CN117903092A - Dihydroxyphthalide compounds and preparation method and use thereof - Google Patents

Abstract

The present invention discloses a class of dihydroxyphthalide compounds (I) and pharmaceutically acceptable salts thereof, a preparation method thereof, a pharmaceutical composition and use thereof in preparing drugs for treating and/or preventing diseases by resisting oxidative stress, inhibiting amyloid protein aggregation, metal ion complexation or resisting neuroinflammation, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, prion disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke and nerve damage caused by brain trauma;

Description

Dihydroxyphthalide compounds and preparation method and use thereof

Technical Field

The present invention belongs to the field of pharmaceutical chemistry and relates to a class of dihydroxyphthalide compounds (I), a preparation method thereof, a pharmaceutical composition and use thereof in preparing drugs for treating and/or preventing diseases related to the nervous system, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, prion disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke and nerve damage caused by brain trauma.

Background Art

Neurodegenerative diseases refer to a general term for diseases caused by chronic progressive degeneration of central nervous system tissues, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). Their pathogenesis is closely related to oxidative stress, neuroinflammation and corresponding damage. Oxidative stress is mediated by reactive oxygen species (ROS) free radicals, including superoxide anions, hydrogen peroxide and hydroxyl free radicals. Under normal physiological conditions, the level of ROS generation is in a dynamic equilibrium with the body’s antioxidant capacity. When the generation of ROS exceeds the antioxidant capacity of cells, oxidative stress will occur. The brain is particularly sensitive to oxidative stress, which can induce a variety of neurological diseases. Other studies have found that vascular dementia, HIV-related dementia, neuropathic pain, ischemic stroke, hemorrhagic stroke, and nerve damage caused by brain trauma are also closely related to the body's oxidative stress and neuroinflammation.

Vascular dementia (VD) is a clinical syndrome of intellectual and cognitive dysfunction caused by various types of cerebrovascular diseases (including ischemic cerebrovascular disease, hemorrhagic cerebrovascular disease, acute and chronic hypoxic cerebrovascular disease, etc.). Due to the complex pathogenesis of vascular dementia, there is currently no drug that can block the development of the disease. Clinical treatment focuses on improving brain blood circulation, brain metabolism, and strengthening brain nutrition.

Alzheimer's disease (AD) is a degenerative disease of the central nervous system characterized by progressive cognitive impairment and memory loss. Its incidence rate is increasing year by year, becoming a high-incidence disease second only to cardiovascular disease and cancer. With the acceleration of the aging process of the global population, its incidence rate is showing a clear upward trend. Since the clinical manifestations of AD are impaired memory, orientation, thinking and judgment, as well as reduced daily living ability, and even abnormal mental and behavioral symptoms, it is difficult to care for patients, which brings a heavy burden to society and families. Currently, the drugs approved for the treatment of mild/moderate AD include acetylcholinesterase (AChE) inhibitors and N-methyl-D-aspartate (NMDA) receptor antagonists for the treatment of severe AD. However, clinical use has shown that these drugs can relieve AD symptoms by increasing the level of acetylcholine in patients or inhibiting the excitotoxicity of excitatory amino acids, but cannot effectively prevent or reverse the course of the disease. In addition, they can cause serious side effects such as hallucinations, confusion, dizziness, nausea, liver toxicity, loss of appetite, and frequent bowel movements, so the long-term efficacy is not ideal. Therefore, there is an urgent clinical need to develop new AD therapeutic drugs that can both improve AD symptoms and change the course of the disease.

AD is a disease caused by multiple factors, with a complex pathogenesis that has not yet been fully elucidated. However, studies have shown that multiple factors, such as decreased acetylcholine levels in the patient's brain, excessive production and deposition of β-amyloid protein, platelet aggregation in cerebral blood vessels, metal ion metabolism disorders, Ca ²⁺ imbalance, neurofibrillary tangles caused by excessive phosphorylation of tau protein, excessive glutamate receptor activity, oxidative stress producing a large number of reactive oxygen species (ROS) and free radicals, and neuroinflammatory reactions, play an important role in the pathogenesis of AD. In response to the above-mentioned pathogenic factors, researchers have adopted the traditional "one drug, one target" drug design strategy and discovered a large number of drugs with high activity and selectivity for a certain target, such as cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. However, these drugs have problems such as a single target, more toxic and side effects in clinical use, and poor long-term efficacy for AD patients.

In recent years, with the continuous clarification of the pathogenic mechanism of neurodegenerative diseases, it has been found that the occurrence and development of neurodegenerative diseases are characterized by multiple mechanisms and multiple factors. Different mechanisms are interrelated and influence each other, forming a complex network regulation system in the occurrence and development of such diseases. Obviously, the development of therapeutic drugs that can act on multiple links in the pathological process of neurodegenerative diseases at the same time is an inevitable choice at present. Based on the above results, researchers proposed a "multi-target-oriented drug" strategy to develop drugs against neurodegenerative diseases. The so-called "multi-target drug" refers to a single chemical entity that acts on multiple targets in the disease network at the same time, and the effect on each target can produce a synergistic effect, so that the total effect is greater than the sum of each single effect. The main difference between multi-target drugs and multi-drug combined use and compound drugs is that they can reduce the dosage, improve the therapeutic effect, avoid interactions between drugs and the resulting toxic and side effects, have uniform pharmacokinetic properties, and are easy to use. Therefore, the development of anti-neurodegenerative disease therapeutic drugs with new chemical structures, new mechanisms of action, multi-target effects, and low toxic and side effects is an important direction at present.

Summary of the invention

The present invention aims to disclose a class of dihydroxyphthalide compounds (I).

Another object of the present invention is to disclose a method for preparing the dihydroxyphthalide compound (I).

Another object of the present invention is to disclose a pharmaceutical composition comprising the dihydroxyphthalide compound (I).

Another object of the present invention is to disclose that the dihydroxyphthalide compound (I) has multi-target effects and can be used for preparing drugs for treating and/or preventing diseases related to the nervous system, including but not limited to vascular dementia, Alzheimer's disease, frontotemporal dementia, prion disease, Lewy body dementia, Parkinson's disease, Huntington's disease, HIV-related dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke and nerve damage caused by brain trauma.

The chemical structure of the dihydroxyphthalide compound (I) provided by the present invention is as follows:

In the formula: A represents O, S, Se or NR ₁ , R ₁ represents H, C ₁ ～C ₆ alkyl; R represents C ₁ ～C ₈ alkyl, Benzyl, substituted benzyl, phenethyl, substituted phenethyl; n represents 1-6; the "substituted benzyl" or "substituted phenethyl" refers to a benzyl or phenethyl group substituted on the benzene ring by 1-4 groups selected from the following groups: F, Cl, Br, I, C _1-4 alkyl, C _1-4 alkoxy, N(CH ₃ ) ₂ , trifluoromethyl, trifluoromethoxy, amino, hydroxyl, cyano; the compound is R configuration, S configuration, or a mixture of R configuration and S configuration in any proportion.

The dihydroxyphthalide compound (I) disclosed in the present invention can be prepared by the following method, and the reaction formula is as follows:

Wherein: the definitions of A and R are the same as those of the general chemical structure formula of the dihydroxyphthalide compound (I).

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

The corresponding trimethoxyphthalide compound (1) is used as a raw material, and selective demethylation is carried out in the presence of hydrobromic acid and a solvent to obtain the corresponding dihydroxyphthalide compound (I); wherein the solvent used in the reaction is: water, C _2-6 fatty acid, benzene, toluene or chlorobenzene; the molar feed ratio of the trimethoxyphthalide compound (1): hydrogen bromide in the hydrobromic acid is 1.0:3.0-100.0, preferably 1.0:3.0-50.0; the reaction temperature is 10°C-160°C, preferably 20°C-130°C; the reaction time is 2-120 hours, preferably 5-24 hours.

The starting material of the present invention, trimethoxyphthalide compound (1), can be prepared by common techniques in the art, including but not limited to the method disclosed in the following document: Reddy, S.R. et al. WO 2013102935.

The pharmaceutical composition disclosed in the present invention comprises a therapeutically effective amount of one or more dihydroxyphthalide compounds (I), and the pharmaceutical composition may further contain 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 in a tissue, system or animal targeted by a researcher or doctor; the "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 carrier, such as: a liquid or solid filler, diluent, excipient, solvent or encapsulating substance, which carries or transports a certain chemical substance. The ideal ratio of the pharmaceutical composition provided by the present invention is that the dihydroxyphthalide compound (I) as an active ingredient accounts for 2% to 99.5% of the total weight.

The dihydroxyphthalide compounds (I) disclosed in the present invention were screened for the following biological activities:

(1) Antioxidant activity of dihydroxyphthalide compounds (I) (ORAC-FL method)

The determination was carried out according to the method reported in the literature (Qiang, X.M. et al. Eur. J Med. Chem. 2014, 76, 314-331), namely: 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) was prepared into a 10-80 μmol/L solution with a pH 7.4 PBS buffer, fluorescein was prepared into a 250 nmol/L solution with a pH 7.4 PBS buffer, and 2,2′-azobisisobutylamidine dihydrochloride (AAPH) was prepared into a 40 mmol/L solution with a pH 7.4 PBS buffer before use. Add 50-10 μmol/L compound solution and fluorescein solution to a 96-well plate, mix well, incubate at 37°C for 15 min, add AAPH solution to make the total volume of each well 200 μL, mix well, and immediately place in Varioskan Flash Multimode Reader (ThermoScientific) instrument, and continuously measure for 90 min at an excitation wavelength of 485 nm and an emission wavelength of 535 nm. Calculate the area under the fluorescence decay curve AUC, where 1-8 μmol/L Trolox is used as the standard, and no sample is added as the blank. The antioxidant activity results of the compound are expressed as the equivalent of Trolox, and the calculation formula is: [(AUC Sample-AUC blank)/(AUCTrolox-AUC blank)]×[(concentration of Trolox/concentration of sample)], each compound is measured in 3 replicates each time, and each group of experiments is repeated three times independently. The results of the test show that the antioxidant activity of the dihydroxyphthalide compounds (I) disclosed in the examples of the present invention is 2.23 to 3.46 times that of Trolox, indicating that the compounds have strong antioxidant activity. Further structure-activity relationship studies have found that the antioxidant activity of the starting material trimethoxyphthalide compounds (1) used in the examples of the present invention is relatively weak, and its activity is 0.28 to 0.80 times that of Trolox; and the corresponding dimethoxyphthalide compounds obtained by removing a methyl group on the phthalide mother nucleus of the trimethoxyphthalide compounds (1) have certain antioxidant activity, and its activity is 0.96 to 2.08 times that of Trolox, but weaker than that of the dihydroxyphthalide compounds (I); the antioxidant activity of butylphthalide is 0.22 times that of Trolox. The above studies show that the presence of two phenolic hydroxyl groups in the molecules of the dihydroxyphthalide compounds (I) is critical to enhancing the antioxidant activity of the compounds.

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

The determination was carried out according to the method reported in the literature (Qiang, XM et al. Eur. J Med. Chem. 2014, 76, 314-331), that is: the pretreated Aβ _1-42 was prepared into a stock solution with DMSO, and diluted to 50 μM with pH 7.4 PBS buffer before use; the test compound was prepared into a 2.5 mM stock solution with DMSO, and diluted to the corresponding concentration with pH 7.4 PBS buffer before use, 20 μL of Aβ _1-42 solution + 20 μL of the test compound solution, 20 μL of Aβ _{Aβ 1-42} solution + 20 μL PBS buffer (containing 2% DMSO) was placed in a 96-well plate and incubated at 37° C. for 24 h. Then, 160 μL of 50 mM glycine-NaOH buffer (pH=8.5) containing 5 μM thioflavin T was added. After shaking for 5 seconds, the fluorescence value was immediately measured using a multifunctional microplate reader at an excitation wavelength of 446 nm and an emission wavelength of 490 nm. The fluorescence value of Aβ _1-42 + the test compound was recorded as IF _i , the fluorescence value of Aβ _1-42 + PBS buffer was recorded as IF _c , and the fluorescence value of only PBS buffer was recorded as IF _0. The inhibition rate of the compound in inhibiting Aβ _1-42 self-aggregation was: 100-(IF _i -IF ₀ )/(IF _c -IF ₀ )*100. Five to six concentrations of the compound were selected to determine their inhibition rates. Each compound and each concentration were tested three times, with curcumin as a positive control. The measurement results show that the dihydroxyphthalide compounds (I) disclosed in the examples of the present invention have significant inhibitory activity on Aβ1-42 self-aggregation, and the inhibition rate of Aβ1-42 self-aggregation at a concentration of 25.0 μM is between 75.0% and 93.8% (the inhibition rate of curcumin is 41.2%), such as: the inhibition rates of Example compounds 1-3, 1-4, 1-5, 1-7 and 1-8 are 78.8%, 90.2%, 83.3%, 80.5% and 82.0%, respectively; the inhibition rates of Example compounds 2-10, 2-11 and 2-18 are 81.8%, 83.6% and 89.5%, respectively; the inhibition rates of Example compounds 3-11, 3-18, 4-20 and 5-18 are 77.0%, 84.2%, 86.3% and 84.5%, respectively. Further structure-activity relationship studies revealed that the starting material used in the examples of the present invention, the trimethoxyphthalide compound (1), the corresponding dimethoxyphthalide compound obtained by removing a methyl group from the phthalide nucleus of the trimethoxyphthalide compound (1), and butylphthalide all had an inhibition rate of less than 40.0% on Aβ1-42 self-aggregation at a concentration of 25.0 μM.

(3) Determination of the complexation between dihydroxyphthalide compounds (I) and metal ions

CuCl ₂ ·2H ₂ O, ZnCl ₂ , FeSO ₄ , AlCl ₃ and the test compound were dissolved in methanol to prepare a 75 μmol/L solution. 100 μL of the test compound solution and 100 μL of the metal ion solution were added to a 96-well plate, mixed, and allowed to stand at room temperature for 30 min. The ultraviolet absorption curve of the mixture in the range of 200-600 nm was recorded on a Varioskan Flash Multimode Reader. The red shift phenomenon and the intensity of the maximum absorption peak of the metal ion and the test compound mixture were observed with 100 μL of the test compound solution and 100 μL of the methanol mixture as controls. The measurement results show that the dihydroxyphthalide compounds (I) disclosed in the examples of the present invention all exhibit complexation with the above metal ions; while the starting material trimethoxyphthalide compounds (1) used in the examples of the present invention have almost no complexation with the above metal ions (the maximum absorption peak intensity of the test compound and the metal ion mixture has no obvious change, and the maximum absorption peak has no red shift phenomenon). This study shows that the two phenolic hydroxyl groups in the dihydroxyphthalide compound (I) have a significant effect on the metal ion complexation of the compound.

(4) Inhibitory activity of dihydroxyphthalide compounds (I) on neuroinflammation (a) Effects of compounds and lipopolysaccharide (LPS) on the activity of BV-2 cells

BV-2 cells in the logarithmic growth phase were prepared into a cell suspension and inoculated into a 96-well plate, and cultured in a cell culture incubator at 37°C and 5% _CO2 for 24 hours. After the cells adhered to the wall, 90 μL of fresh serum-free culture medium was replaced, and 10 μL of the test compound at each concentration was added for pre-incubation for 30 minutes. Three parallel wells were set for each concentration, and a blank control group was set up at the same time; then LPS was added or not, and cultured in a cell culture incubator at 37°C and 5% _CO2 for 24 hours. MTT solution was added, and incubated at 37°C for 4 hours. The supernatant was discarded, and 200 μL of DMSO solution was added to each well. After slight shaking for 10 minutes, the OD value was measured at 490 nm using an enzyme marker, and the mean OD value measured at different concentrations of each test sample was calculated, and the cell survival rate was calculated according to the following company: Cell survival rate (%) = mean OD value of the treatment group/mean OD value of the control group × 100%. The test results show that all the dihydroxyphthalide compounds (I) disclosed in the examples of the present invention, the starting material used - trimethoxyphthalide compounds (1), butylphthalide and LPS did not show cytotoxicity (inhibition rate less than <10%) at a concentration not exceeding 25 μM.

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

BV-2 cells in the logarithmic growth phase were prepared into a cell suspension and inoculated into a 96-well plate, and cultured in a cell culture incubator at 37°C and 5% _CO2 for 24 hours. After the cells adhered to the wall, 90 μL of fresh serum-free culture medium was replaced, and 10 μL of the test compound at each concentration was added for pre-incubation for 30 minutes, with 3 parallel wells for each concentration, and a blank control group was set up at the same time; then LPS was added for stimulation, and the cells were cultured in a cell culture incubator at 37°C and 5% _CO2 for 24 hours. The cell culture supernatants of different treatment groups were taken, and an equal volume of Griess reagent I and an equal volume of Griess reagent II were added. The plates were reacted at room temperature in the dark for 10 minutes, and the absorbance was measured at 540 nm to detect the NO level in the cell supernatant (the specific operation was carried out according to the instructions of the NO detection kit). The test results show that all the dihydroxyphthalide compounds (I) disclosed in the examples of the present invention show strong inhibitory effects on LPS-induced NO production in BV-2 cells in the concentration range of 0.5 μM to 10 μM (the inhibition rate at a concentration of 10.0 μM is more than 68.0%), and have a significant dose-effect relationship; while the inhibition rate of butylphthalide at a concentration of 10.0 μM is 45.5%. The study also found that the starting material used in the examples of the present invention, trimethoxyphthalide compounds (1), also have anti-neuroinflammatory activity (the inhibition rate of LPS-induced NO production in BV-2 cells at a concentration of 10.0 μM is 38.0% to 59.5%).

DETAILED DESCRIPTION

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

Example 1 General method for preparing dihydroxyphthalide compound (I)

Trimethoxyphthalide compound (1) (4.0 mmol), hydrobromic acid (150.0 mmol) and acetic acid 20 ml were added to a reaction flask, and the mixture was stirred under reflux for 5 to 24 hours (the reaction progress was followed by TLC); after the reaction, the pH of the solution was adjusted to about 4.0 with sodium bicarbonate, and then extracted with ethyl acetate (90 ml) three times, the organic layers were combined, washed with saturated sodium chloride aqueous solution, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (eluent: petroleum ether-acetone = 3:1 v/v) to obtain the corresponding dihydroxyphthalide compound (I) with a yield of 35.3%-62.8%. The chemical structures of the compounds were confirmed by ¹ H-NMR, ¹³ C-NMR, NOE and ESI-MS; the purity of the target compound was greater than 96.0% as determined by HPLC. The target compound prepared by the above general method has the following structure:

The ¹ H-NMR, ¹³ C-NMR and high-resolution mass spectrometry data of some compounds are as follows:

¹ HNMR (400MHz, CDCl ₃ ): 6.46 (s, 1H), 5.40 (brs, 2H), 3.99 (s, 3H), 2.18-1.99 (m, 1H), 1.74-1.68 (m, 1H), 1.42- 1.29 (m, 4H), 0.93 (t, J = 6.8Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ ): 171.8, 153.7, 142.5, 141.9, 132.8, 105.7, 96.1, 82.4, 56.7, 34.6, 26.8, 22.4,13.9; HRMS(ESI)calcd for C ₁₃ H ₁₇ O ₅ [M+H] ⁺ m/z:253.1071, found 253.1072;

¹ HNMR (400MHz, CDCl ₃ ): 6.46 (s, 1H), 5.39 (brs, 2H), 3.99 (s, 3H), 1.98 (brs, 1H), 1.73 (brs, 1H), 1.45-1.29 (m, 6H), 0.89 (t, J = 6.8Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ ): 171.8, 153.7, 142.5, 142.0, 132.8, 105.7, 96.1, 82.4, 56.7, 34.9, 31.5, 24.4, 22.4, 14.0; HRMS(ESI)calcd for C ₁₄ H ₁₉ O ₅ [M+H] ⁺ m/z:267.1227, found 267.1224;

¹ HNMR (400MHz, CDCl ₃ ): 6.46 (s, 1H), 5.39 (brs, 2H), 3.99 (s, 3H), 2.18-1.97 (m, 1H), 1.72-1.69 (m, 1H), 1.43- 1.29 (m, 8H), 0.88 (t, J = 6.8Hz, 3H); ¹³ CNMR (100MHz, CDCl ₃ ): 171.8, 153.7, 142.5, 142.0, 132.8, 105.7, 96.1, 82.4, 56.7, 34.9, 31.6, 29.0,24.7,22.5,14.0；HRMS(ESI)calcd for C ₁₅ H ₂₁ O ₅ [M+H] ⁺ m/z:281.1384, found 281.1386.

Claims (10)

1. The dihydroxyphthalide compound is characterized in that the chemical structural general formula of the compound is shown as (I):

wherein: a represents O, S, se or NR ₁,R₁ represents H, C ₁～C₆ alkyl; r represents a C ₁～C₈ alkyl group, Benzyl, substituted benzyl, phenethyl, substituted phenethyl; n represents 1 to 6; the "substituted benzyl" or "substituted phenethyl" refers to a benzyl or phenethyl group substituted on the benzene ring with 1 to 4 groups selected from the group consisting of: F. cl, br, I, C _1-4 alkyl, C _1-4 alkoxy, N (CH ₃)₂, trifluoromethyl, trifluoromethoxy, amino, hydroxy, cyano).

2. The dihydroxyphthalide compound according to claim 1, wherein a represents O, R represents methyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, allyl, 1-cycloheptyl, 3-dimethyl-1-allyl, 2-methyl-2-allyl, propargyl, benzyl, phenethyl, (3-methoxy) benzyl, (4-methoxy) phenethyl, 4-fluorobenzyl, 4-fluorophenethyl, (4-dimethylamino) benzyl, (3-dimethylamino) phenethyl, (4-trifluoromethoxy) benzyl, (4-trifluoromethoxy) phenethyl, (4-methyl) benzyl, (4-trifluoromethyl) phenethyl, (4-cyano) benzyl, (3, 4-difluoro) benzyl.

3. The dihydroxyphthalide compound according to claim 1, wherein a represents S, R represents methyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, allyl, 1-cycloheptyl, 3-dimethyl-1-allyl, 2-methyl-2-allyl, propargyl, benzyl, phenethyl, (3-methoxy) benzyl, (4-methoxy) phenethyl, 4-fluorobenzyl, 4-fluorophenethyl, (4-dimethylamino) benzyl, (3-dimethylamino) phenethyl, (4-trifluoromethoxy) benzyl, (4-trifluoromethoxy) phenethyl, (4-methyl) benzyl, (4-trifluoromethyl) phenethyl, (4-cyano) benzyl, (3, 4-difluoro) benzyl.

4. The dihydroxyphthalide compound according to claim 1, wherein a represents Se, R represents methyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, allyl, 1-cycloheptyl, 3-dimethyl-1-allyl, 2-methyl-2-allyl, propargyl, benzyl, phenethyl, (3-methoxy) benzyl, (4-methoxy) phenethyl, 4-fluorobenzyl, 4-fluorophenethyl, (4-dimethylamino) benzyl, (3-dimethylamino) phenethyl, (4-trifluoromethoxy) benzyl, (4-trifluoromethoxy) phenethyl, (4-methyl) benzyl, (4-trifluoromethyl) phenethyl, (4-cyano) benzyl, (3, 4-difluoro) benzyl.

5. A dihydroxyphthalide compound according to claim 1, wherein A represents NH or NCH ₃, R represents methyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, allyl, 1-cycloheptyl, 3-dimethyl-1-allyl, 2-methyl-2-allyl, propargyl, benzyl, phenethyl, (3-methoxy) benzyl, (4-methoxy) phenethyl, 4-fluorobenzyl, 4-fluorophenethyl, (4-dimethylamino) benzyl, (3-dimethylamino) phenethyl, (4-trifluoromethoxy) benzyl, (4-trifluoromethoxy) phenethyl, (4-methyl) benzyl, (4-trifluoromethyl) phenethyl, (4-cyano) benzyl, (3, 4-difluoro) benzyl.

6. A process for the preparation of a dihydroxyphthalide compound according to any one of claims 1 to 5, wherein the compound is prepared by:

wherein: the definition of A and R is the same as the chemical structural general formula of the dihydroxyphthalide compound (I);

The corresponding trimethoxy phthalide compound (1) is used as a raw material, and the corresponding dihydroxyphthalide compound (I) is obtained by selectively demethylating in the presence of hydrobromic acid and a solvent.

7. The method for preparing the dihydroxyphthalide compound according to claim 6, wherein the solvent used in the reaction is: water, C _2-6 fatty acids, benzene, toluene or chlorobenzene; trimethoxyphthalide compound (1): the molar feed ratio of hydrogen bromide in hydrobromic acid is 1.0:3.0 to 100.0; the reaction temperature is 10-160 ℃; the reaction time is 2 to 120 hours.

8. A pharmaceutical composition comprising a dihydroxyphthalide compound according to any one of claims 1 to 5 and one or more pharmaceutically acceptable carriers or excipients.

9. Use of a dihydroxyphthalide compound according to any one of claims 1 to 5 for the preparation of a medicament for the treatment and/or prophylaxis of diseases by anti-oxidative stress, inhibition of amyloid aggregation, metal ion complexation or anti-neuroinflammation.

10. Use of a dihydroxyphthalide compound according to claim 9, wherein the disease is: vascular dementia, alzheimer's disease, frontotemporal dementia, prion's disease, dementia with Lewy bodies, parkinson's disease, huntington's disease, HIV-associated dementia, multiple sclerosis, amyotrophic lateral sclerosis, neuropathic pain, ischemic stroke, hemorrhagic stroke or nerve damage caused by brain trauma.