A resveratrol derivative and its application

Abstract

This invention discloses a resveratrol derivative or its pharmaceutical salt, with the following general structural formula:; This invention also discloses the application of the resveratrol derivative or its pharmaceutical salt in the preparation of drugs for preventing and treating premature birth.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically, it relates to a resveratrol derivative and its application. Background Technology

[0002] Resveratrol (3,5,4'-trihydroxystilbene) is a class of natural non-flavonoid polyphenols found in plants such as Polygonum cuspidatum and grapes. It has various biological activities such as anti-oxidation, anti-cancer, anti-inflammatory, and anti-shock, and has good protective effects on the cardiovascular and nervous systems. It has attracted much attention in current research on anti-tumor, anti-inflammatory, and antioxidant effects, and is a lead compound with development potential.

[0003] Preterm birth refers to delivery before 37 weeks of gestation. Preterm birth is not only the leading cause of death in infants under 5 years old, but also has a high rate of disability. Reducing the incidence of preterm birth and improving the level of clinical intervention for preterm birth are urgent needs facing the obstetrics community. Because the etiology of preterm birth is difficult to elucidate, various prevention and treatment methods still have significant limitations. Currently, there is an urgent need to explore the molecular mechanisms initiating preterm / normal delivery in order to identify key therapeutic targets. Summary of the Invention

[0004] The first objective of this invention is to provide a resveratrol derivative.

[0005] A second objective of this invention is to provide the application of the resveratrol derivative in the preparation of a drug for preventing and treating premature birth.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a resveratrol derivative or a pharmaceutical salt thereof, with the following general structural formula:

[0008] ;

[0009] in,

[0010] L is selected from ;

[0011] R1 is selected from hydrogen and C1~C10 alkyl groups;

[0012] R2 is selected from hydrogen and C1~C10 alkyl groups;

[0013] R3 is selected from hydrogen;

[0014] R4 is selected from hydrogen;

[0015] X is selected from N and C;

[0016] When X is selected from C, the following groups are attached: ;

[0017] R5 is selected from hydrogen and C1~C10 alkyl groups;

[0018] R6 is selected from hydrogen or phenyl;

[0019] Furthermore, R1, R2, and R5 are not all hydrogen at the same time;

[0020] Alternatively, R4 and R6 can form a six-membered ring with carbon (e.g., (The dashed line indicates the connection point).

[0021] Alternatively, R3 and R4 can form a six-membered ring with carbon (e.g., (The dashed line indicates the connection point).

[0022] Preferably, in the resveratrol derivative,

[0023] L is selected from ;

[0024] R1 is selected from hydrogen, methyl, ethyl, and n-propyl;

[0025] R2 is selected from hydrogen, methyl, ethyl, and n-propyl;

[0026] R3 is selected from hydrogen;

[0027] R4 is selected from hydrogen;

[0028] X is selected from N and C;

[0029] When X is selected from C, the following groups are attached: ;

[0030] R5 is selected from hydrogen, methyl, ethyl, and n-propyl;

[0031] R6 is selected from hydrogen or phenyl;

[0032] Furthermore, R1, R2, and R5 are not all hydrogen at the same time;

[0033] Alternatively, R4, R6, and carbon can form a mixture. The dashed line indicates the connection point;

[0034] Alternatively, R3, R4, and carbon can form a mixture. The dashed line indicates the connection point.

[0035] Most preferably, the structure of the resveratrol derivative is selected from one of the following structures:

[0036]

[0037] In a second aspect, the present invention provides the use of the resveratrol derivative or its pharmaceutical salt in the preparation of a medicament for preventing and treating premature birth.

[0038] A third aspect of the present invention provides a pharmaceutical preparation made from the resveratrol derivative or its pharmaceutical salt and medically acceptable excipients.

[0039] The pharmaceutical preparation is selected from oral liquids, injections, and tablets.

[0040] The drug formulation can be administered orally or intravenously.

[0041] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the resveratrol derivative or a pharmaceutically acceptable salt thereof, a pharmaceutically acceptable carrier, and other drugs for the prevention and treatment of premature birth.

[0042] By adopting the above technical solution, the present invention has the following advantages and beneficial effects:

[0043] This invention synthesizes a novel compound using resveratrol as a lead compound. The resveratrol derivative prepared by this invention exhibits improved molecular activity and drug-like properties, which can, to some extent, compensate for the low bioavailability of resveratrol. The resveratrol derivative prepared by this invention can significantly reduce the rates of preterm birth and stillbirth, and can be applied clinically to prevent and treat preterm birth. Attached Figure Description

[0044] Figure 1 This is a schematic diagram showing the activity results of resveratrol derivatives.

[0045] Figure 2 This is a schematic diagram showing the pharmacodynamic effects of highly active resveratrol derivative compounds 4, 6, 10, and 13.

[0046] Figure 3 This is a schematic diagram showing the results of the anti-preterm birth ability of compound 10. Detailed Implementation

[0047] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0048] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions or as recommended by the manufacturer. Reagents used in the synthesis experiments were purchased from Aladdin, Exploration Platform, Adamas, Bid Chemical, and Anengji, and all reagents were analytical or chemically pure. The NMR spectrometer was 400 / 500 / 600 MHz, with tetramethylsilane (TMS) as the internal standard and DMSO-d6 as the solvent. Chemical shift (δ) and coupling constant (J) are expressed in ppm and Hz, respectively. Column chromatography used 200-400 mesh silica gel (Qingdao Ocean Chemical Co., Ltd.), and thin-layer chromatography (TLC) analysis used GF254 silica gel plates (manufactured by Qingdao Ocean Chemical, China). ESI mass spectrometry was performed using an API-3000LC-MS mass spectrometer. High performance liquid chromatography (HPLC) was performed using a Shimadzu CTO-20A model column (YMC-Pack ODS-A, S-5 μm, 250×10.0 mm l.D), with a mobile phase of 90% methanol / 10% water, a flow rate of 2 mL / min, and a purity greater than 95%.

[0049] The synthetic route for intermediate P3 is shown below:

[0050]

[0051] Compound P1 was purchased from Adamas Reagents Ltd.

[0052] 10 g of compound P1 was dissolved in 50 ml of anhydrous DMF. 6 ml of triethylamine was added, and 8.55 ml of thionyl chloride was slowly added dropwise using a constant-pressure dropping funnel. Care was taken to add the chloride dropwise slowly, as rapid addition could produce a black byproduct. After the addition was complete, the mixture was stirred at room temperature for 4–5 h, monitored by a TCL (Total Chromatography-Concentration Liquid). After the reaction was complete, the reaction solution was quenched in ice water, and the pH was adjusted to 8 with saturated sodium bicarbonate solution. Extraction was performed with dichloromethane, and the organic phase was dried over anhydrous sodium sulfate. The mixture was filtered, and rotary evaporation yielded a brownish-yellow oil. Column chromatography (PE:EA = 24:1) was performed, and rotary evaporation yielded a colorless oil. Ice water was added to precipitate a white solid, which was filtered. The filtered solid was dried in a 37°C oven to obtain compound P2. Actual weight: 9.2 g, theoretical weight: 11.06 g, yield: 83.2%.

[0053]

[0054] 3.7 g of compound P2 and 7 ml of triethyl phosphite were placed in a 25 ml round-bottom flask and heated to 150 °C under nitrogen protection, then refluxed for 5 h. TLC monitoring was performed. After the reaction was complete, wet column chromatography (petroleum ether:ethyl acetate = 10:1) was used to obtain a colorless oily liquid, compound P3. Actual weight 4.9 g, theoretical weight 5.73 g, yield 85.5%.

[0055]

[0056] Example 1

[0057] The preparation of compound 8 is shown below:

[0058]

[0059] Under nitrogen protection and an ice bath, compound P3 (1.00 g, 3.47 mmol) and sodium ethoxide (0.71 g, 10.41 mmol) were dissolved in 10 mL of DMF and stirred for 30 min. 2-Naphthaldehyde (0.45 g, 2.9 mmol) was dissolved in 2 mL of DMF and slowly added to the above mixture. The mixture was then transferred to room temperature and stirred overnight. The reaction was quenched with 10 mL of ice water and extracted with ethyl acetate (20 mL × 3). The organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5:1) to give compound 8 as a white solid (0.67 g, 2.3 mmol, 80% yield).

[0060]

[0061] Example 2

[0062] The preparation of compound 13 is shown below:

[0063]

[0064] In Example 1, 2-naphthaldehyde was replaced with p-phenylbenzaldehyde, and all other steps were the same as in Example 1, to obtain compound 13, a white solid (0.78 g, 2.46 mmol, yield 85%).

[0065]

[0066] Example 3

[0067] The preparation of compound 12 is shown below:

[0068]

[0069] In Example 1, 2-naphthaldehyde was replaced with 4-pyridinecarboxaldehyde, and all other steps were the same as in Example 1, to obtain compound 12, a white solid (0.504 g, 2.09 mmol, yield 72%).

[0070]

[0071] Example 4

[0072] The preparation of compound 10 is shown below:

[0073]

[0074] In Example 1, 2-naphthoaldehyde was replaced with 1-naphthoaldehyde, and everything else was the same as in Example 1, to obtain compound 10, a white solid (0.7 g, 2.415 mmol, yield 84%).

[0075]

[0076] Example 5

[0077] The preparation of compound 11 is shown below:

[0078]

[0079] Resveratrol (1 g, 4.38 mmol) was dissolved in 10 mL of tetrahydrofuran, and 10% palladium on carbon (0.1 g, 0.94 mmol) was added. The gas was replaced three times with a hydrogen balloon, and the mixture was stirred at room temperature for 6 h. The reaction solution was filtered through diatomaceous earth, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (CH2Cl2 / MeOH = 10:1) to give compound 11, a white solid (0.79 g, 3.43 mmol, yield 79%).

[0080]

[0081] Example 6

[0082] The preparation of compounds 6 and 7 is shown below:

[0083]

[0084] Compound 8 (1.93 g, 6.65 mmol) was dissolved in 15 mL of dichloromethane. The gas was purged three times with a nitrogen balloon. The reaction was carried out in a cryogenic reactor at -78 °C. A boron tribromide-dichloromethane mixture (10 mL, boron tribromide concentration 2 M / L) was slowly added dropwise. The mixture was slowly heated to 0 °C and stirred overnight. The reaction was quenched with ice water and extracted with dichloromethane (50 mL × 3). The organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5:1) to give compound 6, a white solid (0.64 g, 2.32 mmol, yield 35%). Compound 7, a white solid (0.83 g, 3.16 mmol, yield 48%).

[0085]

[0086] Example 7

[0087] The preparation of compound 9 is shown below:

[0088]

[0089] In Example 6, compound 8 was replaced with compound 10, and all other steps were the same as in Example 6, to obtain compound 9, a white solid (0.73 g, 2.65 mmol, yield 40%).

[0090]

[0091] Example 8

[0092] The preparation of compounds 1, 2, 3, 4, and 5 is shown below:

[0093]

[0094] Under nitrogen protection and at a temperature of -5°C, resveratrol (2 g, 8.76 mmol) was dissolved in 15 mL of DMF, and sodium hydride (0.32 g, 13.2 mmol) was slowly added in portions. The mixture was stirred for 10 minutes, and iodomethane (1.87 g, 13.14 mmol) was slowly added dropwise. The mixture was then transferred to room temperature and allowed to react overnight. Extracted with ethyl acetate (50 mL × 3), the organic phase was dried over sodium sulfate, filtered, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (PE / EA = 5:1) to give compound 2, white solid (0.12 g, 0.5 mmol, yield 5.6%); compound 1, white solid (0.22 g, 0.91 mmol, yield 10.2%); compound 3, white solid (0.4 g, 1.56 mmol, yield 17.8%); compound 4, white solid (0.3 g, 1.11 mmol, yield 14%); and compound 5, white solid (0.5 g, 2.32 mmol, yield 14%).

[0095]

[0096] Example 9

[0097] To investigate the in vitro activity of resveratrol derivatives, the experiment was divided into three groups: CTRL, model, and drug. Stimulation with DMSO, LPS, and drugs was performed for 6 and 24 hours, respectively. Quantitative polymerase chain reaction (PCR) was used to detect changes in the RNA levels of inflammatory factors COX2, IL6, and IL-1β. First, a preterm birth inflammation model was established on amniotic fibroblasts using different concentrations of LPS for 24 hours. 2016;126(8):2941-2954.), by comparing changes in COX2 gene expression at the RNA level, upregulation of gene expression was considered a successful model. The results showed that LPS 10 ng / mL was the optimal modeling condition, with COX2 gene expression upregulated by 2.5 times and p value less than 0.05. Based on the model, resveratrol and Nfκb inhibitors were used as positive control drugs, and the IL6 and COX2 genes were detected after stimulation for 6 and 24 hours. At the same time, compounds with significant downregulation trends (compound 4, compound 6, compound 10, compound 13) were studied by real-time quantitative PCR experiments, based on IC50. 50 The efficacy of the four compounds was studied by setting up concentration gradients. It was found that resveratrol significantly downregulated COX2 and IL-1β expression at 10 μM compared with the model group. Compound 4 showed the best downregulation effect at 25 μM. The optimal concentrations for downregulating COX2 expression of compounds 6, 10, and 13 were all around 5 μM, while the optimal concentration for IL-1β expression was around 10 μM.

[0098] Amniotic fibroblasts were stimulated with different concentrations of the compound for 12 h, and then incubated with 100 μL / well CCK8 solution for 1 h to determine the OD value, thus characterizing cytotoxicity. The results showed that resveratrol had an IC50 of [missing value]. 50 It is 12.59 μM, IC50 of compound 4 50 At around 10.8 μM, the IC50 of compound 6 is... 50 The IC50 value for compound 10 is 13.07 μM. 50 It is 8.52 μM, IC50 of compound 13 50 The concentration was around 12.66 μM. These results indicate that compound 10 exhibited the strongest cytotoxicity, resveratrol and compound 13 showed similar cytotoxicity, and compound 6 exhibited the weakest cytotoxicity. Due to the strong structural novelty of compounds 10 and 13, they are considered candidate compounds for further investigation.

[0099] Apoptosis assays were used to verify the apoptosis rate before and after drug administration in a model to determine the optimal candidate drug. The cells were divided into three groups: CTRL, model group, and drug group, and administered 1 μL DMSO, 10 ng / mL LPS, and 10 μM of drug (resveratrol, compound 10, and compound 13), respectively. The apoptosis rate in the CTRL group was approximately 3.3%, while the apoptosis rate in the model group was 7.06%, significantly higher than that in the CTRL group. The apoptosis rate in the resveratrol group was lower than that in the model group but close to that in the CTRL group. The results for compounds 10 and 13 were not significantly different from those for resveratrol. These results suggest that resveratrol can effectively reduce infectious inflammation, and compounds 10 and 13 can also reduce infectious inflammation.

[0100] To verify the anti-preterm birth efficacy of resveratrol derivative compound 10, spontaneous preterm birth animal models were established by subcutaneous injection of different concentrations of IL-1β in mice at 16.5 days post-pregnancy. 2023;98:104865.). A control group and a treatment group were set up. The control group received PBS, while the treatment group (resveratrol and compound 10 dissolved in 10% ethanol-corn oil) received two doses (20 mg / kg and 40 mg / kg) of resveratrol and compound 10 via gavage at 13.5 days, 14.5 days, and 16.5 days (0 h and 4 h after IL-1β administration), respectively. (2015;22(12):1561-1568.) The efficacy of resveratrol in treating preterm birth was characterized by the number of mice born prematurely and the number of survivors. Immunohistochemical staining was used to analyze the distribution of COX2, a factor associated with preterm birth. The results showed that resveratrol at 20 mg / kg and compound 10 at 40 mg / kg were the optimal experimental conditions for treating preterm birth. Compound 10 showed the greatest therapeutic effect at a dose of 40 mg / kg, with a preterm birth rate as low as 12.5% ​​and a survival rate as high as 71.2%. Immunohistochemical results showed that compound 10 significantly enhanced COX2 levels in amniotic cells, gradually approaching the control group, while its effect was less pronounced in decidual cells. These findings suggest that compound 10 holds promise for addressing the problem of premature birth caused by amniotic membranes.

[0101] Experimental results are as follows Figure 1 As shown, Figure 1 The diagrams show the activity results of resveratrol derivatives. In diagram a, the amniotic fibroblast inflammation model was established, and the COX2 gene was detected after 24 hours of stimulation with different concentrations of LPS. At 10 ng / mL LPS, the COX2 gene was upregulated 2.5-fold. Diagrams b and c show the expression of COX2 and IL6 after 24 hours and 6 hours of stimulation with resveratrol derivatives, respectively. The results show that compounds 3, 4, 6, 8, 9, 10, and 13 showed a downregulation trend in COX2 gene expression, while compounds 4, 5, 6, 9, 10, 11, and 13 showed similar or downregulation effects on IL6 and IL-1β genes as the positive control drug. Diagrams d and e show the expression of COX2 and IL6 after 24 hours and 6 hours of stimulation with highly active resveratrol derivatives, respectively. Diagram f shows the expression of IL-1β after 24 hours of stimulation with highly active resveratrol derivatives. Diagrams d, e, and f indicate that compounds 4, 6, 10, and 13 showed a downregulation trend in all three genes.

[0102] Figure 2 This is a schematic diagram showing the pharmacodynamic effects of highly active resveratrol derivative compounds 4, 6, 10, and 13. a and b are schematic diagrams showing the gene expression after stimulating different concentrations of the highly active resveratrol derivatives at 24 h and 6 h, respectively; compound 10 shows the most significant pharmacodynamic effect. c and d are schematic diagrams showing the effects of compound 10 based on IC50 values. 50A concentration gradient was set up, and the diagram shows the stimulation at 24h and 6h respectively; e, f, g, h, and i are the IC50 values ​​of the highly active resveratrol derivative. 50 Schematic diagram; j, k, l, m, and n represent the apoptosis results of CTRL, LPS, RSV, compound 10, and compound 13, respectively. The results showed that compounds 10 and 13 could reduce inflammation-induced apoptosis.

[0103] Figure 3 The diagram below shows the results of the anti-preterm birth ability of compound 10. a) is a schematic diagram of the modeling concentration for preterm birth rate, with 5 μg representing the optimal modeling condition, at which the preterm birth rate was 62.5%. b and c are schematic diagrams of the preterm birth rate at different doses of resveratrol and compound 10, respectively. At low doses, the preterm birth rate of compound 10 was higher than that of resveratrol, but at high doses it was much lower, and the preterm birth rate gradually decreased to 12.5%. d) is a schematic diagram of the modeling concentration for preterm birth rate, with a stillbirth rate of 54.5% under the 5 μg modeling condition. e and f are schematic diagrams of the stillbirth rate at different doses of resveratrol and compound 10, respectively. At 40 mg / kg, the stillbirth rate of compound 10 decreased to 28.8%. g) is a schematic diagram of the immunohistochemical detection results of COX2 in amniotic membrane and decidual cells in different groups. The figure shows that the COX2 content of compound 10 in amniotic membrane cells was low and gradually approached that of the control group, indicating that compound 10 has a good ability to prevent and treat preterm birth.

[0104] In summary, among the resveratrol derivatives prepared in this invention, compounds 10 and 13 can reduce the expression of inflammatory factors at the RNA level. Compound 10, in animal studies, significantly reduced preterm birth and stillbirth rates at a concentration of 40 mg / kg. Immunohistochemical results showed that compound 10 can reduce preterm birth inflammation in amniotic cells, indicating that compound 10 can be applied clinically for the prevention and treatment of preterm birth.

[0105] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A resveratrol derivative or a pharmaceutically acceptable salt thereof, characterized by, The general structural formula is as follows: ； in, L is selected from ; R1 is selected from hydrogen and C1~C10 alkyl groups; R2 is selected from hydrogen and C1~C10 alkyl groups; R3 is selected from hydrogen; R4 is selected from hydrogen; X is selected from N and C; when X is selected from C, the following groups are connected: ; R5 is selected from hydrogen and C1~C10 alkyl groups; R6 is selected from hydrogen or phenyl; Furthermore, R1, R2, and R5 are not all hydrogen at the same time; Alternatively, R4 and R6 can form a six-membered ring with carbon. Alternatively, R3 and R4 can form a six-membered ring with carbon.

2. The resveratrol derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized by, Among the resveratrol derivatives L is selected from ; R1 is selected from hydrogen, methyl, ethyl, and n-propyl; R2 is selected from hydrogen, methyl, ethyl, and n-propyl; R3 is selected from hydrogen; R4 is selected from hydrogen; X is selected from N and C; When X is selected from C, the following groups are attached: ; R5 is selected from hydrogen, methyl, ethyl, and n-propyl; R6 is selected from hydrogen or phenyl; Furthermore, R1, R2, and R5 are not all hydrogen at the same time; Alternatively, R4, R6, and carbon can form a mixture. The dashed line indicates the connection point; Alternatively, R3, R4, and carbon can form a mixture. The dashed line indicates the connection point.

3. The resveratrol derivative or its pharmaceutical salt according to claim 2, characterized in that, The structure of the resveratrol derivative is selected from one of the following structures: 。 4. The use of a resveratrol derivative or its pharmaceutical salt as described in any one of claims 1 to 3 in the preparation of a medicament for preventing and treating premature birth.

5. A pharmaceutical preparation, characterized in that, It is made from the resveratrol derivative as described in any one of claims 1 to 3 or its pharmaceutical salt and medically acceptable excipients.

6. The pharmaceutical preparation according to claim 5, characterized in that, The pharmaceutical preparation is selected from oral liquids, injections, and tablets.

7. The pharmaceutical preparation according to claim 5, characterized in that, The drug formulation can be administered orally or intravenously.

8. A pharmaceutical composition, characterized in that, It is made from the resveratrol derivative or its pharmaceutical salt as described in any one of claims 1 to 3, a pharmaceutically acceptable carrier, and other drugs for the prevention and treatment of premature birth.

Images