Preparation method of abietane diterpene compound and application thereof in preparation of anti-diabetic drugs

Abstract

The application belongs to the technical field of medicine, and discloses a preparation method of a cembranoid diterpene compound and application of the cembranoid diterpene compound in preparation of an anti-diabetic drug. The application uses various separation technologies and means to take the Xinjiang Sabina fruit as a separation target, so as to realize enrichment of terpenoids. A cembranoid diterpene compound is obtained through silica gel column chromatography and ODS column chromatography and through a recrystallization method. Through an in-vitro PTP1B inhibition experiment, it is proved that the compound formula I has significant hypoglycemic activity, can be used as an active ingredient of an anti-diabetic drug, and has wide application.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to a method for preparing a rosinane-type diterpenoid compound and its application in the preparation of antidiabetic drugs. Background Technology

[0002] Diabetes, particularly type 2 diabetes, and its closely related obesity, has become a major global public health challenge. Developing novel, safe, and effective therapeutic drugs with novel mechanisms of action is an urgent research need. In recent years, protein tyrosine phosphatase 1B (PTP1B) has been identified as a key new target for combating type 2 diabetes and obesity. PTP1B belongs to the protein tyrosine phosphatase (PTP) family, and its core function is to negatively regulate the insulin signaling pathway by specifically dephosphorylating tyrosine residues on the insulin receptor (IR) and its substrates (such as IRS-1). Overexpression of PTP1B in tissues or cells significantly weakens the activity of receptor tyrosine kinase (PTK), leading to impaired insulin receptor binding, insulin resistance, and ultimately contributing to the development and progression of type 2 diabetes. Therefore, developing highly effective and selective PTP1B inhibitors is considered an important strategy for restoring insulin sensitivity and treating type 2 diabetes. Summary of the Invention

[0003] In view of this, the object of the present invention is to provide a rosinane-type diterpenoid compound or a pharmaceutically formable salt thereof and a method for preparing the same, the compound being used to prepare an antidiabetic drug.

[0004] To solve the above problems, the technical solution provided by the present invention is as follows:

[0005] In a first aspect, the present invention provides a method for preparing a rosinane-type diterpenoid compound, the method comprising the following steps:

[0006] S1. Crush the juniper fruit, extract it by reflux with ethanol, and concentrate the resulting extract under vacuum until no ethanol remains to obtain the total extract;

[0007] S2. The total extract is suspended in water and extracted with ethyl acetate to obtain the extract;

[0008] S3. The extract was separated by silica gel column chromatography to obtain fractions Fr.1-5, and Fr.4 was identified as the target fraction;

[0009] S4. The target fraction is separated by ODS-C18 column chromatography to obtain subfractions Fr.4a-4e;

[0010] S5. After the subfluid Fr.4a-4e was precipitated in methanol solution, it was recrystallized to obtain a rosinane-type diterpenoid compound with the structure shown in Formula I;

[0011]

[0012] In some embodiments of the present invention, in step S3 of the above preparation method, the silica gel column chromatography separation is performed by eluting with petroleum ether / ethyl acetate in volume ratios of 50:1, 25:1, 10:1, 2:1, and 1:1.

[0013] In some embodiments of the present invention, in step S4 of the above preparation method, the ODS-C18 column chromatography separation is performed by sequentially eluting with methanol / water at a volume ratio of 60:40, methanol / water at a volume ratio of 70:30, methanol / water at a volume ratio of 80:20, methanol / water at a volume ratio of 90:10, and methanol / water at a volume ratio of 100:0.

[0014] In some embodiments of the present invention, in step S5 of the above preparation method, the recrystallization is carried out using one of the following solvent systems: methanol / water system, methanol / dichloromethane system, and deuterated methanol / deuterated dichloromethane system.

[0015] In some embodiments of the present invention, in step S5 of the above preparation method, the recrystallization is carried out using a deuterated methanol / deuterated dichloromethane system with a volume ratio of 1:1.

[0016] In a second aspect, the present invention provides the use of the above-mentioned rosinane-type diterpenoid compound as an active ingredient in the preparation of a medicament for treating diabetes.

[0017] In a third aspect, the present invention provides a medicament comprising the above-described rosinane-type diterpenoid compound or a pharmaceutically acceptable salt thereof.

[0018] In some embodiments of the present invention, the medicament further includes pharmaceutically acceptable excipients and carriers.

[0019] In some embodiments of the present invention, the dosage form of the drug is one of granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels, and injections.

[0020] Compared with existing technologies, this invention selects mature and dried Xinjiang juniper fruit, extracts it by reflux with 95% ethanol, and then extracts it multiple times with ethyl acetate. Through specific separation and recrystallization steps, it efficiently obtains sufficient amount of high-purity rosinane-type diterpenoid compounds. For the first time, it discovers and verifies that this compound has significant PTP1B inhibitory activity, thus providing key activity evidence for its development into a novel antidiabetic drug. It has good development prospects and application potential in the preparation of antidiabetic drugs. Attached Figure Description

[0021] Figure 1 For Equation I 1 H NMR spectrum;

[0022] Figure 2 For Equation I 13 C NMR spectrum. Detailed Implementation

[0023] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Where specific conditions are not specified in the following embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0024] Example 1: Preparation of a compound with the structural formula of formula I

[0025] This invention utilizes mature Xinjiang juniper seeds, extracted by reflux with 95% (v / v) ethanol-water, and prepares the compound of this invention through extraction, enrichment, and separation. The Xinjiang juniper seeds are collected from Xinjiang.

[0026] Taking the fruit of *Juniperus chinensis* from Xinjiang as an example, the process for preparing the compound with structural formula I is as follows:

[0027] 1) Extraction, enrichment, and initial separation

[0028] Mature, dried Xinjiang juniper seeds (2.3 kg) were crushed and extracted three times by reflux with 95% ethanol, followed by vacuum concentration until alcohol-free. The extract was then mixed with hot water (60 °C) and repeatedly extracted with ethyl acetate in equal proportions to obtain 106 g of ethyl acetate extract. The extract layer (106.0 g) was subjected to silica gel column chromatography, eluting sequentially with petroleum ether / ethyl acetate at volume ratios of 50:1, 25:1, 10:1, 2:1, and 1:1 to obtain fraction Fr.1-5. The fraction was analyzed by HPLC and... 1 ¹H NMR analysis showed that Fr.4 contained a large amount of rosinane-type diterpenes, which were identified as the target fraction.

[0029] 2) ODS-C18 column chromatography separation

[0030] The target fraction Fr.4 was separated by ODS-C18 column chromatography. Subfractions Fr.4a~4e were obtained by sequentially using methanol / water at a volume ratio of 60:40, 70:30, 80:20, 90:10, and 100:0.

[0031] 3) Recrystallization

[0032] Components Fr.4a~4e precipitated as needle-like crystals in a methanol / water system, and the crystals were eluted with methanol. HPLC analysis revealed the presence of impurities. To obtain a high-purity compound, the eluted crystals were recrystallized sequentially using methanol / water (100:1 v / v), methanol / dichloromethane (1:1 v / v), methanol / dichloromethane (2:1 v / v), deuterated methanol / deuterated chloroform (2:1 v / v), and deuterated methanol / deuterated chloroform (1:1 v / v). HPLC analysis revealed that the purity of the crystals precipitated in the methanol / water (100:1 v / v) system was 90%, in the methanol / dichloromethane (1:1 v / v) system it reached 92%, in the methanol / dichloromethane (2:1 v / v) system it reached 93%, in the deuterated methanol / deuterated chloroform (2:1 v / v) system it reached 95%, and in the deuterated methanol / deuterated chloroform (1:1) system it reached 99%. Finally, 20 g of compound I was obtained. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 1 H NMR image as follows Figure 1 As shown, Equation I 13 C NMR spectrum as shown Figure 2 As shown.

[0033] Example 2: Hypoglycemic Activity of Compound I

[0034] 1) Experimental materials

[0035] Instruments and reagents: Incubator, MD 384 plus microplate reader, micropipette, pH meter, 96-well cell culture plate (COSTAR), Jasco 810 circular dichroism chromatograph. PTP1B enzyme, disodium 4-nitrophenyl phosphate (PNPP), and ursolic acid were purchased from Sigma-Aldrich; citrate monohydrate and trisodium citrate dihydrate were products of Guangzhou Chemical Reagent Factory; ultrapure water was used; other analytical grade reagents were products of Jiangsu Hanbang Technology Co., Ltd.

[0036] 2) Experimental methods

[0037] Add 140 μL of pH 6.8 citrate buffer (0.05 mol / L), 10 μL of LPTP1B enzyme solution (0.5 U / mL), and 2 μL of sample solutions of different concentrations sequentially to a 96-well microplate, and incubate at 37°C for 10 minutes. Then add 48 μL of PNPP solution (0.2 mM), react again for half an hour, and measure the absorbance of the reaction mixture over time at 405 nm. Each experiment was repeated three times. IC50 was used as the IC50 reading. 50 The values ​​were taken as their average. Ursolic acid was used as a positive control.

[0038] Inhibition rate (%) = 100 × (A) control - A sample ) / A control

[0039] 3) Experimental Results

[0040] The above method was used for testing, and the IC50 of sample inhibition of PTP1B was calculated using Formula I. 50 The values ​​are shown in Table 2.

[0041] Table 2. Screening results of test samples inhibiting PTP1B enzyme activity

[0042] compound Inhibition rate <![CDATA[IC 50 (μM)]]> Ursolic acid 85.69 ± 9.44 17.13 ± 3.02 Compound I 79.07 ± 3.60 27.2 ± 5.80

[0043] The results show that compound I of the present invention has good hypoglycemic activity and can be used as an active ingredient in antidiabetic drugs.

[0044] While some embodiments of the present general inventive concept have been shown and described, those skilled in the art will understand that changes may be made to these embodiments without departing from the principles and spirit of the present general inventive concept, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a rosinane-type diterpenoid compound, characterized in that, The preparation method includes the following steps: S1. Crush the juniper fruit, extract it by reflux with ethanol, and concentrate the resulting extract under vacuum until no ethanol remains to obtain the total extract; S2. The total extract is suspended in water and extracted with ethyl acetate to obtain the extract; S3. The extract was separated by silica gel column chromatography to obtain fractions Fr.1-5, and Fr.4 was identified as the target fraction; S4. The target fraction is separated by ODS-C18 column chromatography to obtain subfractions Fr.4a-4e; S5. After the subfluid Fr.4a-4e was precipitated in methanol solution, it was recrystallized to obtain a rosinane-type diterpenoid compound with the structure shown in Formula I; 2. The preparation method according to claim 1, characterized in that, In step S3, the silica gel column chromatography separation is performed by eluting with petroleum ether / ethyl acetate in volume ratios of 50:1, 25:1, 10:1, 2:1, and 1:1, respectively.

3. According to the preparation method of claim 1, in step S4, the ODS-C18 column chromatography separation is performed by sequentially eluting with methanol / water at a volume ratio of 60:40, methanol / water at a volume ratio of 70:30, methanol / water at a volume ratio of 80:20, methanol / water at a volume ratio of 90:10, and methanol / water at a volume ratio of 100:

0.

4. The preparation method according to claim 1, characterized in that, In step S5, the recrystallization is carried out using one of the following solvent systems: methanol / water system, methanol / dichloromethane system, and deuterated methanol / deuterated dichloromethane system.

5. The preparation method according to claim 1, characterized in that, In step S5, the recrystallization is carried out using a deuterated methanol / deuterated dichloromethane system with a volume ratio of 1:

1.

6. The use of a rosinane-type diterpenoid compound prepared by the preparation method according to any one of claims 1-5 as an active ingredient in the preparation of a medicament for treating diabetes.

7. A drug, characterized in that, The drug comprises a rosinane-type diterpenoid compound or a pharmaceutically acceptable salt thereof prepared by the preparation method according to any one of claims 1-5.

8. The medicament according to claim 7, characterized in that, The drug also includes pharmaceutically acceptable excipients and carriers.

9. The drug according to claim 7, characterized in that, The dosage form of the drug is one of granules, tablets, pills, solutions, capsules, films, tinctures, creams, ointments, aerosols, suppositories, liniments, gels, and injections.

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