Application of indole alkaloid in preparation of ferroptosis inhibitor

Abstract

The invention relates to application of indole alkaloids in preparation of ferroptosis inhibitors. The indole alkaloid has a structure as shown in a formula (I): # imgabs0 #, wherein R1, R2 and R3 are independently H, C1-6 alkyl, C1-6 alkoxy or C1-6 carboxylic ester. The specific indole alkaloid disclosed by the invention has a remarkable inhibition effect on ferroptosis of neuronal cells, is low in cytotoxicity, and can be used for preparing medicines for treating and / or preventing neurodegenerative diseases and ischemic cerebral apoplexy caused by ferroptosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and more specifically, to the use of indole alkaloids in the preparation of ferroptosis inhibitors. Background Art

[0002] Iron is the most common transition metal in the brain and is involved in many key cellular functions, such as neurotransmitter synthesis (such as serotonin, norepinephrine, and dopamine), myelination, and neuronal development. Under normal circumstances, iron is essential for brain function, however, excessive accumulation of iron can lead to oxidative stress and lipid peroxidation, thereby damaging neurons. Excessive Fe 2+ It can react with hydrogen peroxide through the Fenton reaction to generate highly reactive hydroxyl radicals. These hydroxyl radicals react with polyunsaturated fatty acids, leading to non-enzymatic lipid peroxidation. Furthermore, iron accumulation activates iron-dependent metabolic enzymes such as lipoxygenase and cytochrome P450 oxidoreductase, further exacerbating redox homeostasis imbalance and lipid peroxidation. This oxidative damage cascade triggered by iron overload may ultimately activate a novel cell death program called ferroptosis.

[0003] As a typical example of iron-dependent cell death, ferroptosis is characterized by oxidative stress collapse caused by the abnormal accumulation of intracellular iron ions and lipid-induced reactive oxygen species. Its molecular mechanisms involve multiple pathological links, including impaired iron metabolism, glutathione (GSH) depletion, and inactivation of glutathione peroxidase 4 (GPx4). Notably, this type of ferroptosis is closely associated with neurodegenerative diseases and neurological disorders such as ischemic stroke, making ferroptosis inhibitors highly promising therapeutic targets.

[0004] In order to inhibit ferroptosis, researchers have developed a variety of ferroptosis inhibitors with different mechanisms of action, which have shown good therapeutic effects in various disease models. Existing ferroptosis inhibitors are mainly divided into several categories: (1) free radical trapping antioxidants (RTAs), such as Fer-1 and Lip-1, whose mechanism of inhibiting ferroptosis is to directly react with peroxyl radicals to block lipid peroxidation; (2) iron chelators, such as deferoxamine (DFO), whose mechanism of inhibiting ferroptosis is to chelate iron ions, reduce intracellular iron content, and prevent the Fenton reaction from forming reactive oxygen species (ROS); (3) lipoxygenase inhibitors, such as N-hydroxy-N-(1-benzothiophen-2-yl-ethyl) urea (Zileuton), whose mechanism of inhibiting ferroptosis is to inhibit lipoxygenases (LOXs); (4) ACSL4 inhibitors, such as pioglitazone, whose mechanism of inhibiting ferroptosis is to inhibit ACSL4 and thus reduce the level of lipid peroxidation substrates. However, the above-mentioned ferroptosis inhibitors have defects such as high toxicity, which limits their clinical application. Therefore, there is an urgent need to develop more ferroptosis inhibitors to meet clinical needs. Summary of the Invention

[0005] The primary objective of the present invention is to overcome the high toxicity of existing ferroptosis inhibitors and to provide the use of indole alkaloids in the preparation of ferroptosis inhibitors. These indole alkaloids have a significant inhibitory effect on neuronal cell ferroptosis and exhibit low cytotoxicity. They can be used to prepare drugs for treating and / or preventing neurodegenerative diseases and / or ischemic stroke caused by ferroptosis.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The use of indole alkaloids in the preparation of ferroptosis inhibitors, wherein the indole alkaloids have a structure as shown in formula (I):

[0008]

[0009] Wherein, R1, R2, and R3 are independently H, C 1～6 Alkyl, C 1～6 Alkoxy or C 1～6 Carboxylate.

[0010] The inventors of the present invention have discovered that specific indole alkaloids have a significant inhibitory effect on ferroptosis of neuronal cells and have low cytotoxicity, and can be used to prepare drugs for treating and / or preventing neurodegenerative diseases and / or ischemic stroke caused by ferroptosis.

[0011] Preferably, the R1 is C 1～6 Carboxylate (such as methyl formate, ethyl formate, propyl formate, butyl formate, pentyl formate, hexyl formate), wherein R2 is hydrogen or C 1～6 Alkoxy (such as methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy), wherein R3 is C 1～6 Alkyl (such as methyl, ethyl, propyl, butyl, pentyl, hexyl).

[0012] More preferably, the structure of the indole alkaloid is shown in one of the following formulas:

[0013]

[0014] Further preferably, the structure of the indole alkaloid is shown below:

[0015]

[0016] The inventors of the present invention have further discovered that the indole alkaloids of the present invention have the following structure: When the ferroptosis inhibitory activity on neuronal cells is stronger.

[0017] Preferably, the ferroptosis inhibitor is a drug for treating and / or preventing neurodegenerative diseases and / or ischemic stroke caused by ferroptosis.

[0018] More preferably, the neurodegenerative disease is at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease or amyotrophic lateral sclerosis.

[0019] More preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] More preferably, the excipient is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.

[0021] More preferably, the dosage form of the drug is injection, tablet, oral solution, granule or capsule.

[0022] Preferably, the ferroptosis inhibitor is an inhibitor of RSL3-induced neuronal cell ferroptosis.

[0023] Preferably, the indole alkaloids are deuterium or tritium isotope variants of indole alkaloids.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The specific indole alkaloids of the present invention have a significant inhibitory effect on neuronal cell ferroptosis and have low cytotoxicity, and can be used to prepare drugs for treating and / or preventing neurodegenerative diseases and ischemic stroke caused by ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the cytotoxicity experiment of the indole alkaloids of Example 1 and Example 2.

[0027] Figure 2 EC is the ferroptosis inhibition activity test of indole alkaloids in Example 1 50 curve.

[0028] Figure 3 EC of the ferroptosis inhibition activity test of indole alkaloids in Example 2 50 curve. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific embodiments, but the examples do not limit the present invention in any form. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present invention more thorough and comprehensive. It should also be understood that the present invention can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the present invention, and the equivalent forms obtained also fall within the scope of protection of the present invention. For example, the features described or described as part of one embodiment can be combined in another embodiment in a suitable manner to produce a new embodiment. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present invention. It should be understood that the present invention can be implemented without one or more of these details. Unless otherwise stated, the raw materials and reagents used in the examples of the present invention are conventionally purchased raw materials and reagents. The mass or weight of the relevant components mentioned in the description of the examples of the present invention can not only refer to the specific content of each component, but also represent the proportional relationship of mass or weight between the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the description of the examples of the present invention, it is within the scope disclosed in the description of the examples of the present invention.

[0030] Example 1

[0031] This example provides the use of an indole alkaloid in the preparation of a ferroptosis inhibitor. The chemical name of the indole alkaloid in this example is aricin (CAS 482-91-7), and its structure is as follows:

[0032]

[0033] 1. Culture of HT22 hippocampal neurons

[0034] HT22 cells (a mouse hippocampal neuronal cell line) were cultured in DMEM high-glucose medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a cell culture incubator containing 5% carbon dioxide. In all experiments, the number of cell passages did not exceed 15.

[0035] 2. Cytotoxicity assay

[0036] (1) Cell inoculation and grouping

[0037] Well-growing HT22 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3,000 cells / well. An equal volume of cell-free culture medium was added to the blank wells and cultured in a CO2 incubator for 24 hours. The cells were divided into a control group and a treatment group, with three replicates per group.

[0038] (2) Compound treatment

[0039] Drug-administered group: After the cells were cultured in an incubator until adherent, different concentrations (10 μM, 20 μM, 30 μM, 40 μM) of the indole alkaloids of this example were added and incubated for 24 h.

[0040] Control group: The treatment method of the control group was basically the same as that of the drug-treated group, except that the indole alkaloids of this example were not added, and an equal amount of DMSO was added and incubated for 24 hours.

[0041] (3) Test methods and result processing

[0042] After the compound intervention, 15 μL of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution diluted with PBS buffer to a concentration of 5 mg / mL was added to each well and cultured in a cell culture incubator at 37°C for 4 hours. The culture medium was discarded, and 100 μL of analytical grade DMSO was added to each well and shaken on a shaker for 10 minutes. Finally, the absorbance (OD value) was measured at a wavelength of 492 nm using a microplate reader. The cell viability was calculated according to the following formula: Cell viability (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%, each experiment was repeated 3 times independently. 样品 is the OD value of the drug-treated group, OD 对照 is the OD value of the control group, OD 空白 Calculate the OD value of only 100 μl of analytical pure DMSO added to the wells.

[0043] 3. Ferroptosis Inhibitory Activity Experiment

[0044] (1) Cell inoculation and grouping

[0045] Well-growing HT22 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3,000 cells / well. An equal volume of cell-free culture medium was added to the blank wells and cultured in a CO2 incubator for 24 hours. The cells were divided into a control group and a treatment group, with three replicates per group.

[0046] (2) Compound treatment

[0047] Drug-treated group: After the cells were cultured in an incubator until they adhered to the wall, ferroptosis inducer (1 μM RSL3) and indole alkaloids at different concentrations (0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 20 μM) were added and incubated for 24 h.

[0048] Control group: The treatment method of the control group was basically the same as that of the drug-treated group, except that the ferroptosis inducer and the indole alkaloids of this example were not added, and an equal amount of DMSO was added and incubated for 24 hours.

[0049] (3) Test methods and result processing

[0050] After the compound intervention, 15 μL of 5 mg / mL DMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution diluted with PBS buffer was added to each well and cultured in a cell culture incubator at 37°C for 4 hours. The culture medium was discarded, and 100 μL of analytical grade DMSO was added to each well and shaken on a shaker for 10 minutes. Finally, the absorbance (OD value) was measured at a wavelength of 492 nm using a microplate reader. The cell viability was calculated according to the following formula: Cell viability (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%, and then GraphPad Prism 8 software was used to fit the curve of cell viability and compound concentration to obtain the EC50 value of the compound. Each experiment was repeated 3 times independently. 样品 is the OD value of the drug-treated group, OD 对照 is the OD value of the control group, OD 空白 EC50 is the concentration for 50% effect, which is the concentration that can induce 50% of the individual effects.

[0051] Example 2

[0052] This example provides the use of an indole alkaloid in the preparation of a ferroptosis inhibitor. The indole alkaloid in this example is acumin (CSA No. 642-17-1), and its structure is as follows:

[0053]

[0054] The culturing method and cytotoxicity test method of HT22 hippocampal neuronal cells in this example are the same as those in Example 1, except that the indole alkaloids in Example 1 are replaced by the indole alkaloids in this example.

[0055] The experimental method for ferroptosis inhibition activity in this example is as follows:

[0056] (1) Cell inoculation and grouping

[0057] Well-growing HT22 cells in the logarithmic growth phase were seeded into 96-well plates at a density of 3,000 cells / well. An equal volume of cell-free culture medium was added to the blank wells and cultured in a CO2 incubator for 24 hours. The cells were divided into a control group and a treatment group, with three replicates per group.

[0058] (2) Compound treatment

[0059] Drug-treated group: After the cells were cultured in an incubator until adherent, a ferroptosis inducer (1 μM RSL3) and different concentrations (0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, 20 μM, 30 μM and 40 μM) of the indole alkaloids of this example were added and incubated for 24 h.

[0060] Control group: The treatment method of the control group was basically the same as that of the drug-treated group, except that the ferroptosis inducer and the indole alkaloids of this example were not added, and an equal amount of DMSO was added and incubated for 24 hours.

[0061] (3) Test methods and result processing

[0062] After the compound intervention, 15 μL of 5 mg / mL DMTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] solution diluted with PBS buffer was added to each well and cultured in a cell culture incubator at 37°C for 4 hours. The culture medium was discarded, and 100 μL of analytical grade DMSO was added to each well and shaken on a shaker for 10 minutes. Finally, the absorbance (OD value) was measured at a wavelength of 492 nm using a microplate reader. The cell viability was calculated according to the following formula: Cell viability (%) = (OD 样品 -OD 空白 ) / (OD 对照 -OD 空白 )×100%, and then GraphPad Prism 8 software was used to fit the curve of cell viability and compound concentration to obtain the EC50 value of the compound. Each experiment was repeated 3 times independently. 样品 is the OD value of the drug-treated group, OD 对照 is the OD value of the control group, OD 空白 EC50 is the concentration for 50% effect, which is the concentration that can induce 50% of the individual effects.

[0063] Example 3

[0064] This embodiment provides an injection for inhibiting ferroptosis, which is prepared by the following process: mixing the indole alkaloid of Example 1 with water for injection, fine filtering, encapsulating, and sterilizing to prepare the injection.

[0065] Example 4

[0066] This embodiment provides a tablet for inhibiting ferroptosis, which is prepared by the following process: mixing the indole alkaloid of Example 2 with an excipient (such as starch slurry), and then pressing the mixture into tablets.

[0067] Test results

[0068] Figure 1 These are the test data of the cytotoxicity experiments of indole alkaloids in Examples 1 and 2. Figures 2-3 The EC values of the ferroptosis inhibition activity experiments of the indole alkaloids in Examples 1 and 2 are respectively 50 Graph. Figure 1 It can be seen that the indole alkaloids of Examples 1 to 2 have almost no toxicity to neuronal cells at 10 to 50 μM, which indicates that the specific indole alkaloids of the present invention have low toxicity to neuronal cells. Figure 2 and Figure 3 It can be seen that the ferroptosis inhibitory activity of the indole alkaloids of Examples 1 and 2 is EC 50 The concentrations of the indole alkaloids were 3.51 μM and 20.01 μM, respectively, which indicated that the specific indole alkaloids of the present invention had a significant inhibitory effect on RSL3-induced neuronal cell ferroptosis, had good neuroprotective activity, and could be used to prepare ferroptosis inhibitors.

[0069] Obviously, the above embodiments of the present invention are merely examples for the purpose of clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims of the present invention.

Claims

1. The use of indole alkaloids in the preparation of ferroptosis inhibitors, characterized in that: The indole alkaloid has a structure as shown in formula (I): Wherein, R1, R2, and R3 are independently H, C 1～6 Alkyl, C 1～6 Alkoxy or C 1～6 Carboxylate.

2. The use according to claim 1, characterized in that The R1 is C 1～6 Carboxylate, wherein R2 is hydrogen or C 1～6 Alkoxy, wherein R3 is C 1～6 alkyl.

3. The use according to claim 2, characterized in that The structure of the indole alkaloids is shown in one of the following formulas:

4. The use according to claim 1, characterized in that The ferroptosis inhibitor is a drug for treating and / or preventing neurodegenerative diseases and / or ischemic stroke caused by ferroptosis.

5. The use according to claim 4, characterized in that The neurodegenerative disease is at least one of Parkinson's disease, Alzheimer's disease, Huntington's disease or amyotrophic lateral sclerosis.

6. The use according to claim 4, characterized in that The drug also includes pharmaceutically acceptable excipients.

7. The use according to claim 6, characterized in that The auxiliary material is at least one of a lubricant, a filler, a binder, a disintegrant, a surfactant, an antioxidant or a pH regulator.

8. The use according to claim 4, characterized in that The dosage form of the medicine is injection, tablet, oral solution, granule or capsule.

9. The use according to claim 1, characterized in that The ferroptosis inhibitor is an inhibitor of RSL3-induced neuronal cell ferroptosis.

10. The use according to claim 1, characterized in that The indole alkaloids are deuterium and tritium isotope variants of indole alkaloids.

Images