Application of tetra-substituted pyrrole derivative in preparation of product for treating neurodegenerative diseases

Abstract

The invention relates to the technical field of medicines, and discloses an application of a tetra-substituted pyrrole derivative in preparation of a product for treating neurodegenerative diseases. According to the tetra-substituted pyrrole derivative or the pharmaceutically acceptable application of the tetra-substituted pyrrole derivative in preparation of products for treating neurodegenerative diseases, the tetra-substituted pyrrole derivative is high in cell safety, and the neuroprotective effect on glutamic acid injury is far better than that of atrasentan and clinical standard drugs edaravone and riluzole; not only can the nucleoplasm dislocation of the TDP-43 be effectively corrected, but also the capability of removing insoluble pathological aggregates, which is not possessed by the atrasentan, is achieved; the co-localization of the TDP-43 and the stress particle protein G3BP1 can be more effectively inhibited, and the formation of pathological particles is intervened from the source; on the whole animal model, the tetra-substituted pyrrole derivative successfully converts the advantage of the cellular level into remarkable survival benefit, and the life of the disease model fruit flies is remarkably prolonged.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of a tetrasubstituted pyrrole derivative in the preparation of products for treating neurodegenerative diseases. Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease influenced by multiple pathological mechanisms, including glutamate-induced excitotoxicity, cell death, mitochondrial dysfunction, oxidative stress, protein misfolding, neuroinflammation, altered RNA metabolism, and neurofilament abnormalities. It is primarily characterized by the degeneration and loss of upper and lower motor neurons in the brain and spinal cord. ALS is an orphan disease; in most countries, its prevalence is approximately 5 cases per 100,000 people, and about 80% of patients die within 5 years of diagnosis, usually from respiratory failure.

[0003] There is currently no cure for amyotrophic lateral sclerosis (ALS). Treatment can only slow disease progression, prolong patient survival, and improve quality of life. Currently, the FDA has only approved three drugs for treating ALS: riluzole (RL), edaravone (Ed), and AMX0035. In addition, many small molecules are in clinical trials, targeting various hypothetical mechanisms, including autophagy, glutamate-mediated excitotoxicity, oxidative stress, and neuroinflammation. Therefore, the development of drugs for neurodegenerative diseases is of great significance for the prevention and treatment of these diseases in clinical practice.

[0004] TDP-43 is an RNA-binding protein primarily located in the cell nucleus, shuttling between the cytoplasm and the nucleus. In neurons and glial cells of over 90% of amyotrophic lateral sclerosis (ALS) and 45% of frontotemporal dementia (FTD) cases, TDP-43 forms abnormal cytoplasmic aggregates and is widely recognized as a pathological marker of most forms of ALS and the most common subtype of FTD. Furthermore, the mislocalization of TDP-43 in the cytoplasm also represents a key pathological feature of other major neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease.

[0005] Vimentin is an important member of the cytoskeleton, widely distributed in various non-epithelial cells. It is a key component in maintaining cell structure and tissue integrity, and plays an important role in cell movement, cell signal transduction, and cell cycle regulation. CN 115990159 A discloses atrasentan, a selective endothelin A receptor antagonist, which can bind to vimentin protein, inhibit the formation of the vimentin-Beclin1-14-3-3 ternary complex, promote neuronal autophagy, help clear mislocalized TDP-43 protein in neurons, and improve the locomotion ability of Drosophila overexpressing TDP-43 protein, reduce the formation of black spots in the eyes of Drosophila, and increase the survival time of Drosophila under high temperature and normal conditions. However, atrasentan has three chiral centers, resulting in high synthesis cost and difficult processing, and its anti-neurodegenerative activity still has room for improvement. Summary of the Invention

[0006] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide the use of a tetrasubstituted pyrrole derivative of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of products for treating neurodegenerative diseases. The specific preparation method of the tetrasubstituted pyrrole derivative of formula (I) or formula (II) is fully described in Chinese Patent Application No. 2025111860118, the entire contents of which are incorporated herein by reference.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] This invention provides the use of tetrasubstituted pyrrole derivatives of formula (I) or formula (II) or pharmaceutically acceptable salts thereof in the preparation of products for treating neurodegenerative diseases:

[0009]

[0010] In equations (Ⅰ) and (Ⅱ),

[0011] R 1 Selected independently from hydrogen and C 1-4 The alkyl group, benzoyl group or its derivative, and -CT are selected from the group consisting of the carboxyl group, ester group, substituted or unsubstituted amide group;

[0012] R 2 Each is independently selected from hydrogen, hydroxyl, and C. 1-4 Alkyl, methoxy, halogen, amino, or nitro groups;

[0013] R 3Each group is independently selected from ester, carboxyl, or substituted amide groups;

[0014] R 4 Each group is independently selected from hydrogen, hydroxyl, methoxy, halogen, oxygen-containing heterocycle, amino, nitro, or cyano.

[0015] In some embodiments of the present invention, in the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 1 Each is independently selected from hydrogen, methyl, benzoyl, 4-methylbenzoyl, -C-COOR 5 or -C-CONR 6 R 7 , where R 5 Selected from hydrogen, straight-chain or branched alkyl groups; R 6 and R 7 Selected independently from hydrogen and C 2-8 Straight-chain alkyl, -C2H4- heterocyclic or dimethylaminoalkyl.

[0016] In some preferred embodiments of the present invention, in the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 1 Each is independently selected from hydrogen, methyl, benzoyl, 4-methylbenzoyl, -C-COOR 5 OR-C-CONR 6 R 7 , where R 5 Selected from hydrogen or -CH3; R 6 and R 7 Selected independently from hydrogen and C 2-8 Straight-chain alkyl, -C2H4-nitrogen five-membered heterocycle, -C2H4-nitrogen six-membered heterocycle, -C2H4-nitrogen-oxygen six-membered heterocycle or dimethylaminoalkyl.

[0017] In some embodiments of the present invention, in the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 2 Each is independently selected from hydrogen, hydroxyl, methyl, methoxy, or halogen.

[0018] In some embodiments of the present invention, in the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 3 Selected independently from -COOR 8 or -CONR 9 R 10 , where R 8 Selected from hydrogen, straight-chain or branched alkyl groups; R 9 and R 10 Each is independently selected from hydrogen, propargyl, or C. 1-4 Straight-chain alkyl groups.

[0019] In some embodiments of the present invention, in the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 4 Each is independently selected from hydrogen, hydroxyl, methoxy, halogen, or oxygen-containing heterocycles.

[0020] In some embodiments of the present invention, the tetrasubstituted pyrrole derivative is selected from at least one of formulas 1-72:

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038] .

[0039] In some preferred embodiments of the present invention, the tetrasubstituted pyrrole derivative is .

[0040] In some embodiments of the present invention, the neurodegenerative disease includes amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, or Huntington's disease.

[0041] In some preferred embodiments of the present invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0042] In some embodiments of the present invention, the product for treating neurodegenerative diseases comprises at least one of (a)-(c):

[0043] (a) Products that reduce glutamate-induced excitotoxicity;

[0044] (b) Products that reduce mislocalization of the TDP-43 protein;

[0045] (c) Products that inhibit the co-localization of TDP-43 protein with G3BP1.

[0046] In some embodiments of the present invention, the product includes a drug or a functional food.

[0047] In some embodiments of the present invention, the medicament further comprises pharmaceutically acceptable excipients.

[0048] In some embodiments of the present invention, the pharmaceutically acceptable excipient is selected from at least one of disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, surfactants, and preservatives.

[0049] In some embodiments of the present invention, the disintegrant is selected from at least one of corn starch, potato starch, crosporovinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, crosporovinyl cellulose sodium, carboxymethyl cellulose, calcium carboxymethyl cellulose, and alginic acid.

[0050] In some embodiments of the present invention, the diluent is selected from at least one of lactose, sucrose, mannitol, corn starch, potato starch, calcium phosphate, calcium citrate, and crystalline cellulose.

[0051] In some embodiments of the present invention, the lubricant is selected from at least one of micronized silica gel, magnesium stearate, calcium stearate, stearic acid, talc, and anhydrous silica gel.

[0052] In some embodiments of the present invention, the adhesive is selected from at least one of gum arabic, gelatin, dextrin, hydroxypropyl cellulose, methylcellulose, and polyvinylpyrrolidone.

[0053] In some embodiments of the present invention, the wetting agent is selected from sodium dodecyl sulfate.

[0054] In some embodiments of the present invention, the flavoring agent is selected from at least one of aspartame, steviol, sucrose, maltitol, and citric acid.

[0055] In some embodiments of the present invention, the suspending agent is selected from at least one of gum arabic, gelatin, methylcellulose, sodium carboxymethylcellulose, hydroxymethylcellulose, and aluminum stearate gel.

[0056] In some embodiments of the present invention, the surfactant is selected from at least one of lecithin, sorbitan monooleate, and glyceryl monostearate.

[0057] In some embodiments of the present invention, the preservative is selected from at least one of methylparaben or propylparaben.

[0058] In some embodiments of the present invention, the dosage form of the drug is at least one of solid dosage form, liquid dosage form, and semi-solid dosage form.

[0059] In some embodiments of the present invention, the solid dosage form includes tablets, granules, powders, or capsules.

[0060] In some embodiments of the present invention, the liquid formulation includes an injectable formulation.

[0061] In some embodiments of the present invention, the semi-solid formulation includes ointments and creams.

[0062] Compared with the prior art, the beneficial effects of the present invention are:

[0063] The present invention provides a tetrasubstituted pyrrole derivative or its pharmaceutically acceptable application in the preparation of products for the treatment of neurodegenerative diseases using salt. The tetrasubstituted pyrrole derivative has high cellular safety and its neuroprotective effect against glutamate damage far exceeds that of atrasentan and the clinical standard drugs edaravone and riluzole. It can not only effectively correct TDP-43 nucleocytoplasmic misalignment, but also has the ability to clear its insoluble pathological aggregates, which atrasentan does not possess. It can more effectively inhibit the co-localization of TDP-43 and stress granule protein G3BP1, intervening in the formation of pathological granules from the source. In whole animal models, the tetrasubstituted pyrrole derivative successfully translates the advantages at the cellular level into significant survival benefits, significantly prolonging the lifespan of the disease model fruit fly. Attached Figure Description

[0064] Figure 1 The results of the 24-hour safety evaluation of each compound in Example 1 on HT-22 cells;

[0065] Figure 2 The results of the 48-hour safety evaluation of each compound in Example 1 on HT-22 cells;

[0066] Figure 3 This demonstrates the neuroprotective effects of the compounds in Example 2 on HT-22 cells;

[0067] Figure 4 The effect of each compound in Example 3 on the distribution ratio of TDP-43 in the cytoplasm / nucleus of HT-22 cells under stress conditions;

[0068] Figure 5 The effect of each compound in Example 3 on the co-localization of TDP-43 and G3BP1 under stress conditions;

[0069] Figure 6 The images show the immunofluorescence of DAPI, TDP-43, and G3BP1 in HT-22 cells under stress conditions for each compound in Example 3.

[0070] Figure 7 The effect of each compound on the nucleoplasmic distribution of TDP-43 was verified by Western Blot in Example 4;

[0071] Figure 8 The effects of each compound on the insoluble aggregates of TDP-43 were verified by Western Blot in Example 5;

[0072] Figure 9 The effect of each compound in Example 6 on the survival time of the diseased fruit fly model. Detailed Implementation

[0073] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0074] The tetrasubstituted pyrrole derivatives involved in the following examples are numbered and structured as follows. Their detailed synthesis steps and compound characterization data (such as 1H NMR spectra) have been disclosed in Chinese patent application No. 2025111860118.

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089] Example 1

[0090] This embodiment evaluates the safety of the above-mentioned tetrasubstituted pyrrole derivatives in the mouse hippocampal neuron cell line (HT-22):

[0091] 1) Preparation of test samples: Dissolve the above tetrasubstituted pyrrole derivatives in dimethyl sulfoxide to prepare 10 mmol / L stock solutions;

[0092] 2) HT-22 cells in the logarithmic growth phase were seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. A blank control group (CTRL) and a drug treatment group were set up, with 3 replicates in each group. The blank control group was in complete culture medium, and the drug treatment groups were in complete culture medium containing 10 μmol / L tetrasubstituted pyrrole derivative, atrasentan (At), or edaravone (Ed), respectively. The cells were cultured for another 24 h (or 48 h). Then, high-content imaging was performed, and the number of cells in each group under different compound treatment conditions was analyzed and counted. The cell viability under different derivative treatment conditions was then calculated using the formula: Cell viability = Number of cells in the drug treatment group / Number of cells in the control group * 100%.

[0093] Figure 1 The results show the 24-hour safety evaluation of each compound in Example 1 on HT-22 cells. Figure 2 The results of the 48-hour safety evaluation of each compound in Example 1 on HT-22 cells are provided by [the relevant authority / organization]. Figure 1 and Figure 2 It was found that, under different treatment times, the cell viability of HT-22 cells was greater than 90% after treatment with atrasentan (At) and edaravone (Ed), while the cell viability of HT-22 cells was also generally greater than 90% after treatment with tetrasubstituted pyrrole derivatives, indicating that tetrasubstituted pyrrole derivatives have low toxicity to HT-22 cells and have good safety.

[0094] Example 2

[0095] This embodiment provides a preliminary in vitro evaluation of the anti-glutamate activity of the aforementioned tetrasubstituted pyrrole derivatives:

[0096] Glutamate is a major excitatory neurotransmitter in the central nervous system, playing a crucial role in neuronal communication. Excessive glutamate binds to and activates ionotropic glutamate receptors, leading to activation of various cellular signaling pathways, oxidative stress, mitochondrial dysfunction, and ultimately cell death. This phenomenon is associated with the pathogenesis of various central nervous system diseases, including stroke, epilepsy, traumatic brain injury, neurodegenerative diseases, and other neuroinflammatory disorders.

[0097] Mouse hippocampal neuronal cell lines are highly sensitive to glutamate and are frequently used as model systems to study glutamate-induced neuronal cytotoxicity. This example investigated the cell viability of HT-22 cells treated with a 10 mmol / L tetrasubstituted pyrrole derivative for 24 hours at a monosodium glutamate (MSG) concentration of 1.25 mmol / L, with edaravone, riluzole, and atrasentan as positive controls, to screen for compounds with good protective effects on neurons.

[0098] 1) Preparation of test samples: Dissolve the above tetrasubstituted pyrrole derivatives in dimethyl sulfoxide to prepare 10 mmol / L stock solutions;

[0099] 2) HT-22 cells in the logarithmic growth phase were seeded in 96-well plates and incubated at 37°C with 5% [unclear text - possibly a specific concentration or concentration]. Cells were cultured in a CO2 incubator for 24 hours. A blank control group (CTRL), a glutamate control group (LGNa), and drug-treated groups were set up, with three replicates in each group. The blank control group used complete culture medium, the glutamate control group used complete culture medium containing 1.25 mmol / L sodium glutamate, and the drug-treated groups used complete culture medium containing 10 μmol / L tetrasubstituted pyrrole derivative and 1.25 mmol / L sodium glutamate, 10 μmol / L atrasentan (At) and 1.25 mmol / L sodium glutamate, 10 μmol / L riluzole (RL) and 1.25 mmol / L sodium glutamate, and 10 μmol / L edaravone (Ed) and 1.25 mmol / L sodium glutamate, respectively. Cells were cultured for another 24 hours. High-content imaging was performed, and the number of cells in each group under different compound treatment conditions was analyzed. Cell viability under different derivative treatment conditions was calculated using the formula: Cell viability = (Number of cells in experimental group / Number of cells in control group) * 100%.

[0100] Figure 3 To demonstrate the neuroprotective effects of the compounds in Example 2 on HT-22 cells, [the following was observed]. Figure 3It was found that under the action of 1.25 mmol / L sodium glutamate, edaravone (Ed), riluzole (RL), and atrasentan (At) could attenuate the neurotoxicity caused by HT-22 induced by glutamate to varying degrees. Among the tetrasubstituted pyrrole derivatives, compounds S8, A6, D2, D4, D5, and D6 could significantly improve the neurotoxicity caused by HT-22 induced by glutamate, and the effect was significantly better than that of edaravone (Ed), riluzole (RL), and atrasentan (At), indicating that tetrasubstituted pyrrole derivatives have the potential to be used in the preparation of products for the treatment of neurodegenerative diseases.

[0101] Example 3

[0102] This embodiment evaluates the performance of the above-mentioned tetrasubstituted pyrrole derivatives in improving the misplaced aggregation of TDP-43 in cells:

[0103] D-Sorbitol is an intermediate in the polyol pathway (an ATP-independent metabolic pathway that produces fructose from glucose). As a novel physiological stressor, it can induce TDP-43 to mislocalize into the cytoplasm, forming TDP-43 aggregates and localizing to stress granules.

[0104] 1) Preparation of test samples: Dissolve the above tetrasubstituted pyrrole derivatives in dimethyl sulfoxide to prepare 10 mmol / L stock solutions;

[0105] 2) HT-22 cells in logarithmic growth phase were seeded in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h. A blank control group (CTRL), an induction group, and a drug treatment group were set up, with three replicates per group. The blank control group was treated with complete culture medium; the induction group was treated with complete culture medium containing 0.25 mol / L D-sorbitol for 1 h; the drug treatment group was pretreated with complete culture medium containing 10 μmol / L tetrasubstituted pyrrole derivatives or atrasentan (At) for 24 h, then replaced with complete culture medium containing 0.25 mol / L D-sorbitol and 10 μmol / L tetrasubstituted pyrrole derivatives, or atrasentan (At) containing 0.25 mol / L D-sorbitol. The samples were incubated with D-sorbitol and 10 μmol / L atrasentan (At) for 1 h. Then, the samples were fixed, permeabilized, blocked, incubated with primary antibodies (TDP-43 and G3BP1), incubated with secondary antibodies, and incubated with DAPI. High-content imaging was performed, and the distribution ratio of TDP-43 in the nucleus and cytoplasm under different compound administration conditions was analyzed and statistically analyzed. The calculation formula was: TDP-43(C / N) = TDP-43 content in cytoplasm / TDP-43 content in nucleus * 100%. The colocalization of TDP-43 and G3BP1 under different compound administration conditions was also analyzed and statistically analyzed.

[0106] Figure 4The effect of each compound in Example 3 on the distribution ratio of TDP-43 in the cytoplasm / nucleus of HT-22 cells under stress conditions reflects the mislocalization of TDP-43 in HT-22 cells. Figure 4 It can be seen that, compared with the induction group, the ratio of TDP-43 content in the cytoplasm to the nucleus was reduced after treatment with atrasentan (At), proving that it can improve the misalignment. The tetrasubstituted pyrrole derivatives A6, D2, D4, etc., compared with atrasentan (At), showed a similar or stronger ability to reduce the ratio of TDP-43 content in the cytoplasm to the nucleus, indicating that they can effectively promote the return of TDP-43 to the nucleus and correct its pathological location.

[0107] Figure 5 The effects of the compounds in Example 3 on the co-localization of TDP-43 and G3BP1 under stress conditions are shown, reflecting the aggregation of TDP-43 into stress particles under stress conditions. Figure 5 It can be seen that after treatment with atrasentan (At), the co-localization coefficient of TDP-43 and G3BP1 decreased, while the effect of the tetrasubstituted pyrrole derivative D5 was extremely outstanding, with a co-localization coefficient significantly lower than that of the atrasentan (At) group. This indicates that the tetrasubstituted pyrrole derivative (especially D5) has a significantly better effect than the parent drug atrasentan (At) in inhibiting the formation of harmful aggregates of TDP-43.

[0108] Figure 6 This is an immunofluorescence image showing the effect of the compounds in Example 3 on DAPI, TDP-43, and G3BP1 proteins in HT-22 cells under stress conditions. Figure 4 and Figure 5 The tetrasubstituted pyrrole derivative D5 was selected, and the effects of atrasentan (At) and the tetrasubstituted pyrrole derivative D5 on the mislocation and aggregation of TDP-43 were visually demonstrated by immunofluorescence. Figure 6It was found that after D-sorbitol treatment, the cell nucleus (DAPI) shrank significantly, the content of TDP-43 in the cytoplasm increased, and a large number of TDP-43 co-localized with the aggregates (G3BP1) generated under stress were observed. After treatment with atrasentan (At), the cell nucleus shrank significantly improved, the content of TDP-43 in the cytoplasm decreased, the number of TDP-43 aggregates in the cytoplasm decreased, and the number of TDP-43 co-localized with G3BP1 decreased, which can effectively improve the mislocalization and aggregation of TDP-43 under stress conditions. After treatment with the tetrasubstituted pyrrole derivative D5, the cell nucleus shrank significantly improved, the content of TDP-43 in the cytoplasm decreased, the number of aggregates decreased, and the number of TDP-43 co-localized with G3BP1 decreased, and the effect was more significant than that of atrasentan (At). This indicates that the tetrasubstituted pyrrole derivative (especially D5) has a significantly better effect than the parent drug atrasentan (At) in inhibiting the mislocalization of TDP-43 and the formation of harmful aggregates.

[0109] Example 4

[0110] This embodiment evaluates the performance of the aforementioned tetrasubstituted pyrrole derivative D5 in improving the mislocalization of intracellular TDP-43:

[0111] 1) Preparation of test samples: Dissolve the above tetrasubstituted pyrrole derivatives in dimethyl sulfoxide to prepare 10 mmol / L stock solutions;

[0112] 2) HT-22 cells in the logarithmic growth phase were seeded in 96-well plates and incubated at 37°C with 5% [unclear text - possibly a specific concentration or concentration]. The cells were cultured in a CO2 incubator for 24 h. A blank control group (CTRL), a D-sorbitol control group, and a drug-treated group were set up, with three replicates per group. The blank control group was cultured in complete medium; the D-sorbitol control group was cultured in complete medium containing 0.25 mol / L D-sorbitol for 1 h; the drug-treated group was pretreated for 24 h with complete medium containing 10 μmol / L tetrasubstituted pyrrole derivative D5, atrasentan (At), or edaravone (Ed), then replaced with medium containing 0.25 mol / L D-sorbitol and 10 μmol / L tetrasubstituted pyrrole derivative, or medium containing 0.25 mol / L D-sorbitol and 10 μmol / L atrasentan (At), or medium containing 0.25 mol / L D-sorbitol and 10 μmol / L edaravone (Ed) for 1 h. Proteins were extracted from the cytoplasm and nucleus of the different experimental groups using a nucleoplasm extraction kit, and protein content was characterized by Western blott assay.

[0113] Figure 7The effects of the compounds verified by Western Blot in Example 4 on the nucleoplasmic distribution of TDP-43 reflect the mislocalization of TDP-43 protein induced by D-sorbitol in HT-22 cells. The levels of TDP-43 in the cytoplasm and nucleus under the action of the compounds edaravone (Ed), the tetrasubstituted pyrrole derivative D5, and atrasentan (At) are shown. Figure 7 It was found that 0.25 mol / L D-sorbitol induced HT-22, resulting in a slight increase in TDP-43 in the cytoplasm and a significant decrease in the nucleus. After treatment with edaravone (Ed) and atrasentan (At), TDP-43 in the cytoplasm decreased slightly, while TDP-43 in the nucleus increased significantly. After treatment with the tetrasubstituted pyrrole derivative D5, TDP-43 in the cytoplasm decreased significantly, while TDP-43 in the nucleus increased significantly. These results indicate that edaravone (Ed), the tetrasubstituted pyrrole derivative D5, and atrasentan (At) can all improve the mislocation state of TDP-43 in D-sorbitol-induced HT-22, and the improvement effect of the tetrasubstituted pyrrole derivative D5 is better than that of edaravone (Ed) and atrasentan (At).

[0114] Example 5

[0115] This embodiment evaluates the performance of the aforementioned tetrasubstituted pyrrole derivative D5 in improving intracellular TDP-43 aggregate protein:

[0116] 1) Preparation of test samples: Dissolve the above tetrasubstituted pyrrole derivatives in dimethyl sulfoxide to prepare 10 mmol / L stock solutions;

[0117] 2) HT-22 cells in the logarithmic growth phase were seeded in 96-well plates and incubated at 37°C with 5% [unclear text - possibly a specific concentration or concentration]. The cultures were incubated in a CO2 incubator for 24 h. A blank control group (CTRL), a D-sorbitol control group, and a drug-treated group were set up, with three replicates for each group. The blank control group was cultured in complete medium; the D-sorbitol control group was cultured in complete medium containing 0.25 mol / L D-sorbitol for 1 h; the drug-treated group was pretreated for 24 h with complete medium containing 10 μmol / L tetrasubstituted pyrrole derivative D5, atrasentan (At), or edaravone (Ed), then replaced with medium containing 0.25 mol / L D-sorbitol and 10 μmol / L tetrasubstituted pyrrole derivative, or 0.25 mol / L D-sorbitol and 10 μmol / L atrasentan (At), or medium containing 0.25 mol / L D-sorbitol and 10 μmol / L edaravone (Ed) for 1 h. Soluble and insoluble proteins were extracted from the different experimental groups, and protein content was characterized by Western blott assay.

[0118] Figure 8The effects of the compounds verified by Western blot in Example 5 on insoluble TDP-43 aggregates reflect the changes in TDP-43 aggregates induced by D-sorbitol in HT-22 cells, and the effects of edaravone (Ed), the tetrasubstituted pyrrole derivative D5, and atrasentan (At) on TDP-43 aggregates. Figure 8 It was found that after induction with 0.25 mol / L D-sorbitol, the soluble components in HT-22 cells remained basically unchanged, while the insoluble components (i.e., TDP-43 aggregates) increased significantly. After treatment with edaravone (Ed) and atrasentan (At), the content of TDP-43 aggregates decreased to varying degrees. After treatment with the tetrasubstituted pyrrole derivative D5, the level of TDP-43 aggregates decreased significantly and was close to that of the blank control group. These results indicate that the tetrasubstituted pyrrole derivative D5 can significantly reduce the level of TDP-43 aggregates under stress conditions, that is, it has the ability to directly clear existing pathological aggregates, which atrasentan (At) does not possess.

[0119] Example 6

[0120] This embodiment investigates the protective effect of the above-mentioned tetrasubstituted pyrrole derivative D5 against diseased fruit flies:

[0121] This embodiment constructed a transgenic fruit fly overexpressing hTDP43 as a model animal to investigate whether hTDP43 overexpression in transgenic fruit flies could affect the survival time and locomotion of fruit flies, as well as neurodegenerative diseases. To assess the survival ability of adult fruit flies, this embodiment conducted experiments under constant temperature and humidity conditions (25±0.5℃, 50±5%). Three to five days after emergence, fruit flies were divided into groups for drug administration experiments. The number of dead individuals in the experimental groups was observed and counted every five days until all individuals died. To ensure the reliability of the data, the sample size in each group was no less than 60, and each individual was recorded as a single death. All experiments were performed in three independent replicates. During the experiment, environmental parameters were strictly controlled, and standardized operating procedures were used for data collection and analysis.

[0122] Figure 9 The effects of each compound in Example 6 on the survival time of the diseased fruit fly model were determined by... Figure 9It was found that the longest survival time of wild-type fruit flies was 50 days; the longest survival time of hTDP-43 overexpressing fruit flies was 25 days; the initial survival rate of overexpressing fruit flies treated with edaravone (Ed) was higher than that of hTDP-43 overexpressing fruit flies, but the longest survival time in the later stage was still 25 days, without significant extension; the survival rate of overexpressing fruit flies treated with atrasentan (At) was higher than that of hTDP-43 overexpressing fruit flies, and the longest survival time was extended to 30 days; the overall survival rate of overexpressing fruit flies treated with tetrasubstituted pyrrole derivative D5 was higher than that of hTDP-43 overexpressing fruit flies, and the longest survival time was extended to 30 days. This indicates that the treatment with tetrasubstituted pyrrole derivative D5 can improve the pathological state of the ALS fruit fly model and prolong its survival time, and the effect is better than that of edaravone (Ed) and atrasentan (At). That is, the tetrasubstituted pyrrole derivative in this application is also effective in complex in vivo environments and can translate the advantages at the cellular level into overall survival benefits for animal models.

Claims

1. Use of the tetrasubstituted pyrrole derivative of formula (I) or formula (II) or a pharmaceutically acceptable salt thereof in the preparation of products for the treatment of neurodegenerative diseases: in, In equations (I) and (II), R 1 Selected independently from hydrogen and C 1-4 The alkyl group, benzoyl group or its derivative, and -CT are selected from the group consisting of the carboxyl group, ester group, substituted or unsubstituted amide group; R 2 Each is independently selected from hydrogen, hydroxyl, and C. 1-4 Alkyl, methoxy, halogen, amino, or nitro groups; R 3 Each group is independently selected from ester, carboxyl, or substituted amide groups; R 4 Each group is independently selected from hydrogen, hydroxyl, methoxy, halogen, oxygen-containing heterocycle, amino, nitro, or cyano.

2. The application according to claim 1, characterized in that, In the tetrasubstituted pyrrole derivatives represented by formula (I) or (II), the R 1 Each is independently selected from hydrogen, methyl, benzoyl, 4-methylbenzoyl, -C-COOR 5 or -C-CONR 6 R 7 , where R 5 Selected from hydrogen, straight-chain or branched alkyl groups; R 6 and R 7 Selected independently from hydrogen and C 2-8 Straight-chain alkyl, -C2H4- heterocyclic or dimethylaminoalkyl; And / or, the R 2 Each is independently selected from hydrogen, hydroxyl, methyl, methoxy, or halogen; And / or, the R 3 Selected independently from -COOR 8 or -CONR 9 R 10 , where R 8 Selected from hydrogen, straight-chain or branched alkyl groups; R 9 and R 10 Each is independently selected from hydrogen, propargyl, or C. 1-4 Straight-chain alkyl; And / or, the R 4 Each is independently selected from hydrogen, hydroxyl, methoxy, halogen, or oxygen-containing heterocycles.

3. The application according to claim 2, characterized in that, The tetrasubstituted pyrrole derivative is selected from at least one of formulas 1-72: 。 4. The application according to claim 1, characterized in that, The neurodegenerative diseases mentioned include amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, Parkinson's disease, or Huntington's disease.

5. The application according to any one of claims 1-4, characterized in that, The product for treating neurodegenerative diseases includes at least one of (a)-(c): (a) Products that reduce glutamate-induced excitotoxicity; (b) Products that reduce mislocalization of the TDP-43 protein; (c) Products that inhibit the co-localization of TDP-43 protein with G3BP1.

6. The application according to claim 5, characterized in that, The products include pharmaceuticals or functional foods.

7. The application according to claim 6, characterized in that, The drug also contains pharmaceutically acceptable excipients.

8. The application according to claim 7, characterized in that, The pharmaceutically acceptable excipients are selected from at least one of the following: disintegrants, diluents, lubricants, binders, humectants, flavoring agents, suspending agents, surfactants, and preservatives.

9. The application according to claim 8, characterized in that, The dosage form of the drug is at least one of solid dosage form, liquid dosage form, and semi-solid dosage form.

10. The application according to claim 9, characterized in that, The solid dosage forms include tablets, granules, powders, or capsules; And / or, the liquid formulation includes an injectable formulation; And / or, the semi-solid formulation includes ointments and creams.

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