3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative, and preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative as represented by formula (I), and a preparation method therefor and the use thereof in the preparation of a drug for preventing or treating a neurodegenerative disease. Compared to 3,5-dihydroxy-4-methoxybenzyl alcohol, the derivative exhibits a stronger molecular polarity, resulting in enhanced enrichment in the brain, and significantly improved brain targeting. Additionally, the derivative has a prolonged in vivo half-life, and reduced cytotoxicity, thereby improving biological safety. R1, R2 and R3 are selected from H or H2PO3, and R1, R2 and R3 cannot all be H.

Description

3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivatives, and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivatives, and preparation method and application thereof. BACKGROUND

[0002] 3,5-dihydroxy-4-methoxybenzyl alcohol, also known as 3,5-dihydroxy-4-methoxybenzyl alcohol (DHMBA), contains three hydroxyl groups, and has a chemical formula of C8H10O4. It is a phenolic organic compound extracted from filter-feeding bivalves, and has strong antioxidant effect. DHMBA is a rare antioxidant that has both immediate direct antioxidant effect and sustainable indirect antioxidant capacity, and active oxygen free radicals in deep cells can also be fully oxidized and eliminated. Studies have shown that the antioxidant activity of DHMBA is about 2.3 times that of representative antioxidant vitamin C, and 6 times that of chlorogenic acid. At the same time, the safety (IC50) of DHMBA is 5 times that of curcumin and lycopene, and it is a safe and stable super antioxidant.

[0003] In terms of physiological function, DHMBA is particularly beneficial to liver health, can protect liver cells from oxidative stress-induced damage and apoptosis, and at the same time, DHMBA helps the normal operation of glutamatergic neuronal activity, helps the memory and learning function of the brain, and reduces emotional anxiety (Ref: Shakushi, Yukina, et al. Biol. Bull. 2015); secondly, it helps to reduce body weight and reduce fatty liver (Ref: Watanabe et al. J Funct Foods. 2016); it also has the functions of accelerating penetration of the blood-brain barrier, promoting sleep, improving memory, delaying senile dementia, promoting skin regeneration, preventing hair loss, etc.

[0004] However, DHMBA has some shortcomings in practical application. First, DHMBA has poor water solubility and needs to be dissolved in a strong acidic environment. Second, the half-life of DHMBA in the body is too short, only half an hour. In the mouse brain, from feeding to complete metabolism, it only takes 3-4 hours. Such a short in vivo residence time means that it is difficult to maintain an effective drug concentration, directly leading to low bioavailability. In order to achieve the expected therapeutic effect, frequent administration (such as multiple times a day) may be required, which not only significantly reduces patient compliance and increases the burden of medication, but also may affect the stability and safety of treatment due to excessive fluctuations in blood drug concentration. Finally, DHMBA has certain cytotoxicity at high concentrations. For example, at 4mM, it will inhibit cell growth. This suggests that its therapeutic window may be narrow. There is a risk of unintended cell damage caused by excessive dosage or local concentration accumulation, which poses a serious challenge to determining a safe and effective clinical dosage and may limit its application in scenarios requiring higher exposure.(Patent Application No. CN202210155421.6 "Synthesis Method and Application of 3,5-Dihydroxy-4-methoxybenzyl Alcohol") SUMMARY

[0005] The purpose of the present application is to provide 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivatives, their preparation methods and applications, solving the problems of poor water solubility, high cytotoxicity and short half-life in the body of 3,5-dihydroxy-4-methoxybenzyl alcohol in the prior art.

[0006] The present application is achieved by the following technical solutions:

[0007] 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivatives represented by Formula I:

[0008] R1, R2, R3 are selected from H or H2PO3, and R1, R2, R3 cannot be H at the same time.

[0009] The hydroxyl groups of 3,5-dihydroxy-4-methoxybenzyl alcohol are selectively phosphated to obtain 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivatives represented by Formula I.

[0010] When R1 is H2PO3 and R2, R3 are both H (1 -phosphorylation), the synthesis method is as follows: the phenolic hydroxyl of 3,5-dihydroxy-4-methoxybenzyl alcohol is selectively protected by benzyl bromide to obtain 3,5-dibenzyl-4-methoxybenzyl alcohol, then the alcohol hydroxyl of 3,5-dibenzyl-4-methoxybenzyl alcohol is phosphorylated by diethyl chlorophosphate, then the benzyl protecting group is removed by hydrogenolysis, and finally 3,5-dihydroxy-4-methoxy-1 -benzyl phosphate is obtained by hydrolysis under alkaline conditions with the addition of trimethylphenyl ammonium bromide.

[0011] Preferably, the specific steps are as follows: 3,5-dihydroxy-4-methoxybenzyl alcohol is dissolved in anhydrous DMF, anhydrous potassium carbonate and potassium iodide are added, benzyl bromide is slowly added under nitrogen protection, the temperature is controlled at 25-30°C, then the temperature is raised to 60°C, the reaction is stirred, and 3,5-dibenzyl-4-methoxybenzyl alcohol is obtained after purification by post-treatment; 3,5-dibenzyl-4-methoxybenzyl alcohol is dissolved in anhydrous THF, cooled to 0°C in an ice bath, triethylamine is slowly added, then diethyl chlorophosphate is added dropwise while keeping the temperature <5°C, purified by post-treatment; then dissolved in methanol, the benzyl protecting group is removed by hydrogenolysis through Pd / C in a hydrogen atmosphere at room temperature, and finally 3,5-dihydroxy-4-methoxy-1 -benzyl phosphate is obtained by hydrolysis under alkaline conditions with the addition of trimethylphenyl ammonium bromide.

[0012] When R1 is H, R2 is H2PO3 and R3 is H (3-phosphorylation), the synthesis method is as follows: the phenolic hydroxyl of 3,5-dihydroxy-4-methoxybenzyl alcohol is phosphorylated by dibenzyl chlorophosphate, and finally 3-phosphorylated-5-hydroxy-4-methoxybenzyl alcohol is obtained by hydrogenolysis of the benzyl protecting group. Preferably, the specific steps are as follows: 3,5-dihydroxy-4-methoxybenzyl alcohol is dissolved in anhydrous dichloromethane under argon protection, cooled to 0°C in an ice bath, triethylamine is added dropwise, and 1.2 equivalents of a dichloromethane solution of dibenzyl chlorophosphate is slowly added dropwise while controlling the dropping speed to maintain the temperature ≤5°C. After dropping, the temperature is raised to room temperature and stirred for 12 hours, purified by post-treatment; then the benzyl protecting group is removed by hydrogenolysis through Pd / C in a hydrogen atmosphere at room temperature, and then purified to obtain.

[0013] When R1, R2, R3 are all H2PO3 (phosphorylated at 1, 3 and 5 positions), the synthesis method is as follows: using tert-butyldimethylsilyl chloride to selectively protect the alcohol hydroxyl group of 3, 5-dihydroxy-4-methoxybenzyl alcohol, then using dibenzyl chlorophosphate to phosphorylate the phenolic hydroxyl group of 3, 5-dihydroxy-4-methoxybenzyl alcohol, then removing the TBDMS protecting group, and finally using dibenzyl chlorophosphate to phosphorylate the alcohol hydroxyl group of 3, 5-dihydroxy-4-methoxybenzyl alcohol, then hydrogenolysis to obtain 3, 5-diphosphoryl-4-methoxy-1-benzylphosphonate. Preferably, the specific steps are as follows: 3, 5-dihydroxy-4-methoxybenzyl alcohol is dissolved in anhydrous DMF, cooled to 0°C in an ice bath, and then imidazole and tert-butyldimethylsilyl chloride (TBDMSCl) are added, followed by stirring at room temperature. After purification by post-treatment, it is dissolved in anhydrous THF, cooled to 0°C in an ice bath, and then DIEA and a THF solution of dibenzyl chlorophosphate are added dropwise while maintaining the temperature <5°C, followed by stirring at room temperature. After purification by post-treatment, it is dissolved in THF, cooled in an ice bath, and then tetrabutylammonium fluoride is added dropwise, followed by stirring at room temperature to remove the TBDMS protecting group. After purification by post-treatment, it is dissolved in anhydrous THF, cooled to 0°C in an ice bath, and then DIEA and a THF solution of dibenzyl chlorophosphate are added dropwise while maintaining the temperature <5°C, followed by stirring at room temperature. After purification by post-treatment, it is hydrogenolysed by Pd(OH) / C at 50°C in a hydrogen atmosphere to remove the benzyl protecting group, and then purified to obtain 3, 5-diphosphoryl-4-methoxy-1-benzylphosphonate.

[0014] The 3, 5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative represented by formula I has stronger molecular polarity than 3, 5-dihydroxy-4-methoxybenzyl alcohol, improves its enrichment in the brain, significantly improves brain targeting, prolongs the in vivo half-life, reduces cytotoxicity, and improves biological safety.

[0015] Therefore, the present application also protects the use of the 3, 5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative represented by formula I in the preparation of a drug for preventing or treating neurodegenerative diseases.

[0016] The drug contains a pharmaceutically acceptable adjuvant and is prepared into various dosage forms, including liquid, solid dosage forms.

[0017] The dosage forms include tablets, capsules, oral liquids, oral preparations, granules, powders, pills, powders, pastes, pills, suspensions, powders, injections, suppositories, sprays, drops, or patches.

[0018] The concentration of the 3, 5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative in the drug is 0.001wt.%-10wt.%.

[0019] The 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative shown in formula I has stronger molecular polarity than 3,5-dihydroxy-4-methoxybenzyl alcohol, improves its enrichment in the brain, significantly improves brain targeting, prolongs the half-life in vivo, and reduces cytotoxicity, thereby improving biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 is a plot of the brain concentration changes of different drugs in Example 4;

[0021] Figure 2 is a plot of the cell safety of different drugs in Example 5. DETAILED DESCRIPTION

[0022] The following is a further description of the present application, but not a limitation of the present application.

[0023] Example 1: Synthesis of 3,5-dihydroxy-4-methoxy-1-phenylmethyl phosphate (phosphorylation of 1 position of 3,5-dihydroxy-4-methoxybenzyl alcohol)

[0024] The synthesis process is as follows:

[0025] Step 1: benzyl protection of phenolic hydroxyl group (synthesis of 3,5-dibenzyl-4-methoxybenzyl alcohol)

[0026] In a dry 500 mL three-necked flask, 3,5-dihydroxy-4-methoxybenzyl alcohol (10.0 g, 49.3 mmol) and anhydrous DMF (150 mL) were added. After stirring and dissolving, anhydrous potassium carbonate (20.4 g, 148 mmol) and potassium iodide (1.64 g, 9.86 mmol) were added.

[0027] Under nitrogen protection, benzyl bromide (17.6 mL, 148 mmol) was slowly added dropwise, and the dropping speed was controlled to maintain the reaction temperature at 25-30°C. After dropping, the temperature was raised to 60°C and the reaction was stirred for 12 hours (TLC monitoring, PE / EA volume ratio = 3:1, raw material point disappeared).

[0028] The reaction was cooled to room temperature, poured into ice water (500 mL), and extracted with ethyl acetate (3 x 200 mL). The organic phase was washed with saturated NaHCO3 solution (200 mL) and saturated brine (200 mL) in sequence. Anhydrous sodium sulfate was added for drying, and the white solid crude product was obtained by concentration under reduced pressure.

[0029] Purification was performed by silica gel column chromatography (eluent: PE / EA volume ratio = 5:1→3:1), the target fraction was collected, and 3,5-dibenzyl-4-methoxybenzyl alcohol (white crystal, yield 85%) was obtained by concentration and drying.

[0030] Step 2: Alcohol hydroxyl phosphonate (synthesis of [3,5-dibenzyloxy-4- methoxyphenyl] methyl phosphonate diethyl ester)

[0031] In a 250 mL three-necked flask under nitrogen protection, 3,5-dibenzyloxy-4- methoxybenzyl alcohol (8.0 g, 20.2 mmol) and anhydrous THF (100 mL) were added and cooled to 0 °C in an ice bath. Triethylamine (6.2 mL, 44.4 mmol) was added slowly dropwise and stirred for 10 min. Diethyl chlorophosphate (3.6 mL, 24.2 mmol) was added dropwise, keeping the temperature < 5 °C. After the addition was completed, the temperature was raised to room temperature and stirred for 6 h (TLC monitoring, PE / EA volume ratio = 2:1, raw material point disappeared).

[0032] The reaction solution was poured into ice water (200 mL) and extracted with dichloromethane (3 x 100 mL). The organic phases were combined and washed successively with 5% HC1 (100 mL), saturated NaHC03(100 mL), and saturated brine (100 mL). After drying over anhydrous sodium sulfate, the solution was concentrated under reduced pressure to give a yellowish oil crude product.

[0033] Purification by silica gel column chromatography (eluent: PE / EA volume ratio = 4:1) gave [3,5-dibenzyloxy-4-methoxyphenyl] methyl phosphonate diethyl ester (colorless oil, yield 78%).

[0034] Step 3: Benzyl deprotection (synthesis of 3,5-dihydroxy-4-methoxybenzyl phosphonate diethyl ester)

[0035] 1. Hydrogenolysis reaction:

[0036] In a hydrogenation flask, the product from the previous step (7.0 g, 13.1 mmol), 10 wt.% Pd / C (1.4 g), and methanol (140 mL) were added. The hydrogen gas was replaced three times and stirred at 1 atm H atmosphere for 24 h at room temperature (TLC monitoring reaction completion).

[0037] 2. Work-up:

[0038] The reaction solution was filtered through celite to remove the catalyst, and the filter cake was washed with methanol (50 mL). The combined filtrate was concentrated under reduced pressure to give a white solid crude product (3,5-dihydroxy-4-methoxybenzyl phosphonate diethyl ester), which was used directly in the next step.

[0039] Step 4: Phosphonate hydrolysis (synthesis of 3,5-dihydroxy-4-methoxy-1- benzyl phosphonate)

[0040] 1. Alkaline hydrolysis:

[0041] The crude product from previous step was dissolved in absolute ethanol (70 mL) and trimethylphenylammonium bromide (0.5 g, phase transfer catalyst) was added. The solution was cooled in an ice bath and 1 M aqueous NaOH (39.3 mL, 39.3 mmol) was added dropwise. The solution was allowed to warm to room temperature and stirred for 8 h.

[0042] 2. Work-up:

[0043] The ethanol was removed under reduced pressure and the remaining aqueous phase was washed with diethyl ether (2 x 50 mL) to remove impurities. The aqueous phase was adjusted to pH 2-3 with 2 M HC1 and a white precipitate was formed.

[0044] 3. Purification:

[0045] The precipitate was collected, washed with cold water and dried under vacuum. Further purification was achieved by preparative HPLC (C18 column, mobile phase: 0.1% TFA water / acetonitrile) and the target product, 3,5-dihydroxy-4-methoxy-1- benzylphosphonate, was obtained as a white solid (total yield 52%) after lyophilization.

[0046] Structural characterization is as follows:

[0047] Structural confirmation: 1 H NMR (D20, 400 MHz) δ: 6.52 (s, 2H, H-2 / H-6), 4.82 (d, J = 8.4 Hz, 2H, CH20P), 3.75 (s, 3H, OCH3).

[0048] 31 P NMR (D20, 162 MHz) δ: -0.85 (s).

[0049] HRMS (ESI-) m / z: [M-H]"calcd for C8H 10 07P": 249.0167, found: 249.0163.

[0050] Example 2: Synthesis of 3-phosphonate-5-hydroxy-4-methoxybenzyl alcohol (3- phosphonate of 3,5-dihydroxy-4-methoxybenzyl alcohol).

[0051] The synthesis process is as follows:

[0052] Step 1: Selective phosphonation (synthesis of 3-(dibenzoyloxyphosphoryloxy)-5- hydroxy-4-methoxybenzyl alcohol)

[0053] In a 250 mL three-necked flask under argon, 3,5-dihydroxy-4-methoxybenzyl alcohol (5.0 g, 24.6 mmol) and anhydrous dichloromethane (80 mL) were added and cooled to 0 °C in an ice bath. Triethylamine (6.9 mL, 49.2 mmol) was added dropwise and the solution was stirred for 10 min to activate the phenolic hydroxyl group.

[0054] A solution of dibenzyl chlorophosphate (8.3 g, 29.5 mmol, 1.2 equiv) in dichloromethane (20 mL) was added slowly, controlling the dropping speed to maintain the temperature < 5 °C. After the addition was completed, the mixture was stirred at room temperature for 12 h (TLC monitoring: PE / EA volume ratio = 1:1, starting material disappeared, new spot Rf= 0.5).

[0055] The reaction solution was quenched with ice water (100 mL), and the mixture was separated. The aqueous phase was back-extracted with dichloromethane (2 x 50 mL). The combined organic phases were washed successively with 5% citric acid (50 mL), saturated NaHCO3(50 mL), and saturated brine (50 mL). The mixture was dried over anhydrous Na2SO4and concentrated under reduced pressure to give the crude product as a light yellow oil.

[0056] Silica gel column chromatography (eluent: PE / EA volume ratio gradient 4:1→2:1) was performed to collect the target fraction, which was concentrated to give the product as a colorless oil (9.2 g, yield 85%).

[0057] Step 2: Benzylic deprotection (synthesis of 3-phospho-5-hydroxy-4-methoxybenzyl alcohol)

[0058] 1. Hydrogenolysis reaction:

[0059] In a hydrogenation flask, the product from the previous step (8.0 g, 18.2 mmol), 10% Pd / C (1.6 g), and a mixture of methanol / THF (1:1, 160 mL) were added. The hydrogen was replaced three times, and the mixture was stirred at 1 atm H for 24 h at room temperature (TLC monitoring: PE / EA volume ratio = 1:2, starting material disappeared).

[0060] 2. Work-up:

[0061] The reaction solution was filtered through celite, and the filter cake was washed with methanol (50 mL). The combined filtrate was concentrated under reduced pressure to give the crude product as a white solid.

[0062] 3. Purification:

[0063] Recrystallization was performed with diethyl ether / n-hexane (volume ratio 1:1) to give the target product as white crystals (4.5 g, yield 82%).

[0064] Final product structure confirmation: 1 H NMR (D2O, 400 MHz) δ: 7.02 (d, J = 2.4 Hz, 1H, H-2), 6.48 (d, J = 2.4 Hz, 1H, H-6), 4.78 (d, J = 8.6 Hz, 2H, -CH2OH), 3.82 (s, 3H, -OCH3).

[0065] 31 P NMR (D2O, 162 MHz) δ: -0.92 (s).

[0066] HRMS (ESI - *m / z*: [M-H]- calcd for C8H 10 O7P - : 249.0167, found: 249.0161.

[0067] Example 3: Synthesis of 3,5-diphosphonate-4-methoxybenzyl phosphate (phosphonation of 1,3,5-positions of 3,5-dihydroxy-4-methoxybenzyl alcohol).

[0068] The synthesis process is as follows:

[0069] Step 1: Alcohol hydroxyl protection (synthesis of 3,5-dihydroxy-4-methoxyphenyl (tert-butyldimethylsilyl) methyl ether)

[0070] In a 250 mL three-necked flask under argon protection, 3,5-dihydroxy-4-methoxybenzyl alcohol (5.0 g, 24.6 mmol) and anhydrous DMF (80 mL) were added and cooled to 0 °C in an ice bath. Imidazole (4.2 g, 61.5 mmol) and tert-butyldimethylsilyl chloride (TBDMSCl, 4.5 g, 29.5 mmol) were added. Stirring at room temperature for 6 hours (TLC monitoring: PE / EA volume ratio = 3:1, raw material point disappeared). The reaction solution was poured into ice water (300 mL) and extracted with ethyl acetate (3 x 100 mL). The organic phase was combined, washed with saturated brine and dried over anhydrous MgSO4.

[0071] Concentrated under reduced pressure and column chromatography on silica gel (PE / EA volume ratio = 10:1) to give the product as colorless oil (7.2 g, yield 90%).

[0072] Step 2: Phenol hydroxyl phosphonation (synthesis of 3,5-bis(dibenzoyloxyphosphoryloxy)-4-methoxyphenyl (TBDMS) methyl ether)

[0073] In a 500 mL three-necked flask under argon protection, the product from the previous step (7.0 g, 21.5 mmol) and anhydrous THF (150 mL) were added and cooled to 0 °C in an ice bath. DIEA (11.2 mL, 64.5 mmol) was added.

[0074] A solution of dibenzyl chlorophosphate (15.3 g, 51.6 mmol) in THF (30 mL) was added dropwise, keeping the temperature <5 °C. Stirring at room temperature for 24 hours (TLC monitoring: PE / EA volume ratio = 2:1, raw material point disappeared).

[0075] The reaction solution was poured into 5% citric acid solution (200 mL) and extracted with ethyl acetate (3 x 150 mL). The organic phase was combined, washed with saturated NaHCO3 and brine, dried over anhydrous MgSO4.

[0076] Silica gel column chromatography (PE / EA = 5:1) gave a light yellow oil (14.8 g, 85% yield).

[0077] Step 3: De-TBDMS protection (synthesis of 3,5-bis(dibenzyl phosphoroyloxy)-4- methoxybenzyl alcohol)

[0078] 1. Deprotection reaction: In a 250 mL flask was added the product from the previous step (14.0 g, 17.3 mmol) and THF (100 mL), and cooled in an ice bath. Tetrabutylammonium fluoride (TBAF, 1 M in THF, 34.6 mL) was added dropwise and stirred at room temperature for 2 hours.

[0079] 2. Work-up: The reaction solution was poured into ice water (200 mL) and extracted with ethyl acetate (3 x 100 mL). The organic phase was washed with saturated NHCI and brine, dried over anhydrous MgSO4.

[0080] 3. Purification: Concentrated under reduced pressure to give a white foamy solid (11.2 g, 95% yield), which was used directly in the next step.

[0081] Step 4: Phosphorylation of alcohol hydroxyl (synthesis of 3,5-bis(dibenzyl phosphoroyloxy)-4-methoxybenzyl dibenzyl phosphate)

[0082] In a 250 mL three-necked flask under argon was added the product from the previous step (10.0 g, 14.7 mmol) and anhydrous THF (100 mL), and cooled in an ice bath to 0 °C. DIEA (7.7 mL, 44.1 mmol) was added. A solution of dibenzyl chlorophosphate (5.2 g, 17.6 mmol) in THF (20 mL) was added dropwise. The reaction was stirred at room temperature for 12 hours (TLC monitored the completion of the reaction). The reaction solution was poured into 5% citric acid solution (200 mL) and extracted with ethyl acetate (3 x 150 mL). The organic phase was combined, washed with saturated NaHCO3 and brine, dried over anhydrous MgSO4. Silica gel column chromatography (PE / EA = 3:1) gave a colorless viscous oil (12.5 g, 90% yield).

[0083] Step 5: Global debenzyl (synthesis of 3,5-bisphosphate-4-methoxybenzyl phosphate)

[0084] 1. Hydrogenolysis reaction:

[0085] In a high pressure hydrogenation vessel, the product from previous step (12.0 g, 12.7 mmol), 20 wt.% Pd(OH) / C (2.4 g) and ethanol / THF (1:1 by volume, 200 mL) were added. Stirring at 50 °C under 3 atm H pressure for 36 h (TLC monitoring).

[0086] 2. Work-up:

[0087] The reaction solution was filtered through celite and the filtrate was concentrated under reduced pressure.

[0088] 3. Purification:

[0089] The crude product was dissolved in water and purified by anion exchange resin (Dowex 1 x 8, HCO type) and lyophilized to give a white powder (5.8 g, 85% yield).

[0090] Final product structure confirmation: 1 H NMR (D2O, 400 MHz) δ: 6.82 (s, 2H, H-2 / H-6), 4.55 (d, J = 8.8 Hz, 2H, -CH2OP), 3.78 (s, 3H, -OCH3).

[0091] 31 P NMR (D2O, 162 MHz) δ: -0.35 (s, 2P, Ar-OPO3 2- ), -1.28 (s, 1P, -CH2OPO3 2- ).

[0092] HRMS (ESI-)*m / z*:[M-H] - calcd for C8H 10 O 10 P3 - : 366.9502, found: 366.9498.

[0093] Example 4:

[0094] Pharmacokinetic profile of 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate in mouse brain: The ability to penetrate the blood-brain barrier and to understand the pharmacokinetic profile in the brain is the first step in assessing the therapeutic effect of a drug in neurological diseases.

[0095] (1) Four experimental groups, each group of 12-month-old natural aging C57 mice were given by drinking water (n = 18), one-time feeding containing 3 mM of 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate obtained in Examples 1, 2, and 3 and 3,5-dihydroxy-4-methoxybenzyl alcohol solution, free drinking, respectively, after feeding 10, 30, 60, 120, 180, 240, 300, 360, 420 min, two mice were taken at each time point for brain sampling;

[0096] (2) The brain tissue samples of the same sampling point were taken together, the brain tissue samples were homogenized, extracted with ethyl acetate, separated using HPLC-C18 reverse phase column (inner diameter 250 x 4.6 mm; SHISEIDO, Tokyo, Japan; eluent acetonitrile / water (v:v = 5:95); temperature 30°C; flow rate 1.0 mL / min; detection wavelength UV 270 nm), the fraction with retention time 8-12 min was collected, freeze-dried, and dissolved in deionized water;

[0097] (3) Quantitative analysis of the extract, specifically:

[0098] (a) Using 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate and DHMBA standard prepared in Examples 1, 2, and 3, prepare aqueous solutions with concentration gradient (0-5000 ppb);

[0099] (b) Take 10 μL of the aqueous solution for determination in the mass spectrometer, respectively, determine the peak area of the measured substance in the standard solution with different concentrations, the quantitative standard curve of mass spectrometry MRM, X-axis is the concentration of DHMBA, y-axis is the peak area of mass spectrometry.

[0100] The results are shown in Figure 1, DHMBA can be transported to the brain within 10-30 min after being ingested by the mice by drinking water administration, and a concentration peak is formed after 30 min, and then the concentration decays with a half-life of about 60 min. The concentration of 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate obtained in Examples 1, 2, and 3 in the brain gradually increased, and the highest concentration was higher than that of DHMBA, and the half-life was longer.

[0101] Example 5

[0102] CCK-8 detection of the cytotoxicity of 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate obtained in Examples 1, 2, and 3 and DHMAB molecules

[0103] (1) The concentration gradient method was used to prepare different concentrations of Example 1, 2, 3 and DHMBA solutions using cell culture medium (DMEM medium containing 10% FBS) as the solvent. HT-22, N2A-sw cells were cultured in cell culture medium, and the culture conditions were 5% CO2, 37°C.

[0104] (2) After culturing, HT-22, N2A-sw cells were cultured in 24-well plates at a cell density of 6x105 per well, and the cell viability was detected using the CCK-8 method. 20 μL of CCK-8 reaction solution was incubated for 1 h, then 50 μL of 1% (w / v) sodium dodecyl sulfate reaction solution was added, the whole reaction was carried out in the dark, and finally the absorption peak was measured at 450 nm (N=6). The results are shown in Figure 2, and compared with 3,5-dihydroxy-4-methoxybenzyl alcohol molecules, 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate has higher cell safety.

[0105] The above embodiments are only some preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art based on the present application are within the scope of the present application.

Claims

1. A 3,5-dihydroxy-4-methoxybenzyl alcohol phosphate derivative of the formula I: ###00001### I R1, R2, R3 are selected from H or H2PO3, and R1, R2, R3 cannot be H at the same time.

2. The method of producing a 3,5-dihydroxy-4-methoxybenzene methanol phosphate derivative according to claim 1, characterized by, Selective phosphoric acid esterification of the hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol.

3. The production method according to claim 2, characterized by, When R1 is H2PO3 and R2, R3 are both H, the synthesis method is as follows: selective protection of the phenolic hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol with benzyl bromide to obtain 3,5-dibenzyl-4-methoxybenzyl alcohol, then phosphoric acid esterification of the alcohol hydroxyl group of 3,5-dibenzyl-4-methoxybenzyl alcohol with diethyl chlorophosphate, then hydrogenolysis to remove the benzyl protecting group, and finally hydrolysis under alkaline conditions by adding trimethylphenyl ammonium bromide to obtain 3,5-dihydroxy-4-methoxy-1-benzyl phosphonate.

4. The production method according to claim 3, characterized by, The specific steps are as follows: 3,5-dihydroxy-4-methoxybenzyl alcohol is dissolved in anhydrous DMF, anhydrous potassium carbonate and potassium iodide are added, and benzyl bromide is slowly added under nitrogen protection, the temperature is controlled at 25-30℃, then the temperature is raised to 60℃, and the reaction is stirred, then after treatment and purification, 3,5-dibenzyl-4-methoxybenzyl alcohol is obtained; 3,5-dibenzyl-4-methoxybenzyl alcohol is dissolved in anhydrous THF, cooled to 0℃ in an ice bath, slowly added with triethylamine, then added with diethyl chlorophosphate while keeping the temperature <5℃, after treatment and purification; then dissolved in methanol, hydrogenolysis to remove the benzyl protecting group in a hydrogen atmosphere through Pd / C at room temperature, and finally hydrolysis under alkaline conditions by adding trimethylphenyl ammonium bromide to obtain 3,5-dihydroxy-4-methoxy-1-benzyl phosphonate.

5. The preparation method according to claim 2, characterized in that When R1 is H, R2 is H2PO3 and R3 is H, the synthesis method is as follows: phosphoric acid esterification of the phenolic hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol with dibenzyl chlorophosphate, and finally hydrogenolysis to remove the benzyl protecting group to obtain 3-phosphonate-5-hydroxy-4-methoxybenzyl alcohol.

6. The preparation method according to claim 2, characterized in that When R1, R2, R3 are all H2PO3, the synthesis method is as follows: selective protection of the alcohol hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol with tert-butyl dimethyl chlorosilane, then phosphoric acid esterification of the phenolic hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol with dibenzyl chlorophosphate, then removal of the TBDMS protecting group, and finally phosphoric acid esterification of the alcohol hydroxyl group of 3,5-dihydroxy-4-methoxybenzyl alcohol with dibenzyl chlorophosphate, and then hydrogenolysis to obtain 3,5-diphosphonate-4-methoxy-1-benzyl phosphonate.

7. Use of the 3,5-dihydroxy-4-methoxybenzyl alcohol phosphonate derivative represented by formula I in claim 1 in the preparation of a medicament for preventing or treating neurodegenerative diseases.

8. Use according to claim 7, characterized in that, The medicament contains a pharmaceutically acceptable adjuvant and is prepared into various dosage forms, including liquid and solid dosage forms.

9. Use according to claim 8, characterized in that, The dosage forms include tablets, capsules, oral liquids, oral preparations, granules, powders, pills, powders, pastes, pills, suspensions, powders, injections, suppositories, sprays, drops or patches.

10. Use according to claim 7, characterized in that, The concentration of the 3,5-dihydroxy-4-methoxybenzyl alcohol phosphonate derivative in the medicament is 0.001wt.%-10wt.%.

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