A porphyrin compound for bioimaging and sonodynamic therapy, and its preparation method and application

Abstract

A porphyrin compound for bioimaging and sonodynamic therapy, as well as its preparation method and application, has a chemical formula shown in Formula 2. The preparation method comprises the following steps: adding p-methylthiobenzaldehyde, a compound of Formula 1, and pyrrole to a reaction flask, evacuating the flask and introducing nitrogen gas, followed by adding anhydrous dichloromethane to obtain a reaction solution; bubbling nitrogen gas through the reaction solution, stirring at 25°C, adding boron trifluoride etherate, maintaining the temperature while continuing to stir, adding DDQ, continuing stirring, adding triethylamine, filtering, concentrating, and purifying to obtain the target product. This method features mild reaction conditions, a short reaction time, and simple operation. The resulting porphyrin compound exhibits excellent sonosensitivity and biocompatibility, can efficiently generate reactive oxygen species under ultrasound, and can produce excellent sonodynamic effects. The compound also exhibits fluorescence emission in the near-infrared region and can be excited by visible light, enabling confocal imaging to detect changes in cell morphology before and after, resulting in excellent bioimaging results. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a porphyrin compound for bioimaging and sonodynamic therapy, and a preparation method and application thereof. Background Art

[0002] Cancer has become one of the diseases that pose the greatest threat to human life and health, and the proportion of cancer deaths in all causes of death in humans has gradually shown an upward trend in recent years. In recent years, photodynamic therapy with photosensitizers has been widely studied and successfully applied to the treatment of superficial tumors. However, due to the limitation of optical penetration depth, photodynamic therapy has poor therapeutic effect on deep tumors, which limits its further application. The development of new non-invasive and efficient therapies for deep tumors is of great significance to solving this problem. With its good focusing performance and strong penetrating ability, ultrasound can achieve a tissue penetration depth of up to ten centimeters. Sonodynamic therapy (SDT) using ultrasound as an excitation source can effectively achieve non-invasive treatment of deep tumors. Therefore, sonodynamic therapy shows great potential and good application prospects in the efficient treatment of deep tumors.

[0003] Researchers have discovered that the photosensitizer hematoporphyrin can produce significant cytotoxicity under ultrasound stimulation. They subsequently named this phenomenon of porphyrin cytotoxicity in ultrasound fields the "sonodynamic approach." Sonodynamic therapy involves the activation of a sonosensitizer by ultrasound waves of appropriate frequency and power, triggering a sonochemical or sonophotochemical reaction and generating reactive oxygen species that kill tumor cells. As the core material foundation of sonodynamic therapy, the development of novel and highly effective sonosensitizers is crucial in this field. Porphyrins are macrocyclic compounds with a conjugated backbone composed of four pyrrole rings connected by methine groups. The eight β-positions and four median hydrogen atoms of the four pyrrole rings in their molecular structure can be substituted with other groups to produce various porphyrin derivatives. Porphyrins possess unique physiological properties and a unique affinity for cancer cells. In medicine, they are not only used as anticancer photosensitizers for photodynamic therapy (PDT), but are also attracting significant attention for their excellent sonodynamic properties. DVDMS has been approved for clinical trials.

[0004] Although sonodynamic therapy has shown great promise in cancer treatment, the development of sonosensitizers is still in its infancy, with only a few porphyrin sonosensitizers approved for clinical trials. Therefore, it is imperative to design and synthesize sonosensitizers with low cytotoxicity, excellent biocompatibility, and superior sonodynamic properties. Summary of the Invention

[0005] The purpose of the present invention is to provide a porphyrin compound for bioimaging and sonodynamic therapy, as well as a preparation method and application thereof. The method has mild reaction conditions, short reaction time, and simple operation. The prepared porphyrin compound has broad application prospects in bioimaging and sonodynamic therapy.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] A porphyrin compound for bioimaging and sonodynamic therapy, whose chemical structure is shown in Formula 2:

[0008]

[0009] In formula 2, R is selected from One of them.

[0010] To achieve the above objectives, the present invention also provides a method for preparing the porphyrin compound for bioimaging and sonodynamic therapy, which comprises the following steps:

[0011] Add p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole to a reaction flask, evacuate the flask, and introduce nitrogen gas, repeating the process several times until the flask is filled with nitrogen gas, and add anhydrous dichloromethane to obtain a reaction solution; bubble nitrogen gas into the reaction solution for 14-16 minutes, then stir at 25°C and add boron trifluoride etherate, keep stirring for 1-2 hours, then add 2,3-dichloro-5,6-dicyano-p-benzoquinone, continue stirring for 20-40 minutes, add triethylamine, continue stirring for 15-25 minutes, filter, concentrate, and purify to obtain a porphyrin compound of formula 2;

[0012] The molar ratio of p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole is 1:(1-3):4;

[0013] The molar ratio of the compound of formula 1, boron trifluoride etherate, and 2,3-dichloro-5,6-dicyano-p-benzoquinone is (4-12):1:8;

[0014] The structural formula of the compound of formula 1 is R-CHO, wherein R is selected from One of them.

[0015] Preferably, the molar volume ratio between the compound of formula 1 and anhydrous dichloromethane is 1 mmol: (250-750) mL.

[0016] Preferably, the molar volume ratio between the compound of formula 1 and triethylamine is 1 mmol: (0.67-2) mL.

[0017] Furthermore, the specific process of filtration, concentration and purification is as follows: the reaction solution is filtered, the filtrate is decompressed to remove dichloromethane, and the crude product is purified by silica gel column chromatography to obtain the porphyrin compound of formula 2.

[0018] Preferably, the eluent used for silica gel column chromatography purification is dichloromethane in a volume ratio or a dichloromethane / petroleum ether mixture in a volume ratio of 1:3.

[0019] To achieve the above objectives, the present invention also provides the use of the above porphyrin compound for bioimaging and sonodynamic therapy as a cell imaging agent.

[0020] To achieve the above objectives, the present invention also provides the use of the above porphyrin compound for bioimaging and sonodynamic therapy as a sonosensitizer for sonodynamic therapy.

[0021] The reaction scheme of the present invention is as follows:

[0022]

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention uses p-methylthiobenzaldehyde, a compound of formula 1, and pyrrole to react to generate a target product. The reaction conditions are mild, the reaction time is short, and the operation is simple. The present invention introduces a 4-thiomethylphenyl unit into the porphyrin skeleton, so that the prepared porphyrin compound has good sonosensitivity and biocompatibility, can efficiently generate reactive oxygen under ultrasound, and can produce excellent sonodynamic effects; in addition, the prepared porphyrin compound has fluorescence emission in the near-infrared region, can be excited by visible light, and can detect changes in the front and back morphology of cells by confocal imaging, with good biological imaging effects. In summary, the porphyrin compound prepared by the present invention has broad application prospects in biological imaging and sonodynamic therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the hydrogen nuclear magnetic resonance spectrum of the porphyrin compound prepared in Example 6;

[0026] Figure 2 The DMSO solution of the porphyrin compound prepared in Example 6 (10 -5 M) UV-visible absorption and emission spectra;

[0027] Figure 3 The DMF solution of the singlet oxygen indicator DPBF in Example 6 (10 -5 M) UV-visible absorption spectra under ultrasound at different time intervals;

[0028] Figure 4The DMF solution of the porphyrin compound prepared in Example 6 and the singlet oxygen indicator DPBF (10 -5 M) UV-visible absorption spectra under ultrasound at different time intervals;

[0029] Figure 5 This is a statistical graph of the CCK8 cytotoxicity test results of the porphyrin compound prepared in Example 6 under light-shielding conditions;

[0030] Figure 6 This is a statistical graph of the CCK8 cytotoxicity test results of the porphyrin compound prepared in Example 6 under ultrasound with a power of 1 W and a frequency of 1 MHz. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and examples. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following examples.

[0032] Example 1: Preparation of 5,10,15-tribenzoic acid-20-(4-methylthiophenyl)porphyrin

[0033]

[0034] The specific preparation process is as follows: p-Methylthiobenzaldehyde (1.0 mmol), 4-carboxybenzaldehyde (3.0 mmol), and pyrrole (4.0 mmol) are added to a reaction flask, evacuated, and nitrogen is introduced three times to fill the reaction flask with nitrogen; then 750 mL of anhydrous dichloromethane is added, and nitrogen is bubbled through the solution for 15 minutes. Subsequently, boron trifluoride etherate (0.25 mmol) is added and stirred at 25°C for 1.5 hours. DDQ (2.0 mmol) is added to the reaction system, stirring is continued for 0.5 hours, and 2 mL of triethylamine is added. Stirring is continued for 20 minutes. The reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane. The crude product is purified by silica gel column chromatography, and the first red band is collected using pure dichloromethane as the eluent to obtain porphyrin compound 2 in a yield of 21%. Its structural characterization data are as follows: 1 HNMR(400MHz, CDCl3)9.15(d,J=8.0Hz,2H), δ=8.92(s,4H),8.68(d,J=8.0Hz,2H),8.32(d,J=7.6.0Hz ,6H),8.06(d,J=7.2.0Hz,6H),7.62(d,J=8.0Hz,2H),7.02(d,J=8.0Hz,2H),2.9(s,3H),-2.5(s,2H).

[0035] Example 2: Preparation of 5,10,15-trimethoxyphenyl-20-(4-methylthiophenyl)porphyrin

[0036]

[0037] The specific preparation process is as follows: p-methylthiobenzaldehyde (1.0 mmol), p-methoxybenzaldehyde (3.0 mmol) of the compound of Formula 1, and pyrrole (4.0 mmol) are added to a reaction flask, vacuumed, and nitrogen is introduced three times to fill the reaction flask with nitrogen. 750 mL of anhydrous dichloromethane is then added, and nitrogen is bubbled through the solution for 15 minutes. Boron trifluoride etherate (0.25 mmol) is then added after stirring at 25°C for 1.5 hours. DDQ (2.0 mmol) is then added, stirring is continued for 0.5 hours, and 2 mL of triethylamine is added. Stirring is continued for 20 minutes. The reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane. The crude product is purified by silica gel column chromatography, using a dichloromethane:petroleum ether volume ratio of 1:3 as the eluent to collect the second red band. The target porphyrin compound 2 is obtained in a 22% yield. Its structural characterization data are as follows: 1 HNMR(400MHz, CDCl3)9.12(d,J=8.0Hz,2H),δ=8.90(s,4H),8.70(d,J=8.0Hz,2H),8.34(d,J=7.6Hz,6H),8 .10(d,J=7.2Hz,6H),7.66(d,J=8.0Hz,2H),7.12(d,J=8.0Hz,2H),3.52(s,9H),2.92(s,3H),-2.52(s,2H).

[0038] Example 3: Preparation of 5,10,15-triiodophenyl-20-(4-methylthiophenyl)porphyrin

[0039]

[0040] The specific preparation process is as follows: p-Methylthiobenzaldehyde (1.0 mmol), p-iodobenzaldehyde (3.0 mmol), and pyrrole (4.0 mmol) are added to a reaction flask, vacuumed, and nitrogen is introduced three times to fill the reaction flask with nitrogen. 750 mL of anhydrous dichloromethane is then added, and nitrogen is bubbled through the solution for 15 minutes. Boron trifluoride etherate (0.25 mmol) is then added, stirred at 25°C for 1.5 hours, and the reaction system is stirred at this temperature. DDQ (2.0 mmol) is then added, stirring is continued for 0.5 hours, and 2 mL of triethylamine is added, stirring is continued for 20 minutes. The reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane. The crude product is purified by silica gel column chromatography, and the second red band is collected using a dichloromethane:petroleum ether volume ratio of 1:3 as the eluent. The target porphyrin compound 2 is obtained in a 21% yield. Its structural characterization data are as follows: 1 HNMR(400MHz, CDCl3)9.16(d,J=8.0Hz,2H), δ=8.93(s,4H),8.70(d,J=8.0Hz,2H),8.42(d,J=7.6Hz, 6H), 8.12 (d, J = 7.2Hz, 6H), 7.60 (d, J = 8.0Hz, 2H), 7.04 (d, J = 8.0Hz, 2H), 2.92 (s, 3H), -2.50 (s, 2H).

[0041] Example 4: Preparation of 5,10,15-(4-fluorophenyl)-20-(4-methylthiophenyl)porphyrin

[0042]

[0043] The specific preparation process is as follows: p-Methylthiobenzaldehyde (1.0 mmol), 4-fluorobenzaldehyde (3.0 mmol), and pyrrole (4.0 mmol) are added to a reaction flask, vacuumed, and nitrogen is introduced three times to fill the reaction flask with nitrogen. 750 mL of anhydrous dichloromethane is then added, and nitrogen is bubbled through the solution for 15 minutes. Boron trifluoride etherate (0.25 mmol) is then added after stirring at 25°C for 1.5 hours. DDQ (2.0 mmol) is then added, stirring is continued for 0.5 hours, and 2 mL of triethylamine is added. Stirring is continued for 20 minutes. The reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane. The crude product is purified by silica gel column chromatography, and the second red band is collected using a dichloromethane:petroleum ether volume ratio of 1:3 as the eluent. The target porphyrin compound is obtained in a yield of 23%. Its structural characterization data are as follows: 1HNMR(400MHz, CDCl3)9.14(d,J=8.0Hz,2H), δ=8.92(s,4H),8.72(d,J=8.0Hz,2H),8.50(d,J=7.6Hz, 6H), 8.18 (d, J = 7.2Hz, 6H), 7.66 (d, J = 8.0Hz, 2H), 7.08 (d, J = 8.0Hz, 2H), 2.90 (s, 3H), -2.50 (s, 2H).

[0044] Example 5: Preparation of 5,10,15-(4-trifluoromethylphenyl)-20-(4-methylthiophenyl)porphyrin

[0045]

[0046] The specific preparation process is as follows: p-Methylthiobenzaldehyde (1.0 mmol), 4-trifluoromethylbenzaldehyde (3.0 mmol), and pyrrole (4.0 mmol) are added to a reaction flask. The flask is evacuated and nitrogen is introduced three times to fill the flask with nitrogen. 750 mL of anhydrous dichloromethane is then added and nitrogen is bubbled through the solution for 15 minutes. Boron trifluoride etherate (0.25 mmol) is then added and stirred at 25°C for 1.5 hours. DDQ (2.0 mmol) is then added and stirred for 0.5 hours. 2 mL of triethylamine is then added and stirred for 20 minutes. The reaction solution is filtered and the dichloromethane is removed from the filtrate under reduced pressure. The crude product is purified by silica gel column chromatography using a dichloromethane:petroleum ether ratio of 1:3 by volume as the eluent to collect the second red band. The target porphyrin compound is obtained in a 21% yield. Its structural characterization data are as follows: 1 H NMR (400MHz, CDCl3) 9.16 (d, J = 8.0 Hz, 2H), δ = 8.91 (s, 4H), 8.70 (d, J = 8.0 Hz, 2H), 8.46 (d, J = 7.6 Hz, 6H), 8.16 (d, J = 7.2Hz, 6H), 7.62 (d, J = 8.0Hz, 2H), 7.02 (d, J = 8.0Hz, 2H), 2.92 (s, 3H), -2.50 (s, 2H).

[0047] Example 6: Preparation of 5,10,15-tripentafluorophenyl-20-(4-methylthiophenyl)porphyrin

[0048]

[0049] The specific preparation process is as follows: p-methylthiobenzaldehyde (1.0mmol), pentafluorobenzaldehyde (1.0mmol) of the compound of formula 1, and pyrrole (4.0mmol) are added to a reaction flask, vacuumed, and nitrogen is introduced three times to fill the reaction flask with nitrogen; then 750mL of anhydrous dichloromethane is added, nitrogen is bubbled into the solution for 15 minutes, and then boron trifluoride ether (0.25mmol) is added after stirring at 25°C. The reaction system is stirred at this temperature for 1.5 hours, after which DDQ (2.0mmol) is added, stirring is continued for 0.5 hours, 2mL of triethylamine is added, stirring is continued for 20 minutes, the reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane. The crude product is purified by silica gel column chromatography, and the second red band is collected with a dichloromethane: petroleum ether volume ratio of 1:3 as the eluent to obtain the target porphyrin compound with a yield of 26%. Its structural characterization is shown in the accompanying figure. Figure 1 The characterization data are: 1 H NMR (400MHz, CDCl3) 9.15 (d, J = 8.0Hz, 2H), δ = 8.92 (s, 4H), 8.82 (d, J = 8.0Hz, 2H), 8.14 (d, J = 8.0Hz, 2H), 7.72 (d, J = 8.0Hz, 2H), 2.90 (s, 3H), -2.62 (s, 2H).

[0050] The porphyrin compound prepared in this example was characterized by ultraviolet absorption and fluorescence emission spectra and the results were as follows:

[0051] The spectrum test concentration used in the present invention is 5 μM, and DMSO is used as the test solvent. The porphyrin compound shows strong absorption in the visible light range of 400-450nm and 500-600nm. The emission spectrum of the sonoporphyrin compound is measured with 560nm as the excitation wavelength. Figure 2 As shown, its emission peak is broad, the emission peak is located at 700nm, and it has strong deep red light emission, which is more conducive to biological imaging.

[0052] The experimental process and results of the porphyrin compound prepared in this example generating singlet oxygen in a DMF solution under ultrasound are as follows:

[0053] DPBF is a commonly used singlet oxygen indicator that can react with singlet oxygen to cause a decrease in the absorption peak. Prepare a DPBF solution of appropriate concentration, add 1 μL of porphyrin compound solution to it, mix well, and place it on the probe of an ultrasonic therapy device for ultrasound. The ultrasound power is 1W and the frequency is 1MHz. The ultraviolet absorption spectrum is tested every 30 seconds. Continuously record the changes in the absorption peak of DPBF. Figure 3It can be seen that the absorbance of ADBA in the DPBF solution without porphyrin compound did not change significantly with the extension of ultrasonic time; Figure 4 As shown in the figure, after the porphyrin compound is added, the absorbance of DPBF decreases rapidly with the extension of the ultrasonic time, which means that the porphyrin compound can effectively generate singlet oxygen under the action of ultrasound.

[0054] The CCK8 cytotoxicity test process and results of the porphyrin compound prepared in this example are as follows:

[0055] The digested cells were seeded in a 96-well plate at a seeding density of 10 4 Each well was cultured for 24 hours at 37°C and 5% CO2. After removing the stale culture medium, the cells were cultured with cell culture medium containing different concentrations of porphyrin compounds (1-20 μM). After 24 hours, the cells were sonicated and cultured for 12 hours. 10 μL CCK8 (5 mg / mL) was added to each well and cultured for 4 hours before terminating the culture. The culture medium was removed and 150 μL DMSO was added to each well. After shaking on a shaker for 10 minutes, the OD570 was measured using a microplate reader. The results of the MTT cytotoxicity assay are shown in the figure. Figure 5 As shown in the figure, under the conditions of light protection and no ultrasound, when the concentration of porphyrin was 1-20 μM, the cell survival rate after 24 hours of culture was greater than 80%, which proved that the sonosensitizer had low dark cytotoxicity and its autofluorescence could be used for cell imaging before sonodynamic therapy. Figure 6 As shown in the figure, when the cells incubated with the sonosensitizer were subjected to ultrasound treatment, the cell survival rate decreased rapidly with the increase of the incubation concentration, indicating that the porphyrin sonosensitizer has an excellent sonodynamic therapeutic effect.

Claims

1. A porphyrin compound for bioimaging and sonodynamic therapy, characterized in that: Its chemical structure is shown in Formula 2: ； In formula 2, R is selected from 、 、 、 、 、 One of them.

2. A method for preparing a porphyrin compound for bioimaging and sonodynamic therapy according to claim 1, characterized in that: The specific steps are: Add p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole to a reaction flask, evacuate the flask, and introduce nitrogen gas, repeating the process several times until the flask is filled with nitrogen gas, and add anhydrous dichloromethane to obtain a reaction solution; bubble nitrogen gas into the reaction solution for 14-16 minutes, then stir at 25°C and add boron trifluoride etherate, keep stirring for 1-2 hours, then add 2,3-dichloro-5,6-dicyano-p-benzoquinone, continue stirring for 20-40 minutes, add triethylamine, continue stirring for 15-25 minutes, filter, concentrate, and purify to obtain a porphyrin compound of formula 2; The molar ratio of p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole is 1:(1-3):4; The molar ratio of the compound of formula 1, boron trifluoride etherate, and 2,3-dichloro-5,6-dicyano-p-benzoquinone is (4-12):1:8; The structural formula of the compound of formula 1 is , where R is selected from 、 、 、 、 、 One of them.

3. The method for preparing a porphyrin compound for bioimaging and sonodynamic therapy according to claim 2, characterized in that: The molar volume ratio between the compound of formula 1 and anhydrous dichloromethane is 1 mmol: (250-750) mL.

4. The method for preparing a porphyrin compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: The molar volume ratio between the compound of formula 1 and triethylamine is 1 mmol: (0.67-2) mL.

5. The method for preparing a porphyrin compound for bioimaging and sonodynamic therapy according to claim 2 or 3, characterized in that: The specific process of filtration, concentration and purification is as follows: the reaction solution is filtered, the filtrate is decompressed to remove dichloromethane, and the crude product is purified by silica gel column chromatography to obtain the porphyrin compound of formula 2.

6. The method for preparing a porphyrin compound for bioimaging and sonodynamic therapy according to claim 5, characterized in that: The eluent used for silica gel column chromatography purification was dichloromethane or a dichloromethane / petroleum ether mixture with a volume ratio of 1:

3.

7. Use of the porphyrin compound according to claim 1 in preparing a cell imaging reagent.

8. Use of the porphyrin compound according to claim 1 in the preparation of a sonosensitizer for sonodynamic therapy.

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