CN107287131A - A kind of micromonospora, its metabolite Macrocyclic lactams compound and the application in antitumor - Google Patents
A kind of micromonospora, its metabolite Macrocyclic lactams compound and the application in antitumor Download PDFInfo
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Abstract
The invention discloses a kind of micromonospora, its metabolite Macrocyclic lactams compound and the application in antitumor.Micromonospora (Micromonospora sp.) FIM05 328 of the present invention, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is：CGMCC NO.13933, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is on March 27th, 2017.Micromonospora (Micromonospora sp.) FIM05 328 of the present invention metabolite can obtain Macrocyclic lactams compound through isolating and purifying, and the Macrocyclic lactams compound can be used for preparing anti-tumor drug.The present invention is isolated and purified by the cytotoxicity reactive compound produced to micromonospora, and compound candidate is provided for the innovative development of antineoplastic, and the drug resource exploitation to microorganism is also significant.
Description
Technical field
The invention belongs to microorganism and its applied technical field of natural products, it is more particularly related to a kind of small
Sporangium (Micromonospora sp.) FIM05-328 and its secondary metabolite isolate and purify obtained Macrocyclic lactams
Compound, and by the compound be applied to it is antitumor in.
Background technology
The incidence of disease and the death rate of tumour are in rising trend in recent years, and cancer turns into seriously endangers the of human health
One killer.The treatment of current tumour is still based on drug therapy, therefore, and the research and development of antineoplastic have before huge market
Scape, the antineoplastic for researching and developing new high-efficiency low-toxicity is significant.
Microbial resources provide infinite source for innovation drug research, and being found from Secondary Metabolites of Microorganisms has
The compound of activity is one of effective way that world pharmaceutical worker generally acknowledges as lead compound Development of New Drugs.Microorganism
Rich and varied secondary metabolite for chemicals new drug research and exploitation provide a large amount of extremely mode configurations of preciousness and
Prodrug small molecule, is the important substance basis of drug discovery source treatment and Continuous Innovation, whole original new drug is ground
Study carefully with decisive significance.
Actinomyces are the main sources of microbial activity secondary metabolite, wherein with streptomyces and some rare put
Based on line bacterium.In rare actinomycete, especially with the novel active of micromonospora (Micromonospora) generation most
It is many, it is only second to streptomyces.At present be found that from micromonospora (Micromonospora) gentamicin, micronomicin,
The weight such as megalomicin, mycinamicin, Dynemicin A, Calicheamicin, ai sibo mycin, kerdarcidin and halomicin
Want antibiotic.
The content of the invention
It is an object of the invention to：According to the medicine of current treatment tumour it is still not comprehensive enough effective the problems such as there is provided one kind
Micromonospora (Micromonospora sp.) FIM05-328 and its secondary metabolite isolate and purify acyl in obtained big ring
Amines, and by the compound be applied to it is antitumor in.
In order to realize foregoing invention purpose, the invention provides a kind of micromonospora (Micromonospora sp.)
FIM05-328, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is：CGMCC
NO.13933, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and preservation date is on March 27th, 2017.
In order to realize foregoing invention purpose, present invention also offers a kind of Macrocyclic lactams compound, the compound be by
Micromonospora (Micromonospora sp.) FIM05-328 secondary metabolite is obtained through isolating and purifying, and its structural formula is such as
Shown in formula (I)：
In order to realize foregoing invention purpose, present invention also offers micromonospora (Micromonospora sp.)
Applications of the FIM05-328 in the Macrocyclic lactams compound is prepared, specifically, the invention provides acyl in above-mentioned big ring
The isolation and purification method of amines, comprises the following steps：
(1) micromonospora (Micromonospora sp.) FIM05-328 described in claim 1 is seeded to culture
Base culture, obtains seed culture fluid；
(2) seed culture fluid is seeded in fermentation culture and carries out fermented and cultured, obtain tunning；
(3) tunning centrifugation is obtained into mycelium and supernatant, mycelium is extracted twice using organic solvent, decompression
Concentration obtains medicinal extract after removing organic solvent；
(4) medicinal extract is merged with supernatant, with after macroporous resin adsorption, eluted respectively with deionized water and methanol, warp
TLC detects that developing agent is that volume ratio is 10:1 chloroform/methanol, Rf values are mixed after sample, dress post for 0.57 component, chlorine is used
Imitative/methanol elution, then detected through TLC, developing agent is that volume ratio is 10:1 chloroform/methanol, Rf values are entered for 0.60 component
Row column chromatography, is eluted with methanol/acetonitrile, obtains the Macrocyclic lactams compound.
Improved as a kind of the of isolation and purification method of Macrocyclic lactams compound of the present invention, in step (1), the training
The pH value for supporting base is 7.5, and every part of culture medium includes following component：Soluble starch 15g, glucose 5g, peptone 5g, yeast
Powder 5g, MgSO47H2O 0.5g, (NH4)2SO40.5g, CaCO31g, seawater 1000mL；It is described culture be 28~30 DEG C,
Shaking table culture 3~5 days under 220~240rpm.
Improved as a kind of the of isolation and purification method of Macrocyclic lactams compound of the present invention, in step (2), the hair
The pH value of ferment nutrient solution is 7.0~7.4, and every part of fermentation culture includes following component：Soluble starch 20g, glucose 5g,
Peptone 5g, Dried Corn Steep Liquor Powder 2.5g, yeast extract 2g, K2HPO4·3H2O 0.5g, CaCO31g, seawater 1000mL；Institute
It is shaking table culture 7~8 days under 22~25 DEG C, 180~200rpm to state fermented and cultured.
Improved as a kind of the of isolation and purification method of Macrocyclic lactams compound of the present invention, in step (2), the kind
The inoculum concentration that sub- nutrient solution is seeded in fermentation culture is 5~10% (v:v).
Understood through antitumor activity test, Macrocyclic lactams compound on tumor cell of the present invention, particularly people's esophageal squamous cell
Cancer cell has significant inhibitory activity, therefore, and Macrocyclic lactams compound of the present invention can be used for the medicine for preparing treatment tumour
Thing, it is highly preferred that Macrocyclic lactams compound of the present invention can be used for the medicine for preparing treatment cancer of the esophagus.
Relative to prior art, the invention has the advantages that：
Present invention finds a kind of new micromonospora (Micromonospora sp.) FIM05-328, by small list
The cytotoxicity reactive compound that spore bacterium (Micromonospora sp.) FIM05-328 is produced is isolated and purified, and is obtained
To a kind of new Macrocyclic lactams compound, the compound has significant inhibiting tumour cells activity, and therefore, the present invention is
The innovative development of antineoplastic provides compound candidate, and the drug resource exploitation to microorganism is also significant.
Micromonospora (Micromonospora sp.) FIM05-328 of the present invention, is preserved in Chinese microorganism strain preservation
Administration committee's common micro-organisms center, deposit number is：CGMCC NO.13933, preservation address is Chaoyang District, Beijing City north
The institute 3 of occasion West Road 1, preservation date is on March 27th, 2017.
Brief description of the drawings
With reference to the accompanying drawings and detailed description, to a kind of micromonospora of the invention, its metabolite Macrocyclic lactams
Compound and application in antitumor and beneficial effect are described in detail.
Fig. 1 is micromonospora of the present invention (Micromonospora sp.) FIM05-328 in Gao Shi-asparagus fern element agar
The form of 28 DEG C of cultures 15 days.
Fig. 2 is that the system of micromonospora of the present invention (Micromonospora sp.) FIM05-328 and its close bacterial strain is entered
Change tree.
Fig. 3 is the ultraviolet absorpting spectrum of Macrocyclic lactams compound of the present invention.
Fig. 4 is the HR-TOF-MS collection of illustrative plates of Macrocyclic lactams compound of the present invention.
Fig. 5 is Macrocyclic lactams compound of the present invention1H-NMR collection of illustrative plates.
Fig. 6 is Macrocyclic lactams compound of the present invention13C-NMR collection of illustrative plates.
Fig. 7 is Macrocyclic lactams compound of the present invention1H-1HCOSY collection of illustrative plates.
Fig. 8 is the hsqc spectrum figure of Macrocyclic lactams compound of the present invention.
Fig. 9 is the HMBC spectrograms of Macrocyclic lactams compound of the present invention.
Figure 10 is the ROESY spectrograms of Macrocyclic lactams compound of the present invention.
Figure 11 for Macrocyclic lactams compound of the present invention HMBC with1H-1H COSY correlation analysis figures.
Embodiment
In order that the purpose of the present invention, technical scheme and advantageous effects become apparent from, it is with reference to embodiments, right
The present invention is further elaborated.It should be appreciated that the embodiment described in this specification is used for the purpose of explaining this
Invention, is not intended to limit the present invention, parameter, the ratio of embodiment etc. can suit measures to local conditions to make a choice and have no reality to result
Matter influences.
Embodiment 1
Bacterial strain FIM 05-0328, are ocean rare actinomycete strain, are obtained from Ningbo of Zhejiang marine clay, in different fine jades
Aerial hyphae is not produced on fat culture medium, substrate mycelium physically well develops, be on inorganic medium it is yellowish to shallow clear, can not
Dissolubility pigment.The color of substrate mycelium arrives orange-yellow to be orange on organic culture medium, and spore color is greyish black to black.It is single
Spore raw on substrate mycelium, spore circle to ellipse, 0.6-1.0 μm of diameter (Fig. 1).20-40 DEG C of growth temperature, most
It is suitable 32 DEG C, grow pH scope 6-12, most suitable 7.0.Do not liquefy gelatin, not solidify milk, nitrate reduction ability weak, in ISP 6
Hydrogen sulfide is not produced on culture medium, melanin is not produced on the culture mediums of ISP 7.Bacterial strain utilization of carbon source situation is shown in Table 1, ISP etc.
Cultural characteristic description is shown in Table 2 on culture medium.
The bacterial strain FIM05-0328 of table 1 utilization of carbon source
The bacterial strain FIM05-0328 of table 2 cultural characteristic
*：G:Growing state；SM：Substrate mycelium；Numeral is color code in color description reference ISCC-NBS, bracket.
16S rRNA genes almost full length sequence is cloned from strain gene group, containing 75 base (NCBI of Isosorbide-5-Nitrae
GenBank sequence numbers：KC622312), Blast comparison results are shown, with micromonospora typical strain Micromonospora
rifamycinica AM105TSequence similarity highest, is 98.58% (1453/1474bp), and structure is with related strain
System chadogram (Fig. 2).
Morphology, physiological and biochemical property and 16S rRNA Genetic homology of carbapenem-resistant results show ocean rare actinomycete
FIM 05-0328 belong to Actinomycetal Micromonosporaceae micromonospora, temporarily name Micromonospora sp.FIM 05-
0328.The homology of 16S rRNA genes and known micromonospora is less than 98.6%, points out micromonospora FIM 05-0328
It is probably a novel species of the category, Classification And Nomenclature：(Micromonospora sp.) FIM05-328, is preserved in China Microbiological
Culture presevation administration committee common micro-organisms center, preserving number is： CGMCC No.13933.Preservation address：Beijing is exposed to the sun
The institute 3 of area North Star West Road 1.Preservation day：On March 27th, 2017.
Micromonospora (Micromonospora sp.) FIM05-328 fermentations isolate and purify Macrocyclic lactams compound：
(1) fermentation condition：
The preparation of culture medium：Soluble starch 15g, glucose 5g, peptone 5g, dusty yeast 5g, MgSO4·7H2O
0.5g, (NH4)2SO40.5g, CaCO31g, seawater 1000mL, pH7.5.121 DEG C of high-temperature sterilization 30min, it is standby.
Micromonospora (Micromonospora sp.) FIM05-328 Gao Shi asparagines agar slant culture is taken to access
In above-mentioned culture medium, 28~30 DEG C, shaking table culture culture 3~5 days, obtain seed culture fluid under the conditions of 220~240rpm.
The preparation of fermentation culture：Soluble starch 20.0g, glucose 5.0g, peptone 5.0g, Dried Corn Steep Liquor Powder
2.5g, yeast extract 2.0g, K2HPO4·3H2O 0.5g, CaCO31.0g, seawater 1000mL, pH 7.0~7.4.121℃
High-temperature sterilization 30min, it is standby.
Seed culture fluid is inoculated into fermentation culture with 5~10% inoculum concentration, 22~25 DEG C, 180~200rpm
Under the conditions of shaking table culture culture 7~8 days, obtain tunning.
(2) separation is extracted：
Tunning (30L) is centrifuged under the conditions of 4500rpm and obtains mycelium and supernatant, mycelium with 1.5~
The methanol/acetone (1 of 2.0 times of volumes：1, v:V) extract twice, extract solution is in removal methanol and the acetone of being concentrated under reduced pressure less than 40 DEG C
Medicinal extract (150g) is obtained, medicinal extract is merged with supernatant, HP-20 macroporous resin adsorptions are used, first with 5 times of column volume deionized waters
Elution, then eluted with 5 times of column volume methanol, TLC detections, developing agent is chloroform/methanol (10:1, v:V), same composition is merged,
Six kinds of components (Rf values are respectively 0.23,0.45,0.57,0.71,0.82 and 0.93) are obtained, (110mg, TLC are detected by component 3
Rf values are 0.57 component) with 100~200 mesh silica gel mixed samples, after dry column-packing, with chloroform/methanol (9:1~1:1, v:V) wash
It is de-, then detected through TLC, developing agent is chloroform/methanol=10:1, merge same composition, obtaining five kinds of components, (Rf values are respectively
0.46th, 0.60,0.71,0.80 and 0.87), component 2 (30mg, TLC detection Rf values for 0.60 component) is again through Sephadex
LH-20 column chromatographies, with methanol/acetonitrile (1:1, v:V) it is eluant, eluent, elution acquisition compound FW05328-1 (6.5mg).
(3) Structural Identification：
The data tests such as mass spectrum, ultraviolet spectra and nuclear magnetic resonance are carried out to FW05328-1 compounds, so that it is determined that
The structure of FW05328-1 compounds.FW05328-1 compounds：Faint yellow unformed powder.UV (MeOH),λmax:294nm
(Fig. 3)；HR-TOF-MS(m/z 484.3058[M+H]+,calcd for 484.3063[M+H]+) (Fig. 4)；Molecular formula is
C29H41NO5；It is 10 to calculate its degree of unsaturation.
Analyze FW05328-1 compounds1H-NMR(400MHz,in DMSO-d6) collection of illustrative plates (Fig. 5), it is seen that high field region shows
Show 2 groups of bimodal methyl hydrogen signals [δ 1.04 (3H, d, J=7.0Hz) and δ 1.20 (3H, d, J=7.0Hz)] and 2 groups of unimodal methyl
Hydrogen signal [δ 1.56 (3H, s) and δ 1.80 (3H, s)], there are multigroup alkene Hydrogen Proton signal and 1 group of active hydrogen proton signal in low field area
δ 7.77 (1H, d, J=7.7Hz).From13C-NMR (100MHz,in DMSO-d6) visible 29 carbon signals of collection of illustrative plates (Fig. 6), with reference to
DEPT135 analysis of spectrums, it is seen that it includes 3 quaternary carbon signal δ 165.3,136.1 and 133.3, wherein δ 165.3 is acyl aminocarbonyl
Carbon signal, δ 136.1,133.3 is alkene quaternary carbon signal；20 methine carbon signal δ 142.7,139.2,138.3,138.2,
136.0,131.4,130.3,130.1,129.6,128.9,128.8,128.8,128.5,126.3,79.0,74.7,68.4,
67.8,46.1 and 39.3, wherein δ 142.7,139.2,138.3,138.2,136.0,131.4,130.3,130.1,129.6,
128.9,128.8,128.8,128.5,126.3 grades 14 are olefinic carbon signal, 79.0,74.7,68.4,67.8,46.1 grades of δ 5
For the carbon signal being connected with hetero atom；2 mesomethylene carbon signal δ 42.8 and 38.6；And 4 methyl carbon signal δ 24.2,
21.0,18.8 with 16.0.From1H-1In HCOSY collection of illustrative plates (Fig. 7), it is seen that (H- of 7.03 (H-3)-δ of δ 5.85 (H-2)-δ 6.33
4)―δ6.55(H-5)―δ6.14(H-6)― δ6.06(H-7)―δ2.51(H-8)―δ3.59(H-9)―δ3.34(H-
10)―δ3.45(H-11)―δ 1.53(H-12)―δ4.24(H-13)―δ5.72(H-14)―δ5.90(H-15)―δ5.88
(H-16) it is sequentially related between, it is seen that (the H- of δ 7.77 (NH-26)-δ 3.65 (H-25)-δ 2.31 (H-24)-δ 5.59
23) it is sequentially related between (the H-21)-δ 6.01 (H-20) of-δ 5.70 (H-22)-δ 6.23, in conjunction with hsqc spectrum figure (Fig. 8),
Following carbon skeleton segment can be contained in pushing-out structure： C2―C3―C4―C5―C6―C7―C8―C9―C10―C11―C12―
C13―C14―C15―C16, C20―C21―C22―C23―C24―C25―NH.In HMBC spectrograms (Fig. 9), it is seen that H-26 (δ 1.20)
With C-24 (δ 38.6), C-25 (δ 46.1)；H-27 (δ 1.04) and C-7 (δ 142.7), C-8 (δ 39.3), C-9 (δ 74.7)；H-
28 (δ 1.56) and C-16 (δ 130.3), C-17 (δ 136.1), C-18 (δ 128.9)；H-29 (δ 1.80) and C-18 (δ 128.9),
C-19 (δ 133.3), C-20 (δ 129.6)；H-10-OH (δ 4.57) and C-10 (δ 79.0), C-11 (δ 68.4)；H-13-OH(δ
4.71) with C-12 (δ 42.8), C-13 (δ 67.8), C-14 (δ 128.5)；H-NH (δ 7.77) and C-1 (δ 165.2), C-25 (δ
46.1) it is, long-range related between C-26 (δ 21.0), along with shared carbon can just connect FW05328-1 compound scaffolds
Get up；
In summary analyze, and combine ROESY spectrograms (Figure 10), obtain FW05328-1 compounds whole carbon signals and
Hydrogen signal belongs to (table 3).The HMBC of FW05328-1 compounds and1H-1Figure 11 is shown in H COSY correlation analyses.Through
Network retrieval, finds no the compound of same structure, thus determines that FW05328-1 compounds are a new constructions
26 yuan of polyene macrocyclic lactam analog compounds.FW05328-1 compounds and compound Salinilactam A structure have one
Similitude is determined, shown in Salinilactam A structure such as formula (II)：
But FW05328-1 compounds and the hydroxyl on compound Salinilactam A lactam nucleus and ethylene linkage position
There are many monomethyl side chains on notable difference, and lactam nucleus of the FW05328-1 compounds than Salinilactam A.
The FW05328-1 compounds of table 31H NMR(in DMSO-d6, 400MHz) and13C NMR(in DMSO-d6,
100MHz) data
Embodiment 2
Respectively by FW05328-1 compounds, Salinilactam A samples and antineoplastic cis-platinum (cis-
Dichlorodiammineplatinum (II), DDP) it is dissolved in DMSO so that solubility reaches 1mg/mL, then dilution makes respectively
Final concentration of 0.75ug/mL, 0.5ug/mL, 0.25ug/mL, 0.125ug/mL, 0.1ug/mL, 0.05ug/mL,
0.005ug/mL、0.0025ug/mL.Take Human esophageal squamous cell cancer cell KYSE30, KYSE180 and EC109 in Exponential growth stage
Plant in 96 orifice plates that (cell concentration is 10 respectively5Individual/mL, 100ul/ hole), culture 24hr makes its adherent, plus 100ul/ pore areas
Medicine fresh culture, each concentration sets 3 multiple holes, continues to cultivate to 72hr, terminates culture.Cis-platinum is clinical conventional chemotherapy
One of medicine, with anticancer spectrum is wide, determined curative effect the features such as, but its side effect is big.
SRB is detected：The cell of culture will be terminated, 10%TCA 50ul are added per hole, 4 DEG C of conditions fix 1hr.Use distilled water
Rinse 5 times, add 4mg/ml SRB solution 50ul after drying naturally per hole, 30min is dyed at room temperature, supernatant is abandoned, 1% second is used
Acid rinses 5 times dyestuffs to remove non-specific binding.150ul 10mM Tris solution is added per hole, is shaken 5 minutes, 540
ELIASA surveys OD values under wavelength, and calculates inhibiting rate.By the conversion to inhibiting rate, IC50 values are calculated using SPSS softwares.
As a result as shown in table 4, FW05328-1 compounds than Salinilactam A to Human esophageal squamous cell cancer cell KYSE30,
KYSE180 and EC109 has more excellent inhibitory activity, compared with Salinilactam A and control group drugs Cisplatin,
FW05328-1 compounds are especially protruded Human esophageal squamous cell cancer cell line EC109 inhibitory activity.
FW05328-1 the and Salinilactam A of table 4 tumor cytotoxic activity
The announcement and teaching of book according to the above description, those skilled in the art in the invention can also be to above-mentioned implementation
Mode carries out appropriate change and modification.Therefore, the invention is not limited in embodiment disclosed and described above,
Some modifications and changes to the present invention should also be as falling into the scope of the claims of the present invention.Although in addition, this theory
Some specific terms have been used in bright book, but these terms are merely for convenience of description, do not constitute any limit to the present invention
System.
Claims (9)
1. a kind of micromonospora (Micromonospora sp.) FIM05-328, is preserved in Chinese microorganism strain preservation management
Committee's common micro-organisms center, deposit number is：CGMCC NO.13933, preservation address is BeiChen West Road, Chaoyang District, BeiJing City
No. 1 institute 3, preservation date is on March 27th, 2017.
2. a kind of Macrocyclic lactams compound, it is characterised in that shown in its structural formula such as formula (I)：
。
3. (Micromonospora sp.) FIM05-328 of micromonospora described in claim 1 is being prepared described in claim 2 greatly
Application in cyclic lactam compound.
4. the isolation and purification method of Macrocyclic lactams compound described in claim 2, it is characterised in that comprise the following steps：
(1) micromonospora (Micromonospora sp.) FIM05-328 described in claim 1 is seeded to culture medium training
Support, obtain seed culture fluid；
(2) seed culture fluid is seeded in fermentation culture and carries out fermented and cultured, obtain tunning；
(3) tunning centrifugation is obtained into mycelium and supernatant, extracts mycelium twice using organic solvent, be concentrated under reduced pressure
Except obtaining medicinal extract after organic solvent；
(4) medicinal extract is merged with supernatant, with after macroporous resin adsorption, is eluted, examined through TLC with deionized water and methanol respectively
Survey, developing agent is that volume ratio is 10:1 chloroform/methanol, Rf values are mixed after sample, dress post for 0.57 component, chloroform/methanol is used
Elution, then detected through TLC, developing agent is that volume ratio is 10:1 chloroform/methanol, post layer is carried out by Rf values for 0.60 component
Analysis, is eluted with methanol/acetonitrile, obtains the Macrocyclic lactams compound.
5. the isolation and purification method of Macrocyclic lactams compound according to claim 4, it is characterised in that in step (1),
The pH value of the culture medium is 7.5, and every part of culture medium includes following component：Soluble starch 15g, glucose 5g, peptone 5g,
Dusty yeast 5g, MgSO47H2O 0.5g, (NH4) 2SO40.5g, CaCO31g, seawater 1000mL；The culture be 28~
30 DEG C, shaking table culture 3~5 days under 220~240rpm.
6. the isolation and purification method of Macrocyclic lactams compound according to claim 4, it is characterised in that in step (2),
The pH value of the fermentation culture is 7.0~7.4, and every part of fermentation culture includes following component：Soluble starch 20g, grape
Sugared 5g, peptone 5g, Dried Corn Steep Liquor Powder 2.5g, yeast extract 2g, K2HPO4·3H2O 0.5g, CaCO31g, seawater
1000mL；The fermented and cultured is shaking table culture 7~8 days under 22~25 DEG C, 180~200rpm.
7. the isolation and purification method of Macrocyclic lactams compound according to claim 4, it is characterised in that in step (2),
The inoculum concentration that the seed culture fluid is seeded in fermentation culture is 5~10 volume %.
8. application of the Macrocyclic lactams compound described in claim 2 in the medicine for preparing treatment tumour.
9. application according to claim 8, it is characterised in that the medicine of the treatment tumour is the medicine for the treatment of cancer of the esophagus
Thing.
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