Phosphate derivatives of scutellarin 7-position, their preparation methods and uses

Abstract

This invention discloses the preparation method and uses of scutellarin 7-phosphate derivatives; belonging to the field of natural medicines and medicinal chemistry. The scutellarin 7-phosphate derivatives and their pharmaceutically acceptable salt structures described in this invention are shown in the following general formula I: By introducing a phosphate group at the 7-position, the scutellarin 7-phosphate derivatives are used in the preparation of drugs for cerebrovascular diseases. They exhibit excellent neuroprotective effects. The scutellarin phosphate derivatives of this invention have good protective activity against H2O2-induced oxidative damage to human neuroblastoma SH-SY5Y cells. Furthermore, the neuroprotective effects of some compounds are significantly stronger than those of the lead compound scutellarin.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical chemistry, in particular to the preparation method and use of scutellarein 7-OH phosphate derivatives. The present application discloses the preparation method of these scutellarein 7-OH phosphate derivatives and their use in the preparation of drugs for treating cerebrovascular diseases. BACKGROUND

[0002] Cerebrovascular diseases include cerebral ischemia, ischemia-reperfusion injury and cerebral embolism. The pathogenesis of cerebrovascular diseases is very complex, but different types of cerebrovascular diseases can cause a large number of nerve cell deaths. Therefore, reducing nerve cell damage is one of the important means for treating cerebrovascular diseases and improving prognosis. It is currently believed that oxidative stress plays an important role in the apoptosis of nerve cells. During the process of oxidative stress, the expression and activity of various enzymes change, such as NADPH oxidase, xanthine oxidase, aldehyde dehydrogenase, superoxide dismutase, etc., leading to the accumulation of ROS. A large dose of ROS can directly cause nerve cell necrosis, and a small dose of ROS can gradually cause nerve cell apoptosis through multiple cell pathways.

[0003] However, so far, the drugs for treating and relieving cerebrovascular diseases are still very limited, and the effect is difficult to satisfy. Natural products are an important source of drug discovery, and many clinically used drugs are directly or indirectly derived from natural products. Therefore, it is crucial to find and obtain candidate compounds with better activity, lower toxicity and more stable properties from natural products to resist oxidative stress damage.

[0004] Scutellarein is a flavonoid compound with medicinal value, which is mainly derived from the traditional Chinese medicinal material of Rabdosia japonica. Modern pharmacological studies have shown that scutellarein can significantly dilate cerebral blood vessels, increase cerebral blood flow, reduce whole blood viscosity, inhibit platelet aggregation, and improve hemorheology, etc. Mechanism studies have shown that scutellarein and its aglycone can increase the activity of the body's antioxidant system, reduce nerve cell apoptosis, and reduce the permeability of the blood-brain barrier, etc. In addition, pharmacological and toxicological experiments have confirmed that scutellarein has very low toxicity to mammals. Therefore, scutellarein is expected to be prepared into a drug for treating cerebrovascular diseases. However, the poor water solubility, low bioavailability and moderate activity of scutellarein limit its clinical application.

[0005] Chemical structural modification is one of the main methods to overcome the pharmacokinetic properties of scutellarin. Currently, structural modifications using scutellarin as a lead compound are mainly divided into two categories: one is direct modification of the scutellarin structure, primarily involving modification of the sugar carboxyl group and the 4′-position phenolic hydroxyl group, including esterification and amidation at the sugar carboxyl site, and etherification and esterification at the 4′-position phenolic hydroxyl group; the other is structural modification of the main in vivo metabolite of scutellarin, scutellarin aglycone (baicalein), mainly involving modification of the phenolic hydroxyl group, such as introducing various pharmacophores or water-soluble groups through etherification and esterification of the 6- and 7-position hydroxyl groups. However, although the above studies have improved the pharmacokinetic properties of scutellarin to some extent, problems still exist in subsequent pharmacological studies, and successful development has not yet been achieved. Summary of the Invention

[0006] Phosphate groups are essential components of nucleic acids, widely present in the human body, and are endogenous substances that are virtually harmless. Introducing a phosphate ester at the 7-OH position of scutellarin aglycone, either directly or through a linking group, facilitates the entry of scutellarin and its aglycone into cells. Simultaneously, introducing the phosphate ester into scutellarin aglycone enhances the neuroprotective activity and improves the pharmacokinetic properties of scutellarin and its aglycone.

[0007] The technical problem to be solved by this invention is to find a pharmaceutical composition with superior anti-oxidative stress damage activity and to further provide a pharmaceutical composition for treating cerebrovascular diseases and other diseases or conditions. This invention uses scutellarin as a lead compound and introduces a phosphate ester at the 7-OH position of scutellarin aglycone by direct attachment or through a linking group, thus synthesizing a phosphate ester derivative of scutellarin aglycone with the general formula I.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0009] General formula I represents the scutellarin aglycone phosphate derivative shown:

[0010]

[0011] Wherein, R1 is hydrogen, a straight-chain or branched alkyl group containing 1-8 carbon atoms; R2 is hydrogen, a straight-chain or branched alkyl group containing 1-8 carbon atoms; and n is an integer from 0 to 8.

[0012] Preferably, R1 is hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, or isobutyl; R2 is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, or hydrogen; and n is an integer from 0 to 6. More preferably, R1 is methyl; R2 is hydrogen; and n is 0, 3, or 5.

[0013] Furthermore, the present invention preferably uses the following compounds:

[0014]

[0015] A pharmaceutical composition of the present invention comprises a therapeutically effective amount of the above-described scutellarin aglycone 7-phosphate ester derivative of general formula I and its pharmaceutically acceptable salt; it also comprises a pharmaceutically acceptable carrier; the carrier refers to a conventional pharmaceutical carrier in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch paste, hydroxypropyl methylcellulose, povidone, syrup, etc., humectants such as ethanol, water, etc., disintegrants such as dry starch, sodium hydroxymethyl starch, low-substituted hydroxypropyl cellulose, effervescent disintegrants, cross-linked povidone, etc., absorption promoters such as calcium sulfate, calcium hydrogen phosphate, light magnesium oxide, calcium carbonate, etc., adsorbent carriers such as chitosan, lubricants such as magnesium stearate, polyethylene glycol, talc, hydrogenated vegetable oil, micronized silica gel, etc., colorants such as titanium dioxide, sunset yellow, methylene blue, pharmaceutical iron oxide red, etc., and coating materials such as acrylic resin, hydroxypropyl methylcellulose, povidone, cellulose acetate, etc. Other excipients such as flavoring agents and sweeteners may also be added to the composition.

[0016] The method for preparing derivatives of general formula I of the present invention is achieved through the following steps:

[0017]

[0018] Ligustrazine (1) reacted with iodomethane under K2CO3 conditions to obtain methylated ligustrazine intermediate (2). Ligustrazine intermediate 2 was hydrolyzed with concentrated HCl under N2 protection to remove the sugar chain, yielding 4′,7-dimethoxyligustrazine aglycone (3).

[0019] 4′,7-Dimethoxysalicylate aglycone (3) reacted with diethyl chlorophosphate under the catalysis of DMAP and triethylamine to give intermediate (4). Intermediate (4) was then deethylated under the conditions of trimethylbromosilane to give the target compound (5).

[0020]

[0021] 4′,7-Dimethoxystilbene aglycone (3) reacts with the corresponding bromoalkane at the 7-position under K2CO3 conditions to introduce the corresponding bromoalkane, yielding intermediates (6a, 6b). Then, it reacts with triethyl phosphite to yield intermediates (7a, 7b). The intermediates (7a, 7b) are further reacted with trimethylbromosilane to remove the diethyl group, yielding the target compound (8a, 8b).

[0022] The use of the 7-phosphate derivatives of scutellarin aglycone shown in Formula I and their pharmaceutically acceptable salts in the preparation of drugs for treating cerebrovascular diseases.

[0023] The above-mentioned pharmaceutical composition is used in the preparation of a drug for treating cerebrovascular diseases.

[0024] The cerebrovascular disease mentioned above refers to cerebral ischemia-reperfusion injury or cerebral infarction.

[0025] Pharmacological tests have demonstrated that the scutellarin phosphate derivatives of the present invention have good protective activity against H2O2-induced oxidative damage to human neuroblastoma SH-SY5Y cells. In addition, the neuroprotective effects of some compounds are significantly stronger than those of the lead compound scutellarin.

[0026] This invention significantly improves the water solubility of scutellarin by introducing a water-soluble phosphate fragment at the 7-OH site of scutellarin aglycone. Furthermore, the introduction of the phosphate fragment also benefits the enhancement of scutellarin aglycone activity, giving this compound the potential to be further developed into a new drug for the treatment of cerebrovascular diseases.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The 7-position phosphate derivative of scutellarin provided by the present invention is a compound with a novel structure.

[0029] (2) The 7-position phosphate derivative of scutellarin aglycone provided by the present invention has significant antioxidant stress damage resistance.

[0030] (3) The chemical structure of the 7-position phosphate derivative of scutellarin aglycone provided by the present invention is stable, which is beneficial to the clinical development of scutellarin.

[0031] (4) The method for preparing the 7-position phosphate derivative of scutellarin aglycone provided by the present invention is simple, easy to implement and low in cost. Detailed Implementation

[0032] Example 1

[0033]

[0034] Dissolve 1.39 g (3 mmol) of scutellarin 1 in 10 mL of DMF, add 1.12 mL (18 mmol) of iodomethane and 2.3 mL (15 mmol) of DBU, and stir at room temperature. Terminate the reaction after 24 h. Pour the reaction mixture into 100 mL of an ice-water mixture, extract with ethyl acetate (50 mL × 3), wash with saturated saline solution, dry over anhydrous sodium sulfate, recover ethyl acetate, and obtain the crude product. Separate by silica gel column chromatography (dichloromethane:methanol 25:1) to give 2635 mg of a pale yellow solid, yield 42%. 1H NMR (DMSO-d6, 400MHz) δ (ppm): 12.94 (s, 1H, 5-OH), 8.06 (d, 2H, J = 9.0Hz, H-2′, 6′), 7.15 (d, 2H, J = 9. 0Hz,H-3′,5′),7.09(s,1H,H-8),6.96(s,1H,H-3),5.61(brs,1H,H-1″),5.50(d,1H,J=5.0Hz,sugar hydroxyl),5.37(d,1H,J=7.3Hz,sugar hydroxyl),5.33(d,1H,J=7.3Hz,sugar hydroxyl),4.21(d,1H,J=9.5Hz,H-5″),3.87(s,3H,-OCH3),3.76(s,3H,-OCH3 ),3.66(s,3H,-OCH3),3.48-3.35(m,3H,H-2″,3″,4″); MS(ESI)m / z:505.1[M+H] + .

[0035] Example 2

[0036]

[0037] Intermediate 2 (5 g, 10 mmol) was added to a mixture of 120 mL anhydrous ethanol, 120 mL concentrated hydrochloric acid, and 10 mL H₂O. The mixture was refluxed under N₂ protection for 36 h. After cooling to room temperature, the reaction solution was poured into an equal volume of water, filtered, washed with water until neutral, dried, and the crude product was separated by silica gel column chromatography (petroleum ether: ethyl acetate 2:1) to give 1.1 g of yellow powder, yield 35%. 1 H NMR (DMSO-d6, 400MHz) δ (ppm): 13.04 (s, 1H, 5-OH), 10.74 (s, 1H, 7-OH), 8.03 (d, 2H, J = 8.9Hz, H-2′, 6′), 7. 10(d,2H,J=8.9Hz,H-3′,5′),6.87(s,1H,H-8),6.61(s,1H,H-3),3.85(s,3H,-OCH3),3.75(s,3H,-OCH3).

[0038] Example 3

[0039]

[0040] Intermediate 3 (315 mg, 1 mmol) was dissolved in 30 mL of THF, and DMAP (367 mg, 3 mmol) and Et3N (416 μL, 3 mmol) were added. The mixture was reacted at 0 °C for 15 min. Diethyl chlorophosphate (580 μL, 4 mmol) was added and refluxed for 8 h. The mixture was concentrated, extracted with ethyl acetate (3 × 20 mL), washed with saturated saline solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Separation was achieved by silica gel column chromatography (petroleum ether: ethyl acetate 1:1) to give 4295 mg of a light yellow powder, with a yield of 66%. Intermediate 4 (90 mg, 0.2 mmol) was dissolved in 10 mL of anhydrous DCM, and TMS-Br (264 μL, 2 mmol) was added. The mixture was reacted at room temperature for 24 h. The filtrate was concentrated and recrystallized from methanol to give 559 mg of a light yellow powder, with a yield of 75%. 1 H NMR (DMSO-d6, 400MHz) δ (ppm): 13.01 (s, 1H, 5-OH), 8.04 (d, 2H, J = 8.7Hz, H-2′, 6′), 7.21 (s, 1H, H-8), 7 .09(d,2H,J=8.9Hz,H-3′,5′),6.96(s,1H,H-3),3.85(s,3H,-OCH3),3.81(s,3H,-OCH3); HRMS(ESI)m / z calcd for C 17 H 15 O9P[M+H] + 395.0525, found 395.0526.

[0041] Example 4

[0042]

[0043] Intermediate 3 (315 mg, 1 mmol) was dissolved in 30 mL of acetone, and K₂CO₃ (417 mg, 3 mmol) and 1,3-dibromopropane (421 μL, 3 mmol) were added and refluxed for 8 h. The mixture was cooled to room temperature, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether: ethyl acetate 6:1) to give 279 mg of a light yellow powder 6a, yield 64%. 6a (86 mg, 0.2 mmol) was dissolved in 10 mL of P(OEt)₃ and reacted overnight at 150 °C. The reaction solution was concentrated, dispersed in water, extracted with ethyl acetate (3 × 20 mL), washed with saturated saline solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give a yellow crude product 7a. Compound 7a was added to 10 mL of acetic acid aqueous solution, refluxed at 170 °C for 1 h, cooled to room temperature, and the reaction solution was poured into 30 mL of H2O. The mixture was extracted with ethyl acetate (3 × 20 mL), washed with saturated saline solution, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated. Separation was performed by silica gel column chromatography (dichloromethane:methanol 20:1) to give 32 mg of the target compound 8a, with a yield of 37%. 1 H NMR (DMSO-d6, 400MHz) δ (ppm): 12.72 (s, 1H, 5-OH), 8.05 (d, 2H, J=8.9Hz, H-2′, 6′), 7.17 (d, 2H, J=8.9Hz, H-3′, 5′), 6.95 (s, 1H, H-8), 6.60 (s,1H,H-3),4.20(t,2H,J=6.2Hz,-CH2-),3.87(s,3H,-OCH3),3.86(s,3H,-OCH3),1.96(m,2H,-CH2-),1.70(m,2H,-CH2-); HRMS(ESI)m / z calcd for C 20 H 21 O9P[M+H] + 437.0979, found 437.0996.

[0044] Example 5

[0045]

[0046] The synthesis method was followed as described in Example 4. The product was a yellow powder, with a yield of 89%. 1H NMR (DMSO-d6, 400MHz) δ (ppm): 12.71 (s, 1H, 5-OH), 8.04 (d, 2H, J=8.9Hz, H-2′, 6′), 7.16 (d, 2H, J=8.9Hz, H-3′, 5′), 6.93 (s, 1H, H-8), 6.58 (s,1H,H-3),4.12(s,2H,-CH2-),3.87(s,3H,-OCH3),3.85(s,3H,-OCH3),1.77(t,2H,J=5.9Hz,-CH2-),1.54(m,6H,-CH2-); HRMS(ESI)m / z calcd for C 22 H 25 O9P[M+H] + 465.1334, found 465.1309.

[0047] The following are the pharmacological experimental results of some compounds in this invention.

[0048] Experimental equipment and reagents

[0049] Clean bench for instruments (Suzhou Cleanroom Group Antai Company)

[0050] Incubator (Thermo Electron Corporation)

[0051] Microplate reader (BioTek Instruments)

[0052] Inverted biological microscope (Olympus)

[0053] Reagents: Cell culture media MEM / F12, DMEM (Jiangsu Kaiji Biotechnology Co., Ltd.)

[0054] Fetal bovine serum (Hangzhou Sijiqing Co., Ltd.)

[0055] CCK8 (Jiangsu Kaiji Biotechnology Co., Ltd.)

[0056] DMSO (Sigma)

[0057] PBS (Jiangsu Kaiji Biotechnology Co., Ltd.)

[0058] FBS (GIBCO)

[0059] Trypsin-EDTA digestion solution (Jiangsu Kaiji Biotechnology Co., Ltd.)

[0060] Penicillin and streptomycin mixture (Jiangsu Kaiji Biotechnology Co., Ltd.)

[0061] Human neuroblastoma cell line SH-SY5Y

[0062] Experimental methods

[0063] Experimental methods for neuroprotective effects

[0064] Cells were cultured routinely in an incubator at 37°C and 5% CO2 saturated humidity. The culture medium was DMEM containing 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. The culture medium was changed after 48 hours. After cell attachment, cells were passaged by digestion with 0.25% trypsin. All cells used in the experiment were in the logarithmic growth phase, and trypan blue exclusion assay showed cell viability >95%.

[0065] Take one flask of cells in good logarithmic growth phase, add digestion solution (0.125% trypsin + 0.01% EDTA) for digestion, and count 5 × 10⁻⁶ cells. 4 Cells were cultured at concentrations of 100 μL / well in a 96-well plate and incubated for 24 hours. The culture medium was removed, and the drug was diluted to the desired concentration with complete medium. 100 μL of the corresponding drug-containing medium was added to each well, and a negative control group was also established. The 96-well cell culture plate was incubated at 37°C in a 5% CO2 incubator for 1 hour, followed by incubation with 400 μM H2O2 solution for another 4 hours. CCK8 was added to the 96-well plate at 10 μL / well and incubated for 2 hours. The plate was then shaken for 10 minutes. Three concentrations of the test substance (2 μM, 10 μM, 20 μM) were investigated. The absorbance of each well was measured at 450 nm using an ELISA reader, and the cell inhibition rate at each concentration was calculated.

[0066] Inhibition rate calculation method:

[0067]

[0068] Relative OD value of drug sensitivity wells = Absolute OD value of drug sensitivity wells - Absolute OD value of blank control wells

[0069] Experimental results

[0070] Table 1. Protective effect of Examples 3-5 on H2O2-induced SH-SY5Y neuronal damage.

[0071]

[0072] Pharmacological tests have demonstrated that the scutellarin aglycone phosphate derivatives of this invention exhibit good protective activity against H2O2-induced oxidative damage to human neuroblastoma SH-SY5Y cells. Furthermore, the neuroprotective effects of some compounds are significantly stronger than those of the lead compound, scutellarin aglycone. These results suggest that the compounds of this invention hold promise for further development into drugs for treating cerebrovascular diseases.

Claims

1. A 7-phosphate ester derivative of scutellarein aglycone of the general formula I: ###0001### I and pharmaceutically acceptable salts thereof. wherein R1 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; R2 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; n is an integer from 0 to 8.

2. The scytovlvinum phosphate derivative of the general formula I according to claim 1, characterized by ###0001### and pharmaceutically acceptable salts thereof. R1 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; R2 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; n is an integer from 0 to 8.

3. The scytovlvinum phosphate ester derivatives of the general formula I according to claim 1, characterized by ###0001### and pharmaceutically acceptable salts thereof. R1 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; R2 is hydrogen, a straight or branched alkyl group containing 1-8 carbon atoms; n is an integer from 0 to 8.

4. The 7-phosphate ester derivative of scutellarein aglycone of the general formula I of claim 1, selected from: ###0003### and pharmaceutically acceptable salts thereof.

5. A pharmaceutical composition comprising the 7-phosphate ester derivative of scutellarein aglycone of the general formula I of any one of claims 1-4 and pharmaceutically acceptable salts thereof.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable carrier.

7. A method for preparing the 7-phosphate ester derivative of scutellarein aglycone of the general formula I of claim 1 and pharmaceutically acceptable salts thereof, characterized by: reacting scutellarein (1) with iodomethane in the presence of K2CO3 to obtain a methylated scutellarein intermediate (2); hydrolyzing the scutellarein intermediate (2) in the presence of concentrated HCl under N2 protection to remove the sugar chain and obtain 4',7-dimethoxy scutellarein aglycone; reacting 4',7-dimethoxy scutellarein aglycone with diethyl chlorophosphate under catalysis of DMAP and triethylamine to obtain intermediate (4), and then removing the diethyl group under the condition of trimethylsilyl bromide to obtain the target compound 5; reacting 4',7-dimethoxy scutellarein aglycone with the corresponding bromoalkane in the presence of K2CO3, then with triethyl phosphite, and then removing the diethyl group under the condition of trimethylsilyl bromide to obtain the target compound (8a, b).

7. A method for preparing the 7-phosphate ester derivative of scutellarein aglycone of the general formula I of claim 1 and pharmaceutically acceptable salts thereof, characterized by: reacting scutellarein (1) with iodomethane in the presence of K2CO3 to obtain a methylated scutellarein intermediate (2); hydrolyzing the scutellarein intermediate (2) in the presence of concentrated HCl under N2 protection to remove the sugar chain and obtain 4',7-dimethoxy scutellarein aglycone; reacting 4',7-dimethoxy scutellarein aglycone with diethyl chlorophosphate under catalysis of DMAP and triethylamine to obtain intermediate (4), and then removing the diethyl group under the condition of trimethylsilyl bromide to obtain the target compound 5; reacting 4',7-dimethoxy scutellarein aglycone with the corresponding bromoalkane in the presence of K2CO3, then with triethyl phosphite, and then removing the diethyl group under the condition of trimethylsilyl bromide to obtain the target compound (8a, b).

8. Use of the 7-phosphate ester derivative of scutellarein aglycone of the general formula I of any one of claims 1-4 and pharmaceutically acceptable salts thereof in the preparation of a medicament for treating cerebrovascular diseases.

9. Use of the pharmaceutical composition of claim 5 in the preparation of a medicament for treating cerebrovascular diseases. The cerebrovascular diseases are cerebral ischemia-reperfusion injury or cerebral infarction. ​ ​ 10. Use according to claim 8 or 9, characterized in that, ​

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