A quinazolinone derivative for inducing ferroptosis and a synthesis method and application thereof

Abstract

The application discloses a quinazolinone derivative for inducing ferroptosis and a synthesis method and application thereof, and belongs to the technical field of biological medicines. The structural feature of the quinazolinone derivative is that specific substituents are introduced into a quinazolinone nucleus, and the quinazolinone derivative belongs to a novel ferroptosis inducer. The C-N bond is constructed by using an innovative ball milling preparation technology, the use of a large amount of organic solvents is avoided, the synthesis steps are greatly simplified, and the preparation cost is saved. In vitro experiments show that the derivative has a significant effect of inducing cell death on kidney cancer, prostate cancer, liver cancer and lung cancer cell lines, is 5 times better than a positive control drug Erastin, and proves the effect of inhibiting tumor growth in animal experiments. Advantages of the application are as follows: 1. The quinazolinone ferroptosis inducer is created for the first time, and the structural limitations of existing Erastin compounds are broken through; 2. The ball milling technology realizes solvent-free and normal-temperature drug synthesis, and meets the green chemistry standard; and 3. The derivative has broad-spectrum antitumor activity and low toxicity, and has clinical transformation potential.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a quinazolinone derivative that induces ferroptosis, its synthesis method, and its application. Background Technology

[0002] Ferroptosis is an iron-dependent, novel programmed cell death mechanism distinct from apoptosis and necrosis, caused by the accumulation of lipid peroxides. It can lead to damage and degenerative diseases in various organs. Furthermore, drug-resistant tumor cells, especially those in a mesenchymal state and prone to metastasis, are highly sensitive to ferroptosis. Therefore, inducing and inhibiting ferroptosis through drugs holds great potential in overcoming tumor drug resistance and treating lipid peroxidation-related degenerative diseases and ischemic organ damage. The cystine transporter SLC7A11 regulates the transport of cystine / glutamate by System Xc- and plays a crucial role in cystine metabolism and ferroptosis regulation. Inhibiting the uptake and transport of extracellular cystine by SLC7A11 prevents the synthesis of glutathione (GSH), a key antioxidant in cells, leading to excessive accumulation of intracellular peroxides and inducing ferroptosis. With the deepening research on ferroptosis, SLC7A11 has been identified as an ideal target for inducing ferroptosis and inhibiting tumor growth. First, SLC7A11 knockout in mice is not lethal, indicating that targeted inhibition of SLC7A11 does not produce excessive side effects on normal tissues and cells. Second, SLC7A11 is highly expressed in various tumors and is negatively regulated by tumor suppressor genes such as p53, BAP1, KEAP1, and ATF3, suggesting its crucial role in tumor development. Third, SLC7A11 is an oxidative stress protein, dynamically regulated by oxidative stress and cytotoxic substances, and may play a key role in specific tumor microenvironments, providing an important window for targeted tumor therapy. Therefore, developing specific small-molecule inhibitors targeting SLC7A11 has broad application prospects in cancer treatment.

[0003] Reported small-molecule inhibitors targeting SLC7A11 are mainly Erastin and its analogues, which inhibit the uptake of extracellular cysteine ​​by SLC7A11, leading to the accumulation of intracellular peroxides and inducing ferroptosis. Erastin suffers from poor metabolic stability and water solubility, limiting its use to in vitro experiments. The derivative of Erastin, IKE (Imidazole ketone erastin), can be used in vivo, but its poor water solubility, poor metabolic stability, and low potency limit its further clinical application. More importantly, IKE has a complex preparation process, low yield, and high cost. Developing novel, specific SLC7A11 inhibitors based on novel chemical synthesis processes and evaluating their cell death-inducing and anti-cancer effects has significant clinical application value. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention aims to provide a novel quinazolinone derivative that induces ferroptosis, thereby solving the technical problems of complex preparation processes, poor water solubility, poor metabolic stability, and low potency of existing small molecule inhibitors of SLC7A11. Simultaneously, the present invention also provides a method for preparing the aforementioned quinazolinone derivative and its isomers, aiming to solve the technical problems of existing technologies requiring synthesis under high temperature and high pressure conditions and insufficient material conversion.

[0005] To achieve the above objectives, the present invention employs the following technical solution:

[0006] This invention discloses a quinazolinone derivative and its isomers that induce ferroptosis, with the structural formula shown in Formula I below:

[0007]

[0008] In Equation I, R1 represents H, D, -OCD3, halogen, and C. 1-4 Alkoxy, C 1-4 Halogenated alkyl groups, deuterated C 1-6 Alkyl group; R2 is D or C 1-4 Alkyl, C 1-4 Halogenated alkyl groups, 3-7 membered cycloalkyl groups, deuterated C 1-6 alkyl;

[0009] R3 can be H, D, -CD3, -OCD3, halogen, or C. 1-4 Alkoxy, C 1-4 Halogenated alkyl groups;

[0010] R4 is D, C 1-4 Alkyl, benzyl, 3-7 membered cycloalkyl, deuterated C 1-6 alkyl;

[0011] W represents the structure shown in equation 1-1 below:

[0012]

[0013] Where Q is C 1-4 Alkyl groups, heteroaryl groups containing -NH, and 3-7 membered cycloalkyl groups;

[0014] Alternatively, Q can be represented by the structure in equation 1-2:

[0015]

[0016] Where n = 1-4; X represents C, O, S, N; R5 represents H, halogen, C 1-4 Alkoxy, deuterated C1-6 Alkyl; or, Q is a structure represented by formulas 1-3 below:

[0017]

[0018] Among them, R6 represents H, D, -OCD3, halogen, and C. 1-4 Alkoxy, deuterated C 1-6 alkyl.

[0019] Preferably, it comprises compounds represented by the following structural formulas A1-A33:

[0020]

[0021]

[0022]

[0023] This invention also discloses a method for preparing the above-mentioned quinazolinone derivatives and their isomers that induce ferroptosis, comprising the following steps:

[0024] 1) Using compound 1 as the starting material, compound 2 was prepared by reacting it in a reaction solvent under reducing agent and acidic conditions;

[0025] 2) Using N,N-diisopropylethylamine as a base, in the presence of a sulfiding agent and a catalyst, compound 2 is reacted to produce compound 3;

[0026] 3) React the base, compound 3, and anthranilic acid derivative, i.e., compound 4, to generate compound 5;

[0027] 4) Compound 5 was subjected to chlorination to obtain intermediate II, i.e., compound 6;

[0028] 5) Under ball milling conditions, in the presence of alkali and grinding aid, compound 6 and compound 7 react to obtain compound I, namely, quinazolinone derivatives and their isomers that induce ferroptosis;

[0029] The structural formulas of compounds 1, 2, 3, 4, 5, and 7 are shown below:

[0030]

[0031] The structural formula of intermediate II, namely compound 6, is shown below:

[0032]

[0033] In the above structural formula:

[0034] R1 represents H, D, -OCD3, halogen, or C.1-4 Alkoxy, C 1-4 Halogenated alkyl deuterated, C 1-6 alkyl;

[0035] R2 represents D and C. 1-4 Alkyl, deuterated C 1-6 Alkyl, C 1-4 Halogenated alkyl or 3-7 membered cycloalkyl;

[0036] R3 represents H, D, -OCD3, halogen, and C. 1-4 Alkoxy, deuterated C 1-6 Alkyl or -C 1-4 Halogenated alkyl groups;

[0037] R4 is D, C 1-4 Alkyl, deuterated C 1-6 Alkyl, benzyl, or 3-7 membered cycloalkyl;

[0038] W represents the structure shown in equation 1-1 below:

[0039]

[0040] Where Q is C 1-4 Alkyl groups, heteroaryl groups containing -NH, and 3-7 membered cycloalkyl groups;

[0041] Alternatively, Q can be represented by the structure in equation 1-2:

[0042]

[0043] Where n = 1-4; X represents C, O, S, N; R5 represents H, D, -OCD3, halogen, C 1-4 Alkoxy, deuterated C 1-6 alkyl;

[0044] Alternatively, Q can be represented by the structure in equation 1-3:

[0045]

[0046] Among them, R6 can be H, D, -OCD3, halogen, or deuterated C. 1-6 Alkyl or C 1-4 Alkyl group.

[0047] Preferably, in step 1), the reducing agent is ferrous chloride, tin dichloride or titanium tetrachloride, the reaction solvent is tetrahydrofuran, and the acidic conditions are provided by a 1-5M hydrochloric acid or sulfuric acid solution, and the volume ratio of the acidic solution to the reaction solvent is 1:1.

[0048] In step 2), the sulfiding agent is CS2, and the catalyst is di-tert-butyl dicarbonate;

[0049] In step 3), the molar ratio of compound 3 to the anthranilic acid derivative is 1:1.2, the base used is triethylamine or N,N-diisopropylethylamine, the reaction temperature is 60-70℃, and the reaction time is 6-12 hours.

[0050] In step 4), thionyl chloride or thioyl chloride is used as the chlorination reagent in the chlorination reaction, and the molar ratio of compound 5 to the chlorination reagent is 1:2; the solvent for the chlorination reaction is tetrahydrofuran or chloroform, the reaction temperature is 50-70℃, and the reaction time is 8-12 hours.

[0051] In step 5), the alkali is cesium fluoride, sodium fluoride or tert-butyllithium, and the grinding aid is dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide. The reaction conditions are 10-30 Hz and the reaction time is 0.5-1 hour.

[0052] Preferably, step 5) specifically includes:

[0053] Compound 6, compound 7, alkali, grinding aid and solvent were ball-milled at 50-70℃ and 10-30Hz for 0.5-1 hours, followed by washing, filtration, concentration and column chromatography to obtain quinazolinone derivatives and their isomers that induce ferroptosis.

[0054] Preferably, the molar amount of compound 7 is 1.2 times the molar amount of compound 6; the base is cesium fluoride, sodium fluoride, or lithium tert-butoxide, and the amount used is 2 times the molar amount of compound 6; the grinding aid is dimethyl sulfoxide, N,N-dimethylformamide, or N,N-dimethylacetamide.

[0055] The present invention also discloses pharmaceutically acceptable salts of the above-mentioned quinazolinone derivatives and their isomers that induce ferroptosis.

[0056] This invention also discloses the use of the above-mentioned quinazolinone derivatives and their isomers or the salts thereof as small molecule inhibitors of SLC7A11 in the preparation of drugs for treating tumors or immune-related diseases.

[0057] Preferably, the tumors include lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, leukemia, colon cancer, non-small cell lung cancer, pancreatic cancer, skin cancer, head and neck cancer, small intestine cancer, rectal cancer, endometrial cancer, vaginal cancer, testicular cancer, esophageal cancer, bile duct cancer, lymphoma, gallbladder cancer, endocrine gland cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brainstem glioma, and pituitary adenoma.

[0058] The immune-related diseases include allergies caused by cell activation and impaired tissue damage repair.

[0059] More preferably, tissue damage such as impaired wound healing.

[0060] Compared with the prior art, the present invention has the following beneficial effects:

[0061] This invention discloses for the first time a class of quinazolinone derivatives and their isomers capable of inducing ferroptosis. By fusing the quinazolinone ring with other groups, a unique heterocyclic structure system is formed, endowing these derivatives with unique biological activities and making them highly promising for applications in anti-tumor and other fields. Experimental verification shows that the quinazolinone compounds of this invention exhibit superior inhibitory effects against various types of cancer cells (including renal cell carcinoma, prostate cancer, liver cancer, and lung cancer) compared to the positive control drugs erastin and IKE, demonstrating broad-spectrum anti-tumor activity and indicating their excellent application potential.

[0062] Regarding the synthesis method, this application effectively shortens the synthesis route, the synthesis method is simple, the key steps can be achieved by ball milling only, without the need for strict nitrogen protection and high temperature and other harsh conditions, the solvent consumption is small, the reaction time is short and the reaction conditions are green and mild, and the yield is as high as 85%. Attached Figure Description

[0063] Figure 1 The effect of compound A15 on the cell viability of different types of cancer cells is shown in Figure A. Figure A shows the cell viability analysis of four cancer types: renal cell carcinoma, prostate cancer, lung cancer, and liver cancer. Figure B shows the cell viability analysis of four specific cell lines: 786-O, DU145, H1299, and Hep3B.

[0064] Figure 2 Electron microscopy observation of mitochondrial morphological changes in cells treated with compound A15;

[0065] Figure 3 To detect the levels of ROS and lipid ROS in DU145 cells after intervention with compound A15;

[0066] Figure 4 To detect the levels of glutamate and GSH in cells after intervention with compound A15;

[0067] Figure 5 The results of in vivo animal experiments to determine the activity of compound A15 are shown in the figure. Among them, A is the morphology of tumors in each group after A15 intervention; B is the tumor growth curve after A15 intervention; C is the weight of tumors in each group after A15 intervention; D is the immunohistochemical staining of tumor tissues in each group, including the oxidative stress cell damage index 4-HNE and Cleaved caspase-3; E is the change of the oxidative stress cell damage index 4-HNE after A15 intervention; and F is the electron micrograph of mitochondria in tumor tissues in each group. Detailed Implementation

[0068] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0069] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0070] The present invention will now be described in further detail with reference to the accompanying drawings:

[0071] This invention focuses on the target SLC7A11 and synthesizes a series of novel SLC7A11 small molecule inhibitors by comparing with its own unique small molecule drug screening database, re-optimizing the design of small molecule structures, and using the general structural formula 1.

[0072]

[0073] Compounds with the general formula 2 are key intermediates in the synthesis process. The synthesis of similar structures reported in the prior art usually requires high-temperature thermal catalysis and high-boiling-point highly polar reaction solvents, resulting in complex post-processing and the generation of unnecessary byproducts.

[0074] Mechanochemistry is a method that induces physical and chemical changes in reactants through grinding, friction, and other methods, leading to alterations in the properties and characteristics of the reactants and solid, liquid, and gaseous substances in the environment. By using mechanical force and fine grinding, not only can high-temperature and high-pressure conditions be avoided, promoting full material conversion, but this method is also green and efficient, reducing organic solvent emissions and reaction time, and improving raw material conversion rates. Therefore, this invention aims to develop a green method for synthesizing key intermediates of quinazoline ketones using mechanochemistry. This method operates under mild conditions, requires no high-temperature catalysis, and consumes little solvent, enabling the efficient and high-yield synthesis of quinazoline derivatives, providing a crucial material basis for their industrial production and related research.

[0075] I. Synthesis Examples

[0076] Example 1

[0077] The synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A1) is shown in the following reaction equation:

[0078]

[0079] The synthesis method includes the following steps:

[0080] Step 1: Synthesis of 3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (3)

[0081] Under nitrogen protection, 1-isopropoxy-2-isothiocyanate benzene (1.9 g, 10.0 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 1.4 g (10.0 mmol) of 2-aminobenzoic acid (1.4 g, 10.0 mmol), and the reaction was carried out at 60 °C for 12 h. After the reaction was completed, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product 3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one (2.5 g, 8.0 mmol) was given as a white solid in 80% yield.

[0082] Step 2: Synthesis of 2-chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (4)

[0083] Under nitrogen protection, SO₂Cl₂ (0.7 mL, 8.0 mmol) was added to a THF solution (40 mL) of 2.5 g (2.5 g, 8.0 mmol) of 3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one, and the reaction was carried out at 60 °C for 8 h. After the reaction was complete, an appropriate amount of dichloromethane was added, and the mixture was washed three times with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 10 / 1). The target product, 2-chloro-3-(2-isopropoxyphenyl)quinazoline-4(3H)-one (2.3 g, 7.2 mmol), was obtained as a white solid in 90% yield.

[0084] Step 3: Synthesis of tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)amino)piperidine-1-carboxylic acid (6)

[0085] 2-Chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3 g, 7.2 mmol), tert-butyl 4-aminopiperidin-1-carboxylate (1.7 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 3 / 1). The target product, tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)amino)piperidin-1-carboxylate (2.9 g, 6.1 mmol), was given as a white solid in 85% yield.

[0086] Step 4: Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A1)

[0087] Under nitrogen protection, tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)amino)piperidine-1-carboxylate (2.9 g, 6.1 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.7 g, 4.9 mmol), was obtained as a white solid in 85% yield. HRMS (ESI): [M+H] + =547.2108.

[0088] 1 H NMR (400MHz, CDCl3) δ8.13(d,J=7.9Hz,1H),7.62(t,J=7.6Hz,1H),7.48(t,J=7.8Hz,1H ),7.42(s,1H),7.25–7.14(m,4H),7.13–7.06(m,2H),6.85(d,J=8.7Hz,2H),4.71–4.58 (m,2H),4.54(dt,J=12.1,6.0Hz,1H),4.30(d,J=28.7Hz,2H),4.02–3.79(m,2H),3.25( t,J=12.4Hz,1H),2.92(dd,J=28.5,15.8Hz,1H),2.23–1.92(m,3H),1.21–1.17(m,6H).

[0089] Example 2

[0090] The synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A2) is shown in the following reaction equation:

[0091]

[0092] 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A1) (0.8 g, 1.5 mmol) was dissolved in DMF (5 mL), sodium tert-butoxide (144.0 mg, 1.5 mmol) was added, and the mixture was stirred for 20 min. Iodomethane (93.0 μL, 1.5 mmol) was added, and the reaction was carried out at room temperature for 2 h. After filtration, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product, 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (0.6 g, 1.0 mmol), was obtained as a white solid in a yield of 68%. HRMS (ESI): [M+H] + =561.2280.

[0093] 1 H NMR (400MHz, CDCl3) δ8.18(d,J=7.8Hz,1H),7.69(t,J=7.4Hz,1H),7.38(t,J=7.8Hz,1H),7.33–7. 22(m,5H),7.05(dd,J=12.0,6.9Hz,2H),6.88(d,J=8.9Hz,2H),4.71–4.58(m,2H),4.55(dt,J=11. 8,5.8Hz,2H),3.94(s,1H),3.66(s,1H),2.90(d,J=11.1Hz,1H),2.62(d,J=15.5Hz,3H),2.39(dd, J=21.1,10.7Hz,1H),1.42(dt,J=12.6,9.4Hz,3H),1.28(t,J=10.0Hz,4H),1.16(d,J=4.3Hz,3H).

[0094] Example 3

[0095] The synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(benzyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A4) is shown in the following reaction equation:

[0096]

[0097] 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (0.8 g, 1.5 mmol) was dissolved in DMF (5 mL), sodium hydride (100.0 mg, 1.5 mmol) was added, and the mixture was stirred for 20 min. Benzyl bromide (180.0 μL, 1.5 mmol) was added, and the reaction was carried out at room temperature for 2 h. After filtration and removal of the solvent under reduced pressure, the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product, 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(benzyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (0.8 g, 1.0 mmol), was obtained as a white solid in 83% yield. HRMS (ESI): [M+H] + =637.2579.

[0098] 1 H NMR (400MHz, CDCl3) δ8.02 (dd, J=7.7, 1.1Hz, 1H), 7.32 (dt, J=14.5, 7.3Hz, 3H), 7.25–7. 16(m,5H),7.12(d,J=8.6Hz,2H),6.97–6.87(m,3H),6.82(d,J=8.5Hz,1H),6.72(dd,J=9 .0,2.9Hz,2H),5.33(d,J=87.7Hz,2H),4.51–4.36(m,3H),3.60–3.23(m,2H),2.90–2.68 (m,3H),1.20(d,J=6.1Hz,4H),1.15(d,J=6.0Hz,3H),1.09(ddd,J=17.3,8.4,4.6Hz,3H).

[0099] Example 4

[0100] The synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropoxyphenyl)quinazolin-4(3H)-one (compound A7) is shown in the following reaction equation:

[0101]

[0102] The synthesis method includes the following steps:

[0103] Step 1: Synthesis of 2-cyclopropoxyaniline (2)

[0104] Under nitrogen protection, 1-nitro-2-cyclopropoxybenzene (3.6 g, 20.0 mmol), ferrous chloride (3.8 g, 30.0 mmol), hydrochloric acid (1.0 M, 50 mL), and THF (50 mL) were mixed and stirred vigorously overnight. After the reaction was complete, sodium hydroxide solution (1.0 M, 50 mL) was added, and the mixture was dried over anhydrous sodium sulfate. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 20 / 1). The target product, 2-cyclopropoxyaniline (1.7 mL, 12.0 mmol), was given as a red liquid in a yield of 60%.

[0105] Step 2: Synthesis of 1-isothiocyano-2-cyclopropoxybenzene (3)

[0106] Under nitrogen protection, 2-cyclopropoxyaniline (1.7 mL, 12.0 mmol), DIPEA (2.7 mL, 15.0 mmol), and THF (50 mL) were mixed and stirred overnight at room temperature. After cooling to 0 °C, CS2 (1.6 mL, 24.0 mmol) and di-tert-butyl dicarbonate (2.0 g, 10.0 mmol) were added, and the mixture was reacted at room temperature for 2 h. After the reaction was complete, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 50 / 1). The target product, 1-isothiocyano-2-cyclopropoxybenzene (1.9 mL, 10.0 mmol), was given as a white solid in 83% yield.

[0107] Step 3: Synthesis of 3-(2-cyclopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (5)

[0108] Under nitrogen protection, 1-isothiocyano-2-cyclopropoxybenzene (1.9 mL, 10.0 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 1-aminobenzoic acid (1.4 g, 10.0 mmol), and the reaction was carried out at 60 °C for 12 h. After the reaction was completed, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product, 3-(2-cyclopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one (2.4 g, 8.0 mmol), was given as a white solid in 80% yield.

[0109] Step 4: Synthesis of 2-chloro-3-(2-cyclopropoxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (6)

[0110] Under nitrogen protection, SO₂Cl₂ (0.7 mL, 8.0 mmol) was added to a THF solution (40 mL) of 2.4 g (8.0 mmol) of 3-(2-cyclopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one, and the reaction was carried out at 60 °C for 8 h. After the reaction was complete, an appropriate amount of dichloromethane was added, and the mixture was washed three times with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 10 / 1). The target product, 2-chloro-3-(2-cyclopropoxyphenyl)-2,3-dihydroquinazoline-4(1H)-one (2.0 g, 7.2 mmol), was obtained as a white solid in 90% yield.

[0111] Step 5: Synthesis of tert-butyl 4-((3-(2-cyclopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylic acid (8)

[0112] 2-Chloro-3-(2-cyclopropoxyphenyl)-2,3-dihydroquinazoline-4(1H)-one (2.0 g, 7.2 mmol), tert-butyl 4-(methylamino)piperidin-1-carboxylate (1.7 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 3 / 1). The target product, tert-butyl 4-((3-(2-cyclopropoxyphenyl)-4-oxo-3,4-dihydroquinazoline-2-yl)(methyl)amino)piperidin-1-carboxylate (2.9 g, 6.1 mmol), was given as a white solid in 85% yield.

[0113] Step 6: Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropoxyphenyl)quinazolin-4(3H)-one (compound A7)

[0114] Under nitrogen protection, tert-butyl 4-((3-(2-cyclopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.9 g, 6.1 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropoxyphenyl)quinazolin-4(3H)-one (2.4 g, 4.3 mmol), was obtained as a white solid in 72% yield. HRMS (ESI): [M+H] + =559.2100.

[0115] 1 H NMR(400MHz, CDCl3)δ8.14(d,J=6.5Hz,1H),7.65(t,J=7.6Hz,1H),7.41–7.35(m,2H) ,7.25–7.16(m,4H),7.11–7.03(m,1H),6.88–6.83(m,2H),4.62(dd,J=13.3,6.9Hz,2H ),4.51(d,J=12.5Hz,1H),3.90(s,1H),3.73(t,J=4.4Hz,1H),3.52(s,1H),2.82(s,1 H),2.34(s,3H),1.41(dd,J=18.8,11.4Hz,3H),1.31–1.18(m,1H),0.96-0.86(m,5H).

[0116] Example 5

[0117] The synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-5-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A15) is shown in the following reaction equation:

[0118]

[0119] Step 1. Synthesis of 8-fluoro-3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (3)

[0120] Under nitrogen protection, 1-isopropoxy-2-isothiocyanate benzene (1.9 g, 10.0 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 1-amino-3-fluorobenzoic acid (1.6 g, 10.0 mmol) and reacted at 60 °C for 12 h. After the reaction was complete, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product, 8-fluoro-3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (2.4 g, 7.3 mmol), was given as a white solid in a yield of 73%.

[0121] Step 2. Synthesis of 2-chloro-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (4)

[0122] Under nitrogen protection, SO₂Cl₂ (0.7 mL, 8.0 mmol) was added to a THF solution (40 mL) of 2.4 g (7.3 mmol) of 8-fluoro-3-(2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one, and the reaction was carried out at 60 °C for 8 h. After the reaction was complete, an appropriate amount of dichloromethane was added, and the mixture was washed three times with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 10 / 1). The target product, 2-chloro-8-fluoro-3-(2-isopropoxyphenyl)quinazoline-4(3H)-one (2.3 g, 7.0 mmol), was obtained as a white solid with a yield of 95%.

[0123] Step 3. Synthesis of tert-butyl piperidine-1-carboxylate (6) ((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate)

[0124] 2-Chloro-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3 g, 7.0 mmol), tert-butyl 4-(methylamino)piperidin-1-carboxylate (1.7 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 3 / 1). The target product, 4-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidin-1-carboxylate (2.9 g, 6.1 mmol), was given as a white solid in 85% yield.

[0125] Step 4.2 Synthesis of 1-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A15)

[0126] Under nitrogen protection, tert-butyl 4-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (3.1 g, 5.9 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.7 g, 4.9 mmol), was obtained as a white solid in 85% yield. HRMS (ESI): [M+H] + =579.2175.

[0127] 1 H NMR (400MHz, CDCl3) δ7.46 (td, J=8.2, 5.6Hz, 1H), 7.30–7.23 (m, 1H), 7.22–7.12 (m, 4H), 6.94 (dd, J= 12.2,8.0Hz,2H),6.79(t,J=9.2Hz,3H),4.60–4.49(m,2H),4.43(dd,J=11.8,4.9Hz,2H),3.84(t,J= 13.3Hz,1H),3.60(t,J=10.1Hz,1H),2.78(t,J=12.9Hz,1H),2.45(s,3H),2.30(q,J=12.6Hz,1H),1. 44–1.20(m,4H),1.17(d,J=6.1Hz,3H),1.07(dt,J=12.3,6.2Hz,3H),1.00(dd,J=27.9,11.7Hz,1H).

[0128] Example 6

[0129] The synthesis of 3-(5-bromo-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (compound A17) is shown in the following reaction equation:

[0130]

[0131] Step 1. Synthesis of 5-bromo-2-isopropoxyaniline (2)

[0132] Under nitrogen protection, 4-bromo-1-isopropoxy-2-nitrobenzene (5.2 g, 20.0 mmol), ferrous chloride (3.8 g, 30.0 mmol), hydrochloric acid (1.0 M, 50 mL), and THF (50 mL) were mixed and stirred vigorously overnight. After the reaction was complete, sodium hydroxide solution (1.0 M, 50 mL) was added, and the mixture was dried over anhydrous sodium sulfate. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 20 / 1). The target product, 5-bromo-2-isopropoxyaniline (2.3 mL, 12.0 mmol), was given as a red liquid in a yield of 60%.

[0133] Step 2. Synthesis of 4-bromo-1-isopropoxy-2-isothiocyanate benzene (3)

[0134] Under nitrogen protection, 5-bromo-2-isopropoxyaniline (2.3 mL, 12.0 mmol), DIPEA (2.7 mL, 15.0 mmol), and THF (50 mL) were mixed and stirred overnight at room temperature. After cooling to 0 °C, CS2 (1.6 mL, 24.0 mmol) and di-tert-butyl dicarbonate (2.0 g, 10.0 mmol) were added, and the mixture was reacted at room temperature for 2 h. After the reaction was complete, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 50 / 1). The target product, 4-bromo-1-isopropoxy-2-isothiocyanate benzene (1.9 mL, 10.0 mmol), was obtained as a white solid in 83% yield.

[0135] Step 3. Synthesis of 3-(5-bromo-2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (5)

[0136] Under nitrogen protection, 1.9 mL of 4-bromo-1-isopropoxy-2-isothiocyanate (10.0 mmol) and 2.1 mL of triethylamine (15.0 mmol) were added to a THF solution (60 mL) of 1.4 g (10.0 mmol) of 2-aminobenzoic acid, and the reaction was carried out at 60 °C for 12 h. After the reaction was completed, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 3 / 1). The target product, 3-(5-bromo-2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one (3.2 g, 8.0 mmol), was given as a white solid in 80% yield.

[0137] Step 4. Synthesis of 3-(5-bromo-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (6)

[0138] Under nitrogen protection, SO₂Cl₂ (0.7 mL, 8.0 mmol) was added to a THF solution (40 mL) of 3-(5-bromo-2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one (3.2 g, 8.0 mmol), and the reaction was carried out at 60 °C for 8 h. After the reaction was complete, an appropriate amount of dichloromethane was added, and the mixture was washed three times with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 10 / 1). The target product, 3-(5-bromo-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (2.8 g, 7.2 mmol), was obtained as a white solid with a yield of 90%.

[0139] Step 5. Synthesis of 3-(5-bromo-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (8)

[0140] 3-(5-bromo-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (2.8 g, 7.2 mmol), tert-butyl 4-(methylamino)piperidin-1-carboxylate (1.7 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 3 / 1). The target product, 4-((3-(5-bromo-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazoline-2-yl)(methyl)amino)piperidin-1-carboxylate (3.5 g, 6.1 mmol), was given as a white solid in 85% yield.

[0141] Step 6.2 Synthesis of 1-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropoxyphenyl)quinazolin-4(3H)-one (compound A17)

[0142] Under nitrogen protection, tert-butyl 4-((3-(5-bromo-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (3.5 g, 6.1 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 3-(5-bromo-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (2.8 g, 4.3 mmol), was obtained as a white solid in 72% yield. HRMS (ESI): [M+H] + =639.1360.

[0143] 1 H NMR(400MHz, CDCl3)δ8.11(dd,J=7.9,1.5Hz,1H),7.60–7.53(m,1H),7.23–7.13(m,3H),7 .03–6.97(m,2H),6.92–6.82(m,3H),6.66(d,J=8.7Hz,1H),5.47–5.32(m,1H),4.67(s,3H) ,4.30(dt,J=11.9,5.9Hz,1H),4.00(d,J=14.7Hz,1H),3.15(t,J=11.7Hz,1H),3.01(s,3H) ,2.92(dd,J=20.6,8.3Hz,2H),2.69(t,J=11.7Hz,1H),1.74(s,2H),0.97(d,J=5.9Hz,7H).

[0144] Example 7

[0145] The synthesis of 3-(5-acetyl-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (compound A18) is shown in the following reaction equation:

[0146]

[0147] Step 1.1 Synthesis of (3-amino-4-isopropoxyphenyl) ethyl-1-one (2)

[0148] Under nitrogen protection, 1-(4-isopropoxy-3-nitrophenyl)ethyl-1-one (4.5 g, 20.0 mmol), ferrous chloride (3.8 g, 30.0 mmol), hydrochloric acid (1.0 M, 50 mL), and THF (50 mL) were mixed and stirred vigorously overnight. After the reaction was complete, sodium hydroxide solution (1.0 M, 50 mL) was added, and the mixture was dried over anhydrous sodium sulfate. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. After filtration and removal of the solvent under reduced pressure, the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 20 / 1). The target product, 1-(3-amino-4-isopropoxyphenyl)ethyl-1-one (1.9 mL, 14.0 mmol), was given as a red liquid in 70% yield.

[0149] Step 2.1 Synthesis of (4-isopropoxy-3-isothiocyanophenyl)ethyl-1-one (3)

[0150] Under nitrogen protection, 1-(3-amino-4-isopropoxyphenyl)ethyl-1-one (1.9 mL, 14.0 mmol), DIPEA (2.7 mL, 15.0 mmol), and THF (50 mL) were mixed and stirred overnight at room temperature. After cooling to 0 °C, CS2 (1.6 mL, 24.0 mmol) and di-tert-butyl dicarbonate (2.0 g, 10.0 mmol) were added, and the mixture was reacted at room temperature for 2 h. After the reaction was complete, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the combined organic phases were dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 50 / 1). The target product, 1-(4-isopropoxy-3-isothiocyanophenyl)ethyl-1-one (1.7 mL, 12.3 mmol), was obtained as a white solid in 88% yield.

[0151] In step 3.3, the synthesis of 1-(4-isopropoxy-3-isothiocyanophenyl)ethyl-1-one (1.7 mL, 12.3 mmol) and triethylamine (2.1 mL, 15.0 mmol) was carried out under nitrogen protection. The reaction was carried out at 60 °C for 12 h. After the reaction was complete, water was added to terminate the reaction. The mixture was extracted twice with dichloromethane, and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 1 / 1). The target product, 3-(5-acetyl-2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (2.7 g, 7.6 mmol), was obtained as a white solid with a yield of 80%.

[0152] Step 4.3 Synthesis of (5-acetyl-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (6)

[0153] Under nitrogen protection, SO₂Cl₂ (0.7 mL, 8.0 mmol) was added to a THF solution (40 mL) of 2.7 g (7.6 mmol) of 3-(5-acetyl-2-isopropoxyphenyl)-2-thio-2,3-dihydroquinazoline-4(1H)-one, and the reaction was carried out at 60 °C for 8 h. After the reaction was complete, an appropriate amount of dichloromethane was added, and the mixture was washed three times with saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 5 / 1). The target product, 3-(5-acetyl-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (2.3 g, 6.4 mmol), was obtained as a white solid in a yield of 84%.

[0154] Step 5.4 Synthesis of tert-butyl piperidine-1-carboxylate (8) ((3-(5-acetyl-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate)

[0155] 3-(5-acetyl-2-isopropoxyphenyl)-2-chloroquinazoline-4(3H)-one (2.3 g, 6.4 mmol), tert-butyl 4-(methylamino)piperidin-1-carboxylate (1.7 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 1 / 1). The target product, 4-((3-(5-acetyl-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazoline-2-yl)(methyl)amino)piperidin-1-carboxylate (2.2 g, 4.2 mmol), was given as a white solid in a yield of 65%.

[0156] Step 6. Synthesis of 3-(5-acetyl-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (compound A18)

[0157] Under nitrogen protection, tert-butyl 4-((3-(5-acetyl-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.2 g, 4.2 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 3-(5-acetyl-2-isopropoxyphenyl)-2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)quinazolin-4(3H)-one (2.3 g, 3.9 mmol), was obtained as a white solid in 93% yield. HRMS (ESI): [M+H] + =603.2372.

[0158] 1H NMR (400MHz, CDCl3) δ7.98 (dd, J=8.7, 1.9Hz, 1H), 7.89 (dd, J=8.9, 5.0Hz, 2H), 7.44–7.33 (m, 1H),7.23–7.11(m,3H),7.05(d,J=8.8Hz,1H),6.84(d,J=8.6Hz,2H),4.74–4.43(m,4H),3.92( t,J=13.7Hz,1H),3.71(d,J=10.0Hz,1H),2.98–2.79(m,1H),2.54(s,3H),2.48(d,J=2.8Hz,3H ),2.36(dd,J=28.5,14.6Hz,1H),1.54–1.34(m,3H),1.27(t,J=6.1Hz,4H),1.25–1.20(m,3H).

[0159] Example 8

[0160] Synthesis of 2-((2-(2-(4-chlorophenoxy)acetyl)-2-azaspiro[3.3]heptane-6-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A21)

[0161]

[0162] Step 1. Synthesis of 6-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (3)

[0163] 2-Chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3 g, 7.2 mmol), 6-(methylamino)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (1.9 g, 8.6 mmol), and 0.5 mL of DMSO were added to a 10 mL ball mill jar, and the reaction was carried out at 30 Hz for 1 h. After the reaction was completed, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 3 / 1). The target product, 6-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (3.1 g, 6.0 mmol), was obtained as a white solid in a yield of 83%.

[0164] Step 2.2 Synthesis of ((2-(2-(4-chlorophenoxy)acetyl)-2-azaspiro[3.3]heptane-6-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A21)

[0165] Under nitrogen protection, tert-butyl 6-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylic acid (3.1 g, 6.0 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), and 2-(4-chlorophenoxy)acetyl chloride (1.5 mL, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting agent: ethyl acetate / petroleum ether = 2 / 1). The target product, 2-((2-(2-(4-chlorophenoxy)acetyl)-2-azaspiro[3.3]heptane-6-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.7 g, 4.7 mmol), was obtained as a white solid in 78% yield. HRMS (ESI): [M+H] + =591.2150.

[0166] 1 H NMR(400MHz, CDCl3)δ7.85(d,J=7.9Hz,1H),7.34–7.25(m,2H),7.22–7.15(m,2 H),7.10(td,J=8.0,4.6Hz,1H),6.98(dd,J=13.0,7.9Hz,2H),6.83–6.56(m,3H ),4.43(s,3H),4.07–3.93(m,1H),3.50–3.41(m,2H),3.37(s,2H),2.42(s,3H) ,2.04–1.65(m,4H),1.19(dd,J=10.7,4.5Hz,3H),1.06(dd,J=17.5,6.0Hz,3H).

[0167] Example 9

[0168] The synthesis of methyl 4-(2-(4-(8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidin-1-yl)-2-oxoethoxy)benzoate (compound A29) is shown in the following reaction equation:

[0169]

[0170] Under nitrogen protection, tert-butyl 4-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (5.1 g, 10.0 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), HATU (5.7 g, 15.0 mmol), and 2-(4-(methoxycarbonyl)phenoxy)acetic acid (2.5 g, 12.0 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, methyl 4-(2-(4-(8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidin-1-yl)-2-oxoethoxy)benzoate (4.0 g, 6.7 mmol), was obtained as a white solid in 67% yield. HRMS (ESI): [M+H] + =603.2615.

[0171] 1 H NMR (400MHz, CDCl3) δ7.91(d,J=8.6Hz,2H),7.85(d,J=7.9Hz,1H),7.30(dd,J=14.9,7.7Hz,2H ),7.18(d,J=7.4Hz,1H),7.14–7.04(m,1H),6.96(t,J=8.8Hz,2H),6.87(d,J=8.6Hz,2H),4.63 (d,J=5.7Hz,2H),4.46(dd,J=11.4,6.0Hz,2H),3.81(s,3H),3.64(s,1H),2.82(s,1H),2.48(s ,3H),2.31(s,1H),1.36(dd,J=13.6,10.1Hz,3H),1.18(d,J=5.8Hz,5H),1.08(d,J=3.3Hz,3H).

[0172] Example 10

[0173] The synthesis of 2-(1-(2-((6-chloropyridin-3-yl)oxy)acetyl)piperidin-4-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (compound A31) is shown in the following reaction equation:

[0174]

[0175] Under nitrogen protection, tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidin-1-carboxylic acid (2.4 g, 6.1 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), HATU (5.7 g, 15.0 mmol), and 2-((6-chloropyridin-3-yl)oxy)acetic acid (2.2 g, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 2-(1-(2-((6-chloropyridin-3-yl)oxy)acetyl)piperidin-4-yl)(methyl)amino)-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (3.6 g, 6.2 mmol), was obtained as a white solid in 62% yield. HRMS (ESI): [M+H] + =580.2110.

[0176] 1 H NMR(400MHz, CDCl3) δ7.99(d,J=2.4Hz,1H),7.86(d,J=8.0Hz,1H),7.34–7.25(m,2H),7.19(dd,J =7.5,3.9Hz,2H),7.15(d,J=8.8Hz,1H),7.09(td,J=7.9,4.6Hz,1H),6.96(t,J=8.4Hz,2H),4.63 (t,J=8.8Hz,2H),4.48(dd,J=11.1,5.5Hz,2H),3.76(s,1H),3.66(t,J=11.3Hz,1H),2.84(s,1H) ,2.51(s,3H),2.34(s,1H),1.40(d,J=12.9Hz,3H),1.19(d,J=5.9Hz,4H),1.09(t,J=6.3Hz,3H).

[0177] Example 11

[0178] The synthesis of 3-(2-isopropoxyphenyl)-2-(methyl(1-(1-methyl-1H-imidazol-4-carbonyl)piperidin-4-yl)amino)quinazolin-4(3H)-one (compound A32) is shown in the following reaction equation:

[0179]

[0180] Under nitrogen protection, tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.4 g, 6.1 mmol) was dissolved in ethyl acetate hydrochloride solution (2.0 M, 10 mL) and stirred at room temperature for 1 h. The solvent was evaporated under reduced pressure, and DCM (20 mL), triethylamine (1.7 mL, 15.0 mmol), HATU (5.7 g, 15.0 mmol), and 1-methyl-1H-imidazolium-4-carboxylic acid (1.2 g, 9.1 mmol) were added sequentially, and the reaction was carried out at room temperature for 3 h. The mixture was washed three times with saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluting medium: ethyl acetate / petroleum ether = 2 / 1). The target product, 3-(2-isopropoxyphenyl)-2-(methyl(1-(1-methyl-1H-imidazol-4-carbonyl)piperidin-4-yl)amino)quinazolin-4(3H)-one (1.7 g, 3.4 mmol), was obtained as a white solid in 55% yield. HRMS (ESI): [M+H] + =501.2605.

[0181] 1 H NMR (400MHz, CDCl3) δ = 8.08 (d, J = 7.9Hz, 1H), 7.58 (t, J = 7.6Hz, 1H), 7.47 (d, J = 7.7Hz, 1H) ,7.37(d,J=15.8Hz,2H),7.27(t,J=7.9Hz,1H),7.19(dd,J=10.7,6.2Hz,2H),6.95(t,J=8. 2Hz,2H),4.69–4.31(m,2H),3.64(s,4H),3.45–3.32(m,1H),2.78(d,J=21.7Hz,1H),2.58( s,3H),2.41(s,1H),1.21(t,J=15.5Hz,6H),1.07(t,J=10.8Hz,4H),0.80(d,J=7.6Hz,1H).

[0182] In addition to the embodiments described above, other schemes exemplified by the present invention were synthesized accordingly. The specific compounds and their corresponding NMR identification data are shown in Table 1 below:

[0183] Table 1

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] II. Synthesis of Important Intermediates

[0190] This invention relates to a mechanically assisted synthesis of key intermediates of quinazoline derivatives at room temperature, the method comprising:

[0191] Add the quinazoline chloride shown in Formula IV, the fatty amine shown in Formula V, the base, and the grinding aid to a ball mill jar; then place the reaction system at a suitable temperature, with a reaction rate of 10-30 Hz and a reaction time of 0.5-1 hour.

[0192] The reaction formula is as follows:

[0193]

[0194] Wherein, R1 is H, halogen, C 1-4 Alkyl, C 1-4 Alkoxy or C 1-4 Halogenated alkyl groups;

[0195] R2 is H, halogen, C1-4 alkoxy or C1-4 haloalkyl;

[0196] The structure of aliphatic amines is shown in the following formula:

[0197]

[0198] Where R3 represents H and C 1-7 Alkyl, benzyl, or 3-7 membered cycloalkyl;

[0199] R4 is C 1-4 Alkyl, benzyl, or 3-7 membered cycloalkyl.

[0200] After the reaction is complete, the ball mill jar is washed three times with dichloromethane. The washing liquid is then filtered, vacuum concentrated, and column chromatography to obtain the product shown in Formula VI.

[0201] Specifically, the intermediate preparation process is as shown in Examples 1, 7, 8 and 18 above, and intermediates with the following structures were obtained respectively.

[0202]

[0203] This disclosure provides a method for synthesizing a key intermediate of a quinazoline derivative, which is further described in detail below with reference to Example 1 of the intermediate:

[0204] The reaction formulas for the preparation of quinazoline chlorides in the following examples are as follows:

[0205]

[0206] Key intermediates for synthesizing a series of quinazoline derivatives from the above raw materials.

[0207]

[0208] Quinazoline chloride 1 (0.2 mmol), various aliphatic amines 2 (0.24 mmol), CsF (0.4 mmol), and 0.12 mL DMSO were added to a 1.5 mL ball mill jar and reacted at 30 Hz for 1 h. After the reaction was complete, the ball mill jar was washed three times with dichloromethane, the organic phases were combined, washed three times with saturated sodium chloride solution, and dried over anhydrous sodium sulfate. The solvent was removed by filtration under reduced pressure, and the product was purified by silica gel column chromatography to obtain the target product 3.

[0209] Specifically, examples of the synthesis of the five important intermediates shown in the following formulas are listed:

[0210]

[0211] Intermediate Synthesis Example 1

[0212]

[0213] 2-(azacyclobutan-1-yl)-3-phenylquinazoline-4(3H)-one (I-3a): White solid, 91% yield (50.4 mg). HRMS (ESI): [M+H] + =278.1291.

[0214] 1 H NMR (400MHz, CDCl3) δ8.12(dd,J=8.0,1.3Hz,1H),7.61(ddd,J=8.5,7.1,1.6Hz,1H),7.54–7.48(m,2H),7.45(ddd,J= 8.8, 2.8, 1.4Hz, 2H), 7.35–7.31 (m, 2H), 7.18 (ddd, J=8.1, 7.2, 1.1Hz, 1H), 3.68–3.52 (m, 4H), 2.02 (t, J=7.7Hz, 2H).

[0215] Intermediate Synthesis Example 2

[0216]

[0217] 2-(azacycloheptan-1-yl)-3-phenylquinazoline-4(3H)-one (I-3d): white solid, 92% yield (58.7 mg). HRMS (ESI): [M+H]+ = 320.1762.

[0218] 1H NMR(400MHz, CDCl3)δ8.14(dd,J=7.9,1.4Hz,1H),7.63(ddd,J=8.4,7.1,1.6Hz,1H),7.51–7.44(m, 3H),7.42–7.37(m,1H),7.36–7.31(m,2H),7.25–7.18(m,1H),3.36–3.21(m,4H),1.50–1.29(m,8H).

[0219] Intermediate Synthesis Example 3

[0220]

[0221] 2-(isopropyl(methyl)amino)-3-phenylquinazoline-4(3H)-one (I-3j): white solid, 91% yield (53.3 mg). HRMS (ESI): [M+H]+ = 294.1058.

[0222] 1H NMR(400MHz, CDCl3)δ8.07(dd,J=7.9,1.3Hz,1H),7.60–7.50(m,1H),7.43–7.33(m,3H),7.27(ddd ,J=9.6,6.8,1.1Hz,3H),7.19–7.09(m,1H),3.89–3.75(m,1H),2.45(s,3H),0.76(d,J=6.7Hz,6H).

[0223] Intermediate Synthesis Example 4

[0224]

[0225] 2-((4-oxo-3-phenyl-3,4-dihydroquinazolin-2-yl)amino)-3-phenylpropionamide (I-3n): White solid, 71% yield (53.7 mg). HRMS (ESI): [M+H] + =385.1661.

[0226] 1H NMR(400MHz, CDCl3)δ8.04(dd,J=7.9,1.1Hz,1H),7.59–7.52(m,1H),7.47( dd,J=9.1,5.5Hz,2H),7.44–7.37(m,2H),7.20–7.11(m,5H),6.96(dd,J=6.4 ,2.8Hz,2H),6.81(d,J=7.6Hz,1H),6.51(s,1H),5.56(s,1H),4.83(dd,J=1 3.9, 6.7Hz, 1H), 4.55 (d, J=6.4Hz, 1H), 2.99 (ddd, J=22.0, 14.2, 7.1Hz, 2H).

[0227] Intermediate Synthesis Example 5

[0228]

[0229] 3-Benzyl-2-(cyclohexyl(methyl)amino)-5,6-dihydropyrimidin-4(3H)-one (I-3q): White solid, 86% yield (51.4 mg). HRMS (ESI): [M+H] + =298.1917.

[0230] 1 H NMR(400MHz, CDCl3)δ8.09(d,J=5.6Hz,1H),7.45(d,J=7.2Hz,2H),7.42–7.29(m,3H),6.01(d,J=5.6Hz,1 H),5.39(s,2H),4.57(s,1H),3.03(s,3H),1.86(d,J=11.7Hz,2H),1.77–1.64(m,3H),1.59–1.33(m,5H).

[0231] III. Activity Test

[0232] 1. In vitro induced ferroptosis activity assay

[0233] Cell viability assay

[0234] The 786-O cell line used in this invention was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. For cell culture, the following were used: RPMI 1640 (Gibco, catalog number 22400-089; batch number 2277021), FBS (Gibco, catalog number 16000-044; batch number 2351176P), penicillin / streptomycin (PS) (Gibco, catalog number 15140-122; batch number 2321114), 75T cell culture flasks (SPL, catalog number 71075), 175T cell culture flasks (SPL, catalog number 71175), 96-well cell culture plates (SPL, catalog number 30096), PBS pH 7.4 (Gibco, catalog number 10010-023; batch number 2085080), and TrypLE™. Express (Gibco, catalog number 12605-010; batch number 2323417), counting chamber (hematocytometer (Marienfeld Superior, catalog number 0650010), 0.4% trypan blue solution (DYNEBIO, catalog number CBT3710; batch number 20211201).

[0235] Methods for processing the compounds of the present invention and for measuring cell viability:

[0236] The compounds from Examples 1-33 were completely dissolved in DMSO (Sigma-Aldrich Cat, catalog number D2438, batch number RNBJ9566) and used in experiments.

[0237] Inoculate 3 × 10⁶ cells per well of a 96-well plate (SPL). 4 Cells were cultured at a total volume of 150 μL. Each compound was diluted 3-fold from a maximum concentration of 3000 nM to a minimum concentration of 0.46 nM. After treating each well with the compound to a total volume of 200 μL, the cells were cultured for 5 days in a CO2 incubator (Thermo Fisher Science, catalog 4111). Then, EZ-Cytox (DOGEN, catalog EZ-3000, lot DLS2109) (20 μL per well) was treated and cultured for 4 hours in a CO2 incubator. The absorbance of the fully cultured samples was measured by setting the wavelength of a plate reader (BMG Labtech, CLARIOstarPlus) to 450 nm, and the plate was shaken in the reader for 3 minutes before measurement. The final measurements were processed using Excel, plotted using Prism-GraphPad, and IC50 was measured. 50 The value was repeated three times. The results are shown in Table 2 below:

[0238] Table 2

[0239]

[0240]

[0241] It can be seen that the quinazolinone compounds and their derivatives and isomers of the present invention exhibit superior anticancer activity compared to the positive control drug IKE in experiments with 786-O cancer cells.

[0242] 2. Effects on the cell viability of different types of cancer cells

[0243] Taking compound A15 as an example, cell viability was measured:

[0244] (1) Cell line culture count

[0245] Cell lines including 786-O (CRL-1932), Hep3B (HB-8064), DU145 (HTB-81), and NCI H1299 (CRL-5803) were all purchased from the ATCC cell bank. For cell culture, DMEM (Gibco, catalog number C11995500BT; batch number 6124563) or RPMI 1640 (Gibco, catalog number 22400-089; batch number 6124215), a counting chamber, and an automated cell counter (LUNA-II) were used. TM Automated Cell Counter, CCK-8 solution (EnoGene, E1CK-000208), multi-functional microplate reader ( 200PRO ELISA reader (TECAN).

[0246] The powdered compounds A15 and IKE were completely dissolved in DMSO to prepare a suitable stock solution, which was then used in the experiments.

[0247] Collect cells in the logarithmic growth phase, and count the number of cells in the prepared cell suspension using an automated cell counter, according to...

[0248] 5×10 3 Inoculate cells with 100 μL of culture medium per well into a 96-well cell culture plate that has been preheated in an incubator, and incubate for 24 h.

[0249] (2) Methods for drug treatment and cell viability assay

[0250] Discard the original culture medium, dilute the stock solution of compound A15 with cell culture medium, and replace the old culture medium in the well plate with 200 μL of culture medium per well to ensure that compound A15 is in full contact with the cells. After 24 hours of treatment, discard the old culture medium, add 100 μL of culture medium (containing 10% CCK-8), place the 96-well cell culture plate in a CO2 incubator and react for 1 hour. Set the wavelength of the microplate reader to 450 nm, shake for 1 minute, and measure the absorbance OD value of each well.

[0251] The sensitivity of various tumor cell lines to compound A15 is shown in the appendix. Figure 1 As shown in Figure A, compound A15 can effectively reduce the activity of various cancer cells.

[0252] 3. Effects of different programmed cell death inhibitors on the inhibition of cancer cell viability by compound A15

[0253] (1) Cell line culture count

[0254] Collect various types of cancer cells in the logarithmic phase, according to 5×10 3 Inoculate cells with 100 μL of culture medium per well into a 96-well cell culture plate that has been preheated in an incubator, and incubate for 24 h.

[0255] (2) By combining various programmed cell death inhibitors with compound A15, we studied the specific types of cancer cell death induced by compound A15.

[0256] Apoptosis inhibitor Z-VAD-FMK (T7020, TargetMol), necroptosis inhibitor Necrostain2racemate (HY-14622A, MCE), ferroptosis inhibitor Liproxstatin-1 (S7699, Selleckchem), and iron chelator Deferoxamine Mesylate (T1637, TargetMol).

[0257] Discard the original culture medium and add compound A15 and the above-mentioned inhibitors to the culture medium. Replace the old culture medium in the well plate with 200 μL of culture medium per well to ensure that the drugs are in full contact with the cells. After 24 hours of treatment, discard the old culture medium and add 100 μL of culture medium (containing 10% CCK-8). Place the 96-well cell culture plate in a CO2 incubator and react for 1 hour. Set the wavelength of the microplate reader to 450 nm, shake for 1 minute, and measure the absorbance OD value of each well.

[0258] The effects of each inhibitor on the inhibitory activity of compound A15 against cancer cells are shown in the table below. Figure 1As shown in Figure B, the inhibitory effect of compound A15 on cancer cell growth can be reversed by Liproxstatin-1 and Deferoxamine Mesylat.

[0259] 4. Mitochondrial electron microscopy observation

[0260] (1) Cell line culture count

[0261] Hep3B cells were seeded at a rate of 1.2 × 10⁶ cells / well into preheated 6-well cell culture plates and cultured for 24 h. After discarding the supernatant, A15 and Liproxstatin-1 were diluted with culture medium and cultured for 24 h.

[0262] (2) Electron microscopy sample collection

[0263] After 24 hours, discard the culture medium, add electron microscopy fixative, and fix at room temperature in the dark for about 5 minutes. Gently scrape off the cells in one direction using a cell scraper. Transfer the cell suspension to a centrifuge tube using a Pasteur pipette, and centrifuge at low speed for about 3-5 minutes. Discard the fixative, add fresh electron microscopy fixative, disperse the cell clumps and resuspend, fix at room temperature in the dark for 30 minutes, then transfer to 4°C for storage before sample preparation and scanning.

[0264] Refer to electron microscope images as follows Figure 2 As shown, treatment with compound A15 can cause significant changes in mitochondrial morphology, including mitochondrial shrinkage, outer membrane rupture, and reduction or disappearance of mitochondrial cristae.

[0265] 5. Detect the levels of ROS and lipid ROS in DU145 cells after A15 intervention.

[0266] (1) Cell line culture count

[0267] DU145 cells were seeded at a rate of 1×10⁵ cells / well into preheated 12-well cell culture plates and cultured for 24 hours. After discarding the supernatant, A15, Liproxstatin-1, and Deferoxamine Mesylate were diluted to working concentrations with culture medium and cultured for 24 hours.

[0268] (2) Detection of intracellular ROS and lipid ROS levels

[0269] Twenty-four hours after drug treatment, the supernatant was discarded. The probes H2DCFDA (for detecting intracellular total oxygen free radicals (ROS)) or BODIPY 581 / 591 (for lipid peroxidation) were diluted to working concentrations with culture medium and cultured at 37°C for 20 minutes. The supernatant was discarded, and the cells were washed with PBS, followed by trypsin digestion. 500 μL of fresh DMEM was added to terminate the digestion, and all cells were transferred to the centrifuge tube by pipetting. The cells were then centrifuged at 3000 rpm for 5 minutes, and the supernatant was gently discarded, leaving the cell pellet. The cell pellet was resuspended in PBS, filtered through a 200-mesh nylon screen into a flow cytometry tube, and then analyzed quantitatively using FlowJO 10.8.1 software.

[0270] The results are as follows Figure 3 As shown, treatment with compound A15 significantly upregulated the levels of total ROS and lipid peroxides in cells, while co-treatment with Liproxstatin-1 significantly reversed the increase in intracellular oxidative stress induced by compound A15.

[0271] 6. Detect the levels of glutamate and GSH in cells after A15 intervention.

[0272] (1) Detection of changes in intracellular glutamate levels after A15 treatment

[0273] Hep3B cells were divided into 5×10 3 Seed cells into 96-well plates and cultured for 24 hours. Afterward, remove the supernatant, wash twice with PBS, and dilute A15 and IKE separately with culture medium. Remove the supernatant, wash cells with pre-cooled PBS, and add 0.6N HCl to lyse the cells. Then add 1M Trisbase to inactivate the cells. Add the prescribed amount of Glutamate-Glo TM The Assay kit (Promega, J7021) provides glutamate dehydrogenase, recombinant luciferase, luciferase substrate, and NAD. Mix thoroughly and allow to stand at room temperature for one hour before using the TECAN. The 200PRO multi-functional microplate reader detects bioluminescent signals.

[0274] (2) Detection of changes in intracellular GSH levels after A15 treatment

[0275] 1) Collect samples

[0276] After seeding Hep3B cells into 6 cm cell culture dishes (density not exceeding 70%), the supernatant was discarded, and the cells were cultured in a medium containing working concentration of A15, the positive control drug IKE, and the GSH synthesis inhibitor Buthionine sulfoximine (pretreated for 10 hours). The cells were washed once with PBS, centrifuged to collect the cells, the supernatant was discarded, and three times the volume of the cell pellet was added to the deproteinizing reagent M (GSH and GSSG detection kit, Beyotime Biotechnology, S0053) and vortexed thoroughly. The cells were subjected to repeated freeze-thaw cycles using liquid nitrogen and a 37°C water bath. After centrifugation at 4°C and 10,000g for 5 minutes, the supernatant was discarded for the determination of total glutathione.

[0277] 2) Determination of GSH

[0278] Based on the principle: glutathione consists of reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG). GSH reacts with DTNB to generate TNB (yellow) and GSSG. When GSSG is reduced to GSH by glutathione reductase, the total glutathione content can be determined by the absorbance of TNB. After removing GSH from the sample, the GSSG content can be measured using the above method, and the difference between total glutathione and GSSG is the GSH content. Prepare the specified concentrations of buffer, glutathione reductase, and DTNB according to the kit instructions, and perform serial dilutions of the standards for subsequent standard curve plotting. Add the prepared liquids sequentially to a 96-well plate, incubate at room temperature for 5 minutes, then add NADPH solution, and use a microplate reader to detect A. 412 The absorbance at a certain point was measured, and then the intracellular GSH content was calculated using a standard curve and protein concentration.

[0279] The results are as follows Figure 4 As shown, similar to the positive control drugs IKE and Erastin, treatment with compound A15 significantly downregulated intracellular GSH levels and increased intracellular glutamate content.

[0280] 7. Animal experiments

[0281] Female Balb / c nude mice aged 4-6 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used for cell xenotransplantation experiments after one week of SPF acclimatization. H1299 cell lines were resuspended in PBS and then... 6 / 200μL was injected subcutaneously into mice, and when the average tumor volume reached approximately 50-100mm², the tumor was treated. 3Mice were randomly divided into three groups and injected intraperitoneally every two days with a solvent: 30 mg / kg A15, 30 mg / kg A15 + 10 mg / kg Liproxstatin-1. Tumor size and animal weight were measured. When the average tumor volume reached 1000 mm², the mice were considered the first group to be treated. 3 Animals were euthanized, tumor size was measured in each group, and samples were collected. Tumor tissue samples were fixed with electron microscopy fixative and 4% paraformaldehyde respectively for subsequent electron microscopy scanning and immunohistochemical staining.

[0282] The results are as follows Figure 5 As shown, the tumor volume and weight in the A15 treatment group were significantly reduced. Figure 5 As shown in Figure AC, intraperitoneal injection of compound A15 effectively inhibits the growth of subcutaneous tumors in mice. Paraffin sections of mouse tumor tissue were prepared and stained, revealing a significant increase in the oxidative stress cell damage marker 4-HNE, while no significant change was observed in the apoptosis marker Cleaved caspase-3. Figure 5 (As shown in DE). Furthermore, electron microscopy revealed that after A15 compound intervention, mitochondria in mouse tumor tissues were significantly swollen, and mitochondrial cristae disappeared, exhibiting obvious ferroptosis morphology. Figure 5 (As shown in F).

[0283] Therefore, it can be confirmed that the compounds of the present invention can effectively inhibit the uptake of extracellular cystine by SLC7A11, inducing ferroptosis. They can be used for the prevention or treatment of tumors or immune-related diseases associated with SLC7A11 inhibitory activity.

[0284] This invention discloses a novel class of quinazolinone derivatives. The structural innovation lies in the introduction of specific substituents (as shown in Formula I) into the quinazolinone core, making it a novel ferroptosis inducer. The quinazolinone derivatives utilize an innovative ball milling technique to construct CN bonds, significantly simplifying the synthesis steps and reducing preparation costs while avoiding the use of large amounts of organic solvents. In vitro experiments show that the quinazolinone derivatives exhibit significant cell death-inducing effects against renal cell carcinoma, prostate cancer, liver cancer, and lung cancer cell lines, outperforming the positive control drug Erastin by 5 times. Animal experiments also demonstrate its inhibitory effect on tumor growth.

[0285] The innovative advantages of this invention are: 1. It is the first to create a class of quinazolinone-based ferroptosis inducers, breaking through the structural limitations of existing Erastin-type compounds; 2. The ball milling technology enables solvent-free, room-temperature drug synthesis, which meets green chemistry standards; 3. It combines broad-spectrum antitumor activity with low toxicity, and has the potential for clinical translation.

[0286] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A quinazolinone derivative that induces ferroptosis, characterized in that, The structural formula is shown in Equation I below: In Equation I, R1 represents H, D, -OCD3, halogen, and C. 1-4 Alkoxy, C 1-4 Halogenated alkyl groups, deuterated C 1-6 alkyl; R2 represents D and C. 1-4 Alkyl, C 1-4 Halogenated alkyl groups, 3-7 membered cycloalkyl groups, deuterated C 1-6 alkyl; R3 can be H, D, -CD3, -OCD3, halogen, or C. 1-4 Alkoxy, C 1-4 Halogenated alkyl groups; R4 is C 1-4 alkyl; W represents the structure shown in equation 1-1 below: 1-1 Where Q is C 1-4 Alkyl groups, 3-7 membered cycloalkyl groups; Alternatively, Q can be represented by the structure in equation 1-2: 1-2 Where n=1; X is O; R5 is H, halogen, C 1-4 Alkyl or deuterated C 1-6 alkyl.

2. A quinazolinone derivative that induces ferroptosis, characterized in that, Including compounds represented by the following structural formulas A1-A33: ； 。 3. The method for preparing the quinazolinone derivative that induces ferroptosis according to claim 1, characterized in that, Includes the following steps: 1) Using compound 1 as the starting material, compound 2 was prepared by reacting it in a reducing agent and acidic solution in a reaction solvent; 2) Using N,N-diisopropylethylamine as a base, in the presence of a sulfiding agent and a catalyst, compound 2 is reacted to produce compound 3; 3) React the base, compound 3, and anthranilic acid derivative, i.e., compound 4, to generate compound 5; 4) Compound 5 was subjected to chlorination to obtain intermediate II, i.e., compound 6; 5) Under ball milling conditions, in the presence of alkali and grinding aid, compound 6 and compound 7 react to obtain compound I, namely a quinazolinone derivative that induces ferroptosis; The structural formulas of compounds 1, 2, 3, 4, 5, and 7 are shown below: The structural formula of intermediate II, namely compound 6, is shown below: In the above structural formula: R1 represents H, D, -OCD3, halogen, or C. 1-4 Alkoxy, C 1-4 Halogenated alkyl deuterated, C 1-6 alkyl; R2 represents D and C. 1-4 Alkyl, deuterated C 1-6 Alkyl, C 1-4 Halogenated alkyl or 3-7 membered cycloalkyl; R3 represents H, D, -OCD3, halogen, and C. 1-4 Alkoxy, deuterated C 1-6 Alkyl or -C 1-4 Halogenated alkyl groups; R4 is C 1-4 alkyl; W represents the structure shown in equation 1-1 below: 1-1 Where Q is C 1-4 Alkyl groups, 3-7 membered cycloalkyl groups; Alternatively, Q can be represented by the structure in equation 1-2: 1-2 Where n=1; X is O; R5 is H, D, -OCD3, halogen, C 1-4 Alkyl or deuterated C 1-6 alkyl.

4. The method for preparing the quinazolinone derivative that induces ferroptosis according to claim 3, characterized in that, In step 1), the reducing agent is ferrous chloride, tin dichloride or titanium tetrachloride, the reaction solvent is tetrahydrofuran, the acidic solution is hydrochloric acid or sulfuric acid, and the volume ratio of the acidic solution to the reaction solvent is 1:

1. In step 2), the sulfiding agent is CS2, and the catalyst is di-tert-butyl dicarbonate; In step 3), the molar ratio of compound 3 to the anthranilic acid derivative is 1:1.2, the base used is triethylamine or N,N-diisopropylethylamine, the reaction temperature is 60-70 °C, and the reaction time is 6-12 hours. In step 4), thionyl chloride or thioyl chloride is used as the chlorination reagent in the chlorination reaction, and the molar ratio of compound 5 to the chlorination reagent is 1:2; the solvent for the chlorination reaction is tetrahydrofuran or chloroform, the reaction temperature is 50-70℃, and the reaction time is 8-12 hours. In step 5), the alkali is cesium fluoride, sodium fluoride or tert-butyllithium, and the grinding aid is dimethyl sulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide. The reaction conditions are 10-30 Hz and the reaction time is 0.5-1 hour.

5. The method for preparing the quinazolinone derivative that induces ferroptosis according to claim 3, characterized in that, Step 5) specifically includes: Compound 6, compound 7, alkali, grinding aid and solvent were ball-milled at 50-70℃ and 10-30Hz for 0.5-1 hours, followed by washing, filtration, concentration and column chromatography to obtain quinazolinone derivatives and their isomers that induce ferroptosis.

6. The method for synthesizing quinazolinone derivatives that induce ferroptosis according to claim 5, characterized in that, The molar amount of compound 7 is 1.2 times that of compound 6; the base is cesium fluoride, sodium fluoride, or lithium tert-butoxide, and the amount used is 2 times that of compound 6; the grinding aid is dimethyl sulfoxide, N,N-dimethylformamide, or N,N-dimethylacetamide.

7. A pharmaceutically acceptable salt of the quinazolinone derivative that induces ferroptosis as described in claim 1 or 2.

8. The use of the quinazolinone derivative of claim 1 or 2 that induces ferroptosis, or the salt of claim 7, as a small molecule inhibitor of SLC7A11 in the preparation of a medicament for treating tumors or immune-related diseases.

9. The application as described in claim 8, characterized in that, The tumors mentioned include lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, leukemia, colon cancer, pancreatic cancer, skin cancer, head and neck cancer, small intestine cancer, rectal cancer, endometrial cancer, vaginal cancer, testicular cancer, esophageal cancer, bile duct cancer, lymphoma, gallbladder cancer, endocrine gland cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brainstem glioma, and pituitary adenoma.

10. The application as described in claim 8, characterized in that, The immune-related diseases include allergies caused by cell activation and impaired tissue damage repair.

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