A beta-galactoside-activated aie-type photosensitizer, and a preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and discloses a beta-galactoside enzyme activated AIE type photosensitizer and a preparation method and application thereof. The present application provides an AIE photosensitizer, which is a compound (1) with a structural formula as shown in formula (1). When the galactose group exists, the power supply group is shielded, the ICT effect is weakened, and the PDT performance is in a closed state. When the galactose group is hydrolyzed by beta-galactosidase, the overall water solubility of the compound (1) from which the galactose group is hydrolyzed becomes poor, and the compound (1) is aggregated. Meanwhile, the power supply group hydroxyl is opened, the ICT effect is enhanced, and the PDT performance is in an open state. The glycosidase activity in ovarian cancer cells is abnormally increased, and the glycosidase in ovarian cancer cells triggers the AIE photosensitizer to be activated in the tumor microenvironment with high glycosidase activity, so that precise positioning is achieved. This selective activation mode significantly reduces the misactivation of the photosensitizer in normal tissues and reduces the damage to healthy tissues.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a β-galactosidase-activated AIE-type photosensitizer, its preparation method, and its application. Background Technology

[0002] Glycosidases are ubiquitous enzymes that play crucial roles in a wide variety of biological events. Abnormal glycosidase activity is associated with multiple diseases; for example, β-galactosidase and α-fucosidase are upregulated in tumors. Elevated glycosidase levels have become a hallmark of cancer, making these enzymes promising targets for tumor diagnosis and treatment. Many tumor cells exhibit high levels of β-galactosidase activity, particularly in cancer types such as ovarian, liver, and colorectal cancer. This characteristic makes them potential targets for tumor diagnosis and treatment, leveraging their high activity for precise tumor localization and targeted therapy.

[0003] Photodynamic therapy (PDT) is a novel clinical treatment option for both cancer and non-malignant diseases. PDT requires a photosensitizer and excitation light. The photosensitizer selectively binds to target cells in healthy tissue, and then the affected area is irradiated with excitation light. Reactive oxygen species (ROS) generated from a series of photochemical reactions (3O2) react with cellular components, namely cell membrane lipids, DNA, and other biomolecules. These reactions lead to loss of biological function and cell death.

[0004] Traditional photosensitizers often lack selectivity, are widely distributed in the body, and may damage healthy tissues. AIE (Aggregation-Induced Emission) photosensitizers are a class of photosensitizers that emit strong fluorescence in their aggregated state. AIE molecules emit light in the aggregated state, and the aggregation-induced emission property means they are almost non-luminescent in solution (when unactivated). This "switching" mechanism can reduce background signal, greatly improving the signal-to-noise ratio of tumor tissue, which is beneficial for clearer and more accurate labeling of tumor tissue and enhancing diagnostic accuracy. In the aggregated state of AIE photosensitizers, intermolecular nonradiative relaxation is reduced, leading to more energy being used for the generation of singlet oxygen and reactive oxygen species (ROS), which can enhance the photosensitizing effect. Summary of the Invention

[0005] To address the current lack of selectivity in photosensitizers, this invention provides a β-galactosidase-activated AIE-type photosensitizer, its preparation method, and its application.

[0006] The specific technical solution of this invention is as follows: This invention provides a β-galactosidase-activated AIE-type photosensitizer, the structural formula of which is shown in formula (1):

[0007] Through research, this invention provides a compound (1) with the structural formula shown in formula (1), which is a β-galactosidase-activated AIE-type photosensitizer. When the galactose group is present, the ICT effect is weakened due to the shielding of the power-donating group, and the PDT performance is in a closed state. When the galactose group is hydrolyzed by β-galactosidase, the overall water solubility of the compound (1) after hydrolysis of the galactose group decreases, and it becomes aggregated. At the same time, the hydroxyl group of the power-donating group is opened, the ICT effect is enhanced, and the PDT performance is in an open state. Glycosidase activity is abnormally increased in ovarian cancer cells. Glycosidase in ovarian cancer cells triggers the AIE photosensitizer to be specifically activated in these tumor microenvironments with high glycosidase activity, achieving precise positioning. This selective activation mode significantly reduces the false activation of the photosensitizer in normal tissues and reduces damage to healthy tissues.

[0008] This invention also provides a method for preparing a β-galactosidase-activated AIE-type photosensitizer, comprising the following steps: Step S1: React compound (2) with malononitrile to obtain compound (3); Step S2: 2,3,4,6-tetraacetoxy-α-D-pyranose bromide was reacted with compound (4) to obtain compound (5); Step S3: React compound (3) and compound (5) to obtain compound (6); Step S4: Deacetylate compound (6); in: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (5) is: The structural formula of compound (6) is:

[0009] As a preferred embodiment of the above preparation method, in step S1, compound (2) and malononitrile are dissolved in toluene and reacted, wherein a catalyst is added to the reaction.

[0010] More preferably, the catalyst is acetic acid and ammonium acetate.

[0011] As a preferred embodiment of the above preparation method, in step S2, 2,3,4,6-tetraacetoxy-α-D-pyranose bromide is reacted with compound (4) in acetonitrile, and the reaction is carried out under anhydrous conditions.

[0012] Further preferred, in step S2, the reaction is carried out with the catalyst cesium carbonate.

[0013] As a preferred embodiment of the above preparation method, in step S3, compound (3) and compound (5) are dissolved in ethanol and reacted, with a catalyst added to the reaction.

[0014] Further preferably, in step S3, the catalyst is piperidine.

[0015] As a preferred embodiment of the above preparation method, in step S4, the method for removing the acetyl group is as follows: dissolve compound (6) in methanol, and then add sodium methoxide / methanol saturated solution to react.

[0016] Based on the above, the present invention also provides the application of the above-mentioned compound (1) AIE type photosensitizer in imaging with ovarian cancer biomarkers.

[0017] Based on the above, the present invention also provides the application of the above-mentioned compound (1) AIE type photosensitizer in the preparation of a drug for treating ovarian cancer.

[0018] Compared with the prior art, the present invention has the following technical effects: 1. This invention provides a compound (1) with the structural formula shown in formula (1), which is a β-galactosidase-activated AIE-type photosensitizer. When the galactose group is present, the ICT effect is weakened due to the shielding of the power-donating group, and the PDT performance is in a closed state. When the galactose group is hydrolyzed by β-galactosidase, the overall water solubility of the compound (1) after hydrolysis of the galactose group decreases, and it becomes aggregated. At the same time, the hydroxyl group of the power-donating group is opened, the ICT effect is enhanced, and the PDT performance is in an open state. Glycosidase activity is abnormally increased in ovarian cancer cells. Glycosidase in ovarian cancer cells triggers the AIE photosensitizer to be specifically activated in these tumor microenvironments with high glycosidase activity, achieving precise positioning. This selective activation method significantly reduces the false activation of the photosensitizer in normal tissues and reduces damage to healthy tissues.

[0019] 2. Compared with traditional photosensitizers, the compound (1) shown in formula (1) does not contain heavy atoms, has less toxic side effects, and is more biocompatible. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of compound (1) prepared in Example 4 of this invention.

[0021] Figure 2 The carbon NMR spectrum of compound (1) prepared in Example 4 of this invention.

[0022] Figure 3The absorption spectra of compound (1) prepared in Example 4 of this invention before and after activation by β-galactosidase are shown.

[0023] Figure 4 The emission spectra of compound (1) prepared in Example 4 of this invention before and after activation by β-galactosidase are shown.

[0024] Figure 5 The results of the study on the specificity of compound (1) prepared in Example 4 of this invention against β-galactosidase are shown.

[0025] Figure 6 This is a confocal micrograph comparing normal cells and β-galactosidase overexpressing cells with compound (1) prepared in Example 4 of this invention.

[0026] Figure 7 This is a comparison diagram of the photodynamic effects of compound (1) prepared in Example 4 of this invention on cells overexpressing β-galactosidase and cells underexpressing β-galactosidase. Detailed Implementation

[0027] The present invention will be further described below with reference to embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0028] In this embodiment of the invention, the structural formula of compound (1) is: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (5) is: The structural formula of compound (6) is:

[0029] Compound (2) is a publicly disclosed compound, and its preparation method can be found in the following references: Zhang, H.; Liu, C., Synthesis and properties of furan / thiophene substituted difluoroboron β-diketonate derivatives bearing a triphenylamine moiety. Dyes and Pigments 2017, 143, 143-150. Example 1 Preparation of compound (3) In this embodiment, compound (3) was prepared. The structural formula of compound (3) is shown in formula (3) as follows:

[0030] The preparation steps in this embodiment are as follows: Compound (2) (1.5 g, 5.0 mmol) and malononitrile (0.327 g, 15.0 mmol) were completely dissolved in a round-bottom flask containing 30 mL of toluene. Then, ammonium acetate (0.77 g, 1.0 mmol) and acetic acid (0.743 g, 5.5 mmol) were added to the reaction system, and the mixture was refluxed for 18 hours. After cooling the reaction system to room temperature, it was extracted with ethyl acetate. The supernatant was washed successively with 20 mL of 1 M HCl, 20 mL of saturated sodium bicarbonate, and 20 mL of saturated brine. The organic phase was concentrated and then purified by column chromatography (eluent: petroleum ether: dichloromethane = 2:1 (v / v)). The eluent containing the target product (brownish-yellow liquid) was collected, concentrated under reduced pressure, and dried to obtain the solid, compound (3), with a yield of 40.38%.

[0031] The obtained solid was subjected to NMR analysis, and the results are as follows: 1H NMR (600MHz, Chloroform-d) δ: 7.56–7.48 (m, 1H), 7.48–7.44 (m, 1H), 7.38–7.27 (m, 1H), 7.09 (td, J=7.4, 1.9Hz, 2H), 6.96–6.92 (m, 3H), 6.90–6.86 (m, 1H), 2.47 (s, 1H).

[0032] In this embodiment, the structural formula of compound (2) is shown in formula (2), as follows:

[0033] Example 2 Preparation of compound (5) In this embodiment, compound (5) was prepared. The structural formula of compound (5) is shown in formula (5) as follows:

[0034] The preparation steps in this embodiment are as follows: 2,3,4,6-Tetraacetoxy-α-D-pyranose bromide (3.7 g, 8.9 mmol) and 8-hydroxyjulonidine-9-carboxaldehyde (1 g, 4.6 mmol) were dissolved in 60 mL of acetonitrile solution, and then cesium carbonate (7.4 g, 22.7 mmol) was added. The reaction was carried out at room temperature under N2 atmosphere for 8 h, and the reaction progress was detected by TLC. After the reaction was completed, the mixture was extracted with ethyl acetate, and the upper layer was washed with 20 mL of saturated brine. The organic phase was concentrated and then purified by column chromatography (eluent: petroleum ether: ethyl acetate = 5:1 (v / v)). The eluent containing the target product was collected (the eluent with pale blue fluorescence was collected), concentrated under reduced pressure, and dried to obtain the solid, namely compound (5), with a yield of 73%.

[0035] The obtained solid was subjected to NMR analysis, and the results are as follows: 1H NMR (600MHz, Chloroform-d) δ: 10.03 (s, 1H), 7.35 (s, 1H), 5.51 (dd, J = 10.4, 8. 0Hz,1H),5.38(d,J=3.4Hz,1H),5.10–5.04(m,1H),4.81(d,J=8.0Hz,1H),4.10– 4.04(m,2H),3.80(td,J=6.8,1.2Hz,1H),3.27(q,J=5.0,4.5Hz,4H),2.81–2.67 (m,4H),2.20(s,3H),2.14(s,3H),2.01(s,3H),1.96(s,3H),1.94–1.86(m,4H).

[0036] Example 3 Preparation of compound (6) In this embodiment, compound (6) was prepared. The structural formula of compound (6) is shown in formula (6) as follows:

[0037] The preparation steps in this embodiment are as follows: Compound (3) (0.10 g, 0.42 mmol), compound (5) (0.13 g, 0.42 mmol), and piperidine (7.1 mg, 0.084 mmol) were added to 4 ml of ethanol solution and refluxed for 2 h. After the reaction was confirmed to be complete by TLC, the reaction solution was extracted with dichloromethane, the lower layer was collected, concentrated under reduced pressure, and subjected to silica gel column chromatography (using a 1:1 mixture of dichloromethane and petroleum ether as the eluent). The eluent containing the target product (purple liquid) was collected, concentrated under reduced pressure, and dried to obtain a solid, which is compound (6).

[0038] The obtained solid was subjected to NMR analysis, and the results are as follows: ¹H NMR (400MHz, DMSO-d6) δ: 7.47–7.41 (m, 4H), 7.39 (d, J = 4.1Hz, 2H), 7.36 (d, J = 8.6Hz, 3H), 7.18–7.14 (m, 5H), 7.14–7.04 (m, 4H), 5.24–5.11 (m, 3H), 4.90 (d, J = 7.8Hz, 1H), 4.13–3.95 (m, 3H), 3.84–3.75 (m, 1H), 3.31 (s, 5H), 2.68 (dt, J = 38.9, 5.6Hz, 4H), 2.08 (s, 3H), 1.95 (s, 3H), 1.88 (s, 7H), 1.70 (s, 3H).

[0039] In this embodiment, the structural formula of compound (3) is shown in formula (3), as follows:

[0040] The structural formula of compound (5) is shown in formula (5), as follows:

[0041] Example 4 Preparation of compound (1) In this embodiment, compound (1) was prepared. The structural formula of compound (1) is shown in formula (1) as follows:

[0042] The preparation steps in this embodiment are as follows: Compound (6) was dissolved in 10 mL of methanol, and 6 drops (approximately 6 μL) of saturated sodium methoxide-methanol solution were added. The reaction was carried out at room temperature for 20 min. The reaction was monitored by TLC, with the polarity being dichloromethane:methanol = 30:1. The solvent methanol was removed by rotary evaporation, and the reaction solution was purified by column chromatography (dichloromethane:methanol 30:1 (v / v)). The eluent containing the target product was collected and concentrated under reduced pressure. The solvent was removed by rotary evaporation, and then dried in an oil pump to obtain a purple oily liquid product, which was compound (1).

[0043] The obtained product was analyzed by NMR, and the results are shown in the figure. Figure 1 and Figure 2 .

[0044] In this embodiment, the structural formula of compound (6) is shown in formula (6), as follows:

[0045] Example 5 This embodiment aims to study the fluorescence absorption and emission spectra of compound (1) (excitation wavelength 540 nm, emission wavelength 640 nm), and is carried out according to the following steps: Compound (1) prepared in Example 4 was weighed and dissolved in dimethyl sulfoxide to prepare a probe stock solution with a concentration of 1 mM. 2 μL of the stock solution was added to 398 μL of PBS buffer, followed by the addition of β-galactosidase (β-Gal). The solution was incubated at 37°C for 30 min, and the absorbance was measured using a UV spectrophotometer. The solution was then transferred to a 96-well plate, and the fluorescence absorption and emission spectra of compound (1) before the addition of β-galactosidase, as well as the fluorescence absorption and emission spectra of compound (1) after incubation with β-galactosidase, were determined. The fluorescence spectra are shown in the attached image. Figure 3 , Figure 4 .

[0046] The results showed that after incubation with β-galactosidase, the fluorescence intensity of compound (1) at 640 nm was enhanced, indicating that the glycosidic bond of the probe was hydrolyzed by β-Gal, and the fluorescence of compound (1) was released.

[0047] Example 6 Take 2 μL of the probe stock solution prepared in Example 4 and add it to 398 μL of PBS buffer to make the concentration of compound (1) in the final test system 5 μM, and set up 12 groups. Then, add a certain substance to each of the 12 groups of systems with a concentration of compound (1) of 5 μM, namely: 1. β-Gal (1 U / mL), 2. β-Glu (1 U / mL), 3. BSA (100 μg / mL), 4. HSA (100 μg / mL), 5. OVA (100 μg / mL), 6. Vc (100 μM), 7. GSH (100 μM), 8. Cys (100 μM), 9. Hcy (100 μM), 10. H2S (100 μM), 11. MgCl2 (100 μM), 12. H2O2 (100 μM).

[0048] Next, it was incubated at 37°C for 30 minutes, and its absorbance was measured using a UV spectrophotometer. A bar chart of its fluorescence intensity at 640 nm was then created. The results are as follows: Figure 5 As shown (λex=540nm).

[0049] Depend on Figure 5 It can be seen that compound (1) only responds to β-Gal. Only after the addition of β-Gal does the fluorescence at 645nm increase significantly. However, after the addition of other interfering substances, the fluorescence intensity at 640nm does not increase significantly. This indicates that the fluorescent probe has high selectivity and specificity and is not affected by other interfering substances in the biological environment.

[0050] Example 7 Compound (1) was used to perform fluorescence imaging on endogenous β-Gal in 4T1 cells, HeLa cells, HEK293 cells, and human ovarian adenocarcinoma cells (SKOV-3 cells). The culture and imaging procedures for 4T1, HeLa, HEK293, and SKOV-3 cells were as follows: 4T1, HeLa, HEK293, and SKOV-3 cells (4×10⁵ / well) were placed in sterile culture dishes at 37°C and cultured in DMEM medium containing 10% embryonic serum for 12 h, maintaining a 5% CO₂ atmosphere. The cells were then transferred to 6-well culture plates and incubated overnight. After washing twice with PBS buffer (pH=7.4), imaging was performed. The fluorescence spectra are shown below. Figure 6 .

[0051] The results showed that after co-incubation with compound (1), the fluorescence of the red channel in SKOV-3 cells gradually increased over time. This was due to the high expression of endogenous β-Gal in human ovarian adenocarcinoma cells (SKOV-3 cells), while no obvious fluorescence signal was observed in other cells with low β-Gal expression. This demonstrates that compound (1) can be an effective tool for imaging endogenous β-Gal in human ovarian adenocarcinoma cells (SKOV-3 cells).

[0052] Example 8 Different concentrations of compound (1) were added to HeLa and SKOV-3 cells, respectively. Four parallel groups were designed, and the cells were treated with light and without light, respectively. Cell viability was measured. The experimental results are shown in […]. Figure 7 .

[0053] from Figure 7 As can be seen, the photosensitizer compound (1) activated by β-galactosidase exhibits strong selective killing of SKOV-3 cells overexpressing β-galactosidase under light conditions, while its killing effect on HeLa cells with low β-galactosidase expression is lower than that on SKOV-3 cells. Therefore, the photosensitizer compound (1) activated by β-galactosidase can serve as a probe for ovarian cancer and selectively eliminate ovarian cancer cells.

[0054] Unless otherwise specified, the raw materials and equipment used in this invention are all commonly used in the field; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A β-galactosidase-activated AIE-type photosensitizer, characterized in that: The structural formula is shown in equation (1):

2. A method for preparing a β-galactosidase-activated AIE-type photosensitizer, characterized in that: Includes the following steps: Step S1: React compound (2) with malononitrile to obtain compound (3); Step S2: 2,3,4,6-tetraacetoxy-α-D-pyranose bromide was reacted with compound (4) to obtain compound (5); Step S3: React compound (3) and compound (5) to obtain compound (6); Step S4: Deacetylate compound (6); in: The structural formula of compound (2) is: The structural formula of compound (3) is: The structural formula of compound (4) is: The structural formula of compound (5) is: The structural formula of compound (6) is:

3. The preparation method according to claim 2, characterized in that: In step S1, compound (2) and malononitrile are dissolved in toluene to carry out the reaction, and a catalyst is added to the reaction.

4. The preparation method according to claim 3, characterized in that: In step S1, the catalyst is acetic acid and ammonium acetate.

5. The preparation method according to claim 2, characterized in that: In step S2, 2,3,4,6-tetraacetoxy-α-D-pyranose bromide is reacted with compound (4) in acetonitrile under anhydrous conditions.

6. The preparation method according to claim 2 or 5, characterized in that: In step S2, cesium carbonate is added as a catalyst to the reaction.

7. The preparation method according to claim 2, characterized in that: In step S3, compounds (3) and (5) are dissolved in ethanol and reacted, with piperidine as a catalyst added to the reaction.

8. The preparation method according to claim 2, characterized in that: In step S4, the method for removing the acetyl group is as follows: dissolve compound (6) in methanol, and then add sodium methoxide / methanol saturated solution to react.

9. The application of the AIE-type photosensitizer as described in claim 1 in imaging ovarian cancer biomarkers.

10. The use of the AIE-type photosensitizer as described in claim 1 in the preparation of a medicament for treating ovarian cancer.

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