NO166800B - PROCEDURE FOR THE PREPARATION OF ANTIBIOTICS X-14868A, X-14868B, X-14868C, AND X-14868D BY PROCESSING NOCARDIA X-14868. - Google Patents

Description

The present invention relates to a method for the production of the polyether compounds antibiotics X-14868A, X-14868B, X-14868C and X-14868D with the general formula

wherein, for antibiotic X-14868A, R 1 is methyl, R 2 is hydrogen, is methyl and R 4 is hydrogen; where, for antibiotic X-14868B, R 1 is methyl, R 2 is hydrogen,

R3 is methyl and R3 is methyl; where, for antibiotic X-14868C, R 1 is hydrogen, R 2 is hydrogen, R 3 is methyl

and R 4 is hydrogen; and, for antibiotic X-14868D, R1

is methyl, R 2 is methyl, R 2 is hydrogen and R 3 is hydrogen, and pharmaceutically acceptable salts thereof.

Furthermore, the preparation of the new antibiotic X-14868C and pharmaceutically acceptable salts thereof, the sodium salt with the following physical characteristics, is included: 1) an infrared spectrum as shown in fig. 3; 2) a melting point of 172-175°C (sodium salt);

3) a specific rotation on

4) a microanalysis of and the preparation of the new antibiotic X-14868D and pharmaceutically acceptable salts thereof, where the sodium salt has the following physical characteristics: 1) an infrared spectrum that appears in fig. 4; 2) a melting point of 194-195°C (sodium salt);

3) a specific rotation on

4) a microanalysis of

These four antibiotic compounds are obtained according to the invention by a method in which a subculture of the strain Nocardia X-14868 ATCC 31585 is grown in an aqueous carbohydrate solution containing a nitrogenous nutrient, at neutral pH and at a temperature of 20-35°C, under submerged aerobic conditions and then separating and isolating the final product from the solution and, if desired, converting these into a pharmaceutically acceptable salt thereof.

In the structural formulas used here, Me means the term me-

shut up.

The organism that produces antibiotics according to the present invention is. a new species called Nocardia sp. X-14868.

A culture of the living organism given the laboratory designation X-14868 has been deposited in the American Type Culture Collection, Rockville, Maryland, USA and added to their permanent collection of microorganisms as ATCC 31585. The culture has been identified as a strain of Nocardia.

The new microorganisms were isolated from a sample collected from a sandy beach in Colloroy, Australia. A representative strain of Nocardia sp. The X-l'4i868 has the following characteristics:

General Characteristics

Nocardia sp. X-14868i is characterized by its lack of air spore formation, fragmentation of gram-positive substrate mycelium after several days of incubation and cell wall composition of meso-diaminopimelic acid, galactose, arabinose and ribose.

Growth characteristics

The organism was grown on standard ISP media (Difco) described by Shirling and Gottlieb "Methods for' Characterization of Streptomyces Species", Intern. J. System. Bacteriol., 16, pp.313-340, 1066, as well as various media that were used to characterize the culture, and which are indicated below:

Yeast extract:

Yeast extract, 1.0%|-glucose, 1.0%^-agar, 1.5%;

pH 6.8

Glucose-yeast-extract-peptone:

Glucose, 0.3%; yeast extract, 0.5%; peptone, 0.5%;

CaCO 3 , 0.75%; agar, 1.5%; pH 7.0.

Glucose-asparagine:

Glucose 1.0%; asparagine, 0.05%;. K2HP0.4, 0.05%;

agar, 1.5%; pH 6.8

Sucrose nitrate:

Sucrose, 1.0%; NaNO^, 0.2%; K2HP04, 0.1%;

MgSO4.7H2O, 0.05%; KC1, 0.05%; agar, 1.5%.

Media used in other experiments were those from the following references:

The experiments were carried out at 28°C and 37°C for almost all media. Color determinations were made after 2 and 4 weeks of incubation.

Carbon consumption was measured by Shirling and Gottlieb's method (above) using ISP-9 (Difco) medium.

A 48 hour old ISP-1 qroot culture of X-14868 was centrifuged and homogenized to give a washed suspension for inoculation.

The ability of the organisms to grow at 10, 28, 36, 45 and 50°C was examined by inoculating slurry of ISP-1 (Difco) medium. Cell wall analysis of the isomer of diaminopimelic acid was carried out according to the method of Becker et al., Appl. microbiol., 12, 421-423, 1964. For the sugar content of the cell wall, the method of Lechevalier and Lechevalier, Actinomycetales, Ed.

H. Prauser, Gustav Fischer, Jena, pp. 311-316, 1970 followed. Nocardomycholic acid assay was performed by a slight modification of the method of Lechevalier, Lechevalier, and Horan, Can. J. Microbiol. 19:965-972, 1973.

Results

Microscopic examination. The strain X-14868 produces a substrate mycelium that fragments after a few days of extensive mycelial development. It produces an aerial mycelium consisting of cord-like tufts, but no spores were found.

Cell wall analysis. The cell wall contains the meso isomer of diaminopimelic acid as well as galactose and arabinose (cell wall type IV in the classification of Lechevalier et al., Adv. Appl. Microbiol., 14, 47-72, 1971). The organism is found to produce nocardomycholic acids. These morphological and chemical criteria refer X-14868 to the family Nocardia.

Macroscopic examination. Table 1 summarizes the growth rate, sporulation rate, aerial mass color, color of reversed substrate mycelium and presence of soluble pigment produced by strain X-14868 on various solid media after 4 weeks of incubation at 28°C. Physiological characteristics. Strain X-14868 hydrolyzed gelatin and casein, but not starch. The culture was resistant to a 10 unit disk of penicillin and 0.005% lysozyme dissolved in the porridge when examined according to the method of Gordon, J. Gen. Microbiol., 45, 355-364, 1966. The strain peptonized completely in litmus milk, no melamine production was detected on ISP 1, 6 or 7.

Table 2 shows the results of carbon consumption on ISP 9 (Difco) of strain X-14868 at 28°C after one month of incubation.

Table 3 is a list of diagnostically important, mostly metabolic characteristics.

Strain X-14868 can be distinguished from other Nocardia species due to the biochemical characteristics listed in Table 4. These species have been selected for comparison because they constitute the only group within the genus Nocardia with which X-14868 has some degree of phenotypic affinity.

It can be seen in table 4 that very little biochemical data is available for four of the species, namely N. transvalensis, N. formica.,N. petroleophila and N. saturnea. However, is

these species very different from X-14868. N. transvalensis is indicated to be very similar to N. brasiliensis, which is included in table 4. N. formica is possibly misplaced in the genus Nocardia and is more similar to the genus Streptomyces. N. petroleophila and N. saturnea are two very different species with little affinity to other Nocardia species. Members of

these two species can live in a carbon-free medium at the expense of atmospheric carbon dioxide, and this is not the case with other species, including X-14868.

The pharmaceutically acceptable salts of antibiotic X-14868 can be prepared by conventional means. These salts are produced from the free acid form of the antibiotic by well-known methods in the field, e.g. by washing the free acid in solution with a suitable base or salt. Examples of such pharmaceutically tolerable basic substances which can form salts for the purpose of the present invention include alkali metal bases, such as sodium hydroxide, potassium hydroxide, lithium hydroxide and the like; alkaline earth metal bases, such as calcium hydroxide, barium hydroxide and the like; and ammonium hydroxide. Alkali metal or alkaline earth metal salts suitable for forming pharmaceutically acceptable salts may include anions such as carbonates, bicarbonates and sulfates.

Nocardia X-14868 produces the compound X-14868A by

cultivation under suitable conditions. A fermentation slurry containing Nocardia X-14868 is prepared by inoculating spores or mycelia of the organism producing compound X-14868A in a suitable medium and then growing under aerobic conditions. For the manufacture of X-14868A

cultivation is possible on a solid medium, while for production in large quantities cultivation in a liquid medium is preferred. The culture temperature can vary over a wide range, 20-35°C, within which the organism can grow, but a temperature of 26-30°C and essentially neutral pH is preferred. In the submerged aerobic fermentation of the organism for the production of the antibiotic X-14868A, the medium may contain as a carbon source a commercially available glyceride oil or a carbohydrate such as glycerol, glucose,

maltose, lactose, dextrin, starch, etc. in pure or raw state and as; nitrogen source, an organic material, such as soybean meal, distiller's solubles, peanut meal, cotton seed meal, meat extract, peptone, fish meal, yeast extract, corn liquor, etc., and if desired, inorganic nitrogen sources such as nitrates and ammonium salts. It may also contain mineral salts such as ammonium sulphate, magnesium sulphate, sodium chloride, potassium chloride, potassium phosphate, calcium carbonate, and the like, and buffering agents such as sodium citrate or phosphate and trace amounts of heavy metal salts. In air-blown submerged cultivation methods, an anti-foam agent such as liquid paraffin, fatty oils or silicone compounds is preferably used. More than one type of carbon source, nitrogen source or anti-foam source can be used for the preparation of antibiotic X-14868A.

The following Table 5 indicates the antimicrobial activity of the antibiotic X-14868A and the three co-components X-14868B, X-14868C and X-14868D. As indicated in Table 5, antibiotic X-14868A and its three co-components have the property of reducing the growth of certain gram-positive bacteria. They will therefore be applicable in washing solutions for sanitary purposes such as when washing hands and cleaning equipment, floors or furnishings in contaminated rooms or laboratories.

The following Table 6 indicates the anticoccidial activity compared to an infected untreated control (IUC) and an uninfected untreated control (UUC) according to the experimental method described below. Experimental methodology. This experiment uses 10 chickens per drug group. 10 chickens are used as weight control and 10 chickens as infected control. The drug is given 48 hours before the infection. 1 g of the test drug is mixed in a mechanical mixer with a sufficient amount of chicken fat to give the desired dosage. The infection lasts approx. 300,000 oo-cytes given orally by pipette of Eimeria acervulina, E. mivati, E. maxima, E. necatrix and E. tenella. The experiment lasts 6 days and then the surviving birds are opened and examined for major damage in the ceca. The experimental birds are graded according to the number of survivors and the number of internal injuries. The results are expressed as average degree of infection (A.D.I.). An average degree of infection of less than 2.5 is considered significant.

The coccidiostatic mixtures according to the present invention contain as active ingredient antibiotic X-14868A or its pharmaceutically acceptable salts or the dried unfiltered porridge is prepared by mixing the active ingredient with an inert ingredient. The inert component can include a filler, filling materials and the like. The term "inert component" means a material that does not act as an antiparasitic agent, e.g. a coccidiostatic is inactive towards the active ingredient, and which can be safely digested by the animals to be treated, and thus such an inert material is one which is inactive for the purpose of the present invention.

When administered orally to livestock susceptible to coccidiosis, especially poultry, such as turkeys and chickens, the active ingredient, as an FDR component, effectively controls the disease, either by preventing it or curing it, after it occurs . Furthermore, the treated poultry either maintain their weight or actually gain weight compared to the controls. The mixtures according to the present invention thus not only control coccidiosis, but also help to improve the efficiency of the transfer of feed to weight gain.

The relevant concentration of the active ingredient in the animal feed can of course be adjusted as a result of individual needs and can vary over a large area. The limiting concentration factor is that the minimum concentration is such that a sufficient amount of active ingredient is provided to effect the desired control of coccidiosis/and the maximum concentration is such that the amount of digested mixture does not lead to any harmful or unwanted side effects.

Thus contains e.g. a pre-mixture or a ready-made sufficiently active ingredient to give from approx. 0.0003 to 0.001% by weight of the daily intake. Preferably, approx. 0.0005% to 0.001% by weight. In general, approx. 0.0005% of the active ingredient sufficient to control and combat coccidiosis. Amounts greater than 0.001%, even if these are active against coccidiosis, generally do not give improved results over 0.001%, and in some cases can negatively affect growth, the degree of effectiveness and mortality.

Although amounts above 0.001% are effective in controlling coccidiosis, this amount is the preferred upper limit for economic reasons, i.e. cost per unit efficiency is the lowest within this range. Amounts lower than 0.0003% are not effective for controlling coccidiosis. A lower limit of 0.0005% is preferred because this ensures business. The most preferred amount, i.e. approx. 0.0005% by weight of the daily poultry consumption is particularly effective as you get the maximum effect with a minimal dose.

The optimal dosage level will of course vary with the size of the animal. When using the antibiotic according to the invention for the treatment or prevention of coccidiosis, it can first be combined with or mixed with a precursor component or carrier for a precursor mixture, a precursor concentrate or a precursor. A pre-addition, concentrate or feed mixture is an article intended to be diluted into a complete feed, i.e. an article intended for direct consumption by an animal or which can be further diluted into a complete feed or which can be digested and used as a supplement to other rations. Four-addition supplements, concentrates and premixes contain a relatively high proportion of coccidiostatics, i.e. the active ingredient in relation to a suitable carrier and which is mixed in a way that provides mainly even distribution of the coccidiostatic in the carrier. Suitable carriers are solid substances which are inert with respect to the active ingredient and which can be safely digested by the animals to be treated. Typical of such carriers are commercial poultry, ground cereal grains, grain by-products, plant protein concentrates (soy, peanuts, etc.), fermentation by-products, salts, lime, inorganic compounds and the like or mixtures thereof. Liquid dispersions can be prepared by using water or vegetable oil, preferably also a surface-active agent, emulsifier and the like in the liquid dispersion, such as ethylenediaminetetraacetic acid, etc., and solubilizers. All suitable carriers or diluents can act as an inert component in the solid formulation of the antiparasitic agent, provided that it is inert to the active material and is not toxic to the animal to which it is to be administered.

The active ingredient can be mixed into a mash/pellet or other desired form with the inert carrier or diluent in the form of solid material by all usual techniques. E.g. mixtures can be formed by finely grinding or pulverizing the active ingredient and the inert ingredient using any commercially available grinding mill or pulverizing apparatus in the presence or absence of precursor material. If raw material is not present when the grinding or pulverizing takes place, the resulting material can be distributed in

any commonly available precursor material. Ty-

whipped poultry meat which can be given together with the active ingredient according to the invention may contain several ingredients, e.g. can they contain cereal products with high energy, so

such as corn, wheat, "wheat red" dog meal, sorghum, oatmeal or similar, medium and low energy grain products, such as oats, barley/wheat flour, bran mixtures, standard bran mixtures etc.

ler similar, stabilized fats, vegetable protein, so

such as soybean flour, corn glutinous flour, peanut flour, or the like; animal protein'such as fishmeal, soluble fish products

ter, meat waste or the like. UGF (unidentified growth factor) sources and other B vitamin carriers such as dry milk products, dried brewer's yeast, soluble stills, soluble mash, or the like, dehydrated alfalfa meal, and various special additives such as additional riboflavin, vitamin B12, calcium pantothenate, niacin, choline, vitamin K and vitamin E or similar, as well as stabilized vitamin A, vitamin

(d-activated animal sterols), potassium and phosphorus additives such as dicalcium phosphate, evaporated bone meal, defluorinated phosphate, lime, or similar, iodized salt, manganese sul-

barrel, zinc carbonate, an antibiotic fertilizer additive, methionine or its hydroxy analog thereof, and an antioxidant.

As can be seen from the above, the coccidiostatic mixtures are intended for oral use. They can be added to the normal feed of the treated animal or can be given by other methods, such as introduction in a tablet, pill or large pill and given to the animal as forced feed. The administration of the active ingredient must be considered depending on the particular animal based on normal livestock breeding practice.

The bicomponents X-14868B, C and D also show anticoccidiostatic activity in in vitro experiments. X-14868B has also been shown to be active in vivo.

A discussion of the mechanism of feed digestion, breakdown and metabolism in a ruminant animal can be found in US patent no. 3,839,557 which describes the use of certain antibiotics to improve the utilization rate of ruminant feed. Economically important ruminants

animals are cattle, sheep and goats.

The efficacy of antibiotic X-14868A by modifying the ratio of. volatile fatty acids produced in the rumen have been detected using the following in vitro

-attempt.

Rumen fluid is obtained from a bull with a fistula rumen. The bull is kept on the following ration:

Grain: 89.9%

Alfalfa meal: 5,000%

Soybean oil meal: 3.00%

Lime: 0.80%

NaCl: 0.60%

Dicalcium phosphate atr. 0.50%

Trace minerals: 0.025%

Vitamin premix additives: 0.1%

Vitamin A, TIU: 4.0003

Vitamin D3, IU: 0.801

Vitamin E, TIU: 3,002

The rumen fluid is immediately passed through a sieve. For each fermentation, 75 ml of the resulting liquid is added to a 250 ml flask containing the following: 1 g of 80%:20% finely ground grain: high ratio; 1 ml of an 18% aqueous glucose solution (1 mmol per flask); 1.5 ml of a 3.1% aqueous urea solution (0.76 mmol per flask); 60 micromoles of each of the four essential amino acids (arginine, histidine, leucine, methionine, threonine, valine, lysine, isoleucine, phenylalanine, tryptophan);

1, ml of an aqueous solution of the test drug to be given either 10 or 25 pq/ml (calculated total volume of fermentation mixture of 80 ml);

All flasks are incubated at 38°C in a shaking water bath equipped with a gas hood. Carbon dioxide is continuously fed through the gas hood. After 4 hours of incubation, a 10 ml quantity of the fermentation liquid is centrifuged at 14,000 rpm.

(approx. 30,000 g) for 20 minutes. 3 ml of the supernatant is added to 1 ml of a 25% metaphosphoric acid solution containing 23 micromoles of 2-methylvaleric acid as an internal standard. It

resulting liquid is allowed to settle at room temperature for 30 minutes. The liquid is filtered through a 0.22 millimicron Milli-pore filter and cooled until gas-liquid chromatographic analyzes for volatile fatty acids.

Gas-liquid chromatographic (GLC) analyzes of four in vitro control fermentations and two fermentations each with 10

and 25 ppm antibiotic X-14868A are listed in the following table.

As shown in Table 7, the ratio of propionate (C 2 ) to acetate and n-butyrate is significantly improved. With the increase of propionates rather than acetates from carbohydrates, the efficiency of carbohydrate and therefore the utilization rate is increased.

Administration of the antibiotic X-14868A (hereafter "antibiotic" or "antibiotic compound") prevents and treats ketosis and improves fci utilization rates.

The causal mechanism for ketosis is a lack of production of propionate compounds. A currently recommended treatment is the administration of propionic acid or for which preferably gives propionates. It is clear that stimulation of propionate production from common precursors will reduce the occurrence of ketosis.

Antibiotic X-14868A has been found to increase the efficiency of feed utilization in ruminants when administered orally to animals. The easiest way to give the antibiotic is to mix it in the animal's feed.

However, the antibiotic can also be given in other ways. F.

e.g. it can be introduced in tablets, soaking liquids, large pills or capsules, and given dosed to animals. Formulations of the antibiotic compound in such dosage forms can be obtained using well-known methods in the veterinary pharmaceutical field.

Capsules are easy to prepare by filling gelatin capsules with any desired form of the desired antibiotics. If desired, the antibiotic may be diluted with an inert powdered diluent such as sugar, starch, or purified crystalline cellulose to increase volume for easier filling into capsules.

Tablets of the antibiotic are prepared by conventional pharmaceutical processes. In addition to the active ingredient, a tablet usually contains a base substance, a disintegrant, an absorbent, a binding agent and a lubricant. Typical bases are lactose, caster sugar, sodium chloride, starch and mannitol. Starch is also a good disintegrant, as is alginic acid. Surfactants such as sodium lauryl sulfate and dioctyl sodium sulfosuccinate are also sometimes used. Commonly used absorbents are again starch and lactose, while magnesium carbonate can also be used for oily substances. Frequently used binders are gelatin, gums, starch, dextrin and various cellulose derivatives. Among the commonly used lubricants are magnesium stearate, talc, paraffin wax, various metal soaps and polyethylene glycol.

The administration of the antibiotic compound can take place by means of a slow-release large pill. Such large pills are made like tablets except that an agent to delay the dissolution of the antibiotic is provided. Large pills are made for release over long periods of time. The slow dissolution is aided by choosing a highly water-insoluble form of the antibiotic. A substance such as iron filings is added to increase the density of the large pellet and keep it static at the bottom of the rumen.

Dissolution of the antibiotic is delayed by using a matrix of insoluble materials in which the drug is embedded. For example substances such as vegetable waxes, purified mineral waxes and water-insoluble polymer materials are applicable.

Soaking liquids of the antibiotic are most easily prepared by choosing a water-soluble form of the antibiotic. If an insoluble form is desired for some reason, a suspension can be made. Alternatively, a soaking liquid can be formulated as a solution in a physiologically acceptable solvent such as a polyethylene glycol.

Suspensions of insoluble forms of the antibiotic can be prepared in non-solvents such as vegetable oils such as peanut, corn, or sesame oil, in a glycol such as propylene glycol or a polyethylene glycol, or in water, depending on the form of the antibiotic selected.

Suitable physiologically tolerable excipients are required to keep the antibiotic suspended. The excipients can be chosen from among thickeners, such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin and the alginates. Many classes of surfactants serve to suspend the antibiotic. E.g. lecithin, alkylphenyl/polyethylene oxide adducts, naphthalene sulfonates, alkylbenzene sulfonates and polyoxyethylene sorbitan esters are useful to produce

still suspensions in liquid non-solvents.

In addition, many compounds that affect the hydrophilicity, density and surface tension of the liquid can help to form suspensions in some cases. E.g. silicone antifoam agents, glycols, sorbitol and sugar can be used as suspending agents.

The suspendable antibiotic can be presented as a suspension or a dry mixture of the antibiotic and excipients, which must be diluted before use.

The antibiotic can also be given in drinking water to ruminants. Introduction into drinking water is carried out by adding a water-soluble or water-suspendable form of the antibiotic to the water in the correct quantity. Formulation of the antibiotic for addition to drinking water follows the same principles as formulation of soaking liquids.

The most practical way to treat animals with the antibiotic compound is through the formulation of the compound in the feed. All types of fodder can be added to the antibiotic compounds, such as normal dry fodder, liquid fodder and pelleted fodder.

The procedures for formulating drugs in animal feed are well known. It is common to prepare a concentrated drug-form mixture as raw material for drug-containing form. E.g. typical drug premixes can contain from approx. 1 to 400 g of drug per .0-, 5 k<J premix. The wide range comes from the wide concentration range of the drug that may be desired in the final feed. Premixes can be either liquid or solid.

The formulation of rumen containing the correct amounts of the antibiotic suitable for treatment is well known. It is only necessary to calculate the amount of compound to be given to each animal, taking into account the quantity per day that the animal eats, and the concentration of the antibiotic compound in the premix to be used, and calculate the correct concentration of antibiotic compound, or premix, in the feed.

All the methods of formulation, mixing and pelleting

of forage that is normally used in the rumen area, is completely suitable for the production of forage containing the antibiotic compound.

As shown, oral administration of the antibiotic beneficially alters the production of propionates relative to the production of acetates in the rumen. It can therefore be postulated that the same treatment would also benefit monogastric animals that ferment vegetable fiber materials in the cecum, as it would be expected that a beneficial change in the propionate/acetate ratio would occur after oral administration of the present antibiotic. Horses, pigs and rabbits are examples of animals that digest part of their feed by cecal fermentation.

Antibiotic X-14868A thus has demonstrable activity as an agent in the treatment or prevention of swine dysentery. The compound was investigated for activity against Treponema hyodysenteriae, the etiological agent of swine dysentery. The results, which are a comparison test according to well-known test methods against a known agent for the treatment and prevention of swine dysentery, are shown in the table below.

Part of the present invention is also the production of the new bicomponents called X-14868B, C and D. These components exhibit in vitro anticoccidiostatic activity against E. tenella as shown below.

The structural formula of the compound X-14868B is as follows:

The infrared absorption spectra of the respective compounds are as follows:

Table 9 indicates physical constants for the various components produced by fermentation of Nocardia X-14868. The following examples will serve to illustrate the invention without limiting it.

EXAMPLE 1

Shake bottle fermentation of Nocardia X- 14868

The antibiotic-producing X-14868A culture is grown and maintained on a starch-casein agar slant with the following composition (g/l distilled water):

Adjust pH to 7.4 with NaOH before autoclaving at 15 pounds pressure for 20 minutes.

The agar slant is inoculated with Nocardia X-14868 culture and incubated at 28°C for 7-14 days. A portion of agar containing mycelium from a well-grown agar slant is then used to inoculate a 500 ml<1>s Erlenmeyer flask containing 100 ml of sterilized inoculum medium with the following composition (g/l distilled water):

Adjust pH to 7.0 with NaOH before sterilization.

The inoculated inoculum medium is incubated at 28°C for 72 hours on a rotary shaker operating at 250 rpm.

A 3 ml portion (3%, vol./vol.) of the resulting culture is then used to inoculate a 500 ml Erlenmeyer flask containing 100 ml of sterilized preparation medium of the following composition (g/l distilled water):

Adjust pH to 6.4 before autoclaving.

The inoculated medium is incubated at 28°C for 5 days on a rotating shaker at 250 rpm. The potency of antibiotic X-14868A in the fermentation broth is measured by its action against Staphylococcus aureus ATCC 6 5 38P using an agar diffusion cup -plate measurement.

EXAMPLE 2

Isolation of antibiotic X-14868A-Na salt and antibiotic X-14868B-Na salt from shake flask fermentation: Step A: The entire mash from 50 1/2-liter Erlenmeyer flasks each containing 100 mL was pooled after 5 days of fermentation (5 liters ) and extracted twice with half the volume of ethyl acetate. After stirring for 1 1/2 hours, the solvent layer was separated and concentrated to an oil (4 g) under reduced pressure. The oil was dissolved in diethyl ether and chromatographed on a 200 g silica gel column packed suspended in diethyl ether. The column was eluted with a gradient between 2 liters of diethyl ether and 2 liters of diethyl ether/acetone (9:1) and then 2 liters of diethyl ether/acetone (1:1) and then 2 liters of methylene chloride/ethyl alcohol (7:3). Fractions of 40 ml each were collected and from fraction numbers 18-75 were pooled, the solvent removed under reduced pressure and the residue (1.66 g) dissolved in ethyl acetate and washed with the same volume of IN HCl twice, followed by washing with the same volume Na2C03 (saturated at room temperature) twice. The solvent phase was dried over Na 2 SO 4 , and by adding n-hexane, crystals of the antibiotic salt X-14868A-Na were obtained. Recrystallization from ethyl acetate/n-hexane gave an analytical sample of antibiotic X-14868A-Na salt. Temp. 193-194°C.

Step B: From fractions number 161-215, after the solvent had been removed under reduced pressure, and the residue dissolved in ethyl acetate and washed with IN HC1, followed by Na2C0.j (saturated at room temperature), washing and drying over Na2SO4, after addition of n-hexane, antibiotic X-14868B-Na salt crystals. Temp. 172.5-174°C.

EXAMPLE 3

Tank fermentation of Nocardia X- 14868

The antibiotic X-14868A-producing culture Nocardia X-14868 is grown and maintained on a starch-casein agar slant with the following composition (g/l distilled water):

Adjust the pH to 7.4 with NaOH before autoclaving.

The agar slant is inoculated with Nocardia X-14868 culture and incubated for 7-14 days at 28°C. A portion of agar containing mycelium from the well-developed agar slant is then used to prepare vegetative inoculum by inoculating a 500 ml<1>s Erlenmeyer flask with 100 ml of inoculum medium of the following composition (g/l distilled water).

The pH is adjusted to 7.0 before autoclaving.

The inoculated medium is incubated for 72 hours at 28°C on a rotating shaking table at 250 rpm.

60 ml (3% v/v) of this growth slurry is used to inoculate a 6-liter Erlenmeyer flask containing 2 liters of inoculum medium of the following composition (g/l distilled water):

The pH is adjusted to 7.0 before autoclaving.

The inoculated medium is incubated for 72 hours at 28°C on a rotating shaking table at 250 rpm.

4 liters of this culture are used to inoculate 230 1 of the following production medium in a 380 l fermenter (g/l tap water):

The pH of the medium is adjusted to 6.4 before sterilization for 1 1/4 hours with 4.2 kg/cm 2 steam.

The inoculated medium is blown through with compressed air at a rate of 90 l/min. and stirred with a stirrer at 280 rpm. The fermentation is carried out at 28°C for 5 days.

The potency of the antibiotic X-148G8A in the fermentation slurry is measured with Staphylococcus aureus ATCC 6538P using the agar diffusion cup-plate method.

EXAMPLE 4

Isolation of antibiotic X-14868A-; C-; and D sodium salts from tank fermentation of Nocardia X-14868.

Step A: After 115 hours of growth, the same volume of ethyl acetate is added to the whole porridge from a 230 liter fermentation as described in example 3. After stirring for 1 hour, the solvent mixture is separated and concentrated to 4.2 liters under reduced pressure. Concentrated solvent extract was washed with the same volume of IN HCl twice followed by washing twice with the same volume of Na 2 CO 3 (saturated at room temperature). The solvent phase is dried over Na2S0^ and concentrated to an oil under reduced pressure. The oil was dissolved in n-hexane and extracted once with acetonitrile followed by two extractions with acetonitrile/methanol (8:2). The acetonitrile and acetonitrile/methanol (8:2) extracts were combined and solvent was removed under reduced pressure. The resulting oil is dissolved in diethyl ether, treated with active carbon, filtered and by adding n-hexane, crude antibiotic X-14868A-Na salt crystals are obtained. The crude antibiotic crystals are dissolved in methylene chloride and chromatographed on a methylene chloride slurry packed 600 g silica gel column. The column is eluted with 4 liters of diethyl ether/acetone/ammonium hydroxide (8/2/0.02). Fractions of 45 ml each are collected and fractions No. 13-15 are combined. The solvent is removed under reduced pressure, the residue is dissolved in ethyl acetate and then washed twice with 1N HCl and twice with Na 2 CO 3 (saturated at room temperature), dried over Na 2 SO 3 . The ethyl acetate phase was concentrated to an oil and dissolved in methylene chloride and by adding n-hexane, crystalline antibiotic X-14868A-Na salt is obtained. Temp. 193-195°C.

Step B; The mother liquor from the crude antibiotic X-14868A-Na salt crystals (described in step A above) is chromatographed on a methylene chloride slurry packed 1 kg silica gel column. The column is eluted with 1 liter of n-hexane and then a gradient between 4 liters of diethyl ether/n-hexane (7:3) to 4 liters of diethyl ether/acetone (8:2). Fractions of 40 ml each are collected and fractions No. 4 3-80 are pooled. The solvent is removed under reduced pressure and the residue is dissolved in ethyl acetate and washed with 1N HCl, followed by an aqueous Na 2 CO 3 ~ (saturated at room temperature) wash and activated carbon treatment. After filtration, the ethyl acetate phase is concentrated to an oil. The oil is dissolved in diethyl ether and the addition of n-hexane gave additional antibiotic X-14868A-Na salt. Temp. 193-195°C.

Step C: Fractions #22-40 from the silica gel column described above in Step A yield crystalline antibiotic X-14868C-Na salt. Temp. 172-175°C.

Step D: The mother liquor from the crystalline antibiotic X-14868A-Na salt described in step B and fractions 16-21 from the silica gel column described in step A were combined, concentrated and chromatographed on a methylene chloride slurry packed 600 g silica gel column. The column is eluted with 4 liters of diethyl ether/hexane (1:1), 4 liters of diethyl ether/acetone/n-hexane/ammonium hydroxide (6:2:2:0.002); 2 liters of diethyl ether/acetone (8:2). Fractions of 40 ml each are collected. Fractions #71-98 are pooled and crystallization provides additional crystalline antibiotic X-14868A-Na salt. Fractions 141-172 are also combined, solvent removed under reduced pressure and crystallization from diethyl ether/n-hexane gives antibiotic X-14868D-Na salt. Temp. 194-195°C.

EXAMPLE 5

Preparation of the thallium salt of the antibiotic X-14868A.

A solution of 51 mg of antibiotic X-14 86 8A-Na salt in methylene chloride is washed with IN HCl, followed by washing with water and then four times with an aqueous solution of thallium carbonate.

■The solvent is separated and concentrated to a small volume under reduced pressure and after the addition of n-hexane, crystalline thallium salt of antibiotic X-14868A was obtained. Recrystallization from diethyl ether/n-hexane gives crystals that were suitable for X-ray analysis.

EXAMPLE 6

Preparation of the calcium salt of antibiotic X-14868A.

A solution of 200 mg of antibiotic X-14868A-Na salt in ethyl acetate was first washed with IN HCl, then three times with an aqueous solution of calcium hydroxide (saturated at room temperature). The solvent was separated and removed under reduced pressure. The calcium salt of antibiotic X-14868A was isolated as a white solid foam.

EXAMPLE 7

Shaker flask fermentation of Nocardia X-14868

Antibiotic X-14868A producing culture was grown and maintained on a starch-casein slant agar with the following composition (g/l distilled water):

The pH was adjusted to 7.4 with NaOH before autoclaving at 15 pounds pressure for 20 minutes.

The agar slant was inoculated with Nocardia X-14868 culture and incubated at 28°C for 7-14 days. : Part of a<3aJ? which contained mycelium from the well-developed agar slant was then used to inoculate a 500 ml Erlenmeyer flask with 100 ml of sterilized inoculum medium with the following composition (g/l distilled water):

The pH was adjusted to 7.0 with NaOH before sterilization.

The inoculated inoculum medium was incubated at 28°C for 72 hours on a rotary shaking table at 250 rpm.

A 3 ml portion (3%, v/v) of the resulting culture is then used to inoculate a 500 ml Erlenmeyer flask with 100 ml of sterilized preparation medium of the following composition (g/l distilled water):

The inoculated medium is incubated at 28°C for 7 days on a rotating shaking table at 250 rpm. The strength of antibiotic X-14868A in the fermentation slurry was measured using Staphylococcus. aureus ATCC 6538P using the agar diffusion cup-plate method.

Claims (2)

1. Procedure for the preparation of the polyether compounds antibiotics X-14863A, X-14863B, X-14868C and X-14868D with the general formula where, for antibiotic X-14868A, R is methyl, R 2 is hydrogen, R 3 is methyl and R 4 is hydrogen; wherein, for antibiotic X-14868B, R 1 is methyl, R 2 is hydrogen, R 2 is methyl and R 3 is methyl; wherein, for antibiotic X-14868C, R 1 is hydrogen, R 2 is hydrogen, R 2 is methyl and R 3 is hydrogen; and, for antibiotic X-14868D, R-^ is methyl, R2 is methyl, R3 is hydrogen and R4 is hydrogen, characterized by growing a subculture of the strain Nocardia X-14868 ATCC 31585 in an aqueous carbohydrate solution containing a nitrogenous nutrient, by neutral pH and at a temperature of 20 - 35UC, under submerged aerobic conditions and then separate and isolate the final product from the solution and, if desired, convert these into a pharmaceutically acceptable salt thereof. 2. Method according to claim 1, characterized in that the polyether compound X-14868A is produced.