HU190430B - Process for producing inlusion complexes of beta-cyclodextrine and 17 hydroxy-7-alpha-mercapto-3-oxo-17-alpha-pregn-4-ene-21-carboxylic acid-gamma-lacton - Google Patents

Abstract

Inclusion complexes of 17-hydroxy-7 alpha -mercapto-3-oxo-17-alpha -pregn.4-ene-21- carboxylic-acid-gamma-lact one and beta-cyclo-dextrin are prepd. by hydrolytic decompsn. of the spirono-lactone in aq. media in the presence of beta-cyclo-dextrin, and complexing of the decompsn. prod. which is by itself labile. The stability, wettability and water solubility of the complex are good. The complex can be used in pharmaceutical prepns..

Description

The present invention relates to a novel pseudoaglycone derivative of antibiotic activity of actaplanin.

Antibiotics are also used in agriculture to stimulate the growth of chickens, pigs, sheep and cattle.

The present invention relates generally to the inventions disclosed in U.S. Patent No. 3,952,095, issued April 20, 1976, to U.S. Patent No. 3,952,095, and U.S. Patent 4,064,233, A-4696, issued April 20, 1976, which discloses a novel antibiotic and a process for its preparation. United States Patent No. 20,115, issued December 20, 1977; U.S. Patent No. 4,115,552, issued September 19, 1978 to A-4696, to A and B; and Manuel Debono, Kurt E. U.S. Patent No. 217,972 to Merkel, Robert E. Weeks, and Herold J. Cole, which is filed concurrently with the present application, wherein the inventors have the antibiotic A-4696 Β, -, B ₂ -, B ₁ -, C _la. -, Cj and Ej components.

The above-mentioned U.S. Patent Nos. 4,064,233 and 4,115,552 disclose an aglycone derivative of antibiotic A-4696 obtained by hydrolysis, but this derivative is not the same as the pseudoaglycone derivative of the present invention. It is possible, but not certain, that the hydrolysis process described in these two patents will result in a very small amount (2% or less) of the pseudo-aglycone derivative of the present invention. However, the conditions of this previously described hydrolysis do not favor the formation of the pseudo-aglycone derivative of the present invention, and even if the pseudo-aglycone derivative of the A-4696 antibiotic of the present invention was formed, it was not previously discovered and detected.

The present invention relates to a process for the preparation of a pseudoaglycone-type hydrolysis derivative of the actaplanin antibiotic complex A-4696. Actaplanin antibiotic A-4696 is a glycopeptide-type antibiotic complex produced by Actinoplanes missouriensis strain ATCC 31,683. When the A-4696 antibiotic complex is subjected to acidic hydrolysis under mild conditions, the pseudo-aglycone derivative of the present invention is formed. The present disclosure does not include degradation products as components of the A-4696 antibiotic complex.

It has now been found that the novel pseudoaglycone derivative of the antibiotic A-4696, or a pharmaceutically active acid addition salt thereof, has an antibiotic effect and can be used to treat tooth decay, gum disease and other diseases caused by pathogenic microorganisms. . Furthermore, the antibiotic of the present invention can also be used in agriculture to stimulate the growth of chickens, pigs, sheep and cattle.

A novel pseudoaglycone derivative of the A-4696 antibiotic complex is prepared by subjecting the A-4696 antibiotic complex to mild acid hydrolysis according to the invention.

The novel pseudoaglycone derivative of antibiotic A-4696 of the present invention is a basic compound that forms salts with suitable acids. For example, the antibiotic of the present invention may conveniently be prepared by hydrolyzing the A-4696 complex with hydrochloric acid under mild conditions to give the dihydrochloride. Other pharmaceutically acceptable salts may be prepared by methods known per se.

When the hydrolysis of the A-4696 antibiotic complex under mild conditions is followed by bioautography, it is observed that the pseudoaglycone derivative of the A-4696 antibiotic complex is rapidly formed during hydrolysis and the starting material disappears. The antimicrobial spectrum of the pseudoaglycone derivative of the present invention is substantially the same as that of the parent compound.

The dihydrochloride of the pseudoaglycone derivative of the antibiotic complex A-4696 is a white crystalline solid which, when heated to about 270 ° C, charred. Water soluble, insoluble in other solvents such as methanol, acetone, diethyl ether, chloroform, pyridine, benzene, aliphatic hydrocarbons and the like. It is stable at a pH in the range of about 1.2 to about 9.5 and in a temperature range of about 35 to about 85.

The elemental dihydrochloride of the pseudoaglycone derivative of the antibiotic complex A-4696 τι α a 1 * 71 ·

CeeHe'o ^ NgCl. 2HCl. 6H ₂ O:

Element Percentage calculated Found carbon 52.75 52.75 hydrogen 5.03 5.15 nitrogen 7.45 7.82 chlorine 7.07 7.17

The maximum ultraviolet absorption of the dihydrochloride of the pseudoaglycone derivative of antibiotic A-4696 in methanol solution is 279 nm, λ max 63.6.

The infrared absorption spectrum of the dihydrochloride of the pseudoaglycone derivative of antibiotic A-4696 in potassium bromide pellet is shown in Figure 1. In the 4000-700 cm- ¹ range, this compound exhibits distinct peaks at the following wavelength values: 3374 wide, 3220, 1728, 1659, 1617, 1592, 1546, 1509, 1489, 1461, 1431, 1355, 1288, 1229, 1208, 1175, 1126, 1103, 1071, 1058, 1014, 986, 897, 880, 843, 816, 767, 731, 710 cm -1.

.From the pseudoaglycone derivative of antibiotic A-4696 with mineral acids per se

190,340

Continuation of Table I Pharmaceutically acceptable salts may be prepared in a manner known per se, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid and the like. With these acids, the salts of the antibiotic are prepared by, for example, acidifying the solution of the free base of the antibiotic with the appropriate acid and precipitating the resulting salt with acetone. In some cases, the salts may be prepared by ion exchange on a column packed with ion exchange resin. Other known methods for preparing the antibiotic salts may also be used.

The novel pseudoaglycone derivative of antibiotic A-4696 inhibits the growth of a number of microorganisms pathogenic to humans, animals or plants, and can thus be used to inhibit the growth of such microorganisms.

The growth inhibitory concentration of the dihydrochloride of the pseudoaglycone derivative of antibiotic A-4696 is shown in Table I below. Concentration values for inhibition were determined by the agar dilution method and the minimum inhibitory concentration (MIC) values in the table are given in micrograms per milliliter.

In the agar dilution assay, the test microorganism is grown on agar plates containing various amounts of the dihydrochloride of the pseudoaglycone derivative of antibiotic A-4696.

Plates are maintained at 37 ° C for 48 hours and the MIC values are read. The MIC is the lowest concentration of antibiotic at which the microorganism under test no longer grows.

Table 1 The antibiotic A-4696 is a pseudoaglycone- Micro-organism derivative minimum inhibitory concentration (MIC, microgram / milliliter) Staphylococcus aureus XI. 1 0.25 Staphylococcus aureus V41 0.25 Staphyloccocus aureus X400 0.5 Staphyloccocus aureus S13E 0.25 Staphyloccocus epidermidis 1 0.5 Staphyloccocus epidermidis 2 0.5 Streptococcus Group A C203 0.25 Streptococcus Group D X66 0.50 Streptococcus Group D 9960 0.50 Streptococcus pneumoniae Park Haemophilus influenzae sens. 0.25 BRUN 16 Haemophilus influenza. 251 32 Shigella sonnei N9 32 E. coli N10 128 E. coli 14 64 E. coli TEM 8

Micro-organism Minimum Inhibitory Concentration (MIC. Micrograms / milliliter) of pseudo-aglycone derivative of antibiotic A-4696 Klebsiella pneumoniae X26 128 Klebsiella pneumoniae K.AE > 128 Enterobacter aerogenes X68 > 128 Enterobacter aerogenes C32 > 128 Enterobacter aerogenes EBI7 > 128 Enterobacter cloacae EB5 > 128 Enterobacter cloacae 265A > 128 Salmonella typhosa X514 > 128 Salmonella typhosa 1335 128 Pseudomonas aeruginosa X528 > 128 Pseudomohas aeruginosa X239 128 Pseudomonas aeruginosa Psl8 128 Serratia marcescens X99 32 Serratia marcescens SE3 128 Proteus morganii PR 15 128 Proteus inconstans PR33 > 128 Proteus rettgeri PR7 32 Proteus rettegri C24 > 128 Citrobacter freundii CF17 > 128 Bordetella bronchiseptica 16 32

As apparent from the above data, the labeled antibiotic A-4696 pseudo-aglycone dihydrochloride salt derivative of effective antibacterial and anti-microbial agent ^35. Accordingly, the incorporation of the pseudoaglycone derivative of antibiotic A-4696, or an acid addition salt thereof, into a suitable toothpaste, gel, powder, or the like, or a suitable mouthwash ⁴⁰ may help to inhibit bacterial action. tooth decay and such damage to the enamel. Alternatively, a solution of the pseudoaglycone derivative of antibiotic A-4696, or one of its ⁴⁵ acid addition salts, may be applied directly to the gums and teeth using a tampon.

The pseudo-aglycone derivative of antibiotic A-4696 further enhances the growth rate of poultry, pigs, ⁵⁰ sheep and cattle and increases feed utilization in these species. For example, when the antibiotic of the present invention is incorporated into poultry and pig feed at a dose of about ⁵⁵ mg / kg to about 25 mg / kg / day, the animals grow faster and have better feed utilization than the same animals but feed with antibiotic-free feed. The term "feed" means complete feed for animals, including various nutrients, concentrates, additives, mineral salts, vitamins, medicated premixes, forage feeds, and the like, which are included in the normal diet of the animals. The ba-31

190 340 typical feeds for rum and pig farming are described in U.S. Patent 4,115,552.

In an important embodiment of the method of the present invention, the pseudo-aglycone derivative of antibiotic A-4696, or a suitable acid addition salt thereof, is orally administered to the animals at about 2 g per tonne of total feed for improved feed utilization and faster growth. about 200 g. Preferably, the active antibiotic of the present invention is added to the animal feed by preparing a suitable premix (such as, for example, U.S. Pat. Contains pseudoaglycone derivative A-4696. The premix thus prepared is then mixed with the feed. Alternatively, the active ingredient may be added to the animal's feed by addition to a concentrate or feed additive.

The pseudo-aglycone derivative of antibiotic A-4696 has several beneficial effects, but is particularly potent. Such materials are always in great need, not only for the treatment of tooth decay and gum disease, but also for other diseases caused by microorganisms in general.

The new pseudo-aglycone derivative of the AZ-4696 antibiotic is most conveniently prepared by subjecting the A-4696 antibiotic complex to mild acid hydrolysis. The A-4696 antibiotic complex can be prepared by culturing a microorganism of the species Actinoplanes in a suitable medium under aerated conditions until the desired growth of the antibiotic in the medium is achieved. The antibiotic thus produced can be isolated and purified by various methods known and known in the art.

The microorganism used as a starting material for the production of the pseudoaglycone derivative of antibiotic A-4696 is a strain of Actinoplanes missouriensis belonging to the family Actinoplanaceae. The Actinoplanaceae family belongs to the Order of Actinomycetales, first described by John N. Couch, Jour. Elisha Mitchell Sci. Soc., 65, 315-318 (1949) and 66, 87-92 (1950), Trans. New York Acad. Sci., 16, 315-318 (1954), Jour. Elisha Mitchell Sci. Soc., 71, 148-155 and 269 (1955), Bergey's Manual of Determinative Bacteriology, 7th Edition, 825-829 (1957) and Jour. Elisha Mitchell Sci. Soc., 79, 53-70 (1963).

The parent strain of Actinoplanes missouriensis producing the antibiotic A-4696 was deposited in the Rockville (Maryland) strain collection available to anyone under ATCC No. 31,683.

The physical and culture properties of Actinoplanes missouriensis ATCC 31,683 are shown below. This strain can be derived from ATCC 23 342 by a series of mutations from strain US 4,115,552. The mycelium of the present strain is similar to the original strain and is essentially morphologically indistinguishable from it. It has no secondary aerial mycelium or sporangium (sporangium) and does not form sporangia even by special methods such as pollen cultivation.

Methods for taxonomic determination of ATCC strain 31 683 are known to those skilled in the art and are largely described in Shirling and Gottlieb, Intern. J. of Systematic Bacteriol. 76 (3), 313-340 (1966)], a method recommended by the International Streptomyces Project (ISP). Enzymes were determined by the method of Blazevic and Ederer (Principles of Biochemical Tests in Diagnostic Microbiology, John Wiley and Sons, Inc., New York, 1975), and the names of the colors, abbreviations and numerals correspond to Kelly and Judd (The ISCC-NBS Centroid Color Charts Standard Sample No. 2106, US Dept. of Commerce, National Bureau of Standards, Washington DC, 1976) according to ISCC-NBS. Lysozyme resistance as well as the degradation of casein, esculin (6,7-dihydroxycoumarin-6-O-glucoside), hypoxanthine, tyrosine and xanthine are described in Berg, Appl. Microbiol. 25, 665-681 (1973)]. The following points system was used in the coal resource utilization studies:

t + = equal to or greater than the control with glucose; positive recovery;

+ = less than glucose but greater than carbonless control; positive recovery;

(+) = doubtful growth; doubtful recovery;

- = no growth; negative recovery.

Accordingly, the following table provides a taxonomic description of the Actinoplanes strain of the present invention, including its breeding and physiological properties.

General breeding and physiological properties of Actinoplanes strain ATCC 31,683

Λ observed property Characteristics

Breeding properties on the following media:

ISP 2. good growth, back

90th gy. Y; no aerial mycelium; no soluble colorant;

ISP 3. good growth, back

93. yGray; no aerial mycelium;

no soluble colorant;

ISP 4. Good Growth, Reverse • 79.1. Eng. YBR; no aerial mycelium; no soluble colorant;

ISP 5. Moderate Growth, Back

90th gy. Y .; no aerial mycelium; no soluble colorant;

ISP 7. Moderate Growth, Back

80. gy.yBr; aerial mycelium

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The observed property Features Bennett's Agar no; soluble coloring agent ^l gosbama; generous growth, backside 93. yGray; no aerial mycelium; no soluble colorant; calcium malt agar good growth, bright, backside Czapek-peptone agar 93. yGray; no aerial mycelium; no soluble colorant; generous growth, backside Glucose-asparagine agar 93. yGray; no aerial mycelium; no soluble colorant; good growth, backside 93. yGray; no aerial mycelium; no soluble colorant; Tomato paste-oatmeal agar good growth, backside Anio-Hensen agar 91st gy.Y; aerial mycelium no; no soluble colorant; moderate growth, backside 53H-agar 91st Eng. Y; aerial mycelium no; no soluble colorant; good growth, backside Czapek-peptone agar 91st gy.Y; aerial mycelium no; no soluble colorant; generous growth, backside casein breakdown 90. gy.Y; no aerial mycelium; no soluble colorant; positive Catalase reaction positive esculin breakdown positive gelatin liquefaction positive (100%) production of hydrogen sulfide traces Hypoxanthine degradation on ISP medium negative lysozyme resistance negative Melanoid synthetic material ISP on Medium 1 negative ISP 6 negative ISP on Medium 7 negative ISP on Medium 7 negative minus triosin sodium chloride tolerance ISP on Medium 2 <2% nitrate reduction negative growth pH range ISP on Medium 2 6 to 8.4 phosphatase production positive skimmed milk reaction negative hydrolysis of starch ISP on Medium 4 negative sucrose tolerance ISP on Medium 2 20% growth temperature Mp 40 ° C range on ISP 2 medium tyrosine breakdown positive urease production negative antibiotic susceptibility: cephalotine (sodium 30 pg) sensitive erythromycin (estolate 15pg) sensitive chloromycetin 30 pg sensitive novobiocin 30 pg sensitive penicillin G 10 units sensitive rifampin 5 pg sensitive streptomycin 10 pg sensitive

The observed property Features tetracycline 30 pg sensitive vancomycin hydrochloride sensitive 30 pgs xanthine production negative coal resource recovery ISP 9. medium * of the following carbon sources: by routine manipulation; without a carbon source - glucose ++ L-arabinose ++ cellobiose, ++ D-fructose ++ D-galactose ++ i-inositol - D-mannitol ++ melibiose + ralim oz + D-rhamnose + D-ribose + salicin + sucrose + D-xylose ++

* The final concentration of sterile carbon source in the medium was 1.0%.

Actinoplanes missouriensis strain ATCC 31,683 can be cultured on a variety of media. However, certain media are preferred for economy of production, maximum production and ease of isolation of the starting antibiotic, for example starch is preferred as carbon source and yeast is preferred as nitrogen source Other useful carbon sources include molasses, glucose, dextrin, glycerol and the like. . Other useful nitrogen sources include amino acid mixtures, peptones, and the like.

The inorganic salts used as nutrients in the culture medium may be conventional salts which provide sodium, potassium, ammonium, calcium, phosphate, chloride, sulfate and the like. In addition, it has a good effect on the production of antibiotic A-4696 if the medium also contains sources that provide growth factors, such as distillation residue or yeast extract.

As required for the cultivation of other microorganisms, the essential medium for the cultivation of the Actinoplanes species used in the method of the present invention must be present. Such trace elements are generally present as an accidental contamination of other media components.

The microorganism producing the antibiotic A-4696 used to prepare the pseudo-aglycone derivative can be cultured over a relatively wide pH range. However, it is desirable that the pH of the medium of the microorganism is about

Between 6.5 and 7.0. As with other microorganisms belonging to the Actinomycetes strain, the pH of the medium gradually changes during culture; by the end of the fermentation period, the pH of the medium is generally between 6.5 and 7.5.

190,340

Preferably, submerged aeration conditions are used to produce the initial A-4696 antibiotic complex. In shake flask cultures, relatively small amounts of antibiotics can be produced; however, to produce larger quantities, it is preferable to cultivate in sterile containers under submerged aeration. The medium in the sterile container is inoculated with a suspension of the mycelial fragment.

Accordingly, it is desirable to prepare a second inoculum from the microorganism by inoculating a relatively small amount of the medium with the mycelial fragment of the microorganism and transferring it under sterile conditions to a larger container. The medium used for the production of the second inoculum may be the same as that used for larger-scale culture, but other media may be used.

The Actinoplanes missouriensis strain ATCC 31,683 grows at temperatures between 20 ° C and 40 ° C, while the initial production of the A-4696 antibiotic is highest at about 30 ° C.

During the submerged aeration culture, sterile air is bubbled into the medium. The amount of air introduced into the medium is between about 0.1 volume per minute and about 1.0 volume per minute per volume of medium. The growth of the microorganism and the production of the antibiotic is most potent if the amount of air introduced into the medium is at least 1/2 volume per minute per volume of medium.

The rate of antibiotic production and the concentration in the medium during culture can be monitored by determining the antibiotic activity of the samples taken from the medium against microorganisms known to be sensitive to the antibiotic produced. An example of a microorganism useful in the assay is Bacillus subtilis. Biological values are measured by known methods or on paper agar plates.

The maximum amount of antibiotic is usually accumulated in shake flasks or submerged aerated culture in about 4-6 days.

The A-4696 antibiotic complex used as starting material for the preparation of the pseudo-aglycone derivative of antibiotic A-4696 can be isolated from the culture medium and separated from any other substances by adsorption and shaking. It is preferable to use adsorption methods, since in this case it is not necessary to work with the large amounts of solvents used in the extraction methods.

The process for the preparation of the pseudo-aglycone derivative of antibiotic A-696 according to the present invention is illustrated by the following non-limiting examples.

Example 1

Preparation of the A-4696 Antibiotic Complex Used as Starting Material for the Pseudo-Aglycone Derivative of the A-4696 Antibody and Some Components of this Complex, Step a) Cultivation in a Shake Flask

The mycelial fragment of Actinoplanes missouriensis strain ATCC 31 683 is inoculated on a slant agar of the following composition:

component Quantity cerelose 0.5% Potato dextrin 2.0% soybean meal 1.5% yeast extract 0.25% calcium carbonate 0.1% agar 2.0% Inoculated strain ATCC 31 683 agar for 6 days at 30 ° C keep at room temperature. The culture does not spore, so the mycelium should be rubbed with a sterile pipette. The rubbed mature culture is covered with sterile distilled water and gently rubbed with a pipette or sterile glass rod to resuspend the mycelium. The resulting suspension was inoculated with 100 ml of sterile vegetative medium of the following composition: Component Quantity cerelose 0.5% Potato dextrin 2.0% Soybean meal * 1.5% yeast extract 0.25% calcium carbonate 0.1% * = Archer Daniels Midland Company, 4666 Faries Parkway, Decatur, Illinois 62,526. The inoculated vegetative medium is rotated at 250 rpm for 48 hours at 30 ° C, shake on a rotary shaker. 10 ml of this vegetative medium was then added. inoculated with 100 ml sterile, of the following composition fermentation broth: Component Quantity cerelose 0.5% yeast 0.25% soybean meal 1.5% cornstarch 2.0% calcium carbonate 0.1% Ság 471 * 0.05%

* = Ság 471 is a well-known silicone-based antifoam available from Union Carbide Company, 120 Riverside Plaza, Chicago, Illinois 60,606.

-6190 340 _

The inoculated main fermentation broth is shaken for 24 hours at 30 ° C on a rotary shaker at 250 rpm.

Then, 0.8 ml portions of this major fermentation broth are inoculated with 100-100 ml portions of a second inoculum medium having the following composition and sterilized in 500 ml Eritermeyer flasks at 121 ° C for half an hour.

component Quantity cerelose 1.25% cornstarch 4.4% sucrose 3.75% molasses 2.0% yeast 1.25% Proflo (cotton seed oil) * 1.25% calcium carbonate 0.25% dibasic potassium phosphate wood 0.625% ammonium sulfate 0.031% Ság 471 0.03%

* = Traders Protein Division, Traders Oil Mill Company, P. O. Box 1837, Fort Worth, Texas 76 101.

The above inoculated media were shaken on a rotary shaker at 250 rpm for about 96 hours at 30 ° C. At the end of this period, the pH of the medium was about 8.0.

Step b) Cultivation in a 40 liter tank fermenter

The inoculum is prepared as described in step a) above by inoculating and incubating the main fermentation broth. 25 liters of the second inoculum medium described above were sterilized in an autoclave for half an hour at 121 ° C and transferred to a 40 liter fermentation vessel. Subsequently, 100 ml of inoculated main fermentation broth is added and cultured for 4 days at 30'C. Meanwhile, the medium is aerated with sterile air, the amount of air per unit volume of medium, half a volume per minute. The medium is agitated with a mixer of appropriate size and speed so that the air is properly distributed in the medium. The pH of the medium increases from an initial value of about 6.5 to about 8.0 by the end of culture.

Step c) Isolation of the A-4696 antibiotic complex

To the fermentation broth obtained above (3800 L) is added 5% by weight Celite 545 filtration aid and the mixture is filtered. The filtered material was resuspended in deionized water (3600 L) and the aqueous suspension was adjusted to pH 10.5 with aqueous sodium hydroxide solution. The solids were filtered off and washed with water. The combined filtrate and washings were acidified to pH 4.5 with 20% aqueous sulfuric acid. The acidic solution was clarified with 1% filtration aid (Celite 545). The clear solution was passed through a column of ⁵ 1.8 x 5 ft. Filled with 350 L of Amberlite IR-116 sodium ion exchange resin and the column was washed with 1200 L of deionized water. The IR-116 resin was removed from its column and batch eluted with pH ¹⁰ aqueous sodium hydroxide solution 10.5 Series. (1000 liters total). The eluate was neutralized to pH = 7 with 20% aqueous sulfuric acid, and the resin was washed three times with 50 ml deionized water. The aqueous washes were also neutralized ¹⁵ sulfate and combined with the neutralized elutummal. The resulting solution was concentrated and the concentrate was freeze-dried. The crude complex thus obtained varies in color from beige to dark brown.

Step d) Removal of salts from the crude A-4696 antibiotic complex

1.0 kg of crude complex slowly portionwise personal ERE ²⁵ was added to 1.5 liter of deionized water. The suspension is stirred for 20 minutes and then neutralized to pH 7 with 10% aqueous ammonium hydroxide. The dried, desalted complex is recovered in 80% yield (based on biological measurement) within about ³ θ of the crude complex.

e) purification of the labeled desalted antibiotic A-4696 complex ³⁵

300 g of dried, desalted complex was suspended in 2 liters of deionized water and the suspension pH was adjusted to 2.7 with ⁴⁰ of 3N aqueous hydrochloric acid. The acidified solution is centrifuged at 2500 rpm for 40 minutes. The supernatant was decanted and applied to a column (8 x 85 cm) filled with 6 liters of clarifying resin (Duolite S761). The active ingredient was eluted at 30 ml / min with ⁴⁵ deionized water. Elution was monitored by TLC. The eluate containing the labeled antibiotic 4696 mm 3 was concentrated under vacuum at 35 ° C, 3 liters of volume, and the concentrate was freeze-dried in ^50. In the purification step, the complex between white and beige is recovered in about 70% yield (based on biological measurement).

Step f) Isolation of individual components of A-4696 antibiotic g Dried clarified A-4696 antibiotic complex is dissolved in about 100 ml of 60 distilled water. The aqueous solution was filtered and applied to a column packed with a reversed phase adsorbent such as Li Chroprep ^R RP-18 * and the column was washed with a gradient elution of aqueous acetonitrile containing triethylamine phosphate. It is obvious to those skilled in the art that ace7

The grandeny concentration of 190,340 thoniyryl should be varied depending on the composition of the product of the given batch; the preferred concentration gradient range is 10 to 40%. Elution is monitored by ultraviolet absorption and the fractions containing the individual components are combined. From the fractions, acetonitrile was distilled off under high vacuum and the remaining aqueous solutions were freeze-dried. The freeze-dried fractions are then redissolved in distilled water and chromatographed on a reverse phase adsorbent such as Sep Pak ^R C18, eluting with aqueous methanol. Aqueous solutions containing each of the components of antibiotic A-4696 are distilled off to obtain purified purified components of antibiotic A-4696 as dry, amorphous solids.

* = E. Merck, Darmstadt, Germany.

** = Waters Associates Inc., Milford, Massachusetts.

Example 2

Preparation of the dihydrochloride of the pseudoaglycone derivative of antibiotic A -4696

2.0 g of the A-4696 antibiotic complex prepared as described in Example 1 a) e) are dissolved in 50 ml of a 5% methanolic hydrochloric acid solution and refluxed for 70 minutes. The solvent was then distilled off under reduced pressure at 35-40 ° C. A small amount of water was added to the residue and the resulting slurry was filtered off. This solid product is air dried and then dissolved in a little methanol and precipitated by adding acetonitrile as a particulate solid. The product thus obtained is filtered off and dried. High performance liquid chromatography (HPLC) of the product showed that it was a single substance. The identity and structure of the product are confirmed by plasma desorption mass spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis.

The dihydrochloride of the pseudo-aglycone derivative of antibiotic A-4696 may also be prepared by subjecting each component of the A-4696 antibiotic complex, or mixtures thereof, to mild acid hydrolysis. 217,962 to Manuel Debono, Kurt E. Merkel, Robert E. Weeks, and Herold J. Cole, filed at the same time as the present application, isolate the individual components of the A-4696 antibiotic A-4696 starting material for the preparation of the pseudo-aglycone derivative of A-4696. US patent application Serial No. contains, especially the A-4696 antibiotic designated B ,, B _2, B _3, C _la and for this, labeled components. An additional possible method for separating and isolating the individual components is given in Example 1 (f) of this specification. The dihydrochloride of the pseudo-aglycone derivative of the A-4696 antibiotic, starting from the individual components of the A-4696 antibiotic complex, or mixtures thereof, is prepared in substantially the same manner as the one described above for the pseudo-aglycone derivative A-4696. starting from the complex.

The pseudo-aglycone derivative obtained from each of the components ^{b of} the antibiotic complex A-4696, or mixtures thereof, is identified by plasma desorption mass spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis.

The dihydrochloride of the pseudo-aglycone derivative of antibiotic A-4696 is a white crystalline solid which charred at about 270 ° C.

Soluble in water, but insoluble in other solvents such as methanol, acetone, diethyl ether, chloroform, pyridine, benzene, aliphatic hydrocarbons and the like. ₂₀ pH range of about 1.2 to about 9.5, and a temperature ranging from about 35 ° C to 85 ° C stable compounds.

The labeled antibiotic A-4696 pseudo-aglycone dihydrochloride derivative of elemental analysis of C ^ H ₆ O ₂₀ N ₂ O ₈ C1.2HC1.6H to:

25_

Element Percentage calculated found carbon 52.75 52.75 hydrogen 5.03 5.15 nitrogen 7.45 7.82 chlorine 7.07 7.17

Ultraviolet defined ₃₅ The labeled antibiotic A-4696 pseudo-agli ^ö concentrates from the dihydrochloride in methanol as solvent, absorption maximum: 279 nm, E | = _m * 63.6.

The infrared absorption spectrum of the dihydrochloride of the pseudo-agly4Q cone derivative of antibiotic A-4696 in potassium bromide pastilles is shown in Figure 1. In the range 4000-700 cm ¹ , the following well-defined absorption maxima can be ^found:

3374 wide, 3220, 1728, 1659, 1617, 1592, 1546, 1509, 1489, 1461, 1431, 1355, 1288, 1229, 1208, 175, 1126, 1103, 1071, 1058, 1014, 986, 897, 880, 843 , 816, 767, 731, 710cm '.

Example 3

A sulfate of the pseudoaglycone derivative of antibiotic A-4696. production

2.0 g of the antibiotic complex A-4696 prepared as described in Example 1 a) e) are dissolved in 50 ml of a 5% solution of sulfuric acid in methanol 50 and heated to reflux for 70 minutes. After distilling off the solvent, a little water was added to the residue and the resulting solid was filtered off. The product was air dried and then dissolved in a small amount of methanol and precipitated by addition of acetonitrile as a 65 particulate solid. This

190 340 the product is filtered off and dried. The product is a homogeneous material by high performance liquid chromatography (HPLC). The identity and structure of the product are confirmed by plasma desorption mass spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis.

The sulfate derivative of the pseudoaglycone derivative of antibiotic A-4696 may also be prepared by subjecting each component of the A-4696 antibiotic complex or mixtures thereof to mild acid hydrolysis. Certain components of the A-4696 antibiotic which may be used to prepare the pseudoaglycone derivative of the A-4696 antibiotic may be isolated as described in Example 1, step f). The sulfate derivative of the pseudoaglycone derivative of antibiotic A-4696 is prepared starting from the individual components of the A-4696 antibiotic complex or mixtures thereof, in the same manner as the anti-A-4696 anti. starting from a biotic complex as described above.

Starting from the A-4696 antibiotic complex designated individual constituents or mixture thereof prepared from ⁵ product identity was confirmed by plasma desorption mass spectroscopy, nuclear magnetic resonance spectroscopy and elemental analysis.

Claims (1)

^A patent claim A process for the formula I-4696 designated pseudo-aglycone antibiotic complex, or a compound prepared at a pharmaceutically acceptable acid addition salt thereof ¹⁵ his passenger, characterized in that the A-4696 labeled antibiotic complex is subjected to mild acid hydrolysis, if desired, the resulting salt and liberating the free compound of formula I.