CN110627715B - Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe - Google Patents
Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe Download PDFInfo
- Publication number
- CN110627715B CN110627715B CN201910798217.4A CN201910798217A CN110627715B CN 110627715 B CN110627715 B CN 110627715B CN 201910798217 A CN201910798217 A CN 201910798217A CN 110627715 B CN110627715 B CN 110627715B
- Authority
- CN
- China
- Prior art keywords
- intermediate product
- quinoline
- aromatic vinyl
- reaction
- vinyl derivative
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
- C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
- C07D215/12—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring 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
- C09B23/14—Styryl dyes
- C09B23/145—Styryl dyes the ethylene chain carrying an heterocyclic residue, e.g. heterocycle-CH=CH-C6H5
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Optics & Photonics (AREA)
- Engineering & Computer Science (AREA)
- Plural Heterocyclic Compounds (AREA)
Abstract
The invention relates to the technical field of nucleic acid detection, in particular to a quinoline aromatic vinyl derivative and application thereof in preparation of a fluorescent dye and a fluorescent probe. The quinoline aromatic vinyl derivative is a compound shown in the formula I or a pharmaceutically acceptable salt thereof, and a solvate, an enantiomer, a diastereoisomer, a tautomer or a mixture of the compound shown in the formula I or the pharmaceutically acceptable salt thereof in any proportion, including a racemic mixture. The compound has the advantages of simple preparation method, higher fluorescence response to nucleic acid, lower biotoxicity and phototoxicity, better water solubility and cell permeability, strong binding constant in the aspect of detection and lower detection limit, and can be applied to preparation of fluorescent dyes and fluorescent probes.
Description
Technical Field
The invention relates to the technical field of nucleic acid detection, in particular to a quinoline aromatic vinyl derivative and application thereof in preparation of a fluorescent dye and a fluorescent probe.
Background
Nucleic acid is not only a basic component of all biological cells, but also plays an important role in the growth, development, reproduction, heredity, variation and other major life phenomena of organisms. Nucleic acid macromolecules are divided into two classes: deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) play a role in storing and transmitting genetic information in the replication and synthesis of proteins.
The G-quadruplex (G-quadruplex) is a specific secondary structure of nucleic acids. Many guanine-rich regions of the human genome have the ability to form this structure, including the terminal guanine repeat of the telomere, as well as promoter regions for a variety of genes, such as the c-kit, c-myc, bc1-2, pdgf, hras, vegf, rb, and insulin groups, among others. The G-quadruplex structure has polymorphism, the number and orientation of chains, the connection mode of loop, the glycoside torsion angle of guanine, metal ions coordinated with a negative charge center of carbonyl and the like, determines the type and conformation of the G-quadruplex, and the differences also provide a plurality of recognition points for proteins and small molecular compounds. G-quadruplexes are classified into three conformations, namely, a positive parallel conformation, a negative parallel conformation and a mixed conformation according to the orientation of the strands.
The formation of the G-quadruplex has a regulatory effect on a series of physiological processes in vivo. Studies have shown that the G-quadruplex structure of certain promoter regions significantly affects the level of transcription and translation of genes, and thus is thought to function as a molecular switch, whose formation and disassembly may involve a series of in vivo important physiological processes, such as signal transduction, apoptosis and cell proliferation. Therefore, the method can specifically detect the existence or formation of the G-quadruplex structure in vivo or in vitro tests, and has very important roles in researching relevant biological functions of the G-quadruplex structure, developing anti-cancer drugs taking the G-quadruplex structure as a target point and the like.
With the development of biotechnology, the conventional method for sequencing DNA molecules by isotope effect cannot meet the requirements; fluorescent labeling has been widely regarded and rapidly developed as a labeling technique with the advantages of high detection speed, good repeatability, small sample size, no radiation, and the like. At present, two types of fluorescent probes have been discovered, one is a fluorescent probe containing an active group, such as rhodamine, fluorescein, acridine and stilbene, and the other is an embedded fluorescent probe which reacts with nucleic acid in a manner of being inserted into a nucleic acid structure with affinity. The rigidity and selectivity of the synthesized stain to G-quadruplexes are not high at present, and the fluorescent probe designed by the inventor has the advantages of rapidness, sensitivity, trace quantity, no fluorescence quenching and the like, and has important significance in researching the structure of the G-quadruplexes in the distribution and biological function mechanism in cells.
Disclosure of Invention
In order to overcome the disadvantages and shortcomings of the prior art, the invention provides a quinoline aromatic vinyl derivative which has high fluorescence response to nucleic acid.
Another object of the present invention is to provide a process for producing the above quinoline arylethylene derivative.
The invention also aims to provide application of the quinoline aromatic vinyl derivative.
The purpose of the invention is realized by the following technical scheme:
a quinoline aromatic vinyl derivative, which is a compound shown in formula I or a pharmaceutically acceptable salt thereof, and a solvate, enantiomer, diastereomer, tautomer or a mixture thereof in any proportion, including a racemic mixture, of the compound shown in formula I or the pharmaceutically acceptable salt thereof;
wherein n is 1 to 10; x is Cl, br, I or benzenesulfonic acid group;
the HA is hydrochloric acid or acetic acid;
the quinoline aromatic vinyl derivative is preferably the following compound:
the X is I, and the HA is hydrochloric acid;
the preparation method of the quinoline aromatic vinyl derivative comprises the following steps:
(1) Mixing 2-methylquinoline, methyl iodide and sulfolane, and reacting at 50-70 ℃; after the reaction is finished, cooling, adding ethyl acetate to precipitate a solid, and filtering to obtain an intermediate product I (1,2-dimethyl-1-iodoquinoline);
(2) Mixing the intermediate product I prepared in the step (1) with a solvent, adding an aromatic aldehyde analogue (R-CHO) and a catalyst, and reacting at 30-60 ℃; after the reaction is finished, cooling, adding petroleum ether to precipitate solid, and filtering to obtain an intermediate product II (1-methyl-2- (substituted aromatic vinyl) -1-iodoquinoline);
(3) Mixing phosphorus oxychloride and N, N-Dimethylformamide (DMF) under the anhydrous and anaerobic conditions, and stirring for 20-40 min at the temperature of 0-5 ℃ to obtain a Vilsmeier reagent; dissolving the intermediate product II prepared in the step (2) in N, N-Dimethylformamide (DMF) to obtain an intermediate product II solution, then dropwise adding the intermediate product II solution into a Vilsmeier reagent, and reacting at 60-80 ℃; adding ice water to quench the reaction after the reaction is finished, adjusting the pH value of the system to be more than 10, and filtering to obtain an intermediate product III (1-methyl-2- (substituted aromatic vinyl) -4-formyl-1-iodoquinoline);
(4) Mixing the intermediate product III prepared in the step (3), 1,3 diaminoguanidine salt and a solvent, then dropwise adding a catalyst, reacting at 30-60 ℃, cooling after the reaction is finished, adding ethyl acetate to precipitate solids, and filtering to obtain a crude product; recrystallizing the crude product with anhydrous ethanol to obtain quinoline aromatic ethylene derivative (1-methyl-2- (substituted aromatic vinyl) -4- (1,3-diamino guanidino) -1-iodo quinoline salt (IV));
the molar ratio of 2-methylquinoline, methyl iodide and sulfolane in step (1) is preferably 1: (2-4): (10-15), the total volume ratio of the ethyl acetate to the 2-methylquinoline, the methyl iodide and the sulfolane is preferably (5-10): 1, the reaction time is preferably 2 to 4 hours;
the molar ratio of the intermediate product I, the solvent, the aromatic aldehyde analog and the catalyst in the step (2) is preferably 1: (30-40): (1.5-2): (0.7-1), the solvent is preferably n-butanol, DMF or dimethyl sulfoxide, and the catalyst is preferably 4-methylpiperidine or triethylamine; the reaction time is preferably 5 to 8 hours;
the molar ratio of the phosphorus oxychloride to the N, N-dimethylformamide in the step (3) is preferably 1: (1-1.3); in the solution of the intermediate product II, the molar ratio of the intermediate product II to the N, N-dimethylformamide is preferably 1: (20-30); the molar ratio of the intermediate product II to the Vilsmeier reagent is preferably 1: (8-10); the reaction time is preferably 4 to 6 hours; the pH is preferably adjusted by adopting NaOH solution;
the molar ratio of the intermediate product III, the salt of 1,3 diaminoguanidine, the solvent and the catalyst described in step (4) is preferably 1:1: (40 to 70): (6-13), the catalyst is concentrated hydrochloric acid or glacial acetic acid, the solvent is preferably at least one of methanol, ethanol, acetonitrile and DMF, and the reaction time is preferably 1-2 h;
the structural formula of the intermediate product I is shown as follows:
the structural formula of the intermediate product II is shown as follows:
the structural formula of the intermediate product III is shown as follows:
the aromatic aldehyde analog is R-CHO, wherein R is
n is 0 to 9;
the synthetic route of the quinoline aromatic vinyl derivative is as follows:
the quinoline aromatic vinyl derivative is applied to the preparation of fluorescent dye;
a fluorescent dye prepared from the quinoline aromatic vinyl derivative;
the quinoline aromatic vinyl derivative is applied to the preparation of a fluorescent probe.
A fluorescent probe, which is prepared from the quinoline aromatic vinyl derivative;
compared with the prior art, the invention has the following advantages and effects:
(1) The invention provides a quinoline aromatic vinyl derivative which has higher fluorescence response to nucleic acid and can be applied to preparation of fluorescent dyes and fluorescent probes.
(2) Compared with the existing embedded fluorescent probe, the fluorescent probe provided by the invention has two characteristics: (1) the introduced amino guanidyl substituent has abundant hydrogen bond donor and acceptor and charge characteristics, and has the possibility of mutual recognition and interaction with structures such as a phosphate skeleton of nucleic acid; (2) the probe has a larger electron conjugated system and plane, the strength of charge transfer effect in the probe molecule can influence the fluorescence emission intensity of the molecule, when the probe is specifically combined with a G-quadruplex and RNA, the flexibility of a rotatable double bond in the molecule is limited, the charge transfer effect is enhanced, and thus the fluorescence intensity is enhanced.
(3) The quinoline aromatic vinyl derivative provided by the invention has lower biotoxicity and phototoxicity as a fluorescent dye and a fluorescent probe, and also has better water solubility and cell permeability, a strong binding constant in the aspect of detection and lower detection limit.
Drawings
FIG. 1 is a graph of fluorescence data fitted to six nucleic acids, compound a fluorescent probe titration 4at, dt21, oxy28, pu27, telo21, RNA.
FIG. 2 is a graph of the fluorescence titration of different nucleic acids by the fluorescent probe of Compound a; wherein, A:4at, B: dt21, C: pu27, D: oxy28, E: telo21, F: RNA.
FIG. 3 shows fluorescence spectra of compound a obtained by titrating G-quadruplex DNA (pu 27) with a fluorescent probe for C and (F-F) 0 )/F 0 Fitted graph.
FIG. 4 is an image of compound a fluorescent probe and dye DAPI counterstained PC3 cells.
FIG. 5 shows the gel electrophoresis of the fluorescent probe of compound a and seven nucleic acids dt21, 4at, ds26, pu27, telo21, oxy28 and RNA.
FIG. 6 is a diagram of the solution of the fluorescent probes 4at, ds26, dt21, pu27, oxy28, telo21, RNA seven nucleic acids and blank control of Compound a under UV light.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art. The reagents, methods and apparatus employed in the present invention are conventional in the art, except as otherwise indicated.
The concentration of concentrated hydrochloric acid in the examples was 11.9mol/L.
Example 1
(1) Dissolving 0.40g (2.28 mmol) of 2-methylquinoline in 2.2ml (23.1 mmol) of sulfolane, dropwise adding 0.42ml (6.84 mmol) of methyl iodide, and heating and stirring at 50 ℃ for 2h; after the reaction is finished, cooling, pouring the reaction solution into 15mL ethyl acetate, standing, and carrying out vacuum filtration to obtain a crude product; the crude product was washed with ethyl acetate to give 0.53g of intermediate I (1,2-dimethyl-1-iodoquinoline) in 83% yield. The results of the nuclear magnetic resonance hydrogen spectrum are as follows: 1H NMR (400mhz, dmso) δ 9.13 (d, J =8.6hz, 1h), 8.61 (d, J =9.0hz, 1h), 8.42 (d, J =8.0hz, 1h), 8.27-8.20 (m, 1H), 8.15 (d, J =8.6hz, 1h), 8.00 (t, J =7.6hz, 1h), 4.47 (s, 3H), 3.12 (s, 3H). The synthesis procedure is as follows:
(2) Mixing 0.39g (1.385 mmol) of intermediate I (1,2-dimethyl-1-iodoquinoline) obtained in step (1) with 4mL (43.7 mmol) of n-butanol, adding 0.282mL (2.077 mmol) of p-dimethylaminobenzaldehyde and 0.20mL (1.18 mmol) of 4-methylpiperidine, followed by stirring at 30 ℃ for 5 hours; after the reaction, 30mL of petroleum ether was added to the reaction mixture, and the mixture was allowed to stand to precipitate a solid, which was then filtered under reduced pressure to obtain 0.49g of intermediate II (1-methyl-2- (4-dimethylaminostyryl) -1-iodoquinoline) in a yield of 85%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows: 1H NMR (400mhz, dmso) δ 8.81 (d, J =9.1hz, 1h), 8.51 (d, J =9.2hz, 1h), 8.43 (d, J =9.0hz, 1h), 8.25 (dd, J =11.4,6.1hz, 2h), 8.08 (t, J =7.9hz, 1h), 7.85 (t, J =8.4hz, 3h), 7.55 (d, J =15.5hz, 1h), 6.83 (d, J =8.9h, 2h), 4.44 (s, 3H), 3.08 (s, 6H). The synthesis procedure is as follows:
(3) Under protection of helium, 1mL (10.72 mmol) of phosphorus oxychloride (POCl) is added to 0.83mL (10.72 mmol) of dry DMF 3 ) Stirring at 0 ℃ for 30min to obtain Vilsmeier reagent (10.72 mmol); 0.56g (1.34 mmol) of intermediate II (1-methyl-2- (4-methylstyryl) -1-iodoquinoline) obtained in step (2) was dissolved in 2.1mL (27 mmol) of dry DMF and added dropwise to Vilsmeier reagent and reacted at 60 ℃ for 4 hours; after the reaction is finished, cooling the reaction liquid to 0 ℃, adding 30mL of ice water for quenching reaction, then adjusting the pH of the reaction liquid to 10 by using 2mol/L NaOH solution, continuing stirring for 30min, performing suction filtration under reduced pressure, and washing a filter cake by using water and methanol respectively to obtain 0.28g of an intermediate product III (1-methyl-2- (4-dimethylaminostyryl) -4-formyl-1-iodoquinoline) with the yield of 46%. The results of the nuclear magnetic resonance hydrogen spectrum are as follows: 1H NMR (400mhz, dmso) δ 10.05 (s, 1H), 8.96 (d, J =9.0hz, 1h), 8.53 (dd, J =16.6,9.0hz, 2h), 8.34-8.24 (m, 3H), 8.14 (dd, J =14.1,7.4hz, 2h), 7.91 (t, J =7.5hz, 1h), 7.76 (d, J =15.7hz, 1h), 7.18 (d, J =8.9hz, 1h), 4.53 (s, 3H), 3.08 (s, 6H) synthesis procedures are as follows:
(4) 0.43g (0.961 mmol) of intermediate III (1-methyl-2- (4-dimethylaminostyryl) -4-formyl-1-iodoquinoline) obtained in step (3) was added to 1.6mL (38.4 mmol) of anhydrous methanol, followed by 0.12g (0,961mmol) of 1,3-diaminoguanidine hydrochloride; then 50 mu L (5.95 mmol) of concentrated hydrochloric acid is added dropwise for catalysis, and then the reaction is carried out for 1h at the temperature of 30 ℃; after the reaction is finished, adding ethyl acetate into the reaction solution, standing to separate out a solid, and performing vacuum filtration to obtain a crude product; the crude product was recrystallized from absolute ethanol to give 0.29g of the compound a 1-methyl-2- (4-dimethylaminostyryl) -4- (1,3-diaminoguanidino) -1-iodoquinoline in 55% yield and the NMR spectrum showed the following: 1H NMR (400MHz, DMSO). Delta.12.37 (s, 1H), 9.01 (d, J =7.2Hz, 1H), 8.60 (dd, J =40.1,28.1Hz, 6H), 8.40-8.14 (m, 4H), 8.07-7.83 (m, 4H), 7.22 (s, 1H), 4.60 (s, 3H), 2.91 (d, J =11.7Hz, 6H) syntheses are as follows:
example 2
(1) Dissolving 0.40g (2.28 mmol) of 2-methylquinoline in 2.7mL (28.5 mmol) of sulfolane, dropwise adding 0.50mL (8 mmol) of methyl iodide, and heating and stirring at 60 ℃ for 3h; after the reaction is finished, cooling, then pouring the reaction liquid into 24mL ethyl acetate for standing, and carrying out vacuum filtration to obtain a crude product; the crude product was washed with ethyl acetate to give 0.58g of intermediate I (1,2-dimethyl-1-iodoquinoline) in 89% yield. The results of NMR spectroscopy are the same as those of example 1.
(2) Mixing 0.39g (1.385 mmol) of intermediate I (1,2-dimethyl-1-iodoquinoline) obtained in step (1) with 4.5mL (49.16 mmol) of n-butanol, adding 0.330mL (2.077 mmol) of p-dimethylaminobenzaldehyde and 0.21mL (1.25 mmol) of 4-methylpiperidine, followed by stirring at 45 ℃ for 6.5h; after the reaction, 30mL of petroleum ether was added to the reaction mixture, and the mixture was allowed to stand to precipitate a solid, which was then filtered under reduced pressure to give 0.52g of intermediate II (1-methyl-2- (4-dimethylaminostyryl) -1-iodoquinoline) in a yield of 91%. The results of NMR spectroscopy are the same as those of example 1.
(3) Under the protection of helium, 1mL (10.72 mmol) of phosphorus oxychloride (POCl) is added to 0.912mL (11.79 mmol) of dry DMF 3 ) Stirring at 2 ℃ for 20min to obtain Vilsmeier reagent (10.72 mmol); 0.49g (1.19 mmol) of intermediate II (1-methyl-2- (4-methylstyryl) -1-iodoquinoline) obtained in step (2) was dissolved in 2.3mL (29.75 mmol) of dry DMF and added dropwise to Vilsmeier reagent and reacted at 70 ℃ for 5 hours; after the reaction is finished, the reaction solution is cooled to 0 ℃, and 30mL of ice water is added for quenchingAfter the reaction is quenched, the pH of the reaction solution is adjusted to 11 by using 2mol/L NaOH solution, the reaction solution is continuously stirred for 30min, then the pressure is reduced and the filtration is carried out, and a filter cake is washed by water and methanol respectively to obtain an intermediate product III (0.22g of 1-methyl-2- (4-dimethylamino styryl) -4-formyl-1-iodoquinoline) with the yield of 49 percent. The result of the NMR spectrum was the same as that of example 1
(4) 0.43g (0.961 mmol) of intermediate III (1-methyl-2- (4-dimethylaminostyryl) -4-formyl-1-iodoquinoline) obtained in step (3) was added to 2.1mL (53 mmol) of anhydrous methanol, followed by 0.12g (0.961 mmol) of 1,3-diaminoguanidine hydrochloride; then 75 mu L (8.9 mmol) of concentrated hydrochloric acid is added dropwise for catalysis, and then the reaction lasts for 1.5h at 45 ℃; after the reaction is finished, adding ethyl acetate into the reaction solution, standing to separate out a solid, and performing vacuum filtration to obtain a crude product; the crude product was recrystallized from absolute ethanol to give 0.31g of the compound a 1-methyl-2- (4-dimethylaminostyryl) -4- (1,3-diaminoguanidino) -1-iodoquinoline in a yield of 59%, and its NMR spectrum showed the same result as in example 1.
Example 3
(1) 0.40g (2.28 mmol) of 2-methylquinoline was dissolved in 3.2mL (34 mmol) of sulfolane, 0.57mL (9.12 mmol) of methyl iodide was added dropwise, and the mixture was stirred at 70 ℃ for 4 hours. After the reaction, the reaction mixture was cooled, and then poured into 40mL of ethyl acetate, and the mixture was allowed to stand, filtered under reduced pressure to obtain a crude product, which was washed with ethyl acetate to obtain 0.57g of intermediate I (1,2-dimethyl-1-iodoquinoline) with a yield of 87%. The result of NMR spectrum was the same as that of example 1.
(2) Mixing 0.39g (1.385 mmol) of intermediate I (1,2-dimethyl-1-iodoquinoline) obtained in step (1) with 5.1mL (55.4 mmol) of n-butanol, adding 0.376mL (2.77 mmol) of p-dimethylaminobenzaldehyde and 0.23mL (1.385 mmol) of 4-methylpiperidine, followed by stirring at 60 ℃ for 8h; after the reaction, 30mL of petroleum ether was added to the reaction mixture, and the mixture was allowed to stand to precipitate a solid, which was then filtered under reduced pressure to obtain 0.51g of intermediate II (1-methyl-2- (4-dimethylaminostyryl) -1-iodoquinoline) in a yield of 88%. The results of NMR spectroscopy are the same as those of example 1.
(3) Under the protection of helium, in the presence of 1mL (13 mmol)1mL (10.72 mmol) of phosphorus oxychloride (POCl) was added to dry DMF 3 ) Stirring at 5 ℃ for 40min to obtain Vilsmeier reagent (10.72 mmol); 0.48g (1.07 mmol) of intermediate II (1-methyl-2- (4-methylstyryl) -1-iodoquinoline) obtained in step (2) was dissolved in 2.8mL (32 mmol) of dry DMF and added dropwise to Vilsmeier reagent and reacted at 80 ℃ for 6 hours; after the reaction is finished, cooling the reaction liquid to 0 ℃, adding 30mL of ice water for quenching reaction, then adjusting the pH of the reaction liquid to 10 by using 2mol/L NaOH solution, continuing stirring for 30min, decompressing and filtering, and washing a filter cake by using water and methanol respectively to obtain 0.21g of an intermediate product III (1-methyl-2- (4-dimethylamino styryl) -4-formyl-1-iodoquinoline) with the yield of 46%. The results of NMR spectroscopy are the same as those of example 1.
(4) 0.43g (0.961 mmol) of intermediate III (1-methyl-2- (4-dimethylaminostyryl) -4-formyl-1-iodoquinoline) obtained in step (3) was added to 2.7mL (67.3 mmol) of anhydrous methanol, followed by 0.12g (0.961 mmol) of 1,3-diaminoguanidine hydrochloride; then 100 mu L (11.9 mmol) of concentrated hydrochloric acid is dripped for catalysis, and then the reaction is carried out for 2h at the temperature of 60 ℃; after the reaction is finished, adding ethyl acetate into the reaction solution, standing to separate out a solid, and performing vacuum filtration to obtain a crude product; the crude product was recrystallized from absolute ethanol to give 0.29g of the compound a 1-methyl-2- (4-dimethylaminostyryl) -4- (1,3-diaminoguanidino) -1-iodoquinoline in a yield of 55%, and its NMR spectrum showed the same result as in example 1.
EXAMPLE 4 fluorescence Spectroscopy experiments with Photoprobes for Compound a on different nucleic acid Selectivity
Weighing 2.6mg of the compound a, and dissolving the compound a in 1mL of DMSO to obtain stock solution of the compound a; weighing KCl 60mmol and Tris 0.6068g, diluting to 500mL with deionized water, and adjusting the final pH to 7.4 with hydrochloric acid to obtain a Tris-HCl solution. The fluorescence intensities of 5mM stock solutions of compound a prepared according to the present invention were measured by a fluorescence spectrophotometer (slit width =10, scanning speed =200nm, ex =495 nm) after being diluted with Tris-HCl solution to a concentration of 5. Mu.M, and different kinds of nucleic acids (Table 1, both synthesized by the same company) were added, and FIG. 1 is a curve fitted to fluorescence data obtained by titrating six kinds of nucleic acids, i.e., 4at, dt21, oxy28, pu27, telo21, and RNA, with the fluorescent probe, pu27 shows a high fluorescence intensity. The fluorescence intensity gradually increases with increasing final concentration of nucleic acid.
TABLE 1 nucleic acid sequences
EXAMPLE 5 determination of detection Limit
The results of diluting 5mM of compound a stock solution (prepared in the same manner as in example 4) to a concentration of 5. Mu.M, scanning the diluted solution with a fluorescence spectrophotometer (slit width =10, scanning speed =200nm, ex =495 nm), and then saturating the diluted solution by slowly adding G-quadruplex DNA (pu 27) thereto are shown in FIGS. 2 to 3, in which FIG. 3 shows the concentration of pu27 and (F-F) in the fluorescence spectrum of compound a probe titration pu27 0 )/F 0 The fitted curve, the abscissa is the final concentration of G-quadruplex DNA (pu 27).
The calculation formula of the detection limit is as follows:
LOD＝K×S b /m
LOD (Compound binding constant), m is the concentration of C and (F-F) 0 )/F 0 Slope of the straight line made, S b For blank deviations from multiple measurements with instrumental blanks, the value of K is usually taken to be 3 as recommended by the international union of pure and applied chemistry. The LOD calculated from FIG. 3 is 8.82nmol/L, and the lower limit of detection indicates that the fluorescent ligand has higher sensitivity and good commercial value.
EXAMPLE 6 cellular imaging assay of Compound a
(1) PC3 cells (commercially available) were seeded in a cell culture solution in a 6-well plate so that the density of the cells was about 5000 cells/mL, and then the CO was 5% at 37 ℃ C 2 Culturing for 70h under the environment;
(2) Discarding the cell culture solution in the 6-well plate in the previous step, washing with pre-cooled 1 XPBS for 3 times, then adding pre-cooled pure methanol 1.5mL, placing for 2min at normal temperature in a dark place, then discarding the pure methanol and washing with pre-cooled 1 XPBS for 3 times;
(3) Adding 1mL of compound a with the concentration of 5 mu M, and then standing for 20min; discard compound a solution in 6-well plate and wash 3 times with pre-cooled 1 × PBS;
(4) Adding 1mL of 1mM DAPI solution into the 6-well plate, standing at 37 deg.C for 2min, discarding the DAPI solution in the 6-well plate, washing with precooled 1 × PBS for 6 times, and soaking for 5min each time; the staining of the cells was observed under an inverted fluorescence microscope.
FIG. 4 is an image of the cell image of the compound a probe and dye DAPI counterstained PC3 cell, and it can be seen from FIG. 4 that the fluorescent probe acts on G-quadruplex in nucleus and cytoplasm, and it is also proved that the fluorescent ligand of the system can image and detect the G-quadruplex in the cell system.
Example 7 nucleic acid gel electrophoresis experiment
(1) Preparation of electrophoresis buffer solution and solution: weighing 27gTris,13.5g boric acid and 2.0811g EDTA, and diluting to 500mL with deionized water to obtain 5 xTBE electric buffer solution; weighing 10g of ammonium persulfate, and using deionized water to fix the volume to 100mL to obtain a 10% ammonium persulfate solution (ready for use); 29g of acrylamide and 1g of N, N-methylenebisamide were weighed and made to 100mL with deionized water to obtain a solution of 29% acrylamide plus 1% of N, N-methylenebisamide.
(2) Preparing glue: preparing the prepared solution into colloidal solution according to a certain proportion, uniformly mixing, beating into a splint, then placing numbers, and standing until the glue is molded.
(3) Sample preparation: the loading buffer-containing DNA was prepared at a concentration of 30. Mu.M and added to the well.
(4) Glue running: adding the solution into a rubber plate runway, putting the rubber plate runway into an electrophoresis tank, and running rubber for 1h at 45 volts by using a1 xTBE solution as an electrophoresis solution, and then running rubber for 2.5h at 100 volts.
(5) Gel imaging: the run-out gel was stained with 5. Mu.M Compound a, placed on a shaker for 20min and finally photographed in a gel imager.
The results are shown in FIG. 5, and FIG. 5 is a gel electrophoresis of compound a probe with seven nucleic acids dt21, 4at, ds26, pu27, telo21, oxy28, and RNA. It can be seen from the figure that the compound a can generate stronger fluorescence phenomenon when combined with pu 27G-quadruplex DNA, and has weaker fluorescence for other G-quadruplex DNA, double-stranded DNA, single-stranded DNA and RNA, which indicates that the compound a has good selectivity for pu 27G-quadruplex.
Example 8 solution System fluorescence visualization experiment
To 1mL of a 5. Mu.M Tris solution of Compound a, 20. Mu.L of 5. Mu.M each of seven nucleic acids, 4at, ds26, dt21, pu27, oxy28, telo21 and RNA, and a blank were added, and photographed under irradiation of ultraviolet light.
The results are shown in FIG. 6, and it can be seen that compound a combines with pu 27G-quadruplex DNA to generate a strong fluorescence phenomenon, which has a little fluorescence on oxy28, and the fluorescence of other nucleic acids is weak, indicating that pu 27G-quadruplex has good selectivity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
SEQUENCE LISTING
<110> Guangdong university of industry
<120> quinoline aromatic vinyl derivative and application thereof in preparation of fluorescent dye and fluorescent probe
<130> 1
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 26
<212> DNA
<213> Artificial
<220>
<223> ds26
<400> 1
caatcggatc gaattcgatc cgattg 26
<210> 2
<211> 28
<212> DNA
<213> Artificial
<220>
<223> oxy28
<400> 2
ggggttttgg ggttttgggg ttttgggg 28
<210> 3
<211> 27
<212> DNA
<213> Artificial
<220>
<223> pu27
<400> 3
tggggagggt ggggagggtg gggaagg 27
<210> 4
<211> 21
<212> DNA
<213> Artificial
<220>
<223> telo21
<400> 4
gggttagggt tagggttagg g 21
<210> 5
<211> 20
<212> DNA
<213> Artificial
<220>
<223> dt21
<400> 5
<210> 6
<211> 12
<212> DNA
<213> Artificial
<220>
<223> 4at
<400> 6
atatatatat at 12
Claims (10)
1. A quinoline aromatic vinyl derivative is characterized in that the derivative is a compound shown in a formula I or a pharmaceutically acceptable salt thereof, and a solvent compound, an enantiomer, a diastereoisomer, a tautomer or a mixture in any proportion of the compound shown in the formula I or the pharmaceutically acceptable salt thereof, and comprises a racemic mixture;
wherein n is 1; x is Cl, br, I or benzenesulfonic acid group;
the HA is hydrochloric acid or acetic acid.
2. The quinoline aromatic vinyl derivative according to claim 1, wherein:
the X is I, and the HA is hydrochloric acid.
3. The process for producing a quinoline arylethylene derivative according to claim 1 or 2, characterized by comprising the steps of:
(1) Mixing 2-methylquinoline, methyl iodide and sulfolane, and reacting at 50-70 ℃; after the reaction is finished, cooling, adding ethyl acetate to precipitate a solid, and filtering to obtain an intermediate product I;
(2) Mixing the intermediate product I prepared in the step (1) with a solvent, adding an aromatic aldehyde analog and a catalyst, and reacting at 30-60 ℃; after the reaction is finished, cooling, adding petroleum ether to precipitate solid, and filtering to obtain an intermediate product II;
(3) Mixing phosphorus oxychloride and N, N-dimethylformamide under the anhydrous and oxygen-free conditions, and stirring for 20-40 min at the temperature of 0-5 ℃ to obtain a Vilsmeier reagent; dissolving the intermediate product II prepared in the step (2) in N, N-dimethylformamide to obtain an intermediate product II solution, then dropwise adding the intermediate product II solution into a Vilsmeier reagent, and reacting at 60-80 ℃; after the reaction is finished, adding ice water to quench the reaction, adjusting the pH value of the system to be more than 10, and filtering to obtain an intermediate product III;
(4) Mixing the intermediate product III prepared in the step (3), 1,3 diaminoguanidine salt and a solvent, then dropwise adding a catalyst, reacting at 30-60 ℃, cooling after the reaction is finished, adding ethyl acetate to precipitate solids, and filtering to obtain a crude product; recrystallizing the crude product by absolute ethyl alcohol to obtain a quinoline aromatic vinyl derivative;
the structural formula of the intermediate product I is shown as follows:
the structural formula of the intermediate product II is shown as follows:
the structural formula of the intermediate product III is shown as follows:
the aromatic aldehyde analog is R-CHO, wherein R is
n is 0.
4. The process for producing a quinoline aromatic vinyl derivative according to claim 3, characterized in that:
the mol ratio of the 2-methylquinoline to the methyl iodide to the sulfolane in the step (1) is 1: (2-4): (10-15).
5. The method for producing a quinoline arylethylene derivative according to claim 3, wherein:
the mol ratio of the intermediate product I, the solvent, the aromatic aldehyde analogue and the catalyst in the step (2) is 1: (30-40): (1.5-2): (0.7-1).
6. The process for producing a quinoline aromatic vinyl derivative according to claim 3, characterized in that:
the molar ratio of the phosphorus oxychloride to the N, N-dimethylformamide in the step (3) is 1: (1-1.3); in the solution of the intermediate product II, the molar ratio of the intermediate product II to the N, N-dimethylformamide is 1: (20 to 30); the molar ratio of the intermediate product II to the Vilsmeier reagent is 1: (8-10).
7. The process for producing a quinoline aromatic vinyl derivative according to claim 3, characterized in that:
the mol ratio of the intermediate product III, the 1,3 diaminoguanidine salt, the solvent and the catalyst in the step (4) is 1:1: (40 to 70): (6-13).
8. Use of the quinoline aromatic vinyl derivative according to claim 1 or 2 for preparing a fluorescent dye of G-quadruplex.
9. The use of the quinoline aromatic vinyl derivative according to claim 1 or 2 for preparing a fluorescent probe for G-quadruplexes.
10. A fluorescent dye or a fluorescent probe of a G-quadruplex, which is prepared from the quinoline aromatic vinyl derivative as claimed in claim 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910798217.4A CN110627715B (en) | 2019-08-27 | 2019-08-27 | Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910798217.4A CN110627715B (en) | 2019-08-27 | 2019-08-27 | Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110627715A CN110627715A (en) | 2019-12-31 |
| CN110627715B true CN110627715B (en) | 2022-11-22 |
Family
ID=68969316
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910798217.4A Active CN110627715B (en) | 2019-08-27 | 2019-08-27 | Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110627715B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112679474B (en) * | 2020-12-08 | 2022-03-04 | 南方医科大学顺德医院(佛山市顺德区第一人民医院) | Ratio type fluorescent probe and preparation method and application thereof |
| CN113773302B (en) * | 2021-08-24 | 2022-12-20 | 广东工业大学 | Quinoline compound and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106833623A (en) * | 2017-02-17 | 2017-06-13 | 广东工业大学 | A kind of fluorescence probe and preparation method thereof |
| CN107266417A (en) * | 2017-06-27 | 2017-10-20 | 广东工业大学 | A kind of indoles ethene substd quinolines analog derivative and its preparation method and application |
| CN109111396A (en) * | 2018-08-28 | 2019-01-01 | 广东工业大学 | A kind of quinoline aromatic ethylene analog derivative and its preparation method and application |
-
2019
- 2019-08-27 CN CN201910798217.4A patent/CN110627715B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106833623A (en) * | 2017-02-17 | 2017-06-13 | 广东工业大学 | A kind of fluorescence probe and preparation method thereof |
| CN107266417A (en) * | 2017-06-27 | 2017-10-20 | 广东工业大学 | A kind of indoles ethene substd quinolines analog derivative and its preparation method and application |
| CN109111396A (en) * | 2018-08-28 | 2019-01-01 | 广东工业大学 | A kind of quinoline aromatic ethylene analog derivative and its preparation method and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110627715A (en) | 2019-12-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106147753B (en) | Thiazole orange styrene compound as G-quadruplex nucleic acid fluorescent probe | |
| Le Droumaguet et al. | Fluorogenic click reaction | |
| CN107266417B (en) | Indole ethylene substituted quinoline derivative and preparation method and application thereof | |
| CN108997312B (en) | RNA fluorescent probe for positioning mitochondria | |
| CN107200718B (en) | Compound, method and kit for detecting DNA G-quadruplex | |
| CN110627715B (en) | Quinoline aromatic ethylene derivative and application thereof in preparation of fluorescent dye and fluorescent probe | |
| CN104449669B (en) | A kind of polyaryl substituted imidazole fluorescence probe and preparation method thereof and the application in detection G-tetra-stranded structure | |
| CN108822081B (en) | Fluorescent probe for simultaneously detecting mitochondria and DNA | |
| CN104151301A (en) | Fluorescent probe, as well as preparation method and applications thereof | |
| CN114014848B (en) | RNA fluorescent probe and preparation method and application thereof | |
| CN107311977B (en) | Indole ethylene compound and preparation method and application thereof | |
| CN111217806B (en) | Hemicyanine compound based on benzothiazole-linked heterocycle and preparation method and application thereof | |
| CN104710977B (en) | A kind of double function probe and preparation method thereof and the application in detecting positive parallel conformation G-tetra-serobila | |
| CN112174885B (en) | P-dimethylamino styrene quinoline derivative, synthetic method thereof and application thereof in nucleic acid fluorescent probe | |
| CN104650610A (en) | Asymmetric near-infrared BODIPY fluorescent dye as well as preparation method and application thereof | |
| CN110606862B (en) | Platinum complex fluorescent probe for specifically detecting RNA G-quadruplex, preparation method and application | |
| CN109913206A (en) | A kind of RNA fluorescence probe and its preparation method and application | |
| CN114133413B (en) | Benzothiazole-triphenylamine compound and preparation method and application thereof | |
| CN114106024B (en) | Fluorescent probe and preparation method and application thereof | |
| CN106397316B (en) | A kind of fluorescence probe and its preparation method and application with charge transfer characteristic | |
| CN113072534B (en) | RNA fluorescent probe and preparation method and application thereof | |
| CN108264502B (en) | Quinoline carbazole fluorescent dye and preparation method and application thereof | |
| CN109232446B (en) | Preparation method of quinoxaline fluorescent probe and application of quinoxaline fluorescent probe in detection of G-quadruplex | |
| KR102637182B1 (en) | Fluorescent dyes, their production methods and applications | |
| CN110183439B (en) | Berberine-Pyridostatin conjugate as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |