A kind of chloroethyl nitrosourea compound containing aromatic group and its preparation method and application

Abstract

The present invention relates to the field of medicine and provides a chloroethylnitrosourea compound containing an aromatic group. The chloroethylnitrosourea compound containing an aromatic group has the following general structural formula: #imgabs0# wherein R is selected from -CH3, -CH2CH3, or #imgabs1#, and Ar is selected from benzene, substituted benzene, #imgabs2#, or #imgabs3#. The chloroethylnitrosourea compound containing an aromatic group of the present invention has a higher ability to penetrate the brain barrier, fully utilizes the effect of the aziridine ion, and has excellent anti-glioma cell activity, and also exhibits anti-tumor cell activity against various tumors.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a chloroethyl nitrosourea compound containing an aromatic group, a preparation method and an application thereof. Background Art

[0002] Gliomas are primary intracranial tumors caused by the cancerous transformation of glial cells in the brain and spinal cord. The World Health Organization divides gliomas into four grades: I and II are low-grade gliomas that are mostly benign, while III and IV are high-grade gliomas. Among them, glioblastoma multiforme (GBM), which belongs to grade IV, is the most common malignant tumor in the central nervous system. Its invasiveness and recurrence are significantly higher than other intracranial tumors, seriously threatening the life and health of patients. The current treatment of GBM is to maximize surgical removal of tumor tissue, supplemented by radiotherapy and chemotherapy after surgery. [1,2] Temozolomide (TMZ) is the first-line chemotherapy drug for GBM patients. It is an alkylating agent of the imidazotetrazine class and has been used clinically since 1999.

[0003] Nitrosoureas are also used in GBM treatment. For example, lomustine combined with temozolomide can improve the survival of patients with newly diagnosed glioblastoma. [3] Small clinical trials have shown that combined use can combine different degrees of DNA damage or potential additive or even synergistic effects, and no typical organ toxicity reactions of nitrosourea compounds were observed during combined use. Dimitrios Pletsas [4] et al. designed and synthesized a new compound 3-(2-phenylaminoethyl) substituted imidazole tetrazine. The biological activity of this compound comes from the active intermediate aziridine ion formed by in vivo conversion. It is mainly used to modify the guanine-N7 site on DNA. This may alleviate the drug resistance problem of GBM chemotherapy drugs to a certain extent. [4] However, the therapeutic effect of 3-(2-phenylaminoethyl)-substituted imidazole tetrazines on brain glioma is still unsatisfactory. Summary of the Invention

[0004] The present invention provides chloroethylnitrosourea compounds containing aromatic groups, which can enhance the ability of drugs to penetrate the brain barrier, give full play to the effects of aziridine ions, and improve the effect of treating brain gliomas.

[0005] The aromatic-containing chloroethyl nitrosourea compound has the following general structural formula:

[0006] Wherein, R is selected from -CH3, -CH2CH3 or Ar is selected from benzene, substituted benzene,

[0007] An object of the present invention is to provide a method for preparing chloroethyl nitrosourea compounds containing aromatic groups.

[0008] The preparation method of the aromatic-containing chloroethyl nitrosourea compound comprises the following steps:

[0009] R 1 -NH-Ar reacts with 2-chloroethanol to generate mono- or di-2-hydroxyethylaniline, the hydroxyl group undergoes halogenation reaction, is substituted with phthalimide, and reacts with hydrazine hydrate to convert into amino group, the amino group reacts with chloroethyl isocyanate to generate chloroethyl urea, and then undergoes nitrosation reaction to generate the aromatic group-containing chloroethyl nitrosourea compound, wherein R 1 is selected from -H, -CH3 or -CH2CH3, Ar is selected from benzene, substituted benzene,

[0010] An object of the present invention is to provide a pharmaceutical composition.

[0011] The pharmaceutical composition comprises the above-mentioned chloroethylnitrosourea compound containing an aromatic group and one or more pharmaceutically acceptable salts of the chloroethylnitrosourea compound containing an aromatic group.

[0012] Furthermore, the pharmaceutical composition is an oral preparation or an injection.

[0013] Furthermore, the injection is an intravenous injection preparation, an intraperitoneal injection or a subcutaneous injection.

[0014] One object of the present invention is to provide a pharmaceutical composition as described above for use in preparing anti-tumor drugs.

[0015] Furthermore, the tumor includes one or more of breast cancer, lung cancer, nasopharyngeal cancer, colon cancer, liver cancer, cervical cancer and brain glioma.

[0016] Beneficial effects:

[0017] The aromatic-containing chloroethyl nitrosourea compound of the present invention has a higher ability of drug penetrating the brain barrier, fully exerts the effect of aziridine ions, has excellent anti-glioma cell activity, and simultaneously exhibits anti-tumor cell activity against various tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The concentrations of TMZ and HJ03 in the blood or brain 30 minutes after administration of 66 mg / Kg provided in Effect Example 3;

[0019] Figure 2 The blood indicators of mice in different groups after 7 days of oral administration provided in Effect Example 4;

[0020] Figure 3 The weight changes of mice during the administration period provided in Effect Example 4;

[0021] Figure 4 This is the in vivo anti-glioma activity evaluation of compound HJ03 provided in Effect Example 5. DETAILED DESCRIPTION

[0022] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but they should not be understood as limiting the scope of implementation of the present invention.

[0023] This study designed a novel aromatic-containing chloroethylnitrosourea compound. The general molecular structure is shown in the figure below. The aromatic-containing chloroethylnitrosourea compound contains one to two β-chloroethylnitrosourea structural units. The β-chloroethyl group can greatly increase the lipophilicity of the drug, allowing the drug to be delivered to the central nervous system through the BBB to a greater extent. Electron-donating groups such as methoxy and methyl or electron-withdrawing groups such as Cl, Br, and -CN are introduced into the 2, 3, and 4 positions of the benzene ring of the aromatic-containing chloroethylnitrosourea compound, and the benzene ring is replaced with other aromatic rings. The active intermediate aziridine ion produced in vivo by the aromatic-containing chloroethylnitrosourea compound can react with DNA, leading to cell apoptosis and achieving the purpose of anti-tumor.

[0024]

[0025] -CH3,-CH2CH3; Ar=substituted phenyl,

[0026] General structural formula of chloroethyl nitrosourea compounds containing aromatic groups

[0027] The present invention provides Example 1, which prepares a series of aromatic-containing chloroethylnitrosourea compounds, named HJ01-HJ22. The Ar and R groups in HJ01-HJ22 are shown in the following table:

[0028]

[0029]

[0030] when When the aromatic group-containing chloroethyl nitrosourea compound is prepared by the following synthetic route:

[0031]

[0032]

[0033] Synthesis Route 1

[0034] When R=-CH3, -CH2CH3, the aromatic-containing chloroethyl nitrosourea compounds are prepared by the following synthetic route:

[0035]

[0036] R 1 Selected from -CH3 or -CH2CH 3。

[0037] Synthesis Route 2

[0038] The difference between Synthesis Route 1 and Synthesis Route 2 is that in Synthesis Route 2, one hydrogen atom on the amino group connected to the Ar group in the starting material xx is replaced with -CH3 or -CH2CH3. After the reaction, the resulting aromatic-containing chloroethylnitrosourea compound has one β-chloroethylnitrosourea structural unit, while the aromatic-containing chloroethylnitrosourea compound produced in Synthesis Route 1 has two β-chloroethylnitrosourea structural units. In the present invention, xx is any number from 01 to 25.

[0039] According to the above synthetic route, Example 1 provides a step-by-step method for preparing chloroethyl nitrosourea compounds containing aromatic groups:

[0040] The preparation method of A01-A25 is the same, comprising the following steps:

[0041] To a sealed tube, 1 mmol of the starting material xx, 2.5 mmol (201 mg) of 2-chloroethanol, and 0.8 mL of aqueous sodium hydroxide solution were added sequentially. After reacting at 50°C for 4 hours, the temperature was raised to 110°C and the reaction was continued with stirring for 20 hours. After cooling to room temperature, 5 mL of water was added, stirred briefly, and extracted with ethyl acetate. The organic layers were combined, extracted again with saturated sodium chloride solution, and dried over anhydrous magnesium sulfate. The mixture was concentrated under reduced pressure and purified by silica gel (100-200 mesh) column chromatography (ethyl acetate:petroleum ether = 1:4) to obtain Axx.

[0042] In the examples provided herein, A02, O3, and O5 were purchased directly from the market. Regardless of whether A02, O3, or O5 was prepared via the aforementioned synthetic route or purchased directly, C02, O3, or O5 can be obtained using the preparation methods of C01-C25 and are all within the scope of protection of the present invention. A02, O3, and O5 are N,N-bis(2-hydroxyethyl)-m-methylaniline, N,N-bis(2-hydroxyethyl)-m-chloroaniline, and N,N-bis(2-hydroxyethyl)-p-methylaniline, respectively.

[0043] The preparation methods of C01-C25 are the same, comprising the following steps:

[0044] In a 25 mL round-bottom flask, 0.44 mmol of Axx was dissolved in 5 mL of acetonitrile. Phosphorus oxychloride (202.4 mg, 1.32 mmol) was added dropwise to the flask in an ice-water bath. After complete addition, the reaction system was heated to reflux at 100°C for 2 hours. After heating was stopped, the mixture was cooled to room temperature, the acetonitrile was removed by rotary evaporation under reduced pressure, 5 mL of cold water was added, and the mixture was stirred for a short time. The mixture was extracted with dichloromethane. The combined organic layers were extracted again with saturated NaHCO₃ solution and dried over anhydrous magnesium sulfate. The crude product Bxx was obtained by concentration under reduced pressure. 0.34 mmol of Bxx and potassium phthalimide (191 mg, 1.03 mmol) were dissolved in 5 mL of DMF and reacted at 100°C overnight. The hot reaction solution was slowly poured into 10 mL of cold water (containing 10 g of ice and 1 g of potassium carbonate). After standing for 1 hour, the mixture was filtered to obtain a yellow precipitate, which was washed with distilled water and dried under vacuum to obtain Cxx as a yellow solid.

[0045] In the embodiments provided herein, B23-B25 are purchased directly from the market. Whether B23-B25 prepared by the above-mentioned synthetic route or directly purchased, C22-C25 can be obtained by the preparation method of C01-C25, and all are within the scope of protection of the present invention. B23-B25 are N-(2-chloroethyl)-N-methylaniline, N-(2-chloroethyl)-N-ethylaniline, and N-(2-chloroethyl)-N,4-dimethylaniline, respectively.

[0046] The preparation method of E01-E25 is the same, comprising the following steps:

[0047] Dissolve 0.2 mmol of Cxx in 5 mL of ethanol, add 100 mg of hydrazine hydrate (2 mmol), and reflux at 90°C for 2 hours. After the reaction is complete, allow to cool to room temperature. Filter off the white precipitate, spin-dry the ethanol in the filtrate, place the flask in an ice bath, add 8 mL of dichloromethane, filter off the white flocculent precipitate, and evaporate the dichloromethane to obtain a clear yellow oil, Dxx. Dissolve 0.18 mmol of Dxx in 3 mL of dichloromethane, dissolve chloroethyl isocyanate in 2 mL of dichloromethane, and add the dichloromethane solution of chloroethyl isocyanate dropwise to the dichloromethane solution of Dxx. Allow to react overnight at room temperature. The resulting white precipitate is washed with DCM, filtered, and dried to obtain compound Exx.

[0048] The preparation methods of HJ01-HJ25 are the same, comprising the following steps:

[0049] Exx and a mixed acid solvent (including acetic acid and acetic anhydride) were weighed into a round-bottom flask. The reaction system was maintained at 0-5°C. 15 mg of sodium nitrite was added three times over 1 hour. The reaction was continued at this temperature for 2 hours. After the reaction, the reaction system was heated to 10-15°C, 5 mL of ice water was added and stirred briefly, and the mixture was extracted with dichloromethane (5 mL x 3). The organic layers were combined, washed with saturated NaHCO₃ solution, and dried over anhydrous magnesium sulfate. HJxx was purified by silica gel (100-200 mesh) column chromatography (eluent: petroleum ether:ethyl acetate = 3:1, v / v) to obtain HJxx. Among them, the ratio of E06, acetic acid and acetic anhydride in the preparation process of HJ06 is 0.1mmol:55.6μL:277.8μL; the ratio of E15, acetic acid and acetic anhydride in the preparation process of HJ15 is 0.1mmol:18.4μL:92.1μL; the ratio of Exx, acetic acid and acetic anhydride in the preparation process of other HJxx is 0.1mmol:50μL:250μL.

[0050] In the synthesis method of HJ01-HJ25, the amount of Exx used is shown in Table 1 below:

[0051] Table 1

[0052]

[0053]

[0054] The aromatic-containing chloroethyl nitrosourea compound HJxx and its corresponding intermediates Axx, Cxx and Exx are shown in Table 2 below:

[0055] Table 2

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064] The mass, yield, and NMR spectrum of Axx are shown in Table 3 below:

[0065] Table 3

[0066]

[0067]

[0068]

[0069] The mass, yield, and NMR spectrum of Cxx are shown in Table 4 below:

[0070] Table 4

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] Exx mass, yield, and NMR spectrum are shown in Table 5 below:

[0077] Table 5

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] The mass, yield, appearance, mass spectrum and nuclear magnetic resonance spectrum of HJxx are shown in Table 6 below:

[0084] Table 6

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092] In order to verify the safety and effectiveness of the aromatic-containing chloroethylnitrosourea compounds provided by the present invention in the treatment of brain glioma, the following effect examples are provided.

[0093] Effect Example 1

[0094] Verification of anti-tumor cell activity

[0095] Methods: (1) Human breast cancer MDA-MB-231 and MCF7 cells, human lung cancer A549 and PC9 cells, human nasopharyngeal carcinoma SUNE-1 cells, human colon cancer HCT116 cells, human liver cancer HepG2 cells, and human cervical cancer Hela cells were obtained and cultured to a density of 90%. The cells were digested with trypsin, harvested, counted, and seeded into 96-well plates at 5,000 cells / well (with three blank control wells). The plates were incubated at 37°C in a 5% CO2 incubator overnight.

[0096] (2) After 24 hours, the HJ01, HJ02, HJ03 and TMZ stock solutions (concentration of 100 mM) were diluted with fresh culture medium to the corresponding final concentrations of drug-containing culture medium and added to the wells. 0.2% DMSO was used as the drug control group.

[0097] (3) After 72 hours, the culture medium was aspirated and 100 μL of complete culture medium and 10 μL of CCK-8 were added to each well. After 2 hours, the OD450 value was measured using a microplate reader. Data analysis: Cell OD value minus the OD value of the blank control well, after subtracting the background OD value, the cell survival rate was calculated. Cell survival rate = OD value of the drug-treated group / OD value of the control group (no drug) × 100%.

[0098] Table 7 Cell survival rates of different cancer cell lines under treatment with TMZ and its analogs

[0099]

[0100]

[0101] The results are shown in Table 7. At a drug concentration of 10 μM, HJ01, HJ02, and HJ03 exhibited superior inhibitory activity against lung cancer cells PC9 and A549, colorectal cancer cells HCT116, cervical cancer cells Hela, nasopharyngeal cancer cells SUNE-1, and liver cancer cells HepG2 compared to 300 μM TMZ. Furthermore, at a drug concentration of 10 μM, HJ02 and HJ03 also exhibited superior inhibitory activity against breast cancer cells MCF7 compared to 300 μM TMZ.

[0102] Effect Example 2

[0103] Verification of anti-glioma cell activity

[0104] Methods: (1) Day 1: Inoculate U251 cells (5000 cells / well) into 96-well plates.

[0105] (2) Day 2: Aspirate the culture medium and replace with drug-containing medium. TMZ and HJ series compounds were diluted into drug-containing medium at a dilution ratio of 1600 → 0 μM and 100 → 0 μM, respectively. Ten drug concentration gradients were set up, with three replicates per group. Three blank control wells were set up in each 96-well plate.

[0106] (3) Day 5: After 72 hours, 10 μL of CCK-8 was added to each well, and after another 2 hours, the OD450 value was measured using a microplate reader.

[0107] (4) Data Analysis: First, subtract the OD value of the cells from the OD value of the blank control wells to obtain the background-subtracted OD value. Then, calculate the cell viability. Cell viability = OD value of the drug-treated group / OD value of the control group (no drug) × 100%. GraphPad was used to calculate the IC50 of the drugs. The results are shown in Table 8.

[0108] Table 8 IC50 of HJ compounds

[0109]

[0110]

[0111] According to the data in Table 8, the IC50 of temozolomide (TMZ) against glioma U251 cells is 357.6±52.1 μM. Most of the compounds of the present invention have an IC50 of <5 μM against glioma U251 cells, and their activity is significantly better than TMZ. For example, the IC50 of compound HJ03 is 1.89±0.01 μM, which is 1 / 189 of that of TMZ.

[0112] Effect Example 3

[0113] Verify the ability of compound HJ03 to penetrate the blood-brain barrier

[0114] Methods: (1) TMZ and HJ03 were prepared into solutions with DMSO; theophylline was prepared into an internal standard solution of 1000 ng / mL with methanol.

[0115] (2) C57BL / 6 mice (6-8 weeks, weighing approximately 20 g) were intraperitoneally injected with a single dose of TMZ (66 mg / kg) or HJ03 (66 mg / kg). Blood was collected from the eyeballs at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, and 3 hours after injection. After blood collection, the mice were sacrificed by cervical dislocation, and the brains were immediately removed. The brain tissue was gently washed with 0.9% sodium chloride solution and immediately frozen at -20°C.

[0116] (3) Blood samples were immediately centrifuged at 3,000 rpm for 15 minutes. 100 μL of plasma was added to 10 μL of 85% orthophosphoric acid, followed by 200 μL of internal standard solution and 200 μL of 10 mM ammonium acetate buffer (pH 3.5). Subsequently, 200 μL of methanol and 200 μL of 100 mM ZnSO₄ were added. After vortex mixing for 1 minute, the sample was centrifuged at 12,000 rpm for 15 minutes, and 5 μL of the supernatant was injected into the LC-MS / MS system.

[0117] LC-MS / MS Parameters: Chromatographic conditions: Elution was performed using a Waters Acquity UPLC BEH C18 column (2.1*100 mm, particle size 1.7 μm) with a linear gradient of (A) (containing 0.1% formic acid) water and (B) (containing 0.1% formic acid) acetonitrile as the mobile phases. The elution program was as follows: 10% Phase B (initial), 10% Phase B (1 min), 10-90% Phase B (1 min), 90% Phase B (2.5 min), 90-10% Phase B (0.1 min), and 10% Phase B (1.4 min), for a total of 6 min. The flow rate was set at 0.3 mL / min, and the injection volume was 5 μL. Mass spectrometry conditions: The ion source was electrospray ionization (ESI), and the detection method was positive ion multiple reaction monitoring (MRM). Precursor ion optimization parameters: Syringe diameter: 4.61 mm, Pump flow rate: 7 uL / min, Scan mode: Q1MS, Scan rate: 200 Da / s, Scan range (Start-Stop): 100 to compound molecular weight (MW) + 30 Da. Observe for the appearance of the expected precursor ion, maintaining a response below 3*e6, and record the response. Product ion optimization parameters: Scan mode: Product Ion (MS2), Scan rate: 200 Da / s, Scan range (Start-Stop): 50 to MW + 20, Collision Energy (CE): 5. Scan at 5 eV intervals until the strongest product ion of the target compound reaches a response greater than three times that of the parent ion. Select the two to three product ions with the strongest responses as candidate products and record their m / z. The mass-to-charge ratio of the selected product ions should differ by at least 20 Da from that of the parent ion. Optimal Declustering Potential (DP) and CE optimization were performed: After entering the precursor and product ion information, data were acquired within the CE range of 5–180 eV with a step of 1 eV. The CE voltage at which the response for each MRM channel reached its maximum was recorded. The DP was then selected and, following the same method, the optimal CE value was fixed and recorded. The ion transitions used for quantitative analysis were: TMZ m / z: 195.1 → 138.1, CE: 12.5, DP: 20; HJ03 m / z: 483.1 → 276.9, CE: 17.94, DP: 27; and the internal standard, theophylline, m / z: 181.1 → 124.1, CE: 24.82, DP: 36.

[0118] (4) The whole brain was homogenized with 100 μL of internal standard solution and 200 μL of 10 mM pH 3.5 ammonium acetate buffer and 400 μL of chromatography-grade methanol. 200 μL of 100 mM ZnSO₄ was added and the mixture was centrifuged at 3,000 rpm for 10 min at 25°C. The supernatant was transferred to a 1.5 mL Eppendorf tube and centrifuged at 12,000 rpm for 15 min. 5 μL of the supernatant was injected into the LC-MS / MS system. The LC-MS / MS system parameters were the same as above.

[0119] The experimental results are shown in Table 9 and Figure 1 , showing that HJ03 and TMZ reached their maximum concentration in the blood 15 minutes after oral administration at a dose of 66 mg / kg, and in the brain at 30 minutes, indicating that HJ03 can penetrate the blood-brain barrier. Although the HJ03 content in the blood is 0.73% of that in TMZ, the content in the brain is 14.7% of that in TMZ, indicating that HJ03 has a stronger ability to penetrate the blood-brain barrier than TMZ ( Figure 1 ).

[0120] Table 9 Pharmacokinetic parameters of TMZ and HJ03

[0121]

[0122] Figure 1 (A, B) Blood-brain concentrations of TMZ and HJ03 (represented by H104 in the figure) 30 minutes after administration of 66 mg / kg. (C) Brain:plasma concentration ratios calculated using the data in (A, B). (Analyzed using a one-way T-test; *P < 0.05 indicates statistically significant, **P < 0.01 indicates statistically significant, and ***P < 0.001 indicates extremely significant.)

[0123] Effect Example 4

[0124] Verification of the biosafety of compound HJ03

[0125] Methods: (1) Grouping: Twenty-five female C57 mice (weighing between 18g and 22g) aged 8 weeks were purchased. After acclimation in the animal room for one week, all mice were randomly divided into a control group and an experimental group. The experimental groups were divided into five groups according to the different doses of HJ03 (132mg / Kg, 66mg / Kg, 20mg / Kg, and 2mg / Kg).

[0126] (2) Administration: HJ03 was prepared into a suspension of corresponding concentration using 0.5% CMC Na and administered orally once a day. The weight of the mice was recorded. The control group was given an equal amount of 0.5% CMC Na solution. The weight changes of the mice are shown in Figure 3 .

[0127] (3) Blood collection: Seven days after administration, collect 200 μL of whole blood using a 15% EDTA-dipotassium vacuum anticoagulation tube and immediately refrigerate (2-8°C). Then collect the remaining blood using an EP tube, let it stand for 30 minutes, centrifuge it at 2000-3000 rpm for 10 minutes, and then collect the serum and refrigerate (2-8°C).

[0128] (4) Testing: The collected blood samples were sent to Zhuhai Baishitong Company for testing of five indicators: white blood cell count (WBC), red blood cell count (RBC), neutrophil (Neu), lymphocyte (Lym), and platelet (PLT). The results were displayed in Figure 2 .

[0129] Figure 2 Results: Oral administration of HJ03 at doses of 2, 20, and 66 mg / kg did not show statistically significant decreases in any blood markers compared to the control group, indicating that oral administration of 66 mg / kg of HJ03 for one week did not cause bone marrow suppression in mice. However, while a dose of 132 mg / kg did not cause a decrease in red blood cell and neutrophil counts in mice, it did significantly reduce the number of white blood cells, lymphocytes, and platelets, indicating some hematotoxicity in mice.

[0130] Figure 2 The blood parameters of mice in different groups after 7 days of oral administration (analyzed by one-way T test, *P<0.05 indicates statistical difference, **P<0.01 indicates significant statistical difference, ***P<0.001 indicates extremely significant statistical difference).

[0131] Figure 3 The results showed that compared with the control group, after 7 days of continuous oral administration of HJ03, the body weight of mice in the 66 mg / Kg and 132 mg / Kg doses decreased, while the body weight of mice in the other dosing groups did not change significantly.

[0132] Effect Example 5

[0133] Verification of the in vivo antitumor activity of compound HJ03

[0134] Methods: (1) Cell culture: CT2A-luc cells were cultured in DMEM medium containing 10% FBS and 1% double antibody at 37°C and 5% CO2. When the cells grew to 90% density, they were digested with 0.25% trypsin and the cell fluid was collected. After centrifugation, the cell suspension was prepared with PBS solution.

[0135] (2) Purchase of Animals: C57BL / 6 female mice aged 6–8 weeks, weighing 17–23 g, were purchased and acclimated to the animal room for one week. Mice were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. All experiments were performed in accordance with the protocols of the Animal Care and Use Ethics Committee of the School of Medicine, Shenzhen University.

[0136] (3) Cell injection: First, the mouse was anesthetized with isoflurane gas. After anesthesia, the hair on the top of the mouse's head was removed with a skin preparation knife, and then the mouse was fixed on the operating table of the brain positioning instrument. A 3-4 mm incision was made at the midline of the mouse's brain, and the coronal suture and sagittal suture were found to determine the anterior fontanelle. The anterior fontanelle was marked as the coordinate origin, and the coordinates of the posterior fontanelle were used to adjust the front and back height of the mouse's head so that the front and back of the mouse's head remained level; then two points were marked 2 mm to the left and right of the origin, and the coordinates of these two points were used to correct the left and right height of the mouse's head so that the sides of the mouse's head also remained level. 5×10 4 CT2A-luc cells were injected at a rate of 0.5 μl / min into the mouse, 1.5 mm to the right and 1 mm below the origin, at a depth of 3.2 mm. After the injection, the mouse was weighed after 5 minutes and then placed in a thermostatic chamber to maintain warmth until awake.

[0137] (4) Tumor fluorescence imaging monitoring: Seven days after the mice were injected with CT2A-luc cells, a luciferase substrate stock solution was prepared in DPBS at a concentration of 15 mg / mL. The mice were intraperitoneally injected with the luciferase substrate stock solution at a dose of 10 μL / g. After waiting for 10 minutes, the mice were anesthetized and placed in an imaging chamber for imaging. The imaging results were used to determine whether the in situ glioma model was successfully established.

[0138] (5) Grouping and drug administration: After fluorescence imaging, all mice with successful tumor formation were randomly divided into control group,

[0139] Four groups were administered: TMZ (66 mg / kg) and HJ03 (20 mg / kg and 2 mg / kg). Both TMZ and HJ03 were suspended in 0.5% sodium carboxymethylcellulose and administered orally five days a week for four consecutive weeks. A control group received an equal volume of sodium carboxymethylcellulose solution. Mouse weights were recorded daily, and growth was observed. Tumor growth was monitored using an IVIS Spectrum imaging system, and photographs were taken to plot tumor growth curves based on the intensity of bioluminescence from the mouse heads.

[0140] (6) Survival analysis: After drug administration, the survival of mice was recorded until they died or developed severe brain damage. The number of days of survival of each deceased mouse was recorded and a survival curve was plotted.

[0141] Results: Compared with the control group, the HJ03 (20 mg / kg) group could significantly inhibit the growth of tumors and prolong the survival of mice (P=0.0008). Compared with the TMZ group (P=0.0172) and the low-dose HJ03 (2 mg / kg) (P=0.009), the HJ03 (20 mg / kg) group could significantly prolong the survival of mice ( Figure 4 A, C). As time went on, the body weight loss of mice in the HJ03 (2 mg / kg) and HJ03 (20 mg / kg) groups was less than that in the control group and TMZ group ( Figure 4 B).

[0142] (A) Bioluminescence imaging was used to measure tumor volume and growth in an orthotopic CT2A-luc cell model. (B) Mouse body weight during treatment. (C) The efficacy of HJ03 treatment was assessed using Kaplan-Meier survival curves. Statistical analysis was performed using the Log-rank test, and P values were calculated.

[0143] References

[0144] [1]Stupp Roger MD,Mason Warren P.MD,van den Bent MartinJ.Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma[J].Cancer / Radiothérapie,2005,9(3):196-197.

[0145] [2]Stupp R, Taillibert S, Kanner A, et al. Effect of Tumor-TreatingFields Plus Maintenance Temozolomide vs Maintenance Temozolomide Alone onSurvival in Patients With Glioblastoma[J]. JAMA, 2017, 318(23):2306.

[0146] [3]Herrlinger U,Tzaridis T,Mack F,et al.Lomustine-temozolomidecombination therapy versus standard temozolomide therapy in patients withnewly diagnosed glioblastoma with methylated MGMT promoter(CeTeG / NOA-09):arandomised,open-label,pHase 3trial[J].The Lancet,2019,393(10172):678-688.

[0147] [4]Pletsas D,Garelnabi EA,Li L,et al.Synthesis and quantitativestructure-activity relationship of imidazotetrazine prodrugs with activityindependent of O6-methylguanine-DNA-methyltransferase,DNA mismatch repair,andp53[J].J Med Chem,2013,56(17):7120-7132.

Claims

1. A chloroethyl nitrosourea compound containing an aromatic group, characterized in that: It has the following general structural formula: Wherein, R is selected from -CH3, -CH2CH3 or Ar is selected from benzene, substituted benzene, The substituted benzene is selected from one of 3-methylphenyl, 3-chlorophenyl, 4-isopropylphenyl, 4-methylphenyl, 4-methoxyphenyl, 3-fluorophenyl, 4-fluorophenyl, 4-chlorophenyl, 2-fluorophenyl, 3-bromophenyl, 4-bromophenyl, 4-tert-butylphenyl, 3,5-dimethylphenyl, 3-cyanophenyl, 3-isopropylphenyl, 4-ethylphenyl, 3-ethylphenyl, 3-fluoro-5-methylphenyl, and 3-fluoro-4-methylphenyl.

2. The method for preparing an aromatic-containing chloroethyl nitrosourea compound according to claim 1, wherein: The following steps are involved: R 1 -NH-Ar reacts with 2-chloroethanol to generate mono- or di-2-hydroxyethylaniline, the hydroxyl group undergoes halogenation reaction, is substituted with phthalimide, and reacts with hydrazine hydrate to convert into amino group, the amino group reacts with chloroethyl isocyanate to generate chloroethyl urea, and then undergoes nitrosation reaction to generate the aromatic group-containing chloroethyl nitrosourea compound, wherein R 1 is selected from -H, -CH3 or -CH2CH3, Ar is selected from benzene, substituted benzene, 3. A pharmaceutical composition, characterized in that The invention comprises one or more of the aromatic-containing chloroethyl nitrosourea compound and the pharmaceutically acceptable salts of the aromatic-containing chloroethyl nitrosourea compound as claimed in claim 1.

4. The pharmaceutical composition according to claim 3, wherein The pharmaceutical composition is an oral preparation or an injection.

5. The pharmaceutical composition according to claim 4, wherein The injection is an intravenous injection preparation, an intraperitoneal injection or a subcutaneous injection.

6. Use of the pharmaceutical composition according to claim 3 in preparing anti-tumor drugs.

7. Use of the pharmaceutical composition as claimed in claim 6 for preparing a drug for treating anti-tumor, characterized in that: The tumor includes one or more of breast cancer, lung cancer, nasopharyngeal cancer, colon cancer, liver cancer, cervical cancer and brain glioma.

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