A prunolide analogue and use thereof

Abstract

The application provides a prunol analog and application thereof, and in-vivo uterine adhesion treatment test results show that the novel prunol analog provided by the application has excellent uterine adhesion treatment effect and has good medicinal prospects.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical chemistry, and particularly relates to a prunus mume analog and application thereof. BACKGROUND

[0002] Due to no relatively obvious early clinical symptoms, mild IUA is only manifested as periodic abdominal pain and menstrual abnormalities, and most IUA patients cannot receive timely treatment. After diagnosis, a comprehensive treatment mainly including hysteroscopic surgery separation is adopted to remove scar tissue and restore the shape of the uterine cavity. However, the hysteroscopic surgery separation cannot avoid the problems of tissue proliferation and inflammation. When moderate and severe IUA occurs, a large area of surgical wound may increase the exudation of inflammatory factors and adhesion-related factors, and increase the risk of postoperative re-adhesion and proliferation. 3h-2d after IUA separation is the time period with the most serious wound exudation and the most exudate, and this is the peak period of inflammatory reaction and the best opportunity to prevent adhesion. Therefore, the treatment of uterine cavity adhesion is often assisted by an anti-inflammatory treatment strategy in the clinic.

[0003] Prunus mume is a medicinal active ingredient extracted and refined from prunus mume, is one of the most natural products with the most characteristics in flavonoids, and is one of the most studied compounds in phenols. In plants, it exists in the form of glycosylation or aglycone. According to research reports, prunus mume has strong antifungal activity, anti-inflammatory and antioxidant effects, and can improve LPS-induced acute lung injury. In recent years, it has been reported that it also plays an important role in anticancer. However, there is still no relevant research report on the treatment of uterine cavity adhesion by prunus mume. Based on this, the application provides a new type of prunus mume analog, which has good treatment effect on uterine cavity adhesion. SUMMARY

[0004] The purpose of the application is to provide a new type of prunus mume analog and application thereof, and the prunus mume analog has good treatment effect on uterine cavity adhesion. The technical scheme adopted by the application is as follows:

[0005] A prunus mume analog, the structure of which is shown in formula (I):

[0006] (I)

[0007] Based on the structure skeleton of prunus mume, a new type of prunus mume analog compound is designed by a drug design method. The rat uterine cavity adhesion test results of the prunus mume analog show that the prunus mume analog has good treatment effect on uterine cavity adhesion.

[0008] The application further provides application of the prunus mume analog, and the prunus mume analog is used for preparing a uterine cavity adhesion treatment drug.

[0009] As a preference, the cherry blossom analogues treat intrauterine adhesions and diseases related thereto by inhibiting the expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β, etc.).

[0010] The present application also provides a pharmaceutical preparation comprising an effective component and pharmaceutical excipients, wherein the effective component comprises the cherry blossom analogue.

[0011] As a preference, the pharmaceutical preparation is any one of an injection, a gel, an ointment, a controlled-release preparation, a sustained-release preparation, or a nano-preparation.

[0012] Compared with the prior art, the present application has the beneficial effects that the present application designs and synthesizes a novel cherry blossom analogue based on the structural skeleton of cherry blossom through a drug design method. The novel cherry blossom analogue has excellent effects of inhibiting the expression of proinflammatory cytokines (TNF-α, IL-6, IL-1β, etc.), thereby having excellent intrauterine adhesion treatment effects and good pharmaceutical prospects. BRIEF DESCRIPTION OF DRAWINGS

[0013] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings obtained according to these drawings without creative labor are still within the scope of the present application.

[0014] Figure 1 It is a preparation process schematic diagram of Example 1;

[0015] Figure 2 It is a cherry blossom analogue 1 H NMR spectrum;

[0016] Figure 3 It is a cherry blossom analogue 13 C NMR spectrum;

[0017] Figure 4 It is a recovery of the uterine cavity size of the synthetic compound in Test Example 1 in rats;

[0018] Figure 5 It is a relieving effect of the synthetic compound in Test Example 2 on the inflammatory reaction of intrauterine adhesions in rats;

[0019] Figure 6 It is a recovery effect of the synthetic compound in Test Example 3 on the number of uterine adhesions in rats. DETAILED DESCRIPTION

[0020] For the purposes of making the objects, technical solutions and advantages of the present application clearer, the present application will be described in further detail below with reference to the drawings.

[0021] Example 1 Preparation of a prunusflavone analogue

[0022] The preparation process is as shown in Figure 1 , Figure 1 , wherein a) K2CO3, MOMCl, acetone; b) DMS, NaOH, TBA-Br, CH2Cl2: H2O = 2: 1; c) HCl, EtOH; d) 3-methyl-2-butenal, EDDA, xylene; e) aldehyde, KOH, EtOH; f) I2, DMSO.

[0023] Specifically:

[0024] a) Dilute 1.00 mmol of 2',4',6'-trihydroxybenzaldehyde and 4.00 mmol of potassium carbonate in 40 mL of acetone and heat to 60 o C. Add 2.50 mmol of methoxymethyl bromide dropwise to the reaction mixture over 30 minutes and reflux the mixture for 3 hours. After cooling to room temperature, filter the reaction mixture and remove the solvent under reduced pressure. After column chromatography, the product is obtained as a white oil; yield 55%.

[0025] b) Dissolve 1.00 mmol of the protected trihydroxyacetophenone in 40 mL of dichloromethane / water (3:2), add 1.50 mmol of sodium hydroxide and 0.10 mmol of tetrabutylammonium bromide. After adding 2.20 mmol of dimethyl sulfate dropwise, stir at room temperature for 2 hours. Then heat the mixture at 60 o C for 30 minutes to destroy the remaining dimethyl sulfate, then extract with dichloromethane, dry the organic layer with anhydrous magnesium sulfate, remove the solvent under reduced pressure, and purify the product as a white oil by column chromatography on silica gel; yield 80%.

[0026] c) Dissolve 1.00 mmol of the methylated acetophenone in 40 mL of ethanol solution, add 1.00 mmol of hydrochloric acid solution (3M), heat to 50 o C and maintain for 3 hours. After cooling, pour the reaction mixture into water, extract with ethyl acetate 3 times, dry the combined organic layers with anhydrous magnesium sulfate and concentrate under vacuum, and purify the product as a white solid by column chromatography on silica gel; yield 85%.

[0027] d) A solution of 2',4'-dihydroxy-6'-methoxyacetophenone (0.55 mmol), aldehyde (0.64 mmol) and ethylenediamine diacetate (0.05 mmol) in xylene (5 mL) was reacted at 120 °C overnight. After cooling, the reaction mixture was poured into water and extracted with ethyl acetate, the combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo, and the yellow solid product was purified by silica gel column chromatography with a yield of 65%. o C. After cooling, the reaction mixture was poured into water and extracted with ethyl acetate, the combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo, and the yellow solid product was purified by silica gel column chromatography with a yield of 65%.

[0028] e) 1.5 mL of aqueous potassium hydroxide solution (40%), 1.00 mmol of acetophenone and 1.20 mmol of the corresponding benzaldehyde were added to an ethanol solution. The reaction mixture was reacted at room temperature for 48 hours. After the reaction was completed, the reaction mixture was poured into 20 ml of water and extracted with ethyl acetate three times, the combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo, and the yellow solid product was purified by silica gel column chromatography with a yield of 45%.

[0029] f) 1.00 mmol of chalcone was added to DMSO, followed by the addition of I2(0.05 mmol), and stirred at 120 °C for 1.5 h. After the reaction was completed, the reaction was directly poured into saturated sodium thiosulfate solution, extracted with ethyl acetate three times, the combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo, and the white solid product - sakuranetin analog was purified by silica gel column chromatography with a yield of 75%. o C. After cooling, the reaction mixture was poured into water and extracted with ethyl acetate, the combined organic layers were dried over anhydrous magnesium sulfate and concentrated in vacuo, and the yellow solid product was purified by silica gel column chromatography with a yield of 65%.

[0030] As shown in Figures 2-3 , the spectral data of the prepared sakuranetin analogs are as follows:

[0031] 1 H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.6 Hz,2H), 6.83 (d, J = 10.0 Hz, 1H), 6.65 (s, 1H), 6.36 (s, 1H), 5.66 (d, J = 10.0 Hz,1H), 3.98 (s, 3H), 1.53 (s, 6H).

[0032] 13C NMR (101 MHz, CDCl3) δ 177.56, 160.73, 159.30, 158.23, 153.92,137.52, 130.20, 129.40, 127.79, 127.22, 115.17, 108.96, 108.74, 102.6 9,78.27, 96.86, 56.55, 28.29.

[0033] Test Example 1: Recovery of uterine cavity size in rats by cherry blossom analogs

[0034] Experimental animals: 5 SD female rats, weighing between 240-280 g.

[0035] Day 1: Fasting for 12 h before modeling, and applying depilatory cream on the abdomen of the rats, and removing the hair with a shaver.

[0036] Day 2: Weighing, recording body weight. Prepare the required drugs and cherry blossom analogs in advance. Anesthetize the rats with 10% chloral hydrate (0.3 ml / 100 g) intraperitoneally. Disinfect the abdominal skin with iodophor, then make an incision along the midline of the abdomen (about 2 cm) to open the abdominal cavity, and gently lift out the "Y" shaped uterus with a non-damaging forceps. Stretch the uterus, make a small incision near the lower end of the "Y" shape, and insert a toothed forceps from the incision to perform a physical curettage. The toothed end of the forceps is repeatedly rubbed against the endometrium until a rough feeling is produced on the smooth endometrium. The simple modeling group is given normal saline, and the other groups are given the corresponding drugs. After treatment, the incision is sutured with 7-0 suture thread, the uterus is slowly returned to the abdominal cavity, and the abdominal cavity is thoroughly flushed with normal saline. The incision is sutured layer by layer with 4-0 suture thread, and the wound is disinfected with iodophor after suture. The postoperative rats are returned to the cage, and the diet is restored. They are raised for more than 2 weeks.

[0037] The uterus tissue is quickly removed after euthanizing the rats, and the anatomical morphology of the rat uterus is observed, as shown in Table 1. Figure 4 The uterine wall of the normal group of rats is smooth and has strong toughness, the thickness is uniform, and the uterus is rich in elasticity. The uterine elasticity of the IUA group of rats is poor, the uterus is red, and obvious blood clots can be observed in the uterine cavity. The uterine elasticity and color of the rats in the cherry blossom analogs treatment group gradually recover to be consistent with the control group, and no blood clots are observed. This indicates that the cherry blossom analogs can effectively repair the shape of the uterine tissue.

[0038] Test Example 2: Effect of cherry blossom analogs on relieving inflammation of uterine cavity adhesion in rats

[0039] The immunohistochemical steps are as follows: 1. Take the rat uterine tissue section in the constant temperature box at 65°C for 4 h; 2. Dewaxing and hydration of the tissue; 3. Distilled water is washed for 2 times, each time for 3 min; 4. High-pressure antigen repair: citric acid buffer is boiled in the microwave oven, the section is immersed in it, and is boiled for 15 min at low and medium fire, and is cooled to room temperature; 5. 3% hydrogen peroxide, 37°C, treatment for 10 min, eliminating endogenous enzymes; 6. Blocking: PBS is washed for 3 times, 5% BSA is added, and is blocked at room temperature for 30 min, and the non-specific protein is blocked; 7. Diluted first antibody (TNF-α and IL-6 dilution ratio is 1:400) is added dropwise, is placed in a wet box, and is incubated at 4°C overnight; 8. After PBS is washed for 3 times, horseradish peroxidase-labeled second antibody is added, and is incubated at room temperature for 1 h; 9. DAB coloration: after PBS is washed for 3 times, DAB coloration solution is added and is incubated for 5 min, 10. Hematoxylin is used to dye the nucleus for 1 min; the staining degree is observed under a microscope; and the results are as shown in Figure 5 .

[0040] The above-mentioned immunohistochemical method is used to detect the alleviating effect of the sakuranetin analog on the inflammatory reaction of the rat intrauterine adhesion. The results show that the use of the sakuranetin analog can significantly inhibit the expression of the pro-inflammatory cytokines TNF-α and IL-6 of IUA ( Figure 5 ), and effectively alleviates the inflammatory reaction of IUA.

[0041] Test Example 3: Recovery effect of sakuranetin analog on the number of rat intrauterine adhesion uterine glands

[0042] The steps of hematoxylin-eosin staining method are as follows: 1. Take the rat uterine tissue section in the constant temperature box at 65°C for 4 h, hematoxylin is dyed for 5 min, distilled water is washed, eosin is dyed for 1 min, and distilled water is washed; 2. Hydrated for 10 min; 3. 85% ethanol is soaked for 5 min; 4. 95% ethanol is soaked for 5 min; 5. 100% ethanol is soaked for 5 min; 6. Xylene I and xylene II are passed once; 7. Dry, and the section is mounted with neutral resin. The results are as shown in Figure 6 .

[0043] The above-mentioned hematoxylin-eosin staining method (H&E) is used to detect the recovery effect of the sakuranetin analog on the number of rat intrauterine adhesion uterine glands. The experimental results show that, compared with the IUA group, the use of the sakuranetin analog can effectively recover the number of rat intrauterine adhesion uterine glands, so that the number of glands is equivalent to that of the control group ( Figure 6 ), indicating that the function of the uterus is effectively repaired.

[0044] The above-mentioned only discloses the preferred embodiments of the present application, and of course cannot limit the scope of the present application, so the equivalent changes made according to the claims of the present application still fall within the scope of the present application.

Claims

1. A cherry blossom extract analogue, characterized in that, The structure is shown in equation (I): （I）。 2. The application of a cherry blossom extract analogue as described in claim 1, characterized in that, The cherry blossom extract analogue is used to prepare a drug for treating intrauterine adhesions.

3. The application of the cherry blossom analogue according to claim 2, characterized in that, The cherry blossom analogue is used to prepare a drug for treating intrauterine adhesions by inhibiting the expression of inflammatory factors, including TNF-α, IL-6, and IL-1β.

4. A pharmaceutical preparation comprising an active ingredient and pharmaceutical excipients, characterized in that, The active ingredient includes the cherry blossom extract analogue as described in claim 1.

5. The pharmaceutical preparation according to claim 4, characterized in that, The pharmaceutical preparation is any one of an injection, gel, ointment, controlled-release agent, sustained-release agent, or nano-formulation.

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