Synthesis method of quinazolinyl-aryl urea derivative A-2 and application of quinazolinyl-aryl urea derivative A-2 in preparation of bladder cancer resisting medicine

Abstract

The invention discloses a synthesis method of a quinazolinyl-aryl urea derivative A-2 and application of the quinazolinyl-aryl urea derivative A-2 in preparation of bladder cancer resistant drugs, and the synthesis method of the compound A-2 comprises the following steps: (1) reducing nitryl by adopting an ammonium chloride aqueous solution and activated iron powder, and (2) reacting an intermediate II with 2, 6-diethyl phenyl isocyanate to obtain the quinazolinyl-aryl urea derivative A-2, according to the application of the compound A-2 in preparation of the bladder cancer resisting medicine, an oral preparation of the compound A-2 can be applied to treatment of middle and high risk non-muscular layer invasive bladder cancer, and the oral preparation of the compound A-2 can be independently used and can also be used before or after an operation, so that the treatment effect is achieved, the recurrence rate is effectively reduced, and the compliance of a patient is improved; the post-treatment process of the synthesis method of the compound A-2 is simple and easy to operate, and the yield of the intermediate II is as high as 94%.

Description

Technical Field

[0001] This invention relates to synthetic pharmaceutical chemistry and pharmaceutical formulation technology, specifically a method for synthesizing a quinazolinyl-arylurea derivative A-2 and its application in the preparation of anti-bladder cancer drugs. Background Technology

[0002] Bladder cancer (BLCA) is a common malignant tumor of the urinary system. With the aging population, changes in lifestyle, and increased environmental pollution, the incidence of bladder cancer is increasing year by year, posing a serious threat to the quality of life of patients. Based on whether it invades the muscle layer, bladder cancer is divided into non-muscleinvasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC) (Lobo N, Afferi L, Moschini M, et al. Eur Urol Oncol, 2022, 5(6): 628-639). Non-malignant bladder cancer (NMIBC) refers to malignant tumors confined to the bladder mucosa, formerly known as superficial bladder cancer. Approximately 75% of newly diagnosed bladder cancers are NMIBC (Sanli O, Dobruch J, Knowles MA, et al. Nat Rev DisPrimers, 2017, 3: 17022; Lenis AT, Lec PM, Chamie K, et al. JAMA, 2020, 324(19): 1980-1991). NMIBC includes tumors in the Ta, Tis, and T1 stages of the TNM staging system. Ta stage papillary urothelial carcinoma presents as papillary structures confined to the bladder mucosa. Although these tumors have a tendency to recur, they usually do not have the characteristic of invading the muscle layer. Tis stage corresponds to carcinoma in situ (CIS), which is a high-grade lesion confined to the bladder mucosa and has a high risk of invading the muscle layer. T1 stage is when the tumor further invades the submucosa of the bladder and may even reach the lamina propria. Although it has not yet invaded the muscle layer, its invasiveness and progression risk are higher than Ta and CIS due to the rich blood supply in the lamina propria. Based on the different risks of recurrence and prognosis, NMIBC can be divided into three categories: low, intermediate and high risk. Some guidelines further subdivide the high-risk category into an extremely high-risk subclass (Babjuk M, Burger M, Capoun O, et al. EurUrol, 2022, 81(1): 75-94).

[0003] Transurethral resection of bladder tumor (TURBT) is the preferred treatment for non-MIBC (Babjuk M, Burger M, Capoun O, et al. Eur Urol, 2022, 81(1): 75-94). However, the high recurrence rate after the procedure seriously affects the prognosis of patients. If patients do not continue treatment after TURBT, about 60% of them will relapse and 10% will progress to MIBC (Lenis AT, Lec PM, Chamie K, et al. JAMA, 2020, 324(19): 1980-1991). For intermediate- to high-risk NMIBC patients, TURBT combined with intravesical adjuvant therapy is a common treatment option. Adjuvant therapy includes: (1) immunotherapy, such as Bacillus Calmette-Guérin (BCG); (2) cytotoxic drug therapy, such as mitomycin C, epirubicin, gemcitabine, pirarubicin, 5-fluorouracil, docetaxel, hydroxycamptothecin, etc.; (3) cytotoxic drugs combined with hyperthermia (Peyton CC, Chipollini J, Azizi M, et al. World J Urol, 2019, 37(10): 2017-2029). TURBT combined with postoperative intravesical BCG instillation is currently the preferred treatment for intermediate- to high-risk MNIBC (Lopez-Beltran A, Cookson MS, Guercio BJ, et al. BMJ, 2024, 384: e076743), but this does not mean that this option is suitable for all intermediate- to high-risk MNIBC patients. Studies have found that 20% to 50% of patients eventually relapse after BCG instillation, and 10% of patients experience progression, eventually requiring radical cystectomy (Cambier S, Sylvester RJ, Collette L, et al. Eur Urol, 2016, 69(1): 60-69).In addition, cystitis and systemic toxicity often occur after BCG instillation (Yates DR, Brausi MA, Catto JW, et al. Eur Urol, 2012, 62(6): 1088-1096); in recent years, its supply has been continuously scarce, the price is expensive, and the access channels are limited (Bandari J, Maganty A, MacLeod LC, et al. Eur Urol Focus, 2018, 4(4): 481-484), and many patients have to give up treatment (Ojea A, Nogueira JL, Solsona E, et al. Eur Urol, 2007, 52(5): 1398-1406).

[0004] The following is a brief introduction to three types of human bladder cancer cells (5637, RT4, and J82). According to literature reports (Earl J, Rico D, Carrillo-de-Santa-Pau E, et al. BMC Genomics, 2015, 16(1): 403; Zuiverloon TCM, de Jong FC, Costello JC, et al. Bladder Cancer, 2018, 4(2):169-183), 5637 and RT4 cells are highly differentiated, grow slowly, have a low risk of metastasis, and are of low malignancy. Among them, the bladder cancer patients who provided 5637 cells had a pathological malignancy grade of G2, which is a moderately differentiated tumor. RT4 cells also have a low malignancy grade of G1 and a pathological stage of pT1. Unlike the former two, patients who provided J82 cells had a high degree of tumor malignancy, grade G3, and a pathological stage of pT3, indicating that the primary tumor was large or had broken through the boundary of the primary organ / tissue and invaded adjacent structures. 5637 cells to some extent represent high-risk NMIBC, while J82 cells represent metastatic bladder cancer. In April 2019, the US FDA approved the first small molecule targeted drug, erdafitinib, for patients with locally advanced or metastatic bladder cancer carrying FGFR3 or FGFR2 mutations who have progressed during or after at least one cycle of prior platinum-based chemotherapy (Loriot Y, Necchi A, Park SH, et al. N Engl J Med, 2019, 381(4): 338-348). Erdafitinib has poor water solubility and is currently only available in oral solid dosage form.

[0005] Due to the lack of effective conservative treatment options, radical cystectomy (RC) remains the standard surgical procedure for patients with recurrent bladder cancer who have failed instillation therapy (Carando R, Shariat SF, Moschini M, et al. Curr Opin Urol, 2020, 30(3): 441-448). However, a significant proportion of recurrent patients refuse radical cystectomy in clinical practice, primarily due to the high risk of complications such as urinary fistula, bladder contracture, and voiding dysfunction. Postoperatively, long-term indwelling catheters or urine bags may be necessary, leading to severe psychological burden and emotional problems that affect quality of life. Therefore, for intermediate- to high-risk NMIBC patients who are insensitive to bladder instillation drugs such as BCG and are unwilling or unable to undergo cystectomy, there is an urgent need to find alternative treatment options that are well-compliant, can not only treat bladder cancer but also effectively prevent and control recurrence. Are there any options such as: gradually shrinking or even eliminating the tumor through oral medication while preserving the bladder, or combining oral medication with surgery to treat intermediate- to high-risk NMIBC? Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a method for synthesizing a quinazolinyl-arylurea derivative A-2 and its applications. This synthetic method is simple and efficient. The synthesized quinazolinyl-arylurea derivative A-2 can be used to prepare oral formulations with anti-bladder cancer activity. These formulations are suitable for intermediate- and high-risk non-malignant bladder cancer (NMIBC), and can not only exert a therapeutic effect but also reduce the recurrence rate of bladder cancer and improve patient treatment compliance.

[0007] The technical solution to achieve the objective of this invention is:

[0008] A method for synthesizing a quinazolinyl-arylurea derivative A-2, wherein the structural formula of the quinazolinyl-arylurea derivative A-2 is as follows: A-2, The method is characterized in that it comprises: (1) Raw material I undergoes a nitro reduction reaction under reducing agent and heating conditions to obtain intermediate II. The structural formula of raw material I is: , The structural formula of intermediate II is: , Among them, the preparation of raw material I adopts the synthetic method of compound 7 described in "He Yuezhen. Design and synthesis of 6-pyridylpiperazine modified quinazolinyl-arylurea derivatives and their anti-bladder cancer activity. 2022. Guangxi Normal University, MA thesis. doi:10.27036 / d.cnki.ggxsu.2022.002175"; (2) Intermediate II was reacted with 2,6-diethylisocyanate to obtain quinazolinyl-arylurea derivative A-2, which is the target product.

[0009] The reducing agent mentioned in step (1) is a combination of activated iron powder and ammonium chloride aqueous solution with a concentration of 5wt%~20wt%; the molar ratio of raw material I, activated iron powder and ammonium chloride is 1:(5~10):(1~2.5), the reaction system also includes solvent methanol, the reaction temperature is 55~65 ℃ and the reaction time is 8~16 h, wherein the activated iron powder is obtained by treating reduced iron powder with 10wt%~20wt% hydrochloric acid aqueous solution.

[0010] The above-mentioned method for synthesizing the quinazolinyl-arylurea derivative A-2 and its application in the preparation of anti-bladder cancer drugs, wherein the application is to prepare an oral formulation with anti-bladder cancer activity, wherein the oral formulation is composed of A-2 as the main drug and suitable excipients; suitable excipients include pharmaceutically acceptable excipients and / or additives, wherein pharmaceutically acceptable excipients may be at least one of diluents, binders, disintegrants, and lubricants; and pharmaceutically acceptable additives may be at least one of preservatives, cosolvents, solubilizers, suspending agents, and emulsifiers.

[0011] The above-mentioned quinazolinyl-arylurea derivative A-2 is used in the preparation of anti-bladder cancer drugs, wherein the oral formulation with anti-bladder cancer activity is one of tablets, capsules, suspensions, solutions, emulsions, or syrups.

[0012] The application of the above-mentioned quinazolinyl-arylurea derivative A-2 in the preparation of anti-bladder cancer drugs is characterized by the application of the oral formulation with anti-bladder cancer activity in the treatment of non-muscle-invasive bladder cancer.

[0013] The application of the above-mentioned quinazolinyl-arylurea derivative A-2 in the preparation of anti-bladder cancer drugs is characterized by the application of the oral formulation with anti-bladder cancer activity in the treatment of non-muscle-invasive bladder cancer.

[0014] In this technical solution, the diluent is at least one of starch, pregelatinized starch, microcrystalline cellulose, dextrin, sucrose, lactose, mannitol, sorbitol, and calcium sulfate dihydrate. The adhesive is one of the following: starch paste, sodium carboxymethyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, gelatin, polyvinylpyrrolidone, and sodium alginate; The disintegrant is one of dry starch, sodium carboxymethyl starch, sodium croscarmellose, low-substituted hydroxypropyl cellulose and croscarmellose. The lubricant is one of magnesium stearate, micronized silica gel, talc, polyethylene glycol, and sodium dodecyl sulfate; In this technical solution, the dispersion medium in the suspension and solution is at least one of polyethylene glycol 300 (PEG300), PEG400, N,N-dimethylacetamide, dimethyl sulfoxide, purified water, physiological saline, and 5% glucose aqueous solution; The preservatives used in suspensions and solutions are at least one of methylparaben, ethylparaben, propylparaben, butylparaben, benzoic acid, sodium benzoate, sorbic acid, benzyl alcohol, chlorobutanol and chlorocresol. If the oral formulation in this technical solution is unstable under gastric conditions, it can be made into enteric-coated tablets or enteric-coated capsules.

[0015] The oral formulation with A-2 as the main drug in this technical solution is for bladder cancer in malignant tumors, especially intermediate- and high-risk NMIBC. The dosing regimen is to administer the A-2 formulation orally alone, or to administer the A-2 formulation before or after bladder tumor resection.

[0016] The prior art is the published thesis "He Yuezhen. Design and synthesis of 6-pyridylpiperazine-modified quinazolinyl-arylurea derivatives and their anti-bladder cancer activity. Guangxi Normal University, MA thesis. 2022." The document doi:10.27036 / d.cnki.ggxsu.2022.002175 describes compound 9g (i.e. compound A-2 in this technical solution), whose synthesis is divided into 7 steps, which are relatively complex. The synthesis method of raw material I (i.e. compound 7 in the above dissertation) has been described in detail, so it will not be repeated here. How to synthesize intermediate II (i.e. compound 8 in the above dissertation) more efficiently from raw material I can be compared with the following two reduction methods: (1) combination of activated iron powder and 15wt% hydrochloric acid; (2) combination of activated iron powder and 10wt% ammonium chloride aqueous solution. The second method is significantly better than the first method because the post-processing of the first method is more troublesome. A large amount of alkali is required when adjusting the pH value. The improved method (2), i.e., 10wt% ammonium chloride solution instead of dilute hydrochloric acid, can be used to carry out the reaction in a milder, weakly acidic environment. After the reaction, only a small amount of weak alkali (e.g., sodium carbonate) aqueous solution needs to be added to neutralize it. Compared with the first method, the amount of alkali used is significantly reduced, and the post-processing steps become easier. Intermediate II reacts with phenyl 2,6-diethylisocyanate to give the target product A-2.

[0017] Compared with existing technologies, this technical solution has the following advantages:

[0018] 1. This technical solution simplifies the post-processing of intermediate II by following the improved synthesis method; 2. Compound A-2 in this technical solution can be easily formulated into an oral preparation. This route of administration can replace the bladder instillation method that is widely used in clinical practice, greatly improving patient medication compliance. 3. The synthesized quinazolinyl-arylurea derivative A-2 is used to prepare a pharmaceutical formulation with anti-bladder cancer activity, which is suitable for the treatment of intermediate- to high-risk NMIBC; the formulation can be used alone or before bladder tumor resection to reduce tumor size, or after bladder tumor resection to reduce recurrence rate.

[0019] This synthetic method is simple and efficient. The synthesized quinazolinyl-arylurea derivative A-2 is used to prepare an oral formulation with anti-bladder cancer activity. This formulation is suitable for intermediate- and high-risk NMIBC and can not only exert a therapeutic effect but also reduce the recurrence rate of bladder cancer and improve patient treatment compliance. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the synthetic route for compound A-2 in the examples;

[0021] Figure 2For example, raw material I 1 H NMR spectrum;

[0022] Figure 3 For intermediate II in the embodiment 1 H NMR spectrum;

[0023] Figure 4 For intermediate II in the embodiment 13 C NMR spectrum;

[0024] Figure 5 The HRMS spectrum of raw material I in the example is shown below;

[0025] Figure 6 The HRMS spectrum of intermediate II in the embodiment is shown below.

[0026] Figure 7 The image shows the HRMS spectrum of compound A-2 in the examples;

[0027] Figure 8 The graph shows the proliferation inhibition activity of compound A-2 after incubation with four types of tumor cells for different times in the examples.

[0028] Figure 9 The diagram shows the cell cycle arrest induced by compound A-2 in bladder cancer 5637 cells in the example.

[0029] Figure 10 In this example, compound A-2 inhibited tumor growth in a nude mouse xenograft model of bladder cancer (5637 model). Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this is not intended to limit the scope of the invention.

[0031] Example:

[0032] The synthetic method of N-(2,6-diethylphenyl)-N'-(3-(((7-methoxy-6-((6-(4-(pyridin-2-yl)piperazin-1-yl)hexyl)oxy)quinazolin-4-yl)amino)methyl)phenyl)urea (compound A-2), such as Figure 1 As shown, it includes the following steps:

[0033] (1) The structural formula of N-(3-aminobenzyl)-7-methoxy-6-((6-(4-(pyridin-2-yl)piperazin-1-yl)hexyl)oxy)quinazolin-4-amine (intermediate II) is: The synthesis is as follows: A 10% (w / w) ammonium chloride aqueous solution is prepared beforehand: 0.56 g (10.5 mmol) of ammonium chloride is dissolved in 5.04 g of deionized water. The reduced iron powder needs to be activated: after removing the iron powder, a 15% (w / w) hydrochloric acid aqueous solution is added, stirred, and the supernatant is slowly poured off. The resulting solid is activated iron powder (2.74 g, 49 mmol). In a round-bottom flask, raw material I (4.00 g, 7.0 mmol) and methanol (95 mL) are added sequentially, stirred to dissolve, and then the activated iron powder and ammonium chloride aqueous solution are added. The mixture is stirred for 5 min, then heated to 60 °C and stirred for 12 h. After the reaction is complete, the mixture is cooled to room temperature, filtered to remove insoluble matter, and the residue is repeatedly washed with methanol. The filtrate is collected, concentrated, and the pH is adjusted to 9-10 with sodium carbonate aqueous solution. The mixture is extracted three times with ethyl acetate, and the organic phases are combined. After removing the solvent, the crude product is obtained. The crude product was purified by column chromatography to obtain a white solid, namely intermediate II (3.56 g, yield: 94%), which is the product of raw material I. 1 The H NMR and HRMS spectra are as follows: Figure 2 and Figure 5 As shown; intermediate II 1 H NMR, 13 The C NMR and HRMS spectra are as follows: Figure 3 , Figure 4 and Figure 6 As shown; (2) The synthesis of N-(2,6-diethylphenyl)-N'-(3-(((7-methoxy-6-((6-(4-(pyridin-2-yl)piperazin-1-yl)hexyl)oxy)quinazolin-4-yl)amino)methyl)phenyl)urea (target compound A-2) was as follows: Dry intermediate II (189.6 mg, 0.35 mmol), 20 mL of dichloromethane, 2,6-diethylisocyanate (245 mg, 1.4 mmol) and triethylamine (0.5 mL) were added sequentially to a round-bottom flask and stirred at room temperature for 48 h. After the reaction was completed, the insoluble matter was removed by filtration, the mixture was concentrated by vacuum distillation, and purified by column chromatography to obtain a white solid, namely compound A-2 (151 mg, yield 60%). The characterization of compound A-2 was as follows: 1H NMR (400 MHz, CD3OD) δ 8.40 (s, 1H), 8.16–8.12 (m, 1H), 7.71 (s,1H), 7.66–7.63 (m, 1H), 7.62–7.58 (m, 1H), 7.31–7.20 (m, 2H), 7.19–7.13 (m,1H), 7.12–7.01 (m, 4H), 6.91 (dt, J = 8.6, 0.9 Hz, 1H), 6.81–6.74 (m, 1H), 4.73 (s, 2H), 4.21–4.10 (m, 1H), 4.04–3.97 (m, 2H), 3.95 (s, 3H), 3.83 (s,3H), 3.76–3.63 (m, 1H), 3.62–3.46 (m, 1H), 3.42–3.32 (m, 5H), 3.17–3.11 (m,2H), 2.68–2.52 (m, 4H), 1.91–1.80 (m, 2H), 1.57–1.43 (m, 4H), 1.42–1.34 (m,2H), 1.12 (t, J = 7.6 Hz, 6H). 13 C NMR (100 MHz, CD3OD) δ 158.29, 156.23,153.97, 149.65, 147.33, 146.90, 145.27, 142.41, 138.91, 138.08, 135.37,133.94, 130.64, 128.71, 126.06, 123.80, 121.70, 119.34, 119.00, 114.57,109.17, 108.04, 103.60, 67.76, 56.73, 55.67, 54.02, 51.37, 42.62, 28.27,25.93, 25.18, 24.65, 23.60, 16.44. HRMS(m / z): calcd. for C 42 H 52 N8O3 [M+H] + :717.4241; found: 717.4231. The HRMS spectrum of compound A-2 is as follows: Figure 7 As shown.

[0034] Experimental Example 1:

[0035] Evaluation of the inhibitory activity of compound A-2 on tumor cell proliferation:

[0036] For compound A-2, sensitive cells were screened using the following method: All cells used in this experiment were human-derived, including: bladder cancer cells (5637, RT4, J82), cervical cancer cells HeLa, osteosarcoma cells 143B, ovarian cancer cells SK-OV-3, gastric cancer cells MGC-803, and normal cells (bladder epithelial cells HCV-29, embryonic lung fibroblasts WI-38, and bronchial epithelial cells BEAS-2B).

[0037] Evaluation method for cell proliferation inhibition activity: The CCK-8 assay (CCK-8 full name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonylbenzene)-2H-tetrazole monosodium salt) was used. Cells in the logarithmic growth phase were collected, and after adjusting the cell density, they were seeded into 96-well plates with approximately 5000 cells per well. A zero-adjustment well (containing only basal medium, CCK-8, and DMSO) was also set up. Cells were incubated in a 5% CO2 incubator at 37 ℃. After the cells reached 70% confluence, the sample (compound A-2 or erdatinib) was added at final concentrations of 1, 2, 4, 6, 8, 12, 16, 24, and 30 μM, with three replicates for each concentration. The sample was pre-prepared as a sterile DMSO stock solution, then diluted with medium, and 20 μL was added to each well. After incubation for the set time, 10 µL of CCK-8 solution was added to each well, and incubation continued for another 40 minutes. Incubation time was 1 minute to 2 hours (incubation time varies depending on the cell type), and the culture was terminated. The OD value of each well was measured at 450 nm using a multi-functional microplate reader. Based on the measured OD values, the inhibition rate was calculated using the formula: Inhibition rate = (OD value of control group - OD value of drug-treated group) / OD value of control group × 100%. Based on the inhibition rate at each concentration, the half-maximal inhibitory concentration (IC50) was calculated using Origin v8.0 software. 50 value);

[0038] The experimental results are shown in Tables 1, 2, and 3. Figure 8 As shown, bladder cancer cells 5637 and RT4 are sensitive to compound A-2, especially after co-incubation of A-2 with 5637 cells for 48 hours, its IC50 value is significantly reduced. 50 The IC50 value was very low, at 1.80 ± 0.22 μM, demonstrating that 5637 cells were particularly sensitive to the target compound A-2; moreover, the IC50 value decreased with prolonged incubation time. 50 The value decreases, showing a time-dependent effect. Figure 8Compound A-2 showed significantly stronger inhibitory activity against proliferation than the positive control erdatinib. Compared to the two cell types mentioned above, J82 cells showed lower sensitivity to A-2. Besides 5637 cells, 143B cells were also sensitive to compound A-2, and its inhibitory activity was significantly stronger than erdatinib. Compared to 5637 and 143B cells, HeLa cells were more sensitive to compound A-2. Furthermore, compound A-2 exhibited weak inhibitory activity against tumor cells SK-OV-3 and MGC-803, as well as three normal cell types (HCV-29, WI-38, and BEAS-2B). In summary, compound A-2 demonstrated excellent inhibitory activity against proliferation in 5637 cells, and this activity showed high selectivity.

[0039] Table 1. Inhibitory activity (IC50) of compounds A-2 and erdatinib against three types of bladder cancer cells (5637, RT4, and J82). 50 ± standard deviation; unit: μM) a 5637 RT4 J82 Compound A-2 1.80±0.22 3.68±0.48 5.98±0.43 Erdatinib 12.32±0.70 9.84±0.42 19.35±1.18

[0040] a With half-maximal inhibitory concentration (IC50) 50 The IC50 value describes the inhibitory activity of compounds A-2 and erdatinib against the proliferation of tumor (or normal) cells and is expressed as an IC50 value. 50 Mean ± standard deviation (IC 50 (± standard deviation), unit: μM; unless otherwise specified, the co-incubation time of the compound with cells is 48 h, the same below;

[0041] Table 2. Inhibitory activity of compounds A-2 and erdatinib against the proliferation of HeLa, 143B, SK-OV-3, and MGC-803 cells (IC50) 50 ± standard deviation; unit: μM) HeLa 143B SK-OV-3 MGC-803 Compound A-2 5.99±0.85 2.86±0.26 15.38±0.19 21.34±1.50 Erdatinib <![CDATA[– b ]]> 7.42±0.61 26.69±2.47 19.35±1.33

[0042] b "–" indicates that the proliferation-inhibiting activity of this compound was not detected, and the same applies below;

[0043] Table 3. Inhibitory activity (IC50) of compounds A-2 and erdatinib against three normal cell types (WI-38, HCV-29, and BEAS-2B). 50 ± standard deviation; unit: μM) WI-38 HCV-29 BEAS-2B Compound A-2 15.66±1.16 17.17±1.83 19.73±2.84 Erdatinib – – 12.17±1.66 .

[0044] Experimental Example 2:

[0045] Effect of compound A-2 on cell cycle distribution in 5637 cells:

[0046] 5637 cells in logarithmic growth phase were seeded into cell culture dishes. After 70% confluence, the culture medium was replaced, and compound A-2 or the cell cycle arrestor paclitaxel was added. Incubation was stopped after the specified time. Cells were digested, collected, centrifuged, and washed twice with pre-chilled phosphate-buffered saline (PBS). Pre-chilled 75% ethanol was added dropwise under vortex conditions for fixation, and the cells were incubated overnight at -20 °C. The cells were centrifuged for 10 min, and the supernatant was discarded. Cells were washed again with PBS and centrifuged. Following the instructions of the kit (Shanghai Beyotime Biotechnology Co., Ltd., product number: C1052), each sample required 0.5 mL staining buffer, 25 μL propidium iodide staining solution, and 10 μL RNase A. After adding these solutions to each cell sample tube, the cells were incubated at 37 °C in the dark for 30 min, with gentle shaking every 10 min. After incubation, the cells were filtered through a 200-mesh filter and transferred to flow cytometry tubes. BD... FACSVerse flow cytometry analysis was performed, and each experiment was repeated three times.

[0047] Experimental results are as follows Figure 9 As shown, Figure 9 The asterisks indicate the percentage of cells in the S phase compared to the control group. *p < 0.05, **p < 0.01. Therefore, compound A-2 arrests the cell cycle of 5637 cells in the S phase, while paclitaxel arrests the cell cycle in the G2 / M phase. Compound A-2 has a synergistic effect when used in combination with paclitaxel. Thus, compound A-2 can inhibit the proliferation of 5637 cells by arresting the cell cycle.

[0048] Experimental Example 3:

[0049] Evaluation of the tumor-suppressive activity of compound A-2 in the 5637 nude mouse xenograft model of bladder cancer:

[0050] Experimental methods: 5637 cells in logarithmic growth phase were digested with trypsin, collected, centrifuged, and the original culture medium was discarded. The cells were resuspended in serum-free DMEM medium and the cell density was adjusted. Five-week-old BALB / c-nu mice (half male, half female, weighing 18-20 g) were used. 0.2 mL of the cell suspension was subcutaneously inoculated into the nude mice in a sterile laminar flow hood. After inoculation, bleeding at the inoculation site was observed. The inoculation was continued until the tumor grew to 30-50 mm. 3Nude mice with good tumor growth were randomly divided into 5 groups. The tumor volume and weight were recorded and marked as day 0. There were 6 or 7 mice in each group during the experiment. The administration method was gavage. The grouping and dosage were as follows: (1) Blank control group, given physiological saline; (2) Positive control group, given a mixture of erdatinib polyethylene glycol 400 (PEG400) and physiological saline (volume ratio 3:1), dose: 24 mg / kg; (3) Low dose group of compound A-2, 12 mg / kg; (4) Medium dose group of compound A-2, 24 mg / kg; (5) High dose group of compound A-2, 36 mg / kg. Each group was given the drug once every 2 days. After the drug administration was completed, the nude mice were observed for symptoms such as diarrhea, loss of appetite, anorexia, and allergies (e.g., skin itching). The nude mice were weighed and the long and short diameters of the tumors were measured with calipers. The observation was continued for 60 days. The formulas for calculating tumor volume and growth inhibition rate are as follows: (1) Tumor volume = 1 / 2 × major diameter × (minor diameter) 2 (2) Tumor growth inhibition rate = (average tumor weight of negative control group - average tumor weight of treatment group) / average tumor weight of negative control group × 100%. Experimental results are as follows: Figure 10 As shown in the figure, the asterisks represent: *p < 0.05, ***p < 0.001, compared to the blank control group. Preparation of compound A-2 and erdatinib oral administration formulation: Weigh the above compounds, add pharmaceutical grade PEG400, sonicate, then add physiological saline (PEG400 to physiological saline volume ratio 3:1), and then sterilize under ultraviolet light for 30 min. Experimental results and conclusions: (1) Compound A-2 showed good antitumor activity when administered by gavage at a dose of 24 mg / kg or above. In particular, after continuous administration of high dose (36 mg / kg), the tumors showed a gradual shrinking trend. After 60 days of administration, the tumors of 3 out of 6 nude mice were not visible to the naked eye, showing excellent antitumor activity. After dissection, no tumor migration or metastasis was found in the A-2 administration group, while the tumors in the erdatinib administration group continued to grow, indicating that the activity of compound A-2 in inhibiting tumor growth was significantly stronger than that of erdatinib. (2) Compound A-2 did not cause symptoms such as decreased appetite, diarrhea, or itching during administration, indicating that nude mice tolerated the above-mentioned dosage well. (3) Based on the results of this group, combined with literature reports (Earl J, Rico D, Carrillo-de-Santa-Pau E, et al. BMC Genomics, 2015, 16(1): 403; Zuiverloon TCM, de Jong FC, Costello JC, et al. Bladder Cancer, 2018, 4(2): 403), (169-183) Speculation: Compound A-2 holds promise as a treatment for intermediate- to high-risk NMIBC patients. For example, by formulating compound A-2 into an oral formulation and administering it continuously to intermediate- to high-risk NMIBC patients, the tumor could gradually shrink and even disappear. This would be a novel treatment option with the advantages of preserving the bladder and avoiding surgery, replacing the traditional "surgical treatment + bladder instillation" approach. This alternative would significantly improve patient compliance, reduce the burden on patients, and improve their quality of life. Another application of the oral formulation of A-2 is preoperative administration, which helps reduce the tumor size and facilitates subsequent surgical treatment. A third application is for patients who do not respond to or relapse after bladder instillation with drugs such as BCG, combining surgical treatment with oral A-2 can reduce the recurrence rate of bladder tumors. In addition, experiments have shown that compound A-2 has significantly higher solubility in various mixed solvents (e.g., PEG400 + saline, PEG400 + PBS, PEG400 + 5% glucose injection) than the positive control erdatinib; erdatinib has poor solubility in the aforementioned solvents.

[0051] Experiment Example 4:

[0052] Formulation and preparation of A-2 ordinary compressed tablets (1000 tablets)

[0053]

prescription

[0054] A-2 100 g

[0055] 170 g lactose

[0056] 200 g of microcrystalline cellulose

[0057] Sodium carboxymethyl starch 20 g

[0058] 10 g of micronized silica

[0059] [Preparation] The powder direct compression method is adopted: A-2, lactose, microcrystalline cellulose and sodium carboxymethyl starch are pulverized and sieved according to the prescription amount, and mixed evenly; finally, micronized silica gel is added, mixed evenly again, and compressed into tablets to obtain the product.

Claims

1. A method for synthesizing quinazolinyl-arylurea derivative A-2, wherein the structural formula of quinazolinyl-arylurea derivative A-2 is: A-2， Its features are, The synthesis method includes: (1) Raw material I undergoes a nitro reduction reaction under reducing agent and heating conditions to obtain intermediate II. The structural formula of raw material I is: , The structural formula of intermediate II is: , Among them, the preparation of raw material I adopts the synthetic method of compound 7 described in "He Yuezhen. Design and synthesis of 6-pyridylpiperazine modified quinazolinyl-arylurea derivatives and their anti-bladder cancer activity. 2022. Guangxi Normal University, MA thesis. doi:10.27036 / d.cnki.ggxsu.2022.002175"; (2) Intermediate II was reacted with 2,6-diethylisocyanate to obtain quinazolinyl-arylurea derivative A-2, which is the target product.

2. The method for synthesizing the quinazolinyl-arylurea derivative A-2 according to claim 1, characterized in that, The reducing agent mentioned in step (1) is a combination of activated iron powder and ammonium chloride aqueous solution with a concentration of 5wt%~20wt%. The molar ratio of raw material I, activated iron powder and ammonium chloride is 1:(5~10):(1~2.5). The reaction system also includes methanol as a solvent. The reaction temperature is 55~65 ℃ and the reaction time is 8~16 h. The activated iron powder is obtained by treating reduced iron powder with 10wt%~20wt% hydrochloric acid aqueous solution.

3. The use of the quinazolinyl-arylurea derivative A-2 prepared by the synthetic method of any one of claims 1 or 2 in the preparation of anti-bladder cancer drugs, characterized in that, The application is to prepare an oral formulation with anti-bladder cancer activity, wherein the oral formulation is composed of A-2 as the main drug and suitable excipients; suitable excipients include pharmaceutically acceptable excipients and / or additives, and pharmaceutically acceptable excipients may be at least one of diluents, binders, disintegrants, and lubricants; pharmaceutically acceptable additives may be at least one of preservatives, cosolvents, solubilizers, suspending agents, and emulsifiers.

4. The use of the quinazolinyl-arylurea derivative A-2 according to claim 3 in the preparation of an anti-bladder cancer drug, characterized in that, The oral preparation with anti-bladder cancer activity is one of the following: tablets, capsules, suspensions, solutions, emulsions, or syrups.

5. The use of the quinazolinyl-arylurea derivative A-2 according to any one of claim 3 or claim 4 in the preparation of an anti-bladder cancer drug, characterized in that, The application of the oral formulation with anti-bladder cancer activity in the treatment of non-muscle-invasive bladder cancer.

6. The use of the quinazolinyl-arylurea derivative A-2 according to any one of claim 3 or claim 4 in the preparation of an anti-bladder cancer drug, characterized in that, The application of the oral formulation with anti-bladder cancer activity in the treatment of intermediate- to high-risk non-muscle-invasive bladder cancer.

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