CN114524809B - Coumarin derivative and preparation method and application thereof - Google Patents
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Abstract
The invention provides a coumarin derivative, and a preparation method and application thereof. The coumarin derivative has a structural formula shown in a formula (I):
the excitation and emission spectrum of the coumarin derivative has high selectivity, high sensitivity and high chemical stability in a visible light region when being used for tin ion fluorescence detection; the coumarin derivative also has good water solubility, can be used for detecting tin ions in an aqueous environment system, and has low toxicity to cells. Can be used for fluorescence detection of tin ions.
Description
Technical Field
The invention belongs to the technical field of organic synthesis, and particularly relates to a coumarin derivative, and a preparation method and application thereof.
Background
Tin ion is one of trace elements necessary for organisms, has important physiological functions, and the main physiological functions are in anti-tumor aspect, because tin can generate anti-tumor tin compounds in thymus of human body, and inhibit the generation of cancer cells. Tin also promotes the synthesis of protein and nucleic acid, which is beneficial to the growth and development of the body; and the composition of a plurality of enzymes and the participation of biological reactions of flavin enzyme can enhance the stability of in vivo environment and the like. The fluorescent probe can detect the concentration, distribution and other information of the target in the organism in real time. For the above reasons, sn excellent in synthesis performance was designed 4+ Fluorescent probes are hot spots for research and have very important significance in the fields of biology, chemistry, clinical medicine and the like.
The organic small molecule fluorescent probe has a structure which can be modified,The method has the advantages of low cost, simple operation, high sensitivity, good selectivity, noninvasive visual analysis, real-time monitoring and the like, and is widely focused in metal ion detection and focused by scientific researchers in aspects of disease diagnosis, surgical navigation and the like. The fluorescence enhancement sensing material can reduce the detection error rate, maintain the detection accuracy of a complex system, and simultaneously detect different analytes by using a plurality of detection objects. However, currently reported Sn 4+ Fluorescent chemical probes are still limited in practical applications, such as: some are difficult to synthesize and have complex structures; some membranes have poor permeation properties; some of the metal ions are not enough in specificity and are easy to interfere with other metal ions; the organic solvent is needed to assist dissolution in the tin ion detection process, so that the toxicity of a detection system is increased, and the application of the fluorescent probe in a biological system is limited.
Therefore, there is a need to develop Sn with high sensitivity, rapid response and low cytotoxicity 4+ Is provided.
Disclosure of Invention
The present invention aims to solve at least one of the above technical problems in the prior art. To this end, the invention provides a coumarin derivative.
The invention also provides a preparation method of the coumarin derivative.
The invention also provides application of the coumarin derivative.
The invention also provides a fluorescent probe.
In a first aspect, the invention provides a coumarin derivative, which has a structural formula shown in formula (I):
the invention relates to one of the technical schemes of coumarin derivatives, which has at least the following beneficial effects:
the excitation and emission spectrum of the coumarin derivative has high selectivity, high sensitivity and high chemical stability in a visible light region when being used for tin ion fluorescence detection; the coumarin derivative also has good water solubility, can be used for detecting tin ions in an aqueous environment system, and has low toxicity to cells.
In a second aspect, the present invention provides a process for the preparation of coumarin derivatives, comprising the steps of:
mixing N-phenethyl benzothiazole salt, 4-hydroxycoumarin, alkali and solvent, and reacting at 20-160 ℃ to obtain coumarin derivatives, wherein the N-phenethyl benzothiazole salt has a structural formula shown in a formula (II):
wherein X is bromine, chlorine or iodine.
The invention relates to a technical scheme in a coumarin derivative preparation method, which has at least the following beneficial effects:
the preparation method takes N-phenethyl benzothiazole salt and 4-hydroxycoumarin as main raw materials to synthesize the fluorescent probe compound, and has the advantages of simple synthesis steps, safe method operation, nontoxic raw materials and low cost. In addition, the preparation method can realize the reaction without using a catalyst, thereby reducing the production cost.
According to some embodiments of the invention, the N-phenethyl benzothiazole salt is prepared by: benzothiazole and
mixing and reacting to obtain the N-phenethyl benzothiazole salt.
Wherein X is bromine, chlorine or iodine.
According to some embodiments of the invention, the molar ratio of the N-phenethylbenzothiazole salt to the 4-hydroxycoumarin is 1: (1-2).
According to some embodiments of the invention, the base is present in an amount of 10 to 100% by mass of the N-phenethyl benzothiazole salt.
According to some embodiments of the invention, the volumetric molar ratio of the solvent to the N-phenethyl benzothiazole salt is (2-5) mL:1mmol.
According to some embodiments of the invention, the base is an inorganic base and/or an organic base.
According to some embodiments of the invention, the inorganic base comprises at least one of potassium hydroxide, calcium hydroxide, or sodium hydroxide.
According to some embodiments of the invention, the organic base comprises at least one of sodium acetate, sodium methoxide, sodium tert-butoxide, potassium tert-butoxide, or triethylamine.
According to some embodiments of the invention, the solvent comprises at least one of ethanol, ethyl acetate, isopropanol, ethylene glycol, acetonitrile, toluene, 1, 4-dioxane, 1, 2-dichloroethane, or water.
According to some embodiments of the invention, the reaction time is 2 to 16 hours.
According to some embodiments of the invention, the reaction further comprises purification after completion.
According to some embodiments of the invention, the purification is a column chromatography purification.
According to some embodiments of the invention, the eluent of the column chromatography purification is petroleum ether: dichloromethane: ethyl acetate volume ratio of 0.5-5: 0 to 20: 1.
In a third aspect, the invention provides the use of a coumarin derivative in a fluorescent probe, a high sensitivity sensor or a tin ion detector.
According to some embodiments of the invention, the coumarin derivative is used as a tin ion fluorescent probe in the preparation of living cell imaging reagents.
In a fourth aspect, the present invention provides a fluorescent probe comprising the coumarin derivative; or coumarin derivatives prepared by the above method.
Drawings
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of N-phenethyl benzothiazole salt in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance carbon spectrum of N-phenethyl benzothiazole salt in example 1 of the present invention;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of coumarin derivatives of example 1 of the present invention;
FIG. 4 is a nuclear magnetic resonance chromatogram of coumarin derivatives of example 1 of the present invention;
FIG. 5 is a graph showing the results of fluorescence performance tests of coumarin derivatives of example 1 of the present invention under various metal ion conditions;
FIG. 6 is a graph of test results for verifying the specificity of coumarin derivatives to tin ions in example 1 of the present invention;
FIG. 7 shows the coumarin derivatives of example 1 of the present invention in different Sn 4+ A fluorescence performance test result graph at concentration;
FIG. 8 shows the fluorescence intensity of coumarin derivatives as a function of Sn in example 1 4+ A plot of mole fraction change as a function of;
FIG. 9 is a diagram showing the coordination structure of coumarin derivatives in example 1 of the present invention;
FIG. 10 shows the coumarin derivatives of example 1 of the present invention in different Sn 4 Toxicity test results for fluorescent cells at +concentration;
FIG. 11 is a confocal fluorescence image of HepG2 cells stained with the coumarin derivative solution of example 1 of the present invention, wherein A is a dark field; b is bright field; c is the superimposed image of A and B.
Detailed Description
Technical solutions in the embodiments of the present invention will be clearly and completely described below, but the embodiments of the present invention are not limited thereto.
The reagents, methods and apparatus employed in the present invention, unless otherwise specified, are all conventional in the art.
The following examples and comparative examples were prepared from the following raw materials:
HepG2 cells: purchased from Shanghai Biyun biotechnology Co.
Nuclear magnetic resonance spectral data were determined by Bruker Avance 500MHz Nuclear magnetic resonance spectrometer as CDCl 3 ，DMSO-d6，CD 3 OD or d 6-acetone as solventThe agent (in ppm) was referenced to TMS (0 ppm) or chloroform (7.26 ppm). When multiple peaks occur, the following abbreviations will be used: s (single, singlet), s (single, singlet, doublet), d (doublet ), t (triplet), m (multiplet ), br (broadened, broad), dd (doublet of doublets, doublet), ddd (doublet of doublet of doublets, doublet), dt (doublet of triplets, doublet), ddt (doublet of doublet of triplets, doublet), td (triplet of doublets, doublet), br.s (broadened singlet, broad doublet). Coupling constant J, in units of hertz (Hz).
Example 1
Firstly, preparing N-phenethyl benzothiazole salt, wherein the structural formula and the preparation method are as follows:
0.945g benzothiazole (7 mmol) and 1.417g beta-bromostyrene (7.7 mmol) are evenly mixed and stirred at 60 ℃ for reaction for 12h to obtain a crude product; washing and drying the crude product by diethyl ether to obtain pure N-phenethyl benzothiazole salt; the yield of the preparation method is 80%.
The nuclear magnetic resonance hydrogen spectrum and the carbon spectrum of the obtained N-phenethyl benzothiazole salt are shown in fig. 1 and 2, and the structural characterization data are as follows:
nuclear magnetic resonance hydrogen spectrum data: 1 H NMR(500MHz,DMSO-d6)δ10.49(s,1H),8.55(dd,J＝8.4,1.2Hz,1H),8.48(d,J＝8.5Hz,1H),7.93(dd,J＝8.5,7.2,1.3Hz,1H),7.86(dd,J＝8.2,7.2,1.0Hz,1H),7.30–7.21(m,5H),5.16(dd,J＝8.2,6.7Hz,2H),3.30(t,J＝7.4Hz,2H).
carbon spectrum data: 13 C NMR(126MHz,DMSO-d6)δ165.15,140.49,136.84,131.92,130.08,129.41,129.07,128.90,127.56,125.82,117.82,53.71,34.92.
example 1 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione having the structural formula (I), prepared by:
uniformly mixing 0.319g N-phenethyl benzothiazole salt (1 mmol) obtained by the preparation, 0.243g 4-hydroxycoumarin (1.5 mmol), 0.032g NaOH (10% of N-phenethyl benzothiazole salt mass) and 1mL ethyl acetate, and stirring at 80 ℃ for reaction for 8 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of the preparation method is 86%.
And (3) structural confirmation: the nuclear magnetic resonance hydrogen spectrum and the carbon spectrum of the coumarin derivative are shown in fig. 3 and 4, and the structural characterization data are as follows:
nuclear magnetic resonance hydrogen spectrum data: 1 H NMR(500MHz，CDCl 3 )δ8.12(dd,J＝8.1，1.7Hz，1H)，7.86(dd，J＝8.0，1.1Hz，1H)，7.69(d，J＝8.4Hz，1H)，7.58(qd,J＝7.6，1.5Hz，2H)，7.49(t，J＝7.6Hz，1H)，7.31–7.27(m，3H)，7.08–7.01(m，3H)，6.84(dd，J＝7.2，2.2Hz，2H)，5.03(t，J＝7.2Hz，2H)，3.01(t，J＝7.1Hz，2H)。
carbon spectrum data: 13 C NMR(126MHz，CDCl 3 )δ175.62，171.26，161.45，153.72，139.79，136.39，133.38，129.97，128.67，128.44，127.67，127.20，126.27，126.04，123.72，122.73，120.78，116.61，114.67，94.23，53.16，34.36。
high resolution mass spectrometry (electrospray ionization mass spectrometry): c (C) 24 H 18 NO 3 S[M+H] + Is a theoretical calculation of (a): 400.1002; test data: 400.0998.
example 2
Example 2 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione prepared by a process comprising the steps of:
uniformly mixing 0.319-g N-phenethyl benzothiazole salt (1 mmol), 0.244g of 4-hydroxycoumarin (1.5 mmol), 0.135g of KOH (50% of the mass of N-phenethyl benzothiazole salt) and 2mL of toluene, and stirring at 20 ℃ for reaction for 24 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of the preparation was 63% and the characterization was the same as in example 1.
Example 3
Example 3 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione prepared by a process comprising the steps of:
0.160. 0.160g N-phenethylbenzothiazole salt (0.5 mmol), 0.162g 4-hydroxycoumarin (1 mmol), 0.160g NaCO 3 (100% of the mass of N-phenethyl benzothiazole salt) and 2.5mL of acetonitrile are uniformly mixed and stirred at 80 ℃ for reaction for 2 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of the preparation was 54% and the characterization was the same as in example 1.
Example 4
Example 4 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione prepared by a process comprising the steps of:
0.239. 0.239g N-phenethylbenzothiazole salt (0.75 mmol), 121g 4-hydroxycoumarin (0.75 mmol), 0.179g Ca (OH) 2 (75% of the mass of N-phenethyl benzothiazole salt) and 3mL of ethanol are uniformly mixed and stirred at 70 ℃ for reaction for 5 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of this preparation was 56% and the characterization of coumarin derivatives was the same as in example 1.
Example 5
Example 5 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione prepared by a process comprising the steps of:
uniformly mixing 0.080g N-phenethyl benzothiazole salt (0.25 mmol), 0.081g 4-hydroxycoumarin (0.5 mmol), 0.08g triethylamine (10% of the mass of N-phenethyl benzothiazole salt) and 3mL water, and stirring at 100 ℃ for reaction for 16 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of the preparation was 59% and the characterization was the same as in example 1.
Example 6
Example 6 provides a coumarin derivative, (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione prepared by a process comprising the steps of
Uniformly mixing 0.255g N-phenethyl benzothiazole salt (0.8 mmol), 0.162g 4-hydroxycoumarin (1 mmol), 0.127g NaOH (50% of the mass of N-phenethyl benzothiazole salt) and 4mL glycol, and stirring at 160 ℃ for reacting for 12 hours to obtain a crude product; purifying the crude product by column chromatography to obtain coumarin derivatives; the yield of the preparation was 68% and the characterization was the same as in example 1.
The structural formula and the preparation method of the N-phenethyl benzothiazole salt in examples 2 to 6 are the same as those in example 1.
Performance testing
Preparing a probe solution: a100. Mu.M methanol solution of (E) -3- (3-phenethylbenzothiazol-2 (3H) -ylidene) chroman-2, 4-dione of example 1 was prepared as a probe solution and stored at room temperature.
Preparing a metal ion solution: the metal ions include: fe (Fe) 2+ 、Mg 2+ 、Cu + 、Cu 2+ 、Ni 2+ 、Mn 2+ 、Co 2+ 、Li + 、K + 、Ba 2+ 、Cd 2+ 、Ca 2+ 、Hg 2+ 、Al 3+ 、Fe 3+ And Sn (Sn) 4+ The method comprises the steps of carrying out a first treatment on the surface of the Solutions thereof were prepared from the corresponding hydrochloride salts, respectively. Weighing a certain amount of metal salt respectively, dissolving in 10mL distilled water, and preparing into 10 -2 And (5) preserving the metal ion solution with mol/L for standby.
Fluorescence Performance test
Experimental example 1
Preparing a liquid to be tested: taking 0.5mL of prepared probe solution and 0.5mL of prepared different metal ion solution, and 4mL of CH 3 OH-H 2 O(v:v＝1:1)And mixing the solutions to obtain the to-be-detected solution of different metal ions.
Preparing blank liquid: 0.5mL of the prepared probe solution was mixed with 2.5mL of water and 2mL of methanol solution.
The fluorescence intensity of the liquid to be measured was analyzed by fluorescence spectrum, and the analysis result is shown in fig. 5.
As can be seen from FIG. 5, in the sample solution, when the metal ion is Fe 2+ 、Mg 2+ 、Cu + 、Cu 2+ 、Ni 2+ 、Mn 2+ 、Co 2+ 、Li + 、K + 、Ba 2+ 、Cd 2+ 、Ca 2+ 、Hg 2+ 、Al 3+ 、Fe 3+ The fluorescence intensity varies to different extents. But Sn is 4+ The fluorescence intensity of the liquid to be measured shows obvious fluorescence attenuation (each liquid to be measured is uniformly marked as 'coumarin derivative+M', F0 is blank liquid fluorescence, F is liquid to be measured fluorescence, ultraviolet absorption at 254nm wavelength is measured, and the ratio of F to F0 is taken as intensity change).
Experimental example 2
In order to further verify the specificity of the coumarin derivatives to tin ions, competition experiments were performed. Sn is mixed with 4+ Adding the solution (10 μm) and any one of the other metal ion solutions with the same concentration into the prepared probe solution, and testing the other competing ion pair coumarin derivatives Sn respectively 4+ The effect of selectivity and test results are shown in FIG. 6 (each test solution is collectively labeled "coumarin derivative +M+Sn"). It can be seen that the coumarin derivatives are specific to Sn before and after the addition of other competing ions 4+ Almost no change in detection of (2) indicates that the designed coumarin derivative probe pair Sn 4+ Has strong selectivity and can meet the actual application requirements.
Experimental example 3
Adding Sn with different concentrations into the prepared probe solution 4+ Testing the fluorescence properties to determine Sn of the coumarin derivatives 4+ Detection range and detection limit. As a result of the test, as shown in FIG. 7, sn 4+ The concentration is 0, 5 multiplied by 10 in turn -9 M、5×10 -8 M、5×10 -6 M、5×10 -5 M、6×10 -5 M、7×10 -5 M、8×10 -5 M、9×10 -5 M、1×10 -4 M, and the fluorescence intensity is correspondingly and sequentially reduced from top to bottom, which shows that the fluorescence intensity of the fluorescent probe compound follows Sn 4+ The increase in concentration gradually decreases as Sn 4+ The concentration reaches 1X 10 -4 At M, the fluorescence intensity of the compound is greatly attenuated. The compound is to Sn 4+ The detection range is 5×10 -9 M to 9X 10 -5 M, the detection limit is 5×10 -9 M indicates that the compound is against Sn 4+ The detection capability of the sensor is better, and the sensor has higher practical application value.
Experimental example 4
To further confirm the mechanism of action between the probe and the metal ion, we performed a preliminary analysis using Job's plot. The specific operation method is as follows: under the condition of ensuring the constant total concentration (10 mu M), testing the fluorescence emission spectrum of 426nm of the probe and the metal ions in different molar ratios, and plotting the fluorescence intensity with Sn according to the result 4+ A plot of the mole fraction change as a function of the mole fraction change is shown in fig. 8.
As can be seen from fig. 8, when the mole fraction of tin ions reaches 0.3, an inflection point appears, indicating that the mole fraction of tin ions and coumarin derivatives is 1:2, and the relationship of the two groups is coordinated. From the above results, we speculate on one possible coordination structure (structural formula shown in FIG. 9).
Experimental example 5
To explore the toxicity of probes to fluorescent cells during use, we performed cell imaging and cytotoxicity experiments. MTT was used to evaluate the toxicity of the probes, and the effect of different concentrations of probes on the cell viability of HepG2 cells at 37℃is shown in FIG. 10. It is apparent that there is no significant cytotoxic response (cell viability ≡80%) with increasing probe concentration. This indicates that the probe shows lower toxicity for fluorescent cell imaging under practical conditions.
Next, the viability of the fluorescent probe compounds was examined by inverted fluorescent microscopy. HepG2 cells were stained with 10. Mu.M fluorescent probe solution at 37℃for 15 minutes at the selected excitation wavelength of 426nm, and the observed intracellular fluorescence was shown in FIG. 11. Bright field experiments indicate that cells are truly present throughout the imaging process. It is to be understood that the above examples of the present invention are provided by way of illustration only and not by way of limitation of the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (13)
1. A coumarin derivative, which is characterized by having a structural formula shown in formula (i):
formula (I).
2. A process for the preparation of coumarin derivatives according to claim 1, characterized in that it comprises the following steps:
mixing N-phenethyl benzothiazole salt, 4-hydroxycoumarin, alkali and a solvent, and reacting at 20-160 ℃ to obtain a coumarin derivative, wherein the N-phenethyl benzothiazole salt has a structural formula shown as a formula (II):
(II)
Wherein X is bromine, chlorine or iodine.
3. The method for producing coumarin derivatives according to claim 2, wherein the molar ratio of the N-phenethyl benzothiazole salt to the 4-hydroxycoumarin is 1: (1-2).
4. The method for producing a coumarin derivative according to claim 2, wherein the mass of the base is 10 to 100% of the mass of the N-phenethyl benzothiazole salt.
5. The method for producing coumarin derivatives according to claim 2, wherein the volume molar ratio of the solvent to the N-phenethyl benzothiazole salt is (2-5) mL:1mmol.
6. A process for the preparation of coumarin derivatives according to claim 2, characterized in that the base is an inorganic base and/or an organic base.
7. The method for producing coumarin derivatives according to claim 6, wherein the inorganic base is at least one selected from potassium hydroxide, calcium hydroxide and sodium hydroxide.
8. The method for producing coumarin derivatives according to claim 6, wherein the organic base is at least one selected from sodium acetate, sodium methoxide, sodium tert-butoxide, potassium tert-butoxide and triethylamine.
9. The method for producing coumarin derivatives according to claim 2, wherein the solvent is at least one selected from ethanol, ethyl acetate, isopropyl alcohol, ethylene glycol, acetonitrile, toluene, 1, 4-dioxane, 1, 2-dichloroethane and water.
10. The method for producing coumarin derivatives according to claim 2, wherein the reaction time is 2 to 16 hours.
11. Use of a coumarin derivative according to claim 1 in the preparation of a fluorescent probe, a high sensitivity sensor or a tin ion detector.
12. Use according to claim 11, of the coumarin derivative as a tin ion fluorescent probe for the preparation of live cell imaging reagents.
13. A fluorescent probe comprising the coumarin derivative of claim 1; or coumarin derivatives prepared by the method of any one of claims 2 to 10.
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| CN103852459A (en) * | 2013-12-18 | 2014-06-11 | 南京理工大学 | Application of rhodamine B-based fluorescence sensor |
| CN107857750A (en) * | 2016-11-18 | 2018-03-30 | 南京工业大学 | A kind of fluorescent probe compounds and its preparation and application |
| CN109535147A (en) * | 2018-12-21 | 2019-03-29 | 大连理工大学 | A kind of formaldehyde fluorescence probe of quick response and its preparation method and application |
| CN110229147A (en) * | 2019-06-21 | 2019-09-13 | 安徽大学 | A kind of cumarin-benzothiazolyl hydrazone compounds and its preparation method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103852459A (en) * | 2013-12-18 | 2014-06-11 | 南京理工大学 | Application of rhodamine B-based fluorescence sensor |
| CN107857750A (en) * | 2016-11-18 | 2018-03-30 | 南京工业大学 | A kind of fluorescent probe compounds and its preparation and application |
| CN109535147A (en) * | 2018-12-21 | 2019-03-29 | 大连理工大学 | A kind of formaldehyde fluorescence probe of quick response and its preparation method and application |
| CN110229147A (en) * | 2019-06-21 | 2019-09-13 | 安徽大学 | A kind of cumarin-benzothiazolyl hydrazone compounds and its preparation method and application |
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