CN113004237A - Spiro compound and preparation method and application thereof - Google Patents
Spiro compound and preparation method and application thereof Download PDFInfo
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- CN113004237A CN113004237A CN202110158957.9A CN202110158957A CN113004237A CN 113004237 A CN113004237 A CN 113004237A CN 202110158957 A CN202110158957 A CN 202110158957A CN 113004237 A CN113004237 A CN 113004237A
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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Abstract
The invention belongs to the technical field of biological medicines, and relates to a spiro compound, and a preparation method and application thereof. The invention provides a novel spiro compound amonocoelolide A, which can be prepared by a biological fermentation method, has short production period and is green and environment-friendly; meanwhile, the compound has anti-influenza virus activity, can be used for preparing antiviral drugs, and has extremely high medical and commercial values.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to a spiro compound, and a preparation method and application thereof.
Background
Spirocyclic compounds are polycyclic compounds in which two single rings share a common carbon atom. The spiro compound has a rigid structure and a stable structure, and the chiral ligand of the spiro compound has larger specific optical rotation, so the spiro compound has important application value in the fields of biology and medicine, and the source of the spiro compound at present mainly depends on a natural way.
Yunnan herba Clinopodii (Gmelina arborea) is half-fallen leaf arbor of Clinopodium of Verbenaceae. The traditional Chinese medicine is in a rare state at present, is mainly distributed in provinces of south China, Bengal and some countries of southeast Asia, has application reports on the aspects of treating chronic fever, bleeding, urinary tract infection, anuria and the like, and can also be used for treating fever, dyspepsia and the like. The Yunnan catalpa ovata has higher medicinal and economic values, but the research on the plant is mainly focused on the aspects of the biological activity and the chemical components of the extracts of leaves, flowers, fruits, stem barks and the like at present. The research report about the Yunnan shicatalpa endophytic fungi is not found at home and abroad.
The fungus Amorocoeloma genus is a genus newly established by Jayasiri et al in 2019, 2 months, and belongs to the family Amonosaceae of Ascomycota (Ascomycota) Ascomycota (dothideomyces) Ascomycetes (Pleosporales) Geospora (Pleosporales). The family Amorosiaceae was subdivided by Thambagala et al in 2015 from the family Pyrenophoraceae (Lophiostomataceae). At that time, the family Amoorosiaceae contained 2 genera Amoorosia and Angustimasharina.
Disclosure of Invention
The invention mainly aims to provide a spiro compound and a preparation method and application thereof. The invention provides a novel spiro compound amonocoelolide A, which can be prepared by a biological fermentation method, has short production period and is green and environment-friendly; meanwhile, the compound has anti-influenza virus activity, can be used for preparing antiviral drugs, and has extremely high medical and commercial values.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a spiro compound amonocoelolide A, which has a structural formula shown as the following formula (I):
the present invention also provides a process for the preparation of the above mentioned spiro compound amonocoelolide a, comprising the steps of:
fermenting fungus Amonocoelophoma sp, and respectively collecting fermentation liquor and mycelium;
extracting fermentation liquor and mycelia with organic solvent respectively, mixing extractive solutions, and concentrating under reduced pressure to obtain fermented crude extract;
performing chromatographic separation on the crude fermentation extract by using a silica gel column, eluting by using a petroleum ether-acetone system, and collecting a crude component;
eluting the crude component with petroleum ether-ethyl acetate system, and recrystallizing to obtain the final product.
Further, the fungus Amorocoelophoma sp.is Amorocoelophoma sp.YE3351, and the strain is preserved in China Center for Type Culture Collection (CCTCC) No. M2020407 in 8-7.2020.
Further, the culture medium used for fermentation is PDB culture medium, wherein the concentration of potato in the PDB culture medium is 0.2-0.4g/mL, and the concentration of glucose is 0.02-0.05 g/mL.
Further, the fermentation broth was extracted with ethyl acetate, and the mycelia were extracted with methanol.
Further, gradient elution is carried out by using a petroleum ether-acetone system from 4:1 to 1:1 in volume ratio, and crude components obtained by elution of the petroleum ether-acetone system from 7:3 in volume ratio are collected.
Further, the obtained crude component was eluted with petroleum ether and ethyl acetate at a volume ratio of 4: 1.
Further, the mixture was recrystallized from a mixed solvent of chloroform and methanol at a volume ratio of 5: 1.
The invention also provides application of the spiro compound amonocoelolide A in preparation of anti-influenza virus drugs.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a novel spiro compound amonocoelolide A, which has good anti-influenza virus activity and provides a new choice for developing anti-influenza virus medicaments.
The compound can be prepared by microbial fermentation, and the method has the advantages of short period, mild culture conditions, few byproducts, strong stereoselectivity and low cost, has higher economic value, low cost and simple and convenient operation, is easy for large-scale production, and provides a new way for obtaining the spiro lactone derivative from natural sources.
The endophytic fungus Amorocoelophoma sp.YE3351 used by the invention is preserved in China center for type culture Collection in 8.7.2020 at the address of China Wuhan university with the preservation number of CCTCC NO.M 2020407; taxonomic designation Amorocoelophoma sp.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a drawing of amonocoelolide A, a compound of the present invention1H-NMR spectrum;
FIG. 2 is a drawing of amonocoelolide A, a compound of the present invention13C-NMR and DEPT spectra;
FIG. 3 is a ROESY spectrum of amonocoelolide A, a compound of the present invention;
FIG. 4 shows the preparation of amonocoelolide A, a compound of the present invention1H-1H COSY spectrum;
FIG. 5 is an HSQC spectrum of amonocoelolide A, a compound of the present invention;
FIG. 6 is an HMBC spectrum of amonocoelolide A, a compound of the present invention;
FIG. 7 is a crystal structure diagram of the X-single crystal diffraction of amonocoelolide A, a compound of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Isolation and preparation of spiro compound amonocoelolide A
(1) Preparing a PDB culture medium: weighing 200g of peeled potato, cutting into small pieces, adding 1000mL of distilled water, boiling, filtering with gauze to obtain a potato filtrate, adding 20g of glucose, naturally adjusting pH, and sterilizing at 121 ℃ for 30min to obtain a PDB culture medium for later use, wherein the concentration of the potato in the PDB culture medium is 0.2g/mL, and the concentration of the glucose is 0.02 g/mL.
(2) Inoculating Amorocoelophoma sp.YE3351 strain into PDB culture medium, shake culturing at 28 + -2 deg.C and 200r/min for 4 days to obtain seed liquid, inoculating the prepared seed liquid into fermentation culture medium according to 10% of inoculation amount, shake culturing at 28 + -2 deg.C and 200r/min for 7-9 days to obtain fermentation product.
(3) Filtering the fermentation product obtained in the step (2) by using gauze to obtain fermentation liquor and mycelium, extracting the fermentation liquor by using ethyl acetate, extracting the mycelium by using methanol, combining extracting solutions, and concentrating under reduced pressure to obtain a fermentation crude extract.
(4) Performing chromatographic separation on the crude fermentation extract by using a silica gel column (200-300 meshes), performing gradient elution by using a petroleum ether-acetone system according to the volume ratio of 4:1 to 1:1, and collecting a crude component eluted by the petroleum ether-acetone system according to the volume ratio of 7: 3; eluting the obtained crude component by using a petroleum ether-ethyl acetate solvent system according to the volume ratio of 4:1, and recrystallizing in a mixed solvent of chloroform and methanol according to the volume ratio of 5:1 to prepare the spiro compound amonocoelolide A.
Example 2
Structural identification of spiro compound amorocoelolide A
The compound amonocoelolide a prepared in example 1 was subjected to 1D/2D NMR (one-dimensional nuclear magnetic resonance spectrum and two-dimensional nuclear magnetic resonance spectrum) and HRESI-MS (high resolution electrospray ionization mass spectrometry) to identify its structure. The molecular formula of amorocoelolide A is C11H14O6；HRESIMS m/z 265.0687[M+Na]+(calcd for C11H14O6Na,265.0688), unsaturation 5.
1The H-NMR spectrum shows that two methyl protons are respectively positioned at deltaH1.36(d, J ═ 7.3Hz) and δH1.62(s), five methine protons at δH 2.85(m)、δH 4.35(s)、δH 4.68(s)、δH6.03(d) and δH 6.96(dd)。13The C DEPT NMR spectrum showed that this compound contained 2 methyl groups, 3 saturated methine groups (two of which are attached to an oxygen atom), 2 olefinic methine groups, 4 quaternary carbons (including 1 unsaturated quaternary carbon, 1 quaternary carbon with oxygen, 2 carbonyl groups). The compound amonocoelolide a has 5 unsaturations and should have two rings in addition to two carbonyl groups and one double bond. From1H-1In the H COSY map, a structural unit of C6-C8/C12 can be deduced. The mode of attachment of the building block to the carbonyl and quaternary carbon was determined by HMBC mapping. From HMBC spectra, it can be observed that the methyl proton H3-12(δH1.36, d, J ═ 7.3Hz) and C-5, C-6, C-7, H-10 and carbonyl C-9, from which the structural unit of cyclohexenone can be determined. The chemical shift value of C-3 is deltaC104.3, are more shifted to the lower field than conventional vicinal quaternary carbons, presumably to join two oxygen atoms. Proton of methyl group H3-11(δH1.62) and quaternary carbon with oxygen C-3 (. delta.))C104.3) and hypoxymethylene C-4 (. delta.))C76.6) has HMBC related points, H-4 and C-5, C-6, C-10, C-11 show HMBC related points, H-10 and C-1, C-4, C-5, C-9 have HMBC related points, and the compound amonocoelolide A can be determined to form a spiro structure by the connection of cyclohexenone and five-membered cyclic lactone through C-5. The compound amonocoelolide a was identified as a novel compound by SciFinder search. As the compound amorocoelolide A has the chemical structure with 5 chiral carbon atoms of C-3, C-4, C-5, C-6 and C-10, a single crystal is further obtained in a mixed solvent with the volume ratio of chloroform to methanol of 5:1 by a method of culturing the single crystal. By X-single crystal diffraction Cu target analysis, the absolute configurations of 5 chiral carbon atoms in the compound amonocoelolide A are determined to be 3R, 4R, 5S, 6R and 10S respectively. The structural formula is shown as the following formula (I):
example 3
Determination of the anti-influenza Virus Activity of the Compound amonocoelolide A
(1) The antiviral activity of the compound amonocoelolide a was evaluated using cytopathic effect (CPE). MDCK cells were collected and counted, and 20ml of 10 cells were prepared with 2% DMEM5/ml cells, MDCK cells at 2X 104Individual cells/well density were seeded into 96-well plates and incubated at 37 ℃ in 5% CO before infection2Standing for 24 h. Two-fold serial dilutions of the compound amonocoelolide A were combined with influenza A virus (100 TCID)50) Mix and incubate at 37 ℃ for 30min, then transfer the mixture to cells in 96-well plates and incubate for another 30 min. After incubation, MDCK cells were washed twice with PBS to remove unabsorbed virus, and then DMEM supplemented with 1 μ g/ml TPCK-trypsin and 0.2% BSA was added. After 48h incubation, antiviral effects were observed under the microscope. The data was further confirmed using MTT assay, the experiment was repeated at least three times, and oseltamivir was used as a positive control.
(2) Toxicity of the compound amonocoelolide A on MDCK cells by MTT method
The concentration is 1 x 104MDCK cells/well were grown in 96-well plates for 24h, then two serial dilutions of the compound amonocoelolide A were added to MDCK cells, followed by 5% CO in a humidified atmosphere at 37 deg.C2And (5) incubating for 48 h. After incubation, the viability of the cells was determined by the MTT assay. Mu.l MTT at a concentration of 0.5mg/ml in DMEM was added to each well and incubated for an additional 4h at 37 ℃. Subsequently, 150 μ l/well of DMSO was added to the plate to extract the heat-treated MTT, and then the absorbance of the extract at a wavelength of 570nm was measured on a microtiter plate reader. The cytotoxicity of amonocoelolide a was determined by comparing the cell viability of cells treated with the compound amonocoelolide a with cells treated with solvent (0.3% methanol).
The experimental result shows that the IC of the spiro compound amonocoelolide A on H1N1 influenza virus50The value (median inhibitory concentration) was 0.29. + -. 0.14. mu.g/ml, CC50The value (median toxic concentration) was 204.2. + -. 2.03. mu.g/ml. IC of compound amonocoelolide A50Positive control drug oseltamivir (IC) of value ratio500.52 plus or minus 0.21 mu g/ml), shows the characteristics of high efficiency and low toxicity, and has potential application in preparing novel anti-influenza virus medicaments.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (9)
1. A spiro compound amonocoelolide a characterized by: the structural formula is shown as the following formula (I):
2. a process for the preparation of the spiro compound amonocoelolide a according to claim 1, comprising the steps of:
fermenting fungus Amonocoelophoma sp, and respectively collecting fermentation liquor and mycelium;
extracting fermentation liquor and mycelia with organic solvent respectively, mixing extractive solutions, and concentrating under reduced pressure to obtain fermented crude extract;
performing chromatographic separation on the crude fermentation extract by using a silica gel column, eluting by using a petroleum ether-acetone system, and collecting a crude component;
eluting the crude component with petroleum ether-ethyl acetate system, and recrystallizing to obtain the final product.
3. The method according to claim 2, wherein the fungus Amonocolophoma sp is Amonocolophoma sp.YE3351 which has been deposited in China Center for Type Culture Collection (CCTCC) No. M2020407 at 8/7 of 2020.
4. The process according to claim 2, wherein the culture medium used for the fermentation is PDB culture medium, wherein the concentration of potato in the PDB culture medium is 0.2-0.4g/mL and the concentration of glucose in the PDB culture medium is 0.02-0.05 g/mL.
5. The method according to claim 2, wherein the fermentation broth is extracted with ethyl acetate and the mycelia are extracted with methanol.
6. The preparation method according to claim 2, characterized in that the crude component eluted from the petroleum ether-acetone system at a volume ratio of 7:3 is collected by gradient elution with the petroleum ether-acetone system from 4:1 to 1: 1.
7. The method according to claim 2, wherein the crude fraction is eluted with petroleum ether and ethyl acetate at a volume ratio of 4: 1.
8. The method according to claim 2, wherein the recrystallization is carried out in a mixed solvent of chloroform and methanol at a volume ratio of 5: 1.
9. Use of the spirocyclic compound amonocoelolide a according to claim 1 for the preparation of a medicament against influenza viruses.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961783A (en) * | 2021-02-04 | 2021-06-15 | 云南大学 | Plant endophytic fungus and application thereof in preparation of spironolactone derivative |
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| CN107602515A (en) * | 2017-10-13 | 2018-01-19 | 南开大学 | One kind 5A5B6CTricyclic spiro lactone derivatives and its production and use |
| CN112961783A (en) * | 2021-02-04 | 2021-06-15 | 云南大学 | Plant endophytic fungus and application thereof in preparation of spironolactone derivative |
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| CN107602515A (en) * | 2017-10-13 | 2018-01-19 | 南开大学 | One kind 5A5B6CTricyclic spiro lactone derivatives and its production and use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112961783A (en) * | 2021-02-04 | 2021-06-15 | 云南大学 | Plant endophytic fungus and application thereof in preparation of spironolactone derivative |
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