CN1880309A - Novel compound, bacteria strain and method for producing novel compound using the strain - Google Patents
Novel compound, bacteria strain and method for producing novel compound using the strain Download PDFInfo
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Abstract
This invention relates to a new compound indicated by formula(I) and a new strain for making same, Altemaria alternate var.monosporus ST-026-R CGMCC No.0899. The process comprises: culture the invented strain in the culture media to produce and accumulate the new compound indicated by formula(I) in the said strain cells and culture media and then recover and purify the invented new compound indicated by formula(I) from the said cells and culture media. This compound has good biological activities of anticancer, antimycotics and antiviral.
Description
Technical field
The present invention relates to a kind of new compound, a kind of new bacterial strain that can produce this new compound, and use the method that this new strain fermentation is produced this new compound.
Technical background
It is following formula (a) plant-growth regulator that the flat 5-247077 of Japanese Patent Application Laid-Open discloses a kind of structural formula, and molecular weight is 352.It can be produced by the shake flask fermentation cladosporium (Cladosporium) 101 in potato extract and dextrose culture-medium.By the 7.2L fermented supernatant fluid, can obtain reclaiming this compound of 73mg by extraction and chromatographic separation.People (Tetrahedron Letters such as Youji Sakagami, Vol.36, No.9, pp.1469-1472,1995) by isolating active substance cladosporium alcohol (cladosporol) (1) in cladosporium (Cladosporium cladosporioides) fungi, its molecular formula is C
20H
16O
6, structural formula is following formula (b), and thinks that it is a kind of β-1,3-dextran (β-1,3-glucan) biosynthesis inhibitor.Structural formula (a) is the two dimensional structure formula of structural formula (b).
Summary of the invention
The object of the present invention is to provide a kind of new compound with pharmaceutical use.
Another object of the present invention is to provide a kind of new bacterial strain that can produce this new compound.
Another object of the present invention is to provide a kind of application method that this new strain fermentation is produced this new compound.
The inventor is through study in a large number for many years deeply and carefully, and discovery, cultivation, mutagenesis go out a kind of new bacterial classification, and it can produce a kind of new compound, and the inventor goes out to utilize this novel bacterial to come the method for this new compound of fermentative production through a large amount of experimental studies.
The invention provides the new compound of following structural formula (I).
Outward appearance: white crystal
Fusing point: 187～191 ℃
Specific rotatory power: 213.01 ° (acetone soln 1.230mg/ml)
Molecular weight: 352
Molecular formula: C
20H
16O
6
UV λ
MaxNm (ε): 212nm (see figure 6)
IR spectrum: (see figure 7)
MS collection of illustrative plates: (see figure 8)
Series NMR spectrum:
1H-NMR (Fig. 9 A),
13C-NMR (Fig. 9 B) and
1H-
1H COSY composes (Fig. 9 C)
X-ray diffracting spectrum: (see figure 10)
The space structure of new compound of the present invention is different with the space structure of the existing compound of aforementioned structural formula (b), and the oxirane ring of new compound of the present invention is outside, and the oxirane ring of the known compound of aforementioned structural formula (b) is inside.Because of above space structure difference, it is active that new compound of the present invention has unique biological, it has very strong biologic activity to multiple cancers such as cancer of the stomach, leukemia, mammary cancer, ovarian cancers, simultaneously, this material also has efficient antifungal, antiviral biological activity, can be developed into cancer therapy drug, antifungal drug and the antiviral of a new generation, have a extensive future.
The novel bacterial of a kind of energy production formula (I) compound provided by the invention is chain lattice spore monospore mutation (Alternaria alternate var.monosporus) ST-026-R CGMCC No.0899.
The inventor is from 310 of the branch of taxusyunnanensis (Taxus yunnanensis) tree of 7 strains from the theropencedrymion of 2500～3000 meters of the old Jun Mountain height above sea level of Lijiang Prefecture, Yunnan Province more than 300 years, bark, leaf and Gen Pi collected specimens.With bark under aseptic condition, use 60% alcohol disinfecting, aseptic water washing, blot excessive moisture, phloem is torn into thin slice from level to level, again sample is cut into small pieces, press endophloem to the epidermal area series arrangement, be seeded on the water agar, under 25 ℃ of constant temperatures, cultivated 7～15 days, grow dissimilar microorganisms in succession, the bacterium colony of different shape, different colours is carried out fermenting experiment after purified, therefrom select and to produce the higher bacterial strain of new compound of the present invention and taxol and productive rate as starting strain.This bacterial strain is carried out ultraviolet mutagenesis, and the ultraviolet mutagenesis number gets the morphologic variation bacterial strain after generation, inherit mutagenesis as starting strain again, must produce the more stable single-ascospore strain of new compound of the present invention and taxol, above morphologic variation is more stable for cultivating for Fu through number, obtains new single-ascospore strain of the present invention.According to this bacterial strain called after chain lattice spore monospore mutation of plant rules of nomenclature, formal name used at school is Alternaria alernata var.monosporus, code name is ST-026-R, culture presevation is at China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 13, Zhongguancun N 1st Lane, Beijing City, preserving number is: CGMCC No.0899.
New bacterial strain CGMCC 0899 provided by the invention has following Microbiological Characteristics:
The hyphal cell length and width approximately equate or grow up in wide about 2 times, hang in the middle of the cell and contract, and expand at two ends, also hangs at the tabula place and contract, and the whole piece mycelia is like chain form.The mycelia branch is short, is acute angle-shaped branch.Conidia chain is short, is multiple dichotomy, and it is unicellular that spore mostly is, and size is about 20-25um, water white transparency, and no wart-like lump, avette or club-like, the spore trace is clear.
Bacterial strain of the present invention on the PDA nutrient agar, sprawl, fine and close, circular, the significantly two-layer line of taking turns, neat in edge, internal layer chocolate, edge canescence arranged.Hyphae colorless or olive brown, branch, tool diaphragm.Two kinds of mycelia of tool thickness, obviously.Mycelial growth is fast, and spore is abundant, can cover with the media surface of 9cm in five days.Mycelia forms lignitoid's lump in media surface.Normal transparent, long cryptomere or the beads shape gemma that forms single or bunchiness of children mycelia is thicker more than 2～3 times than mycelia.Gemma can be sprouted into new mycelia.The mycelia top is a point, thin, transparent growing cells.
Bacterial strain of the present invention produces three kinds of spores, first kind is conidium (gas is given birth to spore), grow spore chain by mycelia branch top, each branch top all can form spore chain, spore chain generally is made up of 4～6 spores, minority can reach 8～10, and conidiophore mostly is 4 cells, cylindrical, transparent, Dan Sheng, upright.The children conidium is obovate or ellipse, shows the particulate state point line that gathers, and color is dark, does not see separation, about 21～25 * 11～15um of size, and mycelia is wide to be 4um, the long 2.0um of beak.From the spore top or the side form spore chain again.Sophisticated conidium is an obovate, and look shallow, and about 25～15um has 4～6～8 tabulas, 1～2 mediastinum.Having between spore cell hangs contracts, the long 2.0um of beak.Second kind is gemma, produces in mycelial growth initial stage and liquid nutrient medium.Should be the peculiar spore of endogenetic fungus, long cryptomere or spherical shape can be sprouted the formation mycelia from the side.The third spore is first discovery, still is not reported, and promptly two mycelia parallel, and produce a joint pipe, produces transparent, a beige oblong spore at joint pipe middle part, and sophisticated spore is by 4～6 tabulas, 2～3 mediastinums.Its growth pattern is similar to the zygogamy of algae water silk floss, but is different from the syngenesis of head mold.
New bacterial strain CGMCC 0899 decapacitation of the present invention produces outside the new compound of the present invention, can also produce anti-cancer medicine paclitaxel.
A kind of method that new bacterial strain CGMCC 0899 of the present invention produces formula of the present invention (I) new compound of using provided by the invention, it is included in the substratum cultivates bacterial strain CGMCC 0899 of the present invention so that produce in described strain cell and in the described substratum and assemble formula of the present invention (I) new compound, and reclaims and purification formula (I) new compound in described cell and the substratum.
Described substratum can be the conventional or known substratum in this area.Described substratum can contain carbon source, nitrogen source.Can also in substratum, add other organic or inorganic material to promote the productive rate of microbial growth and raising formula of the present invention (I) new compound.Described carbon source includes but not limited to glucose, sucrose, maltose, fructose, glycerine, starch, lactose, semi-lactosi.Spendable nitrogenous source includes but not limited to peanut powder, analysis for soybean powder, corn steep liquor, yeast powder, peptone, beef extract, yeast extract, ammonium nitrate, ammonium chloride.The ratio of carbon source and nitrogenous source can be preferably 150: 1～40: 1.Described inorganics includes but not limited to phosphoric acid salt, magnesium salts, and sal epsom for example, molysite, for example ferrous sulfate, iron trichloride, sodium salt is as Seignette salt.Described substratum can include but not limited to trace element, for example boric acid, potassiumiodide, cobalt dichloride, zinc sulfate, manganous sulfate, elicitor, for example methyl jasmonate (mj), arachidonic acid, ammonium citrate, ceric ammonium nitrate, potassium permanganate, pyruvic acid, right-coumaric acid, Vanadosulfuric acid, urobenzoic acid, α-Nai Yisuan, 6-benzyl aminopurine, Silver Nitrate, styracin etc., precursor substance, for example phenylalanine, benzamide, Sodium Benzoate, sodium acetate, ethanamide, propionic acid amide, phenylformic acid, ammonium acetate etc.
Described cultivation can be carried out under the routine of this area or known conditions.Described fermentation can be under aerobic conditions, and temperature can be 23～29 ℃, and the fermentation initial pH value can be 5.5～11.0, and is preferred 6.0～7.0, and can regulate the pH value in the fermentation middle and later periods is 6.0～7.5.Incubation time is decided according to culture condition, and for example, fermentation can be carried out 6～9 days.In described fermenting process, preferably when inoculation, add elicitor methyl jasmonate (mj), arachidonic acid, ammonium citrate, ceric ammonium nitrate, potassium permanganate; Preferred its concentration is (accounting for substratum) 0.005%～0.1%.In described fermenting process, thalli growth can add materials such as precursor substance phenylalanine, benzamide, Sodium Benzoate, sodium acetate, ethanamide to logarithmic growth during the later stage; Preferred its concentration is (accounting for substratum) 0.005%～0.1%.Available during the fermentation basic solution is regulated the pH value.
Described fermenting process can carry out under this area routine or known devices and condition, for example can use to shake bottle and carry out under the conventional or known rotating speed in this area; Also can in the fermentor tank of routine, carry out, for example 7L, 50L fermentor tank.
When needing to use 7L, 50L fermentor tank in described fermenting process, stream adds supplementary carbon source during the fermentation, need not regulate the pH value at earlier fermentation, and can regulate the pH value in the fermentation middle and later periods is 6.0～7.0.Can adopt mycelium to advance the vaccination ways of jar.Can use big air flow early stage to thalli growth to platform after inoculation, and air flow can reduce after plateau.
In order from substratum, to isolate formula of the present invention (I) new compound, can use the conventional or known any separation method that is used for separating metabolite in this area from the substratum of microorganism.For example, mycelium can be separated from fermented liquid by centrifugal or filtration, and formula of the present invention (I) new compound can not extract from filtrate with the organic solvent (for example halon such as methylene dichloride, trichloromethane etc.) that water mixes with one or more.On the other hand, formula of the present invention (I) new compound that is comprised in the isolated mycelium can extract from mycelium by for example using one or more organic solvents (for example acetone, ethyl acetate or methyl alcohol).Gained formula of the present invention (I) new compound crude product can use this area conventional or known purification process purifying, for example chromatography, crystallization process.
The method of extraction and purifying formula of the present invention (I) new compound can may further comprise the steps from described cell and substratum: (1) isolates fermentation thalline and fermented supernatant fluid with described culturing process gained nutrient solution; (2) with the fermentation thalline of the first organic solvent extraction gained, the fermented supernatant fluid with the second organic solvent extraction gained volatilizes solvent; (3) with method and the crystalline method purifying of (2) step gains, get product formula of the present invention (I) new compound with chromatogram purification.
After described fermenting process finishes, can under room temperature or subambient temperature, preferably be lower than room temperature; the pH value of regulating the gained fermented liquid with diluted acid or dilute alkaline soln is 2～9; under the normal or lucifuge, preferred lucifuge carries out carrying out described sepn process again after the pre-treatment.Can separate by this area routine or known method in described (1) step, for example, the centrifugal or filtration with the gained fermented liquid.
Can use organic solvent extraction again with after the dry pulverizing of gained zymophyte soma in described (2) step.Described drying can be according to this area conventional or known method carry out, the oven dry under for example seasoning, the 60 ℃ of following temperature, vacuum lyophilization etc., preferred vacuum lyophilization is preferably carried out under the lucifuge condition.Described first organic solvent that extraction formula of the present invention (I) new compound is used from the fermentation thalline can be the conventional or known solvent in this area, and for example acetone, ethyl acetate, methyl alcohol, ethanol, methylene dichloride, trichloromethane etc. are preferably used ethyl acetate.Described second organic solvent that extraction formula of the present invention (I) new compound is used from fermented supernatant fluid can be conventional or known solvent, for example methylene dichloride, a trichloromethane etc. of this area.Described volatilize solvent can be according to this area conventional or known method carry out for example volatilization, concentrating under reduced pressure etc. naturally.
Chromatogram purification described in described (3) step can carry out according to the conventional or known method of this area, chromatographic column filler can be with silica gel or aluminum oxide etc., moving phase can be used n-hexane/ethyl acetate, ethanol/methylene, ethanol/dichloromethane, acetone/methylene dichloride etc., also can use more than one chromatographic column.
Described crystalline method is that this area routine or known method are carried out.Described crystallisation process can be: gains are dissolved in the methyl alcohol equal solvent, add a certain proportion of pure water then, put in 4～10 ℃ of refrigerators 1～12 hour, formula of the present invention (I) new compound crystal is separated out, filter.Repeat aforementioned crystallisation process, up to reaching certain purity, filter, vacuum-drying promptly gets the pure product of formula of the present invention (I) new compound.Described crystallisation process also can be: gains are dissolved in the ethyl acetate equal solvent, add the sherwood oil equal solvent, be cooled to 4～10 ℃, the crystallization of formula of the present invention (I) new compound is separated out, filter, repeat aforementioned crystallisation process, up to reaching certain purity, filtration obtains crystal, and drying under reduced pressure promptly gets purified product.
The active determination in vitro result of formula of the present invention (I) compound shows that this compound has biologic activity such as antitumor, antimycotic and antiviral.
Therefore, the present invention also provides a kind of anti-tumor drug, and it contains formula of the present invention (I) compound.
The present invention also provides a kind of antifungal medicament, and it contains formula of the present invention (I) compound.
The present invention also provides a kind of antiviral drug, and it contains formula of the present invention (I) compound.
The present invention also provides the application of formula of the present invention (I) compound in preparing antitumor, antimycotic and/or antiviral.
The present invention also provides a kind of pharmaceutical composition, and it contains formula of the present invention (I) compound as effective constituent, and contains conventional or known pharmaceutically acceptable pharmaceutical carrier.Pharmaceutical composition of the present invention can be prepared into various routines or known formulation.
Because unique space structure of new compound of the present invention, make its have significantly different with the existing compound of aforementioned structural formula (b) and can't suspect by the known performance of the existing compound of aforementioned structural formula (b), the unique biological activity.The existing compound of structural formula (b) is considered to a kind of β-1, and (the existing compound of structural formula (a) is considered to a plant growth regulators to the 3-dextran for β-1,3-glucan) biosynthesis inhibitor.New compound of the present invention then has very strong biologic activity to multiple cancers such as cancer of the stomach, leukemia, ovarian cancers, simultaneously, this material also has efficient antifungal (for example pathogenic bacterium such as trichophyton, alpha fungus, Candida albicans, Bacillus subtilus), antiviral biological activity, cancer therapy drug, antifungal drug and antiviral as a new generation have a extensive future.
Description of drawings
What Fig. 1,2 showed is the spore of new bacterial strain of the present invention.
What Fig. 3 showed is the mycelium of new bacterial strain of the present invention.
Fig. 4 is the TLC figure of formula (I) compound sample that makes of the inventive method.
Fig. 5 is the HPLC collection of illustrative plates of formula (I) compound sample that makes of the inventive method.
Fig. 6 is the UV collection of illustrative plates of formula (I) compound sample that makes of the inventive method.
Fig. 7 is the IR collection of illustrative plates of formula (I) compound sample that makes of the inventive method.
Fig. 8 is the FAB-MS collection of illustrative plates of formula (I) compound sample that makes of the inventive method.
Fig. 9 is the serial NMR collection of illustrative plates of formula (I) compound sample that makes of the inventive method: comprise
1H-NMR (Fig. 9 A),
13C-NMR (Fig. 9 B) and
1H-
1H COSY composes (Fig. 9 C).
Figure 10 is the x-ray diffraction pattern of formula (I) compound that makes of the inventive method.
Figure 11 is that formula of the present invention (I) compound is to different cell killing percentage curves.
Figure 12 is that formula of the present invention (I) compound is to people's Gastric Cancer MGC-803 Nude Mice Growth Inhibition curve.
Embodiment
Below will the invention will be further described by embodiment, these descriptions are not that content of the present invention is done further to limit.One skilled in the art will understand that to be equal to replacement to what the technology of the present invention feature did, or corresponding the improvement, still belong within protection scope of the present invention.
The branch of our taxusyunnanensis (Taxus yunnanensis) tree of 7 strains from the theropencedrymion of 2500～3000 meters of the old Jun Mountain height above sea level of Lijiang Prefecture, Yunnan Province more than 300 years, bark, 310 of leaf and Gen Pi collected specimens, respectively be cut into the fritter of 1cm * 1cm size, through 50～90% alcohol disinfectings after 5 minutes, use homogenizer homogenate, afterwards, coat on the PDA substratum, cultivate after 4-5 days for 25 ℃, the mycelia that grows is transferred to the inclined-plane, cultivated 4 days for 20 ℃～35 ℃, get the 1cm2 thalline and be inoculated in (250ml triangular flask loading amount 100ml in the fermention medium, culture medium prescription: glucose 2%, soybean cake powder 0.5%, groundnut meal 0.2%, MgSO4:0.01%, KH2PO4:0.05%), 25 ℃, 300rpm cultivated 16 days in shaking table, filter with double gauze, get wet thallus, wet thallus is (60 ℃) oven dry in baking oven, use ethyl acetate lixiviate (20 gram dry myceliums add the 100ml chloroform) 12 hours again, vacuum concentration to do the medicinal extract sample, add the 1ml methanol solution, get the 5ul point sample on silica gel plate, in three kinds of different layers analysis systems, launch (ethyl acetate: Virahol=95: 5v/v, chloroform: methyl alcohol=7: 3v/v, chloroform: acetonitrile=7: 3v/v) afterwards 60 ℃, oven dry in 30 minutes, develop the color with 5% vitriolic ethanolic soln (v/v), select the pairing thalline of spot with formula (I) compound and the identical mobility of taxol, further identify, obtain isolated microbial strains (bacterium in the Chinese yew, fungi), the final evaluation, select the endogenetic fungus of energy presentation (I) compound and taxol.
The purifying of the new bacterial strain of embodiment 2 presentations (I) compound is identified
With this bacterial strain is that starting strain carries out ultraviolet mutagenesis, gets a morphologic variation bacterial strain UV-012 through the mutagenesis of 12 generations, is that starting strain is inherited mutagenesis again with UV-012, gets a presentation (I) compound and the more stable single-ascospore strain of taxol, and code name is ST-026.Above morphologic variation through 5 generations go down to posterity cultivate more stable.According to the mutation of plant rules of nomenclature called after chain lattice spore monospore, formal name used at school is Alternaria alernatavar.monosporus, code name is ST-026-R, culture presevation is at China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 13, North of Zhongguancun, Beijing one bridge, preserving number is: CGMCC No.0899.
Embodiment 3 seed culture
Table 1: seed culture medium
| Composition | ％ |
| Glucose soybean cake powder groundnut meal MgSO
4 NaH
2PO
4Sterilized water is regulated | 2 0.5 0.2 0.01 0.05 100ml 7.0～7.2 |
Embodiment 4 shake flask fermentations
Loading amount is that 100ml/250ml triangular flask, culture temperature are 25 ℃, rotating speed 150rpm.The inoculation back added 0.005% benzamide, 0.01% phenylalanine, 0.04% sodium acetate on the 4th day.Fermentation period is 9 days.Method purified product according to embodiment 24.The result: the thalline biomass is 50g/L, and the output of target product formula (I) compound is 780mg/L.
Embodiment 5 to 8 shake flask fermentations
Method according to embodiment 4 is carried out shake flask fermentation, and just substratum is respectively:
Embodiment 5: sucrose 50g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 1.2g/L, vitamins B
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH 6.8.The result: the thalline biomass is 45g/L, and the output of target product formula (I) compound is 700mg/L.
Embodiment 6: glucose 10g/L, sucrose 30g/L, soybean cake powder 10g/L, yeast extract 0.5g/L, sal epsom 0.1g/L, vitamins B
110mg/L, dipotassium hydrogen phosphate 0.56g/L, potassium primary phosphate 0.35g/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH 6.8.The result: the thalline biomass is 48g/L, and the output of target product formula (I) compound is 750mg/L.
Embodiment 7: sucrose 40g/L, soybean cake powder 5g/L, yeast extract 0.5g/L, sal epsom 1g/L, dipotassium hydrogen phosphate 0.6g/L, Seignette salt 5g/L, vitamins B
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH 6.8.The result: the thalline biomass is 50g/L, and the output of target product formula (I) compound is 760mg/L.
Embodiment 8: glucose 5g/L, sucrose 20g/L, Zulkovsky starch 10g/L, soybean cake powder 3g/L, yeast extract 0.5g/L, sal epsom 0.1g/L, vitamins B
110mg/L, dipotassium hydrogen phosphate 0.56g/L, potassium primary phosphate 0.35g/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH 6.8.The result: the thalline biomass is 47g/L, and the output of target product formula (I) compound is 760mg/L.
The amount that adds elicitor during inoculation is respectively:
Embodiment 5: arachidonic acid 0.005%, ceric ammonium nitrate 0.06%, potassium permanganate 0.01%;
Embodiment 6: methyl jasmonate (mj) 0.01%, ceric ammonium nitrate 0.006, potassium permanganate 0.02%;
Embodiment 7: arachidonic acid 0.05%, ammonium citrate 0.005%, potassium permanganate 0.008%;
Embodiment 8: ammonium citrate 0.05%, ceric ammonium nitrate 0.02%.
The prerequisite material that the inoculation back added on the 4th day is respectively:
Embodiment 5: benzamide 0.008%, phenylalanine 0.02%, sodium acetate 0.02%;
Embodiment 6: phenylalanine 0.007%, Sodium Benzoate 0.05%, ethanamide 0.01%;
Embodiment 7: benzamide 0.02%, sodium acetate 0.03%, ethanamide 0.005%;
Embodiment 8: phenylalanine 0.002%, sodium acetate 0.01%, ethanamide 0.09%.
The fermentation of embodiment 9 7L jars
According to the method for embodiment 3, just temperature is 25 ℃, 150rpm, cultivation 24 hours, obtain the mycelium of first order seed, adopt mycelium to advance a jar method, inoculum size 10%, be inoculated into substratum (the glucose 10g/L in the 7L fermentor tank, sucrose 20g/L, soybean cake powder 2.5g/L, yeast powder 0.5g/L, sal epsom 0.3g/L, potassium primary phosphate 0.35g/L, dipotassium hydrogen phosphate 0.56g/L, VB
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8) in.Add elicitor simultaneously in inoculation: arachidonic acid (accounting for substratum concentration 0.01%), ceric ammonium nitrate (0.02%), potassium permanganate (0.02%).Fermenting process mid-early stage pH value nature (need not control), thalli growth reached after plateau after 24 hours, with the sodium hydroxide solution control pH value of 2N between 6.0～7.0.In earlier stage air flow requirement is bigger to thalli growth to platform after inoculation, for example controls oxygen dissolving value and reaches more than 30%, and air flow reduces after plateau, for example controls oxygen dissolving value and reaches below 20%, helps the synthetic of formula (I) new compound.In back 36 hours sucrose of inoculation, continue 24 hours with feeding method adding 1%.In the sucrose of inoculation back disposable adding 2% in the 6th day and 2% maltose.Fermenting by 96 hours disposable adding precursor substance 0.005% benzamide, 0.01% phenylalanine, 0.04% sodium acetate.Fermentation period is 9 days.The result: the output of end product formula (I) new compound is 805mg/L.
The fermentation of embodiment 10 7L jars
Bacterial classification is embodiment 2 gained bacterial classifications with conventional spore substratum (rice, glucose, agar) in 23～29 ℃ of strain inclined planes of cultivating 5～9 days, and inoculum size is 2 * 107 spores/1L substratum.The employing spore directly advances the method for jar, with substratum (glucose 10g/L, sucrose 20g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L sal epsom 0.3g/L, potassium primary phosphate 0.35g/L, dipotassium hydrogen phosphate 0.56g/L, the vitamins B of gained spore inoculating to the 7L fermentor tank
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8) in, add 0.01% ethanamide during inoculation.Whole fermentation process arriving the growth platform later stage after the inoculation by adjusting air flow control oxygen dissolving value more than 30%, later stage after is controlled oxygen dissolving value 20% below in growth platform with hydrochloric acid control pH6.0～7.0 of sodium hydroxide and the 2N of 2N.And in back 36 hours of inoculation, the method that adds with stream added 1% sucrose, continued 24 hours; Add 1% substratum in the 6th day stream in inoculation back, continue 24 hours, add the ethanamide of precursor 0.1% fermenting to the growth platform later stage.Fermentation period is 9 days.The result: end product formula (I) new compound output is 760mg/L.
The fermentation of embodiment 11 7L jars
According to the method fermentation of embodiment 10, just fermention medium is: sucrose 50g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 3.0g/L, vitamins B
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8.Adding elicitor during inoculation is: ethanamide (accounting for substratum concentration 0.09%), potassium permanganate (0.01%), ceric ammonium nitrate (0.01%), arachidonic acid (0.005%).Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage, and in disposable precursor 0.1% ethanamide that adds of growth platform later stage, fermentation period is 9 days.The result: the output of end product formula (I) new compound is 755mg/L.
The fermentation of embodiment 12 7L jars
Method according to embodiment 3 is cultivated seed, and seed culture medium is: glucose 20g/L, soybean cake powder 5g/L, groundnut meal 2g/L, sal epsom 0.1g/L, SODIUM PHOSPHATE, MONOBASIC 0.5g/L, pH6.8.Seed is in 25 ℃, and 250rpm cultivated 16 hours.
Adopt conventional mycelium to advance a jar method, the gained seed is inoculated in the 7L fermentor tank, inoculum size is 15%, and fermention medium is (sucrose 20g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 3.0g/L, vitamins B
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8).Add elicitor during inoculation: ethanamide (accounting for substratum concentration 0.01%), potassium permanganate (0.002%), ceric ammonium nitrate (0.005%), arachidonic acid (0.005%).Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage.Inoculate back 36 hours and begin stream and add 2% sucrose, continue 24 hours, disposable adding precursor 0.1% ethanamide in 96 hours, stream adds 1% substratum simultaneously, continues 12 hours.Fermentation period is 9 days, the result: the output of end product formula (I) new compound is 758mg/L.
The fermentation of embodiment 13 7L jars
Carry out the fermentation of 7L jar according to the method for embodiment 12, add elicitor when just inoculating: potassium permanganate (0.02%), ceric ammonium nitrate (0.05%), arachidonic acid (0.005%); Inoculate back 36 hours and begin stream and add 2% sucrose, continue 24 hours, disposable adding precursor 0.01% phenylalanine, 0.04% Sodium Benzoate in 96 hours, 0.01% ethanamide, stream adds 1% substratum simultaneously, fermentation period is 9 days, the result: the output of end product formula (I) new compound is 765mg/L.
The fermentation of embodiment 14 50L jars
Method according to embodiment 3 is cultivated seed, and the gained first order seed is inoculated into substratum (glucose 10g/L, sucrose 20g/L, soybean cake powder 2.5g/L, yeast powder 0.5g/L, sal epsom 0.3g/L, potassium primary phosphate 0.35g/L, dipotassium hydrogen phosphate 0.56g/L, VB
110mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8) in.Add elicitor simultaneously in inoculation: arachidonic acid (0.01%), ceric ammonium nitrate (0.05%), potassium permanganate (0.02%).Fermenting process mid-early stage pH nature (8.0).In inoculation back 36 hours sucrose and 0.2% yeast powders, continue 24 hours with feeding method adding 1%.In the sucrose of inoculation back disposable adding 2% in the 6th day and 2% maltose.Fermenting by 96 hours disposable adding precursor substance 0.005% benzamide, 0.01% phenylalanine, 0.04% sodium acetate.After adding precursor, control the pH value between 6.0～7.0 with the sodium hydroxide solution of 2N.Fermentation period is 9 days.In earlier stage air flow requirement is bigger to thalli growth to platform after inoculation, reaches more than 30% as the control oxygen dissolving value, and air flow reduces after plateau, reaches below 20% as the control oxygen dissolving value, helps synthesizing of formula (I) new compound.The result: the output of end product formula (I) new compound is 780mg/L.
The fermentation of embodiment 15 50L jars
Carry out the fermentation of 50L jar according to the method for embodiment 14, following difference just arranged:
Seed culture medium is: glucose 20g/L, soybean cake powder 5g/L, groundnut meal 2g/L, sal epsom 0.1g/L, SODIUM PHOSPHATE, MONOBASIC 0.5g/L, pH6.8.Seed is in 25 ℃, and 250rpm cultivated 16 hours.
Adopt conventional mycelium to advance a jar method, the gained mycelium is inoculated in the 50L fermentor tank, inoculum size is 15%, fermention medium be (glucose 10g/L, sucrose 20g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 3.0g/L, VB11 0mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8).Add elicitor during inoculation: ethanamide (0.01%), potassium permanganate (0.02%), ceric ammonium nitrate (0.05%), arachidonic acid (0.005%); Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage.Inoculate back 36 hours and begin stream and add 2% sucrose+0.1% yeast extract, continue 24 hours, disposable adding precursor 0.1% ethanamide in 96 hours, stream adds 1% substratum simultaneously, continues 12 hours.Fermentation period is 9 days, the result: the output of end product formula (I) new compound is 740mg/L.
The fermentation of embodiment 16 50L jars
Carry out the fermentation of 50L jar according to the method for embodiment 14, following difference just arranged:
Seed culture medium is: glucose 10g/L, sucrose 30g/L, soybean cake powder 5g/L, groundnut meal 2g/L, sal epsom 0.1g/L, SODIUM PHOSPHATE, MONOBASIC 0.5g/L, pH6.8.Seed is in 25 ℃, and 250rpm cultivated 16 hours.
Adopt conventional mycelium to advance a jar method, the gained mycelium is inoculated in the 50L fermentor tank, inoculum size is 15%, fermention medium be (glucose 10g/L, sucrose 40g/L, soybean cake powder 5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 3.0g/L, VB11 0mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8).Add elicitor during inoculation: ethanamide (0.01%), potassium permanganate (0.02%), ceric ammonium nitrate (0.05%), arachidonic acid (0.005%); Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage.Disposable adding precursor 0.1% ethanamide in 96 hours, stream adds 1% substratum simultaneously, continues 12 hours.Fermentation period is 9 days, the result: the output of end product formula (I) new compound is 738mg/L.
The fermentation of embodiment 17 50L jars
Carry out the fermentation of 50L jar according to the method for embodiment 14, following difference just arranged:
Seed culture medium is: glucose 10g/L, sucrose 30g/L, soybean cake powder 5g/L, sal epsom 0.1g/L, SODIUM PHOSPHATE, MONOBASIC 0.5g/L, pH6.8 also can be conventional seed culture medium.Seed is in 25 ℃, and 250rpm cultivated 16 hours.
Adopt conventional mycelium to advance a jar method, the gained mycelium is inoculated in the 50L fermentor tank, inoculum size is 15%, fermention medium be (sucrose 50g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, lime carbonate 3.0g/L, VB11 0mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8).Add elicitor during inoculation: ethanamide (0.03%); Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage.Disposable adding precursor 0.1% ethanamide in 96 hours, stream adds 1% substratum simultaneously, continues 12 hours.Fermentation period is 9 days, the result: the output of end product formula (I) new compound is 725mg/L.
The fermentation of embodiment 18 50L jars
Carry out the fermentation of 50L jar according to the method for embodiment 14, following difference just arranged:
Seed culture medium is: glucose 20g/L, soybean cake powder 5g/L, groundnut meal 2g/L, sal epsom 0.1g/L, SODIUM PHOSPHATE, MONOBASIC 0.5g/L, pH6.8.Seed is in 25 ℃, and 250rpm cultivated 16 hours.
Adopt conventional mycelium to advance a jar method, the gained mycelium is inoculated in the 50L fermentor tank, inoculum size is 15%, fermention medium be (glucose 10g/L, sucrose 20g/L, soybean cake powder 2.5g/L, yeast extract 0.5g/L, sal epsom 0.06g/L, dipotassium hydrogen phosphate 0.56g/L, potassium primary phosphate 0.35g/L, VB11 0mg/L, iron trichloride 2mg/L, manganous sulfate 5mg/L, zinc sulfate 2.5mg/L, potassiumiodide 0.7mg/L, boric acid 1.4mg/L, pH6.8).Control between pH6.0～7.0 with 2N sodium hydroxide and 2N hydrochloric acid in the fermenting process.To the growth platform later stage, oxygen dissolving value is controlled at more than 30% after inoculation, and platform is controlled oxygen dissolving value below 20% after the later stage.Inoculate back 36 hours and begin stream and add 2% sucrose+0.1% yeast extract, continue 24 hours, disposable adding precursor 0.01% phenylalanine, 0.04% Sodium Benzoate, 0.05% sodium acetate in 96 hours, stream adds 1% substratum simultaneously, continues 12 hours.Fermentation period is 9 days, the result: the output of end product formula (I) new compound is 730mg/L.
The separation and the purifying of embodiment 19 to 23 products
Embodiment 9 to 13 gained fermented liquids are filtered with plate-and-frame filter press, collect the fermentation thalline and the fermented supernatant fluid that filter out.With the gained thalline respectively by dry up naturally, vacuum lyophilization, 60 ℃ of oven dry, 80 ℃, 100 ℃ oven dry, respectively dry thalline 151,146,145,143,142g/7L, dry thalline is pulverized, add (mycelium: solvent=1: 20) ethyl acetate, acetone, ethanol, methyl alcohol, ethyl acetate respectively, extracted respectively 24,26,30,30,25 hours, decompression rotary evaporation solvent gets medicinal extract.The TLC figure that embodiment 20 gained extract medicinal extract sees Fig. 4, and the HPLC collection of illustrative plates is seen Fig. 5.Formula (I) new compound that contains quite big content as seen from the figure in the sample.Spot that the arrow table marks among Fig. 4,5 or peak are the spot or the peaks of formula (I) new compound.
The fermented supernatant fluid that gained is filtered in the front adds the trichloromethane of equal volume or methylene dichloride, shaking out 1～2 hour, and high speed centrifugation (8000rpm, 10～30min), left standstill 10 minutes, isolate organic solvent layer, concentrating under reduced pressure gets medicinal extract.
With after the moving phase dissolving, solution is respectively charged into chromatographic column with the front gains: in silica gel or the aluminum oxide (200～400 orders, high (CM) 5 * 40 in post footpath), moving phase is respectively: 1%～10% ethanol/CH
2Cl
2Or methyl alcohol/CH
2Cl
2, 1%～30% acetone/CH
2Cl
2, in depress gradient elution, flow velocity 10～40ml/min collects purified product, concentrating under reduced pressure, formula (I) new compound of purity about 92%.
Formula (I) new compound of gained purity about 92% is dissolved in the methyl alcohol, slowly adds pure water, be heated to 40～60 ℃,, stop to add water, solution slowly is cooled to 4 ℃～10 ℃, filter when formula (I) when new compound is crystal formation.Drying under reduced pressure promptly gets purity and is 98% formula (I) new compound.Repeat aforementioned recrystallization process 2 to 3 times, filter, drying under reduced pressure promptly gets purity and is 99.5% formula (I) new compound.
The separation and the purifying of embodiment 24-25 product
With embodiment 14,15 gained fermented liquid centrifugations, collect fermentation thalline and fermented supernatant fluid.With the gained thalline lyophilize of fermenting, pulverize through pulverizer and to be that bacterium powder, dry bacterial powder add in the ethyl acetate, soak jolting 12 hours, filter extracting solution, rotary evaporation in vacuo gets medicinal extract.
The gained fermented supernatant fluid is added the trichloromethane or the methylene dichloride of equal volume respectively, and organic solvent layer is isolated in shaking out 1～2 hour behind the high speed centrifugation, and the decompression rotary evaporation gets medicinal extract.
With the front by thalline or fermented supernatant fluid gains medicinal extract with the moving phase dissolving after, solution is respectively charged into chromatographic column: in silica gel or the aluminum oxide (200～400 orders, high (CM) 5 * 40 in post footpath), moving phase is respectively: 1%～10% acetone/CH
2Cl
2, in depress gradient elution, flow velocity 20～60ml/min collects purified product, drying under reduced pressure, formula (I) new compound of purity about 92%.
Formula (I) new compound of gained purity about 92% is dissolved in the ethyl acetate, the sherwood oil that slowly adds 1.5～2 times of volumes, when the crystallization of formula (I) new compound occurs, solution slowly is cooled to 4 ℃～10 ℃, filter, repeat aforementioned recrystallization process 2～4 times, filter, drying under reduced pressure promptly gets purity and is formula (I) new compound more than 99.5%.
Embodiment 26
According to the method for embodiment 24, embodiment 4 to 8 and 16 to 18 gains are carried out separation and purification, get formula (I) new compound, total yield is 78%.
Embodiment 27
Method according to embodiment 19-26, embodiment 10 to 17 gains are carried out separation and purification, just before embodiment 10 to 17 gained fermented liquids separate, slowly splash in the fermented liquid with the HCl of 1mol/L or the NaOH solution of 1mol/L, constantly stir, transfer its pH to be respectively 6.8,6.5 (lucifuge is carried out during operation), 5.5,4.5 (lucifuge is carried out during operation), 3.0, (7.5 lucifuge is carried out during operation), 8.0,6.0 (lucifuge is carried out during operation).Get formula (I) new compound of purifying, total yield is 82%.
The evaluation of embodiment 28 products
Embodiment 19 to 27 gained formula (I) new compounds are carried out UV spectrum (UV), infrared spectra (IR), mass spectrum (MS), nucleus magnetic resonance (NMR), X light diffracting analysis, the results are shown in Figure 6 to Fig. 9.Formula (I) compound that makes of the inventive method has the structure of formula (I) as seen from the figure.
The external anticancer cell pharmacodynamic study of embodiment 29 formulas (I) compound
It below is test to the external anticancer cell pharmacodynamic study of formula (I) compound.
1. detection method: mtt assay
2. substratum: contain 2% calf serum RPMI-1640 substratum
3. instrument: (1) .CO
2Incubator: the U.S. thanks and steps on
(2) .318MC type microplate reader: SANCO Shanghai Sanke Instrument Co., Ltd
4. cell culture condition: temperature: 37 ℃; CO
2Concentration: 4.7%; Incubation time: 24-48 hour.
Primary dcreening operation the results are shown in Table 1 and Figure 11.Figure 11 has shown that formula (I) compound is to different cell killing percentage curves.
The result shows: embodiment 4 makes formula (I) compound sample under the concentration of 5ug/ml, L1210 (mouse lymph leukemia cell), B16 (mouse melanin tumor cell), A2780 (Proliferation of Human Ovarian Cell) and MGC (gastric carcinoma cells) had very high activity, its activity reaches 97.00%, 72.53%, 90.91%, 85.52% respectively, Eca-109 (esophageal cancer cell) there is not activity substantially, this illustrates that formula of the present invention (I) compound has selectivity to the kill capability of different cancer cells, rather than toxicity, can be developed into cancer therapy drug.
Table 1. formula (I) compound is to different cell killing percentage
| L1210 mouse lymph leukemia cell | The Eca-109 esophageal cancer cell | The B16 mouse melanin tumor cell | The A2780 Proliferation of Human Ovarian Cell | The MGC gastric carcinoma cells | |
| The methyl alcohol contrast | 4.28％ | 0.00％ | 0.00％ | 4.00％ | 14.9％ |
| 5ug/ml | 97.00％ | 0.18％ | 72.53％ | 90.91％ | 85.52％ |
| 2.5ug/ml | 94.18％ | 0.00％ | 51.75％ | 73.08％ | 64.37％ |
| 1.25ug/ml | 17.02％ | 0.00％ | 0.00％ | 46.50％ | 42.32％ |
| 625ng/ml | 0.00％ | 0.00％ | 6.37％ | 35.31％ | 40.98％ |
| 312.5ng/ml | 0.00％ | 0.00％ | 1.06％ | 0.00％ | 0.00％ |
Remarks: microplate reader detects wavelength and is: 546nm
Experiment purpose: observe formula of the present invention (I) compound to people's Gastric Cancer MGC 803 nude mice xenografts of human and action intensity.
Tried thing: get formula of the present invention (I) the compound 1.5g that embodiment 4 makes and put in the liquid mixing bottle, add an amount of polyoxyethylene fatty acid ester and N.F,USP MANNITOL, add water for injection again, fully stirring is dissolved it fully, and heat sterilization is sub-packed in 100 cillin bottles, freeze-drying in vacuum freeze drier, the freeze-dried preparation of formula (I) compound, the 15mg/ bottle faces with preceding usefulness 0.9% physiological saline and is diluted to desired concn.
Dosage is provided with: the dosage of formula (I) compound is 15mg/kg, 10mg/kg and 5mg/kg.
Animal: the BALB/cA nude mouse, female, 40-45 age in days, body weight 18 ± 2g.Every treated animal number: 12 of negative control group, 6 of administration groups.
Transplanted tumor: people's Gastric Cancer MGC-803 Nude Mice, it is subcutaneous and set up to be inoculated in nude mouse by the MGC-803 cell strain.The cell inoculation amount is 5 * 106, and after inoculation formed transplanted tumor, biography was used after 3 generations in the nude mouse body.
Experimental technique: the tumor tissue of getting the growth animated period cuts into about 1.5mm3, and under aseptic condition, it is subcutaneous to be inoculated in nude mouse right side armpit.Nude Mice is with vernier caliper measurement transplanted tumor diameter, treat tumor growth to the 100-200mm3 with the animal random packet.Each experimental group was in the 1st, the 6th day intravenous administration.Control group is given equivalent physiological saline.Measure diameter of tumor 2 times weekly, claim the mouse body weight simultaneously.Gross tumor volume (tumor volume, calculation formula TV) is:
TV＝1/2×a×b
2
Wherein a, b represent length and width respectively.(relative tumor volume, RTV), calculation formula is: RTV=V to calculate relative tumour volume according to the result who measures
t/ V
0V wherein
0(d during for minute cage administration
0) measurement gained gross tumor volume, V
tGross tumor volume when measuring each time.The evaluation index of anti-tumor activity is relative tumor proliferation rate T/C (%), and calculation formula is as follows:
T/C(％)＝(T
RTV/C
RTV)×100
T
RTV: treatment group RTV; C
RTV: negative control group RTV.
Therapeutic evaluation standard: T/C (%)＞60% is invalid, T/C (%)＜=60%, and processing p＜0.05 is effective by statistics.
Result and conclusion: the lyophilized injectable powder of formula (I) compound sees Table 2 and Figure 12 to the experimental treatment result of people's Gastric Cancer MGC-803 Nude Mice.Experimental result shows: with the lyophilized injectable powder 15mg/kg of formula (I) compound in the 1st, the 6th day intravenous administration, very obvious to people's Gastric Cancer MGC-803 nude mice xenografts of human, the T/C value is 0.18%, and three week of treatment, a back tumor-bearing mice tumour disappeared fully.
The lyophilized injectable powder of table 2 formula (I) compound is to people's Gastric Cancer MGC-803 Nude Mice
Experimental treatment result
| Group | Dosage | Administering mode | Number of animals | Body weight (g) | TV(mm 3) | RTV | T/C (％) | |||
| Beginning | At last | Beginning | At last | d 0 | d 21 | |||||
| Negative control group | 0.2ml/ only | i.v. | 12 | 12 | 19.0 | 19.8 | 107±48 | 923±475 | 10.5±8.0 | |
| Formula (I) compound | 15mg/kg， d1，d6 | i.v. | 6 | 6 | 18.3 | 17.2 | 102±22 | 2.2±4.3 | 0.02±0.04 | 0.18 |
Ex vivo method (ex vivo) the mouse contrast experiment of embodiment 31 formulas (I) compound lyophilized injectable powder
Control group: 6 of BDF-1 mouse ♂, abdominal injection L1210 (mouse lymph leukemia cell) (lyophilized injectable powder without formula (I) compound is handled), 1 * 10
5Individual/as only, to the results are shown in Table 3.
Ex vivo method (ex vivo) experimental group: 6 of BDF-1 mouse ♂, the L1210 (mouse lymph leukemia cell) that abdominal injection was handled through the lyophilized injectable powder of external use formula (I) compound, 1 * 10
5Individual/only, no longer administration in the body.Cell in vitro treatment process: the lyophilized injectable powder 7.5mg/ml of formula (I) compound, sucking-off 20ul+0.98ml physiological saline=150ug/ml, sucking-off 67ul (pastille 10ug)+L12101.5ml+ salt solution 0.433ml therefrom, every ml L1210 cell contains 5ug formula (I) compound, puts the room temperature effect 2 hours.The results are shown in Table 4.
Table 3 control group: abdominal injection L1210 cell (without formula (I) compound treatment)
| BDF-1♂ | Mean body weight | Time to live | Dead quantity | Remarks |
| 6 | 20.8g/ only | 12 days | 3 | |
| 13 | 1 | |||
| 15 | 2 |
Table 4 ex vivo method (ex vivo) experimental group: abdominal injection L1210 cell (through formula (I) compound treatment)
| BDF-1♂ | Mean body weight | Time to live | Remarks |
| 6 | 20.9g/ only | Still healthy survival in 60 days | Do not see disease symptom |
Above test-results shows: all be killed at the external L1210 (mouse lymph leukemia cell) that crosses through formula (I) compound treatment, the BDF-1 mouse is caused a disease.
Embodiment 32 formulas (I) compound lyophilized injectable powder is to the experimental therapy effect of mouse lymph leukemia cell L1210 transplantation model
Experiment purpose: observation type (I) compound is transplanted the prolongation effect of the time to live of animal model to mouse lymph leukemia cell L1210.
Experimental technique: get 18 of BDF-1 mouse ♀, abdominal injection L1210 (mouse lymph leukemia cell), 1 * 105/only, divide 3 groups at random, 6/group, each experimental group was in subcutaneous injection administration in the 1st, the 6th day.Control group is given equivalent physiological saline.
Tried thing: the lyophilized injectable powder of formula (I) compound, the 15mg/ bottle faces with preceding usefulness 0.9% physiological saline and is diluted to desired concn.
Dosage is provided with: test 1 group: subcutaneous injection, the lyophilized injectable powder of 50mg/kg formula (I) compound.
Test 3 groups: subcutaneous injection, the lyophilized injectable powder of 12.5mg/kg formula (I) compound.
Result and conclusion:
The lyophilized injectable powder of formula (I) compound sees Table 5 to the prolongation exercising result that mouse lymph leukemia cell L1210 transplants the animal model time to live.Experimental result shows: in subcutaneous injection administration in the 1st, the 6th day, the prolongation effect of mouse lymph leukemia cell L1210 being transplanted the animal model time to live was very obvious with the lyophilized injectable powder 50mg/kg of formula (I) compound, and rate elongation reaches 87%.
The lyophilized injectable powder of table 5 formula (I) compound is to mouse lymph leukemia cell L1210 transplantation model
The prolongation effect of time to live
| Group | Dosage | Administering mode | Time to live (on average) | The prolongation time to live (my god) | Time to live rate elongation (%) |
| Negative control group is tested 1 group and is tested 2 groups and test 3 groups | 0，d1，d6 50mg/kg，d1，d6 25mg/kg，d1，d6 12.5mg/kg，d1，d6 | Skin is annotated skin and is annotated. and skin is annotated. and skin is annotated. | 15 days 28 days 22 | - 13 7 5 | - 87％ 47％ 33％ |
The extracorporeal antivirus effect pharmacodynamic study of embodiment 33 formulas (I) compound
Below to the extracorporeal antivirus effect pharmacodynamic study of formula (I) compound.
Test material and method:
Virus strain: coxsackie B 3 viruses.
Sample preparation: formula (I) compound sample that embodiment 4 is made faces with before being dissolved in DMSO and is made into proper concn, makes 3 times of dilutions, totally 8 extent of dilution with nutrient solution.
Positive control drug: virazole (RBV)
Testing method: Veo cell kind 96 well culture plates, infect coxsackie B 3 viruses 1/210 respectively after 24 hours
-5(316TCID50 infective dose) absorption 2 hours, abandon viral liquid, add sample, establish cell control well and virus control hole simultaneously by above extent of dilution, 36 hours observation of cell lesion degrees (CPE) are with the half-inhibition concentration (IC50) of Reed-Muench method difference calculation sample to coxsackie B 3 viruses.
Test result sees Table 6.
Table 6
| Sample number into spectrum | TC 50(ug/ml) | Experiment starting point concentration (ug/ml) | IC 50(ug/ml) | SI |
| Formula (I) compound R BV | 24.7 ＞1000 | 1000 1000 | 8.56 447.8 | 2.88 ＞2.23 |
Annotate: "-" expression sample is at maximal non-toxic dosage nonreactive coxsackie B 3 virus activities in (1) table.
(2) TC
50: the poisonous concentration of half; IC
50: to viral half-inhibition concentration; SI: selectivity index, SI=TC
50/ IC
50
By table as seen, test result shows that the present invention makes the new compound sample and has anti-coxsackie B 3 virus functions.
The external antimycotic drug effect research of embodiment 34 formulas (I) compound
Below to the external antimycotic drug effect research of formula (I) compound.
Experiment purpose: observation type (I) compound is to the lethal effect of pathogenic bacterium such as trichophyton, alpha fungus, Candida albicans, Bacillus subtilus.
Experimental technique and operation: mtt assay.
The preparation of bacteria suspension: making bacterium dense with the medium preparation spore suspension is 2 * 103/ml.Spores such as trichophyton, alpha fungus, Candida albicans, Bacillus subtilus, the substratum of trichophyton, alpha fungus, Candida albicans is " sabouraud culture medium " (Huankai Microbiological Science ﹠ Technolgy Co., Guangdong), and the substratum of Bacillus subtilus is " bacteria culture medium " (Huankai Microbiological Science ﹠ Technolgy Co., Guangdong).
Sample concentration and dilution process:
Formula (I) the compound sample concentration of embodiment 4 preparation: 1. 2mg/ml; 2. 1mg/ml; 3. 0.5mg/ml; 4. 0.25mg/ml.
Sample usage quantity: every hole 50ul.
Result and conclusion:
Formula (I) compound all has a significant germicidal action to pathogenic bacterium such as trichophyton, alpha fungus, Candida albicans, Bacillus subtilus.The results are shown in Table 7.Experimental result shows: during with formula (I) compound amount 〉=50ug/ hole, its germicidal action reaches more than 99%.
Table 7 formula (I) compound to trichophyton, alpha fungus, Candida albicans,
The lethal effect of pathogenic bacterium such as Bacillus subtilus
Claims (17)
1. compound with following structural formula (I).
2. the microorganism of an energy production claim 1 described (I) compound, chain lattice spore monospore mutation (Alternaria alternate var.monosporus) ST-026-R CGMCC No.0899.
3. method for preparing the described formula of claim 1 (I) compound, it comprises: cultivate the described microorganism of claim 2 so that produce and focus type (I) compound in substratum in described microorganism cells and in the described substratum, and reclaim and purification formula (I) compound in described cell and the substratum.
4. method according to claim 3, it is characterized in that described substratum contains in the conventional or known carbon source material that glucose, sucrose, maltose, fructose, glycerine, starch, lactose, semi-lactosi and other help described microorganism growth one or more carbon source material, and help in the routine of described microorganism growth or the nitrogen sources known material one or more nitrogen source in peanut powder, analysis for soybean powder, corn steep liquor, yeast powder, peptone, beef extract, yeast extract, ammonium nitrate, ammonium chloride and other.
5. method according to claim 4 is characterized in that described substratum also contains phosphoric acid salt, magnesium salts, molysite, sodium salt and/or other help conventional or the known inorganic or organic salt material of described microorganism growth; And/or boric acid, potassiumiodide, cobalt dichloride, zinc sulfate, manganous sulfate and other help one or more the trace element in the conventional or known trace element of described microorganism growth.
6. method according to claim 4 is characterized in that described substratum also contains one or more the elicitor material in the conventional or known elicitor material that methyl jasmonate (mj), arachidonic acid, ammonium citrate, ceric ammonium nitrate, potassium permanganate, pyruvic acid, right-coumaric acid, Vanadosulfuric acid, urobenzoic acid, α-Nai Yisuan, 6-benzyl aminopurine, Silver Nitrate, styracin and other help described microorganism growth; And/or phenylalanine, benzamide, Sodium Benzoate, sodium acetate, ethanamide, propionic acid amide, phenylformic acid, ammonium acetate and other help the routine of described microorganism growth or one or more the precursor substance in the known precursors material.
7. method according to claim 3 is characterized in that described substratum contains:
Be selected from glucose, sucrose, maltose, fructose, glycerine, starch, lactose, the semi-lactosi one or more carbon source material;
Be selected from peanut powder, analysis for soybean powder, corn steep liquor, yeast powder, peptone, beef extract, yeast extract, ammonium nitrate, the ammonium chloride one or more nitrogen source;
Phosphoric acid salt, magnesium salts, molysite and/or sodium salt;
Be selected from one or more the trace element in boric acid, potassiumiodide, cobalt dichloride, zinc sulfate, the manganous sulfate;
Be selected from one or more the elicitor material in methyl jasmonate (mj), arachidonic acid, ammonium citrate, ceric ammonium nitrate, potassium permanganate, pyruvic acid, right-coumaric acid, Vanadosulfuric acid, urobenzoic acid, α-Nai Yisuan, 6-benzyl aminopurine, Silver Nitrate, the styracin; And
Be selected from one or more the precursor substance in phenylalanine, benzamide, Sodium Benzoate, sodium acetate, ethanamide, propionic acid amide, phenylformic acid, the ammonium acetate.
8. according to one of any described method in the claim 3 to 7, it is characterized in that described cultivation under aerobic conditions carries out, temperature is 23～29 ℃, and the fermentation initial pH value is 5.5～11.0, and regulating the pH value in the fermentation middle and later periods is 6.0～7.5; If described microorganism is in 7L, 50L or other suitable conventional or known fermentation cylinder for fermentation, vaccination ways adopts routine or mycelium to advance the vaccination ways of jar, stream adds supplementary carbon source and/or nitrogenous source in described fermenting process, need not regulate the pH value at earlier fermentation.
9. according to claim 6 or 7 described methods, it is characterized in that described elicitor material is to add when inoculation, its concentration is (accounting for substratum) 0.005%～0.1%; Described precursor substance is in described fermentation process, and thalli growth added to the logarithmic growth later stage, and its concentration is (accounting for substratum) 0.005%～0.1%.
10. according to one of any described method in the claim 3 to 7, it is characterized in that the step of described recovery and purification formula (I) compound may further comprise the steps: (1) isolates fermentation thalline and fermented supernatant fluid with described culturing process gained nutrient solution; (2) with the fermentation thalline of the first organic solvent extraction gained, the fermented supernatant fluid with the second organic solvent extraction gained volatilizes solvent; (3) with method and the crystalline method purifying of (2) step gains, get product formula (I) compound with chromatogram purification;
Use one or more chromatographic column in the chromatogram purification method described in described (3) step, chromatographic column filler comprises silica gel or aluminum oxide, and moving phase comprises n-hexane/ethyl acetate, ethanol/methylene, ethanol/dichloromethane, acetone/methylene dichloride;
Crystallization method described in described (3) step is: thing to be purified is dissolved in the methanol solvate, adds entry, put in 4～10 ℃ of refrigerators 1～12 hour, formula (I) compound crystal is separated out, filter; Or thing to be purified is dissolved in the ethyl acetate solvent, add sherwood oil, be cooled to 4～10 ℃, formula (I) compound crystal is separated out, filter; Aforesaid crystallisation process carries out once or once; Drying promptly gets pure formula (I) compound.
11. method according to claim 10, the pH value that it is characterized in that before described (1) step regulating with acid or alkaline solution described culturing process gained nutrient solution is 2～9.
12. contain the pharmaceutical composition of the described formula of claim 1 (I) compound.
13. an anti-tumor drug, it contains the described formula of claim 1 (I) compound.
14. an antifungal medicament, it contains the described formula of claim 1 (I) compound.
15. an antiviral drug, it contains the described formula of claim 1 (I) compound.
16. the application of the described formula of claim 1 (I) compound in preparing antitumor, antimycotic and/or antiviral.
17. the described formula of claim 1 (I) compound comprises the medicine of diseases such as leukemic lymphoblastoid, melanoma cell, ovarian cancer, cancer of the stomach in preparation treatment, and the application in the medicine of anti-coxsackie B 3 viruses, trichophyton, alpha fungus, Candida albicans, Bacillus subtilus etc.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174607A (en) * | 2011-02-25 | 2011-09-07 | 中山大学 | Method for increasing output of active metabolites of microorganisms |
| CN102191184A (en) * | 2011-04-08 | 2011-09-21 | 中国计量学院 | Biocontrol endophytic fungi-Alternaria alternata |
| CN102204570A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of alternaria alternate metabolic products in cucumber rhizoctonia solani prevention and control |
| CN104059950A (en) * | 2014-04-02 | 2014-09-24 | 广东双骏生物科技有限公司 | Preparation method for compound |
| CN113243391A (en) * | 2021-05-21 | 2021-08-13 | 山东农业大学 | Sterilization composition containing Taishan mycin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996032490A1 (en) * | 1995-04-14 | 1996-10-17 | Novopharm Limited | Fermentation for taxol production |
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2005
- 2005-09-29 CN CNB2005101056645A patent/CN100420679C/en active Active
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174607A (en) * | 2011-02-25 | 2011-09-07 | 中山大学 | Method for increasing output of active metabolites of microorganisms |
| CN102191184A (en) * | 2011-04-08 | 2011-09-21 | 中国计量学院 | Biocontrol endophytic fungi-Alternaria alternata |
| CN102204570A (en) * | 2011-04-08 | 2011-10-05 | 中国计量学院 | Application of alternaria alternate metabolic products in cucumber rhizoctonia solani prevention and control |
| CN102191184B (en) * | 2011-04-08 | 2012-09-12 | 中国计量学院 | Biocontrol endophytic fungi-Alternaria alternata |
| CN102204570B (en) * | 2011-04-08 | 2013-01-30 | 中国计量学院 | Application of alternaria alternate metabolic products in cucumber rhizoctonia solani prevention and control |
| CN104059950A (en) * | 2014-04-02 | 2014-09-24 | 广东双骏生物科技有限公司 | Preparation method for compound |
| CN104059950B (en) * | 2014-04-02 | 2017-01-04 | 广东双骏生物科技有限公司 | A kind of preparation method of compound |
| CN113243391A (en) * | 2021-05-21 | 2021-08-13 | 山东农业大学 | Sterilization composition containing Taishan mycin |
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| CN100420679C (en) | 2008-09-24 |
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