Alkaloid compound as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to an alkaloid compound as well as a preparation method and application thereof. The alkaloid compound is separated from a fermentation extract of soil fungus aspergillus ustus. Results of a PTPs activity inhibition test, an antibacterial activity test and a PTP1B molecular docking test show that the two alkaloid compounds obtained by the invention have a good inhibition effect on PTP1B, an alternative compound is provided for developing a new PTP1B inhibitor, and the alkaloid compounds have a relatively good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to an alkaloid compound, a preparation method and application thereof. Background Art

[0002] Microbial metabolites are characterized by novel and diverse chemical structures, broad biological activities, and good drugability, making them an important source of lead compounds with potential for pharmaceutical development. Fungi, particularly filamentous fungi, are one of the main sources of microbial metabolites.

[0003] Alkaloids are a group of natural compounds primarily containing basic nitrogen atoms and exhibit a wide range of pharmacological activities, including antimalarial, anti-asthmatic, anticancer, cholinergic, vasodilatory, antiarrhythmic, analgesic, antibacterial, and antidiabetic activities. They are widely found in bacteria, fungi, and plants. For example, Chinese invention patent publication number CN 118108727A discloses a carbazole alkaloid compound, a preparation method, and applications thereof. The carbazole alkaloid compound is extracted and isolated from the condiment Murraya odorifera. In another example, Chinese invention patent publication number CN 118005644A discloses a decahydrofluorene alkaloid, a preparation method, and applications thereof in the preparation of antibacterial drugs. The decahydrofluorene alkaloid is isolated from the mycelium of the fungus Microascus sp. SCSIO41821GDMCC64206.

[0004] However, there are relatively few reports on the presence of alkaloids in fungal metabolites. Based on this, the present application provides an alkaloid compound and its preparation method and application, which is produced by the soil fungus Aspergillus niger ( Aspergillus ustus ), which provides a new direction for the full development of alkaloid compounds from fungi. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide an alkaloid compound and a preparation method and application thereof.

[0006] In order to solve the above problems, the technical solution adopted by the present invention is: In a first aspect, an alkaloid compound, a pharmaceutically acceptable salt thereof, or a stereoisomer thereof is provided, wherein the structure of the alkaloid compound is shown in formula (I) or (II): .

[0007] As an embodiment of the present invention, the alkaloid compound is obtained by removing the soil fungus Aspergillus pyroxene ( Aspergillus ustus ) was isolated from the fermentation extract of NCC3968.

[0008] In a second aspect, a method for preparing the alkaloid compound according to the first aspect is provided, the method comprising: Step A, preparing a solid fermentation product of soil fungus Aspergillus pyroxene; Step B, soaking the solid fermentation product obtained in step A in 95% ethanol overnight, concentrating under reduced pressure, and extracting with ethyl acetate to obtain a crude fermentation extract; Step C, subjecting the crude fermentation extract obtained in Step B to silica gel column chromatography, using a dichloromethane-methanol mixture for gradient elution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5; Step D: Dissolve fraction Fr.3 in methanol, and separate the solution by Sephadex LH 20 gel column chromatography at a flow rate of 15 s / drop, isocratically eluting with methanol. After TLC analysis and combination, fractions Fr.31, Fr.32, and Fr.33 are obtained. Step E: Take fraction Fr.3 2 and separate it by preparative liquid chromatography. R The compound 1 corresponding to formula (I) was obtained in 9.8 min. R The compound 2 corresponding to formula (II) was obtained in 12.9 min.

[0009] As an embodiment of the present invention, step A includes: (1) Inoculate Aspergillus fusus into PDA medium and activate the culture for 7 days; (2) The activated cultured slant spores were rinsed with 15% glycerol and inoculated into a seed culture medium to prepare a seed culture solution. The formula of the seed culture medium was as follows: glucose 1.0%, starch 2.0%, maltose 0.6%, yeast powder 0.3%, hot-rolled soybean cake powder 0.2%, NaCl 0.2%, MgSO4·7H2O 0.1%, CaCO3 0.2%, and the balance was water, pH = 7.0.

[0010] (3) The seed culture solution was inoculated into a rice hot-pressed soybean cake powder water culture medium at an inoculum rate of 5% for fermentation. The formula of the hot-pressed soybean cake powder water culture medium was: 100 g rice, 2.5 g hot-pressed soybean cake powder, and 15 g tap water.

[0011] As an embodiment of the present invention, in step (1), the activation culture temperature is 26°C; in step (2), the culture is inoculated into a seed culture medium and cultured on a shaking table at 26°C and 200 rpm for 3 days; in step (3), the fermentation temperature is 26°C and fermented under static conditions for 14 days.

[0012] As an embodiment of the present invention, in step C, the extract after fermentation extraction and concentration and 200-300 mesh normal phase silica gel are prepared into powder in equal mass ratios; during gradient elution, dichloromethane-methanol are sequentially used in a gradient elution ratio of 100:0→0:100, and each gradient is washed for 3 column volumes.

[0013] As an embodiment of the present invention, in step E, fraction Fr.3-2 is separated by preparative HPLC at a flow rate of 20 mL / min and isocratic elution with an acetonitrile-water volume ratio of 40:60 to obtain compounds of formula (I) and (II).

[0014] In a third aspect, a pharmaceutical composition is provided, comprising the alkaloid compound as described in the first aspect, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, and a pharmaceutically acceptable carrier, adjuvant or excipient.

[0015] In a fourth aspect, there is provided a use of the alkaloid compound as described in the first aspect, a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a protein tyrosine phosphatase inhibitor.

[0016] As a further improvement of the present invention, the protein tyrosine phosphatase is selected from one or more of T cell protein tyrosine phosphatase (TCPTP), SH2 domain-containing protein tyrosine phosphatase 1 (SHP1), protein tyrosine phosphatase 1B (PTP1B), and receptor-type protein tyrosine phosphatase C (CD45).

[0017] As a further improvement of the present invention, the drug is used to prevent, alleviate and / or treat one or more of type 2 diabetes, obesity, autoimmune diseases, immunodeficiency, malignant tumors, leukemia, lymphoma, cancer, Alzheimer's disease, and Parkinson's disease.

[0018] As a further improvement of the present invention, the protein tyrosine phosphatase is selected from one or more of T cell protein tyrosine phosphatase (TCPTP), SH2 domain-containing protein tyrosine phosphatase 1 (SHP1), protein tyrosine phosphatase 1B (PTP1B), and receptor-type protein tyrosine phosphatase C (CD45).

[0019] In a fifth aspect, there is provided a use of the alkaloid compound, a pharmaceutically acceptable salt or a stereoisomer thereof as described in the first aspect in the preparation of an antibacterial drug.

[0020] The beneficial effects of adopting the above technical solution are: The alkaloid compounds disclosed in the present invention are identified by comprehensively using NMR magnetic resonance (one-dimensional and two-dimensional), mass spectrometry (ESI-MS) methods and techniques and quantum chemical calculation technology to identify their chemical structures, and are found to be new compounds after retrieval.

[0021] The alkaloid compounds disclosed in the present invention are separated from the fungus Aspergillus niger by using a combination of silica gel column chromatography, Sephadex LH-20 gel column chromatography, reverse phase medium pressure column chromatography, preparative thin layer chromatography and preparative HPLC. Aspergillus ustus Isolated from fermentation extract.

[0022] The alkaloid compounds disclosed in the present invention were subjected to PTPs activity inhibition, antibacterial activity and PTP1B molecular docking experiments. The results showed that these two compounds have a good inhibitory effect on PTP1B, providing alternative compounds for the development of new PTP1B inhibitors and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 For compound 1 1 H-NMR spectra (solvent: DMSO - d 6).

[0024] Figure 2 For compound 1 13 C-NMR spectra (solvent: DMSO - d 6).

[0025] Figure 3 The HSQC diagram of compound 1 (the solvent is DMSO - d 6).

[0026] Figure 4 is the HMBC diagram of compound 1 (the solvent is DMSO - d 6).

[0027] Figure 5 This is the ESI-MS spectrum of compound 1.

[0028] Figure 6 Molecular docking diagram of compound 1 and PTP1B.

[0029] Figure 7 This is the 3D docking diagram of compound 1 and PTP1B protein.

[0030] Figure 8 This is a locally enlarged image of the 3D docking of compound 1 and PTP1B protein.

[0031] Figure 9 For compound 2 1 H-NMR spectra (solvent: DMSO - d 6).

[0032] Figure 10 For compound 2 13 C-NMR spectra (solvent: DMSO - d 6).

[0033] Figure 11 The HSQC diagram of compound 2 (the solvent is DMSO - d 6).

[0034] Figure 12 The HMBC diagram of compound 2 (the solvent is DMSO - d 6).

[0035] Figure 13 This is the ESI-MS spectrum of compound 2.

[0036] Figure 14 This is the molecular docking diagram of compound 2 and PTP1B.

[0037] Figure 15 This is the 3D docking diagram of compound 2 and PTP1B protein.

[0038] Figure 16 This is a locally enlarged image of the 3D docking of compound 2 and PTP1B protein. DETAILED DESCRIPTION

[0039] In order to make the objectives, technical solutions and advantages of the present invention more clear, the invention is clearly and completely described below in conjunction with specific embodiments.

[0040] The present invention uses the following method to prepare alkaloid compounds 1 and 2, soil fungi ( Aspergillus ustus ) was provided by North China Pharmaceutical Group New Drug Research and Development Co., Ltd., National Engineering Research Center for Microbiology.

[0041] Example 1 Preparation of the compound of the present invention The alkaloid compounds were prepared as follows: Step S1, preparing a fermentation product of soil fungus Aspergillus niger; Frozen fungi Aspergillus ustus The slant spores were inoculated into a sterilized PDA slant culture medium and placed in a 26°C incubator for activation. The activated slant spores were rinsed with 15% glycerol and prepared into a 10 mg / mL spore solution. The solution was inoculated into the seed culture medium at an inoculum size of 5% of the volume of the seed culture medium. The culture was cultured in a shaking incubator at 26°C and 200 rpm for 3 days to obtain a seed culture solution. The culture solution was inoculated into a solid culture medium at an inoculum size of 5% and cultured at 26°C under static conditions. The fermentation product and mycelium were obtained after 14 days.

[0042] Among them, the formula of the seed culture medium is as follows, by weight percentage: glucose 1.0%, starch 2.0%, maltose 0.6%, yeast powder 0.3%, hot-rolled soybean cake powder 0.2%, NaCl 0.2%, MgSO4•7H2O 0.1%, CaCO3 0.2%, and the balance is water, pH = 7.0.

[0043] The solid culture medium is a rice hot-pressed soybean cake powder water culture medium, and its formula is: 100g rice, 2.5g hot-pressed soybean cake powder, and 15g tap water.

[0044] Step S2: preparing a fermentation crude extract: 12 kg of the fermented product was soaked overnight in 12 L of 95% ethanol. The culture was stirred evenly, broken by ultrasonication for 30 minutes, and filtered under reduced pressure to collect the filtrate. The filtrate was concentrated under reduced pressure to remove the ethanol. The resulting aqueous phase was extracted three times with equal volumes of ethyl acetate. The combined extracts were concentrated and drained to yield 34.4 g of a dark brown crude extract. Step S3, preparing fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5: 200-300 mesh normal phase silica gel was soaked in dichloromethane overnight and wet packed with a column volume of 3.0 L. The extract after fermentation extraction and concentration was dissolved in methanol and mixed with 200-300 mesh normal phase silica gel in an equal mass ratio to form a powder. The sample was loaded by dry method and gradient eluted with dichloromethane-methanol in a ratio of 100:0→0:100. Each gradient was washed for 3 column volumes to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5.

[0045] Step S4: preparing components Fr.3-1, Fr.3-2 and Fr.3-3: Fraction Fr.3 was dissolved in methanol and the solution was separated by Sephadex LH-20 gel column chromatography at a flow rate of 15 s / drop, eluted isocratically with methanol, and after TLC identification and combination, components Fr.3-1, Fr.3-2 and Fr.3-3 were obtained.

[0046] Step S5: Preparation of Compound 1 and Compound 2 Fraction Fr.3-2 was separated by preparative HPLC at a flow rate of 20 mL / min and eluted isocratically with acetonitrile-water volume ratio of 40:60. R 5.4 mg of compound of formula (I) was obtained in 9.8 min. R The reaction mixture was heated to 12.9 min to give 4.3 mg of the compound of formula (II).

[0047] (1) Structural identification of chemical 1 Compound 1: pale yellow solid powder, LC-ESIMS gave m / z :166 [M+H] + , combined with 1 H-NMR and 13 C-NMR and the nitrogen rule determined the molecular formula to be C8H7NO3. The molecular formula has a degree of unsaturation of 6. Combined with the 7 olefinic carbon signals in the low-field region of the C spectrum, it is speculated that the structure contains 1 benzene ring, 1 double bond (C=O or C=N) and 1 ring. Based on the molecular formula composition, it can be inferred that there is at least 1 free hydroxyl group. The H spectrum is deuterated by heavy water exchange. δ H 9.03 (1H, s) does not disappear, so the structure is confirmed to contain one free hydroxyl group.

[0048] 1 There are 5 hydrogen signals in the H-NMR spectrum. δ H 9.84 (1H, s), 9.03 (1H, s), 7.78 (1H, br s),7.40 (1H, br s), 2.25 (3H, s), combined with HSQC, δ H 9.84 (1H, s) is the active hydrogen signal; δ H 7.78 (1H, br s), 7.40 (1H, br s) are the proton signals on the benzene ring, so it can be known that the structural skeleton is a 1,2,4,5-tetrasubstituted benzene ring derivative. Further HMBC spectrum shows that H-8 [ δ H 2.25 (3H, s)] are correlated with the olefinic carbons C-5, C-4, and C-6 of the benzene ring, respectively. Combined with the key correlations of H-3 [7.40 (1H, br s)] / C-5, C-1, and C-4 and H-6 [7.78 (1H, br s)] / C-2, C-4, and C-8, and H-7 [9.03 (1H, s)] / C-2, C-1 (4 J), and C-3 (4 J), the free hydroxyl group was finally determined. δ H 9.84 (1H, s) is attributed to the C-4 position of the benzene ring, and the methyl group is located at the C-5 position of the benzene ring. The heavy water exchange experiment of the H spectrum proves that δ H 9.03 (1H, s) is not an active hydrogen, so the possibility of an aldehyde group is ruled out. Therefore, the parent nucleus is a six-membered ring of a benzene ring and a nitrogen-oxygen hybrid. Finally, compound 1 was determined to be 7-Methylbenzo[e][1,4,2]dioxazin-6-ol. After database search, it was confirmed to be a new compound and named Aspergustin A. 1 H-NMR,13 The C-NMR data assignment table is shown in Table 1.

[0049] Table 1 Compound 1 1 H-NMR and 13 C-NMR data (2) Structural identification of chemical 2 Compound 2: pale yellow solid powder, LC-ESIMS gave m / z : 166[M+H] + and 164[MH] - , combined with 1 H-NMR and 13 C-NMR and the nitrogen rule determined the molecular formula to be C8H7NO3. The molecular formula has a degree of unsaturation of 6. Combined with the 7 olefinic carbon signals in the low-field region of the C spectrum, it is speculated that the structure contains 1 benzene ring, 1 double bond (C=O or C=N), and 1 ring. Based on the molecular formula composition, it can be inferred that there is at least 1 free hydroxyl group. After deuterium exchange in the H spectrum, δ H 9.03 (1H, s) does not disappear, so the structure is confirmed to contain one free hydroxyl group.

[0050] observe 1 H-NMR and 13 In the C-NMR spectrum, the signal composition of spectra 1 and 2 is the same. There is a significant difference in the chemical shift of the benzene ring protons and the high-field methyl hydrogen in the hydrogen spectrum. It is speculated that the two are isomers. A careful comparison of the H spectrum reveals that the two groups of olefin hydrogen signals are δ H 7.23(1H, d, J =2.4 Hz), 6.82 (1H, d, J = 2.4 Hz) shows that the two olefinic hydrogens are located at the meta position of the benzene ring, while the two olefinic hydrogens in structure 1 are located at the para position. Combining HSQC and HMBC spectra, we can see that H-8 [ δ H 2.59 (3H, s)] are related to C-6, C-5, and C-1 on the benzene ring, respectively, and combined with H-3 [7.23 (1H, d, J = 2.4 Hz)] / C-5, C-1, C-4 and H-5 [6.82 (1H, d, J =2.4 Hz)] / C-1, C-3, C-4, C-8 and H-7 [9.03 (1H, s)] / C-2, C-1 ( 4 J )、C-3( 4 J) and other key correlations, and finally determined that compound 2 was 8-Methylbenzo[e][1,4,2]dioxazin-6-ol. After database search, it was determined that 2 was a new compound and named Aspergustin B. 1 H-NMR, 13 The C-NMR data assignment table is shown in Table 2.

[0051] Table 2 Compound 2 1 H-NMR and 13 C-NMR data Example 2 Determination of protein tyrosine phosphatase (PTPs) inhibitory activity of compound 1 and compound 2 The human recombinant TCPTP, SHP1, PTP1B, and CD45 used in this example were provided by the laboratory of North China Pharmaceutical New Drug Research and Development Center, and were expressed in Escherichia coli and isolated and purified using genetic engineering technology.

[0052] Determination method: Test compound 1 and test compound 2 were dissolved in DMSO and prepared to 10 mg / mL for use. All compounds were diluted to 8 gradient concentrations by halving the concentration, namely 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.3125 mg / mL, 0.15625 mg / mL, and 0.078125 mg / mL.

[0053] They were divided into four groups, and the inhibitory activities of human recombinant TCPTP, SHP1, PTP1B, and CD45 were measured respectively.

[0054] 2µL of each concentration was added to a 96-well plate. Then, 50µL of a reaction solution containing 0.05µg to 0.01µg / µL of enzyme (the enzymes are human recombinant TCPTP, SHP1, PTP1B, and CD45, and the reaction solution consists of 50mM hydroxyethylpiperazine ethanesulfonic acid (HEPES), 100mM NaCl, 1mM EDTA, and 1mM dithiothreitol (DTT)) was added to each well. After pre-incubation at room temperature for 15min, 50µL of a reaction solution containing 50mM disodium p-nitrophenylphosphonate (p-NPP) was added to each well. After the addition was complete, the 96-well plate was placed in a 37°C incubator and incubated for 60min.

[0055] Afterwards, 10 μL of 10 N sodium hydroxide aqueous solution was added to each well to quench the reaction. The OD value at 405 nm was measured using a microplate reader. The protein inhibition rate was calculated using the following formula, and the corresponding IC was calculated. 50 value.

[0056] Inhibition rate (%) = [1-(OD value of experimental group-OD value of blank group) / (OD value of control group-OD value of blank group)] × 100%.

[0057] For the above steps, experimental group 1 (OD value is the OD value of the protein after adding test compound 1 to react, then adding substrate p-NPP, and testing OD value), experimental group 2 (OD value is the OD value of the protein after adding test compound 2 to react, then adding substrate p-NPP, and testing OD value) and blank group (OD value is the OD value of the blank solution DMSO added to substrate p-NPP, and testing OD value) were set up respectively. The results are shown in Table 3.

[0058] Table 3 IC values ​​of active compounds 50 value As shown in Table 3, both compound 1 and compound 2 showed a certain degree of inhibitory activity against the four PTPs (IC 50 >75µM).

[0059] Example 3 Antibacterial activity test of compound 1 and compound 2 Preparation of compound stock solutions: Dissolve the isolated compounds 1 and 2 in 400µL of methanol (add a small amount of DMSO to enhance solubility for samples with low solubility) and sonicate to prepare a stock solution at a concentration of 100µg / mL. This stock solution was then serially diluted with methanol to obtain sample solutions with concentrations of 50µg / mL, 20µg / mL, 10µg / mL, 5µg / mL, and 2µg / mL. A blank control consisted of 50µL of methanol and 50µL of DMSO.

[0060] Determination method: (1) Four strains of pear pathogens (Anthracnose ( Colletotrichum fructicola )、Ring rot fungus ( Botryosphaeria dothidea )、Artes fruit rot fungus ( Athelia bombacina ), Fusarium ( Fusarium proliferatum )) are activated in corresponding liquid culture medium and made into pathogen spore suspension for later use.

[0061] (2) Punch holes on the sterilized PDA culture medium plate. Punch four identical circular holes in each culture dish, with the four holes equidistant from the center of the culture medium circle.

[0062] (3) Take 10uL (the concentration of pathogen spores is about 10 4 The pathogen suspension (100 μg / mL) was dropped into the center of the culture dish.

[0063] (4) Add 50 μL of sample solution to the corresponding wells. Repeat for each compound in duplicate. Label the compound number. For blank controls, add 50 μL of methanol and 50 μL of DMSO to the corresponding wells.

[0064] (5) All culture dishes were placed in a biochemical incubator at 26°C for 3 days, and observations were made regularly every day.

[0065] (6) Experimental results: After 72 hours of culture, if it is observed that the central strain in some culture dishes grows to or passes through the drug hole during growth, it means that the drug at this concentration has no inhibitory effect on the pathogens; if it is observed that the central strain in some culture dishes cannot grow to the drug hole, it means that the compound at this concentration has an inhibitory effect on the pathogens, and the farther the growth edge of the central strain is from the drug hole, the stronger the inhibitory effect of the compound on the pathogens.

[0066] The results are shown in Table 4.

[0067] Table 4 IC values ​​of active compounds 50 As shown in the blank control culture dishes in Table 4, the four pathogens can all grow to and pass through the wells where methanol or DMSO was added, proving that methanol and DMSO have no inhibitory effect on the four pathogens. This example uses the hyphal growth rate method (GB / T38480-2020) to determine the IC values ​​of compound 1 and compound 2 against the four pathogens. 50 The values ​​were all between 35.17 and 49.02 μM, indicating that both compounds 1 and 2 exhibited certain inhibitory activity against the four pathogens.

[0068] Example 4 Docking study of compound 1 and compound 2 with PTP1B This example demonstrates the inhibitory effect of the compounds on PTP1B activity through molecular docking experiments with PTP1B. The active site of PTP1B includes the primary catalytic site, the P loop (His214-Arg221), the WPD loop (Trp179-Val184), and a secondary binding site (including Arg24, Arg254, Met258, Gly259, and Gln262).

[0069] The PTP1B crystal structure was obtained from the Protein Data Bank (PDB) with the extraction code 1NZ7. Molecular docking experiments were performed using the AutoDock 4.2 program. The Lamarckian genetic algorithm was used, the number of docking cycles was set to 50, and all other default settings were used. The conformation with the highest number of clusters and the lowest free energy was selected. The molecular docking diagram of compound 1 and PTP1B is shown in the figure Figure 6After molecular docking calculations, it was found that the hydroxyl group (-OH) of compound 1 can form a hydrogen bond with SER (serine) at position 216 of the PTP1B protein (purple solid arrow), which is equivalent to binding to the protein.

[0070] 3D docking diagram of compound 1 and PTP1B protein ( Figure 7 and Figure 8 ), it can be seen that small molecule 1 can generate hydrogen bonds or van der Waals forces with SER (serine) at position 216, ALA (alanine) at position 217, and ARG (arginine) at position 221 in the PTP1B enzyme protein, making small molecule 1 stably bound to the protein, thereby affecting the activity of the PTP1B enzyme.

[0071] The molecular docking diagram of compound 2 and PTP1B is shown in the figure Figure 14 After molecular docking calculations, it was found that the hydroxyl group (-OH) of compound 2 can form a hydrogen bond with GLY (glycine) at position 220 of the PTP1B protein (purple solid arrow), which is equivalent to binding to the protein.

[0072] 3D docking diagram of compound 2 and PTP1B protein ( Figure 15 and Figure 16 ), it can be seen that small molecule 2 can generate a hydrogen bond with GLY (glycine) at position 220 in the PTP1B enzyme protein, making small molecule 2 stably bound to the protein, thereby affecting the activity of the PTP1B enzyme.

[0073] The results of molecular docking calculation experiments are shown in Table 5.

[0074] Table 5 Experimental results As shown in Table 5, the docking score of compound 1 is -2.859 kcal / moL, and that of compound 2 is -2.291 kcal / moL, indicating that these two compounds interact with the protein and have an inhibitory effect on PTP1B activity.

[0075] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art may still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An alkaloid compound, a pharmaceutically acceptable salt thereof or a stereoisomer thereof, characterized in that: The structure of the alkaloid compound is shown in formula (I) or (II): 。 2. An alkaloid compound, a pharmaceutically acceptable salt thereof or a stereoisomer thereof according to claim 1, characterized in that: The alkaloid compounds are obtained from the soil fungus Aspergillus niger ( Aspergillus ustus ) was isolated from the fermentation extract.

3. A method for preparing an alkaloid compound according to claim 1 or 2, characterized in that: The method comprises: Step A, preparing a solid fermentation product of soil fungus Aspergillus pyroxene; Step B, soaking the solid fermentation product obtained in step A in 95% ethanol for 24 hours, concentrating under reduced pressure, and extracting with ethyl acetate to obtain a crude fermentation extract; Step C, subjecting the crude fermentation extract obtained in Step B to silica gel column chromatography, using a dichloromethane-methanol mixture for gradient elution to obtain fractions Fr.1, Fr.2, Fr.3, Fr.4 and Fr.5; Step D: Dissolve fraction Fr.3 in methanol, and separate the solution by Sephadex LH-20 gel column chromatography at a flow rate of 15 s / drop, isocratically eluting with methanol. After TLC analysis and combination, fractions Fr.3-1, Fr.3-2, and Fr.3-3 are obtained. Step E: fraction Fr.3-2 was separated by preparative liquid chromatography. R The compound 1 corresponding to formula (I) was obtained in 9.8 min. R The compound 2 corresponding to formula (II) was obtained in 12.9 min.

4. The method for preparing an alkaloid compound according to claim 3, wherein Step A includes: (1) Inoculate the Aspergillus fusus spore suspension into PDA slant culture medium and activate the culture for 7 days; (2) The activated cultured slant spores were rinsed with 15% glycerol and inoculated into a seed culture medium to prepare a seed culture solution. The seed culture medium was formulated as follows by weight: glucose 1.0%, starch 2.0%, maltose 0.6%, yeast powder 0.3%, hot-rolled soybean cake powder 0.2%, NaCl 0.2%, MgSO4·7H2O 0.1%, CaCO3 0.2%, and the balance was water. The pH was 7.

0. (3) The seed culture solution was inoculated into a rice hot-pressed soybean cake powder water culture medium at an inoculum rate of 5% for fermentation. The formula of the hot-pressed soybean cake powder water culture medium was: 100 g rice, 2.5 g hot-pressed soybean cake powder, and 15 g tap water.

5. The method for preparing an alkaloid compound according to claim 4, wherein In step (1), the activation culture temperature is 26°C; Step (2) comprises: washing the activated slant spores in step (1) with 15% glycerol to prepare a spore suspension with a concentration of 10 mg / mL, inoculating the spore suspension into a seed culture medium at an inoculum size of 5%, and culturing at 26°C and 200 rpm on a shaker for 3 days; In step (3), the fermentation temperature is 26° C., and the fermentation is carried out under static conditions for 14 days.

6. The method for preparing an alkaloid compound according to claim 3, wherein: In step C, the extract concentrated after fermentation extraction and 200-300 mesh normal phase silica gel are mixed into powder in equal mass proportions; during gradient elution, dichloromethane-methanol are sequentially used in a gradient elution ratio of 100:0→0:100, and each gradient is washed for 3 column volumes.

7. The method for preparing an alkaloid compound according to claim 3, wherein: In step E, fraction Fr.3-2 was separated by preparative HPLC at a flow rate of 20 mL / min and isocratic elution with an acetonitrile-water volume ratio of 40:60 to obtain the compound of formula (I) and the compound of formula (II).

8. A pharmaceutical composition comprising the alkaloid compound according to claims 1 and 2, a pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable carrier, adjuvant or vehicle.

9. Use of the alkaloid compound according to claim 1 or 2, a pharmaceutically acceptable salt thereof or a stereoisomer thereof in the preparation of a protein tyrosine phosphatase inhibitor, wherein the medicament is used to prevent, alleviate and / or treat one or more of type 2 diabetes, obesity, autoimmune diseases, immunodeficiency, malignant tumors, leukemia, lymphoma, cancer, Alzheimer's disease, and Parkinson's disease.

10. Use of the alkaloid compound according to claim 1 or 2, its pharmaceutically acceptable salt or stereoisomer thereof in the preparation of antibacterial drugs.

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