Application of temozolomide bromide as sound-sensitive agent

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of temozolomide bromide as a sound-sensitive agent, and the temozolomide bromide has a structure shown in a formula (I). The invention provides a novel preparation and purification scheme of the sound-sensitive agent temozolomide bromide, and compared with traditional temozolomide, the product has a better killing effect on tumor cells, and has an extremely remarkable killing effect on the tumor cells and cell cycle arrest under the ultrasonic synergistic effect. The temozolomide derivative has a low toxicity effect similar to that of temozolomide on normal neuronal cells at low concentration, and animal experiments prove that the temozolomide derivative can penetrate through a blood-brain barrier. In addition, the invention has immeasurable clinical application value in the treatment of tumors, especially glioma and / or melanoma.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine, and particularly relates to an application of temozolomide bromide as a sonosensitizer. Background Art

[0002] Gliomas are the most common malignant tumors of the central nervous system, accounting for approximately 50% of intracranial tumors. They are highly invasive, rapidly growing, and have a high recurrence rate, posing a serious threat to patients' health and life. Currently, surgical resection combined with chemoradiotherapy is the main treatment option, but the prognosis remains poor. The median survival for patients with high-grade gliomas, such as glioblastoma, is typically less than 15 months.

[0003] Temozolomide, a first-line chemotherapy agent for gliomas, belongs to the imidazotetrazine class of alkylating agents. Its mechanism of action is that upon entry into the body, it spontaneously degrades to produce active metabolites, which then alkylate guanine residues on tumor cell DNA, inducing DNA cross-linking damage, ultimately inhibiting tumor cell proliferation and inducing apoptosis. This drug has the advantages of high oral bioavailability and blood-brain barrier permeability, making it a key component of comprehensive glioma treatment.

[0004] Sonodynamic therapy (SDT) is a noninvasive tumor treatment technology that combines ultrasound with sonosensitizers. It precisely kills tumor cells by generating reactive oxygen species (ROS). Its core advantages include: deep penetration and non-invasiveness: Ultrasound can penetrate up to 10 cm, allowing the treatment of deep-seated tumors such as gliomas without the need for craniotomy, thus avoiding surgical trauma. Targetedness and safety: Sonosensitizers accumulate within tumor cells, and upon ultrasound activation, they only locally generate ROS, minimizing damage to normal tissue. Repeated treatments are also possible, avoiding the cumulative toxicity of radiotherapy. Combination therapy potential: Synergistically combining sonosensitizers with chemoradiotherapy has shown significant tumor remission in patients with brainstem gliomas. Sonosensitizers are key to the effectiveness of sonodynamic therapy, but currently, there is a lack of sonosensitizers suitable for gliomas in clinical practice. Therefore, the development of highly effective, safe, blood-brain barrier-penetrating, and highly targeted sonosensitizers is a pressing need in the field of glioma treatment. Summary of the Invention

[0005] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a use of temozolomide bromide as a sonosensitizer.

[0006] To achieve the above object, the technical solution adopted by the present invention is: A use of temozolomide bromide as a sonosensitizer, wherein the temozolomide bromide has a structure of formula (I); (I).

[0007] Specifically, ultrasound is used in conjunction with temozolomide for sonodynamic therapy of tumors.

[0008] The excited ultrasonic frequency is 20kHz-1MHz; the ultrasonic intensity is 0.5W / cm 2 -2W / cm 2 ; The ultrasound duty cycle is 10%-50%, and the ultrasound time is 30s-10min.

[0009] Preferably, the excited ultrasonic frequency is 1 MHz; the ultrasonic intensity is 1 W / cm 2 ; Ultrasonic duty cycle 20%; Ultrasonic time 1 min.

[0010] The tumor is a glioma and / or a melanoma.

[0011] Temozolomide bromide is prepared using formula (II): (II).

[0012] The method specifically comprises the following steps: adding a Dess-Martin periodinane to a solvent, adding a bromination reagent in batches, mixing uniformly, adding TMZ-COOH, and reacting at 30-80° C. to obtain a product TMZ-Br.

[0013] The bromination reagent is tetraethylammonium bromide (TEAB); the molar ratio of TMZ-COOH to the bromination reagent is 1:(1-5); the molar ratio of TMZ-COOH to the Dess-Martin oxidant is 1:(1-5); and the solvent is at least one of acetonitrile, DMF, and THF.

[0014] The method also includes a post-processing process, which specifically includes the following steps: cooling the reaction liquid to 20-30°C, filtering, eluting the filter cake with acetonitrile, and sanding the filtrate through a column. The eluting liquid is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 5:1.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a sonosensitizer, temozolomide bromide, which exhibits excellent sonosensitivity and blood-brain barrier penetration. Under ultrasound synergy, it can enhance the drug's toxicity against glioma and melanoma cells by inducing the generation of singlet oxygen. Compared to conventional temozolomide, this product exhibits a superior tumor cell-killing effect and, at low concentrations, exhibits similarly low toxicity to normal neurons as temozolomide. Animal experiments have confirmed that it can penetrate the blood-brain barrier. This invention will have invaluable clinical application value for the treatment of tumors, particularly gliomas and / or melanomas. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is to characterize the hydrogen spectrum of temozolomide bromide; Figure 2 It is to characterize the carbon spectrum of temozolomide bromide; Figure 3 It is a graph characterizing the molecular weight of temozolomide bromide; Figure 4 This is the mass spectrum of drug distribution in different samples after intraperitoneal injection of temozolomide bromide into mice; Figure 5 is a graph showing the detection of singlet oxygen generation at different time points of ultrasound-activated temozolomide bromide and temozolomide and sonosensitizer titanium dioxide; Figure 6 This is a graph showing the detection of singlet oxygen generation by ultrasound-activated temozolomide bromide at different concentrations; Figure 7 This is the IC50 result after temozolomide bromide and temozolomide act on glioma cells; Figure 8 This is a flow cytometric graph of the cell cycle arrest of tumor cells synergistically with temozolomide bromide and temozolomide; Figure 9 This is a graph showing the killing effects of temozolomide bromide and temozolomide on tumor cells under different ultrasound intensities; Figure 10 This is a flow cytometric graph of the effects of temozolomide bromide and temozolomide on tumor cell apoptosis under ultrasound synergy; Figure 11 These are the results of toxicity test experiments on temozolomide bromide and temozolomide on normal neuronal cells. DETAILED DESCRIPTION

[0017] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below with reference to the accompanying drawings and the best embodiments.

[0018] Example 1: Synthesis of Temozolomide Bromide is shown in formula (II): (II); The specific steps include: 1. Synthesis of TMZ-COOH: Dissolve temozolomide (9.7 g) in concentrated sulfuric acid (50 ml) to form a yellow solution; cool to 0°C under nitrogen, and slowly add sodium nitrite (6.9 g, 50 ml) in water dropwise to catalyze the reaction; return the mixture to room temperature, stir in the dark for 24 h, and then cool to 0°C. Quench with ice (100 g) and continue stirring to produce a white solid precipitate. Vacuum filter, wash with cold water, and vacuum dry to obtain carboxylated temozolomide (TMZ-COOH). 2. Synthesis of TMZ-Br: Add Dess-Martin periodinane (95.64 g, 225.484 mmol, 2.2 eq) to a 1 L four-necked flask, followed by acetonitrile (ACN) (400 mL). After addition, add the brominating reagent, tetraethylammonium bromide (TEAB) (47.39 g, 225.484 mmol, 2.2 eq) in portions. Stir at 20°C for 0.5 h. Then add TMZ-COOH (20.00 g, 102.493 mmol, 1.0 eq). After addition, heat to 50°C and react for 3 h. TLC confirms complete reaction. The reaction mixture is cooled to 20-30°C and filtered. The filter cake is rinsed with 200 mL of ACN. The filtrate is then filtered through a column with a 5:1 ratio of petroleum ether (PE) to ethyl acetate (EA) to give 11.60 g of a white powdery solid (yield 46.658%). 11.60 g of white powdery solid was put into storage, which was TMZ-Br.

[0019] Product confirmation testing: Nuclear magnetic resonance hydrogen spectrum, carbon spectrum, and liquid chromatography-mass spectrometry are used to characterize temozolomide bromide.

[0020] H NMR spectroscopy ( Figure 1 ) 1 H NMR (CHLOROFORM-d, 400 MHz) δ 8.37 (s, 1H), 3.99 (s, 3H); C NMR ( Figure 2 ) 13 C NMR (CHLOROFORM-d, 101 MHz) δ 138.6, 133.1, 128.4, 116.9, 36.3.

[0021] Temozolomide bromide was prepared into a solution and analyzed by liquid chromatography-mass spectrometry. According to the molecular formula provided, [M+H] + is 230.020 ( Figure 3 ), the measured value and the theoretical value are within the allowable error range; In summary, combined with the results of H NMR, C NMR, and LC-MS, it can be determined that the product structure conforms to the structure shown in (I); (I).

[0022] Example 2: Product application test: 1. Analyze the blood-brain barrier passage of temozolomide bromide. The specific steps are as follows: After intraperitoneal injection of temozolomide bromide into mice, about 2 hours later, peripheral blood, cerebrospinal fluid, and brain tissue of the mice were collected. After purification, grinding, and fixation, liquid chromatography-mass spectrometry analysis was performed. The results were as follows: Figure 4As shown, the product of the present invention can be detected in the peripheral blood, cerebrospinal fluid and brain tissue of mice.

[0023] 2. Analyze the singlet oxygen generation detection diagram and time dependence under the action of temozolomide bromide. The specific steps are as follows: Use PBS to prepare temozolomide and temozolomide bromide solutions with a concentration of 200 μM, shake and mix, and then stand at room temperature in the dark for 1 hour. Add singlet oxygen detection reagent SOSG to the above reagents at a ratio of 1:1000, shake and mix, and then place on the ultrasound probe. The ultrasound parameters (1 MHz, 1 W / cm 2 , 20%). 200 μl of liquid was taken out at 0, 2, 4, 6, 8, and 10 minutes of ultrasonication, diluted with PBS at a ratio of 1:10, and the ultraviolet absorption of the solution at an excitation wavelength of Ex = 504 nm and an emission wavelength of Em = 525 nm was measured in a spectrophotometer to obtain the absorbance value. The results are as follows Figure 5 As shown in the figure, the sonodynamic force of temozolomide bromide began to be stronger than that of temozolomide at 2 minutes of ultrasound, and gradually increased with the extension of ultrasound time, and was close to that of the traditional sonosensitizer titanium dioxide, indicating that the drug has a strong sonosensitizing effect.

[0024] 3. Analyze the relationship between the sonodynamic intensity and drug concentration of temozolomide bromide. The specific steps are as follows: 20, 50, 100, 200, 400, and 800 μM temozolomide bromide solutions were prepared using PBS, and singlet oxygen detection reagent SOSG was added at a ratio of 1:1000. Then, ultrasound stimulation was applied (parameters: 1 MHz, 1 W / cm 2 , 20%, 1min). The absorbance of each group was measured using a spectrophotometer. The results are shown in Figure 6 It shows that the sonodynamic intensity of temozolomide bromide is positively correlated with the drug concentration.

[0025] 4. Analyze the killing effect of temozolomide bromide on glioma cells and use the CCK-8 method to detect the growth of glioma cells. Specific steps: (1) Observe the growth status of glioma cells. When the growth status is appropriate, digest, count and plate the cells. For a 96-well plate, the number of cells per well is approximately 3,000. Set up three replicate wells for each group.

[0026] (2) 24 hours after inoculation, the cells were treated according to different groups. The cells were divided into two groups: temozolomide group and temozolomide bromide group. The drugs were added using a gradient method, with concentrations of 3000μM, 1000μM, 333.3μM, 111.1μM, 37.07μM, 12.34μM, 4.11μM, 1.37μM, 0.45μM, and 0μM, respectively.

[0027] (3) After 24 hours, add CCK-8 reagent and incubate for 1-4 hours before detection using an enzyme-labeled instrument at a wavelength of 450 nm.

[0028] The results are as follows Figure 7 The figure shows that the IC50 (half inhibitory concentration) of temozolomide bromide in the LN229 cell line is 345.2μM, and the IC50 of temozolomide is 1050μM. It can be seen that the half inhibitory concentration of temozolomide bromide in the glioma cell line is lower than that of temozolomide.

[0029] 5. Analyze the cell cycle arrest effect of temozolomide bromide on glioma cells under ultrasound synergy. Specific steps: (1) Observe the growth status of glioma cells. When the growth status is appropriate, digest, count and plate the cells. For a 12-well plate, the number of cells per well is about 5x10 5 Divided into control group ( Figure 8 blank group), temozolomide group, temozolomide bromide group, simple ultrasound group ( Figure 8 There were 6 groups in total (single ultrasound group), temozolomide ultrasound group, and temozolomide bromide ultrasound group, with 3 replicates in each group.

[0030] (2) After culturing for 24 hours, the cells adhered to the wall and were treated with the culture medium containing the present invention and temozolomide at a concentration of 100 μM. Repeated wells and blank controls were set up. The ultrasound group was treated with ultrasound and cultured for 24 hours.

[0031] (3) Collect cells, wash with PBS, digest with trypsin, centrifuge, and fix with 70% pre-cooled ethanol overnight.

[0032] (4) After washing with PBS, incubate with a staining solution containing PI and RNase A at room temperature in the dark for 30 minutes, and analyze the ratio of each cycle using flow cytometry.

[0033] The results are as follows Figure 8 As shown in the figure, the cell cycle arrest effect of temozolomide bromide on glioma cells was analyzed. It can be seen that after 24 hours of drug treatment, the cell cycle arrest effect of temozolomide bromide on glioma cells was more significant than that of temozolomide, and the flow cytometry results showed that the tumor cell cycle arrest in the temozolomide bromide ultrasound treatment group was significantly increased, indicating that combined ultrasound treatment can further increase the cell cycle arrest rate of temozolomide bromide.

[0034] 6. Analyze the killing effect of temozolomide bromide on glioma cells under different ultrasound intensities. The specific steps are as follows: Observe the growth state of glioma cells. When the growth state is appropriate, digest, count and plate. For a 12-well plate, the number of cells per well is about 5x10 5There were 4 groups with ultrasound duty ratios of 0%, 10%, 20% and 50%, with 3 replicates in each group. The other ultrasound parameters were: 1 MHz, 1 W / cm 2 , act for 1 minute, culture in a constant temperature box for 24 hours, add CCK-8 reagent, culture for 1-4 hours and then detect with an enzyme marker at a wavelength of 450nm.

[0035] The results are as follows Figure 9 It can be seen that with the increase of ultrasound intensity, the killing effect of temozolomide bromide on tumor cells is more significant.

[0036] 7. Analyze the effect of temozolomide bromide on the apoptosis of glioma cells under ultrasound synergy. The specific steps are as follows: (1) Observe the growth status of glioma cells. When the growth status is appropriate, digest, count and plate the cells. For a 12-well plate, the number of cells per well is about 5x10 5 , divided into control group (corresponding to Figure 10 Middle A), temozolomide group, temozolomide bromide group, simple ultrasound group (corresponding Figure 10 There were 6 groups in total (middle D), temozolomide ultrasound group, and temozolomide bromide ultrasound group, with 3 replicates in each group.

[0037] (2) 24 hours after inoculation, the cells were treated with the present invention and temozolomide at a concentration of 100 μM, and the ultrasound group was additionally treated with ultrasound.

[0038] (3) After 8 hours, digest the cells and transfer them to 2 mL EP tubes. Add Annexin-FITC reagent to different EP tubes according to the instructions. Incubate in the dark for 15 minutes, then use flow cytometer to collect data and use BD FlowJo software for data analysis.

[0039] The results are as follows Figure 10 As shown, Figure 10 A is the blank control group, B is the temozolomide group, C is the temozolomide bromide group, D is the simple ultrasound group, E is the temozolomide ultrasound group, and F is the temozolomide bromide ultrasound group; the effect of temozolomide bromide on the apoptosis of glioma cells was analyzed. The apoptosis rate of the temozolomide group was 10.09%, the apoptosis rate of the temozolomide bromide group was 39.44%, the apoptosis rate of the temozolomide ultrasound group was 12.45%, and the apoptosis rate of the temozolomide bromide ultrasound group was 60.96%. It can be seen that the killing effect of temozolomide bromide on glioma cells is better than that of temozolomide, and combined with ultrasound treatment can significantly enhance the killing effect.

[0040] 8. Analyze the toxic effect of temozolomide bromide on normal neuronal cells and use the CCK-8 method to detect neuronal cell viability, specifically including the following steps: (1) Observe the growth of neuronal cells. When the growth state is appropriate, digest, count, and plate the cells. For a 96-well plate, the number of cells per well is approximately 3,000. Set up three replicate wells for each group.

[0041] (2) 24 hours after inoculation, cells were treated according to different groups. The treatment groups were divided into three groups: the neurotoxic drug carmustine, temozolomide, and temozolomide bromide. The drugs were added using a gradient method, with concentrations of 1600, 800, 400, 200, 100, 50, 25, and 0 μM, respectively.

[0042] (3) After 24 hours, add CCK-8 reagent and incubate for 1-4 hours before detection using an enzyme-labeled instrument at a wavelength of 450 nm.

[0043] The results are as follows Figure 11 It is shown that in normal neuronal cells, at low concentrations, both temozolomide bromide and temozolomide exhibit low cytotoxicity.

[0044] In summary, the present invention provides a novel preparation and purification scheme for the sonosensitizer temozolomide bromide. Compared to conventional temozolomide, this product exhibits superior tumor cell killing effects and significantly enhances cell cycle arrest under the synergistic effect of ultrasound. It also exhibits similar low toxicity to normal neuronal cells as temozolomide at low concentrations, and has been shown in animal studies to penetrate the blood-brain barrier. Furthermore, this invention has inestimable clinical value in the treatment of tumors, particularly gliomas and / or melanomas.

[0045] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A use of temozolomide bromide as a sonosensitizer, characterized in that: Temozolomide bromide has the structure of formula (I); （I）。 2. The use of temozolomide bromide as a sonosensitizer according to claim 1, characterized in that: Using ultrasound in combination with temozolomide for sonodynamic therapy of tumors.

3. The use of temozolomide bromide as a sonosensitizer according to claim 2, characterized in that: The excited ultrasonic frequency is 20kHz-1MHz; the ultrasonic intensity is 0.5W / cm 2 -2W / cm 2 ; The ultrasound duty cycle is 10%-50%, and the ultrasound time is 30s-10min.

4. The use of temozolomide bromide as a sonosensitizer according to claim 2, characterized in that: The excited ultrasonic frequency is 1 MHz; the ultrasonic intensity is 1 W / cm 2 ; Ultrasonic duty cycle 20%; Ultrasonic time 1 min.

5. The use of temozolomide bromide as a sonosensitizer according to claim 2, characterized in that: The tumor is a glioma and / or a melanoma.

6. The use of temozolomide bromide as a sonosensitizer according to claim 1, characterized in that: Temozolomide bromide is prepared using formula (II): (II)。 7. The use of temozolomide bromide as a sonosensitizer according to claim 6, characterized in that: The method specifically comprises the following steps: adding a Dess-Martin oxidant to a solvent, adding a bromination reagent, mixing uniformly, adding TMZ-COOH, and reacting at 30-80° C. to obtain a product TMZ-Br.

8. The use of temozolomide bromide as a sonosensitizer according to claim 7, characterized in that: The bromination reagent is tetraethylammonium bromide (TEAB); the molar ratio of TMZ-COOH to the bromination reagent is 1:(1-5); the molar ratio of TMZ-COOH to the Dess-Martin oxidant is 1:(1-5); and the solvent is at least one of acetonitrile, DMF, and THF.

9. The use of temozolomide bromide as a sonosensitizer according to claim 7, characterized in that: The method also includes a post-processing process, which specifically includes the following steps: cooling the reaction liquid to 20-30°C, filtering, eluting the filter cake with acetonitrile, and sanding the filtrate through a column. The eluting liquid is a mixture of petroleum ether and ethyl acetate, and the volume ratio of petroleum ether to ethyl acetate is 5:1.

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