Application of 3,4-dihydro-2H-benzo-[1,4]oxazine drugs or their salts in the preparation of drugs for inhibiting ferroptosis

Abstract

The present invention relates to the use of 3,4-dihydro-2H-benzo-[1,4]oxazine drugs or salts thereof in the preparation of drugs for inhibiting ferroptosis, in particular to the use of 3,4-dihydro-2H-benzo-[1,4]oxazine compounds or salts thereof in the preparation of drugs for treating ferroptosis-related diseases, wherein the ferroptosis-related diseases include brain trauma, stroke, cardiovascular disease, liver and kidney failure, inflammation and neurodegenerative diseases, such as Parkinson's disease and schizophrenia.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, it relates to the use of 3,4-dihydro-2H-benzo-[1,4]oxazine drugs or salts thereof in the preparation of drugs for treating diseases related to ferroptosis. More specifically, it relates to the use of 3,4-dihydro-2H-benzo-[1,4]oxazine compounds or salts thereof in the preparation of drugs for treating diseases including brain trauma, stroke, cardiovascular disease, liver and kidney failure, inflammation, and neurodegenerative diseases. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Ferroptosis is a recently discovered form of programmed cell death that differs from apoptosis, necrosis, and autophagy in morphology, biochemistry, and genetics. Because this process depends on the presence of iron ions, it is called ferroptosis. Its mechanism is an imbalance between the generation and degradation of reactive oxygen species in intracellular membrane lipids, leading to an iron-dependent, oxidative, non-apoptotic programmed cell death. Typical features include reduced mitochondrial size, increased bilayer membrane density, and an increase in reactive oxygen species free radicals in cell membrane lipids.

[0004] Parkinson's disease (PD) is a typical neurodegenerative disorder characterized by the degeneration and death of dopaminergic neurons in the substantia nigra and overexpression of α-synuclein. Recent studies have revealed that ferroptosis plays a key role in neuronal damage in PD. While therapeutic drugs, such as levodopa and its derivatives, can control clinical symptoms, they cannot control disease progression, making many therapeutic goals difficult to achieve, including neuroprotection, treatment of motor complications, and psychosis. Therefore, the development of new therapeutic agents targeting its pathogenesis is urgently needed.

[0005] In addition, more and more studies have confirmed that the occurrence and development of diseases such as neurodegeneration, tissue ischemia-reperfusion injury, stroke, cardiovascular disease, liver and kidney failure, inflammation, and diabetic complications are closely related to cellular ferroptosis. The typical feature of these diseases is cell death, and current treatment methods mainly improve the function and cannot fundamentally alleviate the progression of these diseases. Therefore, ferroptosis inhibitors are considered to be potential drugs for the treatment of these diseases.

[0006] Ferrostatin-1 and lipoxstatin-1 are small-molecule ferroptosis inhibitors identified through high-throughput screening. They function by scavenging lipid free radical damage to cell membranes and blocking the onset of ferroptosis. These inhibitors have a well-defined mechanism of action and structure-activity relationship. However, these two compounds suffer from short half-lives and high toxicity, respectively.

[0007] Summary of the Invention

[0008] To overcome the above problems, the present invention, based on the study of the mechanism of cell ferroptosis, quantitatively calculated and analyzed the degree of conjugation of common bicyclic compounds containing p-π* conjugated structures and combined with in vitro activity evaluation, discovered that several 3,4-dihydro-2H-benzo-[1,4]oxazine drugs are inhibitors of cell ferroptosis, and have new uses in the treatment of certain neurodegenerative diseases, liver and kidney failure, and other diseases.

[0009] In order to achieve the above technical objectives, the technical solutions adopted by the present invention are as follows:

[0010] A pharmaceutical composition for inhibiting cell ferroptosis and / or cell membrane lipid free radical damage, the active ingredient comprising: a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a pharmaceutically acceptable salt or ester thereof, wherein the 3,4-dihydro-2H-benzo-[1,4]oxazine compound is a compound having the following structure:

[0011]

[0012] After extensive and in-depth research, the inventors, through a combination of quantitative calculations and experimental verification, discovered for the first time that 3,4-dihydro-2H-benzo-[1,4]oxazine drugs have good therapeutic prospects for certain neurodegenerative diseases and liver and kidney failure by effectively scavenging membrane lipid peroxyl radicals. Experiments have shown that 3,4-dihydro-2H-benzo-[1,4]oxazine compounds can block the chain reaction of membrane lipid free radicals, reduce the damage of free radicals to cell membranes, prevent the occurrence of cell ferroptosis, and protect cells, thereby having a therapeutic effect on the above-mentioned diseases. In addition, cytotoxicity experiments have also confirmed that 3,4-dihydro-2H-benzo-[1,4]oxazine drugs at therapeutic doses have no obvious toxicity when used to treat related tumors and heart failure. On this basis, the present invention was completed.

[0013] The present invention also provides a pharmaceutical preparation for inhibiting cell ferroptosis and / or cell membrane lipid free radical damage, comprising: the above-mentioned pharmaceutical composition and excipients. The pharmaceutical preparation has high activity and can be stably present.

[0014] The present application does not impose any particular limitation on the specific type of preparation. In some embodiments, the preparation is a targeted preparation, a radioactive agent, a controlled-release agent or a solution to improve the absorption efficiency and therapeutic effect of the drug.

[0015] The present invention also provides a pharmaceutical composition for treating nervous system diseases or liver and kidney failure, the active ingredient comprising: a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a pharmaceutically acceptable salt or ester thereof, wherein the 3,4-dihydro-2H-benzo-[1,4]oxazine compound is a compound having the following structure:

[0016]

[0017] The present invention also provides a pharmaceutical preparation for treating nervous system diseases or liver and kidney failure, comprising: the above-mentioned pharmaceutical composition and excipients. The drug can effectively treat the above-mentioned diseases without toxic side effects.

[0018] The present application does not impose any particular limitation on the specific type of preparation. In some embodiments, the preparation is a targeted preparation, a radioactive agent, a controlled-release agent or a solution to improve the absorption efficiency and therapeutic effect of the drug.

[0019] The present invention also provides a use of a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof in the preparation of a drug for inhibiting ferroptosis and / or cell membrane lipid free radical damage, wherein the 3,4-dihydro-2H-benzo-[1,4]oxazine compound is

[0020]

[0021] The present invention also provides a use of a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof in the preparation of a drug for treating a disease associated with ferroptosis, wherein the 3,4-dihydro-2H-benzo-[1,4]oxazine compound has the following structure:

[0022]

[0023] Studies have found that the occurrence of cell ferroptosis is ultimately caused by lipid peroxidation damage to the cell membrane, and scavenging lipid free radicals has been proven in a large number of in vitro and in vivo models to effectively block the occurrence of cell ferroptosis. The present invention conducts theoretical research on Liproxstatin-1 type ferroptosis inhibitors based on density functional theory, and finds that the aromatic amine group (-NH) group plays a role in scavenging lipid peroxyl free radicals in the structure. Based on reaction thermodynamic calculations and frontier orbital analysis, the structure-activity relationship of Liproxstatin-1 type inhibitors is established. The results show that the extensive conjugated system within the molecule is a necessary structure for this type of inhibitor to have high activity, among which the π-π* effect of the double bond-benzene ring significantly increases the conjugation effect of the molecule. It is well known that p-π* conjugation is another method to increase the conjugation of molecules. Therefore, the present invention combines quantitative calculations with experimental verification to discover that 3,4-dihydro-2H-benzo-[1,4]oxazine drugs may be used to treat related diseases.

[0024] In some embodiments, the disease associated with ferroptosis is selected from neurodegenerative diseases, brain trauma, stroke, cardiovascular disease, liver and kidney failure, and inflammation. By administering a safe and effective amount of the above-mentioned 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof to a target subject, a corresponding therapeutic effect can be achieved.

[0025] In some embodiments, the neurodegenerative disease is selected from Parkinson's disease and schizophrenia.

[0026] The beneficial effects of the present invention are:

[0027] (1) The effect is definite and is applicable to diseases related to ferroptosis, including brain trauma, stroke, cardiovascular disease, liver and kidney failure, inflammation and neurodegenerative diseases, such as Parkinson's disease and schizophrenia. Theoretical and experimental studies of the present invention have shown that 3,4-dihydro-2H-benzo-[1,4]oxazine compounds have good activity in scavenging membrane lipid free radicals and have a good therapeutic effect on diseases related to ferroptosis.

[0028] (2) The drug source is reliable, stable, and highly safe: the above compounds were all purchased from reagent companies, and the toxicology of 3,4-dihydro-2H-benzo-[1,4]oxazine compounds has been experimentally confirmed. Cell toxicity tests have confirmed that such drugs at therapeutic doses have no obvious toxicity when used to treat diseases related to ferroptosis. DETAILED DESCRIPTION

[0029] It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs.

[0030] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0031] As described in the background art, current small molecule ferroptosis inhibitors have the disadvantages of short half-life and high toxicity. Therefore, in the first aspect of the present invention, a novel structural entity is screened and obtained by combining theoretical calculation with experimental verification to obtain active compounds targeting cell ferroptosis. The active compounds are 3,4-dihydro-2H-benzo-[1,4]oxazine compounds listed in Table 1, and the mechanism of action of the active compounds is to block cell membrane lipid peroxyl radical damage.

[0032] Table 1: 3,4-Dihydro-2H-benzo-[1,4]oxazine compounds

[0033]

[0034] The present invention adopts a quantitative calculation method for screening. By optimizing bicyclic compounds containing p-π* conjugation and calculating the energy barrier of the reaction of such compounds with lipid peroxyl radicals, the energy barrier is sorted and verified by biological experiments. On this basis, biological verification of more candidate compounds is carried out, and finally the 12 active compounds listed in Table 1 are screened out.

[0035] According to a second aspect of the present invention, there is provided a use of the 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof in the preparation of a medicament for a disease associated with ferroptosis. Preferably, the disease associated with ferroptosis is selected from neurodegenerative diseases, brain trauma, stroke, cardiovascular disease, liver and kidney failure, and inflammation.

[0036] In another preferred embodiment, the neurodegenerative disease is selected from Parkinson's disease and schizophrenia.

[0037] According to a third aspect of the present invention, an in vitro method for blocking cell ferroptosis and preventing cell damage is provided, wherein a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof described in Table 1 is added to inhibit cell membrane lipid free radical damage.

[0038] According to a fourth aspect of the present invention, a method for treating nervous system diseases or liver and kidney failure is provided, comprising the steps of administering a safe and effective amount of one or more 3,4-dihydro-2H-benzo-[1,4]oxazine compounds or salts thereof to a subject in need.

[0039] As used herein, "pharmaceutically acceptable salts" refer to salts of compounds that, within the scope of reliable pharmaceutical evaluation, are suitable for contact with human or lower animal tissues without undue toxicity, irritation, or allergic reaction, possess a reasonable benefit / risk ratio, are generally water- or oil-soluble or dispersible, and are effective for their intended use. These salts include pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts, which are compatible with the intended use and the chemical properties of the 3,4-dihydro-2H-benzo-[1,4]oxazine compounds.

[0040] The technical solution of this application is described below through specific embodiments.

[0041] Example 1: Quantitative calculation of 3,4-dihydro-2H-benzo-[1,4]oxazine drugs:

[0042] The study found that conjugation is beneficial to the antioxidant activity of amine-containing compounds. Therefore, the inventors purchased amine-containing compounds with different p-π* conjugations from a reagent company. The compounds were structurally optimized using Gaussian software, and natural bonding orbital analysis of the optimized structures was performed using NBO6.0W. The results showed that the oxygen lone pair electrons had the strongest conjugation effect on the benzene ring, with E(2) values ​​of 27.05 and 28.18 Kcal / mol, respectively; significantly higher than the E(2) values ​​of sulfur (18.34 and 19.46 Kcal / mol, respectively). However, the carbon atom had no p-π* conjugation effect due to the lack of lone pairs of electrons. The thermodynamic parameters of the reactions of these compounds with lipid free radicals were calculated using Gaussian software. The results showed that the energy barrier required for the reaction of 3,4-dihydro-2H-benzo-[1,4]oxazine compounds with free radicals was the lowest, with values ​​of 10.85 and 11.19 Kcal / mol, respectively, significantly lower than those of other structures. It can be seen that there is an obvious correlation between the degree of p-π* conjugation of the molecule and the reaction energy barrier, that is, the higher the p-π* conjugation of the molecule, the easier it is for the compound to react with free radicals.

[0043] Table 2 Relationship between the conjugation of amino-containing compounds and their antioxidant activity

[0044]

[0045]

[0046] At the same time, the inventors conducted activity tests on the above compounds. The results showed that 3,4-dihydro-2H-benzo-[1,4]oxazine compounds had the best activity, and their half effective concentration (EC50) for anti-ferroptosis was 1.377mg / L. 50) were 215 and 250 nM, respectively, indicating 20 times the activity of 3,4-dihydro-2H-benzo[1,4]thiazine compounds, while tetrahydroquinoline compounds were inactive at the experimental concentrations. This suggests that the intramolecular p-π* conjugation strength of bicyclic amine antioxidants is positively correlated with their ferroptosis inhibitory activity, and that 3,4-dihydro-2H-benzo-[1,4]oxazine structures are novel ferroptosis inhibitors.

[0047] Example 2: In vitro anti-ferroptosis activity test and cytotoxicity test of target compounds

[0048] In vitro anti-ferroptosis activity test method:

[0049] Erastin was used to induce human fibrosarcoma cells HT-1080 to establish a cell ferroptosis model and to determine the inhibitory activity of the compounds on cell ferroptosis. Briefly, HT-1080 was treated with a lethal concentration of Erastin (10 μM) in the presence of different concentrations of compounds (1-12). Cell viability was detected by MTT assay after 48 hours of continuous culture. Six replicate wells were set up at the same concentration for each experiment and repeated three times independently. The effective concentration of the compound to inhibit ferroptosis (EC 50 ) was defined as the concentration at which the cell viability decreased by 50% compared with the control group. The activity results are shown in Table 3.

[0050] Cytotoxicity assay methods

[0051] HepG2 liver cancer cells were cultured in the presence of different concentrations of compounds for 48 h. Cell viability was determined by MTT assay. The cytotoxic concentration of the compound (IC 50 ) is the concentration of the target compound required to induce 50% cell death. The toxicity results are shown in Table 3.

[0052] Table 3 Ferroptosis inhibition activity and cytotoxicity results of 3,4-dihydro-2H-benzo-[1,4]oxazine drugs

[0053]

[0054]

[0055] EC 50 : The concentration of compound that protects 50% of cells from Erastin-induced ferroptosis.

[0056] IC 50 : The concentration of compound required to induce 50% cell death.

[0057] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent replacements for parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the scope of protection of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. Use of a 3,4-dihydro-2H-benzo-[1,4]oxazine compound or a salt thereof in the preparation of a cell ferroptosis inhibitor and / or a cell membrane lipid free radical damage inhibitor, characterized in that: The 3,4-dihydro-2H-benzo-[1,4]oxazine compound is 。 2. The use according to claim 1, characterized in that The inhibitor comprises: the compound according to claim 1 and auxiliary materials.

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