A novel three-membered fused-ring pyrone compound and its preparation method and use

Abstract

The present invention relates to the field of biomedicine, specifically to a novel three-membered fused-ring pyrone compound, its preparation method, and use. The compound is a three-membered ring fused skeleton, or an isomer, salt, or pharmaceutically acceptable solvate and prodrug thereof. The compounds of the present invention are prepared using the endophytic fungus Penicillium brevis F4a, and their use in anti-tumor treatment. The novel anti-tumor lead compounds provided by the present invention have significant inhibitory activity against a variety of tumor cells and can serve as ideal candidate anti-tumor compounds. The lead compounds can be produced by fungal fermentation, and their preparation method is simple and efficient, and the resulting product has high purity. The invention has good application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a novel three-membered fused-ring pyrone compound, a preparation method thereof, and uses thereof. Background Art

[0002] Cancer is one of the leading causes of death worldwide and a major obstacle to improving life expectancy. In 2020, there were 19.29 million new cancer cases and 9.96 million cancer deaths worldwide. Of these, 4.57 million new cancer cases, or 23.7% of the global total, and 3 million cancer deaths, or 30% of the global total, occurred in China. China ranks first in the world in both cancer incidence and mortality rates. Therefore, the development of new and highly effective anticancer drugs is urgent.

[0003] Plant endophytes have long been widely used in the discovery of anticancer drugs. Since 1993, numerous anticancer drugs have been isolated from endophytes, including paclitaxel, camptothecin, diosgenin, hypericin, podophyllotoxin, and vinblastine. It is well known that the vast diversity of plants has shaped a rich and unique diversity of endophytes. These endophytes are believed to be the base for producing secondary metabolites with anti-tumor cell proliferation activity, making them a research hotspot and a continuous contributor to the discovery of lead anticancer drugs.

[0004] Despite significant progress in anti-tumor treatment in recent years, tumor cells mutate at a rate far exceeding that of normal cells, easily leading to treatment failure or the development of drug-resistant tumor cells. Therefore, novel anticancer agents with distinct mechanisms from existing anti-tumor drugs are a significant need for the treatment of tumors. This invention provides a novel, previously unreported, three-membered ring-fused framework comprising two general formulae: cyclopenteno[d]pyrano[4,3-b]pyrone-like structures and cyclopentano[4,5]furo[3,2-c]pyrone-like structures. The invention also discloses their preparation methods and their inhibitory effects on various tumor cells. Summary of the Invention

[0005] The present invention aims to provide a novel three-membered fused-ring pyrone compound and a preparation method and use thereof.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A novel three-membered fused-ring pyrone compound having a three-membered ring fused skeleton, or an isomer or salt of the above-mentioned compound; or a pharmaceutically acceptable solvate and a prodrug thereof.

[0008] Preferably, the compound is a cyclopentenone-[d]pyrano[4,3-b]pyrone compound having a 6 / 6 / 5 three-membered ring fused skeleton as shown in the general formula I, or a cyclopenta-[4,5]furo-[3,2-c]pyrone compound having a 6 / 5 / 5 three-membered ring fused skeleton as shown in the general formula II; or an isomer, salt, or pharmaceutically acceptable solvate and prodrug thereof of the above compounds;

[0009]

[0010] In the general formula,

[0011] R is hydroxy, benzyloxy, carbonyl, aldehyde, nitro, amino, carboxyl, halogen, C1-C8 alkoxy, C3-C8 cycloalkane, C2-C8 alkene, C3-C8 heterocycle, C3-C8 heteroaryl which are unsubstituted or substituted with the following groups, the following substituents are halogen, oxygen, C1-C8 alkyl, hydroxy C1-C8 alkyl;

[0012] R1 is hydrogen, hydroxy, C1-C8 alkyl, C2-C8 alkenyl, C1-C8 alkyl, C1-C8 alkoxy, carbonyl, aldehyde, halogen, carboxyl, nitro, amino, benzyl, C3-C8 heterocycle, C3-C8 heteroaryl;

[0013] R2 is hydrogen, hydroxy, keto, aldehyde, carboxyl, C2-C8 alkenyl, halogen, nitro, amine, benzyl, C3-C8 heterocycle, or C3-C8 heteroaryl;

[0014] The heteroatoms are N, S, and O.

[0015] Further preferably, the compound is a compound represented by the following general formula I or II, or an isomer, salt, or pharmaceutically acceptable solvate or prodrug thereof of the corresponding compound;

[0016] In the general formula I, R is hydroxy, methoxy, ethoxy, or 2-hydroxymethyl-3-methyl-cyclopentanone;

[0017] In the general formula II, R1 is hydrogen, hydroxymethyl or vinyl;

[0018] R2 is a hydroxyl group or a keto group.

[0019] More preferably, the compound is the following compound and its or the isomer, salt, or pharmaceutically acceptable solvate and prodrug thereof of the corresponding compound;

[0020]

[0021] A method for preparing the novel three-membered fused-ring pyrone compound, comprising separating and purifying the fermentation broth of the plant endophytic fungus F4a after culturing to obtain the compound;

[0022] The plant endophytic fungus F4a is Eupenicillium brefeldianum F4a, which was deposited in the China Center for Type Culture Collection on December 18, 2012, with the deposit number: CCTCC No: M 2012531, and the deposit address is Wuhan, China;

[0023] Further,

[0024] 1) Inoculating the plant endophytic fungus F4a onto PSA solid medium and culturing for 48-96 hours to activate it, inoculating the activated culture into liquid medium and culturing at 28-32°C for 48-72 hours to obtain a seed solution; mixing the obtained seed solution with the liquid medium at a volume ratio of 1:28-1:12, and culturing with shaking at 24-32°C for 5-10 days;

[0025] 2) The fermentation broth is centrifuged to remove bacterial cells to obtain a fermentation supernatant, and the active ingredients in the fermentation broth are solid-phase adsorbed using a macroporous adsorption resin HP20 at a resin to fermentation broth volume ratio of 1:13 to 1:27; adsorption is carried out for 1-3 hours, and after adsorption, a crude extract is obtained by washing; the separated bacterial cells are extracted with acetone to obtain a crude extract, and the crude extracts are combined to obtain a total extract A;

[0026] 3) The total extract A was separated by silica gel column chromatography, and the components were collected according to a gradient elution of dichloromethane:methanol (v / v) ratio of 100:0 to 0:100 to obtain the target secondary metabolite enrichment product.

[0027] 4) The above enriched product was separated by silica gel column chromatography, and pure dichloromethane was eluted to obtain fraction F. a , dichloromethane: methanol (v / v) ratio is 100:1-100:3 and eluted as fraction F b , dichloromethane: methanol (v / v) ratio is 100:4-100:6 and eluted as fraction F c , dichloromethane: methanol (v / v) ratio is 100:7-10:9 eluted as fraction F d , dichloromethane: methanol (v / v) ratio is 100:10-100:15 and eluted as fraction F e .

[0028] Fraction F b Separate with gel LH20 and divide into 4 sub-fractions according to the volume of eluted methanol. Receive 50-70 mL of methanol as sub-fraction F according to the elution order. b4 Receive 80-120 mL of methanol as sub-fraction F b3 Receive 120-140 mL of methanol as sub-fraction F b2 Receive 145-160 mL of methanol as sub-fraction F b1 . The sub-flow fraction F b4After ODS reverse phase column chromatography and methanol-water gradient separation, a total of 4 sub-fractions (F b4a -F b4d ), where 30-40% methanol-water elution is F b4a , 42-45% methanol water elution is F b4b , 46-50% methanol water elution is F b4c , 52-55% methanol-water elution is F b4d . Fraction F b4a Purification was performed by semi-preparative HPLC, eluting with 30-50% aqueous methanol at a flow rate of 2.0 mL / min to obtain compounds 1 (peniapyrone A), 8 (peniapyrone H) and 9 (peniapyrone I); F b4d Purify by semi-preparative HPLC, eluting with 45-65% aqueous methanol at a flow rate of 2.0 mL / min to obtain compound 2-5 (peniapyrones BE);

[0029] Fraction F c Separate with gel LH20 and collect two sub-fractions according to the volume of eluted methanol. Collect 60-80 mL of methanol as sub-fraction F according to the elution order. c2 , collect 80-120 mL of methanol as sub-fraction F c1 . Will get F c2 Six sub-fractions (F c2a -F c2f ), of which 35-42% methanol-water elution is F c2a , 43-46% methanol-water elution is F c2b , 48-52% methanol-water elution is F c2c , 53-57% methanol-water elution is F c2d , 58-62% methanol-water elution is F c2e , 63-66% methanol-water elution is F c2f . The sub-flow fraction F c2a The products were purified by semi-preparative HPLC with 30-50% methanol in water at a flow rate of 2.0 mL / min to obtain compounds 6 (peniapyrone F) and 7 (peniapyrone G).

[0030] The liquid culture medium contains 10-15g of starch, 15-25g of glucose, 0-3g of yeast extract, 0-2g of soybean cake powder, 2-4g of barley extract, 0-1g of ammonium sulfate, 0-2g of potassium nitrate, 2-4g of calcium carbonate, 2-5g of magnesium sulfate heptahydrate, 1-3g of potassium dihydrogen phosphate, and 0-1g of copper sulfate per liter of water, with a pH of 6.0-7.5.

[0031] An application of the novel three-membered fused-ring pyrone compound, and an application of the three-membered fused-ring pyrone compound in the preparation of drugs for tumor-related diseases.

[0032] The tumor-related disease is one or more of pancreatic cancer, breast cancer, and liver cancer.

[0033] The advantages of the present invention are:

[0034] The present invention provides a novel three-membered ring fused skeleton which has not been reported yet, including a cyclopentenone-[d]pyrano[4,3-b]pyrone structure and a cyclopenta[4,5]furo[3,2-c]pyrone structure.

[0035] The novel three-membered ring fused skeleton in the present invention is produced by fungal fermentation and is obtained through a specific separation process.

[0036] The novel three-membered ring fused skeleton compounds obtained in the present invention have significant anti-tumor activity and can be used as ideal lead anti-tumor drugs, especially showing significant inhibitory activity against AsPC-1, CRL-2234 and MCF-7 tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Compounds 1-9 provided in the embodiments of the present invention 1 H- 1 Significant correlation between H COSY and HMBC.

[0038] Figure 2 The important NOESY correlations of compounds 1-9 provided in the examples of the present invention.

[0039] Figure 3 Experimental ECD spectra and calculated ECD spectra of compounds 1-8 in CH3OH provided in the examples of the present invention.

[0040] Figure 4 NMR calculation and DP4+ analysis of compounds 4-8 provided in the examples of the present invention. DETAILED DESCRIPTION

[0041] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments, but the protection content of this patent is not limited to this.

[0042] Example 1 Preparation Method of a Novel Three-Membered Ring Fused Skeleton Produced by Fermentation of Plant Endophytic Fungus F4a

[0043] The liquid culture medium used was 10 g starch, 20 g glucose, 1 g yeast extract, 0.5 g soybean cake powder, 2.5 g barley extract, 1 g ammonium sulfate, 0.8 g potassium nitrate, 4 g calcium carbonate, 2.5 g magnesium sulfate heptahydrate, 3 g potassium dihydrogen phosphate, 0.02 g copper sulfate per liter of water, pH 7.0.

[0044] Endophytic fungus F4a (CCTCCM 2012531) was inoculated onto PSA solid medium for 48 hours of activation. The inoculation was then carried out on the aforementioned liquid medium and incubated at 28°C for 72 hours to prepare a seed solution. The resulting seed solution was then inoculated into the same fermentation medium at a volume ratio of 1:18 and cultured at 28°C for 7 days. The fermentation broth was centrifuged to remove the bacterial cells to obtain the fermentation supernatant. The active components were then adsorbed onto the fermentation broth using a macroporous adsorption resin, HP20, at a resin:broth volume ratio of 1:25. After 2 hours of adsorption, the resin was washed with distilled water and then with a 15% methanol-water solution, followed by desorption with methanol. The crude extract was recovered after recovering the methanol solvent. The bacterial cells were sonicated in acetone for 30 minutes, and the crude extract was recovered after recovering the acetone solvent. The two crude extracts were combined to obtain total extract A.

[0045] Extract A was separated by flash silica gel column chromatography and eluted with a gradient of dichloromethane:methanol (v / v) from 100:0 to 0:100 to obtain 5 fractions (F a -F e ), wherein pure dichloromethane is eluted as fraction F a , dichloromethane: methanol (v / v) ratio is 100:1-100:3 and eluted as fraction F b , dichloromethane: methanol (v / v) ratio of 100:4-100:6 is fraction F c , dichloromethane: methanol (v / v) ratio is 100:7-10:9, and fraction F d , dichloromethane: methanol (v / v) ratio of 100:10-100:15 is fraction F e .

[0046] The collected fraction F b Separate with gel LH20 and divide into 4 sub-fractions according to the volume of eluted methanol. Receive 40-60 mL of methanol as sub-fraction F according to the elution order. b4 Receive 62-100 mL of methanol as sub-fraction F b3 Receive 102-120 mL of methanol as sub-fraction F b2 Receive 125-140 mL of methanol as sub-fraction F b1 . The sub-flow fraction F b4 After ODS reverse phase column chromatography and methanol-water gradient separation, a total of 4 sub-fractions (F b4a -F b4d), where 30-40% methanol-water elution is F b4a , 42-45% methanol water elution is F b4b , 46-50% methanol water elution is F b4c , 52-55% methanol-water elution is F b4d . Fraction F b4a The compound peniapyrone A (1, 12.0 mg, t R :13.8min), peniapyrone H(8, 2.0mg, t R : 15.6min) and peniapyrone I (9, 3.1mg, t R :13.2min); F b4d The compound peniapyrone B (2, 4.2 mg, t R :18.4min), peniapyrone C(3, 3.4mg, t R :19.5min), peniapyrone D(4, 8.2mg, t R :20.7min) and peniapyrone E (5, 5.8mg, t R :21.0min).

[0047] Fraction F c Separate with gel LH20 and collect two sub-fractions according to the volume of eluted methanol. Collect 50-78 mL of methanol as sub-fraction F according to the elution order. c2 , collect 80-100 mL of methanol as sub-fraction F c1 . Will get F c2 Six sub-fractions (F c2a -F c2f ), of which 35-42% methanol-water elution is F c2a , 43-46% methanol-water elution is F c2b , 48-52% methanol-water elution is F c2c , 53-57% methanol-water elution is F c2d , 58-62% methanol-water elution is F c2e , 63-66% methanol-water elution is F c2f . The sub-flow fraction F c2aThe compound peniapyrone F (6, 5.7 mg, t R :13.8min) and peniapyroneG (7, 6.8mg, t R :13.1min).

[0048] The structural analysis of the novel three-membered ring fused skeleton compound peniapyrones obtained above (see Figure 1-4 ):

[0049] Compound 1 is a colorless, transparent oil. Its UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 298 nm. HRESIMS data show an m / z 249.0763 [M+H] + (calcd for C 13 H 13 O5, 249.0763), and its molecular formula is speculated to be C 13 H 12 O5, unsaturation is 8. 1 The H NMR (600 MHz, DMSO-d6) spectrum (Table 1) shows the presence of three olefinic hydrogen proton signals δ H 8.02 (1H, d, J = 5.6 Hz), 6.08 (1H, d, J = 5.6 Hz) and 5.98 (1H, s); one oxygen-containing methine proton signal δ H 5.74 (1H, s); one methine proton signal δ H 2.85 (1H, d, J = 1.7 Hz); a methyl proton signal connected to a double bond δ H 2.13 (3H, s) and one aliphatic methyl proton signal δ H 1.64 (3H, s). 13 The C NMR (150 MHz, DMSO-d6) spectrum and HSQC spectrum (Table 2) show 13 carbon signals, including 1 keto carbonyl carbon signal, 7 unsaturated carbon signals, 2 methine carbon signals, 1 quaternary carbon signal and 2 methyl carbon signals. 2 The hybridized carbon signal can be inferred that there is a trisubstituted α-pyrone, a double bond and a keto carbonyl in the structure of compound 1. Combined with the unsaturation degree of 8, it can be judged that compound 1 is a three-membered ring α-pyrone skeleton. The planar structure can be determined by 1 H- 1 H COSY and HMBC spectra confirmed ( Figure 1). Through the correlation between H-5 and C-3 and C-13 in the HMBC spectrum, it can be inferred that there is a 6-methyl-α-pyrone skeleton fragment (ring A); through the correlation between H-11 and C-3, and H-12 and C-4 and C-7 in the HMBC spectrum, it can be inferred that there is a dihydropyran-2-ol fragment (ring B); through the correlation between H-11 and C-3 and H-12 and C-4 in the HMBC spectrum, it can be inferred that rings A and B are linked through C-3 / C-4. 1 H- 1 The H COSY spectrum's H-8 / H-9 correlations suggest the presence of a CH2=CH2 spin system. The HMBC spectrum's correlations of H-8 with C-10 and C-11, H-9 with C-7 and C-11, H-11 with C-14, and H-14 with C-8 suggest the presence of a 4-methylcyclopent-2-en-1-one fragment (ring C). The HMBC spectrum's correlations of H-12 with C-7 and H-14 with C-3 suggest that rings B and C are linked via C-7 / C-11. In summary, the planar structure of compound 1 was characterized as a previously unreported 6 / 6 / 5-membered fused heterocyclic α-pyrone skeleton.

[0050] Compounds 2 and 3 are both colorless and transparent oils. Their UV spectra show typical 4-hydroxy-2-pyrone absorption at 298 nm. HRESIMS data show that the molecular formulas of compounds 2 and 3 are C 14 H 14 O5 and C 15 H 16 O5, with molecular weight differences of 14 and 28 Da, respectively, from compound 1. Comparison of the 1D NMR data of compounds 2 and 3 with compound 1 revealed that the 12-OH of compound 1 was replaced by -OCH3 in compound 2 and by -OCH2CH3 in compound 3 (Tables 1 and 2), which was further supported by the HMBC correlation between H-1′ and C-12 ( Figure 1 ).

[0051] The relative configurations of C-7, C-11, and C-12 in compounds 1-3 can be determined by NOESY data and 1 H- 1 H coupling constants were determined by analysis (Table 1 and Figure 2 ). The related signals of H-14 / H-11 / H-12 can be observed in the NOESY spectrum, and the small coupling constant between H-11 and H-12 also indicates that they are in a cis relationship. Based on this, H-11, H-12 and H-14 are all classified as α orientation. The calculated ECD curve is fitted with the experimental ECD curve, and the absolute configurations of compounds 1-3 are determined to be 7R, 11S, and 12R ( Figure 3 ).

[0052] Compounds 4 and 5 are both colorless and transparent oils. The UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 298 nm. HRESIMS data show that the molecular formula of compounds 4 and 5 is C 20 H 22 O6, unsaturation 10. Comparison of the 1D NMR data of compounds 4 and 5 with compounds 1-3 revealed that they have the same 6 / 6 / 5-membered fused α-pyrrolidone skeleton (rings A, B, C) (Tables 1 and 2). From the 10 unsaturations, in addition to the 8 unsaturations in the skeleton and 1 ketone carbonyl carbon, 4 and 5 each have an additional D ring. The planar structure of the D ring can be determined by 1 H- 1 H COSY and HMBC spectra confirmed ( Figure 1 ).pass 1 H- 1 The correlation of H-4′ / H-5′ / H-6′( / H-7′) / H-2′ / H-1′ in the HCOSY spectrum and the correlation of H-1′ with C-3′ and C-6′ in the HMBC spectrum, the correlation of H-4′ with C-2′ and C-6′, the correlation of H-5′ with C-2′ and C-3′, and the correlation of H-7′ with C-2′ and C-5′ in the HMBC spectrum suggest the presence of a 2-hydroxymethyl-3-methyl-1-cyclopentanone fragment (ring D). The correlation of H-1′ with C-12 in the HMBC spectrum suggests that ring D is connected to the parent nucleus skeleton via C-12. The relative configurations of C-7, C-11, and C-12 in compounds 4 and 5 can be determined by NOESY data and 1 H- 1 The H coupling constant analysis determined that the orientation was α ( Figure 2 However, the relative configurations of C-2′ and C-6′ were not determined due to the overlap of the signals of H-2′ and H-6′. In order to determine the relative configurations of 4 and 5, NMR calculations were performed in combination with the DP4+ probability ( Figure 4 ). The following four configurations were calculated: (7R*,11S*,12R*,2′S*,6′S*)-4a / 5a, (7R*,11S*,12R*,2′S*,6′R*)-4b / 5b, (7R*,11S*,12R*,2′R*,6′S*)-4c / 5c, and (7R*,11S*,12R*,2′R*,6′R*)-4d / 5d. Combined with DP4+ analysis, the relative configuration of compound 4 is 4a (96.41%), and the relative configuration of compound 5 is 5d (99.86%) ( Figure 4 The calculated ECD curve was fitted with the experimental ECD curve, and the absolute configurations of compounds 4 and 5 were determined to be 7R, 11S, 12R, 2′S, 6′S and 7S, 11R, 12S, 2′S, 6′S, respectively ( Figure 3). Therefore, compounds 4 and 5 are diastereomers.

[0053] Compound 6 is a colorless, transparent oil. Its UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 293 nm. HRESIMS data show an m / z 223.0961 [M+H] + (calcd for C 12 H 15 O4, 223.0970), and its molecular formula is speculated to be C 12 H 14 O4, unsaturation is 6. Comparison of 1D NMR data of compound 6 with compound 1-5 revealed that it has the same α-pyrone skeleton (ring A) (Table 1-4). From the analysis of 6 unsaturation, compound 6 also has a three-membered ring fused α-pyrone skeleton. Its planar structure can be 1 H- 1 H COSY and HMBC spectra confirmed ( Figure 1 ).pass 1 H- 1 The correlation of H-8 / H-9 / H-10 / H-11 in the H COSY spectrum and the correlation of H-8 with C-10 and C-12 in the HMBC spectrum, and the correlation of H-11 with C-8, C-9, and C-12, inferred that there was a 3-methyl-1-cyclopentanol (ring C) in the structure of compound 6. Based on the correlation of H-8 with C-3, H-11 with C-4, and H-12 with C-3 in the HMBC spectrum, it was inferred that ring C was connected to ring A via C-3-C-7 and C-4-OC-11. Therefore, the planar structure of compound 6 was characterized as an unprecedented 6 / 5 / 5 three-membered fused heterocyclic α-pyrone skeleton. NOESY data confirmed that C-7 and C-11 of compound 6 were coplanar ( Figure 2 However, since H-9 has no related signals with H-11 and H-12 in the NOESY spectrum, the relative configuration of C-9 cannot be determined. In order to determine the relative configuration of compound 6, the two possible configurations (7R * , 9S * , 11R * )-6a and (7R * ,9R * , 11R * )-6b NMR was compared with the experimental NMR ( Figure 4 ), combined with DP4+ analysis, the probability of 6a is 100.00%, so the relative configuration of compound 6 is 7R * , 9S * , 11R * The absolute configuration of compound 6 was determined to be 7R, 9S, 11R by ECD calculation.

[0054] Compound 7 is a colorless, transparent oil. Its UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 293 nm. The HRESIMS data suggest that its molecular formula is C 13 H 16 O5, which is 30Da larger than the molecular weight of compound 6. Comparison of 1D and 2D NMR of compound 7 and compound 6 revealed that the C-8 of compound 7 was substituted by a -CH2OH group. 1 H- 1 The correlation of H-11 / H-10 / H-9 / H-8 / H-14 in the HCOSY spectrum and the correlation of H-9 with C-14 and H-14 with C-7 in the HMBC spectrum were used to support the results (Table 3 and Figure 1 The correlation between H-11 and H-12, and between H-12 and H-14 in the NOESY spectrum confirmed that H-7, H-12, and H-14 in compound 7 were coplanar ( Figure 2 However, the NOESY spectrum could not determine the relative configuration of C-9. Therefore, NMR calculations were performed in conjunction with DP4+ probability analysis, and the following two configurations were calculated: (7R * ,8R * , 9S * , 11R * )-7a and (7R * ,8R * ,9R * , 11R * )-7b. Combined with DP4+ analysis, the relative configuration of compound 7 is 7a (100.00%) ( Figure 4 The calculated ECD curve was fitted with the experimental ECD curve, and the absolute configuration of compound 7 was determined to be 7a ( Figure 3 ).

[0055] Compound 8 is a colorless, transparent oil. Its UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 293 nm. The HRESIMS data suggest that its molecular formula is C 13 H 14 O5, the degree of unsaturation is 7. Comparison of 1D and 2D NMR of compound 8 and compound 6 revealed that the two parent nuclei are similar (Table 3-4). Combining the 7 unsaturations and the correlation of H-10 with C-8, H-11 with C-8, and H-14 with C-7 and C-9 in the HMBC spectrum, it is speculated that there is a vinyl substitution at C-8 ( Figure 1 The relative configuration of compound 8 was confirmed to be 7R by NOESY spectrum and computational NMR combined with DP4+ analysis. * ,9S * ,11R * ( Figure 2 and 4The calculated ECD curve was fitted with the experimental ECD curve, and the absolute configuration of compound 8 was determined to be 7R, 9S, 11R ( Figure 3 ).

[0056] Compound 9 is a colorless, transparent oil. Its UV spectrum shows a typical 4-hydroxy-2-pyrone absorption at 293 nm. The HRESIMS data suggest that its molecular formula is C 12 H 12 O4, the degree of unsaturation is 7. Comparison of 1D and 2D NMR of compound 9 and compound 6 revealed that the biggest difference between the two is the presence of a ketocarbonyl substitution on the ring C of compound 9 (Table 3-4 and Figure 1 ).pass 1 H- 1 The correlation between H-10 / H-11 in the H COSY spectrum and the correlation between H-11 and C-9 in the HMBC spectrum, as well as the 7 degrees of unsaturation, all confirm that the ketocarbonyl group is substituted at the C-9 position of compound 9 ( Figure 1 The relative configuration of C-7 and C-11 of compound 9 can be determined by NOESY as (7S*, 11S*) ( Figure 2 ). HPLC-DAD analysis of compound 9 by chiral chromatography column showed that 9 was a pair of optically pure enantiomers (+)-9 and (-)-9.

[0057] Table 1 Hydrogen spectrum of compound 1-5 (600 MHz, DMSO-d6)

[0058]

[0059] Table 2 Carbon spectrum of compounds 1-5 (150 MHz, DMSO-d6)

[0060]

[0061] Table 3 Proton spectrum of compound 6-9 (600 MHz, DMSO-d6)

[0062]

[0063]

[0064] Table 4 Carbon spectrum of compound 6-9 (150 MHz, DMSO-d6)

[0065]

[0066] Example 2 Antitumor activity of novel three-membered ring fused skeleton compounds

[0067] Three different human cancer cell lines (including human metastatic pancreatic cancer AsPC-1 cells, human liver cancer CRL-2234 cells and human breast cancer MCF-7 cells) were selected as cell lines for evaluating antitumor activity.

[0068] Assay method: Place 100 μL of cell suspension in a 96-well plate and pre-incubate the plate in an incubator for 24 hours (37°C, 5% CO2). Add 10 μL of the above-obtained compound at concentrations of 40 μM, 5 μM, 1.25 μM, 0.625 μM, and 0.3125 μM to the plate. Incubate the plate in an incubator for 12-48 hours. Add 10 μL of CCK8 solution to each well. Incubate the plate in an incubator for 2 hours. Measure the absorbance at 450 nm using a microplate reader, and finally obtain the IC 50 value.

[0069] Experimental results: All the compounds obtained above have certain effects, and the results further recorded in Table 5 show that compounds 1, 3-9 have significant cytotoxic activity against AsPC-1, CRL-2234 and MCF-7 tumor cells. Among them, compounds 1, 3, 6, 8 and 9 (IC 50 =1.52~2.89μM) had a higher inhibitory effect on AsPC-1 cells than the positive control dasatinib (IC 50 =3.25 μM), compounds 1 and 3-9 (IC 50 =0.23~12.68μM) had a higher inhibitory effect on CRL-2234 and MCF-7 tumor cells than the positive controls dasatinib and gemcitabine (IC 50 >20 μM), while compound 2 showed weak inhibitory activity against MCF-7 tumor cells (IC 50 =23.35 μM).

[0070] Notably, AsPC-1 cells are KRAS G12D Mutated cancer cells, KRAS is an important therapeutic target for malignant tumors. In the past three decades, only two KRAS inhibitors, AMG 510 and MRTX849, have been approved by the US FDA, but both are used for KRAS G12C However, KRAS G12D Inhibitors have not yet been found. Therefore, compounds 1, 3, 6, 8 and 9 are expected to be further developed for the treatment of KRAS G12D Lead compounds for new anticancer drugs targeting mutant tumors.

[0071] Table 5 Cytotoxic activity of compounds 1, 3-9 against three tumor cell lines (IC 50 ,μM)

[0072]

Claims

1. A three-membered fused-ring pyrone compound, characterized in that: The compound structure is 2. A method for preparing the ternary fused-ring pyrone compound according to claim 1, characterized in that: 1) Inoculating the plant endophytic fungus F4a onto PSA solid medium and culturing for 48-96 hours to activate it, inoculating the activated culture into liquid medium and culturing at 28-32°C for 48-72 hours to obtain a seed solution; mixing the obtained seed solution with the liquid medium at a volume ratio of 1:28-1:12, and culturing with shaking at 24-32°C for 5-10 days; 2) The fermentation broth is centrifuged to remove bacterial cells to obtain a fermentation supernatant, and the active ingredients in the fermentation broth are solid-phase adsorbed using a macroporous adsorption resin HP20 at a resin to fermentation broth volume ratio of 1:13 to 1:27; adsorption is carried out for 1-3 hours, and after adsorption, a crude extract is obtained by washing; the separated bacterial cells are extracted with acetone to obtain a crude extract, and the crude extracts are combined to obtain a total extract A; 3) The total extract A was separated by silica gel column chromatography, and the fractions were collected according to a gradient elution of dichloromethane:methanol (v / v) from 100:0 to 0:100 to obtain the target secondary metabolite enrichment product; 4) The above enriched product was separated by silica gel column chromatography, and pure dichloromethane was eluted to obtain fraction F. a , dichloromethane: methanol (v / v) ratio is 100:1-100:3 and eluted as fraction F b , dichloromethane: methanol (v / v) ratio is 100:4-100:6 and eluted as fraction F c , dichloromethane: methanol (v / v) ratio is 100:7-10:9 eluted as fraction F d , dichloromethane: methanol (v / v) ratio is 100:10-100:15 and eluted as fraction F e ; Fraction F b Separate with gel LH20 and divide into 4 sub-fractions according to the volume of eluted methanol. Receive 50-70 mL of methanol as sub-fraction F according to the elution order. b4 Receive 80-120 mL of methanol as sub-fraction F b3 Receive 120-140 mL of methanol as sub-fraction F b2 Receive 145-160 mL of methanol as sub-fraction F b1 , the substream F b4 After ODS reverse phase column chromatography and methanol-water gradient separation, a total of 4 sub-fractions (F b4a -F b4d ), where 30-40% methanol-water elution is F b4a , 42-45% methanol water elution is F b4b , 46-50% methanol water elution is F b4c , 52-55% methanol-water elution is F b4d , fraction F b4a Purification was performed by semi-preparative HPLC, eluting with 30-50% aqueous methanol at a flow rate of 2.0 mL / min to obtain compounds 1 (peniapyrone A), 8 (peniapyrone H) and 9 (peniapyrone I); F b4d Purify by semi-preparative HPLC, eluting with 45-65% aqueous methanol at a flow rate of 2.0 mL / min to obtain compound 2-5 (peniapyrones BE); Fraction F c Separate with gel LH20 and collect two sub-fractions according to the volume of eluted methanol. Collect 60-80 mL of methanol as sub-fraction F according to the elution order. c2 , collect 80-120 mL of methanol as sub-fraction F c1 , will get F c2 Six sub-fractions (F c2a -F c2f ), of which 35-42% methanol-water elution is F c2a , 43-46% methanol-water elution is F c2b , 48-52% methanol-water elution is F c2c , 53-57% methanol-water elution is F c2d , 58-62% methanol-water elution is F c2e , 63-66% methanol-water elution is F c2f , the substream F c2a Purification was performed by semi-preparative HPLC, eluting with 30-50% aqueous methanol at a flow rate of 2.0 mL / min, to obtain compounds 6 (peniapyrone F) and 7 (peniapyrone G); The liquid culture medium contains 10-15g starch, 15-25g glucose, 0-3g yeast extract, 0-2g soybean meal, 2-4g barley extract, 0-1g ammonium sulfate, 0-2g potassium nitrate, 2-4g calcium carbonate, 2-5g magnesium sulfate heptahydrate, 1-3g potassium dihydrogen phosphate, and 0-1g copper sulfate per liter of water, with a pH of 6.0-7.5; The plant endophytic fungus F4a is Eupenicillium brefeldianum F4a, which was deposited in the China Center for Type Culture Collection on December 18, 2012, with a deposit number of CCTCC No: M 2012531, and the deposit address is Wuhan, China.

3. An application of the three-membered fused-ring pyrone compound according to claim 1, characterized in that: Application of compounds 1, 3, 5-9 in the preparation of drugs for treating pancreatic cancer, breast cancer and liver cancer; The use of compound 4 in the preparation of anti-pancreatic cancer and breast cancer drugs; The application of the compound 2 in the preparation of anti-breast cancer drugs.

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