Application of magnolol nitrone derivative in preparation of medicine for preventing or treating amyotrophic lateral sclerosis

Abstract

The invention belongs to the technical field of medicines, and discloses application of magnolol nitrone derivatives in preparation of medicines for preventing or treating amyotrophic lateral sclerosis. Compared with an empty vector control cell group, the survival rates of SOD1G93A cells and TDP43M337V cells are remarkably reduced, and the survival rates of the SOD1G93A cells and the TDP43M337V cells can be remarkably improved 24 hours after 0.1 mu M of HL-3 is given.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of magnolol nitroketone derivatives in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS). Background Technology

[0002] Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of unknown etiology that primarily affects motor neurons in the cerebral cortex, brainstem, and spinal cord. Clinical manifestations are mainly progressive skeletal muscle atrophy, weakness, fasciculations, bulbar palsy, and pyramidal tract signs. The disease has an insidious onset, and patients often experience respiratory distress in the later stages. The average survival time is 3-5 years. Therefore, ALS is a disease with a short survival period and poor prognosis.

[0003] Currently, the U.S. Food and Drug Administration (FDA) has approved four drugs for the treatment of ALS: riluzole, edaravone, relyvrio, and toferson. Riluzole and edaravone were approved by the FDA in 1996 and 2017, respectively. Riluzole is a glutamate antagonist that can extend the survival of ALS patients by approximately three months. Edaravone is a free radical / reactive oxygen species scavenger that can slightly improve patient symptoms. Relyvrio and toferson are drugs approved by the FDA in the last two years. Relyvrio works by improving endoplasmic reticulum health and delaying nerve cell death. According to its Phase II clinical trial results, relyvrio can extend the survival of ALS patients by 6.5 months. Toferson is only suitable for ALS patients with SOD1 mutations, but the efficacy of both requires further confirmation in Phase III clinical trials. Therefore, there is an urgent need for effective drugs to treat ALS.

[0004] ALS is divided into two types: familial ALS (fALS) and sporadic ALS. fALS accounts for 5-10% of all ALS patients, while the rest are sporadic. However, research has confirmed that gene mutations are associated with the development of both familial and sporadic ALS. Superoxide dismutase 1 (SOD1) was the first identified mutated gene, and its mutation is associated with approximately 20% of fALS patients and 2% of sporadic ALS patients. In recent years, further research has shown that mutations in genes such as 43kDa TAR DNA-binding protein 43 (TDP43) are also closely related to the development of ALS.

[0005] Nitroketones are a class of compounds with strong free radical scavenging capabilities, exhibiting potent scavenging effects against a variety of reactive free radicals. Studies have found that nitroketones have certain therapeutic effects on various free radical-induced diseases (such as cancer, stroke, and Parkinson's disease). We have creatively synthesized a number of magnolol and magnolol nitroketone derivatives, speculating that they may play a certain preventive or therapeutic role in ALS.

[0006] Therefore, the main purpose of this invention is to use SOD1 G93A stable transfected cells and TDP43 M337V stable transfected cells as models to study whether magnolol nitroketone derivatives can improve the pathological damage caused by SOD1 G93A or TDP43 M337V, so as to provide effective data support for their treatment and intervention of amyotrophic lateral sclerosis. Summary of the Invention

[0007] The technical problem solved by this invention is the application of magnolol nitroketone derivatives in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis.

[0008] To address the technical problem of this invention, the present invention provides the following technical solution:

[0009] The first aspect of the present invention is to provide the use of magnolol nitroketone derivatives and pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis.

[0010] This invention relates to magnolol nitroketone derivatives of general formula I or pharmaceutically acceptable salts thereof:

[0011]

[0012] R1 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3;

[0013] R2 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn;

[0014] This invention relates to the magnolol nitroketone derivatives of formula II or pharmaceutically acceptable salts thereof:

[0015]

[0016] R3 and R4 are independent of each other and can be selected from H or H respectively. Furthermore, R3 and R4 are not both H; R5 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, and Bn;

[0017] The structures and numbers of some compounds in this invention are as follows:

[0018]

[0019]

[0020] The compounds represented by general formula I of this invention can be prepared by the following methods:

[0021] The compounds represented by general formula II of this invention can be prepared by the following methods:

[0022]

[0023] The reaction conditions were as follows: (a) Paraformaldehyde, magnesium chloride, triethylamine, anhydrous tetrahydrofuran solvent, heated to 60°C; (b) R₂NHOH, triethylamine, sodium sulfate, ethanol solvent, heated to 90°C; (c) RI, potassium carbonate, tetrahydrofuran solvent, room temperature; (d) i: reactant hexamethylenetetramine, glacial acetic acid solvent, heated to 130°C; II: 33% sulfuric acid, heated to 100°C; (e) 35% sodium hydroxide, chloroform solvent, heated to 60°C.

[0024] In the above compounds, the definitions of R1 and R2 are the same as those for R1 and R2 in general formula I.

[0025] I(a) includes compounds such as HK-1, HK-2, HK-3, HK-4, and HK-5.

[0026] I(b) includes compounds such as HK-6, HK-7, HK-8, HK-9, and HK-10.

[0027] II(a) includes compounds such as HL-1, HL-2, HL-3, HL-4, and HL-5.

[0028] II(b) includes compounds such as HL-6, HL-7, HL-8, HL-9, and HL-10.

[0029] II(c) includes compounds such as HL-11 and HL-12.

[0030] Another aspect of the present invention relates to pharmaceutical compositions in which the compounds of the present invention are used as active ingredients. These pharmaceutical compositions are prepared according to methods known in the art. They can be formulated into any dosage form suitable for human or animal use by combining the compounds of the present invention with one or more pharmaceutically acceptable solid or liquid excipients and / or adjuvants. The content of the compounds of the present invention in their pharmaceutical compositions is typically 0.1-95% by weight.

[0031] The compounds of this invention or pharmaceutical compositions containing them can be administered in unit dose form via enteral or non-enteric routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oral mucosa, eye, lung and respiratory tract, skin, vagina, rectum, etc.

[0032] Dosage forms can be liquid, solid, or semi-solid. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including o / w, w / o, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc.

[0033] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0034] To formulate the compounds of the present invention into tablets, a wide variety of excipients known in the art can be used, including diluents, binders, wetting agents, disintegrants, lubricants, and flow aids. Diluents can be starch, dextrin, sucrose, glucose, lactose, mannose, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, dicalcium phosphate, calcium carbonate, etc.; wetting agents can be water, ethanol, isopropanol, etc.; binders can be starch paste, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, gum arabic paste, gelatin paste, sodium carboxymethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; disintegrants can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, cross-linked sodium carboxymethyl cellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfonate, etc.; lubricants and flow aids can be talc, silica, stearate, tartaric acid, liquid paraffin, polyethylene glycol, etc.

[0035] Tablets can also be further processed into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bilayer and multilayer tablets.

[0036] To formulate the drug delivery unit into capsules, the active ingredient, the compound of the present invention, can be mixed with a diluent and a disintegrant, and the mixture can be placed directly into hard or soft capsules. Alternatively, the active ingredient, the compound of the present invention, can be first formed into granules or microspheres with a diluent, binder, and disintegrant, and then placed into hard or soft capsules. Various diluents, binders, wetting agents, disintegrants, and disintegrants used to prepare tablets of the compound of the present invention can also be used to prepare capsules of the compound of the present invention.

[0037] To prepare the compounds of this invention into injectable formulations, water, ethanol, isopropanol, propylene glycol, or mixtures thereof can be used as solvents, and appropriate amounts of commonly used solubilizers, co-solvents, pH adjusters, and osmotic pressure adjusters can be added. Solubilizers or co-solvents can be poloxamer, lecithin, hydroxypropyl-β-cyclodextrin, etc. pH adjusters can be phosphates, acetates, hydrochloric acid, sodium hydroxide, etc.; osmotic pressure adjusters can be sodium chloride, mannitol, glucose, phosphates, acetates, etc. If preparing lyophilized powder injections, mannitol, glucose, etc., can also be added as supporting agents.

[0038] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0039] To achieve the purpose of medication and enhance the therapeutic effect, the drug or drug composition of the present invention can be administered using any known method of administration.

[0040] The dosage of the pharmaceutical compositions of the present invention can vary widely depending on the nature and severity of the disease to be prevented or treated, the individual condition of the patient or animal, the route of administration, and the dosage form. Generally, the suitable daily dose range of the compounds of the present invention is 0.001-150 mg / kg body weight, preferably 0.1-100 mg / kg body weight, more preferably 1-60 mg / kg body weight, and most preferably 2-30 mg / kg body weight. The above doses can be administered as a single dose unit or divided into several dose units, depending on the physician's clinical experience and the administration regimen, including the use of other treatment methods.

[0041] The compounds or compositions of the present invention can be taken alone or in combination with other therapeutic or symptomatic drugs. When the compounds of the present invention have a synergistic effect with other therapeutic drugs, their dosage should be adjusted according to the actual situation.

[0042] Beneficial technical effects:

[0043] Compared with their respective empty vector control cell groups (pEGFP and pCI), the survival of SOD1 G93A cells and TDP43 M337V cells was significantly reduced. Administration of 0.1 μM HL-3 significantly improved the survival of SOD1 G93A cells and TDP43 M337V cells. Attached Figure Description

[0044] Figure 1 0.1-10 μM HL-3 improved the survival of SOD1 G93A and TDP43 M337V stable cells. A: The MTT assay was used to detect the difference in proliferation between empty vector control pEGFP stable cells and SOD1 G93A stable cells, and the effect of administering 0.1-10 μM HL-3 to SOD1 G93A stable cells for 24 hours on their survival. B: The MTT assay was used to detect the difference in proliferation between empty vector control pCI stable cells and TDP43 M337V stable cells, and the effect of administering 0.1-10 μM HL-3 to TDP43 M337V stable cells for 24 hours on their survival. "#" indicates comparison with the empty vector group, where #P<0.05, ##P<0.01, and "*" indicates comparison with the model group, where *P<0.05. Detailed Implementation

[0045] Example 1. HL-3 has a protective effect on cells stably transfected with SOD1 G93A and TDP43 M337V.

[0046] This study found that compared with the corresponding empty vector control group cells, SOD1 G93A and TDP43 M337V stable transgenic cells significantly reduced cell survival. HL-3 treatment in SOD1 G93A and TDP43 M337V stable transgenic cells for 24 hours showed a significant protective effect, specifically, 0.1 μM HL-3 significantly improved the cell survival rate.

[0047] 1. Cells and Plasmids

[0048] Cell lines:

[0049] The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0050] The SOD1 G93A stable transfected cell line and the pEGFP empty vector control cell line were constructed in our laboratory.

[0051] The TDP43 M337V stably transfected cell line and the pCI empty vector control cell line were constructed in our laboratory.

[0052] 2. Methods

[0053] 2.1 MTT assay for cell viability

[0054] pEGFP empty vector control cells and SOD1 G93A stable cells were cultured at a ratio of 5*10. 4Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated. After 24 hours of incubation, the medium was changed, and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pEGFP empty vector control cells and the SOD1 G93A model group cells; 10 μL of medium containing HL-3 was added to the wells of each SOD1G93A treatment group cells, bringing the final concentrations in each well to 0.1, 1, and 10 μM, respectively. After 4 hours and 24 hours of incubation, 10 μL of MTT was added to each well. After another 4 hours of incubation, 100 μL of triplet solution was added to each well, and the cells were incubated overnight at 37°C to dissolve. The absorbance was then measured at 570 nm.

[0055] pCI empty vector control cells and TDP43 M337V stable cells were divided into 5*10 cells. 4 Cells were seeded at a density of 100 μL / mL in 96-well plates and incubated. After 24 hours of incubation, the medium was changed, and 90 μL of blank medium was added to each well. 10 μL of blank medium was added to the wells of the pCI empty vector control cells and the TDP43 M337V model group cells; 10 μL of medium containing HL-3 was added to the wells of each TDP43M337V treatment group cells, bringing the final concentrations in each well to 0.1, 1, and 10 μM, respectively. After incubation for 24 hours, 10 μL of MTT was added to each well, followed by another 4 hours of incubation. Then, 100 μL of triplet solution was added to each well, and the cells were incubated overnight at 37°C to dissolve. The absorbance was then measured at 570 nm.

[0056] 2.2 Data Statistical Analysis

[0057] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates that the model group cells were compared with their respective empty vector control groups (pEGFP and pCI). # P<0.05, ## P<0.01, "*" indicates that the cells in each drug-treated group were compared with their respective model group cells. * P<0.05.

[0058] 3. Results

[0059] 3.1HL-3 enhances the survival of SOD1 G93A cells and TDP43 M337V cells.

[0060] The results of this embodiment show that, compared with the empty vector control cell group, the survival rates of SOD1 G93A cells and TDP43 M337V cells were significantly reduced. Figure 1 -A and Figure 1-B), while administration of 0.1 μM HL-3 significantly improved the survival rate of SOD1 G93A cells and TDP43M337V cells (-B). Figure 1 -A and Figure 1 -B).

[0061] The above embodiments are merely illustrative of the technical concept and features of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made according to the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. The use of magnolol nitroketone derivatives as shown in general formula (I) and their pharmaceutically acceptable salts in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS): R1 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3; R2 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, Bn.

2. The use of magnolol nitroketone derivatives as shown in general formula (II) and their pharmaceutically acceptable salts in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS): R3 and R4 are independent of each other and can be selected from H or H respectively. Furthermore, R3 and R4 are not both H; R5 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, t-Bu, Ph, and Bn.

3. The application according to any one of claims 1 and 2, characterized in that, The compounds are selected from the following group:

4. The application according to any one of claims 1-3, characterized in that, The pharmaceutically acceptable salt is selected from organic or inorganic acid salts, including hydrochloride, hydrobromide, sulfate, phosphate, acetate, citrate, malate, fumarate, tartrate, methanesulfonate, carbonate, oxalate, lactate, succinate, or gluconate.

5. The use of a pharmaceutical composition in the preparation of a drug for the prevention or treatment of amyotrophic lateral sclerosis (ALS), characterized in that, The pharmaceutical composition comprises the magnolol nitroketone derivative as described in any one of claims 1-3, its pharmaceutically acceptable salt, and a pharmaceutically acceptable carrier.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable carriers are selected from lipid carriers, polymer carriers, protein and peptide carriers, inorganic nanocarriers, carbon-based nanomaterials, hydrogels, and biodegradable materials.

7. The application according to claim 5, characterized in that, The pharmaceutical composition is selected from solutions, emulsions, suspensions, injections, eye drops, nasal drops, lotions, and liniments; solid dosage forms may be tablets, capsules, granules, powders, microcapsules, droplets, suppositories, films, patches, aerosols, powder sprays, and sprays; semi-solid dosage forms may be ointments, gels, and pastes.

8. The application according to claim 7, characterized in that, The solutions include true solutions and colloidal solutions; the emulsions include o / w, w / o, and double emulsions; the injections include aqueous injections, powder injections, and infusions; the tablets include regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets; and the capsules include hard capsules, soft capsules, and enteric-coated capsules.

9. The application according to any one of claims 1-3, characterized in that, The preparation method of the magnolol nitroketone derivative is as follows: The compounds represented by general formula I of this invention can be prepared by the following methods: The compounds represented by general formula II of this invention can be prepared by the following methods: The reaction conditions were as follows: (a) Paraformaldehyde, magnesium chloride, triethylamine, anhydrous tetrahydrofuran solvent, heated to 60°C; (b) R₂NHOH, triethylamine, sodium sulfate, ethanol solvent, heated to 90°C; (c) RI, potassium carbonate, tetrahydrofuran solvent, room temperature; (d) i: reactant hexamethylenetetramine, glacial acetic acid solvent, heated to 130°C; II: 33% sulfuric acid, heated to 100°C; (e) 35% sodium hydroxide, chloroform solvent, heated to 60°C. In the above compounds, the definitions of R1 and R2 are the same as those for R1 and R2 in general formula I; I(a) includes the compounds HK-1, HK-2, HK-3, HK-4, and HK-5; I(b) includes the compounds HK-6, HK-7, HK-8, HK-9, and HK-10; II(a) includes compounds HL-1, HL-2, HL-3, HL-4, and HL-5; II(b) includes compounds HL-6, HL-7, HL-8, HL-9, and HL-10; II(c) includes compounds HL-11 and HL-12.

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