An ester compound, a synthesis method and an extraction method thereof, and an application thereof

Abstract

This invention belongs to the field of anticancer drug technology, specifically relating to an ester compound, its synthesis and extraction methods, and its applications. This invention provides an ester compound having the structure shown in Formula I. Compound I provided by this invention can target and inhibit the activation of downstream EGFR signaling pathways, including the MAPK and HIPPO signaling pathways. The HIPPO pathway regulates tumor EMT processes, thereby inhibiting cell proliferation, migration, invasion, and tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of anticancer drug technology, specifically relating to an ester compound, its synthesis method, extraction method, and application. Background Technology

[0002] Cancer is one of the major health problems facing the world, and it has now become the second leading cause of death globally. Cancer has a high incidence and mortality rate, and these rates are increasing year by year due to changes in lifestyle and environmental pollution. Cancer treatment has become a major focus of current medical research and clinical practice.

[0003] Currently, cancer treatment mainly includes traditional surgical treatment, chemotherapy, and radiotherapy, as well as novel treatment methods such as targeted therapy, immunotherapy, and cell therapy that have developed rapidly in recent years. However, some types of cancer still lack effective targeted therapies and still rely on conventional treatments such as radiotherapy and chemotherapy. Existing anti-tumor drugs still cannot meet clinical needs. Natural products, due to their low toxicity, are gradually attracting researchers' attention in the development of anti-tumor drugs, and more and more researchers are obtaining promising anti-tumor candidate compounds from medicinal plants.

[0004] Edible and medicinal fungi rich in bioactive components have long been used to treat or prevent a variety of diseases. They have been shown to exhibit broad-spectrum pharmacological activities, such as immunomodulatory, antitumor, anti-inflammatory, and antioxidant activities. Some polysaccharide components obtained from fungi have been used as pharmaceuticals and functional foods for various purposes.

[0005] Phellinus linteus is a well-known medicinal fungus belonging to the Polyporaceae family, growing on mulberry trees. Pharmacological studies have shown that Phellinus linteus possesses antioxidant, anti-infective, anti-cancer, and immunomodulatory effects. Currently, various secondary metabolites of Phellinus linteus have been identified, including phenolic compounds, polyketides, terpenes, steroids, and polysaccharides. Polyphenols derived from Phellinus linteus have attracted considerable interest due to their potential anti-cancer properties. For example, they can improve the human immune system, reduce the side effects of anticancer drugs, and alleviate patients' responses to radiotherapy or chemotherapy. Some scholars have also pointed out that phenolic compounds extracted from Phellinus linteus can exert anti-tumor effects by inducing apoptosis in cancer cells and inhibiting their migration. Furthermore, Phellinus linteus extract may induce G0 / G1 cell cycle arrest and apoptosis in human leukemia, nasopharyngeal carcinoma, and hepatocellular carcinoma cells, and can also enhance the expression of the cancer suppressor gene p53 in lung cancer cells. Phellinus linteus extract also plays a role in inducing autophagy and metastasis in cancer cells. However, no compounds extracted from Phellinus linteus with inhibitory effects on pancreatic cancer and oral cancer cell lines have been reported to date. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide an ester compound, its synthesis method and extraction method, and its application. The ester compound provided by the present invention has a significant inhibitory effect on the migration, invasion and proliferation of pancreatic cancer and oral cancer cell lines.

[0007] To achieve the above objectives, the present invention provides an ester compound having the structure shown in Formula I:

[0008] Formula I.

[0009] This invention provides a method for extracting the ester compounds, comprising the following steps:

[0010] The fruiting bodies of Phellinus linteus were successively subjected to alcohol extraction and concentration. The crude extract was then extracted with ethyl acetate to obtain an ethyl acetate extract.

[0011] The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-acetone system. Eight fractions were obtained according to the elution order of the components and named Fr1 to Fr8. The volume ratio of petroleum ether to acetone in the petroleum ether-acetone system was 100:0 to 30.

[0012] The Fr4 fraction was separated by reverse-phase medium-pressure liquid phase separation using a methanol-water gradient elution system to obtain seven fractions, named Fr. 4-1 ~Fr 4-7 ;

[0013] The volume ratio of methanol to water in the methanol-water system is 0.75:0.25~0.90:0.10;

[0014] Fr 4-1 The fraction was separated by reversed-phase HPLC and eluted with a 75% (v / v) methanol aqueous solution to obtain the ester compounds.

[0015] Preferably, the reagent for the alcohol extraction is methanol; the alcohol extraction is a reflux alcohol extraction; and the alcohol extraction is performed three times.

[0016] Preferably, the gradient elution using the petroleum ether-acetone system involves eluting with petroleum ether-acetone systems of volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30.

[0017] Preferably, the gradient elution using a methanol-water system involves sequentially eluting with methanol and water in volume ratios of 0.75:0.25, 0.85:0.15, and 0.90:0.10.

[0018] This invention provides a method for synthesizing the aforementioned ester compounds, characterized by comprising the following steps:

[0019] Compound I-1, having the structure of formula I-1, and ethylene glycol were dissolved in a benzene solvent and carbonyl protection was performed under acidic conditions to obtain compound I-2, having the structure of formula I-2.

[0020] Compound I-2, a nucleophilic reducing agent, and diethyl ether were mixed and reduced to obtain compound I-3 having the structure of formula I-3;

[0021] Compound I-3 was dissolved in acetonitrile and deprotected under acidic conditions to obtain compound I-4 having the structure of formula I-4;

[0022] Compound I-4 and acetic anhydride were dissolved in dichloromethane and subjected to transesterification under pyridine catalyst conditions to obtain the ester compound;

[0023] Formula I-1; Formula I-2; Formula I-3; Formula I-4.

[0024] Preferably, the mass ratio of compound I-2 to the nucleophilic reducing agent is 50:8~10; the reduction reaction temperature is -5~0℃, and the time is 10~15min.

[0025] Preferably, the molar ratio of compound I-4 to acetic anhydride is 5~5.5:9.0~9.5, and the transesterification reaction is carried out at a temperature of 20~30℃ for 3.5~4.5h.

[0026] This invention provides the application of the ester compounds described herein, or the ester compounds prepared by the extraction method described herein, or the ester compounds prepared by the synthesis method described herein, in the preparation of antitumor drugs.

[0027] Preferably, the antitumor drug includes drugs that inhibit pancreatic cancer and / or oral cancer tumors.

[0028] This invention provides an ester compound having the structure shown in Formula I. Compound I provided by this invention can target and inhibit the activation of downstream signaling pathways of EGFR (epidermal growth factor receptor), including the MAPK signaling pathway and the HIPPO signaling pathway. The HIPPO pathway regulates the tumor EMT (epithelial-mesenchymal transition) process, thereby inhibiting cell proliferation, migration and invasion. Attached Figure Description

[0029] Figure 1 Compound I prepared in Example 1 1 H NMR spectrum;

[0030] Figure 2 Compound I prepared in Example 1 13 C NMR spectrum;

[0031] Figure 3 The DEPT135 spectrum of compound I prepared in Example 1;

[0032] Figure 4 The HMQC spectrum of compound I prepared in Example 1;

[0033] Figure 5 The HMBC spectrum of compound I prepared in Example 1;

[0034] Figure 6 Compound I prepared in Example 1 1 H- 1 H COSY spectrum;

[0035] Figure 7 The HRESIMS spectrum of compound I prepared in Example 1;

[0036] Figure 8 The graph shows the inhibitory effect of different concentrations of compound I on the proliferation of pancreatic cancer cells (PANC-1).

[0037] Figure 9 The graph shows the inhibitory effect of different concentrations of compound I on the proliferation of oral cancer cells (CAL-27).

[0038] Figure 10 The graph shows the inhibitory effect of different concentrations of compound I on the colony-forming ability of pancreatic cancer cells (PANC-1).

[0039] Figure 11 The graph shows the inhibitory effect of different concentrations of compound I on the clonogenic ability of oral cancer cells (CAL-27).

[0040] Figure 12 The inhibitory effect of different concentrations of compound I on the migration ability of pancreatic cancer cells (PANC-1);

[0041] Figure 13 The graph shows the inhibitory effect of different concentrations of compound I on the migration ability of oral cancer cells (CAL-27).

[0042] Figure 14 The graph shows the inhibitory effect of different concentrations of compound I on the invasive ability of pancreatic cancer cells (PANC-1).

[0043] Figure 15The graph shows the inhibitory effect of different concentrations of compound I on the invasive ability of oral cancer cells (CAL-27).

[0044] Figure 16 The study demonstrated the inhibitory effect of compound I on pancreatic cancer tumor growth in vivo.

[0045] Figure 17 The study demonstrated the inhibitory effect of compound I on oral cancer tumor growth in vivo. Detailed Implementation

[0046] This invention provides ester compounds having the structure shown in Formula I:

[0047] Formula I.

[0048] The present invention also provides a method for extracting the aforementioned ester compounds, comprising the following steps:

[0049] The fruiting bodies of Phellinus linteus were successively subjected to alcohol extraction and concentration. The crude extract was then extracted with ethyl acetate to obtain an ethyl acetate extract.

[0050] The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-acetone system. Eight fractions were obtained according to the elution order of the components and named Fr1 to Fr8. The volume ratio of petroleum ether to acetone in the petroleum ether-acetone system was 100:0 to 30.

[0051] The Fr4 fraction was separated by reverse-phase medium-pressure liquid phase separation using a methanol-water gradient elution system to obtain seven fractions, named Fr. 4-1 ~Fr 4-7 ;

[0052] The volume ratio of methanol to water in the methanol-water system is 0.75:0.25~0.90:0.10;

[0053] Fr 4-1 The fraction was separated by reversed-phase HPLC and eluted with a 75% (v / v) methanol aqueous solution to obtain the ester compounds.

[0054] In this invention, the fruiting bodies of Phellinus linteus are sequentially subjected to alcohol extraction and concentration, and the crude extract is extracted with ethyl acetate to obtain an ethyl acetate extract.

[0055] In this invention, the reagent used for alcohol extraction is preferably methanol, and the alcohol extraction is preferably reflux extraction; the alcohol extraction is performed three times. In this invention, the first reflux extraction time is preferably 2 hours, the second reflux extraction time is preferably 1.5 hours, and the third reflux extraction time is preferably 1.5 hours.

[0056] In this invention, the concentration is preferably vacuum concentration. This invention does not specifically limit vacuum concentration; it can be concentrated to a paste state using an operation well known to those skilled in the art.

[0057] In this invention, the extraction is preferably carried out by suspending the crude extract in water and then extracting it with ethyl acetate to obtain an ethyl acetate extract.

[0058] After obtaining the ethyl acetate extract, the present invention subjected the ethyl acetate extract to silica gel column chromatography, using a petroleum ether-acetone system for gradient elution. Eight fractions were obtained according to the elution order, named Fr1 to Fr8. In the present invention, the volume ratio of petroleum ether to acetone in the petroleum ether-acetone system is 100:0 to 30.

[0059] In this invention, the gradient elution using a petroleum ether-acetone system is preferably performed by sequentially using petroleum ether-acetone systems with volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:30.

[0060] In this invention, the Fr4 fraction is an elution fraction with a volume ratio of petroleum ether to acetone of 100:1 and 100:2.

[0061] After obtaining the Fr4 fraction, the present invention performs reverse-phase medium-pressure liquid phase separation on the Fr4 fraction using a methanol-water system for gradient elution, yielding 7 fractions, named Fr4. 4-1 ~Fr 4-7 .

[0062] In this invention, the volume ratio of methanol to water in the methanol-water system is 0.75:0.25 to 0.90:0.10. In this invention, the gradient elution of the reverse-phase medium-pressure liquid phase separation is preferably performed using methanol-water systems with volume ratios of 0.75:0.25:0.85:0.15 and 0.90:0.10, respectively.

[0063] In this invention, the Fr 4-1 The distillate fraction is an elution fraction with a methanol-water volume ratio of 0.75:0.25.

[0064] Get Fr 4-1 After distillation, the present invention will remove Fr 4-1 The fraction was separated by reversed-phase HPLC and eluted with a 75% (v / v) methanol aqueous solution to obtain the ester compounds.

[0065] This invention also provides a method for synthesizing the ester compounds described in the above technical solution, comprising the following steps:

[0066] Compound I-1, having the structure of formula I-1, and ethylene glycol were dissolved in a benzene solvent and carbonyl protection was performed under acidic conditions to obtain compound I-2, having the structure of formula I-2.

[0067] Compound I-2 and a nucleophilic reducing agent were dissolved in diethyl ether and reduced to obtain compound I-3 having the structure of formula I-3;

[0068] Compound I-3 was dissolved in acetonitrile and deprotected under acidic conditions to obtain compound I-4 having the structure of formula I-4;

[0069] Compound I-4 and acetic anhydride were dissolved in dichloromethane and subjected to transesterification under pyridine catalyst conditions to obtain the ester compound;

[0070] Formula I-1; Formula I-2; Formula I-3; Formula I-4.

[0071] In this invention, compound I-1 having the structure of formula I-1 and ethylene glycol are dissolved in a benzene-based solvent and carbonyl protection is performed under p-toluenesulfonic acid conditions to obtain compound I-2 having the structure of formula I-2.

[0072] In this invention, the benzene solvent is preferably toluene.

[0073] In this invention, the mass ratio of compound I-1 to ethylene glycol is preferably 1.5~2:1.5~2, more preferably 1.7~1.8:1.5~1.6. In this invention, the acidic conditions are preferably provided by p-benzenesulfonic acid. In this invention, the pH of the acidic conditions is preferably 2~4, more preferably 2.5~3. In this invention, the mass ratio of compound I-1 to toluene is preferably 1.5~2 g:50 mL, more preferably 1.7~1.8 g:50 mL.

[0074] In this invention, the carbonyl protection temperature is preferably 80~150℃, more preferably 100~120℃, and the time is preferably 10~12h, more preferably 11h. In this invention, the carbonyl protection is preferably carried out under stirring conditions.

[0075] In this invention, when the carbonyl protection termination reaction is performed, it is preferable to first cool to room temperature and then terminate the reaction with a cold NaHCO3 solution.

[0076] In this invention, the reaction process for carbonyl protection is as follows:

[0077] .

[0078] In this invention, after carbonyl protection, the product obtained from the carbonyl protection reaction is preferably subjected to ethyl acetate extraction, washing, drying, filtration and concentration in sequence, and the concentrated product is subjected to silica gel column chromatography.

[0079] In this invention, the extraction is preferably performed three times, and the ethyl acetate phases obtained from the three extractions are preferably combined for further processing. In this invention, the washing is preferably performed with water and NaCl solution; the number of times each water washing and NaCl solution washing is performed is preferably 2-3 times. In this invention, the drying is preferably performed with a desiccant, preferably anhydrous Na₂SO₄. 4。 In this invention, the filtration and concentration are not specifically limited, and operations well known to those skilled in the art can be used. In this invention, the eluent for the silica gel column chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is preferably 1:25.

[0080] After obtaining compound I-2, the present invention mixes compound I-2, a nucleophilic reducing agent and diethyl ether, and carries out a reduction reaction to obtain compound I-3 having the structure of formula I-3.

[0081] In this invention, the nucleophilic reducing agent is preferably LiAlH4. In this invention, the mass ratio of compound I-2 to the nucleophilic reducing agent is preferably 50:8~10, more preferably 50:9.

[0082] In this invention, the mixing is preferably carried out by dissolving compound I-2 in diethyl ether to obtain an ether solution of compound I-2; dissolving the nucleophilic reducing agent in diethyl ether to obtain an ether suspension of the nucleophilic reducing agent; and adding the ether solution of compound I-2 dropwise to the ether suspension of the nucleophilic reducing agent.

[0083] In this invention, the concentration of compound I-2 in the ether solution of compound I-2 is preferably 0.6~0.7 mol / L, more preferably 0.62~0.65 mol / L; the concentration of nucleophilic reducing agent in the ether suspension of nucleophilic reducing agent is preferably 0.15~0.2 mol / L, more preferably 0.16~0.19 mol / L.

[0084] In this invention, the temperature of the reduction reaction is preferably -5 to 0°C, and the time is preferably 10 to 15 minutes, more preferably 12 to 13 minutes. In this invention, the reduction reaction is preferably carried out under stirring conditions.

[0085] In this invention, the reduction reaction process is as follows:

[0086]

[0087] In this invention, after the reduction reaction, it is preferable to further cool, wash, dry, filter and concentrate the system obtained from the reduction reaction in sequence, and then perform silica gel column chromatography to obtain compound I-3.

[0088] In this invention, the cooling is preferably to room temperature; the drying reagent is preferably MgSO4. The concentration operation is not specifically limited in this invention; any operation well-known to those skilled in the art can be used. In this invention, the eluent for silica gel column chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is preferably 4:1.

[0089] After obtaining compound I-3, the present invention dissolves compound I-3 in acetonitrile and performs deprotection under acidic conditions to obtain compound I-4 having the structure of formula I-4.

[0090] In this invention, the mass ratio of compound I-3 to acetonitrile is preferably 2-3 g:60 mL, more preferably 2.1-2.5 g:60 mL. In this invention, the acidic conditions are preferably provided by an HCl solution, and the concentration of the hydrochloric acid solution is preferably 2 mol / L.

[0091] In this invention, the deprotection temperature is preferably 50~60℃, more preferably 55℃, and the time is preferably 5.5~6.5h, more preferably 6h.

[0092] In this invention, the deprotection reaction process is as follows:

[0093]

[0094] In this invention, after deprotection, the process preferably further includes extracting the deprotected system, washing, drying, filtering and concentrating the extracted organic phase, and then performing silica gel column chromatography on the concentrated product to obtain compound I-4.

[0095] In this invention, the extraction is preferably performed by mixing the deprotected system with water and ethyl acetate. The extraction is preferably performed three times, and the ethyl acetate phases obtained from the extraction are combined and processed. In this invention, the washing is preferably performed with NaHCO3 solution and NaCl solution. In this invention, the drying reagent is preferably anhydrous Na2SO4. In this invention, the filtration is not specifically limited, as long as it removes the desiccant. In this invention, the eluent for the silica gel column chromatography is preferably ethyl acetate and petroleum ether; the volume ratio of ethyl acetate to petroleum ether is preferably 1:5.

[0096] After obtaining compound I-4, the present invention dissolves compound I-4 and acetic anhydride in dichloromethane and carries out transesterification reaction under pyridine catalyst conditions to obtain the ester compound.

[0097] In this invention, the pyridine catalyst is preferably 4-dimethylaminopyridine and pyridine; the molar ratio of 4-dimethylaminopyridine to pyridine in the pyridine catalyst is preferably 0.6:8.5-9, more preferably 0.6:8.6-8.8. In this invention, the mass ratio of compound I-4 to the volume ratio of dichloromethane is preferably 0.8-1.0 g:50 mL, more preferably 0.95-0.99 g:50 mL. In this invention, the molar ratio of compound I-4 to acetic anhydride is preferably 5-5.5:9.0-9.5, more preferably 5.3-5.4:9.0-9.3.

[0098] In this invention, the temperature of the transesterification reaction is preferably 20-30°C, more preferably 25°C; the time is preferably 3.5-4.5 h, more preferably 4.0 h. In this invention, the transesterification reaction is preferably carried out under stirring conditions.

[0099] In this invention, the reaction process for the transesterification is as follows:

[0100]

[0101] In this invention, after the transesterification reaction, the product obtained from the transesterification reaction is preferably subjected to vacuum concentration, drying, and silica gel column chromatography in sequence. In this invention, the eluent for the silica gel column chromatography is preferably ethyl acetate and petroleum ether, and the volume ratio of ethyl acetate to petroleum ether is preferably 1:25.

[0102] This invention also provides the application of the ester compounds described herein, or the ester compounds prepared by the extraction method described herein, or the ester compounds prepared by the synthesis method described herein, in the preparation of antitumor drugs. In this invention, the antitumor drugs preferably include drugs that inhibit pancreatic cancer and oral cancer tumors.

[0103] In this invention, the antitumor drug preferably comprises the ester compounds described above and pharmaceutically acceptable carriers and / or excipients.

[0104] In this invention, the antitumor drug is preferably an oral dosage form or a parenteral dosage form; the oral dosage form is preferably an oral liquid, syrup, tablet, capsule, chewable tablet, pill, or granule; the parenteral dosage form is preferably an intravenous, intramuscular, intradermal, subcutaneous, or spinal injection.

[0105] To further illustrate the present invention, the following detailed description of the embodiments is provided in conjunction with the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0106] Example 1

[0107] (1) 9.17 kg of poplar fruiting bodies were refluxed with anhydrous methanol three times. The first reflux extraction time was 2 h, the second reflux extraction time was 1.5 h, and the third reflux extraction time was 1.5 h. The extracts were combined and concentrated under reduced pressure to obtain 1.31 kg of methanol extract.

[0108] (2) The extract obtained in step (1) was suspended in water and extracted with ethyl acetate until the organic layer was clear and transparent. The extracts were combined and concentrated under reduced pressure to obtain the ethyl acetate extract phase (165 g).

[0109] (3) Separate the extract obtained in step (2) by silica gel column chromatography using gradient elution with petroleum ether:acetone (100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20 and 100:30, V:V).

[0110] (4) The fractions eluted from petroleum ether:acetone 100:1 and 100:2 in step (3) are separated by reversed-phase medium-pressure liquid phase elution with 75%, 85% and 90% methanol-water systems.

[0111] The fraction eluted from the 75% methanol-water system in step (3) was separated by HPLC, and ester compounds with the structure shown in Formula I were obtained by elution with the 75% methanol-water system. The obtained compounds were then subjected to... 1 H NMR spectrum, 13 C NMR spectrum, DEPT135 spectrum, HMQC spectrum, HMBC spectrum, 1 H- 1 Comprehensive analysis of H COSY and HRESIMS spectra (detection results are shown in [link to relevant documentation]). Figures 1-7 The structure of the compound was determined and designated as compound I, proving that it is a new compound and belongs to the class of benzenepentane derivatives.

[0112] The structure of compound I is as follows:

[0113] Formula I;

[0114] Figure 1 Compound I prepared in Example 1 1 H NMR spectrum Figure 2 Compound I prepared in Example 1 13 C NMR spectrum, from Figures 1-2 It can be seen that, 1 H NMR spectrum and 13 The 13C NMR spectrum information is shown in Table 1. The high-resolution mass spectrometry detection results of compound I are as follows:

[0115] HRESIMS m / z 243.0995 [M + Na] + (calcd for C 13 H 16 O3Na 243.0997).

[0116] Table 1 1 H NMR data and 13 C NMR data

[0117]

[0118] The present invention also performed infrared spectroscopy on compound I prepared in the examples, and the test information is as follows:

[0119] IR (KBr) ν max 3647, 3081, 3059, 3025, 3001, 2920, 2848, 1942, 1869,1801, 1746, 1669, 1600, 1583, 1541, 1492, 1451, 1370, 1327, 1181, 1154, 1068,1027, 979, 963, 942, 905, 841, 755, 695, 535 cm -1 .

[0120] Example 2

[0121] Ethyl 3-oxoylide-5-phenylpentyl ester (compound I-1) (CAS: 17071-29-3, 1.76 g, 8 mM), ethylene glycol (1.4 mL, 24 mM), and p-toluenesulfonic acid (15 mg, 0.08 mM) were dissolved in 50 mL of toluene. The mixture was heated to reflux and dehydrated using a Dean-Stark separator. After stirring for 11 h, the mixture was cooled to room temperature, and the experiment was terminated with 50 mL of cold NaHCO3 solution. The mixture was extracted three times with 40 mL of ethyl acetate. The ethyl acetate phases were combined and washed with 20 mL of water and 20 mL of NaCl solution, respectively. The washed ethyl acetate phases were then dried over anhydrous Na2SO4, filtered, and concentrated to dryness under evaporation. The reaction product was purified by silica gel column chromatography (ethyl acetate / petroleum ether 1:25, V:V) to give 1.06 g of colorless oily product compound I-2 (yield 50%).

[0122] The present invention detects compound I-2, and its NMR data are as follows:

[0123] 1 H NMR (400 MHz, CDCl3) δ H7.31-7.15 (m, 5H), 4.16 (q, J = 7.1 Hz, 2H),4.08 – 3.99 (m, 4H), 2.77 – 2.71 (m, 2H), 2.70 (s, 2H), 2.18 – 2.12 (m, 2H),1.27 (t, J = 7.1 Hz, 3H).

[0124] Compound I-2 (500 mg, 1.9 mM) was dissolved in 3 mL of diethyl ether and slowly added to 15 mL of a 90 mg, 2.73 mM LiAlH4 suspension in diethyl ether. The mixture was stirred at 0 °C for 10 min, then heated under reflux for 1 h. After cooling to room temperature, 0.3 mL of water and 0.5 mL of sodium hydroxide (6 M) were added. The mixture was dried over MgSO4, filtered, and concentrated to dryness. The reaction product was purified by silica gel column chromatography (ethyl acetate / petroleum ether 4:1, V:V) to give 350 mg of a colorless oily product, compound I-3 (yield 83%).

[0125] The present invention detected compound I-3, and its NMR data are as follows:

[0126] 1 H NMR (400 MHz, CDCl3) δ H 7.31-7.25 (m, 2H), 7.22-7.16 (m, 3H), 4.07 –3.99 (m, 4H), 3.80-3.75 (m, 2H), 2.72-2.66 (m, 2H), 2.02-1.94 (m, 4H).

[0127] Compound I-3 (2.22 g, 10 mM) was dissolved in 60 mL of acetonitrile, and 60 mL of 2 mol / L HCl was slowly added. The mixture was stirred at 55 °C for 6 h. The reaction solution was then diluted with 30 mL of water and 60 mL of ethyl acetate, and extracted to obtain an ethyl acetate phase and an aqueous phase. The aqueous phase was extracted twice more with 30 mL of ethyl acetate. The combined ethyl acetate phases were washed with 30 mL of NaHCO3 and 30 mL of brine, respectively, dried over Na2SO4, filtered, and concentrated to dryness under rotary evaporation. The reaction product was purified by silica gel column chromatography (ethyl acetate / petroleum ether 1:5) to give a colorless oily product, compound I-4 (1.24 g, 70%).

[0128] The NMR data for compound I-4 are as follows:

[0129] 1 H NMR (400 MHz, CDCl3) δ H7.30-7.24 (m, 2H), 7.22-7.15 (m, 3H), 3.83(t, J = 5.5 Hz, 2H), 2.90 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.64 (t, J =5.5 Hz, 2H).

[0130] Compound I-4 (0.96 g, 5.39 mM) was dissolved in 50 mL of CH2Cl2, and 4-dimethylaminopyridine (73 mg, 0.6 mM), pyridine (0.705 mL, 8.76 mM), and acetic anhydride (0.877 mL, 9.34 mM) were added. The mixture was stirred at 25 °C for 4 h, and the reaction product was concentrated and dried under reduced pressure. The reaction product was purified by silica gel column chromatography (eluting ethyl acetate / petroleum ether 1:25, V:V) to give 1.18 g of the ester compound as a colorless oil (yield 85%).

[0131] The NMR data of the ester compounds prepared in Example 2 are as follows:

[0132] 1 H NMR (400 MHz, CDCl3) δ H 7.31-7.26 (m, 2H), 7.22-7.16 (m, 3H), 4.32(t, J = 6.2 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 2.77 (t, J = 7.5 Hz, 2H), 2.72 (t, J =6.2 Hz, 2H), 2.01 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ C 207.0, 171.0, 140.9, 128.7, 128.4, 126.3, 77.5, 77.2, 76.8, 59.4, 44.8, 41.6, 29.6, 21.0.

[0133] Its NMR data are basically consistent with those of the isolated natural product compound I.

[0134] Example 3

[0135] The examples studied the effect of compound I provided by the present invention on the proliferation of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 at the in vitro cell level. The effect of compound I on the proliferation ability of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 was evaluated by the MTT proliferation curve method.

[0136] The specific implementation method is as follows: CAL-27 cells and PANC-1 cells were seeded into 96-well plates and cultured into monolayers. After cell adhesion, the culture medium was discarded, and cell culture medium prepared with compound I obtained in Example 2 at concentrations of 5, 10, and 20 μM was added respectively. Control wells without drugs were also set up. The wells were incubated at 37°C and CO2 (5%) for 0, 24, 48, and 72 h. The culture medium was discarded, and MTT solution at a concentration of 5 mg / ml was added. The absorbance value of each well was detected at a wavelength of 570 nm using an ELISA reader.

[0137] The cell survival rate represented by the absorbance of the control group was 100%, and the absorbance of the other drug-treated wells / absorbance of the control group represented the cell survival rate after being affected by the drug. Cell proliferation curves were plotted using Graphpad Prism 5 software, and all indicators were expressed as mean ± standard deviation.

[0138] Figure 8 and Figure 9 The graphs show the inhibitory effects of different concentrations of compound I on the proliferation of pancreatic cancer cells (PANC-1) and oral cancer cells (CAL-27); from Figures 8-9 It can be seen that compound I has a significant inhibitory effect on the proliferation of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27, and this effect is time- and dose-dependent.

[0139] Example 4

[0140] The effects of compound I of the present invention on the colony formation of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 at the in vitro cellular level were evaluated using the colony formation assay.

[0141] The specific implementation method is as follows: 200-500 cells are seeded in 6-well plates and cultured. After the cells adhere, the culture medium is discarded, and cell culture medium prepared by compound I obtained in Example 2 at concentrations of 5, 10, and 20 μM is added respectively. Control wells without drugs are also set up. The cells are cultured in a 37°C, CO2 (5%) incubator for 7-14 days. The culture medium is discarded, and the cells are washed with PBS and fixed with cold methanol for 10 min. Then, they are stained with crystal violet for 15 min. After washing away excess dye with PBS, the cells are photographed and the number of clones is counted.

[0142] Figure 10 and Figure 11 The graphs show the inhibitory effects of different concentrations of compound I on the colony-forming ability of pancreatic cancer cells (PANC-1) and CAL-27 cells; from Figures 10-11 It was found that compound I significantly inhibited the colony formation of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 in a dose-dependent manner.

[0143] Example 5

[0144] The efficacy of compound I in inhibiting the migration of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 at the in vitro cellular level was evaluated using the cell scratch assay.

[0145] The specific implementation method is as follows: cells are seeded in 24-well plates and cultured into a monolayer of cells until 100%. A 100 μm scratch blank area is made using a sterile pipette tip. The liquid is discarded, and the cells are washed twice with 1 × PBS. Cell culture medium with concentrations of 0, 5, 10, and 20 μM of compound 5 is added respectively. Control wells without the drug are also set up. The cells are incubated in a 37℃, CO2 (5%) incubator for 48 h. At 0, 24, and 48 h, samples are taken at fixed positions under an optical microscope, photographs are taken, and the changes in the intercellular distance at both ends of the scratch are recorded.

[0146] The ratio to 0h relative to the starting position is calculated as Sxh / S0h. Cell migration distance-time curves were plotted using Graphpad Prism 5 software, and all indicators are expressed as mean ± standard deviation.

[0147] Figures 12-13 The graphs show the inhibitory effects of different concentrations of compound I on the migration ability of pancreatic cancer cells (PANC-1) and oral cancer cell line CAL-27, respectively. Figures 12-13 It is known that compound I has a significant inhibitory effect on the migration ability of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27, and this effect is time- and dose-dependent.

[0148] Example 6

[0149] This study investigated the effect of compound I on the invasion of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 at the in vitro cellular level. The effect of compound I on the invasive ability of pancreatic cancer cells and oral cancer cells was evaluated using the transwell assay.

[0150] The specific experimental steps were as follows: PANC-1 and CAL-27 cells were implanted into chambers (BD, USA) coated with matrix material and inserted into the wells of a 24-well plate. The 24-well plate was filled with complete culture medium. Different concentrations of compound 5 were added to the chambers. After 24 h, the PANC-1 and CAL-27 cells on the lower surface of the chambers were fixed and stained with crystal violet.

[0151] Figures 14-15 The graph shows the inhibitory effects of different concentrations of compound I on the invasive ability of pancreatic cancer cells (PANC-1) and oral cancer cell line CAL-27. Figures 14-15 It was found that compound I significantly inhibited the invasion ability of pancreatic cancer cell line PANC-1 and oral cancer cell line CAL-27 in a dose-dependent manner.

[0152] Example 7

[0153] This embodiment studies the in vivo antitumor efficacy of compound I in pancreatic cancer. The antitumor efficacy of compound I and the positive control compound cisplatin in a nude mouse xenograft model was tested. The specific procedure is as follows:

[0154] All Balbc / nude mice used in this experiment were purchased from Beijing Vital River Pharmaceutical Co., Ltd. After expansion culture of pancreatic cancer PANC-1 cells, each mouse was injected with 1×10⁻⁶ cells. 7 The seeding volume (cell suspension: Matrigel = 1:1) was 100 μL of cell suspension injected into the axilla of the forelimb of mice; the inoculation continued until the average tumor volume exceeded 100 mm². 3 Mice were randomly divided into four groups: a control group, a cisplatin 1.5 mg / kg group, a compound I 50 mg / kg group, and a cisplatin 1.5 mg / kg combined with compound I 25 mg / kg group, with five mice in each group. Cisplatin was administered intraperitoneally every two days, and compound I was administered intraperitoneally once a day until the end of the experiment.

[0155] After the experiment, tumor tissue was collected, and the tumor volume and weight were measured and statistically analyzed to calculate the tumor inhibition rate. (Tumor inhibition rate = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%)

[0156] Experimental results are as follows Figure 16 As shown, from Figure 16It was found that compound I at a dose of 50 mg / kg could inhibit the growth of pancreatic cancer PANC-1 tumors with an inhibition rate of 41.8%. The positive control drug cisplatin at 1.5 mg / kg also had a significant inhibitory effect on the growth of pancreatic cancer PANC-1 tumors with an inhibition rate of 63.5%. The inhibitory effect of compound I was not as strong as that of the positive control drug cisplatin. The combination of cisplatin and compound 5 could significantly inhibit the growth of pancreatic cancer tumors with an inhibition rate of 69.6%, which was 6.1% higher than that of cisplatin alone. This shows that compound I can exert a synergistic effect when used in combination with platinum-based chemotherapy drugs.

[0157] Example 8

[0158] This embodiment studies the in vivo antitumor efficacy of compound I in an oral cancer PDX model. The antitumor efficacy of compound I and the positive control compound cisplatin in the oral cancer PDX model was tested. The specific procedure is as follows:

[0159] The Balbc / nude mice used in this experiment were all purchased from Vital River Pharmaceuticals, Beijing. Clinical oral cancer tissue samples were transplanted into the axilla of the forelimbs of the mice; tumors were transplanted until the average volume exceeded 1000 mmHg. 3 At that time, second-generation tumor-bearing tumors were carried out until the average volume of the second-generation tumors exceeded 100 mm. 3 Mice were randomly divided into four groups: a control group, a cisplatin 1.5 mg / kg group, a compound I 50 mg / kg group, and a cisplatin 1.5 mg / kg combined with compound I 50 mg / kg group, with four mice in each group. Cisplatin was administered intraperitoneally every two days, and compound I was administered intraperitoneally once a day until the end of the experiment.

[0160] After the experiment, tumor tissue was collected, and the tumor volume and weight were measured and statistically analyzed to calculate the tumor inhibition rate. (Tumor inhibition rate = (1 - tumor weight in the treatment group / tumor weight in the control group) × 100%)

[0161] Experimental results are as follows Figure 17 As shown, from Figure 17 It was found that compound I at a dose of 50 mg / kg significantly inhibited the growth of oral cancer tumors, with an inhibition rate of 60.93%. The positive control drug cisplatin at 1.5 mg / kg also showed a significant inhibitory effect on the growth of oral cancer tumors, with an inhibition rate of 60.09%. The combination of cisplatin and compound I significantly inhibited the growth of pancreatic cancer tumors, with an inhibition rate of 67.94%, which was 7.85% higher than that of the cisplatin monotherapy group.

[0162] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for extracting ester compounds, characterized in that, Includes the following steps: The fruiting bodies of Phellinus linteus were successively subjected to alcohol extraction and concentration. The crude extract was then extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was subjected to silica gel column chromatography with gradient elution using a petroleum ether-acetone system. Eight fractions were obtained according to the elution order of the components and named Fr1 to Fr8. The volume ratio of petroleum ether to acetone in the petroleum ether-acetone system was 100:0 to 30. The Fr4 fraction was separated by reverse-phase medium-pressure liquid phase separation using a methanol-water gradient elution system to obtain seven fractions, named Fr. 4-1 ~Fr 4-7 ; The volume ratio of methanol to water in the methanol-water system is 0.75:0.25~0.90:0.10; Fr 4-1 The fraction was separated by reversed-phase HPLC and eluted with a 75% (v / v) methanol-water solution to obtain the ester compounds; The ester compound has the structure shown in Formula I: Equation I.

2. The method for extracting ester compounds according to claim 1, characterized in that, The reagent used for the alcohol extraction is methanol; the alcohol extraction is a reflux alcohol extraction; and the alcohol extraction is performed three times.

3. The extraction method according to claim 1, characterized in that, The gradient elution using the petroleum ether-acetone system involves sequentially eluting with petroleum ether and acetone in volume ratios of 100:0, 100:1, 100:2, 100:4, 100:6, 100:9, 100:14, 100:20, and 100:

30.

4. The extraction method according to claim 1, characterized in that, The gradient elution using a methanol-water system involves sequentially eluting with methanol and water in volume ratios of 0.75:0.25, 0.85:0.15, and 0.90:0.

10.

5. The use of the ester compounds prepared by the extraction method according to any one of claims 1 to 4 in the preparation of antitumor drugs.

6. The application according to claim 5, characterized in that, The antitumor drugs include those that inhibit pancreatic cancer and / or oral cancer tumors.

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