3-substituted-3-(piperidinoalkylthio or seleno) phthalides, processes for their preparation and uses thereof

Abstract

This invention discloses a class of 3-substituted-3-(piperidinethio or selenyl)phthalide compounds (I) and their pharmaceutically acceptable salts, their preparation methods, pharmaceutical compositions, and their use in the preparation of drugs for the treatment and / or prevention of neurological diseases, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, Prion's disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, and neurological damage caused by traumatic brain injury;

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to a class of 3-substituted-3-(piperidinethio or selenyl)phthalides and their pharmaceutically acceptable salts, their preparation methods, pharmaceutical compositions, and their use in the preparation of drugs for the treatment and / or prevention of neurological diseases, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, Prion's disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, and neurological damage caused by traumatic brain injury. Background Technology

[0002] Neurodegenerative diseases are a general term for diseases caused by chronic, progressive degeneration of central nervous system tissues. These include Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). Their pathogenesis is closely related to oxidative stress, neuroinflammation, and related damage. Oxidative stress is mediated by reactive oxygen species (ROS) free radicals, including superoxide anions, hydrogen peroxide, and hydroxyl radicals. Under normal physiological conditions, ROS production levels are in dynamic equilibrium with the body's antioxidant capacity. When ROS production exceeds the cellular antioxidant capacity, oxidative stress occurs. The brain is particularly sensitive to oxidative stress, thereby inducing various neurological diseases. Other studies have found that vascular dementia, HIV-related dementia, neuropathic pain, ischemic stroke, hemorrhagic stroke, and nerve damage caused by traumatic brain injury are also closely related to oxidative stress and neuroinflammation in the body.

[0003] Vascular dementia (VD) is a clinical syndrome characterized by intellectual and cognitive impairment caused by various types of cerebrovascular diseases, including ischemic cerebrovascular disease, hemorrhagic cerebrovascular disease, and acute and chronic hypoxic cerebrovascular disease. Due to its complex pathogenesis, there are currently no drugs that can halt the progression of vascular dementia; clinical treatment focuses on improving cerebral blood circulation, brain metabolism, and enhancing brain nutrition.

[0004] Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by cognitive impairment and memory loss. Its incidence is rising annually, making it the third most prevalent disease after cardiovascular disease and cancer. With the accelerating aging of the global population, its incidence is showing a significant upward trend. It is estimated that more than 55 million people worldwide currently suffer from dementia, and the total cost of its treatment and care exceeded US$1.2 trillion in 2020. This number is projected to increase to 152 million by 2050. Because AD clinically manifests as a decline in memory, orientation, thinking, and judgment, as well as reduced daily living abilities and even abnormal mental and behavioral symptoms, patient care is challenging, placing a heavy burden on society and families. Currently approved drugs for treating mild / moderate AD include acetylcholinesterase (AChE) inhibitors, and N-methyl-D-aspartate (NMDA) receptor antagonists for treating severe AD. However, clinical use has shown that while these drugs can alleviate Alzheimer's disease (AD) symptoms by increasing acetylcholine levels or inhibiting the excitatory toxicity of excitatory amino acids, they cannot effectively stop or reverse the disease progression. Furthermore, they can cause serious side effects such as hallucinations, confusion, dizziness, nausea, liver toxicity, loss of appetite, and frequent bowel movements, resulting in unsatisfactory long-term efficacy. Therefore, there is an urgent clinical need to develop novel AD treatments that can both improve AD symptoms and alter the disease progression.

[0005] Alzheimer's disease (AD) is caused by multiple factors and has a complex pathogenesis, which is not yet fully understood. However, studies have shown that decreased acetylcholine levels in the brain, excessive production and deposition of β-amyloid protein, platelet aggregation in cerebral blood vessels, metal ion metabolism disorders, and calcium deficiency are contributing factors. 2+ Multiple factors play important roles in the pathogenesis of Alzheimer's disease (AD), including imbalance, neurofibrillary tangles caused by tau-protein hyperphosphorylation, hyperactive glutamate receptors, oxidative stress leading to large amounts of reactive oxygen species (ROS) and free radicals, and neuroinflammatory responses. To address these pathogenic factors, researchers have employed the traditional "one drug, one target" drug design strategy, discovering numerous drugs with high activity and selectivity for specific targets, such as cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. However, these drugs suffer from limitations such as single-target action, numerous toxic side effects during clinical use, and poor long-term efficacy in AD patients.

[0006] In recent years, with the continuous elucidation of the pathogenesis of neurological diseases, it has been discovered that the occurrence and development of these diseases are characterized by multiple mechanisms and factors, with different mechanisms interconnected and influencing each other, forming a complex network regulatory system in the occurrence and development of these diseases. Clearly, developing therapeutic drugs that can simultaneously act on multiple stages of the pathological process of neurological diseases is an inevitable choice. Based on these findings, researchers have proposed a "multi-target drug" strategy for developing drugs against neurological diseases. A "multi-target drug" refers to a single chemical entity that simultaneously acts on multiple targets in the disease network. Simultaneous action on each target can produce a synergistic effect, making the total effect greater than the sum of the individual effects. The main differences between "multi-target drugs" and "multi-drug combination therapy" and "compound drugs" are: reduced dosage, improved therapeutic effect, avoidance of drug interactions and resulting toxic side effects, uniform pharmacokinetic characteristics, and ease of use. Therefore, developing anti-neurological disease drugs with novel structures and mechanisms, multiple targets, and low toxicity is an important current direction.

[0007] Currently, although numerous studies have published research on designing drugs for neurological diseases using a "multi-target drug" strategy, most of the reported active molecules suffer from drawbacks such as significant differences in activity intensity across different targets. This fails to meet the requirement of adaptability of multi-target drugs to the efficacy intensity of each target, resulting in poor multifunctional synergistic effects and hindering their entry into later-stage clinical development. Therefore, there is an urgent need in this field to discover highly efficient and balanced multifunctional active molecules to enhance their drug-like properties.

[0008] Butylphthalide (trade name: Butylphthalide is a Chinese-developed drug for treating cerebral ischemia-reperfusion injury. It possesses various biological activities, including antiplatelet aggregation, antithrombosis, reduction of cerebral edema, reduction of infarct area, improvement of mitochondrial function and cerebral microcirculation, and inhibition of neuronal apoptosis. Recent studies have also found that butylphthalide has certain therapeutic effects on central nervous system diseases such as vascular dementia, Alzheimer's disease, epilepsy, and Parkinson's disease. Mechanism studies have shown that butylphthalide has a certain protective effect against Aβ-induced neurotoxicity, Aβ or lipopolysaccharide (LPS)-mediated neuroinflammation, and oxidative stress damage to nerve cells, indicating its potential in the treatment of neurodegenerative diseases. However, studies have also shown that although butylphthalide has diverse biological activities, these activities (especially: anti-oxidative stress, anti-neuroinflammatory, and inhibition of Aβ aggregation) are all weak, and it lacks AChE inhibitory activity (inhibition of this enzyme is crucial for improving symptoms in patients with AD and VD), which may be the main reason for the drug's poor clinical efficacy. In addition, the drug is an oily liquid at room temperature and is extremely difficult to dissolve in water due to its high lipid solubility, which further limits its widespread clinical application. Although invention patent CN201810285403.3 discloses "a class of piperidine alkylphthalides, their preparation methods and uses", and discloses that such compounds have acetylcholinesterase inhibitory activity, antioxidant activity, Aβ self-aggregation inhibition activity and anti-platelet aggregation activity, the balance of their multifunctional pharmacological effects is obviously defective. Furthermore, subsequent structure-activity relationship studies have found that the strength of the antioxidant activity of such compounds is closely related to the type of substituent on the phthalide core. When the phthalide core is substituted with "OH", the antioxidant activity of the corresponding compound is 0.8 to 2.0 times that of Trolox. However, when "OH" is replaced with "H", "OCH3", "SCH3", "CH3", "dimethylamino", "tetrahydropyrrole", or "halogen", the antioxidant activity of the corresponding compound decreases by at least 2.0 to 3.5 times. Further pharmacokinetic studies in mice revealed that compounds with "OH" substitution on the phthalide core are easily metabolized and excreted in the peripheral system, resulting in lower blood drug concentrations and shorter half-lives. Furthermore, compounds with "OH" substitution, due to their higher molecular polarity, also cross the blood-brain barrier in smaller quantities, leading to lower brain drug concentrations and poor in vivo efficacy. These studies indicate that while compounds with "OH" substitution on the phthalide core exhibit strong antioxidant activity, their drug-likeness is poor. In addition, invention patent CN202111022118.0 discloses "3-(Amide alkylthio)phthalide compounds, their preparation methods and uses," and discloses that these compounds possess acetylcholinesterase inhibitory activity, antioxidant activity, Aβ self-aggregation inhibition activity, and anti-neuroinflammatory activity. However, further drug-likeness studies found that when these compounds were administered to mice via gavage, they were easily metabolized and inactivated in vivo, resulting in a short half-life (t). 1 / 2The low plasma and cerebral drug concentrations (<2.5h) resulted in poor in vivo efficacy, and the formation of potentially toxic phthalic acid metabolites was also observed. Clearly, enhancing the balance of the compound's multifunctionality and drug-like properties through further structural modifications using butylphthalide as a lead compound remains a challenge. Summary of the Invention

[0009] The purpose of this invention is to disclose a class of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and its pharmaceutically acceptable salts.

[0010] Another object of the present invention is to disclose a method for preparing this type of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and its pharmaceutically acceptable salts.

[0011] Another object of the present invention is to disclose pharmaceutical compositions comprising such 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and its pharmaceutically acceptable salts.

[0012] Another objective of this invention is to disclose that this type of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and its pharmaceutically acceptable salts have multi-target effects, and simultaneously possess AChE inhibition, antioxidant stress, neuroprotective and anti-neuroinflammatory activities. They can be used in the preparation of drugs for the treatment and / or prevention of neurological diseases, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, Prion's disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, and neurological damage caused by traumatic brain injury.

[0013] The general chemical structural formula of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) disclosed in this invention is as follows: In the formula: n represents 0-5; X represents S or Se; R1 and R2 each independently represent H, C1-C6 alkyl, C1-C6 alkoxy, halogen, CN or NR5R6, R5 and R6 each independently represent H, C1-C6 alkyl, or when NR5R6 is cyclic, represent tetrahydropyrrolyl, morpholinyl or piperidinyl; R1 and R2 are in any possible position on their respective benzene rings; R3 represents C1-C6 alkyl, (2-pyridine)methyl, (3-pyridine)methyl, (4-pyridine)methyl, benzyl or substituted benzyl; R4 represents C1-C6 alkyl, C2-C7 alkenyl, wherein the alkenyl bond in the alkenyl group is in any possible position on R4; the "substituted benzyl" refers to a benzyl group on the benzene ring that is substituted by 1-4 groups selected from the following group: halogen, C 1-4 Alkyl, C 1-4Alkoxy, N(CH3)2, CF3, CF3O, NHCOCH3, or CN, these substituents are located at any possible position on the benzylbenzene ring; the "halogen" refers to F, Cl, Br, or I.

[0014] The 3-substituted-3-(piperidinethioyl or selenyl)phthalide (I) disclosed in this invention can be prepared by the following method: using the corresponding o-acylbenzoic acid compound (1) as the starting material, it is directly condensed with the corresponding piperidinethioyl or selenyl alcohol compound (2) under argon protection, appropriate solvent and acid action to obtain the corresponding 3-substituted-3-(piperidinethioyl or selenyl)phthalide (I); the reaction formula is as follows: In the formula, R1, R2, R3, R4, X and n are defined in the same way as the general chemical structure of 3-substituted-3-(piperidinethio or selenyl)phthalide (I).

[0015] The specific preparation method for the above synthetic route is described below: An ortho-acylbenzoic acid compound (1) condenses with the corresponding piperidinyl thio or selenol compound (2) under argon protection, in a suitable solvent, and with the action of an acid, to give the corresponding 3-substituted-3-(piperidinyl thio or selenyl)phthalide (I); wherein the solvent used in the reaction is: C 2-6 Fatty acids, C 2-6 Fatty acids and C 1-6 Esters formed from fatty alcohols, chloroform, dichloromethane, 1,2-dichloroethane, chlorobenzene, o-dichlorobenzene, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, benzene, toluene, acetonitrile, 1,4-dioxane, ethylene glycol dimethyl ether, methanesulfonic acid, ethanesulfonic acid, or C 5-8 Alkanes, preferably solvents such as toluene, chloroform, dichloromethane, acetic acid, ethyl acetate, methanesulfonic acid, ethanesulfonic acid, or acetonitrile; acids used include hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, and C6424 ... 1-6 Alkyl sulfonic acid, phosphoric acid, perchloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid or nitric acid, preferably sulfuric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or trifluoroacetic acid; when the reaction solvent is methanesulfonic acid or ethanesulfonic acid, no additional acid is required; the molar ratio of o-acylbenzoic acid compound (1): piperidinyl sulfide or selenol compound (2): acid is 1.0:0.9~4.0:0~100.0, preferably 1.0:1.0~2.5:1.0~10.0; the reaction temperature is 0℃~150℃, preferably room temperature~100℃; the reaction time is 1~60 hours, preferably 2~48 hours.

[0016] The 3-substituted-3-(piperidinethio or selenyl)phthalide (I) obtained according to the above method is reacted with any suitable acid to prepare its pharmaceutically acceptable salt by pharmaceutically conventional salt-forming methods, wherein the acid is: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, aminosulfonic acid, C 1-6 Fatty carboxylic acids (such as formic acid, acetic acid, propionic acid, etc.), trifluoroacetic acid, stearic acid, acetic acid, oxalic acid, benzoic acid, phenylacetic acid, salicylic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, malic acid, lactic acid, hydroxymaleic acid, pyruvic acid, glutamic acid, ascorbic acid, lipoic acid, C 1-6 Alkyl sulfonic acids (such as methanesulfonic acid, ethyl sulfonic acid, etc.), camphor sulfonic acid, naphthalene sulfonic acid, benzene sulfonic acid, p-toluene sulfonic acid, or 1,4-butanedisulfonic acid.

[0017] The pharmaceutical compositions disclosed in this invention comprise a therapeutically effective amount of one or more 3-substituted-3-(piperidinethio or selenyl)phthalide (I) or a pharmaceutically acceptable salt thereof, and may further contain one or more pharmaceutically acceptable carriers or excipients. The "therapeutically effective amount" refers to the amount of drug or agent that elicits a biological or pharmaceutical response in a tissue, system, or animal targeted by an investigator or physician; the "composition" refers to a product obtained by mixing one or more substances or components; the "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance that carries or transports a chemical substance. Ideally, the active ingredient in the pharmaceutical compositions provided by this invention is 3-substituted-3-(piperidinethio or selenyl)phthalide (I) or a pharmaceutically acceptable salt thereof, comprising 2% to 99.5% of the total weight.

[0018] The 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and its pharmaceutically acceptable salts disclosed in this invention were subjected to the following bioactivity screening: (1) Inhibitory activity of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) against cholinesterase Add 30 μL of 1.0 mmol / L acetylcholine iodide or butyryl iodide, 40 μL of PBS buffer (pH 7.4), 20 μL of the test compound solution (DMSO content less than 1%), and 10 μL of acetylcholinesterase (5% supernatant of rat cerebral cortex homogenate, homogenized in phosphate buffer (pH 7.4) or butyrylcholinesterase (25% supernatant of rat serum, homogenized in phosphate buffer (pH 7.4)) solution sequentially to each well. After adding the solutions, mix well and incubate at 37°C for 15 min. Then add 0.2 μL of PBS buffer to each well. 30 μL of 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) solution was used for color development. The optical density (OD value) of each well at 405 nm was measured using a microplate reader. The results were compared with the blank wells without the test sample to calculate the inhibition rate of the compound on the enzyme (enzyme inhibition rate (%) = (1 - OD value of sample group / OD value of blank group) × 100%). Five to six concentrations of the compound were selected, and their enzyme inhibition rates were measured. A linear regression was performed between the negative logarithm of the molar concentration of the compound and the enzyme inhibition rate to obtain the molar concentration at which 50% inhibition rate was achieved, which is the IC50 of the compound. 50 The assay results show that the 3-substituted-3-(piperidinethio or selenyl)phthalide (I) disclosed in the embodiments of the present invention has a significant inhibitory effect on acetylcholinesterase, and its IC50 value is [missing information]. 50 The concentration range was 1.5 nM to 15.8 μM; and the inhibitory activity of these compounds against acetylcholinesterase was significantly higher than that against butyrylcholinesterase (selectivity greater than 10-fold). Furthermore, the results showed that donepezil and rivastigmine, used clinically, had lower IC50 values ​​for AChE inhibition. 50 The IC50 values ​​for inhibition of butyrylcholinesterase were 37.2 nM and 16.0 μM, respectively. 50 The concentrations were 6.3 μM and 5.9 μM, respectively; and the inhibitory activity of the control compound butylphthalide and the starting materials used in the embodiments of the present invention—o-acylbenzoic acid compound (1) and piperidinyl thio or selenool compound (2)—on acetylcholinesterase was weak (their IC50 values ​​were 6.3 μM and 5.9 μM, respectively). 50 All values ​​are greater than 50 μM. Further research revealed that replacing the "X" in the general chemical formula of 3-substituted-3-(piperidinyl thio or selenyl)phthalide (I) disclosed in this invention with "O" or "NH" significantly reduced the inhibitory activity of the corresponding 3-substituted-3-(piperidinyl oxy or amino)phthalide compounds against acetylcholinesterase (its IC50 value is greater than 50 μM). 50 The values ​​all increased by more than 1.8 times.

[0019] (2) 3-substituted-3-(piperidinethio or selenyl)phthalide (I) for Aβ 1-42 Inhibitory activity of oligomers Preprocessed Aβ 1-42Prepare a stock solution using DMSO, and dilute to 20 μM with PBS buffer (pH 7.4) before use. Prepare a 2.5 mM stock solution of the analyte compound using DMSO, and dilute to the appropriate concentration with PBS buffer (pH 7.4) before use. Take 20 μL of Aβ... 1-42 Solution + 20 μL of the analyte solution, 20 μL of Aβ 1-42 Add 20 μL of PBS buffer (containing 2% DMSO) to a 96-well plate and incubate at 37°C for 24 h. Then add 160 μL of 50 mM glycine-NaOH buffer (pH = 8.5) containing 5 μM thioflavone T, shake for 5 s, and immediately measure the fluorescence value using a multi-functional microplate reader at an excitation wavelength of 446 nm and an emission wavelength of 490 nm. Aβ 1-42 +The fluorescence value of the compound to be tested is denoted as IF. i , Aβ 1-42 The fluorescence value of PBS buffer is denoted as IFc, and the fluorescence value of PBS buffer alone is denoted as IF0. The compound inhibits Aβ. 1-42 The inhibition rate of self-aggregation is: 100 - (IF) i -IF0) / (IFc-IF0)*100; select five to six concentrations of the compound and determine its inhibition rate; repeat the test three times for each compound at each concentration, using curcumin as a positive control. The results show that all 3-substituted-3-(piperidinylthio or selenyl)phthalide (I) disclosed in the embodiments of the present invention are effective against Aβ. 1-42 It exhibits strong inhibitory activity against oligomer formation, showing a significant effect at a concentration of 20.0 μM on Aβ. 1-42 The inhibition rate of oligomer formation ranged from 45.2% to 88.0%; curcumin showed an inhibition rate of 42.8% at this concentration; while the control compound butylphthalide and o-acylbenzoic acid compound (1) showed an inhibition rate of 42.8% against Aβ at a concentration of 20.0 μM. 1-42 The inhibition rate of oligomer formation was less than 20.0%. Further research revealed that when the R4 substituent in the general chemical formula of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) disclosed in this invention was replaced with H, the resulting compounds showed improved resistance to Aβ. 1-42 The inhibition rate of oligomer formation will be significantly reduced by 1.5-2.0 times.

[0020] (3) Inhibitory activity of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) against neuroinflammation (a) Effects of compounds and lipopolysaccharide (LPS) on BV-2 cell viability Logarithmic growth phase BV-2 cells were prepared into cell suspensions and seeded into 96-well plates. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours until cell attachment. Then, 90 μL of serum-free fresh culture medium was added, and 10 μL of each concentration of the test compound was added for pre-incubation for 30 minutes. Three parallel wells were used for each concentration, with a blank control group included. LPS was added or not added, and the plates were incubated at 37°C in a 5% CO2 incubator for another 24 hours. MTT solution was added, and the plates were incubated at 37°C for 4 hours. The supernatant was discarded, and 200 μL of LDMSO solution was added to each well. After gentle shaking for 10 minutes, the OD value was measured at 490 nm using a microplate reader. The mean OD value of each test sample at different concentrations was calculated, and cell viability was calculated as follows: Cell viability (%) = Mean OD value of the drug group / Mean OD value of the control group × 100%. Test results show that all 3-substituted-3-(piperidinethio or selenyl)phthalide (I) and the control compound butylphthalide disclosed in the embodiments of the present invention did not show cytotoxicity (inhibition rate less than <15%) at concentrations not exceeding 30 μM.

[0021] (b) Effect of 3-substituted-3-(piperidinylthio or selenyl)phthalide (I) on LPS-induced NO release from BV-2 cells Log-phase BV-2 cells were prepared into a cell suspension and seeded into 96-well plates. The plates were incubated at 37°C in a 5% CO2 incubator for 24 hours until cell attachment. The medium was then replaced with 90 μL of serum-free fresh culture medium. 10 μL of each concentration of the test compound was added for pre-incubation for 30 minutes, with three parallel wells per concentration. A blank control group was also included. LPS stimulation was then added, and the plates were incubated at 37°C in a 5% CO2 incubator for another 24 hours. The cell culture supernatant from each treatment group was collected, and equal volumes of Griess reagent I and Griess reagent II were added. The reaction was carried out at room temperature in the dark for 10 minutes. The absorbance was measured at 540 nm to detect the NO level in the cell supernatant (the specific procedure was performed according to the NO detection kit instructions). Test results show that all 3-substituted-3-(piperidinyl thio or selenyl)phthalide (I) disclosed in the embodiments of this invention exhibit strong inhibitory effects on LPS-induced NO production in BV-2 cells within the concentration range of 0.5 μM to 25 μM (inhibition rates all exceeding 40.6% at 5.0 μM concentration), and show a clear dose-response relationship; moreover, their inhibitory activity is significantly stronger than that of the control compound butylphthalide and o-acylbenzoic acid compound (1) at the same concentration (inhibition rates all less than 20.0% at 5.0 μM concentration), indicating that the 3-substituted-3-(piperidinyl thio or selenyl)phthalide (I) disclosed in the embodiments of this invention has significant anti-neuroinflammatory activity. Further studies found that when the R4 substituent in the general chemical formula of the 3-substituted-3-(piperidinyl thio or selenyl)phthalide (I) disclosed in this invention is replaced with H, the inhibition rate of LPS-induced NO production in BV-2 cells of the corresponding compounds is reduced by 1.3-1.8 times.

[0022] (4) Protection of H2O2-mediated PC12 cell damage by 3-substituted-3-(piperidinethio or selenyl)phthalide (I) Oxidative stress refers to cytotoxic damage caused by endogenous reactive oxygen species (ROS), which participates in multiple stages of the development of most nervous system diseases and is one of the important causes of neuronal apoptosis. Therefore, this invention uses the H2O2-mediated PC12 cell oxidative damage model to evaluate the antioxidant activity of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) to determine the neuroprotective effect of this type of compound.

[0023] (a) Effects of the compound on PC12 cell viability PC12 cells were prepared into cell suspensions and seeded into 96-well plates. After 24 hours of culture in a 37°C, 5% CO2 incubator, the suspension was replaced with serum-free fresh culture medium. Each concentration of the test compound was added and pre-incubated for 30 minutes, with three parallel wells for each concentration. A blank control group was also included. Cells were then cultured for another 24 hours, followed by the addition of MTT solution and incubation at 37°C for 4 hours. The supernatant was discarded, and DMSO solution was added to each well. After gentle shaking for 10 minutes, the OD value was measured at 490 nm using a microplate reader. The mean OD value of each test sample at different concentrations was calculated, and cell viability was calculated as follows: Cell viability (%) = Mean OD value of the drug group / Mean OD value of the control group × 100%. The test results showed that all 3-substituted-3-(piperidinylthio or selenyl)phthalide (I) disclosed in the embodiments of this invention did not exhibit cytotoxicity (inhibition rate <10.0%) at concentrations not exceeding 20 μM.

[0024] (b) Protective effect of the compound against H2O2-mediated PC12 cell damage The test compounds were dissolved in PBS and diluted to different concentrations with DMEM culture medium before use. PC12 cells were cultured in DMEM medium containing 10% fetal bovine serum at a density of 2 × 10⁶ cells / mL. 5Cells were seeded at 100 μL / well in 96-well plates and incubated at 37°C for 24 h in a 5% CO2 incubator. The culture medium was then replaced with fresh DMEM free of serum and phenol red. Different concentrations of the test compound diluent were added to the drug-treated group (PBS was added to the control and damage groups respectively), and incubation continued for 2 h. Then, 150 μM H2O2 damaging agent was added to both the drug-treated and damage groups (PBS was added to the control group), and incubation continued for another 24 h. MTT solution was then added to each well, and the plates were incubated at 37°C for 4 h. The supernatant was discarded, and DMSO solution was added to each well. After gentle shaking for 10 min, the OD value of each well was measured at 490 nm using a microplate reader, and cell viability was calculated. The control group's cell viability was considered 100%, and the drug-treated and damage groups were expressed as a percentage of the control group. Each experiment was repeated three times independently, with Trolox as a positive control. The results showed that all 3-substituted-3-(piperidinethio or selenyl)phthalide (I) disclosed in the embodiments of the present invention exhibited significant neuroprotective activity at concentrations of 10.0 μM and 2.0 μM, with cell viability rates of 73.0%–96.2% and 60.5%–81.0%, respectively. The cell viability rate of the Trolox (10 μM) group was 67.0%, while the cell viability rate of the damaged group without the test compound was only 49.7%, indicating that 3-substituted-3-(piperidinethio or selenyl)phthalide (I) has strong antioxidant activity. Further research revealed that replacing the "X" group in the general chemical formula of 3-substituted-3-(piperidine thio or selenyl)phthalide (I) disclosed in this invention with "CH2", "O" or "NH" significantly reduced the antioxidant activity of the corresponding 3-substituted-3-(piperidine / oxy / amino)phthalide compounds (cell viability was less than 65.0% at a concentration of 10.0 μM).

[0025] (5) Effects of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) on NaNO2-induced learning and memory consolidation impairment in mice Sodium nitrite (NaNO2) can oxidize hemoglobin in red blood cells to methemoglobin. High doses of NaNO2 can significantly reduce the levels of reduced small molecules (GSH) and reductase systems (SOD, GPx, GR) in the body, thereby causing lipid peroxidation and protein carbonylation, leading to oxidative stress. Therefore, NaNO2-induced mouse models are often used for in vivo activity screening of candidate drugs for anti-oxidative stress.

[0026] SPF-grade ICR mice, with an initial weight of 18-22 grams, were randomly divided into: normal group, model group, positive control group (donepezil hydrochloride), and high, medium and low dose groups of the test drug, with 6 mice in each group. Before the platform jump test, mice in each group were administered the corresponding compound by gavage (once daily for 4 days). Mice in the normal and model groups were administered the same volume of 0.5% CMC-Na solution by gavage. The high- and low-dose groups of the test drug were administered the corresponding drug saline solution (60.0 mg / kg and 10.0 mg / kg), respectively. One hour after administration on the third day, the mice were placed on the platform jump device for 3 minutes to adapt, and then placed on a circular platform and trained with 36V AC current for 5 minutes. The time when the mouse first jumped off the platform was recorded as the training latency. After training, mice in all groups except the normal group were subcutaneously injected with NaNO2 saline solution (90.0 mg / kg). One hour after the last administration the following day, the mice were tested again using the platform jump device. The time when the mouse first jumped off the platform was recorded as the test latency, and the number of times the mouse was shocked after jumping off the platform within 5 minutes was recorded as the number of errors. After the behavioral tests, the mice were decapitated and their brains were removed. The cerebral cortex of the mice was then separated on ice and homogenized according to the test requirements. The supernatant of the homogenate was used to determine the malondialdehyde (MDA) and superoxide dismutase (SOD) content in the mouse cerebral cortex. The blood drug and brain drug concentrations and major metabolites were determined by HPLC / MS.

[0027] The results showed that the tested 3-substituted-3-(piperidinethio or selenyl)phthalide (I) (compounds 1-2-4, 1-2-25, 3-2-14, and 4-1-11 in the examples) improved the learning and memory consolidation impairment induced by NaNO2 in mice at both high and low doses (prolonging the latency and reducing the number of errors), and there were statistically significant differences compared with the model group (p<0.001). Moreover, the activity was significantly higher than that of donepezil hydrochloride at the same dose in clinical use (p<0.01). The results also showed that the tested 3-substituted-3-(piperidinylthio or selenophthalide)phthalide (I) could reduce the MDA content in the mouse cerebral cortex to varying degrees and increase SOD activity at both high and low doses, exhibiting a dose-dependent effect. Its effect was also significantly higher than that of donepezil hydrochloride at the same dose (p<0.01). Therefore, the 3-substituted-3-(piperidinylthio or selenophthalide)phthalide (I) disclosed in this invention can alleviate central oxidative stress in mice induced by NaNO2. Furthermore, brain drug concentration tests showed that in the high-dose group (60.0 mg / kg), the brain C of the above-mentioned test compound... max and brain AUC 0-tThe levels were 1.8-4.0 times higher than those of the corresponding reference standard, 3-substituted-3-(piperidinethio or selenyl)phthalide (I), when R4=H (all other substituents are exactly the same); and no phthalic acid metabolites were detected in vivo for the tested 3-substituted-3-(piperidinethio or selenyl)phthalide (I). Detailed Implementation

[0028] The present invention can be further described through the following embodiments; however, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope of the present invention.

[0029] Example 1: General method for the preparation of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) (Method 1) Under argon protection, 2.0 mmol of the corresponding o-acylbenzoic acid compound (1), 2.6 mmol of piperidinyl sulfonate or selenool compound (2), 3.0 mmol of p-toluenesulfonic acid, and 40 mL of toluene were added to a reaction flask. The mixture was heated to 50-60 °C and stirred for 4.0–30.0 hours (the reaction progress was monitored by TLC). After the reaction was completed, the reaction solution was cooled to room temperature, 40 mL of deionized water was added, and the pH of the reaction solution was adjusted to alkaline with saturated sodium carbonate aqueous solution. The mixture was extracted three times with 120 mL of dichloromethane. The organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30–10 / 1 v / v) to obtain the corresponding target compounds (yield: 30.5%–78.0%). The structures of the compounds were determined by TLC. 1 H-NMR, 13 Confirmed by C-NMR and ESI-MS; the purity of the compounds was greater than 96.0% as determined by HPLC.

[0030] Example 2: General method for the preparation of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) (Method 2) Under argon protection, 2.0 mmol of the corresponding o-acylbenzoic acid compound (1), 2.2 mmol of piperidinyl thiosulfate or selenool compound (2), and 12 mL of methanesulfonic acid were added to a reaction flask and stirred at room temperature for 3.0–48.0 hours (the reaction progress was monitored by TLC). After the reaction was completed, 60 mL of deionized water was added, and the pH of the reaction solution was adjusted to alkaline with saturated sodium carbonate aqueous solution. The mixture was extracted three times with 120 mL of dichloromethane. The organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by vacuum distillation. The residue was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 30–10 / 1 v / v) to obtain the corresponding target compounds (yield: 32.0%–81.8%). The structures of the compounds were determined by [further details needed]. 1 H-NMR, 13 Confirmed by C-NMR and ESI-MS; the purity of the compounds was greater than 96.0% as determined by HPLC.

[0031] The structures of the target compounds prepared using the two general methods described above are as follows: The NMR data for some compounds are as follows: 1H NMR(CDCl3):7.35-7.24(m,5H),7.23(s,1H),6.90(s,1H),4.00(s,3H),3.94(s,3H),3.50(s,2H),2.87(d,J=12.0Hz,2H),2.28-2 .22(m,1H),2.12-2.05(m,1H),1.96-1.90(m,2H),1.92(s,3H),1.55(t,J=12.0Hz,2H),1.41(p,J=8.0Hz,2H),1.24-1.04(m,5H); 13 CNMR(CDCl3):168.8,155.3,150.9,146.5,137.7,129.4(2C),128.2(2C),127.1,117.7, 105.5,103.2,92.1,63.3,56.6,56.4,53.7(2C),35.6,35.1,31.9(2C),29.5,27.6,26.2.

[0032] Example 3 General method for preparing salts of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) with acids 1.2 mmol of 3-substituted-3-(piperidinethio or selenyl)phthalide (I) obtained according to Example 1 or Example 2 above and 25 ml of methanol were added to a reaction flask. After stirring evenly, 2.0 mmol of the corresponding acid was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was then removed by vacuum distillation. The residue was recrystallized to obtain the salt of 3-substituted-3-(piperidinethio or selenyl)phthalide, the chemical structure of which was determined by... 1 Confirmed by H NMR and ESI-MS.

Claims

1. A class of 3-substituted-3-(piperidinethio or selenyl)phthalides or pharmaceutically acceptable salts thereof, characterized in that... The general chemical structural formula of this type of compound is shown in (I): In the formula: n represents 0-5; X represents S or Se; R1 and R2 each independently represent H, C1-C6 alkyl, C1-C6 alkoxy, halogen, CN or NR5R6, R5 and R6 each independently represent H, C1-C6 alkyl, or when NR5R6 is cyclic, represent tetrahydropyrrolyl, morpholinyl or piperidinyl; R1 and R2 are in any possible position on their respective benzene rings; R3 represents C1-C6 alkyl, (2-pyridine)methyl, (3-pyridine)methyl, (4-pyridine)methyl, benzyl or substituted benzyl; R4 represents C1-C6 alkyl, C2-C7 alkenyl, wherein the alkenyl bond in the alkenyl group is in any possible position on R4; the "substituted benzyl" refers to a benzyl group on the benzene ring that is substituted by 1-4 groups selected from the following group: halogen, C 1-4 Alkyl, C 1-4 Alkoxy, N(CH3)2, CF3, CF3O, NHCOCH3, or CN, these substituents are located at any possible position on the benzylbenzene ring; the "halogen" refers to F, Cl, Br, or I.

2. The 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that... R1 and R2 each independently represent H, methyl, methoxy, F, Cl, Br, CN, dimethylamino, tetrahydropyrrolyl, morpholinyl, or piperidinyl, and R1 and R2 can be positioned at any possible position on their respective benzene rings.

3. The 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that... R3 represents methyl, ethyl, isopropyl, benzyl, 2-fluorobenzyl, 3-fluorobenzyl, 4-fluorobenzyl, 2,4-difluorobenzyl, 2-chlorobenzyl, 3-chlorobenzyl, 4-chlorobenzyl, 2-bromobenzyl, 3-bromobenzyl, 2-(acetamido)benzyl, 3-(acetamido)benzyl, 4-(acetamido)benzyl, 2-(methoxy)benzyl, 3-(methoxy)benzyl, 4-(methoxy)benzyl, 2-(methyl)benzyl, 4-(methyl)benzyl, 2-(dimethylamino)benzyl, 4-(dimethylamino)benzyl, 2-(cyano)benzyl, 4-(cyano)benzyl, (2-pyridine)methyl, (3-pyridine)methyl, and (4-pyridine)methyl.

4. The 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in claim 1, characterized in that... R4 represents methyl, n-butyl, -CH2CH=CH2, -(CH2)2CH=CH2, -CH2CH=C(CH3)2, -C(CH3)2CH=CH2, and -(CH2)5CH=CH2.

5. The 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-4, characterized in that... The pharmaceutically acceptable salts are those of 3-substituted-3-(piperidinethio or selenyl)phthalides reacting with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, aminosulfonic acid, and C. 1-6 Fatty acids, trifluoroacetic acid, stearic acid, tartaric acid, oxalic acid, benzoic acid, phenylacetic acid, salicylic acid, maleic acid, fumaric acid, succinic acid, tartaric acid, citric acid, malic acid, lactic acid, hydroxymaleic acid, pyruvic acid, glutamic acid, ascorbic acid, lipoic acid, C 1-6 Salts of alkyl sulfonic acids, camphor sulfonic acids, naphthalene sulfonic acids, benzene sulfonic acids, p-toluene sulfonic acids, or 1,4-butanedisulfonic acids.

6. A method for preparing the 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5, characterized in that... The compound can be prepared by the following method: In the formula: R1, R2, R3, R4, X and n are defined in the same way as the general chemical structure of 3-substituted-3-(piperidinethio or selenyl)phthalide (I); Using the corresponding o-acylbenzoic acid compound (1) as the starting material, it is directly condensed with the corresponding piperidinyl thio or selenol compound (2) under argon protection, appropriate solvent and acid to obtain the corresponding 3-substituted-3-(piperidine thio or selenyl)phthalide (I); then, the salt of 3-substituted-3-(piperidine thio or selenyl)phthalide (I) can be obtained by conventional salting method with acid.

7. The method for preparing the 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in claim 6, characterized in that... The solvent used in the reaction is: C 2-6 Fatty acids, C 2-6 Fatty acids and C 1-6 Esters formed from fatty alcohols, chloroform, dichloromethane, 1,2-dichloroethane, chlorobenzene, o-dichlorobenzene, N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, benzene, toluene, acetonitrile, 1,4-dioxane, ethylene glycol dimethyl ether, methanesulfonic acid, ethanesulfonic acid, or C 5-8 Alkanes; the acids used are hydrochloric acid, sulfuric acid, benzenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, and C. 1-6 Alkyl sulfonic acid, phosphoric acid, perchloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid or nitric acid; when the reaction solvent is methanesulfonic acid or ethanesulfonic acid, no additional acid is required; o-acylbenzoic acid compounds (1): piperidinyl sulfonate or selenol compounds (2): the molar ratio of acid is 1.0:0.9~4.0:0~100.0; the reaction temperature is 0℃~150℃; the reaction time is 1~60 hours.

8. A class of pharmaceutical compositions, characterized in that... It comprises a 3-substituted-3-(piperidinethio or selenyl)phthalide compound as described in any one of claims 1-5, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

9. Use of the 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5 in the preparation of medicaments for the treatment and / or prevention of diseases by inhibiting acetylcholinesterase, resisting oxidative stress, inhibiting Aβ protein aggregation, neuroprotection, or anti-neuroinflammatory purposes.

10. Use of the 3-substituted-3-(piperidinethio or selenyl)phthalide compound or a pharmaceutically acceptable salt thereof as described in any one of claims 1-5 in the preparation of medicaments for the treatment and / or prevention of neurological diseases, including: vascular dementia, Alzheimer's disease, frontotemporal dementia, Prion's disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, and neurological damage caused by traumatic brain injury.

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