Trifluoromethylated temozolomide and derivatives thereof and methods of preparation and use

Abstract

The application belongs to the field of antitumor drugs, and particularly relates to trifluoromethylated temozolomide and derivatives thereof, a preparation method and application. The trifluoromethylated temozolomide has a better killing effect on tumor cells relative to traditional temozolomide, has a lower toxic effect on normal neuron cells at a low concentration relative to temozolomide, and can penetrate the blood-brain barrier through animal experiments.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drugs, specifically relating to a trifluoromethylated temozolomide and its derivatives, their preparation methods, and their applications. Background Technology

[0002] Malignant tumors continue to threaten human health and survival, with gliomas (especially glioblastoma, GBM) and melanomas being particularly challenging due to their high invasiveness and resistance to radiotherapy and chemotherapy. Temozolomide, a lipophilic imidazole tetrazine drug, rapidly hydrolyzes under alkaline or near-alkaline conditions (the pH of brain tumor tissue is higher than other tissues) into the metabolite 5-(3-methyltriazol-1-yl)imidazol-4-carboxamide, which further hydrolyzes into the metabolites aminoimidazol-4-carboxamide (AIC) and diazomethane. These are electrophilic methylation molecules that react with DNA. The negatively charged DNA acts as a nucleophile, while the methyl group of diazomethane transfers to the DNA, leading to the formation of multiple DNA adducts. This creates opportunities for mismatched base pairing, ultimately resulting in cytotoxicity. This is how temozolomide exerts its chemotherapeutic effect. Temozolomide (TMZ), a new generation of small molecule alkylating agent prodrug, has a molecular weight of only 194.15 Da, good lipid solubility, a blood-brain barrier penetration rate of approximately 30%-40%, and near 100% oral bioavailability. It is an alkylating agent that easily crosses the blood-brain barrier and is used as a first-line chemotherapy drug for glioma treatment. However, more than 50% of patients treated with temozolomide exhibit congenital or acquired chemotherapy resistance. When residual tumor cells develop resistance to treatment, gliomas may relapse. For relapsed or advanced gliomas, there is still a lack of highly effective second-line treatments. Therefore, the development of new drugs with high anti-tumor activity, low resistance, and good safety profiles has become an urgent need in the current field of glioma treatment. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a trifluoromethylated temozolomide and its derivatives, a preparation method, and applications.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A trifluoromethylated temozolomide having the following structure (I);

[0006] (I).

[0007] The present invention also includes a derivative of the aforementioned trifluoromethylated temozolomide, comprising the parent ring structure of the aforementioned trifluoromethylated temozolomide.

[0008] This invention also includes a method for preparing the aforementioned trifluoromethylated temozolomide, using the following formula (II):

[0009] (II).

[0010] Specifically, the steps include: placing TMZ-COOH in a pressure vessel, purging with nitrogen, cooling to below -40°C, introducing SF4, stirring, slowly heating to 0-30°C, preferably 25°C, and maintaining the temperature to obtain the product TMZ-CF3.

[0011] The molar ratio of TMZ-COOH to SF4 is 1:(1-10); preferably 1:7.5.

[0012] It also includes a post-processing procedure: the pH of the reaction solution is adjusted to neutral with ammonia, the aqueous phase is extracted with dichloromethane, the organic phase is dried, filtered and evaporated to dryness to obtain a crude product, which is dissolved in tetrahydrofuran, precipitated and column filtered, and the eluent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 2:1.

[0013] The present invention also includes the application of the aforementioned trifluoromethylated temozolomide.

[0014] It is used in the preparation of antitumor drugs; the tumors are gliomas and / or melanomas.

[0015] Preferably, it is used as a sonosensitive agent in combination with ultrasound for sonodynamic treatment of tumors.

[0016] Preferably, the excitation ultrasonic frequency is 20 kHz-1 MHz; the ultrasonic intensity is 0.5 W / cm². 2 -2W / cm 2 The ultrasound duty cycle is 10%-50%, and the ultrasound time is 30s-10min.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] This invention provides a novel substance, trifluoromethylated temozolomide, which exhibits better killing effects on tumor cells compared to traditional temozolomide. At low concentrations, it also shows lower toxicity to normal neurons than temozolomide. Animal experiments have verified that it can cross the blood-brain barrier and is used to treat tumors, especially gliomas and / or melanomas. As a preferred form, its combination with ultrasound for sonodynamic therapy will have immeasurable clinical application value in tumor treatment. Attached Figure Description

[0019] Figure 1 This is the characterization of the 1H NMR spectrum of trifluoromethylated temozolomide;

[0020] Figure 2 This is the carbon spectrum characterizing trifluoromethylated temozolomide;

[0021] Figure 3This is a molecular weight diagram characterizing trifluoromethylated temozolomide;

[0022] Figure 4 This is a mass spectrum of drug distribution in different samples after intraperitoneal injection of trifluoromethylated temozolomide into mice;

[0023] Figure 5 The IC50 results of trifluoromethylated temozolomide and temozolomide in different glioma cell lines are shown; among them, Figure 5 In the diagram, A represents the LN229 cell line, and B represents the U251 cell line.

[0024] Figure 6 Singlet oxygen generation detection graphs at various time points for ultrasonically activated trifluoromethylated temozolomide and temozolomide and titanium dioxide as a sonic sensitizer.

[0025] Figure 7 This is a detection graph of singlet oxygen generation by ultrasonic activation of different concentrations of trifluoromethylated temozolomide;

[0026] Figure 8 This is a flow cytometry image showing the effect of ultrasound-assisted trifluoromethylated temozolomide and temozolomide on apoptosis in glioma cell lines.

[0027] Figure 9 The images show the killing effects of trifluoromethylated temozolomide and temozolomide on tumor cells under different ultrasound intensities.

[0028] Figure 10 This is a flow cytometry image of the effects of trifluoromethylated temozolomide and temozolomide on tumor cell apoptosis under ultrasound synergy.

[0029] Figure 11 This is a diagram showing the experimental results of toxicity tests on normal neurons using trifluoromethylated temozolomide and temozolomide. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments.

[0031] Example 1: The synthesis of trifluoromethylated temozolomide (hereinafter referred to as fluorinated temozolomide) is shown in formula (II):

[0032] (II);

[0033] Specifically, the following steps are included:

[0034] 1. Synthesis of TMZ-COOH: Temozolomide (9.7 g) was dissolved in concentrated sulfuric acid (50 ml) to form a yellow solution; the solution was cooled to 0°C under nitrogen, and sodium nitrite (6.9 g, 50 ml) aqueous solution was slowly added dropwise to catalyze the reaction; the mixture was warmed to room temperature, stirred in the dark for 24 h, cooled to 0°C, and quenched with ice (100 g). Stirring continued to produce a white solid precipitate, which was then vacuum filtered, washed with cold water, and vacuum dried to obtain carboxylated temozolomide (TMZ-COOH);

[0035] 2. Synthesis of TMZ-CF3: TMZ-COOH (10.00 g, 51.246 mmol, 1.0 eq) was added to a 250 mL pressure vessel. The pressure vessel was sealed, purged with nitrogen, and cooled to below -40℃. SF4 (41.53 g, 384.349 mmol, 7.5 eq) was added, and the mixture was stirred for 0.5 h. Then, the temperature was slowly raised to 25℃ and maintained for 36 h. TLC was used to confirm the complete reaction of the starting materials. The pH of the reaction solution was adjusted to 7 with 50 mL of ammonia. The aqueous phase was extracted with dichloromethane (DCM) (100 mL * 2). The organic phase was dried, filtered, and evaporated to dryness to obtain 2.8 g of crude product. This crude product was dissolved in 100 mL of THF, prepared by column chromatography (PE:EA = 2:1), yielding 1.42 g of white powdery solid (yield 12.645%). The 1.42 g of white powdery solid was stored as TMZ-CF3.

[0036] Product verification testing: Characterization of trifluoromethylated temozolomide using 1H NMR spectroscopy and liquid chromatography-mass spectrometry.

[0037] 1H NMR spectrum ( Figure 1 ) 1 H NMR (CHLOROFORM-d, 400 MHz) δ 8.50 (s, 1H), 4.08 (s, 3H);

[0038] Nuclear magnetic resonance fluorine spectroscopy (NMR fluorine spectroscopy) Figure 2 ) 19 F NMR (CHLOROFORM-d, 376 MHz) δ -61.46.

[0039] Fluorinated temozolomide was prepared into a solution and analyzed using liquid chromatography-mass spectrometry. According to the provided molecular formula, [M+H] + It is 219.120 ( Figure 3 The measured values ​​and theoretical values ​​are within the allowable error range.

[0040] In summary, by combining the results of 1H NMR spectroscopy, fluorine spectroscopy, and liquid chromatography-mass spectrometry, the product structure can be determined to conform to structure (I).

[0041] (I);

[0042] 2. Product application testing:

[0043] 1. Analysis of the blood-brain barrier crossing ability of fluorinated temozolomide: The specific steps are as follows: Fluorinated temozolomide was injected intraperitoneally into mice. Approximately 2 hours later, peripheral blood, cerebrospinal fluid, and brain tissue were collected from the mice. After purification, grinding, and fixation, the samples were analyzed using liquid chromatography-mass spectrometry (LC-MS). The results are as follows: Figure 4 As shown, the product of this invention can be detected in the peripheral blood, cerebrospinal fluid, and brain tissue of mice.

[0044] 2. To analyze the killing effect of fluorinated temozolomide on glioma cells, the CCK-8 assay was used to detect glioma cell growth; specific steps:

[0045] (1) Observe the growth status of glioma cells. When the growth status is suitable, digest, count and plate them. For 96-well plates, the number of cells per well is about 3000. Set up 3 replicates for each group.

[0046] (2) Twenty-four hours after inoculation, the cells were treated according to different groups. The treatment was divided into two groups: the TMZ group and the TMZ-Br group. The drugs were added using a gradient method, with concentrations of 3000 μM, 1000 μM, 333.3 μM, 111.1 μM, 37.07 μM, 12.34 μM, 4.11 μM, 1.37 μM, 0.45 μM, and 0 μM, respectively.

[0047] (3) After 24 hours, add CCK-8 reagent, incubate for 1-4 hours and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the virus at a wavelength of 450 nm.

[0048] The results are as follows Figure 5 As shown, Figure 5 In the LN229 cell line, the IC50 (half-maximum inhibitory concentration) of temozolomide fluoride was 387.1 μM; in the LN229 cell line, the IC50 was 1050 μM; and in the U251 cell line, the IC50 was 452.0 μM. Figure 5 In the U251 cell line (B), the IC50 of temozolomide was 822.9 μM, indicating that the half-maximal inhibitory concentration of this invention was lower than that of temozolomide in different glioma cell lines.

[0049] 3. Analyze the detection curve and time dependence of singlet oxygen generation under the action of temozolomide fluoride. The specific steps are as follows: Prepare 200 μM solutions of temozolomide and temozolomide fluoride using PBS. After shaking and mixing, let stand at room temperature in the dark for 1 hour. Add SOSG singlet oxygen detection reagent at a ratio of 1:1000 to the above reagents, shake and mix, and then place on an ultrasonic probe. Ultrasonic parameters (1 MHz, 1 W / cm²) are used. 2(20%). At minutes 0, 2, 4, 6, 8, and 10 of the ultrasound, 200 μl of the liquid was collected, diluted with PBS at a ratio of 1:10, and the absorbance of the solution at an excitation wavelength Ex = 504 nm and an emission wavelength Em = 525 nm was measured using a spectrophotometer to obtain the absorbance values. The results are as follows: Figure 6 The results showed that after 2 minutes of ultrasound, the acoustic dynamics of fluorinated temozolomide began to be stronger than that of temozolomide, and gradually increased with the extension of ultrasound time, and were similar to those of the traditional acoustic sensitizer titanium dioxide, indicating that the drug has strong acoustic sensitivity.

[0050] 4. The relationship between the sonodynamic intensity and drug concentration of temozolomide fluoride was analyzed. The specific steps are as follows: Temozolomide fluoride solutions with concentrations of 20, 50, 100, 200, 400, and 800 μM were prepared using PBS. Singlet oxygen detection reagent SOSG was added at a ratio of 1:1000, followed by ultrasonic stimulation (parameters: 1 MHz, 1 W / cm²). 2 (20%, 1 min). The absorbance of each group was measured using a spectrophotometer. The results are as follows: Figure 7 It was found that the sonodynamic intensity of temozolomide fluoride was positively correlated with the drug concentration.

[0051] 5. Analyze the cell cycle arrest effect of fluorinated temozolomide on glioma cells under ultrasound synergy. Specific steps:

[0052] (1) Observe the growth status of glioma cells. When the growth status is suitable, digest, count, and plate them. For a 12-well plate, the number of cells per well is approximately 5 x 10. 5 There were [number] individuals. The control group was divided into [number] and [number] groups. Figure 8 (blank group), temozolomide group, fluorinated temozolomide group, ultrasound-only group ( Figure 8 There are 6 groups in total: single ultrasound group, temozolomide ultrasound group, and fluorinated temozolomide ultrasound group; each group has 3 replicates.

[0053] (2) After culturing for 24 hours, the culture medium was replaced with the culture medium of the present invention and temozolomide at a concentration of 200 μM. A duplicate well and a blank control were set up and cultured for 48 hours. The ultrasonic group was subjected to additional ultrasonic treatment.

[0054] (3) Collect cells, wash with PBS, digest with trypsin, centrifuge, and fix overnight with 70% pre-cooled ethanol.

[0055] (4) After washing with PBS, incubate with staining solution containing PI and RNase A at room temperature in the dark for 30 min, and then detect by flow cytometry to analyze the proportion of each cycle.

[0056] The results are as follows Figure 8As shown, after 24 hours of drug treatment, trifluoromethylated temozolomide had a more significant effect on cell cycle arrest in glioma cells compared to temozolomide. The cell cycle arrest in the ultrasound-treated group with trifluoromethylated temozolomide was significantly increased, indicating that combined ultrasound treatment can further increase the cell cycle arrest rate of fluorinated temozolomide.

[0057] 6. The killing effect of temozolomide fluoride on glioma cells under different ultrasound intensities was analyzed. The specific steps were as follows: Observe the growth status of glioma cells. When the growth status was suitable, digestion, counting, and plating were performed. For 12-well plates, the number of cells per well was approximately 5 x 10-1. 5 Four groups were constructed with ultrasonic duty cycles of 0%, 10%, 20%, and 50%, with three replicates in each group. Other ultrasonic parameters were: 1MHz, 1W / cm². 2 After incubating for 1 minute in a constant temperature incubator for 24 hours, add CCK-8 reagent and incubate for 1-4 hours. Then, use an ELISA reader to detect the enzyme at a wavelength of 450 nm.

[0058] The results are as follows Figure 9 This indicates that as the intensity of ultrasound increases, the killing effect of fluorinated temozolomide on tumor cells is significantly enhanced.

[0059] 7. The effects of fluorinated temozolomide on glioma cell apoptosis under ultrasound synergy were analyzed. The specific steps are as follows:

[0060] (1) Observe the growth status of glioma cells. When the growth status is suitable, digest, count, and plate them. For a 12-well plate, the number of cells per well is approximately 5 x 10. 5 Single. Divided into control group ( Figure 10 (blank group), temozolomide group, fluorinated temozolomide group, ultrasound-only group ( Figure 10 There are a total of 6 groups: single ultrasound group, temozolomide ultrasound group, and fluorinated temozolomide ultrasound group, with 3 replicates in each group.

[0061] (2) 24 hours after inoculation, the present invention and temozolomide were treated with cells at a concentration of 200 μM, and the ultrasound group was additionally treated with ultrasound.

[0062] (3) After 8 hours, digestion was performed and the contents were transferred to 2 mL EP tubes. Annexin-FITC reagent was added to different EP tubes according to the instructions. After incubation in the dark for 15 minutes, data were collected using a flow cytometer and analyzed using BD FlowJo software.

[0063] like Figure 10 In the table, A represents the control group, B represents the temozolomide group, C represents the fluorinated temozolomide group, D represents the ultrasound-only group, E represents the temozolomide ultrasound group, and F represents the fluorinated temozolomide ultrasound group. Results Figure 10As shown, the apoptosis rate (Q2+Q3) of the temozolomide group was 9.36%, and the apoptosis rate (Q2+Q3) of the fluorinated temozolomide group was 28.11%. It can be seen that the killing effect of the present invention on glioma cells is significantly better than that of temozolomide, and the combination of ultrasound treatment can significantly enhance the killing effect and increase the apoptosis rate.

[0064] 8. Analyze the toxic effects of temozolomide fluoride on normal neurons, and detect neuronal cell viability using the CCK-8 assay:

[0065] (1) Observe the growth status of neuronal cells. When the growth status is suitable, digest, count and plate them. For a 96-well plate, the number of cells per well is about 3000. Set up 3 replicates for each group.

[0066] (2) Twenty-four hours after inoculation, the cells were treated according to different groups. The treatment was divided into three groups: the neurotoxic drug carmustine, temozolomide, and fluorinated temozolomide groups. The drugs were added using a gradient method at concentrations of 1600, 800, 400, 200, 100, 50, 25, and 0 μM, respectively.

[0067] (3) After 24 hours, add CCK-8 reagent, incubate for 1-4 hours and then use an enzyme-linked immunosorbent assay (ELISA) reader to detect the virus at a wavelength of 450 nm.

[0068] The results are as follows Figure 11 It was indicated that, in normal neuronal cells, temozolomide fluoride exhibited lower cytotoxicity compared to temozolomide.

[0069] In summary, this invention provides a novel substance, trifluoromethylated temozolomide, which exhibits better tumor cell killing activity compared to traditional temozolomide, and shows lower toxicity to normal neurons at low concentrations. Animal experiments have also demonstrated its ability to cross the blood-brain barrier. This invention will have immeasurable clinical application value for the treatment of tumors, especially gliomas and / or melanomas.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A trifluoromethylated temozolomide, characterized in that, It has the structure of formula (I); （I）。 2. A method for preparing trifluoromethylated temozolomide according to claim 1, characterized in that, Preparation is carried out using reaction (II): (II)。 3. The method for preparing trifluoromethylated temozolomide according to claim 2, characterized in that, Specifically, the steps include: placing TMZ-COOH in a pressure vessel, purging it with nitrogen, cooling it to below -40℃, introducing SF4, stirring, slowly heating it to 0-30℃, maintaining the temperature for reaction, and obtaining the product TMZ-CF3.

4. The method for preparing trifluoromethylated temozolomide according to claim 3, characterized in that, The molar ratio of TMZ-COOH to SF4 is 1:(1-10).

5. The method for preparing trifluoromethylated temozolomide according to claim 3, characterized in that, It also includes a post-processing procedure: the pH of the reaction solution is adjusted to neutral with ammonia, the aqueous phase is extracted with dichloromethane, the organic phase is dried, filtered and evaporated to dryness to obtain a crude product, which is dissolved in tetrahydrofuran, precipitated and column filtered, and the eluent is a mixture of petroleum ether and ethyl acetate with a volume ratio of 2:

1.

6. The application of the trifluoromethylated temozolomide according to claim 1, characterized in that, It is used in the preparation of anti-tumor drugs; the tumor is a glioma.

7. The application of trifluoromethylated temozolomide according to claim 6, characterized in that, It is used as a sonosensitive agent in combination with ultrasound for sonodynamic treatment of tumors.

8. The application of trifluoromethylated temozolomide according to claim 7, characterized in that, The excitation ultrasonic frequency is 20kHz-1MHz; the ultrasonic intensity is 0.5W / cm². 2 -2W / cm 2 The ultrasound duty cycle is 10%-50%, and the ultrasound time is 30s-10min.

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