Paeonol phosphate and its preparation method and use

Abstract

The present invention provides a paeonol phosphate, a preparation method, and uses thereof, relating to the fields of medicine and cosmetics. The paeonol phosphate of the present invention is a novel compound with a simple preparation method and easy operation. By esterifying the phenolic hydroxyl group in the paeonol structure, the peculiar odor of small molecule phenolic compounds is avoided, and the disadvantage of paeonol's poor solubility in cold water is also addressed. Research has found that the paeonol phosphate provided by the present invention has stronger anti-inflammatory activity than paeonol and can be used to prepare anti-inflammatory drugs or cosmetics.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine and cosmetics, and in particular to paeonol phosphate, a preparation method and application thereof. Background Art

[0002] Paeonol is a kind of main active ingredient extracted from the root bark of tree peony or the whole herb of Cynanchum chinense. Its properties are white to slightly yellowish shiny needle-shaped crystals with biological activities such as antipyretic and analgesic, antibacterial and anti-inflammatory, and anti-oxidation. Paeonol has been applied to some daily necessities such as cosmetics, toothpaste, and perfumed soap as the main additive. Tablets and external-use ointments made with paeonol monomer as the main ingredient have also been applied clinically. The paeonol preparations that have been listed include paeonol tablets, paeonol anti-allergic dermatitis ointment, paeonol cyclodextrin injection and oral solution (Int J Nanomedicine, 2011, 6:1603-1610). However, paeonol belongs to small molecule phenolic compounds, has special smell, and its anti-drug activity is not strong. Compared with other non-steroidal anti-inflammatory drugs such as ibuprofen and celecoxib, clinical application is not extensive. Therefore, it is of great significance to use paeonol as a lead compound and obtain paeonol derivatives with strong anti-inflammatory activity, no special odor and no skin irritation through structural optimization. Summary of the Invention

[0003] In view of the deficiencies in the prior art, the first object of the present invention is to provide a paeonol phosphate and a preparation method and use thereof.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] On the one hand, a paeonol phosphate, whose structural formula is shown in the following formula (I):

[0006]

[0007] On the other hand, a method for preparing paeonol phosphate comprises the following steps:

[0008] In an organic solvent, paeonol reacts with dibenzyl phosphite or diethyl phosphite in the presence of an organic base and DMAP to generate an intermediate, which is then debenzylated or deethylated to generate paeonol phosphate.

[0009] Furthermore, the organic solvent is selected from any one or a combination of carbon tetrachloride, acetonitrile, tetrahydrofuran, and dioxane.

[0010] Furthermore, the amount of dibenzyl phosphite or diethyl phosphite used is 1.2 to 1.8 equivalents of paeonol.

[0011] Furthermore, the organic base is selected from any one or a combination of triethylamine, DBU, DIEPA, and pyridine, and the amount of the organic base used is 2.5 to 6 equivalents of paeonol.

[0012] Furthermore, the reaction temperature is 0-25° C., and the reaction time is 30-150 min.

[0013] Furthermore, the debenzylation process is carried out under the action of catalyst / hydrogen; the catalyst is any one of Pd / C and PtO2 or a combination of two.

[0014] Furthermore, the deethylation process is carried out under the action of TMSX; the TMSX is selected from any one or a combination of TMSCl, TMSBr, and TMSI.

[0015] On the other hand, a paeonol phosphate salt is any pharmaceutically acceptable salt of the paeonol phosphate represented by the above formula (I).

[0016] Furthermore, the paeonol phosphate salt includes but is not limited to sodium salt and potassium salt of paeonol phosphate.

[0017] Furthermore, the paeonol phosphate salt is prepared from paeonol phosphate represented by formula (I) and a corresponding inorganic base.

[0018] In another aspect, the above-mentioned paeonol phosphate or paeonol phosphate salt is used in the preparation of anti-inflammatory drugs.

[0019] In another aspect, the above-mentioned paeonol phosphate or paeonol phosphate salt is used in the preparation of cosmetics.

[0020] Furthermore, the cosmetics include but are not limited to any one of anti-inflammatory, soothing, whitening, anti-aging, and repairing cosmetics.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The paeonol phosphate provided by the present invention is a new compound. The present invention is the first to design and successfully synthesize paeonol phosphate.

[0023] 2. The paeonol phosphate provided by the present invention avoids the special odor and skin irritation of small molecule phenol compounds by esterifying the phenolic hydroxyl group in the paeonol structure. In addition, paeonol has poor solubility in cold water, and the solubility of paeonol in 100 mL of cold water is less than 0.12 g. The present invention introduces a hydrophilic phosphate group into the paeonol structure, which can also solve the shortcoming of poor solubility of paeonol in cold water. The solubility of paeonol phosphate in 100 mL of cold water increases to 7.8-8.2 g, and the solubility of paeonol phosphate salt in 100 mL of cold water is greater than 10 g.

[0024] 3. The paeonol phosphate provided by the present invention has stronger anti-inflammatory activity than paeonol and can be used to prepare anti-inflammatory drugs or cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the cytotoxicity test results of paeonol phosphate of the present invention.

[0026] Figure 2 This is the single-dose anti-inflammatory activity test result of the paeonol phosphate of the present invention.

[0027] Figure 3 This is the multi-dose anti-inflammatory activity test result of the paeonol phosphate of the present invention.

[0028] In the figure, P0 is paeonol and P8 is paeonol phosphate. DETAILED DESCRIPTION

[0029] The following non-limiting examples are provided to enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way. The following is merely an illustrative description of the scope of the present invention, and those skilled in the art may make various changes and modifications to the present invention based on the disclosed content, which should also fall within the scope of the present invention.

[0030] In the present invention, some commonly used abbreviations have the following meanings:

[0031] DBU 1,8-diazabicyclo[5.4.0]undec-7-ene

[0032] DIPEA N,N-Diisopropylethylamine

[0033] DMAP 4-dimethylaminopyridine

[0034] TMSX trimethylsilyl reagent

[0035] TMSCl trimethylsilyl chloride

[0036] TMSBr trimethylsilyl bromide

[0037] TMSI trimethylsilyl iodide

[0038] Unless otherwise specified, the various chemical reagents used in the examples of the present invention were obtained through conventional commercial channels.

[0039] The present invention will be further described below by way of specific embodiments.

[0040] Example 1

[0041] Preparation of intermediate paeonol dibenzyl phosphate

[0042]

[0043] At 0°C, dibenzyl phosphite (10.4g, 1.2eq.) was slowly added dropwise to a mixed solution of paeonol (5.5g, 1.0eq.), DIPEA (19.2g, 4.5eq.), and DMAP (50mg) in CCl4 (30mL) / MeCN (60mL), controlling the rate of 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 2.5h. The residual liquid was rotary evaporated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1 to obtain dibenzyl phosphite of paeonol as a colorless liquid weighing 10.0g, with a yield of 71%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.77(d,J=9.6Hz,1H),7.33-7.31(m,10H),6.93(d,J=1.2Hz,1H ), 6.74(d,J=9.6,1.2Hz,1H),5.16(dd,J=8.8,2.0Hz,4H),3.73(s,,3H),2.52(s,3H).

[0044] Example 2

[0045] Preparation of intermediate paeonol dibenzyl phosphate

[0046]

[0047] At 0°C, dibenzyl phosphite (15.6g, 1.8eq.) was slowly added dropwise to a mixed solution of paeonol (5.5g, 1.0eq.), DIPEA (19.2g, 4.5eq.), and DMAP (50mg) in CCl4 (30mL) / MeCN (60mL), controlling the rate of 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 0.5h. The residual liquid was rotary evaporated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1 to obtain dibenzyl phosphite as a colorless liquid weighing 11.7g, with a yield of 83%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.77(d,J=9.6Hz,1H),7.33-7.31(m,10H),6.93(d,J=1.2Hz,1H ), 6.74(d,J=9.6,1.2Hz,1H),5.16(dd,J=8.8,2.0Hz,4H),3.73(s,,3H),2.52(s,3H).

[0048] Example 3

[0049] Preparation of intermediate paeonol dibenzyl phosphate

[0050]

[0051] At 0°C, dibenzyl phosphite (13.1g, 1.5eq.) was slowly added dropwise to a mixed solution of paeonol (5.5g, 1.0eq.), DIPEA (10.6g, 2.5eq.), and DMAP (50mg) in CCl4 (30mL) / MeCN (60mL), controlling the rate of 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 0.5h. The residual liquid was rotary evaporated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1 to obtain dibenzyl phosphite as a colorless liquid weighing 10.3g, with a yield of 73%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.77(d,J=9.6Hz,1H),7.33-7.31(m,10H),6.93(d,J=1.2Hz,1H ), 6.74(d,J=9.6,1.2Hz,1H),5.16(dd,J=8.8,2.0Hz,4H),3.73(s,,3H),2.52(s,3H).

[0052] Example 4

[0053] Preparation of intermediate paeonol dibenzyl phosphate

[0054]

[0055] At 0°C, dibenzyl phosphite (13.1g, 1.5eq.) was slowly added dropwise to a mixed solution of paeonol (5.5g, 1.0eq.), DIPEA (25.5g, 6eq.), and DMAP (50mg) in CCl4 (30mL) / MeCN (60mL), controlling the rate of 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 0.5h. The residual liquid was rotary evaporated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1 to obtain dibenzyl phosphite as a colorless liquid weighing 11.5g, with a yield of 82%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.77(d,J=9.6Hz,1H),7.33-7.31(m,10H),6.93(d,J=1.2Hz,1H ), 6.74(d,J=9.6,1.2Hz,1H),5.16(dd,J=8.8,2.0Hz,4H),3.73(s,,3H),2.52(s,3H).

[0056] Example 5

[0057] Preparation of intermediate paeonol diethyl phosphate

[0058]

[0059] At 0°C, diethyl phosphite (2.07g, 1.5eq.) was slowly added dropwise to a mixed solution of paeonol (1.66g, 1.0eq.), DIPEA (3.23g, 2.5eq.), and DMAP (12mg) in CCl4 (5mL) / MeCN (10mL), controlling the rate at 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 1h. The reaction solution was concentrated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1. Paeonol diethyl phosphate was obtained as a colorless liquid weighing 2.48g, with a yield of 82%. After nuclear magnetic resonance (NMR) analysis, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.76 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H), 6.74 (d, J = 8.8 Hz, 1H), 4.23 (m, 4H), 3.84 (s,, 3H), 2.59 (s, 3H), 1.34 (m, 6H).

[0060] Example 6

[0061] Preparation of intermediate paeonol diethyl phosphate

[0062]

[0063] At 0°C, diethyl phosphite (2.07g, 1.5eq.) was slowly added dropwise to a mixed solution of paeonol (1.66g, 1.0eq.), Et3N (5.06g, 5.0eq.), and DMAP (12mg) in CCl4 (5mL) / MeCN (10mL), controlling the rate at 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 0.5h. The reaction solution was concentrated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1. Paeonol diethyl phosphate was obtained as a colorless liquid weighing 2.90g, with a yield of 96%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.76 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H), 6.74 (d, J = 8.8 Hz, 1H), 4.23 (m, 4H), 3.84 (s,, 3H), 2.59 (s, 3H), 1.34 (m, 6H).

[0064] Example 7

[0065] Preparation of intermediate paeonol diethyl phosphate

[0066]

[0067] At 0°C, diethyl phosphite (2.07g, 1.5eq.) was slowly added dropwise to a mixed solution of paeonol (1.66g, 1.0eq.), DIPEA (6.45g, 5.0eq.), and DMAP (12mg) in CCl4 (5mL) / MeCN (10mL), controlling the rate at 1-2 drops / s. After completion of the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and the reaction was continued for 0.5h. The reaction solution was concentrated and wet-loaded for column chromatography with an eluent of PE / EA 3:1 to 2:1. Paeonol diethyl phosphate was obtained as a colorless liquid weighing 2.93g, with a yield of 97%. After nuclear magnetic resonance detection, the specific test results were as follows: 1 H NMR (400MHz, CDCl3) δ7.76 (d, J = 8.8 Hz, 1H), 6.99 (s, 1H), 6.74 (d, J = 8.8 Hz, 1H), 4.23 (m, 4H), 3.84 (s,, 3H), 2.59 (s, 3H), 1.34 (m, 6H).

[0068] Example 8

[0069] Preparation of Paeonol Phosphate

[0070]

[0071] Paeonol dibenzyl phosphate (10 g, 0.023 mol) was dissolved in 30 mL of methanol. After replacing the nitrogen, 5% Pd / C (1 g) was added. H was introduced overnight under normal pressure. The Pd / C was removed by filtration, and the filtrate was dried to obtain 5.48 g of paeonol phosphate as an off-white solid with a yield of 95% and no special odor. After nuclear magnetic resonance (NMR) detection, the specific test results were as follows: 1 H NMR (400MHz, DMSO-d6) δ7.68(dd,J=8.8,1.1Hz,1H),6.98(dd,J=2.8,1.1Hz,1H),6.83(dd,J=8.5,2.8Hz,1H),3.81(s,3H),2.54(s,3H); 13 C NMR (100MHz, DMSO-d6) δ196.68,163.67,152.91,131.85,123.72,110.29,106.83,56.17,31.68.

[0072] Example 9

[0073] Preparation of Paeonol Phosphate

[0074]

[0075] Paeonol diethyl phosphate (10 g, 1.0 eq.) was dissolved in 250 mL of dry dichloromethane. TMSBr (250 mL) was slowly added dropwise under nitrogen. After the addition was complete, the mixture was naturally warmed to room temperature and stirred for 14 h. After rotary evaporation and concentration, methanol / water = 9 / 150 mL was added. After stirring for 1 h, the mixture was rotary concentrated. After concentration, an orange-red solid was obtained. After washing with 30 mL of ethyl acetate, the solid was dissolved in a solution of anhydrous ethanol (10 mL / g crude product) / (0.2 mL / mL anhydrous ethanol). 10% medicinal activated carbon was added and the mixture was heated at 50 ° C for 0.5 h. The filtrate was filtered through a sand core funnel. The filtrate was colorless. After concentration, 6.84 g of an off-white solid was obtained with a yield of 84%. There was no special odor. After nuclear magnetic resonance examination, the specific test results were as follows: 1 H NMR (400MHz, DMSO-d6) δ7.68(dd,J=8.8,1.1Hz,1H),6.98(dd,J=2.8,1.1Hz,1H),6.83(dd,J=8.5,2.8Hz,1H),3.81(s,3H),2.54(s,3H); 13 C NMR (100MHz, DMSO-d6) δ196.68,163.67,152.91,131.85,123.72,110.29,106.83,56.17,31.68. 1 H NMR (400MHz, D2O) δ7.63 (d, J = 8.8, 1H), 6.90 (s, 1H), 6.74 (d, J = 8.8 1H), 3.80 (s, 3H), 2.57 (s, 3H).

[0076] Example 10

[0077]

[0078] Paeonol phosphate (368 mg, 1.5 mmol) and sodium hydroxide (120 mg, 3.0 mmol) were added to a round-bottom flask. A 1:1 methanol / water mixture was added until the raw materials were completely dissolved (approximately 10 mL). The mixture was reacted at room temperature for 1 hour. Stirring was stopped, and the mixture was filtered. The filtrate was recovered and the solvent was recovered to obtain 413 mg of a white solid with a filtration yield of 95%. The melting point was >230°C. 1 H NMR (400MHz, D2O) δ7.64 (d, J = 8.8, 1H), 7.14 (s, 1H), 6.65 (d, J = 8.8 1H), 3.84 (s, 3H), 2.65 (s, 3H).

[0079] Example 11

[0080]

[0081] Paeonol phosphate (368 mg, 1.5 mmol) was dissolved in 10 mL of a 1:1 ethanol / water mixture. Sodium hydroxide (120 mg, 3.0 mmol) was added and allowed to react at room temperature for 1 hour. Stirring was stopped and the mixture was filtered. After recovering most of the solvent from the filtrate, 15 mL of ethanol was added and filtered to obtain 410 mg of a white solid with a filtration yield of 94%. Melting point > 230°C. 1 H NMR (400MHz, D2O) δ7.64 (d, J = 8.8, 1H), 7.14 (s, 1H), 6.65 (d, J = 8.8 1H), 3.84 (s, 3H), 2.65 (s, 3H).

[0082] Experimental Example 1

[0083] Cytotoxicity assay

[0084] RAW264.7 cells in good growth condition were seeded in 96-well plates. The experiment was divided into blank group, control group, paeonol group and paeonol phosphate group. Each drug group was set with 5 concentration gradients and three replicates were set in each well. 12 hours after cell inoculation, drugs were added according to the concentration gradient for 8 hours. After 8 hours of drug treatment, 10μL CCK-8 solution was added to each well and incubated in the cell culture incubator for another 1 hour. The absorbance was detected at 450nm. The results are as follows Figure 1 shown.

[0085] like Figure 1 As shown in the results, paeonol (P0) had significant cytotoxicity at a concentration of 400 μM (p<0.001), while paeonol phosphate (P8) showed cytotoxicity only at a concentration of 400 μM (p<0.01).

[0086] Experimental Example 2

[0087] Anti-inflammatory activity experiment

[0088] Single-dose anti-inflammatory activity screening: RAW264.7 cells were seeded at an appropriate density in 12-well plates. 12 hours after seeding, a single dose of paeonol and its derivatives (final concentration 50 μM) was added. After 2 hours of drug treatment, LPS (final concentration 500 ng / ml) was used to stimulate the cells to induce inflammation. After 6 hours of treatment, the cells were collected to extract RNA, and RT-qPCR was performed to detect the inflammatory factors iNOS and IL-6. The results are shown in the figure below. Figure 2 The results showed that at 50 μM, paeonol had no significant inhibitory effect on iNOS and IL-6 inflammatory factors, while paeonol phosphate had a significant inhibitory effect on iNOS and IL-6.

[0089] Multi-dose anti-inflammatory activity screening: RAW264.7 cells were seeded at an appropriate density in a cell plate. 12 hours after seeding, paeonol (P0) and paeonol phosphate (P8) were diluted to the appropriate concentration in PBS, the cell supernatant was discarded, and 300uL was added to each well. 2 hours later, LPS (final concentration 500ng / ml) was used to stimulate the cells to induce inflammation for 24 hours. The cell supernatant was then collected and IL-6 and iNOS were detected using ELISA kits. iNOS was detected using a dedicated detection kit according to the kit instructions. Figure 3 The results showed that the expression of inflammatory factor IL-6 in the cell supernatant was significantly reduced (p<0.05) when the concentration of paeonol phosphate (P8) was 25 μM, and it showed obvious dose dependence at 12.5-200 μM, with the lowest expression at 200 μM; at a concentration of 200 μM, the amount of IL-6 in the supernatant after the action of paeonol phosphate was lower than that of paeonol (P0) at the same dose. Figure 3 The results showed that the expression of iNOS was significantly reduced when the concentration of paeonol phosphate (P8) was 25 μM (p<0.05), and showed obvious dose dependence at 12.5-200 μM, with the lowest expression at 200 μM; at a concentration of 200 μM, the amount of iNOS in the supernatant after the action of paeonol phosphate was lower than that of the positive drug paeonol (P0) at the same dose.

[0090] In summary, the anti-inflammatory activity results of single dose and multiple doses all show that the paeonol phosphate provided by the present invention has good anti-inflammatory activity.

[0091] The above description of the embodiments is intended to facilitate understanding and application of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A paeonol phosphate, characterized in that Its structural formula is shown in the following formula (I):

2. The method for preparing paeonol phosphate according to claim 1, wherein The method comprises the following steps: in an organic solvent, paeonol reacts with dibenzyl phosphite or diethyl phosphite under the action of an organic base and DMAP to generate an intermediate, and then debenzylates or removes the ethyl group to generate paeonol phosphate.

3. The method for preparing paeonol phosphate according to claim 2, wherein The amount of dibenzyl phosphite or diethyl phosphite used is 1.2 to 1.8 equivalents of paeonol.

4. The method for preparing paeonol phosphate according to claim 2, wherein The organic base is selected from any one or a combination of triethylamine, DBU, DIEPA, and pyridine, and the amount of the organic base used is 2.5 to 6 equivalents of paeonol.

5. The method for preparing paeonol phosphate according to claim 2, wherein The reaction temperature is 0-30° C., and the reaction time is 75-90 min.

6. The method for preparing paeonol phosphate according to claim 2, wherein The debenzylation process is carried out under the action of catalyst / hydrogen; the catalyst is any one of Pd / C and PtO2 or a combination of the two; The deethylation process is carried out under the action of TMSX; the TMSX is selected from any one or a combination of TMSCl, TMSBr, and TMSI.

7. A paeonol phosphate salt, characterized in that: It is any pharmaceutically acceptable salt of the paeonol phosphate shown in claim 1; the paeonol phosphate salt comprises the sodium salt or potassium salt of the paeonol phosphate.

8. The method for preparing the paeonol phosphate according to claim 7, characterized in that: The paeonol phosphate salt is prepared from paeonol phosphate and corresponding inorganic base.

9. Use of the paeonol phosphate according to claim 1 or the paeonol phosphate salt according to claim 7 in the preparation of anti-inflammatory drugs.

10. Use of the paeonol phosphate according to claim 1 or the paeonol phosphate salt according to claim 7 in the preparation of cosmetics.

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