Preparation and application of NCOA4-targeting benzofuran [2, 3-b] pyridine derivative

Abstract

The invention belongs to the technical field of medicinal chemistry, and discloses a preparation method and application of a benzofuran [2, 3-b] pyridine derivative of a targeted nuclear receptor co-activator 4 (NCOA4). The derivative has a structural general formula as shown in the following formula (I), wherein R1, R2 and R3 substituent groups can be combined at will. The compounds are combined with NCOA4 to interfere the cell iron autophagy process and effectively inhibit the ferroptosis of various cells induced by RSL3 or Erastin. Particularly, the binding affinity of the compound 4k and NCOA4 is optimal (Kd is equal to 0.78 mu M), and the compound 4k has remarkable ferroptosis inhibition activity and can be used for research and development of medicines for ferroptosis-related diseases. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of medicinal chemistry, and particularly relates to a preparation method and application of a benzofuran[2,3-b]pyridine derivative targeting nuclear receptor coactivator 4 (NCOA4). Background Art

[0002] Ferroptosis is an iron-dependent, novel form of programmed cell death distinct from apoptosis, necrosis, and autophagy. It is characterized by the accumulation of intracellular lipid peroxidation products, leading to cell death. It plays a key role in various physiological and pathological processes, including neurodegenerative diseases, ischemia-reperfusion injury, and tumors. Its mechanisms primarily involve abnormal iron metabolism, lipid peroxidation, and related signaling pathways. For example, nuclear receptor coactivator 4 (NCOA4) mediates ferritin autophagy, increasing iron release. Imbalanced regulation of ferroptosis can lead to intracellular iron overload, triggering lipid peroxidation and, in turn, inducing ferroptosis. Transferrin receptor 1 (TfR1) mediates cellular iron uptake, and inhibition of its function can reduce intracellular iron levels. The cystine / glutamate antiporter (system Xc-) is responsible for taking up extracellular cystine and converting it to cysteine (a precursor for glutathione (GSH) synthesis). Inhibition of this transporter can reduce GSH levels and indirectly promote lipid peroxidation. Furthermore, pathways such as abnormal mitochondrial metabolism are also involved in the regulation of ferroptosis. Therefore, regulating ferroptosis is a potentially important strategy for treating a variety of diseases. Currently, ferroptosis inhibitors mainly include two categories: one is lipophilic antioxidants based on antioxidant mechanisms (such as vitamin E and its derivatives, which react with lipid peroxide radicals by donating hydrogen atoms to interrupt the chain reaction); the other is compounds that inhibit iron metabolism (such as iron chelators deferoxamine and deferiprone, as well as protein regulators that inhibit TfR1 expression or function to reduce intracellular iron entry). However, the research on ferroptosis inhibitors still faces many challenges: including off-target effects caused by insufficient specificity, tumor cells may develop resistance to ferroptosis inhibitors through various mechanisms, poor stability of some inhibitors, and unsatisfactory pharmacokinetic properties. These problems have greatly limited the clinical application of ferroptosis inhibitors. Therefore, the development of new ferroptosis inhibitors with strong specificity and excellent pharmacokinetic properties has become a hot topic in the current field of drug research and development, and is expected to provide new means for the treatment of many major diseases. Summary of the Invention

[0003] In order to improve the deficiencies of the above-mentioned technology, the present invention provides a preparation method and use of benzofuran[2,3-b]pyridine derivatives targeting NCOA4. This type of compound can effectively bind to NCOA4, resist cell ferroptosis by inhibiting iron autophagy, and show significant ferroptosis inhibition activity on a variety of cells.

[0004] To achieve the above technical effects, the present invention is implemented through the following technical solutions:

[0005] A benzofurano[2,3-b]pyridine derivative targeting NCOA4, wherein the derivative has the general structural formula shown in the following formula (I):

[0006]

[0007] Wherein, R1 can be selected from phenyl groups, ester groups, and alkyl groups with different substitutions.

[0008] The R2 group can be selected from any of the following substituent groups: The wavy line indicates the position of the substituent.

[0009]

[0010] The R3 group is selected from hydrogen (-H), methyl (-Me), ethyl (-Et) or tert-butyl (- t Bu).

[0011] The above R1, R2 and R3 groups can be combined arbitrarily.

[0012] Furthermore, the benzofuran[2,3-b]pyridine derivative targeting NCOA4 small molecule inhibitor is one of compounds 4a-4r, and the R1, R2, and R3 groups in 4a-4r are shown in the following table.

[0013]

[0014] The present invention also provides a method for preparing the above-mentioned benzofurano[2,3-b]pyridine derivative targeting NCOA4, comprising the following steps:

[0015] General method for the synthesis of compounds 4a-4r: The synthesis of compounds 4a-4r includes the following "one-pot" reaction steps. Acrylamide 1 (0.3mmol, 1.5 equivalents), alkynol 2 (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%), NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle in sequence. The reaction mixture was stirred at room temperature for 1 hour, and the completion of the reaction was monitored by thin layer chromatography (TLC). The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3. The crude product 3 was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, which was monitored by TLC, the mixture was filtered through celite, and the filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent to obtain target compounds 4a-4r as white solids. The total yields of the compounds obtained by the one-pot reaction ranged from 14% to 57%.

[0016] The present invention provides a use of a benzofuran[2,3-b]pyridine derivative targeting NCOA4, wherein the use is used in the preparation of a ferroptosis inhibitor.

[0017] The benzofuran[2,3-b]pyridine derivative of the present invention exhibits a wide range of anti-ferroptosis activities. As a further preferred embodiment, the benzofuran[2,3-b]pyridine derivative is compound 4k.

[0018] Compared with existing technologies, the present invention provides a novel ferroptosis inhibitor targeting NCOA4 and a highly efficient and simple synthesis method for this class of compounds. In particular, compound 4k exhibits significant ferroptosis inhibitory activity against a variety of cell types. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Flow chart of the preparation process of compounds 4a-4r

[0020] Figure 2 The graph shows the inhibitory activity of compound 4k on ferroptosis in different cells.

[0021] Figure 3 Binding affinity diagram of compound 4k to NCOA4 protein DETAILED DESCRIPTION

[0022] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0023] The present invention is a benzofuran [2,3-b] pyridine derivative targeting NCOA4, which has the following structural formula (I). The preparation process flow chart of the structure is shown in the attached specification. Figure 1 .

[0024]

[0025] The present invention adopts the following general characterization method for the following examples:

[0026] The following product testing conditions are as follows: The examples were characterized using 600 Hz nuclear magnetic resonance spectroscopy (Bruker AVANCE III 600) with deuterated chloroform or deuterated dimethyl sulfoxide as solvents and tetramethylsilane (TMS) as the internal standard. The NMR data for the example compounds are reported below: 1 H NMR chemical shift, in δ; peak shape: s = singlet, d = doublet, t = triplet, m = multiplet, coupling constant J (in Hz); 13 C NMR data are reported as chemical shifts; the example compounds were further confirmed by high-resolution mass spectrometry (HRMS, instrument model Vanquish flex+oribitrap exploris 120, Thermo Fisher Scientific, USA).

[0027] Example 1

[0028] Compound 4a of this example: 2,4-diphenylbenzofurano[2,3-b]pyridin-7-ol

[0029] The specific structural formula of compound 4a is: The preparation method of compound 4a is as follows:

[0030] 2-Phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3a. The crude product 3a was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as the eluent. The target compound 4a was obtained as a white solid in a one-pot reaction with a total yield of 45%. Its structural characterization data are as follows: 1H NMR (600MHz, DMSO-d6)δ=10.20(s,1H),8.25–8.19(m,2H),7.91(s,1H),7.83–7.79(m,2H),7.68–7.62(m,2H),7.62–7 .57(m,1H),7.51(m,2H),7.47–7.43(m,1H),7.42(d,J=8.4Hz,1H),7.10(d,J=2.4Hz,1H),6.76(dd,J=8.4,2.4Hz,1H). 13 C NMR (101MHz, DMSO-D6) δ = 159.3, 156.4, 152.2, 145.5, 138.5, 137.7, 129.8, 129.7, 129. 6,129.4,129.1,127.3,123.3,117.1,113.6,113.4,112.9,98.9.HRMS(ESI)calculated for C 23 H 16 NO2 + ([M+H] + ):338.1181,found:338.1188.

[0031] Example 2

[0032] Compound 4b of this example: 2-(naphthalen-1-yl)-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0033] The specific structural formula of compound 4b is:

[0034] The preparation method of compound 4b is as follows:

[0035] 2-(Naphthalene-1-yl)-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3b. The crude product 3b was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4b was obtained as a white solid in a one-pot reaction with a total yield of 32%. Its structural characterization data are as follows: 1 H NMR (400MHz, DMSO-d6) δ=10.30(s,1H),8.32–8.24(m,1H),8.05–8.00(m,2H),7.83–7.80(m,2H),7.78(dd,J= 7.2,1.2Hz,1H),7.65–7.54(m,6H),7.49(d,J=8.8Hz,1H),7.14(d,J=2.4Hz,1H),6.80(dd,J=8.8,2.4Hz,1H). 13 C NMR (101MHz, DMSO-D6) δ = 159.36, 156.42, 154.10, 145.18, 137.67, 137.56, 129.84, 129.61, 129.51, 129.03, 128. 91,128.67,127.21,126.60,126.03,123.44,121.67,121.62,113.50,113.17,112.95.HRMS(ESI)calculatedfor C 27 H 18 NO2 + ([M+H] + ):388.1338,found:388.1330.

[0036] Example 3

[0037] Compound 4c of this example: 4-(4-fluorophenyl)-2-phenylbenzofuro[2,3-b]pyridin-7-ol

[0038] The specific structural formula of compound 4c is:

[0039] The preparation method of compound 4c is as follows:

[0040] 2-Phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-((4-fluorophenyl)ethynyl)-4-hydroxycyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3c. The crude product 3c was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4c was obtained as a white solid in a one-pot reaction with a total yield of 34%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6)δ=10.22(s,1H),8.24–8.19(m,2H),7.91(s,1H),7.88–7.83(m,2H),7 .53–7.44(m,5H),7.41(d,J=8.6Hz,1H),7.10(d,J=2.2Hz,1H),6.78(dd,J=8.6,2.2Hz,1H). 13 C NMR (150MHz, DMSO-d6)δ=163.5,163.1,161.8,158.8,155.9,151.7,143.9,138.0,133.6,133.6,130.9,1 30.8,129.2,128.9,126.8,122.8,116.6,116.1,115.9,113.0,112.9,112.4,98.4.HRMS(ESI)calculated for C 23 H 15 FNO2 + ([M+H] + ):356.1087,found:356.1096.

[0041] Example 4

[0042] Compound 4d of this example: 4-(4-methoxyphenyl)-2-phenylbenzofuro[2,3-b]pyridin-7-ol

[0043] The specific structural formula of compound 4d is: The preparation method of compound 4d is as follows:

[0044] 2-Phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-((4-methoxyphenyl)ethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3d. The crude product 3d was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4d was obtained as a white solid in a one-pot reaction with a total yield of 36%. Its structural characterization data are as follows: 1 H NMR (400MHz, DMSO-d6) δ=10.26(s,1H),8.17–8.12(m,3H),7.90(d,J=8.5Hz,1H),7.56–7.50(m,2H),7.48–7.43(m,1H),7.36– 7.31(m,2H),7.18–7.13(m,2H),7.11(d,J=2.1Hz,1H),6.98(tt,J=7.3,1.1Hz,1H),6.91(dd,J=8.5,2.2Hz,1H),3.38(s,3H). 13 C NMR (101MHz, DMSO-d6)δ=163.18,158.8,157.9,155.8,151.67,141.3,138.0,129.7,129.3,129 .0,126.6,124.6,121.3,115.0,114.9,113.8,113.2,112.8,98.3,66.8.HRMS(ESI)calculated for C 24 H 18 NO3 + ([M+H] + ):368.1287,found:368.1297.

[0045] Example 5

[0046] Compound 4e of this example: 2-phenyl-4-(thiophen-2-yl)benzofuro[2,3-b]pyridin-7-ol

[0047] The specific structural formula of compound 4e is:

[0048] The preparation method of compound 4e is as follows:

[0049] 2-Phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(thiophene-2-ethylidene)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3e. The crude product 3e was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4e was obtained as a white solid in a one-pot reaction with a total yield of 34%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ=10.28(s,1H),8.23–8.14(m,2H),7.93–7.87(m,2H),7.84(d,J=8.7Hz,1H),7.75(dd,J=3.5,1.2Hz,1 H),7.52(t,J=7.5Hz,2H),7.48–7.41(m,1H),7.37(dd,J=5.1,3.6Hz,1H),7.11(d,J=2.1Hz,1H),6.83(dd,J=8.7,2.1Hz,1H). 13 C NMR (150MHz, DMSO-d6)δ=163.3,158.9,155.9,151.5,138.2,137.7,137.6,129.3,128.9,1 28.7,128.6,128.4,126.7,123.0,116.6,112.9,112.6,112.4,98.4.HRMS(ESI)calculated for C 21H 14 NO2S + ([M+H] + ):344.0745,found:344.0749.

[0050] Example 6

[0051] Compound 4f of this example: 4-methyl-2-phenylbenzofurano[2,3-b]pyridin-7-ol

[0052] The specific structural formula of compound 4f is: The preparation method of compound 4f is as follows:

[0053] 2-phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(prop-1-yn-1-yl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3f. The crude product 3f was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4f was obtained as a white solid in a one-pot reaction with a total yield of 27%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ=10.14(s,1H),8.14(dd,J=8.4,1.2Hz,2H),7.88(d,J=8.4Hz,1H),7.85(d,J=0.9Hz,1H),7.5 1(dd,J=8.4,7.2Hz,2H),7.45–7.40(m,1H),7.09(d,J=2.1Hz,1H),6.91(dd,J=8.4,2.1Hz,1H),2.78(d,J=0.6Hz,3H). 13C NMR (101MHz, DMSO-d6)δ=162.8,158.4,155.5,151.3,142.9,138.3,129.1,128.9,126.6,123.6,117.6,114.6,114.1,112.5,98.5,19.1.HRMS(ESI)calculated for C 18 H 14 NO2 + ([M+H] + ):276.1025,found:276.1020.

[0054] Example 7

[0055] Compound 4g of this example: 2-isopropyl-4-phenylbenzofurano[2,3-b]pyridin-7-ol

[0056] The specific structural formula of compound 4g is: The preparation method of compound 4g is as follows:

[0057] N-(pivaloyloxy)methacrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3g. The crude product 3g was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent to obtain 4 g of the target compound as a white solid. The total yield of the compound obtained by the one-pot reaction was 32%. Its structural characterization data are as follows: 1H NMR (600MHz, DMSO-d6) δ=10.09(s,1H),7.74–7.68(m,2H),7.64–7.59(m,2H),7.59–7.54(m,1H),7.37(d,J=8.4H z,1H),7.23(s,1H),7.04(d,J=1.8Hz,1H),6.72(dd,J=8.4,1.8Hz,1H),3.23–3.12(m,1H),1.32(d,J=6.6Hz,6H). 13 C NMR(151MHz,DMSO-d6)δ=163.3,163.2,158.7,155.8,144.9,137.8,129.5,129.5, 128.8,122.9,117.6,113.7,112.5,112.0,98.8,35.7,23.1.HRMS(ESI)calculated for C 20 H 18 NO2 + ([M+H] + ):304.1338,found:304.1344.

[0058] Example 8

[0059] Compound 4h of this example: 2-cyclopentyl-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0060] The specific structural formula of compound 4h is:

[0061] The preparation method of compound 4h is as follows:

[0062] 2-Cyclopentyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour, and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3h. The crude product 3h was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4h was obtained as a white solid in a one-pot reaction with a total yield of 46%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ=10.08(s,1H),7.73–7.68(m,2H),7.64–7.59(m,2H),7.59–7.54(m,1H),7.36(d,J=9.0Hz,1H),7.23(s,1H ),7.04(d,J=2.4Hz,1H),6.72(dd,J=8.4,2.4Hz,1H),3.32–3.27(m,1H),2.10–2.02(m,2H),1.85–1.78(m,4H),1.71–1.64(m,2H). 13 C NMR (151MHz, DMSO-d6)δ=163.4,161.6,158.7,155.8,144.7,137.8,129.5,129.5,128 .8,122.9,118.6,113.7,112.4,111.9,98.8,47.2,33.8,25.9.HRMS(ESI)calculated for C 22 H 20 NO2 + ([M+H] + ):330.1494,found:330.1499.

[0063] Example 9

[0064] Compound 4i of this example: 2-(4-hydroxybenzyl)-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0065] The specific structural formula of compound 4i is:

[0066] The preparation method of compound 4i is as follows:

[0067] 2-(4-Hydroxybenzyl)-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3i. The crude product 3i was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4i was obtained as a white solid in a one-pot reaction with a total yield of 14%. Its structural characterization data are as follows: 1 H NMR (400MHz, DMSO-d6) δ=7.68–7.64(m,1H),7.62–7.54(m,1H),7.36(d,J=8.8Hz,1H),7.22(s,1H) ,7.17–7.13(m,1H),7.05(d,J=2.0Hz,1H),6.75–6.69(m,1H),6.68(d,J=2.0Hz,1H),4.07(s,1H). 13 C NMR (101MHz, DMSO-D6)δ=163.3,158.9,157.7,155.9,137.7,130.5,130.4,129.6,128.8,115.7,113.6,112.6,43.0.HRMS(ESI)calculated forC 24 H 18 NO3 + ([M+H] + ):368.1287,found:368.1281.

[0068] Example 10

[0069] Compound 4j of this example: 6-(tert-butyl)-2-(4-hydroxybenzyl)-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0070] The specific structural formula of compound 4j is: The preparation method of compound 4j is as follows:

[0071] 2-(4-Hydroxybenzyl)-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3j. The crude product 3j was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4j was obtained as a white solid in a one-pot reaction with a total yield of 35%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ=7.71–7.67(m,2H),7.64–7.60(m,2H),7.57(d,J=7.8Hz,1H),7.45(s, 1H),7.23(s,1H),7.17–7.14(m,2H),7.06(s,1H),6.71–6.67(m,2H),4.07(s,2H),1.27(s,9H). 13 C NMR (151MHz, DMSO-d6) δ = 163.3, 157.6, 157.3, 156.2, 153.9, 144.6, 137.6, 132.5, 130.4, 130. 3,129.6,129.3,128.9,119.8,119.1,115.7,112.8,112.4,99.1,29.7.HRMS(ESI)calculated for C 28 H 26 NO3 + ([M+H] + ):424.1913,found:424.1911.

[0072] Example 11

[0073] Compound 4k of this example: 6-(tert-butyl)-2,4-diphenylbenzofurano[2,3-b]pyridin-7-ol

[0074] The specific structural formula of compound 4k is: The preparation method of compound 4k is as follows:

[0075] 2-phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3k. The crude product 3k was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4k was obtained as a white solid in a one-pot reaction with a total yield of 57%. Its structural characterization data are as follows: 1 H NMR (400MHz, DMSO-d6) δ = 10.30 (s, 1H), 8.26–8.19 (m, 2H), 7.93 (s, 1H), 7.87–7.81 (m, 2H) ),7.69–7.59(m,3H),7.55–7.48(m,3H),7.46(d,J=7.2Hz,1H),7.11(s,1H),1.29(s,9H). 13 C NMR (150MHz, CDCl3) δ=164.0,155.7,154.4,152.8,145.5,138.8,138.0,132.9,129.2,129.1,1 28.9,128.8,128.7,127.2,120.6,116.9,114.7,113.9,99.8,34.9,29.7.HRMS(ESI)calculated for C 27 H 24 NO2 + ([M+H] + ):394.1807,found:394.1800.

[0076] Example 12

[0077] Compound 41 of this example: 6-(tert-butyl)-4-phenyl-2-(o-tolyl)benzofurano[2,3-b]pyridin-7-ol

[0078] The specific structural formula of compound 41 is: The preparation method of compound 41 is as follows:

[0079] N-(pivaloyloxy)-2-(o-tolyl)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3l. The crude product 3l was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 41 was obtained as a white solid in a one-pot reaction with a total yield of 24%. Its structural characterization data are as follows: 1 H NMR(600MHz, CDCl3)δ=7.79–7.75(m,2H),7.62(s,1H),7.61–7.57(m,2H),7.56 –7.52(m,2H),7.39(s,1H),7.31(m,1H),7.18(s,1H),2.49(s,3H),1.33(s,9H). 13 C NMR (151MHz, CDCl3) δ = 163.3, 156.3, 154.7, 154.4, 145.0, 139.5, 137.8, 136.2, 133.0, 130.8, 130.1, 1 29.1,128.8,128.7,128.4,125.9,120.4,114.1,113.7,99.8,34.9,29.6,20.6.HRMS(ESI)calculated for C 28 H 26 NO2 + ([M+H] + ):408.1964,found:408.1969.

[0080] Example 13

[0081] Compound 4m of this example: N-(pivaloyloxy)-2-(p-tolyl)acrylamide

[0082] The specific structural formula of compound 4m is: The preparation method of compound 4m is as follows:

[0083] N-(pivaloyloxy)-2-(p-tolyl)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3m. The crude product 3m was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-methylenediaminetetraacetic acid (DMSO-4-methylpropional) and 4-methylenediaminetetraacetic acid (DMSO-4-methylpropional). After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4m was obtained as a white solid in a one-pot reaction with a total yield of 28%. Its structural characterization data are as follows: 1 HNMR(600MHz, DMSO-d6)δ=10.24(s,1H),8.14–8.04(m,2H),7.87(d,J=7.8Hz,1H),7.85–7.76(m,2H),7 .69–7.57(m,2H),7.48(s,1H),7.31(d,J=7.8Hz,2H),7.10(s,1H),2.36(d,J=4.8Hz,3H),1.29(s,9H). 13 C NMR (151MHz, DMSO-d6)δ=163.6,157.9,154.4,151.9,145.0,139.1,137.7,135.8,132.7,129.9,129.6,129 .6,129.2,129.1,128.3,127.1,120.0,116.3,113.4,112.9,99.1,35.0,29.7,21.3.HRMS(ESI)calculated forC 28 H 26 NO2+ ([M+H] + ):408.1964,found:408.1971.

[0084] Example 14

[0085] Compound 4n of this example: 6-(tert-butyl)-2-(3-fluorophenyl)-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0086] The specific structural formula of compound 4n is: The preparation method of compound 4n is as follows:

[0087] 2-(3-Fluorophenyl)-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3n. The crude product 3n was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4n was obtained as a white solid in a one-pot reaction with a total yield of 15%. Its structural characterization data are as follows: 1 HNMR(600MHz, DMSO-d6)δ=10.30(s,1H),8.07(dt,J=7.8,1.2Hz,1H),8.02(m,1H),7.97(s,1H),7.83–7.79(m,2H ),7.67–7.62(m,2H),7.61–7.57(m,1H),7.54(m,1H),7.50(s,1H),7.29–7.21(m,1H),7.10(s,1H),1.28(s,9H). 13C NMR (151MHz, DMSO-d6)δ=164.0,163.5,162.4,158.2,154.6,150.2,150.1,145.1,141.1,141.1,137.5,132.9,131.2,131.2,129 .7,129.2,129.2,123.2,123.1,120.2,117.1,116.2,116.1,114.4,113.8,113.6,112.7,99.1,35.1,29.7.HRMS(ESI)calculated for C 27 H 23 FNO2 + ([M+H] + ):412.1713,found:412.1716.

[0088] Example 15

[0089] Compound 4o of this example: 6-(tert-butyl)-2-(naphthalen-1-yl)-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0090] The specific structural formula of compound 4o is: The preparation method of compound 4o is as follows:

[0091] 2-(Naphthalene-1-yl)-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(phenylethynyl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3o. The crude product 3o was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4o was obtained as a white solid in a one-pot reaction with a total yield of 40%. Its structural characterization data are as follows: 1H NMR(600MHz, DMSO-d6)δ=10.30(s,1H),8.33–8.28(m,1H),8.03(m,2H),7.91–7.83(m,2H),7 .80(dd,J=7.2,1.2Hz,1H),7.68–7.60(m,4H),7.61–7.53(m,43H),7.15(s,1H),1.31(s,9H). 13 C NMR (151MHz, DMSO-d6)δ=163.2,158.1,154.5,153.7,144.8,137.7,137.5,134.0,132.9,131.1,129.8,129.4,129.3, 129.2,128.9,128.6,127.1,126.5,126.0,125.9,121.3,120.2,113.6,112.7,99.2,35.1,29.8.HRMS(ESI)calculated for C 31 H 26 NO2 + ([M+H] + ):444.1964,found:444.1974.

[0092] Example 16

[0093] Compound 4p of this example: 6-(tert-butyl)-4-methyl-2-phenylbenzofuro[2,3-b]pyridin-7-ol

[0094] The specific structural formula of compound 4p is: The preparation method of compound 4p is as follows:

[0095] 2-phenyl-N-(pivaloyloxy)acrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(prop-1-yn-1-yl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3p. The crude product 3p was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4p was obtained as a white solid in a one-pot reaction with a total yield of 24%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ = 10.16 (s, 1H), 7.73–7.69 (m, 2H), 7.62 (t, J = 7.2Hz, 2H), 7.57 (d, J=7.2Hz,1H),7.44(s,1H),7.24(d,J=0.6Hz,1H),7.06(s,1H),2.59(s,3H),1.27(s,9H). 13 C NMR (151MHz, DMSO-d6) δ = 162.9, 157.0, 153.4, 153.3, 144.1, 137.2, 132.1, 129.1, 128. 8,128.5,119.3,119.1,112.5,111.6,98.7,34.6,29.3,23.8.HRMS(ESI)calculatedfor C 22 H 22 NO2 + ([M+H] + ):332.1651,found:332.1661.

[0096] Example 17

[0097] Compound 4q of this example: 6-(tert-butyl)-2-methyl-4-phenylbenzofuro[2,3-b]pyridin-7-ol

[0098] The specific structural formula of compound 4q is: The preparation method of compound 4q is as follows:

[0099] N-(pivaloyloxy)methacrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(prop-1-yn-1-yl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3q. The crude product 3q was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4q was obtained as a white solid in a one-pot reaction with a total yield of 22%. Its structural characterization data are as follows: 1 H NMR (600MHz, DMSO-d6) δ = 10.20 (s, 1H), 8.15–8.12 (m, 2H), 7.83 (d, J = 0.9Hz, 1H), 7.78 (s, 1H), 7 .50(dd,J=8.4,7.2Hz,2H),7.45–7.40(m,1H),7.11(s,1H),2.80(d,J=0.8Hz,3H),1.46(s,9H). 13 C NMR (151MHz, DMSO-d6)δ=162.7,157.1,153.5,150.9,142.4,138.3,132.6,128.9,128 .8,126.5,119.9,117.4,114.9,113.5,98.8,34.6,29.5,19.1.HRMS(ESI)calculated for C 22 H 22 NO2 + ([M+H] + ):332.1651,found:332.1660.

[0100] Example 18

[0101] Compound 4r of this example: 6-ethyl-4-methyl-2-phenylbenzofurano[2,3-b]pyridin-7-ol

[0102] The specific structural formula of compound 4r is:

[0103] The preparation method of compound 4r is as follows:

[0104] N-(pivaloyloxy)methacrylamide (0.3mmol, 1.5 equivalents), 2-(tert-butyl)-4-hydroxy-4-(prop-1-yn-1-yl)cyclohexa-2,5-diene-1-one (0.2mmol, 1.0 equivalents), [Cp*RhCl2]2 (5mol%) and NaOPiv (0.4mmol, 2.0 equivalents) and THF solvent (2.0mL) were added to a sealed reaction bottle (10mL) in sequence. The reaction mixture was stirred at room temperature for 1 hour and the reaction was monitored by TLC. The reaction solvent was then removed under reduced pressure to obtain a crude product of compound 3r. The crude product 3r was dissolved in anhydrous CH2Cl2 (5mL), followed by the addition of TsOH·H2O (0.084mmol, 1.5 equivalents) and The mixture was stirred overnight (approximately 12 hours) at 65°C with 5% zeolite (about 5 mg) of 4-molecular sieves. After completion of the reaction, monitored by TLC, the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography using petroleum ether / ethyl acetate (v / v = 10:1 to 5:1) as eluent. The target compound 4r was obtained as a white solid in a one-pot reaction with a total yield of 33%. Its structural characterization data are as follows: 1 H NMR (400MHz, CDCl3) δ=8.13–8.08(m,2H),7.83(s,1H),7.56(d,J=0.8Hz,1H),7.49(t,J=7.4 Hz,2H),7.42(d,J=7.2Hz,1H),7.16(s,1H),2.84(d,J=0.8Hz,3H),1.34(s,2H),1.28(s,3H). 13 C NMR (101MHz, CDCl3)δ=155.5,154.1,138.6,133.3,129.1,128.9,127.3,124.6,124.2,120.2,117.9,115.6,100.2,30.3,29.9,19.8.HRMS(ESI)calculated forC 20 H 18 NO2 + ([M+H] + ):304.1338,found:304.1345.

[0105] Example 19

[0106] In this example, the anti-ferroptosis activity of the compounds obtained in Examples 1 to 18 was tested. (1) Cells and culture medium: HT-1080, 786-O, and MDA-MB-231 cell lines were purchased from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagle's medium (ExCell Bio) supplemented with 10% fetal bovine serum, 100 U·ml-1 penicillin, and 100 μg·ml-1 streptomycin in a humidified incubator at 37°C and 5% CO2. HT-1080GPX4 KO cells were grown in DMEM supplemented with 1 μM Fer-1. (2) Cell viability test: First, cell plating was performed. Cells were taken from a 10 cm dish, washed once with 4 mL of PBS, and 1 mL of trypsin was added. Subsequently, digestion was performed at room temperature for 1 min, and 4 mL of culture medium was added to terminate the digestion. The cells were gently pipetted to fully disperse the cells. The cells were collected into a 15 mL centrifuge tube and centrifuged at 800 rpm for 3 min. After centrifugation, discard the supernatant, add 2 mL of culture medium to resuspend, and mix the cell suspension by pipetting. Finally, take 10 μL of the resuspended liquid from the mixed cell suspension to fill the pool, count the cells, and distribute the cells into 96-well plates according to the number of cells, so that each well has 190 μL of culture medium and 1×10 4 Cells were cultured in a 37°C constant temperature CO2 (5%) incubator. The compound stock solution was taken out from the -20°C refrigerator and diluted 50 times with culture medium. Subsequently, the diluted compound was added to the corresponding wells with a dispenser (10 μL added to 190 μL) until the dilution was 1000 times, and the concentration of DMSO in each well was 0.1%. The 96-well plate was placed in a 37°C constant temperature CO2 (5%) incubator for 72 hours. After 72 hours, the CellTiter-Glo reagent was taken out from the -20°C refrigerator and thawed in a 37°C water bath. The 96-well plate was removed from the incubator and 100 μL of the supernatant in each well was aspirated with a dispenser. 20 μL of CellTiter-Glo reagent was added to each well with a dispenser, the container was covered, and it was placed on a shaker device and incubated for 10 minutes at room temperature and in the dark. The 96-well plate was placed in an enzyme reader, the parameters were set, and the chemiluminescence signal was measured. Data were collected and analyzed using GraphPad Prims 8 software. The results are shown in the appendix of the manual. Figure 2 .

[0107] Example 20

[0108] This example verifies the binding of compound 4k obtained in Example 11 above to NCOA4 protein. The following experiments are included: (1) NCOA4 383-522 Protein purification. Human NCOA4 383-522The fragment was cloned into the pET28a-his-sumo vector (containing a His-SUMO tag). It was expressed in Escherichia coli BL21 (DE3) at 16°C overnight under the induction of 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG). The cells were then collected by centrifugation, resuspended in lysis buffer (50 mM Tris-HCl (pH 7.4), 500 mM NaCl) and ultrasonically disrupted. The protein was purified using Ni-NTA resin (Smart Life Sciences, China), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) showed that the protein purity was >90%; (2) Microthermophoresis (MST) assay: Microthermophoresis (MST) was used to detect the protein 4k and NCOA4 383-522 The brief process is as follows: His tag fused NCOA4 383-522 After the protein was labeled with RED-tris-NTA fluorescent dye (NanoTemper, Germany, catalog number MO-L018), 10 μL of labeled protein (concentration 200 nM) was mixed with equal volumes of 4k solution with gradient concentrations (100 μM to 0.001 μM). After incubation at room temperature for 30 minutes, the mixture was added to a capillary tube and analyzed using a Monolith NT.115 microthermophoresis instrument (NanoTemper, Germany). Binding affinity (Kd) was calculated using MO.affinity analysis software. Test results are attached to the instruction manual. Figure 3 .

Claims

1. A benzofurano[2,3-b]pyridine derivative targeting NCOA4, characterized in that: The derivative has the general structural formula shown in the following formula (I): Wherein, R1 can be selected from phenyl groups, ester groups, and alkyl groups with different substitutions. The R2 group can be selected from any of the following substituent groups: The wavy line indicates the position of the substituent. The R3 group is selected from hydrogen (-H), methyl (-Me), ethyl (-Et) or tert-butyl (- t Bu). The above R1, R2 and R3 groups can be combined arbitrarily.

2. A benzofuran[2,3-b]pyridine derivative targeting NCOA4, characterized in that: One of the following compounds:

3. The method for preparing a benzofurano[2,3-b]pyridine derivative targeting NCOA4 according to claim 1, characterized in that The following steps are included: The "one-pot" reaction steps of compounds 4a-4r are as follows: acrylamide 1 (0.3 mmol, 1.5 equivalents), alkynol 2 (0.2 mmol, 1.0 equivalents), [Cp*RhCl2]2 (5 mol%), NaOPiv (0.4 mmol, 2.0 equivalents) and THF solvent (2.0 mL) were added to a sealed reaction bottle. The reaction mixture was stirred at room temperature for 1 hour, and the completion of the reaction was monitored by thin layer chromatography (TLC). The reaction solvent was then removed under reduced pressure to obtain the crude product of compound 3. The crude product 3 was dissolved in anhydrous CH2Cl2 (5 mL), and then TsOH·H2O (0.084 mmol, 1.5 equivalents) and The mixture was stirred with 500 μg of zeolite (about 5 mg) at reflux at 65°C overnight (about 12 hours). After completion of the reaction, monitored by thin-layer chromatography (TLC), the mixture was filtered through celite. The filtrate was purified by silica gel column chromatography to obtain target compounds 4a-4r as white solids. The total yields of the compounds obtained by the one-pot reaction ranged from 14% to 57%.

4. The method for preparing a benzofurano[2,3-b]pyridine derivative targeting NCOA4 according to claim 3, characterized in that: The compounds 4a-4r in the step are separated and purified by silica gel column chromatography, and the eluent is petroleum ether / ethyl acetate with a gradient volume ratio of 10:1 to 5:

1.

5. The intermediate compound 3 synthesized by the one-pot reaction step of compounds 4a-4r according to claim 3 is characterized in that: No separation or purification is required.

6. The use of the benzofurano[2,3-b]pyridine derivative targeting NCOA4 according to claim 1, characterized in that: The use is in the research and development of drugs that inhibit ferroptosis-related diseases.

7. The use according to claim 6, wherein the compound inhibits ferroptosis-related diseases comprising: Neurodegenerative diseases (Alzheimer's disease, Parkinson's disease), ischemic stroke, myocardial ischemia-reperfusion injury, acute liver injury, acute kidney injury.

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