Coumarin-TB-pyridinium derivative as well as synthesis method and application thereof

Abstract

The invention provides a coumarin-TB-pyridinium derivative as well as a synthesis method and application thereof, 4-bromoaniline, 4-bromo-3-methoxyaniline, paraformaldehyde, pyridine-4-boric acid, 4-(diethylamino) salicylaldehyde and the like are selected as raw materials, and compounds TB-CM-1 and TB-CM-2 are synthesized through a multi-step reaction, so that the coumarin-TB-pyridinium derivative is obtained. The structure is as follows: the compounds TB-CM-1 and TB-CM-2 both have excellent photophysical properties, viscosity response, aggregation-induced emission characteristics and good biocompatibility, can efficiently generate type I active oxygen, and have good application prospects in an anoxic environment. The TB-CM-2 also has photodynamic antibacterial activity on a staphylococcus aureus biological membrane. The two compounds have strong photodynamic anti-tumor activity and large difference of light toxicity and dark toxicity, wherein the IC50 of the TB-CM-2 on three tested tumor cells is greater than 100 [mu] g.mL <-1 > in the absence of illumination, and the IC50 of the TB-CM-2 on MCF-7, A549 and HepG2 cells is respectively reduced to 0.40 [mu] g.mL <-1 >, 0.13 [mu] g.mL <-1 > and 1.58 ng.mL <-1 > under illumination. The invention provides a new thought for design and synthesis of a novel efficient photodynamic antibacterial / antitumor photosensitizer.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical synthesis, and particularly relates to a synthesis method of a coumarin-Chogel base Tröger's base (TB)-pyridinium derivative and application thereof in the field of photodynamic antibacterial / antitumor. BACKGROUND

[0002] Pathogenic bacteria infection is one of the biggest threats to human beings. The discovery and development of antibiotics such as penicillin have saved hundreds of millions of lives. However, with the abuse of antibiotics, the problem of bacterial drug resistance is becoming increasingly serious, and photodynamic antibacterial therapy (aPDT) has emerged as the times require. The mechanism of aPDT is to use the toxic reactive oxygen species (ROS) generated by photosensitizer (PS) under irradiation of specific wavelength light to destroy the internal and external structures of bacteria, which is an oxidative stress bactericidal method and will not weaken the bactericidal effect due to mutation or evolution of bacteria. In addition, aPDT is increasingly attracting people's attention due to its low toxicity, high efficiency of spatiotemporal selectivity, and advantages such as being applicable to fluorescence tracking imaging.

[0003] Coumarin, α is a class of natural compounds with various pharmacological activities such as anticoagulation, antibacterial, anti-inflammatory, antidiabetic, anticancer, anticonvulsant, and antiproliferative activities. Coumarin has a large conjugated system and n, π electrons in its structure, and compounds containing this fragment have long-wave emission, high fluorescence quantum yield, and large Stokes shift, and are widely used in the fields of dye solar cells, cell imaging, and fluorescent probes. The benzene ring can be connected to various electron donors (D) or acceptors (A). Therefore, coumarin and its derivatives have great application prospects in the design and synthesis of photosensitizers.

[0004] Pyridinium ion has the advantages of good water solubility and high reactivity. Its strong electron-withdrawing ability can enhance the D-A effect on the photosensitizer backbone, reduce the energy gap between the ground state and the excited state, and play the role of increasing the emission wavelength and Stokes shift, improving the signal-to-noise ratio, reducing background interference, and improving the fluorescence imaging ability of the photosensitizer. Since the bacterial cell membrane is negatively charged, the introduction of a pyridinium ion fragment into the photosensitizer molecule can enhance the binding force between the photosensitizer and the bacterial cell membrane, thereby enhancing the aPDT activity of the photosensitizer.

[0005] Tröger's base (TB) possesses a V-shaped, non-planar, rigid structure, preventing intermolecular π-π stacking; it has eight π electrons and two lone pairs, making it an excellent electron donor. Theoretical calculations show that the TB framework has multiple triplet levels (T1) with energies close to the singlet level (S1). n The potential for intersystem crossing is high, leading to a high probability of ROS generation. Therefore, theoretically, TB possesses both aggregation-induced emission (AIE) properties and high ROS generation efficiency, making it an advantageous framework for designing and synthesizing highly efficient aPDT photosensitizers. However, to date, there are no reports of AIE-based photosensitizers using TB as the framework.

[0006] Therefore, this invention synthesized two coumarin-Tröger's base (TB)-pyridinium type I photosensitizers using coumarin as the electron donor (Donor, D) fragment, the TB backbone as the D fragment and bridging group, and pyridinium as the electron acceptor (Acceptor, A) fragment. The optical properties, viscosity response properties, AIE properties, and ROS generation capacity of the products were determined. The aPDT activity of these products under low-energy LED white light irradiation against Staphylococcus aureus, MRSA, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus biofilms was investigated. By examining the effects of the photosensitizers on bacteria before and after aPDT (such as changes in bacterial surface zeta potential, bacterial morphology (SEM), and intrabacterial ROS levels (CLSM), and considering the lipid-water partition coefficient (LogP) of the photosensitizers, the mechanism by which the products generate aPDT activity was explored. The photodynamic antitumor activity of this drug against human non-small cell lung cancer cells (A549), human breast cancer cells (MCF-7), and human liver cancer cells (HepG2) was tested. Summary of the Invention

[0007] Technical Problem: The purpose of this invention is to provide a class of coumarin-Tröger's base-pyridinium derivatives, their synthesis methods, and applications. By introducing coumarin and pyridinium groups into the TB backbone, two coumarin-Tröger's base-pyridinium derivatives were designed and synthesized, and applied to fields such as photodynamic antibacterial and photodynamic antitumor activity. The excellent optical properties, aPDT activity against drug-resistant bacteria, and PDT antitumor activity of the two products make them of great development value in fields such as hospital wastewater treatment, non-invasive antitumor drugs, viscosity probes, cell imaging, and organelle localization.

[0008] Technical solution: The structural formula of a class of coumarin-TB-pyridinium derivatives of the present invention is shown as either the first derivative TB-CM-1 or the second derivative TB-CM-2: .

[0009] The method for synthesizing the coumarin-TB-pyridinium derivative of the present invention includes the following steps: Step 1: 4-Bromoaniline and 4-Bromo-3-methoxyaniline react with paraformaldehyde 2 to obtain the first intermediate and the second intermediate, respectively, as shown in the following reaction formulas: ; Step 2: The first and second intermediates react with N,N-dimethylformamide (DMF) to obtain the third and fourth intermediates, respectively, as shown in the following reaction formulas: ; Step 3: The third and fourth intermediates react with pyridine-4-boronic acid to obtain the fifth and sixth intermediates, respectively, as shown in the following reaction formulas: ; Step 4: 4-(diethylamino)salicylaldehyde reacts with ethyl acetoacetate to give the seventh intermediate, 3-acetyl-7-(diethylamino)coumarin, as shown in the following reaction formula: ; Step 5: The fifth and sixth intermediates react with the seventh intermediate, 3-acetyl-7-(diethylamino)coumarin, to obtain the eighth and ninth intermediates, respectively, as shown in the following reaction formulas: ; Step 6: The eighth intermediate reacts with bromoethane to give the first derivative TB-CM-1, as shown in the following reaction formula: ; Step 7: The ninth intermediate reacts with bromoethane to give the second derivative TB-CM-2, as shown in the following reaction formula: .

[0010] The application of the first derivative TB-CM-1 or the second derivative TB-CM-2 in the preparation of viscosity probes.

[0011] The application of the first derivative TB-CM-1 or the second derivative TB-CM-2 in the preparation of aggregation-induced emission materials.

[0012] The application of the first derivative TB-CM-1 or the second derivative TB-CM-2 in the preparation of photodynamic antibacterial drugs.

[0013] The application of the first derivative TB-CM-1 or the second derivative TB-CM-2 in the preparation of photodynamic antitumor drugs.

[0014] The antibacterial properties described are inhibition of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA).

[0015] The anti-tumor effect is an inhibition of MCF-7, HepG2, and A549 cells.

[0016] Compounds TB-CM-1 and TB-CM-2 both exhibit excellent photophysical properties, viscosity response, aggregation-induced emission (AIE), and good biocompatibility. Both compounds can efficiently generate type I reactive oxygen species (ROS), showing promising application prospects in anoxic environments. Both compounds are effective against tested Gram-positive bacteria (G7). + The bacteria exhibited good aPDT activity at 2 µmol·L⁻¹. -1 For three types of G + The antibacterial rate of both compounds was greater than 96%; TB-CM-2 also exhibited photodynamic antibacterial activity against Staphylococcus aureus biofilms. Both compounds showed strong photodynamic antitumor activity with significant differences in phototoxicity between light and dark: in the absence of light, TB-CM-2 showed an IC50 of 96% against three tested tumor cell lines. 50 >100 μg·mL -1 IC50 of MCF-7, A549 and HepG2 cells under light irradiation 50 The concentrations decreased to 0.40 μg·mL, respectively. -1 0.13 μg·mL -1 and 1.58 ng·mL -1 This invention provides new ideas for the design and synthesis of novel, highly efficient type I photodynamic antibacterial / antitumor photosensitizers.

[0017] Beneficial effects: The coumarin-Tröger's base-pyridinium derivative of the present invention has the following advantages: 1. For the first time, coumarin-Tröger's base-pyridinium derivative photosensitizers TB-CM-1 and TB-CM-2 were synthesized. The synthesis method is simple and the post-processing is convenient.

[0018] 2. The product has good viscosity response capability and has the potential to become a viscosity response probe; it has significant AIE properties and is expected to realize imaging-guided PDT.

[0019] 3. The synthesized photosensitizers TB-CM-1 and TB-CM-2 have a stronger ROS generation capacity than commercially available photosensitizers, and only generate type I ROS. They have superior photodynamic therapy (PDT) potential in hypoxic microenvironments such as tumor cells, overcoming the problem of low PDT activity of type II photosensitizers that are highly dependent on oxygen concentration in hypoxic microenvironments.

[0020] 4. The synthesized photosensitizers TB-CM-1 and TB-CM-2 exhibit strong light absorption and conversion efficiencies, enabling them to be used in LED white light with light intensities far below the literature average. Achieving G under the stimulation + It exhibits highly effective inhibition of bacteria and possesses high biosafety.

[0021] 5. TB-CM-1 and TB-CM-2 against G + The bacteria exhibited good aPDT effect under low-power illumination (LED white light, 1.89 mW / cm²) far below the literature values. 2 Under these conditions, 2 μmol / L photosensitizer drug was effective against three tested G... + All bacteria showed an inhibition rate of over 96%, including methicillin-resistant Staphylococcus aureus (MRSA).

[0022] 6. Both TB-CM-1 and TB-CM-2 exhibit strong photodynamic antitumor activity with significant differences in phototoxicity between light and dark: TB-CM-2 showed an IC50 of 1,500 against three tested tumor cell types in the absence of light. 50 >100 μg·mL -1 IC50 of MCF-7, A549 and HepG2 cells under light irradiation 50 The concentrations decreased to 0.40 μg·mL, respectively. -1 0.13 μg·mL -1 and 1.58 ng·mL -1 PDT exhibits antitumor activity far exceeding that of most type I antitumor photosensitizers currently reported, and has significant potential for further research.

[0023] 7. The excellent optical properties, drug-resistant aPDT activity, and PDT antitumor activity of the two products make them of great development value in the fields of non-invasive antitumor drugs, viscosity probes, cell imaging, and organelle localization. Attached Figure Description

[0024] Figure 1 The UV absorption spectrum (a) and fluorescence emission spectrum (b) of TB-CM-1 in different solvents are shown. Figure 2 The UV absorption spectrum (a) and fluorescence emission spectrum (b) of TB-CM-2 in different solvents are shown. Figure 3 The fluorescence emission spectra (a) and broken line graph (b) of TB-CM-1 at different viscosities are shown. Figure 4 The fluorescence emission spectra (a) and broken line graph (b) of TB-CM-2 at different viscosities are shown. Figure 5 The fluorescence emission spectra (a) and broken line graphs (b) of TB-CM-1 at different ratios of DMSO / toluene are shown. Figure 6 The fluorescence emission spectra (a) and broken line graphs (b) of TB-CM-2 at different ratios of DMSO / toluene are shown. Figure 7 This is a graph showing the total ROS generation capability of TB-CM-1 and TB-CM-2; Figure 8 It is singlet oxygen of TB-CM-1 and TB-CM-2 ( 1 O2) Generate capability map; Figure 9 It is the superoxide anion (O2) of TB-CM-1 and TB-CM-2 ·- Generate capability maps; Figure 10 The bactericidal ability of different concentrations of TB-CM-1 against Staphylococcus aureus (A), Escherichia coli (B), and MRSA (C) is shown. Figure 11 The results show the bactericidal ability of different concentrations of TB-CM-2 against Staphylococcus aureus (A), Escherichia coli (B), and MRSA (C). Figure 12 It is the fifth intermediate 1 H NMR spectrum; Figure 13 It is the fifth intermediate 13 C NMR spectrum; Figure 14 It is the sixth intermediate 1 H NMR spectrum; Figure 15 It is the sixth intermediate 13 C NMR spectrum; Figure 16 It is the eighth intermediate 1 H NMR spectrum; Figure 17 It is the eighth intermediate 13 C NMR spectrum; Figure 18 It is the ninth intermediate 1 H NMR spectrum; Figure 19 It is the ninth intermediate13 C NMR spectrum; Figure 20 It is TB-CM-1 1 H NMR spectrum; Figure 21 It is TB-CM-1 13 C NMR spectrum; Figure 22 It is TB-CM-2 1 H NMR spectrum; Figure 23 It is TB-CM-2 13 C10 NMR spectrum. Detailed Implementation

[0025] The present invention will be further described below with reference to the embodiments.

[0026] This invention designs and synthesizes coumarin-Tröger's base (TB)-pyridonium derivatives by introducing coumarin and pyridonium groups into the TB backbone, and applies them to the preparation of viscosity probes, aggregation-induced emission materials, photodynamic antibacterial drugs, and photodynamic antitumor drugs. The antibacterial effect is the inhibition of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). The antitumor effect is the inhibition of MCF-7, HepG2, and A549 cells. The structural formulas of the two synthesized coumarin-Tröger's base-pyridonium derivatives are shown in Table 1:

[0027] Table 1. Structural formulas of compounds TB-CM-1 and TB-CM-2

[0028] 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 should not be construed as limiting the present invention. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0029] Example 1: Synthesis of TB-CM-1 and TB-CM-2 This invention provides a method for preparing the above-mentioned novel coumarin-TB-pyridinium derivative, comprising: In this embodiment, 4-bromoaniline, 4-bromo-3-methoxyaniline, paraformaldehyde, pyridine-4-boronic acid, and 4-(diethylamino)salicylaldehyde were selected as raw materials. Coumarin and pyridinium groups were introduced onto the pre-synthesized TB skeleton through a simple reaction to synthesize compounds TB-CM-1 and TB-CM-2, including the following steps: 1. Synthesis of first intermediate 3 and second intermediate 5: 4-Bromoaniline (1, 50.0 mmol), 4-bromo-3-methoxyaniline (4, 50.0 mmol), and paraformaldehyde (2, 100.0 mmol) were added sequentially to a 200.0 mL round-bottom flask. The flask was placed in a cryogenic bath and the temperature was adjusted to -15 °C. Trifluoroacetic acid (100.0 mL, added over approximately 30 min) was slowly added dropwise with stirring. The reaction was allowed to proceed at room temperature for 7 days. After the reaction was complete (tracked by TLC), the mixture was poured into ice water, the pH was adjusted to 7 with ammonia, and the mixture was cooled to room temperature. The mixture was then extracted with dichloromethane. The crude product was obtained by rotary evaporation. Acetone was added and heated until the crude product was completely dissolved. The product was recrystallized at room temperature, filtered, and washed with acetone to obtain the first intermediate 3 and the second intermediate 5, respectively.

[0030]

[0031] 2. Synthesis of the third intermediate 6 and the fourth intermediate 7: Weigh 10.0 mmol of either intermediate 3 or intermediate 5 into a 250 mL round-bottom flask (prepared by igniting with a three-wick lamp to remove water before use). After purging with argon three times, add 40 mL of freshly distilled tetrahydrofuran (THF). Transfer the reaction mixture to a Dewar flask at -78°C (dry ice and acetone). Under argon protection, add 10.0 mmol of n-butyllithium. After reacting for 0.5 h, add 10.0 mmol of DMF. Remove the Dewar flask and stir at room temperature for 12 h. Monitor the reaction by TLC until complete. After quenching with water, extract with DCM. The organic phase was collected, and the crude product was separated and purified by column chromatography (PE:EA = 2:1) to obtain the third intermediate 6 or the fourth intermediate 7.

[0032]

[0033] 3. Synthesis of intermediate 9 (fifth) and intermediate 10 (sixth): Weigh 1.0 mmol of third intermediate 6 (or fourth intermediate 7), 2.0 mmol of pyridine-4-boronic acid 2, 0.2 mmol of tetrakis(triphenylphosphine)palladium and 5.0 mmol of anhydrous potassium carbonate into a 50 mL two-necked flask (prepared by igniting with a three-wick lamp to remove water before use, the same applies below). Add 16 mL of toluene, 4 mL of ethanol and 2 mL of deionized water as a mixed solvent. After purging with argon gas three times, place the flask at 80°C. o The reaction was carried out in an oil bath for 12 hours, and TLC was monitored until the reaction was complete. After quenching with water, the reaction was extracted with DCM. The organic phase was collected, and the fifth intermediate 9 (or the sixth intermediate 10) was purified by column chromatography (PE:EA = 1:1). After drying, the products were weighed, and the yields were the fifth intermediate 9 (61%) and the sixth intermediate 10 (55%), respectively.

[0034]

[0035] 4. Synthesis of the seventh intermediate 13: Weigh 5.0 mmol of 4-(diethylamino)salicylaldehyde 11 into a 50 mL double-necked flask, dissolve it in 15 mL of ethanol, add 15.0 mmol of ethyl acetoacetate 12, and react at 80 °C. During the reaction, a yellow crystalline solid is formed. Monitor the reaction by TLC until it is complete. After cooling, filter the mixture and wash the filter cake with 20 mL of ethanol to obtain pure seventh intermediate 3-acetyl-7-(diethylamino)coumarin. After drying, weigh the product. Yield: 84%.

[0036]

[0037] 5. Synthesis of intermediate 14 (eighth) and intermediate 15 (ninth): Weigh 0.5 mmol of the fifth intermediate 9 into a 50 mL double-necked flask, then add 0.6 mmol of the seventh intermediate 13 and 10 mL of ethanol. After heating to dissolve, piperidine is added dropwise, and the mixture is placed in an oil bath at 80 °C for 36 h. During the reaction, solids continuously precipitate out. The reaction is monitored by TLC until it is complete. After the system cools, it is filtered. The filter cake is washed with ethanol to obtain the pure product, the eighth intermediate 14. After drying, it is weighed. Yield: 51%.

[0038] Weigh 0.5 mmol of the sixth intermediate 10 into a 50 mL two-necked flask, then add 0.6 mmol of the seventh intermediate 13 and 10 mL of ethanol. After heating to dissolve, piperidine is added dropwise, and the mixture is placed in an oil bath at 80 °C for 36 h. The reaction is monitored by TLC until complete, then deionized water is added to quench the reaction, and the mixture is extracted with DCM. The organic phase was then collected and purified by column chromatography (PE:EA = 1:2) to obtain the ninth intermediate 15. After drying, it was weighed, with a yield of 37%.

[0039]

[0040] 6. Synthesis of TB-CM-1 and TB-CM-2: Weigh 0.3 mmol of intermediate 14 (or intermediate 15) into a 50 mL double-necked flask, add 6 mL of acetonitrile to dissolve it (if the dissolution is incomplete, add about 0.5 mL of DCM to promote dissolution), slowly add 1.0 mmol of bromoethane 16 in an oil bath at 80 °C with stirring, monitor the reaction by TLC, and after the system has cooled naturally for 12 h, add an appropriate amount of diethyl ether to precipitate the precipitate, stir for another 1 h, filter, wash the filter cake with diethyl ether, dry, and weigh to obtain product TB-CM-1 or TB-CM-2, yield: 46% (32%).

[0041]

[0042] Product TB-CM-1 The chemical formula is: C 38 H 37 BrN4O3 The Chinese name is: 4-(8-(3-(7-(diethylamino)-2-oxo-2- H -chromen-3-yl)-3-oxopropyl-1-en-1-yl)-6 H 12 H -5,11-methyldibenzo[ b, f [1,5]diazoazine-2-yl)-1-ethylpyridine-1-ammonium bromide The English name is: 4-(8-(3-(7-(diethylamino)-2-oxo-2 H -chromen-3-yl)-3-oxoprop-1-en-1-yl)-6 H ,12 H -5,11-methanodibenzo[ b , f ][1,5]diazocin-2-yl)-1-ethylpyridin-1-iumbromide (TB-CM-1) Appearance: Red solid Melting point: >255℃ 1H NMR spectrum: 1 H NMR (400 MHz, DMSO- d 6) δ 9.05 (d, J = 8.0 Hz, 2H, N + -α-H), 8.57 (s, 1H, C=CH in Coumarin), 8.40 (d, J = 8.0 Hz, 2H, N +-β-H), 7.91(d, J = 8.0 Hz, 1H, Ar-H), 7.80 (d, J = 8.0 Hz, 1H, Ar-H), 7.69 (m, 2H, Ar-H), 7.60 (d, J = 12.0 Hz, 1H, C=CH), 7.53 (d, J = 12.0 Hz, 1H, Ar-H), 7.42(d, J = 16.0 Hz, 1H, Ar-H), 7.37 (d, J = 8.0 Hz, 1H, Ar-H), 7.24 (d, J = 8.0Hz, 1H, Ar-H), 6.81 (d, J = 8.0 Hz, 1H, Ar-H), 6.60 (s, 1H, C=CH), 4.77 (q, J = 8.0 Hz, 2H, N + -CH2-), 4.57 (q, J = 8.0 Hz, 2H, bridge -CH2-), 4.44 - 4.23(m, 4H, -CH2-), 3.53 - 3.47 (m, 4H, -CH2-), 1.53 (t, J = 8.0 Hz, 3H, -CH3),1.14 (t, J = 8.0 Hz, 6H, -CH3). Nuclear magnetic resonance: 13 C NMR (100 MHz, CD3OD) δ 186.81, 160.15, 158.73,155.84, 153.76, 153.58, 152.42, 149.95, 148.79, 143.85, 142.95, 142.52,137.59, 132.05, 131.35, 129.59, 129.05, 127.13, 126.86, 126.09, 124.07,123.64, 122.86, 121.45, 115.51, 110.42, 108.51, 95.95, 66.30, 58.39, 58.26,55.96, 44.77, 15.30, 11.39. Mass spectrometry: HRMS (ESI) m / z : calcd for [C 38 H 37 N4O3] + [M] + found (expected):597.2865 (597.2866). Product TB-CM-2 The chemical formula is: C 40 H 41 BrN4O5 The Chinese name is: 4-(8-(3-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-3-oxoprop-1-en-1-yl)-3,9-dimethoxy-6 H ,12 H -5,11-methyldinitrobenzo[ b, f [1,5]diazozinin-2-yl)-1-ethylpyridine-1-ammonium bromide The English name is: 4-(8-(3-(7-(diethylamino)-2-oxo-2 H -chromen-3-yl)-3-oxoprop-1-en-1-yl)-3,9-dimethoxy-6 H ,12 H -5,11-methanodibenzo[ b , f ][1,5]diazocin-2-yl)-1-ethylpyridin-1-ium bromide Appearance: Red solid Melting point: 216.3-217.1 o C, 1H NMR spectrum: 1 H NMR (400 MHz, CD3OD): δ 8.80 (d, J = 8.0 Hz, 2H, N + -α-H), 8.49 (s, 1H, C=CH in Coumarin), 8.22 (d, J = 8.0 Hz, 2H, N + -β-H), 7.95(s, 2H, Ar-H), 7.53 (d, J= 12.0 Hz, 1H, C=CH), 7.32 (s, 2H, Ar-H), 7.00 (s,1H, Ar-H), 6.81 (m, 2H, Ar-H), 6.54 (s, 1H, C=CH), 4.72 (dd, J 1 = 16.0 Hz, J 2= ​​4.0 Hz, 2H, bridge -CH2-), 4.59 (q, J = 8.0 Hz, 2H, N + -CH2-), 4.38 (s, 2H, -CH2-), 4.34 (s, 2H, -CH2-), 3.93 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 3.54 (q, J = 8.0 Hz, 4H, -CH2-), 1.64 (t, J = 8.0 Hz, 3H, -CH3), 1.25 (t, J = 8.0 Hz, 6H, -CH3). Carbon NMR spectrum: 13 C NMR (100 MHz, CD3OD) δ 187.14, 161.29, 158.66, 158.43, 157.14, 154.93, 153.66, 153.30, 151.42, 148.66, 142.91, 137.79, 131.98, 129.61, 127.02, 126.93, 123.65, 121.29, 120.77, 120.04, 119.54, 115.73, 110.36, 108.49, 108.02, 107.16, 95.91, 66.26, 57.88, 57.70, 55.96, 55.18, 54.96, 44.75, 15.33, 11.40. Mass spectrometry: HRMS (ESI) m / z : calcd for [C 40 H 41 N4O5] + [M] + found (expected):657.3076 (657.3077). Example 2 Optical properties of TB-CM-1 or TB-CM-2 of the present invention The UV absorption and fluorescence emission curves of the compounds of this invention in different polar solvents were tested. The specific experimental scheme is as follows: Transfer 1.0 mL of TB-CM-1 or TB-CM-2 working solution (1 × 10⁻⁶). -4 mol·L -1 The solution was placed in nine 10.0 mL volumetric flasks and diluted with dichloromethane (DCM). N , N Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), toluene, methanol (MeOH), acetonitrile (MeCN), n-hexane, or deionized water (H2O) were diluted to volume, and their ultraviolet absorption and fluorescence emission spectra were measured. Figure 1 , Figure 2 ).

[0043] Depend on Figure 1 and 2 It can be seen that the ultraviolet absorption wavelengths λ of compounds TB-CM-1 and TB-CM-2 in various solvents are... abs Within the 450-500 nm range, the maximum fluorescence emission wavelength λ em Within the 525-575 nm range, the fluorescence intensity of both compounds is higher in solvents with low to medium polarity, decreasing with increasing solvent polarity. This may be because the interaction between the compounds and solvent molecules is stronger in highly polar solvents, leading to a shorter excited-state lifetime, a faster transition rate, and thus a decrease in fluorescence intensity. 。

[0044] Taking into account solvent solubility, λ abs , λ em The optical and photophysical properties of TB-CM-1 or TB-CM-2 were tested using DMSO as a solvent, taking into account factors such as these (Table 2).

[0045] Table 2 Optical properties (DMSO) of TB-CM-1 and TB-CM-2

[0046] Example 3: Viscosity response of TB-CM-1 or TB-CM-2 of the present invention Abnormal cells often have higher viscosity than normal cells. This high intracellular viscosity restricts the movement of chemical bonds, making molecules more prone to aggregation. Therefore, photosensitizers with viscosity-responsive properties may also possess AIE (Advanced Ionization Regulator) properties. The specific experimental protocol is as follows: Take 1.0 mL of a concentration of 1×10 -4 mol·L -1The working solutions of TB-CM-1 or TB-CM-2 were diluted to 10.0 mL with glycerol:water in ratios of 1:9 and 9:1, respectively, and their fluorescence emission spectra were measured. Figure 3 , Figure 4 ).

[0047] Depend on Figure 3 and Figure 4 It can be seen that with the increase of viscosity, the relative fluorescence intensity (RFI) of TB-CM-1 and TB-CM-2 increased by 31.4 times and 7.0 times, respectively, indicating that they both exhibit viscosity responsiveness. This is because the increase of viscosity increases the degree of restriction of chemical bond motion, reduces vibrational and rotational degrees of freedom, and transforms nonradiative energy decay into radiative decay. The level of response to viscosity suggests that the three products may have AIE properties.

[0048] Example 4: AIE characteristics of TB-CM-1 or TB-CM-2 of the present invention The response levels to viscosity suggest that the three products may possess AIE properties; therefore, we investigated the AIE properties of the products. The specific experimental protocol is as follows: Take 1.0 mL of solution with a concentration of 1.0 × 10⁻⁶. -4 mol·L -1 The working solutions were diluted to 10.0 mL with DMSO:toluene solutions in ratios of 9:1 to 1:99, respectively, and their fluorescence emission spectra were measured. Figure 5 , Figure 6 ).

[0049] Depend on Figure 5 and Figure 6 It was found that the RFI of both TB-CM-1 and TB-CM-2 reached its maximum at a toluene content of 99%. Compared with the RFI at a toluene content of 10%, the RFI of TB-CM-1 was increased by 1.26 times, and that of TB-CM-2 by 2.8 times. The presence of methoxy groups may have affected the aggregation mode and strength of TB-CM-2, thus giving it slightly stronger AIE properties. These results indicate that both TB-CM-1 and TB-CM-2 possess AIE properties and may have good ROS generation capabilities.

[0050] Example 5: ROS generation capability of TB-CM-1 and TB-CM-2 of the present invention Viscosity and AIE experimental results showed that both products TB-CM-1 and TB-CM-2 have viscosity response capability and AIE property. Since AIE property can enhance the ROS generation capability of products, we used fluorescent probes to test the total ROS generation capability of TB-CM-1 and TB-CM-2.

[0051] 1. Total ROS generation capacity 2,7-Dichlorodihydrofluorescein (DCFH) is readily oxidized by ROS to form 2,7-dichlorofluorescein (DCF). DCF emits fluorescence at 532 nm under 488 nm light excitation. A white LED lamp (1.89 mW·cm⁻¹) was used. -2 The light source was used for irradiation, and the fluorescence emission spectra of TB-CM-1 and TB-CM solutions were measured sequentially for irradiation times of 1, 2, 3, 4, and 5 min. Figure 7 ).

[0052] Depend on Figure 7 It was found that the results were compared with those of commercially available photosensitizers rose red (RB) and crystal violet (CV). Figure 7 It can be seen that both compounds can generate ROS, and their total ROS generation capacity is much higher than that of commercially available photosensitizers RB and CV. After 5 min of light irradiation, the RFI of TB-CM-1 was 7.5 times higher than that of CV, while that of TB-CM-2 was 3.0 times higher. The total ROS production of TB-CM-1 was greater than that of TB-CM-2.

[0053] 2. Singlet oxygen ( 1 O2 generation capacity Detection using 9,10-anthratridiyl-bis(methylene)dimalonic acid (ABDA) 1 O2: Under LED white light irradiation (1.89mW·cm⁻¹) -1 Under irradiation, the absorbance of ABDA at 380 nm was monitored at different time intervals. The change in absorbance at 378 nm was recorded every 1 minute of irradiation, indicating... 1 The O2 generation rate, the entire experiment was irradiated for 5 minutes ( Figure 8 ).

[0054] Depend on Figure 8 It can be seen that neither compound can produce 1 The presence of O2 indicates that the ROS it produces is likely type I.

[0055] 3. Superoxide anion (O2) ·- Generation capability O2 was detected using dihydrorhodamine 123 (DHR123) as a fluorescent probe. •- : In LED white light (1.89 mW·cm -2 Irradiation was performed under 495 nm excitation. Fluorescence signals around 534 nm were measured. Fluorescence intensity around 534 nm was recorded every minute of irradiation to represent O2. •- The generation rate, after irradiation for 5 min ( Figure 9 ).

[0056] Depend on Figure 9It can be seen that both compounds can generate superoxide anions, and their ability to generate superoxide anions is stronger than that of the commercially available type I photosensitizer CV (compared to CV, TB-CM-1 has a 2.65-fold increase in RFI, and TB-CM-2 has a 1.5-fold increase), which is consistent with the total ROS measurement results.

[0057] Example 6: In vitro photodynamic antibacterial activity of TB-CM-1 and TB-CM-2 of the present invention. The above experimental results show that the designed and synthesized compounds TB-CM-1 and TB-CM-2 have excellent ROS generation capabilities. Therefore, we tested the in vitro aPDT activity of the two compounds against five bacteria using the plate colony counting method (Tables 3-4). Figures 10-12 ).

[0058] Table 3. Inhibition rate (%) of compound TB-CM-1 against five bacteria under dark or light conditions. a

[0059] a Experimental conditions: LED white light (1.89 mW·cm²) -2 ) irradiation for 20 minutes; b It has no antibacterial activity.

[0060] Table 4. Inhibition rate (%) of compound TB-CM-2 against five bacteria under dark or light conditions. a

[0061] a Experimental conditions: LED white light (1.89 mW·cm²) -2 ) irradiation for 20 minutes; b It has no antibacterial activity.

[0062] The results show that: 1. Under dark conditions, the two compounds showed very low inhibitory activity against the five tested bacteria, indicating that they have low dark toxicity and high biosafety.

[0063] 2. The two compounds affect three test Gs + All bacteria (including drug-resistant MRSA) exhibited aPDT activity. The antibacterial effect of TB-CM-2 was slightly stronger than that of TB-CM-1, which contradicted the ROS detection results and ESP calculation results. This indicates that the aPDT activity of the product depends not only on the ROS generation capacity of the photosensitizer but also on other factors, such as the ability of the photosensitizer to bind to bacteria.

[0064] 3. The two compounds in 2 µmol·L -1 For three types of G+ The antibacterial rate of the bacteria was greater than 96%, indicating that the design of the photosensitizer was reasonable.

[0065] 4. Both compounds can produce LED white light with energy far below the literature average (1.89 mW·cm⁻¹). -2 Achieving G under the stimulation of ) + The high efficiency of bacterial inhibition further demonstrates its high biosafety.

[0066] 5. The two compounds affect two test Gs - The inhibitory activity against bacteria (Escherichia coli and Pseudomonas aeruginosa) was low, which is due to G... - The cell membrane of the G is a double-layered structure, with a greater membrane thickness compared to G. + It is more difficult to penetrate and its binding force with positive ions is not as strong as that of G. + Large. To develop it for G... - Further optimization of the structure of highly efficient aPDT photosensitizers is needed.

[0067] Example 7: In vitro photodynamic antitumor activity of TB-CM-1 and TB-CM-2 of the present invention. Given the excellent ROS generation capacity of the two compounds, their PDT antitumor activity against MCF-7, HepG2 and A549 cells was tested (Table 5).

[0068] Table 5. IC50 of the two compounds on test cells under dark or light conditions. 50 Value (μg·mL) -1 )

[0069] Table 5 shows that TB-CM-2 exhibits significant differences in phototoxicity and dark toxicity, with IC50 values ​​for the three tested tumor cell types in the absence of light. 50 >100 μg·mL -1 IC50 of A549 cells under light irradiation 50 0.13 μg·mL -1 IC50 in HepG2 cells 50 It then decreased to 1.58 ng·mL -1 The phototoxicity of TB-CM-2 to normal cells should not be ignored. How to reduce its phototoxicity to normal cells (e.g., reduce or remove groups on the photosensitizer that can target normal cells) is also a key research focus in future work.

[0070] These results confirm that the type I ROS photosensitizer obtained by combining coumarin with the TB backbone can indeed demonstrate significant efficacy in photodynamic antitumor applications, and its further research is of great value. Future work will also test its PDT activity against other tumor cells and further investigate its antitumor mechanism, laying the foundation for its application in the field of PDT antitumor therapy.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A class of coumarin-TB-pyridinium derivatives, characterized in that, The derivative is shown as a first derivative TB-CM-1 or a second derivative TB-CM-2 as shown in the following formula: 。 2. A method of synthesizing the coumarin-TB-pyridinium derivative according to claim 1, characterized by, The method comprises the following steps: Step 1: 4-bromoaniline (1), 4-bromo-3-methoxyaniline (4) are respectively reacted with paraformaldehyde (2) to obtain a first intermediate (3) and a second intermediate (5), and the reaction formula is as follows: ； Step 2: The first intermediate (3) and the second intermediate (5) are respectively reacted with N,N dimethylformamide DMF to obtain a third intermediate (6) and a fourth intermediate (7), and the reaction formula is as follows: ； Step 3: The third intermediate (6) and the fourth intermediate (7) are respectively reacted with pyridine-4-boronic acid (8) to obtain a fifth intermediate (9) and a sixth intermediate (10), and the reaction formula is as follows: ； Step 4: 4-(diethylamino)salicylaldehyde (11) is reacted with ethyl acetoacetate (12) to obtain a seventh intermediate (13) 3-acetyl-7-(diethylamino)coumarin, and the reaction formula is as follows: ； Step 5: The fifth intermediate (9) and the sixth intermediate (10) are respectively reacted with the seventh intermediate (13) 3-acetyl-7-(diethylamino)coumarin to obtain an eighth intermediate (14) and a ninth intermediate (15), and the reaction formula is as follows: ； Step 6: The eighth intermediate (14) is reacted with bromoethane (16) to obtain the first derivative TB-CM-1, and the reaction formula is as follows: ； Step 7: The ninth intermediate (15) is reacted with bromoethane (16) to obtain the second derivative TB-CM-2, and the reaction formula is as follows: 。 3. Use of a coumarin-TB-pyridinium derivative according to claim 1, characterized in that The first derivative TB-CM-1 or the second derivative TB-CM-2 is used for preparing a viscosity probe.

4. Use of a coumarin-TB-pyridinium derivative according to claim 1, characterized in that The first derivative TB-CM-1 or the second derivative TB-CM-2 is used for preparing an aggregation-induced emission material.

5. Use of a coumarin-TB-pyridinium derivative according to claim 1, characterized in that The first derivative TB-CM-1 or the second derivative TB-CM-2 is used for preparing a photodynamic antibacterial drug.

6. Use of a coumarin-TB-pyridinium derivative according to claim 1, characterized in that The first derivative TB-CM-1 or the second derivative TB-CM-2 is used for preparing a photodynamic antitumor drug.

7. Use of a coumarin-TB-pyridinium derivative according to claim 5, characterized in that, The antibacterial is inhibition of Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus MRSA.

8. Use of a coumarin-TB-pyridinium derivative according to claim 6, characterized in that, The antitumor is inhibition of MCF-7, HepG2 and A549 cells.

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