KR820002138B1 - Process for preparing a-40104 antibiotics - Google Patents

Abstract

No content.

Description

A-40104 Preparation of Antibiotic

Figure 1: Infrared Absorption Spectrum of A-40104 Factor A

Figure 2: Infrared Absorption Spectrum of B-40104 Factor B

The present invention relates to a method for producing an A-40104 antibiotic complex composed of activators A, B, C, and D by deep aerobic fermentation of Clitopilus pseudo-pinsitus. Individual factors A, B and C have already been isolated, and A-40104 factor C is known as the antibiotic fluromityline. A-40104 Factors A and B are novel antibiotics associated with fluoromethillin, A-40104 Factor A is a major novel factor, and is the D-xylose acetal derivative of Pluromityrin. A-40104 Factors A and B, 19, 20-dihydro derivatives thereof and family of factor A and B (C ₂ -C ₆ ) alkane oil derivatives, family of 19, 20-dihydro derivatives of factor A and B ( C ₂ -C ₆ ) Alkin oil derivatives are all effective against Gram-negative and Gram-positive bacteria, anaerobic bacteria and Mycoplasma. The antibiotic fluromityline was isolated in 1951 by Kavanagh et al. [Proc. Natl. Acad. Soc. 37, 570-574 (1951), as later known, the structural formula is as follows.

Alkali hydrolysis of fluoromerthline yields a compound known as merthylene of the following structural formula.

Many kinds of fluoromerthline derivatives have been prepared: Swiss Federal Patent No. 572,894 (Derwent No. 26553 X) Dutch Kingdom Patent No. 69,11083 (Derwent No, 40,642); Knauseder et al., US Pat. No. 3,716,579; Egger et al., US Pat. No. 3,919,290; Brandl et al., US Pat. No. 3,949,079; Riedl, US Patent No. 3,979,423; Baughn et al., US Pat. No. 3,987,194; Egger et al., US Pat. No. 4,032,530; K. Riedl, '' Studies on Pleuromutilin and Some of Its Derivatives, '' J. Antibiotics 29, 132-139 (1976); H. Egger and H. Reinshagen, "New Pleuromutilin) Derivatives With Enhanced Antimicr-obial Activity. I. Synthesis, '' J. Antibiotics 29, 915-922 (1976) and" II. Structure-Activity Correlations, '' Ibid., 923-927 (1976); F. Knauseder and E. Brandl, "Pleuromutilins: Fermentation, Structure and Bio-Synthesis, '' J. Antibiotics 29, 125-131 (1976); J. Drews et a1.," Antimcrobial Activities of 81. 723 hfu, a New Pleuromutilin Derivative, '' Antimicrob. Agents and Chemotherapy 7, 507-516 (1975).

In the present invention, two novel antibiotics of the fluoromertiline series have been found, but a new method of preparing the fluoromertiline has also been found.

The present invention provides novel preparations of the novel antibiotics A-40104 Factors A, B and D and the antibiotics A-40104 Factors A, B, C and D. These factors have useful antibiotic spectra that include gram-negative, gram-positive and anaerobic bacteria as well as mycoplasma microorganisms. These substances have been shown to be effective in the treatment of dysentery in pigs caused by mycoplasma disease, such as livestock, pigs, and cattle, and treponema hyoidogeneria.

The present invention,

a) The mutant species producing Crytophilus pseudo-pincitus NRRL 11179 or A-40104 are produced in the presence of full-scale antibiotics under deep aerobic fermentation conditions in culture medium containing carbohydrates, anabolic sources of nitrogen and inorganic salts. Incubate until

b) optionally, separating the A-40104 antibiotic mixture from the medium;

c) optionally, separating antibiotic A-40104 factor A, antibiotic A-40104 factor B, fluomerthiline, or antibiotic A-40104 factor D from this antibiotic mixture;

d) optionally, hydrogenating antibiotic A-40104 factor A or antibiotic A-40104 factor B to obtain 19, 20 dihydro A-40104 factor A or 19, 20-dihydro A-40104 factor B;

e) optionally acylating antibiotic A-40104 factor A, 19, 20-dihydro A-40104 factor A, antibiotic A-40104 factor B or 19, 20-dihydro A-40104 factor B 40104 Factor A or 19, 20-DihydroA-40 104 Tetra- (C ₂ -C ₆ ) -alkanoyl derivative of Factor A or A-40104 Factor B or 19, 20-dihydro. A-40104 to prepare a tri- (C ₂ -C ₆ ) -alkanoyl derivative of Factor B,

Antibiotic factors A, B, D, and fluomerthyline; Tetra- (C ₂ -C ₆ )-of antibiotic A-40104-factor A, 19, 20-dihydro A-40104 factor A, and A-40104 factor A and 19, 20-dihydro A-40104 factor A. Alkanoil derivatives; Tri- (C ₂ -C ₆ ) -alkanyls of the antibiotics A-40104 factor B, 19, 20-dihydro A-40104 factor B, and A-40104 factor B and 19, 20 dihydro A-40104 factor B Derivatives; fluoromertiline; And it provides a method for producing A-40104 antibiotic mixture consisting of antibiotic A-40104 factor D.

The present invention also provides Crytopyrus Pseudo-Pincitus NRRL 11179 microorganisms. The infrared absorption spectrum (KBr disk method) of factors A-40104 is shown in the accompanying drawings:

Figure 1 A-40104 Factor A Figure 2 A-40104 Factor B

A-40104 Factors A and B are similar in structure to each other, and are similar to the structure of A-40104 Factor C, an antibiotic known as fluoromerthline. At least four antibiotic factors are produced together during the fermentation process according to the present invention. Fluorothymline and A-40104 factor A can be separated from the fermented broth filtered by a suitable extraction process, and the microfactor A-40104. Factor B can be separated by chromatography, and another trace factor, A-40104, can be separated by chromatography, but has not yet separated enough to be analyzed.

The following relates to the physical and spectral properties of A-40104 Factors A and B.

A-40104 Factor A

A-40104 Factor A- is crystallized from chloroform, its melting point is about 117 to 120 ° C, the elemental analysis shows that the empirical formula is C ₂₇ H ₄₂ O ₉ and the molecular weight is 510 as measured by long-desorption mass spectrometry.

In trifluoroethanol, the ultraviolet absorption spectrum of A-40104 Factor A is measured and shows the maximum absorption at 284 nm (ε32). The maximum polarization dichroism spectrum of A-40104 in trifluoroethanol was found at 200 nm (Δε = + 2.84) and 295 nm (Δ = + 1.72). α] = -14 ° (C1.0, C ₂ H ₅ OH).

When A-40104 Factor A was electrolyzed in 66% aqueous dimethylformamide, no suitable group existed. Infrared absorption spectra were measured by potassium bromide disk method, and the results were as shown in Fig. 1. . Absorption maxima were shown at the following frequencies (m ^-1 ):

3590, 3500, 3450 (broad), 3280 (broad), 2990, 2980, 2960, 2940, 2920, 2890, 2860, 1755, 1735, 1645, 1465, 1447, 1420, 1380, 1330, 1310, 1274, 1240, 1220, 1160, 1117, 1093, 1070, 1060, 1040, 1014, 984, 955, 940, 921, 905, 865, 760, 742, 698, 674, 603, 530 and 442.

A-40104 Factor A is soluble in alcohols such as methanol and ethanol, partially soluble in chloroform and insoluble in water. In view of the physicochemical properties of this compound, factor A-40104 is considered to be a β-D-xylopyranosyl acetal derivative of fluromerthyl having the structure

Factor A-40104 is a white, amorphous compound that is present in trace amounts in the A-40104 antibiotic complex. It is present only 0.01 to 0.1% of the A-40104 antibiotic complex activity. According to the electron-impact mass spectrometry, the molecular weight of factor A-40104 B is 394. Combining the peaks of molecular ions and other fragment ions, it can be seen that the empirical formula of A-40104 Factor B is C ₂₂ H ₃₄ O ₆ . Mass spectrometry of factor B shows that the peak at m / e 318 (C ₂₀ H ₃₀ O ₃ ) is similar to the peak at m / e 303 at the time of spectrometry of fluomertiline, thereby adding an additional presence in factor B. It can be seen that oxygen is not present in the side chain but in the nucleus. M / e 163, on the other hand, is common to both factor B and fluomerthillin. The small peak in m / e245 (C ₁₇ H ₂₅ O), shown in the spectrum of fluoromerlin, shifted to m / e261 (C ₁₇ H ₂₅ O ₂ ) in the spectrum of factor B. Factor B has three hydroxyl groups capable of acylating. For example, treating A-40104 factor B with pyridine and acetic anhydride results in the formation of a triacetyl derivative to give a compound having a composition of C ₂₈ H ₄₀ O ₉ (molecular weight 520), thereby adding the addition present in A-40104 factor B It can be seen that oxygen is present in the hydroxyl group.

The infrared absorption spectrum of the potassium bromide disk method of factor A-40104 is shown in Fig. 2, and the main absorption maximum peak appears at the following frequency (cm ⁻¹ ):

3430 (Broad), 2940, 2880, 1735, 1660, 1452, 1385, 1375, 1305, 1280, 1233, 1152, 1094, 1020, 997

967, 933, 912, 888, 752 and 660.

From the physical and chemical properties of this compound, A-40104 factor B is considered to have the following structural formula.

As is well known, both A-40104 factors A and B contain vinyl groups, which are reduced by standard methods to give the corresponding 19, 20-dihydro derivatives. The 19, 20-dihydro-derivatives of factors A-40104 A and B are also effective as antimicrobial agents and are part of the present invention.

A-40104 Factors A and B and their 19,20-dihydro derivatives have hydroxyl groups that can be acylated by standard processes (Factor A compounds have four hydroxyl groups and factor B compounds Has three hydroxyl groups). It can be seen that the hyperacylated derivatives obtained have identical acyl groups at individual positions (ie, four positions of the Factor A compound and three positions of the Factor B compound) in which acylation can occur. Such hyperacylated derivatives are also effective as antibacterial agents, among which the (C ₂ -C ₆ ) -alkanoyl derivatives of A-40104 Factors A and B and their 19,20-dihydro derivatives are preferred compounds. .

Four individual factors of the A-40104 complex can be separated and identified by chromatography. These factors can be separated by thin layer chromatography, for example using silica gel (Merck Darmstadt). In the solvent system, chloroform: methanol (= 9: 1) and micrococcus luteus were used as an indicator.

Approximate Rf values for Factors A-40104 are shown in Table I.

TABLE I

These factors can also be separated by paper chromatography using untreated paper (whatman No. 1), water saturated with butanol as solvent system, and Micrococcus luteus as the detecting microorganism. Approximate Rf values for A-40104 A to D in the solvent system are as shown in Table II.

TABLE II

A-40104 Preparation of Antibiotic Complex

The present invention relates to a method for preparing A-40104 antibiotic complex, from which A-40104 Factors A and B and known antibiotics fluromitylin are prepared, using a suitable medium under deep aerobic conditions. Until the material is produced, a method of culturing Crytophilus pseudo-pincitus strain of basidiomycete producing A-40104. This antibiotic complex and individual antibiotics can be recovered by various separation and purification methods commonly used in the art.

Crytophilus pseudo-pincitus strains useful for producing A-40104 antibiotics were obtained from Centra-albureau voor Schimmelcultures (CBS) in the Netherlands, and CBS cultures (CBS 270.36) were obtained from Octojuga pseudo-pinsita. Are classified as). The genus Crytophilus is synonymous with Octojuga and is used in the tinnitus to refer to microorganisms. It was not known that this culture was capable of producing antibiotics, but it was found that the present invention has the ability to produce A-40104 antibiotics by extensive screening.

A-40104 Crytophilus pseudo-pincitus, a strain useful for producing Factors A and B, and Pluromitylin, is already a collection of storage cultures (Northern Marketing and Nutrition Research Division, US Department of Agriculture, Agricultural Research Service, Peoria, Illinois 61604). And NRRL 11179 is available as accession number.

As with other microorganisms, it is possible to vary the properties of Crytophilus pseudo-pincitus NRRL11179, a culture producing A-40104. For example, artificial strains and mutants of strain NRRL 11179 can be obtained by treatment with mutagens such as UV, X-ray, radio frequency, radiation, and chemicals, and have the same properties as Crytophilus pseudo-pincitus NRRL 11179. All natural and artificial variants and mutants with A-40104 antibiotics can be used in the present invention.

The medium used for culturing Crytophilus pseudo-pincitus NRRL 11179 may be any of many kinds of media, but several mediums are preferable in consideration of economy, optimum yield and ease of separation of the product. Thus, for example, glucose, tapioca dextrin, starch, corn oil are preferred as carbohydrate sources used in large-scale fermentation methods, and glycerol, maltose, fructose and the like are also used as carbohydrate sources. Olate, laurate and lecithin are also used as other useful carbon sources. Preferred nitrogen sources are soybean mill, soybean flour and dry-dog food-yeast mixture. Cottonseeds, peanuts, meat peptones, and dry dog food can also be used as nitrogen sources. In the production and cultivation of antibiotics, nourishing inorganic salt is not an essential condition, but it is preferable to add soluble salts capable of producing sodium, magnesium, calcium, ammonium, chlorine ions, carbonate ions, sulfate ions and the like. For example, addition of 0.05% MgSO ₄ · 7H ₂ O and 0.2% CaCO ₃ in the medium promotes the production of antibiotics.

Essential trace elements essential for the growth and development of microorganisms must be included in the medium, and these trace elements are usually present in the medium to meet the growth requirements of the microbial fire as impurities in the other components of the medium.

When foaming causes a problem, a small amount of antifoaming agent, such as polypropylene glycol, needs to be added (eg 0.2 ml / l) in large-scale fermentation broth.

In order to produce the actual mass of A-40104 antibiotics, deep aerobic fermentation is preferred in tanks. A small amount of A-40104 antibiotics can be obtained by shaking flask culture. Since inoculation of a large tank with a small amount of culture delays the production of antibiotics, it is preferable to use an inoculum containing a large amount of cells in the actively growing stage. A small amount of medium is inoculated with mycelial fragments of the microorganisms to prepare a culture hybrid to obtain a new, vigorously growing microbial culture. Transfer the inoculum to a larger tank. The medium used for the culture of the inoculum uses the same medium as that used in the large scale fermentation method, and other mediums may be used.

Crytophilus pseudo-pincitus producing A-40104 can grow at temperatures between 20 ° C. and 33 ° C. and the optimum temperature for A-40104 production is about 30 ° C.

As in conventional deep aerobic fermentation, sterile air is blown into the shaken medium. In order to grow microorganisms effectively, they must be supplied through a sufficient amount of air and shaken so that the concentration of dissolved oxygen is as close as possible to 80%. Lower concentrations of dissolved oxygen inhibit production of A-40104 Factors A and C, particularly with a greater effect on Factor A.

In fermentation, A-40104 factor C is produced first, and then A-40104 factor A is produced. The preferred incubation time for the production of A-40104 Factor A is about 12 days, and the addition of corn oil in the medium during fermentation facilitates the conversion of Factor C to Factor A. A-40104 Under conditions most optimal for the production of factor A, 70 to 90% of the antibiotics produced is factor A.

After producing A-40104 antibiotics under deep aerobic fermentation conditions, they can be recovered from the fermentation medium by methods used in the fermentation field, and the antibiotic activity produced during fermentation is mainly present in the broth. Therefore, A-40104 antibiotics can be recovered to the maximum by a combination of filtration, extraction of filtered broth and adsorption chromatography. A particularly preferred method is to extract the filtered broth first with toluene and then with chloroform. A-40104 factor C is isolated from toluene extract and A-40104 factor A is isolated from chloroform extract. Trace factor A-40104 Factor B is separated by absorption chromatography during purification of Factor A-40104 A.

Alternatively, the cultured solid material including the medium component and the mycelium may be used as a raw material of A-40104 antibiotic after removing water, preferably, without undergoing extraction or separation. For example, after A-40104 antibiotic activity is produced, the culture medium is lyophilized and directly mixed with the feed premix. Another method is to produce A-40104 activity in the culture medium, isolate the mycelium and freeze-dried the filtered broth. A-40104 The antibiotic complex is obtained in a form that can be used directly in feed premixes.

While producing antibiotics and isolating and isolating individual antibiotic factors, it is desirable to monitor the production and separation processes using chromatographic processes. For example, a CHCl ₃ : CH ₃ OH (9: 1) solvent system, a silica gel adsorbent, and a TLC system using Micrococcus Luteus as biomarkers are preferred.

A-40104 Antibiotic Activity

A-40104 antibiotic complexes and individual A-40104 factors A and B inhibit the growth of pathogens, in particular Gram-positive bacteria. In particular, A-40104 factor A is effective as an antimicrobial agent, and the minimum inhibitory concentration (MlC'S) of the test bacteria of A-40104 factor A measured by the standard agar dilution method is shown in Table III.

TABLE III

Of particular note in the above activity is that A-40104 antibiotic inhibits the growth of bacteria resistant to other antibiotics. Table III shows the activity by standard disk-plate-test of A-40104 factors A and B against representative pathogens. Activity is measured by observing the inhibitor and its diameter (mm), in which case the diameter of the disk used is 6.35 mm.

Table IV

1 Penicillin-G-sensitive bacteria 2 Penicillin-G-resistant bacteria 3 Methicillin-resistant bacteria 4 groups A 5 groups D

Table V shows the minimum inhibitory concentration of A-40104 factor A compared to ampicillin in an in vitro experiment using plate agar-gradient dilution.

TABLE V

A-40104 Factor A and 19, 20-Dihydro A-40104 Factor A (Dihydro-A-40104A) show antimicrobial effects on experimentally induced bacterial infections in vivo. The effects observed when two doses of these compounds were administered to experimentally infected mice were measured at ED ₅₀ values. [ED ₅₀ -mg / kg effective amount to protect 50% of experimental animals; See warren wick, et al., J. Bacteriol, 81, 233-235 (1961). The ED ₅₀ values of these compounds observed are given in Tables IV and VIII.

Table VI

Activity of Factor A

[Table VII]

Activity of Dihydro-A-40104 A

A-40104 antibiotics also inhibit the growth of many basic bacteria. Table VII shows the activity of A-40104 Factor A measured by standard agar-dilution method.

TABLE III

* MlC is measured by agar dilution method.

The endpoint is determined 24 hours after incubation.

A-40104 The special advantage of antibiotics is that they are relatively nontoxic. For example, LD ₅₀ when intraperitoneally injected mice with tetraacetyl derivatives of A-40104 factor A and A-40104 factor A exceeds 300 mg / kg.

The activity against mycoplasma is very important in view of the antimicrobial effect of A-40104 antibiotic.

Mycoplasma is a pathogen that causes disease in humans and animals, and an effective therapeutic agent capable of combating mycoplasma is essential for the prevention and treatment of mycoplasma diseases in poultry, pigs and cattle.

Table IX shows A-40104 Factor A measured by in vitro gravy-dilution method; 19, 20-dihydro-A-40 104 Factor A; The minimum inhibitory concentration (MIC'S) for mycoplasma of the tetraacetyl derivative of A-40104 A is shown.

TABLE IX

An important point of the present invention is that A-40104 antibiotic is used to treat dysentery in pigs. As described by W. E. Brown et al., Fluromertiline is effective for treating dysentery in swine (US Pat. No. 4,041,75).

The A-40104 antibiotic according to the present invention was found to be effective against treponema hyodigeneria, the microorganism most closely associated with dysentery in pigs. This activity was determined through in vitro experiments by incorporating the compound at concentrations of 50, 5.0 and 0.05 mcg / m1 in trypticase soybean agar plates containing bovine 5% defibrinated blood. Tie the agar surface. Inoculated with 0.1 ml of a 10-fold dilution suspension in high-diseanteria and incubated for 4 days under anaerobic conditions, it is determined whether hemolytic treponema is grown.

In this experiment, A-40104 factor A and 19, 20-dihydro A-40104 factor A were found to be thyroid at 50, 5.0 and 0.5 mcg / ml (agar). We suppressed growth to high Daigen teria.

When used for the treatment of dysentery in pigs, A-40104 is administered orally to infected pigs in the form of antibiotic tablets, capsules and powders. A veterinary composition comprising an A-40104 antibiotic as an inactive ingredient and an active ingredient may be prepared.

However, A-40104 antibiotic factor A is preferably administered in combination with pig feed.

Another advantage as the veterinary medicament of the present invention is the activity of A-40104 antibiotic against Pasteurella species. For example, blood. Multocida is the causative agent of respiratory infections in cattle, poultry and pigs. Haemolytica is a major cause of bovine respiratory disease. Table VII shows the activity of A-40104 Factor A and its dihydro derivatives against Pastella, as determined by standard in vitro experiments.

TABLE X

A pharmaceutical composition containing the antibiotic of A-40104 as an inactive ingredient and an active ingredient can be administered to humans to have an effect on pathogenic bacteria that are susceptible.

Another important aspect of the present invention is that the A-40104 antibiotic is active against spiroplasma.

Spiroplasma citrus causes citrus stubborn disease; Another spiroplasm, corn dwarf spiroplasma, inhibits corn growth. In vitro experiments on spiroplasma citrine, A-40104 factor A and its dehydroderivatives inhibit the growth of spiroplasma citries even at low concentrations of 0.01 ppm.

A-40104 antibiotics are also effective against phytopathogens. For example, spraying A-40104 factor A on leaves or drenching the soil can control powdery mildew.

Experiments with soybeans and barley have shown that A-40104 Factor A can be treated with 100ppm concentration in soil to combat powdery mildew. Penetrating effect of these antibiotics is particularly effective in inhibiting phytopathogens or spiroplasma.

The following examples illustrate the invention in more detail.

Example 1

A. Shake Flask Fermentation Method of A-40104

The aged slope agar culture of Crytophilus pseudo-pincitus NRRL11179 is scraped with a sterile tool and aseptically transferred to the slope agar of the following composition.

Add 1 liter of deionized water.

N-Z Case, Humko Sheffield, Norwich, N. Y.

The inoculated slope agar medium is incubated at about 30 ° C for about 14 days. Mature tetracultures are scraped with a sterile tool and inoculated into 50 ml of culture medium of the following composition using mycelium from about 5 cm ² of the agar surface.

Add 1 liter of tap water.

The pH of the medium is about 5.3 and the pH is adjusted to 6.9 with 5N NaOH.

Stadex 11, A.E. Staley, Decatur I11.

** HySoy T, Humko Sheffield, Norwich, N. Y.

*** Amber BYE300, Amber Laboratories, Juneau, Wisc.

The inoculated culture medium was incubated for 96 hours at 25 ° C. on a shaker rotating at 250 RPM in a 250 ml Erlenmeyer flask.

Such inoculation culture medium may be used directly to inoculate the two-stage culture medium, and preferably the culture may be stored in the vapor of liquid nitrogen for later use. For such storage purposes, the culture is prepared in sovial form as follows. Each vial is filled with 4 ml of inoculation culture medium and 2 ml of glycerol-lactose solution having the following composition.

Add 1 liter of deionized water.

The prepared suspension is stored in liquid nitrogen vapor.

Using the prepared storage suspension (1ml) inoculate 50ml of the first stage culture medium having the same composition as the culture medium.

The inoculated one-stage culture medium is incubated at 250 ° C for about 4 days on a shaker rotating at 250 RPM in a 250-mm wide-angle Erlenmeyer flask.

B. Tank Fermentation

In order to obtain a large amount of inoculum, 400 ml of the two-stage culture medium having the same composition as the above culture medium was inoculated into 10 ml of the one-stage culture medium incubated above, and the two-stage medium was 25 ° C in a 2-liter Erlenmeyer flask. Incubate for approximately 48 hours (on a shaker rotating at 250 RPM with an arc of 2 inches in diameter).

Inoculate 100 liters of sterile production medium having the following composition using a two-stage culture medium (800 ml) prepared as described above.

Deionized water is added to make 1 liter.

Composition is adjusted to pH 5.0 with 4N HCl.

* Purina Dog Chow, Ralston Purina Co., St. Louis, MO. 63188

The inoculated production medium is fermented for 12 days at a temperature of about 30 ° C. in a 165 L fermentation tank, with sterilizing air supplied through the fermentation broth while maintaining the concentration of dissolved oxygen at about 80%.

Example 2

Fermentation is carried out in the same manner as in Example 1 using a culture medium having the following composition.

Add 1 liter of deionized water.

The pH of the medium is about 5.7, adjusted to pH 5.0 with 4N HC1.

* purina Dog Chow

Example 3

Fermentation medium having the following composition is used to ferment in the same manner as in Example 1.

Add 1 liter of deionized water.

The pH of the medium is about 7.4 at which time it is fixed at pH 6.5 using 4N HCl.

Example 4

A-40104Isolation of Factors A and C

The fermented broth (1200 gallons) obtained by the method in Example 3 was filtered using a filter (Hyflo Super-cel, diatomaceous earth, John-Manville Products Corp.) to recover 3640 L of the filtrate, and then the mycelia cake was deionized with water. Washing further recovers 1820 L of filtrate. This filtrate was combined (5460 L) and extracted with toluene (1/2 volume) for 1 hour at juicy pH to give 2500 L toluene extract. This toluene extract mainly contains A-40104 factor C (fluromitylin). The toluene extract was concentrated to about 23 L, and the obtained primary product (1943 g) of A-40104 Factor C was crystallized and then recovered by filtration. The mother liquor was concentrated to about 8 liters and then the second yield (282 g) of Factor C was isolated. The mother liquor is re-concentrated while cooling over 72 hours at a temperature of about 4 ° C. to about 6 liters, whereby factor C is recovered again. Concentration of the mother liquor of this tertiary yield yields a gum oil which is dissolved in diethyl ether (6 L) and cooled at 4 ° C. for 72 hours to give a quaternary yield of factor C (112 g).

After extraction with toluene, the remaining aqueous solution is extracted with chloroform (1/2 volume) for 1 hour, and then the chloroform extract is concentrated to about 10 L and cooled at 4 ° C. for 72 hours. When the crystals precipitate, they are separated by filtration. When dried, 588 g of A-40104 Factor A is obtained.

Example 5

A-40104 Isolation of Complexes and Individual Factor B

The whole fermented broth (214 L) obtained according to the method of Example 1 was filtered through a filter to obtain 119 L filtrate. Mycelium cake was washed with water to recover 80 l more of the filtrate and combined with the above filtrate (199 l) and extracted twice with ethyl acetate (80 l each time) at juicy pH, and the combined ethyl acetate extracts were concentrated under reduced pressure to about 2 l. Hexane (about 20 L) is added and the active complex drops to precipitation. This precipitate is isolated by filtration, redissolved in ethyl acetate (about 300 ml) and reprecipitated with hexane. The precipitate was washed with hexane and dried to give 27.3 g of A-40104 antibiotic complex (containing factors A, B and C).

A portion (5 g) of the complex obtained above is dissolved in chloroform (40 ml), the filtrate is filtered and the filtrate is chromatographed on an open glass column (5.6 x 28 cm) on silica gel (woelm). The column is slurried by filling with chloroform and 20 ml fractions are collected while eluting with chloroform at a rate of 2 ml / min. In fraction 7 the elution solvent is changed to CHCl ₃ : CH ₃ OH (95: 5) and in fraction 28 the elution solvent is changed back to CHC1 ₃ : CH ₃ OH (9: 1). Fractions 72 to 120 are collected and evaporated under vacuum to yield anhydrous powder (4 g).

Another fermentation broth is treated in the same way to further yield 6 g of the material. The obtained sample is collected (10 g), and chloroform (100 ml) is added, and an insoluble substance (1.6 g of A-40104 factor A) is separated by filtration in chloroform. The filtrate is chromatographed on an open glass column (5.8 x 48 cm) on silica gel (woelm). Fill the column with chloroform, wash and collect 20 ml fractions, eluting with CHCl ₃ : CH ₃ OH (97.5: 2.5) at 4 ml / min. Collect fractions 156-190 and evaporate to dryness under reduced pressure. The residue is dissolved in a small amount of chloroform and hexane is added to this solution to form a precipitate. The precipitate was isolated by filtration and dried to yield 99-mg of A-40104 Factor B.

Example 6

19,20-Dihydro-A-40104 Preparation of Factor A

Antibiotic A-40104 Factor A (10.15 g, 0.0199 mole) is dissolved in tetrahydrofuran (188 ml) and 5% pd / c (2.5 g) is added. The reaction mixture is hydrogenated at room temperature for 4 hours and filtered through a sinter-glass funnel with a celite layer. The filtrate was evaporated to dryness in vacuo and the residue was crystallized from ethyl acetate: methanol (2: 1) to afford 7.0447 g (0.01375 mole, 69% yield) of 19, 20-dihydro A-40104 Factor A.

Example 7

19,20-Dihydro-A-40104 Preparation of Factor B

Hydrogenation of Factor A-40104 B under the same conditions as in Example 6 yielded 19, 20-dihydro-A-40104 Factor B.

Example 8

Preparation of Tetraacetyl A-40104 Factor A

-22.2°(C1.0, C₂H₅OH): 실험식 C₃₅H₅₀O₁₃질량분광분석법으로 측정한 분자량은 678.325이다.Factor A (2 g) is dissolved in anhydrous pyridine (5 ml) and acetic acid anhydride (5 ml) is added. Once a clear solution is obtained, it is left at room temperature for 120 hours and added in ethanol (20 ml). The solution is evaporated under vacuum until traces of pyridine and acetic acid are removed to give 2.1 g of tetraacetyl derivative of A-40104 Factor A:

-22.2 ° (C1.0, C ₂ H ₅ OH): The molecular weight measured by Empirical Formula C ₃₅ H ₅₀ O ₁₃ Mass Spectrometry is 678.325.

Elemental Analysis for C ₃₅ H ₅₀ O ₁₃

Theoretic value: C 62.00; H 7.40; O 30.60

Found: C 62.06; H 7.32; O 30.19

[Examples 9 to 14]

Triacetyl derivatives thereof are prepared from A-40104 Factor B in a similar manner as in Example 8.

19, 20-dihydro-A-40104 Factor A was reacted according to the method of Example 8 to prepare a detraacetyl derivative of 19, 20-dihydro-A40104 Factor A.

A triacetyl derivative of 19, 20-dihydro-A-40104 Factor B was prepared by reacting 19, 20-dihydro-A-40104 Factor B according to the method of Example 8.

A-40104 Factor A is reacted with n-butyric anhydride according to the method of Example 8 to prepare a tetra (n-butyryl) derivative of A-40104 Factor A.

A-40104 Factor B is reacted with propionic anhydride according to the method of Example 8 to prepare a tripropionyl derivative of A-40104 Factor-B.

19, 20-dihydro-A-40104 Factor A was reacted with valeric anhydride according to the method of Example 8 to detra (n-valeryl) derivative of 19, 20-dihydro-A-40104-Factor A To prepare.

Claims (1)

Cultivation of antibiotic A-40104 producing bacteria belonging to the species Citopi1us pseudo pinsitus was cultured in a culture medium until deep production of antibiotic A-40104 factor A under deep aerobic fermentation conditions. Method for producing antibiotic A-40104 Factor A, characterized in that.

Images