CN105218447B - Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent - Google Patents
Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent Download PDFInfo
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Abstract
A kind of Sclerotiorin derivatives and preparation method thereof and the application as anti-influenza A H 1 N 1 virus agent, Spawn incubation first is carried out to fungi Penicillium sp. (TA33 1) during preparation, fermented and cultured is carried out to the fungi again, gained mycelium chloroform methanol mixed liquor (1: 1) is extracted 3 times after being concentrated under reduced pressure, coarse extract is extracted with ethyl acetate 3 times to obtain；Normal-phase silica gel column chromatography, the gel filtration chromatographies of Sephadex LH 20, HPLC high performance liquid chromatography are carried out successively, obtains yellow powder, are (+) Sclerotiorin；In the dichloromethane solution dissolved with (+) Sclerotiorin, organic primary amine and potassium carbonate are added, compound of formula I is obtained after reaction.The present invention provides a kind of anti-influenza A H 1 N 1 virus agent, it is characterised in that with the compound of formula I or its pharmaceutically acceptable salt of the present invention, for treating influenza caused by A (H 1 N 1) virus.
Description
Technical field
The present invention relates to a kind of Sclerotiorin derivatives and preparation method and application, more particularly to a kind of to first
Type H1N1 influenza viruses have Sclerotiorin derivatives of extremely strong inhibitory activity and preparation method and application.
Background technology
H1N1virus is influenza A, carries H1N1 hypotype swine influenza virus strains, includes fowl stream
Sense, the nbccs gene segment of three kinds of influenza viruses of swine flu and human influenza, while possess Asia swine flu and African pig stream
Influenza Virus feature.From on March 18th, 2009 Mexico break out H1N1virus since, successively find infection,
Death.Therefore, finding safe and efficient anti-influenza A H 1 N 1 virus agent turns into the problem for being badly in need of solving in the world.Ocean
Natural products is considered as the important sources of novel antiviral agent.Marine microorganism due to can send out on a large scale in the lab
Ferment, survivable natural environment, and as the most important source of activity marine compound.However, in recent years there is not yet directly
Application of the native compound in source or derivatives thereof as anti-influenza A H 1 N 1 virus agent from marine microorganism.
(Centers for Disease Control and Prevention.Update：novel influenza A
(H1N1) virus infections-worldwide, May 6,2009.MMWR Morb Mortal Wkly Rep.2009,
58,453-458；Scalera, N.M.；Mossad, S.B.Postgrad Med.2009,121,43-47；Medina, R.A.；
Garcia-Sastre, A.Nat.Rev.Microbiol.2011,9,590-603.).
The content of the invention
It is an object of the invention to provide a kind of preparation method of the Sclerotiorin derivatives from marine fungi
With the application as anti-influenza A H 1 N 1 virus agent, it can meet the demand of prior art.Culture presevation information：Preservation
Organization：China Committee for Culture Collection of Microorganisms's common micro-organisms center；Depositary institution address：Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica of institute of North Star West Road 1；Preservation date：On April 3rd, 2014；Deposit number：CGMCC
No.8994；Classification And Nomenclature：Penicillium sp..
The present invention provides compound of formula I or its pharmaceutically acceptable salt：
Or its pharmaceutically acceptable salt.R is in Formulas I The present invention provides the preparation method of compound of formula I, it is characterised in that first in strain
Spawn incubation is carried out to the endogenetic fungus Penicillium sp. (TA33-1) for being isolated from gorgonian in culture medium, then fermented
Fermented and cultured is carried out to the fungi in culture medium, gained mycelium is dense with chloroform-methanol mixed liquor (1: 1) 3 decompressions of extraction
After contracting, coarse extract is extracted with ethyl acetate 3 times to obtain；Ethyl acetate coarse extract carry out respectively normal-phase silica gel column chromatography,
After Sephadex LH-20 gel filtration chromatographies, then through HPLC high performance liquid preparative chromatographies, gained eluent is concentrated, obtains yellow powder
End, it is (+)-Sclerotiorin；In the dichloromethane solution dissolved with (+)-Sclerotiorin, add organic primary amine and
Potassium carbonate, compound of formula I is obtained after reaction.
Bacterium culture medium preferably comprises glucose 0.1%-5.0% (percentage by weight, similarly hereinafter), ferment in above-mentioned preparation method
Female cream 0.01%-1%, peptone 0.01%-1%, agar 0.1%-3.0%, sodium chloride 0.05%-5%, remaining is water, training
Foster temperature is preferably 0-30 DEG C, and incubation time is preferably 3-15 days；Fermentation medium preferably comprises rice 1.0%-80.0% (weights
Measure percentage, similarly hereinafter), sodium chloride 0.05%-5%, remaining is water, and cultivation temperature is preferably 0-30 DEG C, and incubation time is preferably
10-60 days；The preferred 200-300 mesh silica gel of stationary phase that described normal-phase silica gel column chromatography uses, mobile phase preferred volume ratio are
15%-60% ethyl acetate-light petrol mixed solvent；The mobile phase that the Sephadex LH-20 gel filtration chromatographies use is excellent
It is petroleum ether: chloroform: methanol=2: 1: 1 mixed solvent to select volume ratio；Used in the HPLC high performance liquid preparative chromatographies
Chromatographic column is this area routine ODS C18 posts, and preferably 10 × 250mm of Kromasil, 5 μm, flow velocity is preferably 1.0-5.0mL/
Min, mobile phase preferred volume ratio are 50%-80% Methanol+Water；Described organic primary amine is
The Sclerotiorin derivatives that the present invention obtains from marine fungi have extremely strong to H1N1virus
Inhibitory activity, available for exploitation anti-influenza A H 1 N 1 virus agent, have a extensive future.
Another embodiment of the present invention provides compound of formula I or its pharmaceutically acceptable salt is preparing anti-H 1 N 1
Application in influenza virus agent.
Term " pharmaceutically acceptable salt " refers to the addition of atoxic inorganic or organic acid and/or alkali in the present invention
Salt.Reference can be made to " Salt selection for basic drugs ", Int.J.Pharm. (1986), 33,201-217.
Embodiment
For the ease of a further understanding of the present invention, examples provided below has done more detailed description to it.But
It is that these embodiments only are not used for limiting the scope of the present invention or implementation principle, reality of the invention for being better understood from inventing
The mode of applying is not limited to herein below.
Embodiment 1
(1) gorgonian endogenetic fungus Penicillium sp. (TA33-1) Spawn incubation
Culture medium used in Spawn incubation contains glucose 1.0% (percentage by weight, similarly hereinafter), yeast extract 0.2%, albumen
Peptone 0.2%, agar 1.0%, sodium chloride 3.0%, remaining is water, test tube slant is made during use, fungal bacterial strain is trained at 30 DEG C
Support 3 days.
(2) gorgonian endogenetic fungus Penicillium sp. (TA33-1) fermentation
Culture medium used in fermented and cultured contains rice 40.0% (percentage by weight, similarly hereinafter), sodium chloride 3.0%, remaining
For water；Fungal bacterial strain is cultivated 30 days in 28 DEG C.
(3) (+)-Sclerotiorin extraction separation
The mycelium for taking 60 bottles of steps (2) to obtain, extracted 3 times after being concentrated under reduced pressure, used with chloroform-methanol mixed liquor (1: 1)
Ethyl acetate extracts 3 times to obtain coarse extract；Ethyl acetate coarse extract carries out normal-phase silica gel column chromatography (stationary phase 200- respectively
300 mesh silica gel；Mobile phase is 30% ethyl acetate/petroleum ether mixed solvent, volume ratio), Sephadex LH-20 gel column layers
After analysing (petroleum ether: chloroform: methanol=2: 1: 1 mixed solvent, volume ratio), then through the separation of HPLC high performance liquid preparative chromatographies
(chromatographic column is 10 × 250mm of Kromasil, and 5 μm, flow velocity 2.0mL/min, the methanol-water mixing that mobile phase is 65% is molten
Agent, volume ratio), gained eluent is concentrated, obtains yellow powder, is (+)-Sclerotiorin.
Embodiment 2
(1) gorgonian endogenetic fungus Penicillium sp. (TA33-1) Spawn incubation
Culture medium used in Spawn incubation contains glucose 0.1%-5.0% (percentage by weight, similarly hereinafter), yeast extract
0.01%-1%, peptone 0.01%-1%, agar 0.1%-3.0%, sodium chloride 0.05%-5%, remaining is water, during use
Test tube slant is made, fungal bacterial strain is cultivated 3-15 days at 0-30 DEG C.
(2) gorgonian endogenetic fungus Penicillium sp. (TA33-1) fermentation
Culture medium used in fermented and cultured contains rice 1.0%-80.0% (percentage by weight, similarly hereinafter), sodium chloride
0.05%-5%, remaining is water, and fungal bacterial strain is cultivated 10-60 days in 0-30 DEG C.
(3) the extraction separation of (+)-Sclerotiorin compounds
Take and gained mycelium chloroform-methanol mixed liquor (1: 1) is extracted to 3 decompressions obtained by 10-300 bottles step (2)
After concentration, coarse extract is extracted with ethyl acetate 3 times to obtain；Normal-phase silica gel column chromatography is carried out respectively after the concentration of ethyl acetate coarse extract
(stationary phase is this area routine purification on normal-phase silica gel, and mobile phase is 15%-40% ethyl acetate-light petrol mixed solvent, volume
Than), (mobile phase is petroleum ether to Sephadex LH-20 gel filtration chromatographies: chloroform: methanol=2: 1: 1 mixed solvent, volume
Than) after, then (chromatographic column is this area routine ODS C18 posts, flow velocity 1.0-5.0mL/ through HPLC high performance liquid preparative chromatographies
Min, mobile phase be 50%-80% Methanol+Water, volume ratio), gained eluent is concentrated, yellow powder is obtained and consolidates
Body, it is (+)-Sclerotiorin compounds.
Other Spawn incubations, the fermentation condition not particularly pointed out in embodiment 1-2, and normal phase silica gel column chromatography separation,
Other experimental operating conditions such as the separation of Sephadex LH-20 gel filtration chromatographies, high performance liquid chromatography separation are this area routine
Experimental operating conditions, those skilled in the art can reasonably be selected according to being actually needed.
Embodiment 3
Above-mentioned dried (+)-Sclerotiorin (0.1mol) is weighed to be dissolved in dichloromethane, under normal temperature condition,
Be sufficiently stirred it is lower organic primary amine (0.12mol) is added drop-wise in reaction solution dropwise, reaction 1 hour after, into reactant plus
Enter distilled water (200mL) and carry out terminating reaction, extracted with ethyl acetate (500mL), after extract is concentrated, carry out post layer
Analysis, is eluted with ethyl acetate, eluent concentration, and compound of formula I is obtained after recrystallization.
Other organic chemical reactionses conditions not particularly pointed out in embodiment 3, and normal phase silica gel column chromatography separation etc. its
His experimental operating conditions are the conventional experimental operating conditions in this area, those skilled in the art can according to being actually needed,
Reasonably selected.
The particular compound structure example of compound of formula I：
The structural identification data of compound of formula I：
1：1H NMR (600MHz, CDCl3)δH7.84 (1H, s, H-1), 7.16 (1H, s, H-4), 6.98 (1H, d, J=
15.6Hz, H-10), 6.58 (1H, t, J=2.4Hz), 6.41 (2H, d, J=1.8Hz), 5.70 (1H, d, J=15.6Hz, H-
9), 5.68 (1H, d, J=9.6Hz, H-12), 3.83 (6H, s), 2.42 (1H, m, H-13), 2.18 (3H, s, H-20), 1.59
(3H, s, H-17), 1.57 (3H, s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14), 1.00 (3H, d, J=
6.6Hz, H-16), 0.85 (3H, t, J=7.2Hz, H-15)13C NMR (150MHz, CDCl3)δC193.9 (C-6), 184.7
(C-8), 170.3 (COCH3), 161.8,161.8,147.9 (C-1), 147.4,144.2,143.2,141.8,141.1,132.0
(C-11), 116.2 (C-9), 114.3 (C-5), 109.6,109.6,109.6,103.3 (C-8a), 101.8 (C-4), 84.9 (C-
7), 56.0 (- OCH3), 55.9 (- OCH3), 35.1 (C-13), 30.1 (C-14), 23.3 (C-18), 20.4 (C-16), 20.3
(COCH3), 12.5 (C-17), 12.1 (C-15) .ESIMSm/z 526.05/528.05 [M+H]+/[M+H+2]+(3: 1),
547.99/550.01[M+Na]+/[M+Na+2]+(3: 1), 1072.84/1072.77 [2M+Na]+/[2M+Na+2]+(3∶1)
.HRESIMSm/z526.1981[M+H]+(calcd for C29H33O6NCl, 526.1991)
2：1H NMR (600MHz, CDCl3)δH7.85 (1H, s, H-1), 7.16 (1H, s, H-4), 6.99 (1H, d, J=
15.6Hz, H-10), 6.50 (2H, s), 5.68 (2H, oVerlapped, H-9and H-12), 3.91 (3H, s), 3.87 (6H,
S), 2.42 (1H, m, H-13), 2.19 (3H, s, H-20), 1.59 (3H, s, H-17), 1.57 (3H, s, H-18), 1.42 (1H, m,
H-14), 1.32 (1H, m, H-14), 1.00 (3H, d, J=6.6Hz, H-16), 0.85 (3H, t, J=7.2Hz, H-15)13C
NMR (150MHz, CDCl3)δC193.9 (C-6), 184.7 (C-8), 170.4 (COCH3), 154.1,148.0,148.0,
147.6,144.1,143.3,141.2,141.2,139.2,135.8,131.9 (C-11), 116.2,114.3 (C-5), 109.6,
104.2,103.4 (C-8a), 84.8 (C-7), 61.2 (- OCH3), 56.7 (- OCH3), 56.6 (- OCH3), 35.1 (C-13),
30.1 (C-14), 23.3 (C-18), 20.4 (C-16), 20.3 (COCH3), 12.5 (C-17), 12.0 (C-15) .ESIMSm/
z556.07/558.06[M+H]+/[M+H+2]+(3: 1), 578.05/580.04 [M+Na]+/[M+Na+2]+(3: 1), 1132.83
[2M+Na]+.HRESIMSm/z556.2087[M+H]+(calcd for C30H35O7NCl, 556.2097)
3：1H NMR (600MHz, CDCl3)δH8.84 (1H, d, J=2.4Hz), 8.24 (1H, d, J=8.4Hz), 8.20
(1H, d, J=2.4Hz), 7.94 (1H, d, J=7.8Hz), 7.91 (1H, ddd, J=8.4,6.6,1.2Hz), 7.88 (1H, s,
H-1), 7.74 (1H, ddd, J=7.8,7.2,1.2Hz), 7.19 (1H, s, H-4), 7.04 (1H, d, J=15.6Hz, H-10),
5.70 (1H, d, J=9.6Hz, H-12), 5.58 (1H, d, J=15.6Hz, H-9), 2.38 (1H, m, H-13), 2.19 (3H, s,
H-20), 1.61 (3H, s, H-17), 1.46 (3H, s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14), 0.98
(3H, d, J=6.6Hz, H-16), 0.84 (3H, t, J=7.2Hz, H-15)；13C NMR (150MHz, CDCl3)δC 193.6(C-
6), 185.1 (C-8), 170.4 (COCH3), 148.7,147.9,147.6,147.4,144.3,143.5,141.1,134.1,
133.1,131.9,131.7,129.9,128.9,128.2,127.3,115.7,115.1 (C-5), 110.4,104.6,84.8
(C-7), 35.1 (C-13), 30.1 (C-14), 23.2 (C-18), 20.4 (C-16), 20.3 (COCH3), 12.5 (C-17), 12.1
(C-15).ESIMSm/z 517.05/519.10[M+H]+/[M+H+2]+(3: 1), 539.01/541.01 [M+Na]+/[M+Na+
2]+(3: 1), 1054.85 [2M+Na]+.HRESIMSm/z517.1885[M+H]+(calcd for C30H30O4N2Cl,
517.1889).
4：1H NMR (600MHz, CDCl3)δH7.88 (2H, d, J=8.4Hz), 7.76 (1H, s, H-1), 7.49 (2H, d,
J=8.4Hz), 7.13 (1H, s, H-4), 6.98 (1H, d, J=15.6Hz, H-10), 5.71 (1H, d, J=9.6Hz, H-12),
5.49 (1H, d, J=15.6Hz, H-9), 2.42 (1H, m, H-13), 2.18 (3H, s, H-20), 1.58 (3H, s, H-17), 1.54
(3H, s, H-18), 1.42 (1H, m, H-14), 1.32 (1H, m, H-14), 1.00 (3H, d, J=6.6Hz, H-16), 0.86 (3H,
T, J=7.2Hz, H-15)13C NMR (150MHz, CDCl3)δC193.4 (C-6), 185.1 (C-8), 170.3 (COCH3),
148.7,146.6,144.0,143.9,143.1,140.2,134.2,134.2,131.7,127.7,127.7,117.2,
115.8,114.9,114.1,110.5,104.9,84.8 (C-7), 35.1 (C-13), 30.1 (C-14), 23.1 (C-18), 20.3
(C-16), 20.3 (COCH3), 12.4 (C-17), 12.1 (C-15) .ESIMSm/z 491.04/493.07 [M+H]+/[M+H+2]+
(3: 1), 513.04/514.99 [M+Na]+/[M+Na+2]+(3: 1), 1118.99 [2M+Na]+；HRESIMSm/z 491.1728
[M+H]+(calcd for C28H28O4N2Cl, 491.1732)
5：1H NMR (600MHz, CDCl3)δH7.89 (1H, s, H-1), 7.00 (1H, s, H-4), 6.96 (1H, d, J=
15.6Hz, H-10), 6.31 (1H, d, J=15.6Hz, H-9), 5.72 (1H, d, J=9.6Hz, H-12), 4.60 (2H, s, H-
21), 2.63 (1H, t, 2.4Hz, H-23), 2.50 (1H, m, H-13), 2.17 (3H, s, H-20), 1.88 (3H, s, H-17),
1.55 (3H, s, H-18), 1.45 (1H, m, H-14), 1.35 (1H, m, H-14), 1.03 (3H, d, J=6.6Hz, H-16),
0.89 (3H, t, J=7.2Hz, H-15)13C NMR (150MHz, CDCl3)δC193.5 (C-6), 184.7 (C-8), 170.2
(COCH3), 148.4,147.7,145.4,144.2,140.8,132.0,114.9,114.6,111.4,103.0,85.0 (C-
7), 77.5,75.3,43.8,35.2 (C-13), 30.1 (C-14), 23.3 (C-18), 20.4 (C-16), 20.3 (COCH3),
12.7 (C-17), 12.1 (C-15) .ESIMS m/z 428.03/430.09 [M+H]+/[M+H+2]+(3: 1), 450.01/
452.02[M+Na]+/[M+Na+2]+(3: 1), 876.80/878.80 [2M+Na]+/[2M+Na+2]+.HRESIMS m/z
428.1621[M+H]+(calcd for C24H27O4NCl, 428.1623)
Embodiment 4
The compound of formula I of the present invention is as follows to the inhibitory activity method of testing of H1N1virus：By using thin
Born of the same parents' lesion inhibitory action (CPE) carries out antiviral activity research.Test Virus is：H1N1 (influenza virus), wherein MDCK
(MDCK) it is H1N1 sensitive cells.After the mdck cell for being trained individual layer is digested with pancreatin, it is inoculated in 96 orifice plates, grows up to list
Layer is standby.H1N1 diseases are inoculated on mdck cell, add that 2% serum 1640 culture medium is rearmounted 37 DEG C, 5%CO2Under the conditions of cultivate,
After there is more than 90% lesion, centrifugation is blown and beaten after multigelation 3 times, quantitative separating, -80 DEG C of refrigerators freeze standby.Test sample
Product are after often pipe is dissolved with 10 μ L DMSO, add 200 μ L 2%1640 nutrient solutions, and continuous 10 2 doubling dilutions, totally 10 it is dilute
Degree of releasing, then laterally it is inoculated on the cell monolayer in 96 plate holes, 11 are classified as virus control, 12 are classified as cell controls, 37 DEG C,
5%CO2Culture, observes lesion, Continuous Observation 24h per hour.After more than 90% lesion occurs in virus control, by liquid in plate hole
Body, which is inhaled, to be abandoned, and adds 1% neutral red staining, and OD values are determined in 540nm wavelength, and it is effective to calculate medicine half with Reed-Muench methods
Concentration (IC50)。
The compound of formula I of the present invention shows important inhibitory activity to H1N1virus, much stronger than positive drug profit
Ba Weilin.This shows that compound of formula I or its pharmaceutically acceptable salt can be used for the antivirotic for preparing high-efficiency low-toxicity, and sea
Foreign source fungi Penicillium sp. (TA33-1) can carry out large scale fermentation production, ensure that the source of compound of formula I,
Have broad application prospects.
Claims (7)
1. a kind of Selerotiorin derivatives or its pharmaceutically acceptable salt, it is characterised in that there is the structure shown in Formulas I：
Wherein R is
2. the preparation method of the compound of formula I described in claim 1, it is characterised in that first to fungi in bacterium culture medium
Penicillium sp. carry out Spawn incubation, then carry out fermented and cultured to the fungi in the fermentation medium, by gained mycelium
Extracted 3 times with chloroform-methanol mixed liquor after being concentrated under reduced pressure, coarse extract is extracted with ethyl acetate 3 times to obtain；Ethyl acetate coarse extract
, will after carrying out normal-phase silica gel column chromatography, Sephadex LH-20 gel filtration chromatographies respectively, then through HPLC high performance liquid preparative chromatographies
Gained eluent concentrates, and obtains yellow powder, is (+)-Sclerotiorin；In the dichloromethane dissolved with (+)-Sclerotiorin
In alkane solution, organic primary amine and potassium carbonate are added, Formulas I chloro polyketide is obtained after reaction；Wherein described bacterium culture medium
In containing glucose, yeast extract, peptone, agar, coarse sea salt, water；In described fermentation medium containing rice, coarse sea salt,
Water；Described chromatographic isolation is normal phase silica gel column chromatography separation, gel filtration chromatography separation and high performance liquid chromatography separation；Described
Organic primary amine is
3. preparation method as claimed in claim 2, it is characterised in that the bacterium culture medium contains glucose 0.1%-
5.0%th, yeast extract 0.01%-1%, peptone 0.01%-1%, agar 0.1%-3.0%, sodium chloride 0.05%-5%, remaining
For water, above-mentioned percentage composition is weight percentage, and cultivation temperature is 0-30 DEG C, and incubation time is 3-15 days.
4. preparation method as claimed in claim 2, it is characterised in that the fermentation medium contain rice 1.0%-80.0%,
Sodium chloride 0.05%-5%, remaining is water, and above-mentioned percentage composition is weight percentage, and cultivation temperature is 0-30 DEG C, during culture
Between be 10-60 days.
5. the preparation method as described in claim any one of 2-4, it is characterised in that what described normal-phase silica gel column chromatography used
Stationary phase is 200-300 mesh silica gel, and mobile phase is the ethyl acetate-light petrol mixed solvent that volume ratio is 15%-60%；It is described
The mobile phase that Sephadex LH-20 gel filtration chromatographies use is petroleum ether for volume ratio: chloroform: methanol=2: 1: 1 mixing is molten
Agent；The chromatographic column used in the HPLC high performance liquid preparative chromatographies is 10 × 250mm of Kromasil, 5 μm, flow velocity 1.0-
5.0mL/min, mobile phase are volume ratio 50%-80% Methanol+Water.
6. a kind of anti-influenza A H 1 N 1 virus agent, it is characterised in that it contains the compound of formula I or its medicine described in claim 1
Acceptable salt is as active ingredient on.
7. the compound of formula I or its pharmaceutically acceptable salt described in claim 1 are preparing anti-influenza A H 1 N 1 virus agent
In application.
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| CN105586373A (en) * | 2015-06-24 | 2016-05-18 | 中国海洋大学 | Sclerotiorin derivative, preparation method thereof and application thereof as marine anti-fouling agent |
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| CN111205252B (en) * | 2020-02-10 | 2021-10-29 | 杭州科兴生物化工有限公司 | SEK15 polyketide compound with neuraminidase inhibition effect and preparation method and application thereof |
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| CN105586373A (en) * | 2015-06-24 | 2016-05-18 | 中国海洋大学 | Sclerotiorin derivative, preparation method thereof and application thereof as marine anti-fouling agent |
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