CN110698445A - A kind of 3-amine alkyl phthalide compound, its preparation method and use - Google Patents

Abstract

The invention disclosesA novel 3-amine alkyl phthalide compound (I) and pharmaceutically acceptable salts thereof, a preparation method thereof, a pharmaceutical composition and application thereof in preparing medicaments for treating and/or preventing related diseases of the nervous system, wherein the diseases include but are not limited to vascular dementia, Alzheimer disease, Parkinson disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, glaucoma, ischemic stroke, hemorrhagic stroke, nerve injury caused by brain trauma and the like.

Description

A kind of 3-amine alkyl phthalide compound, its preparation method and use

technical field

The invention belongs to the field of medicinal chemistry, and relates to a new type of 3-aminoalkylphthalide compound (I), its preparation method, pharmaceutical composition and use in the preparation of medicines for treating and/or preventing nervous system-related diseases, including but not limited to vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, glaucoma, ischemic brain Stroke, hemorrhagic stroke, and neurological damage caused by traumatic brain injury.

Background technique

Alzheimer's disease (AD, senile dementia) is a degenerative disease of the central nervous system characterized by progressive cognitive impairment and memory impairment. The high incidence of vascular disease and cancer has risen to the fourth leading cause of death in developed countries such as Europe and the United States. According to the report of the World Health Organization, 10% of people over the age of 65 in the world have intellectual disabilities, one-half of them have dementia, and the incidence rate of people over the age of 85 is nearly 50%. The number of AD patients in my country is about 6-7 million, and the incidence rate exceeds 5%. With the acceleration of the aging process of the global population, its incidence is showing a clear upward trend. According to the "Global Impact of Alzheimer's Disease: 2013-2050" report published by Alzheimer's Disease International in December 2013, AD will become a The biggest health challenge facing the world in the coming decades, the number of patients will rise from 44 million in 2013 to 76 million by 2030, and a staggering 135 million by 2050. The clinical manifestations of AD are the decline of memory ability, orientation ability, thinking and judgment ability, as well as the ability of daily living, and even abnormal mental and behavioral symptoms. Drugs currently approved for the treatment of mild/moderate AD include acetylcholinesterase (AChE) inhibitors, and N -methyl- D -aspartate (NMDA) receptor antagonists for the treatment of severe AD, but Clinical use has shown that these drugs can relieve AD symptoms by increasing acetylcholine levels in patients or inhibiting the excitotoxicity of excitatory amino acids, but cannot effectively prevent or reverse the course of the disease, and can also cause hallucinations, confusion, dizziness, headache, nausea, liver Toxicity, loss of appetite and frequent defecation and other serious side effects, so the long-term efficacy is not ideal. Therefore, there is an urgent need to develop new AD therapeutic drugs that can improve AD symptoms and change the course of the disease.

AD is a disease caused by a variety of factors, the pathogenesis is complex, and its pathogenesis has not yet been fully elucidated. However, studies have shown that the decrease of acetylcholine level in the brain of patients, the excessive production and deposition of β -amyloid, the aggregation of platelets in cerebral blood vessels, the disorder of metal ion metabolism, the imbalance of Ca ²⁺ balance, and the neurological effects caused by the hyperphosphorylation of tau -protein. Various factors, such as fibrillary tangles, hyperactivity of glutamate receptors, oxidative stress producing a large amount of reactive oxygen species (ROS) and free radicals, and neuroinflammation play an important role in the pathogenesis of AD. In response to the above pathogenic factors, researchers adopted the traditional "one drug, one target" drug design strategy, and found a large number of drugs with high activity and high selectivity for a certain target, such as: cholinesterase inhibitors and N -methyl - D -aspartate receptor antagonists, etc. However, these drugs have problems such as single target of action, many toxic and side effects in clinical use, and poor long-term efficacy in AD patients.

In recent years, with the continuous elucidation of the pathogenic mechanism of AD, it has been found that the occurrence and development of AD is characterized by multi-mechanism and multi-factor effects. network control system. Obviously, the development of therapeutic drugs that can simultaneously act on multiple links in the pathological process of AD is an inevitable choice at present. Based on the above results, the researchers proposed a "Multitarget-directed Ligands" (MTDLs) strategy to develop anti-neurodegenerative disease drugs. The so-called "multi-target ligand" means that a single chemical entity acts on multiple targets in the disease network at the same time, and the effect on each target can produce a synergistic effect, so that the total effect is greater than the sum of each single effect. Drugs called "Multifunctional" or "Multipotential". The main difference between multi-target drugs and multi-drug combination and compound drugs is that they can reduce the dosage, improve the therapeutic effect, avoid the interaction between drugs and the resulting toxic and side effects, uniform pharmacokinetic properties, Ease of use, etc. Therefore, the research and development of anti-neurodegenerative disease therapeutic drugs with novel chemical structures, novel mechanisms of action, multi-target effects, and low toxicity and side effects not only meets the urgent needs of the aging society, but also has good market prospects. A large number of clinical studies have confirmed that AChE inhibitors can effectively relieve the symptoms of AD patients, and the short-term treatment effect is positive; therefore, when designing multi-targeted anti-AD drugs, it is usually necessary to retain the AChE inhibitory activity of the compounds (inhibition of this enzyme can improve the symptoms of AD patients to On this basis, one or more other targets or functions with pharmacological synergy are added to achieve multi-target AD treatment effect [1, Huang Shufang et al. Research on multi-target Alzheimer's disease treatment drugs Progress. Chinese Journal of Medicinal Chemistry 2011, 21(6): 433-434; 2. Luo Wen et al. Research progress of multi-targeted small molecule inhibitors in the treatment of Alzheimer's disease. Chinese Journal of Medicinal Chemistry 2011, 21(6) : 442-443]. Obviously, it is still important to design and discover multi-targeted AD therapeutics that simultaneously inhibit acetylcholinesterase, inhibit β -amyloid overproduction and deposition, anti-oxidative stress, anti-platelet aggregation, and anti-neuroinflammatory response. research direction.

SUMMARY OF THE INVENTION

The purpose of the present invention is to disclose a class of 3-aminoalkylphthalide compounds (I) and pharmaceutically acceptable salts thereof;

Another object of the present invention is to disclose the preparation method of such 3-aminoalkylphthalide compounds (I) and pharmaceutically acceptable salts thereof;

Another object of the present invention is to disclose a pharmaceutical composition comprising the 3-aminoalkylphthalide compound (I) and a pharmaceutically acceptable salt thereof;

Another object of the present invention is to disclose that such 3-aminoalkylphthalide compounds (I) and their pharmaceutically acceptable salts have multi-target effects, and can be used in the preparation of medicaments for the treatment and/or prevention of diseases related to the nervous system. Uses, including but not limited to vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, glaucoma, deficiency Hemorrhagic stroke, hemorrhagic stroke, and nerve damage caused by traumatic brain injury.

The general chemical structure of the 3-amine alkylphthalide compound (I) provided by the present invention is:

In the formula: X represents O, NR ₇ or S; A ₁ -A ₂ represents CH-CH ₂ or C=CH; when A ₁ -A ₂ represents C=CH, the compound is in Z -form configuration, E -form configuration, or any ratio mixture of Z -form and E -form configuration; when A ₁ -A ₂ represents CH-CH ₂ , the compound is R configuration, S configuration, or R configuration and Any ratio mixture of S configuration; n represents 1-12; R ₃ and R ₄ each independently represent H, OH, SH, C ₁ ~C ₁₂ alkyl, C ₁ ~C ₁₂ alkoxy, CN, halogen, NR ₅ R ₆ or C ₁ -C ₁₂ alkylthio; R ₅ and R ₆ each independently represent H, C ₁ -C ₁₂ alkyl; NR ₅ R ₆ also represents tetrahydropyrrolyl, morpholinyl or piperidine R ₁ represents H, C ₁ ～C ₁₂ alkyl; R ₂ represents C ₁ ～C ₁₂ alkyl, benzyl or substituted benzyl; NR ₁ R ₂ also represents tetrahydropyrrolyl, morpholinyl, piperidine base, piperidinyl substituted by C ₁ ~C ₁₂ alkyl at 4-position, piperidinyl substituted by benzyl or substituted benzyl at 4-position, piperazinyl, 4-position by C ₁ ~C ₁₂ Piperazinyl substituted by alkyl, piperazinyl substituted by benzyl or substituted benzyl at 4-position; R ₇ represents H, C ₁ ~C ₁₂ alkyl, phenyl or substituted phenyl, benzyl or substituted Benzyl; the above term "halogen" refers to F, Cl, Br, or I; "substituted phenyl" or "substituted benzyl" refers to the benzene ring substituted by 1-4 groups selected from the group below Benzyl: F, Cl, Br, I, C _1-4 alkyl, C _1-4 alkoxy, NR ₈ R ₉ , trifluoromethyl, trifluoromethoxy, nitro, carboxyl, hydroxyl, cyano base, R ₈ and R ₉ each independently represent H or C ₁ ～C ₁₂ alkyl group, NR ₈ R ₉ also represents tetrahydropyrrolyl, morpholinyl or piperidinyl; these substituents can be in any possibility of the benzene ring Location. However, the 3-aminoalkylphthalide compound (I) proposed by the present invention does not include the following compounds:

。

.

The 3-aminoalkylphthalide compound (I) proposed by the present invention can be prepared by the following method:

Using the corresponding 3-bromide (1) as the starting material, react with triphenylphosphine in a suitable solvent to obtain the corresponding 3-triphenylphosphine salt compound (2); the obtained compound 2 is reacted with amine alkyl aldehyde Compounds (3) are subjected to Wittig reaction under appropriate solvent and basic conditions to obtain the E/Z configuration mixture of the corresponding compound (4) (that is, the compound I mixture of E/Z configuration); the mixture of compound (4) can be used In a conventional method, the corresponding compounds of E -form or Z -form configuration can be obtained by separation and purification by silica gel column chromatography; the mixture of the obtained compound (4) can also be directly separated and purified in a suitable solvent by catalytic hydrogenation to separate the double bond. Reduction to obtain the corresponding 3-aminoalkylphthalide compound (I) racemate; use conventional chiral chromatography to separate the corresponding 3-aminoalkylphthalide compound (I) racemate, then The corresponding optical isomers are obtained; its reaction formula is as follows:

In the formula: the definitions of X, R ₁ , R ₂ , R ₃ , R ₄ and n are the same as the general chemical structure of the 3-aminoalkylphthalide compound (I).

For above-mentioned synthetic route, its concrete preparation method is described as follows:

Step A): 3-bromide (1) and triphenylphosphine are reacted in a suitable solvent to obtain the corresponding 3-triphenylphosphine salt compound (2); wherein, the solvent used in the reaction is: C _3-8 aliphatic Ketone, N,N -dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1,4-dioxane, ethylene glycol dimethyl ether or _{C5 -8} alkane, the preferred solvent is 2-methyltetrahydrofuran, ethyl acetate, acetonitrile, toluene, or 1,4-dioxane; the molar feeding ratio of 3-bromide (1): triphenylphosphine is 1.0: 1.0～10.0, the preferred molar charging ratio is 1.0:1.0～5.0; the reaction temperature is 40～150 ℃, and the preferred reaction temperature is 60～120 ℃; the reaction time is 1～120 hours, and the preferred reaction time is 2～72 hours.

Step B): The 3-triphenylphosphine salt compound (2) obtained in step A) and the amine alkyl aldehyde compound (3) are subjected to Wittig reaction under appropriate solvent and basic conditions to obtain the corresponding compound (4). E/Z configuration mixture; wherein, the solvent used in the reaction is: C _1-8 aliphatic alcohol, C _3-8 aliphatic ketone, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, N,N -dimethylformamide, dimethylformamide sulfoxide, dichloromethane, 1,4-dioxane, benzene, toluene, acetonitrile or C _5-8 alkane, the preferred solvent is: chloroform, dichloromethane, acetone, acetonitrile, tetrahydrofuran or toluene; used in the reaction The bases are: alkali metal hydroxides, alkaline earth metal hydroxides, alkali metal carbonates, alkaline earth metal carbonates, alkali metal hydrogen carbonates, alkaline earth metal hydrogen carbonates, alkali metal salts of C _1-8 alcohols, organic Tertiary amines or quaternary ammonium bases (eg: triethylamine, tributylamine, trioctylamine, pyridine, N -methylmorpholine, N -methylpiperidine, triethylenediamine, tetrabutylammonium hydroxide), The preferred base is: potassium hydroxide, sodium hydroxide, potassium carbonate, triethylamine, pyridine or sodium methoxide; the molar feeding ratio of compound (2): compound (3): base is 1.0:1.0~10.0:1.0~10.0, The preferred molar feeding ratio is 1.0:1.0~3.0:1.0~5.0; the reaction temperature is 0~120 ° C, the preferred reaction temperature is room temperature ~ 100 ° C; the reaction time is 20 minutes ~ 48 hours, and the preferred reaction time is 1 ~ 24 hours.

Step C): The mixture of compound (4) obtained in step B) is directly reduced by catalytic hydrogenation in a suitable solvent without separation and purification to obtain the corresponding 3-aminoalkylphthalide compound (I) racemization wherein, the solvent used in the reaction is: C _1-6 fatty alcohols, C _3-8 fatty ketones, C _1-6 fatty acids, C _1-6 fatty acids and C _1-6 fatty alcohols form esters, ethers (such as ether , isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, etc.), benzene, toluene or xylene, aliphatic hydrocarbons (such as: hexane, heptane, octane, etc. ), the preferred solvent is: tetrahydrofuran, methanol, ethanol or isopropanol; the catalyst used in the catalytic hydrogenation is: Raney Ni, PtO ₂ , 1%～30% Pd-C, 1%～30% Pd(OH) ₂ -C, The preferred catalysts are: Raney Ni, PtO ₂ , 5%~20% Pd-C; the mass ratio of compound (4) to the catalyst is 1.0:0.01~1.0; the reaction pressure is normal pressure~10.0 MPa, preferably normal pressure~2.0 MPa; the reaction temperature is room temperature to 150° C., preferably room temperature to 80° C.; the reaction time is 1 to 96 hours, preferably 2 to 50 hours.

The 3-aminoalkylphthalide compound (I) obtained by the above method contains an amino group in the molecule, the amino group is basic, and can be prepared with any suitable acid by a conventional pharmaceutically acceptable salt-forming method. The salt, the acid is: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, sulfamic acid, C _1-6 aliphatic carboxylic acid (such as: formic acid, acetic acid, propionic acid, etc.), trifluoroacetic acid, hard Fatty acid, pamoic 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 acid (such as: methylsulfonic acid, ethylsulfonic acid, etc.), camphorsulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or 1,4- Butanedisulfonic acid.

The starting materials of the present invention - 3-bromo compound (1) and amine alkyl aldehyde compound (3) can be prepared by techniques common in the art, including but not limited to the methods disclosed in the following documents: 1. Guidong Z. et al. WO2011130478A1; 2. Sakamoto F. et al. Chem. Pharm. Bull. 1983, 31(8), 2698-2707; 3. Chunzhi Z. et al. Chinese Journal of Organic Chemistry 2014, 34, 1881-1888; 4. Sugimoto H. et al. US 5100901; 5. Mingyu W. et al. European Journal of Medicinal Chemistry 2016, 121, 864-879.

The pharmaceutical composition disclosed in the present invention comprises a therapeutically effective amount of one or more 3-aminoalkylphthalide compounds (I) or a pharmaceutically acceptable salt thereof, and the pharmaceutical composition may further contain one or more A pharmaceutically acceptable carrier or excipient. The "therapeutically effective amount" refers to the amount of a drug or agent that induces a biological or medical response in a tissue, system or animal targeted by a researcher or doctor; the "composition" refers to a combination of more than one substance or group Parts mixed products; the "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable substances, compositions or carriers, such as: liquid or solid fillers, diluents, excipients, solvents or encapsulations Substances that carry or transport a chemical substance. The ideal ratio of the pharmaceutical composition provided by the present invention is that the 3-aminoalkylphthalide compound (I) or its pharmaceutically acceptable salt as the active ingredient accounts for 2% to 99.5% of the total weight.

The 3-aminoalkylphthalide compounds (I) disclosed in the present invention and their pharmaceutically acceptable salts have been screened for the following biological activities:

(1) Inhibitory activity of 3-aminoalkylphthalide compounds (I) on acetylcholinesterase and butyrylcholinesterase

Add 1.0 mmol/L thioacetylcholine iodide or thiobutyrylcholine iodide (both purchased from Sigma) 30 μL, pH 7.4 PBS buffer 40 μL, and test compound solution 20 μL to the 96-well plate in turn. μL (DMSO content is less than 1%) and 10 μL acetylcholinesterase (5% homogenate supernatant of rat cerebral cortex, pH 7.4 phosphate buffer as homogenization medium) or butyrylcholinesterase (rat serum 25 % supernatant, pH 7.4 phosphate buffer as homogenization medium) solution, after adding and mixing, incubate at 37 °C for 15 min, and add 0.2% 5,5'-dithio-bis(2- Nitrobenzoic acid) (DTNB, purchased from Sigma company) solution 30 μL color developed, the optical density (OD value) of each well at 405nm was measured with a microplate reader, compared with the blank well without the sample to be tested, and the compound pair was calculated. Enzyme inhibition rate (enzyme inhibition rate (%)=(1-sample group OD value/blank group OD value) × 100%); select five to six concentrations of the compound, determine the enzyme inhibition rate, and use the compound mole The negative logarithm of the concentration is linearly regressed with the inhibition rate of the enzyme, and the molar concentration when the inhibition rate of 50% is obtained is the IC ₅₀ of the compound. The measurement results show that the 3-aminoalkylphthalide compounds (I) disclosed in the examples of the present invention [including: the E -form and Z -form configuration targets of the compound (4)] have both acetylcholinesterase properties. Significant inhibitory effect with IC ₅₀ of 1.2×10 ^-3 nM~20.0 µM. Further structure-activity relationship analysis found that under the condition of a certain carbon chain length, the E and Z configurations of the 3-aminoalkylphthalide compounds (I) have little difference in the inhibitory activity of acetylcholinesterase, but After the double bond in the molecule is reduced, the inhibitory activity is reduced; in addition, the chiral configuration of the target has a certain influence on the activity of inhibiting acetylcholinesterase, but they still have significant acetylcholinesterase inhibitory activity, and their IC ₅₀ are both. less than 20.0 µM. The measurement results also show that the inhibitory activity of 3-aminoalkylphthalide compound (I) on acetylcholinesterase is significantly higher than that on butyrylcholinesterase (selectivity is greater than 100 times), indicating that the present invention The disclosed compounds exhibit selective inhibition of acetylcholinesterase, indicating that such compounds are less toxic to the peripheral system. In addition, the assay results also show that the clinically used rivastigmine has an _IC50 of 10.5 µM for AChE inhibition and an _IC50 for butyrylcholinesterase inhibition of 2.6 µM; and the control compound (II) shown below (Y in its chemical structural formula represents O, NH or S, respectively) and the control compound (III) had an IC ₅₀ for inhibition of acetylcholinesterase greater than 150µM;

。

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(2) Inhibitory activity of 3-aminoalkylphthalide compounds (I) on Aβ _1-42 self-aggregation

Refer to the method reported in the literature (Qiang, XM et al. Eur. J Med. Chem. 2014, 76, 314-331) for the determination, that is: the pretreated Aβ _1-42 was made into a stock solution with DMSO, using Before use, dilute to 50 μM with PBS buffer at pH 7.4; the compounds to be tested are made into 2.5 mM stock solution with DMSO, and diluted to the corresponding concentration with PBS buffer at pH 7.4 before use. Take 20 _{μL of Aβ1-42} solution + 20 μL of the test compound solution, 20 _{μL of Aβ1-42} solution + 20 μL of PBS buffer (containing 2% DMSO) were incubated in a 96-well plate at 37°C for 24 h, and then 160 μL of 50 mM glycine containing 5 μM Thioflavin T was added -NaOH buffer (pH=8.5), immediately after shaking for 5s, use a multi-plate reader to measure the fluorescence value at the excitation wavelength of 446 nm and the emission wavelength of 490 nm; the fluorescence value of A β _1-42 + the compound to be tested is marked as IF _i , the fluorescence value of A β _1-42 +PBS buffer was recorded as IF _c , and the fluorescence value only containing PBS buffer was recorded as IF ₀ , the inhibition rate of compound inhibiting the self-aggregation of A β _1-42 was: 100-( IF _i -IF ₀ )/(IF _c -IF ₀ )*100; five to six concentrations of the compound were selected to determine the inhibition rate; each compound was tested three times for each concentration, and curcumin was used as a positive control. The measurement results show that the 3-aminoalkylphthalide compounds (I) disclosed in the examples of the present invention [including: the E -form and Z -form configuration targets of the compound (4)] can self-aggregate on Aβ _1-42 All have significant inhibitory activity, and the inhibition rate of Aβ _1-42 self-aggregation at 25.0µM concentration is between 20.0% and 55.0%; and widely used clinical anti-AD drugs: donepezil, rivastigmine, memantine hydrochloride Amine, as well as the above-mentioned control compound (II) (Y in its chemical structural formula represents O, NH or S, respectively) and control compound (III) at a concentration of 25.0 µM all inhibited the self-aggregation of Aβ _1-42 by less than 10% .

(3) Antiplatelet aggregation activity of 3-aminoalkylphthalide compounds (I)

Three male rabbits were taken, local anesthetized with lidocaine, the common carotid artery was surgically separated and blood was collected, anticoagulated with 3.8% sodium citrate 1:9, centrifuged at 500 r/min for 10 minutes to prepare platelet-rich plasma (PRP). ), the remaining part was centrifuged at 3000 r/min to prepare platelet-poor plasma (PPP), and the platelet aggregation experiment was carried out according to the turbidimetric method. Add 240 μL of PRP and 30 μL of test drugs at different concentrations to the assay tube, incubate for 5 minutes, and then add 30 μL of adenosine diphosphate (ADP) (final concentration of 10 μmol/L) and 30 μL of thrombin (final concentration of 0.5 U/mL) and 30 μL arachidonic acid (AA) (final concentration of 1.0 mmol/L) as inducers, observe and record the maximum aggregation rate within 5 minutes. Using normal saline (NS) as a control, the inhibition rate (%) of each test compound was calculated. The assay results show that the 3-aminoalkylphthalide compounds (I) disclosed in the examples of the present invention [including: the E- and Z -form configuration targets of compound (4)] have an effect on ADP induction, thrombin induction and AA-induced platelet aggregation has a significant inhibitory effect, and the inhibition rate at 50.0 µM concentration is greater than 25.0%. The widely used anti-AD drugs in clinic: donepezil, rivastigmine, memantine hydrochloride, and the above-mentioned control compound (II) (Y in its chemical structural formula represents O, NH or S, respectively) and control compound (III) are in At the same concentration, the inhibition rate of platelet aggregation was less than 15.0%.

(4) Inhibitory activity of 3-aminoalkylphthalide compound (I) on neuroinflammation

(a) Effects of compounds and lipopolysaccharide (LPS) on BV-2 cell viability

The BV-2 cells in logarithmic growth phase were taken to form a cell suspension and inoculated into a 96-well plate, and incubated at 37°C in a 5% CO ₂ cell incubator for 24 h. After the cells adhered, they were replaced with fresh serum-free culture medium 90 10 μL of each concentration of the compound to be tested was added for 30 min of pre-incubation, 3 parallel wells for each concentration, and a blank control group was set at the same time; then with or without LPS, set it in a 37°C, 5% CO ₂ cell incubator to continue Incubate for 24 h, add MTT solution, incubate at 37 °C for 4 h, discard the supernatant, add 200 μL DMSO solution to each well, shake slightly for 10 min, measure the OD value at 490 nm with a microplate reader, and calculate each sample. The average value of OD values measured at different concentrations of the product was calculated, and the cell survival rate was calculated according to the following company: cell survival rate (%) = mean OD value of the drug group/mean OD value of the control group × 100%. The test results show that all 3-aminoalkylphthalide compounds (I) [including: E and Z configuration targets of compound (4)] and LPS disclosed in the examples of the present invention do not exceed 25 μM None of the concentrations showed cytotoxicity (inhibition rate less than <5%).

(b) Effects of 3-aminoalkylphthalide compounds (I) on LPS-induced NO release from BV-2 cells

The BV-2 cells in logarithmic growth phase were taken to form a cell suspension and inoculated into a 96-well plate, and incubated at 37°C in a 5% CO ₂ cell incubator for 24 h. After the cells adhered, they were replaced with fresh serum-free culture medium 90 10 μL of each concentration of the compound to be tested was added to pre-incubate for 30 min, 3 parallel wells for each concentration, and a blank control group was set at the same time; then LPS was added to stimulate, and the culture was continued in a 37°C, 5% CO ₂ cell incubator for 24 hours. h, take the cell culture supernatant of different treatment groups, add equal volume of Griess reagent I and equal volume of Griess reagent II, react in the dark at room temperature for 10 min, and measure the absorbance at 540 nm to detect the level of NO in the cell supernatant ( The specific operation was carried out according to the instructions of the NO detection kit). The test results show that all the 3-aminoalkylphthalide compounds (I) disclosed in the examples of the present invention [including: the E -form and Z -form configuration targets of compound (4)] are in the concentration range of 0.5 μM to 25 μM Both showed strong inhibition of LPS-induced NO production in BV-2 cells (the inhibition rate at 2.5 μM concentration was more than 20.0%), and had an obvious dose-effect relationship; and their inhibitory activity was higher than that under the same concentration. The above-mentioned control compound (II) (Y in its chemical structural formula represents O, NH or S, respectively) and control compound (III) significantly enhanced (n=6, P <0.05), indicating that the 3 -Aminoalkylphthalides (I) have significant anti-neuroinflammatory activity. The experiment further found that the chiral center of compound (I) had no significant effect on the anti-neuroinflammatory activity of the compound.

Detailed ways

The present invention can be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications can be made in the present invention without departing from the spirit and scope of the inventions.

Example 1 General method for the preparation of compound (4)

2.0 mmol of the corresponding 3-bromide (1), 2.4 mmol of triphenylphosphine and 20 ml of toluene were added to the reaction flask, and the reaction was stirred under reflux for 12 to 24.0 hours (the reaction progress was tracked by TLC). The liquid was cooled to room temperature, filtered with suction, the filter cake was washed with toluene and petroleum ether in turn, and dried to obtain the corresponding 3-triphenylphosphine salt compound (2) in a yield of 60.0% ^-88.0 %. Confirmed by H-NMR;

Add 1.0 mmol of 3-triphenylphosphine salt compound (2) prepared in the previous step, 1.3 mmol of amine alkyl aldehyde compound (3) and 30 ml of dichloromethane into the reaction flask, stir evenly, and then add triethylamine 1.5 mmol, and then the reaction was stirred at room temperature for 12-24.0 hours (the reaction progress was tracked by TLC); after the reaction was completed, the solvent was evaporated under reduced pressure, 30 mL of deionized water was added to the residue, and the pH of the reaction solution was adjusted to strong with 10% aqueous hydrochloric acid Acidic, then adjusted the pH of the reaction solution to weakly alkaline with saturated aqueous sodium bicarbonate solution, extracted three times with 120 mL of dichloromethane, combined the organic layers, washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and reduced The solvent was evaporated under pressure, the residue was the E/Z configuration mixture of compound (4), yield: 35.6%-85.0%); the obtained mixture was separated and purified by silica gel column chromatography to obtain the corresponding E -formula or Z -configuration compounds, and their chemical structures were confirmed by ¹ H-NMR and ESI-MS.

Example 2 General method for the preparation of 3-aminoalkylphthalide compounds (I)

Add 1.0 mmol of the compound (4) mixture prepared according to the method in Example 1 and 25 ml of ethanol into the reaction flask. After stirring evenly, 40 mg of 10% Pd/C was added. The reaction was stirred with hydrogen for 2.0 to 24.0 hours (the progress of the reaction was followed by TLC). After the reaction, the solvent was evaporated under reduced pressure, and the residue was purified by silica gel column chromatography (eluent: dichloromethane: methanol = 20 to 30: 1 v /v), the corresponding 3-aminoalkylphthalide compounds (I) were obtained with a yield of 60.5%-92.0%, and their chemical structures were confirmed by ¹ H-NMR, ¹³ C-NMR and ESI-MS; the obtained target The purity of the compounds were all greater than 97.0% as determined by HPLC. The structure of the target product prepared by the above general method is as follows:

；

;

Note: When R ₁ and R ₂ share a cell in the table, it means the substituent "-NR ₁ R ₂ ";

The ¹ H-NMR data of some compounds are as follows:

¹ H NMR (CDCl ₃ ): 7.35-7.27 (m, 4H), 7.26-7.24 (m, 2H), 6.96 (s, 1H), 5.46 (t, J = 7.6 Hz, 1H), 4.00 (s, 3H) ), 3.95 (s, 3H), 3.55 (s, 2H), 2.50 (q, J =7.6 Hz, 2H), 2.49 (t, J = 7.6 Hz, 2H), 2.21 (s,3H), 1.81−1.75 (m, 2H);

¹ H NMR (CDCl ₃ ): 7.30 (s, 1H), 7.29-7.26 (m, 5H), 7.21 (s, 1H), 5.71 (t, J = 8.4 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.57 (s, 2H), 2.60 (q, J = 8.4 Hz, 2H), 2.57-2.53 (m, 2H), 2.26 (s, 3H), 1.84 (t, J = 7.2 Hz , 2H);

¹ H NMR (CDCl ₃ ): 7.32-7.25 (m, 6H), 6.80 (s, 1H), 5.37 (dd, J = 3.6, 7.6Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H) ), 3.52 (s, 2H), 2.41 (t, J = 6.8 Hz, 2H), 2.22 (s, 3H), 2.04-1.99 (m, 1H), 1.73-1.69 (m, 1H), 1.62-1.58 ( m, 2H), 1.54-1.47 (m, 2H);

¹ H NMR (CDCl ₃ ): 7.67 (d, J = 7.6 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H), 7.24 (s, 1H), 7.04 (s, 1H), 6.98 (t, J = 7.6 Hz, 1H), 6.89 (d, J = 7.6 Hz, 1H), 5.53 (t, J = 8.0 Hz, 1H), 4.24 (s, 2H), 4.03 (s, 3H), 3.96 (s, 3H), 3.86 (s, 3H), 3.07 (q, J = 8.0 Hz, 2H), 2.96 (t, J = 6.8 Hz, 2H) , 2.48 (q, J = 7.2 Hz, 2H), 2.21-2.17 ( m, 2H), 1.44 (t, J = 7.2 Hz, 3H);

¹ H NMR (CDCl ₃ ): 7.46 (d, J = 7.6 Hz, 1H), 7.29 (s, 1H), 7.25 (t, J = 7.6 Hz, 1H), 7.20 (s, 1H), 6.90 (t, J = 7.6 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 5.67 (t, J = 8.0 Hz, 1H), 3.97 (s, 3H), 3.93 (s, 3H), 3.78 (s, 5H), 2.78-2.70(m, 4H), 2.62 (q, J = 7.6 Hz, 2H), 1.98-1.88 (m, 2H), 1.16 (t, J = 7.6 Hz, 3H);

¹ H NMR (CDCl ₃ ): 7.44 (d, J = 7.6 Hz, 1H), 7.27 (s, 1H), 7.23 (t, J = 7.6 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H) , 6.86 (d, J = 7.6 Hz, 1H), 6.81 (s, 1H), 5.35 (dd, J = 3.6, 7.6 Hz, 1H), 3.97 (s, 3H), 3.94 (s, 3H), 3.82 ( s, 3H), 3.71 (s, 2H), 2.63 (q, J = 6.8 Hz, 2H), 2.55 (t, J = 7.2 Hz, 2H), 2.04-1.99(m, 1H), 1.73-1.64 (m , 3H), 1.56-1.47 (m, 2H), 1.12 (t, J = 6.8 Hz, 3H);

¹ H NMR (CDCl ₃ ): 7.24 (s, 1H), 7.02 (s, 1H), 5.52 (t, J = 7.6 Hz, 1H), 4.01 (s, 3H), 3.95 (s, 3H), 2.62- 2.55 (m, 6H), 2.49 (q, J = 7.6 Hz, 2H), 1.92-1.85 (m, 2H), 1.75-1.72 (m, 4H), 1.55-1.50 (m, 2H);

¹ H NMR (CDCl ₃ ): 7.30 (s, 1H), 7.22 (s, 1H), 5.73 (t, J = 8.0 Hz, 1H), 4.03 (s, 3H), 3.97 (s, 3H), 2.59 ( q, J = 7.6 Hz, 2H), 2.47 (t, J = 7.2 Hz, 6H), 1.87-1.80 (m, 2H), 1.64-1.60 (m, 4H), 1.48-1.45 (m, 2H);

¹ H NMR (CDCl ₃ ): 7.26 (s, 1H), 6.89 (s, 1H), 5.40 (dd, J = 3.2, 8.0 Hz, 1H), 4.01 (s, 3H), 3.94 (s, 3H), 2.95-2.90 (m, 4H), 2.78 (t, J = 8.0 Hz, 2H), 2.20-2.12 (m, 1H), 1.95-1.88 (m, 6H), 1.80-1.71 (m, 1H), 1.61- 1.45 (m, 4H).

Example 3 General method for preparing salts of 3-aminoalkylphthalide compounds (I) and acids

Add 1.0 mmol of 3-aminoalkylphthalide compound (I) and 25 ml of acetone obtained in Example 1 and Example 2 into the reaction flask, stir evenly, add 2.5 mmol of the corresponding acid, heat up and reflux and stir for 20 After the reaction was completed, it was cooled to room temperature, the solvent was evaporated under reduced pressure, and the salt of 3-aminoalkylphthalide compound (I) was obtained by separation and purification by conventional methods. Its chemical structure was confirmed by ¹ H NMR and ESI-MS.

Claims (8)

1. A3-amine alkyl phthalide compound or pharmaceutically acceptable salt thereof is characterized in that the chemical structural general formula of the compound is shown as (I):

in the formula: x represents O, NR₇Or S; a. the₁-A₂Represents CH-CH₂Or C = CH; when A is₁-A₂When C = CH, the compound isZ-configuration,EA configuration of the formula orZA formula andE-mixtures of formula (la) configuration in any ratio; when A is₁-A₂Represents CH-CH₂When the compound isRThe configuration,SConfiguration, orRConfiguration andSa mixture of the configurations in any ratio; n represents 1 to 12; r₃And R₄Each independently represents H, OH, SH, C₁~C₁₂Alkyl radical, C₁~C₁₂Alkoxy, CN, halogen, NR₅R₆Or C₁~C₁₂An alkylthio group; r₅And R₆Each independently representing H, C₁~C₁₂An alkyl group; NR (nitrogen to noise ratio)₅R₆Also represents tetrahydropyrrolyl, morpholinyl or piperidinyl; r₁Representation H, C₁~C₁₂An alkyl group; r₂Is represented by C₁~C₁₂Alkyl, benzyl or substituted benzyl; NR (nitrogen to noise ratio)₁R₂Also represents tetrahydropyrrolyl, morpholinyl, piperidinyl, 4-position by C₁~C₁₂Piperidinyl substituted by alkyl, piperidinyl substituted by benzyl or substituted benzyl in the 4-position, piperazinyl, piperidinyl substituted by C in the 4-position₁~C₁₂Piperazinyl substituted with alkyl, piperazinyl substituted at the 4-position with benzyl or substituted benzyl; r₇Representation H, C₁~C₁₂Alkyl, phenyl or substituted phenyl, benzyl or substituted benzyl; the term "halogen" as defined above means F, Cl, Br, or I; "substituted phenyl" or "substituted benzyl" refers to a benzyl group on the phenyl ring substituted with 1-4 groups selected from the group consisting of: F. cl, Br, I, C_1-4Alkyl radical, C_1-4Alkoxy, NR₈R₉Trifluoromethyl, trifluoromethoxy, nitro, carboxyl, hydroxyl, cyano, R₈And R₉Each independently represents H or C₁~C₁₂Alkyl radical, NR₈R₉Also represents tetrahydropyrrolyl, morpholinyl or piperidinyl; these substituents may be in any possible position of the phenyl ring;

however, the 3-aminoalkylphthalides (I) do not include the following compounds:

。

2. the 3-aminoalkylphthalides according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is a mixture of the 3-aminoalkylphthalides with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, sulfamic acid, C_1-6Aliphatic carboxylic acid, trifluoroacetic acid, stearic acid, pamoic 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_1-6Salts of alkylsulfonic acids, camphorsulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or 1, 4-butanedisulfonic acid.

3. A process for the preparation of a 3-aminoalkylphthalide compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2, wherein the compound is prepared by:

in the formula: x, R₁、R₂、R₃、R₄And n is defined as the same as the general chemical structure formula of the 3-amine alkyl phthalide compound (I);

step A): taking corresponding 3-bromide (1) as an initial raw material, and reacting with triphenylphosphine in a proper solvent to obtain a corresponding 3-triphenylphosphine salt compound (2);

step B): the 3-triphenylphosphine salt compound (2) obtained in the step A) and the corresponding amino alkyl aldehyde compound (3) are subjected to Wittig reaction in a solvent under the alkaline condition to obtain the corresponding compound (4)E/ZA mixture of configurations; the mixture is separated and purified by silica gel column chromatography by conventional method to respectively obtain correspondingEIs of the formulaZ-a compound of formula (la);

step C): reducing the double bond of the compound (4) obtained in the step B) in a proper solvent through catalytic hydrogenation to obtain a corresponding 3-amine alkyl phthalide compound (I) racemate; separating the corresponding 3-aminoalkyl phthalide compound (I) racemate by using a conventional chiral chromatography to obtain a corresponding optical isomer;

the 3-amine alkyl phthalide compound (I) obtained by the method contains amino which is basic, and can be prepared into pharmaceutically acceptable salts with any suitable acid by a pharmaceutically conventional salt forming method.

4. The method for preparing 3-aminoalkylphthalide compounds or pharmaceutically acceptable salts thereof according to claim 3, wherein the solvent used in the reaction in step A) is: c_3-8An aliphatic ketone,N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C_5-8The molar charge ratio of alkane, 3-bromide (1) and triphenylphosphine is 1.0: 1.0 ~ 10.0.0, the reaction temperature is 40 ~ 150 ℃ and 150 ℃, and the reaction time is 1 ~ 120 hours and 120 hours.

5. The method for preparing 3-aminoalkylphthalide compounds or pharmaceutically acceptable salts thereof according to claim 3, wherein the solvent used in the reaction in step B) is: c_1-8Fatty alcohol, C_3-8Aliphatic ketone, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C_5-8An alkane; the base used in the reaction is: alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, alkaline earth metal carbonate, alkali metal bicarbonate, alkaline earth metal bicarbonate, C_1-8Alkali metal salts of alcohols, triethylamine, tributylamine, trioctylamine, pyridine,N-methylmorpholine,NMethylpiperidine, triethylenediamine, tetrabutylammonium hydroxide, compound (2), compound (3), and base in a molar ratio of 1.0: 1.0 ~ 10.0.0: 1.0 ~ 10.0.0, and a reaction temperatureThe reaction temperature is 0 ~ 120 deg.C, and the reaction time is 20 min ~ 48 h.

6. The method for preparing 3-aminoalkylphthalide compounds or pharmaceutically acceptable salts thereof according to claim 3, wherein the solvent used in the reaction in step C) is: c_1-6Fatty alcohol, C_3-8Aliphatic ketones, C_1-6Fatty acid, C_1-6Fatty acids with C_1-6Esters formed from fatty alcohols, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, benzene, toluene or xylene, hexane, heptane, octane; the catalysts used for the catalytic hydrogenation were: raney Ni, PtO₂、1%~30%Pd-C、1%~30% Pd(OH)₂The mass ratio of the compound (4) to the catalyst is 1.0: 0.01 ~ 1.0.0, the reaction pressure is ~ 10.0.0 MPa at normal pressure, the reaction temperature is ~ 150 ℃ at room temperature, and the reaction time is 1 ~ 96 hours.

7. A pharmaceutical composition comprising a 3-aminoalkylphthalide compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2, together with one or more pharmaceutically acceptable carriers or excipients.

8. Use of a 3-aminoalkylphthalide compound or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 2 in the manufacture of a medicament for the treatment and/or prophylaxis of neurological-related disorders: vascular dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, glaucoma, ischemic stroke, hemorrhagic stroke, and nerve damage due to brain trauma.