SU518142A3 - The method of obtaining 7 (d-5-amino-5-carboxivalamido (-3-) -methoxy-psulfooxycinnamoyloxymethyl) 7-methoxy-3-cephem-4-carboxylic acid and 7- (d-5-amino-5carboxiveramido (-3- ) methoxy-p-hydroxycinamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid - Google Patents

Description

(54) METHOD OF OBTAINING 7-p- {0-5-AMINO-5KARBOXIVALARIDO) -3- (a-METHOXY-pULFOOXYCINNAMOYLOXYMETHYL) -7-METHOXY-3C, EFEM-4-CARBONIC ACID-7-7-CH-I-7-METOXY-3C, EFEM-4-CARBONIC ACID-7-METOXI-3C, EFEM-4-CARBONIC ACID-7-METHOXI-3C -AMINO-5KARBOXIVALERAMIDO) -3- (a-METHOXY-p-OXYCINNAMOYLOXIMETHYL) -7-METHOXI-3-CEPHEM-4-CARBON

ACIDS center and stronger along edges. Soluble pigment is light, reddish yellow. Agar from egg white. Substrate mycelium is greyish-brown-brown. Aerial mycelium is reddish-brown-yellow with a greenish tinge, the edge is brownish-reddish-yellow, the growth is weak in the center and stronger than ON the edges. Dissolving pigment is light reddish-yellow. Gelatin oblique agar. Grew in the form of sediment on the bottom of the vessel, layered, painted in cream color. Nutritious gelatin agar vegetative mycelium cream. Aerial mycelium is pale, reddish yellow. Soluble pigment em, gelatin liquefaction is good. Litmus milk. Incomplete ring. Vegetative mycelium brownish. The air-mycelium is soft, whitish. Unloaded milk. Incomplete ring. Vegetative mycelium brown. There is no aerial mycelium. Soluble pigment is light brown. Starch agar spit to. Cream vegetative mycelium. Aerial mycelium fawn. Soluble pigment pet. Good hydrolysis. Potato mass. Vegetative mycelium is light yellow. Aerial mycelium is average. Weak potato darkening. Calcium Calcium Agar. The vegetative mycelium is flat translucent and colorless along the edges of the dark and cream-colored in the center. Aerial mycelium from cream to yellow. Kra reddish yellow. There is no soluble pigment. Tyrosine agar spit k. Autonomic mycelium cream. Aerial mycelium is yellowish-red with a greenish hue, the edges are reddish-yellow. Soluble pigment is very light brown. Peptone-iron-yeast extract agar. Vegetative mycelium cream. There is no aerial mycelium. There is no soluble pigment. Hydrogen sulfide does not form. Well absorbs glucose, arabinose, xylose, maltose, mannose, mannitol, lactose; poorly absorbs: mnosite, sucrose, rhamnose, raffinose; ie absorbs: fructose and cellulose. In addition to the above culture (MA-2837), an additional twenty-five cultures were identified as antibiotic producers. These include: three Cult of the species of Streptomyces griseus, eleven cultures of Streptomyces viridochromogenes, five cultures of Steptomyces fimbriatus, three cultures of Str. halstedii, one culture of Streptomyces rochei, one culture of Streptomyces cinnamaneusis, and one culture of Streptomyces chartreneusis. These stamps of the Streptomyces genus are identified as cultures MA-4160, MA-4174, MA-4171, MA-4177, MA-4178, MA-4180, MA64, MA-4165, MA-4166, MA-4167, MA-2892, - 3265, MA-4162, MA-4163, MA-4159, MA69, MA-4170, L1A-4179, MA-4161, MA-4168, -4175, MA-4181, MA-2938, MA-4176 and MA73 in the collection Crops firm Me sk and Co., c. Rahway New Jersey. These cultures are permanently stored with the collection of Northeru Utilization Research and Development Brauch of theU. US Department of Commerce in Peoria, Illinois. The following Streptomyces griseus MA-4160NRRL3951 MA-4174NRRL3953 MA-4171NRRL3952 Streptomyces viridochromogenes MA-4177 NRRL3970 MA-4178MRRL3971 MA-4180NRRL3972 MA-4164NRRL3966 numbers were assigned to the lthor. MA-4165NRRL3967. MA-4166NRRL3968 MA-4167NRRL3969 MA-2892NRRL3962 MA-3265NRRL3963 MA-4162NRRL3964 MA-4163NRRL3965 Streptomyces fimbriatus MA-4159 NRRL3954 MA-4169NRRL3956 MA-4170KRRL3957 MA-4179NRRL3958 MA-4161NRRL3955 Sireptomyces halstedii MA-4168 NRRL3959 MA-4175NRRL3960 MA-4181NRRL3961 Streptomyces rochei MA-2938NRRL3973 Streptomyces cinnamoneusis MA-4176NRRL3974 Streptomyces chartreusis MA-4173 NRRL3975 example 1. ulturu Str. griseus is grown on a medium with the following composition: reda 1: Difco yeast extract 10, 0 g Glucose 10.0 g Phosphate buffer 2.0 ml MgSO4-7 Ngb 0.05 g Distilled water 1000.0 ml Difco agar 25.0 g Phosphate buffer: KH2P0491 , 0 g NasHPO95.0 g Distilled water 1000.0 ml wasp agar is prepared by pouring the medium ml into test tubes. The tubes are then closed with stoppers and sterilized at 120 ° C for 15 minutes, so that it is inclined. The inoculated agar is kept at 28 ° C for one week and then stored at 4 ° C until used. Culture on one of these agars is used to inoculate in Erlenmeyer flasks (250 ml) containing 50 ml of medium II. 5 ml of sterilized medium is added to the tubes, the culture is collected from the surface of the agar, and 1 ml of the culture is carefully added dropwise to each of the three flasks. Medium II has the following composition, g: Bulk extract 3.0 NZ amine (enzymatic casein hydrolyzate) 10.0 Dextrose 10.0 NaCl5.0 Distilled water 1000 pH neutralizes to 7.2 with NaOH. Inoculated tubes are shaken using a rotary pump. at 220 rpm for three days. The culture tubes are then used for inoculation in Erlenmeyer flasks with a capacity of 2 liters, each containing 350 ml of medium III and using 2-3% culture. The medium III has the following composition, g: Dextrose 10.0 Asparagin1.0 K2HPO40.1 MgSO4 -7H2O0.5 Yeast extract0.5 Mixture element indicator No. 210.0 ml Distilled water1000.0 ml Mixture number indicator composition No. 2: FeSO4-7H201.0 g MnS04-H2O1.0 g CuCl2-2H2O25.0 mg Caci100.0 mg HZVOZ56.0 mg (LH4) bMo7O24-4H2019.0 mg ZnSO4-7H20200.0 mg Distilled water 1000.0 mg pH adjusted to 7.2 with NaOH solution. The tubes are shaken with Chalki at a speed of 135-150 rev / mip for 4 days at 28 ° C. At the end of the incubation period, the contents of the eleven tubes are drained and examined. The displaced, centrifuged broth has a protective zone against Proteus Vulgaris, equal to 22 ml in the study on Petri discs with discs. This mixture of antibiotic includes 7 | 5-O-5-amino-5-carboxivalamide-) -3- (a - methoxy - / g - sulfooxycinnamoyloxymethyl) - 7-methoxy-3-cephem-4-carboxylic acid and 7p- (B -5-amino-5-carboxivalamido-) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid (1a). Step 1. The contents of the lyophilized tube (MA-2837) are suspended in 2 ml of medium 1 / ohm, example 1), and the resulting inoculum is lyophilized by culture MA-2857 inoculated with solid agar medium 1 (see example 1). These bevels were incubated at 28 ° C for 5 days or until spore was formed, and then 10 ml of medium VIII was added to the bevels. Medium VIII, composition,%: Mineral extract0.3 NaCl0.5 NZ Amine1.0 Dextrose 1.0 pH7.0 The culture on each agar is scraped off and the resulting suspension is used as inocula in step 2. Step 2. The suspension obtained in step 1 , is used to inoculate an Erlenmeyer flask containing 50 ml of sterilized medium VIII (see step 1). The incubated flask is placed for 48 hours at 28 ° C in a rotary shaker (220 rpm). EtacH. The contents of the inoculated flask from step 2 are used to inoculate an Erletsmeyer two-liter flask containing 500 ml of medium VIII (see step 1). The monochromatic flask is placed for 48 hours at 28 ° C in a rotary shaker (220 rpm). Step 4. The inoculum obtained in step 3 is used to inoculate a stainless steel fermenter with a capacity of 757 liters, which contains 467 liters of sterile medium VIII. The fermentation is carried out at a temperature of 28 ° C with displacement (130 rpm) for 65 hours while simultaneously blowing air. During the fermentation, a small amount of polyglycol 200 defoamer is used. Step 5. The 378 l of the obtained stage 4 inoculum is used to inoculate 4542 l of medium IX contained in a 5680 l stainless fermentor. Wednesday IX, composition,%: Liquid for soaking grain 4.0 Dextrose 2.0 pH adjusted to 7.2 with NaOH Fermentation was carried out at a temperature of 28 ° C and moving (1200 rpm), passing air through the apparatus for 30- 36 hours. Small amounts of polyglycol 200 are used as an antifoaming agent. The broth is collected and its activity is determined using Petri dishes disks, and a protective zone is obtained against Proteus vulgaris MB-838. equal to 32.5 mm at the age of culture at 31 h. The selection of antibiotic. Filtered culture fluid

(40,688 l), crawl in step A, step 5, is collected after 36 hours and the pH is adjusted to 7-8 to 3.0 in the fermenter by the addition of phosphoric acid. When the culture fluid is passed through a plate-type filter press, mycelium is removed. The filtered broth is then passed through a filtering layer of the adsorbing resin (Amberlite XAD2) with a volume of 378.5 liters at a speed of 37.85 l / min. Spent broth is tested and discarded, and the resin filter bed is washed with two volumes of water. The antibiotic is washed out of the resin filter layer with a 60% methanol solution in water, the solution flow rate is 19 l / min. Forty fractions, each 19 l each, are collected and examined.

Fractions 2-10 are combined and the methanol is removed by vacuum evaporation. The pH of the final concentrate (157 L) was adjusted to 3.5 with ammonium hydroxide and kept chilled.

Samples are bioassayed against Proteus vulgaris by a conventional method using disc Petri dishes. Spent broth and rinse:

10 fractions of 378.5 liters each showed the absence of protective zones without breaking down, just as there was no wash water.

The total weight of the solid investigated protukta:

Filtered

blylon119 kg

738 liters of combined

eluate 7.23 kg

157 l. Of concentrated eluate7.2 kg

The resulting concentrate (78.5 L) is diluted with water up to 117 L and adsorbed onto a filter bed of 22.5 L, consisting of a weakly basic anion exchange resin. (Amberlite IPA-68 resin on the chlorine ring) at a pH of 4 and a flow rate of 2 gallons / min. The filter layer is then washed with 45 liters of washing water, and then eluted with a solution of 1 N sodium nitrate and 0.1 M sodium acetate at pH 7.5 and a flow rate of 1.5 liters / min. Collect 10 eluate fractions of 18.9 liters each and adjust the pH to 4 with hydrochloric acid.

All fractions are subjected to a bioassay using a standard method using Petri dish discs against Proteus vulgaris. The obtained fractions 1–10 are combined and adsorb onto Amberlite XAD-2 adsorbing resin, forming a filtering layer with a volume of 45 liters (at pH 3 and flow rate of 5 liters / min). The filter layer is washed with 90 liters of water at the same rate. The antibiotic is then eluted from the resin with a 25% aqueous solution of acetone at a flow rate of 5 liters / min. Collect 16 fractions of 18.9 liters.

All fractions are bioassayed by the usual method against Proteus vulgaris. When researching the nutrient solution (190l) .- -,:. .

The fractions of the 2-2-16 sluate are combined and the acetone is evaporated in vacuo, eventually giving 17.4l of the concentrate, then the concentration of the concentrate is adjusted to 4 with ammonium hydroxide and 620 g of the antibiotic 810A, which is a mixture, are obtained during the subsequent freezing phase. consisting of 7) 3 (O-5-amino-5-carboxivalamido) -3- (a-methoxy- "- sulfooxycinnamoxymethyl) -7 methoxy-3-cephem-4-carboxylic acid n (O-5-amino-5- carboxyvaleramido) -3 - (a-methoxy.-hydroxy / g-oxycinnaamoyloxy and methyl) -7-methoxy-3-cephem-4-: carboxylic acid. The obtained dry product of a concentration of 300 μg / ml gives 25 mm to the protective zone.

The chromatography column is filled with a filter layer 100 cm high consisting of DEAE Sephadeko A-25 anion exchange resin. Antibiotic 81OA (10 g) is dissolved in 18 ml of a solution consisting of 0.5 moles.

ammonium bromide and 0.05 moles of acetic acid, and the column is filled with this mixture. Then, an elution solution is pumped through the filtering layer at a rate of 81 mmol / h and 10 ml eluate fractions are collected. Eluate

They are investigated using a differential refractometer, which registered mass peaks in tubes 19, 36, 79, 109 and 206. Every third fraction is examined. Against Proteus vulgaris (MB-838) using 0.5-inch disks with an RP of 7.

The retest is conducted and fractions 82-130 and 180-234 are pooled.

Fractions containing the first active component of the two extracts shown

100 ml of Amberlite XAD-2 resin are combined and adsorbed on the filter layer. The filter layer is washed with one volume of water and then eluted with three volumes of a 90% aqueous solution of methanol. Methanol

evaporated in vacuo and the aqueous concentrate was subjected to freeze-drying to obtain 810 mg of a product consisting of 7 (3-0-5-amino-5-carboxivalamido) -3- (ametoxy- "- oxycinnamyloxymethyl) -7 - methoxy-3- cephem-4-carboxylic acid. The biological activity of this product, determined using anti-Proteus vulgaris discs at a concentration of 18 µg / ml, was 25 mm (diameter of the protective zone). Analysis

UV absorption gave the following results:

UV absorption in 0.1 n. HC1 Lmax - 305

0.1 n. NaOH K,

328

mako.

The fractions obtained in two series of experiments on Sephadex-A-25, containing the second active ingredient, are combined and adsorbed onto 100 ml of a filtered layer of Amberlite XAD-2 resin. The filter bed is washed with one volume of water and then the eluates are combined, and the methanol is distilled off, evaporating it in vacuum. The aqueous concentrate is freeze dried and 720 mg of 7- | 3- (B-5-amino-5-carboxivalramido) -3- (a - methoxy-p-sulfooxycinnamoxymethyl) - - methoxy-3-cephem-4-carboxylic acid (1c) is obtained. The ultraviolet absorption analysis gave the following results:

UV absorption in 0.1 n. HC1 Kmaks 287 mm 432

UV absorption in 0.1 n. NaOH 280 mm E- „432

Claims (3)

1. A process for the preparation of 7-p- (0-5-amino-5-carboxivalamido-) -3 (a-methoxy-p-sulfooxycine-amino-oxymethyl) -7-methoxy-3-cephem-4-carboxylic acid and 7p- (B-5- amino-5-carboxivalamido) -3- (a-methoxy-p-hydroxycinnamoyloxymethyl) -7-methoxy-3-cephem-4-carboxylic acid, characterized in that a producing culture from the group of actinomycetes, for example Streptomyces griseus MA2837, is grown in aerobic conditions of the medium containing the sources of carbon, nitrogen and mineral salts, followed by separation of the target product from the filtrate of the culture fluid known in Mami. 2. The method according to claim 1, characterized in that the cultivation is carried out in a medium containing 1-6% carbohydrate and 0.2-6.0% assimilated nitrogen. 3. Method according to paragraphs. 1-2, that is, with the fact that the growth is carried out at 20-37 ° C and pH 5.5-8.0.