A class of pyridine-TB derivatives and their synthesis method and application

Abstract

The present invention provides a class of pyridine-TB derivatives and their synthesis methods and applications. 4-bromo-3-methoxyaniline, pyridine-4-boric acid, paraformaldehyde, n-butyllithium, ethyl bromide, etc. are selected as raw materials and prepared through a multi-step reaction: #imgabs0# The product has a large Stokes shift (both greater than 100 nm) in both solution and solid state; Compound 7 has significant AIE properties; has a wide pH range of application and can be used in human physiological environments; has good viscosity response, can detect changes in egg white protein, and can be used as a fluorescent probe for detecting changes in protein viscosity; all easily enter living A549 cells, and Compound 7 has a strong targeting ability to the endoplasmic reticulum of A549 cells (Pearson coefficient Pr is 0.76); Al 3+ With recognition ability, it is expected to become an excellent Al 3+ Fluorescent probe; has good photodynamic therapy (PDT) effect on HpeG2 cells and A549 cells.

Description

Technical Field

[0001] The present invention belongs to the fields of organic synthesis, analytical chemistry and bioimaging, and specifically relates to a class of pyridine- Synthesis of base (TB) derivatives and their applications in metal ion recognition, photodynamic therapy, viscosity response and endoplasmic reticulum targeting. Background Art

[0002] The endoplasmic reticulum (ER) is composed of a single membrane layer in the cytoplasm, forming a three-dimensional network of flat membrane sacs, membrane tubes, and membrane vesicles. As a subcellular organelle, it plays a vital role in the life activities of mammalian cells. The development of new and efficient ER-targeted fluorescent probes and the visualization of the ER are of great significance for studying its physiological morphology and monitoring the impact of active substances, microenvironment, and physiological processes on it. It also provides important guidance for the diagnosis and treatment of certain metabolic diseases.

[0003] In recent years, the design and synthesis of ER-targeted fluorescent probes have garnered increasing attention. ER fluorescent probes can be divided into two categories: single-function ER-localizing probes and multifunctional ER probes, which possess multiple functions, including localizing and detecting active species, ER morphology, and the environment within the ER. Currently reported ER-targeted fluorescent probes cover a wide range of applications, including simple ER imaging, metal ions, small molecules, macromolecules, and the microenvironment.

[0004] However, the development of ER fluorescent probes faces several challenges: their underlying molecular mechanisms are unclear and their variety is limited. Therefore, developing diverse, highly selective, highly effective, and low-toxic ER fluorescent probes remains both a challenge and a key focus in this field. Furthermore, the sensitivity of small-molecule fluorescent probes targeting microenvironments such as viscosity, polarity, and membrane fluidity within the ER is generally low, significantly limiting their development and representing a weak link in the current biomedical and diagnostic fields.

[0005] Pyridine is a six-membered heterocyclic compound with planar aromaticity and containing a nitrogen heteroatom. Its π-plane expansion is conducive to enhancing the electron flow between D and A, while increasing intermolecular interactions and providing a well-ordered crystal structure. The pyridine group is easy to modify, and as an electron acceptor, it can enable the material to maintain a high luminescence efficiency; it has a large T2-T1 energy difference, which can reduce the conversion rate within the triplet exciton, adjust the energy gap, and have excellent optical properties. The introduction of positive charge can further increase its electron mobility and charge separation, and optimize the optical properties. Therefore, compounds containing pyridinium groups have begun to be used in fluorescent probes, ion recognition, organic light-emitting diodes, solar cells and other fields. Moreover, after converting it into a pyridinium group, the binding degree of the molecule to tumor cells can be increased (the membrane potential of tumor cells is lower than that of normal cells), thereby improving the photodynamic therapy effect of the molecule on tumor cells.

[79] .

[0006] Base (TB) and its derivatives have a unique V-shaped backbone and a long conjugated structure. They exhibit multiple transition modes (π-π*, n-π*, and spatial transitions) under photon excitation, theoretically exhibiting a high molar absorptivity, making them excellent as the backbone for ultraviolet light-absorbing materials. Therefore, this invention introduces a pyridine group into the TB backbone to design and synthesize a class of pyridine-TB derivatives, which have applications in metal ion recognition, photodynamic therapy, viscosity response, and endoplasmic reticulum targeting. Summary of the Invention

[0007] Technical problem: The purpose of the present invention is to provide a class of pyridine-TB derivatives, their synthesis method and application. By introducing a pyridine group into the TB skeleton, a class of pyridine-TB derivatives is designed and synthesized, and applied to the fields of metal ion recognition, photodynamic therapy, viscosity response and endoplasmic reticulum targeting.

[0008] Technical solution: A class of pyridine-TB derivatives of the present invention has the following structural formulas: the first derivative 5 and the second derivative 7:

[0009]

[0010] The synthesis method of the pyridine-TB derivative of the present invention comprises the following steps:

[0011] Step 1, 3-methoxy-3-bromoaniline 1 reacts with paraformaldehyde 2 to obtain the first intermediate 3, and the reaction formula is as follows:

[0012]

[0013] Step 2: The first intermediate 3 is reacted with pyridine-4-boronic acid 4 by coupling reaction to obtain the first derivative 5. The reaction formula is as follows:

[0014]

[0015] Step 3: The first derivative 5 reacts with bromoethane 6 to obtain the second derivative 7. The reaction formula is as follows:

[0016]

[0017] The present invention discloses a class of pyridine-TB derivatives, wherein the second derivative 7 is used as Al 3+ Application of fluorescent probes.

[0018] The present invention provides an application of a class of pyridine-TB derivatives, and an application of the second derivative 7 in the preparation of cancer photodynamic therapy drugs.

[0019] The application of a class of pyridine-TB derivatives described in the present invention, and the application of the second derivative 7 in the preparation of viscosity probes.

[0020] The application of a class of pyridine-TB derivatives described in the present invention, and the application of the second derivative 7 in the preparation of endoplasmic reticulum targeting probes.

[0021] The application of a class of pyridine-TB derivatives described in the present invention in the preparation of cancer photodynamic therapy drugs is aimed at inhibiting human liver cancer HpeG2 cells and human lung cancer A549 cells.

[0022] The application of the pyridine-TB derivatives described in the present invention is to locate the endoplasmic reticulum of human lung cancer A549 cells in the endoplasmic reticulum targeting probe.

[0023] Beneficial effects:

[0024] 1. The synthesis method is simple and the post-processing is convenient;

[0025] 2. The product has excellent luminescence properties: large Stokes shift, high fluorescence brightness, significant AIE properties and excellent solid-state luminescence.

[0026] 3. The product has a wide pH range and can be used in human physiological environments.

[0027] 4. The product easily enters living A549 cells and has a strong targeting ability to the endoplasmic reticulum (Pearson coefficient Pr is 0.76).

[0028] 5. The product has the ability to target the endoplasmic reticulum and also responds effectively to viscosity, which is of great value for further research;

[0029] 6. Product to Al 3+ With recognition ability, it is expected to become an excellent Al 3+ Fluorescent probes;

[0030] 7. It has good photodynamic therapy effect on A549 and HepG-2 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 :(a) UV absorption and (b) fluorescence emission spectra of the first derivative 5 in different solvents; (c) UV absorption and (d) fluorescence emission spectra of the second derivative 7 in different solvents

[0032] Figure 2 : (a) UV absorption and (b) fluorescence emission spectra of the first intermediate 3, the first derivative 5 and the second derivative 7 in solution

[0033] Figure 3 : Solid-state fluorescence emission spectra of the first intermediate 3, the first derivative 5 and the second derivative 7

[0034] Figure 4 :(a) Fluorescence emission spectra of the first derivative 5 at different viscosities and (b) line graph; (c) Fluorescence emission spectra of the second derivative 7 at different viscosities and (d) line graph

[0035] Figure 5 :(a) Changes in fluorescence intensity of the second derivative 7 at different temperatures; (b) Fluorescence emission spectra of the second derivative 7 before and after protein denaturation

[0036] Figure 6 :(a) Fluorescence emission spectra of the first derivative 5 at different pH values ​​and (b) line graph; (c) Fluorescence emission spectra of the second derivative 7 at different pH values ​​and (d) line graph

[0037] Figure 7 : (a) Fluorescence emission spectra of the first derivative 5 in different THF / H2O ratios and (b) line graph; (c) Fluorescence emission spectra of the second derivative 7 in different DMSO / H2O ratios and (d) line graph (the inset is a photo of 10% and 90% water content under UV light)

[0038] Figure 8 : Scanning electron micrograph of the second derivative 7 at 90% water content

[0039] Figure 9 :(a) Fluorescence emission spectra of the second derivative 7 in the presence of different metal ions and (b) bar graph

[0040] Figure 10 :(a) The second derivative 7 at different concentrations of Al 3+ Fluorescence emission spectra in the presence of (b) and standard curve

[0041] Figure 11 : The second derivative 7-Al 3+ Job's curve of the system

[0042] Figure 12 :(a) Survival rate of A549 cells or (b) HepG-2 cells incubated with different concentrations of the second derivative 7 in the dark or under illumination for 30 minutes

[0043] Figure 13 :Co-localization imaging of the first derivative 5 on the endoplasmic reticulum of A549 cells DETAILED DESCRIPTION

[0044] The present invention will be further described below with reference to the embodiments.

[0045] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and are not to be construed as limiting the present invention. It will be understood by those skilled in the art that various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention.

[0046] The present invention designs and synthesizes a class of pyridine-TB derivatives by introducing a pyridine group into the TB skeleton, and applies them to the fields of metal ion recognition, photodynamic therapy, viscosity response and endoplasmic reticulum targeting.

[0047] The structural formula of pyridine-TB derivatives is shown in Table 1:

[0048] Table 1 Synthesis of the first derivative 5 and the second derivative 7

[0049]

[0050]

[0051] In this embodiment, 4-bromo-3-methoxyaniline, pyridine-4-boric acid, paraformaldehyde, n-butyl lithium, ethyl bromide, etc. are used as raw materials to prepare the product through a multi-step reaction. The steps include:

[0052] Compound 1 reacts with paraformaldehyde to obtain a first intermediate 3, the first intermediate 3 reacts with pyridine-4-boronic acid to obtain a first derivative 5 through a coupling reaction, and the first derivative 5 reacts with bromoethane to obtain a second derivative 7.

[0053] The compounds of the following examples were prepared by the above synthesis method:

[0054] (1) A 250 mL round-bottom flask was charged with 1 (60 mmol) and 2 (150 mmol). The flask was placed in a low-temperature tank and the temperature was adjusted to -15°C. 120 mL of trifluoroacetic acid was slowly added dropwise through a constant pressure dropping funnel. After about 30 minutes of complete addition, the reaction system was moved to a 25°C environment and reacted for 7 days. After the reaction was complete (TLC tracking), it was quenched with ice water, the pH was adjusted to 7 with ammonia water, and the mixture was cooled to room temperature, filtered, washed three times with purified water, and recrystallized from acetone to obtain the first intermediate 3 (yield 65%).

[0055]

[0056] Synthesis of the first intermediate 3 of Formula 1

[0057] (2) The first intermediate 3 (2.0 mmol), pyridine-4-boronic acid 4 (4.2 mmol), tetrakis(triphenylphosphine)palladium (13.0 mmol) and potassium carbonate (0.2 mmol) were added to a 100 mL round-bottom flask in sequence. 14 mL of toluene, 4 mL of ethanol and 2 mL of distilled water were added under argon protection and reacted at 90°C for 12 h. After the reaction was complete (TLC tracking), the mixture was extracted with ethyl acetate, and the organic phase was dried over anhydrous sodium sulfate and dried to obtain a crude product, which was separated and purified by column chromatography (V 石油醚 :V 乙酸乙酯 =1:1), and recrystallized from acetone to obtain the first derivative 5 (yield 58%).

[0058]

[0059] Synthesis of the first derivative 5 of Equation 2

[0060] (3) The first derivative 5 (0.5 mmol) was placed in a 50 mL round-bottom flask, 10 mL of acetonitrile was added, and bromoethane 6 (0.2 mL) was slowly added dropwise with stirring at 25°C. After the addition was complete, the temperature was raised to 82°C and the mixture was refluxed for 6 h. After the reaction was complete (TLC tracking), the reaction system was cooled to room temperature, and diethyl ether was added dropwise until a precipitate formed. The precipitate was filtered, washed, and dried to obtain the second derivative 7 (88%).

[0061]

[0062] Synthesis of the second derivative 7 of Equation 3

[0063] 3,9-dimethoxy-2,8-di(pyridin-4-yl)-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine(5)

[0064]

[0065] 3.78(s,6H). 13 C NMR (100MHz, CDCl3) δ156.13,149.80,149.44,145.95,128.83,124.29,124.15,120.03,107.76,66.86,58.13,55.75.

[0066] 4,4'-(3,9-dimethoxy-6H,12H-5,11-methanodibenzo[b,f][1,5]diazocine-2,8-diyl)bis(1-ethyl pyridin-1-ium)bromide(7)

[0067]

[0068] 4.40(d,J=16.6Hz,2H),4.32(s,2H),3.86(s,6H),1.53(t,J=7.2Hz,6H). 13 C NMR (100MHz, DMSO-d6) δ156.97,153.81,153.41,144.01,130.32,127.16,121.65,119.48,108.79,57.95,56.68,55.90,16.77.

[0069] Solvation effect

[0070] The first derivative 5 and the second derivative 7 were prepared with n-hexane (n-Hexane), toluene (Toluene), tetrahydrofuran (THF), chloroform (CHCl3), ethyl acetate (EA), acetonitrile (MeCN), methanol (MeOH) and dimethyl sulfoxide (DMSO) to a concentration of 1×10 -5 mol·L -1 The UV absorption and fluorescence emission spectra of the solution were tested. Figure 1 ).

[0071] Depend on Figure 1 (a) It can be seen that the λ of the first derivative 5 abs At around 310nm, it is attributed to the π-π long conjugated system in the molecule * The B-band absorption caused by the transition (also indicates that there is a chromophore on the long conjugated system). The absorbance of the first derivative 5 in n-hexane is the lowest, which may be due to its poor solubility in n-hexane. Figure 1 In (c), the second derivative 7 has a absAt around 370 nm, it is larger than that of the first derivative 5. This is likely due to the positive ions in the second derivative 7 molecules increasing the degree of charge separation, reducing ΔE, and making electronic transitions easier and smoother. Due to its poor solubility, the second derivative 7 has almost no absorption in n-hexane, toluene, tetrahydrofuran, and ethyl acetate.

[0072] Figure 1 In (b), the relative fluorescence intensity (RFI) of the first derivative 5 is larger in medium and small polar solvents, and smaller in high polar solvents, but λ em A significant red shift occurs with increasing solvent polarity. Figure 1 In (d), the second derivative 7 has a larger RFI in a highly polar solvent, and λ em There is a certain red shift, which may be due to the greater polarity of the second derivative 7, so it has a greater solubility in highly polar solvents. Compared with the first derivative 5, the λ em This is also due to the fact that the second derivative 7 has positive ions, which increases the degree of charge separation, reduces the ΔE value, and makes the electronic transition easier and smoother.

[0073] Photophysical properties

[0074] The photophysical properties of the first intermediate 3, the first derivative 5, and the second derivative 7 in solution were investigated. The specific experimental scheme is as follows:

[0075] Weigh 10 -5 mol of the first intermediate 3, the first derivative 5, and the second derivative 7 were diluted with a solution to a concentration of 1×10 -5 mol / L, and tested its UV absorption, fluorescence emission and solid-state fluorescence emission spectrum (1×10 -5 mol·L -1 , Figure 2 ).

[0076] The spectral data of the first intermediate 3, the first derivative 5 and the second derivative 7 are shown in Table 2.

[0077] Table 2 Spectral data of the first intermediate 3, the first derivative 5 and the second derivative 7

[0078]

[0079] a UV absorption wavelength in solution; b Molar extinction coefficient ε = A / bC, unit is 1×10 5 L·mol -1 cm -1 ; c Fluorescence emission wavelength in solution; d Stokes shift in solution;e Relative fluorescence quantum yield (reference: quinine sulfate); f Fluorescence brightness FB = ε*Φ, unit is L·mol -1 cm -1 ; g solid-state excitation wavelength; h solid-state fluorescence emission wavelength; i Solid-state Stokes shift.

[0080] Depend on Figure 2 (a) and Table 2 show that compared with the first intermediate 3, the first derivative 5 and the second derivative 7 in the solution have the highest λ abs There is a significant red shift and ε is enhanced, which may be due to the introduction of pyridine increasing the length of the conjugated system and the n electron cloud density. As mentioned above, the positive ion of the second derivative 7 significantly increases the degree of charge separation, reduces ΔE, and makes the intramolecular charge transfer (ICT) easier and smoother, so its λ abs Compared with the first derivative 5, it is obviously red-shifted.

[0081] Depend on Figure 2 (b) Figure 3 As shown in Table 2, compared with the first intermediate 3, the fluorescence properties of the product have the following changes: the solution and solid λ of the first derivative 5 and the second derivative 7 em Both of them showed obvious red shift, and the Stokes shift of the solution increased significantly; the Φ of the first derivative 5 and the second derivative 7 increased significantly, and the FB was significantly enhanced. The Φ of the second derivative 7 was slightly smaller than that of the first derivative 5. The possible reason is that the second derivative 7 has a positive charge and the electron cloud density is reduced. Therefore, the Φ of the second derivative 7 is slightly smaller than that of the first derivative 5, which is consistent with the theoretical law.

[0082] In summary, introducing a pyridine ring into the TB skeleton can increase the length of the conjugated system and the n-electron cloud density. Modifying the pyridine ring into pyridinium can significantly increase the degree of charge separation, reduce ΔE, enhance the intramolecular charge transfer effect, and gradually improve the luminescence performance of the compound.

[0083] These results demonstrate that combining the TB backbone with the pyridinium group can amplify the advantages of both in luminescence performance, offering a new approach to obtaining products with superior luminescence properties. These results also confirm theoretical calculations and demonstrate the rationality of this design.

[0084] Viscosity response

[0085] The first derivative 5 and the second derivative 7 were prepared with methanol as solvent to a concentration of 1×10 -4 mol·L -1Take five 10 mL volumetric flasks, pipette 1.0 mL of the working solution into each volumetric flask, add 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, and 8.0 mL of glycerol, respectively, and dilute to volume with methanol to make the concentration of the first derivative 5 or the second derivative 7 1×10 - 5 mol·L -1 , and their fluorescence emission spectra (λ ex =310nm and 360nm, slit: 5 / 5nm, Figure 4 ).

[0086] Depend on Figure 4 As can be seen, the fluorescence intensity of both the first derivative 5 and the second derivative 7 increases with increasing viscosity. This may be because at low viscosity, the degree of intramolecular rotation is high and the fluorescence intensity is low; however, as the viscosity increases, the movement of submolecular atomic groups is hindered, the degree of restriction of intramolecular motion (RIM) increases, and the molecules release energy mainly through radiation, which increases the fluorescence intensity.

[0087] The above results indicate that the first derivative 5 and the second derivative 7 have the potential to become viscosity-responsive fluorescent probes. Therefore, we took the second derivative 7 as an example and conducted a protein aggregation experiment using egg white as a sample: First, the stability of the second derivative 7 was studied. The second derivative 7 was prepared with DMSO as the solvent at a concentration of 1×10 -4 mol·L -1 Take five 10 mL volumetric flasks, pipette 1.0 mL of the working solution of the second derivative 7 and 1.0 mL of PBS buffer solution into each volumetric flask, and adjust the volume with DMSO to make the concentration of 1 × 10 -5 mol·L -1 Each volumetric flask was placed in a water bath at 20℃, 40℃, 60℃, 80℃ and 100℃ for five minutes, and the changes in fluorescence intensity (λ ex =360nm, slit: 5 / 5nm, Figure 5 a).

[0088] Then, the second derivative 7 was prepared with DMSO as solvent to a concentration of 1×10 -4 mol·L -1 The working solution was prepared with PBS to a concentration of 1 × 10 -4 mol·L -1 Take two 10 mL volumetric flasks, pipette 1.0 mL of the working solution of the second derivative 7 and 1.0 mL of the egg white solution into each volumetric flask, and adjust the volume with DMSO so that the concentrations of the second derivative 7 and egg white in the system are both 1×10 -5mol·L -1 Each volumetric flask was placed in a water bath at 25°C and 95°C for five minutes, and its fluorescence emission spectra (λ ex =360nm, slit: 10 / 10nm, Figure 5 b).

[0089] Depend on Figure 5 (a) It can be seen that as the temperature increases, the fluorescence intensity of the second derivative 7 does not change much, indicating that the second derivative 7 has excellent thermal stability. Figure 5 (b) As can be seen, the fluorescence of the second derivative 7 is weak at 25°C. This is likely due to the unique V-shaped molecular configuration of the TB backbone and its high molecular rigidity, which hinders the binding of the pyridinium ions at both ends of the second derivative 7 to the receptor sites on the protein. However, after protein denaturation (95°C), the fluorescence intensity of the second derivative 7 is significantly enhanced. This is mainly because the increased temperature causes protein denaturation, providing a channel for the probe to reach the binding site, and the second derivative 7 is distributed along the unfolded protein molecule, forming a "necklace beads" structure. This V-shaped pyridinium salt structure of the second derivative 7 binds to the protein through non-covalent interactions (such as hydrophobic and electrostatic interactions), reducing the conformational freedom of the probe molecules and thus inducing them to emit strong fluorescence as aggregates. These results suggest that the second derivative 7 has the potential to produce a high-level response to changes in ER viscosity and is suitable for further study as a versatile ER fluorescent probe.

[0090] pH response

[0091] The first derivative 5 and the second derivative 7 were prepared with DMSO as solvent at a concentration of 1×10 -4 mol·L -1 1.0 mL of the working solution was measured and placed in a 10 mL volumetric flask, followed by the addition of 1.0 mL of a buffer solution with a pH value of 3-10 (citric acid / disodium hydrogen phosphate system was selected when the pH value was 3-8, and sodium bicarbonate / sodium carbonate system was selected when the pH value was 9-10), and DMSO was used to adjust the volume so that the concentration of the first derivative 5 or the second derivative 7 was 1×10 -5 mol L -1 , the fluorescence emission spectrum (λ ex =310nm and 360nm, slit: 2.5 / 5nm, Figure 6 ).

[0092] Depend on Figure 6 It can be seen that when the solution pH is 3-10, the fluorescence emission spectra of the first derivative 5 and the second derivative 7 do not change significantly, indicating that the first derivative 5 and the second derivative 7 have a wide pH application range and are expected to be applied in the human physiological environment.

[0093] AIE effect

[0094] Taking the first derivative 5 as an example, 5 was prepared with THF as solvent to a concentration of 1×10 -4 mol·L -1 Take 10 10mL volumetric flasks, transfer 1.0mL of the working solution into each volumetric flask, add 0.0-9.0mL of distilled water and THF to make the volume constant, so that the concentration of the first derivative 5 is 1×10 -5 mol L -1 , the fluorescence emission spectrum (λ ex =310nm and 360nm, slit: 2.5 / 5nm, Figure 7 ).

[0095] Depend on Figure 7 It can be seen that with the increase of water content, the fluorescence intensity of the first derivative 5 gradually decreases, and it does not have AIE properties. However, the fluorescence of the second derivative 7 changes significantly with the increase of water content. When the dilute DMSO solution of the second derivative 7 is excited at 360nm, almost no fluorescence signal is recorded; however, when a large amount of water is added to the DMSO solution, a significant enhancement of luminescence is observed in the DMSO / water mixture, and the fluorescence of the second derivative 7 is turned on, showing AIE activity. Since the second derivative 7 is insoluble in water, it will aggregate in the DMSO / water mixture, and the system of 90% DMSO / water "solution" becomes turbid, that is, the "effective concentration" of the solution is reduced. In addition, the positively charged pyridine ring and the N on the N bridge can act as electron acceptors and electron donors, respectively, thereby polarizing the entire molecule, and the strong push-pull interaction enhances intramolecular charge transfer (ICT). Therefore, the enhanced fluorescence emission of the second derivative 7 may be the result of the synergistic effect of its twisted geometry and partial coplanarity.

[0096] from Figure 7 (d) Fluorescence photograph and Figure 8 It can be seen from the scanning electron microscope image that when the water content is 90%, the molecules are obviously aggregated and emit blue fluorescence, and the fluorescence brightness increases.

[0097] Recognition of metal ions

[0098] The second derivative 7 was tested with Na + , K + Mg 2+ , Ca 2+ 、Fe 2+ 、Cu 2+ 、Zn 2+ 、Al 3+ 、Fe 3+ The fluorescence emission spectrum (λ ex =360nm, slit: 5 / 10nm, Figure 9 ).

[0099] Depend on Figure 9 It can be seen that compared with other metal ions, the addition of Al 3+ After that, the fluorescence intensity of the second derivative 7 is greatly enhanced, indicating that the second derivative 7 has a strong affinity for Al 3+ It has obvious identification function.

[0100] The changes in fluorescence intensity of the second derivative 7 after adding metal ions are shown in Table 3.

[0101] Table 3 Effects of the second derivative 7 on different metal ions

[0102]

[0103]

[0104] a The change rate of the fluorescence intensity of the compound after adding metal ions is η = (I-I0) / I0×100%. "-" means no

[0105] Combine Figure 9 As can be seen from Table 3, the recognition efficiency has little to do with the change of ion radius.

[0106] Then Al 3+ Fluorescence titration experiment of the second derivative 7 (λ ex =360nm, slit: 5 / 10nm, Figure 10 ).

[0107] Depend on Figure 10 It can be seen that when Al 3+ The concentration is 1×10 -5 -6×10 -5 mol·L -1 Within the range, as Al 3+ As the concentration increases, the fluorescence intensity of the second derivative 7 at 473 nm gradually increases. 3+ The concentration is 6×10 -5 mol·L -1 When the fluorescence enhancement rate of the first derivative 5 reaches 135%, the fluorescence intensity is similar to that of Al 3+ The concentration has a good linear relationship, R 2 is 0.99428, and the linear equation y = 36.28571 × 10 5 x+1079.33333. The calculation shows that the second derivative 7 has a 3+ The detection limit was 8.9×10 - 7 mol·L -1 , which is expected to be used to detect Al in drinking water3+ The content (7.4×10 -6 mol·L -1 ).

[0108] Then the second derivative 7-Al was drawn 3+ The Job's curve of the system (λ ex =360nm, slit: 5 / 10nm, concentration: 1×10 -5 mol·L -1 , Figure 11 ).

[0109] Depend on Figure 11 It can be seen that the second derivative 7-Al 3+ The Job's curve has no obvious inflection point, indicating that the second derivative 7 and Al 3+ No stable complex was formed, and the specific coordination mode needs further confirmation.

[0110] Extracorporeal photodynamic therapy

[0111] The dark toxicity and phototoxicity of the second derivative 7 to human non-small lung cancer cells (A549) and human liver cancer cells (HepG-2) were tested by standard MTT assay. Figure 12 ).

[0112] like Figure 12 As shown in the figure, when no light was applied, both A549 and HepG-2 cells treated with the second derivative 7 showed high survival rates, indicating that the second derivative 7 had negligible dark toxicity; however, after light exposure, the survival rates of A549 and HepG-2 cells decreased significantly, and with the increase of the concentration of the second derivative 7, the cell survival rates gradually decreased, showing a clear concentration dependence. The concentration of the second derivative 7 was 100.0 μmol·L -1 When the cells were treated with PBS, the survival rates of A549 and HepG-2 cells were only about 10%.

[0113] Table 4 Half inhibition rate (IC) of the first derivative 5 and the second derivative 7 on two cell lines 50 )

[0114]

[0115] IC of the first derivative 5 and the second derivative 7 on two cell lines 50 The values ​​are shown in Table 4. The results showed that the first derivative 5 had lower dark toxicity to both cells, indicating that the first derivative 5 had good biocompatibility; the second derivative 7 had lower dark toxicity and higher phototoxicity to A549 and HepG-2 cells, with high PDT efficiency, and was worthy of further research.

[0116] Endoplasmic reticulum colocalization imaging

[0117] Based on the good optical properties, lipid solubility, pH adaptability and low dark toxicity of the first derivative 5, a co-localization experiment was conducted on the endoplasmic reticulum of A549 cells by co-staining method ( Figure 13 ), and the commercial endoplasmic reticulum green localization probe ER-Green was selected as the colocalization reagent.

[0118] Depend on Figure 13 It can be seen that the first derivative 5 emits blue fluorescence (blue channel), and the luminescence position overlaps well with the position of the ER-Green green channel, with a colocalization coefficient of 0.76. These results indicate that the first derivative 5 is mainly located in the endoplasmic reticulum of the cell.

[0119] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A pyridine-TB derivative in the preparation and detection of Al 3+ The application of fluorescent probe is characterized by: The pyridine-TB derivative is:

2. Use of a pyridine-TB derivative in the preparation of a cancer photodynamic therapy drug, characterized in that: The cancers are human liver cancer and human lung cancer, and the pyridine-TB derivative is 3. Use of a pyridine-TB derivative in preparing an endoplasmic reticulum targeting probe, characterized in that: The application is directed to endoplasmic reticulum localization in human lung cancer A549 cells, and the pyridine-TB derivative is

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