Cyclohexanone polyketides, methods of making and anti-influenza virus applications thereof

Abstract

This invention belongs to the field of biomedical technology and discloses a cyclohexanone-derived polyketide compound, its preparation method and application. It provides ten cyclohexanone polyketide compounds, including: amorocoelone A (1), amorocoelone C (2), (4R,5S,6R,9R,10R)-4,9,10-trihydroxy-6-methyl-3-methylene-2-oxaspiro[4.5]dec-7-en-1-one (3), massarilactone A (4), massarilactone C (5), massarilactone E (6), amorocoelone H (7), amorocoelone I (8), (R)-1-hydroxy-1-(5-hydroxy-2-methylphenyl)propan-2-one (9), paraverrucsin A (10), wherein compounds (1), (2), (7) and (8) are new compounds. These compounds can be prepared by bio-fermentation, which has a short production cycle and is environmentally friendly. At the same time, the compounds have anti-influenza virus activity and can be used to prepare antiviral drugs, which has extremely high medical and commercial value. The method has the advantages of short cycle, mild culture conditions, few by-products, strong stereoselectivity, and low cost. It also has high economic value, is low-cost, easy to operate, and easy to produce on a large scale, providing a new way to obtain polyketide compounds containing cyclohexanone structural units from natural sources.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a cyclohexanone polyketide compound, its preparation method, and its application. Background Technology

[0002] Cyclohexanone polyketides are a class of natural products with cyclohexanone as their basic backbone, synthesized through a series of condensation reactions of short-chain acyl units catalyzed by polyketide synthases. These compounds are mainly derived from secondary metabolic pathways in microorganisms and plants, and are characterized by their diverse structures, unique origins, and varied biological activities, making them of significant value in the biomedical field.

[0003] *Gmelina arborea*, a semi-deciduous tree belonging to the genus *Gmelina* in the family Verbenaceae, is currently rare. It is mainly distributed in southern provinces of China, Bangladesh, and some Southeast Asian countries. Its applications have been reported in treating chronic fever, bleeding, urinary tract infections, and dysuria, and it can also be used to treat fever and indigestion. *Gmelina arborea* has high medicinal and economic value, but current research on this plant mainly focuses on the bioactivity and chemical composition of extracts from its leaves, flowers, fruits, and stem bark. No research reports on endophytic fungi in *Gmelina arborea* have been found domestically or internationally.

[0004] The genus *Amorocoelophoma* is a newly established genus of fungi, belonging to the kingdom Mycota, phylum Ascomycota, class Dothideomycetes, order Pleosporales, and family Amorosiaceae. Amorosiaceae was subdivided from Lophiostomataceae by Thambugala et al. in 2015. Currently, the genus *Amorocoelophoma* includes seven species: *A. cassiae*, *A. camelliae*, *A. neoregeliae*, *A. sinensis*, *A. hydei*, *A. soli*, and *A. dicranopteridis*. Research on strains of this genus is limited to taxonomy; no reports on the metabolites of these strains have been published. Summary of the Invention

[0005] The main objective of this invention is to provide a cyclohexanone polyketide compound, its preparation method, and its application. Ten cyclohexanone compounds are provided: amorocoelone A (1), amorocoelone C (2), (4R,5S,6R,9R,10R)-4,9,10-trihydroxy-6-methyl-3-methylene-2-oxaspiro[4.5]dec-7-en-1-one (3), massarilactone A (4), massarilactone C (5), massarilactone E (6), amorocoelone H (7), amorocoelone I (8), (R)-1-hydroxy-1-(5-hydroxy-2-methylphenyl)propan-2-one (9), and paraverrucsin A (10), wherein compounds (1), (2), (7), and (8) are novel compounds. These compounds are prepared through bio-fermentation, which has a short production cycle and is environmentally friendly. At the same time, these compounds have anti-influenza virus activity and can be used to prepare antiviral drugs, thus having extremely high medical and commercial value.

[0006] To achieve the above objectives, the present invention provides the following technical solution: providing ten cyclohexanone polyketide compounds including: amorocoelone A (1), amorocoelone C (2), (4R,5S,6R,9R,10R)-4,9,10-trihydroxy-6-methyl-3-methylene-2-oxaspiro[4.5]dec-7-en-1-one (3), massarilactone A (4), massarilactone C (5), massarilactone E (6), amorocoelone H (7), amorocoelone I (8), (R)-1-hydroxy-1-(5-hydroxy-2-methylphenyl)propan-2-one (9), paraverrucsin A (10).

[0007] A method for preparing cyclohexanone polyketide compounds 1-10, comprising the following steps:

[0008] The fungus Amorocoelophoma sp. was fermented, and the fermentation broth was collected;

[0009] The fermentation broth was extracted with an organic solvent, the extracts were combined, and the crude fermentation extract was concentrated under reduced pressure.

[0010] The crude fermentation extract was separated by silica gel column chromatography, eluted with a chloroform-methanol system, and the crude fraction was collected.

[0011] The crude fraction obtained is eluted by a petroleum ether-ethyl acetate, petroleum ether-acetone, chloroform-methanol, chloroform-acetone, ethyl acetate-acetone, or methanol-water system, or by recrystallization, to obtain the final product.

[0012] Preferably, the fungus Amorocoelophoma sp. is Amorocoelophoma sp. YE3351, which was deposited at the China Center for Type Culture Collection on August 7, 2020, with accession number CCTCC NO.M 2020407.

[0013] Preferably, the fermentation medium is SDB medium, wherein the concentration of peptone in SDB medium is 0.01 g / mL and the concentration of glucose is 0.04 g / mL.

[0014] Preferably, the fermentation broth is extracted with ethyl acetate.

[0015] Preferably, a gradient elution is performed using a chloroform-methanol system with a volume ratio from 1:0 to 1:1, and the crude fractions obtained by elution from the chloroform-methanol system at different volume ratios are collected.

[0016] Preferably, the obtained crude component is eluted with petroleum ether-ethyl acetate, petroleum ether-acetone, chloroform-methanol, chloroform-acetone, ethyl acetate-acetone, or methanol-water in different volume ratios, or recrystallized.

[0017] Application of cyclohexanone polyketides and their preparation methods in the preparation of anti-influenza virus drugs.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. This invention provides ten cyclohexanone compounds 1-10, which have good anti-influenza virus activity, providing a new option for the development of anti-influenza virus drugs.

[0020] 2. The compounds of the present invention are prepared by microbial fermentation. This method has the advantages of short cycle, mild culture conditions, few by-products, strong stereoselectivity and low cost. It also has high economic value, is low in cost, simple to operate and easy to produce on a large scale, and provides a new way to obtain polyketide compounds containing cyclohexanone structural units from natural sources.

[0021] 3. The plant endophytic fungus Amorocoelophoma sp. YE3351 used in this invention was deposited on August 7, 2020, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCCNO.M 2020407; its taxonomic name is Amorocoelophoma sp. Attached Figure Description

[0022] Figure 1 Compound 1 of the present invention 1 Schematic diagram of H-NMR spectrum;

[0023] Figure 2 Compound 1 of the present invention 13 Schematic diagrams of C-NMR and DEPT spectra;

[0024] Figure 3 This is an X-ray single-crystal diffraction crystal structure diagram of compound 1 of the present invention;

[0025] Figure 4 Compound 2 of the present invention 1 Schematic diagram of H-NMR spectrum;

[0026] Figure 5 Compound 2 of the present invention 13 Schematic diagrams of C-NMR and DEPT spectra;

[0027] Figure 6 This is an X-ray single-crystal diffraction crystal structure diagram of compound 2 of the present invention;

[0028] Figure 7 Compound 7 of the present invention 1 Schematic diagram of H-NMR spectrum;

[0029] Figure 8 Compound 7 of the present invention 13 Schematic diagrams of C-NMR and DEPT spectra;

[0030] Figure 9 This is a schematic diagram of the ECD spectrum of compound 7 of the present invention;

[0031] Figure 10 This is a Graph showing the DP4+ analysis results of compound 7 of the present invention;

[0032] Figure 11 Compound 8 of the present invention 1 Schematic diagram of H-NMR spectrum;

[0033] Figure 12 Compound 8 of the present invention 13 Schematic diagrams of C-NMR and DEPT spectra;

[0034] Figure 13 This is a schematic diagram of the ECD spectrum of compound 8 of the present invention;

[0035] Figure 14 This is a graph showing the DP4+ analysis results of compound 8 of the present invention;

[0036] Figure 15 The diagram shows the structural formulas of ten cyclohexanone polyketone compounds of this invention. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] Example 1

[0039] Isolation and preparation of cyclohexanone compounds 1-10

[0040] (1) Preparation of PDB medium: Weigh 200 g of peeled potatoes, cut them into small pieces, add 1000 mL of water and boil for 20 min. Filter with gauze to obtain potato filtrate. Add 20 g of glucose, set the pH to natural, and sterilize at 121 ℃ for 30 min to obtain PDB medium for later use. The concentration of potato in PDB medium is 0.2 g / mL, and the concentration of glucose is 0.02 g / mL. Preparation of SDB medium: Weigh 10 g of peptone and 40 g of glucose, add 1 L of water, set the pH to natural, and sterilize at 121 ℃ for 30 min to obtain SDB medium for later use. The concentration of peptone in SDB medium is 0.01 g / mL, and the concentration of glucose is 0.04 g / mL.

[0041] (2) Inoculate Amorocoelophoma sp. YE3351 into PDB medium and culture it in a shaker at 28±2 ℃ and 200 r / min for 3 days to obtain seed liquid. Then, inoculate the prepared seed liquid into SDB fermentation medium at an inoculation rate of 10% and culture it in a shaker at 28±2 ℃ and 200 r / min for 7~8 days to obtain fermentation product.

[0042] (3) The fermentation product obtained in step (2) is filtered with gauze to obtain fermentation broth. The fermentation broth is extracted with ethyl acetate and concentrated under reduced pressure to obtain crude fermentation extract.

[0043] (4) The crude fermentation extract was separated by chromatography using a normal-phase silica gel column (200-300 mesh) and an RP-18 reversed-phase silica gel column. The crude fractions (Frs. 1–9) obtained by elution with a chloroform-methanol system at volume ratios from 1:0 to 1:1 were collected. Fr. 2 was eluted by normal-phase silica gel column chromatography (petroleum ether-acetone, 9:1 to 1:1) to obtain Frs. 2.1–2.5. Fr. 2.2 was recrystallized to obtain compound (6). Fr. 2.4 was eluted by normal-phase silica gel column chromatography (petroleum ether-ethyl acetate, 4:1 to 3:2) to obtain compounds (1) and (8). Fr. 3 was eluted by normal-phase silica gel column chromatography (chloroform-acetone, 9:1 to 1:1) to give Frs. 3.1–3.6. Fr. 3.5 was eluted by gradient elution by normal-phase silica gel column chromatography (petroleum ether-ethyl acetate, 9:1 to 2:3) to give compounds (4) and (5). Fr. 3.6 was eluted by gradient elution by RP-18 reversed-phase silica gel column chromatography (methanol-water, 1:4, 3:7, 2:3) to give compound (7). Fr. 4 was eluted by gradient elution by normal-phase silica gel column chromatography (petroleum ether-ethyl acetate, 9:1 to 1:1) to give compound (9). Fr. 6 was eluted by gradient elution with normal-phase silica gel column chromatography (ethyl acetate-acetone, 9:1 to 2:3) to give Frs. 6.1–6.4. Fr. 6.3 was eluted by gradient elution with RP-18 reversed-phase silica gel column chromatography (methanol-water, 1:1, 3:2, 7:3) to give compound (10). Fr. 6.4 was eluted by gradient elution with normal-phase silica gel column chromatography (chloroform-acetone, 4:1, 1:1) to give compound (3). Fr. 7 was eluted by gradient elution with normal-phase silica gel column chromatography (chloroform-methanol, 5:1, 4:1) to give compound (2).

[0044] Example 2

[0045] Structural identification of cyclohexanone compounds 1-10

[0046] like Figure 15 As shown, compounds 1-10 prepared in Example 1 were identified by 1D / 2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy), HRESI-MS (high-resolution electrospray ionization mass spectrometry), IR (infrared spectroscopy), X-ray (X-single crystal diffraction), UV (ultraviolet spectroscopy), OR (specific rotation), ECD calculation, NMR calculation, and DP4+ analysis. The new compounds amorocoelone A (1), amorocoelone C (2), amorocoelone H (7), and amorocoelone I (8) were identified as follows:

[0047] amorocoelone A (1)

[0048] Compound 1 is a colorless crystal with the molecular formula C. 21 H 26 O9. HRESI-MS m / z 440.1906 [M+NH4] ＋ (calcd for C 21 H 30 NO9, 440.1915), with an unsaturation degree of 9. , MeOH); UV(MeOH) λ max (log ε): 223 (14.43) nm; IR (KBr) ν max 3596, 3469, 2917, 1748,1725, 1688, 1617, 1243, 1152, 1125, 1067, . 1 H NMR (600 MHz, acetone-d6): δ 1.09 (3H, d, J = 7.3 Hz, H-1), 1.21 (3H, d, J = 7.0 Hz, H-21), 2.13 (3H, s, H-20), 2.36 (3H, s, H-10), 2.63 (1H, m, H-7), 2.64 (1H, d, J =3.3 Hz, H-15α), 2.80 (1H, d, J = 16.8 Hz, H-15β), 3.04 (1H, m, H-13), 3.06(1H, m, H-2), 4.10 (1H, d, J = 10.0 Hz, H-6), 4.40 (1H, dd, J = 2.1, 3.2 Hz,H-14), 4.46 (1H, s, H-18), 4.56 (1H, d, J = 3.2 Hz, 6-OH), 5.05 (1H, d, J =3.7 Hz, H-17), 5.26 (1H, d, J = 3.4 Hz, 17-OH), 5.72 (1H, s, H-8), 6.01 (1H,dd, J = 2.9, 10.0 Hz, H-4), 6.85 (1H, dd, J = 2.2, 10.1 Hz, H-3). 13C NMR (150MHz, acetone-d6): 13.8 (C-21), 18.1 (C-1), 26.3 (C-10), 28.5 (C-20), 32.8 (C-2), 45.6 (C-13), 46.8 (C-15), 51.0 (C-7), 67.0 (C-12), 72.0 (C-6), 77.3 (C-8), 79.7 (C-17), 81.7 (C-14), 81.8 (C-18), 124.8 (C-4), 156.3 (C-3), 168.3(C-11), 198.5 (C-5), 202.7 (C-19), 206.4 (C-16), 206.6 (C-9).

[0049] 1 H-NMR spectrum ( Figure 1 The results show that compound 1 has two bimodal methyl proton δ peaks. H 1.09 (3H, d, J = 7.3Hz), δ H 1.21 (3H, d, J = 7.0 Hz); 2 singlet methyl proton δ H 2.13 (3H, s), δ H 2.36 (3H,s); 5 unsaturated methine or methylene protons, namely δ H 2.63 (1H, m), δ H 2.64 (1H, d, J = 3.3Hz), δ H 2.80 (1H, d, J = 16.8 Hz), δ H 3.04 (1H, m), δ H 3.06 (1H, m); 7 methine protons or active hydrogen protons, namely δ H 4.10 (1H, d, J = 10.0 Hz), δ H 4.40 (1H, dd, J = 2.1, 3.2Hz), δ H 4.46 (1H, s), δ H 4.56 (1H, s), δ H 5.05 (1H, d, J = 3.7 Hz), δ H 5.26 (1H,d, J = 3.4 Hz), δ H 5.72 (1H, s); 2 olefin protons δ H6.01 (1H, dd, J = 2.9,10.0 Hz), δ H 6.85 (1H, dd, J = 2.2, 10.1 Hz). 13 C10 DEPT-NMR spectrum ( Figure 2 The results show that compound 1 contains one aprotic quaternary carbon signal, four methyl groups, four carbonyl groups, one ester group, two sp² hybridized methine groups, three sp³ hybridized methine groups, five oxygen-substituted methine groups, and one methylene group. Compound 1 has an unsaturation degree of 9 and, in addition to the four carbonyl groups, one double bond, and one ester group, should also have three rings.

[0050] HSQC spectra show that δ H 4.56 (1H, d, J = 3.2 Hz), δ H 5.26 (1H, d, J = 3.4 Hz) The hydrogen proton is active hydrogen, meaning this compound contains two hydroxyl groups. According to... 1 H-NMR, 13 C-NMR and HSQC spectra were used to determine δ H 2.64 (1H, d, J = 3.3 Hz), δ H 2.80 (1H, d, J = 16.8 Hz) represents the methylene proton. 1 H-NMR spectra show that the methyl proton H3-1 (δ H 1.09, d, J = 7.3 Hz) and H3-21 (δ H The splitting of the peak at 1.21 (d, J = 7.0 Hz) into a d peak indicates that the two methyl groups reacted with two methine groups (H-2, δ) respectively. H 3.06 and H-13, δ H 3.04) Connection; H-4 (δ) H 6.01) and H-3 (δ) H The coupling constant (6.85) is 10.0 Hz, indicating the presence of a cis-1,2-disubstituted double bond in the compound's structure. According to... 1 H- 1 The H COSY plot clearly shows the linear coupling relationships between H3-1 / H-2, H-3 / H-4, and 6-OH / H-6 / H-7 / H-8. Although H-2 / H-3 is missing... 1 H- 1 H COSY related points, but methyl proton H3-1 (δ H 1.09) and tertiary carbon C-2 (δ C 32.8), unsaturated tertiary carbon C-3 (δ C156.3), tertiary carbon C-7 (δ C 51.0) has an HMBC correlation point, indicating that C-2 and C-3 are connected. Additionally, the HMBC spectrum shows that H-3 (δ) H 6.85) and carbonyl carbon C-5 (δ C 198.5), H-4 (δ H 6.01) and tertiary oxygen carbon C-6 (δ C 72.0) The presence of HMBC correlation indicates that compound 1 contains a 5-substituted 4-methyl-6-hydroxy-α,β-unsaturated cyclohexenone structural unit. H-10 (δ H 2.36) with carbonyl carbon C-9 (δ C 206.6), methyl carbon C-8 (δ C 77.3) It has HMBC relevance, which proves that the side chain α-acetoxypropionate group (C-8 (–C-11) –C-9–C-10) is a structural unit. 1 H- 1 The H COSY plot shows three spin systems: H3-21 / H-13, H-14 / H-15, and 17-OH / H-17. H-13 / H-14 is also missing. 1 H- 1 HCOSY related points, but methyl proton H-21 (δ H 1.21) and tertiary carbon C-13 (δ C 45.6), Tertiary oxygen carbon C-14 (δ C 81.7) has a correlation point, indicating that C-13 and C-14 are connected. In the HMBC spectrum, H-14, H-15 are related to the carbonyl carbon C-16 (δ). C 206.4), H-17 (δ) H 5.05) and C-16, saturated quaternary carbon C-12 (δ C 67.0), C-13 (δ) C 45.6), C-11 (δ) C 168.3) and tertiary oxygen carbon C-18 (δ C 81.8) shows an HMBC correlation, indicating the presence of a 3,3-disubstituted 2-hydroxy-4-methylcyclohexane-1-one structural unit. Furthermore, H3-20 (δ) H 2.13) with carbonyl carbon C-19 (δ C 202.7), tertiary oxygen carbon C-18 has HMBC correlation points, and the structural unit is C-20–C-19–C-18; H-14 (δ H4.40) The presence of an HMBC correlation point with C-18 indicates that C-14 is linked to C-18 via a C–O–C bond. The formation of this bond results in 3,3-disubstituted 2-hydroxy-4-methylcyclohexanone sharing C-18 and C-14 with the oxacycle to form a 6-oxabicyclic [3.2.1]octane skeleton. H-8 (δ H 5.72) shows an HMBC correlation with the ester group carbon C-11, indicating that the cyclohexenone moiety and the bicyclic [3.2.1]octane moiety are linked by COC to form a dimer structure. Based on the above analysis, the planar structure of compound 1 was determined. This structure was not found in SciFinder and was identified as a new compound with a specific rotation of -14.48.

[0051] NOESY spectra show that H-13 and H-15β (δ H 2.80) is correlated, but not with H-15α (δ H 2.64) There is no correlation, indicating that H-13 and H-15β are located on the same side of the cyclohexanone plane, and H-15α is located on opposite sides of the cyclohexanone plane.

[0052] Compound 1 has nine chiral carbon atoms: C-12, C-13, C-14, C-17, C-18, C-2, C-6, C-7, and C-8. Its relative and absolute configuration cannot be determined solely by NMR spectroscopy. Colorless single crystals were obtained by culturing in acetone, and X-ray Cu target analysis revealed the spatial structure of compound 1, as shown below. Figure 3 As shown, the absolute configurations of its nine chiral carbon atoms are 2R, 6R, 7S, 8S, 12S, 13S, 14R, 17R, and 18S. This compound is a complex polyketide molecule composed of a propyl backbone, a cyclohexenone ring, and a bicyclic carboxylic acid ester group containing a 6-oxabicyclo[3.2.1]octane skeleton. It also contains multiple chiral centers and functional groups, and is named amorocoelone A.

[0053] amorocoelone C (2)

[0054] Compound 2 is a colorless crystal with the molecular formula C. 10 H 14 O5. HRESI-MS m / z 259.0824 [M+COOH] – (calcd for C 11 H 15 O 7, 259.0823), with an unsaturation degree of 4. , MeOH), IR (KBr)ν max3410, 2934, 2648, 2537, 1731, 1418, 1310, 1203, 1099, ; CD (c0.05, MeOH) λ max (Δε) 219 (–10.13), 354 (–0.04), 323 (+1.26), 333 (+1.21) nm. 1 H NMR (600 MHz, acetone-d6): δ 1.16 (3H, d, J = 7.4 Hz, H-1), 1.20 (3H, s,H-10), 2.15 (1H, m, H-2), 2.65 (1H, t, J = 2.8 Hz, H-7), 2.78 (1H, d, J = 3.2Hz, H-4), 4.07 (1H, dd, J = 0.9, 3.3 Hz, H-6), 4.22 (1H, td, J = 0.6, 3.4 Hz,H-3); 13 C NMR (150 MHz, acetone-d6): 18.4 (C-1), 23.8 (C-10), 39.8 (C-2), 58.2(C-7), 65.1 (C-4), 70.5 (C-3), 71.3 (C-9), 73.6 (C-6), 208.9 (C-5), 211.8 (C-8).

[0055] 1 H-NMR spectrum ( Figure 4 The results show that compound 2 contains two methyl protons δ. H 1.16 (3H, d, J = 7.4 Hz), δ H 1.20 (3H, s), 3 methine protons δ H 2.15 (1H, m), δ H 2.65 (1H, t, J = 2.8 Hz), δ H 2.78 (1H, d, J = 3.2 Hz), 2 δ-oxomethyl protons H 4.07 (1H, dd, J = 0.9, 3.3 Hz),δ H 4.22 (1H, td, J = 0.6, 3.4 Hz). 13 C10 DEPT-NMR spectrum ( Figure 5The data shows that compound 2 has one saturated quaternary carbon, two methyl groups, two carbonyl groups, one methine group, two methine groups with strongly electron-withdrawing groups, and two methine groups with moderately electron-withdrawing groups. Compound 2 has an unsaturation degree of 4 and, in addition to containing two carbonyl groups, also has two rings. According to... 1 H- 1 The H COSY spectrum clearly shows the linear coupling relationship between H3-1 / H-2 / H-3 / H-4. Further analysis of the HMBC correlation signal indicates that H3-1 (δ H 1.16) and C-7(δ) C 58.2), H-7 (δ) H 2.65) and C-2 (δ C 39.8), C-3 (δ) C 70.5), C-8 (δ) C 211.8), C-9 (δ) C 71.3), H3-10 (δ) H 1.20) and C-9, C-4 (δ) C 65.1), C-8, H-4 (δ) H 2.78) exhibits long-range coupling with C-9, C-10, C-8, C-3, and C-2, confirming the presence of a 2,5-dimethylcyclohexane-1-one structural unit in the molecule. Furthermore, the coupling signals between H-6 and H-7 in the ¹H-¹HCOSY spectrum, and between H-4 and C-5 (δ) in the HMBC spectrum... C 208.9), C-6 (δ) C 73.6), H-7 has a long-range correlation with C-5 and C-6, leading to the deduction of the planar structure of this compound. The core skeleton of this compound is a bicyclic [2.2.2]octane, with three bridgehead carbon atoms connected by three two-carbon bridges, forming a bicyclic bridged structure derived from cyclohexanone. Its structure was not found in SciFinder, thus identifying it as a new compound.

[0056] Because compound 2 contains six chiral carbon atoms (C-2, C-3, C-4, C-6, C-7, C-9), its relative and absolute configuration cannot be determined by NMR alone. Colorless crystals were obtained by culturing in a petroleum ether-acetone system, and X-ray Cu target analysis was performed. The crystal structure is shown below. Figure 6 As shown, the absolute configurations of the nine chiral carbon atoms in compound 2 are 2S, 3R, 4R, 6R, 7R, and 9S. This compound is named amorocoelone C.

[0057] amorocoelone H (7)

[0058] Compound 7 is a yellow oily substance with the molecular formula C. 10 H14 O4. HRESI-MS m / z 199.0968 [M+H] + (calcd for C 10 H 15 O4 (199.0965), with an unsaturation degree of 4. , MeOH); UV(MeOH) λ max (log ε): 228 (26.03) nm; IR (KBr) ν max 3445, 2977, 2936, 2879, 1716, 1686, 1265, ; CD (c 0.06, MeOH) λ max (Δε) 223 (–13.03), 320(+1.25) nm; 1 H NMR (500 MHz, CD3OD): δ 1.06 (3H, d, J = 7.4 Hz, H-1), 1.35 (3H,d, J = 7.0 Hz, H-10), 3.12 (1H, m, H-2), 3.69 (1H, dd, J = 5.5, 12.4 Hz, H-7), 4.34 (1H, d, J = 7.0 Hz, H-9), 4.62 (1H, d, J = 12.4 Hz, H-6), 5.97 (1H,dd, J = 1.0, 10.0 Hz, H-4), 7.02 (1H, dd, J = 5.6, 10.0 Hz, H-3); 13 C NMR (150MHz, CD3OD): 13.9 (C-1), 18.1 (C-10), 32.7 (C-2), 52.3 (C-7), 68.3 (C-6), 72.4 (C-9), 124.8 (C-4), 154.0 (C-3), 200.1 (C-5), 210.6 (C-8).

[0059] according to 1 H-NMR ( Figure 7 ), 13 C DEPT-NMR ( Figure 8 ) and HSQC NMR spectra, compound 7 contains 2 methyl groups (δ) H 1.06, d, J = 7.4 Hz, δ C 13.9; δ H 1.35, d, J = 7.0 Hz, δC 18.1), 2 carbonyl groups (δ C 200.1, δ C 210.6), 1 cis-1,2-disubstituted olefin (δ H 7.02, dd, J = 5.6, 10.0 Hz, δ C 154.0; δ H 5.97, dd, J = 1.0, 10.0 Hz, δ C 124.8), 2 methines (δ H 3.12, δ C 32.7; δ H 3.69, dd, J = 5.5, 12.4 Hz, δ C 52.3), 2 hydroxymethyl groups (δ H 4.63, d, J = 12.4 Hz, δ C 68.3; δ H 4.34, d, δ C 72.4). Compound 7 has four degrees of unsaturation, and in addition to two carbonyl groups and one double bond, it also has one ring. Compound 7's... 1 H-NMR and 13 The C-NMR spectrum of compound 7 is very similar to that of the known compound arthropsadiol C. Furthermore, TLC spotting (using dichloromethane and acetone as developing solvents in a 9:1 ratio) shows that the Rf value of this compound is slightly larger than that of arthropsadiol C. Therefore, compound 7 is identified as an isomer of arthropsadiol C.

[0060] The planar structure of compound 7 was further confirmed by 2D NMR spectra. 1 H- 1 According to the H COSY spectrum, coupling exists between H3-1 / H-2 / H-3 / H-4, H-2 / H-7 / H-6 / 6-OH, and H-9 / H3-10; according to the HMBC spectrum, H-4 (δ H 5.97) and C-2(δ C 32.7), C-6 (δ) C 68.3), H-6 (δ) H 4.63) and C-2, C-7 (δ) C 52.3), C-5 (δ) C 200.1), H-7(δ) H 3.69) and C-1 (δ C 13.9), C-2, C-6, C-5, C-8 (δC 210.6) A relevant point exists, suggesting that compound 7 contains a 5-substituted 6-hydroxy-4-methyl-α,β-unsaturated cyclohexenone. Furthermore, H-6, H-7, and H-9 (δ) H 4.34), H3-10 (δ) H 1.35) shows an HMBC correlation with the carbonyl carbon C-8, suggesting a hydroxypropionyl side chain linked to C-5. This analysis further confirms that compound 7 has the exact same planar structure as arthropsadiol C.

[0061] Compound 7 has four chiral carbon atoms: C-2, C-6, C-7, and C-9. According to the NOESY spectrum, H-6 is correlated with H3-1 but not with H-2, indicating that H-6 is on the same side as H3-1 and in the opposite direction to H-2. 1 In the H-NMR spectrum, H-7(δ H 3.69) The split peaks are dd, with coupling constants of 5.5 and 12.4 Hz. They show a NOE correlation with H-2 and a weak NOE correlation with H-6, indicating that H-7 is on the same side as H-2 and in the opposite direction to H-6. H-6 and H-7 show a weak NOE correlation, and their coupling constant is 12.4 Hz, indicating that H-6 and H-7 are in opposite directions. Since the relative configuration of C-9 could not be determined by NMR spectra, the specific rotation of this compound was further measured, and the specific rotation was -159.07. Furthermore, to determine the absolute configuration of the compound, its CD value was measured, and ECD quantum chemical calculations were performed. The ECD spectrum showed that compound 7 exhibits a negative "Cotton" effect at 223 nm (CD value -13.03) and a positive "Cotton" effect at 320 nm (CD value 1.25). ECD calculations were performed on two possible structures of compound 7: (2R, 6S, 7R, 9S)-7a and (2R, 6S, 7R, 9R)-7b. The ECD spectra ( Figure 9 As can be seen, the ECD curve of compound 7 fits well with those of 7a and 7b, confirming the absolute configurations of C-2, C-6, and C-7 as 2R, 6S, and 7R, respectively. However, due to the very similarity of the ECD curves of 7a and 7b, the absolute configuration of C-9 cannot be accurately determined. Therefore, DP4+ probability analysis was performed on 7a and 7b.

[0062] NMR calculations were performed using Gaussian 09 software for two possible configurations: (2R, 6S, 7R, 9S)-7a (isomer 1) and (2R, 6S, 7R, 9R)-7b (isomer 2). Furthermore, DP4+ analysis indicated that the absolute configuration of compound 13 is (2R, 6S, 7R, 9R)-7b (isomer 2), with a probability of [insert probability here]. 13 C DP4+: 66.70%, 1 H DP4+: 82.45%, 13 C+ 1 H DP4+: 90.40%. DP4+ calculation results are as follows: Figure 10 As shown. SciFinder did not find its structure, identifying it as a new compound, and named it amorocoelone H.

[0063] amorocoelone I (8)

[0064] Compound 8 is a colorless oil with the molecular formula C. 10 H 14 O4. HRESI-MS m / z 221.0784 [M+Na] + (calcd for C 10 H 14 O4Na (221.0790), with an unsaturation degree of 4. , MeOH); UV(MeOH) λ max (log ε): 227 (23.79) nm; IR (KBr) ν max 3446, 2971, 2881, 1711, 1686, 1359, ; CD (c 0.05, MeOH) λ max (Δε) 225 (–23.91), 317 (–3.57), 270 (+1.03) nm. 1H NMR (600 MHz, CDCl3): δ 1.34 (3H, d, J = 7.3 Hz, H-1), 2.25 (3H, d, J = 2.3 Hz, H-10), 2.32 (1H, m, H-7), 2.99 (1H, m, H-2), 3.60 (1H, d, J = 2.1 Hz, 6-OH), 4.08 (1H, d, J = 3.4 Hz, 8-OH), 4.23 (1H, s ,H-8), 4.33 (1H, dd, J = 1.9, 12.3 Hz, H-6), 6.08 (1H, dd, J = 3.0, 10.0 Hz,H-4), 6.84 (1H, dd, J = 2.1, 10.0 Hz, H-3); 13 C NMR (150 MHz, CDCl3): 18.3 (C-1), 25.1 (C-10), 33.3 (C-2), 53.3 (C-7), 71.1 (C-6), 74.9 (C-8), 124.8 (C-4), 156.8 (C-3), 200.3 (C-5), 208.0 (C-9).

[0065] according to 1 H-NMR ( Figure 11 ), 13 C DEPT-NMR ( Figure 12 The compound contains two methyl groups (δ¹²) and HSQC NMR spectra. H 1.34, d, J = 7.3 Hz, δ C 18.3; δ H 2.25, d, J = 2.3 Hz, δ C 25.1), 2 carbonyl groups (δ) C 200.3, δ C 208.0), 1 cis-1,2-disubstituted olefin (δ H 6.84, dd, J = 2.1, 10.0 Hz, δ C 156.8; δ H 6.08, dd, J = 3.0, 10.0 Hz, δ C 124.8), 2 methines (δ) H 2.99, δ C 33.3; δ H 2.32, m, δ C53.3), 2 hydroxymethyl groups (δ) H 4.33, dd, J = 1.9, 12.3 Hz, δ C 71.1; δ H 4.23, s, δ C 74.9) and 2 hydroxyl groups (δ) H 3.60, d, J = 2.1 Hz; δ H 4.08, d, J = 3.4 Hz). Compound 8 should have 4 degrees of unsaturation, 2 carbonyl groups, 1 double bond, and 1 ring. Compound 8 is similar to compound arthropsadiol D. 1 H and 13 The C-shifts of compound 8 show a high degree of similarity, and they share the same molecular formula. Further comparative analysis revealed that, compared to compound arthropsadiol D, compound 8 exhibits a 3.52 ppm shift in the C-9 chemical shift towards the higher field and a 1.87 ppm shift in the C-2 chemical shift towards the lower field. Furthermore, TLC (chloroform:acetone = 95:5) comparisons showed that compound 8 has a slightly larger Rf value than compound arthropsadiol D, thus identifying this compound as an isomer of arthropsadiol D.

[0066] The planar structure of compound 8 was further confirmed by 2D NMR spectra. 1 H- 1 In the H COSY spectrum, H3-1 / H-2 / H-3 / H-4, H-2 / H-7 / H-6, H-7 / H-8, 6-OH (δ H 3.60) / H-6 are correlated; and H-3 (δ) H 6.84) and C-1(δ) C 18.3), C-2 (δ) C 33.3), C-7 (δ) C 53.3), C-5 (δ) C 200.3), H-4 (δ) H 6.08) and C-2, C-6 (δ) C 71.1), H-6 (δ) H 4.33) shows a correlation with C-2, C-7, and C-5, indicating the presence of a 5-substituted 4-methyl-6-hydroxy-α,β-unsaturated cyclohexenone in the structure of compound 8. Furthermore, H-7 (δ... H 2.32) / H-8 (δ H 4.23) / 8-OH (δ H 4.08) exists1 H- 1 H COSY related points, H3-10 (δ) H 2.25) and C-9, C-8 (δ) C 74.9) The presence of HMBC correlation allowed for the identification of the hydroxypropionyl side chain structural unit in compound 8. The above analysis confirmed that compound 8 shares the same planar structure as compound arthropsadiol D.

[0067] This compound has four chiral carbon atoms: C-2, C-6, C-7, and C-8. In the NOESY spectrum, H-6 correlates with H-2 but not with H3-1, indicating that H-6 is located on the same side as H-2 and in the opposite direction to H3-1. 1 The H-NMR spectrum shows that H-6 (δ) H 4.33) The split peaks are dd peaks with coupling constants of 1.9 and 12.3 Hz. They show a NOE correlation with 6-OH but not with H-7, indicating that H-6 and H-7 are located in opposite directions. H-7 shows a NOE correlation with H-1 but not with H-2, indicating that H-7 is located in opposite directions to H-1 and on the same side as H-2.

[0068] Since the relative configuration of C-8 could not be determined by NMR spectra, the specific rotation of the compound was further measured, and the specific rotation was -40.48. In addition, the CD value of the compound was measured, and ECD calculations were performed. The ECD spectrum showed that compound 8 exhibited a negative "Cotton" effect at 225 nm and 317 nm, with CD values ​​of -23.91 and -3.57, respectively; and a positive "Cotton" effect at 270 nm, with a CD value of 1.03. ECD quantum chemical calculations were performed on the two possible structures of the compound, (2R, 6R, 7R, 8S)-8a and (2R, 6R, 7R, 8R)-8b. The results were obtained from the ECD spectra (…). Figure 13 As can be seen, the experimental ECD curve of compound 8 fits well with the calculated values ​​of 8a and 8b. However, because the chiral center of C-8 contributes very little to the ECD, the ECD curves of 8a and 8b are very similar. Therefore, we can only determine the absolute configurations of C-2, C-6, and C-7 as 2R, 6R, and 7R, but not the absolute configuration of C-8. We have isolated compound arthropsadiol D, whose configuration is 2R, 6R, 7R, 8R. Since arthropsadiol D has different Rf values ​​and significant differences in specific rotation compared to compound 8, we speculate that the absolute configuration of compound 8 is 2R, 6R, 7R, 8S.

[0069] To further determine the absolute configuration of the chiral carbon atom C-8 in compound 8, NMR calculations were performed on two possible configurations, (2R, 6R, 7R, 8S)-8a (isomer 1) and (2R, 6R, 7R, 8R)-8b (isomer 2), using the DP4+ method. DP4+ analysis showed that the experimental chemical shift of compound 8 is consistent with the calculated chemical shift of (2R, 6R, 7R, 8S)-11a (isomer 1), with a probability of [missing information]. 13 C DP4+: 99.94%, 1 H DP4+: 98.50%, 13 C+ 1 H DP4+: 100.0%. DP4+ calculation results are as follows: Figure 14 As shown above, the absolute configuration of compound 8 is 2R, 6R, 7R, 8S, which also confirms the above speculation. SciFinder did not find its structure, thus identifying it as a new compound, and named it amorocoelone I.

[0070] Example 3

[0071] Assay of anti-influenza virus activity of compounds 1-10

[0072] (1) The antiviral activity of compounds 1-10 was assessed using the cytopathic effect (CPE) assay. MDCK cells were collected and counted, and 20 ml of 10 was prepared with 2% DMEM. 5 / ml cells, MDCK cells at 2×10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and incubated at 37°C for 24 h in 5% CO2 before infection. The test compound was then seeded at a serially diluted two-fold concentration with influenza A virus (100 TCID50). 50 The mixture was prepared and incubated at 37°C for 30 min. The mixture was then transferred to cells in 96-well plates and incubated for another 30 min. After incubation, MDCK cells were washed twice with PBS to remove unabsorbed virus, and then DMEM supplemented with 1 μg / ml TPCK-trypsin and 0.2% BSA was added. Antiviral activity was observed under a microscope after 48 h of incubation. Data were further confirmed using MTT assay. The experiment was repeated at least three times, with arbidol hydrochloride used as a positive control.

[0073] (2) The toxicity of compounds 1-10 to MDCK cells was detected by the MTT assay.

[0074] With a concentration of 1×10 4MDCK cells were grown in 96-well plates for 24 h, then twice-diluted test compounds were added to the cells, followed by incubation at 37°C in humidified 5% CO2 for 48 h. Cell viability was assessed by MTT assay after incubation. 100 μl of 0.5 mg / ml MTT in DMEM was added to each well and incubated at 37°C for an additional 4 h. Subsequently, 150 μl / well of DMSO was added to the plate to extract the heat-treated MTT, and the absorbance of the extract at 570 nm was measured on a microtiter plate reader. The cytotoxicity of the test compound was determined by comparing cell viability with that of cells treated with the test compound versus those treated with a solvent (0.3% methanol).

[0075] Experimental results show that cyclohexanone compounds 1-10 have an IC50 of 1-10 against H1N1 influenza virus. 50 Value (half-maximal inhibitory concentration) and CC 50 value (Hydrotoxic concentration) is shown in Table 1. IC50 of compounds 1-10 50 Values ​​compared to the positive control drug Arbidol (IC50) 50 The values ​​were comparable to those of compounds 1-6 and 8-10 (0.44 ± 0.13 μg / ml), and the selectivity indices (SI) of compounds 1-6 and 8-10 were significantly higher than those of the positive control drug Arbidol (SI 0.44 ± 0.13 μg / ml). = 80.5), showing the characteristics of high efficiency and low toxicity, and has the potential use as a new anti-influenza virus drug.

[0076] Table 1. Inhibitory effects of compounds 1-10 on H1N1 influenza A virus and their toxicity to MDCK cells.

[0077]

[0078] a IC 50 The half-maximal inhibitory concentration (IC50) is the concentration measured using IC50. 50 (MD) means: ;

[0079] b CC 50 The median cytotoxic concentration (50%) is measured in CC. 50 (MD) indicates; c SI refers to the Selectivity Index, and its value is CC. 50 / IC 50 .

[0080] In summary, ten cyclohexanone compounds 1-10 are provided. The compounds described in this invention have good anti-influenza virus activity, providing new options for the development of anti-influenza virus drugs.

[0081] The compound can be prepared by microbial fermentation. This method has the advantages of short cycle, mild culture conditions, few by-products, strong stereoselectivity and low cost. It also has high economic value. The low cost, simple operation and easy large-scale production provide a new way to obtain polyketide compounds containing cyclohexanone structural units from natural sources.

[0082] The plant endophytic fungus used was Amorocoelophoma sp. YE3351, which was deposited on August 7, 2020, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO.M2020407; its taxonomic name is Amorocoelophoma sp.

[0083] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0084] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. Cyclohexanone polyketide compounds, characterized in that: The ten cyclohexanone compounds include: amorocoelone A (1), amorocoelone C (2), (4R,5S,6R,9R,10R)-4,9,10-trihydroxy-6-methyl-3-methylene-2-oxaspiro[4.5]dec-7-en-1-one (3), massarilactone A (4), massarilactone C (5), massarilactone E (6), amorocoelone H (7), amorocoelone I (8), (R)-1-hydroxy-1-(5-hydroxy-2-methylphenyl)propan-2-one (9), and paraverrucsin A (10), of which compounds (1), (2), (7) and (8) are new compounds.

2. A method for preparing cyclohexanone polyketide compounds 1-10 as described in claim 1, characterized in that: Includes the following steps: Step 1: Ferment the fungus Amorocoelophoma sp. and collect the fermentation broth; Step 2: Extract the fermentation broth with an organic solvent, combine the extracts, and concentrate under reduced pressure to obtain the crude fermentation extract; Step 3: Separate the crude fermentation extract by silica gel column chromatography, elute with chloroform-methanol system, and collect the crude fraction; Step 4: Elute the obtained crude component with a petroleum ether-ethyl acetate, petroleum ether-acetone, chloroform-methanol, chloroform-acetone, ethyl acetate-acetone, or methanol-water system, or recrystallize it to obtain the final product.

3. The preparation method according to claim 2, characterized in that, The fungus described is Amorocoelophoma sp. YE3351, which was deposited at the China Center for Type Culture Collection on August 7, 2020, with accession number CCTCC NO.M 2020407.

4. The preparation method according to claim 2, characterized in that, The fermentation medium used was SDB medium, in which the concentration of peptone was 0.01 g / mL and the concentration of glucose was 0.04 g / mL.

5. The preparation method according to claim 2, characterized in that, The fermentation broth was extracted with ethyl acetate.

6. The preparation method according to claim 2, characterized in that, Gradient elution was performed using a chloroform-methanol system from a volume ratio of 1:0 to 1:1, and the crude fractions obtained from elution at different volume ratios of the chloroform-methanol system were collected as Frs. 1–9.

7. The preparation method according to claim 2, characterized in that, The crude fraction Fr. 2 was eluted by normal-phase silica gel column chromatography with petroleum ether-acetone at a ratio of 9:1 to 1:1 to give Frs. 2.1–2.

5. Fr. 2.2 was recrystallized to give compound (6). Fr. 2.4 was eluted by normal-phase silica gel column chromatography with petroleum ether-ethyl acetate at a ratio of 4:1 to 3:2 to give compounds (1) and (8). The crude fraction Fr. 3 was eluted by normal-phase silica gel column chromatography with chloroform-acetone at a ratio of 9:1 to 1:1 to give Frs. 3.1–3.

6. Fr. 3.5 was eluted by normal-phase silica gel column chromatography with petroleum ether-ethyl acetate at a ratio of 9:1 to 2:3 to give compounds (4) and (5). Fr. 3.6 was eluted by RP-18 reversed-phase silica gel column chromatography with methanol-water at a ratio of 1:4, 3:7, 2:3 to give compound (7). Compound (9) was obtained by normal-phase silica gel column chromatography with petroleum ether-ethyl acetate gradient elution from 9:1 to 1:

1. The crude fraction Fr. 6 was obtained by normal-phase silica gel column chromatography with ethyl acetate-acetone gradient elution from 9:1 to 2:3 to Frs. 6.1–6.

4. Fr. 6.3 was obtained by RP-18 reversed-phase silica gel column chromatography with methanol-water gradient elution from 1:1, 3:2, and 7:3 to Frs. 6.4 was obtained by normal-phase silica gel column chromatography with chloroform-acetone gradient elution from 4:1 and 1:1 to Frs. 6.

4. The crude fraction Fr. 7 was obtained by normal-phase silica gel column chromatography with chloroform-methanol gradient elution from 5:1 and 4:1 to Frs. 6.

4.

8. The application of a cyclohexanone polyketide compound as described in claims 1-7 and its preparation method in the preparation of anti-influenza virus drugs.

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