Endoplasmic reticulum targeting ferroptosis inhibitor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicines, in particular to an endoplasmic reticulum targeting ferroptosis inhibitor as well as a preparation method and application thereof. Specifically, by targeting the endoplasmic reticulum which is a key starting part of ferroptosis, the chain reaction of lipid peroxidation of the endoplasmic reticulum is blocked, the levels of alanine transaminase (ALT) and aspartate transaminase (AST) in serum are remarkably reduced, infiltration of inflammatory cells to the liver is reduced, and liver tissue damage is repaired. The innovation point of the invention lies in that the core effect of the endoplasmic reticulum in a ferroptosis mechanism is determined for the first time, and a new strategy for intervening ferroptosis by targeting the endoplasmic reticulum is provided, so that a theoretical basis is provided for treating acute drug-induced liver injury by taking ferroptosis as a target spot; and meanwhile, a new direction and scientific support are provided for clinical application of APAP and ferroptosis inhibitor combined medication, and remarkable technical innovation and clinical value are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an endoplasmic reticulum-targeted ferroptosis inhibitor and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not be necessarily 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] Acute liver injury (ALI) is a common acute liver disease with complex etiologies, including drug toxicity (such as acetaminophen overdose), viral infection, ischemia-reperfusion injury, metabolic diseases, and immune-mediated pathological processes. Its clinical manifestations include large-area necrosis of hepatocytes, aggravated inflammatory response, and a sharp decline in liver function. In severe cases, it can develop into acute liver failure (ALF).

[0004] At present, the clinical treatments for acute liver injury mainly include the following categories: ① symptomatic supportive treatment: use of liver-protecting drugs (such as reduced glutathione, adenosine methionine, etc.) to reduce the degree of liver cell damage; ② anti-inflammatory treatment: alleviate the disease process by inhibiting excessive inflammatory response; ③ liver transplantation: used for severe patients who are difficult to control with drug treatment. However, the shortage of donor resources and high surgical costs limit the popularity of liver transplantation. Although the above treatment methods have delayed the progression of the disease to a certain extent, the efficacy is limited and there are many side effects. There is currently no specific therapeutic drug for acute liver injury. Therefore, exploring new molecular mechanisms of acute liver injury and developing precision targeted drugs have become medical problems that need to be solved urgently.

[0005] Ferroptosis is a new type of programmed cell death that is different from apoptosis, necrosis and autophagy in terms of morphology, biochemistry and genetics. Because this process depends on the presence of iron ions, it is called ferroptosis. Its mechanism of occurrence is: the balance between the generation and degradation of reactive oxygen species in intracellular membrane lipids is unbalanced, and cells undergo iron-dependent, oxidative, non-apoptotic programmed cell death. Typical characteristics are: mitochondrial size decreases, double membrane density increases, and at the same time, there is an increase in reactive oxygen free radicals in cell membrane lipids.

[0006] The core mechanism of ferroptosis includes lipid peroxidation catalyzed by iron ions and the failure of antioxidant defense systems (such as glutathione peroxidase 4, GPX4). This mode of cell death is closely related to cellular metabolic disorders, inflammation-mediated oxidative stress and hepatocyte necrosis in acute liver injury. However, although ferroptosis has been widely reported as a target for acute liver injury, there are still technical bottlenecks in how to achieve precise regulation of the ferroptosis process.

[0007] Studies have shown that the endoplasmic reticulum (ER) is a key regulatory node for ferroptosis and the starting site of lipid peroxidation. The importance of the endoplasmic reticulum in lipid metabolism and calcium homeostasis makes it the main site for lipid peroxidation accumulation. During the induction of ferroptosis, polyunsaturated fatty acids in the endoplasmic reticulum membrane are easily oxidized to generate lipid peroxidation products (such as malondialdehyde and 4-hydroxynonenal). Studies have further pointed out that the initiation of ferroptosis usually starts with lipid peroxidation in the endoplasmic reticulum membrane and then extends to other cell membranes such as mitochondrial membrane and plasma membrane. The dynamic process of lipid peroxidation extending from the endoplasmic reticulum to the plasma membrane is considered to be an important stage in the process of ferroptosis. Studies have found that the occurrence of ferroptosis can be effectively inhibited by blocking endoplasmic reticulum lipid peroxidation.

[0008] Although existing inhibitors of ferroptosis (such as Ferrostatin-1 and Liproxstatin-1) have shown certain therapeutic potential, their mechanism of action is relatively generalized and lacks specific targeted regulation of the endoplasmic reticulum, the starting point of ferroptosis. In addition, existing drugs mostly focus on global antioxidant effects, which may interfere with normal cell metabolic functions and increase the risk of side effects. Summary of the invention

[0009] In order to overcome the above problems, the present invention provides a ferroptosis inhibitor targeting the endoplasmic reticulum and a preparation method and application thereof.

[0010] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0011] The first aspect of the present invention provides a compound having a chemical structure as shown in the following formula (I) or a stereoisomer thereof or a physiologically acceptable salt thereof:

[0012]

[0013] The second aspect of the present invention provides a method for preparing the compound of the first aspect, comprising the following steps:

[0014] In an alkaline environment, N-(2-chloroethyl)-4-methylbenzenesulfonamide reacts with 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole to synthesize a compound represented by formula (I).

[0015] The third aspect of the present invention provides the use of the compound described in the first aspect in the preparation of a ferroptosis inhibitor.

[0016] The fourth aspect of the present invention provides the use of the compound described in the first aspect in the preparation of a drug for preventing, improving or treating acute drug-induced liver injury.

[0017] The fifth aspect of the present invention provides use of the compound described in the first aspect in the preparation of an endoplasmic reticulum lipid oxidation inhibitor.

[0018] The sixth aspect of the present invention provides a pharmaceutical preparation, comprising the compound described in the first aspect.

[0019] The seventh aspect of the present invention provides a pharmaceutical composition, comprising the compound described in the first aspect.

[0020] The beneficial effects of the present invention are:

[0021] The present invention relates to the field of biomedicine technology, and specifically to a ferroptosis inhibitor targeting the endoplasmic reticulum, and a preparation method and application thereof. Specifically, the present invention blocks the chain reaction of lipid peroxidation of the endoplasmic reticulum membrane by targeting the endoplasmic reticulum, a key starting site of ferroptosis, significantly reduces the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, reduces the infiltration of inflammatory cells into the liver, and repairs liver tissue damage. The innovation of the present invention is to clarify for the first time the core role of the endoplasmic reticulum in the ferroptosis mechanism, and propose a new strategy for intervening ferroptosis by targeting the endoplasmic reticulum, which provides a theoretical basis for the treatment of acute drug-induced liver injury with ferroptosis as the target, and provides a new direction and scientific support for the clinical application of APAP combined with ferroptosis inhibitors, which has significant technical innovation and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 The compound represented by formula (I) has a significant inhibitory effect on cell ferroptosis induced by 6 ferroptosis inducers;

[0024] Figure 2 The compound represented by formula (I) targets and inhibits RSL3-induced endoplasmic reticulum lipid peroxidation;

[0025] Figure 3The liver damage of mice treated with the compound of formula (I); wherein A is the result of biochemical detection of aspartate aminotransferase ALT, and B is the result of biochemical detection of alanine aminotransferase AST;

[0026] Figure 4 This is the liver damage of mice treated with the compound represented by formula (Ⅰ) (HE staining). DETAILED DESCRIPTION

[0027] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0028] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. 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 "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0029] A first typical embodiment of the present invention provides a compound having a chemical structure as shown in the following formula (I) or a stereoisomer thereof or a physiologically acceptable salt thereof:

[0030]

[0031] Physiologically acceptable salts refer to organic and inorganic salts of the compound represented by formula (I). Physiologically acceptable salts are well known to those skilled in the art. Physiologically acceptable salts include, but are not limited to, inorganic salts such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, etc., and organic salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, etc., or salts obtained by other methods described in the literature, such as ion exchange methods.

[0032] A second typical embodiment of the present invention provides a method for preparing the compound of the first aspect, comprising the following steps:

[0033] In an alkaline environment, N-(2-chloroethyl)-4-methylbenzenesulfonamide reacts with 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole to synthesize a compound represented by formula (I).

[0034] In one or more embodiments, in an alkaline environment, N-(2-chloroethyl)-4-methylbenzenesulfonamide and 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole are stirred in an organic solvent to react to synthesize the compound represented by formula (I).

[0035] Preferably, the molar ratio of N-(2-chloroethyl)-4-methylbenzenesulfonamide to 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole is (0.9-1.2):1, preferably 1:1.

[0036] Preferably, the organic solvent is an alcohol solvent, and the alcohol solvent is preferably methanol.

[0037] Preferably, the concentration of N-(2-chloroethyl)-4-methylbenzenesulfonamide in the organic solvent is 0.18-0.24 mol / L, preferably 0.2 mol / L.

[0038] A third typical embodiment of the present invention provides use of the compound described in the first aspect in the preparation of a ferroptosis inhibitor.

[0039] A fourth typical embodiment of the present invention provides use of the compound described in the first aspect in the preparation of a drug for preventing, improving or treating acute drug-induced liver injury.

[0040] In one or more embodiments, the acute drug-induced liver injury is acetaminophen-induced acute liver injury.

[0041] A fifth typical embodiment of the present invention provides use of the compound described in the first aspect in the preparation of an endoplasmic reticulum lipid oxidation inhibitor.

[0042] Studies have shown that the compound represented by formula (I) can target endoplasmic reticulum membrane lipids, block the extension of free radicals on lipid chains, and reduce the damage of free radicals to the endoplasmic reticulum membrane and cell membrane, thereby effectively preventing the occurrence of ferroptosis, protecting liver cells and repairing acute liver damage caused by ferroptosis.

[0043] A sixth typical embodiment of the present invention provides a pharmaceutical preparation comprising the compound described in the first aspect.

[0044] A seventh typical embodiment of the present invention provides a pharmaceutical composition comprising the compound described in the first aspect.

[0045] In the above-mentioned pharmaceutical composition, the dosage of the compound can be routinely adjusted according to factors such as the purpose of drug administration, the subject's condition, etc. In the above-mentioned pharmaceutical composition, the dosage of the above-mentioned compound should be an effective dosage, and the pharmaceutical composition adopts a dosage form that is easy to accurately administer.

[0046] When the pharmaceutical composition is used to prepare a pharmaceutical preparation for in vivo administration, the pharmaceutical preparation should be sterile, and methods for achieving sterility of the pharmaceutical preparation should be known to those skilled in the art, for example, it can be achieved by filtering through a sterile filter membrane, etc. Those skilled in the art can also select a suitable pharmaceutically acceptable carrier according to the desired dosage form of the pharmaceutical composition to prepare it into different dosage forms, such as a dosage form for gastrointestinal administration or a dosage form for parenteral administration.

[0047] Preferably, the above-mentioned dosage forms for administration via the gastrointestinal tract include powders, tablets, granules, capsules, sustained-release preparations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, and the like.

[0048] Preferably, parenteral dosage forms include injection dosage forms (e.g., injections, including intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, intracavitary injections, and other injections); respiratory tract dosage forms (e.g., sprays, aerosols, powder aerosols, etc.); skin dosage forms (e.g., external solutions, lotions, liniments, ointments, plasters, pastes, patches, etc.); mucosal dosage forms (e.g., eye drops, nasal drops, eye ointments, gargles, sublingual tablets, adhesive tablets, patches, etc.); cavity dosage forms (e.g., suppositories, aerosols, effervescent tablets, drops, pills, etc., used in the rectum, vagina, urethra, nasal cavity, ear canal, etc.).

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0050] N-(2-chloroethyl)-4-methylbenzenesulfonamide was purchased from Shanghai Bid Pharmaceutical Technology Co., Ltd.; Ferrostatin-1, an inhibitor of ferroptosis, Erastin, RSL3, ML210, FIN56, DL-buthionine-(S,R)sulfoximine (BSO), and sulfasalazine were purchased from Cayman Company and dissolved in sterile dimethyl sulfoxide (DMSO) to prepare the required concentration. Fluorescent probe C11-BODIPY 581 / 591 ER-Tracker Blue-White was purchased from Invitrogen.

[0051] HT-1080 human fibrosarcoma cell line culture conditions: DMEM high glucose medium (GIBCO) containing 10% FBS (GIBCO), 37°C, 5% CO 2 Saturated humidity incubator.

[0052] The statistical analysis was performed using R software, and the experimental data were expressed as Mean ± SEM. Tukey's test (ANOVA) was used for intergroup comparisons in cell and animal experiments, and Student's t-test was used for comparisons between two groups. P < 0.05 was considered statistically significant, and different letters indicate P < 0.05.

[0053] Example 1

[0054] At room temperature, N-(2-chloroethyl)-4-methylbenzenesulfonamide (233.7 mg, 1 mmol) was added to a solution of 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole (209 mg, 1 mmol) and KOH (56 mg, 1 mmol) in methanol (5 mL), and the mixture was stirred at room temperature for 4 hours. After removing the solvent, the crude product was purified by flash column chromatography to obtain 361 mg of the compound shown in formula (I) with a yield of 87.3% as a white solid.

[0055] Wherein, the structural formula of N-(2-chloroethyl)-4-methylbenzenesulfonamide is as follows:

[0056]

[0057] The structural formula of 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole is as follows:

[0058]

[0059] 1 H NMR(500MHz,Chloroform-d)δ9.46(s,1H),7.86(dd,J=9.9,1.1Hz,1H),7.75–7.68(m,2H),7.34–7.28(m,2H),7.24(t,J=5.8Hz,1 H),7.12–7.04(m,1H),6.86(dd,J=8.3,1.2Hz,1H),5.59(s,2H),3.42(t,J=5.0Hz,2H),3.22(dt,J=5.7,5.0Hz,2H),2.41(s,3H).

[0060] 13 C NMR (100MHz, DMSO-d6) δ162.36,160.60,144.99,143.79,139.03,136.59,129.80,127.22,124.13,119.85,118.32,115.97,43.96,29.67,21.54.

[0061] HRMS(ESI)m / z:calcd for C 15 H 13 N 3 O 2 S:[M+H]406.4806; found:406.4789.

[0062] HPLC purity = 96.3%.

[0063] Example 2

[0064] The broad-spectrum anti-ferroptosis effect of the compound represented by formula (I):

[0065] After HT-1080 human fibrosarcoma cells adhered to the wall, DMSO, six ferroptosis inducers, and six ferroptosis inducers + the compound shown in formula (I) (1 μM) were added to the culture medium. After 24 hours of treatment, cell viability was detected by CCK8. The experiment was repeated 3 times. The six inducers were RSL3, Erastin, sulfasalazine, ML210, FIN56, and BSO. The final concentrations used in the experiment were 3 μM, 10 μM, 250 μM, 90 μM, 8 μM, and 800 μM, respectively. Cell viability assay (CCK8): HT-1080 cells treated with drugs for 24 hours were tested for cell viability by CCK8 colorimetry. The CCK8 kit was purchased from Beijing Solebow Technology Co., Ltd.

[0066] The experimental results showed that compared with the control group, the six ferroptosis inducers could significantly kill cells; and the compound represented by formula (I) could significantly inhibit the ferroptosis of cells induced by the six different inducers (p<0.01). Figure 1 It is shown that the compound shown in formula (I) has a broad-spectrum anti-ferroptosis effect and is a promising new compound targeting ferroptosis.

[0067] Example 3

[0068] The compound represented by formula (I) targets and inhibits RSL3-induced endoplasmic reticulum lipid peroxidation:

[0069] After HT-1080 human fibrosarcoma cells adhered to the wall, DMSO, RSL3 (3 μM, final concentration), RSL3 (3 μM) + the compound shown in formula (I) (1 μM), and RSL3 (3 μM) + Ferrostatin-1 (1 μM) were added to the culture medium. After 12 hours of drug treatment, HT-1080 cells were digested with trypsin to form a single cell suspension. C11-BODIPY 581 / 591(1 μM), ER-TrackerBlue-White (1 μM), incubated at room temperature in the dark for 5 min, observed continuously under a confocal laser scanning microscope, and photographed for analysis.

[0070] ER-Tracker Blue-White probe can locate the location of the endoplasmic reticulum, C11-BODIPY 581 / 591 The level of lipid peroxidation can be measured. The experimental results showed that after the cells were induced by RSL3, obvious lipid peroxidation first appeared on the endoplasmic reticulum membrane, and then the lipid peroxidation level on the mitochondria and cell membrane also increased, and finally led to the occurrence of cell ferroptosis, which is consistent with the literature reports. However, the compound treatment group ( Figure 2 The lipid peroxidation level of the endoplasmic reticulum membrane did not increase, and the lipid peroxidation level of the mitochondrial membrane and cell membrane did not increase. This shows that the compound shown in formula (I) targets the endoplasmic reticulum and effectively removes endoplasmic reticulum lipid free radicals, thereby preventing the occurrence of cell ferroptosis. C11 BODIPY 581 / 591 Correlation of oxidation signal with ERTracker Blue-White (expressed as Manders coefficient). Data are presented as mean ± SEM, and each point represents an image containing multiple cells.

[0071] Example 4

[0072] The compound represented by formula (I) has an alleviating effect on acute liver injury induced by acetaminophen (APAP) in mice:

[0073] Animal grouping: 40 C57BL / 6 mice aged 8 to 10 weeks were selected and randomly divided into the following 4 groups, half of which were male and half were female. They were randomly divided into a control group; an APAP group (350 mg / kg); a low-dose group of the compound represented by formula (I) (5 mg / kg) + APAP group (350 mg / kg), and a high-dose group of the compound represented by formula (I) (10 mg / kg) + APAP group (350 mg / kg).

[0074] Animal treatment: The acute liver injury model in mice was induced by APAP (350 mg / kg), which was dissolved in physiological saline. The compound represented by formula (I) was dissolved in DMSO and injected intraperitoneally 1 hour before the injection of APAP. 9 hours after the intraperitoneal injection of APAP, blood samples were collected from the retro-orbital venous plexus for liver function tests. The mice were then dissected and the liver tissue was immediately removed for subsequent routine HE staining.

[0075] Serum and liver were obtained for analysis.

[0076] Liver function test: Collect the whole blood of mice in each group, let it stand for 1 to 2 hours, and directly centrifuge it at low speed to separate the serum for use. Dilute the separated serum sample 5 times, and operate the samples according to the product instructions of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits. The aspartate aminotransferase (AST) and alanine aminotransferase (ALT) detection kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.

[0077] The experimental results found that: Figure 3 , Figure 4 The results showed that compared with the APAP treatment group, the necrotic area in the compound treatment group shown in formula (I) was significantly reduced, and the ALT and AST levels in the serum of the compound treatment group shown in formula (I) were significantly reduced ( Figure 3 ), therefore, the compound represented by formula (I) has an alleviating effect on APAP-induced acute drug-induced liver injury in mice ( Figure 4 ).

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A compound, characterized in that The compound is a chemical structure represented by the following formula (I) or a stereoisomer thereof or a physiologically acceptable salt thereof:

2. The method for preparing the compound according to claim 1, characterized in that: The steps include: In an alkaline environment, N-(2-chloroethyl)-4-methylbenzenesulfonamide reacts with 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole to synthesize a compound represented by formula (I).

3. The preparation method according to claim 2, characterized in that: In an alkaline environment, N-(2-chloroethyl)-4-methylbenzenesulfonamide and 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole are stirred and reacted in an organic solvent to synthesize a compound represented by formula (I).

4. The preparation method according to claim 3, characterized in that: The molar ratio of N-(2-chloroethyl)-4-methylbenzenesulfonamide to 2-(2-amino-3-hydroxyphenyl)-5-mercapto-1,3,4-oxadiazole is (0.9-1.2): 1 The organic solvent is an alcohol solvent; The concentration of the N-(2-chloroethyl)-4-methylbenzenesulfonamide in the organic solvent is 0.18-0.24 mol / L.

5. Use of the compound according to claim 1 in the preparation of a ferroptosis inhibitor.

6. Use of the compound according to claim 1 in the preparation of a drug for preventing, improving or treating acute drug-induced liver injury.

7. The use according to claim 6, characterized in that The acute drug-induced liver injury is acute liver injury induced by acetaminophen.

8. Use of the compound according to claim 1 in the preparation of an endoplasmic reticulum lipid oxidation inhibitor.

9. A pharmaceutical preparation, characterized in that Comprising the compound described in claim 1.

10. A pharmaceutical composition, characterized in that Comprising the compound described in claim 1.

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