Porphyrin compound for biological imaging and sonodynamic therapy as well as preparation method and application of porphyrin compound

Abstract

The invention discloses a porphyrin compound for biological imaging and sonodynamic therapy as well as a preparation method and application thereof. The chemical structural formula of the compound is shown as a formula 2; the preparation method comprises the following steps: adding p-methylthiobenzaldehyde, a compound as shown in a formula 1 and pyrrole into a reaction bottle, vacuumizing, introducing nitrogen, and adding anhydrous dichloromethane to obtain a reaction solution; introducing nitrogen into the reaction liquid, stirring at 25 DEG C, adding boron trifluoride diethyl etherate, keeping the temperature, continuously stirring, adding DDQ, continuously stirring, adding triethylamine, filtering, concentrating, and purifying to obtain the target product. The method is mild in reaction condition, short in reaction time and easy to operate, the obtained porphyrin compound has good acoustic sensitivity and biocompatibility, active oxygen can be efficiently generated under the ultrasonic action, and the excellent sonodynamic effect can be generated; the compound also has fluorescence emission in a near-infrared region, can be excited by visible light, can detect the change of the front and back morphologies of the cells through confocal imaging, and has a better biological imaging effect. # 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 good focusing performance and strong penetration 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] Some scholars have found that the photosensitizer hematoporphyrin can produce obvious cytotoxicity under the stimulation of ultrasound. Since then, they have named the phenomenon of porphyrin cytotoxicity in the ultrasound field as "Sonodynamic approach". Sonodynamic therapy uses the sonosensitizer to induce sonochemical or sonophotochemical reactions and produce reactive oxygen species to kill tumor cells under the stimulation of ultrasound of appropriate frequency and power. As the core material basis of sonodynamic therapy, the development of new and efficient sonosensitizers has become the key in this field. Porphyrin is a class of conjugated skeleton macrocyclic compounds formed by four pyrrole rings connected by methine groups. The eight β-positions and four median hydrogen atoms of the four pyrrole rings in the molecular structure can be replaced by other groups to generate various porphyrin derivatives. It not only has unique physiological properties, but also has a unique affinity with cancer cells. In medicine, it is not only used as an anticancer photosensitizer for photodynamic therapy (PDT), but also has attracted widespread attention due to its excellent sonodynamic performance. Among them, DVDMS has been approved for clinical trials.

[0004] Although sonodynamic therapy has shown great promise in tumor treatment, the development of sonosensitizers is still in its infancy, with only a few porphyrin sonosensitizers approved for clinical trials. Therefore, it is very necessary to design and synthesize sonosensitizers with low cytotoxicity, good biocompatibility, and excellent sonodynamic performance. Summary of the invention

[0005] The purpose of the present invention is to provide a porphyrin compound for bioimaging and sonodynamic therapy, and 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, the chemical structure of which is shown in Formula 2:

[0008]

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

[0010] To achieve the above object, the present invention also provides a method for preparing the porphyrin compound for bioimaging and sonodynamic therapy, the specific steps of which are:

[0011] Add p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole to a reaction bottle, evacuate the bottle, and introduce nitrogen, repeat several times to fill the reaction bottle with nitrogen, and add anhydrous dichloromethane to obtain a reaction solution; introduce nitrogen into the reaction solution for 14-16 minutes, then stir at 25°C and add boron trifluoride etherate, keep warm and continue 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 ethyl ether, 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 filtering, concentrating and purifying 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, and has 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 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;

[0028] Figure 4The DMF solution (10 -5 M) UV-visible absorption spectra under ultrasound at different time;

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

[0030] Figure 6 This is a statistical chart 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 embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, 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 (1.0mmol), 4-carboxybenzaldehyde (3.0mmol) of the compound of formula 1, and pyrrole (4.0mmol) are added to a reaction flask, vacuumized, and nitrogen is introduced, and the reaction flask is filled with nitrogen. Then 750mL of anhydrous dichloromethane is added, and nitrogen is introduced into the solution for 15min, and then stirred at 25°C and boron trifluoride ether (0.25mmol) is added. The reaction system is stirred for 1.5h at this temperature, and then DDQ (2.0mmol) is added, and stirring is continued for 0.5h. 2mL of triethylamine is added, and stirring is continued for 20min. The reaction solution is filtered, and dichloromethane is removed by decompression of the filtrate. The crude product is purified by silica gel column chromatography, and the first red band is collected with pure dichloromethane as the eluent to obtain porphyrin compound 2 with 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 (1.0mmol), p-methoxybenzaldehyde (3.0mmol) of the compound of formula 1, and pyrrole (4.0mmol) are added to a reaction bottle, vacuumed, and nitrogen is introduced, and repeated three times to fill the reaction bottle with nitrogen; then 750mL of anhydrous dichloromethane is added, and nitrogen is introduced into the solution for 15min, and then stirred at 25°C and boron trifluoride ether (0.25mmol) is added, and the reaction system is stirred for 1.5h at this temperature, and then DDQ (2.0mmol) is added, and stirring is continued for 0.5h, and 2mL of triethylamine is added, and stirring is continued for 20min, and the reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane, and 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 an eluent, and the target porphyrin compound 2 is obtained, and the yield is 22%. Its structural characterization data are: 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 (1.0mmol), p-iodobenzaldehyde (3.0mmol) of the compound of formula 1, and pyrrole (4.0mmol) are added to a reaction bottle, vacuumed, and nitrogen is introduced, and repeated three times to fill the reaction bottle with nitrogen; then 750mL of anhydrous dichloromethane is added, and nitrogen is introduced into the solution for 15min, and then stirred at 25°C and boron trifluoride ether (0.25mmol) is added, and the reaction system is stirred for 1.5h at this temperature, and then DDQ (2.0mmol) is added, and stirring is continued for 0.5h, and 2mL of triethylamine is added, and stirring is continued for 20min, and the reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane, and 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 an eluent, and the target porphyrin compound 2 is obtained, and the yield is 21%. Its structural characterization data are: 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 (1.0mmol), 4-fluorobenzaldehyde (3.0mmol), and pyrrole (4.0mmol) are added to a reaction bottle, vacuumed, and nitrogen is introduced, and repeated three times to fill the reaction bottle with nitrogen; then 750mL of anhydrous dichloromethane is added, and nitrogen is introduced into the solution for 15min, and then stirred at 25°C and boron trifluoride ether (0.25mmol) is added, and the reaction system is stirred for 1.5h at this temperature, and then DDQ (2.0mmol) is added, and stirring is continued for 0.5h, and 2mL of triethylamine is added, and stirring is continued for 20min, and the reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane, and 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 an eluent, and the target porphyrin compound can be obtained, and the yield is 23%. Its structural characterization data are: 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 (1.0mmol), 4-trifluoromethylbenzaldehyde (3.0mmol), and pyrrole (4.0mmol) are added to a reaction bottle, vacuumed, and nitrogen is introduced, and repeated three times to fill the reaction bottle with nitrogen; then 750mL of anhydrous dichloromethane is added, and nitrogen is introduced into the solution for 15min, and then stirred at 25°C and boron trifluoride ether (0.25mmol) is added, and the reaction system is stirred for 1.5h at this temperature, and then DDQ (2.0mmol) is added, and stirring is continued for 0.5h, and 2mL of triethylamine is added, and stirring is continued for 20min, and the reaction solution is filtered, and the filtrate is decompressed to remove dichloromethane, and 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 an eluent, and the target porphyrin compound can be obtained, and the yield is 21%. Its structural characterization data are: 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: add p-methylthiobenzaldehyde (1.0mmol), pentafluorobenzaldehyde (1.0mmol), and pyrrole (4.0mmol) to a reaction bottle, evacuate, introduce nitrogen, repeat three times, and fill the reaction bottle with nitrogen; then add 750mL of anhydrous dichloromethane, blow nitrogen into the solution for 15min, then stir at 25°C and add boron trifluoride ether (0.25mmol), continue stirring at this temperature for 1.5h, add DDQ (2.0mmol), continue stirring for 0.5h, add 2mL of triethylamine, continue stirring for 20min, filter the reaction solution, remove dichloromethane from the filtrate under reduced pressure, purify the crude product by silica gel column chromatography, collect the second red band with a dichloromethane: petroleum ether volume ratio of 1:3 as the eluent, and obtain the target porphyrin compound with a yield of 26%. Its structural characterization is shown in the attached 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 UV absorption and fluorescence emission spectrum characterization process and results of the porphyrin compound prepared in this example are 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 region 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 relatively 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, which 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 compounds did not change significantly with the extension of ultrasonic time; Figure 4 As shown, 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 produce 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 added with 10 μL CCK8 (5 mg / mL) and cultured for 4 hours. 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 tested using a microplate reader. The results of the MTT cytotoxicity test are shown in the figure. Figure 5 As shown in the figure, under the condition 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 own fluorescence 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 as claimed in claim 1, characterized in that: The specific steps are: Add p-methylthiobenzaldehyde, the compound of formula 1, and pyrrole to a reaction bottle, evacuate the bottle, and introduce nitrogen, repeat several times to fill the reaction bottle with nitrogen, and add anhydrous dichloromethane to obtain a reaction solution; introduce nitrogen into the reaction solution for 14-16 minutes, then stir at 25°C and add boron trifluoride etherate, keep warm and continue 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 ethyl ether, and 2,3-dichloro-5,6-dicyano-p-benzoquinone is (4-12):1:8; The structural formula of the compound of formula 1 is R-CHO, wherein 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 by suction, 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 is dichloromethane or a dichloromethane / petroleum ether mixture with a volume ratio of 1:

3.

7. Use of the porphyrin compound according to claim 1 as a cell imaging agent.

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

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