A pyridinecarboxylic acid alkaloid, its preparation method and application

Abstract

The present invention belongs to the field of biomedical technology, and particularly relates to a pyridinecarboxylic acid alkaloid, its preparation method and application. This compound can be obtained by using a fungus derived from sea lilies in the South China Sea through modern microbial fermentation engineering technology, and it is easy to achieve large-scale industrial production. Moreover, it is found through research that the pyridinecarboxylic acid alkaloid compound has a significant inhibitory effect on gastric cancer cells, can significantly inhibit the activity and clone formation of gastric cancer cells, promote the apoptosis of gastric cancer cells, and reduce the invasion of gastric cancer cells; when the pyridinecarboxylic acid alkaloid compound and a first-line clinical chemotherapy drug such as 5-fluorouracil act on gastric cancer cells in combination, the cell inhibition effect is also significantly improved, showing a synergistic anti-gastric cancer effect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine. More specifically, it relates to a pyridinecarboxylic acid alkaloid, its preparation method and application. Background Art

[0002] Gastric cancer is one of the most common malignant tumors in the world, ranking fifth in incidence and fourth in mortality among all tumors. Moreover, approximately 60% of gastric cancer patients are diagnosed with local or distant metastases at the initial diagnosis, and the 5-year overall survival rates of these patients are only about 30% and 5% respectively. Surgical resection and perioperative adjuvant chemotherapy have improved the survival rate of locally advanced gastric cancer, but the treatment strategies for patients with local or distant metastases are still limited. Targeted therapy in the form of small molecule inhibitors and monoclonal antibodies has become an important method for multimodal treatment of gastric cancer. For example, trastuzumab, as a HER2 monoclonal antibody, has been approved for first-line treatment of advanced HER2-positive gastric cancer (for example, Chinese Patent Application CN104045714A discloses trastuzumab mutants IgG and their applications). In addition, ramucirumab (an antibody targeting vascular endothelial growth factor receptor 2 (VEGFR-2)) and nivolumab or pembrolizumab (antibodies targeting programmed cell death protein 1 (PD-1)) as second-line and third-line treatments have also significantly improved the progression-free survival and overall survival of gastric cancer patients. However, in clinical treatment, the therapeutic drugs for gastric cancer patients are still relatively scarce. Therefore, it is urgent to explore more new and effective drugs for gastric cancer patients to improve the effectiveness of clinical treatment. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the relatively scarce therapeutic drugs for existing gastric cancer patients, and provide a pyridinecarboxylic acid alkaloid with a significant inhibitory effect on gastric cancer.

[0004] The object of the present invention is to provide a preparation method of the pyridinecarboxylic acid alkaloid.

[0005] Another object of the present invention is to provide the application of the pyridinecarboxylic acid alkaloid in the preparation of anti-gastric cancer drugs.

[0006] Another object of the present invention is to provide a pharmaceutical composition for anti-gastric cancer.

[0007] The above objects of the present invention are achieved by the following technical solutions:

[0008] A pyridinecarboxylic acid alkaloid having the structures of formula (I) and (II):

[0009]

[0010] Alkaloids are a class of nitrogen-containing organic compounds that are mostly alkaline and widely distributed in plants. The vast majority are found in higher plants and have complex structures and significant biological activities. Many alkaloids have also been found to have anti-tumor activities. Marine organisms have developed unique metabolic methods in special environments. Many literatures have proven that fungi derived from marine organisms can produce various secondary metabolites with novel structures and significant physiological activities, and have various medicinal values such as antibacterial, anti-tumor, immunomodulatory, and enzyme inhibitory effects. The pyridinecarboxylic acid alkaloids provided by the present invention are extracted, separated, and purified from the fermentation products of marine microorganisms.

[0011] Therefore, the present invention also provides a preparation method of the pyridinecarboxylic acid alkaloids, which are obtained by extracting, separating, and purifying from the fermentation products of fungi derived from sea lilies in the South China Sea.

[0012] Furthermore, the fungus derived from sea lilies in the South China Sea is named Penicillium brocae SYSU-CJ-17, deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, the deposition date is May 9, 2023, and the deposition number is GDMCC No: 63444.

[0013] Even further, the preparation method specifically includes the following steps:

[0014] S1. Activate and expand the culture of the fungus derived from sea lilies in the South China Sea to obtain a fermented product;

[0015] S2. Soak and extract the fermented product obtained in step S1 with ethyl acetate, and concentrate the extract to obtain a crude extract;

[0016] S3. Perform silica gel column chromatography on the crude extract obtained in step S2, use petroleum ether - ethyl acetate as the eluent for gradient elution, and collect the fraction with a volume ratio of petroleum ether - ethyl acetate of 1:1;

[0017] S4. Perform separation and purification on the fraction with a volume ratio of petroleum ether - ethyl acetate of 1:1 obtained in step S3 by high performance liquid chromatography to obtain pyridinecarboxylic acid alkaloids.

[0018] Further, in step S3, the gradient elution is carried out with petroleum ether - ethyl acetate as the eluent, and the gradient elution is carried out in sequence according to the volume ratios of petroleum ether - ethyl acetate of 5:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:4, 0:1.

[0019] Furthermore, when preparing the pyridinecarboxylic acid alkaloid compound (I), the high-performance liquid conditions in step S4 are as follows: the mobile phase is 50-70% methanol / water (specifically, 50-70% methanol and 30-50% water, with the sum of the two being 100%), the flow rate is 2-3 mL / min, the detection wavelength is 230 nm or 275 nm, and the retention time is 10-20 min.

[0020] Furthermore, when preparing the pyridinecarboxylic acid alkaloid compound (II), the high-performance liquid conditions in step S4 are as follows: the mobile phase is 50-70% methanol / water (specifically, 50-70% methanol and 30-50% water, with the sum of the two being 100%), the flow rate is 2-3 mL / min, the detection wavelength is 230 nm or 275 nm, and the retention time is 5-15 min.

[0021] Preferably, the pyridinecarboxylic acid alkaloid compounds can be distinguished by the order of elution peaks using the method of the present invention; among them, the pyridinecarboxylic acid alkaloid compound (II) elutes first.

[0022] Preferably, the chromatographic column used in high-performance liquid chromatography is a semi-preparative phenyl column; specifically, it can be a semi-preparative column Ultimate XB-Phenyl, 10×250 mm, 5 μm; the instrument is Essentia LC-16.

[0023] Furthermore, in step S1, the activation and scale-up culture are carried out by first preparing a seed culture solution and then performing fermentation culture.

[0024] Specifically, the medium used for preparing the seed culture solution is a seed medium containing 25-35 g / L of sea salt and 20-30 g / L of PDB medium powder; the culture conditions are a shaking speed of 100-150 rpm and constant temperature culture at 25-28 °C for 3-5 days.

[0025] The medium used for fermentation culture is a rice medium. Among them, the rice medium is 40-50 g of rice and 50 mL of 2-3% brine. After being placed in a culture flask, mixed well and sealed, it is sterilized at 121 °C (0.1 MPa) in a high-temperature sterilizer for 25 min and cooled to room temperature, and then left for 2 days to obtain; preferably, the conditions for the fermentation culture are static culture at 25-28 °C for 28-32 days.

[0026] It has been found that the pyridinecarboxylic acid alkaloid provided by the present invention or its pharmaceutically acceptable salt has a significant inhibitory effect on gastric cancer. Therefore, the present invention also claims the use of the pyridinecarboxylic acid alkaloid in the preparation of anti-gastric cancer drugs.

[0027] Furthermore, the gastric cancer is caused by gastric cancer cell lines such as MGC803, MKN45, HGC27, AGS, SNU1 or KATO3.

[0028] In addition, the present invention also provides a pharmaceutical composition for anti-gastric cancer, which composition comprises the pyridinecarboxylic acid alkaloid or a pharmaceutically acceptable salt thereof and a first-line clinical chemotherapy drug; preferably, the first-line clinical chemotherapy drug can be 5-fluorouracil. Experiments have proved that when the pyridinecarboxylic acid alkaloid and 5-fluorouracil (5-Fu) are used in combination on gastric cancer cells, they can significantly inhibit the cell viability of gastric cancer cells and the colony formation of gastric cancer cells, and have a significant synergistic anti-cancer effect.

[0029] The present invention has the following beneficial effects:

[0030] The present invention provides a pyridinecarboxylic acid alkaloid compound, which can be obtained by using a fungus derived from Metacrinus rotundus in the South China Sea through modern microbial fermentation engineering technology, and is easy to realize large-scale industrial production. And it is found that the pyridinecarboxylic acid alkaloid compound has a significant inhibitory effect on gastric cancer cells, can significantly inhibit the activity and colony formation of gastric cancer cells, promote the apoptosis of gastric cancer cells, and reduce the invasion of gastric cancer cells; when the pyridinecarboxylic acid alkaloid compound and 5-fluorouracil act on gastric cancer cells in combination, the cell inhibition effect is also significantly improved, and it has a synergistic anti-gastric cancer effect. Description of the Drawings

[0031] Figure 1 1H NMR spectrum of the pyridinecarboxylic acid alkaloid compound (I) obtained in Example 1 of the present invention.

[0032] Figure 2 13C NMR spectrum of the pyridinecarboxylic acid alkaloid compound (I) obtained in Example 1 of the present invention.

[0033] Figure 3 HRESIMS mass spectrum of the pyridinecarboxylic acid alkaloid compound (I) obtained in Example 1 of the present invention.

[0034] Figure 4 1H NMR spectrum of the pyridinecarboxylic acid alkaloid compound (II) obtained in Example 2 of the present invention.

[0035] Figure 5 13C NMR spectrum of the pyridinecarboxylic acid alkaloid compound (II) obtained in Example 2 of the present invention.

[0036] Figure 6 HRESIMS mass spectrum of the pyridinecarboxylic acid alkaloid compound (II) obtained in Example 2 of the present invention.

[0037] Figure 7Statistical chart of the inhibitory activity screening data of the pyridinecarboxylic acid alkaloid compounds (I) or (II) obtained in the present invention against various gastric cancer cell lines (MGC803, MKN45, HGC27, AGS, SNU1, KATO3).

[0038] Figure 8 Statistical chart of the inhibitory activity data of pyridinecarboxylic acid alkaloid compound (II) at different concentrations against various gastric cancer cell lines.

[0039] Figure 9 Microscopic images and statistical charts of the colony formation of pyridinecarboxylic acid alkaloid compound (II) at different concentrations in gastric cancer cells.

[0040] Figure 10 WB images of the effects of pyridinecarboxylic acid alkaloid compound (II) at different concentrations on apoptosis-related proteins (PARP-1, cPARP-1, c-Caspase7) and proliferation-related proteins (c-Myc, cyclinD) in different gastric cancer cell lines (MGC803, MKN45).

[0041] Figure 11 Microscopic images and statistical charts of the invasion of pyridinecarboxylic acid alkaloid compound (II) at different concentrations in gastric cancer cells.

[0042] Figure 12 Statistical chart of the inhibitory effect on the cell viability of pyridinecarboxylic acid alkaloid compound (II) and 5-Fu alone or in combination on gastric cancer cells.

[0043] Figure 13 Microscopic images of the colony formation of pyridinecarboxylic acid alkaloid compound (II) and 5-Fu alone or in combination on gastric cancer cells. Detailed implementation mode

[0044] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0045] The fungus used in the embodiments of the present invention is a marine fungus, which is a fungus isolated from a sea lily in the South China Sea. Through molecular biological identification, its classification is Penicillium brocae, and the fungus is named Penicillium brocae SYSU-CJ-17. It is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms. The preservation date is May 9, 2023, and the preservation number is GDMCC No: 63444.

[0046] Seed culture medium: Prepared by adding 30 g of sea salt and 24 g of PDB culture medium powder to each liter of water, evenly distributed into 4 1-L conical flasks, sterilized in a high-temperature sterilizer at 121 °C (0.1 MPa) for 25 min, cooled to room temperature, and left standing for 24 hours to obtain.

[0047] Rice culture medium: 50 g of rice, 50 mL of 3% brine, placed in a culture flask, mixed well and sealed, sterilized in a high-temperature sterilizer at 121 °C (0.1 MPa) for 25 min, cooled to room temperature, and left standing for 2 days to obtain.

[0048] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0049] Example 1 Extraction and Characterization of Pyridinecarboxylic Acid Alkaloid Compound (I)

[0050] 1. The extraction of pyridinecarboxylic acid alkaloid compound (I) specifically includes the following steps:

[0051] S1. Obtaining the seed culture solution: Inoculate the fungus Penicillium brocae SYSU-CJ-17 derived from sea lilies in the South China Sea into the seed culture medium, place the inoculated conical flask on a shaker, with the shaker speed at 100 - 150 rpm, and cultivate at a constant temperature of 25 °C for 72 hours to obtain the seed culture solution.

[0052] S2. Fermentation culture: Select the culture medium in the flask without contamination for inoculation, inoculate 10 mL of the strain (the seed culture solution obtained in step S1) into each bottle, a total of 200 bottles are inoculated, and leave it standing for 30 days to obtain the fermentation product.

[0053] S3. Extraction and separation: After fermentation culture, soak and extract the fermentation product obtained in S2 with ethyl acetate, and concentrate the extract under reduced pressure to obtain a crude extract; use petroleum ether - ethyl acetate with different volume ratios as the eluent, and perform gradient elution with petroleum ether - ethyl acetate with volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:4, 0:1 in sequence, and perform column chromatography separation on the crude extract using a silica gel column to obtain different polar components.

[0054] S4. High-performance liquid separation and purification: Separate and purify the elution part with a petroleum ether - ethyl acetate volume ratio of 1:1 obtained in step S3 by high-performance liquid chromatography. The conditions of the high-performance liquid chromatography are as follows: Mobile phase: 60% MeOH - 40% H2O; Flow rate: 3 mL / min, Chromatographic column: Semi-preparative column Ultimate XB-Phenyl, 10 × 250 mm, 5 μm; Instrument: Essentia LC-16, Detection wavelength: 230 nm or 275 nm, and a white powder is obtained at a retention time of 12 min.

[0055] 2. Characterization of pyridinecarboxylic acid alkaloid compound (I):

[0056] The obtained white powder was subjected to nuclear magnetic resonance and mass spectrometry tests, and the resulting spectra are as shown in Figures 1 to 3 After analysis and testing, the physicochemical property data of the compound structure are as follows:

[0057] UV(MeOH)λ max (logε) 229.6 (4.08), 269.4 (3.78);

[0058] IR(neat)ν max 3367cm -1 , 2917cm -1 , 2844cm -1 , 1720cm -1 , 1574cm -1 , 1431cm -1 , 1389cm -1 , 1312cm -1 , 1254cm -1 , 1216cm -1 , 1154cm -1 , 1119cm -1 , 1077cm -1 , 1016cm -1 , 869, 796cm -1 , 708cm -1 , 630cm -1 ;

[0059] HR-ESIMS m / s 515.2752 [M + H] + (calcd for C 28 H 39 N2O7, 515.2752), and the detailed information is shown in Table 1.

[0060] Table 1 HR-ESIMS information of pyridinecarboxylic acid alkaloid compound (I)

[0061]

[0062] NMR nuclear magnetic data:

[0063] 13 C NMR(150MHz, CDCl3)δ C171.11C, 165.92C, 165.06C, 150.12CH, 149.75CH, 145.58C, 144.93C, 142.96C, 142.35C, 137.23CH, 136.80CH, 125.46CH, 125.04CH, 67.85CH, 67.13CH2, 65.08CH2, 52.88CH3, 33.15CH2, 33.12CH2, 30.92CH2, 30.90CH2, 29.52CH2, 29.40CH2, 29.17CH2, 29.14CH2, 20.93CH2.

[0064] 1 1H NMR (600 MHz, CDCl3) δ H 8.54 s, 8.53 s, 8.06 d, 8.04 d, 7.65 dd, 7.62 dd, 4.47 dd, 4.41 dd, 4.29 - 4.25 m, 4.22 d, 3.98 s, 2.67 td, 2.07 s, 1.61 q, 1.33 - 1.20 m。

[0065] From the analysis of the results of mass spectrometry and nuclear magnetic resonance, it can be determined that the molecular formula of compound (Ι) is C 28 H 38 N2O7。

[0066] From the planar structure, a chiral center C-19 of compound (I) is found. Due to the presence of a flexible long chain in this compound, the accurate configuration cannot be obtained by ECD calculation. However, the chirality of this compound with a monoglycerol fragment and only one chiral center can be determined by optical rotation. If the optical rotation value is negative, the chiral center is in the S configuration; if it is positive, it is in the R configuration.

[0067] Therefore, the optical rotation of the white powder was measured, and the optical rotation data of this compound were obtained as It can be judged that the configuration of compound (I) is 19R.

[0068] In summary, the obtained white powder is the pyridinecarboxylic acid alkaloid compound (I), and the specific structural formula is as follows:

[0069]

[0070] Example 2 Extraction and Characterization of Pyridinecarboxylic Acid Alkaloid Compound (II)

[0071] 1. The extraction of pyridinecarboxylic acid alkaloid compound (II) specifically includes the following steps:

[0072] S1. Obtaining the seed culture medium: The fungus Penicillium brocae SYSU-CJ-17 derived from Metacrinus rotundus was inoculated into the seed culture medium. The inoculated conical flask was placed on a shaker, and the shaker speed was 100 - 150 rpm. It was cultured at a constant temperature of 25 °C for 72 hours to obtain the seed culture medium.

[0073] S2. Fermentation culture: Select those without contamination in the culture medium in the flask for inoculation. 10 mL of the bacterial strain (the seed culture medium obtained in step S1) was inoculated into each bottle, and a total of 200 bottles were inoculated. It was statically cultured for 30 days to obtain the fermentation product.

[0074] S3. Extraction and separation: After fermentation culture, the fermentation product obtained in S2 was soaked and extracted with ethyl acetate, and the extract was concentrated under reduced pressure to obtain a crude extract. Petroleum ether - ethyl acetate with different volume ratios was used as the eluent in turn, and gradient elution was carried out with petroleum ether - ethyl acetate with volume ratios of 5:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:4, 0:1. The crude extract was separated by silica gel column chromatography to obtain different polar components.

[0075] S4. High - performance liquid separation and purification: The elution part with a petroleum ether - ethyl acetate volume ratio of 1:1 obtained in step S3 was separated and purified by high - performance liquid chromatography. The conditions of the high - performance liquid chromatography were as follows: mobile phase: 60% MeOH - 40% H2O; flow rate: 3 mL / min, chromatographic column: semi - preparative column Ultimate XB - Phenyl, 10×250 mm, 5 μm; instrument Essentia LC - 16, detection wavelength was 230 nm or 275 nm, and a pale yellow amorphous substance was obtained at a retention time of 8.5 min.

[0076] 2. Characterization of pyridinecarboxylic acid alkaloid compound (II):

[0077] The obtained pale yellow amorphous substance was detected by nuclear magnetic resonance and mass spectrometry. The obtained spectra are as Figures 4 to 6 shown. After analysis and detection, the physicochemical property data of the compound structure are as follows:

[0078] UV(MeOH)λ max (logε)229.8(4.07),268.7(3.78)nm;

[0079] IR(neat)ν max 3352cm -1 ,2913cm -1 ,2852cm -1 ,1704cm -1 ,1655cm -1 ,1570cm -1 ,1428cm-1 , 1389 cm -1 , 1312 cm -1 , 1246 cm -1 , 1204 cm -1 , 1116 cm -1 , 1023 cm -1 , 862 cm -1 , 773 cm -1 , 696 cm -1 , 626 cm -1 ;

[0080] HR-ESIMS m / s 473.2650 [M+H] + (calcd for C 26 H 37 N2O6, 473.2646), as shown in Table 2 for details.

[0081] Table 2 HR-ESIMS information of pyridinecarboxylic acid alkaloid compounds (II)

[0082]

[0083] NMR nuclear magnetic data:

[0084] 13 C NMR (150 MHz, CD3OD) δ C 166.57 C, 165.95 C, 150.71 CH, 150.67 CH, 146.27 C, 146.24 C, 144.54 C, 144.40 C, 138.85 CH, 138.75 CH, 126.42 CH, 126.18 CH, 71.13 CH, 68.15 CH2, 63.88 CH2, 53.10 CH3, 33.76 CH2, 31.97 CH2, 31.95 CH2, 30.52 CH2, 30.38 CH2, 30.17 CH2.

[0085] 1 H NMR (600 MHz, CD3OD) δ H 8.51 dd, 8.12 d, 8.06 d, 7.83 ddd, 4.46 dd 4.33 dd, 4.00 dt, 3.95 s, 3.65 d, 2.73 q, 1.68 - 1.61 m, 1.35 - 1.25 m.

[0086] From the analysis of the results of mass spectrometry and nuclear magnetic resonance, it can be determined that the molecular formula of compound (II) is C 26 H 36 N2O6.

[0087] A chiral center C-19 of compound (II) was found from the planar structure. Since this compound has a flexible long chain, the accurate configuration cannot be obtained by ECD calculation. However, the chirality of this compound with a monoglycerol fragment and only one chiral center can be judged by optical rotation. If the optical rotation value is negative, the chiral center is in the S configuration; if it is positive, it is in the R configuration.

[0088] Therefore, the optical rotation of the obtained pale yellow amorphous substance was measured, and the optical rotation data of this compound was It can be judged that the chirality of this compound is 19S.

[0089] To sum up, the obtained pale yellow amorphous substance is a pyridinecarboxylic acid alkaloid compound (II), and the specific structural formula is as follows:

[0090]

[0091] Anti-cancer activity test of the compound in Example 3

[0092] 1. Cell culture

[0093] In this example, the gastric cancer cell lines (MGC803, MKN45, HGC27, AGS, SNU1, and KATO3) used were cultured in RPIM-1640 medium (hereinafter referred to as the medium) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The cells were placed in a cell incubator containing 5% CO2 at a temperature of 37 °C for culture.

[0094] 2. Determination of cell viability by CCK8 method

[0095] 500 cells were evenly diluted in 100 μL of the medium and seeded in a 96-well plate. After 24 h of cell culture, compound (I) or compound (II) was diluted into different concentrations and added to each well with 50 μL of the medium; after continuous 4-day cell incubation, CCK8 reagent was added. The 96-well plate was further incubated in an incubator containing 5% CO2 at 37 °C for 1-2 hours; the absorbance was measured at 450 nm according to the instructions of the CCK8 kit. The final result was shown as a percentage, and the in vitro IC 50 value was calculated using GraphPad Prism 7 software. The inhibition rate calculation formula is [OD (drug addition group) - OD (control group)] / [OD (control group) - OD (blank group)] × 100%.

[0096] The results are shown in Figures 7 - 8 . As can be seen from the figure, compound (I) or (II) can inhibit the proliferation activity of gastric cancer cells in a dose-dependent manner.

[0097] 3. Plate cloning experiment - compound (II) acting alone

[0098] Cells in the logarithmic growth phase were evenly seeded into a six-well plate at 500 cells per well; after culturing the cells for 24 h, different concentrations of the drug were added, and the six-well plate was continuously incubated in an incubator at 37 °C. Three replicate wells were set for each group; fresh medium was changed every three days, and different concentrations of compound (II) were added again; after culturing for 10 - 12 days, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 minutes. Finally, the cells were stained with crystal violet solution in the dark for 20 minutes, and after washing the crystal violet solution with PBS, the cell colonies were counted. The experiment was repeated 3 times.

[0099] The results are shown in Figure 9 , and it can be seen from the figure that compound (II) can inhibit the proliferation of gastric cancer cells by inhibiting the formation of clones.

[0100] 4. Western Blot

[0101] After treating the cells with the drug for 48 h, the cells were collected. The collected cells were treated with cell lysate RIPA for about 15 minutes, and then the lysate was centrifuged at 15000 rpm and 4 °C for 15 min. The supernatant was collected and transferred, and the protein concentration was quantified by the BCA protein assay method. Subsequently, 5×loading buffer was added, and the protein supernatant was boiled for denaturation. An appropriate amount of the protein supernatant was added to a 6% - 15% polyacrylamide gel, and the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for about 100 min, and then transferred to a nitrocellulose membrane (PVDF membrane). The membrane was blocked with skim milk powder at room temperature for 1 h, then washed 3 times with TBS-T solution, and incubated overnight at 4 °C with the corresponding primary antibody. The next day, it was incubated with rabbit secondary antibody at room temperature for 1 h. Finally, the protein bands on the membrane were visualized and analyzed using a developer.

[0102] The results are shown in Figure 10 , and it can be seen from the figure that compound (II) can upregulate the apoptotic proteins PARP-1 and c-caspase-7 in a dose-dependent manner and inhibit the expression of the proliferation proteins c-myc and cyclin D.

[0103] 5. Transwell chamber invasion assay

[0104] Cell invasion was detected using a 24-well plate. Serum-free medium was added, and the membrane of the chamber was soaked in the serum-free medium and maintained in an incubator at 37 °C for 1 h or overnight to activate the chamber; the cells treated with different concentrations of the drug in the six-well plate for 48 h were digested and counted, and 2×10 4300 μL of serum-free medium for a single cell was evenly added to the upper chamber of the Transwell chamber, and 500 μL of medium containing 10% fetal bovine serum was added to the lower chamber. The chamber was placed in an incubator with 5% CO2 at 37 °C and cultured overnight; after incubation for 24 h, the cells were fixed with 4% paraformaldehyde for 15 min and stained with crystal violet in the dark for 30 min; the crystal violet staining solution was gently rinsed off with PBS, and the cells on the upper layer of the chamber were gently scraped off with a cotton swab; after the chamber was dried, it was photographed under a microscope for counting and statistics.

[0105] The results are shown in Figure 11 , as can be seen from the figure, compound (II) can dose-dependently inhibit the invasion ability of gastric cancer cells.

[0106] 6. Colony formation assay - combined effect of compound (II) and 5-fluorouracil (5-Fu)

[0107] Refer to point 3 of the colony formation assay to test the effect of the combination of compound (II) and 5-fluorouracil (5-Fu) on cells.

[0108] The results are shown in Figures 12 - 13 , as can be seen from the figure, compound (II) (Pen-I in the figure) can synergistically enhance the significant inhibitory effect of the first-line clinical chemotherapy drug 5-fluorouracil (5-Fu) on the cell colony formation of two gastric cancer cell lines, and there is a good synergistic effect between them.

[0109] The above embodiments are the preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A pyridinecarboxylic acid alkaloid, characterized in that, The pyridinecarboxylic acid alkaloids have the structures of formula (I) and (II): （I）； (II).

2. The preparation method of the pyridinecarboxylic acid alkaloid according to claim 1, characterized in that, The pyridinecarboxylic acid alkaloids are obtained by extraction, separation and purification from the fermentation products of fungi derived from Metacrinus rotundus in the South China Sea.

3. The preparation method according to claim 2, wherein The fungus derived from the sea lily in the South China Sea is named Penicillium brocae SYSU-CJ-17, which is deposited in the Guangdong Microbial Culture Collection Center. The deposit date is May 9, 2023, and the deposit number is GDMCC No: 63444.

4. The preparation method according to claim 3, characterized in that The preparation method specifically includes the following steps: S1. Activate and expand the culture of the fungi derived from Metacrinus rotundus in the South China Sea to obtain a fermentation product; S2. Immerse and extract the fermentation product obtained in step S1 with ethyl acetate, and concentrate the extract to obtain a crude extract; S3. Perform silica gel column chromatography on the crude extract obtained in step S2, use petroleum ether - ethyl acetate as the eluent for gradient elution, and collect the fraction with a volume ratio of petroleum ether to ethyl acetate of 1:1; S4. Perform separation and purification on the fraction with a volume ratio of petroleum ether to ethyl acetate of 1:1 obtained in step S3 by high performance liquid chromatography to obtain pyridinecarboxylic acid alkaloids.

5. The preparation method according to claim 4, characterized in that, In step S3, the gradient elution is carried out with petroleum ether - ethyl acetate as the eluent, and the gradient elution is carried out in sequence according to the volume ratios of petroleum ether to ethyl acetate of 5:1, 4:1, 3:1, 2:1, 1:1, 2:3, 1:4, 0:

1.

6. The preparation method according to claim 4, wherein When preparing the pyridinecarboxylic acid alkaloid compound (I), the high performance liquid chromatography conditions in step S4 are that the mobile phase is 50 - 70% methanol / water, the flow rate is 2 - 3 mL / min, the detection wavelength is 230 nm or 275 nm, and the retention time is 10 - 20 min.

7. The preparation method according to claim 4, characterized in that, When preparing the pyridinecarboxylic acid alkaloid compound (II), the high performance liquid chromatography conditions in step S4 are that the mobile phase is 50 - 70% methanol / water, the flow rate is 2 - 3 mL / min, the detection wavelength is 230 nm or 275 nm, and the retention time is 5 - 15 min.

8. Use of the pyridinecarboxylic acid alkaloid according to claim 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti - gastric cancer drug.

9. The application according to claim 8, wherein The gastric cancer is caused by gastric cancer cell lines such as MGC803, MKN45, HGC27, AGS, SNU1 or KATO3.

10. A pharmaceutical composition for treating gastric cancer, characterized in that, The composition includes the pyridinecarboxylic acid alkaloid according to claim 1 or a pharmaceutically acceptable salt thereof and a first - line clinical chemotherapy drug; the first - line clinical chemotherapy drug is selected from 5 - fluorouracil.

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