A small molecule pyroptosis inducer and its application as a tumor sonodynamic therapeutic agent

Abstract

This invention discloses a small-molecule pyroptosis inducer for tumor cells. It utilizes an N,N-diethylimine group as an electron acceptor (A), a rhodamine scaffold as a π-conjugated system, thiophene and triphenylamine groups as the first electron donor (D1) and second electron donor (D2), respectively, and a rotatable vinyl and C-C single bond to increase intramolecular motion, forming a novel A-π-D1-D2 type small-molecule pyroptosis inducer, Rd-TTPA, with enhanced D-A structure and intramolecular motion. This small-molecule pyroptosis inducer can efficiently target tumor cell mitochondria, inducing pyroptosis through ultrasound-mediated reactive oxygen species production. It can serve as an enhanced sonodynamic deep tumor therapeutic agent guided by near-infrared II (NIR-II) fluorescence imaging.

Description

Technical Field

[0001] This invention belongs to the field of biochemical engineering, specifically relating to the application of a small molecule pyroptosis inducer and its enhanced sonodynamic therapy reagent for tumors. Background Technology

[0002] Immunotherapy has become a major clinical treatment for patients with intermediate and advanced cancer. However, the low response rate of immunotherapy due to tumor antigen deficiency, low efficiency of adaptive immune response initiation, and lack of inflammatory cell infiltration remains a significant challenge in clinical treatment. Pyroptosis is a novel form of programmed necrosis mediated by gasdermin. Unlike apoptosis and necrosis, pyroptotic cells produce a large number of inflammatory factors and release tumor antigens, activating antigen-presenting cells and initiating adaptive immune responses. Therefore, developing effective strategies to induce pyroptosis is of paramount importance for enhancing tumor immunotherapy.

[0003] Currently, effective methods for inducing pyroptosis mainly rely on chemotherapy drugs, such as doxorubicin, paclitaxel, and topotecan. However, chemotherapy drugs can lead to tumor resistance and cause serious toxic side effects such as bone marrow suppression, neurotoxicity, and cardiovascular toxicity, which limits their widespread use in cancer treatment. Furthermore, poor penetration into deep tissues (mm) and the inherent phototoxicity of photosensitizers also hinder the killing efficiency of photodynamic therapy against deep tumors and can cause adverse side effects on normal tissues.

[0004] In recent years, some pyroptosis inducers have been developed to induce pyroptosis in tumor cells. However, these pyroptosis inducers often lack tumor cell targeting ability and cannot be monitored for retention at the tumor site or distribution in the body. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a small molecule pyroptosis inducer that can efficiently target the mitochondria of tumor cells and induce pyroptosis through ultrasound-mediated generation of reactive oxygen species. It can be used as an enhanced sonodynamic deep tumor therapeutic agent guided by near-infrared II (NIR-II) fluorescence imaging.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is as follows:

[0007] A small molecule pyroptosis inducer (Rd-TTPA) has the chemical structural formula shown in Formula 1:

[0008]

[0009] This invention provides a method for preparing the above-mentioned small molecule pyroptosis inducer (Rd-TTPA), the main steps of which are as follows:

[0010] 4) Under ice-water bath conditions, freshly distilled cyclohexanone was added dropwise to 95%-98% concentrated sulfuric acid. Under stirring conditions, 4-diethylaminoketo acid was added, and the reaction was carried out at 80-100℃ for 1-2 hours. After the reaction was completed, the mixture was poured into ice and 70%-72% perchloric acid solution was added to precipitate the product, yielding compound 1.

[0011] 5) Add 4-bromotriphenylamine and tetra(triphenylphosphine)palladium (Pd(PPh3)4) to dry toluene, then add it to a methanol solution of 5-formylthiophene-2-boric acid and potassium carbonate (i.e., 5-formylthiophene-2-boric acid and potassium carbonate are dissolved in methanol). React under reflux at 110-120℃ for 20-30 h. After extraction with water and dichloromethane (CH2Cl2), compound 2 is obtained by column chromatography.

[0012] 6) Compound 1 and Compound 2 were dissolved in acetic acid and reacted under reflux at 110-120℃ for 20-30 h. The crude product was obtained by extraction with water and dichloromethane (CH2Cl2), and then purified by column chromatography, rotary evaporation and vacuum drying to obtain the target product, namely the small molecule pyroptosis inducer Rd-TTPA.

[0013] The chemical structural formulas of compounds 1, 2, and the small molecule pyroptosis inducer Rd-TTPA are shown below:

[0014]

[0015] According to the above scheme, in step 1), the ratio between the amount of cyclohexanone and the volume of concentrated sulfuric acid is 12 mmol:(10-20) mL; the ratio between the amount of 4-diethylaminoketo acid and cyclohexanone is 1:(1.5-2.5); and the ratio between the volume of perchloric acid solution and the amount of cyclohexanone is (1.5-2.5) mL:12 mmol.

[0016] According to the above scheme, in step 2), the molar ratio of 4-bromotriphenylamine to Pd(PPh3)4 is (5-15):1, and the ratio of the amount of 4-bromotriphenylamine to the volume of toluene is 1 mmol: (5-15) mL; the molar ratio of 5-formylthiophene-2-boronic acid to 4-bromotriphenylamine is (1.5-2.5):1, the molar ratio of K2CO3 to 4-bromotriphenylamine is (4.5-5.5):1, and the ratio of the volume of methanol solvent to the amount of 4-bromotriphenylamine is (10-20) mL: 1 mmol; the column chromatography purification elution agent is a mixed solvent composed of petroleum ether (PE) and ethyl acetate (EA) in a volume ratio of (10-20):1.

[0017] According to the above scheme, in step 3), the molar ratio of compound 1 to compound 2 is (1.0-2.0):1, the molar ratio of acetic acid to compound 1 is 0.3 mmol:(5-15) mL, and the concentration of acetic acid is 99%; the column chromatography purification rinsing agent is a mixed solvent composed of dichloromethane (CH2Cl2) and methanol in a volume ratio of 200:1 to 100:1.

[0018] The synthetic route for the aforementioned small molecule pyroptosis inducer (Rd-TTPA) is shown below:

[0019]

[0020] The aforementioned small molecule pyroptosis inducer Rd-TTPA is an ultrasound-mediated tumor cell pyroptosis inducer. It can be used as an in vitro pyroptosis inducer, as an in vivo bioimaging agent, or as an in vivo tumor inhibitor. The application conditions are: low-frequency ultrasound irradiation (1.0-3.0 MHz).

[0021] The technical concept of this invention is as follows:

[0022] This invention uses an N,N-diethylimine group as an electron acceptor (A), a rhodamine scaffold as a π-conjugated system, a thiophene group and a triphenylamine group as the first electron donor (D1) and the second electron donor (D2), respectively, and a rotatable vinyl and C-C single bond to increase intramolecular motion, forming a novel A-π-D1-D2 type small molecule pyroptosis inducer Rd-TTPA with enhanced DA structure and intramolecular motion.

[0023] The small molecule pyroptosis inducer Rd-TTPA of this invention actively targets the mitochondria of tumor cells by utilizing its positive charge property, effectively increasing its accumulation at the tumor site and reducing toxic side effects on normal tissues. Furthermore, it can induce pyroptosis by efficiently generating a large amount of reactive oxygen species (ROS) in situ under ultrasound-mediated (low-frequency ultrasound irradiation), thereby achieving efficient tumor inhibition and ensuring its biocompatibility in vivo. Therefore, it can be used as an in vitro pyroptosis inducer, as well as as an in vivo bioimaging reagent or an in vivo biological tumor inhibitor.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The small molecule pyroptosis inducer Rd-TTPA constructed in this invention is a novel A-π-D1-D2 small molecule with an enhanced DA structure and intramolecular motion. This novel D-π-A structure is beneficial for the generation of reactive oxygen species in Rd-TTPA under ultrasound irradiation. Furthermore, Rd-TTPA with its enhanced D-π-A structure also exhibits near-infrared II emission properties and a large Stokes shift, which is advantageous for its imaging and monitoring in vivo, enabling enhanced sonodynamic therapy of deep tumors guided by near-infrared II (NIR-II) fluorescence imaging.

[0026] 2. The small molecule pyroptosis inducer Rd-TTPA constructed in this invention has a simple synthesis process, low synthesis cost, and high synthesis yield, and has high practical application value and economic benefits.

[0027] 3. The small molecule pyroptosis inducer constructed in this invention utilizes low-intensity ultrasound instead of light as an excitation source to activate and generate reactive oxygen species (ROS). It can efficiently target the mitochondria of tumor cells and induce pyroptosis through ultrasound-mediated generation of ROS. This achieves the effect of pyroptosis-enhanced sonodynamic therapy to damage tumor tissue, thereby inhibiting tumor tissue. It also has low toxicity to normal tissues and good biocompatibility.

[0028] 4. The small molecule pyroptosis inducer constructed in this invention is achieved through sonodynamic therapy. Sonodynamic therapy is non-invasive and has specific spatiotemporal selectivity. It can also penetrate deep tissues (10-20cm) and ablate deep tumor tissues through ultrasound excitation. It has great potential in the clinical translation of tumor treatment. Attached Figure Description

[0029] Figure 1 The UV absorption and fluorescence emission spectra of the small molecule pyroptosis inducer Rd-TTPA in Example 1 are shown.

[0030] Figure 2 The image shows the fluorescence changes of DCFH in aqueous solution under different treatment conditions in Example 1. The Control group is the blank group, the Rd-TTPA group is the Rd-TTPA control group, the US group is the ultrasound irradiation control group, and the Rd-TTPA+US group is the Rd-TTPA treatment group under ultrasound irradiation.

[0031] Figure 3 (a) is a graph showing the fluorescence changes of DCFH-DA in cells under different treatment conditions in Example 1; 3(b) is a statistical graph showing the fluorescence changes of DCFH-DA in cells under different treatment conditions in Example 1.

[0032] Figure 4 This is a laser confocal fluorescence image of Rd-TTPA-induced pyroptosis in Example 1.

[0033] Figure 5 (a) is a cytotoxicity experiment diagram of Rd-TTPA in Example 1; 5(b) is a live-dead cell staining experiment diagram of Rd-TTPA in Example 1.

[0034] Figure 6 (a) is a near-infrared II fluorescence imaging image of Rd-TTPA in vivo in Example 1; 6(b) is a statistical graph of fluorescence intensity changes of Rd-TTPA in near-infrared II imaging in vivo.

[0035] Figure 7 (a) is a graph showing the changes in tumor volume of 4T1 subcutaneous tumors under different treatment conditions in Example 1; Figure 7 (b) is a graph showing the changes in body weight of tumor-bearing mice under different treatment conditions in Example 1; Figure 7 (c) shows HE staining and Ki67 staining of tumor tissue from the 4T1 subcutaneous tumor in Example 1 under different treatment conditions.

[0036] Figure 8 The images show the 1H NMR spectrum of compounds 1 and 2, the final product Rd-TTPA, and the mass spectrum of the final product Rd-TTPA in Example 1. Detailed Implementation

[0037] To better understand the present invention, the following embodiments further illustrate the content of the present invention, but the present invention is not limited to the following embodiments.

[0038] In the following examples, the tumor cells used were murine breast cancer cells (4T1 cells).

[0039] Example 1

[0040] The structure and synthetic route of a small molecule pyroptosis inducer Rd-TTPA are shown below:

[0041]

[0042] The specific steps for preparing the aforementioned small molecule pyroptosis inducer Rd-TTPA are as follows:

[0043] (1) Synthesis of Compound 1: Freshly distilled cyclohexanone (1.18 mL, 12 mmol) was added to 98% concentrated H2SO4 (15 mL) at 0 °C. 4-Diethylaminoketo acid (1.88 g, 6.0 mmol) was added under stirring. After reacting at 90 °C for 1.5 h, the mixture was poured into ice (150 g), and then perchloric acid (70%, 2.0 mL) was added to precipitate the product. Finally, the precipitate was filtered, washed with cold water, and vacuum dried to obtain red, flaky compound 1.

[0044] (2) Synthesis of Compound 2: 4-Bromotriphenylamine (324 mg, 1.0 mmol) and Pd(PPh3)4 (115 mg, 0.1 mmol) were added to 10 mL of dry toluene. The reaction mixture was then added to a methanol solution (15 mL) of 5-formylthiophene-2-boronic acid (312 mg, 2.0 mmol) and K2CO3 (0.79 g, 5.0 mmol), and the mixture was refluxed at 110 °C for 24 h. After extraction with water and CH2Cl2, the crude product was purified by column chromatography (PE / EA, v:v = 16:1) to obtain compound 2 as a yellow powder.

[0045] (3) Synthesis of Rd-TTPA: Compound 1 (112.8 mg, 0.3 mmol) and compound 2 (71 mg, 0.2 mmol) were dissolved in 10.0 mL of 99% acetic acid and reacted under reflux at 110 °C overnight (about 24 h). After extraction with water and CH2Cl2, the crude product was purified by column chromatography (CH2Cl2 / MeOH, v:v = 200 / 1 to 100 / 1) to obtain blue powder compound Rd-TTPA.

[0046] 1. Compound 1 and Compound 2 obtained in steps (1) and (2) respectively, as well as the final product Rd-TTPA, were subjected to 1H NMR spectroscopy (e.g., Figure 8 As shown in the image, the test results are as follows:

[0047] NMR data for compound 1: 1 H NMR (400MHz, DMSO-d6) δ12.57(s,1H),8.20(dd,J=7.9,1.3Hz,1H),7.87(td,J=7. 5,1.4Hz,1H),7.77(td,J=7.7,1.3Hz,1H),7.36–7.27(m,2H),7.19(d,J=6.0Hz,1 H),7.02(d,J=9.5Hz,1H),3.66(q,J=7.2Hz,4H),3.18–3.04(m,2H),2.14(dt,J=1 2.1,5.9Hz,2H),1.87(p,J=6.4Hz,2H),1.74–1.64(m,2H),1.20(t,J=7.0Hz,6H).

[0048] NMR data for compound 2: 1H NMR (400MHz, DMSO-d6) δ9.87(s,1H),8.00(d,J=4.0Hz,1H),7.70(s,1H),7.68(s,1H),7.61(d,J=4 .0Hz,1H),7.38(s,1H),7.36(d,J=1.1Hz,2H),7.34(s,1H),7.14–7.08(m,6H),6.98–6.94(m,2H).

[0049] NMR data of the final product Rd-TTPA: 1 H NMR (400MHz, Chloroform-d) δ8.47 (s, 1H), 8.28 (dd, J=7.9, 1.3Hz, 1H), 7.83 (d, J= 4.0Hz,1H),7.73(td,J=7.5,1.3Hz,1H),7.64(td,J=7.7,1.2Hz,1H),7.53–7.44(m ,2H),7.36–7.27(m,5H),7.22–6.99(m,10H),6.95–6.83(m,2H),3.61(q,J=7.4Hz, 4H),2.87(d,J=6.9Hz,2H),2.45–2.21(m,2H),1.85(s,2H),1.27(d,J=9.0Hz,6H).

[0050] In addition, the small molecule pyroptosis inducer Rd-TTPA prepared in this embodiment was also subjected to mass spectrometry analysis. The test results are as follows: ESI-MS: for C 47 H 41 N2O3S + :expected m / z = 713.28324 [M] + ;found

[0051] m / z = 713.28353 [M] + .

[0052] Based on the above NMR and mass spectrometry test results, it can be determined that the target compound Rd-TTPA prepared in this embodiment is consistent with the structure shown in Formula 1.

[0053] 2. Determination of absorption and fluorescence spectra of the small molecule pyroptosis inducer Rd-TTPA

[0054] (1) Absorption spectroscopy determination of the small molecule pyroptosis inducer Rd-TTPA: The absorption spectrum of Rd-TTPA was determined in CH2Cl2 solvent. For example... Figure 1 As shown, Rd-TTPA exhibits a strongest absorption peak at 670 nm.

[0055] (2) Fluorescence spectroscopy determination of the small molecule pyroptosis inducer Rd-TTPA: The fluorescence spectrum of Rd-TTPA was determined in CH2Cl2 solvent. For example... Figure 1 As shown, Rd-TTPA exhibits a peak emission at 995 nm.

[0056] 3. Determination of ROS generation in solution by the small molecule pyroptosis inducer Rd-TTPA

[0057] The ROS generation capacity of Rd-TTPA in PBS solution under ultrasonic irradiation was determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein (DCFH). Rd-TTPA was added to 1 mL of DCFH in PBS solution, and the mixture was sonicated (3.0 MHz, 1.0 mW / cm²). 2 Irradiation for 0-10 minutes resulted in a final concentration of 10 μM for both DCFH and Rd-TTPA. Figure 2 As shown, under ultrasonic irradiation, in the presence of 10 μM Rd-TTPA, the fluorescence intensity of 10 μM DCFH solution at 530 nm increased with increasing ultrasonic irradiation time. Within 10 min of ultrasonic irradiation, the fluorescence intensity of DCFH increased by 6.8 times, proving that Rd-TTPA is a good acoustic sensitizer.

[0058] 4. Determination of ROS production in cells by the pyroptosis inducer Rd-TTPA

[0059] The ROS production capacity of Rd-TTPA in 4T1 cells (mouse breast cancer cells) under ultrasound irradiation was determined using the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). 4T1 cells were irradiated at a concentration of 1 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density in 35 mm glass dishes and divided into: (1) blank group; (2) Rd-TTPA control group; (3) ultrasound irradiation control group (US); and (4) Rd-TTPA+US group. After the cells adhered overnight, the culture medium was discarded. 1 mL of fresh culture medium was added to groups (1) and (3), and 1 mL of fresh culture medium containing Rd-TTPA was added to groups (2) and (4). The final concentration of Rd-TTPA was 10 μM. The cells were incubated for 30 min. Then, groups (3) and (4) were subjected to ultrasound (3.0 MHz, 1.0 W / cm²). 2 Irradiate for 10 minutes. Then, all four groups are washed with PBS 2-3 times, incubated with DCFH-DA for 30 minutes (final concentration of DCFH-DA: 5 μM), and washed with PBS 3 times. Figure 3As shown, under ultrasound irradiation, 4T1 cells exhibited obvious green fluorescence in the presence of 10 μM Rd-TTPA, while other control and blank groups showed no obvious green fluorescence, proving that Rd-TTPA is a good sonosensitive agent.

[0060] 5. The small molecule pyroptosis inducer Rd-TTPA induces pyroptosis.

[0061] To investigate Rd-TTPA-induced pyroptosis, this invention utilized a cell membrane green fluorescence kit (DIO) to observe the morphological changes of Rd-TTPA-treated 4T1 cells under ultrasound irradiation using a laser confocal microscope. 4T1 cells were stained with 1×10⁻⁶ cells... 5 Cells were seeded at a density in 35 mm glass dishes and divided into: (1) blank group; (2) Rd-TTPA control group; (3) ultrasound irradiation control group (US); and (4) Rd-TTPA+US group. After the cells adhered overnight, the culture medium was discarded. 1 mL of fresh culture medium was added to groups (1) and (3), and 1 mL of fresh culture medium containing Rd-TTPA was added to groups (2) and (4), respectively. The final concentration of Rd-TTPA was 10 μM. The cells were incubated for 30 min. Then, groups (3) and (4) were subjected to ultrasound (3.0 MHz, 1.0 W / cm²). 2 Irradiate for 10 min. Then, all four groups are washed 2-3 times with PBS, and Hoechst 33342 and DIO are added. The final concentration of DIO is 1 μM, and the final concentration of Hoechst 33342 is 5 μM. Incubate for 10 min, then wash 3 times with PBS. Figure 4 As shown, under ultrasound irradiation, in the presence of 10 μM Rd-TTPA, 4T1 cells exhibited vesicular protrusions (showing green fluorescence), while the cell nuclei showed no obvious nuclear condensation or nuclear rupture, which proves that Rd-TTPA can induce pyroptosis.

[0062] 6. In vitro antitumor activity and cytotoxicity experiments of the small molecule pyroptosis inducer Rd-TTPA

[0063] (1) The in vitro antitumor effect of Rd-TTPA was evaluated using the MTT cytotoxicity assay kit. 4T1 cells were inoculated at 5 × 10⁻⁶ cells per cell line. 3Cells were seeded at a density in 96-well plates and divided into: (1) blank group; (2) Rd-TTPA control group; (3) ultrasound irradiation control group (US); (4) Rd-TTPA+US group; each group was further divided into 7 concentration groups, and each concentration group had 5 replicates. After the cells adhered overnight, 100 μL of fresh culture medium was added to groups (1) and (3), and 100 μL of fresh culture medium containing different concentrations (0 μM, 5 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM) of Rd-TTPA was added to groups (2) and (4), respectively, and incubated for 12 h. Then, groups (3) and (4) were subjected to ultrasound (3.0 MHz, 1.0 W / cm²). 2 Irradiate for 10 min, wash 2-3 times with PBS, add 100 μL of fresh medium containing MTT (5 mg / mL) and incubate for 4 h. Discard the medium, add 150 μL of LDMSO and incubate with shaking for 10 min. Measure the absorbance of each well at 490 nm using a microplate reader. The concentration of Rd-TTPA is 0-30 μM, and the final concentration of MTT is 0.5 mg / mL. Figure 5 As shown in Figure a, the toxicity of Rd-TTPA is negligible without ultrasound irradiation, and the cell viability can still reach 80% when the Rd-TTPA concentration reaches 20 μM. However, under ultrasound irradiation, the activity of 4T1 cells decreases with increasing Rd-TTPA concentration.

[0064] (2) This invention further demonstrates that Rd-TTPA possesses good antitumor activity in vitro through live cell and dead cell staining (Calcein-AM and PI). 4T1 cells were stained at 1×10⁻⁶... 5 Cells were seeded at a density in 35 mm glass dishes and divided into: (1) blank group; (2) Rd-TTPA control group; (3) ultrasound irradiation control group (US); and (4) Rd-TTPA+US group. After overnight cell adhesion, the culture medium was discarded. 1 mL of fresh culture medium was added to groups (1) and (3), and 1 mL of fresh culture medium containing Rd-TTPA was added to groups (2) and (4), respectively. The final concentration of Rd-TTPA was 10 μM. Cells were incubated for 30 min. Then, groups (3) and (4) were subjected to ultrasound (3.0 MHz, 1.0 W / cm²). 2 Irradiate for 10 min. All four groups were then washed with PBS 2-3 times, incubated with Calcein-AM and PI for 40 min, and washed with PBS 2-3 times; the final concentration of Calcein-AM and PI was 1 μM. Figure 5As shown in b, only green fluorescence signals were observed in the blank group, Rd-TTPA control group, and ultrasound irradiation control group, indicating that the toxicity of Rd-TTPA is negligible and the ultrasound used in the experiment had no significant effect on cell viability. However, half of the cells in the Rd-TTPA+US group showed strong red fluorescence signals, while the green fluorescence signal was significantly weakened, indicating that Rd-TTPA can induce cell death as a sonosensitive agent, further demonstrating that Rd-TTPA has excellent sonodynamic antitumor activity in vitro.

[0065] 7. In vivo tumor imaging of the small molecule pyroptosis inducer Rd-TTPA

[0066] Based on the excellent near-infrared absorption and near-infrared II fluorescence emission of Rd-TTPA in vitro, this invention utilizes a NIR-II in vivo imaging system to investigate the imaging capability of Rd-TTPA in 4T1 tumor-bearing mice. 4T1 cells were cultured to the logarithmic growth phase and prepared with sterile PBS to a cell number of 1.0 × 10⁻⁶ cells. 7 A cell suspension of 100 μL was injected subcutaneously into the right hind limb of three female Balb / c nude mice (20 ± 2 g each). The tumor size was increased to 100 mm. 3 At approximately 10:00 AM, Rd-TTPA (1.0 mM, 50 μL, dissolved in 1% DMSO) was injected into Balb / c nude mice via the tail vein, and NIR-II fluorescence imaging was performed on each mouse at different time points (0, 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96 h). Figure 6 As shown, after intravenous administration of Rd-TTPA, the fluorescence intensity at the tumor site increased with time, reaching its maximum at 8 hours and remaining bright at 96 hours. This indicates that Rd-TTPA can target tumor tissue and has a long retention time, which is beneficial for in vivo NIR-II fluorescence imaging.

[0067] 8. In vivo tumor inhibition experiment of the small molecule pyroptosis inducer Rd-TTPA

[0068] Given the excellent properties of Rd-TTPA in cell studies, this invention further investigated its antitumor activity in vivo. Balb / c nude mice (female, 20±2g) were randomly divided into 4 groups of 3 mice each. 4T1 cells were cultured to the logarithmic growth phase and prepared into cells with a cell count of 1.0 × 10⁻⁶ using sterile PBS. 7 A cell suspension of 100 μL / mL was injected subcutaneously into the right hind limb of each mouse. When the tumor size reached 100 mm... 3At approximately 10:00 AM, tumor-bearing nude mice were divided into four groups: (1) blank group; (2) Rd-TTPA control group; (3) ultrasound irradiation control group (US); and (4) Rd-TTPA+US group. Each group of nude mice was injected intratumorally with 50 μL of PBS solution according to the above groupings (1) and (3), and Rd-TTPA (0.1 mM, 50 μL, dissolved in 1% DMSO) was injected into groups (2) and (4). Changes in mouse body weight and tumor size were monitored and recorded every two days. Ultrasound irradiation conditions for groups (3) and (4) were: 3.0 MHz, 1.0 W / cm². 2 10 minutes. Figure 7 As shown in Figure a, under ultrasound irradiation, the tumors in the Rd-TTPA group showed no significant growth within 14 days compared to the other control and blank groups, indicating that Rd-TTPA can effectively inhibit tumors through sonodynamic therapy. Furthermore, as... Figure 7 As shown in b, the body weight of nude mice in each group did not change significantly within 14 days of treatment, indicating that Rd-TTPA has good biocompatibility.

[0069] Subsequently, the antitumor effect of Rd-TTPA was confirmed histologically. After 14 days of treatment, mice were euthanized, and tumor tissues from each group, as well as heart, liver, spleen, lung, and kidney tissues, were fixed, embedded, sectioned, and analyzed by H&E staining and Ki-67 staining. Figure 7 As shown in Figure c, the tissue morphology of the control group and the blank group was normal, while the tumor tissue in the Rd-TTPA group under ultrasound irradiation showed significant destruction. Furthermore, the morphology of the heart, liver, spleen, lungs, and kidneys in the Rd-TTPA group under ultrasound irradiation was not significantly different from that of the control group and the blank group, further demonstrating that Rd-TTPA has good biocompatibility. The experimental results indicate that the small molecule pyroptosis inducer Rd-TTPA has effective tumor inhibitory effects in vivo and exhibits good biocompatibility.

[0070] This invention is not limited to the above embodiments. For those skilled in the art, several improvements and modifications can be made without departing from the principle of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention.

Claims

1. A small molecule pyroptosis inducer for tumor cells, characterized in that, The chemical structural formula is shown in Formula 1: Formula 1.

2. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 1, characterized in that, The main steps are as follows: 1) Cyclohexanone was added dropwise to concentrated sulfuric acid under ice-water bath conditions. 4-Diethylaminoketo acid was added while stirring, and the mixture was reacted at 80-100 °C for 1-2 h. After the reaction was completed, the mixture was poured into ice and perchloric acid solution was added to precipitate the product, yielding compound 1. 2) 4-Bromotriphenylamine and tetra(triphenylphosphine)palladium were added to toluene, and then added to a methanol solution of 5-formylthiophene-2-boronic acid and potassium carbonate. The reaction was refluxed at 100-120 °C for 20-30 h. The mixture was extracted with water and dichloromethane and purified by column chromatography to obtain compound 2. 3) Compound 1 and compound 2 were dissolved in acetic acid and reacted under reflux at 100-120 °C for 20-30 h. The crude product was obtained by extraction with water and dichloromethane, and then purified by column chromatography and dried to obtain a small molecule pyroptosis inducer. The chemical structural formulas of compounds 1 and 2 are shown below: 。 3. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, In step 1), the ratio between the amount of cyclohexanone and the volume of concentrated sulfuric acid is 12 mmol: (10-20) mL, and the ratio of the amount of 4-diethylaminoketo acid to cyclohexanone is 1: (1.5-2.5).

4. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, In step 1), the concentration of concentrated sulfuric acid is 95%-98%; the ratio between the volume of perchloric acid solution and the amount of cyclohexanone is (1.5-2.5) mL: 12 mmol; and the concentration of perchloric acid solution is 70%-72%.

5. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, In step 2), the molar ratio of 4-bromotriphenylamine to Pd(PPh3)4 is (5-15):1, the molar ratio of 5-formylthiophene-2-boronic acid to 4-bromotriphenylamine is (1.5-2.5):1, and the molar ratio of K2CO3 to 4-bromotriphenylamine is (4.5-5.5):

1.

6. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, In step 2), the ratio between the amount of 4-bromotriphenylamine and the volume of toluene is 1 mmol: (5-15) mL; the ratio between the volume of the solvent methanol and the amount of 4-bromotriphenylamine is (10-20) mL: 1 mmol; the column chromatography purification rinsing agent is a mixed solvent composed of petroleum ether and ethyl acetate in a volume ratio of (10-20):

1.

7. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, Step 3), the molar ratio of compound 1 to compound 2 is (1.0-2.0): 1, and the molar ratio of compound 1 to acetic acid is 0.3 mmol: (5-15) mL.

8. The method for preparing a small molecule pyroptosis inducer for tumor cells according to claim 2, characterized in that, In step 3), the column chromatography purification rinsing agent is a mixed solvent composed of dichloromethane and methanol in a volume ratio of 200:1 to 100:

1.

9. The application of the small molecule pyroptosis inducer described in claim 1 in the preparation of sonodynamic therapy reagents or imaging reagents for tumors.

10. The application according to claim 9, characterized in that, The application conditions for the sonodynamic tumor treatment reagent are: 1.0-3.0 MHz low-frequency ultrasound irradiation.

Images