CN102643765B - Streptomyces, anti-tumor compound (Spiro-Indimycin A-D) as well as preparation method and application thereof - Google Patents
Streptomyces, anti-tumor compound (Spiro-Indimycin A-D) as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a streptomyces, anti-tumor compound (Spiro-Indimycin A-D) as well as preparation and application thereof. The streptomyces sp. SCSIO 03032 is preserved in China Center for Type Culture Collection (CCTCC) on July 18, 2011, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 2011258. The invention provides a new streptomyces sp. SCSIO 03032, which can be used for producing the compound Spiro-Indimycin A, B, C and D with novel structure and antineoplastic activity; and the compound Spiro-Indimycin A, B, C and D not only has a rare spiral structure, but also has selective and efficient activity for tumour cells, and is an ideal candidate compound for being developed to be the efficient and anti-tumor compound with low toxicity.
Description
Technical field:
The invention belongs to the industrial microorganism field, be specifically related to a kind of new streptomycete (Streptomyces sp.) SCSIO 03032 that can produce multiple antineoplastic compound, and the antineoplastic compound Spiro-Indimycin A-D of this bacterium production and its preparation method and application.
Background technology:
In recent years, find novel sea actinomycetes resource, and therefrom screen novel active secondary metabolite and become the important directions of marine microorganism research in the world.Find novel structure from marine actinomycete, meta-bolites efficient, low toxicity becomes the new source that medicine is found gradually.
Summary of the invention:
First purpose of the present invention is to provide a kind of new streptomycete (Streptomyces sp.) SCSIO 03032, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC) on July 18th, 2011, address: Wuhan, China city Wuhan University, its deposit number is CCTCC NO:M 2011258.
New streptomycete of the present invention (Streptomyces sp.) SCSIO 03032 separates and obtains in the marine bottom sediment of 3412 meters of (87 ° 59.7 of E ', 9 ° 59.3 of N ') depth of waters from the Indian Ocean.
1, morphological feature and physiological and biochemical analysis
Streptomycete (Streptomyces sp.) SCSIO 03032 belongs to Gram-positive, aerobic actinomycetes, and the micro-yellow branch of base silk, gas silk canescence branch also is divided into curling spore chain; Spore ellipsoid shape (Fig. 2), long 0.4-0.7 μ m, smooth surface.On the PDA substratum growing way a little less than, on ISP5, growth has soluble pigment to produce.Catalase, urase, milk solidify and the trunk reacting positive, gelatine liquefication, melanochrome production and oxidation enzyme reaction feminine gender.Energy hydrolyzed starch, Mierocrystalline cellulose, polysorbas20,40,80; H
2s produces and the nitrate reduction reaction negative; Can utilize D-R, the D-cellobiose, fructose, inositol, Sodium.alpha.-ketopropionate, the D-raffinose, the L-rhamnosyl, PEARLITOL 25C, D-Glucose, D-sorbose and Xylitol, be sole carbon source and energy growth.And can not utilize the D-semi-lactosi, D-lactose, D-MANNOSE, D-ribose, D-trehalose and D-wood sugar; To Sulfamonomethoxine and lincomycin sensitivity.The tolerance range of pH, salt concn and temperature is respectively pH 6.0-9.0,0-9% and 15-40 ℃.Cell walls contains L-DAP.The advantage quinone is MK-9 (H6) and MK-11; Main fatty acid is i-C
15:0(8%), i-C
16:0(24%), i-C
16:1g (14%), ai-C
17:0and ai-C (15%)
17:1a (12%).The G+C molar content is 71.6 (± 0.5) %.
2, molecular biology identification
The 16S rDNA of conventional pcr amplification bacterial strain SCSIO 03032 order-checking, its sequence, as shown in SEQ ID NO.1, then is submitted in GenBank, and 16S rDNA nucleotide sequence is carried out to the BLAST analysis, result shows, this bacterial strain and Streptomyces specialis GW41-1564
tsimilarity be 97%, rebuild this genus phylogenetic tree and show that bacterial strain SCSIO 03032 gathers at this monoid, illustrate that this bacterial strain SCSIO 03032 is for streptomyces.As shown in Figure 1, clearly disclose the Phylogenetic of this bacterial strain and one group of streptomyces species by adjacent method, shown a kind of in streptomyces of this Pseudomonas.
According to analyses such as above morphology, physiology, chemistry, itself and known immediate bacterial strain and Streptomyces specialis GW41-1564
tlarger difference is arranged, and genomic hybridization shows that its hybridization value is 23%, far below 70% (Stackebrandt, E.& of intraspecies variation standard; Ebers, J.Taxonomic parameters revisited:tarnished gold standards.Microbiol Today. (2006) .33,152-155.); Therefore, the multinomial grouped data of analysis-by-synthesis, identify the novel species that this bacterial strain is streptomyces (Streptomyces), called after: streptomycete (Streptomyces sp.) SCSIO 03032, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC) on July 18th, 2011, address: Wuhan, China city Wuhan University, its deposit number is CCTCC NO:M 2011258.
Streptomycete of the present invention (Streptomyces sp.) SCSIO 03032 can produce 4 kinds of antineoplastic compound Spiro-Indimycin A-D with novel structure.
Therefore second purpose of the present invention is to provide compound S piro-Indimycin A-D, and its structural formula is suc as formula shown in (I):
The inventor found through experiments, compound S piro-Indimycin A-D comprises mammary cancer MCF-7 to human cancer cell, human pancreas cancer 1990, people's liver cancer HepG2, human cervical carcinoma Hale, Humanmachine tumour B16, people's lung cancer H460 and the selective cytotoxic activity of people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM, the wherein IC of Spiro-Indimycin B to Humanmachine tumour B16 cell, people's lung cancer H460 cell and people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM
50respectively 11 μ M, 36.6 μ M and 4.5 μ M, and to other subject cell strain without obvious growth inhibitory activity; The IC of Spiro-Indimycin C to human liver cancer cell HepG2 and people's lung cancer H460 cell
50respectively 14.1 μ M and 35.4 μ M, and to other subject cell strain without obvious growth inhibitory activity; The IC of Spiro-Indimycin D to human liver cancer cell HepG2, Humanmachine tumour B16 cell and people's lung cancer H460 cell
50respectively 44.4 μ M, 40.4 μ M and 36.3 μ M, and to other subject cell strain without obvious growth inhibitory activity, the IC of Spiro-Indimycin A to people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM
50be 44.4 μ M, and to other subject cell strain without obvious growth inhibitory activity (table 3).
The 3rd purpose of the present invention is to provide the application of compound S piro-Indimycin A-D in preparing antitumor drug.
Preferably, the application of described compound S piro-Indimycin A in preparation treatment people acute lymphoblastic leukemia medicine.
Preferably, the application of described compound S piro-Indimycin B in preparation treatment Humanmachine tumour, people's lung cancer or people's acute lymphoblastic leukemia medicine.
Preferably, the application of described compound S piro-Indimycin C in preparation treatment people's liver cancer or people's lung-cancer medicament.
Preferably, the application of described compound S piro-Indimycin D in preparation treatment Humanmachine tumour, people's liver cancer or people's lung-cancer medicament.
The 4th purpose of the present invention is to provide streptomycete (Streptomyces sp.) SCSIO 03032 in the application for the preparation of compound S piro-Indimycin A, B, C or D.
The 5th purpose of the present invention is to provide the preparation method of Spiro-Indimycin A, B, C or D, it is characterized in that, described compound S piro-Indimycin A, B, C or D are that the preparation separation obtains from the fermenting culture of streptomycete (Streptomyces sp.) SCSIO 03032.
Preferably, be to prepare compound S piro-Indimycin A, B, C or D by the following method from the fermenting culture of streptomycete (Streptomyces sp.) SCSIO 03032, concrete steps are as follows:
A) prepare the fermenting culture of streptomycete (Streptomyces sp.) SCSIO 03032, the fermented liquid of this fermenting culture and mycelium are separated, fermented liquid is through macroporous resin adsorption, rear with acetone wash-out macroporous resin, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration; Mycelium is first used acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains medicinal extract B after distillation and concentration;
B) crude extract extractum A and medicinal extract B merged is through silica gel column chromatography, by chloroform/methanol as eluent, within 100: 0～0: 100, carry out gradient elution from volume ratio, collect the cut Fr.1 that 95: 5 gradient elutions of chloroform/methanol volume ratio get off, by the anti-phase medium pressure liquid chromatography of ODS, water/methyl alcohol is as eluent, within 100: 0～0: 100, carry out gradient elution from volume ratio, collect the cut Fr.1-1 that water/methyl alcohol volume ratio 30: 70 gradient elution gets off, after the LH-20 gel column, using chloroform/methanol volume ratio 1: 1 as the moving phase wash-out, follow the trail of and separate with thin-layer chromatography, collecting on thin layer plate by the chloroform/acetone volume ratio is the cut that 9: 1 values of Rf during as developping agent are 0.8, be Spiro-Indimycin B, the cut that the Rf value is 0.6, be Spiro-Indimycin A, collecting on thin layer plate by the petrol ether/ethyl acetate volume ratio is the cut that 40: 60 values of Rf during as developping agent are 0.8, is Spiro-Indimycin C, collecting on thin layer plate by the petrol ether/ethyl acetate volume ratio is the cut that 50: 50 values of Rf during as developping agent are 0.8, is Spiro-Indimycin D.
The fermenting culture of the preparation streptomycete of described a) step (Streptomyces sp.) SCSIO 03032 is preparation by the following method preferably: by streptomycete (Streptomyces sp.) SCSIO of activation 03032 access seed culture medium, 28 ℃, 200rpm, cultivate 48h and obtain seed liquor, seed liquor is linked in fermention medium with 10% inoculum size, 28 ℃, 200rpm, shaking culture 120h, and make fermenting culture, the formula of described seed culture medium and fermention medium is all to contain in every liter of substratum: starch 10g, yeast powder 5g, peptone 4g, CaCO
32g, thick sea salt 30g, surplus is water, pH 7.2.
The invention provides a kind of new streptomycete (Streptomyces sp.) SCSIO 03032, this bacterium can the production structure novelty compound S piro-Indimycin A, B, C and D with anti-tumor activity, compound S piro-Indimycin A, B, C and D are except having rare spirane structure, also to tumour cell selectively, efficiently active, the desirable candidate compound of antineoplastic compound efficient, low toxicity that is that exploitation becomes.
Streptomycete of the present invention (Streptomyces sp.) SCSIO 03032 is preserved in Chinese Typical Representative culture collection center (CCTCC) on July 18th, 2011, address: Wuhan, China city Wuhan University, its deposit number is CCTCC NO:M 2011258.
The accompanying drawing explanation:
Fig. 1 is the phylogenetic tree of relation between the nearest kind of the sibship with it of the adjacent method reconstruction of streptomycete (Streptomyces sp.) SCSIO 03032 based on 16S rDNA sequence;
Fig. 2 is the scanning electron microscope microscopic morphology of streptomycete (Streptomyces sp.) SCSIO 03032;
Fig. 3 is the extract-extractum A of supernatant liquor and the high-efficient liquid phase chromatogram of mycelial extract-medicinal extract B;
High performance liquid chromatography (HPLC) condition: chromatographic column is phenomex 150 * 4.6mm (SphereClone SAX), moving phase comprises flow A phase and mobile B phase, mobile phase A phase: the trifluoroacetic acid of the acetonitrile of 10% (volume fraction)+0.08% (volume fraction), solvent is water, the B phase flows: the acetonitrile of 90% (volume fraction), and solvent is water; Sample introduction program: 0-20min, mobile phase ratio is A phase/B phase (volume ratio): 95: 5-0: 100,20-21min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100,21-22min, mobile phase ratio is A phase/B phase (volume ratio): 0: 100-95: 5,22-30min, mobile phase ratio is A phase/B phase (volume ratio): 95: 5, detect wavelength 254nm, flow velocity 1ml/min, wherein 1 representation compound 1,2 representation compound 2,3 representation compounds 3 and 4 representation compounds 4.
Fig. 4 is the X-ray structure iron of compound 1;
Fig. 5 is the X-ray structure iron of compound 2.
Embodiment:
Following examples are to further illustrate of the present invention, rather than limitation of the present invention.
Embodiment 1:
One, separation and the evaluation of streptomycete (Streptomyces sp.) SCSIO 03032
Streptomycete of the present invention (Streptomyces sp.) SCSIO 03032 is from the Indian Ocean: separate to obtain in the marine bottom sediment that (87 ° 59.7 of E ', 9 ° 59.3 of N ') depth of water is 3412 meters.Isolation medium is the ISP2 substratum of optimizing in prior art, and every liter contains yeast extract powder 2.0g, Fructus Hordei Germinatus extract powder 2.0g, and glucose 1.0g, agar 20.0g, distilled water 500ml, natural sea-water 500ml, pH 7.2.The separation and Culture condition is: 28 ℃, and 14 days.Obtain a bacterial strain SCSIO 03032 (streptomycete Streptomyces sp.SCSIO 03032) from the marine bottom sediment separation and purification thus.
1, morphological feature and physiological and biochemical analysis
This Pseudomonas is in Gram-positive, aerobic actinomycetes, and the micro-yellow branch of base silk, gas silk canescence branch also is divided into curling spore chain; Spore ellipsoid shape (Fig. 2), long 0.4-0.7 μ m, smooth surface.On the PDA substratum growing way a little less than, on ISP5, growth has soluble pigment to produce.Catalase, urase, milk solidify and the trunk reacting positive, gelatine liquefication, melanochrome production and oxidation enzyme reaction feminine gender.Energy hydrolyzed starch, Mierocrystalline cellulose, polysorbas20,40,80; H
2s produces and the nitrate reduction reaction negative; Can utilize D-R, the D-cellobiose, fructose, inositol, Sodium.alpha.-ketopropionate, the D-raffinose, the L-rhamnosyl, PEARLITOL 25C, D-Glucose, D-sorbose and Xylitol, be sole carbon source and energy growth.And can not utilize the D-semi-lactosi, D-lactose, D-MANNOSE, D-ribose, D-trehalose and D-wood sugar; To Sulfamonomethoxine and lincomycin sensitivity.The tolerance range of pH, salt concn and temperature is respectively pH 6.0-9.0,0-9% and 15-40 ℃.Cell walls contains L-DAP.The advantage quinone is MK-9 (H6) and MK-11; Main fatty acid is i-C
15:0(8%), i-C
16:0(24%), i-C
16:1g (14%), ai-C
17:0and ai-C (15%)
17:1a (12%).The G+C molar content is 71.6 (± 0.5) %.
2, molecular biology identification
The extraction of the genomic dna related to during streptomycete (Streptomyces sp.) SCSIO 03032 identifies, the pcr amplification of 16S rDNA, the structure of sequence alignment and systematic evolution tree and physiology, chemistry and Morphological Identification etc., equal reference [Tian, X.P., Zhi, X.Y., Qiu, Y.Q., Zhang, Y.Q., Tang, S.K., Xu, L.H., Zhang, S., Li, W.J.Sciscionella marina gen.nov., sp.nov., a marine actinomycete isolated from a sediment in the northern South China Sea.Int J Syst Evol Microbiol, 2009, 59 (Pt 2): 222-228].
Extract the genomic dna of bacterial strain SCSIO 03032 according to the method in reference, the 16S rDNA of this bacterial strain of pcr amplification order-checking, obtain 1430 bases, its sequence is as shown in SEQ ID NO.1, then be submitted in GenBank, 16S rDNA nucleotide sequence is carried out to the BLAST analysis, and result shows, this bacterial strain and Streptomyces specialis GW41-1564
tsimilarity be 97%, rebuild this genus phylogenetic tree and show that bacterial strain SCSIO 03032 gathers at this monoid, illustrate that this bacterial strain SCSIO 03032 is for streptomyces.As shown in Figure 1, clearly disclosed the Phylogenetic of this bacterial strain SCSIO03032 and one group of streptomyces species by adjacent method, show a kind of in streptomyces of this Pseudomonas, by the genomic hybridization methods and results, show bacterial strain SCSIO 03032 and the most similar bacterial classification Streptomyces specialis GW41-1564
tthe hybridization value be 23%, this hybridization value is far below 70% (Stackebrandt, E.& of intraspecies variation standard; Ebers, J.Taxonomic parameters revisited:tarnished gold standards.Microbiol Today. (2006) .33,152-155.); To the most similar known bacterial strain, larger difference is all arranged at aspects such as morphology, physiology, cytochemistries simultaneously, therefore, the multinomial grouped data of analysis-by-synthesis, identify the novel species that this bacterial strain is streptomyces (Streptomyces), called after: streptomycete (Streptomyces sp.) SCSIO 03032, this bacterium is preserved in Chinese Typical Representative culture collection center (CCTCC) on July 18th, 2011, address: Wuhan, China city Wuhan University, its deposit number is CCTCC NO:M 2011258.
Two, the separation of active metabolite Spiro-Indimycin A-D and preparation
1, substratum (seed culture and fermention medium): every liter contains starch 10g, yeast powder 5g, peptone 4g, CaCO
32g, sea salt 30g, surplus is water, pH 7.2.121 ℃, sterilizing 30min;
2, fermentation
2.1, seed culture: single bacterium colony of streptomycete (Streptomyces sp.) SCSIO 03032 that activates on culture dish is accessed respectively to 18 bottles, in the taper culturing bottle of every bottle of 250mL that contains the 50mL substratum, 28 ℃, 200rmin
-1, cultivate 48h, make seed liquor 900mL.
2.2, fermentation culture: seed liquor is linked in 9L fermention medium (be placed in the taper culturing bottle of 250mL, every bottle contains the 50ml substratum, 180 bottles) with 10% inoculum size (volume percent) totally, 28 ℃, 200rmin
-1, shaking culture 120h, obtain the 9L fermenting culture.
3, extraction: fermenting culture first carries out centrifugation (3500rmin
-1, 8min), obtain supernatant liquor (fermented liquid) and the mycelium of 9L volume.Fermented liquid is through macroporous resin XAD16 Solid-Phase Extraction, and rear with acetone wash-out macroporous resin 3 times, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate 3 times, and ethyl acetate layer obtains supernatant liquor extract-extractum A (4.1g) after distillation and concentration; At room temperature lixiviate of 2L acetone 3 times for mycelium, each 3 hours, united extraction liquid, 6L ethyl acetate extraction for the residue water mixed liquid after reclaim under reduced pressure acetone, the ethyl acetate layer underpressure distillation obtains mycelium extract-medicinal extract B (0.9g).
4, the extraction of active compound separates and identifies
4.1 the extraction of compound S piro-Indimycin A-D separates and identifies: the sample of the extractum A that takes a morsel respectively and medicinal extract B is dissolved in 100 μ l methyl alcohol, centrifuging and taking supernatant 10 μ l, through the HPLC sample detection, show, contain compound 1 (1) and 2 (2) in extractum A, contain compound 3 (3) and 4 (4) in medicinal extract B (Fig. 3), extractum A and medicinal extract B are merged.By the crude extract of this merging through silicagel column (300-400 order) chromatography, by chloroform/methanol as eluent, within 100: 0～0: 100, carry out gradient elution from volume ratio, collect the cut Fr.1 (3.03g) that 95: 5 gradient elutions of chloroform/methanol volume ratio get off, evaporate to dryness, through the anti-phase medium pressure liquid chromatography of ODS (YMC*GEL ODS-A ball-shaped filling material, the 120A aperture, the 50um particle diameter, 240 * 4.0cm I.D.), flow velocity is 25ml/min, water/methyl alcohol is as eluent, within 100: 0～0: 100, carry out gradient elution from volume ratio, collect the cut Fr.1-1 (1.57g) that water/methyl alcohol volume ratio 30: 70 gradient elution gets off, after LH-20 gel column (2.5*100cm), using chloroform/methanol volume ratio 1: 1 as the moving phase wash-out, flow velocity is 1ml/min, every 100ml collects and merges into portion, order obtains Fr.1-1-A-G 7 duplicate samples, Fr.1-1-C (331mg) wherein, Fr.1-1-D (626mg), Fr.1-1-E (27mg).Fr.1-1-C with the chloroform/acetone volume ratio be 9: 1 as developping agent, through thin layer preparation, the band that scraping Rf value is 0.8, eluent ethyl acetate obtains compound 2 (18.0mg) (Spiro-Indimycin B); The band that scraping Rf value is 0.6, eluent ethyl acetate obtains compound 1 (5.0mg) (Spiro-Indimycin A); Fr.1-1-D with the petrol ether/ethyl acetate volume ratio be 40: 60 as developping agent, through thin layer preparation, the band that scraping Rf value is 0.8, eluent ethyl acetate obtains compound 3 (22.0mg) (Spiro-Indimycin C); Fr.1-1-E with the petrol ether/ethyl acetate volume ratio be 50: 50 as developping agent, through thin layer preparation, the band that scraping Rf value is 0.8, eluent ethyl acetate obtains compound 4 (6.6mg) (Spiro-Indimycin D).
By structural analysis, 4 the compound 1-4-compound S piro-Indimycin A-D (formula (II)) that prepare from the fermenting culture of streptomycete (Streptomyces sp.) SCSIO 03032 of the present invention are identified it, and its qualification result is as follows:
Compound 1: white crystal, its nuclear magnetic data ownership is as shown in table 1,
uV (MeOH) λ
max(log ε) 272nm (4.05); 227nm (4.40); IR (KBr) λ
max3443,1725,1229cm
-1;
1h NMR (500MHz, CD
3oD) in Table 1,
13c NMR (125MHz, CD
3oD) in Table 2.High resolution mass spectrum HRESIMS m/z C
24h
15cl
2n
3o
3(measured value [M-H]
-478.0353 calculated value is 478.0361), degree of unsaturation is 18.The hydrogen spectrum of compound 1 shows 2 methoxyl groups [δ H 3.53 (3H, s), 4.00 (3H, s)], 7 fragrant protons, 2 reactive hydrogens [δ H 11.47 (1H, s), 12.20 (1H, brs)].
13c NMR and DEPT NMR show 2 carbonyl carbon [δ C 159.6 (s), 160.3 (s)], 13 quaternary carbons, 7 methine carbons, 2 methyl carbon.The hydrogen spectrum C spectrum of compound 1 is compared [McArthur, K.A. with known compound Lynamicin D; Mitchell, S.S.; Tsueng, G.; Rheingold, A.; White, D.J.; Grodberg, J.; Lam, K.S.; Potts, B.C.Lynamicins A-E, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete, J Nat Prod, 2008,71,1732-7.], find that the two is extremely similar, difference is: compound 1 is few 2 hydrogen in molecular formula, simultaneously more degrees of unsaturation; Many 1 sp in the carbon spectrum of compound 1
3hydridization quaternary carbon, a few sp in Lynamicin D simultaneously
2the quaternary carbon of hydridization; The chemical shift of C-2 ' in compound 1 is the low (δ of deflection obviously
c169.2).Infer the C-3 ' of compound 1 and C-5 " be connected and be connected into ring, the conjugation of former C-2 ' and between C-3 ' existence shifts simultaneously, becomes the conjugation of between C-2 ' and N-1 ' according to above evidence.Above supposition further proves by the X-ray (Fig. 4) of compound 1.And the chlorine atom existed in molecule, make X-ray can clearly the absolute configuration of compound 1 be defined as to C-3 ' S configuration.The structure of compound 1 is suc as formula shown in (II), called after Spiro-Indimycin A.
Compound 2: white crystal.[α]
20 D+169.2(c?0.67，MeOH)；UV(MeOH)λ
max(logε)252nm(4.77)；209nm(4.66)；IR(KBr)λ
max3418，1695，1284cm
-1。The negative ion high resolution mass spectrum provides [M-H] at 436.0602 places
-peak, (calculated value is 436.0620), show that in conjunction with carbon spectrum, hydrogen spectrum the molecular formula of this compound is C
23h
16cl
2n
3o
2, include 16 degrees of unsaturation.The 1D NMR of compound (
1hNMR (500MHz, CD
3oD) in Table 1,
13c NMR (125MHz, CD
3oD) in Table 2.) there is following signal in prompting hydrogen spectrum: 1 n-formyl sarcolysine base [δ
h(2.88 1H, s)], 1 oxygen methyl [δ
h(3.36 3H, s)], 7 methine protons, 1 methene proton [δ
ha(3.63 1H, d, J=8.5Hz) and δ
hb(4.06 1H, d, J=8.5Hz)] and 2 tradable protons; There is following signal in the carbon spectrum: 1 carbonyl carbon, 13 quaternary carbons (comprising the quaternary carbon of 12 sp2 hydridization and the quaternary carbon of 1 sp3 hydridization), 7 methine carbons, 1 mesomethylene carbon, 1 oxygen methyl carbon and 1 n-formyl sarcolysine base carbon.Comparative compound 2 and known compound Lynamicins A[McArthur, K.A.; Mitchell, S.S.; Tsueng, G.; Rheingold, A.; White, D.J.; Grodberg, J.; Lam, K.S.; Potts, B.C.Lynamicins A-E, chlorinated bisindole pyrrole antibiotics from a novel marine actinomycete, J Nat Prod, 2008,71,1732-7.] hydrogen spectrum carbon spectrum data, find that the two is similar, prompting compound 2 and compound L ynamicins A similar, also belong to bisindole alkaloid.In the hydrogen of compound 2 spectrum, exist two ABX coupling systems (H-5 '/H-7 '/H-8 '; H-5 "/H-7 "/H-8 "), similar to Lynamicins A, show to exist two 1,2 in compound 2,4-trisubstituted benzene ring, be attributed to it respectively 1a and 1b (formula (III)).Further, the 1a fragment is set up by following HMBC is relevant: from H-5 ' to C-6 '/and C-7 '/C-9 '; From H-7 ' to C-5 '/C-9 '; From H-8 ' to C-6 '/C-4 (above being correlated with shows that chlorine replaces in C-6 ' position) simultaneously and from H-2 ' to C-3 '/C-4 '/C-9 '.The nitrogen methyl proton of H-10 ' position [δ H 2.88 (s)] is to the relevant N-1 ' that this n-formyl sarcolysine base is navigated to the 1a fragment of HMBC of C-2 ' [δ C 64.3 (t)] and C-9 ' [δ C 151.9 (s)].The 1b fragment obtains by following HMBC: from H-5 " to C-3 "/C-7 "/C-6 "/C-9 ", from H-7 " to C-5 "/C-6 "/, and from H-8 " to C-3 "/C-5 "/C-4 "/C-6 "/C-9 ".The 1c fragment is relevant by following HMBC: H-2 is to relevant the obtaining of COSY of C-3/C-4/C-5 and H-2/NH-1.Except this, from H-2 to C-3 " the relevant prompting of weak HMBC methoxy acyl group in the C-5 position, replace.Different from known compound Lynamicins A is, compound 2 forms 5/5 a rare volution, crucial HMBC is relevant: H-2 ' [δ H 3.63 (d)] is to C-3[δ C 140.1 (s)], H-2 ' [δ H 4.06 (d)] supports the formation of spirane structure to C-2 " [δ C 154.5 (s)] and H-2[δ H6.91 (d)] to C-3 " [δ C 112.1 (s)].The above structure to compound 2 is inferred by X-ray (Fig. 5) further to be proved, and the chlorine atom existed in molecule, makes X-ray clearly the absolute configuration of compound 2C-3 ' to be defined as to the R configuration.The structure of compound 2 is suc as formula shown in (II), called after Spiro-Indimycin B.
Compound 3: white solid, its nuclear magnetic data ownership is as shown in table 1.
uV (MeOH) λ
max(log ε) 251nm (4.44); 210nm (4.63); IR (KBr) λ
max3417,1701,1285cm
-1;
1h NMR (500MHz, CD
3oD) in Table 1,
13c NMR (125MHz, CD
3oD) in Table 2.HRESIMS m/z[M-H]
-422.0499 (calcd for C
22h
14cl
2n
3o
2422.0463), its molecular formula and compound 2 differ a methyl, carefully analyze the H-spectrum, C-spectrum, HSQC and HMBC spectrum, draw compound 3 be compound 2 disappearances 1 '-homologue of N methyl, its structure is suc as formula shown in (II), called after Spiro-Indimycin C.
Compound 4: white solid, its nuclear magnetic data ownership is as shown in table 1.
uV (MeOH) λ
max(ε) 266nm (4.23); 243nm (26373); 208nm (4.48); IR (KBr) λ
max3307,1718,1268cm
-1;
1h NMR (500MHz, CD
3oD) in Table 1,
13c NMR (125MHz, CD
3oD) in Table 2.HRESIMS?m/z[M-H]
-494.0661，(calcd?for?C
25H
18Cl
2N
3O
4，494.0674)。The hydrogen of compound 4 spectrum is compared with compound 2 with the carbon spectrum, found than more than 2 methyl esters groups of compound, also find the sp of the C-2 position in 2
2the methine carbon of hydridization [δ H 6.91 (d, J=3.0Hz, H-2); δ C 110.3 (d, C-2)] by sp
2the quaternary carbon of hydridization [δ C 111.5 (s, C-2)] replaces, and 2 methyl esters that this explanation compound 4 is compounds 2 replace analogs, and its structure is suc as formula shown in (II), called after Spiro-Indimycin D.
Table 1: compound 1-4's
1h-NMR nuclear magnetic data ownership (data are measured in 500MHz, are coupling constant (Hz) in bracket)
asample is dissolved in DMSO and detects;
bsample is dissolved in CDCl
3detect
Table 2: compound 1-4's
13c-NMR nuclear magnetic data ownership (data are measured in 125MHz)
asample is dissolved in DMSO and detects;
bsample is dissolved in CDCl
3detect
The determination of cytotoxic activity of embodiment 3:Spiro-Indimycin A-D
Spiro-Indimycin A-D is for seven kinds of tumor cell lines: MCF-7 Human Breast Cancer Cells, human pancreas cancer 1990, human liver cancer cell HepG2, human cervical carcinoma Hale, Humanmachine tumour B16 cell, people's lung cancer H460 cell and people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM have carried out determination of activity, experimental technique reference [Wu, Z.C.; Li, D.L.; Chen, Y.C.; Zhang, W.M., A new isofuranonaphthalenone and benzopyrans from the endophytic fungus Nodulisporium sp.A4 from Aquilaria sinensis.Helv.Chim.Acta 2010,93, (5), 920-924.], with Staurosporine in contrast.
Its result is as shown in table 3, result shows the selective cytotoxic activity of Spiro-Indimycin A-D, wherein the IC of Spiro-Indimycin B to Humanmachine tumour B16 cell, people's lung cancer H460 cell and people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM
50respectively 11 μ M, 36.6 μ M and 4.5 μ M, and to other subject cell strain without obvious growth inhibitory activity; The IC of Spiro-Indimycin C to human liver cancer cell HepG2 and people's lung cancer H460 cell
50respectively 14.1 μ M and 35.4 μ M, and to other subject cell strain without obvious growth inhibitory activity; Spiro-Indimycin D is to human liver cancer cell HepG2, the IC of Humanmachine tumour B16 cell and people's lung cancer H460 cell
50respectively 44.4 μ M, 40.4 μ M and 36.3 μ M, and to other subject cell strain without obvious growth inhibitory activity; The IC of Spiro-IndimycinA to people's acute lymphoblastic leukemia T lymphocyte CCRF-CEM
50be 44.4 μ M, to other subject cell strain without obvious growth inhibitory activity; With positive control, Staurosporine compares, show as the optionally restraining effect of Spiro-Indimycin A-D to seven kinds of surveyed cells, and its activity is stronger, and being expected exploitation by biotechnology or chemically modified becomes anti-cancer agent.
The cytotoxic assay of table 3:Spiro-Indimycin A-D
Annotate :-IC
50>100 μ g/ml
Claims (10)
1. streptomycete (Streptomyces sp.) SCSIO03032, its deposit number is CCTCC NO:M2011258.
2. compound S piro-Indimycin A-D, its structural formula is as shown in formula I:
3. the application of compound S piro-Indimycin A-D claimed in claim 2 in preparing antitumor drug.
4. application according to claim 3, is characterized in that, the application of described compound S piro-Indimycin A in preparation treatment people acute lymphoblastic leukemia medicine.
5. application according to claim 3, is characterized in that, the application of described compound S piro-Indimycin B in preparation treatment Humanmachine tumour, people's lung cancer or people's acute lymphoblastic leukemia medicine.
6. application according to claim 3, is characterized in that, the application of described compound S piro-Indimycin C in preparation treatment people's liver cancer or people's lung-cancer medicament.
7. application according to claim 3, is characterized in that, the application of described compound S piro-Indimycin D in preparation treatment Humanmachine tumour, people's liver cancer or people's lung-cancer medicament.
8. streptomycete claimed in claim 1 (Streptomyces sp.) SCSIO03032 is in the application for the preparation of compound S piro-Indimycin A claimed in claim 2, B, C or D.
9. the preparation method of a compound S piro-Indimycin A claimed in claim 2, B, C or D, it is characterized in that, described compound S piro-Indimycin A, B, C or D are that in the fermenting culture of the described streptomycete of Accessory Right requirement 1 (Streptomyces sp.) SCSIO03032, preparation separates and obtains.
10. preparation method according to claim 9, is characterized in that, concrete steps are as follows:
A) prepare the fermenting culture of streptomycete (Streptomyces sp.) SCSIO03032, the fermented liquid of this fermenting culture and mycelium are separated, fermented liquid is through macroporous resin adsorption, rear with acetone wash-out macroporous resin, after elutriant reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration; Mycelium is first used acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains medicinal extract B after distillation and concentration;
B) crude extract extractum A and medicinal extract B merged is through silica gel column chromatography, by chloroform/methanol as eluent, carry out gradient elution from volume ratio 100:0～0:100, collect the cut Fr.1 that chloroform/methanol volume ratio 95:5 gradient elution gets off, by the anti-phase medium pressure liquid chromatography of ODS, water/methyl alcohol is as eluent, carry out gradient elution from volume ratio 100:0～0:100, collect the cut Fr.1-1 that water/methyl alcohol volume ratio 30:70 gradient elution gets off, after the LH-20 gel column, using chloroform/methanol volume ratio 1:1 as the moving phase wash-out, follow the trail of and separate with thin-layer chromatography, collecting on thin layer plate by the chloroform/acetone volume ratio is the 9:1 cut that the Rf value is 0.8 during as developping agent, be Spiro-Indimycin B, the cut that the Rf value is 0.6, be Spiro-Indimycin A, collecting on thin layer plate by the petrol ether/ethyl acetate volume ratio is the 40:60 cut that the Rf value is 0.8 during as developping agent, is Spiro-Indimycin C, collecting on thin layer plate by the petrol ether/ethyl acetate volume ratio is the 50:50 cut that the Rf value is 0.8 during as developping agent, is Spiro-Indimycin D.
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| JP2014541509A JP5826406B2 (en) | 2012-03-28 | 2012-04-13 | Streptomyces, antitumor compound Spiro-Indymycin AD, production method and use thereof, and antitumor agent and drug containing spiroindimycin |
| PCT/CN2012/074013 WO2013143184A1 (en) | 2012-03-28 | 2012-04-13 | Streptomyces, and anti-tumor compounds spiro-indimycin a-d and preparation method and application thereof |
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| CN101126074A (en) * | 2007-07-13 | 2008-02-20 | 东南大学 | Marine streptomycete with antineoplastic activity and culture method thereof |
| CN102295562A (en) * | 2011-07-01 | 2011-12-28 | 中国科学院微生物研究所 | Antineoplastic compound, its preparation method and applications |
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| CN101126074A (en) * | 2007-07-13 | 2008-02-20 | 东南大学 | Marine streptomycete with antineoplastic activity and culture method thereof |
| CN102295562A (en) * | 2011-07-01 | 2011-12-28 | 中国科学院微生物研究所 | Antineoplastic compound, its preparation method and applications |
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| Lynamicins A-E, Chlorinated Bisindole Pyrrole Antibiotics from a Novel Marine Actinomycete;Katherine A. McArthur et al.;《J. Nat. Prod》;20081231;全文 * |
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