example 9 Compound I blue-green light can no Example 10 Compound J blue-green light can no Example 11 Compound K blue-green light can no Example 12 Compound L blue-green light can no Example 13 Compound M blue-green light can no Example 14 Compound N blue-green light can no
  • the cyanine compounds of the present invention can be used as dyes, especially as fluorescent dyes, as referred to in the second aspect of the present invention.
  • the above-mentioned fluorescent dye containing the cyanine compound of the present invention can be used for quantitative detection of nucleic acid and/or biological staining.
  • it can be used in live cell staining.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Plural Heterocyclic Compounds (AREA)

Abstract

L'invention concerne un composé de cyanine, un colorant contenant le composé de cyanine et l'utilisation du composé de cyanine. Le composé de cyanine présente une structure de formule générale I, dans laquelle X est choisi dans un groupe constitué par C(CH3)2, O, S et Se ; R1 et R2 sont chacun indépendamment choisis dans le groupe constitué par H, un groupe alkyle en C1-C18, phényle, OR6 et un atome d'halogène ; et R3 et R4 sont chacun indépendamment choisis dans le groupe constitué par un alkyle en C1-C18, un carboxyle en C1-C18, un hydroxyle en C1-C18, C1-C18 NR5R6, benzyle et un benzyle substitué, un substituant du benzyle substitué étant choisi dans un groupe constitué par un alkyle en C1-C18}, CN, COOH, NH2, NO2, OH, SH, un alcoxy en C1-C6, un alkylamino en C1-C6, un acylamino en C1-C6, un atome d'halogène et un alkyle halogéné en C1-C6. R5 et R6 sont chacun indépendamment choisis dans le groupe constitué par H et un alkyle en C1-C18. Y- est un anion. Le composé de cyanine selon la présente invention présente une bonne perméabilité aux cellules vivantes et est capable de pénétrer dans une cellule sans endommager la membrane cellulaire et de colorer l'acide nucléique ; et la lumière d'excitation est une lumière bleu-vert à petite longueur d'onde.
PCT/CN2021/130477 2020-11-13 2021-11-12 Composé de cyanine, colorant contenant le composé de cyanine et utilisation du composé de cyanine Ceased WO2022100716A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202180075741.4A CN116490500A (zh) 2020-11-13 2021-11-12 菁类化合物、含菁类化合物的染料以及菁类化合物的应用

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PCT/CN2020/128838 WO2022099658A1 (fr) 2020-11-13 2020-11-13 Composé de cyanine, colorant contenant un composé de cyanine et application d'un composé de cyanine
CNPCT/CN2020/128838 2020-11-13

Publications (1)

Publication Number Publication Date
WO2022100716A1 true WO2022100716A1 (fr) 2022-05-19

Family

ID=81600788

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2020/128838 Ceased WO2022099658A1 (fr) 2020-11-13 2020-11-13 Composé de cyanine, colorant contenant un composé de cyanine et application d'un composé de cyanine
PCT/CN2021/130477 Ceased WO2022100716A1 (fr) 2020-11-13 2021-11-12 Composé de cyanine, colorant contenant le composé de cyanine et utilisation du composé de cyanine

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/128838 Ceased WO2022099658A1 (fr) 2020-11-13 2020-11-13 Composé de cyanine, colorant contenant un composé de cyanine et application d'un composé de cyanine

Country Status (2)

Country Link
CN (1) CN116490500A (fr)
WO (2) WO2022099658A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658751A (en) * 1993-04-13 1997-08-19 Molecular Probes, Inc. Substituted unsymmetrical cyanine dyes with selected permeability
CN1226928A (zh) * 1996-05-31 1999-08-25 米凯尔·库比斯塔 核酸分析的探针
CN102112875A (zh) * 2008-07-30 2011-06-29 希森美康株式会社 宫颈部异常细胞检测用试剂及用该试剂检测宫颈部异常细胞的方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1242962A (fr) * 1959-08-25 1960-10-07 Kodak Pathe Nouveau procédé pour la préparation de colorants sensibilisateurs de la série des cyanines et des mérocyanines et produits obtenus
US5321130A (en) * 1992-02-10 1994-06-14 Molecular Probes, Inc. Unsymmetrical cyanine dyes with a cationic side chain
US5534416A (en) * 1993-04-13 1996-07-09 Molecular Probes, Inc. Fluorescent viability assay using cyclic-substituted unsymmetrical cyanine dyes
JP5306811B2 (ja) * 2005-05-11 2013-10-02 ライフ テクノロジーズ コーポレーション ２本鎖ｄｎａへの高い選択性を有する蛍光化学物質及びそれらの使用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5658751A (en) * 1993-04-13 1997-08-19 Molecular Probes, Inc. Substituted unsymmetrical cyanine dyes with selected permeability
CN1226928A (zh) * 1996-05-31 1999-08-25 米凯尔·库比斯塔 核酸分析的探针
CN102112875A (zh) * 2008-07-30 2011-06-29 希森美康株式会社 宫颈部异常细胞检测用试剂及用该试剂检测宫颈部异常细胞的方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIVER, T. BOGGIONI, A. SECCO, F. TURRIANI, E. VENTURINI, M. YARMOLUK, S.: "Influence of cyanine dye structure on self-aggregation and interaction with nucleic acids: A kinetic approach to TO and BO binding", ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ACADEMIC PRESS, US, vol. 465, no. 1, 23 August 2007 (2007-08-23), US , pages 90 - 100, XP022211409, ISSN: 0003-9861, DOI: 10.1016/j.abb.2007.04.034 *
LEE L. G., CHEN C.-H., CHIU L. A.: "THIAZOLE ORANGE: A NEW DYE FOR RETICULOCYTE ANALYSIS.", CYTOMETRY, ALAN LISS, NEW YORK., US, vol. 07., 1 January 1986 (1986-01-01), US , pages 508 - 517., XP000613662, ISSN: 0196-4763, DOI: 10.1002/cyto.990070603 *
NETZEL THOMAS L., NAFISI KAMBIZ, ZHAO MIN, LENHARD JEROME R., JOHNSON IAIN: "Base-Content Dependence of Emission Enhancements, Quantum Yields, and Lifetimes for Cyanine Dyes Bound to Double-Strand DNA: Photophysical Properties of Monomeric and Bichromomphoric DNA Stains", THE JOURNAL OF PHYSICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY, vol. 99, no. 51, 1 December 1995 (1995-12-01), pages 17936 - 17947, XP055929526, ISSN: 0022-3654, DOI: 10.1021/j100051a019 *

Also Published As

Publication number Publication date
WO2022099658A1 (fr) 2022-05-19
CN116490500A (zh) 2023-07-25

Similar Documents

Publication Publication Date Title
EP1678258B1 (fr) Reactifs de marquage a colorants de cyanine
Ernst et al. Cyanine dye labeling reagents for sulfhydryl groups
CN102115610B (zh) 一类荧光染料、制备方法及其应用
Kim et al. Far-red/near-infrared emitting, two-photon absorbing, and bio-stable amino-Si-pyronin dyes
WO2020124688A1 (fr) Colorants fluorescents à haute stabilité, à haute luminosité et à spectre complet, et synthèse et application de ceux-ci
CS273617B2 (en) Method of resorufine's derivatives production
WO1996000902A1 (fr) Colorants a base de cyanine substitues par des ions n-heteroaromatiques et par des ions iminium, utilises comme marques fluorescentes
US6403625B1 (en) Fluorescent labeling reagents
WO2011059457A1 (fr) Composés luminescents haute performance
CN108504130B (zh) 一种花菁类荧光染料及其合成方法
CN105623647B (zh) 一种检测细胞内co的荧光探针及其制备方法和应用
CN102702769B (zh) 一类绿光荧光菁染料、制备方法及其应用
CN113999254A (zh) 一种苯并噻二唑并咪唑类荧光染料及其合成方法
JP5730311B2 (ja) 反応性シアニン化合物
CN102807588B (zh) 一种原位检测生物体内相邻巯基蛋白质的化合物及其方法
JP2010505991A (ja) フルオロ置換ベンゾオキサゾールポリメチン色素
CN112341453A (zh) 一种基于香豆素的荧光探针及其制备方法和应用
EP2157088A1 (fr) Colorants d'indocyanine fluorescents et leurs dérivés destinés à l'analyse de macromolécules biologiques
WO2022100716A1 (fr) Composé de cyanine, colorant contenant le composé de cyanine et utilisation du composé de cyanine
CN114702447A (zh) 一种萘酰亚胺衍生物及其制备方法与应用
CN116239893B (zh) 一种季铵盐修饰的花菁类荧光染料及其合成方法和应用
CN112939960A (zh) 羰基氮杂环丁烷取代的nbd类荧光染料及其合成方法和应用
CN111333621B (zh) 一种488nm激发的免洗Halo-tag探针及其合成和生物应用
WO2020186483A1 (fr) Colorant de pentaméthine cyanine et son procédé de préparation
CN111333618A (zh) 一种488nm激发的免洗SNAP-tag探针及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21891236

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202180075741.4

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21891236

Country of ref document: EP

Kind code of ref document: A1