CN114805263A - 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 thereof, a pharmaceutical composition and application thereof in preparing medicaments for treating and/or preventing related diseases of a nervous system, wherein the diseases comprise but are not limited to vascular dementia, Alzheimer disease, Parkinson disease, Huntington disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, nerve injury caused by brain trauma and the like;
Description
Technical Field
The invention belongs to the field of medicinal chemistry, and relates to a 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof, a preparation method thereof, a medicinal composition and application thereof in preparing medicaments for treating and/or preventing related diseases of a nervous system, wherein the diseases comprise but are not limited to vascular dementia, Alzheimer disease, Parkinson disease, Huntington's disease, HIV (human immunodeficiency Virus) related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, nerve injury caused by brain trauma and the like.
Background
Neurodegenerative diseases refer to a general term for diseases caused by chronic progressive degeneration of central nervous tissue, including Alzheimer's Disease (AD), Parkinson's Disease (PD), Huntington's Disease (HD), Amyotrophic Lateral Sclerosis (ALS), and Multiple Sclerosis (MS), and the pathogenesis of which is closely related to oxidative stress, neuroinflammation, and corresponding injury. Oxidative stress is mediated by Reactive Oxygen Species (ROS) radicals, including superoxide anions, hydrogen peroxide, and hydroxyl radicals, among others. Under normal physiological conditions, the ROS production level and the body antioxidant capacity are in a dynamic balance state, when the ROS production exceeds the cell antioxidant capacity, Oxidative stress (Oxidative stress) occurs, and the brain is particularly sensitive to the Oxidative stress, so that various nervous system diseases are induced. In addition, researches show that vascular dementia, HIV-related dementia, neuropathic pain, ischemic stroke, hemorrhagic stroke, nerve injury caused by brain trauma and the like are closely related to oxidative stress and neuroinflammation of the body.
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 disease development exists at present, and the clinical treatment mainly aims at improving the blood circulation and the brain metabolism of the brain and strengthening the brain nutrition. Recent studies have shown that VD patients exhibit impaired cognitive function and are often accompanied by abnormalities in the cholinergic system. The density of ChAT positive neurons and fibers in the hippocampal region of a 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 reduction of the concentration is positively correlated with the severity of dementia; cerebral ischemia can cause the activity of acetylcholinesterase in brain to rise; meanwhile, the acetylcholinesterase inhibitors are found to protect neuron damage caused by ischemia and promote nerve damage and brain function recovery after cerebral ischemia.
Alzheimer's disease (senile dementia, AD) is a degenerative disease of the central nervous system mainly involving progressive cognitive impairment and memory impairment, and its incidence rate is on the rise year by year, and it is a high-grade disease second to cardiovascular disease and cancer. With the accelerated aging process of the global population, the incidence rate of the disease is in a remarkably rising trend. It is estimated that more than 5000 million people suffer from dementia worldwide, the total amount of treatment and care cost exceeds 1 trillion dollars in 2018, and the number of patients will increase to 1.52 billion by 2050. Because AD is clinically manifested as hypomnesis, orientation ability, thinking and judgment ability, reduction of daily life ability, even abnormal mental behavior symptoms, and the like, the nursing difficulty of patients is large, and the heavy burden is brought to the society and families. Currently approved drugs for the treatment of light/moderate AD are acetylcholinesterase (AChE) inhibitors, and for the treatment of severe ADN-methyl-D-an aspartate (NMDA) receptor antagonist. Clinical use has shown that these drugs can increase acetylcholine levels or inhibit excitatory ammonia in patientsThe excitotoxicity of amino acid is used for relieving AD symptoms, but the disease course cannot be effectively prevented or reversed, and severe toxic and side effects such as hallucinations, consciousness chaos, dizziness, nausea, hepatotoxicity, inappetence, frequent defecation and the like can be caused, so that the long-term curative effect is not ideal. Therefore, there is an urgent clinical need to develop a novel therapeutic agent for AD that has both improved symptoms and altered course of disease.
AD is a disease caused by various factors, the pathogenesis of the AD is complex, and the pathogenesis of the AD is not completely clarified so far. However, studies have shown that the patient has a decreased acetylcholine level in the brain,βOverproduction and deposition of amyloid, platelet aggregation in cerebral vessels, metabolic disorders of metal ions, Ca 2+ Imbalance of balance,tauNeurofibrillary tangles caused by protein hyperphosphorylation, glutamate receptor hyperactivity, large amounts of Reactive Oxygen Species (ROS) and free radicals produced by oxidative stress, and various factors such as neuroinflammatory responses play important roles in the pathogenesis of AD. In view of the above pathogenic factors, researchers have found a large number of drugs with high activity and high selectivity to a target by using the traditional "one drug one target" drug design strategy, such as: cholinesterase inhibitors andN-methyl-DAspartate receptor antagonists and the like. However, the drugs have the problems of single action target, more toxic and side effects in clinical use, poor long-term curative effect on AD patients and the like.
In recent years, with the continuous elucidation of the pathogenic mechanism of AD, the occurrence and development of AD have the characteristics of multi-mechanism and multi-factor action, and different mechanisms are mutually associated and influenced to form a complex network regulation and control system in the occurrence and development process of AD. Based on the above results, researchers have proposed a "multi-target drug" strategy to develop anti-neurodegenerative drugs. By "multi-target drug" is meant that a single chemical entity can act on multiple targets in the disease network that are closely related to treatment, and the effect on each target can produce a synergistic effect, such that the total effect is greater than the sum of the individual effects, and such compounds are also referred to as "Multifunctional" or "multi-potential" drugs. The main differences between the multi-target medicine and the multi-medicine combined application and the compound medicine are as follows: can reduce dosage and improve therapeutic effectThe medicine has the advantages of good curative effect, avoidance of interaction between medicines and toxic and side effects caused by the interaction, uniform pharmacokinetic property, convenience in use and the like. Therefore, research and development of neurodegenerative disease-resistant therapeutic drugs with novel chemical structures, novel action mechanisms, multi-target effects and low toxic and side effects are currently important directions. A large number of clinical studies have proved that AChE inhibitors can effectively relieve the symptoms of patients with dementia, and the short-term treatment effect is positive; therefore, in designing multi-target anti-dementia drugs, it is usually necessary to retain the AChE inhibitory activity of the compound (inhibiting the enzyme is crucial to improving the symptoms of dementia patients), and to add one or more other targets or functions with pharmacological synergistic effects on the basis of the AChE inhibitory activity, so as to achieve multi-target anti-dementia therapeutic effects. Obviously, the design and the discovery have the effects of inhibiting acetylcholinesterase and inhibitingβThe overproduction and deposition of amyloid, oxidative stress, metal ion complexation and multi-target anti-dementia drugs against neuritis may make a breakthrough in the treatment of associated dementia.
Disclosure of Invention
The invention aims to disclose a 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salt thereof.
The invention also aims to disclose a preparation method of the 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof.
The invention also aims to disclose a pharmaceutical composition containing the 3- (hydroxybenzyl) phthalide compound (I) and pharmaceutically acceptable salts thereof.
The invention also aims to disclose that the 3- (hydroxybenzyl) phthalide compound (I) and the pharmaceutically acceptable salt thereof have multi-target effect, and can be used for preparing the medicine for treating and/or preventing related diseases of the nervous system, including but not limited to vascular dementia, Alzheimer disease, Parkinson disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke, nerve injury caused by brain trauma and the like.
The general chemical structure formula of the 3- (hydroxybenzyl) phthalide compound (I) disclosed by the invention is as follows:
in the formula:
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-configuration,EA configuration of the formula orZA formula andE-mixtures of formula (la) configuration in any ratio; when A is 1 -A 2 Represents CH-CH 2 When the compound isRThe configuration,SConfiguration or racemate;
R 1 and R 2 Each independently represents H, OH, CF 3 O、C 1 ~C 6 Alkyl radical, C 1 ~C 6 Alkoxy radical, C 1 ~C 6 Alkylthio, halogen or NR 7 R 8 ，R 7 And R 8 Each independently representing H, C 1 ~C 6 An alkyl group;
R 4 and R 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 Further represents tetrahydropyrrolyl, morpholinyl, piperidinyl, piperazinyl, 4-substituted by C 1 ~C 6 Piperazinyl substituted with alkyl, 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 phenyl ring;
the term "halogen" as defined above means F, Cl, Br or I; "substituted benzyl" or "substituted phenyl" refers to benzyl or phenyl groups on the phenyl ring substituted with 1-4 groups selected from: F. cl, Br, I, C 1-4 Alkyl radical, C 1-4 Alkoxy, dimethylamino, cyanoAnd the substituents are in any possible positions of the benzene ring.
The 3- (hydroxybenzyl) phthalide compound (I) disclosed by the invention can be prepared by the following method:
taking the corresponding 3-bromophenylphthalide (1) as an initial raw material, and reacting with triphenylphosphine in a proper solvent to obtain a corresponding 3-triphenylphosphine salt compound (2); the obtained compound 2 and a hydroxy (aminomethyl) benzaldehyde compound (3) are subjected to Wittig reaction in a proper solvent under the alkaline condition to obtain a corresponding compound (4)E/ZMixture of configurations (i.e.E/ZCompound I mixture of configuration); separating and purifying the compound (4) mixture by silica gel column chromatography to respectively obtain corresponding compoundsEIs of the formulaZ-a compound of formula (la); the mixture of the compound (4) can be directly reduced with double bonds in a proper solvent through reduction reaction without separation and purification to obtain a corresponding 3- (hydroxybenzyl) phthalide compound (I) racemate; separating the corresponding 3- (hydroxybenzyl) phthalide compound (I) racemate by using a conventional chiral chromatography method to obtain a corresponding optical isomer; the reaction formula is as follows:
in the formula: x, R 1 、R 2 、R 4 And R 5 The definition of (A) is the same as 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-bromophenylphthalate 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 3-8 An aliphatic ketone,N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C 5-8 Alkanes, preferably solvents such as 2-methyltetrahydrofuran, ethyl acetate, acetonitrile, toluene or 1, 4-dioxane; 3-bromophenylphthalides(1): the molar charge ratio of triphenylphosphine is 1.0: 1.0-10.0, and preferably, the molar feed ratio is 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 1-120 hours, and the preferable reaction time is 2-72 hours.
Step B): the 3-triphenylphosphine salt compound (2) obtained in the step A) and a hydroxyl (aminomethyl) benzaldehyde compound (3) are subjected to Wittig reaction in a proper solvent under the alkaline condition to obtain a corresponding compound (4)E/ZA mixture of configurations; wherein, the solvent used in the reaction is: c 1-8 Fatty alcohol, C 3-8 Aliphatic ketone, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C 5-8 Alkanes, preferred solvents are: chloroform, dichloromethane, acetone, acetonitrile, tetrahydrofuran or toluene; 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 1-8 Alkali metal salts of alcohols, organic tertiary or quaternary amines (e.g. triethylamine, tributylamine, trioctylamine, pyridine, 4-dimethylaminopyridine, tert-butyl amine, octyl amine, pyridine, 4-butyl amine, tert-butyl amine, octyl,N-methylmorpholine,NMethylpiperidine, triethylenediamine, tetrabutylammonium hydroxide), the preferred bases being: sodium hydroxide, potassium carbonate, triethylamine, pyridine or sodium methoxide; compound (2): compound (3): the molar charge ratio of alkali is 1.0: 1.0-10.0: 1.0-10.0, and preferably, the molar feed ratio is 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, and the preferable reaction time is 1 to 24 hours.
Step C): directly reducing double bonds in a proper solvent through reduction reaction without separating and purifying the compound (4) mixture obtained in the step B) to obtain a corresponding 3- (hydroxybenzyl) phthalide compound (I) racemate; wherein, the solvent used in the reaction is: c 1-6 Fatty alcohol, C 3-8 Aliphatic ketones, C 2-6 Fatty acid, C 2-6 Fatty acids with C 1-6 Esters and ethers of aliphatic alcohols (e.g. diethyl 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.), preferred solvents are: tetrahydrofuran, methanol, ethanol or isopropanol; the reduction reaction is preferably reduced by catalytic hydrogenation using the following catalysts: raney Ni, PtO 2 、1%~30% Pd-C、1%~30% Pd(OH) 2 -C, preferably the catalyst is: raney Ni, PtO 2 5% -20% of Pd-C; the mass ratio of the compound (4) to the catalyst was 1.0: 0.01 to 1.0; the reaction pressure is normal pressure to 10.0 MPa, preferably normal pressure to 2.0 MPa; the reaction temperature is room temperature-150 ℃, and preferably room temperature-80 ℃; the reaction time is 1 to 96 hours, preferably 2 to 50 hours.
The 3- (hydroxybenzyl) phthalide compound (I) obtained by the above method can be prepared into pharmaceutically acceptable salts thereof by a pharmaceutically conventional salt-forming method with any suitable acid, wherein the acid is: hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, sulfamic acid, C 1-6 Aliphatic 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 Alkyl sulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, etc.), camphorsulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, or 1, 4-butanedisulfonic acid.
The starting materials of the present invention, 3-bromophenylphthalide (1) and hydroxy (aminomethyl) benzaldehyde (3), may be prepared by techniques common in the art, including but not limited to the methods disclosed in the following references: 1. guidong 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 pharmaceutical composition disclosed by the invention comprises one or more 3- (hydroxybenzyl) phthalides (I) or pharmaceutically acceptable salts thereof with a therapeutically effective amount, and the pharmaceutical composition can further contain one or more pharmaceutically acceptable carriers or excipients. The "therapeutically effective amount" refers to the amount of a drug or agent that elicits a biological or medicinal response in a tissue, system, or animal targeted by a researcher or physician; the term "composition" refers to a product formed by mixing more than one substance or component; the "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable substance, composition or vehicle, such as: liquid or solid fillers, diluents, excipients, solvents or encapsulating substances, which carry or transport certain chemical substances. The ideal proportion of the pharmaceutical composition provided by the invention is that the 3- (hydroxybenzyl) phthalide compound (I) or the pharmaceutically acceptable salt thereof is taken as an active component and accounts for 2-99.5% of the total weight.
The 3- (hydroxybenzyl) phthalide compound (I) and the pharmaceutically acceptable salt thereof disclosed by the invention are subjected to the following biological activity screening:
(1) inhibitory activity of 3- (hydroxybenzyl) phthalide compound (I) on acetylcholinesterase and butyrylcholinesterase
Adding 1.0 mmol/L thioacetyl choline iodide or thiobutyrylcholine iodide 30 μ L, PBS buffer solution of pH7.4 40 μ L, test compound solution 20 μ L (DMSO content is less than 1%) and acetylcholinesterase 10 μ L (rat brain cortex 5% homogenate supernatant, phosphate buffer solution of pH7.4 as homogenate medium) or butyrylcholinesterase (rat serum 25% supernatant, pH7.4 phosphate buffer solution as homogenate medium) into 96-well plate, mixing, incubating at 37 deg.C for 15min, adding 0.2% 5, 5' -dithio-bis (2-nitrobenzoic acid) (DTNB) solution into each well for 30 μ L color development, measuring optical density (OD value) of each well at 405nm with enzyme labeling instrument, the inhibition rate of the compound to the enzyme (enzyme inhibition (%) = (1-sample group OD value/blank group OD value) × 100%) was calculated as compared with the blank wells to which the sample to be tested was not added; selectingSelecting five to six concentrations of the compound, measuring enzyme inhibition rate, and performing linear regression by using the negative logarithm of the molar concentration of the compound and the inhibition rate of the enzyme to obtain the molar concentration when 50% of the inhibition rate is obtained as the IC of the compound 50 . The measurement result shows that the 3- (hydroxybenzyl) phthalide compound (I) (including the compound (4)) disclosed in the embodiment of the inventionEIs of the formulaZTarget compound with-type configuration) has significant inhibition effect on acetylcholinesterase and IC 50 Is 3.50X 10 -3 nM to 10.0 mu M; wherein, the compounds of the examples: (Z) IC of (E) -2-1-3 50 72.0 nM; compound (A) to (B)E) IC of (E) -2-1-3 50 26.0 nM; IC of Compound 2-1-3 50 2210.0 nM; compound (A) to (B)Z) IC of (E) -2-1-4 50 31.0 nM; compound (A) to (B)E) IC of (E) -2-1-4 50 660.0 nM; IC of Compounds 2-1-4 50 960.0 nM; compound (A) to (B)Z) IC of (E) -2-1-5 50 66.0 nM; compound (A) to (B)E) IC of (E) -2-1-5 50 8.83 nM; IC of Compounds 2-1-5 50 33.0 nM; compound (A) to (B)Z) IC of (1) - (5) 50 0.092 nM; compound (A) to (B)E) IC of (1) - (5) 50 It was 0.071 nM. Further structural-activity relationship analysis shows that when A is used 1 -A 2 Represents CH-CH 2 When the chiral configuration does not significantly affect the activity of inhibiting acetylcholinesterase; the determination result also shows that the inhibitory activity of the 3- (hydroxybenzyl) phthalide compound (I) on acetylcholinesterase is obviously higher than that of butyrylcholinesterase (the selectivity is more than 100 times), which indicates that the compound disclosed by the invention has a selective inhibitory effect on acetylcholinesterase and indicates that the compound has low toxicity on peripheral systems. In addition, the measurement results also show that the IC of AChE inhibition by the clinically used rivastigmine 50 IC for butyrylcholinesterase inhibition at 12.3 μ M 50 Is 3.0 mu M; and the control Compound (II) (corresponding 3- (hydroxybenzyl) phthalide Compound (I) — CH) 2 NR 4 R 5 Compound obtained after replacing structural fragment with' -H) and hydroxyl (aminomethyl) benzaldehyde compound (3) IC for inhibiting acetylcholinesterase 50 Are all larger than 100 mu M;
(2) antioxidant activity of 3- (hydroxybenzyl) phthalides (I) (ORAC-FL method)
Reference (Qiang, X.M.et al.Eur. J Med. Chem.2014, 76, 314-: 6-hydroxy-2, 5,7, 8-tetramethylchromane-2-carboxylic acid (C)Trolox) The solution was adjusted to 10-80. mu. mol/L with PBS buffer solution of pH7.4, the solution was adjusted to 250 nmol/L with PBS buffer solution of pH7.4 for fluorescein (fluorescein), and the solution was adjusted to 40 mmol/L with PBS buffer solution of pH7.4 for 2, 2' -azobisisobutylamidine dihydrochloride (AAPH) before use. The compound solution and the fluorescein solution were added to a 96-well plate at 50-10. mu. mol/L, mixed, incubated at 37 ℃ for 15min, and AAPH solution was added to make the total volume per well 200. mu.L, mixed, immediately placed in a Varioskan Flash Multimode Reader (Thermo Scientific) instrument, and continuously measured at 485 nm excitation wavelength and 535 nm emission wavelength for 90 min. Calculating the area AUC under the fluorescence decay curve, wherein the area AUC is 1-8 mu mol/LTroloxAs a standard, taking a sample not to be tested as a blank, and expressing the antioxidant activity result of the compound asTroloxThe formula of the equivalent of (a) is: [ (AUC Sample-AUC blank)/(AUCTrolox-AUC blank)] ×[(concentration of Trolox/concentration of sample)]Each compound was assayed in 3 replicates each, each set of experiments was independently repeated three times. The measurement result shows that the 3- (hydroxybenzyl) phthalide compound (I) (including the compound (4)) disclosed in the embodiment of the inventionEIs of the formulaZTarget of formula (II) isTrolox0.95-3.5 times of the total amount of the compound, which shows that the compound has stronger antioxidant activity. Further structural-activity relationship research shows that the ' phenolic hydroxyl ' in the 3-position side chain of the 3- (hydroxybenzyl) phthalide compound (I) is replaced by ' H ', and ' -CH (hydroxyl-benzyl) is reserved 2 NR 4 R 5 The antioxidant activity of the obtained compound is obviously reduced (less than that of the structural fragment)Trolox0.40 times of); and the aboveThe antioxidant activity of the control compound (II) is obviously reduced compared with that of the 3- (hydroxybenzyl) phthalide compound (I), the phenolic hydroxyl in the side chain at the 3-position in the control compound (II) is further replaced by H, and the obtained compound (without the phenolic hydroxyl or CH) 2 NR 4 R 5 ") has less antioxidant activity thanTrolox0.40 times of.
(3) 3- (hydroxybenzyl) phthalide compounds (I) p Aβ 1-42 Inhibitory Activity of self-aggregation
Reference (Qiang, X.M.et al.Eur. J Med. Chem.2014, 76, 314-: pretreated Aβ 1-42 Stock solutions were prepared in DMSO, and diluted to 50. mu.M in PBS buffer, pH7.4, before use; the test compound was diluted to a concentration of 2.5 mM in DMSO, and 20. mu.L of A was added to the stock solution before use, which was diluted with PBS (pH7.4)β 1-42 Solution + 20. mu.L of test Compound solution, 20. mu.L of Aβ 1-42 Solution +20 μ L PBS buffer (containing 2% DMSO) in 96-well plates, incubated at 37 ℃ for 24h, then 160 μ L of 50mM glycine-NaOH buffer (pH = 8.5) containing 5 μ M thioflavin T was added, and fluorescence was measured immediately after shaking for 5s with a multifunctional plate reader at 446nm excitation wavelength and 490nm emission wavelength; a. theβ 1-42 + fluorescence value of test Compound recorded as IF i ，Aβ 1-42 The fluorescence value of + PBS buffer was designated as IF c The fluorescence value of the buffer solution containing only PBS was designated as IF 0 Compounds inhibiting Aβ 1-42 The inhibition rate of self-aggregation is: 100- (IF) i -IF 0 )/(IF c -IF 0 ) 100, x; selecting five to six concentrations of the compound, and determining the inhibition rate; each compound was tested in triplicate at each concentration, with curcumin as a positive control. The measurement result shows that the 3- (hydroxybenzyl) phthalide compound (I) (including the compound (4)) disclosed in the embodiment of the inventionEIs of the formulaZTarget of formula (II) to Aβ 1-42 The self-aggregation has obvious inhibitory 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; but widely used clinicallyanti-AD agents used: donepezil, rivastigmine, memantine hydrochloride, and "-CH of the above-mentioned control Compound (II) (corresponding 3- (hydroxybenzyl) phthalide Compound (I)) 2 NR 4 R 5 The compound obtained after replacing the structural fragment with' -H) and the hydroxy (aminomethyl) benzaldehyde compound (3) were added to A at a concentration of 25.0. mu.Mβ 1-42 The inhibition rate of self-aggregation is less than 15.0 percent.
(4) Determination of complexation of 3- (hydroxybenzyl) phthalide compound (I) and metal ion
Dissolving CuCl with methanol 2 、ZnCl 2 、FeSO 4 、AlCl 3 And a to-be-detected compound, preparing a solution of 75 mu mol/L, adding 100 mu L of the to-be-detected compound solution and 100 mu L of the metal ion solution into a 96-well plate, uniformly mixing, standing for 30 min at room temperature, recording an ultraviolet absorption curve of the mixture in the range of 200-600 nm on a Varioskan Flash Multimode Reader (Thermo Scientific) instrument, and observing the red shift phenomenon of the maximum absorption peak and the intensity of the maximum absorption peak of the mixed solution of the metal ions and the to-be-detected compound by taking 100 mu L of the to-be-detected compound solution and 100 mu L of methanol mixed solution as references. The measurement result shows that the 3- (hydroxybenzyl) phthalide compound (I) (including the compound (4)) disclosed in the embodiment of the inventionEIs of the formulaZTarget of formula (II) all exhibit p-Cu 2+ And Fe 2+ Selective complexation; and control Compound (II) (corresponding 3- (hydroxybenzyl) phthalide Compound (I) — CH 2 NR 4 R 5 The compound obtained after replacing the structural fragment with' -H) has no complexing effect on the four metal ions.
(5) 3- (hydroxybenzyl) phthalide compound (I) to Cu 2+ Induced Aβ 1-42 Inhibitory Activity of aggregation
Adding CuCl 2 75 μ M solution was prepared using HEPES buffer, and compound stock (2.5 mM) and 200 μ M A were combined using HEPES bufferβ 1-42 The 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.LL Cu 2+ Solution + 20. mu. L Aβ 1-42 Solution +20 μ L HEPES buffer and 60 μ L HEPES buffer in 96-well plate, mixed, incubated at 37 ℃ for 24h, then 190 μ L glycine-NaOH buffer (pH = 8.5) of 50mM containing 5 μ M thioflavin T was added, and fluorescence was measured with multifunctional microplate reader at 446nm excitation wavelength and 490nm emission wavelength immediately after shaking for 5 s; cu 2+ +Aβ 1-42 + fluorescence value of test Compound recorded as IF i ，Cu 2+ +Aβ 1-42 The fluorescence value of + HEPES buffer was recorded as IF c The fluorescence value of the buffer containing only HEPES was recorded as IF 0 Compound pair Cu 2+ Induced Aβ 1-42 The inhibition of aggregation was: 100- (IF) i -IF 0 )/(IF c -IF 0 )*100. Triplicate wells were assayed per concentration of each compound, with curcumin as a positive control. The measurement result shows that the 3- (hydroxybenzyl) phthalide compound (I) (including the compound (4)) disclosed in the embodiment of the inventionEIs of the formulaZ-type configuration target) at a concentration of 25.0 μ M for Cu 2+ Induced Aβ 1-42 The inhibition rate of aggregation is more than 45.0 percent; and the aforementioned 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) under the concentration of 25.0 mu M is less than 20.0%.
(6) Inhibitory activity of 3- (hydroxybenzyl) phthalide compound (I) on neuroinflammation
(a) Effect of Compounds and Lipopolysaccharide (LPS) on BV-2 cell Activity
Preparing BV-2 cells in logarithmic growth phase into cell suspension, inoculating the cell suspension in a 96-well plate, placing the plate at 37 ℃ and 5% CO 2 Culturing for 24h in a cell culture box, changing to 90 μ L of fresh serum-free culture solution after the cells adhere to the wall, respectively adding 10 μ L of each concentration compound to be tested, pre-incubating for 30 min, and setting a blank control group for each concentration of 3 parallel holes; then, with or without LPS, the mixture was left at 37 ℃ with 5% CO 2 Continuously culturing in a cell culture box for 24h, adding MTT solution, incubating at 37 ℃ for 4h,discarding the supernatant, adding 200 mu LDMSO solution into each well, slightly shaking for 10min, measuring OD (optical density) at 490nm by using a microplate reader, calculating the average value of the measured OD values of different concentrations of each test sample, and calculating the cell survival rate according to the following companies: cell survival (%) = administration group OD mean/control group OD mean × 100%. The test results show that all 3- (hydroxybenzyl) phthalides (I) and LPS disclosed in the examples of the present invention do not show cytotoxicity (inhibition rate less than that of less than 25 μ M) at a concentration of not more than 25 μ M<5%）。
(b) Influence of 3- (hydroxybenzyl) phthalide compound (I) on NO release of LPS-induced BV-2 cells
Preparing BV-2 cells in logarithmic growth phase into cell suspension, inoculating the cell suspension in a 96-well plate, placing the plate at 37 ℃ and 5% CO 2 Culturing for 24h in a cell culture box, changing to 90 μ L of fresh serum-free culture solution after the cells adhere to the wall, respectively adding 10 μ L of each concentration compound to be tested, pre-incubating for 30 min, and setting a blank control group for each concentration of 3 parallel holes; then LPS stimulation was added and the mixture was left at 37 ℃ with 5% CO 2 And (3) continuously culturing for 24h in the cell culture box, taking cell culture supernatants of different treatment groups, adding a Griess reagent I with the same volume and a Griess reagent II with the same volume, carrying out a dark reaction at room temperature for 10min, and measuring absorbance at 540 nm to detect the level of NO in the cell supernatants (the specific operation is carried out according to the instruction of the NO detection kit). The test results show that all 3- (hydroxybenzyl) phthalides (I) (including compound (4)) disclosed in the examples of the present inventionEIs of the formulaZThe formula configuration target substance) shows stronger inhibition of BV-2 cell NO generation induced by LPS in the concentration range of 0.5 mu M to 25 mu M (the inhibition rate under the concentration of 2.5 mu M is more than 25.0 percent), and has obvious dose-effect relationship; and their inhibitory activity was significantly stronger than "-CH in control compound (II) (corresponding 3- (hydroxybenzyl) phthalide compound (I)) at the same concentration 2 NR 4 R 5 The compound obtained after replacing the structural fragment with "-H") and the hydroxy (aminomethyl) benzaldehyde compound (3) (the inhibition rate under 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 the advantages ofHas remarkable anti-neuritis activity.
(7) Influence of 3- (hydroxybenzyl) phthalide compound (I) on mouse memory acquisition disorder caused by scopolamine
SPF grade ICR male mice, 25-30g, randomly divided into: normal group, model group, positive control group, test drug high-low dose group (15.0 mg/kg, 2.5 mg/kg), each group of 10 animals. The tested medicine is given by one-time intragastric administration, the solvent of 0.5 percent CMC-Na is given to the blank group and the model group, and the administration volumes are both 0.1ml/10 g; 45 min after administration, normal mice were injected with normal saline in the abdominal cavity, and the other animals were injected with scopolamine (3.0 mg/kg) in an administration volume of 0.1ml/10 g; after 30 min of molding, the mice were placed in the non-electrostimulated Y maze for behavioral testing. During testing, a mouse is placed at the tail end of one arm, the mouse freely passes 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 rate% = [ alternation times/(total number of entries-2)]X 100, results are expressed as mean ± standard deviation, and differences between groups were analyzed using one-way anova. The results of the measurements show that under the experimental conditions, the tested 3- (hydroxybenzyl) phthalide compound (I) (example compound: (A)Z) -1-1-5, Compound (A)E) -1-1-5) has a dose-dependent improvement effect on mouse acquired dysmnesia caused by scopolamine, and has statistical difference with a model group (p<0.001) and the activity is obviously higher than that of the clinical medicine rivastigmine (at the same molar concentration)p<0.01) and also stronger than the clinical drug donepezil at the same molar concentration (p<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. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
Example 1A 1 -A 2 General method for preparing 3- (hydroxybenzyl) phthalide compound (I) when C = CH
5.0 mmol of the corresponding 3-bromo compoundAdding the phthalide (1), 6.0 mmol of triphenylphosphine and 80 ml of toluene into a reaction bottle, heating, refluxing and stirring for reaction for 10-30.0 hours (tracking the reaction process 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 the corresponding 3-triphenylphosphine salt compound (2), wherein the yield is 56.0-82.6%, and the chemical structures are all subjected to treatment 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 ml of dichloromethane prepared in the previous step into a reaction bottle, stirring uniformly, adding 1.5 mmol of triethylamine, and stirring at room temperature for reacting for 8.0-28.0 hours (tracking the reaction process by TLC); after the reaction, the solvent was distilled off under reduced pressure, 40 mL of deionized water was added to the residue, the pH of the reaction solution was adjusted to strong acidity with 10% hydrochloric acid aqueous solution, the pH of the reaction solution was adjusted to weak alkalinity with saturated sodium bicarbonate aqueous solution, three times of extraction was performed with 150 mL of dichloromethane, the organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate and filtered, the solvent was distilled off under reduced pressure, and the residue was the compound (4) as a residueE/ZA mixture of configurations; purifying the mixture by silica gel column chromatography (eluent: dichloromethane: methanol = 25-80: 1 v/v) to obtain the correspondingZ-3- (hydroxybenzyl) phthalide compound (I) (yield: 18.5% -26.2%) andE-3- (hydroxybenzyl) phthalide compound (I) (yield: 12.3% -22.5%), the chemical structure of which is shown in 1 H-NMR、 13 C-NMR and ESI-MS confirmation; the purity of the obtained compound is more than 96.0% by HPLC.
Example 2A 1 -A 2 Represents CH-CH 2 General method for preparing 3- (hydroxybenzyl) phthalide compound (I)
Compound (4) (i.e., A in the general chemical structure) prepared according to the method of example 1 1 -A 2 Representing C = CHE/ZConfiguration mixture) 1.0 mmol and 30 ml of ethanol are added into a reaction bottle, after being stirred uniformly, 35.0 mg of 10 percent Pd/C is added, after being introduced with hydrogen for three times of replacement, hydrogen is introduced at room temperature and normal pressure, and the mixture is stirred and reacted for 3.5 to 28.0 hours (the reaction process is tracked by TLC),after the reaction is finished, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel column chromatography (eluent: dichloromethane: methanol = 25-80: 1 v/v) to obtain the corresponding 3- (hydroxybenzyl) phthalide compound (I) (namely, A in the chemical structural general formula) 1 -A 2 Represents CH-CH 2 ) The yield is 60.3% -90.0%; its chemical structure is all through 1 H-NMR、 13 The purity of the obtained target substance is more than 96.0% by HPLC (high performance liquid chromatography) confirmed by C-NMR and ESI-MS; the target prepared by the method has the following structure:
；
NMR data for some of the 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 general procedure for salt formation of (hydroxybenzyl) phthalides (I) with acid
Adding 1.0 mmol of 3- (hydroxybenzyl) phthalide compound (I) obtained in the above example 1 or 2 and 30 ml of acetone into a reaction bottle, stirring uniformly, adding 3.0 mmol of corresponding acid, heating, refluxing, stirring, reacting for 20 min, cooling to room temperature after reaction, removing solvent by reduced pressure evaporation, recrystallizing the residue with acetone, and filtering to obtain solid, i.e. salt of 3- (hydroxybenzyl) phthalide compound (I), wherein the chemical structure of the salt is obtained by 1 H NMR and ESI-MS.
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 isZ-configuration,EA configuration of the formula orZA formula andE-mixtures of formula (la) configuration in any ratio; when A is 1 -A 2 Represents CH-CH 2 When the compound isRThe configuration,SConfiguration or racemate;
R 1 and R 2 Each independently represents H, OH, CF 3 O、C 1 ~C 6 Alkyl radical, C 1 ~C 6 Alkoxy radical, C 1 ~C 6 Alkylthio, halogen or NR 7 R 8 ，R 7 And R 8 Each independently representing H, C 1 ~C 6 An alkyl group;
R 4 and R 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 Further represents tetrahydropyrrolyl, morpholinyl, piperidinyl, piperazinyl, 4-substituted by C 1 ~C 6 Piperazinyl substituted with alkyl, 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 phenyl ring;
the term "halogen" as defined above means F, Cl, Br or I; "substituted benzyl" or "substituted phenyl" refers to benzyl or phenyl groups on the phenyl ring substituted with 1-4 groups selected from: F. cl, Br, I, C 1-4 Alkyl radical, C 1-4 Alkoxy, dimethylamino, cyano, these substituents being in any possible position of the phenyl ring.
2. The 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein R is 1 And R 2 Preferably 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 is 4 And R 5 Preferably selected from: methyl or ethyl; NR (nitrogen to noise ratio) 4 R 5 When in ring formation, the ring is preferably selected from: tetrahydropyrrolyl, piperidinyl, morpholinyl, piperazinyl, 4-methylpiperazinyl, 4-benzylpiperazinyl, 4- (3-fluorobenzyl) piperazinyl, 4- (2-methoxybenzyl) piperazinyl.
4. 3- (hydroxybenzyl) phthalide according to any of claims 1 to 3 or a pharmaceutically acceptable salt thereof, characterized in that the pharmaceutically acceptable salt is a salt of such a 3- (hydroxybenzyl) phthalide with hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid, phosphoric acid, amidosulphonic acid, C 1-6 Aliphatic 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 acids, camphorsulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or 1, 4-butanedisulfonic acid.
5. The process for preparing 3- (hydroxybenzyl) phthalide or a pharmaceutically acceptable salt thereof according to any of claims 1 to 4, wherein the compound is prepared by:
in the formula: x, R 1 、R 2 、R 4 And R 5 The definition of (A) is the same as the chemical structural general formula of the 3- (hydroxybenzyl) phthalide compound (I);
step A): reacting the 3-bromophenylphthalide (1) 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 a hydroxyl (aminomethyl) benzaldehyde compound (3) are subjected to Wittig reaction in a proper solvent under the alkaline condition to obtain a corresponding compound (4)E/ZThe mixture is separated and purified to obtain the 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 reduction reaction to obtain a corresponding 3- (hydroxybenzyl) phthalide compound (I) racemate; then separating by conventional chromatography to obtain the corresponding optical isomer.
6. The method for preparing 3- (hydroxybenzyl) phthalide or a pharmaceutically acceptable salt thereof according to claim 5, wherein the solvent used in the reaction in step A) is: c 3-8 An aliphatic ketone,N,N-dimethylformamide, tetrahydrofuran, 2-methyltetrahydrofuran, ethyl acetate, diethyl ether, benzene, toluene, acetonitrile, 1, 4-dioxane, ethylene glycol dimethyl ether or C 5-8 An alkane; 3-bromophenylphthalide (1): the molar charge ratio of triphenylphosphine is 1.0: 1.0 to 10.0; the reaction temperature is 40-150 ℃; the reaction time is 1-120 hours.
7. The method for preparing 3- (hydroxybenzyl) phthalide or a pharmaceutically acceptable salt thereof according to claim 5, wherein in step B), the solvent used for the reaction is: c 1-8 Fatty alcohol, C 3-8 Aliphatic ketone, diethyl ether, tetrahydrofuran, 2-methyltetrahydrofuran,N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, 1, 4-dioxane, benzene, toluene, acetonitrile or C 5-8 An 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 1-8 Alkali metal salts of alcohols, triethylamine, tributylamine, trioctylamine, pyridine, 4-dimethylaminopyridine, pyridine, and the like,N-methylmorpholine,N-methylpiperidine, triethylenediamine or tetrabutylammonium hydroxide; compound (2): compound (3): the molar charge ratio of alkali is 1.0: 1.0-10.0: 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 or a pharmaceutically acceptable salt thereof according to claim 5, wherein in step C), the solvent used for the reaction is: c 1-6 Fatty alcohol, C 3-8 Aliphatic ketones, C 2-6 Fatty acid, C 2-6 Fatty acids with C 1-6 Esters 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 or octane; the reduction reaction is preferably carried out using catalytic hydrogenation using the following catalysts: raney Ni, PtO 2 、1%~30% Pd-C、1%~30% Pd(OH) 2 -C; the mass ratio of the compound (4) to the catalyst was 1.0: 0.01 to 1.0; the reaction pressure is normal pressure to 10.0 MPa; the reaction temperature is room temperature-150 ℃; the reaction time is 1-96 hours.
9. A pharmaceutical composition comprising a 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 and one or more pharmaceutically acceptable carriers or excipients.
10. Use of the 3- (hydroxybenzyl) phthalide compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the manufacture of a medicament for the treatment and/or prevention of a nervous system related disease which is: 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 nerve damage due to brain trauma.
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