Quinazolinone derivative capable of inducing ferroptosis as well as synthesis method and application of quinazolinone derivative

Abstract

The invention discloses quinazolinone derivatives capable of inducing ferroptosis as well as a synthesis method and application of the quinazolinone derivatives, and belongs to the technical field of biological medicines. The quinazolinone derivative is structurally characterized in that a specific substituent group is introduced into a quinazolinone parent nucleus, and the quinazolinone derivative belongs to a novel ferroptosis inducer. According to the invention, a C-N bond is constructed through an innovative ball-milling preparation technology, so that the use of a large amount of organic solvent is avoided, the synthesis steps are greatly simplified, and the preparation cost is saved. In-vitro experiments show that the derivative has a remarkable cell death inducing effect on kidney cancer, prostatic cancer, liver cancer and lung cancer cell lines, the effect is 5 times better than that of a positive control drug Erastatin, and the tumor growth inhibiting effect of the derivative is proved in animal experiments. The quinazolinone ferroptosis inducer has the advantages that the quinazolinone ferroptosis inducer is created for the first time, and the structural limitation of existing Erastin compounds is broken through; 2, solvent-free and normal-temperature drug synthesis is realized by a ball milling technology, and the green chemical standard is met; 3, broad-spectrum anti-tumor activity and low toxicity are combined, and clinical transformation potential is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to a quinazolinone derivative inducing ferroptosis, a synthesis method and application thereof. Background Art

[0002] Ferroptosis is an iron-dependent, novel cell programmed death caused by the accumulation of lipid peroxides, which is different from apoptosis and necrosis. It can cause multiple organ damage and degenerative lesions. In addition, drug-resistant tumor cells, especially those in the mesenchymal state and prone to metastasis, are very sensitive to ferroptosis. Therefore, drug-induced and inhibited ferroptosis has great potential in overcoming tumor resistance and treating degenerative diseases and ischemic organ damage associated with lipid peroxidation. The cystine transporter SLC7A11 can regulate the transport of cystine / glutamate by System Xc-, and plays a key role in cystine metabolism and ferroptosis regulation. Inhibiting the uptake and transport of extracellular cystine by SLC7A11 can prevent the synthesis of glutathione (GSH), a key antioxidant substance in cells, leading to excessive accumulation of peroxides in cells and inducing ferroptosis. With the gradual deepening of ferroptosis research, studies have found that SLC7A11 is an ideal target for inducing ferroptosis and inhibiting tumors. First, SLC7A11 knockout mice are 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 a variety of tumors and is negatively regulated by tumor suppressor genes such as p53, BAP1, KEAP1, ATF3, etc., suggesting that it plays a key role in tumor development. Third, SLC7A11 is an oxidative stress protein that is dynamically regulated by oxidative stress, cytotoxic substances, etc. It may play a key role in specific tumor microenvironments and provide an important window for tumor targeted therapy. Therefore, the development of small molecule inhibitors specific for SLC7A11 has broad application prospects in cancer treatment.

[0003] The reported small molecule inhibitors for SLC7A11 are mainly Erastin and its analogs, which can inhibit the uptake of extracellular cystine by SLC7A11, causing the accumulation of intracellular peroxides to induce ferroptosis. Erastin has poor metabolic stability and water solubility and can only be used for in vitro experiments. Erastin's derivative IKE (Imidazole ketone erastin) can be used in vivo, but it has problems such as poor water solubility, poor metabolic stability and low potency, which limits its further clinical application. More importantly, the preparation process of IKE is complicated, the yield is low, and the cost is high. The development of a new specific targeted SLC7A11 inhibitor based on a new chemical synthesis process and the detection of its effects on inducing cell death and suppressing tumors have important clinical application value. Summary of the invention

[0004] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a new quinazolinone derivative that induces ferroptosis, so as to solve the technical problems of the existing small molecule inhibitors of SLC7A11, such as complex preparation process, poor water solubility, poor metabolic stability and low potency; at the same time, the present invention also provides a method for preparing the above-mentioned quinazolinone derivatives that induce ferroptosis and their isomers, aiming to solve the technical problems that the prior art must be synthesized under high temperature and high pressure conditions and the material conversion is insufficient.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention discloses a quinazolinone derivative and an isomer thereof that induces ferroptosis, and the structural formula is shown in the following formula I:

[0007]

[0008] In formula I, R1 is H, D, -OCD3, halogen, C 1-4 Alkoxy, C 1-4 Haloalkyl, deuterated C 1-6 Alkyl; R2 is D, C 1-4 Alkyl, C 1-4 Haloalkyl, 3-7 membered cycloalkyl, deuterated C 1-6 alkyl;

[0009] R3 is H, D, -CD3, -OCD3, halogen, C 1-4 Alkoxy, C 1-4 Haloalkyl;

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

[0011] W is a structure represented by the following formula 1-1:

[0012]

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

[0014] Alternatively, Q is a structure represented by the following formula 1-2:

[0015]

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

[0017]

[0018] Wherein, R6 is H, D, -OCD3, halogen, C 1-4 Alkoxy, deuterated C 1-6 alkyl.

[0019] Preferably, the compound includes the following structural formula A1-A33:

[0020]

[0021]

[0022]

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

[0024] 1) Compound 1 is used as a starting material, and reacts in a reaction solvent under a reducing agent and acidic conditions to obtain compound 2;

[0025] 2) using N,N-diisopropylethylamine as a base, in the presence of a sulfiding agent and a catalyst, reacting with compound 2 to generate compound 3;

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

[0027] 4) subjecting compound 5 to chlorination reaction to obtain intermediate II, i.e. compound 6;

[0028] 5) Under ball milling conditions, in the presence of a base and a grinding aid, compound 6 and compound 7 are reacted to obtain compound I, i.e., quinazolinone derivatives and isomers thereof that induce ferroptosis;

[0029] Wherein: the structural formulas of compound 1, compound 2, compound 3, compound 4, compound 5 and compound 7 are shown below:

[0030]

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

[0032]

[0033] In the above structural formula:

[0034] R1 is H, D, -OCD3, halogen, C1-4 Alkoxy, C 1-4 Deuterated alkyl halide, C 1-6 alkyl;

[0035] R2 is D, C 1-4 Alkyl, deuterated C 1-6 Alkyl, C 1-4 Haloalkyl or 3-7 membered cycloalkyl;

[0036] R3 is H, D, -OCD3, halogen, C 1-4 Alkoxy, deuterated C 1-6 Alkyl or -C 1-4 Haloalkyl;

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

[0038] W is a structure represented by the following formula 1-1:

[0039]

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

[0041] Alternatively, Q is a structure represented by the following formula 1-2:

[0042]

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

[0044] Alternatively, Q is a structure represented by the following formula 1-3:

[0045]

[0046] Wherein, R6 is H, D, -OCD3, halogen, deuterated C 1-6 Alkyl or C 1-4 Alkoxy.

[0047] Preferably, in step 1), the reducing agent is ferrous chloride, tin dichloride or titanium tetrachloride, the reaction solvent is tetrahydrofuran, the acidic condition is provided by 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 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;

[0050] In step 4), the chlorination reaction uses thionyl chloride or sulfuryl chloride as the chlorination reagent, 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° C., and the reaction time is 8-12 hours;

[0051] In step 5), the base is cesium fluoride, sodium fluoride or tert-butyl lithium, 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] Under ball milling conditions, compound 6, compound 7, a base, a grinding aid and a solvent are ball milled at 50-70°C and 10-30Hz for 0.5-1 hour, and then washed, filtered, concentrated and subjected to column chromatography to obtain quinazolinone derivatives and isomers thereof that induce ferroptosis.

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

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

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

[0057] Preferably, the tumor includes lung cancer, liver cancer, gastric cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, blood cancer, 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, lymph node cancer, gallbladder cancer, endocrine gland cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brain stem glioma and pituitary adenoma.

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

[0059] Further preferably, the tissue damage is such as a wound healing disorder.

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

[0061] The present invention discloses for the first time a class of quinazolinone derivatives and isomers thereof capable of inducing ferroptosis, and forms a special heterocyclic structure system by fusing the quinazolinone ring with other groups, which endows the derivatives with unique biological activity and provides them with very good application prospects in anti-tumor and other aspects. Experimental verification shows that the quinazolinone compounds of the present invention show a better inhibitory effect than the positive drugs erastin and IKE on various types of cancer cells (including kidney cancer, prostate cancer, liver cancer and lung cancer), and have a broad-spectrum anti-tumor activity, which illustrates their excellent application potential.

[0062] In terms of the synthesis method, the present application effectively shortens the synthesis path. The synthesis method is simple, and the key steps can be achieved by ball milling alone. There is no need for strict nitrogen protection and harsh conditions such as high temperature. The amount of solvent used is small, the reaction time is short, the reaction conditions are green and mild, and the yield is as high as 85%. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The effect of compound A15 on the cell viability of different types of cancer cells; A is a cell viability analysis chart of four cancer types, namely, kidney cancer, prostate cancer, lung cancer and liver cancer; B is a cell viability analysis chart of four specific cell lines, namely, 786-O, DU145, H1299 and Hep3B;

[0064] Figure 2 This is the electron microscopic observation result of the mitochondrial morphology changes in cells treated with compound A15;

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

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

[0067] Figure 5 The figure is a result diagram of the activity of compound A15 measured in in vivo animal experiments; wherein, 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, which includes oxidative stress cell damage indicators 4-HNE and Cleaved caspase-3; E is the change of oxidative stress cell damage indicator 4-HNE after A15 intervention; and F is an electron microscopic image of mitochondria in tumor tissues in each group. DETAILED DESCRIPTION

[0068] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0070] The present invention is further described in detail below in conjunction with the accompanying drawings:

[0071] The present invention focuses on the target of SLC7A11, and through comparison with its own unique small molecule drug screening database and re-optimization and design of small molecule structures, a series of new SLC7A11 small molecule inhibitors are synthesized, whose general structural formula is 1.

[0072]

[0073] The compound of general structural formula 2 is a key intermediate in the synthesis process. The synthesis of similar structures reported in the prior art usually requires high-temperature thermal catalysis and high-boiling-point polar reaction solvents, resulting in a complicated post-processing process and the generation of unnecessary by-products.

[0074] Mechanochemistry is a method of inducing physical and chemical changes in reactants by grinding, friction and other methods, thereby causing changes in the properties and performance of reactants and solids, liquids, gases, etc. in the environment. By supplying energy through mechanical force and fine grinding, not only can high temperature and high pressure conditions be avoided, and the material is fully converted, but also it is green and efficient, reduces organic solvent emissions and reaction time, and improves the conversion rate of raw materials. Therefore, the present invention aims to develop a green method for synthesizing key intermediates of quinazolinones using mechanochemistry, which has mild conditions, does not require high temperature catalysis, consumes less solvent, can effectively achieve high yield synthesis of quinazoline derivatives, and provides a key material basis for its industrial production and related research.

[0075] 1. Synthesis Example

[0076] Example 1

[0077] Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (Compound A1), the reaction equation is as follows:

[0078]

[0079] The synthesis method comprises the following steps:

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

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

[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 3-(2-isopropoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (2.5 g, 8.0 mmol), and the mixture was reacted at 60°C for 8 h. After the reaction was completed, an appropriate amount of dichloromethane was added, and the mixture was washed three times with a saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 10 / 1). The target product 2-chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3 g, 7.2 mmol) was obtained as a white solid with a yield of 90%.

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

[0085] 2-Chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3g, 7.2mmol), tert-butyl 4-aminopiperidine-1-carboxylate (1.7g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=3 / 1). The target product tert-butyl 4-((3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)amino)piperidine-1-carboxylate (2.9g, 6.1mmol) was obtained as a white solid with a yield of 85%.

[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 a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 with a yield of 85%. 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] Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (Compound A2), the reaction equation is as follows:

[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, stirred for 20 min, iodomethane (93.0 μL, 1.5 mmol) was added, and the mixture was reacted at room temperature for 2 h. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluent: 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 with 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] Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(benzyl)amino)-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (Compound A4), the reaction equation is as follows:

[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, stirred for 20 min, benzyl bromide (180.0 μL, 1.5 mmol) was added, and the mixture was reacted at room temperature for 2 h. After filtration, the solvent was removed under reduced pressure, and the mixture was purified by silica gel column chromatography (eluent: 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 with a yield of 83%. 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] Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropyloxyphenyl)quinazolin-4(3H)-one (Compound A7), the reaction equation is as follows:

[0101]

[0102] The synthesis method comprises the following steps:

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

[0104] Under nitrogen protection, 1-nitro-2-cyclopropyloxybenzene (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 completed, sodium hydroxide solution (1.0 M, 50 mL) was added and dried over anhydrous sodium sulfate. Dichloromethane was used to extract twice, and the organic phases were combined and dried over anhydrous sodium sulfate. After filtering, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=20 / 1). The target product 2-cyclopropyloxyaniline (1.7 mL, 12.0 mmol) was obtained as a red liquid with a yield of 60%.

[0105] Step 2: Synthesis of 1-isothiocyanato-2-cyclopropyloxybenzene (3)

[0106] Under nitrogen protection, 2-cyclopropyloxyaniline (1.7 mL, 12.0 mmol), DIPEA (2.7 mL, 15.0 mmol) and THF (50 mL) were mixed and stirred at room temperature overnight. The mixture was cooled 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 completed, water was added to terminate the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined 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 (eluent: ethyl acetate / petroleum ether = 50 / 1). The target product 1-isothiocyanato-2-cyclopropyloxybenzene (1.9 mL, 10.0 mmol) was obtained as a white solid with a yield of 83%.

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

[0108] Under nitrogen protection, 1-isothiocyanato-2-cyclopropyloxybenzene (1.9 mL, 10.0 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 2-aminobenzoic acid (1.4 g, 10.0 mmol) and the mixture was reacted at 60 °C for 12 h. After the reaction was completed, water was added to terminate the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined 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 (eluent: ethyl acetate / petroleum ether = 3 / 1). The target product 3-(2-cyclopropyloxyphenyl)-2-thio-2,3-dihydroquinazolin-4(1H)-one (2.4 g, 8.0 mmol) was obtained as a white solid with a yield of 80%.

[0109] Step 4: Synthesis of 2-chloro-3-(2-cyclopropyloxyphenyl)-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 3-(2-cyclopropyloxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (2.4 g, 8.0 mmol), and the mixture was reacted at 60°C for 8 h. After the reaction was completed, an appropriate amount of dichloromethane was added, and the mixture was washed three times with a saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 10 / 1). The target product 2-chloro-3-(2-cyclopropyloxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (2.0 g, 7.2 mmol) was obtained as a white solid with a yield of 90%.

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

[0112] 2-Chloro-3-(2-cyclopropyloxyphenyl)-2,3-dihydroquinazolin-4(1H)-one (2.0g, 7.2mmol), tert-butyl 4-(methylamino)piperidine-1-carboxylate (1.7g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=3 / 1). The target product tert-butyl 4-((3-(2-cyclopropyloxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.9g, 6.1mmol) was obtained as a white solid with a yield of 85%.

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

[0114] Under nitrogen protection, tert-butyl 4-((3-(2-cyclopropyloxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.9 g, 6.1 mmol) was dissolved in a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 2 / 1). The target product 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropyloxyphenyl)quinazolin-4(3H)-one (2.4 g, 4.3 mmol) was obtained as a white solid with a yield of 72%. 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] Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-5-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (Compound A15), the reaction equation is as follows:

[0118]

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

[0120] Under nitrogen protection, 1-isopropoxy-2-isothiocyanatobenzene (1.9 g, 10.0 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 2-amino-3-fluorobenzoic acid (1.6 g, 10.0 mmol) and the mixture was reacted at 60 °C for 12 h. After the reaction was completed, water was added to terminate the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 obtained as a white solid with 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 8-fluoro-3-(2-isopropoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (2.4 g, 7.3 mmol), and the mixture was reacted at 60°C for 8 h. After the reaction was completed, an appropriate amount of dichloromethane was added, and the mixture was washed three times with a saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 10 / 1). The target product 2-chloro-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-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 4-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (6)

[0124] 2-Chloro-8-fluoro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3g, 7.0mmol), tert-butyl 4-(methylamino)piperidine-1-carboxylate (1.7g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=3 / 1). The target product tert-butyl 4-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylate (2.9g, 6.1mmol) was obtained as a white solid with a yield of 85%.

[0125] Step 4. Synthesis of 2-(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 a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 with a yield of 85%. 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 as follows:

[0130]

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

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

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

[0134] Under nitrogen protection, 5-bromo-2-isopropoxyaniline (2.3mL, 12.0mmol), DIPEA (2.7mL, 15.0mmol) and THF (50mL) were mixed and stirred at room temperature overnight. The mixture was cooled to 0°C, CS2 (1.6mL, 24.0mmol) and di-tert-butyl dicarbonate (2.0g, 10.0mmol) were added, and the mixture was reacted at room temperature for 2h. After the reaction was completed, water was added to terminate the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined 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 (eluent: ethyl acetate / petroleum ether=50 / 1). The target product 4-bromo-1-isopropoxy-2-isothiocyanatobenzene (1.9mL, 10.0mmol) was obtained as a white solid with a yield of 83%.

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

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

[0137] Step 4. Synthesis of 3-(5-bromo-2-isopropoxyphenyl)-2-chloroquinazolin-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-dihydroquinazolin-4(1H)-one (3.2 g, 8.0 mmol), and the mixture was reacted at 60°C for 8 h. After the reaction was completed, an appropriate amount of dichloromethane was added, and the mixture was washed three times with a saturated sodium bicarbonate solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 10 / 1). The target product 3-(5-bromo-2-isopropoxyphenyl)-2-chloroquinazolin-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 (2.8g, 7.2mmol), tert-butyl 4-(methylamino)piperidine-1-carboxylate (1.7g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether=3 / 1). The target product tert-butyl 4-((3-(5-bromo-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazoline-2-yl)(methyl)amino)piperidine-1-carboxylate (3.5g, 6.1mmol) was obtained as a white solid with a yield of 85%.

[0141] Step 6. Synthesis of 2-(1-(2-(4-chlorophenoxy)acetyl)piperidin-4-yl)(methyl)amino)-3-(2-cyclopropyloxyphenyl)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 a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 a yield of 72%. 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 as follows:

[0146]

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

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

[0149] Step 2. Synthesis of 1-(4-isopropoxy-3-isothiocyanatophenyl)ethan-1-one (3)

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

[0151] Step 3. Synthesis of 3-(5-acetyl-2-isopropoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (5) Under nitrogen protection, 1-(4-isopropoxy-3-isothiocyanatophenyl)ethan-1-one (1.7 mL, 12.3 mmol) and triethylamine (2.1 mL, 15.0 mmol) were added to a THF solution (60 mL) of 2-aminobenzoic acid (1.4 g, 10.0 mmol) and reacted at 60°C for 12 h. After the reaction was completed, water was added to terminate the reaction, and the mixture was extracted twice with dichloromethane. The organic phases were combined 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 (eluent: ethyl acetate / petroleum ether = 1 / 1). The target product 3-(5-acetyl-2-isopropoxyphenyl)-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (2.7 g, 7.6 mmol) was obtained as a white solid in a yield of 80%.

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

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

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

[0155] 3-(5-acetyl-2-isopropoxyphenyl)-2-chloroquinazolin-4(3H)-one (2.3g, 6.4mmol), 4-(methylamino)piperidine-1-carboxylic acid tert-butyl ester (1.7g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 1 / 1). The target product 4-((3-(5-acetyl-2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)piperidine-1-carboxylic acid tert-butyl ester (2.2g, 4.2mmol) was obtained as a white solid with 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 a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 with a yield of 93%. 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 tert-butyl 6-((8-fluoro-3-(2-isopropoxyphenyl)-4-oxo-3,4-dihydroquinazolin-2-yl)(methyl)amino)-2-azaspiro[3.3]heptane-2-carboxylate (3)

[0163] 2-Chloro-3-(2-isopropoxyphenyl)quinazolin-4(3H)-one (2.3g, 7.2mmol), 6-(methylamino)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (1.9g, 8.6mmol), and 0.5mL DMSO were added to a 10mL ball mill and reacted at 30Hz for 1h. After the reaction was completed, the ball mill was washed three times with dichloromethane, the organic phase was 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 purified by silica gel column chromatography (eluent: 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 (3.1g, 6.0mmol) was obtained as a white solid with a yield of 83%.

[0164] Step 2. 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)

[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-carboxylate (3.1 g, 6.0 mmol) was dissolved in a hydrochloric acid ethyl acetate 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 in sequence and reacted 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 purified by silica gel column chromatography (eluent: 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 a yield of 78%. 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] 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), the reaction equation is as follows:

[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 a hydrochloric acid ethyl acetate 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 in sequence and reacted 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 purified by silica gel column chromatography (eluent: 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 a yield of 67%. 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] 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), the reaction equation is as follows:

[0174]

[0175] 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 a hydrochloric acid ethyl acetate 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 in sequence and reacted at room temperature for 3 h. The mixture was washed three times with a saturated sodium chloride solution and dried over anhydrous sodium sulfate. After filtration, the solvent was removed under reduced pressure and purified by silica gel column chromatography (eluent: 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 a yield of 62%. 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] Embodiment 11

[0178] The synthesis of 3-(2-isopropoxyphenyl)-2-(methyl(1-(1-methyl-1H-imidazole-4-carbonyl)piperidin-4-yl)amino)quinazolin-4(3H)-one (Compound A32) is as follows:

[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 a hydrochloric acid ethyl acetate 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-imidazole-4-carboxylic acid (1.2 g, 9.1 mmol) were added in sequence and reacted 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 purified by silica gel column chromatography (eluent: ethyl acetate / petroleum ether = 2 / 1). The target product 3-(2-isopropoxyphenyl)-2-(methyl(1-(1-methyl-1H-imidazole-4-carbonyl)piperidin-4-yl)amino)quinazolin-4(3H)-one (1.7 g, 3.4 mmol) was obtained as a white solid in a yield of 55%. 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 above embodiments, other schemes exemplified in the present invention were synthesized accordingly, and the specific compounds and their corresponding NMR identification data are shown in Table 1 below:

[0183] Table 1

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] 2. Synthesis of important intermediates

[0190] The present invention synthesizes a key intermediate of a quinazoline derivative at room temperature with the assistance of mechanical force, and the method comprises:

[0191] Add quinazoline chloride represented by formula IV, fatty amine represented by formula V, base and grinding aid into a ball mill; then place the reaction system at a suitable temperature, reaction conditions are 10-30Hz, and the time is 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 Haloalkyl;

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

[0196] The structure of fatty amines is shown below:

[0197]

[0198] Wherein, R3 is H, C 1-7 Alkyl, benzyl or 3-7 membered cycloalkyl;

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

[0200] When the reaction is complete, the ball mill is washed with dichloromethane for three times, and the washing liquid is filtered, vacuum concentrated, and column chromatographed to obtain the product shown in Formula VI.

[0201] Specifically, the intermediate preparation process is as shown in the above-mentioned Example 1, Example 7, Example 8 and Example 18, and the intermediates shown in the following structures are respectively prepared.

[0202]

[0203] The present disclosure provides a method for synthesizing a key intermediate of a quinazoline derivative, which is further described below in conjunction with Intermediate Example 1:

[0204] The reaction formula for preparing the series of quinazoline chlorides in the following examples is as follows:

[0205]

[0206] From the above raw materials, a series of key intermediates of quinazoline derivatives are synthesized

[0207]

[0208] Quinazoline chloride 1 (0.2 mmol), various fatty amines 2 (0.24 mmol), CsF (0.4 mmol) and 0.12 mL DMSO were added to a 1.5 mL ball mill and reacted at 30 Hz for 1 h. After the reaction was completed, the ball mill 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 filtering, the solvent was removed under reduced pressure and purified by silica gel column chromatography. The target product 3 was obtained.

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

[0210]

[0211] Intermediate Synthesis Example 1

[0212]

[0213] 2-(Azetidin-1-yl)-3-phenylquinazolin-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-(Azepan-1-yl)-3-phenylquinazolin-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-phenylquinazolin-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-phenylpropanamide (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] 3. Activity Test

[0232] 1. In vitro ferroptosis-inducing activity assay

[0233] Cell viability assay

[0234] The 786-O cell line used in the present invention was purchased from the cell line bank of Shanghai Chinese Academy of Sciences. For cell culture, RPMI1640 (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 flask (SPL, catalog number 71075), 175T cell culture flask (SPL, catalog number 71175), 96-well cell culture plate (SPL, catalog number 30096), PBS pH 7.4 (Gibco, catalog number 10010-023; batch number 2085080), TrypLETM Express (Gibco, catalog number 12605-010; batch number 2323417), counting chamber (Hematocytometer (MarienfeldSuperior, catalog number 0650010), 0.4% Trypan blue solution (DYNEBIO, catalog number CBT3710; batch number 20211201).

[0235] Methods of treatment with compounds of the present invention and cell viability assay:

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

[0237] 3 × 10 cells were seeded in each well of a 96-well plate (SPL). 4 Cells were added and the cells were cultured in a total volume of 150 μL. Each compound was diluted 3 times from the highest concentration of 3000 nM to the lowest concentration of 0.46 nM. After the compound was treated to each well to make the total volume 200 μL, it was cultured in a CO2 incubator (Thermo Fisher Science, catalog number 4111) for 5 days. Then, after treating EZ-Cytox (DOGEN, catalog number EZ-3000, batch number DLS2109) (20 μL per well), it was cultured in a CO2 incubator for 4 hours. The absorbance of the fully cultured sample was measured by setting the wavelength of the plate reader (BMG Labtech, CLARIOstarPlus) to 450 nm, and the measurement was performed after shaking in the plate reader for 3 minutes before measurement. The final measurement values ​​were sorted with the Excel program, the charts were displayed by the Prism-GraphPad program, and the IC 50 The results were 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 show better anticancer activity than the positive drug IKE in the 786-O cancer cell experiment.

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

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

[0244] (1) Cell line culture count

[0245] Cell lines such as 786-O (CRL-1932), Hep3B (HB-8064), DU145 (HTB-81), and NCI H1299 (CRL-5803) were purchased from 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) was used, counting chamber and automatic counter (LUNA-II TM Automated Cell Counter), CCK-8 solution (EnoGene, E1CK-000208), multifunctional microplate reader ( 200PRO microplate reader, TECAN).

[0246] Powdered compound A15 and IKE were completely dissolved in DMSO to prepare appropriate stock solutions, which were then used in the experiments.

[0247] Collect logarithmic phase cells and count the number of cells in the prepared cell suspension using an automatic cell counter.

[0248] 5×10 3 The cells were inoculated into a 96-well cell culture plate preheated in the incubator at a concentration of 1 cell / 100 μL culture medium / well and cultured for 24 h.

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

[0250] The original culture medium was discarded, the mother solution of compound A15 was diluted with cell culture medium, and the old culture medium in the well plate was replaced at 200 μL culture medium / well to allow compound A15 to fully contact with the cells. After 24 hours of treatment, the old culture medium was discarded, 100 μL culture medium (containing 10% CCK-8) was added, and the 96-well cell culture plate was placed in a CO2 incubator for reaction for 1 hour. The wavelength of the microplate reader was set to 450 nm, and the plate was shaken for 1 minute and the absorbance OD value of each well was measured.

[0251] The sensitivity of various types of tumor cell lines to compound A15 is shown in the attached Figure 1 As shown in Figure A, it can be seen that 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 and calculate the number of cells to be 5×10 3 The cells were inoculated into a 96-well cell culture plate preheated in the incubator at a concentration of 1 cell / 100 μL culture medium / well and cultured for 24 h.

[0255] (2) The specific type of cancer cell death induced by Compound A15 was studied by combining treatment with various programmed cell death inhibitors and Compound A15.

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

[0257] The original culture medium was discarded, compound A15 and the above inhibitors were added to the culture medium, and the old culture medium in the well plate was replaced at a rate of 200 μL culture medium / well to allow each drug to fully contact the cells. After 24 hours of treatment, the old culture medium was discarded, and 100 μL culture medium (containing 10% CCK-8) was added. The 96-well cell culture plate was placed in a CO2 incubator for reaction for 1 hour, the wavelength of the microplate reader was set to 450 nm, the plate was shaken for 1 minute, and the absorbance OD value of each well was measured.

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

[0259] 4. Electron microscopic observation of mitochondria

[0260] (1) Cell line culture count

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

[0262] (2) Electron microscopy sample collection

[0263] After 24 hours, discard the culture medium, add electron microscopy fixative, fix for about 5 minutes at room temperature and away from light, and gently scrape the cells in one direction with a cell scraper. Use a Pasteur pipette to suck the cell fluid into a centrifuge tube, put it into a centrifuge, and centrifuge it at low speed for about 3-5 minutes. After discarding the fixative, add new electron microscopy fixative, blow off the cell clusters and resuspend them, fix for 30 minutes at room temperature and away from light, and then transfer to 4° for storage and sample preparation and scanning.

[0264] Refer to the electron microscope pictures as Figure 2 As shown, it can be seen that compound A15 treatment can cause significant changes in mitochondrial morphology, mitochondrial shrinkage, outer membrane rupture, and reduction or disappearance of mitochondrial cristae.

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

[0266] (1) Cell line culture count

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

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

[0269] After 24 hours of drug treatment, the supernatant was discarded, and the probe H2DCFDA for detecting total intracellular oxygen free radicals (ROS) or the fluorescent probe BODIPY 581 / 591 for lipid photooxidation (lipidperoxidation) was diluted to the working concentration with culture medium, and the cells were cultured at 37°C for 20 minutes. The supernatant was discarded, and the cells were washed with PBS and digested with trypsin. 500uL of new DMEM was added to terminate the digestion, and all the cells were transferred to the centrifuge tube just now by blowing with a pipette. Then centrifuged at 3000rpm for 5min, and the supernatant was gently discarded, leaving the cell pellet. After resuspending the cell pellet with PBS, it was filtered into a flow tube with a 200-mesh nylon mesh, and then put on the machine, and the results were quantitatively analyzed using the software flowjo10.8.1.

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

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

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

[0273] Hep3B cells were cultured at 5×10 3 / well inoculated into 96-well plates, cultured for 24 hours, removed supernatant, washed twice with PBS, and cultured with A15 and IKE diluted with culture medium. Remove supernatant, wash cells with pre-cooled PBS, add 0.6N HCL to lyse cells, and then add 1M Trisbase to inactivate the lysate. Add the specified amount of Glutamate-Glo TM The glutamate dehydrogenase, recombinant luciferase, luciferin reductase substrate, and NAD provided in the assay kit (Promega, J7021) were mixed thoroughly and waited for one hour at room temperature before using TECAN Bioluminescent signals were detected using 200PRO multi-function microplate reader.

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

[0275] 1) Collect samples

[0276] After Hep3B cells were inoculated into a 6 cm cell culture dish (density not exceeding 70%), the supernatant was discarded and the cells were cultured with a medium containing working concentrations of A15 and positive drug IKE, as well as GSH synthesis inhibitor Buthionine sulfoximine (pretreatment 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 with protein removal reagent M (GSH and GSSG detection kit, Beyotime Biotechnology, S0053) and fully vortexed. The cells were repeatedly frozen and thawed using liquid nitrogen and a 37°C water bath, and centrifuged at 4°C, 10,000 g for 5 minutes, and the supernatant was removed for the determination of total glutathione.

[0277] 2) Determination of GSH

[0278] According to the principle: Glutathione is composed 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 measured based on 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 buffer, glutathione reductase and DNTB of the specified concentration according to the instructions of the kit, and perform gradient dilution of the standard for subsequent drawing of the standard curve. Add the above-prepared liquids to a 96-well plate in sequence, incubate at room temperature for 5 minutes, then add the NADPH solution, and use an enzyme reader to detect A 412 The absorbance at 400 nm was measured, and the intracellular GSH content was calculated using the standard curve and protein concentration.

[0279] The results are as follows Figure 4 As shown, like the positive drugs IKE and Erastin, compound A15 significantly downregulated the level of intracellular GSH and increased the content of intracellular Glutamate after treatment.

[0280] 7. Animal Experimentation

[0281] Female Balb / c nude mice aged 4-6 weeks (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were used for cell xenotransplantation experiments after adapting to the SPF environment for one week. H1299 cells were resuspended in PBS and incubated at 5×10 6 / 200μL was inoculated into mice subcutaneously. When the average tumor volume reached about 50-100mm 3The mice were randomly divided into three groups and injected intraperitoneally with solvent, 30 mg / kg A15, and 30 mg / kg A15 + 10 mg / kg Liproxstatin-1 every two days. The tumor size and animal body weight were measured. When the average tumor volume reached 1000 mm 3 The animals were killed at 4 pm, the tumor size of each group was measured and samples were collected. The tumor tissue samples were fixed with electron microscope fixative and 4% paraformaldehyde respectively for subsequent electron microscope scanning and immunohistochemical staining.

[0282] The results are as follows Figure 5 As shown, the tumor volume and tumor weight of the A15-treated group were significantly reduced ( Figure 5 The results are shown in Figure 5A-C, indicating that intraperitoneal injection of compound A15 can effectively inhibit the growth of subcutaneous tumors in mice. Paraffin sections of tumor tissues were made from mice for staining, and it was found that 4-HNE, an indicator of oxidative stress cell damage, increased significantly, while Cleaved caspase-3, a marker of cell apoptosis, did not change significantly ( Figure 5 In addition, electron microscopy showed that after the intervention of compound A15, the mitochondria in the mouse tumor tissue were significantly swollen, and the mitochondrial cristae disappeared, showing 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 and induce ferroptosis, and can be used to prevent or treat tumors or immune-related diseases related to the inhibitory activity of SLC7A11.

[0284] The present invention discloses a novel class of quinazolinone derivatives, the structural innovation of which is the introduction of specific substituents (shown in Formula I) into the quinazolinone nucleus, which is a novel ferroptosis inducer. The quinazolinone derivatives construct CN bonds through innovative ball milling preparation technology, which greatly simplifies the synthesis steps while avoiding the use of a large amount of organic solvents, saving preparation costs. In vitro experiments show that the quinazolinone derivatives show a significant effect of inducing cell death in renal cancer, prostate cancer, liver cancer and lung cancer cell lines, which is 5 times better than the positive control drug Erastin, and its effect of inhibiting tumor growth has been demonstrated in animal experiments.

[0285] The innovative advantages of the present invention are: 1. It is the first quinazolinone-type ferroptosis inducer, breaking through the structural limitations of existing Erastin compounds; 2. Ball milling technology enables solvent-free, room-temperature drug synthesis, which meets green chemistry standards; 3. It has both broad-spectrum anti-tumor activity and low toxicity, and has the potential for clinical transformation.

[0286] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A quinazolinone derivative and isomer thereof that induces ferroptosis, characterized in that: The structural formula is shown in Formula I below: In formula I, R1 is H, D, -OCD3, halogen, C 1-4 Alkoxy, C 1-4 Haloalkyl, deuterated C 1-6 alkyl; R2 is D, C 1-4 Alkyl, C 1-4 Haloalkyl, 3-7 membered cycloalkyl, deuterated C 1-6 alkyl; R3 is H, D, -CD3, -OCD3, halogen, C 1-4 Alkoxy, C 1-4 Haloalkyl; R4 is D, C 1-4 Alkyl, benzyl, 3-7 membered cycloalkyl, deuterated C 1-6 alkyl; W is a structure represented by the following formula 1-1: Where Q is C 1-4 Alkyl, heteroaryl containing -NH, 3-7 membered cycloalkyl; Alternatively, Q is a structure represented by the following formula 1-2: Wherein, n=1-4; X is C, O, S, N; R5 is H, halogen, C 1-4 Alkoxy or deuterated C 1-6 alkyl; Alternatively, Q is a structure represented by the following formula 1-3: Wherein, R6 is H, D, -OCD3, halogen, C 1-4 Alkoxy or deuterated C 1-6 alkyl.

2. The quinazolinone derivatives and isomers thereof that induce ferroptosis according to claim 1, characterized in that: Including compounds represented by the following structural formulas A1-A33:

3. The method for preparing the quinazolinone derivatives and isomers thereof that induce ferroptosis according to claim 1 or 2, characterized in that: The following steps are involved: 1) Compound 1 is used as a starting material, and reacts in a reaction solvent under a reducing agent and acidic conditions to obtain compound 2; 2) using N,N-diisopropylethylamine as a base, in the presence of a sulfiding agent and a catalyst, reacting with compound 2 to generate compound 3; 3) reacting a base, compound 3 and an anthranilic acid derivative, i.e., compound 4, to generate compound 5; 4) subjecting compound 5 to chlorination reaction to obtain intermediate II, i.e. compound 6; 5) Under ball milling conditions, in the presence of a base and a grinding aid, compound 6 and compound 7 are reacted to obtain compound I, i.e., quinazolinone derivatives and isomers thereof that induce ferroptosis; Wherein: the structural formulas of compound 1, compound 2, compound 3, compound 4, compound 5 and compound 7 are shown below: The structural formula of intermediate II, i.e. compound 6, is shown below: In the above structural formula: R1 is H, D, -OCD3, halogen, C 1-4 Alkoxy, C 1-4 Deuterated alkyl halide, C 1-6 alkyl; R2 is D, C 1-4 Alkyl, deuterated C 1-6 Alkyl, C 1-4 Haloalkyl or 3-7 membered cycloalkyl; R3 is H, D, -OCD3, halogen, C 1-4 Alkoxy, deuterated C 1-6 Alkyl or -C 1-4 Haloalkyl; R4 is D, C 1-4 Alkyl, deuterated C 1-6 Alkyl, benzyl or 3-7 membered cycloalkyl; W is a structure represented by the following formula 1-1: Where Q is C 1-4 Alkyl, heteroaryl containing -NH, 3-7 membered cycloalkyl; Alternatively, Q is a structure represented by the following formula 1-2: Wherein, n=1-4; X is C, O, S, N; R5 is H, D, -OCD3, halogen, C 1-4 Alkoxy or deuterated C 1-6 alkyl; Alternatively, Q is a structure represented by the following formula 1-3: Wherein, R6 is H, D, -OCD3, halogen, deuterated C 1-6 Alkyl or C 1-4 Alkoxy.

4. The method for preparing quinazolinone derivatives and isomers thereof that induce 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 condition is provided by hydrochloric acid or sulfuric acid solution, 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 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), the chlorination reaction uses thionyl chloride or sulfuryl chloride as the chlorination reagent, 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° C., and the reaction time is 8-12 hours; In step 5), the base is cesium fluoride, sodium fluoride or tert-butyl lithium, 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 quinazolinone derivatives and isomers thereof that induce ferroptosis according to claim 3, characterized in that: Step 5) specifically includes: Under ball milling conditions, compound 6, compound 7, a base, a grinding aid and a solvent are ball milled at 50-70°C and 10-30Hz for 0.5-1 hour, and then washed, filtered, concentrated and subjected to column chromatography to obtain quinazolinone derivatives and isomers thereof that induce ferroptosis.

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

7. The quinazolinone derivatives inducing ferroptosis and pharmaceutically acceptable salts of their isomers as claimed in claim 1 or 2.

8. Use of the quinazolinone derivatives and isomers thereof that induce ferroptosis according to claim 1 or 2 or the salts according to claim 7 as small molecule inhibitors of SLC7A11 in the preparation of drugs for treating tumors or immune-related diseases.

9. The use according to claim 8, characterized in that The tumors include lung cancer, liver cancer, stomach cancer, colorectal cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, blood cancer, 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, lymph node cancer, gallbladder cancer, endocrine gland cancer, adrenal cancer, lymphoma, multiple myeloma, thymoma, mesothelioma, kidney cancer, brain cancer, central nervous system tumors, brain stem glioma and pituitary adenoma.

10. The use according to claim 8, characterized in that The immune-related diseases include allergies caused by cell activation and tissue damage and repair disorders.

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