N-alkyl pyrrole derivative as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of medicines, and relates to N-alkyl pyrrole derivatives as well as a preparation method and application thereof. The N-alkyl pyrrole derivative provided by the invention has a structure as shown in a formula I or a formula II, and the N-alkyl pyrrole derivative is obtained by fermenting and extracting cedar plant endophytic fungi Penicillium oxalicum, has relatively good anti-tumor activity and can be used as an active component of an anti-tumor drug.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a class of N-alkyl pyrrole derivatives and a preparation method and application thereof. Background Art

[0002] In recent years, plant endophytic fungi have become an important source of natural active compounds due to their unique living environment and metabolic pathways. Studies have shown that plant endophytic fungi can produce secondary metabolites with novel structures and diverse biological activities through long-term co-evolution with the host, including antibacterial, anti-inflammatory, and anti-tumor active ingredients. Among them, natural products with cytotoxic activity have attracted much attention due to their potential in the development of anti-tumor drugs. However, existing anticancer drugs generally face problems such as drug resistance, large toxic side effects, and limited sources. There is an urgent need to discover highly effective and low-toxic cytotoxic compounds from new natural products.

[0003] Active compounds isolated from plant endophytic fungi have demonstrated significant anticancer potential, but most compounds with known structures have limited activity profiles or face production bottlenecks. Furthermore, the metabolites of endophytic fungi are highly strain-specific, leaving significant room for further exploration of endophytic fungi from diverse host species. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a novel N-alkylpyrrole derivative and its preparation method and application. The N-alkylpyrrole derivative has good in vitro cytotoxic activity and can be used as an active ingredient in anti-tumor drugs with a wide range of uses.

[0005] In a first aspect of the present invention, an N-alkylpyrrole derivative or an acceptable salt thereof is provided, wherein the N-alkylpyrrole derivative is 2-[2-(2-hydroxyacetyl)-1H-pyrrol-1-yl]ethyl acetate, having the structure shown in Formula I:

[0006]

[0007] In a second aspect of the present invention, an N-alkylpyrrole derivative or an acceptable salt thereof is provided, wherein the N-alkylpyrrole derivative is 2-(1-(2-hydroxyethyl)-1H-pyrrol-2-yl)-2-oxoethyl acetate, having the structure shown in Formula II:

[0008]

[0009] The third aspect of the present invention provides a method for preparing the above-mentioned N-alkylpyrrole derivatives, wherein the method comprises fermenting and extracting the endophytic fungus Penicillium oxalicum of cedar plants to obtain the N-alkylpyrrole derivatives.

[0010] In some embodiments of the present invention, the preparation method comprises the following steps:

[0011] 1) Primary extraction: extracting a rice culture of the cedar plant endophytic fungus Penicillium oxalicum with an organic solvent, and concentrating to dryness under reduced pressure at room temperature to obtain an organic solvent extract;

[0012] 2) Extraction: The organic solvent extract obtained in step 1) is extracted with a methanol-water-cyclohexane system, and the methanol-water portion is concentrated to dryness under reduced pressure at room temperature to obtain a crude extract;

[0013] 3) Normal phase open chromatography column purification: the crude extract obtained in step 2) was added to a chromatographic column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and a petroleum ether-ethyl acetate solvent with a volume ratio of 0:100, and the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:30 was taken as the target fraction;

[0014] 4) Reverse-phase high performance liquid chromatography purification: The target fraction obtained in step 3) is separated and purified by reverse-phase high performance liquid chromatography to obtain the N-alkylpyrrole derivative.

[0015] In some embodiments of the present invention, the organic solvent is ethyl acetate.

[0016] In some embodiments of the present invention, the methanol-water-cyclohexane system is a mixed solution of methanol-water solution and cyclohexane, and the volume percentage of methanol in the methanol-water solution is 90%.

[0017] In some embodiments of the present invention, in the methanol-water-cyclohexane system, the volume ratio of methanol-water to cyclohexane is 1:1.

[0018] In some embodiments of the present invention, the reversed-phase high performance liquid phase conditions are: a methanol-water system with a volume ratio of 55:45.

[0019] The present invention also provides a use of the above-mentioned N-alkylpyrrole derivatives or acceptable salts thereof as active ingredients in the preparation of anti-tumor drugs.

[0020] In some embodiments of the present invention, the tumor is one of lung cancer, ovarian adenocarcinoma, cervical cancer, central nervous system tumor and human colon cancer.

[0021] The compound of the present invention is derived from a fermentation extract of the cedar plant endophytic fungus Penicillium oxalicum. Its chemical structure was identified as a novel pyrrole alkaloid compound by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HR-MS). In vitro cytotoxicity experiments demonstrated that the compound exhibited significant inhibitory activity against tumor cells and good selectivity. This discovery not only provides a new candidate molecule for the development of anti-tumor drugs but also provides a scientific basis for studying the metabolic mechanisms of plant-endophytic fungi interactions, possessing significant application value and industrialization prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the hydrogen spectrum data diagram of compound 1 obtained in Example 1 of the present invention;

[0023] Figure 2 This is the carbon spectrum data of compound 1 obtained in Example 1 of the present invention;

[0024] Figure 3 This is a two-dimensional nuclear magnetic resonance data diagram (HSQC) of compound 1 obtained in Example 1 of the present invention;

[0025] Figure 4 This is a two-dimensional nuclear magnetic resonance data diagram (HMBC) of compound 1 obtained in Example 1 of the present invention;

[0026] Figure 5 This is a high-resolution mass spectrometry (HRESIMS) data diagram of compound 1 obtained in Example 1 of the present invention;

[0027] Figure 6 This is the hydrogen spectrum data diagram of compound II obtained in Example 1 of the present invention;

[0028] Figure 7 This is the carbon spectrum data of compound II obtained in Example 1 of the present invention;

[0029] Figure 8 This is a two-dimensional nuclear magnetic resonance data diagram (HSQC) of compound II obtained in Example 1 of the present invention;

[0030] Figure 9 This is a two-dimensional nuclear magnetic resonance data diagram (HMBC) of compound II obtained in Example 1 of the present invention;

[0031] Figure 10 This is a high-resolution mass spectrum (HRESIMS) data diagram of compound II obtained in Example 1 of the present invention. DETAILED DESCRIPTION

[0032] To further illustrate the present invention, the N-alkylpyrrole derivatives and the preparation method thereof provided by the present invention are described in detail below with reference to the examples.

[0033] Example 1. Preparation of compounds of formula I and formula II

[0034] The present invention adopts the steps of extraction and purification under conventional laboratory culture conditions to prepare the compound of the present invention, wherein the microorganism used is Penicillium oxalicum.

[0035] The process for a specific compound is as follows:

[0036] 1) Cultivation

[0037] The cedar endophytic fungus Penicillium oxalicum was cultured in a rice solid medium at 30°C for 21 days. The rice solid medium contained 1g of potassium dihydrogen phosphate, 0.5g of magnesium sulfate, 0.5g of potassium nitrate, and 100ml of distilled water per 200g of rice.

[0038] 2) Extraction

[0039] The culture obtained in step 1) was soaked in 10 liters of ethyl acetate for extraction (at room temperature), and the ethyl acetate layer was taken and concentrated to dryness under reduced pressure to obtain 18.1 g of ethyl acetate extract.

[0040] 3) Extraction

[0041] The crude ethyl acetate extract was extracted with an equal volume of 90% methanol-water-cyclohexane system, and the 90% methanol-water portion was concentrated and dried to obtain 7.4 g of crude extract.

[0042] 4) Normal phase silica gel column chromatography separation

[0043] The crude extract was loaded onto silica gel and added to a chromatographic column containing 200 g of normal phase silica gel (200-300 mesh). Gradient elution was performed with a petroleum ether-ethyl acetate system (100:10-0:100). The eluted fractions were collected, and the petroleum ether-ethyl acetate component with a volume ratio of 100:30 was the main collected fraction.

[0044] 5) Reverse-phase high performance liquid chromatography purification

[0045] The selected fractions were analyzed and purified by reverse phase high performance liquid chromatography. ’ sphere ODS-H80 reverse phase chromatography column (250×10mm, 4μm, ), using a methanol-water isocratic elution system (methanol-water (55:45, v / v)) at a flow rate of 3.0 mL / min. Detection wavelength was 203 nm, and the injection volume was 50 μL. Compounds I and II were obtained as pale yellow powders.

[0046] like Figure 1-2 As shown, the H NMR spectrum and C NMR spectrum data of compound I are shown in Table 1 (Table 1), and the two-dimensional NMR data (HSQC) of compound I is shown in Figure 3 As shown, the two-dimensional nuclear magnetic resonance data (HMBC) is as follows Figure 4 As shown, the high resolution mass spectrometry (HRESIMS) data is shown in Figure 5 As shown, high resolution ESI mass spectrum data: (HRESIMS) m / z 210.0762 [MH] - It can be seen that compound I is an N-alkylpyrrole derivative 2-[2-(2-hydroxyacetyl)-1H-pyrrol-1-yl]ethylacetate. The structural formula of compound I is:

[0047]

[0048] like Figure 6-7 As shown, the H NMR and C NMR data of compound II are shown in Table 1 (Table 1), and the two-dimensional NMR data (HSQC) of compound II is shown in Figure 8 As shown, the two-dimensional nuclear magnetic resonance data (HMBC) is as follows Figure 9 As shown, the high resolution mass spectrometry (HRESIMS) data is shown in Figure 10 As shown, high-resolution ESI mass spectrum data: (HRESIMS) m / z 234.0742 ([M+Na]+. Compound II is an N-alkylpyrrole derivative 2-(1-(2-hydroxyethyl)-1H-pyrrol-2-yl)-2-oxoethyl acetate. The structural formula of compound II is:

[0049]

[0050] Table 1. 1 H and 13 CNMR data for compounds I and II(CD3OD,500MHz)

[0051]

[0052] Example 2: Cytotoxic activity of compounds I and II of the present invention

[0053] (1) Experimental materials

[0054] Instruments and reagents: Cell lines: A549 (human lung cancer cell line), SK-OV-3 (human ovarian adenocarcinoma cells), HeLa (human cervical cancer cells), XF498 (central nervous system tumor cells), HCT115 (human colon cancer cells); complete culture medium (DMEM + fetal bovine serum + double antibody); MTT reagent (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide).

[0055] Bio-Rad 680 microplate reader, Shimadzu UV-2401PC spectrophotometer.

[0056] Test samples: Compound I and Compound II, the compounds were dissolved in dimethyl sulfoxide and then diluted.

[0057] (2) Experimental methods

[0058] 1) Sample preparation: Compounds I and II were accurately weighed and dissolved in dimethyl sulfoxide solution to prepare a sample with an initial concentration of 1 mg / mL. Cell culture medium was then added to dilute the sample to different concentrations for later use.

[0059] 2) MTT (cytotoxicity) activity test: The cultured tumor cells were suspended in culture medium to prepare a cell suspension, and the cells were evenly seeded in a 96-well plate (5×10 4 Cells / well) were incubated for 24 hours. 2 μL of the prepared samples of Compounds I and II were added to each well (to final concentrations of 3.0625-100 μM, respectively). After 24 hours of incubation, 20 μL of the prepared MTT reagent was added to each well. The cells were incubated in the dark at 37°C for 4 hours. After aspirating the supernatant, 150 μL of DMSO was added to each well. The cells were shaken to mix and the absorbance was measured at 490 nm. The relative inhibition rate of Compounds I and II on tumor cell growth and proliferation was calculated. Three parallel experiments were performed for each sample group. The results are shown in Table 2.

[0060] Table 2.Cytotoxicities of compounds I and II(IC 50 ,ηM)

[0061]

[0062] cis-Platinum: positive control.

[0063] The results show that compounds I and II of the present invention have strong killing activity against lung cancer, ovarian adenocarcinoma, cervical cancer, central nervous system tumors and human colon cancer, can be used as active ingredients of natural anti-tumor drugs, and have wide applications in biomedicine and food industries.

[0064] 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. An N-alkylpyrrole derivative or an acceptable salt thereof, characterized in that: The N-alkylpyrrole derivative is 2-[2-(2-hydroxyacetyl)-1H-pyrrol-1-yl]ethyl acetate, which has the structure shown in Formula I:

2. An N-alkylpyrrole derivative or an acceptable salt thereof, characterized in that: The N-alkylpyrrole derivative is 2-(1-(2-hydroxyethyl)-1H-pyrrol-2-yl)-2-oxoethyl acetate, which has the structure shown in Formula II:

3. A method for preparing the N-alkylpyrrole derivative according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: fermenting and extracting the endophytic fungus Penicillium oxalicum of cedar plants to obtain the N-alkyl pyrrole derivatives.

4. The preparation method according to claim 3, characterized in that The preparation method comprises the following steps: 1) Primary extraction: extracting a rice culture of the cedar plant endophytic fungus Penicillium oxalicum with an organic solvent, and concentrating to dryness under reduced pressure at room temperature to obtain an organic solvent extract; 2) Extraction: The organic solvent extract obtained in step 1) is extracted with a methanol-water-cyclohexane system, and the methanol-water portion is concentrated to dryness under reduced pressure at room temperature to obtain a crude extract; 3) Normal phase open chromatography column purification: the crude extract obtained in step 2) was added to a chromatographic column filled with normal phase silica gel, and eluted with a petroleum ether-ethyl acetate solvent with a volume ratio of 100:10, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:20, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:30, a petroleum ether-ethyl acetate solvent with a volume ratio of 100:100, and a petroleum ether-ethyl acetate solvent with a volume ratio of 0:100, and the fraction eluted with the petroleum ether-ethyl acetate solvent with a volume ratio of 100:30 was taken as the target fraction; 4) Reverse-phase high performance liquid chromatography purification: The target fraction obtained in step 3) is separated and purified by reverse-phase high performance liquid chromatography to obtain the N-alkylpyrrole derivative.

5. The preparation method according to claim 4, characterized in that The organic solvent is ethyl acetate.

6. The preparation method according to claim 4, characterized in that The methanol water-cyclohexane system is a mixed solution of methanol water solution and cyclohexane, and the volume percentage of methanol in the methanol water solution is 90%.

7. The preparation method according to claim 4, characterized in that In the methanol water-cyclohexane system, the volume ratio of the methanol solution to the cyclohexane is 1:

1.

8. The preparation method according to claim 4, characterized in that The reversed-phase high performance liquid phase conditions are: a methanol-water system with a volume ratio of 55:

45.

9. Use of the N-alkylpyrrole derivative or an acceptable salt thereof according to claim 1 or 2 as an active ingredient in the preparation of an anti-tumor drug.

10. The use according to claim 9, characterized in that The tumor is one of lung cancer, ovarian adenocarcinoma, cervical cancer, central nervous system tumor and human colon cancer.

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