Thiophene-substituted aza-BODIPY compound, nano-particles of compound, and preparation methods and applications of thiophene-substituted aza-BODIPY compound and nano-particles

Abstract

The invention discloses a thiophene-substituted aza-boron-dipyrromethene compound, nanoparticles of the compound, and preparation methods and applications of the thiophene-substituted aza-boron-dipyrromethene compound and the nanoparticles. According to the thiophene-substituted aza-BODIPY compound designed and synthesized by the invention, due to the introduction of a thiophene group, the absorption spectrum of aza-BODIPY can be remarkably subjected to red shift, and is successfully subjected to red shift to a near-infrared region of 810 nm, so that the application of the thiophene-substituted aza-BODIPY compound in a living body and in the aspect of photothermal therapy is facilitated. Nanoparticles prepared by taking the compound as an inner core can be effectively distributed to visceral adipose tissues after local abdominal cavity administration, selectively target the visceral adipose tissues, have good biocompatibility and relatively high photothermal conversion efficiency, can realize temperature regulation and control by adjusting the concentration and 808nm laser power, and can be applied to the field of intraperitoneal administration. The material can be used for photo-thermal obesity treatment.

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 diseases. 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 into devastating diseases.

[0003] At present, the treatment options for obesity mainly include drug therapy, lifestyle intervention, bariatric surgery and liposuction, but most of the intervention measures are not ideal. Among them, long-term use of drugs based on organic small molecules will produce serious off-target side effects, and are often accompanied by adverse reactions, damaging organs such as the intestines, liver and kidneys. Therefore, there is an urgent need to develop targeted treatment strategies for obesity with good efficacy and less toxicity. Summary of the invention

[0004] The main purpose of the present invention is to provide a thiophene-substituted aza-fluoro-boron dipyrrole compound and nanoparticles of the compound, and a preparation method and application thereof, 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]thiophene-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutane-1-one to a cyclization coupling reaction with 3-(benzo[b]thiophene-2-yl)-1-(4-methoxyphenyl)-4-nitrobutane-1-one and ammonium acetate to obtain an intermediate, and then subjecting the intermediate to a condensation reaction with etherified boron trifluoride 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-nitrobutane-1-one to 3-(benzo[b]thiophen-2-yl)-1-(4-methoxyphenyl)-4-nitrobutane-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-fluoro-boron dipyrrole compound, or the preparation conditions.

[0015] The present invention also provides the use of the above-mentioned thiophene-substituted aza-fluoro-boron dipyrrole compound and / or the above-mentioned nanoparticle 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-fluoro-boron 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, and the preparation method is simple. The nanoparticles of the present invention can be effectively distributed to the visceral adipose tissue after local intraperitoneal administration, selectively target the 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. 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 with different power densities.

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

[0023] Figure 5 This is a graph showing the toxicity analysis 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 2 shows the in vitro biodistribution of BDP NPs in various organs and white adipose tissue of normal and obese mice 24 h after intraperitoneal injection.

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

[0027] Fig. 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 scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0029] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or can be obtained 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 (BDP)

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

[0033]

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

[0035]

[0036] The preparation process is as follows:

[0037] (1) In a 100 mL flask, 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, and the mixture was heated to reflux at 115° C. for 12 h. After the reaction was completed, the mixture was cooled to room temperature and then filtered. The filtrate was washed with saturated brine, and the organic phase was collected and dried over anhydrous sodium sulfate, and then concentrated under reduced pressure to obtain an 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 then washed with saturated brine, and the organic phase was taken out and 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 structure of BDP is characterized as follows: 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 ultrasonication was performed for 10 min at 100 W power in an ultrasonic disruptor (equipment model SCIENTZ-IID, brand Ningbo Xinzhi, the same below) to prepare BDP NPs with a particle size of 20 nm, which was recorded as NP-20.

[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 ultrasonic crusher was ultrasonicated for 120s at a power of 40W to prepare BDP NPs with a particle size of 60nm, 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 the ultrasonic crusher was ultrasonicated for 60s at a power of 12W to prepare BDP NPs with a particle size of 120nm, 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 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 analyzes 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 with different power densities (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 placed in a six-well plate and exposed to 808nm laser irradiation with different power densities (0.2-1.2W / cm 2 ) for 5 minutes. 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 3 It can be seen that with the increase of power density, the final temperature of BDPNPs gradually increases, indicating that the three prepared BDP NPs have good photothermal properties. These temperatures can effectively cause adipocyte apoptosis. Importantly, this process can be adjusted by changing the concentration of BDP NPs and the 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 cooled to room temperature, and repeated five times. The temperature at the center of the sample was monitored in real time using an infrared thermal imager, and the temperature change was recorded 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 in five heating and cooling cycles, indicating that all three BDP NPs have good photothermal stability.

[0052] Experimental Example 2

[0053] Study of BDP NPs in living cells

[0054] In this experimental example, various in vivo studies were conducted on one of the BDP NPs (NP-60) prepared in Example 2, and 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 inoculated into a 96-well plate, and the 96-well plate was placed in a cell culture incubator under the following culture conditions: 37°C, 5% CO2, saturated humidity, and cultured for 24 hours to allow them to completely adhere to the wall. After that, fresh culture medium was replaced, and different concentrations (0-100 μM) of BDP NPs were added. After 12 hours of culture, MTT solution (5 mg / mL) was added to each well, and culture was continued for 4 hours. Then, 100 μL DMSO was added to each well, and the 96-well plate was placed on a horizontal shaking shaker 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%. See the results 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 The cells were inoculated into a 96-well plate and placed in a cell culture incubator at 37°C, 5% CO2, and saturated humidity for 24 h to allow them to adhere completely to the wall. Fresh culture medium was then replaced and different concentrations (0-50 μM) of BDP NPs were added. After 12 h of culture, the 96-well plate was 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 待测组 -OD 空白组 ) / (OD 细胞组 -OD 空白组 )×100%. See the results Figure 6 ,from Figure 6 It can be seen that BDP NPs showed 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 (50μM, 100μL) was intraperitoneally injected into the inguinal white adipose tissue area of ​​normal mice and obese mice, respectively. The mice were euthanized 24 hours after the injection, and the main organs (heart, liver, spleen, lung, kidney) and white adipose tissue (subcutaneous adipose tissue and visceral adipose tissue such as left and right perirenal, left and right gonads, and mesentery) were sampled to evaluate its in vivo targeting effect under the small animal in vivo imaging system. Results can be found in 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 ) for 5 min to evaluate the photothermal therapeutic effect in mice. The weight changes of mice in each group were recorded every day during the treatment. Fig. 9 ,from Fig. 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 thiophene-substituted azafluoroborane dipyrrole compounds prepared by the present invention have a size of about 60 nm, a maximum absorption peak at 810 nm, and selectively target visceral adipose tissue after intraperitoneal injection. They 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 regulation 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 principle of the present invention should be included in the protection scope 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:

2. The method for preparing the thiophene-substituted aza-fluoro-boron-dipyrrole compound according to claim 1, characterized in that: The following steps are involved: First, 3-(benzo[b]thiophene-2-yl)-1-(4-(diethylamino)phenyl)-4-nitrobutane-1-one, 3-(benzo[b]thiophene-2-yl)-1-(4-methoxyphenyl)-4-nitrobutane-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 etherified boron trifluoride in the presence of a catalyst to obtain the thiophene-substituted azafluoroborane dipyrrole compound.

3. The method for preparing the thiophene-substituted aza-fluoro-boron-dipyrrole compound according to claim 2, characterized in that: 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, characterized in that: 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, characterized in that: The catalyst is N, N-diisopropylethylamine, and the molar ratio of the intermediate to 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, characterized in that: The reaction solvent of the condensation reaction is dichloromethane, the reaction temperature is 40-50° C., and the reaction time is 10-12 hours.

7. A nanoparticle of a thiophene-substituted aza-fluoro-boron 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 as claimed in claim 1 which is encapsulated in the encapsulation layer.

8. The method for preparing nanoparticles according to claim 7, characterized in that: The method comprises the following steps: mixing an encapsulating matrix and a thiophene-substituted aza-fluoro-boron dipyrrole compound, and preparing the nanoparticles by a nanoprecipitation method.

9. The method for preparing nanoparticles according to claim 8, characterized in that: 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-fluoro-boron dipyrrole compound or the preparation conditions.

10. Use of the thiophene-substituted aza-fluoro-boron dipyrrole compound according to claim 1 and / or the nanoparticle according to claim 7 in the preparation of adipose tissue-targeted drugs for photothermal treatment of obesity.

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