CN101054403A - Novel antibiotic Chemomycin A, B, C, D and preparation method thereof - Google Patents

Abstract

The present invention relates to a group of novel compound Chemomicin B,C,D and its preparation method, pharmaceutical composition containing Chemomicin B,C,D as active ingredients and its uses of Chemomicin B,C,D in preparing antineoplastic drug. Chemomicin B,C,D is Angucyclinone antibiotic separated from Nocardia Mediterranei Var.Kanglensis 1747-64 culture. In vitro activity test prove Chemomicin B,C,D has strong suppressing activity for human oesophagus cancer cell KYSE150 and has good development prospect as a novel antineoplastic drug.

Description

The new antibiotic card is mycin (Chemomicin) B, C, D and manufacture method thereof not

Technical field:

The present invention relates to one group of new angle anthracycline antibiotics (Angucyclinone): block not mycin B, C, D and preparation method thereof, mycin B, C, D are the pharmaceutical composition of activeconstituents and block not mycin B, C, the application of D in the preparation antitumor drug to block not.

Background technology:

Kind surplus the microbiotic of finding to derive from microorganism has so far surpassed ten thousand, wherein many as medicine, agricultural chemicals is used widely.Angucyclinone class microbiotic is the microbiotic that the class found the sixties in 20th century has extensive biologic activity, and its structure and anthracycline antibiotics are more close, and wherein A encircles and is the form at angle and on anthracene nucleus.Most of Angucyclinone microbiotic all has anti-tumor activity, to the activity of gram-positive microorganism and Gram-negative bacteria a little less than, this type of microbiotic also has inhibitor activity, antiviral activity, receptor antagonist activity and immunosuppressive activity individually.Because this class microbiotic has extensive biologic activity, has caused many investigators' attention in the world, and this class microbiotic has been carried out deep research.Up to the present, existing more than 100 [Rohr, the J.NatRep.9 (2): 103-137,1992] of this class microbiotic of the Angucyclinone that has been found that.

Along with the extensive screening study of new antibiotic constantly develops in depth, content height, segregative major constituent microbiotic are separated mostly in the microbial fermentation solution, find that the difficulty of new antibiotic is increasing.To further find to have the microbiotic of notable biological activity, need new research strategy, as selecting rare actinomycete, extreme environment microorganism etc. on the bacterial classification; Chemically adopt modern chromatogram-spectrum coupling technique to realize the early stage structure decision of little fast standard, separate minor component or the like.

This laboratory is from rare actinomycete screening antitumor antibiotics process, separate the Angucyclinone microbiotic-Ka Mo mycin B, C, the D (ChemomicinB, C, D) that obtain having anti-tumor activity, learn data analysis through UV spectrum, infrared spectra, mass spectrum and nucleus magnetic resonance POP, determine that mycin B, C, D (ChemomicinB, C, D) are not the microbiotic of three new textures to card.This structural formula is different with all Angucyclinone class microbiotic of known structure so far, has not yet to see with mycin B, C, D do not have same structure and active relevant report.Up to now, in the Angucyclinone class microbiotic that has been found that, also not seeing Angucyclinone class microbiotic becomes antitumor drug listing.Therefore, blocking not, mycin B, C, D (Chemomicin B, C, D) have good research and development application prospect as the new texture antitumor antibiotics.

One of purpose of the present invention is to provide suc as formula blocking the not structure of mycin B, C, D (ChemomicinB, C, D) shown in (1), (2), (3);

Two of purpose of the present invention is that the not preparation method of mycin B, C, D (Chemomicin B, C, D) of card is provided;

Three of purpose of the present invention is, provides that mycin B, C, D (Chemomicin B, C, D) are the pharmaceutical composition of activeconstituents to block not;

Four of purpose of the present invention is that card is provided, and mycin B, C, D (Chemomicin B, C, D) and composition thereof must not used in the preparation antitumor drug.

Summary of the invention:

The invention provides suc as formula the card of structure shown in (1), (2), (3) not mycin B, C, D (ChemomicineB, C, D):

By a series of spectroscopy analysis, determined to block the not structure of mycin B, C, D (ChemomicinB, C, D).

The said card of the present invention not mycin B is a kind of powdery substance of white, and molecular formula is C ₁₉H ₁₈O ₆, its constitutional features is a kind of Fourth Ring structure with anthraquinone ring, wherein on the C-3 of A ring the methylol structure is arranged.It is soluble in methyl alcohol, ethyl acetate, methyl-sulphoxide equal solvent, is slightly soluble in water, chloroform.When chloroform: during methyl alcohol=9: 1, block the not R of mycin B on silica-gel plate _f=0.35.The said card of the present invention not mycin C is a kind of red granules crystalloid material, and molecular formula is C ₁₉H ₁₆O ₆, its structure to block that mycin B is very not similar, be not the C-6a position of mycin B and the degraded product after the dehydrogenation of C-12a position of card.It is soluble in methyl alcohol, ethyl acetate, and the methyl-sulphoxide equal solvent is slightly soluble in water, chloroform.When chloroform: during methyl alcohol=9: 1, block the not R of mycin C on silica-gel plate _f=0.35.The said card of the present invention not mycin D is a kind of chocolate brown powder shape material, and molecular formula is C ₁₉H ₁₄O ₅, be card not mycin C between C-4a position and the C-12b position dehydration after degraded product, solvability to preceding two very similar.When chloroform: during methyl alcohol=9: 1, block the not R of mycin D on silica-gel plate _f=0.55.

The said card of the present invention not mycin B, C, D is from the process of rare actinomycete screening antitumor antibiotics, is obtained by separation in Nocardia bacteria Mediterranean Sea change strain 1747-64 (the Nocardia Mediterranei Var.Kanglensis 1747-64) culture.Said generation bacterium has been delivered on December 8th, 2005 and has been positioned at the Pekinese " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " carries out preservation, and its deposit number is: CGMCC No.1554.

In order to obtain card not mycin B, C, D and anti-tumor activity thereof, the present invention has taked following technological line and step:

1, blocks not fermentation and the cultivation of mycin B, C, D: at first be that the Nocardia bacteria Mediterranean Sea change strain 1747-64 bacterium that will grow in the inclined-plane is inoculated in seed culture medium, on rotary shaker, cultivate, change fermention medium then over to and on the reciprocating vibration shaking table, cultivate, the results fermented liquid;

2, block the not extraction of mycin B, separate: in the fermenting culture, add weak acid (as the hydrochloric acid of 2N etc.), suction filtration is removed mycelia, obtains supernatant liquor, uses non-polar organic solvent (as ethyl acetate or acetone etc.) extraction again, and concentrating under reduced pressure obtains medicinal extract.Medicinal extract adopts Sephadex LH-20 that the component that contains target compound is implemented further to separate earlier through silica gel column chromatography then, and eluting solvent is a methyl alcohol.Be prepared with polyamide layer at last, methanol-eluted fractions, the concentrate under reduced pressure at low temperature evaporate to dryness obtains not mycin B of white powder target compound card at last;

3, block the not extraction of mycin C, D, separate: will through polyamide layer separates the card that obtains not the pure product of mycin B dissolve with small amount of methanol, add less water then, in 50 ℃ of heating sex change in 48 hours degradeds down, prepare and obtain through efficient thin layer plate;

4, the structure of blocking not mycin B, C, D is identified: according to UV, IR, HR-MS, ¹H-NMR, ¹³C-NMR, DEPT data test and ¹H- ¹The relevant spectrum of H ( ¹H- ¹H COSY), ¹H- ¹³Relevant spectrum of C (HSQC) and inverse detection are long-range ¹H- ¹³The analysis of the relevant spectrum of C heteronuclear multikey (HMBC) has determined to block the not structure of mycin B, C, D;

5, block the not Determination of biological activity of mycin B, C, D: adopt mtt assay to carry out the anti tumor activity in vitro test, selected tumor cell line behaviour esophageal cancer cell KYSE150.Result of study shows, blocks the anti-tumor activity that not mycin B, C, D have highly significant.

The present invention also provides and has blocked the not pharmaceutical composition of mycin B, C, D.Said pharmaceutical composition is that mycin B, C, D are activeconstituents to block not, forms with pharmaceutically acceptable one or more carriers.

More than said pharmaceutically acceptable carrier be meant that the pharmaceutical carrier of pharmaceutical field routine is as thinner, vehicle such as water etc.; Weighting agent such as starch, sucrose etc.; Tackiness agent such as derivatived cellulose, alginate, gelatin and polyvinylpyrrolidone; Wetting agent such as glycerine; Disintegrating agent such as agar, lime carbonate and sodium bicarbonate; Absorption enhancer such as quaternary ammonium compound; Tensio-active agent such as cetyl alcohol; Lubricant such as talcum powder, calcium stearate etc.In addition, also can in composition, add other assistant agent such as flavouring agent, sweeting agent etc.

The present invention also provides and has blocked not mycin B, C, D and composition thereof the application in the preparation antitumor drug.

The invention effect:

Compound shown in structural formula provided by the invention (1), (2), (3) is not mycin B, C, D (Chemomicin B, C, D) of three new Angucyclinone class microbiotic cards, experimental results show that through biologic activity, these three compounds all have very remarkable antitumor effect, and being expected to research and develop from now on becomes new clinical effective antitumour microbiotic.

Description of drawings:

The UV spectrum of accompanying drawing 1-Chemomicin B in MeOH;

The infrared spectra of accompanying drawing 2-Chemomicm B (KBr compressing tablet);

The HR-ESI-MS spectrum of accompanying drawing 3-Chemomicin B;

Accompanying drawing 4-Chemomicin B is at DMSO-d ₆In ¹The H-NMR spectrum;

Accompanying drawing 5-Chemomicin B is at DMSO-d ₆+ D ₂Among the O ¹The H-NMR spectrum;

Accompanying drawing 6-Chemomicin B is at DMSO-d ₆In ¹³The C-NMR spectrum;

Accompanying drawing 7-Chemomicin B is at DMSO-d ₆In DEPT spectrum;

Accompanying drawing 8-Chemomicin B is at DMSO-d ₆In ¹H- ¹H COSY spectrum;

Accompanying drawing 9-Chemomicin B is at DMSO-d ₆In hsqc spectrum;

Accompanying drawing 10-Chemomicin B is at DMSO-d ₆In HMBC spectrum;

The UV spectrum of accompanying drawing 11-Chemomicin C in MeOH;

The infrared spectra of accompanying drawing 12-Chemomicin C (KBr compressing tablet);

The HR-ESI-MS spectrum of accompanying drawing 13-Chemomicin C;

Accompanying drawing 14-Chemomicin C is at DMSO-d ₆In ¹The H-NMR spectrum;

Accompanying drawing 15-Chemomicin C is at CD ₃Among the OD ¹The H-NMR spectrum;

Accompanying drawing 16-Chemomicin C is at CD ₃Among the OD ¹³The C-NMR spectrum;

Accompanying drawing 17-Chemomicin C is at CD ₃DEPT spectrum among the OD;

Accompanying drawing 18-Chemomicin C is at CD ₃Among the OD ¹H- ¹H COSY spectrum;

Accompanying drawing 19-Chemomicin C is at CD ₃Hsqc spectrum among the OD;

Accompanying drawing 20-Chemomicin C is at CD ₃HMBC spectrum among the OD;

The UV spectrum of accompanying drawing 21-Chemomicin D in MeOH;

The infrared spectra of accompanying drawing 22-Chemomicin D (KBr compressing tablet);

The HR-ESI-MS spectrum of accompanying drawing 23-Chemomicin D;

Accompanying drawing 24-Chemomicin D is at CD ₃Among the OD ¹The H-NMR spectrum;

Accompanying drawing 25-Chemomicin D is at CD ₃Among the OD ¹³The C-NMR spectrum;

Accompanying drawing 26-Chemomicin D is at CD ₃DEPT spectrum among the OD;

Accompanying drawing 27-Chemomicin D is at CD ₃Among the OD ¹H- ¹H COSY spectrum;

Accompanying drawing 28-Chemomicin D is at CD ₃Hsqc spectrum among the OD;

Accompanying drawing 29-Chemomicin D is at CD ₃HMBC spectrum among the OD.

Specific embodiments:

Following examples can make those skilled in the art more fully understand the present invention, but do not limit the present invention in any way.

＜embodiment 1 〉: block the not fermentation of mycin B, C, D

(2% Zulkovsky starch, the 1%KNO on inclined-plane will be grown in ₃, 0.05%NaCl, 0.05%K ₂HPO ₄,, 0.001%FeSO ₄7H ₂O,, 0.05%MgSO ₄7H ₂O,, 2% agar, pH7.0) Nocardia MediterraneiVar.Kanglensis 1747-64 bacterium is inoculated in seed culture medium (2% glucose, 1% analysis for soybean powder, 0.5% yeast powder, 0.5%CaCO ₃, pH6.5) 50ml/250ml shake bottle, cultivated 48 hours on rotary shaker at 28 ℃, with 5% inoculum size, change fermention medium (3% glucose, 1% groundnut meal, 1% yeast powder, 0.5% peptone, 0.1%CaCO over to then ₃,, pH6.5) in the triangular flask of 100ml/500ml, on the reciprocating vibration shaking table, cultivating 72 hours under 28 ℃, the results fermented liquid is 20L altogether.

＜embodiment 2 〉: block the not extraction of mycin B, C, D

Above gained fermented liquid is transferred pH to 3～4 with the HCl of 2N, and pad is gone up the filter paper suction filtration in the B, removes mycelia etc., obtains the supernatant fermented liquid.With the ethyl acetate extraction of fermented liquid 1/3 volume three times, after the ethyl acetate that three extractions are obtained merges, add the anhydrous sodium sulfate drying dehydration, left standstill 3～4 hours, concentrating under reduced pressure obtains medicinal extract.

＜embodiment 3 〉: block the not separation of mycin B, C, D

Above gained medicinal extract earlier through silica gel column chromatography with chloroform: the methyl alcohol gradient elution carries out initial gross separation, gradient is respectively chloroform: methyl alcohol=100: 1 (202mL altogether), chloroform: methyl alcohol=20: 1 (300mL altogether), chloroform: methyl alcohol=9: 1 (200mL altogether), collect each cut, the component that will contain target compound-Ka Mo mycin B merges concentrated; Adopt SephadexLH-20 that the component that contains target compound is further separated then, eluting solvent is a methyl alcohol, and flow velocity is 1mL/3 minute, shared methyl alcohol 350mL, collect each component in the elutriant, and the component that will contain target compound (blocking not mycin B) merges and concentrates; And then adopt polyamide layer to prepare (20 * 20cm, chloroform: methyl alcohol: water=9: 1: 0.5), collect R _f=0.65 green fluorescence band (blocking not mycin B) concentrates after the methanol-eluted fractions, carries out column chromatography to remove the impurity in the polyamide layer with Sephadex LH-20 again, low temperature rotation evaporate to dryness final vacuum drying, obtain the not pure product of mycin B of card, purity is 97.5%, altogether 30mg.

To block the not pure product of mycin B, rotation evaporate to dryness under 50 ℃ is to blocking not mycin B sex change degraded.Sample after the degraded is dissolved with small amount of methanol, through efficient TLC plate preparation (chloroform: methyl alcohol=9: 1), collect R _f=0.35 yellow band (blocking not mycin C) and R _f=0.55 dun colour band (blocking not mycin D), use chloroform: the solvent elution of methyl alcohol=19: 1, room temperature rotation evaporate to dryness obtains the not pure product of mycin C of card after the vacuum-drying, and purity is 95.3%, altogether 12mg; Block the not pure product of mycin D, purity is 92.6%, altogether 8mg.

＜embodiment 4 〉: block the not structure evaluation of mycin B, C, D

Blocking not, the physicochemical property of mycin B sees Table 1.According to UV (Fig. 1), IR (Fig. 2), HR-ESI-MS (Fig. 3), ¹H-NMRDMSO-d ₆Be solvent (Fig. 4), DMSO-d ₆+ D ₂O be solvent (Fig. 5), ¹³C-NMR DMSO-d ₆Be solvent (Fig. 6), DEPT DMSO-d ₆For solvent (Fig. 7) data and ¹H- ¹The relevant spectrum of H COSY DMSO-d ₆For solvent (Fig. 8), ¹H- ¹³The relevant spectrum of C HSQC DMSO-d ₆For solvent (Fig. 9) and inverse detection long-range ¹H- ¹³The relevant spectrum of C heteronuclear multikey HMBCDMSO-d ₆Be the analytical results of solvent (Figure 10), determined to block the not structure of mycin B.

Table 1. blocks the not physicochemical property of mycin B

Color and shape	White powder
		Molecular formula and molecular weight HPLC-ESI-MS HRESI-MS (m/z) Found:Calcd:UV λ max MeOHnm(ε) IRυ max(KBr)cm -1R fThe value solvability	C 19H 18O 6 MW＝342 [M-H] -＝341 341.1029[M-H] -341.1031 202 (17,995), 230 (35,263), 345 (6,005) 3436,2926,2860,1646,1577,1455,1,340 0.35 chloroforms: methyl alcohol=be soluble in methyl alcohol at 9: 1, ethyl acetate, DMSO; Be slightly soluble in water, chloroform

The present invention is according to high resolution mass spectrum HR-ESI-MS (Fig. 3), combination simultaneously ¹H-NMR (Fig. 4), ¹³C-NMR (Fig. 6) and DEPT (Fig. 7) spectrum, the molecular formula of having determined Chemomicin B is C ₁₉H ₁₈O ₆, degree of unsaturation N=11.By right ¹H-NMR (Fig. 4), ¹³C-NMR (Fig. 6), the analysis-by-synthesis of DEPT spectrum (Fig. 7) and HSQC (Fig. 9) spectrum, in 19 carbon signals of ChemomicinB 3 carbonyl signals are arranged, 8 aromatic carbons or olefinic carbon signal are (comprising 4 methine carbons, 2 quaternary carbons, 2 quaternary carbons that contain hydroxyl or methylol replacement), 1 quaternary carbon signal that hydroxyl replaces, also have 3 methyne signals in addition, 4 methylene signals (wherein 1 has hydroxyl to replace).3 hydroxyl proton signals, chemical shift are respectively δ 11.83 (forming the phenolic hydroxyl group proton of hydrogen bond), and 5.26 (2 hydroxyl protons) are when adding D ₂These 3 proton signals disappear after the O exchange.

According to IR spectrum (Fig. 2), at 3436cm ^-1The hydroxyl signal is arranged, at 1646cm ^-1Near the carbonyl signal is arranged, this with ¹³Low carbonyl signal δ 204.1 (C-1) in the C-NMR spectrum (Fig. 6), 197.0 (C-7), 195.1 (C-12) match.According to ¹Chemical shift ABX coupling system between 7.0～8.0ppm in the H-NMR spectrum (Fig. 4), simultaneously in conjunction with hsqc spectrum (Fig. 9) and ¹H- ¹HCOSY spectrum (Fig. 8) can be determined, C-9, and C-10, the chemical shift of proton on the C-11 position is respectively δ 7.29,7.74,7.55, and the carbon geochemistry displacement is respectively δ 122.7,136.5,117.6.According to the long-range coupling relation of HMBC spectrum (Figure 10), can determine C-7a, C-8, C-11a, the position of C-12 and chemical displacement value are because 8-OH proton and 7-carbonyl form intramolecular hydrogen bond, thereby the chemical shift that causes the 7-carbonyl carbon to δ 197.0, can be determined the position of C-7 to low field displacement.Because the C-8 position has-the OH replacement, chemical shift is to low field displacement to 159.8.

According to ¹H-NMR spectrum (Fig. 4), ¹H- ¹The analysis of HCOSY spectrum (Fig. 8) and hsqc spectrum (Fig. 9) can be determined other two macrostructure fragments (following figure fragment I and II).Long-range coupling relation according to HMBC spectrum (Figure 10), there are long-range coupling in methine protons δ 3.37 (H-12a) and chemical shift δ 197.0 (C-7), δ 195.1 (C-12), δ 44.9 (C-6a), δ 55.8 (C-12b), methine protons δ 3.55 (H-12b) and δ 71.2 (C-4a), δ 55.8 (C-12a), there is long-range coupling in δ 44.9 (C-6a), can determine C-12a and C-12b position, can couple together with the C ring by bar structure fragment I like this.According to UV spectrum (Fig. 1), near the strong absorption peak the 230nm meets the calculated value of α, beta unsaturated ketone, infers that structure fragment II is connected with carbonyl, determines that like this C-1 is a carbonyl.Proton δ 1.45 (H-5) and δ 20.4 (C-6), δ 44.9 (C-6a), δ 55.8 (C-12b), there is long-range coupling in δ 71.2 (C-4a), δ 2.28 (4-H) and δ 120.1 (C-2), δ 164.2 (C-3), δ 71.2 (C-4a), δ 31.1 (C-5), δ 55.8 (C-12b), δ 63.1 (C-13) has long-range coupling, can derive structure fragment I and II and couple together by C-4a and these two carbon of C-12b, determined the position of C-4a, like this A ring and B ring have been coupled together.Through with the documents and materials of Kanglemycin C relatively, simultaneously in conjunction with the research of HMBC as shown below, determine card not mycin B structure as the formula (1).

Block not mycin B at DMSO-d ₆In the NMR data as shown in table 2.

Table 2. blocks not mycin B at DMSO-d ₆NMR data among the I

Position	δ c a	δ H b(mult，J HZ)	1H- 1H COSY	HMBC
					1 2 3 4 4a-OH 5 6 6a 7 7a 8-OH 9 10 11 11a 12 12a 12b 13-CH 2 13-OH	204.1 120.1 164.2 41.6 71.2 31.1 20.4 44.9 197.0 118.2 159.8 122.7 136.5 117.6 134.9 195.1 43.7 55.8 63.1	5.91(s) 2.28(d，18.0) 2.69(dd，2.5，18.0) 5.26(s) 1.45(m) 1.45(m) 1.99(dd，13.0，3.0) 1.80(m) 2.93(ddd，3.5，3.5，3.0) 11.83(s) 7.29(d，8.5) 7.74(t，7.5，8.0) 7.55(d，7.5) 3.37(dd，3.0，3.0) 3.55(s) 4.06(d，5.0) 4.08(d，5.0) 5.26(s)	4H，13H 4H 2H，4H，13H 13H 6H，12bH 6H，12bH 5H， 5H，6aH 6H，12aH， 10H 9H，11H 10H 6aH，12bH 5H，12aH 2H，13OH，4H 2H，13OH，4H 13H	4，12b，13 2，3，4a，5，12b，13 2，3，4a，5 4a，5，12b 4a，6，6a，12b 4a，6，6a，12b 4a，12a 1，6，12a 7a，8，9 7a，8，11 8，11，11a 7a，9，12 6a，7，12，12b 4a，5，6a，7，12a 2，3 2，3

¹H-NMR(500 M HZ，δin ppm，J in HZ)， ¹³C-NMR(125MHZ，δin ppm)

Block not the physicochemical property of mycin C and see 3.The present invention is according to UV (Figure 11), IR (Figure 12), high resolution mass spectrum HR-ESI-MS (Figure 13), combination simultaneously ¹H-NMR (Figure 15 and Figure 14), ¹³C-NMR (Figure 16) and DEPT (Figure 17) spectrum, the molecular formula of having determined Chemomicin C is C ₁₉H ₁₆O ₆, degree of unsaturation N=12.By right ¹H-NMR (Figure 14 and Figure 15), ¹³C-NMR (Figure 16), the analysis-by-synthesis of DEPT spectrum (Figure 17) and HSQC (Figure 19) spectrum, in 19 carbon signals of ChemomicinC 3 carbonyl signals are arranged, 10 aromatic carbons or olefinic carbon signal are (comprising 4 methine carbons, 4 quaternary carbons, 2 quaternary carbons that contain hydroxyl or methylol replacement), 1 quaternary carbon signal that has hydroxyl to replace, also has 1 methyne signal in addition, 4 methylene signals (wherein 1 has hydroxyl to replace).According to ¹The H-NMR spectrum, solvent is DMSO-d ₆(Figure 14), can observe 3 hydroxyl proton signals, chemical shift is respectively δ 11.91 (forming the phenolic hydroxyl group proton of hydrogen bond), and 5.24 (2 hydroxyl protons) are CD at solvent ₃During OD (Figure 15), these 3 proton signals disappear.

Table 3. blocks the not physicochemical property of mycin C

Color and shape	The crystallization of red granules shape
		Molecular formula and molecular weight HPLC-ESI-MS HRESI-MS (m/z) Found:Calcd:UV λ max MeOH nm(ε) IRυ max(KBr)cm -1R fThe value solvability	C 19H 16O 6 MW＝340 [M+Na] +＝363[2M+Na +]＝703 341.10248[M+H] +341.10196 417 (4,614), 267 (12,182), 230 (28,710), 212 (35,535) 3383,2923,2854,1640,1617,1455,1281,1,128 0.35 chloroforms: methyl alcohol=9: 1 is soluble in methyl alcohol, ethyl acetate, DMSO; Be slightly soluble in water, chloroform

Owing to Chemomicin C is that Chemomicin B sex change degraded after 50 ℃ of heating comes, Chemomicin B becomes Chemomicin C after taking off 2 H.Compare by DEPT spectrum (Fig. 7) with Chemomicin B, discovery is to have lacked 2 methine carbon signals between 40.0～50.0ppm in chemical shift, and between 140.0～150.0ppm many 2 fragrant quaternary carbon signals, prompting thus, 2 H that taken off are positioned on 2 methine carbons, take off to form two keys behind the H.According to ¹H- ¹HCOSY composes (Figure 18), infers that 2 H that taken off is positioned on C-6a and the C-12a, shown in figure below.The research of HMBC spectrum (Figure 20), the comprehensive spectroscopy data of Chemomicin B and Chemomicin C relatively, determine card not mycin C structure as the formula (2).

Block not mycin C at CD ₃ODI and DMSO-d ₆NMR data among the II are as shown in table 4.

Table 4. blocks not mycin C at CD ₃OD (I) and DMSO-d ₆(II) the NMR data in

Position	(I) (II) (I)
						δ c a	δ H b(mult，J Hz)	δ H b(mult，J Hz)	1H- 1H COSY	HMBC
	1 2 3 4 4a-OH 5 6 6a 7 7a 8-OH 9 10 11 11a 12 12a 12b 13-CH 2 13-OH	198.5 121.1 164.9 42.0 71.0 28.7 21.1 144.9 190.8 116.0 162.3 124.6 137.2 119.9 133.5 184.5 143.1 53.5 64.7	5.91(s) 2.81(d，18.0) 2.41(d，18.0) 5.24(s) 1.63(m) 1.63(m) 2.61(m) 2.61(m) 11.91(brs) 7.32(d，9.0) 7.74(t，7.5，8.0) 7.56(d，6.5) 4.03(s) 4.11(s) 4.11(s) 5.24(s)	6.0(s) 2.82(dd，18.0，2.5) 2.45(d，18.0) 1.68(m) 1.77(m) 2.67(m) 2.67(m) 7.20(d，8.0) 7.62(t，8.0，8.0) 7.57(d，8.0) 4.11(s) 4.17(s) 4.17(s)	4H，13H 2H，13H 6H 6H 5H，6H 5H，6H 10H 9H，11H 10H 2H，4H，13H 2H，4H，13H						4，12b，13 1，2，3，4a，5 1，2，3，4a，5，12a，12b，13 4a，6，6a，1 2b 4a，6，6a 1，4a，5，7，6a，12a 1，4a，5，7，6a，12a 7a，8，11 8，11a 7a，9，12 1，4a，5，6a，12，12a 2，3，4 2，3，4

¹H-NMR(500MHz，δin ppm，J in Hz)， ¹³C-NMR(125MHz，δin ppm)

Blocking not, the physicochemical property of mycin D sees Table 5.The present invention is according to UV (Figure 21), IR (Figure 22), high resolution mass spectrum HR-ESI-MS (Figure 23), combination simultaneously ¹H-NMR (Figure 24), ¹³C-NMR (Figure 25) and DEPT (Figure 26) spectrum, the molecular formula of having determined Chemomicin D is C ₁₉H ₁₄O ₅, calculate its degree of unsaturation N=13.By right ¹H-NMR (Figure 24), ¹³The analysis-by-synthesis of C-NMR (Figure 25), DEPT spectrum (Figure 26) and HSQC (Figure 28) spectrum, 2 low carbonyl signal is arranged in 19 carbon signals of ChemomicinD, 14 aromatic carbon signals are (comprising 5 methine carbons, 6 quaternary carbons, 3 quaternary carbons that contain hydroxyl or methylol), 3 methylene signals (wherein 1 has hydroxyl to replace).

Table 5. blocks the not physicochemical property of mycin D

Color and shape	Chocolate brown powder
		Molecular formula and molecular weight HPLC-ESI-MS HRESI-MS (m/z) Found:Calcd:UV λ max MeOHnm(ε) IRυ max(KBr)cm -1R fThe value solvability	C 19H 14O 5 MW＝322 [M+Na] +＝345[2M+Na] +＝667 323.09130[M+H] +323.09140 210 (23,847), 266 (10,783), 446 (4,258) 3262,2923,2853,1613,1456,1,255 0.55 chloroforms: methyl alcohol=9: 1 is soluble in methyl alcohol, ethyl acetate, chloroform, DMSO; Be slightly soluble in water

Owing to Chemomicin D is that Chemomicin C sex change degraded after 50 ℃ of heating comes, become Chemomicin D after the Chemomicin C dehydration.According to ¹³C-NMR spectrum (Figure 25) and DEPT spectrum (Figure 26), spectroscopy data with ChemomicinC compare simultaneously, 1 carbonyl carbon signal that is positioned at low δ 190.0～200.0ppm has been lacked in discovery, 1 methylene signals that is positioned at δ 40.0～50.0ppm, 1 methyne letter and 1 quaternary carbon signal that has hydroxyl to replace that is positioned at δ 60.0～80.0ppm that is positioned at δ 50.0～60.0ppm, and increased by 4 aromatic carbon signals at δ 110.0～160.0ppm more. ¹In the H-NMR spectrum (Figure 24), (H-2 H-4) 2 fragrant proton signals occurred to δ 6.74, and near the olefinic carbon proton signal the δ 6.0 disappears.Can determine that thus after the dehydration, the A ring forms an aromatic nucleus between 4a-and the 12b-.Compare with the chemical structure of ChemomicinC, the carbonyl of C-1 becomes-the OH replacement, and the C-4 methylene radical becomes fragrant methyne, shown in figure below ¹H- ¹HCOSY spectrum (Figure 27) and HMBC compose (Figure 29) and, determine card not mycin D structure as the formula (3).

Block not mycin D at CD ₃NMR data among the OD are as shown in table 6.

Table 6. blocks not mycin D at CD ₃NMR data among the OD

Position	δ c a	δ H b(mult，J Hz)	1H- 1H COSY	HMBC
						1 2 3 4 4a 5 6 6a 7 7a 8-OH 9 10 11 11a 12 12a 12b 13-CH 2	156.9 114.7 147.1 118.2 142.8 29.3 21.6 145.7 189.8 115.9 162.1 124.4 137.2 119.9 135.3 185.2 145.3 117.8 64.6	6.74(s) 6.74(s) 2.65(m) 2.68(m) 2.65(m) 2.68(m) 7.1 8(d，8.5) 7.60(t，8.0，7.5) 7.50(d，7.0) 4.50(m) 4.50(m)	13H 13H 6H 6H 5H 5H 10H，11H 9H，11H 9H，10H 2H，4H 2H， 4H	1，4，12b 2，12b 4，6a，12b 4，6a，12b 4a，6a，7a 4a，6a， 7a 7a，8，11 8， 11a 7a，9，12 2，3，4 2，3，4

¹H-NMR(500MHz，δinppm，J in Hz)， ¹³C-NMR(125MHz，δin ppm)

Embodiment 5: block the not biologic activity experiment of mycin B, C, D

Adopt mtt assay to carry out anti tumor activity in vitro test, the cell strain behaviour esophageal cancer cell KYSE150 that selects for use.The concrete operations step is, mycin B, C, D are not dissolved among the DMSO respectively with above embodiment gained card, concentration range is 0.08ug/ml-10.0ug/ml, people's esophageal cancer cell KYSE150 that will be in logarithmic phase is through 0.125% trysinization, with RPMI RPMI-1640 diluting cells to 5000/hole.Add testing sample in the tumour cell, 37 ℃, 5% CO ₂Cultivated 3 days, every hole adds MTT 20ul, and 37 ℃ are continued to cultivate 4 hours, behind the 100ul nutrient solution, add 10% acidifying SDS liquid (100ul/ hole) in the sucking-off hole, culture plate is placed on the microwell plate vibrator vibrated 10 minutes, make the crystallisate dissolving.Detect each hole absorbance (the OD value detects wavelength 570 nanometers) in microplate reader.Experimental result shows, blocks not mycin B, C, the D IC to people's esophageal cancer cell KYSE150 ₅₀Be respectively 0.49ug/ml, 0.76ug/ml, 0.33ug/ml, all demonstrate very strong antitumor activity, different pharmaceutical concentration is as shown in table 7 to the inhibiting rate of people's esophageal cancer cell KYSE150.

Table 7. different concns card not mycin B, C, D to the inhibiting rate of people's esophageal cancer cell KYSE150

Drug concentration (ug/mL)	Chemomicin B inhibiting rate (%)	Chemomicin C inhibiting rate (%)	Chemomicin D inhibiting rate (%)
				10	99.4	98.9	98.6
5	99.6	99.1	99.0
				2.5	99.0	98.8	98.7
1.25	98.6	98.1	98.4
				0.63	66.1	31.8	58.2
0.31	18.3	0	48.5
				0.16	4.8	0	9.3
0.08	0	0	1.2

Claims (8)

1, not mycin B, C, D (ChemomicinB, C, D) of one group of new antibiotic card, its structure is suc as formula shown in (1), (2), (3):

2, preparation blocks the not method of mycin B, C, D according to claim 1, it is characterized in that said method may further comprise the steps:

1) blocks the not fermentation culture of mycin B, C, D;

2) block the not extraction separation of mycin B, C, D;

3) block the not structure evaluation of mycin B, C, D.

3, preparation method as claimed in claim 2, it is characterized in that Nocardia bacteria Mediterranean Sea health and happiness that fermentation culture system will grow in the inclined-plane become strain 1747-64 bacterium (deposit number into: CGMCC No.1554) be inoculated in seed culture medium, on rotary shaker, cultivate, change fermention medium then over to, on the reciprocating vibration shaking table, cultivate the results fermented liquid.

4, preparation method as claimed in claim 2 is characterized in that extraction separation ties up in the fermentation culture, adds the hydrochloric acid of weak acid such as 2N, suction filtration is removed mycelia, obtain supernatant liquor, use non-polar organic solvent such as ethyl acetate or acetone extract again, concentrating under reduced pressure obtains medicinal extract; Medicinal extract further separates the component that contains target compound with Sephadex LH-20 then earlier through silica gel column chromatography, and eluting solvent adopts methyl alcohol; Be prepared with polyamide layer at last, methanol-eluted fractions, 4 degree are the concentrating under reduced pressure evaporate to dryness down, obtains not mycin B of white powder target compound card; Will through polyamide layer separate the card that obtains not the pure product of mycin B dissolve with small amount of methanol, add less water then, in 50C heating sex change in 48 hours degraded down,, obtain target compound card not mycin C and D through efficient thin layer plate preparation.

5, as claim 2 or 4 described preparation methods, it is characterized in that structure identify system according to UV, IR, HR-MS, molecular formula, ¹H-NMR, ¹³C-NMR, DEPT data test and ¹H- ¹The relevant spectrum of H, ¹H- ¹³Relevant spectrum of C and inverse detection are long-range ¹H- ¹³The not structure of mycin B, C, D of target compound card is determined in the analysis of the relevant spectrum of C heteronuclear multikey.

6, the described card of claim 1 pharmaceutical composition of mycin B, C, D not is characterized in that mycin B, C, D are effective constituent to said combination system to block not, form with one or more pharmaceutically acceptable carriers respectively.

7, not mycin B, C, the application of D in the preparation antitumor drug of the described card of claim 1.

8, the application of the described composition of claim 6 in the preparation antitumor drug.

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