Pyranopyridone alkaloids, methods for preparing the same and use thereof in the preparation of anti-tumor drugs

Abstract

The application discloses pyrano pyridone alkaloids, a preparation method thereof and application thereof in preparation of antitumor drugs. Three pyrano pyridone compounds 1-3 with antitumor activity are prepared from Penicillium sp. SCSIO 41320, and the structural formulae are as shown in formula (I). Experiments prove that the compounds 1-3 have good antitumor activity and can be used for development of antitumor drugs. The application provides alternative compounds for development of new antitumor drugs, and has important significance for development of marine microbial drug resources in China.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine technology, specifically relating to a class of pyranopyridone alkaloids, their preparation methods, and their application in the preparation of antitumor drugs. Background Technology

[0002] Small cell lung cancer (SCLC) is a high-grade neuroendocrine carcinoma, accounting for approximately 15% of all lung cancers. Compared to non-small cell lung cancer (NSCLC), it is characterized by earlier metastasis, rapid development of chemotherapy resistance, and poorer prognosis. Although targeted therapy strategies for NSCLC are becoming increasingly diverse, no novel targeted therapy has yet demonstrated significant efficacy in the clinical treatment of SCLC. Pyranopyridone alkaloids, as an important subclass of 2-pyridone alkaloids, have a structure fused with a 4-hydroxy-2-pyridone core heterocyclic pyran. They are mainly derived from fungal metabolites and artificial synthesis, and possess various biological activities such as antitumor, antibacterial, anti-inflammatory, and hepatoprotective effects. Especially with the deepening research on the secondary metabolic pathways and structural diversity of marine fungi, the pharmacological potential of pyridone compounds is constantly being discovered and validated, increasingly attracting researchers to explore their application prospects in the field of antitumor therapy. Summary of the Invention

[0003] The first objective of this invention is to provide a class of pyranopyridone alkaloid derivatives or their pharmaceutical salts, wherein the structural formulas of the pyranopyridone alkaloid derivatives are shown in Formulas 1-3.

[0004]

[0005] A second objective of this invention is to provide a method for preparing the above-mentioned pyranopyridone alkaloid derivatives, wherein the pyranopyridone alkaloid derivatives are isolated and prepared from the fermentation culture of Penicillium sp. SCSIO 41320.

[0006] Preferably, the preparation method comprises the following steps:

[0007] 1) Preparation of fermentation culture of Penicillium sp. SCSIO 41320;

[0008] 2) The fermentation culture was repeatedly extracted three times with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain a crude extract. The crude extract was separated by normal-phase silica gel column chromatography using a gradient elution of CH2Cl2-MeOH at a volume fraction of 0-100% to obtain six fractions Fr.1 to Fr.6. Fraction Fr.1 eluted at a CH2Cl2:MeOH volume ratio of 100:0 was collected, and fraction Fr.2 eluted at a CH2Cl2:MeOH volume ratio of 99:1 was collected. Fr.1 and Fr.2 were combined and eluted with MeOH-H2O at a volume fraction of 0-100% on an ODS reversed-phase column. According to liquid phase analysis, nine fractions Fr.11 to Fr.19 were obtained. Fraction Fr.18 eluted at a MeOH:H2O volume ratio of 75:25 was collected. Fr.18 was then subjected to half-phase chromatography with methanol:water at a volume ratio of 80:20 and a flow rate of 2.5 mL / min. Preparative high-performance liquid chromatography (HPLC) with isogradient elution was used to collect five fractions, Fr.181–Fr.185, with retention times of 16.5 min for Fr.182, 18.5 min for Fr.183, and 24.5 min for Fr.185. Fr.182 was further separated by isogradient HPLC with an acetonitrile:water ratio of 46:54 at 3 mL / min, and compound 2 (retention time 20.0 min) was collected. Fr.183 was separated by isogradient HPLC with an acetonitrile:water ratio of 48:52 at 2.8 mL / min, and compound 3 (retention time 26.5 min) was collected. Fr.185 was separated by isogradient HPLC with an acetonitrile:water ratio of 60:40 at 2.5 mL / min, and compound 1 (retention time 27.5 min) was collected.

[0009] Preferably, the fermentation culture of Penicillium sp. SCSIO 41320 is obtained by inoculating Penicillium sp. SCSIO 41320 into a rice fermentation medium for fermentation. The rice fermentation medium consists of 120g of rice and 120mL of seawater with a salinity of 3.3% added to each bottle of medium.

[0010] A third objective of this invention is to provide the application of the above-mentioned pyranopyridone alkaloid derivatives or their pharmaceutical salts in the preparation of antitumor drugs.

[0011] Preferably, the antitumor drug is an anti-small cell lung cancer drug.

[0012] Preferably, the administration method of the antitumor drug includes oral administration and injection administration.

[0013] A fourth object of the present invention is to provide an antitumor drug comprising an effective amount of any of the above-described pyranopyridone alkaloid derivatives or pharmaceutical salts thereof as active ingredients, and a pharmaceutically acceptable carrier.

[0014] A fifth objective of this invention is to provide the use of Penicillium sp. SCSIO 41320 in the preparation of pyranopyridone alkaloid derivatives.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This invention relates to a class of pyranopyridone alkaloid derivatives with novel structures, which exhibit significant antitumor activity. Compound 1, in particular, demonstrates significant inhibitory activity against small cell lung cancer cell lines H446, H1048, H69AR, and H82, with an IC50 concentration of [missing value]. 50 The values ​​were 0.53±0.02 μM, 0.72±0.02 μM, 1.20±0.02 μM, and 0.47±0.02 μM, respectively; compound 2 showed significant inhibitory activity against small cell lung cancer cell lines H446, H1048, H69AR, and H82, with IC50 values ​​of 0.53±0.02 μM, 0.72±0.02 μM, 1.20±0.02 μM, and 0.47±0.02 μM, respectively; 50 The values ​​were 4.72±0.24 μM, 4.05±0.24 μM, 7.75±0.49 μM, and 4.23±0.25 μM, respectively; Compound 3 showed significant inhibitory activity against small cell lung cancer cell lines H446, H1048, H69AR, and H82, with IC50 values ​​of 4.72±0.24 μM, 4.05±0.24 μM, 7.75±0.49 μM, and 4.23±0.25 μM, respectively; 50 The values ​​were 0.96±0.05 μM, 1.20±0.06 μM, 2.08±0.06 μM, and 1.68±0.04 μM, respectively. Compounds 1-3 exhibit good antitumor activity and have the potential to serve as lead compounds for the development of antitumor drugs, which is of great value for promoting the development of marine microbial drug resources in my country.

[0017] The Penicillium sp. SCSIO 41320 of this invention was deposited on April 18, 2025 at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences, Postcode: 510070, with accession number GDMCC No: 66161. Attached Figure Description

[0018] Figure 1 The main compounds are compounds 1-3. 1 H- 1 H COSY, HMBC information.

[0019] Figure 2The main NOESY information for compounds 1-3.

[0020] Figure 3 This is the X-ray single crystal image of compound 1.

[0021] Figure 4 The inhibitory activities of compounds 1-3 at different concentrations on the proliferation of four tumor cell lines were investigated.

[0022] Figure 5 The effect of compound 1 on the formation of small cell lung cancer.

[0023] Figure 6 The effect of compound 1 on the growth of SCLC cells in a zebrafish tumor xenograft model. Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to illustrate the content of the present invention and do not constitute a limitation on the scope of protection of the present invention. Unless otherwise stated, the experimental methods used in the embodiments are conventional methods; the raw materials and reagents used, unless otherwise specified, can be obtained commercially.

[0025] Example 1: Preparation and structural identification of compounds 1, 2 and 3

[0026] 1. Preparation of fermentation culture of Penicillium sp. SCSIO 41320

[0027] The rice fermentation medium consisted of 120g of rice and 120mL of seawater with a salinity of 3.3% added to each bottle, and autoclaved at 121℃ for 20min. Penicillium sp. SCSIO 41320 was inoculated into the medium and cultured on a shaker at 28℃ for 3 days to obtain a seed culture. This seed culture was then inoculated into the rice medium at a volume ratio of 5% and cultured on a shaker at 28℃ for 30 days to obtain the fermentation product inoculated with Penicillium sp. SCSIO 41320.

[0028] 2. Isolation and purification of compounds 1-3

[0029] The fermentation culture was repeatedly extracted three times with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain 95.3 g of crude extract, which was then extracted with methanol and petroleum ether to obtain 43.9 g of methanol fraction. The crude methanol fraction was separated by normal-phase silica gel column chromatography. After mixing and dry packing, a CH2Cl2-MeOH (0-100%, v / v) gradient elution was used to obtain six fractions Fr.1–Fr.6. Fraction Fr.1, eluted at a CH2Cl2:MeOH volume ratio of 100:0, and fraction Fr.2, eluted at a CH2Cl2:MeOH volume ratio of 99:1, were collected. Fr.1 and Fr.2 were combined and eluted with MeOH-H2O at a volume fraction of 0-100% on an ODS reversed-phase column. Liquid chromatography analysis yielded nine fractions Fr.11–Fr.19. Fraction Fr.18, eluted at a MeOH:H2O volume ratio of 75:25, was collected. Fr.18 was separated by semi-preparative high performance liquid chromatography with a methanol:water volume ratio of 80:20, 2.5 mL / min, using a NanoChromcolumn (ChromCore 120C18, 250×10 mm ID, S-5 μm) column and isogradient elution. Five fractions, Fr.181 to Fr.185, were collected. The retention times of Fr.182, Fr.183, and Fr.185 were 16.5 min, 18.5 min, and 24.5 min, respectively. Compound 2 (Fr. 182) was separated by isogradient HPLC using an acetonitrile:water ratio of 46:54, 3 mL / min, on a Naphthyl column (ΠNAP, 250×10 mm ID, COSMOSIL), and the compound with a retention time of 20.0 min was collected. Compound 3 (Fr. 183) was separated by isogradient HPLC using an acetonitrile:water ratio of 48:52, 2.8 mL / min, on a Naphthyl column (ΠNAP, 250×10 mm ID, COSMOSIL), and the compound with a retention time of 26.5 min was collected. Compound 1 (Fr. 185) was separated by isogradient HPLC using an acetonitrile:water ratio of 60:40, 2.5 mL / min, on a Naphthyl column (ΠNAP, 250×10 mm ID, COSMOSIL), and the compound with a retention time of 27.5 min was collected.

[0030] 3. Structural identification of compounds 1-3

[0031] The obtained compounds 1-3 were subjected to nuclear magnetic resonance (NMR), mass spectrometry (MS), optical rotation (OR), and single-crystal diffraction (X-Ray) data analysis to determine their chemical structures.

[0032] Structural identification of new compound 1: yellow crystals, high-resolution mass spectrometry (HR-ESI-MS) m / z 334.2019 [M+H]+ (calcd for C 19 H 28 NO4,334.2013), suggested molecular formula is C 19 H 27 NO4; 1 H and 13 The C NMR data are shown in Table 1, and the NMR spectrum of compound 1 is shown in Table 1. Figure 1 Compound 1 is partially similar to the known compound penicipyridone J, with the main difference being the absence of the carbon and hydrogen signals of the hydroxyphenyl group attached at C-8 in penicipyridone J, while compound 1 has a hydrogen signal at the C-8 position. Furthermore, high-resolution analysis confirmed the molecular formula of compound 1 to be C10. 19 H 27 NO4, while the molecular formula of penicipyridone J is C 25 H 31 NO4, therefore it is inferred that the hydroxyphenyl group at the C-8 position of compound 1 is substituted with hydrogen. Regarding the relative configuration of compound 1, the NOESY spectrum ( Figure 2 The determination was made by ) . Compound 1 exhibits Δ1'E and Δ3'E configurations, with the former determined by a larger coupling constant ( 3 J H-1′ / H-2′ =15.6Hz) is inferred, while the latter is derived from H-1'(δ H 5.53) and H-8'(δ) H The NOESY correlation signal of 1.73) is supported. Larger... 3 J H-2 / H-3 The value (11.2 Hz) indicates that H-2 and H-3 are in a trans configuration, while the smaller value indicates a trans configuration. 3 J H-3 / H-4 The value (2.7 Hz) indicates a cis configuration between H-3 and H-4. After crystallization in methanol, a single crystal suitable for X-ray diffraction analysis was obtained. Single-crystal X-ray diffraction experiments were conducted... Figure 3 Using Cu Kα radiation [Flack parameter = -0.03(10)], the absolute configuration of compound 1 was determined to be 1'E, 3'E, 2S, 3S, 4S, 5'R. Upon searching, compound 1 was identified as a novel compound and named oxalicpyridone A.

[0033] Structural identification of new compound 2: Yellow solid, high-resolution mass spectrometry (HR-ESI-MS) m / z 320.1860 [M+H]+ (calcd for C 18 H 26 NO4, 320.1856), suggested molecular formula is C18 H 25 NO4; 1 H and 13 The C10 NMR data are shown in Table 1. The NMR data of compound 2 are very similar to those of compound 1. The significant difference is in the C4 NMR data, including the absence of signals from carbon and hydrogen at position 10 and the presence of an additional proton signal δ. H 5.02, and δ was found in HMBC and COSY. H 5.02 and key correlation signals at C-4. These results indicate that compound 2 has a hydroxyl group attached to C-4, making it an O-demethylated derivative of compound 1. Compound 2 is a novel structure and has been named oxalicpyridone B. Key COSY and HMBC related information for compound 2 can be found in [link to relevant documentation]. Figure 1 NOESY information can be found Figure 2 .

[0034] Structural identification of new compound 3: Yellow solid, high-resolution mass spectrometry (HR-ESI-MS) m / z 334.2016 [M+H]+ (calcd for C 19 H 28 NO4,334.2013), suggested molecular formula is C 19 H 27 NO4; 1 H and 13 The C10 NMR data are shown in Table 1. The NMR data of compound 3 are very similar to those of compound 1, proving that they have the same planar structure. The difference lies in the significant differences in the NMR data at C-2, C-3, and C-4 of the pyran ring segment, indicating that compound 3 is an isomer of compound 1. The absolute configurations of compound 3 at C-2, C-3, and C-4 were determined to be 2S, 3R, and 4R by NOESY, coupling constant, and ECD calculations. Compound 3 is a novel structure and is named oxalicpyridone C. The main COSY and HMBC related information for compound 3 can be found in [Table 1]. Figure 1 NOESY information can be found Figure 2 .

[0035] Table 1: Compounds 1-3 1 H and 13 C NMR data (DMSO-d) 6, 500MHz)

[0036]

[0037]

[0038] Based on the above physicochemical data analysis, the specific structures of compounds 1-3 are shown in formula (I).

[0039]

[0040] Example 2: Detection of antitumor activity of compounds 1-3

[0041] H446, H1048, H69AR, and H82 cell lines were purchased from the American Type Culture Collection (ATCC). H446, H69AR, and H82 cells were cultured in RPMI-1640 medium, while H1048 cells were cultured in DMEM medium. Both media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin antibiotics. All cells were cultured at 37°C and 5% CO2. The GAPDH antibody (catalog number #2118, dilution 1:1000) and PARP-1 antibody (catalog number #9542, dilution 1:1000) used in the experiments were purchased from Cell Signaling Technology, Boston, USA.

[0042] Wild-type zebrafish strain AB were cultured in an automated recirculating aquaculture system under constant temperature of 28±0.5℃ and a diurnal cycle of 14 hours light / 10 hours dark. The zebrafish were fed twice daily with commercially available flake fish food and brine shrimp. Zebrafish embryos were obtained through natural mating of adult fish. Healthy zebrafish juveniles, two days after fertilization, were cultured in E3 buffer (containing NaCl, KCl, MgSO4, and CaCl2) for subsequent experiments.

[0043] The inhibitory activity data of compounds 1-3 against the above four types of tumor cells are shown in Table 2 and... Figure 4 As shown.

[0044] Table 2: Inhibitory effects of compounds 1-3 on the proliferation of four tumor cell lines (IC50) 50 (μM)

[0045]

[0046] Note: DDP: cisplatin; Eto: etoposide.

[0047] When the confluence of small cell lung cancer (SCLC) cells reached 80%, the cells were washed three times with PBS buffer. Following the experimental procedure, the cells were stained with 1 μg / mL CM-Dil (dissolved in PBS) at 37°C for 10 minutes, followed by a further staining at 4°C for 15 minutes. After staining, the cells were washed three times with PBS, digested with 0.25% trypsin for 2 minutes, and then collected. After centrifugation at 2000 rpm, the cells were resuspended in serum-free DMEM medium and the density was adjusted to 10,000 cells / μL. Two days after fertilization (2 dpf) in zebrafish larvae, the egg membrane was removed with 1 mg / mL streptomycin, and the cells were washed three times with fresh E3M buffer. After anesthetizing the larvae with 0.003% tricaine methanesulfonate for 2 minutes, SCLC cells (100 cells / 10 nmL, 5-20 nmL per embryo) were injected into the yolk sac region of the zebrafish using a pneumatic Pico-pump microinjector with a glass microneedle.

[0048] Following drug injection, xenografted zebrafish larvae were co-cultured with the drug for 48 hours. The drug solution was changed every 24 hours, and dead individuals were promptly removed to prevent solution contamination. Forty-eight hours post-injection, tumor growth in the yolk sac region of the zebrafish was observed under a fluorescence microscope, and the fluorescence density of the micrographs was analyzed using ImageJ software to assess the tumor growth inhibition effect of the drug treatment.

[0049] The effect of compound 1 on the formation of small cell lung cancer cell lines H446 and H1048 is shown in the figure. Figure 5 .like Figure 5 As shown in Figure A, with increasing concentrations of compound 1 (0, 0.25, 0.5, 1 μM), the total number of clonal colonies formed by cancer cells H446 and H1048 significantly decreased, indicating that compound 1 inhibited cancer cell proliferation. Figure 5 As shown in B, with the increase of compound 1 concentration (0, 0.25, 0.5, 1 μM), the expression of PARP-1 gradually decreased, while the expression of c-PARP-1 gradually increased, indicating that compound 1 induced apoptosis in cancer cells.

[0050] The effect of compound 1 at different concentrations on the growth of SCLC (small cell lung cancer) cells in a zebrafish tumor xenograft model is shown in the figure. Figure 6 .like Figure 6 As shown, compound 1 exhibited a dose-dependent inhibitory effect on the growth of SCLC cells in a zebrafish tumor xenograft model at different concentrations. With increasing concentration of compound 1, the fluorescence intensity of SCLC cells gradually decreased, indicating that compound 1 enhanced its inhibitory effect on cancer cell growth. The inhibitory effect was most significant at a concentration of 0.5 μM.

[0051] The above experimental results show that compounds 1-3, as novel 4-hydroxy-2-pyridone alkaloid derivatives, have significant antitumor activity; in particular, compound 1 shows significant antitumor activity against small cell lung cancer and has the potential to be a lead compound for the development of antitumor drugs, which is of great value for promoting the development of marine microbial drug resources in my country.

[0052] The embodiments of this invention are merely illustrative of the technical solutions of this invention and are not intended to limit the scope of protection of this invention. Any equivalent modifications or substitutions made based on the technical solutions of this invention, as long as they do not depart from the spirit and scope of the technical solutions of this invention, should be included within the scope of protection of the claims of this invention.

Claims

1. Pyranopyridone alkaloid derivatives or their medicinal salts, with structural formulas as shown in any of formulas 1-3: 。 2. A method for preparing the pyranopyridone alkaloid derivative of claim 1, characterized in that, The pyranopyridone alkaloid derivatives mentioned above are from Penicillium sp. It was isolated and prepared from the fermentation culture of SCSIO 41320. Penicillium sp. SCSIO 41320 is deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 18, 2025, with accession number GDMCC No: 66161.

3. The preparation method according to claim 2, characterized in that, The specific steps are as follows: 1) Preparation Penicillium sp. Fermentation culture of SCSIO 41320; 2) The fermentation culture was repeatedly extracted three times with ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to obtain a crude extract. The crude extract was separated by normal-phase silica gel column chromatography using a gradient elution of CH2Cl2-MeOH at a volume fraction of 0-100% to obtain six fractions Fr.1~Fr.

6. Fraction Fr.1 eluted at a CH2Cl2:MeOH volume ratio of 100:0 was collected, and fraction Fr.2 eluted at a CH2Cl2:MeOH volume ratio of 99:1 was collected. Fr.1 and Fr.2 were combined and eluted with MeOH-H2O at a volume fraction of 0-100% on an ODS reversed-phase column. According to liquid phase analysis, nine fractions Fr.11~Fr.19 were obtained. Fraction Fr.18 eluted at a MeOH:H2O volume ratio of 75:25 was collected. Fr.18 was then eluted with methanol:water at a volume ratio of 80:20 and 2.5... Semi-preparative HPLC was performed at a flow rate of 3 mL / min with an isogradient elution rate to collect five fractions, Fr.181 to Fr.

185. The retention times of Fr.182, Fr.183, and Fr.185 were 16.5 min, 18.5 min, and 24.5 min, respectively. Fr.182 was further separated by acetonitrile:water elution at a flow rate of 46:54 and a flow rate of 3 mL / min, and compound 2 (retention time 20.0 min) was collected. Fr.183 was separated by acetonitrile:water elution at a flow rate of 48:52 and a flow rate of 2.8 mL / min, and compound 3 (retention time 26.5 min) was collected. Fr.185 was separated by acetonitrile:water elution at a flow rate of 60:40 and a flow rate of 2.5 mL / min, and compound 1 (retention time 27.5 min) was collected.

4. The preparation method according to claim 3, characterized in that, The aforementioned Penicillium sp. The fermentation culture of SCSIO41320 is... Penicillium sp. The rice was obtained by fermenting SCSIO 41320 in rice fermentation medium, which consisted of 120 g of rice and 120 mL of seawater with a salinity of 3.3% added to each bottle of medium.

5. The use of the pyranopyridone alkaloid derivative or its pharmaceutical salt as described in claim 1 in the preparation of drugs for treating small cell lung cancer.

6. The application according to claim 5, characterized in that, The administration method of the anti-small cell lung cancer drug is selected from oral administration and injection administration.

7. An antitumor drug, characterized in that, It comprises an effective amount of any of the pyranopyridone alkaloid derivatives or pharmaceutical salts thereof shown in any of formulas 1-3 of claim 1 as the active ingredient, and a pharmaceutically acceptable carrier.

8. The antitumor drug according to claim 7, characterized in that, The anti-tumor drug is a drug for treating small cell lung cancer.

9. The claim 2 Penicillium sp. The use of SCSIO 41320 in the preparation of the pyranopyridone alkaloid derivatives of claim 1.

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