CN1011245B - Process for preparing antibiotics do-248-a and do-248-b - Google Patents

Abstract

Antibiotic DO-248-A and antibiotic DO-248-B disclosed in the following structural formula I has the vitality of anti-acid-resisting bacteria. The DO-248-A, the DO-248-B or medicinal salts of the DO-248-A and the DO-248-B used as an active component of the anti-acid-resisting bacteria are applied to people or animals by oral taking or a parenteral approach. Microorganisms can cultivate streptovericillium and generate the antibiotic DO-248-A or the antibiotic DO-248-B, the two antibiotics or one of the two antibiotics are collected from a culture medium, and the antibiotic DO-248-A and the antibiotic DO-248-B are obtained. A61K31/38.

Description

Process for preparing antibiotics do-248-a and do-248-b

The present invention relates to microbiotic DO-248-A shown in the following formula I and DO-248-B and their pharmaceutically useful salt.

R is ethyl or sec.-propyl in the formula

And then, relate to the active ingredient of these antibiotic preparation process and these microbiotic and/or the anti-aciduric bacteria of its pharmacologically acceptable salt.

Microbiotic is used as the existing history for a long time of antiphthisic medicine clinically.Aminoglycoside particularly, for example: Streptomycin sulphate, kantlex; Also has the semisynthetic macrocyclic lactone that uses as combination drug with other antitubercular agent, as Rifampin.

In addition, also addressed relevant prior art in the 15th the chemistry of peptides symposium compilation that T.shi ba is compiled.Usually, be difficult to prevent because the microorganism that the life-time service microbiotic is produced with resistance.And the treatment tuberculosis that current existing problem is mentioned above to be this generation with resistant microorganism can resist.

Though known microbiotic pheganomycins has α-guanidine radicals-3, the aceticoceptor of 5-dihydroxyphenyl, (Proceeding of the 15th Symposium on Peptide Chemistry 121, (1977)), DO-248-A and DO-248-B then are the derivatives of glycine, and the microbiotic Pheganomycins of this and six peptides or seven peptide derivants has tangible difference on chemical structure.

Microbiotic DO-248-A of the present invention and DO-248-B have acid-fast organism, the antibiotic activity of anti-kalamycin resistance bacterium, anti-rifampicin resistance bacterium.And the pathogenic bacteria of anti-sars type aciduric bacteria property disease is more effective than kantlex.As the drug use of anti-aciduric bacteria composition, microbiotic DO-248-A and DO-248-B can be oral or parenteral injection give the human or animal.Microbiotic DO-248-A and DO-248-B belong to (Streptoverticillium) a kind of microorganism that produces microbiotic DO-248-A and DO-248-B by cultivating streptomyces verticillus, collects from substratum and preparation again.

Fig. 1-4 represents DO-248-A respectively, the ultra-violet absorption spectrum in the aqueous solution, the infrared absorption spectrum in the Potassium Bromide solid dielectric, ¹The H-NMR wave spectrum and ¹³The C-NMR wave spectrum.Represent DO-248-B respectively for Fig. 5-8, the ultra-violet absorption spectrum of the aqueous solution, the infrared absorption spectrum in the Potassium Bromide medium, ¹The H-NMR wave spectrum and ¹³The C-NMR wave spectrum.

Antibiotic DO-248-A of the present invention and DO-248-B physico-chemical property are as follows:

（1）DO-248-A

1) ultimate analysis

Theoretical value (%) C ₁₄H ₂₀N ₄O ₅2H ₂O

Carbon 46.66 hydrogen 6.71 nitrogen 15.58

Experimental value (%): carbon 47.01 hydrogen 6.15 nitrogen 15.44

2) molecular weight (measuring) by secondary ion mass spectrometry (SIMS)

（M+1） ⁺325

3) fusing point

194-200℃

4) specific rotation

(α) ^22.5D+84.3 ± 1.3(C0.966, water)

5) uv-absorbing (see figure 1)

nm（E ^1% _1cm）276（36）

（E ^1% _1cm）283（36）

（E ^1% _1cm）297（74）

6) infrared spectra (see figure 2)

KBr 3350，1660，1605，

ν cm 1433，1390，1305，

max 1280，1130，1080，

1015，823，803，

7) solvability

Water-soluble, be soluble in methyl alcohol, ethanol, dimethyl formamide is slightly soluble in chloroform, ethyl acetate, acetone is insoluble to benzene, ether and hexane.

8) color reaction

Sakaguch reaction: the positive.

9) outward appearance and acid-basicity and neutrality

Colourless powder, the tool amphoteric properties.

10) ¹H-NMR (200MHz.D ₂O, the External Reference standard: tetramethylsilane is (according to the δ value of DSS; 0.65) (see figure 3)

δppm（J-Hz）1.23（d6H J＝7），3.35（m1HJ＝7），3.61（ABdLH J＝17）3.83（ABd 1HJ＝17），

5.10（S1H），6.48（S2H）

11) ¹³C-NMR(δ value 67.4) (see figure 4)

δppm 20.5（q×2）;25.0（d），44.4（t），59.3（d），108.1（d×2），123.8（s），134.8（s），156.4（s×2）157.3）

（s），171.0（s），177.0（s），

12) amino acid analysis

Discovery contains glycine and ammonia.

（2）DO-248-B

1) ultimate analysis

Theoretical value (%) C ₁₃H ₁₈N ₄O ₅2H ₂O

Carbon 45.08, hydrogen 6.40 nitrogen 16.18

Experimental value (%) carbon 45.26 hydrogen 6.13 nitrogen 16.13

2) molecular weight (measuring) by secondary ion mass spectrometry (SIMS)

（M+1） ⁺311

3) fusing point

189-193℃

4) uv-absorbing (see figure 5)

276（36），281.5（36）

^Drip276(36), 281.5(36)

^Drip297(72)

5) infrared absorption spectrum (see figure 6)

KBr 3350，1660，1608，1435，

ν cm 1393，1310，1250，1105，

max 1000。

6) color reaction

The Sakaguchi reaction; Positive.

7) outward appearance and acid-basicity and neutrality

Colourless powder, the tool amphoteric properties.

8) ¹H-NMR(200NHz.D ₂O, External Reference: tetramethylsilane is (according to the δ value of DSS; 0.65)) (see figure 7)

δppm（J＝Hz）1.00（t3H J＝7.3），2.52（q2HJ＝7.3），3.61（ABd 1H J＝17），3.83（ABd 1HJ＝17），5.10（S1H）6.50（S2H）

9) ¹³C-NMR(25.2MHz.D ₂O, internal reference: diox (δ value 67.4))

δ ppm(J=Hz 13.6(q), 16.9(t), 44.5(t), 59.4(d); 107.4(d * 2), 120.1(s), 134.8(s), 156.4(s * 2), 157.3(s), 171.0(s), 177.0(s), (see figure 8)

Physico-chemical property and chemical structure according to above DO-248-A and DO-248-B determine that their structural formulas are as follows: (I)

(R is ethyl or sec.-propyl in the formula)

R group in the DO-248-A structural formula I is a sec.-propyl.Be named as N-(α-guanidine radicals-3,5-dihydroxyl-4-isopropyl benzene acetyl) glycine.DO-248-B R group in the structural formula I is an ethyl, is named as N-(α-guanidine radicals-3,5-dihydroxyl-4-ethylbenzene acetyl) glycine.Therefore DO-248-A and DO-248-B are the new microbiotic of a class.

DO-248-A and DO-248-B utilize a kind of bacterial strain that isolated streptomyces verticillus belongs to from soil according to a conventional method to produce.This microorganism is same species by the sort research evaluation with rose streptomyces verticillus (Streptoverticillium roseoverticillatum) Forina and Locci.

The taxonomic property of this microorganism is as follows:

(1) morphological properties, (cultivating 14 days at 28 ℃) with yeast extract and Fructus Hordei Germinatus extract nutrient agar

This microorganism growth is good and form a piece aerial hyphae, and conidium is adsorbed on these mycelia.Microscopically is observed the aerial hyphae branch and is formed colyliform.Aerial hyphae forms straight spore chain, and the aerial hyphae majority of each chain all is less than 10.Under electron microscope, observe the mycelia smooth surface, do not find sporocyst, tool trichospore and sclerotium.

(2) cultural property:

Table 1

Substratum growing state aerial hyphae substratum gas solubility

The living mycelia pigment of formation situation color

The good pink no toner of sucrose nitrogenous source fine jade does not have

The fat substratum is pink

The glucose aspartoyl is good pink not to be had to azarin

Amine nutrient agar rose pink

The good pink light red nothing of glycerine aspartoyl

The amine nutrient agar

The good dark red nothing of white powder of inorganic salt starch

Nutrient agar is to light red

The good dark red brownish black of rose pink of tyrosine fine jade

The fat substratum is to pink (on a small quantity)

The good taupe brown taupe brown of trophicity fine jade

The fat substratum

The good rose pink of yeast Fructus Hordei Germinatus extracting is to the red tangerine yellowish brown

Thing nutrient agar light brown yellow

The good rose pink of rolled oats fine jade does not have to azarin

Fat substratum light gray

The good dark red yellowish brown of pale pink of Benmef

Nutrient agar is to greenish orange look orange (on a small quantity)

Color instructs (Japanese color institute) to judge according to Standard Colors.

(3) physical properties:

The gelatin liquefaction feminine gender

The melanochrome nucleus formation positive

The tyrosine oxidase reaction is weak positive

Curdled milk effect feminine gender

The milk peptonization is weak positive

The water-disintegrable positive of starch

(4) utilization of carbon source

The carbon source kind growing state

L-arabinose+

The D-wood sugar+

D-glucose ++

D-fructose ++

Sucrose+

Inositol ++

The L-rhamnosyl+

Raffinose+

D-N.F,USP MANNITOL ++

Contrast (no carbohydrate)+

++: the expression well-grown ,+expression growing state is general.

In control group (sugar-free) this microorganism to be confirmed to be growing state general.

(5) growth temperature

This microorganism can be 15-45 ℃ of growth, and the suitableeest growth temperature is 26-32 ℃.

(6) composition of cell walls

Find that the diaminopimelic acid in the cell walls exists with the LL-type.

According to above-mentioned a series of character, this microorganism obviously belongs to streptomyces verticillus and belongs to (Streptovertillium).

Following literature research be and the immediate several microorganisms of this microorganism.

（1）Waxman.S.A：The Actinomycetes vol.2 1961

(document 1) (actinomycin, second volume, 1961)

（2）ElWood B，Shirling and David Gottlieb：International Journal of Systematic Bacteriology vol18 69-189，279-392（1968），Vol 19，391-512（1969）vol.22，265-394（1972）

(document 2) (international system bacteriology yearbook)

18 roll up 69 pages-189 pages 279 pages-392 pages nineteen sixty-eights

19 volume 391-512 pages or leaves 1969

22 volume 265-394 pages or leaves 1972

（3）Bergey′s Manual of Deteminative Bacter ioLogy，the eight edition（1974）

(document 3) (uncle Jie Shi identify bacterium handbook the 8th edition 1974)

(4) other is about the paper of new actinomycin

Analytical results, this microorganism and following three kinds of microorganisms are the most approaching.

(1) streptomyces verticillus hiroshimense(document 2,18 volume 130-134 pages or leaves (nineteen sixty-eight); 3,835 pages in document)

(2) rose wheel silk strepto-(168 pages (1968) of document 2,18 volumes; 3,835 pages in document)

(3) streptomyces verticillus bierticillatus(document 2,18 is rolled up 300 pages (nineteen sixty-eight); 3,834 pages in document)

This microorganism is compared through check and with above-mentioned three kinds of microorganisms, finds that it is similar to above-mentioned three kinds of microorganisms, and with wherein the rose streptomyces verticillus is the most approaching, because these two kinds of microorganisms other characteristic except that the curdled milk different in kind is all the same.Therefore bacterial strain DO-248 is to belong to above-mentioned bacterial classification by evaluation, and called after: rose streptomycete DO-248.This bacterial strain is stored in the Yatabe-machi of Japan, Tsukuba-gun, Ibaraki pref with No. 7561, FERM on March 26th, 1984.Fermentation research institute of industrial science technical division, go into volume by budapest treaty with FERM BP-745 number on March 22nd, 1985 and preserve, and be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) with CGMCC No.0034 on August 6th, 1985.

The present invention includes each step that is subordinated to preparation DO-248-A and DO-248-B in the bacterial strain that streptomyces verticillus can produce (comprising above-mentioned DO-248 bacterial strain) microbiotic DO-248-A and DO-248-B.

The process of preparation DO-248-A and DO-248-B is as follows from the microorganism that can produce microbiotic DO-248-A and DO-248-B.This process can be carried out according to the flow process of general fermentative production.Just produce microorganism aerated culture in containing the substratum of several nutritions of DO-248-A and DO-248-B.The composition of culture condition and substratum is identical with common production microbiotic service condition.Substratum carbonaceous sources, nitrogenous source and inorganic salt.As substratum.Add VITAMIN and metabolite precursor or the like once in a while as required.Carbon source such as glucose, sucrose, starch, dextran, glycerine, molasses, organic acid and their analogue, can be used alone or as a mixture.Nitrogenous source is for example: soybean protein, corn soak juice, meat extract, yeast extract paste, cotton seed meal, peptone, wheat germ, ammonium sulfate, ammonium nitrate etc., can be used alone or as a mixture.Inorganic salt can for example add substratum as required, lime carbonate, sodium-chlor, Repone K, sal epsom, cobalt chloride, several phosphoric acid salt etc.

Cultivation can prepare antibiotic method routinely to be carried out, and among the present invention, liquid culture is desirable especially, and under the liquid level aerated culture to carry out scale operation also fine.The PH scope of substratum is from about 5.5 to about 8.5, and the temperature of fermentation is from about 20 ℃ to about 40 ℃, preferably between 25 ℃ to 32 ℃.Incubation time depends on the scale of fermentation to a large extent, and large scale fermentation is produced incubation time and is preferably in 20-80 hour.

No matter before fermentation or in the fermenting process, all can add defoamer vegetables oil or its analogue as required.

Cultivation finishes, and DO-248-A and DO-248-B be Separation and Recovery from substratum according to conventional methods.For example, can prepare: filter, centrifugal, absorption, desorb, several hypersober chromatographies of use, with several organic solvent extractings or be used in combination as required in order to the below method.

DO-248-A provided by the invention and DO-248-B comprise that for aciduric bacteria the nuclear bacillus is effective.The test-results of description DO-248-A anti-microbial activity.

Test method:

(0.01mg) is as shown in table 2 for acid-fast bacillus, in 2.0ml Dubos tween albumin liquid nutrient medium, cultivate, add the DO-248-A(compd A of the minimum inhibition concentration of determining by double dilution process (MIC μ g/ml) in the substratum), kantlex (KM) and Rifampin (RFP).Mycobacterium is 28 ℃ of cultivations, and other all microorganisms are all 37 ℃ of cultivations.Usually after fortnight, identify, but mycobacterium fortutitum and Mycobacterium chelonei identify that after a week mycobacterium xenopi is identified at the five-pointed star after date.

As shown in table 2, microbiotic DO-248-A of the present invention can suppress to have the bacterium of kantlex and rifampicin resistance effectively, can also more effectively suppress that the mycobacterium mycobacterium avium-it can cause and is difficult for atypical acid resistance disease of curing than kantlex and Rifampin.DO-248-B is identical with the DO-248-A effect, so these microbiotic and its pharmaceutically useful salt, can be used as the active ingredient that suppresses aciduric bacteria, is used for humans and animals.

DO-248-A and DO-248-B or its pharmacologically acceptable salt can be oral or give the human or animal through non-enteron aisle.Medicine can tablet, and forms such as capsule, powder, liquid are oral.Medicine is with common pharmaceutically useful vehicle preparation, as stablizer, flavouring agent, wetting agent, sanitas, tensio-active agent, seasonings, spices or the like.Also can injection, form parenterai administration such as suppository.

Table 2

Test microorganism minimum inhibition concentration (μ g/ml)

Plant based compound A kantlex Rifampin

M. mycobacterium tuberculosis H37Rv 0.05 0.025＜0.001

″ R-15 ^1）0.10 ＜0.001 ＞25

″ R-18 ^2）0.025 ＞25 ＞25

M. mycobacterium tuberculosis var bovis BCG 0.05 0.00156＜0.001

M. bank Sa mycobacterium KMC1101 0.00313 6.25 0.0125

M. mycobacterium marinum KM1202 12.5 3.13 1.56

M. lymphoid tuberculosis mycobacterium KMC2102 0.2 0.0125 0.00313

M. mycobacterium gordonae KMC2201 0.00625 0.39 0.0125

M.szulgai KMC2401 3.13 1.56 0.025

M. mycobacterium xenopi KMC2301 0.39 0.10 0.00625

M. monkey mycobacterium KMC1302 6.25 12.5＞25

M. bird structure mycobacterium KMC3101 0.78 3.13 0.20

M. Mycobacterium intracellulare KMC3209 0.025 0.05＜0.001

M. non-pigmented mycobacterium KMC3602 12.5＞25 25

M. mycobacterium fortutitum KMC4101＞25 25 3.13

M. Mycobacterium chelonei KMC4201＞25＞25 6.25

M. the clinical branch 0.10 6.25 3.13 of mycobacterium tuberculosis in the bird born of the same parents

From product

Be 16

1) rifampicin resistance system

2) block that rhzomorph and rifampicin resistance system

Pharmaceutical dosage is according to the state of an illness, and great changes have taken place for sex, age, body weight, but the about 0.2-8 gram of normal adult consumption every day is for suitable.Anti-aciduric bacteria medicine of the present invention can be with other the medicine of anti-aciduric bacteria with identical method and use.

The preparation method of research material DO-248-A of the present invention and DO-248-B is exemplified below, but this does not should be understood to the present invention and only is confined to this.

Example 1

(a) fermentation step

S-substratum: 0.5% Zulkovsky starch, 0.5% glucose, peptone more than 0.5% (Polypeptone), 0.5% meat extract, 0.25% yeast extract paste, 0.25% sodium-chlor, deionized water (before the sterilization substratum PH being transferred to 7.0).

X-substratum: 2% thick starch, 2% glucose, 5% yeast extract paste, 0.25% sodium-chlor, 0.25% lime carbonate, 0.0005%MgCl4H ₂O, 0.0005%CuSO ₄5H ₂O, 0.0005%ZnSO ₄7H ₂O

With platinum loop rose streptomyces verticillus DO-248 bacterial strain (FERM BP-745) is inoculated in the SakaguchiShi culturing bottle of 500ml, in the bottle the above-mentioned S substratum of 100ml is housed, 28 ℃ of wave and culture 48 hours.The every 4ml of above-mentioned nutrient solution is inoculated in the 500ml SakaguchiShi bottle that the 100mLX-substratum is housed, 28 ℃ of wave and culture 4 days obtain 43.8 liters of nutrient solutions again.

(b) separating step

43.8 liters of nutrient solutions of above process preparation add 1.2 kilograms of flocculating aids Hyflosuper Cel

Jhons-Manville Sales company) removes by filter cell.Filtrate (39 liters) is adsorbed required composition by a post (150-300 μ m, Mitsubishi Chemical Industries company limited) that 4.36 liters of synthetic adsorbent HP-20 are housed.Wash post with water, use the methanol-eluted fractions activeconstituents again, then concentrating under reduced pressure.The first half of active ingredient pollutes a lot of water-soluble impurities, this part is merged active ingredient through the chromatography column purifying (75-150 μ m Mitsubishi Chemical Industries company limited) that 580ml synthetic adsorbent CHP-20P is housed, frost drying becomes 25 gram brown ceramic powder raw product, then 25 gram raw product are adsorbed on the 470 gram silicagel columns, chloroform: methyl alcohol: water (15: 10: 1) comes wash-out to remove impurity, obtains active ingredient with methanol-eluted fractions again; The vacuum-evaporation drying.The tawny evaporate to dryness residue that 3.18 grams is contained activeconstituents is dissolved in the less water, by the Sepha-dex LH-20(Pharmacia AB of 700mL) chromatography column, after the water washing, use methanol-eluted fractions, elutriant vacuum-evaporation drying.Obtain 1.22% powder, be dissolved in a spot of water again, with Toyopearl HW-50C(100-500 micron Togo Soda Mfg company limited) post adsorption activity fraction, use methanol-eluted fractions after washing post with water.Activeconstituents vacuum-evaporation drying (can not remove fully according to this purge process impurity, but DO-248-B can first half obtaining suitable amount as minor constituent).Merge every kind of rough powder by reversed phase chromatography post (Lodar Lichronrep RP-18

, 25 * 310 millimeters Merck Co of 40-63 micron) and use methyl alcohol: water (1: 1) is removed impurity, and activeconstituents dewaters in a vacuum and obtains the 445mg yellow powder.Use Nucleosil at last

10C ₃(10 * 250mm, M.Nargel CO.Ltd.) post of partly collecting of model carries out RPHPLC (reversed-phase high-performance liquid chromatography) product is separated, with 14-15 minute and 7-8 minute wash-out DO-248-A and DO-248-B respectively, flow velocity methyl alcohol with per minute 3ml: water (1: 2) wash-out, used ultraviolet 245nm wavelength detecting.

Elutriant frost drying respectively obtains the colourless DO-248-A powder of 116mg and 16mgDO-248-B colourless powder.

Example 2

1) fermentation step

S substratum: 0.5% Zulkovsky starch, 0.5% glucose, peptone more than 0.5%, 0.5% meat extract, 0.25% yeast extract paste, 0.25%NaCl, deionized water (transferring PH to 0.7 before the sterilization)

The A substratum

2% thick starch 2% glucose 2% defatted soyflour

The 1%Baotosoyton(trade(brand)name) 0.25% sodium-chlor, water (transferring PH to 7.0 before the sterilization)

Tilt to add rose streptomyces verticillus DO-248(FERMPB-745 to 2 liters of Erlenmeyer bottles that the S substratum that 800ml is made up of above-mentioned composition is housed) seed culture fluid, change with per minute 180 at 28 ℃ and to cultivate 24 hours.

In 30 liters bottle, the 20 liters of A substratum formed by mentioned component of packing into, the above-mentioned substratum of just having made of 800ml of packing in each bottle was respectively again cultivated 5 days, and condition is 28 ℃, and per minute rotates 180-300 to be changeed, and ventilates 20 liters/minute.

B) separating step

The substratum that top step provides is transferred to PH3.0, and centrifugal with Scharbles type whizzer, obtains 1001 supernatant liquors, supernatant is with the flow velocity of the per minute 700ml Dowex through 10 liters * 50 * 4(NH ⁺ ₄Type) (US.Dow Chemical Co) chromatography column.Chromatography column washes with water, promptly obtains 35 liters active ingredient then with the ammoniacal liquor wash-out of 0.3N.After removing deammoniation, active ingredient is transferred PH to 9.0, then with the flow velocity of per minute 150mL Dowex 1 * 2(CL through one 5 liters ^-Type) chromatography column.Chromatography column 50% methanol-eluted fractions that contains 5% sodium-chlor, 13 liters the active ingredient that obtains is like this evaporated in a vacuum.Rest solution is transferred PH to 7.0, and through one 2.5 liters HP-20(Mitsubishi Chemical Industries company limited) chromatography column, chromatography column is used 50% methanol-eluted fractions, 2.7 liters of active ingredient revaporization of gained again after washing.Obtain the meal that the product freeze-drying can obtain 2.25 gram DO-248 like this.

B) purification step of DO-248-A

Monitoring in the following DO-248-A purge process and quantitatively be to detect its absorption by high performance liquid chromatography to carry out at 230 millimicrons of places.Used chromatography column is Nucleosil

10C ₈4 * 250 nanometer chromatography columns of (M.Nargel company limited product), solvent is 20mM, and the phosphoric acid buffer of pH7.0 and acetonitrile are with the mixed solution of 5: 1 ratio, and the hold-up volume of DO-248-A is 8.00ml under this condition.

The coarse meal 12.0 that top step (containing 105mg DO-248-A approximately) is provided restrains) be dissolved in the phosphoric acid buffer (PH7.0) of the 200mM of about 40ml, after removing small amount of impurities, with solution process QAE-Sephadex A-25(Pharmacia AB) post (200ml).After crossing post, with this damping fluid of 600ml with contain the same damping fluid of 1M sodium-chlor and carry out the linear concentration gradient wash-out with the phosphoric acid buffer of 200ml20mM.Elutriant detects with above-mentioned high performance liquid chromatography, collects the component that contains DO-248-A.The elutriant of collecting is again through a CHP-20P(Mitsubishi, Chemical Industries company limited) chromatography column (100ml), the methyl alcohol that this post washes with water earlier then with 300ml water and 300ml80% carries out the linear concentration gradient wash-out, elutriant detects with the high performance liquid chromatography post, collects the component that contains DO-248-A.The elutriant of collecting evaporates in a vacuum, and just to obtain 100mg purity be 70% powder in lyophilize then.This dry powder of about 50mg is collected post (50mg Nucl eosil 30C through a high performance liquid chromatography again ₁₈20 * 250mm) also open up layer with phosphoric acid buffer and the methyl alcohol of 50mM PH7.0 with 9: 1 mixed solution, and drip washing partly is used in the absorption detecting at 230nm place and collects each component that contains that peak of desired substance.Collect the methanol-eluted fractions that liquid passes through CHP-20P post (40ml) and water and 50% again, detect, collect the elutriant that contains DO-248-A, evaporate in the vacuum with high performance liquid chromatography, then lyophilize get final product the colourless DO-248-A powder of 60mg.

Claims (6)

1, a kind of microbiotic DO-248-A of structure such as formula I and/or method of DO-248-B of preparing.

Wherein R is sec.-propyl or ethyl, it is included in cultivates rose wheel silk Streptomycin sulphate (Streptoverticillium roseovertic-illatum) CGMCC0034 that can produce microbiotic DO-248-A and/or DO-248-B in the substratum, and reclaims microbiotic DO-248-A and/or DO-248-B from substratum.

2, a kind of method according to claim 1, wherein said substratum is a liquid nutrient medium.

3, a kind of method according to claim 1, the pH of wherein said substratum is transferred to about 5.5-8.5.

4, a kind of method according to claim 1, wherein said substratum are about 20-40 ℃ in temperature and carry out.

5, a kind of method according to claim 1, wherein said cultivation are about 25-32 ℃ in temperature and carry out.

6, a kind of method according to claim 1, wherein said cultivation was carried out 20-80 hour approximately.

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