Application of pyranocarbazole oxime derivatives in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS)

Abstract

The application belongs to the technical field of medicine, and discloses application of a pyrano-carbazole oxime derivative in preparation of a drug for preventing or treating amyotrophic lateral sclerosis. Specifically, the application discloses a pyrano-carbazole compound as shown in a general formula I. The compound is prepared by an artificial synthesis method, contains a pharmaceutical composition thereof, and has application in amyotrophic lateral sclerosis.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the use of pyranocarbazole oxime 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 the motor neurons of the cerebral cortex, brainstem, and spinal cord. Clinical manifestations are mainly progressive skeletal muscle atrophy, weakness, fasciculations, bulbar palsy, and pyramidal tract signs. The incidence of ALS in China is approximately 0.6 per 100,000 people, with a prevalence of about 3.1 per 100,000 people. 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 classified into two types: familial ALS (fALS) and sporadic ALS. fALS accounts for 5-10% of all ALS patients, while the remainder 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 pathogenesis of ALS. Animal and cell models based on SOD1 and TDP43 gene mutations are widely used in the pathogenesis and drug development of ALS.

[0005] Pyranocarbazole alkaloids have a wide range of biological activities. In this invention, oximes and nitroketones were introduced into the pyranocarbazole alkaloid core to obtain a series of derivatives.

[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 pyranocarbazole oxime 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] This invention discloses compounds of general formulas I and II. Pharmacological experiments have shown that these compounds can significantly improve the survival rate of SOD1 G93A and TDP43 M337V cells. Therefore, the compounds of this invention can be used for the prevention or treatment of amyotrophic lateral sclerosis (ALS).

[0008] The use of a class of pyranocarbazole oxime derivatives or pharmaceutically acceptable salts thereof, represented by general formula I, in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS):

[0009]

[0010] R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively.

[0011] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3;

[0012] This invention relates to the use of a class of pyranocarbazole nitroketone derivatives of general formula II or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment of amyotrophic lateral sclerosis (ALS):

[0013]

[0014] R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively.

[0015] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3;

[0016] R4 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn;

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

[0018]

[0019] The compounds represented by general formula I of this invention can be prepared by the following methods:

[0020]

[0021] The reaction conditions (a) were hydroxylamine hydrochloride, 1 mol / L sodium hydroxide solution, ethanol as solvent, and room temperature.

[0022] The compounds represented by general formula II of this invention can be prepared by the following methods:

[0023]

[0024] The reaction conditions (b) were: R4NHOH, triethylamine, sodium sulfate, ethanol solvent, and heating at 90°C.

[0025] For the preparation methods of intermediates 1 and 2, please refer to "European Journal of Medicinal Chemistry. 2020, 190, 112079".

[0026] 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.

[0027] 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.

[0028] 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.

[0029] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] This invention uses an NSC-34 cell model transfected with the SOD1 G93A and TDP43 M337V mutant genes to evaluate the protective effect of this compound against ALS-related cytotoxicity. NSC-34 cells are motor neuron-like cells commonly used in ALS research, and this model simulates the neurotoxic environment induced by ALS-related gene mutations.

[0039] Beneficial technical effects:

[0040] Compared with their respective empty vector control cell groups (pEGFP and pCI), the survival of SOD1 G93A cells and TDP43M337V cells was significantly reduced. Administration of 10 μM CXT-24 and CXT-5 significantly improved the survival of SOD1 G93A cells and TDP43 M337V cells. Attached Figure Description

[0041] Figure 1 0.1-10 μM CXT-24 and CXT-5 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 CXT-24 and CXT-5 to SOD1 G93A stable cells for 24 hours on the survival of SOD1 G93A stable cells. 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 CXT-24 and CXT-5 to TDP43 M337V stable cells for 24 hours on the survival of TDP43 M337V stable cells. "#" indicates a comparison with the unloaded group, where #P<0.05 and ##P<0.01; "*" indicates a comparison with the model group, where *P<0.05. Detailed Implementation

[0042] Example 1. CXT-24 and CXT-5 have a protective effect against SOD1 G93A and TDP43 M337V stably transfected cells.

[0043] This embodiment found that compared with the corresponding empty vector control group cells, stable transfected cells could significantly reduce cell survival. After 24 hours of administration of CXT-24 and CXT-5 to SOD1 G93A and TDP43 M337V stable transfected cells, there was a significant protective effect. Specifically, 10 μM CXT-24 and CXT-5 significantly improved the cell survival rate.

[0044] 1. Cells and Plasmids

[0045] Cell lines: SOD1 G93A and TDP43 M337V

[0046] The NSC-34 cell line was purchased from Shanghai Hongshun Biotechnology Co., Ltd.

[0047] The SOD1 G93A stable transfected cell line and the pEGFP empty vector control cell line were constructed in our laboratory.

[0048] The TDP43 M337V stably transfected cell line and the pCI empty vector control cell line were constructed in our laboratory.

[0049] 2. Methods

[0050] 2.1 MTT assay for cell viability

[0051] 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 CXT-24 and CXT-5 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 dissolved overnight at 37°C. The absorbance was then measured at 570 nm.

[0052] 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 CXT-24 and CXT-5 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.

[0053] 2.2 Data Statistical Analysis

[0054] 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 significantly different from their respective empty vector control groups (pEGFP and pCI), with #P < 0.05 indicating a significant difference.

[0055] ##P<0.01, "*" indicates that the cells in each drug-treated group were compared with their respective model group cells, where *P<0.05,

[0056] **P<0.01.

[0057] 3. Results

[0058] 3.1 CXT-24 and CXT-5 enhance the survival of SOD1 G93A cells and TDP43 M337V cells

[0059] The results of this embodiment show that, compared with the empty vector control cell group, the survival rates of SOD1 G93A cells and TDP43M337V cells were significantly reduced. Figure 1 -A and Figure 1 -B), while administration of 10 μM CXT-24 and CXT-5 significantly improved the survival rate of SOD1 G93A cells and TDP43 M337V cells (-B). Figure 1 -A and Figure 1 -B).

[0060] 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 a class of pyranocarbazole oxime derivatives, as shown in Formula I, or pharmaceutically acceptable salts thereof, in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS): R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively. R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3.

2. The use of a class of pyranocarbazole nitroketone derivatives of general formula II or pharmaceutically acceptable salts thereof in the preparation of drugs for the prevention or treatment of amyotrophic lateral sclerosis (ALS): R1 and R2 are independent of each other and can be selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, C(CH3)3, OCH3, OCH2CH3, OCH2CH2CH3, CF3, and OCF3, respectively. R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3; R4 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, 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 pyranocarbazole oxime derivatives as described in any one of claims 1-3, their pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.

6. The application according to claim 5, characterized in that, The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

7. The application according to claim 5, characterized in that, The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nanosuspensions.

8. The application according to any one of claims 1 and 2, characterized in that, The pyranocarbazooxime compounds mentioned above are artificially synthesized.

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