A terpyridine zinc complex and its preparation method and application

Abstract

The invention discloses a terpyridine zinc complex and a preparation method and application thereof, belonging to the technical field of fluorescent probe material preparation. The preparation method is as follows: a ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol and a zinc salt are mixed, an alcohol solvent is added thereto, and the reaction is heated to reflux; after the reaction is completed, the terpyridine zinc complex is obtained after filtering, washing, and drying. The terpyridine zinc complex of the present invention not only has excellent two-photon absorption properties and photodynamic therapy effects, but also can be used as a mitochondrial targeted fluorescent probe, and is applied to the preparation of mitochondrial analysis and imaging products. The raw materials of the terpyridine zinc complex provided by the present invention are cheap, easy to obtain, safe and low-toxic. 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2) is prepared by using edible flavor 3-ethoxy-4-hydroxybenzaldehyde, and the trace element zinc required by the human body is used as the central atom, and the reaction is one-step synthesis, the process is simple, and industrial production is easy to achieve.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent probe materials, and in particular to a terpyridine zinc complex and a preparation method and application thereof. Background Art

[0002] Mitochondria are an important component in cells and play a vital role in human life activities. They participate in multiple metabolic pathways, control redox homeostasis, signal transduction, innate immunity and apoptosis. Mitochondrial dysfunction plays an important role in metabolism, immunity, aging and disease development. Therefore, by using fluorescent probes targeting mitochondria, accurate tracking and treatment of diseases can be achieved to achieve better treatment effects.

[0003] The Chinese patent document with publication number CN115850311A discloses a mitochondrial-targeted fluorescent probe and its preparation method and application. At present, most of the reported mitochondrial fluorescent probes are organic molecules, including porphyrin, phthalocyanine and its derivatives. However, these molecules often have some inevitable disadvantages, such as complex synthesis, difficult purification and high toxicity.

[0004] A Chinese patent document with publication number CN114437010A discloses a mitochondrial-targeted hypochlorite two-photon fluorescent probe and its preparation method and application. However, there is no obvious donor-acceptor structure in the molecule, which results in a small two-photon absorption cross-section and weak two-photon absorption activity, limiting its application in living cells and tissues. Summary of the invention

[0005] The purpose of the present invention is to provide a terpyridine zinc complex and a preparation method and application thereof. The terpyridine zinc complex prepared by the method has readily available raw materials, simple synthesis, specific targeting to mitochondria, and has the advantages of good biocompatibility, nonlinear optical activity and photodynamic therapy.

[0006] To achieve this object, the technical solution of the present invention is:

[0007] In the first aspect, the present invention provides a terpyridine zinc complex, which can be abbreviated as ZS2, and its general formula is as follows:

[0008]

[0009] Where X is NO3 - , Cl - , CH3COO - Any one of .

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned terpyridine zinc complex, comprising the following steps:

[0011] The ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol and zinc salt are mixed, an alcohol solvent is added thereto, and the mixture is heated under reflux for reaction; after the reaction is completed, the mixture is filtered, washed, and dried to obtain a terpyridine zinc complex.

[0012] .

[0013] Furthermore, the zinc salt is one of zinc nitrate hexahydrate, zinc chloride or zinc acetate; the alcohol solvent is methanol or ethanol; the mass ratio of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol, zinc salt and alcohol solvent is 1.8-3.7:1: 41-66; the heating reflux reaction temperature is 60-80°C and the time is 3-8h.

[0014] Furthermore, the preparation method of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol comprises the following steps: weighing 2-acetylpyridine and KOH, adding water and stirring until the solution turns yellow, then adding 3-ethoxy-4-hydroxybenzaldehyde and ethanol, then adding ammonia water dropwise, heating to react, obtaining a suspension, filtering to obtain a precipitate, and recrystallizing the precipitate to obtain orange needle-shaped crystals of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2).

[0015] Furthermore, the mass ratio of 2-acetylpyridine, KOH and water is 1:0.46-1.16:0.92-4.46, and the mass ratio of 3-ethoxy-4-hydroxybenzaldehyde, ethanol and ammonia water is 1:5-6:20.5-41; the heating reaction temperature is 60-80°C, and the reaction is carried out for 3-7h.

[0016] In a third aspect, the present invention provides an application of a terpyridine zinc complex prepared by the above method, wherein the terpyridine zinc complex is used to prepare mitochondrial analysis and imaging products.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The terpyridine zinc complex provided by the present invention forms a donor-acceptor structure with obvious electron push-pull effect through the introduction of zinc atoms and the synergistic effect of ligands, the intramolecular electron mobility is further improved, and the two-photon absorption activity is significantly improved. In the two-photon Z-scan test experiment, it was found that the two-photon absorption band reached the near-infrared region (850nm), and the two-photon absorption cross section reached 795-873GM, which is an excellent two-photon fluorescent probe.

[0019] 2. The central atom zinc in the terpyridine zinc complex provided by the present invention can promote intersystem crossing through spin-orbit coupling, increase the generation of singlet oxygen, and significantly generate reactive oxygen species after illumination. Reactive oxygen species at low levels participate in cell signal transduction, cell proliferation and homeostasis, etc., and have the effect of treating tumors.

[0020] 3. The terpyridine zinc complex provided by the present invention can adjust the electrostatic interaction between metal ions and counterions after the ligand is coordinated with different zinc salts, thereby regulating its photophysical properties. The terpyridine zinc complex using zinc nitrate hexahydrate as the zinc source has the largest luminescence wavelength and fluorescence quantum yield.

[0021] 4. The terpyridine zinc complex provided by the present invention has low toxicity. Through the cell survival rate experiment, it was found that under dark conditions, when the terpyridine zinc complex was at a concentration of 25 μM, the survival rate of Hela cells reached 90%.

[0022] 5. The terpyridine zinc complex provided by the present invention is localized in mitochondria through electrostatic interaction. In the cell confocal imaging experiment, it was found that the staining location and phenomenon were consistent with the commercial staining of mitochondria, that is, the terpyridine complex showed specific targeting to mitochondria and can be used to prepare mitochondrial analysis and imaging products.

[0023] 6. The raw material of the terpyridine zinc complex provided by the present invention is cheap, easy to obtain, safe and low in toxicity. The edible flavor 3-ethoxy-4-hydroxybenzaldehyde is used to prepare 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2), and zinc, a trace element required by the human body, is used as the central atom. The reaction is synthesized in one step, the process is simple, and industrial production is easy to achieve. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the mass spectrum of the terpyridine zinc complex in Example 1 of the present invention;

[0025] Figure 2 (a) is a two-photon Z-scan test diagram of the terpyridine zinc complex in Example 1 of the present invention under 850nm laser; (b) is a two-photon absorption cross-sectional diagram of the terpyridine zinc complex in Example 1 of the present invention under 700-1000nm laser wavelength;

[0026] Figure 3 This is a graph showing the reactive oxygen species (ROS) of the terpyridine zinc complex in Example 1 of the present invention;

[0027] Figure 4 This is a fluorescence confocal experiment image of live and dead cells of the terpyridine zinc complex in Example 1 of the present invention;

[0028] Figure 5This is a diagram showing the cytotoxicity experiment of the terpyridine zinc complex in Example 1 of the present invention;

[0029] Figure 6 This is a co-localization experiment diagram of staining of the terpyridine zinc complex in Hela cells in Example 1 of the present invention;

[0030] Figure 7 Graph showing the oil-water separation coefficient of the terpyridine zinc complex in Example 1 of the present invention. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. Example 1

[0032] Weigh 6.05 g of 2-acetylpyridine and 2.8 g of KOH, add 5.6 g of deionized water and stir until the solution turns yellow, add 4.15 g of 3-ethoxy-4-hydroxybenzaldehyde and 20.75 g of ethanol, then add 85 g of 25 wt% ammonia water dropwise, heat to react, control the reaction temperature at 60°C, react for 7 h, obtain a suspension after the reaction is completed, filter to obtain a precipitate, and recrystallize the precipitate to obtain orange needle-shaped crystals of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2);

[0033] Weigh 0.55g of ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2) and 0.3g of Zn(NO3)2·6H2O, add 36.8g of methanol to dissolve, heat to reflux, control the reaction temperature to 80℃, and react for 3h; after the reaction is completed, filter, wash, and dry to obtain an orange-yellow solid product, which is the target product, terpyridine zinc complex. The molecular weight of terpyridine zinc complex is 802.22, such as Figure 1 The peak at position 401.43 in the mass spectrum shows that the terpyridine zinc complex was successfully synthesized. ESI-MS: m / z: calcd for [C 46 H 38 N6O4Zn / 2]: 401.11, found: 401.43. The structural formula is as follows:

[0034] . Example 2

[0035] Weigh 10.59 g of 2-acetylpyridine and 7.35 g of KOH, add 19.85 g of deionized water and stir until the solution turns yellow, add 5.81 g of 3-ethoxy-4-hydroxybenzaldehyde and 30.79 g of ethanol, then add 154.7 g of 25 wt% ammonia water dropwise, heat to react, control the reaction temperature at 65 ° C, react for 6 hours, obtain a suspension after the reaction is completed, filter to obtain a precipitate, and recrystallize the precipitate to obtain orange needle-shaped crystals 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2);

[0036] Weigh 0.89g of ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2) and 0.36g of Zn(NO3)2·6H2O, add 55.2g of ethanol to dissolve, heat to reflux reaction, control the reaction temperature at 70℃, and react for 5h; after the reaction is completed, filter, wash, and dry to obtain an orange-yellow solid product, which is the target product terpyridine zinc complex. The structural formula is as follows:

[0037] . Example 3

[0038] Weigh 16.34 g of 2-acetylpyridine and 15.12 g of KOH, add 51.41 g of deionized water and stir until the solution turns yellow, add 7.47 g of 3-ethoxy-4-hydroxybenzaldehyde and 41.83 g of ethanol, then add 244.8 g of 25 wt% ammonia water dropwise, heat to react, control the reaction temperature to 70 ° C, react for 4 h, obtain a suspension after the reaction is completed, filter to obtain a precipitate, and recrystallize the precipitate to obtain orange needle-shaped crystals 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2);

[0039] Weigh 1.29g of ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2) and 0.2g of ZnCl2, add 64.4g of ethanol to dissolve, heat to reflux reaction, control the reaction temperature at 65°C, and react for 7h; after the reaction is completed, filter, wash, and dry to obtain an orange-yellow solid product, which is the target product, terpyridine zinc complex. The structural formula is as follows:

[0040] . Example 4

[0041] Weigh 26.62g of 2-acetylpyridine and 30.8g of KOH, add 123.2g of deionized water and stir until the solution turns yellow, add 9.13g of 3-ethoxy-4-hydroxybenzaldehyde and 54.78g of ethanol, and then add 374g of 25wt% ammonia water dropwise, heat to react, control the reaction temperature to 80°C, react for 3h, obtain a suspension after the reaction, filter to obtain a precipitate, and recrystallize the precipitate to obtain orange needle-shaped crystals of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2);

[0042] Weigh 1.77g of ligand 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol (S2) and 0.3g of Zn(CH3COO)2, add 73.6g of ethanol to dissolve, heat to reflux reaction, control the reaction temperature at 60℃, and react for 8h; after the reaction is completed, filter, wash, and dry to obtain an orange-yellow solid product, which is the target product terpyridine zinc complex. The structural formula is as follows:

[0043] .

[0044] Experimental Example 1

[0045] The terpyridine zinc complexes of Examples 1-4 provided by the present invention were subjected to two-photon fluorescence Z-scan testing using a titanium sapphire femtosecond pulse laser (680-1080 nm, 80 MHz, 140 fs). Using fluorescein as a standard reference sample, the two-photon absorption cross section (2PA) of the terpyridine zinc complex was calculated using the fluorescence contrast method and the effective quantum yield of the complex in the corresponding solvent. Figure 2 As shown in the data in the table below, the present invention forms a donor-acceptor structure with obvious electron push-pull effect by introducing zinc atoms and cooperating with ligands, so that the electron mobility in the molecule is further improved, and the two-photon absorption activity is significantly improved. The two-photon absorption band reaches the near-infrared region at 850nm, and the maximum two-photon absorption cross section is 873GM. It is an excellent two-photon fluorescent probe.

[0046]

[0047] Experimental Example 2

[0048] The terpyridine zinc complex ZS2 provided in Example 1 of the present invention was tested for its ability to generate ROS by a reactive oxygen species (ROS) generation experiment. Figure 3 As shown in the figure, a PBS solution of zinc complex ZS2 (1×10 -3mol / L), the absorbance of ABDA gradually decreased under light conditions, and the survival rate was significantly reduced to 20%, indicating that ZS2 has the effect of treating tumors; it shows that the central atom zinc can promote intersystem crossing through spin-orbit coupling and increase the generation of singlet oxygen.

[0049] Experimental Example 3

[0050] The AM / PI experiment of the terpyridine zinc complex ZS2 provided in Example 1 of the present invention was used to detect its PDT effect. Figure 4 As shown, under dark conditions, 1 μM ZS2 was added to Hela cells and incubated for 15 minutes, then stained with AM / PI, and red fluorescence signals were detected under 850 nm light irradiation. Reactive oxygen species can be significantly produced after illumination, and participate in cell signal transduction, cell proliferation and homeostasis at low levels, indicating that ZS2 can induce apoptosis of cancer cells under light irradiation.

[0051] Experimental Example 4

[0052] The cytotoxicity experiment of the terpyridine zinc complex ZS2 provided in Examples 1-4 of the present invention detected the dark toxicity to Hela cells. Hela cells were seeded in a 96-well plate, with 5×10 3 The experimental group was set to a concentration gradient of 0-25 μM, 100 μL of stock solution was added to each well, and pure culture medium was added to the blank group (0 μM) as a control. After adding the sample, it was placed in a CO2 incubator for 24 h and then detected by MTT colorimetry. Each group of experiments was set to 8 parallels. Figure 5 As shown in the following table, under dark conditions, the survival rate of the terpyridine zinc complex at a high concentration of 25μM can still reach more than 90%. The terpyridine zinc complex obtained by using 3-ethoxy-4-hydroxybenzaldehyde, which is cheap, easy to obtain, safe and low-toxic, as a ligand and zinc, a trace element required by the human body, as the central atom, has low cytotoxicity, indicating that ZS2 can meet the requirements of cell imaging and other aspects.

[0053]

[0054] Experimental Example 5

[0055] Example 1 of the invention provides a cell confocal imaging experiment of the terpyridine zinc complex ZS2. 5 μM ZS2 was used to stain Hela cells, incubated for 40 minutes, and the culture dish was gently washed 3-4 times with PBS buffer. The fluorescent probe was observed by confocal microscopy to pass through the cell membrane and color the cells. In order to confirm the location of the fluorescent probe targeting the cell, the mitochondrial stain MitoTrackerRed was used for co-localization imaging, such as Figure 6 As shown, the coloring location and phenomenon of this fluorescent probe are consistent with those of MitoTrackerRed, indicating that ZS2 can be localized in mitochondria through electrostatic effects, showing specific targeting to mitochondrial bodies, and can be used to prepare mitochondrial analysis and imaging products.

[0056] Experimental Example 6

[0057] The oil-water separation coefficient experiment of the terpyridine zinc complex ZS2 provided in Example 1 of the present invention detected its LogP value. 1 mg of S2 / ZS2 was dissolved in 5 ml of n-octanol, 3 ml of the supernatant was mixed with 3 ml of water, and the obtained solution was shaken for 30 minutes, and the absorbance of the n-octanol and water layers was measured respectively. Figure 7 As shown, the calculated LogP=0.47 indicates that the terpyridine zinc complex ZS2 has good biological affinity and is easier to enter cells.

[0058] Experimental Example 7

[0059] The luminescence wavelength and fluorescence quantum yield test of the terpyridine zinc complexes of Examples 1, 3 and 4 provided by the present invention, in Examples 1, 3 and 4, the electrostatic interaction between the metal ions and the counterions can be adjusted by coordinating the ligands with zinc nitrate hexahydrate, zinc chloride and zinc acetate, respectively, thereby regulating the photophysical properties thereof, obtaining molecules with different luminescence wavelengths, and measuring the fluorescence quantum yield of the terpyridine zinc complex in DMSO solution (1×10 -3 mol / L), as shown in the following table, compared with the terpyridine complexes with zinc chloride and zinc acetate as zinc sources, the terpyridine zinc complex with zinc nitrate hexahydrate as zinc source has the largest luminescence wavelength and fluorescence quantum yield.

Claims

1. A terpyridine zinc complex, characterized in that: The general formula of the terpyridine zinc complex is as follows: ， Where X is NO3 - , Cl - , CH3COO - Any one of .

2. A method for preparing the terpyridine zinc complex as claimed in claim 1, characterized in that: Add water to 2-acetylpyridine and KOH, stir until the solution turns yellow, then add 3-ethoxy-4-hydroxybenzaldehyde and ethanol, then add ammonia water dropwise, heat and react to obtain a suspension, filter to obtain a precipitate, recrystallize the precipitate to obtain orange needle-shaped crystals of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol; mix 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol and zinc salt, then add an alcohol solvent, heat and reflux to react; after the reaction is completed, filter, wash and dry to obtain a terpyridine zinc complex; The zinc salt is one of zinc nitrate hexahydrate, zinc chloride or zinc acetate; the alcohol solvent is methanol or ethanol; the mass ratio of 4-([2,2':6',2''-terpyridine]-4'-yl)-2-ethoxyphenol, zinc salt and alcohol solvent is 1.8-3.7:1:41-66; the heating reflux reaction temperature is 60-80°C and the time is 3-8h.

3. The method for preparing the terpyridine zinc complex according to claim 2, characterized in that: The mass ratio of 2-acetylpyridine, KOH and water is 1:0.46-1.16:0.92-4.46, and the mass ratio of 3-ethoxy-4-hydroxybenzaldehyde, ethanol and ammonia water is 1:5-6:20.5-41; the heating reaction temperature is 60-80°C, and the reaction is carried out for 3-7h.

4. An application of the terpyridine zinc complex as claimed in claim 1, characterized in that: The terpyridine zinc complex is used to prepare mitochondrial analysis and imaging products.

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