A thiophene-substituted aza-fluoroboron dipyrrole compound, nanoparticles of the compound, and their preparation method and application

Abstract

The present invention discloses a thiophene-substituted aza-fluoro-boron-dipyrrole compound, nanoparticles of the compound, and their preparation methods and applications. The thiophene-substituted aza-fluoro-boron-dipyrrole compound designed and synthesized by the present invention, the introduction of the thiophene group can significantly red-shift the absorption spectrum of aza-fluoro-boron-dipyrrole, successfully red-shifting to the 810nm near-infrared region, which is more conducive to its application in vivo and in photothermal therapy. Nanoparticles prepared with this compound as the core can be effectively distributed to visceral adipose tissue after local intraperitoneal administration, selectively targeting visceral adipose tissue, have good biocompatibility and high photothermal conversion efficiency, can achieve temperature regulation by adjusting its concentration and 808nm laser power, and can be used for photothermal treatment of obesity.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a thiophene-substituted aza-fluoro-boron dipyrrole compound, nanoparticles of the compound, and preparation methods and applications thereof. Background Art

[0002] Obesity is a persistent global health problem that is associated with an increased risk of many chronic diseases, such as type 2 diabetes (T2D), hypertension, malignancies, and cardiovascular disease. White adipose tissue (WAT) not only stores energy in the form of triglycerides but also plays a key role in body metabolism by secreting a series of metabolic factors known as adipokines. However, excessive accumulation of WAT or obesity can lead to other systemic metabolic disorders and subsequently develop devastating diseases.

[0003] Current treatment options for obesity primarily include medication, lifestyle interventions, bariatric surgery, and liposuction, but most interventions are suboptimal. Among them, long-term use of drugs based on organic small molecules can produce serious off-target side effects and is often accompanied by adverse reactions, damaging organs such as the intestines, liver, and kidneys. Therefore, there is an urgent need to develop targeted obesity treatment strategies with improved efficacy and reduced toxicity. Summary of the Invention

[0004] The main purpose of the present invention is to provide a thiophene-substituted aza-fluoro-boron dipyrrole compound, nanoparticles of the compound, and their preparation methods and applications, which can be used to prepare adipose tissue-targeted drugs.

[0005] To achieve the above object, the present invention provides a thiophene-substituted aza-fluoro-boron dipyrrole compound, the structural formula of which is shown in Formula 1:

[0006]

[0007] The present invention also provides a method for preparing the above-mentioned thiophene-substituted aza-fluoro-boron-dipyrrole compound, comprising the following steps: firstly subjecting 3-(benzo[b]thiophen-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutan-1-one to a cyclization coupling reaction with 3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutan-1-one and ammonium acetate to obtain an intermediate; and then subjecting the intermediate to a condensation reaction with boron trifluoride etherate in the presence of a catalyst to obtain the thiophene-substituted aza-fluoro-boron-dipyrrole compound.

[0008] Furthermore, the molar ratio of 3-(benzo[b]thiophen-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutan-1-one, 3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutan-1-one and ammonium acetate is 1:1:5.

[0009] Furthermore, the reaction solvent of the cyclization coupling reaction is n-butanol, the reaction temperature is 100-115° C., and the reaction time is 10-12 h.

[0010] Furthermore, the catalyst is N,N-diisopropylethylamine, and the molar ratio of the intermediate to boron trifluoride etherate and the catalyst is 1:15:10.

[0011] Furthermore, the reaction solvent of the condensation reaction is dichloromethane, the reaction temperature is 40-50° C., and the reaction time is 10-12 h.

[0012] The present invention also provides nanoparticles of a thiophene-substituted aza-fluoro-boron-dipyrrole compound, comprising an encapsulation layer formed by an encapsulation matrix and the thiophene-substituted aza-fluoro-boron-dipyrrole compound wrapped in the encapsulation layer.

[0013] The present invention also provides a method for preparing the nanoparticles, comprising the following steps: mixing an encapsulating matrix and a thiophene-substituted aza-fluoro-boron dipyrrole compound, and preparing the nanoparticles by a nanoprecipitation method.

[0014] Furthermore, the size of the nanoparticles can be adjusted by adjusting the type of encapsulating matrix, the ratio of the encapsulating matrix to the thiophene-substituted aza-fluoroboron dipyrrole compound, or the preparation conditions.

[0015] The present invention also provides use of the above-mentioned thiophene-substituted aza-fluoro-boron dipyrrole compound and / or the above-mentioned nanoparticles in the preparation of adipose tissue-targeted drugs for photothermal treatment of obesity.

[0016] The beneficial effects of the present invention are embodied in:

[0017] (1) The present invention designs and synthesizes a thiophene-substituted aza-fluoro-boron-dipyrrole compound. The introduction of the thiophene group can significantly red-shift the absorption spectrum of aza-fluoro-boron-dipyrrole, successfully red-shifting it to the 810nm near-infrared region, which is more conducive to its application in vivo and in photothermal therapy.

[0018] (2) The present invention uses a thiophene-substituted aza-fluoroborane dipyrrole compound as the core to prepare nanoparticles of the compound, which have low cytotoxicity. The size of the nanoparticles can be regulated by adjusting the type of encapsulating matrix, the ratio of the encapsulating matrix to the compound, and the preparation conditions. The preparation method is simple. The nanoparticles of the present invention can be effectively distributed to visceral adipose tissue after local intraperitoneal administration, selectively targeting visceral adipose tissue, have good biocompatibility and high photothermal conversion efficiency, and can be used for photothermal treatment of obesity by adjusting its concentration and 808nm laser power to achieve temperature regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the UV-visible absorption spectrum of BDP in dichloromethane.

[0020] Figure 2 The hydrated particle size diagram and TEM imaging of BDP NPs in PBS solution.

[0021] Figure 3 Real-time temperature change diagram of BDP NPs under 808nm laser irradiation at different power densities.

[0022] Figure 4 Photothermal stability of BDP NPs during five heating-cooling cycles.

[0023] Figure 5 This is a graph analyzing the toxicity of NP-60 on 3T3-L1 cells.

[0024] Figure 6 This is a diagram showing the photothermal killing effect of NP-60 on 3T3-L1 cells.

[0025] Figure 7 Figure 3 shows the in vitro biodistribution of BDP NPs in various organs and white adipose tissue of normal and obese mice 24 hours after intraperitoneal injection.

[0026] Figure 8 is the ratio of the total radiation efficiency of BDP NPs in the liver and visceral adipose tissue of normal and obese mice.

[0027] Figure 9 This is a curve of relative body weight changes of mice in each group during BDP NPs treatment. DETAILED DESCRIPTION

[0028] In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the following examples are given for illustration. It should be noted that the following examples do not limit the scope of protection claimed by the present invention.

[0029] Unless otherwise specified, the raw materials, reagents, or devices used in the following examples can be obtained from conventional commercial channels or by existing known methods; unless otherwise specified, the methods used in the examples of the present invention are methods known to those skilled in the art.

[0030] Example 1

[0031] Synthesis of Thiophene-Substituted Aza-Fluoroboron Dipyrroles (BDPs)

[0032] The structural formula of BDP is shown in Formula 1:

[0033]

[0034] The synthesis reaction equation of BDP is shown in Equation 2:

[0035]

[0036] The preparation process is as follows:

[0037] (1) 3-(Benzo[b]thiophen-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutan-1-one (3.96 g, 10 mmol), 3-(Benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutan-1-one (3.55 g, 10 mmol), ammonium acetate (3.85 g, 50 mmol) and 20 mL of n-butanol were added to a 100 mL flask and heated under reflux at 115°C for 12 h. After the reaction was completed, the mixture was cooled to room temperature and filtered. The filtrate was washed with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain the intermediate.

[0038] (2) The intermediate (0.66 g, 1 mmol), N,N-diisopropylethylamine (1.29 g, 10 mmol), boron trifluoride etherate (2.13 g, 15 mmol) and 10 mL of dichloromethane were added to a 50 mL flask and reacted at room temperature for 12 h under nitrogen protection. After the reaction, the mixture was washed with distilled water, the organic phase was collected and washed with saturated brine, and the organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure and chromatographed on a silica gel column (the eluent was a mixture of dichloromethane and petroleum ether, V 二氯甲烷 :V 石油醚 =3:1), and BDP is obtained. The structural characterization of BDP is: 1 H NMR(400MHz,Chloroform-d)δ8.34(d,J=9.7Hz,2H),8.09(d,J=8.6Hz,2H),7.92-7.79(m,5H),7.68(d,J=8.6Hz,1H), 7.46-7.37(m,4H),7.10-6.94(m,4H),6.78-6.72(m,2H),3.97(s,3H),3.45(q,J=7.2Hz,4H),0.89(t,J=6.8Hz,6H).

[0039] Figure 1 The UV-visible absorption spectrum of BDP in dichloromethane is shown in Figure 2. Figure 1 It can be seen that the maximum absorption peak of BDP is located at 810 nm.

[0040] Example 2

[0041] Preparation of Thiophene-Substituted Aza-Fluoroboron Dipyrrole Nanoparticles (BDP NPs) In this example, three BDP NPs of different sizes were prepared. The preparation process is as follows:

[0042] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-PEG 2000 ) is the encapsulation matrix, DSPE-PEG 2000 The mass ratio of BDP to BDP was 50:1, and the BDP NPs with a particle size of 20 nm were prepared by ultrasonication at a power of 100 W for 10 min in an ultrasonic crusher (equipment model SCIENTZ-IID, brand Ningbo Xinzhi, the same below).

[0043] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000(DSPE-PEG 2000 ) is the encapsulation matrix, DSPE-PEG 2000 The mass ratio of BDP to BDP was 2:1, and the BDP NPs with a particle size of 60 nm were prepared by ultrasonication at a power of 40 W for 120 s, which was recorded as NP-60.

[0044] 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000(DSPE-PEG 5000 ) is the encapsulation matrix, DSPE-PEG 5000 The mass ratio of BDP to BDP was 2:1, and ultrasonication was performed for 60 seconds at a power of 12 W in an ultrasonic crusher to prepare BDP NPs with a particle size of 120 nm, which was recorded as NP-120.

[0045] The longer the PEG segment of the encapsulating matrix, the higher the ratio of BDP to the encapsulating matrix, the larger the diameter of the NPs, and the greater the amount of BDP encapsulated in a single NP. In addition, the power and duration of ultrasound also greatly affect the precipitation process during NP formation and ultimately affect the size of the NPs.

[0046] Figure 2 The hydrated particle size diagram and TEM imaging diagram of BDP NPs in PBS solution (part a in the figure corresponds to NP-20, part b corresponds to NP-60, and part c corresponds to NP-120). Figure 2 It can be seen that the average hydrodynamic diameters of the three BDP NPs are 20 nm, 60 nm and 120 nm, respectively. The field emission transmission electron microscopy images also clearly show the differences in the size of BDP NPs under different preparation conditions.

[0047] Experimental Example 1

[0048] In vitro photothermal performance testing of BDP NPs

[0049] This experimental example analyzed the photothermal properties of the three BDP NPs prepared in Example 2, and the results are as follows:

[0050] Figure 3 Figure 2 shows the real-time temperature change of BDP NPs under 808nm laser irradiation at different power densities (part a corresponds to NP-20, part b corresponds to NP-60, and part c corresponds to NP-120). BDP NPs of the same concentration were placed in a six-well plate and exposed to 808nm laser irradiation at different power densities (0.2-1.2W / cm 2 ) for 5 minutes. Use an infrared thermal imager to monitor the temperature of the sample center in real time and record the temperature change every 30 seconds. Figure 3 As can be seen, the final temperature of the BDPNPs gradually increased with increasing power density, demonstrating that all three prepared BDP NPs possess excellent photothermal properties. These temperatures can effectively induce adipocyte apoptosis. Importantly, this process can be tuned by varying the BDP NP concentration and 808nm laser power density.

[0051] Figure 4 The photothermal stability of BDP NPs during five heating-cooling cycles (part a in the figure corresponds to NP-20, part b corresponds to NP-60, and part c corresponds to NP-120). BDP NPs of the same concentration were exposed to 808 nm laser irradiation (1.0 W / cm 2 ) for 5 minutes, then naturally cool to room temperature, and repeat this cycle five times. Use an infrared thermal imager to monitor the temperature at the center of the sample in real time, and record the temperature change every 30 seconds. Figure 4 It can be seen that after laser irradiation, the maximum temperature difference of BDP NPs remained at a similar level during the five heating and cooling cycles, indicating that all three BDP NPs have good photothermal stability.

[0052] Experimental Example 2

[0053] Investigation of BDP NPs in living cells

[0054] In this experiment, various in vivo studies were conducted on one of the BDP NPs (NP-60) prepared in Example 2. The results are as follows:

[0055] The cytotoxicity of BDP NPs was analyzed by MTT (thiazolyl blue) method. 3T3-L1 cells were plated at 1×10 4Cells were seeded into a 96-well plate and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 24 hours to allow them to adhere completely. Fresh culture medium was then replaced and BDP NPs at different concentrations (0-100 μM) were added. After culturing for 12 hours, MTT solution (5 mg / mL) was added to each well and cultured for another 4 hours. 100 μL of DMSO was then added to each well. The 96-well plate was placed on a horizontal shaking shaker and shaken for 10 minutes. The wavelength was set to 492 nm on the microplate reader, and the absorbance (OD value) of the solution in each well of the 96-well plate was measured. The cell survival rate was calculated according to the following formula: Cell survival rate = (OD 待测组 -OD 空白组 ) / (OD 细胞组 -OD 空白组 )×100%. Figure 5 ,from Figure 5 It can be seen that BDP NPs have almost no cytotoxicity in the range of 0-100 μM and can be used for subsequent biological applications.

[0056] 3T3-L1 cells were plated at 1×10 4 Cells were seeded into 96-well plates and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 24 hours to allow them to adhere completely. Fresh culture medium was then replaced and BDP NPs at different concentrations (0-50 μM) were added. After 12 hours of culture, the 96-well plates were exposed to 808 nm laser irradiation (1.0 W / cm 2 ) for 5 minutes, add MTT solution (5 mg / mL) to each well, continue to culture for 4 hours, then add 100 μL DMSO to each well, place the 96-well plate on a horizontal shaking shaker and shake for 10 minutes. Set the wavelength to 492 nm on the microplate reader, measure the absorbance (OD value) of the solution in each well of the 96-well plate, and calculate the cell survival rate according to the following formula: Cell survival rate = (OD value) 待测组 -OD 空白组 ) / (OD 细胞组 -OD 空白组 )×100%. Figure 6 ,from Figure 6 It can be seen that BDP NPs exhibited good photothermal killing effect in 3T3-L1 cells.

[0057] Experimental Example 3

[0058] In vivo investigation of BDP NPs

[0059] This experimental example conducted various in vivo studies on the three BDP NPs prepared in Example 2, and the results are as follows:

[0060] BDP NPs solution (50 μM, 100 μL) was intraperitoneally injected into the inguinal white adipose tissue area of ​​normal mice and obese mice. 24 hours after injection, the mice were euthanized, and samples were collected from their major organs (heart, liver, spleen, lungs, and kidneys) and white adipose tissue (subcutaneous adipose tissue and visceral adipose tissue such as the left and right perirenal glands, left and right gonads, and mesentery). The targeting effect in vivo was evaluated using a small animal in vivo imaging system. Figure 7-8 ,from Figure 7 、 Figure 8 It can be seen that compared with NP-20 and NP-120, NP-60 showed better targeting effect on visceral adipose tissue in obese mice.

[0061] BDP NPs solution (100 μM, 100 μL) was intraperitoneally injected into the inguinal white adipose tissue area of ​​obese mice. 24 h after injection, the entire abdominal cavity of the mice was exposed to 808 nm laser (1.0 W / cm 2 ) irradiation for 5 minutes to evaluate the photothermal therapeutic effect in mice. The weight changes of mice in each group were recorded every day during the treatment. Figure 9 ,from Figure 9 It can be seen that NP-60 has a better therapeutic effect than NP-20 and NP-120, which may be due to its better targeting to visceral adipose tissue in obese mice.

[0062] In summary, the nanoparticles NP-60 of the thiophene-substituted nitrogen-fluoroboron dipyrrole compound prepared by the present invention have a size of about 60 nm and a maximum absorption peak at 810 nm. After intraperitoneal injection, they selectively target visceral adipose tissue, have good biocompatibility and photothermal stability, and can be used for photothermal treatment of obesity by adjusting the concentration and 808 nm laser power to achieve control of the therapeutic temperature.

[0063] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A thiophene-substituted aza-fluoro-boron dipyrrole compound, characterized in that: Its structural formula is shown in Formula 1: Formula 1.

2. The method for preparing the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 1, wherein: The following steps are involved: First, 3-(benzo[b]thiophen-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutan-1-one, 3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutan-1-one, and ammonium acetate are subjected to a cyclization coupling reaction to obtain an intermediate, and then the intermediate is subjected to a condensation reaction with boron trifluoride ether in the presence of a catalyst to obtain the thiophene-substituted azafluoroborane dipyrrole compound; the catalyst is N,N-diisopropylethylamine, and the structural formula of the intermediate is shown in Formula 2; Formula 2.

3. The method for preparing the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 2, wherein: The molar ratio of 3-(benzo[b]thiophen-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutan-1-one, 3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutan-1-one and ammonium acetate is 1:1:

5.

4. The method for preparing the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 2 or 3, wherein: The reaction solvent of the cyclization coupling reaction is n-butanol, the reaction temperature is 100-115° C., and the reaction time is 10-12 h.

5. The method for preparing the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 2, wherein: The molar ratio of the intermediate, boron trifluoride etherate and the catalyst is 1:15:

10.

6. The method for preparing the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 2 or 5, wherein: The reaction solvent of the condensation reaction is dichloromethane, the reaction temperature is 40-50° C., and the reaction time is 10-12 h.

7. A nanoparticle of a thiophene-substituted aza-fluoroboron dipyrrole compound, characterized in that: The invention comprises an encapsulation layer formed by an encapsulation matrix and the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 1 which is wrapped in the encapsulation layer.

8. The method for preparing nanoparticles according to claim 7, wherein: The following steps are involved: The encapsulating matrix and the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 1 are mixed, and the nanoparticles are prepared by a nanoprecipitation method.

9. The method for preparing nanoparticles according to claim 8, wherein: The size of the nanoparticles can be adjusted by adjusting the type of encapsulating matrix, the ratio of the encapsulating matrix to the thiophene-substituted aza-fluoroboron dipyrrole compound, or the preparation conditions.

10. Use of the thiophene-substituted azafluoroboron dipyrrole compound according to claim 1 and / or the nanoparticles according to claim 7 in the preparation of a drug targeted to adipose tissue for photothermal therapy of obesity.

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