Berberine photosensitizer, preparation method and application thereof

Abstract

The application belongs to the field of tumor photodynamic therapy, and discloses a berberine photosensitizer and a preparation method and application thereof, and particularly relates to a berberine compound shown in the following general formula (I). The compound has an aggregation-induced emission property, can efficiently generate active oxygen to kill tumor cells under light conditions, and has good application potential in the field of imaging-guided photodynamic therapy.

Description

Technical Field

[0001] This invention belongs to the field of tumor photodynamic therapy, specifically relating to a class of berberine-based photosensitizers with aggregation-induced emission properties, their preparation methods, and applications. Background Technology

[0002] Cancer, as a major disease threatening human health, presents a challenging task in terms of precision treatment. Photodynamic therapy (PDT) is a novel therapy that combines photosensitizers (PS) with light and oxygen to selectively treat malignant tumors, vascular lesions, and microbial infections through photodynamic reactions. The principle of PDT is that when the photosensitizer is irradiated by a light source, it absorbs energy and transitions from the ground state (S0) to the first singlet excited state (S1), then through intersystem crossing (ISC) to the first triplet excited state (T1). There, it reacts with surrounding molecules through electron or energy transfer, generating biotoxic reactive oxygen species (ROS), which kill tumor cells, thus treating the tumor. Compared to traditional tumor treatment methods, PDT has advantages such as high spatiotemporal selectivity, no drug resistance, minimal invasiveness, and low intrusion, demonstrating unique advantages in the treatment of solid tumors.

[0003] Several photosensitizers are already in clinical use. For example, verteporfen was approved by the FDA in the United States in 2001 for the treatment of age-related ocular neovascularization, pathological myopia, and ocular histoplasmosis. Given the enormous potential of photodynamic therapy in the field of anti-tumor therapy, developing new structural types of photosensitizers that are more active and safer is of significant research value.

[0004] Berberine's structure can be viewed as a fused pair of isoquinoline rings, exhibiting certain antitumor activity. It exerts its antitumor activity through multiple pathways, including inducing cell cycle arrest, inhibiting telomerase activity, promoting mitochondrial-mediated apoptosis, p53-mediated apoptosis, and mitophagy-dependent necrotizing apoptosis, and inhibiting inflammatory factors. Simultaneously, studies have demonstrated that berberine possesses certain photosensitizing activity, with significantly stronger antitumor activity under light conditions compared to those under light-protected conditions. While berberine shows potential for photodynamic therapy, its weak photosensitizing activity and limited killing effect, coupled with the requirement for ultraviolet light excitation and difficulty in penetrating deep tissues, greatly limit its deeper applications. Reasonable structural modification of berberine to construct a donor-π-receptor (D-π-A) system is expected to extend the absorption / emission wavelength while enhancing its photosensitizing activity. This invention aims to provide a novel class of berberine-based photosensitizers with aggregation-induced luminescence properties and strong reactive oxygen species generation capabilities, and to apply them to the fields of tumor imaging and photodynamic therapy. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to overcome the shortcomings of low photosensitivity and short excitation wavelength of berberine, and to provide a novel berberine photosensitizer with aggregation-induced emission properties, which can effectively generate reactive oxygen species, thereby improving the problems of weak photosensitivity of berberine itself, limited tumor killing effect, and the need for ultraviolet light excitation, which makes it difficult to penetrate deep tissues.

[0006] Another objective of this invention is to disclose a method for preparing this type of photosensitive material, which is highly operable and efficient;

[0007] Another objective of this invention is to elucidate the photophysical properties and reactive oxygen species generation performance of the aforementioned photosensitizers and to apply them to the fields of cell imaging and photodynamic therapy.

[0008] Technical solution: The berberine-based photosensitizer with aggregation-induced emission properties described in this invention comprises compounds with structures as shown in general formula (I):

[0009]

[0010] in,

[0011] X is selected from F, Cl, Br, I;

[0012] R is selected from one of the following structural segments:

[0013]

[0014] A method for preparing compounds of general formula (I) includes the following steps:

[0015] Starting with berberine hydrochloride, a C-12 substituted berberine bromide intermediate 1 was obtained by bromination reaction, and then a Suzuki-Miyaura coupling reaction was carried out with an aromatic group substituted with boric acid or borate ester to obtain compound of general formula (I).

[0016]

[0017] Preferably, the compound is selected from the following compounds:

[0018]

[0019] A preferred method for preparing the compound includes the following steps:

[0020] (1) Synthetic route of compound BBR-1:

[0021] Starting with berberine hydrochloride, a C-12 substituted berberine bromide intermediate 1 was obtained by bromination reaction, and then reacted with triphenylamine 4-boronic acid via Suzuki-Miyaura coupling reaction to obtain compound BBR-1.

[0022]

[0023] Reaction conditions: (a) Bromine, 1,4-dioxane, room temperature, 48 h; (b) Triphenylamine 4-boronic acid, palladium acetate, sodium carbonate, anhydrous ethanol, room temperature, 12 h.

[0024] (2) Synthetic route of compound BBR-2:

[0025] Starting with triphenylamine 4-borate, intermediate 2 was obtained by Suzuki-Miyaura coupling reaction with 4,7-dibromo-2,1,3-benzothiadiazole. Intermediate 3 was then obtained by Suzuki-Miyaura coupling reaction with pinacol diborate. Finally, compound BBR-2 was obtained by Suzuki-Miyaura coupling reaction with berberine bromide 1.

[0026]

[0027] Reaction conditions: (a) 4,7-dibromo-2,1,3-benzothiadiazole, tetratriphenylphosphine palladium, potassium carbonate, tetrahydrofuran, water, argon protection, 90℃, 6h; (b) pinacol diboronate, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, potassium acetate, 1,4-dioxane, 120℃, 5.5h; (c) intermediate 1, palladium acetate, sodium carbonate, anhydrous ethanol, 90℃, 7h.

[0028] (3) Synthetic route of compound BBR-3:

[0029] Starting with 4-ynyltriphenylamine, intermediate 4 was obtained by Suzuki-Miyaura coupling reaction with 4,7-dibromo-2,1,3-benzothiadiazole. Intermediate 5 was then obtained by Suzuki-Miyaura coupling reaction with pinacol diboronate. Finally, compound BBR-3 was obtained by Suzuki-Miyaura coupling reaction with berberine bromide 1.

[0030]

[0031] Reaction conditions: (a) 4,7-dibromo-2,1,3-benzothiadiazole, bis(triphenylphosphine)palladium chloride, triphenylphosphine, cuprous iodide, tetrahydrofuran, triethylamine, argon protection, 70℃, 12h; (b) pinacol diborate, 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride, potassium acetate, tetrahydrofuran, 80℃, 6h; (c) Intermediate 1, palladium acetate, sodium carbonate, anhydrous ethanol, 90℃, 7h.

[0032] The compound exhibits aggregation-induced emission.

[0033] The compound, when used as a photosensitizer and activated by light, can effectively generate reactive oxygen species to kill tumor cells.

[0034] The compound has applications in organelle and tumor imaging and photodynamic therapy for tumors.

[0035] The present invention has the following advantages and beneficial effects:

[0036] 1. The berberine photosensitizer provided by this invention has a non-planar structure, which can effectively inhibit the intermolecular π-π stacking effect, exhibiting aggregation-induced emission effect and improving the ability to generate reactive oxygen species.

[0037] 2. Compared with traditional photosensitizers, the berberine-based photosensitizers provided by this invention introduce a donor group with triphenylamine as the core, which can effectively prolong the conjugated system and achieve a red shift in absorption / emission wavelength, thereby optimizing berberine excitation from ultraviolet light to visible light excitation, improving penetration ability and reducing toxic side effects.

[0038] 3. The berberine-based photosensitizer raw materials provided by this invention are readily available and simple to synthesize. No similar reports have been found, and it has strong commercial value.

[0039] 4. The berberine-based photosensitizers provided by this invention have been successfully applied to the targeted localization and imaging of organelles within living cells, providing a new tool for imaging-guided photodynamic therapy of tumors and possessing great application potential. Attached Figure Description

[0040] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof.

[0041] Figure 1 Emission spectrum of compound BBR-1

[0042] Figure 2 Emission spectrum of compound BBR-2

[0043] Figure 3 Emission spectrum of compound BBR-3

[0044] Figure 4 Determination of the singlet oxygen generation capacity of compound BBR-1 by ABDA method

[0045] Figure 5 Determination of the singlet oxygen generation capacity of compound BBR-2 by ABDA method

[0046] Figure 6 ABDA method for determining the singlet oxygen generation capacity of compound BBR-3

[0047] Figure 7 Cytotoxicity of compound BBR-1 under light conditions

[0048] Figure 8 Cytotoxicity of compound BBR-2 under light conditions

[0049] Figure 9 Cytotoxicity of compound BBR-3 under light conditions

[0050] Figure 10 Intracellular mitochondrial fluorescence imaging of compound BBR-2

[0051] Figure 11 Intracellular Golgi fluorescence imaging of compound BBR-2 Detailed Implementation

[0052] (The embodiments described are for illustrative purposes only and are not intended to limit the scope of the invention.)

[0053] The claims of this invention specifically set forth novel features of the invention. Exemplary embodiments utilizing the principles of the invention are set forth below. The features and advantages of the invention can be better understood by referring to the following description.

[0054] Although preferred embodiments of this application are described herein, these embodiments are provided by way of example only. It should be understood that variations of the embodiments of this application described herein can also be used to implement the technical solutions of this application. Those skilled in the art should understand that various variations, changes, and substitutions may occur without departing from the scope of this application. It should be understood that the scope of protection of each aspect of this application is determined by the claims, and the methods and structures within the scope of these claims, as well as their equivalent methods and structures, are all within the scope covered by the claims of this application.

[0055] The preparation of some of the compounds is as follows:

[0056] ¹H-NMR nuclear magnetic resonance was measured using a JEOL ECZ-400S (400 Hz) NMR spectrometer (TMS was used as an internal standard), and mass spectrometry was performed using a Thermo Q Exactive LC-MS / MS.

[0057] Column chromatography used 200-300 mesh silica gel (Qingdao Ocean Chemical Plant), with petroleum ether-ethyl acetate, dichloromethane-methanol, or petroleum ether-dichloromethane systems as eluents. Thin-layer chromatography (TLC) was performed using GF254 TLC plates (Yantai Jiangyou Silica Gel Development Co., Ltd.); the TLC developing system was petroleum ether-ethyl acetate, dichloromethane-methanol, or petroleum ether-dichloromethane system; TLC was displayed under ZF7 three-way ultraviolet light (Henan Gongyi Yuhua Instrument Co., Ltd.).

[0058] Example 1: Synthesis of Intermediate 1

[0059]

[0060] Berberine hydrochloride (5 g, 13.4 mmol) was dissolved in dioxane (40 mL), and a dioxane solution (20 mL) containing Br2 (20.8 g, 130 mmol) was added dropwise under ice bath conditions. The mixture was stirred at room temperature for 4 days. The precipitate was filtered and washed successively with 10% Na2S2O3 solution, 10% NaHCO3 solution, and saturated NaCl solution to obtain 3.72 g of a yellow solid, with a yield of 56%. 1 H NMR (400MHz, DMSO-d6) δ9.98 (s, 1H), 8.51 (d, J = 7.4Hz, 2H), 7.93 (s, 1H), 7.10 ( s,1H),6.18(s,2H),4.95(t,J=6.3Hz,2H),4.10(s,6H),3.20(t,J=6.3Hz,2H).

[0061] Example 2: Synthesis of Intermediate 2

[0062]

[0063] Triphenylamine 4-borate (2.89 g, 10 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (2.94 g, 10 mmol), tetratriphenylphosphine palladium (1.011 g, 0.875 mmol), and potassium carbonate (6.910 g, 50 mmol) were added to 75 mL of tetrahydrofuran and 15 mL of water. The mixture was refluxed at 90 °C for 6 h under argon protection. The reaction was monitored by TLC until completion. The solvent was removed under reduced pressure at 55 °C, and the product was purified by column chromatography to obtain 3.02 g of orange solid, with a yield of 66%. 1 H NMR (400MHz, CDCl3) δ7.87(d,J=7.6Hz,1H),7.79(d,J=8.7Hz,2H),7.52(d,J=7.6 Hz, 1H), 7.28 (t, J = 7.7Hz, 4H), 7.17 (dd, J = 8.2, 3.7Hz, 6H), 7.06 (t, J = 7.3Hz, 2H).

[0064] Example 3: Synthesis of Intermediate 3

[0065]

[0066] Intermediate 2 (2.54 g, 10 mmol), pinacol diborate (10.2 g, 40 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (0.056 g, 0.075 mmol), and potassium acetate (1.227 g, 12.5 mmol) were added to 80 mL of 1,4-dioxane. The mixture was refluxed at 120 °C for 5.5 h under argon protection. The reaction was monitored by TLC until completion. The solvent was removed under reduced pressure at 65 °C to obtain 1.487 g of the mixture, which was directly added to the next reaction step.

[0067] Example 4: Synthesis of Intermediate 4

[0068]

[0069] 4-ethynyltriphenylamine (5.387 g, 20 mmol), 4,7-dibromo-2,1,3-benzothiadiazole (5.88 g, 20 mmol), bis(triphenylphosphine)palladium chloride (0.14 g, 0.2 mmol), triphenylphosphine (0.05 g, 0.2 mmol), and cuprous iodide (0.076 g, 0.4 mmol) were added to 60 mL of tetrahydrofuran and 30 mL of triethylamine. The mixture was refluxed at 70 °C for 12 h under argon protection. The reaction was monitored by TLC until completion. The solvent was removed under reduced pressure at 55 °C, and the product was purified by column chromatography to obtain 1.745 g of orange solid, with a yield of 36%. 1 H NMR (400MHz, CDCl3) δ7.82(d,J=7.7Hz,1H),7.62(d,J=7.6Hz,1H),7.49(d,J=8.8Hz,2 H),7.33–7.27(m,4H),7.13(d,J=7.6Hz,4H),7.11–7.06(m,2H),7.03(d,J=8.7Hz,2H).

[0070] Example 5: Synthesis of Intermediate 5

[0071]

[0072] Intermediate 4 (0.481 g, 1 mmol), pinacol diborate (1.02 g, 4 mmol), 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (0.24 g, 0.3 mmol), and potassium acetate (0.49 g, 5 mmol) were added to 30 mL of tetrahydrofuran. The mixture was refluxed at 80 °C for 6 h under argon protection. The reaction was monitored by TLC until completion. The solvent was removed under reduced pressure at 65 °C to obtain 1.238 g of the mixture, which was then directly added to the next reaction step.

[0073] Example 6: Synthesis of product BBR-1

[0074]

[0075] Intermediate 1 (0.119 g, 0.24 mmol), triphenylamine 4-borate (0.208 g, 0.72 mmol), palladium acetate (0.005 g, 0.022 mmol), and sodium carbonate (0.051 g, 0.48 mmol) were added to 10 mL of anhydrous ethanol, stirred overnight at room temperature, and the solvent was removed under reduced pressure at 55 °C. The mixture was purified by preparative plate to obtain 52 mg of orange solid, with a yield of 37%. 1 H NMR(400MHz,DMSO-D6)δ9.96(s,1H),8.31(s,1H),8.02(s,1H),7.54–7.49(m,3H),7.38–7.29(m,4H) ,7.15–7.04(m,9H),6.12(s,2H),4.94(d,J=7.0Hz,2H),4.09(d,J=4.1Hz,6H),3.16(t,J=6.2Hz,2H).

[0076] Example 7: Synthesis of product BBR-2

[0077]

[0078] Intermediate 1 (0.223 g, 0.45 mmol), intermediate 3 (0.689 g, 0.72 mmol), palladium acetate (0.0089 g, 0.04 mmol), and sodium carbonate (0.095 g, 0.9 mmol) were added to 15 mL of anhydrous ethanol. The mixture was refluxed at 80 °C for 5 h under argon protection. The solvent was removed under reduced pressure at 55 °C, and the mixture was purified by plate preparation to obtain 132 mg of orange-red solid, with a yield of 41%. 1 H NMR (400MHz, DMSO-D6) δ10.01(s,1H),8.33–8.25(m,2H),8.13–8.04(m,3H),7.99(dd,J=7.3,2.1Hz,1H),7.57–7.50(m,1H),7.46(d,J= 1.2Hz,1H),7.40–7.34(m,3H),7.24–7.01(m,9H),6.06(s,2H),4.97(d,J=6.2Hz,2H),4.19(s,3H),4.09(s,3H),3.19(t,J=6.3Hz,2H).

[0079] Example 8: Synthesis of product BBR-3

[0080]

[0081] Intermediate 1 (0.163 g, 0.45 mmol), intermediate 5 (0.524 g, 0.99 mmol), palladium acetate (0.007 g, 0.033 mmol), and sodium carbonate (0.070 g, 0.66 mmol) were added to 7 mL of anhydrous ethanol. The mixture was refluxed at 80 °C for 7 h under argon protection. The solvent was removed under reduced pressure at 55 °C, and the mixture was purified by a preparative plate to obtain 21 mg of an orange-red solid, with a yield of 9%. 1 H NMR(400MHz,DMSO-D6)δ10.00(d,J=3.0Hz,1H),8.25(s,1H),8.21(s,1H),8.0 7(dd,J=7.1,1.6Hz,1H),7.89(d,J=7.3Hz,1H),7.51(d,J=8.5Hz,2H),7.43(s, 1H),7.35(t,J=7.7Hz,4H),7.15–7.07(m,6H),7.01(s,1H),6.93(d,J=8.4Hz,2 H),6.04(s,2H),4.93(s,2H),4.15(s,3H),4.06(s,3H),3.16(t,J=6.4Hz,2H).

[0082] Experimental Example 1

[0083] 1. Determination of absorption and emission spectra of photosensitizers

[0084] (1) Prepare the DMSO stock solution (10mM) of the photosensitizer in the example, and then dilute it with H2O to a final concentration of 10μM for the photosensitizer molecular test solution.

[0085] (2) Add the above photosensitizer test solution to a 96-well plate, with two replicates for each sample, 200 μL per well, and then use Tecan Spark. TM The 10M Multimode Microplate Reader is used to test the absorption spectra of compounds.

[0086] (3) The DMSO stock solution (10 mM) was diluted to a final concentration of 10 μM with different proportions of THF / water solvent system, wherein the proportions of THF were 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 100% respectively. Based on the maximum absorption wavelength obtained above, the emission spectrum of the photosensitizer in the above system was selected for excitation at 405 nm, as shown below. Figures 1-3 As shown.

[0087] 2. Determination of the singlet oxygen generation capacity of photosensitizers

[0088] (1) Dilute the above photosensitizer stock solution with PBS to a final concentration of 10 μM photosensitizer test solution.

[0089] (2) Using RB as a standard and ABDA (100 μM) as a reactive oxygen species indicator, a photosensitizer (10 μM) was added, and the absorbance curves of ABDA were tested after different illumination times. An I / I0 graph was plotted based on the change in absorbance over time at a fixed wavelength. Figures 4-6 As shown.

[0090] Experiment Example 2

[0091] Photosensitizer cytotoxicity test: HepG2 cells were seeded in 96-well plates (5 × 10⁻⁶ cells / well). 3 Cells were cultured in MEM medium containing 10% fetal bovine serum in a CO2 incubator (37℃, 5% CO2) for 24 h. Then, photosensitizers were added at final concentrations of 100 μmol / L, 50 μmol / L, 10 μmol / L, 1 μmol / L, 100 nmol / L, 10 nmol / L, 1 nmol / L, and 0.1 nmol / L, respectively, and incubated for 4 h. After 15 min of illumination, the cells were cultured for another 24 h. Then, 20 μL / well of MTT (5 mg / mL) was added and the cells were incubated for another 4 h. The medium was removed, and 100 μL of DMSO was added to each well. The OD value at 570 nm was read, and cell viability was calculated. Figures 7-9 As shown.

[0092] Experimental Example 3

[0093] Live cell imaging experiments:

[0094] 1. Mitochondrial imaging: 4T1 cells were seeded in confocal culture dishes (2×10⁻⁶ cells / year). 5 Cells were cultured in a CO2 incubator (37℃, 5% CO2) on 1640 medium containing 10% fetal bovine serum for 24 h. Then, BBR-2 was added to a final concentration of 5 μmol / L and incubated for 1 h. The medium was removed, and the cells were washed three times with PBS (pH 7.4). The cells were then incubated again for 1 h on medium containing Mito-Traker Red to a final concentration of 1 μmol / L. Images were then acquired using a confocal microscope. Figure 10 As shown.

[0095] 2. Golgi apparatus imaging: 4T1 cells were seeded in confocal culture dishes (2×10⁻⁶ cells / mL). 5Cells were cultured in a CO2 incubator (37℃, 5% CO2) with 1640 medium containing 10% fetal bovine serum for 24 h. Then, 5 μL of Glogi-Tracker Red probe diluted with 1 ml of diluent was added and incubated for 0.5 h. The medium was removed, and the cells were washed three times with PBS (pH 7.4). BBR-2 solution was added to a final concentration of 1 μmol / L and incubated for 1 h. The medium was removed, and the cells were washed three times with PBS (pH 7.4). Images were then acquired under a confocal microscope. Figure 11 As shown.

Claims

1. A class of berberine compounds as shown in formula (I) or pharmaceutically acceptable salts thereof: （I） in, X is selected from F, Cl, Br, I; R is selected from one of the following structural segments: 、 、 。 2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, characterized in that, The compound is selected from the following compounds: 。 3. A method for preparing the compound according to claim 1 or 2, characterized in that, Includes the following steps: (1) Synthetic route of compound BBR-1: ； Starting with berberine hydrochloride, a C-12 substituted berberine bromide intermediate 1 was obtained by bromination reaction, and then reacted with triphenylamine 4-boronic acid via Suzuki-Miyaura coupling reaction to obtain compound BBR-1. (2) Synthetic route of compound BBR-2: ； Starting with triphenylamine 4-borate, intermediate 2 was obtained by Suzuki-Miyaura coupling reaction with 4,7-dibromo-2,1,3-benzothiadiazole. Intermediate 3 was then obtained by Suzuki-Miyaura coupling reaction with pinacol diborate. Finally, intermediate BBR-2 was obtained by Suzuki-Miyaura coupling reaction with berberine bromide 1. (3) Synthetic route of compound BBR-3: ； Starting with 4-ynyltriphenylamine, intermediate 4 was obtained by Suzuki-Miyaura coupling reaction with 4,7-dibromo-2,1,3-benzothiadiazole. Intermediate 5 was then obtained by Suzuki-Miyaura coupling reaction with pinacol diboronate. Finally, compound BBR-3 was obtained by Suzuki-Miyaura coupling reaction with berberine bromide 1.

4. A photosensitizer having the structure of the compound of any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.

5. The use of the compound of any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof in the preparation of a photodynamic therapy for tumors.

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