A pyrone derivative and its preparation method and application

Abstract

The present invention relates to a pyrone derivative, its preparation method, and application. The pyrone derivative has the structure shown in Compound 1: #imgabs0# The present invention is the first to isolate a pyrone derivative with novel structure and significant anti-bollworm and anti-nematode activity from the fungus Fusariumsolani sp. QJS4-1-2, derived from Kandelia candel. The derivative is expected to be developed as an insecticide.

Description

Technical Field

[0001] The present invention belongs to the field of marine fungal secondary metabolites, and particularly relates to a pyrone derivative and a preparation method and application thereof. Background Art

[0002] Kandelia candela, a plant of the genus Kandel in the rhizophoraceae family, is a folk medicinal plant. Its bark has hemostatic and antibacterial properties, and an ethanol extract of its roots can treat rheumatoid arthritis. Chemical components isolated from Kandelia candela exhibit pharmacological activities such as antitumor, antioxidant, antibacterial, and hypoglycemic activity. Kandelia candela fungal extracts have been shown to play an important role in the biological control of diseases. A series of novel compounds, including lactones, cyclic peptides, and polyketides, have been isolated from fungal secondary metabolites, exhibiting multiple biological activities such as cytotoxicity, antibacterial activity, and anti-inflammatory activity. The inventors previously isolated a series of active secondary metabolites from the fungus Fusarium solani sp. QJS4-1-2, derived from the mangrove plant Kandelia (Chinese Journal of Marine Drugs, Vol. 43, No. 2, pp. 35-40). Further research on this fungus revealed a novel pyrone derivative with significant anti-insect activity. Summary of the Invention

[0003] The present invention provides a pyrone derivative or a pharmaceutically acceptable salt thereof, characterized in that the pyrone derivative has the structure shown in Compound 1:

[0004]

[0005] Another embodiment of the present invention provides a method for preparing the above-mentioned pyrone derivative, characterized in that it comprises the following steps:

[0006] (1) Prepare seed culture medium, inoculate QJS4-1-2 strain into the seed culture medium, and culture at 28°C for 4 days to obtain seed culture solution;

[0007] (2) inoculating the seed culture solution obtained in step (1) into a fermentation medium, and culturing at room temperature for 35 days to obtain a fermentation product;

[0008] (3) separating the fermentation broth and the bacterial cells from the fermented product obtained in step (2), extracting the fermentation broth and the bacterial cells 2 to 4 times with equal volumes of ethyl acetate, combining the extracts and concentrating under reduced pressure to obtain an extract;

[0009] (4) The extract obtained in step (3) was subjected to vacuum silica gel column chromatography, using petroleum ether-ethyl acetate as eluent for gradient elution, the elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, 0:100, and two column volumes were collected for each gradient. The eluates obtained with the gradients of petroleum ether:ethyl acetate of 80:20 and 70:30 were combined and concentrated, and then chromatographed on a Sephadex LH-20 column with an eluent of chloroform:methanol = 1:1, and then prepared by high performance liquid chromatography HPLC, the chromatographic column was Agilent C18, 9.4×250 mm, 7 μm, the flow rate was 2 mL / min, and the mobile phase was CH3CN:H2O = 35:65, to finally obtain compound 1, i.e., the pyrone derivative.

[0010] The ratio of the eluent or mobile phase is by volume; the seed culture medium is potato dextrose water culture medium (PDB culture medium); the fermentation culture medium is rice solid culture medium (the formula is: each 1L conical flask contains 50g rice, 60g water, 0.5g sea salt and 1.5g peptone); the seed culture medium and fermentation culture medium are both sterilized at 120°C for 25-30 minutes.

[0011] Another embodiment of the present invention provides the use of the above-mentioned compound 1 or a pharmaceutically acceptable salt thereof in the preparation of an anti-nematode and / or anti-bollworm drug.

[0012] The present invention provides a pharmaceutical composition, characterized in that it uses the above-mentioned compound 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[0013] The pharmaceutical composition provided by the present invention is characterized in that it further comprises a pharmaceutically acceptable excipient (preferably a pharmaceutically acceptable carrier, diluent or excipient). The dosage form of the pharmaceutical composition can be a solid preparation, a semi-solid preparation or a liquid preparation.

[0014] The "Kandelion-derived fungus Fusariumsolani sp. QJS4-1-2" described in the present invention, referred to as "QJS4-1-2", was collected by the inventors from the Dongzhaigang Mangrove Nature Reserve in Haikou City. The strain was identified as Fusariumsolani sp. through 18S rRNA amplification and ITS sequencing, based on the morphological characteristics of the fungus and molecular biological methods (ITS-rRNA sequence alignment). The sequence data has been uploaded to GenBank with the accession number OQ137026. QJS4-1-2 has been disclosed in the inventor's previous research paper "Study on the Secondary Metabolites of an Endophytic Fungus Fusariumsolani sp. and Its Antiinsect Activity, Chinese Journal of Marine Drugs, Vol. 43, No. 2, pp. 35-40", and has been preserved in the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education where the applicant is located. The public can obtain QJS4-1-2 according to the method described in the inventor's above-mentioned research paper, or purchase it from the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education where the applicant is located. The applicant promises that the Key Laboratory of Tropical Medicinal Resources Chemistry of the Ministry of Education will provide QJS4-1-2 to the public for 20 years from the date of application of this invention.

[0015] Compared with the prior art, the present invention has the advantages that: the present invention is the first to separate and obtain from QJS4-1-2 a pyrone derivative with a novel structure and significant anti-bollworm and anti-nematode activities. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is the mass spectrum of compound 1.

[0017] Figure 2 is compound 1 1 H NMR spectrum.

[0018] Figure 3 is compound 1 13 C NMR spectrum.

[0019] Figure 4 This is the DEPT diagram of compound 1.

[0020] Figure 5 is the HSQC pattern of compound 1.

[0021] Figure 6 is the HMBC pattern of compound 1.

[0022] Figure 7 is the COSY pattern of compound 1. DETAILED DESCRIPTION

[0023] In order to facilitate further understanding of the present invention, the following examples are provided to illustrate it in more detail. However, these examples are only for better understanding of the invention and are not intended to limit the scope or implementation principles of the present invention. The implementation methods of the present invention are not limited to the following.

[0024] Example 1

[0025] (1) Prepare seed culture medium. Inoculate the QJS4-1-2 strain into a conical flask containing potato dextrose water culture medium (PDB culture medium). Each 1L conical flask is filled with 300mL of PDB culture medium, and a total of 4 bottles of seed liquid are fermented. Place these 4 bottles of seed liquid in a 28°C constant temperature shaker for shaking growth and culture for 4 days (until the QJS4-1-2 strain grows fully in the PDB culture medium) to obtain seed culture liquid.

[0026] (2) The seed culture solution obtained in step (1) was inoculated into a rice solid culture medium (100 bottles, the formula being: each 1 L conical flask contains 50 g of rice, 60 g of water, 0.5 g of sea salt and 1.5 g of peptone), and the culture was allowed to stand at room temperature for 35 days to obtain a fermentation product.

[0027] (3) separating the fermentation broth and the bacterial cells from the fermentation product obtained in step (2), extracting the fermentation broth and the bacterial cells three times with equal volumes of ethyl acetate, combining the extracts and concentrating under reduced pressure to obtain an extract;

[0028] (4) The extract obtained in step (3) was subjected to vacuum silica gel column chromatography using petroleum ether-ethyl acetate as eluent for gradient elution. The elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Two column volumes were collected for each gradient. The eluates obtained with the gradients of petroleum ether:ethyl acetate of 80:20 and 70:30 were combined and concentrated, and then chromatographed on a Sephadex LH-20 column with an eluent of chloroform:methanol = 1:1. The product was then prepared by high performance liquid chromatography (HPLC) using an Agilent C18 column, 9.4×250 mm, 7 μm, a flow rate of 2 mL / min, and a mobile phase of CH3CN:H2O = 35:65, to obtain compound 1 (13.5 mg).

[0029]

[0030] Compound 1: 1H NMR(600MHz,Chloroform-d)δ7.75(d,J=5.6Hz,1H),7.15(dd,J=7.5,1.7Hz,1H),7.13(td,J=7.7,1.7Hz,1H),6. 88(dd,J=8.1,1.2Hz,1H),6.84(td,J=7.4,1.2Hz,1H),6.33(t,J=2.8Hz,1H),6.32(t,J=2.0Hz,2H),3.85(s,2H). 13 C NMR (150MHz, Chloroform-d) δ180.73,169.52,155.78,155.11,131.10,129.18,121.08,120.15,116.40,115.79,114.88,34.79.

[0031] Compound 1 1 H. 13 C NMR (600 MHz, 150 MHz, Chloroform-d) data

[0032] Position δ(C) δ(H) 2 155.78CH 7.75d(5.6) 3 114.88CH 6.32t(2.0) 4 180.73C 5 116.40CH 6.33t(2.8) 6 169.52C 7 <![CDATA[34.79CH2]]> 3.85s 8 121.08C 9 155.11C 10 131.10CH 7.15dd(7.5,1.7) 11 129.18CH 7.13td(7.7,1.7) 12 120.15CH 6.84td(7.4,1.2) 13 115.79CH 6.88dd(8.1,1.2)

[0033] Analysis: C 12 H 10 O3, high resolution mass spectrum is 203.0695. (C 12 H 11 O3 + , calculated value is 203.0703). Ω=1+n C -1 / 2n H =1+12-5=8.

[0034] Compound 4, yellow oil. HR-ESI-MS gave the compound molecular formula as C 12 H 10 O3(m / z203.0695[M+H] + ,calcd 203.0703), the unsaturation is 8. 1 H NMR spectrum showed that the compound had four aromatic hydrogens (10-H, δ H 7.15; 11-H,δ H 7.13; 12-H,δ H 6.84; 13-H, δ H 6.88), 3 alkenyl hydrogens (2-H, δ H 7.75; 3-H,δ H 6.32; 5-H,δ H 6.33) and two methylene hydrogens (7-H, δH 3.85). 13 CNMR spectrum can be used to determine that the compound has a keto carbonyl carbon (C4, δ C 180.73), 10 alkenyl carbons (C2, δ C 155.78; C3,δ C 114.88; C5,δ C 116.40; C6, δ C 169.52; C8, δ C 121.08; C9, δ C 155.11; C10, δ C 131.10;C11,δ C 1129.18; C12, δ C 120.15; C13, δ C 115.19) and one methylene carbon (C7,δ C 34.79). In the COSY spectrum, δ H 7.75 and δ H The correlation peak at 6.32 indicates the presence of a double bond containing two protons. The coupling constant of the other double bond proton (δ H 6.33, td, J = 2.8) suggests that the proton is meta-coupled to the previous double bond proton, combined with the chemical shift of the carbonyl carbon (C4, δ C 180.73), and it was preliminarily determined that the compound was a β-substituted-γ-pyrone derivative[1]. 1 H NMR spectrum low field region δ H The four hydrogen signals at 7.18-6.84 (two groups of dd peaks and two groups of td heavy peaks) indicate the presence of an ortho-disubstituted benzene ring in the molecule; and the COSY spectrum also confirms that these four protons form a coupling system. The HMBC spectrum shows that H-7 has related peaks with C3, C6, C8, C9, and C10, suggesting that the benzene ring is connected to the pyrone through a methylene group. After reviewing the literature, it was found that the NMR data of compound 1 is basically similar to that of compound aspergyllone. The main difference is that C9 in the aspergyllone molecule is an unsubstituted aromatic carbon, while C9 (δ C 155.11) is a quaternary carbon. Its chemical shift indicates that C9 is connected to a hydroxyl group. Finally, the structure of compound 4 was identified as 2-(9-hydroxy)benzyl-4H-pyran-4-one.

[0035] Example 2

[0036] The growth inhibition activity against cotton bollworm larvae was tested according to existing methods. Six grams of feed and cotton bollworm larvae of the same age were evenly distributed in a six-well plate. The test compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 1 mg / mL. A gradient of 200, 100, 50, 25, and 12.5 μL / well was added to the sample group, the negative control group, and azadirachtin in the positive control group. The experiment was repeated three times for an observation period of 7-14 days, during which the growth of the cotton bollworm larvae in the plate was continuously checked. The results are shown in the table below.

[0037]

[0038] Example 3 Determination of Nematode Activity Using the Immersion Method

[0039] Prepare a 100-120 nematode / mL aqueous solution using sterile water. Accurately weigh the test compound, dissolve it in an appropriate amount of DMF, and dilute to 5 mL with 0.5% Tween-80 solution. Use a pipette to pipette 1 mL of the aqueous solution into a 12-well plate. Simultaneously, pipette 1 mL of the prepared 100 μg / mL compound solution into the sample well to create a 50 μg / mL mixed test solution. Gently shake the solution to mix thoroughly and incubate at 28°C in the dark. Use avermectin as a positive control, and sterile water supplemented with equal amounts of DMF and Tween-80 as a blank control. Observe and record nematode mortality after 24, 48, and 72 hours. Identify nematode mortality using body posture: live nematodes are bent and able to swim, while dead nematodes are stiff and motionless. Acupuncture is also used for identification. Three replicates are performed for each concentration, and the experiment is repeated three times. Calculate nematode mortality and corrected mortality.

[0040]

[0041]

[0042]

Claims

1. A pyrone derivative or a pharmaceutically acceptable salt thereof, characterized in that The pyrone derivative has the structure shown in Compound 1: 。 2. The method for preparing the pyrone derivative according to claim 1, characterized in that The steps include: (1) Prepare seed culture medium, inoculate QJS4-1-2 strain into seed culture medium, culture at 28°C for 4 days to obtain seed culture solution; (2) The seed culture solution obtained in step (1) was inoculated into a fermentation medium, and the culture was allowed to stand at room temperature for 35 days to obtain a fermentation product; (3) separating the fermentation broth and the bacterial cells from the fermentation product obtained in step (2), extracting the fermentation broth and the bacterial cells 2 to 4 times with equal volumes of ethyl acetate, combining the extracts and concentrating under reduced pressure to obtain an extract; (4) The extract obtained in step (3) was subjected to vacuum silica gel column chromatography, using petroleum ether-ethyl acetate as the eluent for gradient elution, and the elution gradients were 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, respectively. Two column volumes were collected for each gradient, and the eluates obtained with the gradients of petroleum ether:ethyl acetate of 80:20 and 70:30 were combined and concentrated, and then chromatographed on a Sephadex LH-20 column with an eluent of chloroform:methanol = 1:1, and then prepared by high performance liquid chromatography HPLC, with an Agilent C18 column, 9.4×250 mm, 7 μm, a flow rate of 2 mL / min, and a mobile phase of CH3CN:H2O = 35:65, to finally obtain compound 1, i.e., the pyrone derivative.

3. The preparation method according to claim 2, characterized in that The seed culture medium is potato glucose water culture medium.

4. The preparation method according to any one of claims 2 to 3, characterized in that The fermentation medium is rice solid medium.

5. Use of the pyrone derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-nematode drug.

6. Use of the pyrone derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-cotton bollworm drug.

7. A pharmaceutical composition, characterized in that The pyrone derivative or a pharmaceutically acceptable salt thereof according to claim 1 is used as an active ingredient.

8. The pharmaceutical composition according to claim 7, characterized in that Also contains pharmaceutically acceptable excipients.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable excipient is selected from pharmaceutically acceptable carriers, diluents or excipients.

10. The pharmaceutical composition according to any one of claims 7 to 9, characterized in that The dosage form of the pharmaceutical composition is a solid preparation, a semisolid preparation or a liquid preparation.

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