10H-phenoxazine ferroptosis inhibitor as well as preparation method and application thereof

Abstract

The invention discloses a 10H-phenoxazine compound as well as a preparation method and application thereof. The 10H-phenoxazine compound has a structure as shown in a formula (I). The invention also discloses application of the compounds and pharmaceutically acceptable salts thereof, or crystal forms thereof, or solvates thereof, or stereoisomers thereof in preparation of ferroptosis inhibitors, and application of the compounds and pharmaceutically acceptable salts thereof in preparation of drugs for treating and / or preventing neurodegenerative diseases, tissue ischemia reperfusion injury, cerebral apoplexy, cardiovascular diseases, renal failure, liver injury, diabetic complications, diabetes mellitus and the like. The invention relates to application in medicines for treating pancreatic cancer. # imgabs0 #

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine technology, and in particular to a 10H-phenoxazine ferroptosis inhibitor and a preparation method and application thereof. Background Art

[0002] Ferroptosis is a programmed cell death that is iron-dependent and lipid peroxidation-driven. The morphological characteristics of ferroptosis are mainly manifested in the reduction of mitochondrial volume, increase of mitochondrial membrane density, reduction or disappearance of mitochondrial cristae, rupture of mitochondrial outer membrane, and normal size of nucleus, which are the main morphological characteristics that distinguish ferroptosis from apoptosis, necrosis and autophagy. The biochemical characteristics of ferroptosis are mainly manifested in the accumulation of intracellular iron and ROS, activation of mitogen-activated protein kinase (MAPK) signal transduction system, inhibition of cystine / glutamate transporter system and increased NADPH oxidation.

[0003] More and more studies have confirmed that ferroptosis is closely related to many conditions, disorders and diseases. Dixon et al. first discovered in 2012 that this cell death pattern was associated with small molecule-induced RAS tumor cell death (Scott JDixon et al., Cell. 2012 May 25; 149 (5): 1060-72. doi: 10.1016 / j.cell.2012.03.042). Recent studies have further demonstrated the role of ferroptosis in cancer, organ damage and degenerative diseases (Xuejun Jiang et al., Nat Rev Mol Cell Biol. 2021 April; 22 (4): 266-282). Specifically, it involves the occurrence and development of various cancers, neurodegenerative diseases, cardiovascular and cerebrovascular diseases, immune-related diseases, liver and kidney failure, inflammation, metabolic diseases, etc. In particular, it plays an important role in diseases such as Alzheimer's disease, Parkinson's disease, tumors, stroke, ischemia-reperfusion injury, atherosclerosis, liver and kidney failure, inflammation and diabetic complications. By stimulating or inhibiting the occurrence of ferroptosis, the occurrence and development of related diseases can be tolerated. Therefore, ferroptosis inhibitors are considered to be potential drugs for the treatment of these diseases.

[0004] Ferrostatin-1 (Fer-1) is a known effective and selective ferroptosis inhibitor (Scott J Dixon et al., Cell. 2012 May 25; 149(5): 1060-72. doi: 10.1016 / j.cell.2012.03.042). However, the activity of Ferrostatin-1 is not high, and the IC50 values ​​in various in vitro ferroptosis cell models are all higher than 100 nM. Therefore, how to discover and prepare a more active ferroptosis inhibitor is a problem that needs to be solved urgently.

[0005] Summary of the invention

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In some embodiments of the present invention, a compound as shown in general formula (I), or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof is provided:

[0008]

[0009] in:

[0010] M is selected from: 5-10 yuan aryl or 5-10 yuan heteroaryl, and the aryl or heteroaryl is optionally substituted by one or more substituents selected from halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, amino, hydroxyl, 5-10 yuan cycloalkyl, 5-10 yuan heterocyclyl, 5-10 yuan aryl and 5-10 yuan heteroaryl;

[0011] Y is selected from:

[0012] R1 is selected from the group consisting of a hydrogen atom, a cyano group, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, an amino group, a hydroxyl group, a 5-10-membered cycloalkyl group, a 5-10-membered heterocyclic group, a 5-10-membered aryl group, and a 5-10-membered heteroaryl group. 5-10 membered cycloalkyl, 5-10 membered heterocyclyl, 5-10 membered aryl and 5-10 membered heteroaryl are optionally substituted with one or more substituents selected from halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, amino, hydroxy, 5-10 membered cycloalkyl, 5-10 membered heterocyclyl, 5-10 membered aryl and 5-10 membered heteroaryl;

[0013] The number of heteroatoms contained in the heterocyclic group and heteroaryl group is 1, 2 or 3;

[0014] The heteroatoms of the heterocyclic group and heteroaryl group are selected from one or more of N, O and S;

[0015] The restriction is that when Y is selected from: , R1 does not exist.

[0016] In some embodiments of the present invention, a compound represented by general formula (II) is provided, or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof:

[0017]

[0018] in:

[0019] R2 is selected from: hydrogen atom, halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by one or more substituents selected from halogen, C1-C6 alkyl, 5-10 membered cycloalkyl, 5-10 membered heterocyclic group;

[0020] n is selected from: 0, 1, 2 or 3;

[0021] Y is selected from:

[0022] R1 is selected from: a hydrogen atom, a C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally substituted by one or more substituents selected from halogen, a C1-C6 alkyl group, a 5-10 membered cycloalkyl group, and a 5-10 membered heterocyclic group.

[0023] In some embodiments of the present invention, the compound represented by the above general formula (II) or its pharmaceutically acceptable salt, crystal form, solvate, stereoisomer, wherein preferably: R2 is selected from hydrogen atom, halogen;

[0024] n is selected from: 0 or 1; R1 is selected from: C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by one or more substituents selected from halogen, C1-C6 alkyl, 5-10 membered cycloalkyl, 5-10 membered heterocyclic group; further preferably, R1 is selected from CH3, CH2CF3.

[0025] In some embodiments of the present invention, the compound represented by the above formula (I), or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof, wherein the compound can be selected from the following compounds:

[0026]

[0027] In some embodiments of the present invention, a pharmaceutical composition is provided, which comprises at least one compound of the present invention described herein or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof, and a pharmaceutically acceptable carrier.

[0028] In some embodiments of the present invention, there is provided use of the compounds of the present invention described herein or their pharmaceutically acceptable salts, or their crystal forms, or their solvates, or their stereoisomers, or the above-mentioned pharmaceutical compositions in the preparation of drugs for ferroptosis inhibitors; the ferroptosis inhibitors are targeted drugs for inhibiting cellular ferroptosis.

[0029] In some embodiments of the present invention, there is provided a use of the compounds of the present invention described herein or their pharmaceutically acceptable salts, crystal forms, solvates, stereoisomers, or the above-mentioned pharmaceutical compositions in the preparation of drugs for treating and / or preventing neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver injury, diabetic complications, and pancreatic cancer. The neurodegenerative diseases are Parkinson's syndrome and Alzheimer's disease; the strokes include ischemic strokes and hemorrhagic strokes.

[0030] Definitions and General Terms:

[0031] The following terms and the like are used to describe the present invention. It should be understood that terms that are not specifically defined are given a meaning consistent with the use of the terms in the context of the present invention as understood by those of ordinary skill in the art.

[0032] The term "alkyl" used in the present invention refers to a saturated aliphatic hydrocarbon group, and the term includes straight and branched hydrocarbon groups. Such as C1-C6 alkyl. "C1-C6 alkyl" refers to an alkyl group having 1 to 6 carbon atoms, for example, an alkyl group having 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, including but not limited to methyl, ethyl, propyl (e.g., n-propyl, isopropyl), butyl (e.g., n-butyl, isobutyl, tert-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), hexyl (e.g., n-hexyl), etc.

[0033] The term "halogen" as used herein refers to fluorine, chlorine, bromine or iodine, preferably fluorine or chlorine.

[0034] The term "alkenyl" as used herein refers to an aliphatic hydrocarbon group containing a carbon-carbon double bond, preferably a straight chain or branched chain having 2 to 6 carbon atoms, such as vinyl, 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl and 2-butenyl.

[0035] The term "alkynyl" used in the present invention refers to a straight chain or branched hydrocarbon group having at least one carbon-carbon triple bond, preferably a straight chain or branched hydrocarbon group having 2 to 6 carbon atoms, such as ethynyl, propynyl and butynyl.

[0036] The term "cycloalkyl" as used herein refers to a non-aromatic mono- or polycyclic ring system, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, preferably cyclopropyl.

[0037] The term "heterocyclyl" as used in the present invention refers to a "heterocyclic group" or "heterocycle" which is a saturated or unsaturated monocyclic or polycyclic ring system containing one or more heteroatoms selected from O, N or S, preferably a 5-10 membered monocyclic or polycyclic ring.

[0038] The term "aryl" as used herein refers to an aromatic group such as phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl, preferably phenyl.

[0039] The term "heteroaryl" as used in the present invention refers to an aromatic ring containing one or more heteroatoms selected from O, N or S. The heteroaryl group can be a mono-, bi- or tricyclic ring system, such as: quinolyl, pyrazolyl, pyrrolyl, thienyl, furanyl, pyridyl, pyrimidinyl, pyrazinyl, triazolyl, imidazolyl, oxazolyl, isoxazolyl, pyridazinyl, benzofuranyl, benzothiophenyl, benzoxazole, indolyl, etc.

[0040] The term "pharmaceutically acceptable salt" used in the present invention refers to a salt of the compound of the present invention, which is prepared from the compound having a specific substituent discovered in the present invention and a pharmaceutically acceptable acid or base.

[0041] The term "solvate" as used herein describes a molecular complex comprising a compound of any of the above chemical formulae or a pharmaceutically acceptable salt thereof and one or more pharmaceutically acceptable solvent molecules (eg, ethanol).

[0042] The term "stereoisomers" as used herein refers to compounds which have identical chemical constitution, but differ with regard to the arrangement of the atoms and groups in space. These include enantiomers, diastereomers, geometric isomers, atropisomers or conformational isomers.

[0043] The term "pharmaceutically acceptable carrier" used in the present invention refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient, and representative carriers include water, oil, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity enhancers, transdermal enhancers, etc.

[0044] Beneficial technical effects achieved:

[0045] 1) The compounds of the present invention have excellent biological activity, which is significantly better than the existing positive control compound Ferrostain-1. For example, the biological activity of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7 is 5-15 times that of the control compound Ferrostain-1.

[0046] 2) The preparation method of the compound of the present invention is simple, the reaction conditions are mild, it is easy to operate and control, the energy consumption is small, the yield is high, the cost is low, and it is suitable for industrial production. DETAILED DESCRIPTION

[0047] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

[0048] Example 1

[0049]

[0050] Add formaldehyde aqueous solution (0.672g, 2.0eq) and 1-(4-bromophenyl)piperazine (compound 1a, 1g, 1.0eq) to the reactor in sequence and stir. Add formic acid (0.381g, 2.0eq) dropwise, raise the temperature to 80±5℃, keep the temperature for 1h, TLC shows that the raw material is completely converted. Turn off the heating, when the reaction temperature drops to 30℃, add 20% NaOH aqueous solution to the reaction system to make the system pH = 8-9. Extract with ethyl acetate, combine the organic phases and dry with anhydrous sodium sulfate, concentrate until there is no obvious fraction, transfer the material to a vacuum drying oven at 40℃ and dry, to obtain 0.919g compound 1b, with a yield of 87%.

[0051] Tetrahydrofuran and compound 1b (900 mg, 1.0 eq) were added to the reaction bottle in sequence, cooled to below -60 ° C, n-butyl lithium (1.7 ml, 1.2 eq, 2.5 M / L) was added dropwise, and the mixture was stirred for 2 hours after the addition was completed. Trimethyl borate (550 mg, 1.5 eq, diluted with an equal volume of tetrahydrofuran and added dropwise), the temperature was controlled not to exceed -60 ° C during the addition process, and the mixture was stirred for 2 hours. The mixture was naturally heated to room temperature and reacted for 16 hours. Saturated ammonium chloride solution was added dropwise to make the pH value of the system about 8.3, stirred for 2 hours, filtered, and the filter cake was rinsed with water and n-heptane in sequence. The obtained filter cake was slurried with a mixed solvent of water and ethyl acetate for 2 hours, filtered, and dried in a vacuum oven at 40 ° C to obtain 528 mg of compound 1c, with a yield of 68%.

[0052]

[0053] To the reaction bottle, 2-iodophenol (1.3 g, 1.0 eq), 4-bromo-1-fluoro-2-nitrobenzene (1.3 g, 1.0 eq), potassium carbonate (1.63 g, 2.0 eq) were added in sequence, and then 20 mL of DMSO was added. The temperature was raised to 100 °C and the reaction was carried out for 18 h. TLC showed that the raw material was completely converted. The heating was turned off, the reaction was cooled to room temperature, and water was added to quench the reaction. Then, the reaction was extracted with ethyl acetate, and the organic phases were combined and backwashed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated. The obtained crude product was dissolved in a mixed solvent of ethanol: water = 3:1, and iron powder (1.3 g, 4.0 eq) and ammonium chloride (158 mg, 0.5 eq) were added. The reaction was refluxed for 1 h. TLC showed that the raw material was completely converted. The heating was turned off. After the reaction was cooled to room temperature, it was filtered, the filtrate was concentrated, and a saturated aqueous sodium bicarbonate solution was added. The mixture was then extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column chromatography to obtain 1.82 g of compound 1d. The yield was 79%.

[0054] Compound 1d (1.65 g, 1.0 eq), sodium tert-butoxide (0.57 g, 1.4 eq), Pd2(dba)3 (0.19 g, 0.05 eq), 1,1'-bis(diphenylphosphino)ferrocene (dppf, 0.235 g, 0.1 eq), and 20 mL of toluene were added to the reaction flask in sequence. The mixture was kept at 130 °C for 20 h under nitrogen atmosphere. TLC showed that the raw material was completely converted. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain 0.333 g of compound 1e with a yield of 30%.

[0055] Compound 1e (0.5 g, 1.0 eq), compound 1c (0.504 g, 1.2 eq), Pd(PPh3)4 (110 mg, 0.05 eq), potassium carbonate (0.527 g, 2.0 eq), 10 mL of a 4:1 mixed solvent of 1,4-dioxane and water were added to the reaction flask in sequence, and the mixture was reacted at 100 °C for 15 h under a nitrogen atmosphere. TLC showed that the raw materials were completely converted, and the mixture was cooled to room temperature, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain 238 mg of compound 1, with a yield of 30%. MS m / z (ESI): 357.1848 (M+H) + , 1HNMR (400MHz, DMSO-d6) δ = 7.62 (s, 1H), 7.53–7.51 (m, 2H), 7.25 (d, J = 1.2Hz, 1H), 7.20–7.18 (m, 2H), 7.06–7.04 (m, 2 H),6.98(d,J=5.6Hz,1H),6.95–6.93(m,2H),6.86–6.84(m,1H),3.19(t,J=5.6Hz,4H),2.46-2.43(m,4H),2.22(s,3H).

[0056] Example 2

[0057]

[0058] Add formaldehyde aqueous solution (0.63g, 2.0eq) and 1-(4-bromo-2-fluorophenyl)piperazine (compound 2a, 1g, 1.0eq) to the reaction kettle in sequence and stir. Add formic acid (0.36g, 2.0eq) dropwise, raise the temperature to 80±5℃, keep the temperature for 1h, TLC shows that the raw material is completely converted. Turn off the heating, when the reaction temperature drops to 30℃, add 20% NaOH aqueous solution to the reaction system to make the system pH = 8-9. Extract with ethyl acetate, combine the organic phases and dry with anhydrous sodium sulfate, concentrate until there is no obvious fraction, transfer the material to a vacuum drying oven at 40℃ and dry, to obtain 0.864g of compound 2b, with a yield of 82%.

[0059] Tetrahydrofuran and compound 2b (800 mg, 1.0 eq) were added to the reaction bottle in sequence, the temperature was lowered to below -60 ° C, n-butyl lithium (1.4 ml, 1.2 eq, 2.5 M / L) was added dropwise, and the mixture was stirred for 2 hours after the addition was completed. Trimethyl borate (456.5 mg, 1.5 eq, diluted with an equal volume of tetrahydrofuran and added dropwise), the temperature was controlled not to exceed -60 ° C during the addition process, and the mixture was stirred for 2 hours. The mixture was naturally heated to room temperature and reacted for 16 hours. Saturated ammonium chloride solution was added dropwise to make the pH value of the system about 8.3, stirred for 2 hours, filtered, and the filter cake was rinsed with water and n-heptane in sequence. The obtained filter cake was slurried with a mixed solvent of water and ethyl acetate for 2 hours, filtered, and dried in a vacuum oven at 40 ° C to obtain 384 mg of compound 2c, with a yield of 55%.

[0060]

[0061] Compound 1e (0.5 g, 1.0 eq), compound 2c (0.545 g, 1.2 eq), Pd(PPh3)4 (110 mg, 0.05 eq), potassium carbonate (0.527 g, 2.0 eq), 10 mL of a 4:1 mixed solvent of 1,4-dioxane: water were added to the reaction flask in sequence, and the mixture was reacted at 100 °C for 15 h under a nitrogen atmosphere. TLC showed that the raw materials were completely converted, and the mixture was cooled to room temperature, filtered, and the filtrate was concentrated and purified by silica gel column chromatography to obtain 222 mg of compound 2, with a yield of 26%. MS m / z (ESI): 375.1743 (M+H) +, 1HNMR (400MHz, DMSO-d6) δ = 7.66 (s, 1H), 7.33–7.25 (m, 3H), 7.25–7.19 (m, 2H), 7.08–7.03 (m, 2H), 6.99 (d, J= 6.0Hz,1H),6.90(dd,J=6.0,4.0Hz,1H),6.87–6.82(m,1H),3.08–3.05(m,4H),2.44–2.40(m,4H),2.25(s,3H).

[0062] Example 3

[0063]

[0064] 1-(4-bromophenyl)piperazine (compound 1a, 1.24 g, 1.0 eq), 20 mL of dichloromethane, trifluoroethyl trifluoromethanesulfonate (1.2 mL, 1.4 eq), and cesium carbonate (5.04 g, 3.0 eq) were added to the reaction flask. The reaction was stirred at room temperature for 0.5 h. TLC showed that the raw material was completely converted. Saturated aqueous ammonium chloride was added to quench the reaction, followed by extraction with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by silica gel column chromatography to obtain 1.23 g of compound 3b with a yield of 74%.

[0065] Referring to the synthesis method of compound 1c, compound 3c was obtained by replacing compound 1b with compound 3b.

[0066]

[0067] Referring to the synthesis method of compound 1, compound 1c was replaced by compound 3c to finally obtain compound 3. MS m / z (ESI): 425.1721 (M+H) + ,1HNMR(400MHz,DMSO-d6)δ=7.79(s,1H),7.54–7.52(m,2H),7.26–7.24(m,2H),7.18-7.16(m,1H),7.10–7 .04(m,2H),6.99–6.95(m,3H),6.85-6.83(m,1H),3.32-3.29(m,4H),3.28-3.23(m,2H),2.82-2.79(m,4H).

[0068] Example 4

[0069] Refer to the synthesis method of compound 1, except that the compound Replace with compound Finally, compound 4 was obtained. MS m / z (ESI): 443.1625 (M+H) +, 1HNMR (400MHz, DMSO-d6) δ = 7.82 (s, 1H), 7.32 (dd, J = 6.0, 2.0Hz, 1H), 7.29 (d, J = 1.0Hz, 1H), 7.28–7.24 (m, 2H), 7.23–7.21 (m, 1H), 7. 11–7.05(m,2H),6.99(d,J=6.0Hz,1H),6.91-6.89(m,1H),6.86–6.83(m,1H),3.29-3.20(m,2H),3.21-3.17(m,4H),2.83-2.80(m,4H).

[0070] Example 5

[0071] Refer to the synthesis method of compound 1, except that the compound Replace with compound Finally, compound 5 was obtained. MS m / z (ESI): 369.1843 (M+H) + , 1HNMR (400MHz, DMSO-d6) δ = 7.78 (s, 1H), 7.53–7.50 (m, 2H), 7.25–7.22 (m, 2H), 7.21–7.18 (m, 1H), 7.07–7.03 (m, 2H), 6.99–6.80 (m, 1H),6.97–6.94(m,2H),6.87–6.84(m,1H),3.87–3.82(m,2H),3.17–3.13(m,2H),2.97–2.93(m,2H),2.24(s,3H),2.14–2.11(m,2H).

[0072] Example 6

[0073] Refer to the synthesis method of compound 1, except that the compound Replace with compound Finally, compound 6 was obtained. MS m / z (ESI): 437.1720 (M+H) + , 1HNMR (400MHz, DMSO-d6) δ = 7.79 (s, 1H), 7.54–7.49 (m, 2H), 7.25–7.20 (m, 3H), 7.11–7.05 (m, 2H), 7.0–6.98 (m, 1H) ,6.97–6.94(m,2H),6.84–6.82(m,1H),3.80–3.75(m,2H),3.28–3.16(m,4H),3.06–3.02(m,2H),2.10–2.07(m,2H).

[0074] Example 7

[0075] Refer to the synthesis method of compound 1, except that the compound Replace with compound Finally, compound 7 was obtained. MS m / z (ESI): 369.1842 (M+H) + , 1HNMR (400MHz, DMSO-d6) δ = 7.81 (s, 1H), 7.53–7.51 (m, 2H), 7.25–7.22 (m, 2H), 7.20–7.18 (m, 1H), 7.07–7.03 (m, 2H), 6.99 (d, J =6.0Hz,1H),6.96–6.94(m,2H),6.86–6.84(m,1H),3.54–3.45(m,4H),3.36–3.29(m,2H),2.44–2.42(m,3H),2.11–2.00(m,2H).

[0076] Example 8

[0077] Refer to the synthesis method of compound 1, except that the compound Replace with compound Finally, compound 8 was obtained. MS m / z (ESI): 383.2000 (M+H) + , 1HNMR (400MHz, DMSO-d6) δ = 7.83 (s, 1H), 7.56–7.52 (m, 2H), 7.26–7.18 (m, 3H), 7.10–7.04 (m, 2H), 6.99–6.97 (m, 1H), 6.94 –6.91(m,2H),6.88–6.83(m,1H),3.60–3.48(m,4H),2.97–2.90(m,2H),2.69–2.61(m,2H),2.36–2.30(m,2H),2.27(s,3H).

[0078] Example 9: Study on the Inhibition Rate of Ferroptosis by the Compounds of the Present Invention

[0079] Experimental method: In order to study the inhibition rate of the compounds of the present invention on Ferroptosis, a screening model for ferroptosis was constructed in this embodiment, as follows: The Ferroptosis screening model mainly uses the CCK8 cell viability detection method. First, mouse hippocampal neuron cells HT22 and human neuroblastoma cells SH-SY5Y were cultured in a dish, and cells in the logarithmic growth phase were inoculated in a specific number in a 96-well plate (5000 cells / well), 80 μL per well, and then placed in an incubator at 37°C and 5% CO2 environment for culture to allow the cells to adhere to the wall. After 24 hours, 10 μL of various concentrations of the compounds of the present invention prepared using the specified culture medium and 10 μL of the Ferroptosis inducer Erastin with a final concentration of 10 μM were added, and 3 replicate wells were set for each compound to ensure the accuracy of the results. A positive control group (a certain concentration of the compound Ferrostain-1 and 10 μL of the Ferroptosis inducer Erastin prepared by adding 10 μL of the same culture medium as above, without cells) and a solvent control group (an equal volume of culture medium and DMSO equal to the culture medium as above, with cells) were set up, and 3 replicate wells were set up in each group to ensure the accuracy of the results. After adding the drug, the wells were placed in an incubator and cultured for 24 hours. 10 μL of CCK8 solution was added to each well, and the wells were placed in an incubator for further culture for 2-4 hours, and then the absorbance at 450 nm was detected with an enzyme reader to calculate the inhibition rate of the compound on Ferroptosis. The inhibition rate was calculated using the following formula:

[0080] Inhibition rate (IR) = [1-(A experimental group-A blank) / (A solvent-A blank)]*100%

[0081] GraphPad Prism 6 software was used to fit the inhibition rate curve and calculate the IC 50 .

[0082] Experimental results: See Table 1.

[0083] Table 1. Determination of ferroptosis activity of the compounds of the present invention on HT22 cells and SH-SY5Y cells

[0084]

[0085] A: 1-30nM; B: 50~100nM; C: 150~200nM;

[0086] From the test results shown in Table 1, it can be seen that the compounds of the present invention have excellent biological activity, which is significantly better than the existing positive control compound Ferrostain-1. For example, the biological activity of compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, and compound 7 is 5-15 times that of the control compound Ferrostain-1. It is suggested that the compounds of the present invention can be used to prepare ferroptosis inhibitors as drugs for preparing various diseases related to ferroptosis, such as neurodegenerative diseases, Parkinson's syndrome, stroke, cardiovascular disease, renal failure, diabetic complications, cancer, etc.

Claims

1. A compound represented by general formula (I), or a pharmaceutically acceptable salt, crystal form, solvate, or stereoisomer thereof: in: M is selected from: 5-10 yuan of aryl or 5-10 yuan of heteroaryl, and the aryl or heteroaryl is optionally substituted by one or more substituents selected from halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, amino, hydroxyl, 5-10 yuan of cycloalkyl, 5-10 yuan of heterocyclyl, 5-10 yuan of aryl and 5-10 yuan of heteroaryl; Y is selected from: R1 is selected from the group consisting of a hydrogen atom, a cyano group, a C1-C6 alkyl group, a C2-C6 alkenyl group, a C2-C6 alkynyl group, a C1-C6 alkoxy group, an amino group, a hydroxyl group, a 5-10-membered cycloalkyl group, a 5-10-membered heterocyclic group, a 5-10-membered aryl group, and a 5-10-membered heteroaryl group. 5-10 membered cycloalkyl, 5-10 membered heterocyclyl, 5-10 membered aryl and 5-10 membered heteroaryl are optionally substituted with one or more substituents selected from halogen, cyano, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, amino, hydroxy, 5-10 membered cycloalkyl, 5-10 membered heterocyclyl, 5-10 membered aryl and 5-10 membered heteroaryl; The number of heteroatoms contained in the heterocyclic group and heteroaryl group is 1, 2 or 3; The heteroatoms of the heterocyclic group and heteroaryl group are selected from one or more of N, O and S; The restriction is that when Y is selected from: , R1 does not exist.

2. The compound according to claim 1, or a pharmaceutically acceptable salt, crystal form, solvate, or stereoisomer thereof, characterized in that: The compound is shown in general formula (II): in: R2 is selected from: hydrogen atom, halogen, C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by one or more substituents selected from halogen, C1-C6 alkyl, 5-10 membered cycloalkyl, 5-10 membered heterocyclic group; n is selected from: 0, 1, 2 or 3; Y is selected from: R1 is selected from: a hydrogen atom, a C1-C6 alkyl group, wherein the C1-C6 alkyl group is optionally substituted by one or more substituents selected from halogen, a C1-C6 alkyl group, a 5-10 membered cycloalkyl group, and a 5-10 membered heterocyclic group.

3. The compound according to claim 2, or a pharmaceutically acceptable salt, crystal form, solvate, or stereoisomer thereof, characterized in that: in, R2 is selected from: hydrogen atom, halogen; n is selected from: 0 or 1; R1 is selected from: C1-C6 alkyl, wherein the C1-C6 alkyl is optionally substituted by one or more substituents selected from halogen, C1-C6 alkyl, 5-10 membered cycloalkyl, 5-10 membered heterocyclyl; More preferably, R1 is selected from CH3, CH2CF3.

4. The compound according to any one of claims 1 to 3, or a pharmaceutically acceptable salt, crystal form, solvate, or stereoisomer thereof, characterized in that: The compound is one of the following compounds:

5. A pharmaceutical composition, characterized in that It includes the compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof, and a pharmaceutically acceptable carrier.

6. Use of the compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt, crystal form, solvate, stereoisomer thereof, or the pharmaceutical composition according to claim 5 in the preparation of a medicament for a ferroptosis inhibitor.

7. Use of the compound according to any one of claims 1 to 4, or its pharmaceutically acceptable salt, crystal form, solvate, stereoisomer, or the pharmaceutical composition according to claim 5 in the preparation of drugs for treating and / or preventing neurodegenerative diseases, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, renal failure, liver injury, diabetic complications, and pancreatic cancer.

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