Paeonol phosphate as well as preparation method and application thereof

Abstract

The invention provides paeonol phosphate as well as a preparation method and application thereof, and relates to the technical field of medicines and cosmetics. The paeonol phosphate belongs to a novel compound, the preparation method is simple, and the operation is simple and convenient; phenolic hydroxyl groups in a paeonol structure are esterified, so that the special smell of small-molecular phenol compounds is avoided, and the defect of poor solubility of paeonol in cold water is overcome; research finds that compared with paeonol, the paeonol phosphate provided by the invention has stronger anti-inflammatory activity and can be used for preparing anti-inflammatory drugs or cosmetics.

Description

Technical Field

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

[0002] Paeonol is a kind of main active ingredient extracted from the root bark of peony or the whole herb of Cynanchum chinense, and its properties are white to slightly yellow shiny needle-shaped crystals, with biological activities such as antipyretic and analgesic, antibacterial and anti-inflammatory, and anti-oxidation. Paeonol has been used as the main added component in some daily necessities such as cosmetics, toothpaste, and perfumed soap. Tablets and external ointments made with paeonol monomer as the main ingredient have also been applied in clinical practice, and the paeonol preparations that have been listed include paeonol tablets, paeonol anti-allergic dermatitis ointment, paeonol cyclodextrin injection and oral solution, etc. (Int J Nanomedicine, 2011, 6: 1603-1610). But paeonol belongs to small molecule phenolic compounds, has special smell, and anti-drug activity is not strong, and 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 purpose, the technical solution adopted by the present invention is as follows:

[0005] On the one hand, a paeonol phosphate, the structural formula of which 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 usage of dibenzyl phosphite or diethyl phosphite 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 to 25° C., and the reaction time is 30 to 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 thereof.

[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 the paeonol phosphate ester 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 type of 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 smell and skin irritation of small molecular 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 disadvantage 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 test results of the cytotoxicity experiment 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 test result of the multi-dose anti-inflammatory activity 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 can enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way. The following content is only an exemplary description of the scope of the present invention, and those skilled in the art can make various changes and modifications to the invention of the present invention based on the disclosed content, and they 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 to a mixed solution of CCl4 (30mL) / MeCN (60mL) containing paeonol (5.5g, 1.0eq.), DIPEA (19.2g, 4.5eq.), and DMAP (50mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and continued to react for 2.5h. The residual liquid was subjected to wet evaporation and column chromatography, and the eluent was PE / EA 3:1 to 2:1, and paeonol dibenzyl phosphate was obtained as a colorless liquid, weighing 10.0g, with a yield of 71%. After NMR 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 to a mixed solution of CCl4 (30mL) / MeCN (60mL) containing paeonol (5.5g, 1.0eq.), DIPEA (19.2g, 4.5eq.), and DMAP (50mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and continued to react for 0.5h. The residual liquid was subjected to wet evaporation and column chromatography, and the eluent was PE / EA3:1 to 2:1, and paeonol dibenzyl phosphate was obtained as a colorless liquid, weighing 11.7g, with a yield of 83%. After NMR 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 (13.1 g, 1.5 eq.) was slowly added to a mixed solution of CCl4 (30 mL) / MeCN (60 mL) containing paeonol (5.5 g, 1.0 eq.), DIPEA (10.6 g, 2.5 eq.), and DMAP (50 mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15 min, then returned to room temperature and continued to react for 0.5 h. The residual liquid was subjected to wet evaporation and column chromatography, and the eluent was PE / EA 3:1 to 2:1, and paeonol dibenzyl phosphate was obtained as a colorless liquid, weighing 10.3 g, with a yield of 73%. After NMR 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 (13.1 g, 1.5 eq.) was slowly added to a mixed solution of CCl4 (30 mL) / MeCN (60 mL) containing paeonol (5.5 g, 1.0 eq.), DIPEA (25.5 g, 6 eq.), and DMAP (50 mg) at a rate of 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15 min, then returned to room temperature and continued to react for 0.5 h. The residual liquid was subjected to wet evaporation and column chromatography, with the eluent being PE / EA 3:1 to 2:1, and paeonol dibenzyl phosphate was obtained as a colorless liquid weighing 11.5 g with a yield of 82%. After NMR 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 to a mixed solution of CCl4 (5mL) / MeCN (10mL) containing paeonol (1.66g, 1.0eq.), DIPEA (3.23g, 2.5eq.), and DMAP (12mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15min, and then returned to room temperature to continue the reaction for 1h. After the reaction solution was concentrated, it was wet-loaded for column chromatography, and the eluent was PE / EA 3:1 to 2:1, and paeonol diethyl phosphate was obtained as a colorless liquid, weighing 2.48g, with a yield of 82%. After NMR 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).

[0060] Example 6

[0061] Preparation of intermediate paeonol diethyl phosphate

[0062]

[0063] At 0°C, diethyl phosphite (2.07g, 1.5eq.) was slowly added to a mixed solution of CCl4 (5mL) / MeCN (10mL) containing paeonol (1.66g, 1.0eq.), Et3N (5.06g, 5.0eq.), and DMAP (12mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and continued to react for 0.5h. After the reaction solution was concentrated, wet-loaded for column chromatography, and the eluent was PE / EA 3:1 to 2:1, and paeonol diethyl phosphate was obtained as a colorless liquid, weighing 2.90g, with a yield of 96%. After NMR 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 to a mixed solution of CCl4 (5mL) / MeCN (10mL) containing paeonol (1.66g, 1.0eq.), DIPEA (6.45g, 5.0eq.), and DMAP (12mg), and the rate was controlled at 1-2 drops / s. After the addition, the mixture was stirred at 0°C for 15min, then returned to room temperature and continued to react for 0.5h. After the reaction solution was concentrated, wet-loaded for column chromatography, and the eluent was PE / EA 3:1 to 2:1, and paeonol diethyl phosphate was obtained as a colorless liquid, weighing 2.93g, with a yield of 97%. After NMR 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, and after replacing N2, 5% Pd / C (1 g) was added, and H2 was introduced overnight under normal pressure, and Pd / C was removed by filtration. The filtrate was spin-dried to obtain an off-white solid of paeonol phosphate (5.48 g), with a yield of 95% and no special smell. After nuclear magnetic resonance detection, the specific test results were: 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 (10g, 1.0eq.) was dissolved in 250mL dry dichloromethane, and TMSBr (250mL) was slowly added dropwise under nitrogen. After the addition was completed, the temperature was naturally raised to room temperature, stirred for 14h, and concentrated by rotary evaporation. Methanol / water = 9 / 150mL was added, and stirred for 1h, and then concentrated by rotary evaporation. After concentration, an orange-red solid was obtained. After washing with 30mL ethyl acetate, it was dissolved in a solution of anhydrous ethanol (10mL / g crude product) / (0.2mL / mL anhydrous ethanol), 10% medicinal activated carbon was added, and heated at 50°C for 0.5h. The filtrate was colorless, and the filtrate was concentrated to obtain 6.84g of off-white solid, with a yield of 84%. There was no special smell. After nuclear magnetic resonance detection, the specific test results were: 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, and a 1:1 mixed solvent of methanol / water was added until the raw materials were completely dissolved (about 10 mL). The mixture was reacted at room temperature for 1 hour, and stirring was stopped. The mixture was filtered and the solvent was recovered from the filtrate to obtain 413 mg of a white solid with a filtration rate 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] Embodiment 11

[0080]

[0081] Paeonol phosphate (368 mg, 1.5 mmol) was dissolved in 10 mL of a 1:1 mixed solvent of ethanol / water, and sodium hydroxide (120 mg, 3.0 mmol) was added. The mixture was reacted at room temperature for 1 hour, and stirring was stopped. The mixture was filtered. After the filtrate was used to recover most of the solvent, 15 mL of ethanol was added and filtered to obtain 410 mg of a white solid with a filtration yield of 94%. 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).

[0082] Experimental Example 1

[0083] Cytotoxicity assay

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

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

[0086] Experimental Example 2

[0087] Anti-inflammatory activity test

[0088] Single-dose anti-inflammatory activity screening: RAW264.7 cells were seeded in 12-well plates at an appropriate density. After 12 hours of seeding, a single dose of paeonol and its derivatives (final concentration 50 μM) was added. After 2 hours of drug action, LPS (final concentration 500 ng / ml) was used to stimulate and induce cell inflammation. After 6 hours of action, cells were collected to extract RNA, and RT-qPCR was performed to detect iNOS and IL-6 inflammatory factors. The results are shown in 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] Screening of multi-dose anti-inflammatory activity: RAW264.7 cells were seeded in a cell plate at an appropriate density. 12 hours after seeding, paeonol (P0) and paeonol phosphate (P8) were diluted with PBS to an appropriate concentration, the cell supernatant was discarded, and 300uL was added to each well. 2 hours later, LPS (final concentration 500ng / ml) was used to stimulate and induce cell inflammation for 24 hours, and then the cell supernatant was collected. IL-6 and iNOS were detected using ELISA kits, and 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 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 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 to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and 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, characterized in that: 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 removes the benzyl group or the ethyl group to generate paeonol phosphate.

3. The method for preparing paeonol phosphate according to claim 2, characterized in that: The usage of dibenzyl phosphite or diethyl phosphite is 1.2 to 1.8 equivalents of paeonol.

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

5. The method for preparing paeonol phosphate according to claim 2, characterized in that: The reaction temperature is 0-30°C, and the reaction time is 75-90 minutes.

6. The method for preparing paeonol phosphate according to claim 2, characterized in that: 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 as shown in claim 1; the paeonol phosphate salt comprises a sodium salt or a potassium salt of the paeonol phosphate.

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