CN114805263B - 3- (hydroxybenzyl) phthalide compound, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof, a preparation method, a pharmaceutical composition and application thereof in preparing medicaments 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, ischemic cerebral apoplexy, hemorrhagic cerebral apoplexy, nerve injury caused by cerebral trauma and the like;
Description
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and relates to a 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof, a preparation method, a pharmaceutical composition and application thereof in preparing medicaments for treating and/or preventing nervous system related diseases, including but not limited to vascular dementia, alzheimer disease, parkinson disease, huntington disease, HIV related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic cerebral apoplexy, hemorrhagic cerebral apoplexy, nerve injury caused by cerebral trauma and other diseases.
Background
Neurodegenerative diseases are the general names of diseases caused by chronic progressive degenerative changes of central nervous tissue, and include Alzheimer's Disease (AD), parkinson's Disease (PD), huntington's disease (Huntington disease, HD), amyotrophic lateral sclerosis (Amyotrophic lateral sclerosis, ALS), multiple sclerosis (Multiple sclerosis, MS) and the like, and the pathogenesis thereof is closely related to oxidative stress, neuroinflammation and corresponding injury. Oxidative stress is mediated by reactive oxygen (Reactive oxygen species, ROS) radicals, including superoxide anions, hydrogen peroxide, and hydroxyl radicals, among others. Under normal physiological conditions, ROS production levels are in a state of dynamic equilibrium with the organism's antioxidant capacity, and Oxidative stress (Oxidative stress) occurs when ROS production exceeds the cell's antioxidant capacity, whereas the brain is particularly sensitive to Oxidative stress, thereby inducing various neurological diseases. In addition, it has been found that vascular dementia, HIV-related dementia, neuropathic pain, ischemic stroke, hemorrhagic stroke, and nerve injury caused by brain trauma are also closely related to oxidative stress and nerve inflammation of the body.
Vascular dementia (Vascular Dementia, VD) is a clinical syndrome of intellectual and cognitive dysfunction caused by various types of cerebrovascular diseases including ischemic cerebrovascular diseases, hemorrhagic cerebrovascular diseases, acute and chronic hypoxic cerebrovascular diseases, etc. Due to the complex pathogenesis of vascular dementia, no medicine capable of blocking the development of the disease exists at present, and clinical treatment is mainly performed to improve the blood circulation and the brain metabolism of the brain and strengthen the nutrition of the brain. Recent studies have shown that VD patients exhibit impairment of cognitive function, often accompanied by abnormalities in the cholinergic system. The density of the hippocampal ChAT positive neurons and fibers of the VD patient is reduced, the ChAT activity of different parts in the brain is reduced, the concentration of acetylcholine in cerebrospinal fluid of the VD patient is obviously lower than the normal level, and the degree of the reduction of the concentration is positively related to the severity of dementia; cerebral ischemia can lead to increased activity of acetylcholinesterase in the brain; meanwhile, some acetylcholinesterase inhibitors are found to be capable of protecting neuron injury caused by ischemia and promoting nerve injury and recovery of brain function after cerebral ischemia.
Alzheimer's Disease (AD) is a central nervous system degenerative disease mainly composed of progressive cognitive disorder and memory impairment, and the incidence of which is in an increasing trend year by year, becoming a high-incidence disease next to cardiovascular disease and cancer. With the acceleration of the aging process of the global population, the incidence rate of the disease is obviously increased. It is estimated that over 5000 tens of thousands of people worldwide are currently suffering from dementia, and the total cost of treatment and care is over dollars 1 trillion in 2018, and the number of people suffering from dementia will increase to 1.52 billion by 2050. AD is clinically manifested by reduced memory, orientation, thinking and judgment, reduced daily life, even abnormal mental behavior symptoms, and the like, which makes patient care difficult and places a heavy burden on society and families. Drugs currently approved for the treatment of mild/moderate AD are acetylcholinesterase (AChE) inhibitors, as well as for the treatment of severe ADNMethyl-)D-an aspartate (NMDA) receptor antagonist. Clinical application shows that the medicines can relieve AD symptoms by improving the level of acetylcholine in patients or inhibiting the excitotoxicity of excitatory amino acids, but can not effectively prevent or reverse the course of the disease, and can also cause serious toxic and side effects such as illusion, consciousness chaos, dizziness, nausea, hepatotoxicity, inappetence, frequent stool and the like, so that the long-term curative effect is not ideal. Thus, there is a great clinical need to develop new therapeutic agents for AD that have both symptomatic improvement and altered course of disease.
AD is a disease caused by a number of factors,the pathogenesis is complex and has not yet been fully elucidated. However, studies have shown that the level of acetylcholine in the brain of the patient is reduced,βExcessive production and deposition of amyloid, platelet aggregation in cerebral vessels, metal ion metabolism disorder, ca 2+ Dysbalance of,tauMany factors, such as neurofibrillary tangles, glutamate receptor hyperactivity, oxidative stress to produce large amounts of Reactive Oxygen Species (ROS) and free radicals, and neuroinflammatory reactions, caused by protein hyperphosphorylation play an important role in the pathogenesis of AD. For the above-mentioned pathogenesis, researchers have adopted the traditional "one drug one target" drug design strategy, and found a large number of drugs with high activity and high selectivity to a certain target, such as: cholinesterase inhibitorsNMethyl-)D-aspartate receptor antagonists and the like. However, the medicines have the problems of single action target point, more toxic and side effects in clinical use, poor long-term curative effect on AD patients and the like.
In recent years, along with the continuous elucidation of the pathogenesis of AD, the occurrence and development of AD are found to have the characteristics of multi-mechanism and multi-factor effect, and the different mechanisms are mutually related and influenced, so that a complex network regulation and control system in the occurrence and development process of AD is formed. Based on the above results, researchers have proposed a "multi-target drug" strategy to develop anti-neurodegenerative disease drugs. The expression "multi-target drug" refers to a compound in which a single chemical entity can act on multiple targets closely related to treatment in a network of the disease, and the actions on the targets can produce a synergistic effect so that the total effect is greater than the sum of the single effects, and the compound is also called as a "multi-functional" or "multi-potential" drug. The main differences of the multi-target medicine and multi-medicine combined application and the compound medicine are as follows: can reduce the dosage, improve the treatment effect, avoid the interaction between medicines and the toxic and side effect caused by the interaction, has uniform pharmacokinetic property, is convenient to use, and the like. Therefore, research and development of anti-neurodegenerative disease therapeutic drugs with novel chemical structures, novel action mechanisms, multi-target actions and low toxic and side effects are currently important directions. A large number of clinical studies have demonstrated that AChE inhibitors are effective in alleviating the symptoms of dementia patients and have a positive short-term therapeutic effectThe method comprises the steps of carrying out a first treatment on the surface of the Therefore, it is generally necessary to preserve the AChE inhibitory activity of the compound (inhibiting the enzyme is critical for improving symptoms of dementia patients) and add one or more other targets or functions with pharmacological synergism on the basis of the AChE inhibitory activity to achieve the multi-target anti-dementia therapeutic effect when designing multi-target anti-dementia drugs. Obviously, the design and the discovery have the functions of inhibiting acetylcholinesterase and inhibiting acetylcholinesteraseβThe excessive generation and deposition of amyloid, antioxidant stress, metal ion complexation and anti-neuroinflammation multi-target anti-dementia drugs may break through the treatment of related dementia.
Disclosure of Invention
The invention aims to disclose a 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof.
The invention also aims to disclose a preparation method of the 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof.
It is a further object of the present invention to disclose pharmaceutical compositions comprising such 3- (hydroxybenzyl) phthalides (I) and pharmaceutically acceptable salts thereof.
It is still another object of the present invention to disclose the use of the 3- (hydroxybenzyl) phthalide compound (I) and its pharmaceutically acceptable salts for the preparation of a medicament for the treatment and/or prevention of neurological 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, ischemic stroke, hemorrhagic stroke, and brain trauma-induced nerve injury.
The chemical structural general formula of the 3- (hydroxybenzyl) phthalide compound (I) disclosed by the invention is as follows:
wherein:
x represents O, S or NR 6 ；
A 1 -A 2 Represents CH-CH 2 Or c=ch; when A is 1 -A 2 When c=ch, the compound isZ-a configuration,E-configuration, orZ-form of a lens andE-any ratio mixture of the formula configuration; when A is 1 -A 2 Represents CH-CH 2 In the case of the compounds described areRConfiguration(s),SConfiguration or racemate;
R 1 and R is 2 Each independently represents H, OH, CF 3 O、C 1 ~C 6 Alkyl, C 1 ~C 6 Alkoxy, C 1 ~C 6 Alkylthio, halogen or NR 7 R 8 ，R 7 And R is 8 Each independently represents H, C 1 ~C 6 An alkyl group;
R 4 and R is 5 Each independently represents C 1 ~C 12 An alkyl group;
R 6 representation H, C 1 ~C 6 Alkyl, phenyl, substituted phenyl, benzyl;
NR 4 R 5 and NR 7 R 8 Also represents tetrahydropyrrolyl, morpholinyl, piperidinyl, piperazinyl, C-substituted at the 4-position 1 ~C 6 Piperazinyl substituted by alkyl, piperazinyl substituted by benzyl or substituted benzyl at the 4-position;
R 1 、R 2 、-CH 2 NR 4 R 5 and OH may be in any possible position on its corresponding benzene ring;
the term "halogen" refers to F, cl, br or I; "substituted benzyl" or "substituted phenyl" refers to a benzyl or phenyl group substituted on the phenyl ring with 1 to 4 groups selected from the group consisting of: F. cl, br, I, C 1-4 Alkyl, C 1-4 Alkoxy, dimethylamino, cyano, these substituents being in any possible position of the benzene ring.
The 3- (hydroxybenzyl) phthalide compound (I) disclosed by the invention can be prepared by the following method:
reacting the corresponding 3-bromophthalide compound (1) serving as a starting material with triphenylphosphine in a proper solvent to obtain a corresponding 3-triphenylphosphine salt compound2) The method comprises the steps of carrying out a first treatment on the surface of the The obtained compound 2 and hydroxy (aminomethyl) benzaldehyde compound (3) are reacted under proper solvent and alkaline condition to obtain the corresponding compound (4)E/ZConfigurational mixture (i.eE/ZCompound I mixture of configuration); separating and purifying the mixture of the compound (4) by silica gel column chromatography to obtain corresponding compounds respectivelyE-orZ-a compound of formula (la); the obtained mixture of the compound (4) can be directly reduced by reduction reaction in a proper solvent without separation and purification to obtain the corresponding racemate of the 3- (hydroxybenzyl) phthalide compound (I); separating the corresponding racemate of the 3- (hydroxybenzyl) phthalide compound (I) by utilizing a conventional chiral chromatography to obtain a corresponding optical isomer; the reaction formula is as follows:
wherein: x, R 1 、R 2 、R 4 And R is 5 The definition of the compound is the same as that of the chemical structural general formula of the 3- (hydroxybenzyl) phthalide compound (I).
For the above synthetic route, the specific preparation method is described as follows:
step A): reacting the 3-bromophthalide compound (1) with triphenylphosphine in a proper solvent to obtain a corresponding 3-triphenylphosphine salt compound (2); wherein, the solvent used in the reaction is: c (C) 3-8 Aliphatic ketone,N,NDimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C 5-8 Alkanes, preferably solvents such as 2-methyltetrahydrofuran, ethyl acetate, acetonitrile, toluene or 1, 4-dioxane; 3-bromophthalide (1): the molar feed ratio of triphenylphosphine was 1.0: 1.0-10.0, preferably a molar feed ratio of 1.0:1.0 to 5.0; the reaction temperature is 40-150 ℃, and the preferable reaction temperature is 60-120 ℃; the reaction time is 1 to 120 hours, preferably 2 to 72 hours.
Step B): 3-triphenylphosphine salt compound (2) obtained in the step A) and hydroxy (aminomethyl) benzylThe aldehyde compound (3) is reacted with Wittig under proper solvent and alkaline condition to obtain the corresponding compound (4)E/ZA configurational mixture; wherein, the solvent used in the reaction is: c (C) 1-8 Fatty alcohols, C 3-8 Aliphatic ketone, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,N,NDimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C 5-8 Alkanes, preferably solvents are: chloroform, dichloromethane, acetone, acetonitrile, tetrahydrofuran or toluene; the alkali used in the reaction is as follows: alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, alkaline earth metal carbonate, alkali metal hydrogencarbonate, alkaline earth metal hydrogencarbonate, C 1-8 Alkali metal salts of alcohols, organic tertiary amines or quaternary ammonium bases (e.g. triethylamine, tributylamine, trioctylamine, pyridine, 4-dimethylaminopyridine),N-methylmorpholine,NMethylpiperidine, triethylenediamine, tetrabutylammonium hydroxide), preferably the base is: sodium hydroxide, potassium carbonate, triethylamine, pyridine or sodium methoxide; compound (2): compound (3): the molar feed ratio of the alkali is 1.0:1.0 to 10.0: 1.0-10.0, preferably a molar feed ratio of 1.0:1.0 to 3.0:1.0 to 5.0; the reaction temperature is 0-120 ℃, and the preferable reaction temperature is room temperature-100 ℃; the reaction time is 20 minutes to 48 hours, preferably 1 to 24 hours.
Step C): directly reducing double bonds in a proper solvent through reduction reaction without separating and purifying the mixture of the compound (4) obtained in the step B) to obtain a corresponding raceme of the 3- (hydroxybenzyl) phthalide compound (I); wherein, the solvent used in the reaction is: c (C) 1-6 Fatty alcohols, C 3-8 Aliphatic ketones, C 2-6 Fatty acids, C 2-6 Fatty acid and C 1-6 Esters of fatty alcohols, ethers (e.g., diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, etc.), benzene, toluene or xylene, aliphatic hydrocarbons (e.g., hexane, heptane, octane, etc.), preferred solvents are: tetrahydrofuran, methanol, ethanol or isopropanol; the reduction reaction is preferably catalytic hydrogenation reduction, and the catalyst used in the catalytic hydrogenation is as follows: raney Ni, ptO 2 、1%~30% Pd-C、1%~30% Pd(OH) 2 -C, preferably the catalyst is: raney Ni, ptO 2 5% -20% Pd-C; the mass ratio of the compound (4) to the catalyst is 1.0:0.01 to 1.0; the reaction pressure is normal pressure to 10.0MPa, preferably normal pressure to 2.0 MPa; the reaction temperature is room temperature-150 ℃, preferably room temperature-80 ℃; the reaction time is 1 to 96 hours, preferably 2 to 50 hours.
The 3- (hydroxybenzyl) phthalide compound (I) obtained according to the above method can be prepared into pharmaceutically acceptable salts thereof with any suitable acid by a pharmaceutically conventional salt-forming method, wherein the acid is: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, sulfamic acid, C 1-6 Fatty carboxylic acids (e.g. formic acid, acetic acid, propionic acid, etc.), 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 1-6 Alkylsulfonic acids (e.g., methylsulfonic acid, ethylsulfonic acid, etc.), camphorsulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or 1, 4-butanesulfonic acid.
The starting materials of the present invention, 3-bromophthalide (1) and hydroxy (aminomethyl) benzaldehyde (3), can be prepared by techniques common in the art, including, but not limited to, the methods disclosed in the following documents: 1. guilong Z.et al. WO 2011130478A1；2、Sakamoto F. et al.Chem. Pharm. Bull.1983, 31(8), 2698-2707；3、Chunzhi Z. et al.Chinese Journal of Organic Chemistry2014, 34, 1881-1888；4、Aissaoui H. et al.WO 2014072903；5、Fattorusso C. et al.Journal of Medicinal Chemistry2008, 51(5), 1333-1343；6、Karki S.S. et al.Journal of Medicinal Chemistry2016, 59(2), 763-769；6、Torrente E. et al.Journal of Medicinal Chemistry2015, 58(15), 5900-5915。
The disclosed pharmaceutical compositions comprise a therapeutically effective amount of one or more 3- (hydroxybenzyl) phthalide compounds (I) or pharmaceutically acceptable salts thereof, which may further comprise one or more pharmaceutically acceptable carriers or excipients. The "therapeutically effective amount" refers to the amount of a drug or agent that causes a biological or medical response to a tissue, system or animal targeted by a researcher or doctor; the term "composition" refers to a product formed by mixing more than one substance or component; the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or carrier, such as: liquid or solid fillers, diluents, excipients, solvents or encapsulating substances that carry or transport a chemical substance. The ideal proportion of the pharmaceutical composition provided by the invention is that the 3- (hydroxybenzyl) phthalide compound (I) or pharmaceutically acceptable salt thereof is taken as an active ingredient to account for 2 to 99.5 percent of the total weight.
The 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof disclosed by the invention are subjected to the following biological activity screening:
(1) Inhibitory Activity of 3- (hydroxybenzyl) phthalide Compound (I) against acetylcholinesterase and butyrylcholinesterase
Adding 30 mu L of 1.0 mmol/L of thioacetylcholine iodide or thiobutyrylcholine iodide, 40 mu L of PBS buffer with pH7.4, 20 mu L of compound solution to be tested (DMSO content is less than 1%) and 10 mu L of acetylcholinesterase (rat brain cortex 5% homogenized supernatant, phosphate buffer with pH7.4 is taken as homogenized medium) or butyrylcholinesterase (rat serum 25% supernatant, pH7.4 phosphate buffer is taken as homogenized medium) sequentially into a 96-well plate, incubating for 15min at 37 ℃ after adding, adding 30 mu L of 5,5' -dithio-bis (2-nitrobenzoic acid) (DTNB) solution into each well, developing color, measuring the optical density (OD value) of each well at 405nm by an enzyme marker, and calculating the inhibition rate of the compound to enzyme (enzyme inhibition rate (%) = (1-sample group OD value/blank group OD value) ×100%); selecting five to six concentrations of the compound, measuring the enzyme inhibition rate, and obtaining the molar concentration of the compound which is the IC of the compound when the 50% inhibition rate is obtained by linear regression of the negative logarithm of the molar concentration of the compound and the inhibition rate of the enzyme 50 . The measurement results show that the 3- (hydroxybenzyl) phthalide compound (I) (comprising: compound (4)) disclosed in the examples of the present inventionE-orZ-structural target) has remarkable inhibition effect on acetylcholinesterase and IC thereof 50 Is 3.50X10 -3 nM to 10.0 [ mu ] M; wherein the compound of the examples [ ]Z) IC of 2-1-3 50 72.0. 72.0 nM; compounds [ (]E) IC of 2-1-3 50 26.0. 26.0 nM; IC of Compounds 2-1-3 50 2210.0 nM; compounds [ (]Z) IC of 2-1-4 50 31.0. 31.0 nM; compounds [ (]E) IC of 2-1-4 50 660.0 nM; IC of Compounds 2-1-4 50 960.0 nM; compounds [ (]Z) IC of 2-1-5 50 66.0. 66.0 nM; compounds [ (]E) IC of 2-1-5 50 8.83 nM; IC of Compounds 2-1-5 50 33.0. 33.0 nM; compounds [ (]Z) IC of 1-1-5 50 0.092 nM; compounds [ (]E) IC of 1-1-5 50 0.071 nM. Further analysis of the structure-activity relationship shows that when A 1 -A 2 Represents CH-CH 2 When the chiral configuration has no obvious influence on inhibiting the activity of acetylcholinesterase; the determination result also shows that the inhibition activity of the 3- (hydroxybenzyl) phthalide compound (I) to acetylcholinesterase is obviously higher than that of butyrylcholinesterase (the selectivity is more than 100 times), and the compound disclosed by the invention has a selective inhibition effect on acetylcholinesterase, so that the compound has low toxicity to a peripheral system. In addition, the measurement results also show that the clinically used rivastigmine inhibits the AChE IC 50 IC for butyrylcholinesterase inhibition at 12.3. Mu.M 50 3.0 [ mu ] M; and a control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I)) shown below-CH 2 NR 4 R 5 IC for inhibiting acetylcholinesterase by "compound obtained by replacing structural fragment with" -H "and hydroxy (aminomethyl) benzaldehyde compound (3) 50 Are all larger than 100 mu M;
(2) Antioxidant Activity of 3- (hydroxybenzyl) phthalide (I) (ORAC-FL method)
Reference (Qiang, X.M).et al.Eur. J Med. Chem.2014, 76, 314-331), i.e.: 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acidTrolox) 10-80. Mu. Mol/L of the solution was prepared with PBS buffer pH7.4, 250 nmol/L of fluorescein (fluoroscein) was prepared with PBS buffer pH7.4, and 40 mmol/L of 2,2' -azobisisobutylamidine dihydrochloride (AAPH) was prepared with PBS buffer pH7.4 before use. 50-10 mu mol/L of the compound solution and the fluorescein solution are added into a 96-well plate, uniformly mixed, incubated at 37 ℃ for 15min, AAPH solution is added to ensure that the total volume of each well is 200 mu L, uniformly mixed, immediately placed into a Varioskan Flash Multimode Reader (Thermo Scientific) instrument, and continuously measured at 485 nm excitation wavelength and 535 nm emission wavelength for 90 min. Calculate the area under the fluorescence decay curve AUC, wherein the area under the fluorescence decay curve AUC is 1-8 mu mol/LTroloxAs a standard, the antioxidant activity of the compound was expressed asTroloxThe equivalent weight of (2) is calculated as: [ (AUC Sample-AUC blank)/(AUC)Trolox-AUC blank)] ×[(concentration of Trolox/concentration of sample)]Each compound was assayed 3 replicate wells at a time and each set of experiments was independently repeated three times. The measurement results show that the 3- (hydroxybenzyl) phthalide compound (I) (comprising: compound (4)) disclosed in the examples of the present inventionE-orZ-configurational target) isTrolox0.95-3.5 times of the total weight of the compound, which shows that the compound has stronger antioxidant activity. Further structure-activity relationship studies show that the phenolic hydroxyl group in the 3-side chain of the 3- (hydroxybenzyl) phthalide compound (I) is replaced by H, and the' -CH is reserved 2 NR 4 R 5 Structural fragments, the antioxidant activity of the obtained compound is obviously reduced (smaller thanTrolox0.40 times of (2); the antioxidant activity of the control compound (II) is obviously reduced compared with that of a 3- (hydroxybenzyl) phthalide compound (I), and the phenolic hydroxyl group in the 3-side chain in the control compound (II) is replaced by H, so that the obtained compound (neither phenolic hydroxyl group nor' -CH) 2 NR 4 R 5 ") are less thanTroloxIs 0.40 times that of the above.
(3) 3- (hydroxybenzyl) phthalide compound (I) to Aβ 1-42 Inhibition of self-aggregation
Reference (Qiang, X.M).et al.Eur. J Med. Chem.2014, 76, 314-331), i.e.: pretreated Aβ 1-42 Stock solutions were made up with DMSO and diluted to 50 μm with PBS buffer ph7.4 prior to use; the test compound was prepared as a stock solution of 2.5. 2.5 mM in DMSO, diluted to the corresponding concentration in PBS buffer pH7.4 before use, and 20. Mu.L of A was takenβ 1-42 Solution +20. Mu.L of test compound solution, 20. Mu.L of Aβ 1-42 Solution +20. Mu.L of PBS buffer (containing 2% DMSO) in 96-well plates, incubated at 37℃for 24h, then 160. Mu.L of 50mM glycine-NaOH buffer (pH=8.5) containing 5. Mu.M thioflavin T was added, and immediately after shaking for 5s, the fluorescence value was determined with a multifunctional microplate reader at 446nm excitation wavelength and 490nm emission wavelength; a is thatβ 1-42 The fluorescence value of the +test compound is recorded as IF i ，Aβ 1-42 Fluorescence values of +PBS buffer were recorded as IF c The fluorescence value of the buffer containing PBS alone was recorded as IF 0 Compounds inhibit Aβ 1-42 The inhibition rate of self aggregation is as follows: 100- (IF) i -IF 0 )/(IF c -IF 0 ) 100; selecting five to six concentrations of the compound and determining the inhibition thereof; each concentration of each compound was repeated three times with curcumin as positive control. The measurement results show that the 3- (hydroxybenzyl) phthalide compound (I) (comprising: compound (4)) disclosed in the examples of the present inventionE-orZ-configurational target) pair aβ 1-42 Self-aggregation has obvious inhibition activity on A at the concentration of 20.0 mu Mβ 1-42 The inhibition rate of self aggregation is between 25.0 and 65.0 percent; and the anti-AD drugs widely used clinically: donepezil, rivastigmine, memantine hydrochloride, and the above control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I)) "-CH 2 NR 4 R 5 The compound obtained after the structural fragment is replaced by the '-H' and the hydroxy (aminomethyl) benzaldehyde compound (3) have the concentration of 25.0 mu MLower pair Aβ 1-42 The inhibition rate of self aggregation is less than 15.0 percent.
(4) Determination of complexation of 3- (hydroxybenzyl) phthalide Compound (I) with Metal ion
Dissolving CuCl with methanol 2 、ZnCl 2 、FeSO 4 、AlCl 3 And the compound to be tested is prepared into a 75 mu mol/L solution, 100 mu L of the compound to be tested and 100 mu L of the metal ion solution are added into a 96-well plate, uniformly mixed, kept stand at room temperature for 30 min, an ultraviolet absorption curve of the mixture in the range of 200-600 nm is recorded on a Varioskan Flash Multimode Reader (Thermo Scientific) instrument, 100 mu L of the compound to be tested and 100 mu L of the methanol mixed solution are used as a contrast, and the red shift phenomenon of the maximum absorption peak and the intensity of the maximum absorption peak of the metal ion and the compound to be tested mixed solution are observed. The measurement results show that the 3- (hydroxybenzyl) phthalide compound (I) (comprising: compound (4)) disclosed in the examples of the present inventionE-orZ-configurational targets) all exhibit a specific effect on Cu 2+ And Fe (Fe) 2+ Selective complexation; and control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I)) "-CH 2 NR 4 R 5 The "structural fragment substituted with" -H "is a compound) having no complexing effect on all of the four metal ions.
(5) 3- (hydroxybenzyl) phthalide compound (I) vs. Cu 2+ Induced Aβ 1-42 Aggregation inhibition Activity
CuCl is added 2 A75. Mu.M solution was prepared with HEPES buffer, and a stock solution of the compound (2.5. 2.5 mM) and 200. Mu.M A were prepared with HEPES bufferβ 1-42 Stock solution was diluted to 75. Mu.M, and 20. Mu.L of Cu was taken out 2+ Solution +20 mu L Aβ 1-42 Solution +20. Mu.L of test compound solution, 20. Mu.L of Cu 2+ Solution +20 mu L Aβ 1-42 Solution +20. Mu.L HEPES buffer and 60. Mu.L HEPES buffer in 96-well plates, mixing, incubating at 37℃for 24h, then adding 190. Mu.L glycine-NaOH buffer (pH=8.5) containing 5. Mu.M thioflavin T, shaking for 5s and measuring immediately after shaking with a multifunctional microplate reader at 446nm excitation wavelength and 490nm emission wavelengthFluorescence value; cu (Cu) 2+ +Aβ 1-42 Fluorescence values of the +test compounds were recorded as IF i ，Cu 2+ +Aβ 1-42 Fluorescence values of +HEPES buffer were recorded as IF c Fluorescence values containing HEPES buffer alone were recorded as IF 0 Compound pair Cu 2+ Induced Aβ 1-42 The inhibition rate of aggregation is: 100- (IF) i -IF 0 )/(IF c -IF 0 ) *100. Three duplicate wells were assayed for each compound at each concentration, with curcumin as a positive control. The measurement results show that the 3- (hydroxybenzyl) phthalide compound (I) (comprising: compound (4)) disclosed in the examples of the present inventionE-orZ-configuration target) at a concentration of 25.0 μm for Cu 2+ Induced Aβ 1-42 The inhibition rate of aggregation is more than 45.0 percent; and the control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I) -CH 2 NR 4 R 5 The inhibition rate of the compound obtained after the structural fragment is replaced by the' -H) and the hydroxy (aminomethyl) benzaldehyde compound (3) at the concentration of 25.0 mu M is less than 20.0 percent.
(6) Inhibitory Activity of 3- (hydroxybenzyl) phthalide Compound (I) against neuroinflammation
(a) Effect of Compounds and Lipopolysaccharide (LPS) on BV-2 cell Activity
Inoculating BV-2 cells in logarithmic growth phase into 96-well plate, and placing at 37deg.C and 5% CO 2 Culturing in a cell culture box for 24 hours, changing into 90 mu L of fresh culture solution without serum after cells are attached, respectively adding 10 mu L of each concentration of compound to be tested, pre-incubating for 30 min, setting 3 parallel holes of each concentration, and setting a blank control group; then, with or without LPS, the mixture is placed at 37 ℃ and 5% CO 2 Culturing in a cell culture incubator for 24 hours, adding MTT solution, incubating at 37 ℃ for 4 hours, discarding supernatant, adding 200 mu LDMSO solution into each hole, slightly oscillating for 10 minutes, measuring OD value at 490nm by using an enzyme-labeling instrument, calculating the average value of the OD values measured at different concentrations of each sample, and calculating the cell survival rate according to the following companies: cell viability (%) = mean OD of dosing group/mean OD of control group x 100%. Test results show that the embodiment of the inventionAll 3- (hydroxybenzyl) phthalides (I) and LPS disclosed in (A) showed no cytotoxicity (inhibition ratio less than 25. Mu.M)<5%）。
(b) Effect of 3- (hydroxybenzyl) phthalides (I) on LPS-induced release of NO by BV-2 cells
Inoculating BV-2 cells in logarithmic growth phase into 96-well plate, and placing at 37deg.C and 5% CO 2 Culturing 24-h in a cell culture box, changing into 90 mu L of fresh culture solution without serum after cells are attached, adding 10 mu L of each concentration of compound to be tested, pre-incubating for 30 min, setting 3 parallel holes of each concentration, and setting a blank control group; then LPS is added for stimulation, and the mixture is placed at 37 ℃ and 5 percent CO 2 The cell culture was continued in a cell incubator for 24. 24h, the cell culture supernatants from the different treatment groups were taken, an equal volume of Griess reagent I and an equal volume of Griess reagent II were added, reacted at room temperature in the dark for 10min, and absorbance was measured at 540. 540 nm to detect the NO level in the cell supernatant (specific procedure was performed according to the NO detection kit instructions). The test results show that all 3- (hydroxybenzyl) phthalides (I) (including Compound (4)) disclosed in the examples of the present inventionE-orZ-target of formula configuration) shows strong inhibition of LPS-induced NO production of BV-2 cells in the concentration range of 0.5 mu M to 25 mu M (inhibition rate at the concentration of 2.5 mu M exceeds 25.0%), and has obvious dose-effect relationship; and their inhibitory activity is significantly higher than that of the control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I)) "-CH 2 NR 4 R 5 The compound obtained after the structural fragment is replaced by the' -H) and the hydroxy (aminomethyl) benzaldehyde compound (3) (the inhibition rate at the concentration of 2.5 mu M is less than 15.0 percent) show that the 3- (hydroxybenzyl) phthalide compound (I) disclosed in the embodiment of the invention has obvious anti-neuroinflammation activity.
(7) Effect of 3- (hydroxybenzyl) phthalide Compound (I) on scopolamine-induced mouse memory acquisition disorder
SPF grade ICR male mice, 25-30g, were randomly divided into: normal group, model group, positive control group, high test drug levelLow dose groups (15.0 mg/kg, 2.5 mg/kg) of 10 animals each. The tested medicine is administrated by one-time gastric lavage, and the administration volume of the blank group and the model group is 0.1ml/10g; 45 min after administration, normal mice were intraperitoneally injected with physiological saline, and the other animals were each injected with scopolamine (3.0 mg/kg) at a volume of 0.1ml/10g; after molding for 30 min, mice were placed into a non-electrically stimulated Y maze for behavioral testing. During the test, the mice are placed at the tail end of one arm, allowed to freely pass through the maze for 8 minutes, the times of entering each arm and the alternation times are recorded, and the alternation rate is calculated according to the following formula: alternation ratio% = [ alternation number/(total entry number-2)]X 100, results are expressed as mean ± standard deviation, and the differences between groups are analyzed by one-way variance analysis. The measurement results show that under the experimental conditions, the 3- (hydroxybenzyl) phthalide compound (I) (example compound [ ]Z) -1-1-5, compounds ]E) 1-1-5) dose-dependent improvement of acquired memory impairment in scopolamine mice, statistically different from model groupp<0.001 And the activity is obviously higher than that of the clinical medicine rivastigmine at the same molar concentrationp<0.01 Is also stronger than the clinical medicine donepezil with the same molar concentrationp<0.01）。
Detailed Description
The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof.
Example 1A 1 -A 2 General method for preparing 3- (hydroxybenzyl) phthalide compound (I) when C=CH
5.0 mmol of the corresponding 3-bromophthalide compound (1), 6.0 mmol of triphenylphosphine and 80 ml toluene are added into a reaction bottle, and the mixture is heated, refluxed and stirred for reaction for 10 to 30.0 hours (the reaction progress is tracked by TLC); after the reaction is finished, cooling the reaction liquid to room temperature, carrying out suction filtration, washing a filter cake by toluene and petroleum ether in sequence, and drying to obtain a corresponding 3-triphenylphosphine salt compound (2)The yield is 56.0-82.6%, and the chemical structures are all determined by 1 H-NMR confirmation;
adding 1.0 mmol of the 3-triphenylphosphine salt compound (2), 1.2 mmol of the hydroxy (aminomethyl) benzaldehyde compound (3) and 25. 25 ml of methylene dichloride into a reaction bottle, uniformly stirring, adding 1.5 mmol of triethylamine, and stirring at room temperature for reaction for 8.0-28.0 hours (the reaction progress is tracked by TLC); after the reaction is finished, the solvent is distilled off under reduced pressure, 40 mL deionized water is added into the residue, the pH of the reaction solution is regulated to be strong acid by using 10 percent hydrochloric acid aqueous solution, the pH of the reaction solution is regulated to be weak alkaline by using saturated sodium bicarbonate aqueous solution, the reaction solution is extracted for three times by using 150 mL methylene dichloride, the organic layers are combined and washed by using saturated sodium chloride aqueous solution, the organic layers are dried by anhydrous sodium sulfate and filtered, the solvent is distilled off under reduced pressure, and the residue is the compound (4)E/ZA configurational mixture; purifying the mixture by silica gel column chromatography (eluent: dichloromethane: methanol=25-80:1 v/v) to obtain corresponding productZ-3- (hydroxybenzyl) phthalide compound (I) (yield: 18.5% -26.2%) andEthe chemical structure of the 3- (hydroxybenzyl) phthalide compound (I) (yield: 12.3% -22.5%) is that of 1 H-NMR、 13 C-NMR and ESI-MS corroboration; the purity of the obtained compounds is greater than 96.0% as determined by HPLC.
Example 2A 1 -A 2 Represents CH-CH 2 In the process, 3- (hydroxybenzyl) phthalide compound (I) is prepared by
The compound (4) (i.e., A in the chemical structural formula) prepared according to the method of example 1 1 -A 2 Representing c=chE/ZConfiguration mixture) 1.0 mmol and ethanol 30. 30 ml are added into a reaction bottle, after stirring uniformly, 10% Pd/C35.0 mg is added, after hydrogen replacement is carried out for three times, hydrogen is introduced at room temperature and normal pressure to stir for 3.5-28.0 hours (the reaction progress is tracked by TLC), after the reaction is finished, the solvent is distilled off under reduced pressure, and the residue is purified by silica gel column chromatography (eluent: methylene dichloride: methanol=25-80:1 v/v) to obtain the corresponding 3- (hydroxybenzyl) phthalide compound (I) (i.e.: a in the general chemical structure 1 -A 2 Represents CH-CH 2 ) The yield is 60.3-90.0%; its chemistryAll the structures are warp 1 H-NMR、 13 C-NMR and ESI-MS confirm that the purity of the obtained target substance is more than 96.0% by HPLC measurement; the structure of the target object prepared by the general method is as follows:
；
NMR data for some compounds were as follows:
1 H NMR (CDCl 3 ): 7.40 (d, J = 7.6 Hz, 1H), 7.29 (s, 1H), 7.16 (s, 1H), 7.11 (s, 1H), 7.01 (d, J = 7.6 Hz, 1H), 6.22 (s, 1H), 4.06 (s, 3H), 3.97 (s, 3H), 3.80 (s, 2H), 2.66 (q, J = 7.2 Hz, 4H), 1.13 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 ): 167.2, 158.4, 155.2, 151.5, 144.4, 135.3, 133.5, 128.7, 122.5, 120.7, 117.3, 116.1, 105.8, 105.4, 100.7, 56.8, 56.5, 56.4, 46.3 (2C), 11.1 (2C);
1 H NMR (CDCl 3 ): 7.27 (s, 1H), 7.15 (s, 1H), 7.03 (d, J = 7.8 Hz, 1H), 6.98 (s, 1H), 6.87 (d, J = 7.8 Hz, 1H), 6.75 (s, 1H), 3.95 (s, 3H), 3.83 (s, 2H), 3.74 (s, 3H), 2.66 (q, J = 7.2 Hz, 4H), 1.14 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 ): 167.0, 158.6, 154.2, 151.5, 146.5, 133.6, 132.2, 128.6, 122.3, 119.9, 119.0, 116.7, 111.2, 105.4, 104.5, 56.7, 56.3, 55.9, 46.4 (2C), 11.1 (2C);
1 H NMR (CDCl 3 ): 7.27 (s, 1H), 6.93 (d, J = 7.2 Hz, 1H), 6.71 (s, 1H), 6.68 (d, J = 7.2 Hz, 1H), 6.45 (s, 1H), 5.53 (t, J = 7.2 Hz, 1H), 3.92 (s, 3H), 3.83 (s, 3H), 3.80 (d, J = 13.8 Hz, 1H), 3.75 (d, J = 13.8 Hz, 1H), 3.27 (dd, J = 14.4 Hz, J = 7.2 Hz, 1H), 2.90 (dd, J = 14.4 Hz, J = 7.2 Hz, 1H), 2.67-2.61 (m, 4H), 1.12 (t, J = 7.2 Hz, 6H); 13 C NMR (CDCl 3 ): 170.6, 158.3, 154.2, 150.4, 143.9, 135.9, 128.5, 120.8, 120.4, 118.0, 117.3, 105.9, 104.1, 80.6, 56.4, 56.2, 56.1, 46.2 (2C), 40.9, 11.0 (2C);
1 H NMR (CDCl 3 ): 7.38 (d, J = 7.8 Hz, 1H), 7.29 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.22 (s, 1H), 4.06 (s, 3H), 3.97 (s, 3H), 3.86 (s, 2H), 2.67 (m, 4H), 1.87 (m, 4H); 13 C NMR (CDCl 3 ): 167.3, 158.1, 155.2, 151.5, 144.5, 135.3, 133.7, 128.3, 122.8, 120.8, 117.2, 116.1, 105.8, 105.5, 100.7, 58.6, 56.5, 56.4, 53.5 (2C), 23.7 (2C);
1 H NMR (CDCl 3 ): 7.26 (s, 1H), 7.13 (s, 1H), 7.07 (d, J = 7.2 Hz, 1H), 7.01 (s, 1H), 6.89 (d, J = 7.2 Hz, 1H), 6.74 (s, 1H), 3.95 (s, 3H), 3.91 (s, 2H), 3.75 (s, 3H), 2.71 (m, 4H), 1.90 (m, 4H); 13 C NMR (CDCl 3 ): 167.0, 158.2, 154.2, 151.5, 146.5, 133.9, 132.2, 128.3, 122.4, 120.0, 119.0, 116.8, 111.2, 105.4, 104.5, 58.3, 56.3, 56.0, 53.4 (2C), 23.6 (2C);
1 H NMR (CDCl 3 ): 7.25 (s, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.76 (s, 1H), 6.68 (d, J = 8.0 Hz, 1H), 6.46 (s, 1H), 5.53 (t, J = 6.8 Hz, 1H), 3.92 (s, 3H), 3.86 (m, 2H), 3.83 (s, 3H), 3.25 (dd, J = 13.6 Hz, J = 6.8 Hz, 1H), 2.92 (dd, J = 13.6 Hz, J = 6.8 Hz, 1H), 2.70 (brs, 4H), 1.84 (brs, 4H); 13 C NMR (CDCl 3 ): 170.6, 157.9, 154.2, 150.4, 143.8, 136.2, 128.3, 120.8, 120.5, 118.0, 117.3, 105.9, 104.1, 80.6, 57.8, 56.2, 56.1, 53.3 (2C), 40.9, 23.5 (2C);
1 H NMR (CDCl 3 ): 7.37 (d, J = 8.0 Hz, 1H), 7.28 (s, 1H), 7.20 (s, 1H), 7.11 (s, 1H), 7.01 (d, J = 8.0 Hz, 1H), 6.22 (s, 1H), 4.06 (s, 3H), 3.97 (s, 3H), 3.73 (s, 2H), 2.56 (brs, 4H), 1.67 (m, 4H), 1.52 (brs, 2H); 13 C NMR (CDCl 3 ): 167.2, 158.1, 155.2, 151.5, 144.6, 135.3, 133.8, 129.1, 121.6, 120.8, 117.4, 116.1, 105.7, 105.5, 100.8, 61.5, 56.5, 56.4, 53.8 (2C), 25.6 (2C), 23.8;
1 H NMR (CDCl 3 ): 7.27 (s, 1H), 7.14 (s, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.99 (s, 1H), 6.87 (d, J = 7.6 Hz, 1H), 6.74 (s, 1H), 3.97 (s, 3H), 3.74 (s, 5H), 2.56 (brs, 4H), 1.66 (m, 4H), 1.53 (brs, 2H); 13 C NMR (CDCl 3 ): 167.0, 158.3, 154.2, 151.5, 146.5, 133.6, 132.2, 128.8, 121.7, 119.9, 119.0, 116.7, 111.2, 105.3, 104.4, 61.7, 56.3, 55.9, 53.8 (2C), 25.7 (2C), 23.8;
1 H NMR (CDCl 3 ): 7.26 (s, 1H), 6.93 (d, J = 6.8 Hz, 1H), 6.72 (s, 1H), 6.68 (d, J = 6.8 Hz, 1H), 6.44 (s, 1H), 5.53 (t, J = 6.8 Hz, 1H), 3.92 (s, 3H), 3.83 (s, 3H), 3.71 (d, J = 14.4 Hz, 1H), 3.75 (d, J = 14.4 Hz, 1H), 3.66 (dd, J = 13.6 Hz, J = 6.8 Hz, 1H), 2.90 (dd, J = 13.6 Hz, J = 6.8 Hz, 1H), 2.53 (brs, 4H), 1.65 (m, 4H), 1.51 (brs, 2H); 13 C NMR (CDCl 3 ): 170.6, 158.1, 154.2, 150.4, 143.8, 136.0, 128.8, 120.4, 120.2, 118.0, 117.4, 106.0, 104.1, 80.6, 61.5, 56.2, 56.1, 53.7 (2C), 41.0, 25.6 (2C), 23.8;
1 H NMR (CDCl 3 ): 7.64 (s, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.07 (s, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.20 (s, 1H), 4.04 (s, 3H), 3.96 (s, 3H), 3.76 (s, 2H), 2.65-2.19 (brs, 4H), 1.67 (m, 4H), 1.52 (brs, 2H); 13 C NMR (CDCl 3 ): 167.6, 158.8, 155.3, 151.2, 142.6, 135.7, 131.1, 130.3, 124.5, 121.8, 116.6, 115.6, 106.1, 105.4, 100.4, 61.7, 56.5, 56.4, 53.8 (2C), 25.6 (2C), 23.8;
1 H NMR (CDCl 3 ): 7.33 (d, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.07 (s, 1H), 7.05 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H), 6.71 (s, 1H), 3.95 (s, 3H), 3.73 (s, 3H), 3.72 (s, 2H), 2.63-2.24 (brs, 4H), 1.68 (m, 4H), 1.53 (brs, 2H); 13 C NMR (CDCl 3 ): 167.0, 158.4, 154.2, 151.4, 145.6, 132.3, 129.6, 129.5, 123.6, 121.9, 118.9, 116.2, 111.4, 105.4, 103.8, 61.8, 56.3, 56.0, 53.8 (2C), 25.7 (2C), 23.7。
example 33 (hydroxybenzyl) phthalides (I) and acid salt formation
Adding 1.0 mmol of 3- (hydroxybenzyl) phthalide compound (I) obtained in the embodiment 1 or 2 and acetone 30 ml into a reaction bottle, stirring uniformly, adding 3.0 mmol of corresponding acid, heating, refluxing and stirring for reaction for 20 min, cooling to room temperature after the reaction is finished, decompressing and distilling off the solvent, recrystallizing the residue with acetone, filtering out the separated solid to obtain the salt of the 3- (hydroxybenzyl) phthalide compound (I), wherein the chemical structure is that 1 H NMR and ESI-MS corroborations.
Claims (10)
1. A3- (hydroxybenzyl) phthalide compound or pharmaceutically acceptable salt thereof is characterized in that the chemical structural general formula of the compound is shown as (I):
x represents O, S or NR 6 ；
A 1 -A 2 Represents CH-CH 2 Or c=ch; when A is 1 -A 2 When c=ch, the compound is in the Z-configuration, E-configuration, or any ratio mixture of Z-and E-configurations; when A is 1 -A 2 Represents CH-CH 2 When the compound is in R configuration, S configuration or racemate;
R 1 and R is 2 Each independently represents H, OH, CF 3 O, methyl, methoxy, methylthio, halogen or NR 7 R 8 ，R 7 And R is 8 Each independently represents methyl;
R 4 and R is 5 Each independently represents methyl or ethyl;
R 6 represents H, methyl, phenyl, benzyl;
NR 4 R 5 and NR 7 R 8 Also represents tetrahydropyrrolyl, morpholinyl, piperidinyl, piperazinyl substituted at the 4-position with methyl, piperazinyl substituted at the 4-position with benzyl or substituted benzyl;
R 1 、R 2 、-CH 2 NR 4 R 5 and OH may be in any possible position on its corresponding benzene ring;
halogen refers to F, cl, br or I; the substituted benzyl refers to benzyl substituted by 1-4 groups selected from the following groups on the benzene ring: F. cl, br, I or methoxy, these substituents being in any possible position of the benzene ring.
2. The 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 1 And R is 2 Represents H, OH, methoxy, trifluoromethoxy, dimethylamino, tetrahydropyrrolyl, piperidinyl, methylthio, methyl or Cl.
3. The 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R 4 And R is 5 Represents methyl or ethyl; NR (NR) 4 R 5 Ring-forming means tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl, 4-methylpiperazinyl, 4-benzylpiperazinyl, 4- (3-fluorobenzyl) piperazinyl, 4- (2-methoxybenzyl) piperazinyl.
4. A 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the pharmaceutically acceptable salt is a mixture of the 3- (hydroxybenzyl) phthalide compound with hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, sulfamic acid, C 1-6 Fatty 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 1-6 Salts of alkylsulfonic acid, camphorsulfonic acid, naphthalene sulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or 1, 4-butanesulfonic acid.
5. A process for the preparation of a 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the compound is obtainable by:
wherein: x, R 1 、R 2 、R 4 And R is 5 The definition of the compound is the same as that of a 3- (hydroxybenzyl) phthalide compound (I);
step A): reacting the 3-bromophthalide compound (1) with triphenylphosphine in a proper solvent to obtain a corresponding 3-triphenylphosphine salt compound (2); the suitable solvents are: c (C) 3-8 Aliphatic ketone, N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, tolueneAcetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C 5-8 An alkane;
step B): the 3-triphenylphosphine salt compound (2) obtained in the step A) and the hydroxy (aminomethyl) benzaldehyde compound (3) react under proper solvent and alkaline condition to obtain E/Z configuration mixture of the corresponding compound (4), and the corresponding E-type or Z-type configuration compound is obtained through separation and purification; the suitable solvents are: c (C) 1-8 Fatty alcohols, C 3-8 Aliphatic ketones, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, N-dimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C 5-8 An alkane;
step C): reducing the double bond of the compound (4) obtained in the step B) in a proper solvent through a reduction reaction to obtain a corresponding raceme of the 3- (hydroxybenzyl) phthalide compound (I); separating by chromatography to obtain corresponding optical isomer; the suitable solvents are: c (C) 1-6 Fatty alcohols, C 3-8 Aliphatic ketones, C 2-6 Fatty acids, C 2-6 Fatty acid and C 1-6 Esters of fatty alcohols, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, benzene, toluene or xylene, hexane, heptane or octane.
6. The method for preparing 3- (hydroxybenzyl) phthalide compound or its pharmaceutically acceptable salt according to claim 5, wherein in step A), the solvent used in the reaction is: c (C) 3-8 Aliphatic ketones, N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C 5-8 An alkane; 3-bromophthalide (1): the molar feed ratio of triphenylphosphine was 1.0:1.0 to 10.0; the reaction temperature is 40-150 ℃; the reaction time is 1 to 120 hours.
7. The method for preparing 3- (hydroxybenzyl) phthalide compound or its pharmaceutically acceptable salt according to claim 5, wherein in step B), the solvent used for the reaction is: c (C) 1-8 Fatty alcohols, C 3-8 Aliphatic ketones, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, N-dimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C 5-8 An alkane; the alkali used in the reaction is as follows: alkali metal hydroxide, alkaline earth metal hydroxide, alkali metal carbonate, alkaline earth metal carbonate, alkali metal hydrogencarbonate, alkaline earth metal hydrogencarbonate, C 1-8 Alkali metal salts of alcohols, triethylamine, tributylamine, trioctylamine, pyridine, 4-dimethylaminopyridine, N-methylmorpholine, N-methylpiperidine, triethylenediamine or tetrabutylammonium hydroxide; compound (2): compound (3): the molar feed ratio of the alkali is 1.0:1.0 to 10.0:1.0 to 10.0; the reaction temperature is 0-120 ℃; the reaction time is 20 minutes to 48 hours.
8. The method for preparing 3- (hydroxybenzyl) phthalide compound or its pharmaceutically acceptable salt according to claim 5, wherein in step C), the solvent used in the reaction is: c (C) 1-6 Fatty alcohols, C 3-8 Aliphatic ketones, C 2-6 Fatty acids, C 2-6 Fatty acid and C 1-6 Esters of fatty alcohols, diethyl ether, isopropyl ether, methyl tert-butyl ether, tetrahydrofuran, 2-methyltetrahydrofuran, ethylene glycol dimethyl ether, benzene, toluene or xylene, hexane, heptane or octane; catalytic hydrogenation is used in the reduction reaction, and the catalyst used in the catalytic hydrogenation is as follows: raney Ni, ptO 2 、1％～30％Pd-C、1％～30％Pd(OH) 2 -C; the mass ratio of the compound (4) to the catalyst is 1.0:0.01 to 1.0; the reaction pressure is normal pressure to 10.0MPa; the reaction temperature is between room temperature and 150 ℃; the reaction time is 1-96 hours.
9. A pharmaceutical composition comprising a 3- (hydroxybenzyl) phthalide compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers or excipients.
10. Use of a 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment and/or prevention of a neurological related disorder, said neurological related disorder being: vascular dementia, alzheimer's disease, parkinson's disease, huntington's disease, HIV-associated dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, and nerve damage caused by brain trauma.
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