CS207457B2 - Method of preparation of the mixture of the antibioticum a-35512 - Google Patents

Abstract

The antibiotic mixture A-35512 containing the factors A, B, C, E, F, G and H, antibiotic A-35512 factor A, antibiotic A-35512 factor B, antibiotic A-35512 factor C, antibiotic A-35512 factor E, antibiotic A-35512 factor F, antibiotic A-35512 factor G and antibiotic A-35512 factor H and also antibiotic A-35512 factor B aglycone are prepared. This antibiotic mixture and these factors are obtained by submerse culture under aerobic fermentation conditions of the microorganism Streptomyces candidus NRRL 8156 in a nutrient medium which contains assimilable sources of carbohydrates, nitrogen and inorganic salts until a substantial amount of the antibiotic is formed. The antibiotics obtained can be used as antibacterial agents and as growth-promoting agents for increasing the feed utilisation in ruminants and poultry.

Description

(54) A method of preparing a mixture of antibiotics A-35512

A-35512 antibiotic mixture, including microbially active related factors A, В, С, E, F, G, and H, is prepared by submerged aerobic fermentation of the novel Streptomyces candidus NRRL 8156. Individual A-35512 factors are separated and isolated by chromatography. By moderate acid hydrolysis of A-35512 Factor В, several sugars are cleaved to give the A-35512 Factor B aglycone. In addition, A-35512 factor V is useful in the treatment of periodontitis and pimples.

Antibiotics A 35512 are closely related glycopeptide antibiotics. The antibiotic A-35512 factor в, the most fully characterized member of the A-35512 antibiotic complex, appears to be a new member of the antibiotic-containing peptides family, including vancomycin (US Patent No. 3,067,099), A-4696 А, В and C (US Patent No. 3,952,095), avoparcin (U.S. Patent No. 3,855,410), ristomycline A (Lomakina, N., 7th International Symposium of Chemistry of National Products, Riga, Latvia, p. 625, 1970), and ristocetin A (U.S. Pat. No. 2,990,329). Antibiotics A-35512 differ from known antibiotics in, for example, different mobility in different chromatographic systems and in the content of amino acids and sugars.

Although many antibiotic active substances are known today, the demand for new improved antibiotics is still ongoing. One problem in current antibiotic therapy is that antibiotics differ in efficacy against different pathogenic organisms. Another problem lies in the fact that the organism of strains is still developing resistance to currently used antibiotics. Yet another problem arises from the fact that individual patients often have different responses to specific antibiotics due to hypersensitivity and / or toxic effects. In view of this problem in current therapy, one feature of the invention is that novel antibiotics are prepared that are useful against diseases caused by microorganisms.

Another feature of the invention is to increase food utilization in ruminants and poultry. As demand for food is increasing, increasing usability is of great importance. Diethylstilbestrol and other estrogens increase the usefulness of food in poultry and animals. There is all the danger that these ingredients may be consumed in meat. Thus, there is a great economic need for new pathways to exploit the efficacy of limited food ingredients post-207437

A-35512 factor A used in the production of ruminant and poultry meat. The mixture of A-35512 antibiotics, their factors and their derivatives are beneficial in this regard.

SUMMARY OF THE INVENTION The present invention provides a process for preparing a mixture of antibiotics A-35512 consisting of factors A, В, С, E, F, G and H, and dividing it into individual factors and subsequently preparing factor B aglycone, characterized in that Streptomyces is cultured. candidus NRRL 8156 in a nutrient medium containing assimilable sources of carbohydrates, nitrogen, and inorganic salts, at a temperature of between 20 and 40 ° C under submerged aerobic fermentation conditions, optionally isolating A-35512 antibiotic mixture from A- 35512 antibiotic mixture 35512 Antibiotic A-35512 factors A, В, С, E, F, G and H are isolated and optionally A-35512 Factor B aglycone is prepared from the A-35512 Factor В antibiotic.

The number and ratio of individual factors occurring simultaneously in this antibiotic mixture varies and depends on the fermentation conditions used. The antibiotic compounds prepared according to the invention are referred to herein as A-35512 antibiotics. Factor В is the main factor in this A-35512 mixture. The A-35512 factors present in the A-35512 mixture are isolated as individual components from the mixture as hydrochlorides. Each individual factor can be converted to the free base (free of chlorine ions), for example, by chromatography on a weakly basic ion exchange resin. Other pharmaceutically acceptable salts of A-35512 factors are also useful as the free base and the corresponding hydrochloride. In the utility discussions, the term "A-35512 compound" is used for the sake of simplicity to refer to compounds selected from the group consisting of A-35512 factors A, В, С, E and H and pharmaceutically acceptable salts thereof.

Infrared absorption spectra (in KBr tablets) of the following A-35512 factors are shown in the attached figures.

Giant. Fig. 1 - Factor A dihydrochloride A-35512. Fig. 2 - Factor B dihydrochloride A-35512. Factor H 3-hydrochloride A-35512

Giant. Fig. 4 - Factor C dihydrochloride A-35512. Factor E 5-hydrochloride A-35512

The infrared absorption spectrum of aglycone A-35512 Factor В (in nujol) is shown in Figure 6.

The A-35512 factors of the invention are closely related compounds.

Seven antibiotic factors were isolated in fermentation as a mixture of A-35512 antibiotics. The individual factors are separated from each other and the factors A, V, С, E and H are isolated as individual compounds as described below. The A-35512 mixture is water soluble, especially soluble in alcohols such as methanol and ethanol, but is insoluble in other organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetone, trieth and dioxane.

A-35512 factor A is a white, amorphous basic compound. A-35512 factor A has the following percentage elemental composition:

carbon 54.29%, hydrogen 5.19%, nitrogen 5.58%, oxygen 33.76%, chlorine 1.69%.

Factor A dihydrochloride A-35512 is a white, amorphous, hygroscopic compound. Factor A dihydrochloride A-35512 has the following average percentage composition:

carbon 51.03 % hydrogen 5.10 0/0, nitrogen 4.75 % oxygen 34.20 % chlorine 4.80 %.

The infrared absorption spectrum of dihydro-, chloride A-35512 factor A in KBr tablets is shown in Figure 1. The most important absorption maxima have the following frequencies (cm ^-1 ): 3405 (strong), 3300 (inflex), 2950 (weak), 1750 (weak), 1670 (strong), 1625 (inflex), 1602 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1405 (weak), 1345 (inflex), 1312 (medium), 1225 (medium), 1180 (weak), 1130 (weak), 1080 (strong), and 1020 (inflex).

The ultraviolet (UV) absorption spectra of factor A dihydrochloride A-35512 show an absorption maximum at 282 nm (ε 11700) in acidic and neutral methanol and an absorption maximum at 292 nm in basic methanol. (ε 14,000), calculated using a molecular weight of 2000. The ultraviolet spectra of Factor A dihydrochloride A-35512 also indicate terminal absorption at 225 nm.

13c the nuclear magnetic resonance spectrum of factor A dihydrochloride A-35512 in D2O has the following characteristics: f

Whose. PPM Height (%) 3 175.3 0.8 4 173.0 2.1 5 172.1 2,0 6 171.4 1.5 7 170.9 2.7 8 170.5 . 2.3 9 169.5 1.6 10 159.0 1.3 11 157.9 2.3 12 156.2 2.6 13 155.6 2.3 14 155.3 2.4 15 Dec 154.4 1.1 16 136.3 1.4 17 136.0 1.0 18 135.1 1.4 19 Dec 133.5 1,2 20 May 129.6 1.6

20'7 457 s

0. PPM Height (%) 21 129.1 1.7 22nd 128.7 1,8 23 127.5 1.0 24 126.0 1.4 25 124.3 2.8 26 122.1 1.6 27 Mar: 109.9 1.3 28 107.4 1.6 29 101.7 0.9 30 77.6 3.8 31 76.3 4.6 32 75.5 2.6 33 74.8 2.5 34 74.5 2.4 35 73.4 3.7 36 72.8 6.0 37 72.0 4.4 38 70.9 7.0 39 69.6 2.8 40 67.4 90,9 * 41 65.4 1.7 42 61.7 3.1 43 56.7 1.7 44 55.5 1.3 45 54.7 0.8 46 24.6 0.9 47 19.1 1.7 48 17.9 2,0 49 17.2 1.9 50 16.7 3.2 51 16.2 3.0

* dioxane standard

Dihydrochloride. A-35512 factor A has the following specific rotation:

fajo ²⁵ -100 (c 1, H2O),

Mm ²⁵ -400 (c 1, H 2 O).

Electrometric titration of Factor A dihydrochloride A-35512 in 66% aqueous dimethylformamide shows the presence of four titratable groups with pKa values of approximately 7.35, 9.09, 10.49 and 12.44 (initial pH 6.2).

The approximate molecular weight of factor A dihydrochloride A-35512 determined by titration is about 2106.

The A-35512 factor A hydrochloride is soluble in water, partially soluble in alcohols such as methanol and ethanol, but is generally insoluble in other organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetomiril and dioxane.

Factor A dihydrochloride A-35512 is stable for 7 hours in aqueous solutions with a pH of about 3 to 10.

Amino acid analysis of acid-hydrolysed ^{dihydrochloride} A-35512 factor A shows that A-35512 factor A comprises at least five amino acid residues of which one is glycine.

A-35512 factor B

A-35512 factor B is a white, amorphous basic compound. The approximate empirical formula for A-35512 factor B is

097-99 ^ () 1- ^ 51 ^ 8-904 (^ 48 ^.

A-35512 factor B has the following average percentage composition:

carbon 53.97%, hydrogen 4.75%, nitrogen 5, -25%, oxygen 34.29 ·%, · chlorine 1.59%.

This elemental composition is in accordance with the preferred empirical formula

C98H104N9O47Cl (calculated: C 53.60, H 4.75, N 5.74, O 34.30, Cl 1.61). An alternative preferred empirical formula is

C98H103N8O47Cl (calculated: C 54.00, H 4.75, N 5.15, O 34.50, Cl 1.60).

The ultraviolet absorption spectra of A-35512 factor B show an absorption maximum at 282 nm (ε 15,000) in acidic and neutral methanol and an absorption maximum at 292 nm in alkaline methanol. (ε 16,000), calculated using a molecular weight of 2000. The UV spectra of A-35512 factor B have a terminal absorption at 225 nm.

A-35512 factor B has the following specific rotations:

MD2 ⁵ -123 ° (c = 1, HZO) [Wj365 ²⁵ -446 ° (c = 1, HZO).

The electrometric titration of A-35512 factor B in 66% aqueous dimethylformamide indicates the presence of four titratable groups with pK a of 7.15, 8.81, 10.20, 12.00 and possibly the presence of another group with a pK a greater than 13 , 50.

The molecular weight of A-35512 factor B determined by titration is about 2143.

Factor B dihydrochloride A-35512 is a white crystalline compound (50% aqueous methanol). Although Factor B dihydrochloride A-35512 is a hygroscopic compound and does not exhibit a different melting point, the thermogram shows that weight loss begins at. 25 ° C and 7.4% losses are at 121 ° C, at 135 ° C further losses occur and lead to decomposition.

Factor B dihydrochloride A-35512 has the following elemental percentage composition:

carbon 52.57 O / ₀₎ hydrogen 4,80%, nitrogen 5.66%, oxygen 32,86%, chlorine 4.51%.

This elemental composition is particularly in accordance with another alternative empirical formula

C98H103N9O.17Cl. H, 4.65; N, 5.57; O, 33.20; Cl, 4.65.

The UV spectra of factor B dihydrochloride A-35512 show an absorption maximum at 282 nm (ε = 12,000 in acidic and neutral methanol) and an absorption maximum at 292 (ε = 14,000) in alkaline methanol, calculated using a molecular weight of 2000. Factor B 'dihydrochloride A-35512 also shows terminal absorption at 225 nm.

Factor B dihydrochloride A-35512 has the following specific rotations:

(a] t> 25 -128 ° (c - 1, HZO) [a)]. 3652 ⁵ -475 (c = 1, HZO).

Electrometric titrations of factor B dihydrochloride A-35512 in 66% aqueous dimethylformamide indicate the presence of four titratable groups with a pK a of approximately 7.15, - 8.87, 10.30, and 12.10 and a possible presence of another group with a pK a greater than 13 , 1.

The approximate molecular weight of factor B dihydrochloride A-35512 determined by titration is about 2027.

The 13c nuclear-magnetic resonance spectrum of Factor B dihydrochloride A-35512 has the following characteristics:

No. PPM Height (%)

2 173.0 4.1 3 171.9 3.7 4 171.6 3.3 5 171.0 5.8 6 170.8 5.0 7 169.6 3.6 8 159.0 4.1 9 157.9 4.4 10 157.5 3.7 11 156.6 4.8 12 155.6 6.1 13 155.3 4.2 14 154.9 3.3 15 Dec 154.3 4.2 16 151.7 3.3 17 144.3 3.1 18 136.7 3.5 19 Dec 136.2 4.9 20 May 135.4 4.0 21 135.2 4.4 22nd 133.6 4.2 23 133.3 4.1 24 129.8 1.7 25 129.3 3.0 26 128.8  -2.6 27 Mar: 127.6 1.5 28 126.1 3.9 29 124.2 5.6 30 122.4 1.4 31 122.0 4.4 32 120.7 3.3

C. PPM Height (%) 33 116.5 2.7 34 109.5 0.8 35 108.2 1.1 36 107.7 2.7 37 104.5 1.7 38 101.8 2.9 39 100.9 1.6 40 98.2 1.0 41 76.9 1,2 42 76.1 1,8 43 74.1 2,0 44 73.5 2.7 45 72.7 2.4 46 72.3 4.0 47 71.0 7.1 48 70.3 2.5 49 69.7 2.5 50 67.4 74,7 * 51 64.6 1,2 52 62.0 1.5 53 58.0 1.3 54 56.8 1.7 55 55.4 3.9 56 54.3 2.5 57 24.5 2,0 58 17.9 3.0 59 17.2 2,0 60 16.3 2.5

“Dioxane standard

Factor B dihydrochloride A-35512 crystallized from a mixture of methanol and water has the following X-ray powder diffraction pattern characteristics (Cu ⁺⁺ radiation of 1.5405 λ, nickel filter, d = interplanar spaces in angstroms):

d relative intensity

17.15 100 ALIGN! 12.90 80 10.85 70 9.25 70 8.87 60 8.22 50 7.86 50 6.93 40 6.20 40 5.62 40 5.04 05 / 4.02 02 / 3.54 02 /

The infrared absorption spectrum of factor B dihydrochloride A-35512 in KBr tablets is shown in Figure 2. The most significant absorption maxima are at the frequencies [cm -1]: 3420 (strong), 3300 (inflex), 2950 [weak], 1752 (weak) , 1675 (strong), 1630 (inflex), 1605 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1410 (weak), 1345 (inflex), 1312 [medium], 12225 (medium) , 1180 (weak), 1135 (weak), 1080 (strong and 1020 (weak)).

Amino acid analysis of the acid-hydrolyzed Factor B A-35512 dihydrochloride shows that A-35512 Factor B contains at least five amino acid residues, one of which is glycine. The four remaining amino acid residues in A-35512 factor B are complex and appear to be the same as those found in A-35512 factor A.

The structure of one of the amino acid residues appears to be

Analyzes of these acid hydrolysis products show that Factor B dihydrochloride A-35512 contains the following sugars: glucose, fucose, amnose, rhamnose, and 3-amino-2 (3 (6-trideoxy-3-C-methyl-L-xylohexylpyranose). Slight acid hydrolysis of Factor B dihydrochloride A-35.512 cleaves glucose, fucose, mannose, and rhamnose to give a characteristic aglycone.

Factor B dihydrochloride A-35512 has at least one esterifying hydroxy group.

Factor B dihydrochloride A-35512 is water soluble, partially soluble in alcohols such as methanol and ethanol, and insoluble in other less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, . acetoniiril and dioxane.

Factor B dihydrochloride A-35512 is stable for 7 hours in aqueous solutions of pH 3 to 10.

A -35512 factor C

A-35512 factor C is a white, amorphous basic compound.

A-35512 factor C has the following approximate percentage composition:

carbon 55.90 · "/ o, hydrogen 5 ° 1%, nitrogen 5.88%, oxygen 32.33%, chlorine 1.90% / o.

The elemental composition is in accordance with the preferred empirical formula

C8, H00N8O38Cl.

A-35512 factor C has an approximate molecular weight of 1862.

Factor C dihydrochloride A-35512 is a white amorphous compound. Factor C dihydrochloride A-35512 has the following elemental percentage composition:

carbon 55/76 ° C, hydrogen 5, (07%, nitrogen 5, (íL "), oxygen 30.29%, chlorine 4.88%.

The empirical formula of Factor C dihydrochloride A-35512 is in the range

C83-85H99_iNi037_39Cl3.

The elemental composition is particularly in accordance with. another empirical formula

C84H07NuO38Cl.2 HCl (calculated · C 52.2%, H 5.1%, N 5.8%, 0 31.5 o / o, Cl 5.4%).

The infrared absorption spectrum of factor C dihydrochloride A-35512 in KBr tablets is shown in Figure 4 of the accompanying drawing. The most significant absorption maxima are at the following frequencies (cm ^- 1): 3370. (wide), 3280 ('inflex), 3040 (inflex), 2080 (inflex), 2020 (weak), 1740 (weak), 1658 (strong), 1620 (faint), .1589 (faint), 1503 (faint), 1460 (faint), 1428 (faint), 1385 (faint), 1330 (faint), .1205 (faint), 1210 (faint), 1162 (faint) ), 1120 (weak), 1060 (sine), and 1005 (medium).

The ultraviolet absorption spectra of factor C dihydrochloride A-35512 show an absorption maximum at .282 nm (ε 14,600) in acidic and neutral methanol and an absorption maximum at 202 nm (ε 16,400) in basic methanol, calculated using a molecular weight of 2000.

C13 nuclear magnetic resonance spectrum of dihydrochloride A-35512. Factor C in DaO exhibits the following characteristics:

C. PPM Height (%) 1 172.0 2.5 2 172.2 2,0 3 2.2 4 171.0 3.0 5 560 (6 2,0 6 158.6 1,8 7 157.8 3.0 8 156.5 2.1 0 156.1 2.8 10 155.6 4.2 11 154.6 3.0 12 151.1 1.5 13 143.3 1.4 14 136.0 3.2 15 Dec 135.4 2.7 16 133.2 3.7 17 128.7 2.3 18 126.5 3.1 10 124.6 2.1 20 May 120.3 2.7 21 121.5 2.2

C. PPM Height (%) 22nd 120.1 1,2 23 118.0 1.7 24 116.5 1,2 25 107.8 2.7 26 104.5 1.9 27 Mar: 101.7 1.9 28 94.5 1.0 29 75.9 3.0 30 74.3 2,0 31 73.4 2.3 32 72.1 3.5 33 70.9 4.3 34 68.7 2.9 71,7 * 35 67.4 36 64.3 1.3 37 62.2 1.6 38 56.1 1.4 39 55.2 3.5 40 54.2 2.1 41 24.3 1.9 42 17.9 2.2 43 17.1 2,0 44 16.2 2,0

* dioxane standard

Factor C dihydrochloride A-35512 has the following specific rotations:

[a] ²⁵ = -161 ° (c = 1.05, H2 O], [a:] 365 ²⁵ -6 = 14 ° (C = 1.05, H2 O).

Electrometric titration of A-35512 dihydrochloride factor C at 66o / _o aqueous dimethylformamide indicates the presence of three titratable groups with pKa values of approximately 7.30, 8.92 and 10.99 and the possible presence of two or more groups with pKa values greater than 11, 5.

The approximate molecular weight of factor C dihydrochloride A-35512 determined by titration is about 1982.

The amino acid analysis of the acid hydrolyzed Factor C dihydrochloride A-35512 shows that it contains at least five amino acid residues, one of which is glycine. The four remaining amino acid residues have not yet been identified.

Factor C dihydrochloride A-35512 is soluble in water, dimethylsulfoxide and aqueous dimethylformamide, partially soluble in alcohols such as methanol and ethanol, and is insoluble in other less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene , hexane, acetonitrile and dioxane.

Factor C dihydrochloride A-35512 is stable in aqueous solutions of pH from about 3 to about 10 for 146 hours.

A-35512 Factor E

A-35512 factor E is a white, amorphous basic compound. A-35512 factor E has the following approximate percentage elemental composition:

carbon 54.84%, hydrogen 4.73%, nitrogen 5.26%, oxygen 32.67%, chlorine 1.72%.

Factor E hydrochloride A-35512 is a white, amorphous compound. Factor E hydrochloride A-35512 has the following approximate percentage elemental composition:

carbon 52.67%, hydrogen 4.59%, nitrogen 5.55%, oxygen 33.51%, chlorine 3.62%.

The infrared absorption spectrum of factor E hydrochloride A-35512 in KBr tablets is shown in Figure 5 of the accompanying drawings. The most prominent absorption maxima are at the following frequencies (cm ^-1 ): 33'60 (strong), 3220 (inflex), 2900 (weak), 1725 (weak), 1650 (strong), 1580 (medium), 1498 (strong), 1450 (weak), 1419 (weak), 1295 (medium), 1205 (medium), 1172 (medium), 1110 (weak), 1060 (strong) and 1000 (weak).

The ultraviolet absorption spectra of factor E hydrochloride A-35512 show the following absorption maxima: in neutral methanol 270 nm (inflex) and 359 nm (ε = 16216); in acidic methanol, 286 nm (ε = 18018) and 310 nm (inflex) in basic methanol 270 nm (inflex), 300 nm (ε = 16216) and 354 nm (ε = 17568), calculated using a molecular weight of 2000.

Factor E hydrochloride A-35512 has the following specific rotations:

[α] _D ²⁵ = -108.3 (c = 1 H 2 O).

Electrometric titration of Factor E hydrochloride A-35512 in aqueous 680/0 dimethlformamide indicates the presence of three titratable groups with pKa values of approximately 6.30, 9.09 and 11.6'2 and possibly one or two groups with pKa values of about

12.5 or greater (initial pH 5.45).

The approximate molecular weight of Factor E hydrochloride A-35512 determined by titration is approximately 2018.

A-35512 factor E hydrochloride amino acid analysis shows that A-35512 factor E contains six previously unidentified amino acid residues.

Factor E hydrochloride A-35512 is soluble in water, partially soluble in alcohols such as methanol and ethanol, but insoluble in most organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane .

A-35512 Factor H

A-35512 factor H is a white, amorphous basic compound. A-35512 factor H has the following approximate elemental composition:

carbon 53.76 hydrogen 5.32%, nitrogen 5.53%, oxygen 33.48%, chlorine 1.59%.

A-35512 factor H has an empirical formula in the range

Cs5-87H103-107N8O38-40Cl and a preferred experimental empirical formula

СвбНшsmeОзэС1 and approximate molecular weight 1908.

Factor H hydrochloride A-35512 is a white, amorphous hygroscopic compound. Factor H hydrochloride A-35512 has the following average elemental percentage:

carbon 53.10%, hydrogen 5.37%, nitrogen 5.35%, oxygen 30.120 / 0, chlorine 3.78%.

The infrared absorption spectrum of factor H hydrochloride A-35512 in KBr tablets is shown in Fig. 3 of the accompanying drawings. The most significant absorption peaks have the following frequencies (cm ^-1 ): 3410 (strong), 3240 (inflex), 2940 (weak), 1670 (strong), 1630 (inflex), 1605 (strong), 1520 (strong), 1470 (strong) weak, 1442 (weak), 1400 (weak), 1345 (inflex), 1310 (medium), 1225 (medium), 1180 (weak), 1135 (weak), 1080 (strong) and 1020 (inflex).

The ultraviolet absorption spectra of factor H hydrochloride A-35512 in acid and neutral methanol show an absorption maximum at 282 nm (ε = 13 500) and in basic methanol an absorption maximum at 292 nm (ε 14000), calculated on a molecular weight of 2000. Factor H -35512 also exhibits terminal absorption at 225 nm.

C ¹³ nuclear magnetic resonance spectrum of factor H hydrochloride A-35512 in DzO , 1 has the following characteristics: C. PPM Height (%) 2 177.2 2.7 3 171.6 5.2 4 170.9 5.8 5 169.6 4.7 6 158.9 3.1 7 157.6 4.3 8 156.6 3.8 9 155.6 4.1 10 155.4 3.8 11 154.3 2.4 12 151.3 1.6 13 137.7 2,0 14 136.7 2.2 15 Dec 136.0 4.0

C. PPM -Height (%) 16 135.3 1.9 17 133.5 5.0 18 129.4 3.7 19 Dec 127.3 1.3 20 May 126.1 3.2 21 124.2 6.9 22nd 122.6 4.1 23 107.6 2.7 24 101.8 1,8 25 76.2 2.8 26 73.5 4.4 27 Mar: 72.3 7.4 28 71.0 12.2 29 69.7 4.6 30 67.4 73.1 * 31 61.6 3.5 32 56.8 1,8 33 55.4 2.8 34 55.0 1.5 35 24.5 2.6 36 17.9 4.6 37 17.2 2.6 38 16.3 3.6

* dioxane standard.

Factor H hydrochloride A-35512 has the following specific rotations:

_{[?] D} 25 = -123.5 ° (c = 1, H2 O).

Electrometric titration of A-35512 hydrochloride factor H at 66o / ₀ aqueous dimethylformamide indicates the presence of five titratable groups with pKa values .přibližně 5.0, 7.46, 9.80, 11.43 and 13.02 (initial pH 5.93 ).

The approximate molecular weight of factor H hydrochloride A-35512 is determined by a titration of approximately 1660,

The amino acid analysis of the acid-hydrolyzed Factor H hydrochloride A-35512 contains at least five amino acid residues, one of which is glycine. The four remaining amino acid residues in A-35512 Factor H are identical to those found in A-35512 Factors A and B.

Factor H hydrochloride A-35512 is soluble in water, partially soluble in alcohols such as methanol and ethanol, but insoluble in most organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane .

Factor H hydrochloride A-35512 is stable for 72 hours in aqueous solutions of pH 3 to 10.

A-35512 factors A, В, С, E and H and minor factors F and G are preferably separated by paper chromatography using a solvent mixture of 1-butanol: pyridine: acetic acid: water (15: 10: 3: 12). Bioautography using Sarcina llitea is a preferred detection method. The approximate Rf values of A-35512 factors in this system are shown in Table I.

TABLE I

35512 factor Rf value factor And. 2 HCl 0.21 factor (B) 2 HCl 0.34 factor C. 2 HCl 0.46 factor E. HCl 0.64 factor F 0.81 factor G 0.93 factor H. HCl 0.15

In various systems, A-35512 Factor H has a chromatographic profile very similar to that of antibiotic AM 374 (U.S. Pat. No. 3,700,768). Antibiotic A-35512- factor H can be distinguished from antibiotic AM 374 in at least two chromatographic systems on paper. Rf 'values of A-35512 factor H. . The 2 HCl and AM 374 in the two systems are shown in Table II.

TABLE II

Rf value Solvent system A-35512 H AM 374

CH3OH: 0.1 N HCl (3: 1) 0.47 05 ^ 8 '

1-Propanol: NH 3 OH: H 2 O (6: 3: 1) 0.11 0.20

Several A-35512 factors can be separated by high pressure liquid chromatography using polyamide (Z1PAX, Dupont) as the stationary phase and 1.33 molar aqueous monobasic potassium phosphate solution as the mobile phase using UV light detection (250 nm). . Table III shows the retention times for A-35512 factors by HPLC separation using the following conditions:

column size: 1/8 "- x 6 ', packing: polyamide (Z1PAX, DuPont), solvent: KH2PO4 (327 g) / H2O (1800 ml), flow rate: 0.5 -ml / min, speed: 2" / h, pressure: 1500 PSI

TABLE III

A-35512 - retention factor-time (minutes)

A B C E F

G H

9,375

13,125

26.25

5,625

7.5

7.5

7.5

Agiykon A-35512 Factor B

carbon 54,29%, hydrogen 4,34%, nitrogen 7.40%, chlorine 5,02%, oxygen 28.95% (difference)

The infrared absorption spectrum of aglycone A-35512 factor B hydrochloride in nujol is shown in FIG. 6 of the accompanying drawings. The most significant absorption bands are at the following frequencies (cm ~ 1): 3440 (inflex), 3340 (inflex), 3215 (strong), 2950 (inflex), 2910- (strong), 2840 (strong), 2640 (inflex), 1735 (weak), 1655 (strong), 1590 (medium), 1500 (strong), 1460 (strong), 1378 (medium), 1365 (inflex), 1298 (medium), 1215 (medium), 1155 (medium), 1120 (inflex), 1105 (weak), - 1060 (weak), 1040 (weak), 1008 (medium), 925 (weak), 875 (weak), 765 (inflex) - and 718 - (weak).

The electrometric titration of aglycone A-35512 Factor B hydrochloride in 66% aqueous dimethylformamide indicates the presence of three titratable pKa groups. about 7.5, 9.25 and 11.0, and for the possible presence of two other groups with pKa values greater than 11.0.

Factor B aglycone hydrochloride A-35512 has a molecular weight of about 1282 as determined by titration.

Factor B aglycone hydrochloride A-35512 has the following specific rotation:

(ar _D 25 17178 ° (c = 5, CH 3 OH), ¢¢ 136525-7 16.8 ° (c = 5, CH 3 OH).

The ultraviolet absorption spectra of Aglycon A-35512 Factor B hydrochloride show an absorption maximum at 282 nm (Eld 102.62) in acidic and neutral methanol and an absorption maximum at 302 nm in basic methanol (E J d 182.09).

^{The 13} C nuclear magnetic resonance spectrum of Aglycone A-35512 factor B in DMSO-d6 at 90 ° C has the following characteristics:

PPM Height (-%)

C.

1 187.8 37.2 2 172.0 40.9 3 170.7 45.2 4 170,1 · 46.9 5 169.6 47.7 6 168.4 57.6 7 166.7 52.5 8 157.4 49.9 9 156.6 45.5 10 155.7 55.8 11 155.6 71.5 12 155.4 56.7 13 154.5 50.5 14 149.3 43.0 15 Dec 138.8 38.8 16 136.9 54.3 17 136.3 40.2 18 135.2 31.7 19 Dec 134.7 28.4 20 May 133.8 40.9 21 128.2 102.9

Factor B aglycone hydrochloride A-35512 is a white amorphous compound of approximately the following elemental composition:

PPM Height (θ / ο)

22nd 126.2 77.2 23 123.0 57.5 24 121.3 38.1 25 117.7 44.4 26 117.0 31.5 27 Mar: 108.6 31.0 28 106.7 47.9 29 105.7 81.2 30 103.2 26.5 31 93.6 33.4 32 74.9 39.8 33 71.8 33.9 34 68.9 63.2 40.2 43.1 35 36 60.4 33.6 37 57.1 57.8 38 55.2 33.1 39 53.2 30.1 40 51.8 36.0 41 40.7 43.4 42 39.9 60.9 43 39.1 43.9 44 23.8 55.7 45 17.1 47.8 46 0.0 43.7

* DMSO-de ¹³ C NMR spectrum shows that the aglycone A-35512 of Factor В has retained the 3-amino-2,3,6-.alpha.-β-ω-3-ω-ω-ω-ω-ω-thiogorogonate portion of the molecule (one sugar is present in A) -35512 factor B).

The amino acid analysis of the acid-hydrogenated aglycone A-35512 Factor В shows that it contains glycine and at least three complex amino acid residues. The structure of one of these amino acid residues is

Factor В Aglycone A-35512 hydrochloride has at least 1 esterifiable hydroxy group and is soluble in water and methanol and is insoluble in less polar organic solvents such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile and dioxane .

Factor В-Aglycone A-35512 hydrochloride has an approximate Rf value for cellulose thin layer chromatography (aluminum foil) using 1-butanol-4-pyridine-acetic acid-vocla (15: 10: 3: 12). Bioautography using Sarcina lutea is a preferred detection method.

Factor В of aglycone A-35512 hydrochloride has an approximate Rf value of 0.26 in thin layer silica gel cellulose chromatography using methanol: chloroform: conc. NH 3 OH (3: 2: 1).

Aglycone A-35512 Factor В (free base) is a white amorphous compound with the following approximate elemental percentage (mean):

carbon 52.65 o / o, hydrogen 4,57%, nitrogen 6.91 o / o chlorine 2.94 o / _Oi oxygen 27.04 "/ o, ash 4.70 o / o.

The infrared absorption spectrum of aglycone A-35512 Factor В (free base) in KBr tablets exhibits prominent absorption bands at the following frequencies: (cm ^-1 ): 3360 (strong), 32'60 (inflex), 2940 (inflex), 1735 ( inflex), 1660 (strong), 1598 (medium), 1510 (strong), 1440 (medium), 1295 (weak), 1215 (medium), 1165 (medium), 1122 (weak), 1070 (weak), 1018 (medium) strong), 940 (weak) and 920 (weak).

Electrometric titration of the aglycone A-35512 factor aj (free base) in 66% aqueous dimethylformamide indicates the presence of five titratable groups with pKa values of about 6.2, 8.2, 10.1, 11.4, 12.4 and possibly one or two other groups with pKa values greater than 12.5.

Aglycone A-35512 Factor В (free base) has the following specific rotation:

[α] _D ²⁵ = -64.5 ° (c = 3, DMSO).

The ultraviolet abruption spectra of aglyicoin A-35512 factor V (free base) exhibit an absorption maximum at 282 nm (ε 1 cl 43.65) in neutral and acidic methanol and an absorption maximum at 301 nm in basic methanol (λ nn 67.46).

Aglycone A-35512 Factor В (free base) has the same approximate Rf values previously described for Aglycone A-35512 Factor B Hydrochloride.

In addition to the free base and the hydrochloride, the individual A-35512 factors and the A-35512 factor V aglycone form other pharmaceutically acceptable salts of the A-35512 factors A, V, С, E, H and factor B aglycone, which are also part of the invention. "Pharmaceutically acceptable" salts are salts in which the toxicity of the compounds as such to warm-blooded animals does not increase relative to the free bases. Examples and suitable salts of A-35512 Factors A, V, C, E, and H and A-35512 Factor В Aglycone include those salts formed by standard reaction with inorganic and organic acids such as sulfuric, phosphoric, acetic, succinic, lemon, milk, maleic, fumaric, palmitic, cholic, pamoic, mucosal, D-glutamic, d-camphoric, glutaric, glycolic, phthalic, vinous, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic, sorbic, picrous, benzoic, cinnamic ;

The novel antibiotics of the invention are prepared by culturing a strain of Streptomyces candidus NRRL 8156, producing A-35512 under submerged aerobic conditions in a suitable nutrient medium until sufficient antibiotic activity is produced. Antibiotics are isolated using various isolation and purification procedures commonly used in the preparation of antibiotics by fermentation.

The new organisms used for the preparation of α-35512 A-35512 were isolated from a soil sample collected on Eniwetok Atoll. This organism was classified as a new strain of Streptomyces candidus (Krassilnikov) Waksman, described in E. B. Shirling and D. Gottlieb in the Cooperative Description of Type Cultures of Streptomyces. II. Species Descriptions from Second Study ', Intern. J. Systematic Baoteriol. 18 (4): 297-392 (1968); and S.A. Waksman, "The Actinomycetes. Vol. 2, Classification, Identification, and Descriiptions of Genera and Species, Williams and Wilkins Co, Baltimore, 1961.

This classification is based on the methods recommended by the International Streptomyces Project [E. B. Shirling and D. Gottlieb, "Methods for Characterization of Streptomyces Species," Intern. Bull. Systematic Bacteriol. 16: 313-340 (1966)] and certain additional tests. Color names were assigned according to the ISCC-NBS method (K.L. Kelly and D. B. Judd, "The ISCC-NBS Method of Determining Colors and the Dictionary of Color Names", US Department of Commerce Cir. 553, Washington, D. C., 1955). The numbers in brackets refer to the Tresner and Backus color series (HD Tresner and SJ Backus, "System of Color Wheels for Streptomy.cete Taxonomy", Appl. Microbiol. 11: 335-338 (1963)) and the color code is underlined: o Maerz and Paul colors (A. Maerz and M.R. Paul, "Dictionary of Color", McGraw-IHll, New York, NY 1950) are given in brackets. Cultures were grown at 30 ° C for 14 days. unless otherwise stated.

Characterization of the A-35512 antibiotic producing strain

Morphology

Long, wavy sporophores are produced. Spores occurring in chains of · 10 to 50 are cylindrical and measure from 0.7 to · 1.05 μ x 1.4 to 3.5 μ. Some media have sclerotia-like structures. Sometimes broom-shaped hyphae or tufted hyphae are observed. The surface of the spores was observed by an electron microscope and found to be smooth.

Cultural characteristics on various media:

.........._...........

Medium characteristics

ISP No. 2 (yeast extract, malt extract agar):

abundant growth, gray-yellow reverse [11J5], abundant aerial mycelium and sporulation, bléé (w) a; no soluble pigment.

ISP No. 3 (oatmeal, agar): moderate growth, light yellow-green back [10B1], nice airy mycelium and sporulation, white (w) 13ba, no soluble pigment, sclerotia-like bodies were observed.

ISP No. 4 (inorganic salts - starch agar): growth good, reverse amber white [10C1], good aerial mycelium and sporulation, yellow-gray (GY) 2dc, slightly brown soluble pigment, spheroid-like bodies observed.

ISP No 5 (glycerol asparagine agar):.

growth good, reverse light yellow-green [10B2], good aerial mycelium and sporulation, white (w) and, no soluble pigment, mycelium is tuft.

Emerson agar:

growth good, reverse light olive [14L6], no airy mycelium or sporulation, light brown soluble pigment.

Bennett's modified agar: growth abundant, reverse slightly yellow [11J6], abundant aerial mycelium and sporulation, white (w) and, no soluble pigment.

Czapek solution, agar: growth good, reverse light yellow-green [10B1], good airy mycelium · a sporulation, white · (w) b, no soluble pigment, · mycelium is fluffy.

Tomato paste, porridge, agar: growth · abundant, reverse gray-yellow, [1115], abundant airy mycelium and sporulation, white (w) and, mycelium turns up from agar surface, no soluble pigment.

Nutrient agar:

growth nice to good, reverse light yellow-green (18D2), good aerial mycelium and sporulation, white (w) and, light brown soluble pigment.

Glucose asparagine agar: growth good, reverse light yellow-green [17E1], good aerial mycelium and sporulation, white (w) 13ba, no soluble pigment.

Trypton, yeast extract, agar:

grow nice, rub white [10A1], nice airy mycelium and sporulation, white (w) b, no soluble pigment.

Tyrosine agar:

growth good, reverse white [10B2], good aerial mycelium and sporulation, white (w) and, brown soluble pigment, bodies similar to sclerotia were observed.

Glycerol-glycine agar:

growth good, reverse white [10B2], good aerial mycelium and sporulation, white (w) b, slightly brown soluble pigment.

Calcium maleate agar:

growth good, reverse white [10B1], good aerial mycelium and sporulation, white (w), and, no soluble pigment, medium clears around the eye.

The organism was studied for selected physiological properties under standard conditions. The following properties and characteristics were found:

Observed characteristics of the characteristic

Effect on milk:

coagulation with clarification after 14 days.

Nitrate reduction: positive.

Production of melamine pigment on: sloping soil peptone, iron, agar:

negative.

Tyrosine agar, sloping ground:

weakly positive (pigment after 7 days).

Ttypton, yeast extract, media: negative.

Liquefaction of gelatin:

complete in 14 days.

Temperature requirements:

up to 30 ° C, good growth and sporulation.

(ISP No. 2, medium yeast extract, malt extract, sloping soil):

° C, weak growth, no aerial mycelium or spores, 40 ° C weak vegetative growth, only 45 ° C no growth.

The results of carbon utilization tests were performed with NRRL 8156 and are presented below. The symbols used to indicate growth are:

4- = good growth, positive utilization (-H) = poor to nice growth (-) = poor growth, probably under-utilization - = no growth, no utilization.

Carbon sources none (negative control) (-)

D-glucose (positive control) 4L-arabinose (-) sucrose (-) i-inositol +

D-mannitol +

D-frukto'sa4rhamnosa (-) raffinosa (-)

D-xylose (4-)

Certain characteristics of S. candidus strain NRRL 8156, producing A-35512, are different from those of the organisms described by Shirling and Gottlieb [Elwood B. Shirling and David Gottlieb Cooperative Description of Type Cultures of Streptomyces

III. Additional Species Descriptions from First and Second Studies', Intern. J. Systematic Bacteriol. 18 (2), 69-189 (1968)] and Waksman [S. A. Waksman, 'The Actinomycetes. Classification, Identification and Description of Genera and Species', Vol. 2, The Williams and Wilkins Co., Baltimore, Md., 1961]. These differences are summarized in Table IV.

TABLE IV

Carbon utilization

NRRL 8156

L-arabinose rhamnosa i-inositol

Published description +

+

Gelatin liquefaction Effect on the mill complete in 14 days of coagulation, clarification in 14 days slow no coagulation, good peptonization.

24

The Streptomyces candidus culture used to produce A-35512 antibiotics has been deposited and is available from the Northern Regional Research Center, U. S. Department of Agrisulture, Agricultural Research Service, Peoria, Illinois, 61604, from where it is available. under the general number NRRL 8156.

As with other organisms, the culture characteristics of Streptomyces-candidus NRRL 8156 producing A-35512 are capable of variation. For example, artificial variants and mutants of the NRRL 8156 strain -se can be obtained by the action of various known mutagens such as ultraviolet light, X-rays, radio waves, radioactive radiation, and various chemical compounds. All natural and artificial variants and mutants associated with Streptomyces candidus and producing A-35512 antibiotics can be used in the present invention.

The nutrient medium used to grow Streptomyces candidus 8156 may be any of a variety of media. However, certain nutrient media are preferred for economical production, optimum yield and easy product isolation.

For example, a preferred source of carbohydrates in large-scale fermentation is sucrose, although glucose, tapioca dextrin, fructose, mannitol, maltose, lactose and the like can also be used. A convenient source. Nitrogen is a soluble peptone of meat, although - although soybean - flour, ground pork blood, amino acids such as glutamic acid, etc., can also be used. In the case of nutrient inorganic salts which may be added to the nutrient medium, soluble salts yielding zinc, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate anodes are commonly added.

The essential trace elements necessary for the growth and development of the organism should also be present in the nutrient medium. These trace elements commonly occur as impurities in other media components in an amount sufficient for the growth requirements of the organism.

It may also be necessary to add a small amount (e.g. 0.2 ml / L) of an antifoaming agent such as polypropylene glycol, and especially in large scale production where foaming can cause significant problems.

Submerged aerobic fermentation in tanks is preferred for producing sufficient amounts of A-35512 antibiotics. Small amounts of A-35512 can be obtained from shake flask culture. In view of the prolongation of the inoculation time of large tanks with organism spores, it is preferable to use a vegetative loop. The vegetative seed is prepared by inoculating a small volume of nutrient media with spores or parts of the mycelium of the organism to obtain a fresh, active, growing culture of the organism. The vegetative loop is then transferred to a large tank.

The A-35512 producing organism can be grown at a temperature between 20 ° C and about 40 ° C.

Optimal production is between 30 ° C and 30 ° C.

As is common in the aerobic submerged process, sterile air is introduced into the nutrient medium. For efficient growth of the organism, the volume of air used in tank production is preferably greater than 0.1 volume of air per volume of nutrient medium per minute (v / v / min). For efficient production of A-35512 antibiotics, the volume of air used in the tank production is about 0.25 vol / vol / min.

The production of A-35512 -antibiotics can be monitored during fermentation by testing samples of a nutrient medium or extracts of a solid proportion of mycelium for antibiotic activity against organisms known to be susceptible to these antibiotics. The bioassay is preferably carried out with paper discs on agar plates containing medium with limited nutrients.

After fermentation under submerged aerobic conditions, A-35512 antibiotics can be isolated from the fermentation broth by methods used in fermentations. The antibiotic effect produced during fermentation by the A-35512 producing organism generally occurs in the filtered nutrient medium. The maximum isolation of the A-35512 antibiotics is achieved by initially filtering out the mycelium mass. The filtered medium can then be purified in various ways to obtain a mixture of A-35512 antibiotics. Preferred techniques include adsorption of the filtered medium on a polyamide column and elution of the column with water and an aqueous alcohol mixture. Eluted fractions exhibiting antibiotic activity may be combined to give the A-35512 mixture. Alternatively, using this technique, eluted fractions can be pooled based on thin-layer chromatography behavior to give A-35512 factor B and an enriched mixture of other factors A-35512.

Further purification of individual individual A-35512 factors includes additional adsorption and extraction. Adsorbents such as alumina, silica gel and ion exchange resins may be advantageously used for this purpose.

A-35512 factors - occur in fermentation media as hydrochlorides. The preferred isolation process - on the polyamide - yields A-35512 factor B and the remaining proportions of the A-35512 mixture as hydrochlorides. Each individual factor can then be converted to the free bases (free of chlorine ions) by a suitable method, such as chromatography on a weakly basic ion exchange resin.

Alternatively, solids of the broth, including medium and mycelium, can be used without extraction or separation, but preferably after removal of all water, as a source of the A-35512 antibiotic. For example, after producing A-35512 antibiotic activity, the nutrient medium can be freeze-dried and then mixed directly with the feed mixture.

Aglycone A-35512 factor B is prepared by mild acid hydrolysis of A-35512 factor B. A-35512 factor B is most readily available in the form of the dihydrochloride. Dihydro207457

Thus, A-35512 factor B chloride is a preferred starting material for the preparation of A-35512 factor B aglycone. However, A-35512 factor B or other acid salts of A-35512 factor B may also be used. The acid hydrolysis is carried out according to conventional methods. Although any of the acids can be used for this purpose, hydrochloric acid is the preferred acid for the preparation of factor B aglycone A-35512. If hydrochloric acid is used, then the factor B aglycone A-35512 is isolated as the hydrochloride. The hydrolysis is preferably carried out in boiling water for one to two hours. Longer reaction times lead to aglycone degradation and less active and later inactive products are obtained. The optimal reaction time for specific reaction conditions can be determined by testing the bioactivity of aliquots of the reaction.

The mixture of A-35512 antibiotics, A-35512 antibiotic factors A, B, C, E and H and Aglycone A-35512 factor B inhibit the growth of certain pathogenic organisms, particularly gram-positive bacteria. The minimum inhibitory concentration (MIC) of the various A-35512 factors A, B, C and H inhibits selected bacteria as determined by standard dilution assays, which are included in Table V.

TABLE V

Test organism A-35512 Factor A-2 HCl

Staphylococcus aureus 30556,25

Staphylococcus aureus 307425.0

Streptococcus faecalis6,25

An important aspect of the invention is that A-35512 antibiotics inhibit the growth of organisms that are resistant to other antibiotics. Table VI summarizes the effects determined by the standard target method on

MIC (mcg / ml)

A-35512  Factor B. 2 HCl A-35512 factor C. 2 HCl A-35512 Factor H-HCl 3.12 • 6.25 25.0 6.25 6.25 25.0 3.12 3.12 12.5

Plates for A-35512 Factors A, B, C, E and H (30 mcg / target) against various organisms The activity is measured in mm diameter of the observed zone of inhibition.

TABLE VI

Test organism Zone diameter (mm.)

A-35512 factor A. 2 HCl A-35512 factor B. 2 HCl A-35512 factor C. 2 HCl A-35512 Factor E. HCl A-35512 Factor H. HCl Staphylococcus aureus 3055 (penicillin G sensitive) 13.4 18.4 0 14.4 11.9 Staphylococcus aureus 3074 (penicillin G resistant) 13.2 16.8 0 14.5 11.6 * Staphylocuccus aureus 3130 (meehicШn resistant) 14.4 18.4 0 15.4 12.6 Streptococcus pyogenes (Group A) 17.0 19.6 10.5 17.0 16.0 Streptococcus sp. 9960 (Group D) 15.5 19.4 0 16.4 13.8 Diplococcus pneumoniae - - 11.0 20.0 -

Park I

Table VII gives additional dilution tests on Agar A-35512 Factor V Dihydrochloride and the minimum inhibitory concentrations (MIC) for Group A Streptococci and Diplococcus pneumonia are shown.

TABLE VII

Test organism MIC (mcg / ml) A-35512 Factor В

Streptococcus pyogenes C2031

Streptococcus pyogenes 103891

Streptococcus pyogenes 123448

Streptococcus pyogenes 129611

Streptococcus pyogenes 663-720.5

Streptococcus pyogenes 664-720.5

Streptococcus pyogenes 665-721

Streptococcus pyogenes 132340.5

Streptococcus pyogenes 190350.5

Streptococcus pyogenes M65170.5

Streptococcus pyogenes D277811

Diplococcus pneumoniae

Park I2

Diplococcus pneumoniae type 142

Table VIII lists additional agar dilution assays for Factor V dihydrochloride A against several Streptococci D groups.

TABLE VIII

Test organism MIC (mcg / ml) A-35512 Factor В

Streptococcus sp. D2822

Streptococcus sp. 99012

Streptococcus sp. 99134

Streptococcus sp. 99334

Streptococcus sp. 99604

Streptococcus sp. 12253F4

Streptococcus sp. Shrigley4

Streptococcus sp. Mitis4

Streptococcus sp. 2382

Streptococcus sp. SS9924

Table IX gives additional agar dilution tests for A-35512 Factor В Dihydrochloride against other Gram-positive organisms.

TABLE IX

Test organism MIC (mcg / ml) A-35512 Factor В

Staphylococcus aureus 3123 * 4

Staphylococcus auresu H43 ** 4

Staphylococcus aureus 3124 ** 4

Staphylococcus aureus 3126 ** 2

Staphylococcus auresu 3127 ** 4

Tested organism MIC (mcg / ml) A-35512 factor V

Staphylococcus auresu H57 ** 2

Staphylococcus auresu 3074 ** 4

Staphylococcus aureus 3130 *** 4

Staphylococcus aureus 3131 *** '* 4

Staphylococcus aureus 3133 *** 2

Staphylococcus aureus 3136 *** 2

Staphylococcus aureus 3125 *** 4

Staphylococcus epidermis

3064 ** 2

Staphylococcus epidermis

3078 * 2

Bacillus subtilis ATCC 66332

Sarcina lutea PCI-1001-FDA1 * penicillin G sensitive ** penicillin G resistant *** penicillin G resistant, methicillin resistant **** penicillin G resistant, methicillin resistant, clindamycin resistant.

Antibiotics A 35512 also inhibit the growth of certain anaerobic bacteria. Table X summarizes the activity of factor V dihydrochloride A-35512 against various anaerobic bacteria using a standard dilution test.

TABLE X

Tested organism MIC (mcg / ml) A-35512 factor V Actinomyces israelii 0.5 Clostridium perfringens 2,0 Clostridium septicum 4.0 Eubacterium aerofaciens 2,0 Peptococcus asaccharolyticus 1.0 Peptococcus prevoti 2,0 Peptostreptococcus anaerobius 1.0 Peptostreptococcus intermedius 4.0 Propionibacterium acnes 0.5 Bacteroides fragilis ssip. fra- 64.0 gilis 111 Fusobacterium necrophorum 8.0

Factor V dihydrochloride A-35512 exhibits an in vivo antimicrobial effect against experimental bacterial infections. When two subcutaneous doses of A-35512 factor V are administered to mice with cleared infection, the effect is measured as the ED 50 [effective dose in mg / kg protecting 50% _{of the} test animals (see Warren Wick et al., J. Bacteriol. 81 233-235 (1961)] The ED 50 values observed for A-35512 factor VI dihydrochloride are shown in Table XI.

TABLE XI

Tested organism

Streptococcus pyogenes C203

Diplococcus pneumoniae Park I

Staphylococcus aureus 3055

Factor V dihydrochloride A-35512 also protects guinea pigs that have been infected with Clostridium chauvoei (causing Black Leg disease in cattle). These tests were performed according to U, S. D. A. Suipplemental Assay Method for Potency Testing Clostridium

ED50

Change infection

1.75 260 x LD 50 (ip)

4.3 60.8 x LD 50 (ip)

6.9 206 x LD 50 (ip) chauvo-containing products (SAM 200, U. S. Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Servise Laboratories, Ames Iowa 50010, April 7, 1975, under 9 C.F. 113.91). The test results are shown in Table XII.

TABLE XII

Treatment

Dead / total

Percentage of protection

A-35512 factor B, 10 mg / kg

A-35512 factor V, 1 mg / kg * control * Intramuscular administration.

0/10

3/10

10/10

100 ALIGN!

0

TABLE XIV

The effect of Aglycone A-35512 Factor B, measured by a conventional diffusion test using discs (6 mm in diameter, immersed in solutions containing 1 mg of test compound per ml of solution and placed on agar plates inoculated with the test organism), is summarized in Table XIII.

TABLE XIII

Tested organism

Stahpylococcus aureus

Bacillus subtilis Sarcina lutea

Bacillus subtilis *

Inhibition zone (mm) A-35512 factor V aglycone (free base) * nutrient restricted agar.

Table XIV summarizes the minimum inhibitory concentrations (MIC), using aglycone hydrochloride A-35512 V, is active on selected Staphylococcus aureus strains as determined by standard agar dilution tests.

Tested organism MIC (mcg / ml) Slaphyl-ococcus aureus 3055 0.5 Staphylococcus aureus H290 0.5 Staphylococcus aureuis V92 0.5 Staphylococcus aureus V104 0.25 Staphylococcus aureus 3074 0.25 Staphylococcus aureus H43 ** 0.5 Staphylococcus aureus V57 ** 0.5 Staiphylococcus aureus H356 1.0 Staphylococcus aureus 3130 0.5 Staphylococcus aureus 3131 **** 0.5

* penicillin-G sensitive ** penicillin-G resistant *** penicillin-G resistant, methicillin resistant **** penicillin-G resistant, methicillin resistant, clindamycln resistant

Table XV shows additional agar dilution tests for aglycone A-35512 Factor В hydrochloride against several Streptococcus Group D.

TABLE XV

testovaný organism BALL (mcg / ml) Streptococcus sp. Shrigley 1.0 Streptococcus sp. Mitis 1.0 Streptococcus sp. 12253F 1.0 Streptococcus sp. SS992 1.0 Streptococcus sp. 9933 1.0 Streptococcus sp. 9913 1.0 Streptococcus sp. 282 1.0 Streptococcus sp. 238 1.0

Aglycone A-35512 factor B exerts an in vivo antibacterial effect against experimental infections. When two subcutaneous doses of Factor B aglycone hydrochloride A-35512 are administered to mice with known infection, the observed effect is measured as ED 50 values. The ED 50 values determined for Aglycone A-35512 Factor B hydrochloride are shown in Table XVI.

TABLE XVI

testovaný organism EDso change of infection Streptococcus pyogenes C203 5.8 2570 x LD 50 (lip) Streptococcus pneumoniae 7.0 373 x LDso (iip) Staphylococcus aureus 3055 1.04 370 x LD 50 (ip)

Antibiotics A-35512 are relatively non-toxic. For example, the LDo dihydrochloride of A-35512 Factor A when administered subcutaneously to fasted female Cox and Harlar ICR mice is greater than 8000 mg / kg. The LD 50 of Factor B dihydrochloride · A-35512 in mice by intraperitoneal injection is approximately 1356 mg / kg and by intravenous administration> 1000 <1250 mg / kg. Both Factor C dihydrochloride A-35512 and Factor E hydrochloride A-35512 have both acute toxicity when administered intraperitoneally to mice greater than 300 mg / kg.

Another important property of the A-35512 mixture and compounds is the ability to improve food utilization in animals. This ability has been demonstrated in ruminants that have developed a ruminant stomach.

It is known that the efficacy of carbohydrate utilization in ruminants is enhanced by a treatment in which ruminants are used. it stimulates the flora of the ruminant stomach of the animals to produce. propionate compounds rather than acetate or butyrate compounds (for a more complete discussion, see C.hurch et al. in "Digestive Physiology and Nutrition · of Ruminants", Vol. 2, 1971, pp. 622 and 625 J.).

Efficacy of food use can be demonstrated by in vivo tests in cattle with a stomach outlet by the method described by Arthus P. Raun in U.S. Patent No. 3,794,732 (see in particular Example 8): Table XVII summarizes the results of these tests using dihydrochloride A -35512 factor B, which are reported in mole percent of the concentration of propionic acid in the stomach of ruminants, and the value is the average of five analyzes over the 21 days of the test period.

treatment number of animals TABLE XVII and diameter · mol. % propionic acid molar% increase over control control 5 19.3 - A-35512 factor B 50 g / t 5 26.0 6.7

The mixture of A-35512 compounds and A-35512 compounds are effective to increase propionates and thus increase food use when administered to ruminants orally in an amount of from 0.15 to about 10.0 mg / kg / day. Best results were obtained using amounts of about 1.0 to about 2.0 mg / kg / day. A preferred method of administering the composition or compounds of A-35512 is by mixing with animal feed, but these compounds may also be administered, for example, in the form of tablets, dragees, or capsules. Modifications of these different dosage forms can be achieved using methods well known in veterinary pharmaceutical practice. Each individual dosage unit contains an amount of A-35512 mixture or compound, directly related to the daily dose of the animal to be treated.

Mixture A-35512 or compound A-35512 are also useful as growth promoting agents in animals. When tested in broiler chickens, Factor B dihydrochloride A-35512 is added to the diet at a rate of 20 g per tonne, which increases the weight gain and food utility. Table XVIII summarizes the results of these tests, in which tests using four groups of eight birds were repeated twice.

treatment TABLE XVIII % improvement over control conc. (g / ton) weight% improvement increment (g) against control use of food control - 380 - 1,850 - A-35512 Factor B 20 May 394 3.68 1,776 4.00

Table XIX summarizes the results of these tests using 12 replicates of 80 birds each.

TABLE XIX treatment k.onc. (g / ton) weight increment (g) % improvement over control use of food % improvement over control control A-35512 - 1898 - 2,250 - Factor B 20 May 1956 * 3.06 2,177 * .2,81

* P 0.05

The A-35512 mixture and the A-35512 compound are effective in improving growth in poultry when an amount of from about 1 to about 10 g of the mixture of A-35512 per tonne of food is applied with food.

The invention is illustrated in more detail in the following examples.

Example 1

A. Fermentation of A-35512 in a shake flask

The lyophilized tablet of Streptomyces candidus NRRL 8156 is dissolved in 1-2 ml of sterilized water. This solution was used to inoculate Bacte 'slant agar and malt slant agar (ISP No. 2, Difco Laboratories, Detroit, Michigan).

The inoculated slope was incubated for about seven days at 30 ° C. The grown slant culture was overlaid with water (2 ml) and scraped with a sterile pipette. A portion (0.1 ml) of this aqueous spore suspension was used to inoculate another sloping agar broth with ISP No. 2. This inoculated sloping broth was incubated for about seven days at 30 ° C. The grown sloping soil was covered with water (5 ml) and the spores were scraped off with a sterile pipette. A portion (2.5 ml) of the resulting spore suspension was used to inoculate 50 ml of the vegetative medium of the following composition.

quantity components

Trypticase soy medium (Bartimore Btologcal Laboratories, Cockeyswille Md.) 30 g water (deionized) make up to 1 liter

The inoculated vegetative medium is incubated in a 250 ml Erlenmeyer flask at 30 °

Celsius for 48 hours on a rotary shaker with a movement of 5 cm and 250 revolutions per minute. Such inoculated viral medium (0.5 ml) was used to inoculate 50 ml of production medium of the following composition:

Component Amount (8/1) Tapioca dextrin 25.0 glucose 10.0 NH4NO3 2.5 KC1 1.5 MgSO4 1.1 FeCii. 4HaO 0.03 Zndž 0.03 KH2PO4 0.1 L-glutamic acid 1.0 DL-citrullin 0.1 CaCO3 5.0 deionized water up to 1 liter

The inoculated production medium was incubated in a 250 ml Erlenmeyer flask for 8-10 days at 32 ° C on a shaker with a 5 cm shift at 250 rpm.

B. Fermentation of A-35512 in a tank

To prepare a larger seed volume, 20 ml of seeded vegetative medium prepared as described above was used to inoculate 400 ml of the second stage of the vegetative growth medium having the same composition as the vegetative medium. This second stage medium is incubated for 24 hours in a 2-liter flask at 32 ° C on a 5 cm shaker at 250 rpm.

Incubated second stage vegetative medium (800 ml) prepared in this manner was used to inoculate 100 liters of sterile production medium. The inoculated production medium is fermented in a 1651-liter fermentation tank for 8 to 10 days at 32 ° C. The fermentation medium is aerated with sterile air at rapid rate.

with a 0.25 v / v / min slurry and stir with a conventional stirrer at 200 rpm.

Example 2

Alternatively, the incubated vegetative medium prepared by the procedure described in Example 1 of Section A may be stored for later use by keeping it in the liquid phase of liquid nitrogen according to the following procedure.

In a small (13 x 100 mm) sterile screw-capped tube, place 2 ml of the suspending agent as follows:

Ingredient Quantity glycerol 20% lactose 10 ° / o water (deionized) 70%

To this suspending agent is added 2 ml of vegetative medium incubated for 48 hours, prepared as described above. The mixed solution is frozen and kept in the gas phase in a liquid nitrogen container.

The vegetative medium thus stored is melted for use in shake fermentation or for tank fermentation by placing the tube in a water bath at a temperature of 43 ° C. A portion of the thawed solution (1 ml) from the tube is used to inoculate 50 ml of the vegetative medium of the same composition as described in Example 1, section A. The inoculated vegetative medium is used as described in Example 1 either after fermentation in shake flasks, or to prepare a larger loop for the fermentation tank.

Example 3

The fermentation is carried out according to the procedure of Example 1, but using a production medium for shake flasks or fermentation in a tank as follows

component

Tapioca dextrin molasses soluble peptone from meat

MgSOr. 7H ₂ O

CaCO3 water quantity · (g / 1)

75.0

40.0

15.0

0.5

The 2.0-1 liter described in Example 1 is filtered using a filter aid (Hyflo supercel, diatomaceous earth, Johns-Manville Products Corp.), wherein the pH of the medium is 6.8-7.2. The clear filtrate obtained in this way is passed through a column containing 10 ml of polymeric adsorbent (Amiberlit XAD-4 Rohn and Haas Co.) per 100 ml of the medium filtrate, at a rate of 150 ml per minute. Fractions obtained in this manner are tested for biological activity against Sarcina luitea by a standard method using discs. Biologically inactive eluates are discarded. Wash the column with water (1/8 of the total volume) at a rate of 150 ml / minute. The inactive water is spilled.

The column was then eluted with 50% aqueous methanol (600 L) at a rate of 200 mL per minute. The eluates containing the A-35512 antibiotic mixture are concentrated in vacuo to a volume of 15 liters, containing 200 mg of A-35512 antibiotic mixture per liter each.

Example 5

Isolation of factors from A-35512 antibiotic mixture

The mixture of A-35512 antibiotics (about 3000 g dissolved in 15 liters of methanol), obtained as described in Example 4, is chromatographed on a polyamide column (Woelm, 100 liters). The column is eluted with deionized water at a flow rate of about 80 to 120 ml per minute. .

Fractions were monitored by cellulose thin layer chromatography or paper chromatography in n-butanol / pyridine / acetic acid / water (15: 10: 3: 12) and bioautography using Sarcina lutea.

Discard the first 100 liters of eluate.

The wash rate is then changed to about 160-200 ml / min, and twelve-filter fractions are collected. Twenty factions are collected in this way.

Then the elution solvent was changed to a water-methanol gradient using the following procedure:

A base of 360 liters of methanol with a siphon overflow was added. connect with · a tank containing · 120 liters of water. In a container containing water, the solution was stirred and used to elute the column.

Twenty four fractions (24 liters each) were collected in this manner at a rate of 200-300 ml / min.

Based on the bioautographic results, the fraction groups were combined and evaporated to dryness in vacuo. This yields factor B dihydrochloride A-35512 and the following enriched mixture of factors:

and

Example 4

Isolation of A-35512 antibiotic mixture

Whole fermentation medium (1136 liters) after 207457 fraction volume [liters] factor weight

1 - 10 120 A + H 192 g 11 - 24 216 (B) 269 g 25-31 168 B + C 590 g 32-44 312 C, E, F, G 224 g Example 6 Example 8 Clean by A- ^{35512 f} and ^to B Purification of A-35512 Factor A

The partially purified A-35512 Factor B dihydrochloride (400 g) obtained by the method of Example 5 was dissolved in 1.2 L of 50% aqueous methanol and chromatographed on an aluminum oxide column prepared as follows:

The acidic alumina (10 kg, M. Woelm) was stirred in 50% aqueous methanolic mixture. After standing the mixture, the supernatant was decanted and discarded. The alumina is again stirred in 50% aqueous methanol and then packed into a 13.5 cm diameter column. The alumina column was washed with 50% aqueous methanol until a clear eluate was obtained.

The column was eluted with 50% aqueous methanol at a rate of 8-10 °. ml / min and fractions of 240-300 ml are collected. Fractions were monitored by thin layer bioautography as described in Example 5.

Based on these. data, the fractions were pooled to give purified Factor B dihydrochloride A-35512.

fractions. weight - 21 9,6g - 29 72,0g - 37 117,0g

Each .podíl crystallized from concentrated 50 ^1% aqueous methanolic solution at 49 ° C. The purified Factor B dihydrochloride A-35512 contains about 4.6% chlorine. A solution of factor B dihydrochloride A-35512 in 66% dimethylformamide has a pH of about 6.5.

Example 7

Preparation of A-35512 chlorine-free factor B

Purified Factor B dihydrochloride A-35512 (1 g), obtained as described in Example 6 and dissolved in water (40 mL), is passed through a 2.5 x 18 cm ion exchange column (Bio-Rad AG 3-4 times in OH cycle) The column was eluted with deionized water, the first eluate (5 ml) was discarded, and the following eluate (50 ml) was evaporated to dryness in vacuo to give 0.76 g of A-35512 factor B. % free of chlorine ions in the form of a white powder containing about 1.59% of chlorine A solution of this chlorine-free A-35512 factor B in 60% aqueous dimethylformamide has a pH of 9.13.

Partially purified Factor A dihydrochloride A-35512 (1 g), obtained as described in Example 5 (fractions 1-10), was dissolved in 50% aqueous methanol (5 mL). This solution was chromatographed on an alumina column. 3 x 16 cm (M. Woelm), prepared as described in Example 5. The column was eluted with 50% aqueous methanol at a rate of 0.5 ml / min. Fractions of 5 ml were collected. Based on these tests, fractions 7 to 15 were combined, concentrated to low volume under reduced pressure, and lyophilized to give 0.3 g of A-35512 Factor A dihydrochloride (4.71% chlorine).

Example 9

Preparation of A-35512 chlorine-free factor A

Factor A dihydrochloride A-35512 (200 mg), prepared as described in Example 8, was dissolved in water (10 mL). This solution was passed through an ion exchange column (1.5 x 10 cm) (Bio-Rad AG 3-4 times in an OH cycle) at a rate of 0.5 ml / min, eluting with deionized water. The original eluate (10 mL) was discarded. The following eluate (20 mL) was concentrated to a small volume under reduced pressure and then lyophilized to give 115 mg of A-35512 Factor A free of chlorine ions.

Example 10

Cleaning of A-35512 Factor C .......... ..........................

Partially purified Factor C dihydrochloride A-35512 (15 g), obtained as described in Example 5 (fractions 25-31), was dissolved in deionized water (40 mL). This solution was applied to a 4 x 115 cm polyamide column using MN < 0.07 mm, Brinkman Instruments Inc, prepared and washed overnight with water. The column is eluted with deionized water at a rate of about 3 ml per minute. The first eluate (250 ml) was discarded and 24 ml fractions were collected.

The fractions were monitored by thin layer chromatography bioautography. Chromatographic plate with celuiosis (on an aluminum support

E. Merck W. Germany) using the sec .: pyridine: acetic acid: water mixture (10:

: 10: 3: 8) and using Bacillus subtills as an organism. Based on the results of thin layer chromatography, fractions 1 to 33 are combined, concentrated to 150 ml in vacuo and lyophilized.

Two additional portions of partially purified Factor C dihydrochloride A-35512 are chromatographed under the same conditions. Fractions 1 to 33 are combined, concentrated in vacuo to 150 ml and lyophilized. The three lyophilized samples from the three columns were combined and recovered

12.3 g of partially purified dihydrochloride. of factor C.

Factor C dihydrochloride A-35512 was further purified by chromatography on another polyamide column, as described above, but collecting 15 ml fractions at a rate of 1 ml per minute. The column eluates are again monitored by thin layer bioautography. Fractions 36 to 58 are combined, concentrated in vacuo to a volume. 150 ml and lyophilization yielded 5.3 g of further purified factor A dihydrochloride A-35512.

The final purification of A-35512 Factor C dihydrochloride is performed by column chromatography with 5 x 41 cm alumina (Woelm). The column is packed and washed with 50% aqueous methanol. Once the wash eluate is clear, apply A-35512 Factor C onto the column (5.3 g, dissolved in 30 mL of 50% aqueous methanol). The column was eluted with 50% aqueous methanol at a rate of 1 ml per minute. Fractions of 12 ml were collected and the eluates monitored by thin layer chromatography and bioautography as described above. Fractions 22-74 are combined, concentrated in vacuo to a volume of 250 ml and lyophilized to give 3.86 g of Factor C dihydrochloride A-35512.

Example 11

Preparation of A-35512 chlorine-free factor C

A-35512 Factor C dihydrochloride (200 mg), prepared as described in Example 10, was chromatographed on a weakly basic ion-exchange resin using the procedure outlined in Example 9 to give 156 mg of chlorine-free A-35512 Factor C. This chlorine-free A-35512 factor C contains at least 1.90% of chlorine.

Example 1 2

Purification of A-35512 Factor E

Partially purified Factor E A-35512 hydrochloride (8.1 g), obtained as described in Example 5 (fractions 32-44), was dissolved in deionized water (40 mL). This solution is loaded onto a 5 x 110 cm polyamide column (MN < 0.07 mm, Brinkman Instruments, Inc) prepared and washed with water overnight. The column was then eluted with deionized water at a rate of 20 ml for 15 minutes and fractions of 20 ml were collected. After the 118th fraction, the elution solvent was changed to 50% aqueous · methanol. Fractions were monitored by thin layer chromatography and bioautography as described in Example 10.

Fractions 148 to 195 contain A-35512 factor E. These fractions are combined, concentrated under reduced pressure to a volume of 150 ml and lyophilized to give 2.7 g of A-35512 factor E hydrochloride.

A portion of this partially purified A-35512 Factor E hydrochloride (615 mg) was dissolved in 50% aqueous methanol (5 ml) and loaded onto a 1.5 x 50 cm · acidic alumina column which was prepared and washed with 50% aqueous methanol until clear eluate. The column was then eluted with 50% aqueous methanol at a rate of 1 ml per minute and 10 ml fractions were collected. Again, fractions were monitored by thin layer chromatography and lyophilized. Fractions 5-8 are combined and concentrated under reduced pressure to a volume of about 10 ml. Deionized water (about 50 mL) was then added and the resulting solution was lyophilized to give 480 mg of Factor E hydrochloride A-35512.

Example 13

Preparation of A-35512 chlorine-free factor E

A-35512 Factor E hydrochloride (200 mg), prepared as described in Example 12, is chromatographed on a weakly basic ion-exchange resin using the procedure outlined in Example 9 to yield 170 mg of chlorine-free A-35512 Factor E. The chlorine-free A-35512 factor E contains about 1.72% chlorine.

Example 14

Cleaning A-35512 Factor. H

Partially purified Factor H A-35512 hydrochloride (30 g), obtained as described in Example 5 (fractions 1-10), was dissolved in a minimum amount of ethanol: water (7: 3). The resulting solution is adsorbed on a column of acidic alumina (3 x 60 cm, Woelm, packed in methanol and eluted with methanol until clear eluate). The column was then eluted with methanol at a rate of four ml / min. Fractions were collected in a volume of 24 ml. The eluting solvent was changed to a methanol: water (1: 1) mixture in fraction 59.

Fractions were monitored by thin layer chromatography using chloroform: methanol: ammonium hydroxide (2: 3: 1) and bioautography with Baccius subtilis at alkaline pH.

Fractions 51 to 118 were combined and evaporated in vacuo to give 6.4 g of purified Factor H hydrochloride A-35512.

Example 15

Preparation of A-35512 chlorine-free factor H

A-35512 Factor H hydrochloride (200 mg), prepared as described in Example 14, was chromatographed on a weakly basic ion-exchange resin using the procedure described in Example 9. 143 mg of A-35512 Factor H free of chlorine ions were obtained. This chlorine-free A-35512 factor H contains approximately 1.59% chlorine.

Example 16

Preparation of Aglycone A-35512 Factor B

Factor B dihydrochloride A-35512 (5.0 g), obtained as described in Example 6, was dissolved in water (200 mL). This solution was acidified with 4N hydrochloric acid (14 mL). The resulting solution was heated to reflux for two hours. The solution is then cooled and evaporated in vacuo to about 3/4 of the original volume. Hydrochloric acid (6N) is then added dropwise to this solution until precipitation is complete. The resulting precipitate was filtered off and dried to give 3.56 g of crude A-35512 factor B aglycone hydrochloride.

The filtrate was concentrated and analyzed. The filtrate contains glucose, fucose, mannose, and rhamnose.

The crude A-35512 factor B aglycone was purified by acid-washed alumina (Woelm, activity I) chromatography using water: methanol (1: 9). The eluates from the column are monitored by thin layer chromatography on cellulose. The fractions containing aglycone A-35512 factor B were combined and evaporated in vacuo to give 398 mg of partially purified product. Comparison of chromatographic results using ninhydrin detection shows that inactive impurities are still present in the product.

Part of this partially purified A-35512 Factor B aglycone (100 mg) was further purified by polyamide chromatography (4 g, Machery, Nagel & Co. NM-SC-6, Brinkmann Instruments Co. distribution, <0.07 mm), elution with water. Separation on this column is also monitored by thin-layer cellulose chromatography as described above. The eluted fractions containing Factor B-Aglycone A-35512 were combined and lyophilized to give 64 mg of purified Factor B Aglycone A-35512 hydrochloride. Total yield 5.08% from the Factor A-35512 starting factor.

Example 17

Preparation of A-35512 Factor B Aglycone Free Base

The Factor B aglycone A-35512 hydrochloride (90 mg) obtained in Example 16 was dissolved in 30 mL of 1: 1 methanol / water. This solution was neutralized with an ion-exchange resin [3.5 ml, Bio-Rad AG-3-4X (OH-)). The resulting solution was stirred at room temperature for 15 minutes. The resin is then filtered off. The filtrate is concentrated under vacuum at about one-half the volume under vacuum at about 60 ° C, after which 68 mg of the free base of aglycone A-35512 factor B is freeze-dried.

Claims (10)

SUBJECT A process for the preparation of a mixture of antibiotics A-35512, consisting of factors A, B, C, E, F, GH, and its division into individual factors, and the subsequent preparation of factor B aglycone, characterized by culturing Streptomyces candidus NRRL 8156 · Medium containing assimilable sources of carbohydrates, nitrogen and inorganic salts, at a temperature between 20 to 40 ° C under submersive aerobic fermentation conditions, optionally the mixture of antibiotics A-35512 is isolated from the nutrient medium, or antibiotics A-35512 are isolated from the mixture of antibiotics A-35512 35512 Factors A, B, C, E, F, G, H, and optionally, A-35512 Factor B aglycone is prepared from the A-35512 Factor B antibiotic. 2. A method according to claim 1 for the preparation of the A-35512 antibiotic mixture, characterized in that the A-35512 antibiotic mixture is isolated from the nutrient medium by filtration. 3. A method according to claim 1 for the preparation of A-35512 factor A, characterized in that A-35512 factor A is isolated from the mixture of antibiotics A-35512 by chromatographic separation. 4. The method of item 1 for preparation A -35512 factor B characterized in that A-35512 factor B is isolated from the mixture of antibiotics A-35512 by chromatographic separation. 5. The method of item 1 for the preparation of A-35512 factor C, characterized in that A-35512 factor C is isolated from the mixture of antibiotics A-35512 by chromatographic separation. 6. The method of item 1 for preparing A-35512 factor E, wherein A-35512 factor E is isolated from the A-35512 antibiotic mixture by chromatographic separation. 7. A process according to claim 1 for the preparation of A-35512 factor F, characterized in that A-35512 factor F is isolated from a mixture of antibiotics A-35512 by chromatographic separation on paper. 8. The method of item 1 for preparing A-35512 Factor G, wherein A-35512 Factor G is isolated from A-35512 antibiotic mixtures by chromatographic separation on paper. 9. The method of item 1 for the preparation of A-35512 Factor H, characterized in that A-35512 Factor H is isolated from the mixture of antibiotics A-35512 by chromatographic separation. 10. The process of claim 1 for preparing an Factor В aglycone, wherein z A-35512 Factor В prepares Factor В Aglycone by acid hydrolysis under mild conditions.