Aromatic alkylamine ferroptosis inhibitor based on butylphthalide structure, preparation method therefor, and application thereof

Abstract

Disclosed are an aromatic alkylamine ferroptosis inhibitor based on a butylphthalide structure, a preparation method therefor, and an application thereof. The chemical structural formula of the ferroptosis inhibitor is as shown in formula (1) or formula (2). The ferroptosis inhibitor is capable of inhibiting ferroptosis caused by a ferroptosis inducer, and reduces the level of intracellular reactive oxygen species. The ferroptosis inhibitor reduces neurological damage caused by cerebral ischemia-reperfusion, and alleviates symptoms of neurological disorders such as Alzheimer's disease and Parkinson's disease. Compared to the ferroptosis inhibitor Ferrostatin-1, the arylalkylamine compound exhibits better metabolic stability and is suitable for in-vivo efficacy evaluation. Therefore, the novel arylalkylamine compound provided by the present invention demonstrates great application value in the treatment of ferroptosis-related neurological disorders.

Description

An aromatic alkylamine ferroptosis inhibitor based on a butylphthalide structure and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to an aromatic alkylamine ferroptosis inhibitor based on a butylphthalide structure and a preparation method and application thereof. BACKGROUND

[0002] Cell death can be roughly divided into two categories: uncontrolled cell death caused by excessive cell damage and regulated cell death dependent on strict control of molecular pathways. Apoptosis is the most typical form of regulated cell death, which triggers cell death by activating caspases. Ferroptosis is a new cell death mode discovered in recent years, which is oxidative cell death induced by various causes, and is iron-dependent. Its occurrence is caused by the imbalance between the generation and degradation of intracellular lipid reactive oxygen species (ROS). The control mechanism of ferroptosis elucidated in the first few years of its discovery mainly revolves around cysteine and glutathione metabolism, and phospholipid peroxidase GPX4 prevents the accumulation of peroxidized lipids. Ferroptosis inducers directly or indirectly act on glutathione peroxidase (GPXs) through different pathways, leading to decreased cellular antioxidant capacity, ROS accumulation, and ultimately causing oxidative cell death. The complex interaction between lipid, iron and cysteine metabolism has become an important regulatory factor for this cell death pathway. Recently, the regulation of ferroptosis has become an attractive strategy for intervention in human diseases, including cancer, neurodegenerative diseases and ischemic diseases.

[0003] Ferroptosis can be inhibited by iron chelators, lipophilic antioxidants and / or ferrostatin-1 (fer-1). Fer-1 is an arylalkylamine with antioxidant properties, and is one of the earliest identified ferroptosis inhibitors. As a lipid peroxidation reductant, Fer-1 intercepts and eliminates lipid free radicals through hydrogen atom transfer or direct reduction, and the specific structural formula of Fer-1 is as follows:

[0004] NBP is a racemic compound containing L-butylphthalide and D-butylphthalide, which is originally isolated from celery seeds. As a new drug independently developed by China, NBP has been approved by the State Food and Drug Administration for the clinical treatment of stroke patients in 2002, and has been approved by the US FDA for a multicenter phase II clinical trial of ischemic stroke treatment. Extensive experimental and clinical studies have confirmed that NBP has biological activities such as anti-inflammatory, anti-oxidative stress, protection of blood-brain barrier, improvement of brain microcirculation, promotion of angiogenesis, and protection of nerve cells. In addition, based on the complex pharmacological mechanism of NBP, the clinical application of NBP is also increasing, and research has found that NBP has obvious effect on the treatment of some neurodegenerative diseases such as Alzheimer's disease, vascular dementia, Parkinson's disease and other neurological diseases, and the specific structure of NBP is as follows: SUMMARY

[0005] In view of the shortcomings of the prior art, the purpose of the present application is to provide a butylphthalide structure-based aromatic amine iron death inhibitor and a preparation method and application thereof. The aromatic amine compound has strong anti-iron death activity and can be used for preparing a drug for treating iron death related diseases; and the poor Fer-1 metabolic stability is improved, which can be used for subsequent iron death related pharmacological biological evaluation; such inhibitors can also be used for the treatment of existing NBP symptoms, and provide novel structure molecules for the treatment of neurological diseases and iron death related diseases.

[0006] In order to achieve the above-mentioned purpose of the application, the technical scheme adopted by the present application is as follows:

[0007] A butylphthalide structure-based aromatic amine iron death inhibitor, the structural formula of the aromatic amine iron death inhibitor is as shown in formula 1 or formula 2:

[0008] In the formula, R 1 is selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, amino, phenyl, benzyl, naphthyl, C5-C 10 aromatic heterocyclic group or C3-C7 saturated heterocyclic group;

[0009] R 2 is selected from C0-C8 alkyl, C3-C 12 cycloalkyl, adamantyl or polyalkynyl;

[0010] R 3 is selected from hydrogen, alkyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C 10 cycloalkyl;

[0011] R 4alkyl or haloalkyl selected from RC1-C10, aryl, heteroaryl;

[0012] R 5 C0 to C6 alkyl-C3 to C6 cycloalkyl-C0 to C6 alkyl or C0 to C6 alkyl-C3 to C6 cycloalkyl-C0 to C6 alkyl containing 1-3 substituents independently selected from C0 to C6 alkyl, C0 to C6 alkyl-Z-C0 to C6 alkyl or C0 to C6 alkyl-Z-C0 to C6 alkyl containing 1-3 substituents independently selected from C0 to C6 alkyl, wherein Z is selected from N, NR a , -SO2-, OC, OC=O, CO or C=OO;

[0013] C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, phenyl, benzyl, naphthyl, C5-C 10 aromatic heterocyclyl, C3-C7 saturated heterocyclyl, C3-C 12 cycloalkyl, polyalkynyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenol or C3-C 10 cycloalkyl can be substituted by one or more atoms or groups;

[0014] R a selected from H, CH3, CH2CH3, C3-C6 alkyl, C1-C6 haloalkyl or optionally substituted aryl, alkylaryl, piperazinyl, piperidinyl, morpholinyl, heterocycloalkyl, heteroaryl, C1-C6 alkoxy, NH(C1-C4 alkyl) and N(C1-C4 alkyl)2, wherein the optionally substituted groups are selected from C1-C6 alkyl or C2-C7 propenoic acid group.

[0015] Further, the inhibitor is compound I-1 to I-14, II-1 to II-12, III-1 to III-3, and the structural formula is specifically as follows:

[0016] The application also provides a preparation method of the aromatic alkylamine ferroptosis inhibitor based on the butyl phthalide structure, and the preparation method comprises the following steps:

[0017] The synthesis of the compound I-1 to I-14 is as follows: 5-bromophthalide is used as a raw material, a free radical substitution reaction is generated by NBS under the catalysis of AIBN to obtain compound 2, and then hydrolysis is performed to obtain compound 3; compound 3 is reacted with a corresponding Grignard reagent to obtain compound 4; compound 4 is reacted with potassium nitrate under the condition of concentrated sulfuric acid to generate compound 5; substitution reaction is generated with cyclohexylamine in a mixed solution of DMF / tetrahydrofuran to obtain compound 6; compound 7 is obtained by reducing the nitro group of compound 6 with zinc powder, and then reductive amination reaction is performed with a corresponding aldehyde ketone to obtain compound I-1 to I-14.

[0018] Reagents and reaction conditions: (a) NBS, AIBN, chloroform, reflux; (b) water, 110°C; (c) butyl magnesium bromide, tetrahydrofuran; (d) concentrated sulfuric acid, potassium nitrate; (e) cyclohexylamine, triethylamine, DMF, tetrahydrofuran; (f) zinc powder, hydrochloric acid, methanol; (g) corresponding amine, acetic acid, sodium triacetoxyborohydride, dichloromethane.

[0019] The synthesis of the compounds III-1~III-3 is as follows: compound 10 is synthesized by the method described above, then hydrolysis under basic conditions to obtain compound 11, which is further reacted with acyl chloride to obtain the corresponding ester 12, compound 13 is condensed from 12 and the corresponding alcohol in the presence of HDTU, DIEPA; the target compounds III-1~3 are obtained through reduction reaction.

[0020] Reagents and reaction conditions: (a) NBS, AIBN, chloroform, reflux; (b) water, 110°C; (c) butyl magnesium bromide, tetrahydrofuran; (d) concentrated sulfuric acid, potassium nitrate; (e) cyclohexylamine, triethylamine, DMF, tetrahydrofuran; (h) sodium hydroxide, methanol, water, 50°C; (i) acetyl chloride, triethylamine, dichloromethane, -10°C; (j) HATU, DMAP, dichloromethane, room temperature-50°C; (f) zinc powder, hydrochloric acid, methanol; (g) corresponding amine, acetic acid, sodium triacetoxyborohydride, dichloromethane.

[0021] The application also provides the use of the inhibitor in the preparation of an iron death inhibitor and a butyl phenyl lactone (NBP) related structure drug.

[0022] The application also provides the use of the inhibitor in the preparation of a drug for treating an iron death related disease.

[0023] Further, the iron death related disease includes neurodegenerative, tissue ischemia-reperfusion injury, stroke, Alzheimer's disease, Parkinson's syndrome and other neurodegenerative diseases, cardiovascular, liver and kidney failure, inflammation, diabetic complications.

[0024] Further, the drug further adds a pharmaceutically acceptable salt, carrier or adjuvant.

[0025] Further, the aromatic alkylamine compound can be used to treat iron death, because the compound can be used as a free radical scavenger to reduce active oxygen and lipid peroxide in cells, thereby rescuing iron death caused by active oxygen;

[0026] Further, the aromatic alkylamine compound can be used for treating NBP-related diseases, can resist ischemia-induced brain injury, improve neurodegenerative diseases, can improve intracerebral microcirculation, inhibit platelet aggregation, protect mitochondria, regulate energy metabolism, and reduce oxidative stress damage.

[0027] Further, the aromatic alkylamine compound can be used for treating NBP-related diseases, can resist ischemia-induced brain injury, improve neurodegenerative diseases, can improve intracerebral microcirculation, inhibit platelet aggregation, protect mitochondria, regulate energy metabolism, and reduce oxidative stress damage.

[0028] Compared with the prior art, the application has the advantages and beneficial effects that: the application obtains a kind of aromatic alkylamine compound capable of acting on ferroptosis through reasonable drug design and a variety of synthesis means, enriches the structure of ferroptosis inhibitor. The aromatic alkylamine compound can inhibit ferroptosis caused by ferroptosis inducer and reduce the level of intracellular reactive oxygen species, which is verified by experiments. The metabolic stability of the aromatic alkylamine compound is obviously improved compared with compound Fer-1, which is verified by experiments. Therefore, the aromatic alkylamine provided by the application has good application value for treating ferroptosis and NBP-related diseases. DETAILED DESCRIPTION

[0029] The embodiments of the application are described in detail below, and the embodiments are implemented on the premise of the technical scheme of the application, and detailed implementation modes and specific operation processes are given, but the protection scope of the application is not limited to the following embodiments.

[0030] Embodiment 1

[0031] 1, 3, 5-dibromo-isobenzofuran-1 (3H)-one (compound 2)

[0032] 5-bromo-isobenzofuran-1 (3H)-one (1.0 g, 4.69 mmol), NBS (1.0 g, 5.63 mmol) and AIBN (0.45 g, 0.47 mmol) were added to a flask containing 40 mL of chloroform, and the reaction was carried out at 70°C for 5 h, and TLC detection was carried out until the reaction was completed. The reaction liquid was poured into 50 mL of water, and the water layer was extracted with DCM (50 mL x 3), and the organic phases were combined and concentrated. The mixture was separated by column chromatography (PE:EA = 20:1) to obtain yellow solid compound 2 (1.2 g, 92%). 1 H NMR (400 MHz, Chloroform-d) δ 7.96 (d, J = 8.6 Hz, 1H), 7.69 (dd, J = 2.2, 0.7 Hz, 1H), 7.54 (dd, J = 8.5, 2.3 Hz, 1H), 6.86 (s, 1H).

[0033] 2, 5-Bromo-3-hydroxyisobenzofuran-l(3H)-one (Compound 3)

[0034] 3, 5-Dibromo-isobenzofuran-l(3H)-one (1 g, 3.46 mmol) was added to the reaction bottle, 20 mL of water as solvent, 110°C for 1 h, after the reaction was completed, white solid compound 3 (1 g, 94%) was obtained by suction filtration. 1 H NMR (400 MHz, DMSO-d6) δ 8.25 (d, J = 8.0 Hz, 1H), 7.90 - 7.88 (m, 1H), 7.82 (dd, J = 8.1, 1.7 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 6.61 (s, 1H).

[0035] 3, 5-Bromo-3-hydroxyisobenzofuran-l(3H)-one (Compound 3)

[0036] 5-Bromo-3-hydroxyisobenzofuran-l(3H)-one (1 g, 4.38 mmol) was added to the reaction bottle, 15 mL of tetrahydrofuran as solvent, N2 replaced three times and protected with N2, butyl magnesium bromide (1.4 g, 8.76 mmol) was slowly added to the reaction bottle under ice bath conditions, and stirred at room temperature for 5 h. After the reaction was completed, dilute hydrochloric acid was added dropwise under ice bath conditions, the PH was adjusted to 2, and the stirring was continued at room temperature for 1 h. Then the reaction liquid was poured into 50 mL of water, the water layer was extracted with DCM (50 mL x 3), and the organic phase was combined and concentrated. The mixture was separated by column chromatography (PE:EA = 20:1) to obtain yellow solid compound 4 (0.2 g, 28%) 1 H NMR (400 MHz, Chloroform-d) δ 7.94 (d, J = 8.5 Hz, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.56 (dd, J = 8.5, 2.3 Hz, 1H), 5.48 - 5.42 (m, 1H), 2.02 (tdd, J = 9.1, 4.9, 1.4 Hz, 2H), 1.43 - 1.31 (m, 4H).

[0037] 4, 5-Bromo-3-hydroxyisobenzofuran-l(3H)-one (Compound 3)

[0038] To a reaction vial containing 5-bromo-3-butylisobenzo-furan-l(3H)-one (0.2 g, 0.75 mmol), KNO3(0.37 g, 3.66 mmol) was added slowly under ice bath condition. The reaction was allowed to proceed at room temperature for 2 h. After the reaction was completed, the reaction was quenched by the addition of ice water slowly. The reaction mixture was poured into 20 mL of water and the aqueous layer was extracted with DCM (50 mL x 3). The combined organic phase was concentrated. The mixture was separated by column chromatography (PE:EA = 15: 1) to give compound 5 (0.18 g, 91%) as a white solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.56 (s, 1H), 7.94 (d, J = 0.6 Hz, 1H), 5.47 (dd, J = 5.2, 4.4 Hz, 1H), 2.02 (tdd, J = 9.0, 4.9, 1.7 Hz, 2H), 1.45 - 1.31 (m, 4H).

[0039] 5, 3-Butyl-5-(cyclohexylamino)-6-nitroisobenzo-furan-l(3H)-one (Compound 6)

[0040] To a reaction vial containing 5-bromo-3-butyl-6-nitroisobenzo-furan-l(3H)-one (0.05 g, 0.16 mmol), cyclohexylamine (0.047 g, 0.48 mmol), triethylamine (0.03 g, 0.32 mmol), 0.5 mL of DMF and 1.5 mL of tetrahydrofuran were added as solvents. The reaction was allowed to proceed at 70 °C for 5 h. After the reaction was completed, the reaction mixture was poured into 30 mL of water and the aqueous layer was extracted with DCM (30 mL x 3). The combined organic phase was concentrated. The mixture was separated by column chromatography (PE:EA = 10: 1) to give compound 6 (0.04 g, 83%) as a yellow solid. 1 H NMR (400 MHz, Chloroform-d) δ 8.56 (s, 1H), 7.94 (d, J = 0.6 Hz, 1H), 5.47 (dd, J = 5.2, 4.4 Hz, 1H), 2.02 (tdd, J = 9.0, 4.9, 1.7 Hz, 2H), 1.45 - 1.31 (m, 4H).

[0041] 6, 6-Amino-3-butyl-5-(cyclohexylamino)isobenzo-furan-l(3H)-one (Compound 7)

[0042] Compound 6 (0.04 g, 0.12 mmol) was added to a reaction flask, 5 mL of CH3OH was used as solvent, 1 mL of dilute hydrochloric acid and excess zinc powder were added, and the mixture was reacted at room temperature for 10 minutes. The zinc powder was removed by filtration, and the mixture was separated by column chromatography (PE:EA = 2:1) to obtain black solid compound 7 (0.04 g, 83%). 1 H NMR (400MHz, Methanol-d4) δ7.04(d,J=2.9Hz,1H),6.56(d,J=2.4Hz,1H),5.35(dt,J=6.6,3.0Hz, 1H),3.44(ddt,J=10.8,7.2,3.2Hz,1H),2.15–1.94(m,5H),1.88–1.63(m,7H),1.55–1.41(m,6H).

[0043] 7. 5-(cyclohexylamino)-6-((2,6-dichlorobenzyl)amino)-3-pentylisobenzofuran-1(3H)-one (Compound I-1)

[0044] Compound 7 (0.03 g, 0.099 mmol), cyclohexanone (0.014 g, 0.011 mmol), and NaBH(OAc)3 were added to a reaction flask containing 4 mL of DCE. One drop of acetic acid was added, and the reaction was carried out at room temperature for 5 h. After the reaction was completed, the reaction solution was poured into 30 mL of water, and the aqueous layer was extracted with DCM (30 mL × 3). The organic phases were combined and concentrated. The mixture was separated by column chromatography (PE:EA = 4:1) to give a black solid compound I-1 (0.015 g, 64%). 1 HNMR(400MHz,Chloroform-d)δ7.35(d,J=7.9Hz,2H),7.25–7.18(m,2H),6.44(s,1H),5.31(dd,J=8.0,3.8Hz,1H),4.42(s,2H),3 .33(tt,J=10.1,3.8Hz,1H),2.02(dtd,J=35.1,9.9,8.9,3.9Hz,3H),1.85–1.61(m,5H),1.51–1.32(m,8H),0.91(t,J=7.1Hz,3H). 13 C NMR(101MHz,Chloroform-d)δ168.34,140.49,139.81,137.02,124.99,112.39,109.94,80.10,52 .75,52.70,33.93,33.45,33.41,33.38,27.77,26.18,26.16,25.31,25.29,25.26,22.81,14.09.

[0045] 8. 3-Butyl-5-(cyclohexylamino)-6-((4-nitrobenzyl)amino)isobenzo-furan-1(3H)-one (Compound I-2)

[0046] Synthesis method same as I-1, yellow solid, yield 79%. 1 H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H), 7.60 - 7.54 (m, 2H), 7.14 (s, 1H), 7.09 (s, 1H), 5.91 (t, J = 5.3 Hz, 1H), 5.82 (d, J = 7.8 Hz, 1H), 5.52 - 5.35 (m, 1H), 4.44 (dt, J = 5.3, 1.0 Hz, 2H), 3.55 - 3.36 (m, 1H), 2.02 (tdd, J = 9.0, 4.9, 1.7 Hz, 2H), 1.90 - 1.76 (m, 2H), 1.67 - 1.24 (m, 13H), 0.87 (t, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 146.84, 144.22, 140.70, 139.59, 135.78, 128.65, 125.31, 123.53, 111.39, 109.73, 80.10, 52.70, 48.27, 33.93, 33.38, 27.77, 26.16, 25.26, 22.81, 14.09.

[0047] 9. 3-Butyl-5-(cyclohexylamino)-6-((pyridin-4-ylmethyl)amino)isobenzo-furan-1(3H)-one (Compound I-3)

[0048] Synthesis method same as I-1, yellow solid, yield 59%. 1 H NMR (400 MHz, Chloroform-d) δ 8.17 - 8.10 (m, 2H), 7.50 (dd, J = 8.8, 2.0 Hz, 2H), 6.94 (d, J = 1.2 Hz, 1H), 6.48 (s, 1H), 5.26 (dd, J = 8.0, 3.8 Hz, 1H), 4.41 (s, 2H), 3.35 (tt, J = 10.1, 3.7 Hz, 1H), 2.12 - 2.03 (m, 2H), 2.01 - 1.91 (m, 2H), 1.80 (dp, J = 12.4, 4.3, 3.8 Hz, 3H), 1.73 - 1.65 (m, 2H), 1.44 - 1.24 (m, 10H). 13C NMR (101 MHz, Chloroform-d) δ 171.37, 156.04, 152.54, 150.70, 148.23, 142.55, 136.03, 122.28, 112.99, 112.48, 100.87, 80.81, 51.21, 34.69, 32.78, 32.72, 26.95, 25.68, 24.76, 24.68, 22.53, 13.98.

[0049] 10. 3-Butyl-5-(cyclohexylamino)-6-((4-methylbenzyl)amino)isobenzo-furan-1(3H)-one (Compound 1-4)

[0050] Synthesis method same as 1-1, black solid, yield 54%. 1 H NMR (400 MHz, Chloroform-d) δ 7.28 (d, J = 8.0 Hz, 2H), 7.17 (d, J = 7.7 Hz, 2H), 7.13 (s, 1H), 6.46 (s, 1H), 5.29 (dd, J = 7.9, 3.8 Hz, 1H), 4.22 (s, 2H), 3.41 - 3.24 (m, 1H), 2.35 (s, 3H), 2.10 - 2.01 (m, 2H), 2.01 - 1.92 (m, 1H), 1.79 (dq, J = 12.9, 3.8 Hz, 2H), 1.68 (ddd, J = 14.4, 5.8, 3.5 Hz, 2H), 1.47 - 1.35 (m, 6H), 1.24 (ddd, J = 6.8, 5.2, 2.1 Hz, 3H), 0.90 (t, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 140.70, 139.59, 137.30, 136.45, 135.78, 129.64, 127.84, 125.31, 111.39, 109.73, 80.10, 52.70, 48.27, 33.93, 33.38, 27.77, 26.16, 25.26, 22.81, 21.04, 14.09.

[0051] 11. 3-Butyl-5-(cyclohexylamino)-6-(cyclopentylamino)isobenzofuran-1(3H)-one (Compound 1-5)

[0052] Synthesis method same as 1-1, black solid, yield 64%. 1H NMR (400 MHz, Chloroform-d) δ 7.11 (s, 1H), 6.44 (s, 1H), 5.29 (dd, J = 8.0, 3.8 Hz, 1H), 3.77 (ddd, J = 12.1, 6.8, 5.2 Hz, 1H), 3.31 (td, J = 10.0, 5.0 Hz, 1H), 2.12 - 1.93 (m, 5H), 1.84 - 1.61 (m, 8H), 1.54 - 1.22 (m, 14H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 140.49, 139.81, 136.65, 124.99, 112.39, 109.94, 80.10, 55.47, 52.70, 33.93, 33.38, 33.05, 27.77, 26.16, 25.26, 23.51, 22.81, 14.09.

[0053] 12. 3-Butyl-5-(cyclohexylamino)-6-((naphthalen-1-ylmethyl)amino)isobenzo-furan-1(3H)-one (Compound I-6)

[0054] Synthesis method same as I-1, black solid, yield 64%. 1 H NMR (400 MHz, Chloroform-d) δ 8.11 - 8.06 (m, 1H), 7.93 - 7.83 (m, 2H), 7.55 - 7.46 (m, 4H), 7.29 (s, 1H), 6.49 (s, 1H), 5.33 (dd, J = 7.9, 3.8 Hz, 1H), 4.70 (s, 2H), 3.30 (ddt, J = 10.5, 7.8, 3.8 Hz, 1H), 2.00 (dtt, J = 9.8, 6.3, 2.7 Hz, 3H), 1.80 - 1.59 (m, 5H), 1.53 - 1.32 (m, 6H), 1.27 - 1.11 (m, 4H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 140.70, 139.59, 135.86, 133.57, 133.47, 132.48, 128.81, 128.43, 127.70, 127.47, 126.36, 125.41, 125.31, 124.20, 111.39, 109.73, 80.10, 52.70, 47.04, 33.93, 33.38, 27.77, 26.16, 25.26, 22.81, 14.09.

[0055] 13. 3-Butyl-5-(cyclohexylamino)-6-((thiophen-2-ylmethyl)amino)isobenzo-furan-l(3H)-one (Compound 1-7)

[0056] Synthetic procedure same as 1-1, black solid, yield 32%. 1 H NMR (400 MHz, Chloroform-d) δ 7.17 (s, 1H), 7.04 (dt, J = 3.2, 1.1 Hz, 1H), 6.98 (ddd, J = 6.0, 5.0, 3.5 Hz, 2H), 6.46 (d, J = 0.8 Hz, 1H), 5.32 - 5.27 (m, 1H), 4.46 (d, J = 1.0 Hz, 2H), 3.33 (dq, J = 9.9, 5.0, 4.0 Hz, 1H), 2.10 - 1.92 (m, 4H), 1.84 - 1.64 (m, 6H), 1.50 - 1.34 (m, 9H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 141.72, 140.70, 139.77, 135.50, 127.19, 126.00, 125.66, 125.31, 111.38, 109.73, 80.10, 52.70, 44.16, 33.93, 33.38, 27.77, 26.16, 25.26, 22.81, 14.09.

[0057] 14. 3-Butyl-5-(cyclohexylamino)-6-(phenethylamino)isobenzofuran-l(3H)-one (Compound 1-8)

[0058] Synthetic procedure same as 1-1, black solid, yield 21%. 1 H NMR (400 MHz, Chloroform-d) δ 7.34 - 7.28 (m, 4H), 7.11 (s, 1H), 6.41 (s, 1H), 5.30 - 5.26 (m, 1H), 3.36 (t, J = 6.7 Hz, 2H), 3.29 (t, J = 9.7 Hz, 1H), 2.98 (t, J = 6.8 Hz, 2H), 2.06 - 1.90 (m, 4H), 1.82 - 1.57 (m, 6H), 1.39 (dddd, J = 28.8, 15.2, 6.5, 2.6 Hz, 10H). 13C NMR (101 MHz, Chloroform-d) δ 168.34, 140.89, 139.32, 139.01, 136.89, 128.86, 128.80, 126.65, 125.22, 110.89, 109.86, 80.10, 52.70, 45.32, 35.20, 33.93, 33.38, 27.77, 26.16, 25.26, 22.84, 14.04.

[0059] 15. 6-(Benzylamino)-3-butyl-5-(cyclohexylamino)isobenzo-furan-l(3H)-one (Compound I-9)

[0060] Synthesis method same as I-l, yellow solid, yield 87%. 1 H NMR (500 MHz, Chloroform-d) δ 7.38 - 7.23 (m, 5H), 7.14 (s, 1H), 7.09 (s, 1H), 5.54 - 5.37 (m, 1H), 4.42 (dt, J = 5.3, 0.9 Hz, 2H), 3.45 (dp, J = 7.9, 4.8 Hz, 1H), 2.02 (tdd, J = 9.0, 4.9, 1.7 Hz, 2H), 1.87 - 1.77 (m, 2H), 1.67 - 1.33 (m, 12H), 0.87 (t, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 140.89, 139.32, 139.01, 136.89, 128.86, 128.80, 126.65, 125.22, 110.89, 109.86, 80.10, 52.70, 45.32, 35.20, 33.93, 33.38, 27.77, 26.16, 25.26, 22.84, 14.04.

[0061] 16. 3-Butyl-5-(cyclohexylamino)-6-((2,6-dichlorobenzyl)amino)isobenzo-furan-l(3H)-one (Compound I-10)

[0062] Synthesis method same as I-l, yellow solid, yield 87%. 1H NMR (400 MHz, Chloroform-d) δ 7.35 (d, J = 7.9 Hz, 2H), 7.25 - 7.18 (m, 2H), 6.44 (s, 1H), 5.31 (dd, J = 8.0, 3.8 Hz, 1H), 4.42 (s, 2H), 3.33 (tt, J = 10.1, 3.8 Hz, 1H), 2.02 (dtd, J = 35.1, 9.9, 8.9, 3.9 Hz, 3H), 1.85 - 1.61 (m, 5H), 1.51 - 1.32 (m, 8H), 0.91 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 171.87, 147.82, 146.00, 136.27, 135.15, 134.22, 129.65, 128.58, 113.61, 113.12, 100.53, 80.74, 51.51, 45.98, 34.78, 33.01, 32.93, 29.72, 27.00, 25.79, 24.98, 24.90, 22.56, 14.00.

[0063] 17. 6-((4-bromobenzyl)amino)-3-butyl-5-(cyclohexylamino)isobenzo-furan-l(3H)-one (Compound 1-11)

[0064] Synthesis method same as 1-1, yellow solid, yield 57%. 1 H NMR (400 MHz, Chloroform-d) δ 7.46 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 7.5 Hz, 2H), 7.06 (s, 1H), 6.47 (s, 1H), 5.28 (dd, J = 8.0, 3.8 Hz, 1H), 4.23 (d, J = 5.0 Hz, 2H), 3.34 (dtt, J = 10.1, 6.3, 3.4 Hz, 1H), 2.06 (dt, J = 15.3, 4.5 Hz, 2H), 2.00 - 1.92 (m, 1H), 1.83 - 1.75 (m, 2H), 1.73 - 1.61 (m, 3H), 1.49 - 1.30 (m, 8H), 0.89 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, Chloroform-d) δ 172.02, 145.73, 143.71, 137.54, 136.03, 131.85, 129.65, 121.44, 114.01, 108.54, 101.02, 80.76, 51.59, 48.59, 34.81, 33.10, 33.01, 27.04, 25.78, 24.93, 24.85, 22.55, 13.99.

[0065] 18. 3-Butyl-5-(cyclohexylamino)-6-((4-fluorobenzyl)amino)isobenzo-furan-1 (3H)-one (Compound 1-12)

[0066] Synthesis method same as 1-1, yellow solid, yield 28%. 1 H NMR (400 MHz, Chloroform-d) δ 7.35 (dd, J = 8.6, 5.4 Hz, 2H), 7.1 1 (s, 1 H), 7.05 (t, J = 8.7 Hz, 2H), 6.47 (s, 1 H), 5.32 - 5.27 (m, 1 H), 4.25 (s, 2H), 3.38 - 3.28 (m, 1 H), 2.10 - 1.91 (m, 4H), 1.79 (dd, J = 1 1.6, 6.2 Hz, 2H), 1.73 - 1.48 (m, 5H), 1.48 - 1.33 (m, 8H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 163.01, 160.99, 140.70, 139.59, 135.78, 135.54, 135.51, 130.08, 130.01, 125.31, 1 15.26, 1 15.10, 1 1 1.39, 109.73, 80.10, 52.70, 47.98, 33.93, 33.38, 27.77, 26.16, 25.26, 22.81, 14.09.

[0067] 19. 3-Butyl-5-(cyclohexylamino)-6-((4-methoxybenzyl)amino)isobenzofuran-1 (3H)-one (Compound 1-13)

[0068] Synthesis method same as 1-1, yellow solid, yield 28%. 1H NMR (400 MHz, Chloroform-d) δ 7.31 (d, J = 8.7 Hz, 2H), 7.14 (s, 1H), 6.92 - 6.89 (m, 2H), 6.46 (s, 1H), 5.32 - 5.27 (m, 1H), 4.20 (s, 2H), 3.81 (s, 3H), 3.32 (s, 1H), 2.11 - 1.92 (m, 2H), 1.85 - 1.75 (m, 2H), 1.68 (dd, J = 14.5, 6.1 Hz, 2H), 1.48 - 1.33 (m, 9H), 0.91 (d, J = 7.2 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 168.34, 159.03, 140.70, 139.59, 135.78, 132.87, 129.42, 125.31, 113.71, 111.39, 109.73, 80.10, 55.32, 52.70, 48.61, 33.93, 33.38, 27.77, 26.16, 25.26, 22.78, 14.14.

[0069] 20, 3-Butyl-5-(cyclohexylamino)-6-((pyridin-4-ylmethylidene)amino)isobenzo-furan-1(3H)-one (Compound I-14)

[0070] 1 H NMR (400 MHz, Chloroform-d) δ 8.67 (s, 1H), 8.64 - 8.61 (m, 2H), 7.95 (s, 1H), 7.71 - 7.53 (m, 2H), 7.21 (d, J = 0.6 Hz, 1H), 5.56 - 5.31 (m, 1H), 3.47 (dt, J = 7.0, 4.7 Hz, 1H), 2.02 (tdd, J = 9.0, 4.9, 1.7 Hz, 2H), 1.89 - 1.69 (m, 2H), 1.69 - 1.34 (m, 12H), 0.87 (t, J = 7.0 Hz, 3H). 13 C NMR (101 MHz, Chloroform-d) δ 169.16, 162.18, 150.59, 146.31, 142.64, 139.50, 138.33, 125.64, 123.32, 122.61, 108.45, 80.43, 52.89, 33.93, 33.38, 27.77, 26.13, 25.25, 22.78, 14.07.

[0071] 21, 6-Amino-3-butyl-5-(cyclohexylamino)isobenzo-furan-1(3H)-one (Compound II-1)

[0072] 1 H NMR (400MHz, Methanol-d4) δ7.04(d,J=2.9Hz,1H),6.56(d,J=2.4Hz,1H),5.35(dt,J=6.6,3.0Hz, 1H),3.44(ddt,J=10.8,7.2,3.2Hz,1H),2.15–1.94(m,5H),1.88–1.63(m,7H),1.55–1.41(m,6H). 13 C NMR (125MHz, Common NMR Solvents)δ167.66,138.67,138.51,137.98,126.12,112.66,109.72,80.02,52.88,33.93,33.38,27.77,26.16,25.26,22.81,14.09.

[0073] 22. 6-Amino-3-butyl-5-(ethylamino)isobenzofuran-1(3H)-one (II-2)

[0074] 1 H NMR(400MHz,Chloroform-d)δ7.12(s,1H),6.44(s,1H),5.29(dd,J=7.8,4.0Hz,1H),3.23(q,J= 7.2Hz,2H),2.02–1.91(m,1H),1.75–1.62(m,1H),1.35(t,J=7.2Hz,7H),0.89(t,J=7.2Hz,3H).

[0075] 23. 6-Amino-3-butyl-5-(cyclopropylamino)isobenzofuran-1(3H)-one (II-3)

[0076] 1 H NMR (400MHz, Methanol-d4) δ6.97(s,1H),6.89(s,1H),5.35(dd,J=7.5,3.9Hz,1H),3.18(q,J=7.3Hz,1H),2.0 7–1.93(m,2H),1.72–1.61(m,2H),1.41–1.38(m,2H),0.93–0.90(m,3H),0.83–0.79(m,2H),0.57–0.51(m,2H).

[0077] 24. 6-Amino-3-butyl-5-(cyclopentylamino)isobenzofuran-1(3H)-one (II-4)

[0078] 1 H NMR (400 MHz, Methanol-d4) δ 6.99 (s, 1H), 6.53 (s, 1H), 5.32 (dd, J = 7.5, 3.9 Hz, 1H), 3.93 - 3.86 (m, 1H), 2.10 - 1.95 (m, 4H), 1.81 - 1.71 (m, 3H), 1.68 - 1.54 (m, 7H), 0.89 (s, 3H). 13 C NMR (101 MHz, MeOD) δ 173.36, 145.67, 144.00, 134.59, 112.10, 109.29, 100.79, 81.35, 54.18, 34.30, 32.57, 32.49, 26.56, 23.78, 22.17, 12.94.

[0079] 25. 6-Amino-3-butyl-5-(cycloheptylamino)isobenzo-furan-l(3H)-one (II-5)

[0080] 1 H NMR (400 MHz, Methanol-d4) δ 6.99 (s, 1H), 6.53 (s, 1H), 5.32 (dd, J = 7.5, 3.9 Hz, 1H), 3.93 - 3.86 (m, 1H), 2.10 - 1.95 (m, 4H), 1.81 - 1.71 (m, 3H), 1.68 - 1.54 (m, 7H), 0.89 (s, 3H). 13 C NMR (101 MHz, MeOD) δ 173.36, 145.67, 144.00, 134.59, 112.10, 109.29, 100.79, 81.35, 54.18, 34.30, 32.57, 32.49, 26.56, 23.78, 22.17, 12.94.

[0081] 26. 6-Amino-3-butyl-5-((tetrahydro-2H-pyran-4-yl)amino)isobenzo-furan-l(3H)-one (II-6)

[0082] 1H NMR (400 MHz, Chloroform-d) δ 7.16 (s, 1H), 6.46 (s, 1H), 5.33 - 5.26 (m, 1H), 4.04 (dq, J = 12.2, 4.2 Hz, 2H), 3.62 - 3.54 (m, 3H), 2.11 - 2.04 (m, 2H), 2.02 - 1.93 (m, 1H), 1.73 - 1.55 (m, 4H), 1.48 - 1.38 (m, 3H), 0.90 (t, J = 7.1 Hz, 3H). 13 C NMR (101 MHz, CDC13) δ 171.75, 146.24, 143.50, 133.95, 114.12, 112.71, 101.05, 80.84, 66.66, 66.61, 48.81, 34.80, 33.05, 33.01, 26.98, 22.51, 13.98.

[0083] 27, 5-(((1R, 3S, 5r, 7r)-adamantane-2-yl)amino)-6-amino-3-butylisobenzofuran-1 (3H)-one (II-7)

[0084] 1 H NMR (400 MHz, Chloroform-d) δ 7.10 (s, 1H), 6.89 (d, J = 0.8 Hz, 1H), 5.32 - 5.27 (m, 1H), 3.71 (q, J = 7.4 Hz, 1H), 2.74 (s, 6H), 2.01 - 1.85 (m, 3H), 1.68 (ddd, J = 14.7, 8.1, 6.2 Hz, 3H), 1.26 - 1.23 (m, 8H), 0.91 (d, J = 7.3 Hz, 3H).

[0085] 28, 6-amino-5-(cyclohexylamino)-3-methylisobenzofuran-1 (3H)-one (II-8)

[0086] 1 H NMR (400 MHz, Chloroform-d) δ 7.12 (s, 1H), 6.42 (s, 1H), 5.37 (q, J = 6.6 Hz, 1H), 3.33 (tt, J = 10.1, 3.7 Hz, 1H), 2.06 (dt, J = 11.3, 4.6 Hz, 2H), 1.79 (dq, J = 12.6, 4.1 Hz, 3H), 1.68 (dt, J = 13.0, 4.3 Hz, 2H), 1.55 (d, J = 6.6 Hz, 3H), 1.47 - 1.31 (m, 4H). 13C NMR (101 MHz, CDC13) δ 171.57, 147.71, 144.22, 133.52, 119.55, 112.60, 100.63, 51.45, 33.02, 32.94, 25.77, 24.91, 24.82, 20.79.

[0087] 29. 6-Amino-5-(cyclohexylamino)-3-ethylisobenzofuran-l(3H)-one (II-9)

[0088] 1 H NMR (400 MHz, Chloroform-d) δ 7.12 (s, 1H), 6.43 (s, 1H), 5.30 - 5.27 (m, 1H), 3.39 - 3.28 (m, 1H), 2.13 - 1.90 (m, 5H), 1.80 (s, 1H), 0.96 (t, J = 7.4 Hz, 4H).

[0089] 30. 6-Amino-5-(cyclohexylamino)-3-propylisobenzofuran-l(3H)-one (II-10)

[0090] 1 H NMR (400 MHz, Chloroform-d) δ 7.10 (s, 1H), 6.41 (s, 1H), 5.24 (dd, J = 7.2, 4.0 Hz, 1H), 3.30 (tt, J = 10.0, 3.8 Hz, 1H), 2.03 (ddt, J = 14.5, 7.2, 3.8 Hz, 3H), 1.80 - 1.74 (m, 2H), 1.65 (dd, J = 10.7, 6.0 Hz, 1H), 1.42 - 1.32 (m, 3H), 1.24 (ddt, J = 15.4, 8.4, 3.2 Hz, 4H), 0.95 (td, J = 7.3, 1.5 Hz, 3H). 13 C NMR (101 MHz, CDC13) δ 171.57, 147.71, 144.22, 133.52, 119.55, 112.60, 100.63, 51.45, 33.02, 32.94, 25.77, 24.91, 24.82, 20.79.

[0091] 31. 6-Amino-5-(cyclohexylamino)-3-isobutylisobenzofuran-l(3H)-one (II-11)

[0092] 1H NMR (400 MHz, Chloroform-d) δ 7.13 (s, 1H), 6.43 (s, 1H), 5.34 - 5.23 (m, 1H), 3.32 (d, J = 10.3 Hz, 1H), 2.07 (d, J = 12.1 Hz, 2H), 1.99 - 1.90 (m, 1H), 1.80 (d, J = 13.1 Hz, 2H), 1.67 (p, J = 7.6, 7.1 Hz, 2H), 1.49 - 1.28 (m, 11H), 0.88 (d, J = 6.5 Hz, 3H).

[0093] 32, 6-Amino-5-(cyclohexylamino)-3-pentylisobenzo-furan-l(3H)-one (II-12)

[0094] 1 H NMR (400 MHz, Chloroform-d) δ 7.13 (s, 1H), 6.43 (s, 1H), 5.34 - 5.23 (m, 1H), 3.32 (d, J = 10.3 Hz, 1H), 2.07 (d, J = 12.1 Hz, 2H), 1.99 - 1.90 (m, 1H), 1.80 (d, J = 13.1 Hz, 2H), 1.67 (p, J = 7.6, 7.1 Hz, 2H), 1.49 - 1.28 (m, 11H), 0.88 (d, J = 6.5 Hz, 3H). 13 C NMR (101 MHz, CDC13) δ 171.82, 146.61, 144.09, 133.36, 113.49, 112.82, 100.99, 80.82, 51.50, 35.12, 32.99, 32.92, 31.61, 25.77, 24.90, 24.83, 24.57, 22.52, 14.01.

[0095] Example 2

[0096] The synthesis route of compounds III-1 to 3 is partly the same as Example 1.

[0097] 1, 4-(Cyclohexylamino)-2-(l-hydroxypentyl)-5-nitrobenzoic acid (Compound 11)

[0098] Compound 10 (1.00 g, 3.01 mmol) was taken in a reaction flask containing methanol (50 mL) and water (50 mL) followed by NaOH (0.24 g, 6.02 mmol) under stirring conditions. The reaction solution was heated at 50 °C and stirred for 0.5 h. The solvent was removed under reduced pressure, then dissolved in water (100 mL) and acidified with 5% HC1 at -15-0 °C to pH 2-3. The mixture was then quickly extracted with cold Et20 (50 mL x 3) and dried to obtain compound 1 which was used quickly for the next step without any purification.

[0099] 2, 2-(1 -acetoxy pentyl)-4-(cyclohexylamino)-5-nitrobenzoic acid (Compound 12)

[0100] The solution containing compound 11 (0.50 g, 1.42 mmol) in Et20 was stirred at -15 °C, then a mixture of triethylamine (0.14 g, 1.42 mmol) and DMAP (0.02 g, 0.14 mmol) in dichloromethane was added. After that, acetyl chloride (0.17 g, 2.13 mmol) was dissolved in dry CH2CI2 and added dropwise to the reaction mixture, then the solution was stirred at -15 °C for 5 h. The mixture was acidified with 1 M HC1 to pH 2, then stirred at room temperature for 0.5 h. The organic phase was washed with water, dried over anhydrous Na2S04, then filtered and concentrated. The residue was then purified by column chromatography using EtOAc / pet. ether (1 / 10, v / v) as eluent to obtain compound 12 as a light yellow liquid (62.40%).1H NMR (400 MHz, Chloroform-d) δ 7.80 (s, 1H), 7.38 (s, 1H), 6.55 (dd, J = 8.4, 4.2 Hz, 1H), 3.34-3.21 (m, 1H), 2.10 (s, 3H), 2.12-2.03 (m, 2H), 1.91-1.78 (m, 4H), 1.44-1.32 (m, 8H), 1.30-1.24 (m, 2H), 0.90 (t, J = 7.2 Hz, 3H).

[0101] 3, Methyl 2-(1 -acetoxy pentyl)-4-(cyclohexylamino)-5-nitrobenzoate (Compound 13)

[0102] Compound 12 (0.2 g, 0.50 mmol) was dissolved in dry dichloromethane, followed by the addition of DMAP (0.006 g, 0.05 mmol) and HATU (0.27 g, 0.75 mmol) and allowed to react for half an hour at room temperature. Dry methanol (0.10 mL) was then added and stirred at room temperature for 8 hours. After the reaction was completed, the solution was washed with water, dried over anhydrous Na2S04, then filtered and concentrated. The crude product was purified by column chromatography to obtain a yellow oil in a yield of 70.20%. 1 H NMR (400 MHz, Chloroform-d) δ 7.81 (s, 1H), 7.50 (s, 1H), 6.34 (dd, J = 8.0, 5.2 Hz, 1H), 3.86 (s, 3H), 3.30 - 3.17 (m, 1H), 2.21 - 1.96 (m, 6H) 1.84 - 1.72 (m, 5H), 1.43 - 1.22 (m, 8H), 0.83 (t, J = 6.8 Hz, 3H).

[0103] 4, 5-Amino-4-(cyclohexylamino)-2-(1-hydroxypentyl) benzoic acid (III-1)

[0104] Synthesis method is the same as compound 11. 1 H NMR (400 MHz, Chloroform-d) δ 7.41 (s. 1H), 6.89 (s, 1H), 4.45 - 4.39 (m, 2H), 3.30 - 3.29 (m, 1H), 2.10 - 1.99 (m, 5H), 1.91 - 1.78 (m, 4H), 1.44 - 1.32 (m, 7H), 0.90 (t, J = 7.2 Hz, 3H).

[0105] 5, 5-Amino-4-(cyclohexylamino)-2-(1-hydroxypentyl) benzoic acid methyl ester (III-2)

[0106] Synthesis method is the same as example 1. 1 H NMR (400 MHz, Chloroform-d) δ 7.25 (s, 1H), 6.84 (s, 1H), 4.69 (s, 1H), 4.41 (m, 1H), 3.76 (s, 3H), 3.32 - 3.19 (m, 1H), 2.82 (s, 5H), 2.04 (s, 3H), 1.81 (s, 4H), 1.39 - 1.26 (m, 4H), 0.88 (t, J = 6.4 Hz, 3H).

[0107] 6, 2-(1-Acetyloxy-pentyl)-5-amino-4-(cyclohexylamino) benzoic acid methyl ester (III-3)

[0108] The synthetic method is the same as Example 1. 1 H NMR (400 MHz, Chloroform-d) δ 7.28 (s, 1H), 6.84 (s, 1H), 6.44-6.34 (m, 1H), 3.90 (s, 3H), 3.29-3.16 (m, 1H), 2.00-1.92 (m, 5H), 1.87-1.75 (m, 4H), 1.45-1.31 (m, 3H), 1.28-1.20 (m, 3H), 0.87 (t, J = 6.8 Hz, 3H).

[0109] Example 3: Test of ferroptosis inhibition activity of the compound

[0110] GPX4 inhibitors such as RSL-3 can induce cells to undergo ferroptosis, and this ferroptosis can also be blocked by other small molecules such as lipophilic antioxidants, such as Ferrostatin-1 (fer-1), Liproxstatin, etc. Therefore, the ability of ferroptosis inhibitors to block ferroptosis can be indicated by the reversal of cell ferroptosis induced by ferroptosis inducers.

[0111] Cell lines: human fibrosarcoma cell HT1080 and mouse hippocampal neuron cell HT22 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.

[0112] Method: MTT method, as follows: human renal cancer cell line HT1080 / HT22 in logarithmic growth phase was digested, collected and diluted, about 4000-5000 cells per well were seeded in a 96-well plate, 3 replicate wells were set for each group, and 80 μL per well. Incubate in a 37°C, 5% CO2 incubator overnight. The experiment was set up with DMSO control group and nine different concentrations of compound administration group. Add different concentrations of compounds to the administration group, and set up the DMSO control group (the same dilution multiple as the highest concentration of compound), continue to incubate in a 5% CO2, 37°C incubator for 1 h, then add 1 μM / 100 nM RSL3 to each compound concentration to induce ferroptosis, and set up RSL3 control group and DMSO control group, continue to incubate in a 5% CO2, 37°C incubator for 48 h. Add 5 mg / mL of 20 μL MTT solution to each well, continue to incubate the cells in a 37°C incubator for 2 h, add 100 μL DMSO to each well, and shake for 10 min, mix well. Place on an enzyme-linked immunosorbent assay instrument, detect the optical density value (OD value) of each well at 570 nm wavelength, and repeat the experiment 3 times. Calculate according to the following formula: survival rate % = experimental group OD value / DMSO control group OD value x 100%.

[0113] The results are shown in Table 1, which show that the various compounds of the application can significantly inhibit ferroptosis and have good ferroptosis inhibition activity.

[0114] Table 1 Inhibitory activity of compounds against RSL3-induced ferroptosis in HT22 cells

[0115] In the table: "A" indicates IC 50 ≤ 0.1 μΜ, "B" indicates IC 50 > 0.1 μΜ and ≤ 0.5 μΜ "C" indicates IC 50 > 0.5 μΜ and ≤ 2 μΜ, "D" indicates IC 50 > 2 μΜ.

[0116] Table 2 Inhibitory activity of compounds against RSL3-induced ferroptosis in HT1080 cells

[0117] In the table: "A" indicates IC 50 ≤ 0.1 μΜ, "B" indicates IC 50 > 0.1 μΜ and ≤ 0.5 μΜ "C" indicates IC 50 > 0.5 μΜ and ≤ 2 μΜ, "D" indicates IC 50 > 2 μΜ.

[0118] Example 4: Compounds exhibit good metabolic stability in vitro

[0119] Human / rat plasma and rat liver microsomes samples were stored at -20°C and gradually thawed to room temperature before processing. Compounds were dissolved in acetonitrile to make a stock solution of 4 mg / mL, then added to human / rat plasma or rat microsomes to make a final concentration of 0.4 mg / mL and incubated at 37°C for 12 hours (0, 0.25, 0.5, 1, 2, 4, 8, 12 hours). The incubation was terminated at specific time points by adding acetonitrile. The mixture was vortexed for 30 seconds and centrifuged (12000 rpm / 10 minutes), then filtered through a 0.22 micron filter and injected into an HPLC system (Agilent 1260 HPLC) with Alltima C18 (5 micron, 4.6 mm x 250 mm; mobile phase, methanol (20%) / water (80%) to methanol (100%) to methanol (20%) / water (80%); flow rate, 1.0 mL / min; UV detection wavelength, 254-400 nm; temperature, 25°C; injection volume, 10 μL) for 15 minutes.

[0120] As an example of compound I-1, it can be seen from the results in Table 3 that it exhibits good metabolic stability in human plasma, rat plasma and rat liver microsomes, with a half-life of more than 12 hours and a drug recovery rate of more than 85% at 12 hours, indicating that I-1 has good metabolic stability in vitro. It can be seen that the aromatic alkylamine-based ferroptosis inhibitor based on the butylphthalide structure has good metabolic properties and can be used for further pharmacological evaluation in animals.

[0121] Table 3 Evaluation of in vitro metabolic stability of compounds

[0122] The above merely describes preferred embodiments of the present application and is not intended to limit the present application in any form. Although the present application has been disclosed as above with reference to the preferred embodiments, the present application is not intended to be limited thereto, and any person skilled in the art can make some changes or modifications to the above-mentioned technical content within the scope of the technical solutions of the present application to obtain equivalent embodiments with equivalent changes. Any simple modification, equivalent change and modification made to the above embodiments in accordance with the technical essence of the present application shall still fall within the scope of the present application.

Claims

1. An aromatic alkylamine ferroptosis inhibitor based on a butylphthalide structure, characterized in that, The structural formula of the aromatic alkylamine ferroptosis inhibitor is shown in Formula 1 or Formula 2: In the formula R 1 Selected from hydrogen, C1-C6 alkyl, C1-C6 alkoxy, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, amino, phenyl, benzyl, naphthyl, C5-C 10 Aromatic heterocyclic groups or C3-C7 saturated heterocyclic groups; R 2 Selected from C0-C8 alkyl, C3-C 12 Cycloalkyl, adamantyl, or polyacetylenic; R 3 Selected from hydrogen, alkyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenolic or C3-C4 10 cycloalkyl; R 4 Selected from alkyl or haloalkyl, aryl, and heteroaryl groups of RC1-C10; R 5 Selected from C0 to C6 alkyl-C3 to C6 cycloalkyl-C0 to C6 alkyl or C0 to C6 alkyl-C3 to C6 cycloalkyl-C0 to C6 alkyl, C0 to C6 alkyl-Z-C0 to C6 alkyl or C0 to C6 alkyl-Z-C0 to C6 alkyl containing 1-3 independent substituents; wherein Z is selected from N, NR a , -SO2-, OC, OC=O, CO or C=OO; Among them, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8 cycloalkyl, C3-C8 cycloalkoxy, phenyl, benzyl, naphthyl, C5-C 10 Aromatic heterocyclic groups, C3-C7 saturated heterocyclic groups, C3-C 12 Cycloalkyl, polyynyl, aryl, C1-C6 alkyl-aryl, C1-C6 alkyl-phenolic or C3-C 10 Cycloalkyl groups can be substituted by one or more atoms or groups; R a The group is selected from H, CH3, CH2CH3, C3-C6 alkyl, C1-C6 haloalkyl or optionally substituted aryl, alkylaryl, piperazinyl, piperidinyl, morpholinyl, heterocyclic alkyl, heteroaryl, C1-C6 alkoxy, NH (C1-C4 alkyl) and N (C1-C4 alkyl)2, wherein the optionally substituted group is selected from C1-C6 alkyl or C2-C7 acrylate.

2. The aromatic alkylamine ferroptosis inhibitor based on the butylphthalide structure according to claim 1, characterized in that, The inhibitors are compounds I-1 to I-14, II-1 to II-12, and III-1 to III-3, and their structural formulas are as follows:

3. The preparation method of the aromatic alkylamine ferroptosis inhibitor based on the butylphthalide structure according to claim 2, characterized in that, The preparation method includes the following steps: The synthesis of compounds I-1 to I-14 is as follows: 5-bromophthalide was used as a starting material, and under the catalysis of AIBN, a free radical substitution reaction was carried out via NBS to obtain compound 2, which was then hydrolyzed to obtain compound 3; compound 3 reacted with the corresponding Grignard reagent to obtain compound 4; compound 4 reacted with potassium nitrate under concentrated sulfuric acid conditions to generate compound 5; compound 5 and cyclohexylamine underwent a substitution reaction in a mixed solution of DMF / tetrahydrofuran to obtain compound 6; compound 7 was obtained by reducing the nitro group of compound 6 with zinc powder, and then subjected to a reductive amination reaction with the corresponding aldehydes and ketones to obtain compounds I-1 to I-14, and II-1 to II-12; Reagents and reaction conditions: (a) NBS, AIBN, chloroform, reflux; (b) water, 110℃; (c) butyl magnesium bromide, tetrahydrofuran; (d) concentrated sulfuric acid, potassium nitrate; (e) cyclohexylamine, triethylamine, DMF, tetrahydrofuran; (f) zinc powder, hydrochloric acid, methanol; (g) the corresponding amine, acetic acid, sodium triacetoxyborohydride, dichloromethane.

4. The method for preparing the aromatic alkylamine ferroptosis inhibitor based on the butylphthalide structure according to claim 2, characterized in that, The preparation method includes the following steps: The synthesis of compounds III-1 to III-3 is as follows: compound 10 is hydrolyzed under alkaline conditions to give compound 11, which is further reacted with acyl chloride to give the corresponding ester 12. Compound 13 is formed by the condensation of compound 12 and the corresponding alcohol in the presence of HDTU and DIEPA, and then reduced to give the target compounds III-2 to III-3. Reagents and reaction conditions: (a) NBS, AIBN, chloroform, reflux; (b) water, 110°C; (c) butyl magnesium bromide, tetrahydrofuran; (d) concentrated sulfuric acid, potassium nitrate; (e) cyclohexylamine, triethylamine, DMF, tetrahydrofuran; (h) sodium hydroxide, methanol, water, 50°C; (i) acetyl chloride, triethylamine, dichloromethane, -10°C; (j) HATU, DMAP, dichloromethane, room temperature -50°C; (f) zinc powder, hydrochloric acid, methanol; (g) the corresponding amine, acetic acid, sodium triacetoxyborohydride, dichloromethane.

5. The use of the inhibitor according to claim 1 or 2 in the preparation of ferroptosis inhibitors and / or butylphthalide analog drugs.

6. The use of the inhibitor according to claim 1 or 2 in the preparation of a treatment for ferroptosis-related diseases and / or diseases treated with butylphthalide.

7. The application according to claim 6, characterized in that, The iron death-related diseases include neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular diseases, liver and kidney failure, inflammation, diabetic complications, and cardiovascular diseases.

8. The application according to claim 6, characterized in that, The diseases that butylphthalide can treat include resisting ischemic brain damage, improving neurodegenerative diseases, improving cerebral microcirculation, inhibiting platelet aggregation, protecting mitochondria, regulating energy metabolism, and reducing oxidative stress damage.

9. A drug for treating ferroptosis-related diseases, characterized in that, The drug contains the compound of claim 1 or 2 and a pharmaceutically acceptable salt, carrier, or adjuvant.

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