BODIPY Compounds and Their Preparation Methods and Applications

Abstract

The present invention discloses a BODIPY compound, a preparation method thereof and an application thereof. The structural formula of the compound is shown in formula (I), wherein R1 is hydrogen, halogen, heterocyclic hydrocarbon group, aryl group with 6 to 8 carbon atoms or aliphatic hydrocarbon group with 1 to 10 carbon atoms; R2 is hydrogen, aryl group with 6 to 10 carbon atoms or aliphatic hydrocarbon group with 1 to 6 carbon atoms. In the present invention, a BODIPY dimer is connected through a sulfur bridge bond to obtain a BODIPY tetramer with the structure shown in formula (I), which has a very excellent ability to generate reactive oxygen species, and can effectively inhibit the proliferation of glioma cells under light conditions, and can be used as a potential phototherapeutic drug for treating glioma. #imgabs0#

Description

Technical Field

[0001] The present invention relates to the technical field of phototherapy molecular drugs, and particularly relates to a BODIPY compound, a preparation method thereof and an application thereof. Background Art

[0002] Glioma, abbreviated as glioma, is derived from the malignant transformation of interstitial cells in the central nervous system and mainly occurs in the brain tissue. It is the most common type of human primary brain tumor, accounting for 80% of intracranial malignant tumor cases. Whether the glioma has high or low differentiation, it has malignant biological behavior characteristics such as spontaneity, invasiveness and recurrence. At present, the main clinical treatment method for glioma is mainly surgical resection, combined with comprehensive treatment methods such as radiotherapy, chemotherapy and immunotherapy. Although a variety of treatment schemes have been tried and studied clinically, these treatment methods still have certain limitations in tumor treatment. For example, there is no obvious boundary between glioma and normal brain tissue, and due to the important functions of the brain tissue, it is difficult for surgery to achieve a large-scale radical resection. Especially in the important functional areas and their adjacent parts, surgery can only remove a certain volume of tumor tissue and it is difficult to achieve a true total resection. In addition, chemotherapy and radiotherapy have poor selectivity and large toxic and side effects, bringing great pain to patients. Therefore, exploring new and effective clinical treatment means and combining multiple treatment modes is a practical way to improve the curative effect of tumors and prevent tumor recurrence.

[0003] As a novel clinical medical method, optical therapy of tumors has attracted increasing attention in recent years. Nowadays, people are committed to exploring various diagnostic imaging and treatment modalities to construct a phototherapy system. Phototherapy can not only achieve real-time diagnosis but also perform in-situ treatment, opening up a new way for cancer research. In diagnostic techniques, fluorescence imaging (FLI) technology exhibits obvious advantages, including high sensitivity, rapid response, and non-invasiveness, and can perform real-time monitoring and visualization research on the imaging site. As a promising alternative to traditional tumor treatment, phototherapy has become a novel light-controllable, non-invasive, and effective treatment means, including photodynamic therapy (PDT) and photothermal therapy (PTT). In addition, the thermal signal generated during the PTT process can also be captured by a photothermal imaging system for photothermal imaging (PTI), which has good temperature sensitivity and the ability of real-time monitoring. However, the treatment efficiency of the PDT / PTT single modality is often restricted by the hypoxia and heat shock effects of the tumor microenvironment. The combined treatment of PDT and PTT is considered a breakthrough design strategy, which can achieve a synergistic effect and improve the treatment effect. For example, PTT can improve the hypoxic conditions in tumor tissues by increasing blood flow velocity, thereby promoting PDT, and further eliminating heat-resistant tumor cells in PTT. Therefore, it is crucial to construct a multifunctional phototherapy system for simultaneous multimodal imaging and synergistic phototherapy. Currently, the most commonly used strategy for constructing a multimodal optical diagnosis and treatment system is to combine multiple components with single functions into a nanoplatform. Although this integrated method is effective to some extent, this all-in-one method will inevitably be restricted by complex components, low repeatability, and unclear pharmacokinetics. Compared with the above nanomaterials, organic small molecules will be an ideal substitute for the multifunctional phototherapy system due to their simple composition, convenient preparation, clear structure, excellent biocompatibility, and high repeatability, etc.

[0004] Therefore, in order to realize clinical application as soon as possible, it is very necessary to develop a single organic molecule with multiple functions. Summary of the Invention

[0005] For this reason, the present invention provides a BODIPY-based compound, its preparation method and applications.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] According to the first aspect of the embodiments of the present invention, the present invention provides a BODIPY-based compound, and the structural formula of the compound is shown in formula (I):

[0008]

[0009] Among them, R1 is hydrogen, halogen, heterocyclic hydrocarbon group, aryl group with 6 to 8 carbon atoms or aliphatic hydrocarbon group with 1 to 10 carbon atoms; R2 is hydrogen, aryl group with 6 to 10 carbon atoms or aliphatic hydrocarbon group with 1 to 6 carbon atoms.

[0010] In some preferred embodiments, R1 is hydrogen, five-membered heterocyclic hydrocarbon group or C1-C8 methoxy-substituted aryl group; R2 is hydrogen, C6-C10 alkyl-substituted phenyl or C1-C6 straight-chain alkyl group.

[0011] In some preferred embodiments, R1 is halogen or p-methoxyphenyl, and R2 is 2,4,6-trimethylphenyl.

[0012] In some preferred embodiments, the structure of the compound is as follows:

[0013]

[0014] According to the second aspect of the embodiments of the present invention, the present invention provides a method for preparing the BODIPY compound as described above. Under an inert atmosphere, an intermediate represented by the structural formula (II), a sulfide, and a phase transfer catalyst are reacted in an organic solvent.

[0015]

[0016] Among them, the definitions of R1 and R2 are the same as above.

[0017] Furthermore, the sulfide is selected from one or more of sodium sulfide, (bis(tributyltin)sulfide), sulfur powder, potassium sulfide, and zinc sulfide;

[0018] The phase transfer catalyst is tetrabutylammonium bromide, tetrabutylammonium chloride, or tetrabutylammonium fluoride;

[0019] The molar ratio of the intermediate, the sulfide, and the phase transfer catalyst is 1:0.5-1:0.1-0.3.

[0020] Furthermore, the organic solvent is N,N-dimethylformamide, toluene, tetrahydrofuran, or dichloromethane.

[0021] Furthermore, the temperature of the reaction is 70-90 °C.

[0022] According to the third aspect of the embodiments of the present invention, the present invention provides the application of the BODIPY compound as described above in the preparation of a phototherapy anti-tumor drug.

[0023] Furthermore, the tumor is glioma.

[0024] The present invention has the following advantages:

[0025] In the present invention, a BODIPY dimer is connected through a sulfur bridge bond to obtain a BODIPY tetramer with the structure shown in formula (I). It has very excellent reactive oxygen species generation ability, and can effectively inhibit the proliferation of glioma cells under light conditions, and can be used as a potential phototherapeutic drug for the treatment of glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can also be obtained according to the provided drawings.

[0027] Figure 1 1H NMR spectrum of BODIPY T1 obtained in the present invention;

[0028] Figure 2 13C NMR spectrum of BODIPY T1 obtained in the present invention;

[0029] Figure 3 HRMS mass spectrum of BODIPY T1 obtained in the present invention;

[0030] Figure 4 1H NMR spectrum of BODIPY T2 obtained in the present invention;

[0031] Figure 5 13C NMR spectrum of BODIPY T2 obtained in the present invention;

[0032] Figure 6 HRMS mass spectrum of BODIPY T2 obtained in the present invention;

[0033] Figure 7 Absorption and emission spectrum of BODIPY T2 obtained in the present invention;

[0034] Figure 8 Reactive oxygen species test spectrum of BODIPY T1, T2 and comparative example T3 obtained in the present invention;

[0035] Figure 9 CCK-8 method cytotoxicity test result graph of BDP-T2 NPs against glioma U87 obtained in the present invention;

[0036] Figure 10 EdU proliferation ability test graph of BDP-T2 NPs obtained in the present invention for inhibiting glioma U87 cells. DETAILED DESCRIPTION OF THE INVENTION

[0037] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0038] In the following content, sodium sulfide nonahydrate was purchased from Sinopharm Chemical Reagent Co., Ltd.; tetrabutylammonium bromide was purchased from Energy Chemical; F127, a non-ionic surfactant, is a copolymer of polypropylene glycol and ethylene oxide (polyether), purchased from Sigma-Aldrich; the fluoroboron fluorescent dye BODIPY is one of the compounds with the structures shown in Formulas D1 and D2, synthesized with reference to: Org. Lett. 2021, 23, 7661.

[0039]

[0040] Example 1

[0041]

[0042] In a 10 mL Schlenk reaction tube, D1 (72 mg, 0.1 mmol), nBu4NBr (6 mg, 0.02 mmol) and Na2S·9H2O (13 mg, 0.055 mmol) were weighed and dissolved in tetrahydrofuran (4 mL) solution. The reaction mixture was heated in a metal bath and stirred under argon at 80 °C for 3 hours. After the reaction was completed, it was cooled to room temperature, and then the reaction mixture was poured into water and extracted with dichloromethane (30 mL × 3). The organic layers were combined, dried over anhydrous Na2SO4, and then concentrated under reduced pressure with a vacuum pump to obtain a crude product. The crude product was purified by column chromatography (silica gel, petroleum ether / dichloromethane: 1:2, v / v) to obtain a black solid powder BODIPY T1 (15 mg, 21%).

[0043] The characterization data are as follows:

[0044] Compound shown in Formula (T1): 1 1H NMR (500 MHz, CDCl3) δ: 6.93 (d, J = 2.3 Hz, 8H), 6.54 (d, J = 4.3 Hz, 4H), 6.47 - 6.45 (m, 6H), 6.34 (d, J = 4.3 Hz, 2H), 2.34 (s, 6H), 2.33 (s, 6H), 2.11 (s, 24H), 2.10 (s, 24H). 1313C NMR(126MHz,CDCl3)δ:163.0,162.1,149.0,143.2,143.0,142.0,140.1,139.7,139.1,139.0,137.2,136.8,136.8,134.4,129.2,128.9,128.7,128.5,128.2,126.9,126.5,123.1,118.1,117.8,117.3,21.1,20.1,20.0.HRMS(MALDI-TOF)calcd.for C 72 H 56 B4Cl2F8N8S3[M] + :1394.3445,found 1394.3440.

[0045] Example 2

[0046]

[0047] In a 10 mL Schlenk reaction tube, weigh out compound D2 (79 mg, 0.1 mmol), nBu4NBr (5 mg, 0.02 mmol) and Na2S·9H2O (13 mg, 0.05 mmol), and dissolve them in a mixed solution of toluene (2 mL) and water (0.02 mL). Heat with a liquid metal bath, and stir the reaction mixture under argon at 80 °C for 1 hour. After the reaction is completed, cool to room temperature, then pour the reaction mixture into water and extract with dichloromethane (30 mL × 3). Combine the organic layers, dry over anhydrous Na2SO4, and then concentrate under reduced pressure using a vacuum pump to remove the organic solvent. The crude product is purified by column chromatography (silica gel; petroleum ether / dichloromethane; 1:1, v / v) to obtain a dark purple solid BODIPY T2 (62 mg, 80%).

[0048] The characterization data are as follows:

[0049] Compound shown in formula (T2): 1 1H NMR(500MHz,CDCl3)δ:8.00(d,J=8.9Hz,4H),8.00(d,J=8.9Hz,4H),7.04(d,J=9.0Hz,4H),6.93(d,J=13.4Hz,8H),6.64(q,J=4.4Hz,4H),6.48(d,J=4.3Hz,2H),6.43(d,J=4.2Hz,2H),6.40(d,J=3.8Hz,4H),3.90(s,6H),2.34(d,J=11.2Hz,12H),2.13(d,J=20.1Hz,24H). 1313C NMR(126MHz,CDCl3)δ:163.67,161.94,160.68,158.79,148.47,142.19,141.55,140.50,139.24,139.11,139.02,138.43,137.35,137.28,137.23,131.75,131.22,130.03,129.50,128.56,128.54,128.30,128.05,125.52,124.53,123.04,121.44,116.93,116.15,114.53,55.81,21.51,20.42.HRMS calcd.for C 86 H 70 B4F8N8O2S3[M] + :1538.5028,found 1538.5035.

[0050] Comparative Example 1

[0051]

[0052] Weigh 68 mg (0.1 mmol) of BODIPY D3 into a 25 mL round-bottom flask, add it to 10 mL of dry dichloromethane solution to dissolve it. Then weigh 486 mg (5 mmol) of FeCl3 and dissolve it in 1 mL of dry CH3NO2 solution, and slowly add it dropwise to the above solution. The reaction mixture is stirred at room temperature. Monitor the reaction progress by TLC plate spotting. After the reaction is completed, quench it with saturated aqueous NaHCO3 solution. Dilute the reaction mixture with dichloromethane, wash it twice with water, dry it with anhydrous Na2SO4, and then concentrate it under reduced pressure with a vacuum pump to obtain the crude product. Then purify it by silica gel column chromatography (eluent: petroleum ether / dichloromethane = 2:1, v / v) to obtain black solid BODIPY T3 (24 mg, 36%).

[0053] The characterization data are as follows:

[0054] The compound shown in formula (T3): 1 1H NMR(500MHz,CDCl3)δ:7.61(d,J = 4.5Hz,2H),6.98(s,4H),6.94(s,4H),6.69(d,J = 4.5Hz,2H),6.54(d,J = 4.2Hz,2H),6.50(s,2H),6.48(s,2H),6.32(d,J = 4.2Hz,2H),2.38(s,6H),2.34(s,6H),2.18(s,12H),2.10(s,12H). 1313C NMR(126MHz,CDCl3)δ:163.4,162.7,147.5,143.5,143.4,140.8,140.2,139.6,139.5,139.3,137.3,137.1,134.8,129.7,128.8,128.7,127.3,127.2,124.9,118.5,118.4,117.8,21.6,21.5,20.6,20.3.HRMS(MALDI-TOF)calcd.for C 72 H 56 B4C l2 F8N8S2[M] + :1362.3691,found 1362.3697.

[0055] Example 3 Preparation of Nanoparticles

[0056] The preparation of BDP-T2 nanoparticles (BDP-T2 NPs) refers to Small, 2018, 14, 1802991. BODIPY T2 (374 μL, chloroform solution of 267.6 μM) and F127 (680 μL, chloroform solution of 7.7 mg mL -1 were added to a round-bottom flask containing 5 mL of chloroform. The mixture was sonicated for 20 minutes at room temperature. Then, chloroform was completely removed by concentration under reduced pressure using a vacuum pump, and water (5 mL) was added and shaken. Finally, the liquid was stored at 4 °C for subsequent experiments.

[0057] Test Example 1 Measurement of Reactive Oxygen Species Yield

[0058] The ability of the phototherapeutic molecule to generate reactive oxygen species (ROS) in vitro was tested using the change in the optical absorption value of the characteristic peak of 1,3-diphenylisobenzofuran (DPBF). The decrease in the absorption intensity of DPBF at 416 nm was used to determine the yield of reactive oxygen species generated by the phototherapeutic drug. The specific operation steps were as follows: Using toluene as the test reagent, the mixed solution group of DPBF (0.1 μM) and the BODIPY compound was used as the experimental group, the mixed solution group containing DPBF and the reference aza - BODIPY was used as the control group, and the solution group containing only DPBF was used as the blank control group. The mixed solution was irradiated with a laser, and the ultraviolet-visible absorption spectrum of the mixed solution was measured every 10 s to calculate the ability of the sample to generate reactive oxygen species. The results are shown in Figure 8 .

[0059] The results showed that both BODIPY T1 and T2 had very excellent abilities to generate reactive oxygen species, and their singlet oxygen quantum yields were 84% and 96% respectively. The singlet oxygen quantum yield of compound T3 in Comparative Example 1 was only 12%.

[0060] Test Example 2 CCK-8 Method for Cytotoxicity Test

[0061] Hela cells (Jiangsu Kaygen Biotechnology Co., Ltd.) were seeded into a 96-well plate at a density of 1×10 4 cells per well, with 200 μL of culture medium in each well. The edge wells were filled with sterile phosphate buffer solution (PBS). The cells were cultured in an incubator at 37 °C and 5% CO2 for 24 h. The cell morphology was observed under an inverted microscope to ensure good cell growth. After aspirating the culture medium and rinsing with PBS, 20 μL of aqueous solutions of BDP-T2 NPs with different concentration gradients (20, 40, 60, 80, 100 μM) and 180 μL of DMEM medium were added, and the incubation was continued at 37 °C for 24 h. After the incubation, 200 μL of CCK-8 solution (0.5 mg / mL) was added to each well, and the incubation was carried out at 37 °C for 4 h. The CCK-8 solution was aspirated, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well for dissolution, and the mixture was shaken for 10 min. The 96-well plate was placed on a microplate reader, and the absorbance (OD) values of the solutions in each well were read at a detection wavelength of 570 nm and a reference wavelength of 650 nm. Cell viability (%) = (OD value of the sample / OD value of the blank) × 100%. The results are shown in Figure 9 .

[0062] The results showed that after co-culturing BDP-T2 NPs with cells for 24 h, the cell viability was all greater than 80%, indicating that the BDP-T2 NPs provided by the present invention had low cytotoxicity to cells. Further, a phototoxicity test was carried out using the same test method. The results showed that the phototoxicity IC 50 value was 0.06 μM, indicating that BDP-T2 NPs had obvious phototoxicity to U87 tumor cells.

[0063] Test Example 3 EdU Cell Proliferation Assay

[0064] Glioma cell culture: Glioma U87 cells cryopreserved in a cryogenic refrigerator were quickly thawed in a 37 °C water bath. Then, they were transferred to a 10 mL centrifuge tube and centrifuged for 5 min (rotation speed: 1500 rpm). The supernatant was removed, 1 mL of DMEM medium (containing 10% (v:v) fetal bovine serum and 1% (v:v) penicillin-streptomycin) was added, and the mixture was homogenized. 0.5 mL of the mixture was added to a culture dish containing 10 mL of medium. The cells were cultured in a CO2 incubator for 24 h (ambient atmosphere: 37 °C and 5% CO2). The cells were passaged every 2 - 3 days. Glioma U87 cells and GL-261 cells were passaged with trypsin-EDTA digestive solution (0.25%), and cells in the logarithmic growth phase were collected for experiments.

[0065] Glioma cells U87 were seeded in 24-well cell culture plates. After the cells adhered to the wall, they were grouped and treated with the photodynamic therapy BDP-T2 NPs drug. The experimental groups were as follows: control group (dark condition), low-dose group (0.5 μM + light), and high-dose group (1.0 μM + light). The cell proliferation ability was detected using an EdU kit, and the specific operation was carried out according to the instructions provided by the manufacturer. The results were observed and photographed under a fluorescence inverted microscope (100×, Olympus, Tokyo, Japan). The percentage of EdU-positive stained cells (red) among the total cells (blue) represented the cell proliferation rate. The results are shown in Figure 10 .

[0066] The results showed that BODIPY T2 could effectively inhibit the proliferation of glioma cells, and the inhibitory effect was enhanced with the increase in the concentration of the BODIPY photodynamic therapy molecule or the extension of the light irradiation time.

[0067] Although the present invention has been described in detail with general descriptions and specific examples above, some modifications or improvements can be made based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A BODIPY compound, characterized in that, The structural formula of the said compound is shown as formula (I): Wherein, R1 is halogen or p-methoxyphenyl, and R2 is 2,4,6-trimethylphenyl.

2. The BODIPY compound according to claim 1, characterized in that, The structure of the said compound is as follows:

3. A method for preparing the BODIPY compound according to claim 1, characterized in that, Under an inert atmosphere, react the intermediate shown as formula (II), a sulfide and a phase transfer catalyst in an organic solvent. Wherein, the definitions of R1 and R2 are the same as those in claim 1.

4. The method for preparing the BODIPY compound according to claim 3, characterized in that, The said sulfide is selected from one or more of sodium sulfide, sodium sulfide nonahydrate, (bis(tributyltin)sulfide), sulfur powder, potassium sulfide, zinc sulfide; The said phase transfer catalyst is tetrabutylammonium bromide, tetrabutylammonium chloride or tetrabutylammonium fluoride; The molar ratio of the intermediate, the sulfide and the phase transfer catalyst is 1:0.5 - 1:0.1 - 0.

3.

5. The method for preparing the BODIPY compound according to claim 3, characterized in that, The said organic solvent is N,N-dimethylformamide, toluene, tetrahydrofuran or dichloromethane.

6. The method for preparing the BODIPY compound according to claim 3, characterized in that, The temperature of the said reaction is 70 - 90 °C.

7. Use of the BODIPY compound according to claim 1 in the preparation of a phototherapy anti-glioma drug.

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