Clerodane type diterpenoid dimer compound and preparation method and application thereof

Abstract

The invention discloses a clerodane diterpenoid dimer compound and a preparation method and application thereof, and belongs to the technical field of medical intermediates, sculellaria barbata is cleaned with distilled water, reflux extraction is performed for three times with 95% ethanol, extraction is performed for two hours each time, a solvent is removed by a rotary evaporator to obtain a total extract, the total extract is suspended with ten times of water, and the clerodane diterpenoid dimer compound is obtained. The preparation method comprises the following steps: sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol for three times, removing a solvent through a rotary evaporator, carrying out silica gel column chromatography on an EtOAc extract and carrying out gradient elution with PE-EtOAc to obtain four fractions, carrying out gradient elution with silica gel column chromatography and a mobile phase PE-EtOAc to obtain five fractions, and purifying with preparative HPLC (High Performance Liquid Chromatography) to obtain the clerodane type diterpenoid dimer compound. According to the invention, modern separation and purification, structural analysis and other technologies are utilized to carry out detailed analysis on the whole herb of sculellaria barbata to obtain a novel compound, and the compound has a better antibacterial effect on plant growth.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical intermediates, and in particular relates to a crotan-type diterpene dimer compound and a preparation method and application thereof. Background Art

[0002] Scutellaria barbata (scientific name: Scutellaria barbata D. Don) is a perennial herbaceous plant in the Lamiaceae family. Native to China, it is endemic to the country and primarily distributed in southern, eastern, and southwestern China. Scutellaria barbata typically thrives in humid environments and prefers sunny locations. It is commonly found near waterways, wetlands, and forest edges. Its flowers are typically yellow and bloom in the summer, with beautiful shapes and ornamental value. In traditional Chinese medicine, Scutellaria barbata is considered a medicinal plant, commonly used for clearing heat and detoxifying, reducing swelling, and relieving pain. Its roots, stems, and leaves can all be used as medicine, exhibiting certain pharmacological effects, with the dried whole herb generally used. Overall, Scutellaria barbata holds significant ecological and medicinal value and is also a favorite among gardening enthusiasts for its unique flowers. Traditional studies on the biological activity of crotonane-type diterpenes have revealed antitumor activity. This patent, for the first time, discovers that crotonane-type diterpene dimers also possess significant antibacterial activity, worthy of further development and application. Summary of the Invention

[0003] Technical issues solved:

[0004] In response to the shortcomings of the prior art, such as weak separation specificity of diterpene dimers, this application provides a crotan-type diterpene dimer compound and its preparation method. This type of compound can be used as a pharmaceutical intermediate and can also be used in drugs for treating bacterial infections.

[0005] Technical solution:

[0006] To achieve the above objectives, this application is implemented through the following technical solutions:

[0007] A crotonane-type diterpene dimer compound, Discutebarbatine A, has a structure of Formula I:

[0008]

[0009] The present application also discloses a method for preparing a crotan-type diterpene dimer compound, comprising the following steps:

[0010] The first step is preliminary extraction: 10 kg of the medicinal material Scutellaria barbata was washed with distilled water, then reflux-extracted three times with 95% ethanol (EtOH), each extraction lasting two hours. The solvent was removed using a rotary evaporator to obtain 460 g of a total extract. The total extract was suspended in ten times the amount of water and extracted three times with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (n-Bu). The solvent was removed using a rotary evaporator to obtain 10 g, 90 g, 130 g, and 210 g of extracts in the PE, EtOAc, n-Bu, and HO layers, respectively.

[0011] The second step is purification by reverse phase medium and low pressure chromatography column: the EtOAc extract is subjected to silica gel column chromatography and gradient elution with PE-EtOAc to obtain four fractions Fr.B-1, Fr.B-2, Fr.B-3, and Fr.B-4; Fr.B-2 is subjected to silica gel column chromatography with the mobile phase being PE-EtOAc gradient elution to obtain five fractions Fr.B-2-1, Fr.B-2-2, Fr.B-2-3, Fr.B-2-4, and Fr.B-2-5; Fr.B-2-1 is purified by preparative HPLC using a JAIGEL-ODS-AP-P column to obtain compound Discutebarbatine A.

[0012] Furthermore, the EtOAc extract was subjected to silica gel column chromatography (5 cm×100 cm) with a PE-EtOAc gradient elution of 100:0-0:100, v / v.

[0013] Furthermore, the 11 g of Fr.B-2 was chromatographed on a silica gel column 2 cm × 30 cm with a mobile phase of

[0014] PE-EtOAc gradient elution, 100:0-100:10, v / v.

[0015] Furthermore, the 2 g of Fr.B-2-1 was purified by preparative HPLC using a JAIGEL-ODS-AP-P column to obtain 4 mg of compound Discutebarbatine A.

[0016] Furthermore, the preparative HPLC was MeOH-H2O, 80:20, v / v.

[0017] Furthermore, the JAIGEL-ODS-AP-P chromatographic column is 250 mm×20 mm, 4 μm, and has a flow rate of 5 mL / min.

[0018] Furthermore, the medicinal material Scutellaria barbata is the dried whole herb of Scutellaria barbata.

[0019] Application of a crotan-type diterpene dimer compound in medicine for treating bacterial infection.

[0020] Beneficial effects:

[0021] The present application provides a crotan-type diterpene dimer compound and its preparation method and application, which have the following beneficial effects compared with the prior art:

[0022] 1. The present invention utilizes modern separation and purification technology to systematically separate Scutellaria barbata and obtain a new class of croton-type diterpene dimers;

[0023] 2. In vitro tests have shown that these compounds have good antibacterial activity;

[0024] 3. It can be used as an active ingredient in antibacterial drugs and has a wide range of uses;

[0025] 4. Compared with crotonyl diterpenoid monomers, it has obvious antibacterial activity, which is worthy of development and application of this compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a hydrogen spectrum data diagram of the compound obtained in Example 1 of the present application;

[0027] Figure 2 This is the carbon spectrum data of the compound obtained in Example 1 of the present application;

[0028] Figure 3 is the HSQC chart of the compound obtained in Example 1 of the present application;

[0029] Figure 4 is the HMBC diagram of the compound obtained in Example 1 of the present application;

[0030] Figure 5 The compound obtained in Example 1 of the present application 1 H- 1 H COSY diagram;

[0031] Figure 6 It is the NOESY diagram of the compound obtained in Example 1 of the present application. DETAILED DESCRIPTION

[0032] To further illustrate the present invention, the preparation method of the crotan-type diterpene dimer provided by the present invention is described in detail below with reference to the examples.

[0033] Example 1

[0034] A crotan-type diterpene dimer compound, a crotan-type diterpene dimer compound

[0035] Discutebarbatine A has the structure of Formula I:

[0036]

[0037] The preparation method of the croton-type diterpene dimer compound adopts the steps of extraction and purification under conventional conditions under laboratory culture conditions to prepare the compound of the present invention, wherein the plant used is the dried whole herb of Scutellaria barbata, and specifically comprises the following steps:

[0038] The first step is preliminary extraction: 10 kg of the medicinal material Scutellaria barbata was washed with distilled water, then reflux-extracted three times with 95% ethanol (EtOH), each extraction lasting two hours. The solvent was removed using a rotary evaporator to obtain 460 g of a total extract. The total extract was suspended in ten times the amount of water and extracted three times with petroleum ether (PE), ethyl acetate (EtOAc), and n-butanol (n-Bu). The solvent was removed using a rotary evaporator to obtain 10 g, 90 g, 130 g, and 210 g of extracts in the PE, EtOAc, n-Bu, and HO layers, respectively.

[0039] The second step was purification by reverse phase medium and low pressure chromatography column: the EtOAc extract was chromatographed on a silica gel column 5 cm × 100 cm and eluted with a PE-EtOAc gradient of 100:0-0:100, v / v, to obtain four fractions Fr.B-1, Fr.B-2, Fr.B-3, and Fr.B-4; 11 g of Fr.B-2 was chromatographed on a silica gel column 2 cm × 30 cm with a mobile phase of PE-EtOAc gradient of 100:0-100:10, v / v, to obtain five fractions Fr.B-2-1, Fr.B-2-2, Fr.B-2-3, Fr.B-2-4, and Fr.B-2-5; 2 g of Fr.B-2-1 was purified by preparative HPLC using a JAIGEL-ODS-AP-P column, 250 mm × 20 mm, 4 μm, at a flow rate of 5 mL / min to obtain 4 mg of compound Discutebarbatine A.

[0040] Compound I, Compound 1 is a white powder with a HR-ESI-MS molecular ion peak of m / z 1065.4598 [MH] - (calc.mass 1065.4596for C 58 H 69 N2O 17 - ), indicating that its molecular formula is C 58 H 70 N2O 17， The unsaturation is 25.

[0041] As shown in Table 1, 1 H-NMR (600 MHz, CDCl3) Compound 1 includes eight aromatic ring signals: δ H9.21(1H,br s,H-3″), 9.15(1H,br s,H-3″′), 8.81(2H,br d,J=4.5Hz,H-5″ / 5″′), 8.23(2H,br d, J=7.7Hz, H-7″ / H-7″′), 7.42 (2H, dd, J=4.5, 7.7Hz, H-6″ / H-6″′).

[0042] Correlation analysis by HMBC: δ H 9.21(1H,br s,H-3″) / 164.6(C-1″), 119.1(C-2″), 154.1(C-5″); 9.15(1H,br s,H-3″′) / 164.4(C-1″′), 119.0(C-2″′), 154.1(C-5″′); 8.81(1H,br d,J=4.5Hz,H-5″ / 5″′) / 151.2(C-3″), 123.7(C-6″); 7.42(1H,dd,J=4.5,7.7Hz,H-6″ / ) / 154.1(C-5″), 137.0(C-7″); 8.81(1H,br d, J = 4.5 Hz, 5″′) / 123.7 (6″′); 7.42 (1H, dd, J = 4.5, 7.7 Hz, H-6″′) / 137.1 (C-7″′) indicated the presence of two nicotine groups.

[0043] There are three acetyl groups C 171.0(1′-CO- / 6′-CO-), 170.0(11′-CO-), 21.3((1′-COCH3), 21.0(6′-COCH3 / 11′-COCH3), 2.02(1′-COCH3 / 6′-COCH3 / 11′-COCH3).

[0044] Combine 13 C-NMR data and HSQC data showed 40 other carbon signals, including two carbonyl carbons [δ C 173.7 (C-15), 173.6 (C-15′)], four double-chain carbons [δ C 143.5(C-4), 143.3(C-4′), 143.5(C-4), 143.3(C-4′)], seven oxygenated methane carbon atoms [δ C 76.7 (C-6), 76.6 (C-6′), 74.4 (C-7), 74.2 (C-7′), 74.0 (C-1′), 71.9 (C-11), 71.7 (C-11′)], two oxygen-containing methylene carbons [δ C81.0 (C-16), 76.6 (C-16′)], four oxygen-containing quaternary carbons 81.1 (C-8 / 8′), 76.5 (C-13 / 13′)], four oxygen-containing quaternary carbons [δ C 44.8 (C-5′), 44.5 (C-5), 39.0 (C-9′), 38.8 (C-9)], two sp3 hybridized methyl carbons [δ C 44.4 (C-10′), 43.5 (C-10)], six sp3 hybridized methylene carbons [δ C 44.4 (C-14′), 43.3 (C-14), 33.3 (C-2 / 2′ / 12′), 29.5 (C-12), 28.8 (C-1)], eight methyl carbons [δ C 21.6(C-17), 21.3(C-17′), 20.8(C-18′), 20.3(C-20 / 20′), 20.2(C-18), 17.0(C-19′), 16.8(C-19)].

[0045] Table 1 Compounds 1 H-NMR and 13 C-NMR data summary table

[0046]

[0047]

[0048] The above results show that the structure of the obtained compound is correct.

[0049] Example 2:

[0050] Antibacterial activity of the crotan-type diterpene dimer compound of the present invention

[0051] (1) Experimental materials

[0052] Instruments and reagents: Microbial incubator; microplate reader (Bio-TEK ELx800); LB medium (Shanghai Bioengineering Co., Ltd.). Pathogenic bacterial strains used in the test: Enterobacter cloacae, Escherichia coli, Klebsiella aerogenes, Pseudomonas aeroginosa, Salmonella typhimurium, and Staphylococcus aureus were purchased from the Institute of Microbiology, Chinese Academy of Medical Sciences.

[0053] Test sample: Discutebarbatine A, with a purity of more than 90%; at the same time, streptomycin sulfate was selected as a positive control drug, and each compound was dissolved in DMSO and then diluted.

[0054] (2) Experimental methods

[0055] LB culture medium was prepared and sterilized at 121°C for 25 minutes. The diluted culture was added to a 96-well plate, with 180 μl added to each well. Compounds 1 and 2 were prepared in DMSO at specific concentrations and serially diluted to the wells containing the culture medium, resulting in final concentrations of 64, 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / ml, respectively. The cells were incubated in a 37°C incubator for 24 hours. The absorbance was read at 600 nm using a microplate reader. Streptomycin sulfate was used as a positive control, and a mixture of 180 μl of blank culture medium and 20 μl of DMSO was used as a blank control. The experiment was repeated three times. The minimum inhibitory concentration (MIC) of each test sample against human pathogens was calculated.

[0056] (3) Experimental results

[0057] The results of the in vitro antibacterial activity screening of the test samples are shown in Table 2.

[0058]

[0059] The results show that the compound I of the present invention has good spectrum antibacterial activity and can be used as an active ingredient of antibacterial drugs.

[0060] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A crotan-type diterpene dimer compound, characterized in that: The crotan-type diterpene dimer compound Discutebarbatine A has a structure of formula 1:

2. A method for preparing the crotan-type diterpene dimer compound according to claim 1, characterized in that: The steps include: The first step is preliminary extraction: 10 kg of the medicinal material Scutellaria barbata is cleaned with distilled water, and then refluxed and extracted three times with 95% ethanol EtOH, each extraction lasting two hours; the solvent is removed by a rotary evaporator to obtain 460 g of a total extract; the total extract is suspended with ten times the amount of water, and then extracted three times with petroleum ether PE, ethyl acetate EtOAc and n-butanol n-Bu in sequence; the solvent is removed by a rotary evaporator to obtain 10 g, 90 g, 130 g and 210 g of PE, EtOAc, n-Bu and H2O layer extracts respectively; The second step is purification by reverse phase medium and low pressure chromatography column: the EtOAc extract is subjected to silica gel column chromatography and gradient elution with PE-EtOAc to obtain four fractions Fr.B-1, Fr.B-2, Fr.B-3, and Fr.B-4; Fr.B-2 is subjected to silica gel column chromatography with PE-EtOAc gradient elution as the mobile phase to obtain five fractions Fr.B-2-1, Fr.B-2-2, Fr.B-2-3, Fr.B-2-4, and Fr.B-2-5; Fr.B-2-1 is purified by preparative HPLC using a JAIGEL-ODS-AP-P column to obtain the compound Discutebarbatine A.

3. The method for preparing a crotan-type diterpene dimer compound according to claim 2, characterized in that: The EtOAc extract was subjected to silica gel column chromatography (5 cm×100 cm) with a PE-EtOAc gradient elution of 100:0-0:100, v / v.

4. The method for preparing a crotan-type diterpene dimer compound according to claim 2, characterized in that: The 11gFr.B-2 was chromatographed on a silica gel column 2cm×30cm, and the mobile phase was PE-EtOAc gradient elution, 100:0-100:10, v / v.

5. The method for preparing a crotan-type diterpene dimer compound according to claim 2, characterized in that: The 2gFr.B-2-1 was purified by preparative HPLC using a JAIGEL-ODS-AP-P column to obtain 4 mg of compound Discutebarbatine A.

6. The method for preparing a crotan-type diterpene dimer compound according to claim 5, characterized in that: The preparative HPLC was MeOH-H2O, 80:20, v / v.

7. The method for preparing a crotan-type diterpene dimer compound according to claim 5, characterized in that: The JAIGEL-ODS-AP-P chromatographic column is 250 mm×20 mm, 4 μm, and has a flow rate of 5 mL / min.

8. The method for preparing a crotan-type diterpene dimer compound according to claim 2, characterized in that: The medicinal material Scutellaria barbata is the dried whole herb of Scutellaria barbata.

9. Use of the crotan-type diterpene dimer compound according to claim 1 in a drug for treating bacterial infection.

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