Carboline-fluoropyrrole photosensitizers, preparation and use in anti-breast cancer

Abstract

The application discloses a kind of karyolin-fluorine boron pyrrole photosensitizer and preparation and application in anti-breast cancer, comprising compound with structure as shown in general formula (I). Experimental results show that the photosensitizer has high light stability, high in-vivo and in-vitro singlet oxygen quantum yield;Cell results show that the photosensitizer has high photodynamic activity (10-40nM) and low dark toxicity (>100 μM) to different sources and different types of breast cancer cells, high selectivity index (selectivity index=dark toxicity IC 50 / phototoxicity IC 50 , range in 270-9400), with good treatment window;The photosensitizer also has lipid droplet and endoplasmic reticulum positioning function, can induce breast cancer cells to occur iron death;Mouse breast cancer model also confirms that the photosensitizer can effectively inhibit the tumor growth of breast cancer tumor-bearing mouse.

Description

Technical Field

[0001] This invention belongs to the field of tumor photodynamic therapy, and relates to carboline-BODIPY photosensitizers, their preparation, and their application in anti-breast cancer treatment. It is a small molecule photosensitizer of the carboline-BODIPY class, its preparation method, and its application in the preparation of anti-breast cancer drugs. Background Technology

[0002] Chemotherapy, radiotherapy, and surgery are routine treatments for cancer, but these traditional therapies are often less effective, have higher recurrence rates, and significant side effects. Newer treatments such as immunotherapy and targeted therapy, which have received widespread attention in recent years, also suffer from high costs, low immune response rates or off-target effects, and drug resistance. As a complementary therapy to traditional treatments, photodynamic therapy (PDT) has gained widespread attention due to its advantages such as high precision, non-invasiveness, controllability, low toxicity, and repeatability. Furthermore, PDT can be combined with various other treatment methods to optimize cancer treatment outcomes.

[0003] The core of photodynamic therapy (PDT) is the photosensitizer. Currently, the first-generation hematoporphyrin-based photosensitizers used clinically are mixed formulations, and their large-scale application is limited due to drawbacks such as poor tissue penetration, low reactive oxygen species (ROS) production efficiency, and lack of tissue specificity. Second-generation photosensitizers, such as porphyrins and phthalocyanines, have solved some of the problems of first-generation hematoporphyrin-based photosensitizers, such as single-component formulations, improved photosensitivity efficiency, and extended absorption wavelengths, but they lack tumor specificity and have complex synthesis routes. Third-generation photosensitizers, based on second-generation photosensitizers, combine with biologically specific substances, such as antibodies, targeting peptides, and nanoparticles, improving the tumor tissue selectivity of PDT. Although third-generation photosensitizers have solved the tumor tissue selectivity problem, the highly reactive oxygen species (ROS) produced by the photosensitizers, such as singlet oxygen... 1 O2 has an extremely short lifespan (10-320 ns), with a lifespan of less than 1 μs within cells, and its effective range is limited to 10-300 nm. The anti-tumor effect produced during local photodynamic therapy is relatively weak. Lipid droplets (LDs), as highly dynamic storage organelles derived from the endoplasmic reticulum, are widely distributed in the cytoplasm and are key hubs of cellular metabolism. Singlet oxygen (… 1 O2-induced damage to lipid droplets (LDs) can lead to excessive release of cytotoxic lipids. This excessive release can directly damage organelle function and induce ferroptosis in tumor cells. Achieving specific distribution of photosensitizers within lipid droplets allows for the in-situ generation of singlet oxygen within the droplets. 1O2) can enhance ferroptosis in tumor cells after photodynamic therapy, thereby amplifying the anti-tumor effect of photodynamic therapy. However, currently marketed photosensitizers lack subcellular organelle localization capabilities. Some studies have attempted to assemble photosensitizers into lipid droplet-targeting nanomaterials; however, issues such as absorption and retention of these lipid droplet-targeting photosensitizers in reticuloendothelial tissues and organs limit their future clinical translation. Therefore, developing small-molecule photosensitizers that do not rely on nanopolymers and whose intrinsic molecular properties allow for direct and specific localization of lipid droplets holds promise for overcoming these challenges. Summary of the Invention

[0004] The purpose of this invention is to provide a carboline-BODIPY type photosensitizer, a small molecule photosensitizer of the carboline-BODIPY type, characterized in that it comprises a structure as shown in general formula (I):

[0005]

[0006] In the compound of general formula (I), R1 is selected from hydrogen and alkyl, wherein alkyl is preferably methyl; R2 is selected from hydrogen and ester, wherein ester is preferably methyl formate.

[0007] Another object of the present invention is to provide a method for preparing the Carboline-BODIPY type small molecule photosensitizer, which is achieved through the following steps:

[0008] a) Using L-tryptophan and 2,2-dimethoxyacetaldehyde as starting materials, a β-Carboline core was constructed in one step via Pictet–Spengler reaction (2);

[0009] b) The β-Carboline core (2) is aromatized by potassium permanganate or N-chlorosuccinimide to give intermediate (3);

[0010] c) Methylation of the carboxyl group or nitrogen atom of intermediate (3) with iodomethane to obtain intermediate (4);

[0011] d) The acetal group of intermediate (4) is hydrolyzed to an aldehyde group in an acetone solution of p-toluenesulfonic acid to obtain intermediate (5).

[0012] e) Intermediate (5) and 2,4-dimethylpyrrole were dissolved in solvent IV, and a catalytic amount of trifluoroacetic acid was added. The reaction was carried out overnight at room temperature under an inert gas (e.g., nitrogen) protection. 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) was added to the resulting reaction system to oxidize dipyrrolemethane to dipyridinemethylene. Then, excess triethylamine and boron trifluoride diethyl ether were added to complex the intermediate (6).

[0013] f) Under the action of elemental iodine and iodic acid, intermediate (6) undergoes iodination to obtain the final products CAR-1, CAR-2, and CAR-3.

[0014] The reaction formula is as follows:

[0015]

[0016] Step a) is as follows:

[0017] L-tryptophan was dissolved in solvent I, and an aqueous solution of 2,2-dimethoxyacetaldehyde and trifluoroacetic acid were added. The reaction was carried out at room temperature for 2 ± 0.5 hours. The molar ratio of L-tryptophan to 2-dimethoxyacetaldehyde was 1:1.2. 10 g of L-tryptophan was mixed with 10 ± 2 mL of trifluoroacetic acid. The reaction product was post-treated to obtain the β-Carboline core (2).

[0018] Step b) is as follows:

[0019] β-Carboline nucleus (2) was dissolved in solvent II, and triethylamine, potassium permanganate, or N-chlorosuccinimide were added. The reaction was carried out at room temperature for 3 ± 0.5 h; the molar ratio of β-Carboline nucleus to triethylamine was 1:2.5, and the molar ratio of β-Carboline nucleus to potassium permanganate / N-chlorosuccinimide was 1:2. The reaction product was post-processed to obtain intermediate (3).

[0020] Step c) is as follows:

[0021] Intermediate (3) was dissolved in solvent III, and NaH aqueous solution was added first, and the mixture was stirred at room temperature for 2 hours. Then, iodomethane was added dropwise at room temperature and reacted at room temperature for 5 hours. The molar ratio of intermediate (3) to NaH was 1:3, and the molar ratio of intermediate (3) to iodomethane was 1:1.5. After post-treatment, intermediate (4) was obtained from the reaction product.

[0022] Step d) is as follows:

[0023] Intermediate (4) was dissolved in solvent IV, p-toluenesulfonic acid was added, and the reaction was carried out at room temperature for 24 h. The reaction product was post-treated to obtain intermediate (5); intermediate (4): p-toluenesulfonic acid = 1:2.5.

[0024] Step e) is as follows:

[0025] Intermediate (5) and 2,4-dimethylpyrrole were dissolved in solvent V, and a catalytic amount of trifluoroacetic acid was added. The reaction was carried out overnight at room temperature under nitrogen protection. The molar ratio of intermediate (5) to 2,4-dimethylpyrrole was 1:2. 25±5μL of trifluoroacetic acid was used for every 2.5 mmol of intermediate (5).

[0026] 2,3-Dichloro-5,6-dicyanobenzoquinone (DDQ) was added to the resulting reaction system and stirred overnight at room temperature; the molar ratio of intermediate (5) to 2,3-dichloro-5,6-dicyanobenzoquinone was 1:1.5. Then, Et3N and BF3.OEt2 were added and stirred at room temperature for 2 hours; the molar ratio of intermediate (5) to Et3N was 1:14, and the molar ratio of intermediate (5) to BF3.OEt2 was 1:16.2. The reaction product was post-processed to obtain intermediate (6).

[0027] Step f) is as follows:

[0028] Intermediate (6) and I2 are dissolved in solvent VI, and an aqueous solution of HIO3 is added. The reaction is carried out at 70±10℃ for 40±10 min. The molar ratio of intermediate (6):I2 is 1:2.5, and the molar ratio of intermediate (6):HIO3 is 1:2. The reaction products are post-treated to obtain the final products CAR-1, CAR-2, and CAR-3.

[0029] Solvent I in step a) is dichloromethane; solvent II in step b) is DMF; solvent III in step c) is DMF; solvent IV in step d) is acetone; solvent V in step e) is dichloromethane; and solvent VI in step f) is ethanol.

[0030] Another object of the present invention is to provide the application of Carboline-BODIPY-type small molecule photosensitizers in the preparation of antitumor drugs, wherein the Carboline-BODIPY-type small molecule photosensitizers can kill tumor cells through photodynamic effects and exert antitumor effects. Preferably, the tumor cells include human triple-negative breast cancer cells MDA-MB-231, ER-positive breast cancer cells MCF-7, human breast cancer cells Bcap-37, and mouse triple-negative breast cancer cells 4T1.

[0031] The Carboline-BODIPY type small molecule photosensitizer described in this invention has lipid droplet and endoplasmic reticulum targeting functions.

[0032] The Carboline-BODIPY type small molecule photosensitizer described in this invention has the function of inducing ferroptosis in tumor cells.

[0033] The present invention has the following beneficial effects: 1. Compared with the first to third generation photosensitizers, the Carboline-BODIPY-type small molecule photosensitizers provided by the present invention are simple to synthesize and have low cost. 2. The Carboline-BODIPY-type small molecule photosensitizers provided by the present invention have good photostability, a large molar absorptivity, and high singlet oxygen generation efficiency. 3. The Carboline-BODIPY-type small molecule photosensitizers provided by the present invention have a broad therapeutic window, that is, these photosensitizers exhibit high photodynamic activity and low cell dark toxicity; they have good photodynamic effects on human MDA-MB-231 and mouse 4T1 triple-negative breast cancer cells, ER-positive MCF-7 breast cancer cells, and Bcap-37 human breast cancer cells. 4. The Carboline-BODIPY-type small molecule photosensitizers provided by the present invention can effectively inhibit tumor growth in breast cancer-bearing mice. 5. The Carboline-BODIPY-type small molecule photosensitizers provided by the present invention have lipid droplet and endoplasmic reticulum targeting functions, without the need to introduce additional positioning groups. 6. The Carboline-BODIPY type small molecule photosensitizer provided by this invention can induce ferroptosis in breast cancer cells.

[0034] In summary, the photosensitizers provided by this invention possess high photostability and high in vitro and in vivo singlet oxygen quantum yield. Cellular results show that these photosensitizers exhibit high photodynamic activity (10–40 nM) and low dark toxicity (>100 μM) against breast cancer cells from different sources and of different types, with a high selectivity index (selectivity index = dark toxicity IC50). 50 / Phototoxicity IC 50 The range is 270-9400, which has a good therapeutic window. This type of photosensitizer also has lipid droplet and endoplasmic reticulum localization functions, which can induce ferroptosis in breast cancer cells, solving the problem of subcellular organelle localization of existing photosensitizers. In mouse breast cancer models, it has also been confirmed that this type of photosensitizer can effectively inhibit tumor growth in breast cancer-bearing mice, and has the potential to be further developed into a new type of anti-tumor photosensitizer. Attached Figure Description

[0035] Figure 1 The images show the UV absorption and fluorescence emission spectra of photosensitizers CAR-1, CAR-2, and CAR-3 (10 μM concentration) in different polar solvents.

[0036] Figure 2 This is the excitation and emission spectrum of ethanol.

[0037] Figure 3 The graph shows the singlet oxygen production rate of photosensitizers CAR-1, CAR-2, and CAR-3 after irradiation for different durations in ethanol solutions of different concentrations.

[0038] Figure 4 The photostability diagrams of photosensitizers CAR-1, CAR-2, and CAR-3 (10 μM concentration) in ethanol solution are shown.

[0039] Figure 5 This is a map showing the colocalization of CAR-1 with lipid droplets in 4T1 and MDA-MB-231 cells.

[0040] Figure 6 This is a map showing the colocalization of CAR-1 in the endoplasmic reticulum of 4T1 and MDA-MB-231 cells.

[0041] Figure 7 The fluorescence enhancement of photosensitizer CAR-1 in DCFH-DA as an intracellular reactive oxygen species indicator;

[0042] Figure 8 Bar chart showing the phototoxicity and dark toxicity of photosensitizers CAR-1, CAR-2, and CAR-3 on 4T1, MDA-MB-231, MCF-7, and Bcap-37 breast cancer cells.

[0043] Figure 9 This is an in vivo photodynamic effect diagram of the photosensitizer CAR-3.

[0044] Figure 10 Heatmaps of CAR-1 mechanism validation experiments and graphs of gene expression changes over time. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0046] Example 1: 1-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4λ) 4 ,λ 5 -Dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborane-10-yl)-9H-pyridine[3,4-b]indole (CAR-1)

[0047]

[0048] The synthesis steps are as follows:

[0049] Step 1: Synthesis of (3S)-1-(dimethoxy)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid

[0050] L-Tryptophan (10 g, 48.97 mmol) was dissolved in anhydrous dichloromethane (60 mL). 2,2-Dimethoxyacetaldehyde (60% aqueous solution, 5 mL, 58.76 mmol, 1.2 eq) and a dichloromethane solution of trifluoroacetic acid (10 mL) were added at room temperature. The reaction was allowed to proceed for 2 h at room temperature. During this time, TLC monitoring showed the reaction was complete. Dichloromethane was removed by rotary evaporation. Water (120 mL) was added to the system, and a 10% NaHCO3 aqueous solution was slowly added with stirring at room temperature to adjust the pH to 7. A large amount of gray solid precipitated from the system. The solid was filtered, washed with water, and dried (at 50 °C to constant weight) to obtain a gray solid (3S)-1-(dimethoxy)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid, yield: 92%. (mp>250 ​​°C) 1 H NMR(500MHz,DMSO-d6)δ10.21(s,1H),7.41(dd,J=12.1,8.0Hz,2H),7.06–7 .01(m,1H),6.95(t,J=7.4Hz,1H),4.46(d,J=6.5Hz,1H),4.29(d,J=6.4Hz,1 H),3.58(dd,J=11.3,4.1Hz,1H),3.47(d,J=4.0Hz,6H),3.42(d,J=12.6Hz, 1H),3.03–2.95(m,1H),2.63(ddd,J=14.0,11.4,2.2Hz,1H).HRMS(ESI):m / z calcd for C 15 H 19 N₂O₄[M+H] + :291.1339,found:291.1348.

[0051] Step 2: Synthesis of 2-(dimethoxymethyl)-9H-pyrido[3,4-b]indole

[0052]

[0053] The (3S)-1-(dimethoxy)-2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole-3-carboxylic acid (2 g, 6.89 mmol) obtained in step 1 was dissolved in 50 mL of anhydrous DMF. A DMF solution of triethylamine (2.4 mL, 17.23 mmol, 2.5 eq) and N-chlorosuccinimide (1.9 g, 14.46 mmol, 2 eq) was added at room temperature, and the reaction was allowed to proceed for 3 h at room temperature. After the reaction was complete, the mixture was poured into ice water, extracted with ethyl acetate, and the organic layer was washed with saturated Na₂CO₃ aqueous solution and saturated brine. The mixture was dried over anhydrous Na₂SO₄, and the ethyl acetate was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1). A red oily substance, 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole, was obtained, with a yield of 70%.

[0054] 1 H NMR(500MHz,Chloroform-d)δ9.19(s,1H),8.44(d,J=5.2Hz,1H),8.13(d,J=7.9Hz,1H),7.95(d,J=5.2 Hz,1H),7.59–7.50(m,2H),7.28(ddd,J=8.0,6.7,1.4Hz,1H),5.75(s,1H),3.52(s,6H).HRMS(ESI):m / z calcd for C 14 H 15 N₂O₂[M+H] + :243.1128,found:243.1120.

[0055] Step 3: Synthesis of 3H-pyrido[3,4-b]indole-1-carboxaldehyde

[0056]

[0057] The 1-(dimethoxymethyl)-9H-pyrido[3,4-b]indole (3.9 g, 15.92 mmol) obtained in step 2 was dissolved in anhydrous acetone. After adding p-toluenesulfonic acid (5.48 g, 31.98 mmol), the reaction proceeded at room temperature for 24 h. TLC was used to monitor the reaction progress. The system was then transferred to ice water, extracted with ethyl acetate, and the organic layer was washed with water and saturated brine. The residue was dried over anhydrous Na₂SO₄, and ethyl acetate was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 9:1) to give a yellow solid 9H-pyrido[3,4-b]indole-1-carboxaldehyde, yield: 60%. mp: 201.8–202.5 °C; 1H NMR(500MHz,Chloroform-d)δ10.34(s,1H),10.10(s,1H),8.63(d,J=4.9Hz,1H),8. 18–8.11(m,2H),7.61(dt,J=16.6,8.1Hz,2H),7.36(t,J=7.4Hz,1H).HRMS(ESI):m / z calcd for C 12 H9N2O[M+H] + :197.0709,found:197.0720.

[0058] Step 4: 1-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ) 4 ,λ 5 Synthesis of -dipyrrole[1,2-c:2',1'-f][1,2,3]diazaborane-10-yl)-9H-pyridine[3,4-b]indole

[0059]

[0060] At room temperature, 9H-pyrido[3,4-b]indole-1-carboxaldehyde (490 mg, 2.5 mmol) and 2,4-dimethyl-1H-pyrrole (514 μL, 5 mmol, 2 eq) obtained in step 3 were dissolved in anhydrous DCM (25 mL), and a catalytic amount of trifluoroacetic acid (25 μL) was added dropwise. The reaction was carried out overnight (approximately 12 hours) under nitrogen protection at room temperature. 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (851 mg, 3.75 mmol, 1.5 eq) was added to the above system at room temperature, and the mixture was stirred overnight at room temperature. Then, Et3N (5 mL, 36 mmol, 14 eq) was added to the system, followed by the slow dropwise addition of BF3·OEt2 (5 mL, 40.5 mmol, 16.2 eq). The mixture was stirred at room temperature for 2 hours. The reaction mixture was poured into water, extracted with dichloromethane, and the organic layer was washed with saturated brine. The mixture was dried over anhydrous Na₂SO₄, and dichloromethane was removed by rotary evaporation. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 9:1) to give a reddish-brown solid – 1-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ). 4 ,λ 5 -Dipyrrole[1,2-c:2',1'-f][1,2,3]diazaborane-10-yl)-9H-pyridine[3,4-b]indole, 15% yield.

[0061] mp:>250℃; 1H NMR(500MHz,Chloroform-d)δ8.84(s,1H),8.62(d,J=5.3Hz,1H),8.18(d,J=7.9Hz,1H),8.07(d,J=5.2Hz,1H) ,7.61–7.53(m,2H),7.34(ddd,J=8.0,6.9,1.2Hz,1H),5.94(s,2H),2.51(s,6H),1.11(s,6H).HRMS(ESI):m / z calcd forC 24 H 22 BF2N4[M+H] + :415.1900,found:415.1906.

[0062] Step 5: 1-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4λ) 4 ,λ 5 Synthesis of -dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborane-10-yl)-9H-pyridine[3,4-b]indole (CAR-1)

[0063]

[0064] The 1-(5,5-difluoro-1,3,7,9-tetramethyl-5H-4λ) obtained in step 4 4 ,λ 5 -Dipyrrole[1,2-c:2',1'-f][1,2,3]diazaborane-10-yl)-9H-pyridin[3,4-b]indole (207 mg, 0.5 mmol, 1.0 eq) and I2 (317 mg, 1.25 mmol, 2.5 eq) were dissolved in 15 mL of ethanol. HIO3 (176 mg, 1 mmol, 2.0 eq) was dissolved in a small amount of water. The aqueous solution of HIO3 was slowly added dropwise to the system. After the addition was complete, the reaction was carried out at 70 °C for 40 min. The reaction of the starting material was monitored by TLC until complete. Saturated Na2S2O3 solution was added to the system, and the mixture was extracted with DCM. The organic phase was washed with saturated brine and dried over anhydrous Na2SO4. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 20:1). A rose-red solid 1-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4λ) was obtained. 4 ,λ 5 -Dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborane-10-yl)-9H-pyridine[3,4-b], yield: 70%. mp>250℃. 1H NMR(500MHz,Chloroform-d)δ8.71(s,1H),8.64(d,J=5.3Hz,1H),8.19(dd,J=8.0,1.0Hz,1H),8.12(d,J=5.2Hz,1H) ,7.60(ddd,J=8.2,7.0,1.2Hz,1H),7.56–7.51(m,1H),7.36(ddd,J=8.0,7.0,1.0Hz,1H),2.60(s,6H),1.12(s,6H). 13 CNMR(126MHz,DMSO-d6)δ157.38,144.72,141.59,139.41,136.38,136.34,134.60,131.59,1 29.66,129.42,122.59,120.80,120.44,116.89,112.69,87.18,16.39,15.62.HRMS(ESI):m / z calcd for C 24 H 19 BF2I2N4[M+H] + :666.9833,found:666.9841

[0065] Example 2: 1-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4λ) 4 ,λ 5 Synthesis of -dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborane-10-yl)-9-methyl-9H-pyridine[3,4-b]indole (CAR-2)

[0066]

[0067] The synthesis was the same as in Example 1, except that tryptophan was replaced with N-methyltryptophan, with a yield of 60%. (mp>250℃) 1 HNMR(500MHz,Chloroform-d)δ8.61(d,J=5.1Hz,1H),8.22(d,J=7.9Hz,1H),8.15(d,J=5.1Hz,1H),7.67(dd d,J=8.4,7.2,1.3Hz,1H),7.46(d,J=8.4Hz,1H),7.41–7.34(m,1H),3.73(s,3H),2.68(s,6H),1.11(s,6H). 13C NMR(126MHz,Chloroform-d)δ158.15,145.23,142.41,139.33,136.0,135.63,135.34,132.15 ,130.39,129.52,121.94,120.67,116.01,109.96,85.99,31.46,16.36,16.11.HRMS(ESI):m / z calcd for C 25 H 21 BF2I2N4[M+H] + :680.9989,found:680.9989.

[0068] Example 3: Methyl 1-(5,5-difluoro-2,8-diiodo-1,3,7,9-tetramethyl-5H-4λ) 4 ,λ 5 Synthesis of methyl 3-dipyrrole[1,2-c:2',1'-f][1,3,2]diazaborane-10-yl)-9-methyl-9H-pyridine[3,4-b]indole-3-carboxylic acid (CAR-3)

[0069] The synthesis was the same as in Example 1, except that tryptophan was replaced with N-methyltryptophan methyl ester, with a yield of 64%. mp:>250; 1 H NMR(500MHz,DMSO-d6)δ9.22(s,1H),8.59(d,J=7.9Hz,1H),7.82–7.69(m,2H),7.4 4(ddd,J=7.9,5.4,2.6Hz,1H),3.91(s,3H),3.73(s,3H),2.61(s,6H),0.99(s,6H). 13 C NMR(126MHz,Chloroform-d)δ158.40,145.13,142.78,135.61,135.19,132.23,130.46 ,129.98,122.13,121.69,120.98,118.90,110.39,86.10,53.19,31.69,16.43,16.36.

[0070] HRMS(ESI): m / z calcd for C 27 H 24 BF₂I₂N₄O₂[M+H] + :739.0044,found:739.0043.

[0071] Example 4: Effect of solvent polarity on the UV absorption and fluorescence emission spectra of photosensitizers

[0072] The synthesized photosensitizers CAR-1, CAR-2, and CAR-3 (test concentration 10 μM) were subjected to UV and fluorescence spectra analysis in different polar solvents. The polar solvents were toluene, tetrahydrofuran, ethanol, acetone, acetonitrile, and N,N-dimethylformamide, respectively.

[0073] UV spectrum as shown Figure 1 As shown, solvents of different polarities have no significant effect on the maximum absorption of the photosensitizer, and the maximum absorption range is 540–550 nm.

[0074] Fluorescence spectra such as Figure 1 As shown, fluorescence emission gradually weakens with increasing solvent polarity, and the photosensitizer exhibits the lowest fluorescence emission in DMF; the maximum fluorescence emission range in different solvents is 550–565 nm.

[0075] Figure 2 The images show the excitation and emission spectra of photosensitizers in ethanol solution. It is evident that the Stokes shifts of photosensitizers CAR-1, CAR-2, and CAR-3 can reach 22-24 nm.

[0076] Example 5: In vitro singlet oxygen generation capacity of photosensitizers CAR-1, CAR-2, and CAR-3

[0077] In vitro singlet oxygen detection was performed on the photosensitizers CAR-1, CAR-2, and CAR-3 synthesized in the examples. DPBF (1,3-diphenylisobenzofuran) was used as the singlet oxygen detection reagent. The singlet oxygen rate of CAR-1, CAR-2, and CAR-3 after irradiation for different durations at four different concentrations (7.5 nM, 37.5 nM, 75 nM, and 375 nM) was measured according to... Figure 3 It can be seen that as the concentrations of photosensitizers CAR-1, CAR-2, and CAR-3 increase, the rate of singlet oxygen production accelerates; and as the illumination time prolongs, the amount of singlet oxygen produced increases.

[0078] Example 6: In vitro photostability of photosensitizer in ethanol solution

[0079] The photostability of photosensitizers CAR-1, CAR-2, and CAR-3 (10 μM concentration) was tested. White light was used to irradiate the solutions, and the maximum absorbance of the photosensitizers was measured at different time points. The results are as follows: Figure 4 As shown, the maximum absorption of the compounds did not change significantly within 15 minutes of illumination, indicating that CAR-1, CAR-2, and CAR-3 have good photostability and are not prone to photobleaching.

[0080] Example 7: MTT assay for photodynamic activity and dark toxicity of CAR-1, CAR-2, and CAR-3

[0081] The photosensitizers CAR-1, CAR-2, and CAR-3 synthesized in the above examples were subjected to photodynamic activity and dark toxicity tests on breast cancer cells. Breast cancer cell lines 4T1, MDA-MB-231, MCF-7, and BCAP-37 were selected. Breast cancer cells in logarithmic growth phase were seeded in 96-well plates at 5000 cells per well and cultured at 37°C in 95% humidification and 5% CO2 for 24 h. After cell attachment, the culture medium was aspirated, the cells were washed twice with PBS, and then culture medium containing different concentrations of photosensitizers was added and incubated for 24 h.

[0082] The photodynamic group placed the 96-well plate under white light (20 mW / cm²). 2 Irradiate for 10 minutes and continue incubation under the same conditions for 24 hours.

[0083] The dark poison group was incubated for 24 hours under the same conditions described above. That is, the dark poison group was strictly protected from light throughout the process.

[0084] Then, add 20 μL of MTT solution (5 mg / mL) to the 96-well plate and incubate for another 4 hours. Remove all the culture medium from the 96-well plate, wash twice with PBS, add 150 μL of DMSO to dissolve the formazan, and read the absorbance of each well using a microplate reader to calculate the IC50. 50 Values. The results are shown in Tables 1 and 2 below.

[0085] Table 1 shows the photodynamic activity (IC50) of the photosensitizers CAR-1, CAR-2, and CAR-3 disclosed in Examples 1-3 on breast cancer cell lines. 50 (nM)

[0086] compound 4T1 MDA-MB-231 MCF-7 BCAP-37 CAR-1 23.35±0.6 36.4±2.6 41.2±2.5 25.8±2.6 CAR-2 23.38±3.4 16.25±0.6 18.19±18 14.07±0.28 CAR-3 22.88±3.5 11.65±0.3 11.65±1.69 10.62±0.58

[0087] Table 2 shows the dark toxicity (CC) of the photosensitizers CAR-1, CAR-2, and CAR-3 disclosed in Examples 1-3 to breast cancer cell lines. 50 (μM)

[0088] compound 4T1 MDA-MB-231 MCF-7 BCAP-37 CAR-1 >10 >10 >100 >100 CAR-2 >100 >100 >100 >100 CAR-3 >100 >100 >100 >100

[0089] The results showed that the photosensitizers CAR-1, CAR-2, and CAR-3 all had good photodynamic effects on breast cancer cells from different sources and of different types, and had low dark toxicity.

[0090] Example 8. Organelle Colocalization Experiment

[0091] Organelle colocalization experiments were performed on the photosensitizer CAR-1. Commercial probes BODIPY493 / 503 were selected as lipid droplet colocalization probes, and commercial probe ER tracker Red was selected as endoplasmic reticulum colocalization probes. CAR-1 (concentration 2 μM) was co-incubated with 4T1 and MDA-MB-231 breast cancer cells for 12 h, and then the two probes were added and incubated for another 20-30 min before colocalization detection was performed.

[0092] The results are as follows Figure 5 and 6 As shown, analysis revealed that the photosensitizer CAR-1 exhibited excellent lipid droplet localization capabilities in both types of breast cancer cells, with a Pearson coefficient as high as 0.92. Furthermore, the photosensitizer CAR-1 also showed a certain localization effect on the endoplasmic reticulum, with a Pearson coefficient ranging from 0.7 to 0.8.

[0093] Example 9: Intracellular Reactive Oxygen Species Generation Experiment

[0094] This study investigated the generation of reactive oxygen species (ROS) in MDA-MB-231 cells using the photosensitizer CAR-1. The commercially available probe DCFH-DA was selected as the intracellular ROS detection probe. Different concentrations of CAR-1 (5 nM, 30 nM, and 60 nM) were co-incubated with MDA-MB-231 for 12 h. The 60 nM group was further incubated with sodium azide for 4 h. After incubation, the cells were washed twice with PBS, and DCFH-DA was added for an additional 20 min of incubation. Fluorescence was detected under confocal microscopy after 5 min of white light irradiation. The results are shown below. Figure 7 As shown, with the increase of CAR-1 concentration, the amount of ROS generated gradually increases, which is manifested by enhanced green fluorescence. The group with added sodium azide can quench reactive oxygen species, which is manifested by significantly lower green fluorescence than the group without added sodium azide at the same concentration.

[0095] Example 10: Photodynamic therapy of Carboline-BODIPY small molecule photosensitizers in breast cancer-bearing mice.

[0096] The photosensitizer CAR-3 was selected for in vivo photodynamic antitumor activity evaluation. 5*103 cells were subcutaneously implanted in mice. 5 4T1 breast cancer cells were collected, and the tumor volume was increased to 50 mm. 3 Approximately 30 minutes after administration, mice were randomly divided into three groups. Groups one and two were administered the photosensitizer CAR-3 0.6 mg / kg, while group three was administered an equal volume of PBS solution. Approximately 30 minutes after administration, a green LED light (80 mW / cm²) was used. 2Mice in the first and third groups were irradiated for 15 minutes. Mice in the second group were placed in the dark after administration. They were administered the drug and exposed to light on days one and three, and tumor volume and mouse weight changes were measured every other day. The tumor volume in the experimental group mice was 600 mmHg. 3 Mice were euthanized approximately 15 days later, and the tumors were dissected and weighed. The results were as follows: Figure 9 As shown, CAR-3 exhibits good in vivo antitumor activity.

[0097] Example 10: Mechanism study of Carboline-BODIPY type small molecule photosensitizers.

[0098] Single-cell sequencing (RNA-seq) of the photosensitizer CAR-1 was used to study the gene expression of 4T1 and MDA-MB-231 cells after photodynamic therapy. The results are as follows: Figure 10 As shown in the figure. Analysis of the experimental results revealed that the expression of HAC1 and SLC7A11 genes was upregulated and the expression of RGS4 gene was downregulated in photodynamic therapy cells. Combined with the expression levels of other genes in the heatmap, it can be concluded that the highly expressed signaling pathways in the cells are associated with endoplasmic reticulum and ferroptosis.

[0099] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A carbline-fluoroboron pyrrole photosensitizer, characterized in that, The structural formula is as follows: 。 2. The method for preparing the photosensitizer according to claim 1, characterized in that, This can be achieved through the following steps: a) Using L-tryptophan and 2,2-dimethoxyacetaldehyde as starting materials, β-Carboline core was constructed in one step via Pictet–Spengler reaction (2). b) The β-Carboline core (2) is aromatized by potassium permanganate or N-chlorosuccinimide to obtain the intermediate (3). c) Methylation of the carboxyl group or nitrogen atom of intermediate (3) with iodomethane to obtain intermediate (4); d) The acetal group of intermediate (4) is hydrolyzed into an aldehyde group in an acetone solution of p-toluenesulfonic acid to obtain intermediate (5); e) Intermediate (5) and 2,4-dimethylpyrrole were dissolved in solvent IV, and a catalytic amount of trifluoroacetic acid was added. The reaction was carried out overnight at room temperature under inert gas protection. 2,3-dichloro-5,6-dicyanobenzoquinone was added to the resulting reaction system, followed by the addition of excess triethylamine and boron trifluoride diethyl ether to obtain intermediate (6). f) Under the action of elemental iodine and iodic acid, intermediate (6) undergoes iodination to obtain the final products CAR-1, CAR-2, and CAR-3; The reaction formula is as follows: ； Wherein: R1 is hydrogen or methyl formate; R2 is methyl or hydrogen.

3. The preparation method according to claim 2, characterized in that, Step a): L-tryptophan was dissolved in solvent I, an aqueous solution of 2,2-dimethoxyacetaldehyde was added, and trifluoroacetic acid was added. The reaction was carried out at room temperature for 2 ± 0.5 hours, wherein the molar ratio of L-tryptophan to 2-dimethoxyacetaldehyde was 1:1.2; 10 ± 2 mL of trifluoroacetic acid was used for every 10 g of L-tryptophan; the reaction product was post-treated to obtain the β-Carboline core (2). Step b): The β-Carboline core (2) is dissolved in solvent II, triethylamine is added, and potassium permanganate or N-chlorosuccinimide is added, and the reaction is carried out at room temperature for 3 ± 0.5 h; wherein the molar ratio of β-Carboline core to triethylamine is 1:2.5, and the molar ratio of β-Carboline core to potassium permanganate / N-chlorosuccinimide is 1:2; the reaction product is post-treated to obtain intermediate (3). Step c): Intermediate (3) is dissolved in solvent III, NaH aqueous solution is added first, and the mixture is stirred at room temperature for 2 h; then iodomethane is added dropwise at room temperature and reacted at room temperature for 5 h; wherein the molar ratio of intermediate (3):NaH is 1:3; the molar ratio of intermediate (3):iodomethane is 1:1.5; the reaction product is post-processed to obtain intermediate (4). Step d): Intermediate (4) is dissolved in solvent IV, p-toluenesulfonic acid is added, and the reaction is carried out at room temperature for 24 h; the reaction product is post-treated to obtain intermediate (5); wherein intermediate (4): p-toluenesulfonic acid = 1: 2.5; Step e): Intermediate (5) and 2,4-dimethylpyrrole are dissolved in solvent V, and a catalytic amount of trifluoroacetic acid is added. The reaction is carried out overnight at room temperature under nitrogen protection, wherein the molar ratio of intermediate (5):2,4-dimethylpyrrole is 1:2, and 25±5 μL of trifluoroacetic acid is used for every 2.5 mmol of intermediate (5); 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) is added to the resulting reaction system and stirred overnight at room temperature; the molar ratio of intermediate (5):2,3-dichloro-5,6-dicyanobenzoquinone is 1:1.5; then, Et3N and BF3.OEt2 are added and stirred at room temperature for 2h; the molar ratio of intermediate (5):Et3N is 1:14, and the molar ratio of intermediate (5):BF3.OEt2 is 1:16.2; the reaction product is post-processed to obtain intermediate (6). In step f): intermediate (6) and I2 are dissolved in solvent VI, and an aqueous solution of HIO3 is added. The reaction is carried out at 70±10 °C for 40±10 min. The molar ratio of intermediate (6):I2 is 1:2.5, and the molar ratio of intermediate (6):HIO3 is 1:

2. The reaction products are post-treated to obtain the final products CAR-1, CAR-2, and CAR-3. Solvent I in step a) is dichloromethane; solvent II in step b) is DMF; solvent III in step c) is DMF; solvent IV in step d) is acetone; solvent V in step e) is dichloromethane; and solvent VI in step f) is ethanol.

4. The application of the carboline-fluoroboron pyrrole photosensitizer according to claim 1 in the preparation of antitumor drugs, characterized in that, The carboline-fluoroboron pyrrole photosensitizer kills tumor cells via photodynamic therapy. The tumor cells include human triple-negative breast cancer cells MDA-MB-231, ER-positive breast cancer cells MCF-7, human breast cancer cells Bcap-37, and mouse triple-negative breast cancer cells 4T1.

5. The application according to claim 4, characterized in that, The carboline-fluoroboron pyrrole photosensitizer has the function of targeting lipid droplets and endoplasmic reticulum, and can induce ferroptosis in breast cancer cells.

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