CN104031052B - Antibiotic Indimicins A E and preparation method thereof and the application in preparing antitumor drug - Google Patents

Antibiotic Indimicins A E and preparation method thereof and the application in preparing antitumor drug Download PDF

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CN104031052B
CN104031052B CN201410214540.XA CN201410214540A CN104031052B CN 104031052 B CN104031052 B CN 104031052B CN 201410214540 A CN201410214540 A CN 201410214540A CN 104031052 B CN104031052 B CN 104031052B
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张长生
张文军
马亮
李苏梅
张庆波
张海波
张光涛
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South China Sea Institute of Oceanology of CAS
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Abstract

Do you the invention discloses antibiotic Indimicins? A E and preparation method thereof and the application in preparing antitumor drug. Indimicins? A-E, shown in structural formula such as formula (I), does is it by streptomycete Streptomyces? sp.SCSIO03032 ferments generation, containing novel 6 '-chloro-1 ', 3 '-bis-methyl-2 '-H-indole structure and to MCF-7 Human Breast Cancer Cells, human liver cancer cell HepG2, people's pulmonary carcinoma H460 cell and human glioma cell SF-268 have cytotoxic activity, it is carried out biological engineering transformation or modifying for chemical structure is expected to be developed into anti-cancer agent.

Description

Antibiotic Indimicins A E and preparation method thereof and the application in preparing antitumor drug
Technical field:
The invention belongs to industrial microorganism field, be specifically related to new antibiotic IndimicinsA E and preparation method thereof and the application in preparing antitumor drug.
Background technology:
Bisindole alkaloid various structures and there is multiple biology, pharmacologically active. At present, the analog of some bisindole alkaloids has been used for clinical trial (H.Nakano as the inhibitor of protein kinase K or the inhibitor of DNA topoisomerase I, andS.Omura, ' ChemicalBiologyofNaturalIndolocarbazoleProducts:30Yearss incetheDiscoveryofStaurosporine ', JAntibiot, 62 (2009), 17-26.).
Summary of the invention:
First purpose of the present invention is to provide the bisindole alkaloid indimicinsA E with anti-tumor activity five kinds new.
The bisindole alkaloid indimicinsA E five kinds new of the present invention, shown in its structure such as formula (I):
Wherein work as R1=CH3,R2During=H, for IndimicinsA (1); Work as R1=CH3,R2=CH3Time, for IndimicinsB (2); Work as R1=H, R2=CH3Time, for IndimicinsC (3); Work as R1=H, R2During=H, for IndimicinsD (4); When in indimicinA, C-2 ' and C-2 " between carbon-carbon single bond when disconnecting, for indimicinE (5).
The preparation method that second purpose of the present invention is to provide a kind of bisindole alkaloid IndimicinsA E.
The IndimicinsA E of the present invention is that preparative separation obtains from the fermentation culture medium of streptomycete (Streptomycessp.) SCSIO03032.
The present invention prepares IndimicinsA E preferably by following methods from the fermentation culture medium of streptomycete (Streptomycessp.) SCSIO03032, specifically comprises the following steps that
A, preparation streptomycete (Streptomycessp.) SCSIO03032 fermentation culture medium, the fermentation liquid of this fermentation culture medium and mycelium are separated, fermentation liquor macroporous resin adsorption, afterwards with acetone eluting macroporous resin, after eluent reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration;Mycelium first uses acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains extractum B after distillation and concentration;
B, crude extract extractum A and extractum B merged is through silica gel column chromatography, by chloroform/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2 that chloroform/methanol volume ratio 98:2 gradient elution gets off, by the anti-phase medium pressure liquid chromatography of ODS, with water/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2-3 that water/methanol volume ratio 20:80 gradient elution gets off, after LH-20 gel column, using chloroform/methanol volume ratio 1:1 as mobile phase eluting, eluting fraction separates then through Thin Layer Chromatography, obtain compound indimicinA, indimicinB, indimicinC, indimicinD and indimicinE.
The fermentation culture medium of preparation streptomycete (Streptomycessp.) SCSIO03032 of described a) step is prepared preferably by following methods: streptomycete (Streptomycessp.) SCSIO03032 of activation is accessed seed culture medium, 28 DEG C, 200rpm, cultivate 48h and obtain seed liquor, seed liquor is linked in fermentation medium with the inoculum concentration of 10%, 28 DEG C, 200rpm, shaken cultivation 120h, and prepare fermentation culture medium, described seed culture medium and the formula of fermentation medium are all contain in every liter of culture medium: starch 10g, yeast powder 5g, peptone 4g, CaCO32g, thick sea salt 30g, surplus is water, pH7.2.
3rd purpose of the present invention is to provide streptomycete (Streptomycessp.) SCSIO03032 application in preparing compound indimicinA, indimicinB, indimicinC, indimicinD or indimicinE.
The present inventor is found through experiments, human cancer cell is included breast carcinoma MCF-7 by compound indimicinsA E, people hepatocarcinoma HepG2, people pulmonary carcinoma H460 and human glioma cell SF-268 has cytotoxic activity, and wherein human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by indimicinB50It is 10.0 ��Ms, 13.9 ��Ms, 19.1 ��Ms and 11.5 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by indimicinA50It is 23.7 ��Ms, 22.3 ��Ms, 41.2 ��Ms and 17.9 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinC50It is 21.8 ��Ms, 20.9 ��Ms, 29.7 ��Ms and 23.4 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinD50It is 22.4 ��Ms, 23.9 ��Ms, 21.5 ��Ms and 22.3 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinE50It is 36.4 ��Ms, 32.0 ��Ms, 43.7 ��Ms and 33.4 ��Ms respectively.
Therefore, the 4th purpose of the present invention is to provide compound indimicinA, B, C, D or E application in preparing antitumor drug.
Described antitumor drug is preferably anti-breast cancer, people's hepatocarcinoma, the medicine of people's pulmonary carcinoma or human glioma.
5th purpose of the present invention is to provide a kind of antitumor drug, it is characterised in that comprise indimicinA, B, C, D or E of effective dose as active ingredient.
Described antitumor drug is preferably anti-breast cancer, people's hepatocarcinoma, the medicine of people's pulmonary carcinoma or human glioma.
The present invention separates from streptomycete (Streptomycessp.) SCSIO03032 and obtains 5 new bisindole alkaloid indimicinsA E with anti-tumor activity, it can be used for preparing antitumor drug, is expected to be developed into anti-cancer agent by biological engineering or chemical modification.
Streptomycete (Streptomycessp.) SCSIO03032 of the present invention is preserved in China typical culture collection center (CCTCC) on July 18th, 2011, address: Wuhan University of Wuhan, China city, its deposit number is CCTCCNO:M2011258, it is disclosed in the patent No.: ZL201210087537.7, and denomination of invention is: in the patent of streptomycete, antitumoral compounds Spiro-IndimycinA-D and its preparation method and application.
Accompanying drawing illustrates:
Fig. 1 is the high-efficient liquid phase chromatogram of the extract-extractum A of supernatant and mycelial extract-extractum B;
High performance liquid chromatography (HPLC) condition: chromatographic column is phenomex150 �� 4.6mm (SphereCloneSAX), mobile phase includes A phase and B phase, mobile phase A phase: the formic acid of the acetonitrile+0.08% (volume fraction) of 10% (volume fraction), solvent is water, flowing B phase: the acetonitrile of 90% (volume fraction), solvent is water; Injection procedure: 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, detects wavelength 254nm, flow velocity 1ml/min, wherein 1 representation compound 1,2 representation compound 2,3 representation compound 3,4 representation compound 4,5 representation compounds 5.
The crucial HMBC of Fig. 2 compound 1,1H�C1HCOSY and NOE is relevant and the crystal diffraction structure of compound 1.
The crucial HMBC of Fig. 3 compound 2-4,1H�C1HCOSY and NOE is correlated with.
Fig. 4 compound 2-4 CD collection of illustrative plates in methanol solution.
The crucial HMBC of Fig. 5 compound 5 and1H�C1HCOSY is correlated with, the ECD collection of illustrative plates of (3 ' R)-5 and (3 ' S)-5 of the compound 5 ECD collection of illustrative plates in methanol solution and calculating.
Detailed description of the invention:
Following example are further illustrating the present invention, rather than limitation of the present invention.
Embodiment 1: the separation of active metabolite IndimicinA-E and preparation
1, culture medium (seed culture medium and fermentation medium): every liter contains starch 10g, yeast powder 5g, peptone 4g, CaCO32g, sea salt 30g, surplus is water, pH7.2. 121 DEG C, sterilizing 30min;
2, fermentation
2.1, seed culture: single bacterium colony of streptomycete (Streptomycessp.) SCSIO03032 activated on culture dish is respectively connected to 18 bottles, in the taper culture bottle of every bottle of 250mL containing 50mL seed culture medium, 28 DEG C, 200r min-1, cultivate 48h, prepare seed liquor 900mL.
2.2, fermentation culture: seed liquor is linked into 9L fermentation medium with the inoculum concentration (percent by volume) of 10% and (is placed in the taper culture bottle of 250mL, every bottle contains 50ml fermentation medium, totally 180 bottles) in, 28 DEG C, 200r min-1, shaken cultivation 120h, obtains 9L fermentation culture medium.
3, extract: fermentation culture medium first carries out centrifugation (3500r min-1,8min), obtains supernatant (fermentation liquid) and the mycelium of 9L volume. Fermentation liquor macroporous resin XAD16 Solid-Phase Extraction, use acetone eluting macroporous resin 3 times afterwards, after eluent reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate 3 times, and ethyl acetate layer obtains supernatant extract-extractum A (3.6g) after distillation and concentration; Mycelium 2L acetone at room temperature lixiviate 3 times, each 3 hours, united extraction liquid, remain water mixed liquid 6L extraction into ethyl acetate after recovered under reduced pressure acetone, mycelium extract extractum B (0.7g) is distilled to obtain in ethyl acetate layer decompression.
4, the extraction of reactive compound separates and identifies
The extraction of 4.1 compound IndimicinA-E separates and identifies: the sample taking a small amount of extractum A and extractum B respectively is dissolved in 100 �� l methanol, centrifuging and taking supernatant 10 �� l, through HPLC sample detection, show, containing compound 1 (1), 4 (4) and 5 (5) in extractum A, containing compound 2 (2) and 3 (3) (Fig. 1) in extractum B, extractum A and extractum B are merged. by the crude extract of this merging through silicagel column (300-400 order) chromatography, by chloroform/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2 (2.54g) that chloroform/methanol volume ratio 98:2 gradient elution gets off, it is evaporated, through ODS anti-phase medium pressure liquid chromatography (YMC*GELODS-A ball-shaped filling material, 120A aperture, 50um particle diameter, 240 �� 4.0cmI.D.), flow velocity is 25ml/min, with water/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2-3 (1.41g) that water/methanol volume ratio 20:80 gradient elution gets off, after LH-20 gel column (2.5*100cm), using chloroform/methanol volume ratio 1:1 as mobile phase eluting, every 50ml collects and merges into portion, order obtains five parts of samples of Fr.2-3-A F, Fr.2-3-D (280mg) is prepared with thin layer chromatography, collect thin layer prepare on plate with petrol ether/ethyl acetate volume ratio be 9:1 as developing solvent time Rf value be the fraction of 0.8, it is compound 1 (indimicinA) (100.6mg), Rf value is the fraction of 0.7, is compound 3 (indimicinC) (17.2mg), Rf value is the fraction of 0.6, is compound 5 (indimicinE) (53.5mg). Fr.2-3-C (25mg) is prepared with thin layer chromatography, collect on lamellae with petrol ether/ethyl acetate volume ratio be 9:1 as developing solvent time Rf value be the fraction of 0.8, be compound 2 (indimicinB) (14.3mg). Fr.2-3-E (17mg) is prepared with thin layer chromatography, collect on lamellae with petrol ether/ethyl acetate volume ratio be 75:25 as developing solvent time Rf value be the fraction of 0.8, be compound 4 (indimicinD) (10.6mg). pass through structural analysis, it is identified by 5 the Compound Compound 1-5 (corresponding compound indimycinA-E) (formula (I)) prepared from the fermentation culture medium of streptomycete (Streptomycessp.) SCSIO03032 of the present invention, and its qualification result is as follows:
Compound 1 (IndimicinA): white crystal,UV(MeOH)��max(log ��) 252nm (4.55); 207nm (4.75); ECD (c3.9 �� 10-5M,MeOH)��max(�� ��)-4.43 (251), 8.67 (227), 12.11 (217) nm; IR (KBr) ��max3371,2291,1603,1477cm-1;1HNMR(500MHz,CDCl3) and13CNMR(125MHz,CDCl3) data, in Table 1, table 2; HRESIMSm/z [M-H]-408.1051,(calcdforC23H20Cl2N3,408.1029)��
The positive source high-resolution Electrospray Mass Spectrometry figure of compound 1 shows that its quasi-molecular ion peak is m/z408.1051 [M+H]+, thus it is speculated that its molecular formula is C23H19Cl2N3(value of calculation is 408.1029, and degree of unsaturation is 15), IR composes at 3371cm-1Having strong absorption, there is NH in prompting in the molecule.Analyze1H,13C and HSQC (table 1, table 2) data show that this compound contains three methyl [��H3.60(3H,s,H-10),��C36.3; ��H2.77(3H,s,H-10��),��C34.9; ��H1.50(3H,s,H-11��),��C27.1], eight sp2The methine of hydridization, a sp3Methine [the �� of hydridizationH4.02(1H,s,H-2��),��C72.8], 10 sp2The quaternary carbon of hydridization and a sp3Quaternary carbon [the �� of hydridizationC(45.5 C-3 ')]. Pass through1H-1HCOSY atlas analysis shows containing two typical ABX coupling system [��H7.33 (1H, d, J=2.0Hz), 7.09 (1H, dd, J=8.5,2.0Hz), 6.43 (1H, d, J=8.5Hz) and ��H7.71 (1H, d, J=2.0Hz), 7.18 (1H, dd, J=8.5,2.0Hz), 7.31 (1H, d, J=8.5Hz], this illustrates in compound 1 containing two 1,2,4-trisubstituted phenyl ring. These NMR data features of compound 1 and spiroindimicinsA-D and lynamicinsA-D (Zhang, the W. of bibliographical information; Liu, Z.; Li, S.; Yang, T.; Zhang, Q.; Ma, L.; Tian, X.; Zhang, H.; Huang, C.; Zhang, S.; Ju, J.; Shen, Y.; Zhang, C.Org.Lett.2012,14,3364-3367; McArthur, K.A.; Mitchell, S.S.; Tsueng, G.; Rheingold, A.; White, D.J.; Grodberg, J.; Lam, K.S.; Potts, B.C.J.Nat.Prod.2008,71,1732-1737) similar, prompting compound 1 belongs to single pyrroles double; two indole ring alkaloid family. Carefully analyze1H-1The double; two substituted azole ring (Fig. 2) of 6 "-chloro-2 " in HCOSY and HMBC associated home compound 1,3 "-bis-replacements-1 "-H-indole and 1-methyl-3,4-. With compound lynamicinsA-D the difference is that containing rare 6 '-chloro-1 ', 3 '-bis-methyl-2 '-H-indole in compound 1, this passes through H-10 ' (��H2.77) to C-2 ' (��C72.8) and C-9 ' (��C134.9),H-2��(��H4.02) to C-3 ' (��C45.5), and H-11 ' (��H1.50) to C-2 ' (��C72.8),C-3��(��C45.5) and C-4 ' (��C139.3) HMBC relevant (Fig. 2) be confirmed. Except this, " constituting hexatomic ring, this is by H-2 ' (�� for C-2 ' and C-2 in compound 1H4.02) to C-2 " (��C130.3),C-3��(��C109.2) and C-4 (��C123.1) HMBC relevant (Fig. 2) confirm. So, it is determined that the planar structure of compound 1. H-2 ' and H in compound 13-11 ' cis-configuration be by H-2 ' and H3-11 ' NOESY be correlated with (Fig. 2) of determining. The Cu target diffraction of compound 1 monocrystalline confirms its structure (Fig. 2) further, simultaneously according to the configuration of C-2 ' and C-3 ' in compound 1 is defined as S and R (absolute configuration parameter-0.014 (12)) (Fig. 2). Therefore shown in the 1 of the structure of compound 1 such as formula (I), its R1=CH3,R2=H, called after IndimicinA.
Compound 2 (IndimicinB): white solid,UV(MeOH)��max(log ��) 207nm (4.81); ECD (c2.4 �� 10-5M,MeOH)��max(�� ��)-7.81 (252), 5.94 (233), 34.23 (207) nm; IR (KBr) ��max3339,2922,1600,1476cm-1;1HNMR(500MHz,CDCl3) and13CNMR(125MHz,CDCl3) data are in Table 1, table 2; HRESIMSm/z [M-H]-422.1191,(calcdforC23H20Cl2N3,422.1185)��
The positive source high-resolution Electrospray Mass Spectrometry figure of compound 2 shows that its quasi-molecular ion peak is m/z422.1191 [M+H]+, thus it is speculated that its molecular formula is C24H21Cl2N3(value of calculation is 422.1185). Compound 21H and13C modal data (table 1, table 2) is much like with compound 1. Different places is many methyl in compound 2, from H3-11(��H2.60,3H, s) to C-2 (��C122.0) and C-3 (��C112.5) HMBC methyl is positioned C-2 position. The planar structure of compound 2 confirms (Fig. 3) further by 2DNMR collection of illustrative plates.Therefore shown in the 2 of the structure of compound 2 such as formula (I), its R1=CH3,R2=CH3, called after IndimicinB.
Compound 3 (IndimicinC): anoff-white, opticallyinactivesolid.UV(MeOH)��max(log ��) 204nm (4.51); ECD (c2.4 �� 10-5M,MeOH)��max(�� ��)-9.73 (254), 8.72 (235), 32.37 (208) nm; IR (KBr) ��max3410,2921,1603,1479cm-1;1HNMR(500MHz,CDCl3) and13CNMR(125MHz,CDCl3) data are in Table 1, table 2; HRESIMSm/z [M-H]-408.1039,(calcdforC23H20Cl2N3,408.10295)��
Compound 3 is white solid. The positive source high-resolution Electrospray Mass Spectrometry figure of compound 3 shows that its quasi-molecular ion peak is m/z408.1039 [M+H]+, thus it is speculated that its molecular formula is C24H21Cl2N3(value of calculation is 408.1029). Compound 31H and13Much like (table 1, the table 2) of CNMR data and compound 2. According to the NH-1 (�� in compound 3H7.641H, brs) and H-5 (��H6.341H, d, J=2.0Hz) between exist COSY be correlated with, thus it is speculated that compound 3 lacks the methyl group of N-1 compared with compound 2. The planar structure of compound 3 confirms (Fig. 3) further by 2DNMR collection of illustrative plates. Therefore shown in the 3 of the structure of compound 3 such as formula (I), its R1=H, R2=CH3, called after IndimicinC.
Compound 4 (IndimicinD): white solid.UV(MeOH)��max(log ��) 204nm (4.39); ECD (c2.6 �� 10-5M,MeOH)��max(�� ��)-3.89 (251), 3.67 (228), 7.38 (207) nm; IR (KBr) ��max3278,2924,1604,1481cm-1;1HNMR(500MHz,CDCl3) and13CNMR(125MHz,CDCl3) data are in Table 1, table 2; HRESIMSm/z [M-H]-392.0712,(calcdforC23H20Cl2N3,392.0727)��
Compound 4 is white solid, and the positive source high-resolution Electrospray Mass Spectrometry figure of compound 4 shows that its quasi-molecular ion peak is m/z392.0712 [M+H]+, thus it is speculated that its molecular formula is C22H17Cl2N3(value of calculation is 392.0727). Compound 41H and13CNMR modal data (table 1, table 2) is similar to compound 1. H-2 (�� in compound 4H6.991H, dd, J=1.5,1.5)/NH-1 (��H8.041H, brs), with H-5 (��H6.531H, dd, J=1.5,1.5)/NH-1 (��H8.041H, brs) between1H-1HCOSY is relevant shows that compound 4 lacks N-1 position methyl group. The planar structure of compound 4 confirms (Fig. 3) further by 2DNMR collection of illustrative plates. Identical with compound 1, compound 24 has two chiral carbon in C-2 ' and C-3 ' position, and their relative configuration passes through H3-11 ' relevant with the NOESY of between H-2 ' it is defined as cis-configuration (Fig. 3). By being composed compared with compound 1 (Fig. 4) by their CD, the absolute configuration of C-2 ' and C-3 ' position is defined as S and R. Therefore shown in the 4 of the structure of compound 4 such as formula (I), its R1=H, R2=H, called after IndimicinD.
Compound 5 (IndimicinE): white solid.UV(MeOH)��max(log ��) 203nm (4.50); ECD (c2.4 �� 10-5M,MeOH)��max(�� ��)-4.24 (318), 12.98 (239), 13.80 (206) nm; IR (KBr) ��max3418,2929,1722,1462cm-1;1HNMR(500MHz,CDCl3) and13CNMR(125MHz,CDCl3) in Table 1, table 2; HRESIMSm/z [M-H]-410.1207,(calcdforC23H20Cl2N3,410.1185)��
Compound 5 is white solid, and the positive source high-resolution Electrospray Mass Spectrometry figure of compound 5 shows that its quasi-molecular ion peak is m/z410.1207 [M+H]+, thus it is speculated that its molecular formula is C23H21Cl2N3(value of calculation is 410.1185). Compound 51H,13C and HSQCNMR data (table 1, table 2) show that it has similar structure fragment to compound 1: 6 "-chloro-2 ", 3 "-bis-replacements-1 "-H-indole, 6 '-chloro-1 ', 3 '-bis-methyl-2 '-H-indole and 1-methyl-3, the double; two substituted azole ring of 4-. The difference is that compound 5 lacks a methine contained in compound 1 and a fragrant quaternary carbon, many methylene [��H3.08,3.31, (2H, d, H-2 '); Double bond disconnects, this supposition is by H3-10 ' in compound 5 (�� H2.32) to C-2 ' (�� C69.3) and H2-2 ' (�� H3.03,3.31) relevant to the HMBC of C-3 ' (�� C43.7) and that H-2 " (�� H6.091H; brs) and NH-1 " is between (�� H7.931H, brs) COSY (Fig. 5) confirmation.CD spectrum and the ECD of the 3 ' R calculated and 3 ' S that comparative compound 5 is measured in methanol solution compose, find that the CD spectrum measured is consistent in the region of 200-400nm with the ECD of 3 ' R spectrum, all at 200-240, there is negative Ces in 270-350nm region, has positive Ces (Fig. 5) in 240-260nm region. Thus, it is determined that compound 5 is configured as R C-3 ' position. Therefore, shown in the 5 of the structure of compound 5 such as formula (I), called after IndimicinE, when in indimicinA, C-2 ' and C-2 " between carbon-carbon single bond when disconnecting, for indimicinE.
Table 1. compound IndimicinsA E (1 5)1HNMR (500MHz) nuclear magnetic data belongs to
�� C69.3] and methine [�� H6.09 (1H, s, a H-2 "); �� C125.4]. Speculate C-2 ' and C-2 in compound 5 " between carbon-to-carbon
Table 2. compound IndimicinsA E (1 5)13CNMR (125MHz) nuclear magnetic data belongs to
The determination of cytotoxic activity of embodiment 2:IndimicinsA-E
IndimicinsA-E is for four kinds of tumor cell lines: MCF-7 Human Breast Cancer Cells, human liver cancer cell HepG2, and people's pulmonary carcinoma H460 cell and human glioma cell SF-268 have carried out determination of activity, experimental technique list of references [Wu, Z.C.; Li, D.L.; Chen, Y.C.; Zhang, W.M., Anewisofuranonaphthalenoneandbenzopyransfromtheendophyti cfungusNodulisporiumsp.A4fromAquilariasinensis.Helv.Chim .Acta2010,93, (5), 920-924.], (human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 using cisplatin as comparison50It is 6.0 ��Ms, 1.9 ��Ms, 3.6 ��Ms and 4.1 ��Ms respectively). Result shows that human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by indimicinB50It is 10.0 ��Ms, 13.9 ��Ms, 19.1 ��Ms and 11.5 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by indimicinA50It is 23.7 ��Ms, 22.3 ��Ms, 41.2 ��Ms and 17.9 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinC50It is 21.8 ��Ms, 20.9 ��Ms, 29.7 ��Ms and 23.4 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinD50It is 22.4 ��Ms, 23.9 ��Ms, 21.5 ��Ms and 22.3 ��Ms respectively; Human cancer cell is included the IC of breast carcinoma MCF-7, people's pulmonary carcinoma H460 cell, people hepatocarcinoma HepG2 and human glioma cell SF-268 by IndimicinE50It is 36.4 ��Ms, 32.0 ��Ms, 43.7 ��Ms and 33.4 ��Ms respectively. The IndimicinsA-E four kinds of cells to surveying be presented with cytotoxic activity, be expected to be developed into anti-cancer agent by biological engineering or chemical modification.

Claims (8)

1. compound indimicinsA-E, shown in its structure such as formula (I):
Wherein, compound 1 is indimicinA, its R1=CH3,R2=H; Compound 2 is indimicinB, its R1=CH3,R2=CH3; Compound 3 is indimicinC, its R1=H, R2=CH3; Compound 4 is indimicinD, its R1=H, R2=H; Compound 5 is indimicinE.
2. the preparation method of the compound indimicinsA-E described in a claim 1, it is characterised in that specifically comprise the following steps that
A, preparation streptomycete (Streptomycessp.) SCSIO03032 fermentation culture medium, the fermentation liquid of this fermentation culture medium and mycelium are separated, fermentation liquor macroporous resin adsorption, afterwards with acetone eluting macroporous resin, after eluent reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate, and ethyl acetate layer obtains extractum A after distillation and concentration; Mycelium first uses acetone extraction, and after leaching liquid reclaims acetone, remaining water mixed liquid is extracted with ethyl acetate again, and ethyl acetate layer obtains extractum B after distillation and concentration;
B, crude extract extractum A and extractum B merged is through silica gel column chromatography, by chloroform/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2 that chloroform/methanol volume ratio 98:2 gradient elution gets off, by the anti-phase medium pressure liquid chromatography of ODS, with water/methanol as eluant, gradient elution is carried out from volume ratio 100:0��0:100, collect the fraction Fr.2-3 that water/methanol volume ratio 20:80 gradient elution gets off, after LH-20 gel column, using chloroform/methanol volume ratio 1:1 as mobile phase eluting, eluting fraction separates then through Thin Layer Chromatography, obtain compound indimicinA, indimicinB, indimicinC, indimicinD and indimicinE.
3. preparation method according to claim 2, it is characterized in that, the fermentation culture medium of preparation streptomycete (Streptomycessp.) SCSIO03032 of described a step is to prepare by the following method: streptomycete (Streptomycessp.) SCSIO03032 of activation is accessed seed culture medium, 28 DEG C, 200rpm, cultivate 48h and obtain seed liquor, seed liquor is linked in fermentation medium with the inoculum concentration of 10%, 28 DEG C, 200rpm, shaken cultivation 120h, and prepare fermentation culture medium, described seed culture medium and the formula of fermentation medium are all contain in every liter of culture medium: starch 10g, yeast powder 5g, peptone 4g, CaCO32g, thick sea salt 30g, surplus is water, pH7.2.
4. streptomycete (Streptomycessp.) SCSIO03032 application in compound indimicinA, indimicinB, indimicinC, indimicinD or the indimicinE described in preparation claim 1.
5. the application in preparing antitumor drug of compound indimicinsA, B, C, D or the E described in claim 1.
6. application according to claim 5, it is characterised in that described antitumor drug is anti-breast cancer, people's hepatocarcinoma, the medicine of people's pulmonary carcinoma or human glioma.
7. an antitumor drug, it is characterised in that comprise indimicinA, B, C, D or the E described in the claim 1 of effective dose as active ingredient.
8. antitumor drug according to claim 7, it is characterised in that described antitumor drug is anti-breast cancer, people's hepatocarcinoma, the medicine of people's pulmonary carcinoma or human glioma.
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