Use of pyrrolocarbazole oxime derivatives in the treatment of alzheimer's disease

Abstract

The application belongs to the technical field of medicine, and discloses application of a pyrano-carbazole compound in preventing or treating Alzheimer's disease. 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 is applied in resisting Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to the application of pyranocarbazole oxime derivatives in the treatment of Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by memory loss, cognitive impairment, and behavioral and personality changes. It is the most common type of dementia in the elderly, having a significant impact on patients, their families, and society. Characteristic pathological changes in Alzheimer's disease include neuronal degeneration, the formation of amyloid plaques, and neurofibrillary tangles formed by hyperphosphorylation of tau protein. To date, no drug can completely stop or reverse the progression of AD. The vast majority of drugs used clinically to treat AD are single-target agents, including cholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, and drugs targeting β-amyloid protein (Aβ). Previously, the US FDA approved six drugs for the treatment of AD, including tacrine, donepezil, rivastigmine, galantamine, memantine hydrochloride, and memantine combined with donepezil. Sodium mannoside, approved for marketing in China in 2019, improves cognitive impairment by reshaping the gut microbiota balance and reducing the accumulation of peripheral metabolites phenylalanine / isoleucine, thereby alleviating neuroinflammation in the brain. On July 29, 2024, Alpha Cognition announced that its oral extended-release drug Zunveyl (benzgalantamine) for the treatment of mild to moderate Alzheimer's disease received FDA approval in the United States. While these drugs can alleviate AD symptoms, they are not effective in preventing AD. Therefore, there is an urgent clinical need to develop drugs that have both preventative and therapeutic effects on AD.

[0003] Pyranocarbazole alkaloids possess a wide range of biological activities. This invention introduces oximes and nitrones into the pyranocarbazole alkaloid core to obtain pyranocarbazole oxime derivatives. Pharmacological experiments have confirmed that this compound can significantly improve cognitive dysfunction and alleviate AD-related pathological changes in APP / PS1 mice. Summary of the Invention

[0004] This invention discloses compounds of general formulas I and II. Pharmacological experiments have shown that the compounds and their formulations of this invention have a significant ameliorative effect on cognitive impairment and pathological features in APP / PS1 mice. Therefore, the compounds of this invention can be used for the prevention and / or treatment of Alzheimer's disease.

[0005] The technical problem solved by this invention is to provide the use of pyranocarbazole oxime compounds represented by general formulas I and II and their pharmaceutically acceptable salts in the preparation of drugs for the prevention or treatment of Alzheimer's disease. To solve this technical problem, this invention provides the following technical solution:

[0006] The first aspect of this invention is the use of a class of pyranocarbazole oxime derivatives, as shown in general formula I, or pharmaceutically acceptable salts thereof, in the preparation of drugs for the prevention or treatment of Alzheimer's disease:

[0007]

[0008] 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.

[0009] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2,

[0010] CH(CH3)CH2CH3.

[0011] The first aspect of this invention also provides 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 Alzheimer's disease:

[0012]

[0013] 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.

[0014] R3 is selected from H, CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, CH(CH3)CH2CH3;

[0015] R4 is selected from CH3, CH2CH3, CH2CH2CH3, CH(CH3)2, CH2CH2CH2CH3, CH2CH(CH3)2, C(CH3)3, Ph, Bn.

[0016] The compounds are selected from the following group:

[0017]

[0018]

[0019] 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.

[0020] The pyranocarbazooxime compounds mentioned above are artificially synthesized.

[0021] The second aspect of the present invention is to provide the use of a pharmaceutical composition in the preparation of a drug for the prevention or treatment of Alzheimer's disease, characterized in that the pharmaceutical composition comprises the pyranocarbazole oxime derivatives described in the first aspect of the present invention, their pharmaceutically acceptable salts, and pharmaceutically acceptable carriers.

[0022] The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

[0023] The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nano-suspensions.

[0024] The pharmaceutically acceptable carrier is selected from microcapsules and microspheres, nanoparticles or liposomes.

[0025] The pharmaceutical composition is selected from injections, tablets, capsules, pills, granules, oral liquids, suspensions, sustained-release preparations, subcutaneous implants, and nano-suspensions.

[0026] The pyranocarbazooxime compounds are prepared by extraction from plant materials.

[0027] This pharmaceutical composition is prepared according to methods known in the art. It 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 its pharmaceutical composition is typically 0.1-95% by weight.

[0028] 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.

[0029] 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.

[0030] The compounds of this invention can be formulated into conventional formulations, sustained-release formulations, controlled-release formulations, targeted formulations, and various microparticle delivery systems.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] In addition, colorants, preservatives, flavorings, tasters or other additives may be added to pharmaceutical preparations if necessary.

[0036] 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.

[0037] 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.

[0038] 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. Attached Figure Description

[0039] Figure 1 Compound CXT-24 can improve cognitive impairment in APP / PS1 mice during the Morris water maze and platform jumping tests. A: Effect of CXT-24 on the escape latency of APP / PS1 mice during the navigation phase of the Morris water maze test. B: Effect of CXT-24 on the platform crossing latency of APP / PS1 mice during the spatial exploration phase of the Morris water maze test. C: Effect of CXT-24 on the number of platform crossings in APP / PS1 mice during the spatial exploration phase of the Morris water maze test. D: Effect of CXT-24 on the latency of APP / PS1 mice jumping off the insulated platform in the platform jumping test. E: Effect of CXT-24 on the number of errors in the platform jumping test of APP / PS1 mice.

[0040] Figure 2Compound CXT-24 can alleviate Aβ plaque deposition and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice.

[0041] Figure 3 The compound CXT-24 can reduce the release of inflammatory factors in LPS-stimulated BV2 cells.

[0042] Figure 4 Compound CXT-24 can reduce LPS+Aβ 1-42 Stimulates the release of inflammatory factors from BV2 cells.

[0043] Figure 5 Compound CXT-24 and its derivatives can enhance Aβ 1-42 Survival of SKN cells under injury conditions. Detailed Implementation

[0044] Pharmacological Experiment 1

[0045] The effect of compound CXT-24 on improving cognitive impairment in APP / PS1 mice during the Morris water maze and platform jumping tests.

[0046] 1. Animal grouping and administration

[0047] Animals: APP / PS1 transgenic mice and age-matched control WT mice were bred in our laboratory.

[0048] Grouping and Administration: Patients were divided into four groups: a WT solvent control group (n=8) receiving CMC-Na solvent; an APP / PS1 solvent control group (n=12) receiving CMC-Na solvent; an APP / PS1 CXT-24 10 mg / kg group (n=14) receiving CXT-24 10 mg / kg; and an APP / PS1 CXT-24 20 mg / kg group (n=13) receiving CXT-24 20 mg / kg. All groups were administered the medication by gavage at a volume of 0.1 mL / 10 g, starting at 7 months of age, 6 times per week. The Morris water maze and platform jump tests were performed at week 19 of administration.

[0049] 2. Methods

[0050] 2.1 Morris Water Maze Experiment

[0051] The Morris water maze experiment setup consists of a circular pool, a concealed platform, a camera system, and a trajectory analysis system. The mouse pool is 120 cm in diameter and divided into four quadrants. The concealed platform, 12 cm in diameter, is located in the center of one quadrant, 1 cm below the water surface. During the experiment, the water temperature is maintained at 23 ± 1 °C. Dissolving titanium dioxide in warm water and adding it to the pool turns the surface white, providing a clear contrast to the black mice. Lighting is adjusted to ensure the camera positioning system accurately captures the mice's swimming path. The pool is concealed by curtains, and a number of markers are placed around it to provide reference points for the mice's spatial exploration.

[0052] The Morris water maze experiment consisted of two phases: orientation navigation and spatial exploration. The orientation navigation experiment lasted 7 days, with mice trained 4 times daily. Mice were placed in the water from any quadrant facing the pool wall, and the timer started. If a mouse successfully found a hidden platform within 60 seconds, that time was recorded as the escape latency; otherwise, the latency was calculated as 60 seconds. Regardless of whether the mouse found the platform within 60 seconds, it was allowed to remain on the platform for 30 seconds at the end to reinforce its memory of the platform's location. On the 8th day, the spatial exploration experiment was conducted. The platform was removed, and the mouse entered the water from the quadrant opposite to where the platform was previously located, freely exploring for 60 seconds. The time spent in the original platform quadrant and the opposite quadrant, as well as the number of times the mouse crossed the platform, were recorded.

[0053] After the spatial exploration experiment, a clue-based experiment is conducted. The hidden platform is then returned to the pool, and a small flag is attached to it. The animal is observed to determine if it can locate the platform directly using the flag as a clue. If an animal fails to complete this task more than twice, it may indicate problems with its vision, motor skills, or escape motivation, and that animal is eliminated from the experiment.

[0054] 2.2 Diving Platform Experiment

[0055] The mouse jumping platform apparatus consists of a test box, electric grids, and insulated jumping platforms. The test box comprises several square compartments with a base area of ​​10×10cm. Several electric grids are arranged side-by-side under each compartment and connected to an electrical stimulator. A 4×4×4cm insulated jumping platform is placed in the corner of each compartment for the mouse to avoid the electric shock. The jumping platform experiment is divided into three phases: adaptation, training, and testing. On the first day, during the adaptation phase, the mouse is gently placed on the platform facing the corner and allowed to freely explore the compartment for 5 minutes to adapt to the environment. On the second day, during the training phase, the mouse is again placed on the insulated platform facing the corner, and a 62V, 0.8mA current is applied to the electric grids. When the mouse jumps off the insulated platform and its limbs touch the electric grids, it receives an electric shock. To avoid harm, the mouse must jump back onto the platform. The training phase lasts for 3 minutes, and the number of electric shocks received by each mouse is recorded. If the number is less than 3, the mouse is removed from the platform in the last minute to ensure at least 3 electric shocks for effective training. The third day is the testing period. The mice are then placed stably on the insulated platform again. The time it takes for the mouse to jump off the platform for the first time is recorded as the jumping latency. The number of times the mouse jumps off the platform during the testing period is recorded as the number of errors. After each set of operations, the mouse is sprayed with 75% alcohol and then wiped dry with gauze to remove any odor.

[0056] 2.3 Data Statistical Analysis

[0057] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates a difference compared to the control group, where #p < 0.05 and ##p < 0.01; "*" indicates a difference compared to the model group, where *p < 0.05 and **p < 0.01.

[0058] 3. Experimental Results

[0059] 3.1 Compound CXT-24 can improve cognitive impairment in APP / PS1 mice in the Morris water maze and jumping platform tests.

[0060] The results showed that in the Morris water maze test, compared with the WT Control group, the APP / PS1 Control group exhibited significant spatial memory impairment, with a significantly increased escape latency during the seven-day orientation navigation training, a significantly reduced number of platform crossings during the spatial exploration phase, and a significantly increased platform crossing latency. However, after drug administration, the spatial memory impairment in APP / PS1 mice was somewhat improved, with a significantly reduced escape latency during the seven-day orientation navigation training, a significantly increased number of platform crossings during the spatial exploration phase, and a significantly reduced platform crossing latency. Figure 1In the platform jumping experiment, compared with the WT Control group, the APP / PS1 Control group showed a significantly shorter latency to jump off the insulating platform and a significantly increased number of errors; after drug administration, the APP / PS1 mice showed a significantly increased latency to jump off the insulating platform, a significantly reduced number of errors, and a significant improvement in learning and memory impairment. Figure 1 ).

[0061] In summary, the results of this embodiment suggest that compound CXT-24 can improve cognitive impairment in APP / PS1 mice during the Morris water maze and platform jumping tests.

[0062] Pharmacological Experiment 2

[0063] The effect of compound CXT-24 on improving Aβ plaque deposition and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice.

[0064] 1. Preparation of frozen sections of mouse brain tissue

[0065] After the animal behavior experiment in Example 1 was completed, the following procedures were performed: Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate, their hearts were perfused, and their brains were removed by decapitation. The hemibrain was fixed in 4% PFA solution for 24 hours, followed by dehydration using a gradient of 10%-20%-30% sucrose solution. The soaking volume of PFA and sucrose solutions should be at least 5 times the volume of the mouse hemibrain. After the brain tissue sank to the bottom of the 30% sucrose solution, the hemibrain was retrieved and embedded using OCT embedding medium, then frozen at -80°C. The embedded hemibrain was removed from the -80°C freezer and fixed onto the sample holder of a cryostat using OCT embedding medium. The cryostat chamber temperature, sample holder temperature, and sample holder angle were adjusted, and sagittal sections of the embedded brain tissue were prepared to a thickness of 8 μm. Brain slices from the more complete hippocampal region were collected, mounted flat on glass slides, and frozen at -80°C.

[0066] 2. Methods

[0067] 2.1 Immunofluorescence staining

[0068] Remove frozen sections from -80°C and equilibrate to room temperature for 15 min. Rinse three times with PBS for 3 min each time. Incubate sections for 1 hour with 10% blocking sheep serum (containing 0.5% Triton-X100 to increase membrane permeability) to block non-specific binding sites. Add primary antibody (prepared in PBS) at an appropriate dilution and incubate overnight at 4°C. Discard the primary antibody and rinse sections in PBS for 3 min each time, three times consecutively. Add 1:200 diluted fluorescent secondary antibody and incubate at 37°C in the dark for 1 hour. Discard the secondary antibody and add 1:10000 diluted Hoechst staining solution and incubate at 37°C in the dark for 15 min. Rinse sections in PBS for 3 min each time, three times consecutively. After rinsing once with water, add a few drops of 90% glycerol to the tissue, cover with a coverslip, and mount with nail polish.

[0069] 2.2 Fluorescence microscopy imaging and observation

[0070] Aβ plaques in the hippocampus and cortex of brain tissue sections, as well as the activation of astrocytes and microglia, were observed under 4x and 10x objectives. Frozen sections from 5 animals in each group underwent immunofluorescence staining, and 5 brain slices were taken from each animal. At least 4 fields of view were selected from each brain slice for statistical analysis. The images were analyzed and processed using ImageJ software.

[0071] 2.3 Data Statistical Analysis

[0072] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates a difference compared to the control group, where ###P < 0.001; "*" indicates a difference compared to the model group, where *P < 0.05 and **P < 0.01.

[0073] 3. Experimental Results

[0074] 3.1 Compound CXT-24 can alleviate Aβ plaque deposition and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice.

[0075] The results of this embodiment show that after drug administration, Aβ deposition in the hippocampus and cortex of APP / PS1 mice was reduced, and the expression of astrocyte marker protein GFAP and microglia marker protein Iba-1 was also significantly decreased. Figure 2 ).

[0076] In summary, the results of this embodiment suggest that compound CXT-24 can significantly improve Aβ plaque deposition and neuroinflammation in the cerebral cortex and hippocampus of APP / PS1 mice.

[0077] Pharmacological Experiment 3

[0078] The effect of compound CXT-24 on the release of inflammatory factors in LPS-stimulated BV2 cells

[0079] 1. Cells

[0080] Cells: BV2 cells.

[0081] 2. Methods

[0082] 2.1 Preparation of cell supernatant samples

[0083] When the BV2 cell growth density is 80-90%, the cells are grown at a rate of 2*102 5 Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated for 24 hours. Subsequently, the original complete culture medium was discarded, and low-serum medium containing 2% FBS was added for 6 hours of starvation. The culture plates were then divided into eight groups: Group 1 was the negative control group, receiving only 100 μL of medium containing 2% FBS; Group 2 was the model group, receiving 90 μL of low-serum medium and 10 μL of 10 μg / mL LPS working solution; Groups 3-8 were the drug treatment groups, receiving 80 μL of low-serum medium and 10 μL of 10 μg / mL LPS working solution, and 10 μL of different concentration gradients of CXT-24 working solution (30 nM, 100 nM, 300 nM, 1 μM, 3 μM, or 10 μM) in each well, and incubated for 6 hours. The cell culture supernatant was then collected for the detection of inflammatory factors IL-6 and TNF-α.

[0084] 2.2 ELISA detection of IL-6 and TNF-α secretion levels in supernatant

[0085] The measurement process was performed according to the instructions for the ELISA kit.

[0086] 2.3 Data Statistical Analysis

[0087] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates a comparison with the control group, where ### P < 0.001; "*" indicates a comparison with the model group, where *** P < 0.001.

[0088] 3. Experimental Results

[0089] 3.1 Compound CXT-24 can reduce the release of inflammatory factors from LPS-stimulated BV2 cells.

[0090] The results of this embodiment show that, compared with the control group, the secretion levels of IL-6 and TNF-α in the model group cells were significantly increased. Compared with the model group, CXT-24 administration for 6 hours significantly reduced the secretion levels of IL-6 and TNF-α. Figure 3 ).

[0091] In summary, the results of this embodiment suggest that compound CXT-24 can reduce the release of inflammatory factors in LPS-stimulated BV2 cells.

[0092] Pharmacological Experiment 4

[0093] Compound CXT-24 can reduce LPS+Aβ 1-42 Stimulates the release of inflammatory factors from BV2 cells.

[0094] 1. Cells

[0095] Cells: BV2 cells.

[0096] 2. Methods

[0097] 2.1 Preparation of cell supernatant samples

[0098] When the BV2 cell growth density is 80-90%, the cells are grown at a rate of 2*102 5 Inoculated at a density of 100 cells / mL into 96-well plates and incubated for 24 hours. Then, the original complete culture medium was discarded, and low-serum medium containing 2% FBS was added for 6 hours of starvation. The culture plates were then divided into the following 8 groups: Group 1 was the negative control group, containing 90 μL of medium containing 2% FBS and 10 μL of blank medium; Group 2 was the model group, containing 90 μL of low-serum medium and 10 μL of 1 μg / mL LPS + 100 μM Aβ. 1-42 Mix the working solution; groups 3-8 are the drug administration groups, with 80 μL of low serum culture medium and 10 μL of 1 μg / mL LPS + 100 μM Aβ added to each well. 1-42 Mix the working solution with CXT-24 working solution at different concentration gradients (less than 10 μL): 30 nM, 100 nM, 300 nM, 1 μM, 3 μM, or 10 μM, and incubate for 6 h. Subsequently, aspirate the cell culture supernatant for the detection of inflammatory factors IL-6 and TNF-α.

[0099] 2.2 ELISA detection of IL-6 and TNF-α secretion levels in supernatant

[0100] The measurement process was performed according to the instructions for the ELISA kit.

[0101] 2.3 Data Statistical Analysis

[0102] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates a difference compared to the control group, where ###P < 0.001; "*" indicates a difference compared to the model group, where *P < 0.05, **P < 0.01, and ***P < 0.05.

[0103] 3. Results

[0104] 3.1 Compound CXT-24 can reduce LPS+Aβ 1-42 Stimulates the release of inflammatory factors from BV2 cells.

[0105] The results of this embodiment show that, compared with the control group, the secretion levels of IL-6 and TNF-α in the model group cells were significantly increased. Compared with the model group, CXT-24 administration for 6 hours significantly reduced the secretion levels of IL-6 and TNF-α. Figure 4 ).

[0106] In summary, the results of this embodiment suggest that compound CXT-24 can reduce LPS+Aβ. 1-42 Stimulates the release of inflammatory factors from BV2 cells.

[0107] Pharmacological Experiment 5

[0108] Compound CXT-24 and its derivatives can enhance Aβ 1-42 Survival of SKN cells under damage conditions

[0109] 1. Cells

[0110] Cells: SK-N-SH cells.

[0111] 2. Methods

[0112] 2.1 MTT assay for cell viability

[0113] SK-N-SH cells were used at a rate of 1*10 5 Seeds were planted in 96-well plates at a density of 10 cells / mL, incubated for 24 h, and then 10 μM β-carbohydrate was added. 1-42 Incubate with different concentrations of CXT-24 or 10 μM CXT-24 series derivatives for 24 h, then add 10 μL MTT solution (5 mg / mL) to each well of cultured cells, incubate for 4 hours, discard the supernatant, add 150 μL LDMSO solution to each well, dissolve and measure the absorbance value at 570 nm.

[0114] 2.2 Data Statistical Analysis

[0115] Data are presented as mean ± standard deviation (mean ± SD). Statistical analysis was performed using Student's t-test. "#" indicates a comparison with the control group, where ### P < 0.001; "*" indicates a comparison with the model group, where *** P < 0.001.

[0116] 3. Experimental Results

[0117] 3.1 Compound CXT-24 and its derivatives can enhance Aβ 1-42 Survival of SKN cells under damage conditions

[0118] The results showed that, compared with the control group, the cell survival rate in the model group was significantly reduced, while the cell survival rate was significantly improved after administration of CXT-24 10 μM compared with the model group. Figure 5 Furthermore, as shown in Table 1, many of its derivatives can improve cell survival.

[0119] Table 1. Effects of CXT-24 and its derivatives on Aβ 1-42 The effect of induced neuronal injury on cell survival (n=3)

[0120]

[0121]

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 Alzheimer's disease: 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. 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 Alzheimer's disease: 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 Alzheimer's disease, 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 nano-suspensions.

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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