Preparation method and application of long conjugated pyridinium macrocyclic photosensitizer

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of a long-conjugated pyridinium macrocycle photosensitizer. In order to synthesize a novel pyridinium macrocycle photosensitizer molecule with high singlet oxygen yield, high stability and low toxicity for photodynamic therapy, the application provides a long-conjugated pyridinium macrocycle photosensitizer and a preparation method thereof. The long-conjugated pyridinium macrocycle photosensitizer is simple to prepare and has high yield. Meanwhile, the preparation method of the photosensitizer has the advantages of easy-to-obtain raw materials, good repeatability, high yield, high singlet oxygen generation efficiency, low dark toxicity, high light toxicity and good photodynamic application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a preparation method and application of a long-conjugated pyridinium macrocycle photosensitizer. BACKGROUND

[0002] Due to the high morbidity and mortality of cancer, cancer is still one of the main causes of human death worldwide, so it is imminent to develop effective cancer treatment methods. As a new light therapy, photothermal therapy (PTT) and photodynamic therapy (PDT) have received more and more attention in cancer treatment in recent years. Among them, photodynamic therapy (PDT) refers to the process that under the irradiation of a specific wavelength of light, the photosensitizer absorbs light energy to produce energy level transition, and the generated electrons combine with the surrounding oxygen to generate active oxygen free radicals ROS, which oxidize biological macromolecules such as proteins and DNA in cells, and then induce tumor cell death. Compared with traditional cancer treatment methods (surgery, radiotherapy, chemotherapy, etc.), PTT and PDT have the advantages of low dark toxicity, high light toxicity, high selectivity, non-invasiveness, etc., and show good application prospects.

[0003] Photosensitizers play an important role in PDT and are the key to PDT therapy. However, current photosensitizers still have problems such as low singlet oxygen yield and poor stability. Macrocyclic compounds, especially those containing nitrogen heteroatoms, have good coordination with metal ions, so they not only have good biological stability, but also can adjust the luminescent properties of the synthesized compounds by changing different coordination metal ions, and show good application prospects in photosensitizers. Among them, artificial macrocyclic receptors occupy a unique and important position in the field of supramolecules, but common macrocycles (crown ether, cyclodextrin, cucurbituril, etc.) have only weak absorption in the ultraviolet-visible light region, which seriously hinders their development and application, making them difficult to be used as biologically related photo-luminescent materials. In order to alleviate these problems, some light-responsive macrocycles composed of conjugated components have attracted people's attention because of their great advantages in host-guest chemistry and molecular recognition, such as pyridinium macrocycles. However, how to construct a new generation of pyridinium macrocycles with good optical properties and explore their biocompatibility and phototoxicity is rarely reported.

[0004] Therefore, under this background, it is of great significance to synthesize a new type of pyridinium macrocycle photosensitizer molecule with high singlet oxygen yield, high stability and low toxicity for photodynamic therapy. SUMMARY

[0005] In order to overcome the above-mentioned deficiencies of the prior art, the present application provides a long conjugated pyridinium macrocycle photosensitizer and a preparation method thereof, the preparation method of the photosensitizer is simple, raw materials are easy to obtain, and has good repeatability, high yield, high singlet oxygen production efficiency, low dark toxicity and strong phototoxicity, indicating a good photodynamic application prospect.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] The present application provides a long conjugated pyridinium macrocycle compound in the first aspect, the compound is C3·Br4, the chemical formula is C 48 H 42 N6Br4, the structural formula is as follows:

[0008]

[0009] Preferably, the crystal structure of the compound C3·(PF6)4 is as follows:

[0010]

[0011] The long conjugated pyridinium macrocycle compound can be ion exchanged into a hexafluorophosphate form, which is beneficial to its crystallization and determination of its structure: C 70 H 56 N8P4F 24 , which is a monoclinic system with a space group P21 / c, and the cell parameters are: a=25.5834, b=7.9990, c=38.8007, alpha=gamma=90°, beta=103.31°, and the cell volume is

[0012] The present application provides a preparation method of the long conjugated pyridinium macrocycle compound of the first aspect in the second aspect, and the preparation method comprises the following steps:

[0013] S1, under an inert gas atmosphere, 2,6-dibromoaniline, 4-(4-pyridyl)phenylboronic acid, tetrakis(triphenylphosphine)palladium and K2CO3 are added to an organic solvent, and then heated to reflux, and then the organic layer is collected by extraction, and then the organic layer is washed with water, salt washed and dried, and then filtered to remove the solvent, and finally separated by silica gel column chromatography to obtain an intermediate 1, and the structural formula is as follows:

[0014]

[0015] S2, the intermediate 1 of step S1 is dissolved in an organic solvent, and 2,6-bis(bromomethyl)pyridine is dissolved in an organic solvent, the two solutions are combined and heated to reflux, until a large amount of yellow precipitate is generated, then the precipitate is collected by filtration, washed and dried to obtain the long conjugated pyridinium macrocycle compound C3·Br4.

[0016] Meanwhile, a preparation method of the long conjugated pyridinium macrocycle crystal structure is also provided, that is, the long conjugated pyridinium macrocycle C3·Br4 is dissolved in water, and then an excess of NH4PF6 is added to generate a yellow precipitate immediately, and after filtration, water washing and drying, the pyridinium macrocycle crystal compound C3·(PF6)4 is obtained, and then the C3·(PF6)4 is dissolved in an organic solvent, and then gas phase diffusion is carried out in an ether-containing atmosphere at room temperature, and the long conjugated pyridinium macrocycle crystal structure is obtained through crystallization.

[0017] The preparation method of the long conjugated pyridinium macrocycle compound of the present application has the advantages of simple operation, high yield, easy-to-obtain raw materials, good repeatability, high yield and important production value.

[0018] Preferably, in step S1, the equivalent ratio of the 2,6-dibromoaniline to the 4-(4-pyridyl)phenylboronic acid is 1:3-5 (preferably 1:3); the equivalent of the 2,6-dibromoaniline is 1 equivalent, and the equivalent of the tetrakis(triphenylphosphine)palladium is 0.08-0.1 (preferably 0.08); the equivalent of the 2,6-dibromoaniline is 1 equivalent, and the equivalent of the K2CO3 is 10-15 (preferably 10).

[0019] Preferably, in step S2, the equivalent ratio of the intermediate 1 to the 2,6-bis(bromomethyl)pyridine is 1:1-2, preferably 1:1.

[0020] Preferably, in step S1, the heating reflux reaction is carried out at a temperature of 100-150℃ for 24-48 hours; preferably at 100℃ for 24 hours.

[0021] Preferably, in step S2, the heating reflux reaction is carried out at a temperature of 80-100℃ for 24-48 hours; preferably at 80℃ for 24 hours.

[0022] Preferably, in step S1, the silica gel column chromatography is eluted with PE:EA=2:1, 1:1, 1:2 as the mobile phase, and the eluate of the PE:EA=1:2 part is collected.

[0023] Preferably, in step S1, the silica gel column chromatography is carried out using a silica gel column with a mesh size of 200.

[0024] Preferably, in step S2, the organic solvent is acetonitrile, and the amount thereof is determined according to the complete solubility of the solute.

[0025] Preferably, the amount of water is determined according to the complete solubility of the solute.

[0026] Preferably, the time of the gas phase diffusion is 2-4 days.

[0027] The third aspect of the present application provides the long conjugated pyridinium macrocycle compound of the first aspect in the preparation of PDT photosensitizer.

[0028] The long conjugated pyridinium macrocycle compound of the present application has high singlet oxygen production efficiency, low dark toxicity, high light toxicity, and wide application prospect in photodynamic therapy.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] (1) The preparation method of the long conjugated pyridinium macrocycle compound of the present application is simple to operate, simple in synthesis steps, high in product purity, and saves the complex purification process.

[0031] (2) The preparation method of the long conjugated pyridinium macrocycle compound of the present application is easy to obtain raw materials, has good repeatability, and high yield.

[0032] (3) The long conjugated pyridinium macrocycle compound of the present application has high singlet oxygen production rate, high stability, low dark toxicity and high light toxicity, wide application prospect in photodynamic therapy, and is expected to be used for preparing PDT photosensitizer.

[0033] The present application provides a new idea for the synthesis of new photosensitizer, and expands the application value of pyridinium macrocycle photosensitizer. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is the hydrogen spectrum of intermediate 4,4"-di(pyridin-4-yl)-[1,1':3',1"-terphenyl]-2'-amine;

[0035] Figure 2 is the carbon spectrum of intermediate 4,4"-di(pyridin-4-yl)-[1,1':3',1"-terphenyl]-2'-amine;

[0036] Figure 3 is the mass spectrum of intermediate 4,4"-di(pyridin-4-yl)-[1,1':3',1"-terphenyl]-2'-amine;

[0037] Figure 4 is the hydrogen spectrum of long conjugated pyridinium macrocycle C3·Br4;

[0038] Figure 5 is the carbon spectrum of long conjugated pyridinium macrocycle C3·Br4;

[0039] Figure 6 is the mass spectrum of long conjugated pyridinium macrocycle C3·Br4;

[0040] Figure 7 Crystal structure of C3 · (PF6)4: (a) Ellipsoid plot (ellipsoid 30%); (b) Void size;

[0041] Figure 8 UV-Vis absorption spectrum of long conjugated pyridinium macrocycle photosensitizer provided for Example 1;

[0042] Figure 9 Fluorescence emission spectrum of long conjugated pyridinium macrocycle photosensitizer provided for Example 1;

[0043] Figure 10 Singlet oxygen production efficiency plot of long conjugated pyridinium macrocycle photosensitizer provided for Example 1;

[0044] Figure 11 Phototoxicity vs. dark toxicity plot of long conjugated pyridinium macrocycle photosensitizer provided for Example 1, black (left), light (right);

[0045] Figure 12 Green fluorescence of singlet oxygen fluorescent probe SOSG over time plot of long conjugated pyridinium macrocycle photosensitizer provided for Example 1. DETAILED DESCRIPTION

[0046] The specific embodiments of the present application will be further described with the following non-limiting Examples. It is to be understood that the terminology used herein is for the purpose of describing the present application and is not intended to be limiting. Moreover, the features identified in the respective embodiments of the present application described below can be combined unless the contexts imply otherwise.

[0047] The experimental methods in the following examples are routine unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified.

[0048] Example 1 Preparation of pyridinium macrocycle photosensitizer

[0049] The synthesis process is shown in Formula 1:

[0050]

[0051] The specific synthesis process includes the following steps:

[0052] (1) Intermediate synthesis: 2,6-dibromoaniline (1 g, 3.98 mmol), 4-(4-pyridinyl)benzeneboronic acid (4.74 mg, 11.96 mmol), tetrakis(triphenylphosphine)palladium (0.37 g, 0.32 mmol) and K2CO3(5.50 g, 39.8 mmol) were placed in a 250 mL round bottom flask under N2atmosphere, and then 1,4-dioxane (100 mL) and H2O (20 mL) were added to the above reaction system, which was then refluxed at 100 °C for 24 h. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times (3 x 30 mL), and the combined organic layer was washed with water twice (2 x 30 mL) and then with 10% brine once, and dried over anhydrous Na2SO4. The Na2SO4was then filtered using a Buchner funnel, and the filtrate was concentrated under reduced pressure to remove the solvent, and separated by silica gel column chromatography (PE:EA = 1:2) to obtain the intermediate 1 as a white solid (1017.1 mg, yield: 63.9%).

[0053] The synthetic route of the intermediate 4,4"-di(pyridin-4-yl)-[1,1':3',1"-terphenyl]-2'-amine obtained in step (1) is shown in Formula II, and the hydrogen spectrum is shown in Figure 1 1 H NMR (400 MHz, CDC13) δ 8.70 (d, J = 4.5 Hz, 4H, Ha), 7.77 (d, J = 7.7 Hz, 4H, Hb), 7.67 (d, J = 7.6 Hz, 4H, Hc), 7.58 (d, J = 4.7 Hz, 4H, Hd), 7.20 (d, J = 7.4 Hz, 2H, He), 6.95 (t, J = 7.5 Hz, 1H, Hf), 3.92 (s, 2H, Hg). Figure 2 13 C NMR (101 MHz, CDC13) δ 150.12, 148.06, 140.79, 140.63, 136.99, 130.12, 130.08, 127.55, 127.22, 121.59, 118.53. Figure 3 Figure 3 m / z = [M+H] = 100.1896 quasi-molecular ion peak can be clearly observed from + = 100.1896 quasi-molecular ion peak can be clearly observed from

[0054]

[0055] ​​​(2) Synthesis of long conjugated pyridinium macrocycle: Intermediate 1 (89 mg, 0.22 mmol) was dissolved in acetonitrile (100 mL) in a 500 mL reaction flask. Separately, 2,6-bis(bromomethyl)pyridine (59.2 mg, 0.22 mmol) was dissolved in acetonitrile (30 mL) and the solution was added slowly to the reaction flask using a dropping funnel (50 mL). The reaction was refluxed at 80 °C for 48 h until a large amount of yellow precipitate was formed. After cooling to room temperature, the precipitate was collected by filtration and washed with acetonitrile for 3 times and dried under vacuum to give compound C3·Br4 as a yellow solid (125.8 mg, yield 42.5%).

[0056] The synthetic route of long conjugated pyridinium macrocycle C3·Br4 obtained in step (2) is shown in Formula III, and its hydrogen spectrum is shown in Figure 4 1 H NMR (400 MHz, D20) δ 8.63 (d, J = 6.5 Hz, 8H), 8.16 (d, J = 6.5 Hz, 8H), 8.07 (t, J = 7.8 Hz, 2H), 7.81 (d, J = 8.2 Hz, 8H), 7.65 (d, J = 7.8 Hz, 4H), 7.56 (d, J = 8.1 Hz, 8H), 7.23 (d, J = 7.6 Hz, 4H), 6.93 (t, J = 7.6 Hz, 2H), 5.92 (s, 8H). The carbon spectrum is shown in Figure 5 13 CNMR (151 MHz, D20) δ 155.47, 152.25, 145.42, 142.92, 139.67, 139.00, 131.55, 131.19, 129.97, 128.11, 127.21, 123.54, 122.19, 119.94, 62.99. The mass spectrum is shown in Figure 6 Figure 6 from which the quasi-molecular ion peak of m / z = [M+H] + = 1325.1434 can be obviously observed, and its structure is shown in Formula 2:

[0057]

[0058]

[0059] ​​​Further, to determine the structure of C3 accurately, single crystal X-ray diffraction is the most intuitive method. Therefore, solvent exchange is needed: C3»Br4(198.6 mg, 0.15 mmol) is dissolved in H2O (50 mL), and an excess of NH4PF6 is added thereto, a yellow precipitate is immediately generated, filtered, washed with H2O, and dried in vacuum to obtain compound C3»(PF6)4as a yellow solid (238.2 mg, yield: 82.5%). Subsequently, crystal culture is carried out: C3»(PF6)4solid (2 mg) is dissolved in 0.5 mL of acetonitrile in a liquid phase vial, and the liquid phase vial is placed in a 20 mL glass bottle, 2 mL of isopropyl ether is added, and after sealing, it is placed at room temperature for culture, and yellow needle-shaped crystals, i.e. compound C3»(PF6)4, are obtained after 3 days, and the structure is shown in formula 3:

[0060]

[0061] Single crystal X-ray diffraction analysis is carried out on the crystal obtained by solvent exchange and crystal culture using an Agilent Gemini S ultra CCD diffractometer diffractometer, and OLEX2 modeling and refinement are used. The results show that non-hydrogen atoms are anisotropically refined, and hydrogen atoms are fixed by a riding model, and the chemical formula is C 70 H 56 N8P4F 24 , and the obtained crystal structure is shown in Figure 7 a, C3»(PF6)4is folded into a V-shaped structure to maintain the energy stability of the whole molecule, and the width is ( Figure 7 b). Crystallographic parameters are shown in Table 1.

[0062] Table 1 Crystal data of long conjugated pyridinium macrocyclic photosensitizer

[0063]

[0064]

[0065] Example 2 Preparation of pyridinium macrocyclic photosensitizer

[0066] (1) Synthesis of intermediate: 2,6-dibromoaniline (1 g, 3.98 mmol), 4-(4-pyridyl)benzeneboronic acid (2376.1 mg, 11.94 mmol), tetrakis(triphenylphosphine)palladium (0.37 g, 0.32 mmol) and K2CO3(6050.8 mg, 43.78 mmol) were placed in a 250 mL round bottom flask. Under N2atmosphere, 1,4-dioxane (100 mL) and H2O (20 mL) were added to the above reaction system, which was then refluxed at 110 °C for 28 h. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times (3 x 30 mL), the organic layers were combined and washed with water twice (2 x 30 mL) and then with brine once, and dried over anhydrous Na2SO4. It was then further filtered and the solvent was removed under reduced pressure, and the product was isolated by silica gel column chromatography (PE:EA = 1:2) to obtain the intermediate 1 as a white solid (988.46 mg, yield: 62.1%).

[0067] (2) Synthesis of long conjugated pyridinium macrocycle: Intermediate 1 (89 mg, 0.22 mmol) was dissolved in acetonitrile (100 mL) and placed in a 500 mL reaction bottle. Separately, 2,6-bis(bromomethyl)pyridine (69.9 mg, 0.26 mmol) was dissolved in acetonitrile (30 mL), and the acetonitrile solution of 2,6-bis(bromomethyl)pyridine was slowly added to the reaction bottle using a dropping funnel (50 mL), and refluxed at 80 °C for 24 h until a large amount of yellow precipitate was produced. After cooling to room temperature, the precipitate was collected by filtration and washed with acetonitrile three times, and dried under vacuum to obtain compound C3·Br4as a yellow solid (122.6 mg, yield 41.4%).

[0068] In addition, in order to obtain the crystal structure of C3, solvent exchange was required: C3·Br4(239 mg, 0.18 mmol) was dissolved in H2O (55 mL) and an excess of NH4PF6was added, which immediately produced a yellow precipitate, which was filtered and washed with H2O and dried under vacuum to obtain compound C3·(PF6)4as a yellow solid (244.1 mg, yield: 85.4%). Subsequently, crystal culture was performed: C3·(PF6)4solid (2.5 mg) was dissolved in 0.6 mL of acetonitrile in a liquid phase vial, which was then placed in a 20 mL glass bottle, 3 mL of isopropyl ether was added, and after sealing, it was left to stand at room temperature for culture, and yellow needle-like crystals were obtained after 4 days.

[0069] Structural identification was performed according to the method in Example 1, and the results showed that the composition and structure of the photosensitizer obtained in Example 1 were consistent.

[0070] Example 3 Preparation of pyridinium macrocycle photosensitizer

[0071] (1) Synthesis of intermediate: 2,6-dibromoaniline (1 g, 3.98 mmol), 4-(4-pyridyl)benzeneboronic acid (3168.1 mg, 15.92 mmol), tetrakis(triphenylphosphine)palladium (0.37 g, 0.32 mmol) and K2CO3 (6600.9 mg, 47.76 mmol) were placed in a 250 mL round bottom flask. Under N2 atmosphere, 1,4-dioxane (100 mL) and H2O (20 mL) were added to the above reaction system, and refluxed at 120 °C for 32 h. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times (3 x 30 mL), the organic layers were combined and washed with water twice (2 x 30 mL), and then washed with brine once, dried over anhydrous Na2SO4. It was then further filtered and the solvent was removed under reduced pressure, and column chromatography on silica gel (PE:EA = 1:2) was performed to isolate the intermediate 1 as a white solid (978.9 mg, yield: 61.5%).

[0072] (2) Synthesis of long conjugated pyridinium macrocycle: Intermediate 1 (89 mg, 0.22 mmol) was dissolved in acetonitrile (100 mL), and 2,6-bis(bromomethyl)pyridine (75.8 mg, 0.29 mmol) was dissolved in acetonitrile (30 mL). The acetonitrile solution of 2,6-bis(bromomethyl)pyridine was slowly added to the reaction flask using a dropping funnel (50 mL), and refluxed at 90 °C for 30 h until a large amount of yellow precipitate was produced. After cooling to room temperature, the precipitate was collected by filtration and washed with acetonitrile three times, and dried under vacuum to obtain compound C3·Br4 as a yellow solid (120.2 mg, yield 40.6%).

[0073] In addition, in order to obtain the crystal structure of C3, solvent exchange was performed: C3·Br4 (239 mg, 0.18 mmol) was dissolved in H2O (90 mL), and an excess of NH4PF6 was added thereto, and a yellow precipitate was immediately formed. The precipitate was filtered and washed with H2O, and dried under vacuum to obtain compound C3·(PF6)4 as a yellow solid (253.5 mg, yield: 88.7%). Subsequently, crystal culture was performed: C3·(PF6)4 solid (4 mg) was dissolved in 0.8 mL of acetonitrile in a liquid phase vial, and the liquid phase vial was placed in a 20 mL glass bottle, 3.5 mL of isopropyl ether was added, and after sealing, it was left to stand at room temperature for culture, and yellow needle-shaped crystals were obtained after 5 days.

[0074] Structural identification was performed according to the method in Example 1, and the results showed that the composition and structure of the photosensitizer obtained in Example 1 were consistent.

[0075] Example 4 Preparation of pyridinium macrocycle photosensitizer

[0076] (1) Intermediate synthesis: 2,6-dibromoaniline (1 g, 3.98 mmol), 4-(4-pyridyl)benzeneboronic acid (3960.1 mg, 19.9 mmol), tetrakis(triphenylphosphine)palladium (0.37 g, 0.32 mmol) and K2CO3 (7151.0 mg, 51.74 mmol) were placed in a 250 mL round bottom flask. Under N2 atmosphere, 1,4-dioxane (100 mL) and H2O (20 mL) were added to the above reaction system, and refluxed at 130 °C for 40 h. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times (3 x 30 mL), the combined organic layers were washed with water twice (2 x 30 mL), and then with brine once, dried over anhydrous Na2SO4. It was then further filtered and the solvent was removed under reduced pressure, and column chromatography on silica gel (PE:EA = 1:2) was performed to isolate the intermediate 1 as a white solid (967.7 mg, yield 60.8%).

[0077] (2) Synthesis of long conjugated pyridinium macrocycle: Intermediate 1 (89 mg, 0.22 mmol) was dissolved in acetonitrile (100 mL), and 2,6-bis(bromomethyl)pyridine (81.6 mg, 0.31 mmol) was dissolved in acetonitrile (30 mL). The acetonitrile solution of 2,6-bis(bromomethyl)pyridine was slowly added to the reaction flask using a dropping funnel (50 mL), and refluxed at 100 °C for 35 h until a large amount of yellow precipitate was produced. It was cooled to room temperature, filtered to collect the precipitate, and washed with acetonitrile three times, and dried under vacuum to obtain compound C3·Br4 as a yellow solid (116.9 mg, yield 39.5%).

[0078] In addition, in order to obtain the crystal structure of C3, solvent exchange was performed: C3·Br4 (200 mg, 0.15 mmol) was dissolved in H2O (80 mL), and an excess of NH4PF6 was added thereto, and a yellow precipitate was immediately formed, which was filtered and washed with H2O, and dried under vacuum to obtain compound C3·(PF6)4 as a yellow solid (197.3 mg, yield: 82.5%). Subsequently, crystal culture was performed: C3·(PF6)4 solid (4.5 mg) was dissolved in 1.2 mL of acetonitrile in a liquid phase vial, and the liquid phase vial was placed in a 20 mL glass bottle, 4 mL of isopropyl ether was added, and after sealing, it was left to stand at room temperature for culture, and yellow needle-shaped crystals were obtained after 6 days.

[0079] Structural identification was performed according to the method in Example 1, and the results showed that the composition and structure of the photosensitizer obtained in Example 1 were consistent.

[0080] Example 5 Preparation of pyridinium macrocycle photosensitizer

[0081] (1) Intermediate synthesis: 2,6-dibromoaniline (1 g, 3.98 mmol), 4-(4-pyridyl)benzeneboronic acid (1960.3 mg, 19.9 mmol), tetrakis(triphenylphosphine)palladium (0.37 g, 0.32 mmol) and K2CO3 (8251.1 mg, 59.7 mmol) were placed in a 250 mL round bottom flask. Under N2 atmosphere, 1,4-dioxane (100 mL) and H2O (20 mL) were added to the above reaction system, and refluxed at 150 °C for 48 h. After the reaction was completed, it was cooled to room temperature, extracted with ethyl acetate three times (3 x 30 mL), the organic layers were combined and washed with water twice (2 x 30 mL), and then with brine once, and dried over anhydrous Na2SO4. Further filtration and solvent removal under reduced pressure, and separation by silica gel column chromatography (PE:EA = 1:2) gave the intermediate 1 as a white solid (948.6 mg, yield 59.6%).

[0082] (2) Synthesis of long conjugated pyridinium macrocycle: Intermediate 1 (89 mg, 0.22 mmol) was dissolved in acetonitrile (100 mL), and 2,6-bis(bromomethyl)pyridine (116.6 mg, 0.44 mmol) was dissolved in acetonitrile (30 mL), and the acetonitrile solution of 2,6-bis(bromomethyl)pyridine was slowly added to the reaction bottle through a dropping funnel (50 mL), and refluxed at 100 °C for 48 h until a large amount of yellow precipitate was generated, cooled to room temperature, the precipitate was collected by filtration and washed with acetonitrile three times, and dried under vacuum to obtain compound C3·Br4 as a yellow solid (110.4 mg, yield 37.3%).

[0083] In addition, in order to obtain the crystal structure of C3, solvent exchange was required: C3·Br4 (265 mg, 0.20 mmol) was dissolved in H2O (100 mL), and an excess of NH4PF6 was added, and a yellow precipitate was immediately generated, which was filtered and washed with H2O, and dried under vacuum to obtain compound C3·(PF6)4 as a yellow solid (yield: 90.5%). Subsequently, crystal culture was carried out: C3·(PF6)4 (6 mg) solid was dissolved in 1.5 mL of acetonitrile in a liquid phase vial, and the liquid phase vial was placed in a 20 mL glass bottle, 6 mL of isopropyl ether was added, and after sealing, it was placed at room temperature for culture, and yellow needle-like crystals were obtained after 7 days.

[0084] Structural identification was carried out according to the method in Example 1, and the results showed that the composition and structure of the photosensitizer obtained in Example 1 were consistent.

[0085] Experimental Example 1 Characterization and performance test of pyridinium macrocycle photosensitizer

[0086] (1) UV-Vis absorption spectrum test

[0087] The photosensitizer C3·Br4 prepared in Example 1 was dissolved in water to prepare a 50 μM solution, and 3.0 mL of the C3·Br4 solution was taken in a cuvette for UV-visible absorption spectrum test. As shown in Figure 8 , the synthesized new pyridinium macrocycle photosensitizer C3·Br4 has a characteristic absorption peak at 321 nm in the visible light.

[0088] (2) Fluorescence emission spectrum test

[0089] The photosensitizer C3·Br4 prepared in Example 1 was dissolved in water to prepare a 50 μM solution, and 3.0 mL of the C3·Br4 solution was taken in a cuvette for fluorescence emission spectrum test. As shown in Figure 9 , when the excitation wavelength is 321 nm, the synthesized new pyridinium macrocycle photosensitizer C3·Br4 exhibits strong near-infrared fluorescence emission at 500 nm and 659 nm.

[0090] (3) Singlet oxygen detection

[0091] The singlet oxygen generation efficiency of the pyridinium macrocycle photosensitizer C3·Br4 under white light irradiation (λ>420 nm, 80 mW / cm 2 ) was tested by taking 9,10-anthracenediyl-bis(methylene)dicarboxylic acid (ABDA) as the singlet oxygen indicator and commercial rose Bengal (RB) as the reference reagent of the photosensitizer. The specific operation is as follows:

[0092] 20 μL of ABDA solution (10 mM) was added to 1.98 mL of C3·Br4 solution (10 μM), and then the mixed solution was irradiated under white light (λ>420 nm, 80 mW / cm 2 ). The same irradiation time was used as the control group without macrocycle, and the absorption spectrum and absorbance change at 378 nm of the solution were measured every 20 seconds to obtain the efficiency of generating reactive oxygen species. Rose Bengal (RB) was used as a positive control sample. The following formula was used to calculate 1 the quantum yield of O2:

[0093]

[0094] where KG and KST are the decomposition rate constants of ABDA under the action of the photosensitizer (G) and RB (ST). AG and AST represent the light absorbed by the photosensitizer (G) and RB (ST), respectively, which is determined by the integral of the area under the absorption band in the wavelength range of 400-600 nm. ΦST is the O2 quantum yield of RB, which is 0.75 in water. 1

[0095] Figure 10 ​a is a graph showing the change in the absorption spectrum of ABDA with illumination time in the presence of C3·Br4, obtained from calculations. Figure 10 C3·Br4 in b showed a higher singlet oxygen production efficiency than RB, with a singlet oxygen quantum yield of up to 1.703.

[0096] (4) Phototoxicity test

[0097] HepG2 cells in the logarithmic growth phase were seeded into 96-well plates (1 × 10⁶ cells per well). 4 Cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. The culture medium was then aspirated, and 100 μL of fresh DMEM medium containing different concentrations of C3·Br4 (0, 1, 5, 10, 15, 20 μM) was added. The cells were then cultured in the dark for 24 hours. Afterwards, they were irradiated with white light (λ>420 nm, 80 mW / cm²). 2 10 min. Continue culturing for another h, wash three times with PBS, remove the culture medium, and add fresh culture medium. Simultaneously, add 10 μL of MTT solution (5 mg / mL) to each well, and continue culturing at 37°C for 4 h. Discard the MTT, add 100 μL of DMSO to each well, and shake on a shaker at low speed for 5 min to fully dissolve the crystals. Then, measure the absorbance at 490 nm using an ELISA reader. Each experiment was performed in triplicate. The values ​​here are mean ± standard deviation (SD). All statistical tests were performed using SPSS software. The results are as follows: Figure 11 As shown. By Figure 11 It can be seen that C3·Br4 has low dark toxicity but strong phototoxicity, demonstrating a good photodynamic ability to kill cancer cells.

[0098] (5) Intracellular singlet oxygen measurement experiment

[0099] HepG2 cells in logarithmic growth phase were seeded into 24-well plates and cultured until adherent. Then, 1 mL of 5 μM C3·Br4 was added and the cells were cultured for 12 h. Afterward, the cells were irradiated with white light (λ>420 nm, 80 mW / cm²). 2 After 2 min, 4 min, and 6 min, SOSG (5 μM) was added, and the cells were incubated for 20 min. The cells were then washed three times with PBS solution, followed by the addition of 0.5 mL of PBS solution. Fluorescence images of the cells were then collected using a laser confocal microscope. Red fluorescence of C3·Br4 excited at 325 nm was collected in the 500-750 nm range. Simultaneously, after excitation at 488 nm, green fluorescence in the 500-530 nm range was collected using the singlet oxygen sensor green (SOSG) probe. Results are as follows: Figure 12 As shown, by Figure 12It can be seen that with the increase of illumination time, the green fluorescence is more and more, indicating that the singlet oxygen is more and more.

[0100] The above experimental results show that the photosensitizer prepared by the application has strong singlet oxygen production capacity, small dark toxicity and strong phototoxicity. Meanwhile, the preparation method of the photosensitizer is simple, raw materials are easy to obtain, and has good repeatability, high yield, and wide application prospect in photodynamic therapy.

[0101] The above detailed description of the embodiments of the present application, but the present application is not limited to the described embodiments. For those skilled in the art, without departing from the principles and spirit of the present application, various changes, modifications, replacements and modifications of these embodiments still fall within the scope of the present application.

Claims

1. A long conjugated pyridinium macrocyclic compound, characterized in that, The compound is C3•Br4, with the chemical formula C... 48 H 42 N6Br4, the structural formula is shown below:

2. A long conjugated pyridinium macrocyclic compound, characterized in that, The structure of the compound is shown below:

3. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 1, characterized in that, Includes the following steps: S1. Under an inert gas atmosphere, an organic solvent was added to 2,6-dibromoaniline, 4-(4-pyridyl)phenylboronic acid, tetrakis(triphenylphosphine)palladium, and K2CO3. After heating and reflux, the organic layer was collected by extraction. The organic layer was washed with water, salt, and dried, and then filtered to remove the solvent. Finally, intermediate 1 was obtained by silica gel column chromatography, and its structural formula is shown below: S2. Dissolve intermediate 1 from step S1 in an organic solvent, and dissolve 2,6-bis(bromomethyl)pyridine in an organic solvent. Combine the two solutions and heat under reflux until a large amount of yellow precipitate is produced. Filter and collect the precipitate, and after washing and drying, obtain the long conjugated pyridinium macrocyclic compound.

4. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 2, characterized in that, The long conjugated pyridinium macrocyclic compound of claim 1 was dissolved in water, and then excess NH4PF6 was added, immediately generating a yellow precipitate. After filtration, washing with water, and drying, the long conjugated pyridinium macrocyclic crystalline compound C3•(PF6)4 was obtained. C3•(PF6)4 was then dissolved in an organic solvent, and then gas-phase diffusion was carried out at room temperature in an ether-containing atmosphere. After crystallization, the crystal structure of the long conjugated pyridinium macrocyclic compound was obtained.

5. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 3, characterized in that, In step S1, the equivalent ratio of 2,6-dibromoaniline to 4-(4-pyridyl)phenylboronic acid is 1:3 to 5; with 2,6-dibromoaniline as 1 equivalent, the equivalent of tetra(triphenylphosphine)palladium is 0.08 to 0.1; with 2,6-dibromoaniline as 1 equivalent, the equivalent of K2CO3 is 10 to 15.

6. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 3, characterized in that, In step S2, the equivalent ratio of intermediate 1 to 2,6-bis(bromomethyl)pyridine is 1:1 to 2.

7. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 3, characterized in that, In step S1, the temperature of the heating reflux reaction is 100-150°C, and the time is 24-48 hours.

8. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 3, characterized in that, In step S2, the temperature of the heating reflux reaction is 80-100°C, and the time is 24-48 hours.

9. The method for preparing the long conjugated pyridinium macrocyclic compound according to claim 3, characterized in that, In step S1, the silica gel column chromatography separation is performed by elution with mobile phases of PE:EA = 2:1, 1:1, and 1:2, and the eluent fraction of PE:EA = 1:2 is collected.

10. The use of the long conjugated pyridinium macrocyclic compound of claim 1 in the preparation of a photosensitizer for PDT treatment of liver cancer.

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