Phenoxazine-fluoropyrrole hybrid conjugated organic free radical photothermal agents, methods of making and use thereof

Abstract

The application belongs to the technical field of medical material synthesis and application, and relates to an organic free radical photothermal agent based on phenazine-fluoroboropyrrole hybrid conjugation as well as a preparation method and application thereof. The preparation method of the organic free radical photothermal agent comprises the following steps: (1) in the presence of a first solvent, compound 1 and an acylation reagent are mixed to perform a first reaction to obtain compound 2; (2) in the presence of a second solvent, compound 2, compound 3, acetic acid and piperidine are mixed to perform a second reaction to obtain the organic free radical photothermal agent. The organic small-molecule free radical photothermal agent based on phenazine-fluoroboropyrrole hybrid conjugation provided by the application can realize strong short-wave infrared absorption in an organic solvent through simple functional group regulation, and the maximum absorption wavelength is greater than 1000 nm. The preparation method is simple in synthesis method, low in raw material cost and mild in action condition, and the target product is obtained through a two-step process.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical material synthesis and application, and particularly relates to an organic free radical photothermal agent based on phenazine-fluoropyrrole hybrid conjugation and a preparation method and application thereof. BACKGROUND

[0002] As a non-invasive cancer treatment method, photothermal therapy has been favored by researchers in recent years. Its principle is to convert light energy into heat energy through photothermal materials to raise the temperature at the tumor site to kill cancer cells. The ultraviolet absorption of traditional photothermal materials is mostly located in the near-infrared region, and the penetration depth is shallow and the photothermal conversion efficiency is low. The photothermal materials absorbing in the short-wave infrared region make up for the shortcomings of the near-infrared region absorbing materials, and have the advantages of low phototoxicity and high biological safety, and show significant advantages in the field of photothermal therapy. At present, the photothermal materials absorbing in the short-wave infrared region mainly include inorganic materials and conjugated polymers. The above-mentioned materials have good photothermal therapy effect. However, these materials have obvious shortcomings. For example, inorganic materials are difficult to degrade in vivo, and have potential biological toxicity. The conjugated polymer has poor reproducibility. Compared with the above two, organic small molecules not only have good biological compatibility and reproducibility, but also have easy-to-control performance, showing great potential. Therefore, it is of great significance to develop organic small molecule photothermal agents with short-wave infrared absorption for application in the field of tumor photothermal therapy.

[0003] The current common strategy for promoting wavelength redshift mainly includes adjusting the electron push-pull effect and expanding the conjugated structure, but the above two strategies are difficult to achieve the maximum ultraviolet absorption peak to reach the short-wave infrared region. In recent years, the free radical regulation strategy provides a new idea for realizing the short-wave infrared absorption of organic small molecules. However, due to the lack of suitable skeletons and the instability of free radicals themselves, it is still a challenging task to develop stable short-wave infrared absorbing organic free radical photothermal reagents and successfully apply them in the field of photothermal therapy. SUMMARY

[0004] In order to solve the above problems existing in the prior art, the application provides an organic free radical photothermal agent based on phenazine-fluoropyrrole hybrid conjugation and a preparation method thereof. A kind of photothermal agent with short-wave infrared absorption is synthesized by a simple preparation method and low-cost raw materials. The maximum absorption wavelength of the molecule in dimethyl sulfoxide is 1008 nm.

[0005] In order to achieve the above purpose, the first aspect of the application provides an organic free radical photothermal agent based on phenazine-fluoropyrrole hybrid conjugation, and the structural formula of the photothermal agent is:

[0006] .

[0007] The second aspect of the present application provides a preparation method of the organic free radical photothermal agent, comprising the following steps:

[0008] (1) in the presence of a first solvent, compound 1 and acylation reagent are mixed to carry out a first formylation reaction, and compound 2 is obtained;

[0009] (2) in the presence of a second solvent, compound 2, compound 3, acetic acid and piperidine are mixed to carry out a second condensation reaction, and the organic free radical photothermal agent is obtained;

[0010] The reaction process is as follows:

[0011] .

[0012] The third aspect of the present application provides application of the organic free radical photothermal agent in preparation of an antitumor therapeutic drug.

[0013] The fourth aspect of the present application provides an antitumor therapeutic agent, which is a nanoparticle comprising the organic free radical photothermal agent and a surface coating layer thereof; wherein the material of the coating layer is polyoxyethylene polyoxypropylene ether triblock copolymer; the content of the organic free radical photothermal agent is 0.22%-0.5% and the content of the coating layer is 3.4%-5% based on the total weight of the nanoparticle.

[0014] The fifth aspect of the present application provides a preparation method of the antitumor therapeutic agent, comprising the following steps:

[0015] (a) the organic free radical photothermal agent is dissolved in an organic solvent, a coating layer precursor is added and uniformly mixed, a coating reaction is carried out, the reaction is ended, the organic solvent is removed, and a reaction product is obtained;

[0016] (b) a buffer solution is added to the reaction product of step (a), ultrasonic dispersion is carried out, a reaction is carried out, the reaction is ended, the solvent is removed, and the therapeutic agent is obtained.

[0017] The present application achieves the following beneficial effects:

[0018] 1. The organic small-molecule free radical photothermal agent based on phenazine-fluoroboropyrrole hybrid conjugation provided by the present application is a novel organic small-molecule skeleton, and strong short-wave infrared absorption in an organic solvent can be realized through simple functional group regulation, and the maximum absorption wavelength is greater than 1000 nm.

[0019] 2. The preparation method of the organic small-molecule free radical photothermal agent based on phenazine-fluoroboropyrrole hybrid conjugation provided by the present application has simple synthesis method, low raw material cost and mild reaction conditions, and the target product is obtained through a two-step process.

[0020] 3. The application of the short-wave infrared absorption type organic small molecule radical based on phenazine-fluoropyrrole hybrid conjugated organic small molecule radical photothermal agent, which is coated by a biocompatible amphiphilic polymer to form stable nanoparticles, is applied to photoacoustic imaging guided photothermal therapy in a biological body.

[0021] Other features and advantages of the present application will be described in detail in the following detailed description. BRIEF DESCRIPTION OF DRAWINGS

[0022] The above and other objects, features and advantages of the present application will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings in which like reference characters refer to like parts throughout the figures, and in which:

[0023] Figure 1 NMR spectrum of PZ-BDP in the present application.

[0024] Figure 2 Mass spectrum of PZ-BDP in the present application.

[0025] Figure 3 UV absorption spectrum of PZ-BDP nanoparticles (PZ-BDP NPs) in PBS buffer solution in the present application.

[0026] Figure 4 In vitro photothermal curve comparison chart of PZ-BDP nanoparticle PBS buffer solution (PZ-BDP NPs) and blank PBS buffer solution (PBS) in the present application.

[0027] Figure 5 Thermal imaging chart of breast cancer model mice during anti-tumor treatment in the present application, wherein, from bottom to top, the thermal imaging chart of blank PBS buffer solution (PBS) with 1064 nm laser irradiation time, the thermal imaging chart of 100 μM PZ-BDP nanoparticle PBS buffer solution (100 μM NPs) with 1064 nm laser irradiation time, and the thermal imaging chart of 300 μM PZ-BDP nanoparticle PBS buffer solution (300 μM NPs) with 1064 nm laser irradiation time.

[0028] Figure 6Fig. 1 is a diagram showing the change in tumor volume of the breast cancer model mice in the experimental group and the control group in the treatment process for 12 days in the present application, wherein the experimental group is the 100 μM PZ-BDP nanoparticle PBS buffer solution group (100 μM NPs) and the 100 μM PZ-BDP nanoparticle PBS buffer solution + 1064 nm laser group (100 μM NPs + Light), and the control group is the blank PBS buffer solution group (PBS) and the blank PBS buffer solution + 1064 nm laser group (PBS + Light). DETAILED DESCRIPTION

[0029] The preferred embodiments of the present application will be described in more detail below. Although the preferred embodiments of the present application are described below, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein.

[0030] The first aspect of the present application provides a phenazine-fluoropyrrole hybrid conjugated organic free radical photothermal agent, and the structural formula of the photothermal agent is:

[0031] .

[0032] The second aspect of the present application provides a preparation method of the organic free radical photothermal agent, comprising the following steps:

[0033] (1) mixing compound 1 and acylation reagent in the presence of a first solvent to perform a first step formylation reaction to obtain compound 2;

[0034] (2) mixing compound 2, compound 3, acetic acid and piperidine in the presence of a second solvent to perform a second step condensation reaction to obtain the organic free radical photothermal agent;

[0035] The reaction process is as follows:

[0036] .

[0037] According to the present application, preferably, in step (1), the first solvent is N,N-dimethylformamide; the acylation reagent is phosphorus oxychloride; the concentration of compound 1 in the reaction system is 0.25 mmol / L-0.3 mmol / L; the molar ratio of the use amount of compound 1 to the acylation reagent is 1:1.1~5.0; and the first reaction conditions include that the temperature is 0-40℃ and the time is 0.5-3h.

[0038] According to the present application, preferably, in step (2), the second solvent is anhydrous toluene; and the molar ratio of the use amount of compound 2, piperidine, acetic acid and compound 3 is 1:2.0~3.0:2.0~3.0:0.5~0.8.

[0039] According to the application, preferably, in step (2), the conditions of the second reaction include: temperature is 0-40℃, time is 0.5-3h.

[0040] The third aspect of the application provides the use of the organic free radical photothermal agent in the preparation of an anti-tumor therapeutic drug.

[0041] In the application, the organic free radical photothermal agent can realize photoacoustic imaging guided photothermal therapy in deep tumor tissues of organisms.

[0042] The fourth aspect of the application provides an anti-tumor therapeutic drug, which is a nanoparticle, comprising the organic free radical photothermal agent and a surface coating layer thereof; wherein the material of the coating layer is polyoxyethylene polyoxypropylene ether triblock copolymer; the content of the organic free radical photothermal agent is 0.22%-0.5% and the content of the coating layer is 3.4%-5% based on the total weight of the nanoparticle.

[0043] The fifth aspect of the application provides a preparation method of the anti-tumor therapeutic drug, comprising the following steps:

[0044] (a) dissolving the organic free radical photothermal agent in an organic solvent, uniformly mixing the coating layer precursor, performing a coating reaction, removing the organic solvent after the reaction is completed, and obtaining a reaction product;

[0045] (b) adding a buffer solution to the reaction product of step (a), performing a reaction after ultrasonic dispersion, removing the solvent after the reaction is completed, and obtaining the therapeutic drug.

[0046] According to the application, preferably, in step (a), the organic solvent is chloroform; the concentration of the organic free radical photothermal agent in the reaction system is 1.7 10 -6 mol / L-2 10 -6 mol / L; the addition amount of the organic free radical photothermal agent and the coating layer precursor is such that the content of the organic free radical photothermal agent is 0.22%-0.5% and the content of the coating layer is 3.4%-5% based on the total weight of the nanoparticle; and the conditions of the coating reaction include: temperature is 10-40℃, time is 1-4h.

[0047] According to the application, preferably, in step (b), the buffer solution is a phosphate aqueous solution; the concentration of the buffer in the buffer solution is 0.01 mol / L-0.1 mol / L; the volume ratio of the reaction product of step (b) to the buffer solution is 1:5-10; and the conditions of the reaction include: temperature is 10-40℃, time is 10-14h.

[0048] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0049] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0050] Example 1

[0051] Synthesis of compound 2: Compound 1 (1 g, 3.38 mmol) was added to a 100 mL Shrek tube, followed by the addition of 10 mL of anhydrous... N , N Dissolve dimethylformamide and cool the system in an ice-water bath to 0°C. o C. Phosphorus oxychloride (0.57 g, 3.718 mmol) was slowly added dropwise, followed by stirring at room temperature for 2 hours. The solution was then poured into ice water, and sodium hydroxide aqueous solution was added dropwise until the solution turned orange. Extraction was performed with ethyl acetate, followed by washing with water and drying. Column chromatography was performed using petroleum ether:ethyl acetate = 10:1 as the developing solvent to give 0.765 g of an orange-red solid, in 70% yield.

[0052] Synthesis of an organic free radical photothermal agent based on phenazine-fluoroboron-pyrrole hybrid conjugation (PZ-BDP): Acetic acid (3.0 equiv) and piperidine (2.0 equiv) were added sequentially to a toluene solution (10 mL) containing compound 3 (2.2 equiv) and compound 4 (0.5 mmol). The reaction mixture was refluxed for about 1 hour and then cooled to room temperature. After the toluene was evaporated to dryness, column chromatography was performed using petroleum ether:ethyl acetate = 10:1 as the developing solvent to give 0.17 g of a green solid, with a yield of 36%.

[0053] The reaction process is as follows:

[0054]

[0055] like Figure 1 The NMR spectrum of compound PZ-BDP shown in the figure indicates that... 1 H NMR (600 MHz, C6D6) delta / ppm = 8.19 (d, 2H), 7.28–7.12 (m, 2H), 7.00 (d, 2H), 6.64 (dt, J = 24, 6 Hz, 5H), 6.36 (s, 2H), 6.25 (dd, J = 24, 7.5 Hz, 4H), 6.10 (d, 2H), 3.29 (t, 4H), 3.12 (t, 4H), 2.16 (d, 2H), 1.64 (p, 4H), 1.47 (p, 4H), 1.38–1.14 (m, 40H), 0.98–0.89 (dt, J = 35.16, 7.14, 12H). The PZ-BDP structure can be verified.

[0056] like Figure 2 The high-resolution mass spectrum of compound PZ-BDP shown in the figure indicates that HRMS (ESI) + ): calcd.for C 72 H 94 BF5N5: [M+H] + = 1148.7549.

[0057] Application Example 1

[0058] The phenazine-fluoroboron-pyrrole hybrid conjugated small organic molecule radical (PZ-BDP) obtained in Example 1 was dissolved in chloroform, and Pluronic F-127 was added and stirred for two hours. The organic solvent was evaporated, a phosphate buffer solution was added, and the mixture was ultrasonically dispersed for half an hour, followed by stirring for another 12 hours. The prepared nanoparticles exhibited high photothermal conversion efficiency, reaching up to 81%, in in vitro photothermal tests.

[0059] Application Example 2

[0060] The nanoparticles obtained in Example 1 were applied to the anti-tumor treatment of organisms. They were injected into the tail vein of a breast cancer model mouse, and the tumor site of the mouse was irradiated with a 1064 nm laser for 10 minutes every 48 hours. After 12 days of treatment, the ideal therapeutic effect was achieved.

[0061] like Figure 3 As shown, based on the ultraviolet absorption spectrum, it can be determined that the maximum absorption wavelength of PZ-BDP nanoparticles in PBS buffer solution is 1000 nm.

[0062] like Figure 4 As shown, according to the in vitro photothermal curve, the temperature of the PZ-BDP nanoparticles can rise from 32°C to 75.6°C after 10 minutes of 1064 nm laser irradiation.

[0063] like Figure 5 As shown in the photothermal imaging of PZ-BDP nanoparticles at the breast cancer tumor site, the temperature at the tumor site gradually increased to 55℃ as the illumination time increased.

[0064] like Figure 6 As shown, during the 12-day treatment period, based on the tumor volume change curves of the experimental and control groups, the "nanoparticle + 1064 nm laser" group exhibited a significant anti-tumor effect.

[0065] This invention utilizes phenazine-fluoroboron-pyrrole hybrid conjugation to prepare a class of photothermal agents with strong short-wave infrared absorption. By adjusting the electron push-pull effect, conjugated structure, and free radical generation strategy, the maximum ultraviolet absorption wavelength reaches the short-wave infrared region. We encapsulated this photothermal agent to form water-soluble nanoparticles and investigated its photothermal therapy capabilities in in vivo deep tumor tissues, demonstrating good imaging effects.

[0066] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. An organic free radical photothermal agent based on phenazine-fluoroboron-pyrrole hybrid conjugation, characterized in that, The structural formula of the photothermal agent is: 。 2. The method for preparing the organic free radical photothermal agent according to claim 1, characterized in that, Includes the following steps: (1) In the presence of the first solvent, compound 1 and the acylation reagent were mixed to carry out the first step of the formylation reaction to obtain compound 2; (2) In the presence of the second solvent, compound 2, compound 3, acetic acid and piperidine are mixed to carry out the second condensation reaction to obtain the organic free radical photothermal agent; The reaction process is as follows: 。 3. The preparation method according to claim 2, wherein, In step (1), the first solvent is N,N-dimethylformamide; The acylation reagent is phosphorus oxychloride; In the reaction system, the concentration of compound 1 was 0.25 mmol / L–0.3 mmol / L; The molar ratio of compound 1 to the acylation reagent is 1:1.1~5.0; The conditions for the first step of the formylation reaction include: a temperature of 0-40℃ and a time of 0.5-3h.

4. The preparation method according to claim 2, wherein, In step (2), the second solvent is anhydrous toluene; The molar ratio of compound 2, piperidine, acetic acid and compound 3 is 1:2.0~3.0:2.0~3.0:0.5~0.

8.

5. The preparation method according to claim 2, wherein, In step (2), the conditions for the second condensation reaction include: a temperature of 0-40℃ and a time of 0.5-3h.

6. The use of the organic free radical photothermal agent according to claim 1 in the preparation of an antitumor therapeutic agent, wherein the tumor is breast cancer.

7. An antitumor therapeutic agent, characterized in that, The therapeutic agent is nanoparticles, including the organic free radical photothermal agent and its surface coating layer as described in claim 1; The coating layer is made of polyoxyethylene polyoxypropylene ether triblock copolymer; Based on the total weight of the nanoparticles, the content of the organic free radical photothermal agent is 0.22%-0.5%, and the content of the coating layer is 3.4%-5%.

8. The method for preparing the antitumor therapeutic agent according to claim 7, characterized in that, Includes the following steps: (a) Dissolve the organic free radical photothermal agent in an organic solvent, add the coating layer precursor and mix evenly to carry out the coating reaction. After the reaction is completed, remove the organic solvent to obtain the reaction product. (b) Add a buffer solution to the reaction product of step (a), disperse it by ultrasound, and then carry out the reaction. After the reaction is completed, remove the solvent to obtain the therapeutic agent.

9. The method for preparing the antitumor therapeutic agent according to claim 8, wherein, In step (a), the organic solvent is chloroform; The concentration of the organic free radical photothermal agent in the reaction system is 1.7%. 10 -6 mol / L-2 10 -6 The amount of organic free radical photothermal agent and coating layer precursor added is such that, based on the total weight of the nanoparticles, the content of the organic free radical photothermal agent is 0.22%-0.5%, and the content of the coating layer is 3.4%-5%. The conditions for the coating reaction include: a temperature of 10-40℃ and a time of 1-4h.

10. The method for preparing the antitumor therapeutic agent according to claim 8, wherein, In step (b), the buffer solution is an aqueous phosphate solution; The concentration of the buffer in the buffer solution is 0.01 mol / L-0.1 mol / L; The volume ratio of the reaction product to the buffer solution in step (b) is 1:5-10; The reaction conditions include a temperature of 10-40℃ and a time of 10-14h.

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