CH637159A5 - Preparation of an antibiotic mixture - Google Patents

Description

The invention relates to a process for the preparation of the antibiotic mixture A-35512 with the factors A, B, C, E, F, G and H, the antibiotic A-35512 factor A, the antibiotic A-35512 factor B, the 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 antibiotic A-35512 factor B aglycone.

The method according to the invention is characterized in that the microorganism Streptomyces Candidus NRRL 8156 is submerged in a nutrient medium containing assimilable sources of carbohydrates, nitrogen and inorganic salts under aerobic fermentation conditions until a substantial amount of the antibiotic is formed.

It is possible to use a mutant of the named microorganism Streptomyces Candidus NRRL 8156 in the method according to the invention.

The factors A, B, C, E, F, G and H are microbiologically active and they can be separated and isolated chromatographically. Weak acid hydrolysis of A-35512 factor B gives e.g. removing several sugars A-35512 factor B aglycone. The antibiotic mixture A-35512 is an antibacterial and growth-promoting agent with which the feed conversion in ruminants and poultry can be increased. The antibiotic A-35512 factor B is also suitable for the treatment of dental caries and acne.

The antibiotic mixture A-35512 is a mixture of closely related antibiotic glycopeptides. The antibiotic A-35512 factor B, which is the best characterized link from the antibiotic complex A-35512, appears to be a new representative from the group of peptide-containing antibiotics, including Vancomycin (US Pat. No. 3,067,099), A- 4696 factor A, factor B and factor C (U.S. Patent 3,952,095), avoparcin (U.S. Patent 3,855,410), ristomycin A (N. Lomakina, 7th International Symposium on Chemistry of National Products, Riga, Latvia, page 625 , 1970) and Ristocetin A (U.S. Patent 2,990,329). The antibiotic mixture A-35512 differs from the known antibiotics, for example in the running speed in various chromatographic systems and in the amino acid and sugar content.

3rd

637 159

There are a number of antibacterial agents in existence, but more new and improved antibiotics are still needed. A problem with frequent antibiotic therapy is the fact that the antibiotics differ in their effectiveness against pathogenic organisms. Another problem is that organism strains are developing that are resistant to the antibiotics used today. Another problem lies in the fact that individual patients often react strongly to certain antibiotics due to hypersensitivity and / or toxic effects. Because of these problems with today's therapy, the aim of the invention is to create a method for producing new antibiotics which can be used against diseases caused by microorganisms.

The antibiotics obtained according to the invention can be used in agents with which the feed conversion in ruminants and poultry can be improved, since the need to increase the feed conversion is becoming increasingly important. Such an increase in feed conversion in a number of animals and in poultry can already be achieved, for example, with diethylstilbestrol and other estrogens. However, these feed additives have the disadvantage that they remain in the meat intended for consumption. We are therefore looking for other and new ways to increase feed conversion in order to better utilize the limited amounts of feed when fattening ruminants and poultry. The antibiotic mixture A-35512 with its individual factors and their derivatives represents a step forward in this direction.

The invention is therefore based on the method described above for producing the antibiotic mixture A-35512 with the factors A, B, C, E, F, G and H, the antibiotic A-35512 factor A, the antibiotic A-35512 factor

B, the antibiotic A-35512 factor C, the antibiotic A-35512 factor E, the antibiotic A-35512 factor F, the antibiotic A-35512 factor G and the antibiotic A-35512 factor H as well as the aglycone derivative of factor B.

The antibiotic mixture A-35512 can be separated from the culture medium, the antibiotics A-35512 factors A, B, C, E, F, G and H can be isolated from the antibiotic mixture A-35512 and the antibiotic A from the antibiotic A-35512 -35512 Prepare factor B aglycon.

The number and ratio of the individual factors formed simultaneously with this antibiotic mixture fluctuate depending on the fermentation conditions used. The antibiotically active substances obtained according to the invention are arbitrarily referred to as antibiotics A-35512. The factor B is the predominant factor in the antibiotic mixture A-35512. The various factors of A-35512 occur in the mixture A-35512 and can be obtained individually from this mixture in the form of their hydrochloride salts. Each individual factor can be converted into its free base (namely the ionic chlorine-free form) by, for example, chromatographing it over a weakly acidic ion exchange resin. In addition to the forms of the free base and the hydrochloride salts, other pharmaceutically acceptable salts of the individual factors of the antibiotic A-35512 are also suitable. For the sake of brevity, the term compound A-35512 is simply used in the discussion about the usability of the present antibiotic in order in this way to obtain a compound from group A-35512 factors A, B,

C, E and H and their pharmaceutically acceptable salts.

The infrared absorption spectra (obtained with KBr pellets) of the following factors of the antibiotic A-35512 are shown in the drawing:

Figure 1 - A-35512 factor A dihydrochloride Figure 2 - A-35512 factor B dihydrochloride Figure 3 - A-35512 factor H hydrochloride Figure 4 - A-35512 factor C dihydrochloride 5 Figure 5 - A-35512 factor E hydrochloride

The infrared absorption spectrum of the antibiotic A-35512 factor B aglycone (measured in Nujol-Mull) is shown in FIG. 6.

The A-35512 factors obtained according to the invention are closely related compounds. Up to seven antibiotic factors can be isolated from the antibiotic mixture A-35512 obtained during cultivation. The individual factors can be separated from one another, and the factors A, B, C, E and H are preferably isolated as individual compounds in the manner described below. The antibiotic mixture A-35512 is soluble in water, partially dissolves in alcohols, such as methanol or ethanol, and is completely in other organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane, acetonitrile or dioxane 20 insoluble.

Antibiotic A-35512 factor A

Antibiotic A-35512 factor A is a white amorphous basic compound. The antibiotic A-35512 factor A 25 has an elemental composition of 54.29% carbon, 5.19% hydrogen, 5.58% nitrogen, 33.76% oxygen and 1.69% chlorine.

Antibiotic A-35512 factor A dihydrochloride is a white amorphous hygroscopic compound. The antibiotic 30-A 35512 factor A dihydrochloride has an average elemental composition of 51.03% carbon, 5.10% hydrogen, 4.75% nitrogen, 34.20% oxygen and 4.80% chlorine.

The infrared absorption spectrum of the antibiotic A-35512 factor A dihydrochloride in the form of a KBr-Press-35ling is shown in FIG. 1 of the drawing and has significant absorption maxima at the following frequencies (cm-1): 3405 (strong), 3300 (shoulder), 2950 (weak), 1750 (weak), 1670 (strong), 1625 (shoulder), 1602 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1405 (weak), 1345 40 (shoulder) , 1312 (medium), 1225 (medium), 1180 (weak), 1130 (weak), 1080 (strong) and 1020 (shoulder).

The ultraviolet absorption spectra of the antibiotic A-35512 factor A dihydrochloride show an absorption maximum at 282 nm (epsi-45 ion = 11,700) in acidic and neutral methanol and an absorption maximum at 292 nm (epsilon = 14,000) in basic methanol, calculated on the basis of of a molecular weight of 2000. The ultraviolet spectra of the antibiotic A-35512 factor A dihydrochloride also show a final absorption even at 225 nm.

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

65

No.

ppm

Height (%)

3rd

175.3

0.8

4th

173.0

2.1

5

172.1

2.0

6

171.4

1.5

7

170.9

2.7

8th

170.5

2.3

9

169.5

1.6

10th

159.0

1.3

11

157.9

2.3

12

156.2

2.6

13

155.6

2.3

637 159

4th

No.

ppm

Height (%)

14

155.3

2.4

15

154.4

1.1

16

136.3

1.4

17th

136.0

1.0

18th

135.1

1.4

19th

133.5

1.2

20th

129.6

1.6

21st

129.1

1.7

22

128.7

1.8

23

127.5

1.0

24th

126.0

1.4

25th

124.3

2.8

26

122.1

1.6

27th

109.9

1.3

28

107.4

1.6

29

101.7

0.9

30th

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 as standard

The antibiotic A-35512 factor A dihydrochloride has the following specific rotation values:

[alpha] o = —100 (c = l.mO)

[alpha] 365 = -400 (c = 1, H2O).

The electrometric titration of the antibiotic A-35512 factor A dihydrochloride in 66 percent aqueous dimethylformamide indicates the presence of four titrable groups with pKa values of about 7.35.9.09.10.49 and 12.44 (initial pH Value 6.2).

The apparent molecular weight of the antibiotic A-35512 factor A dihydrochloride, determined by titration, is approximately 2106.

The antibiotic A-35512 factor A dihydrochloride is soluble in water, partially soluble in alcohols, such as methanol or ethanol, and in other organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate,

Toluene, hexane, acetonitrile or dioxane are generally insoluble.

The antibiotic A-35512 factor A dihydrochloride is stable in aqueous solutions with pH values from about 3 to about 10 over a period of 72 hours.

An amino acid analysis of the acid hydrolyzed antibiotic A-35512 factor A dihydrochloride shows that the antibiotic A-35512 factor A contains at least 5 amino acid residues, one of which is glycine.

Antibiotic A-35512 factor B

Antibiotic A-35512 factor B is a white amorphous basic compound. The antibiotic A-35512 factor B has a possible empirical formula from Cgv.çgHioi-iosNg.gC ^ e-5 48C1. Antibiotic A-35512 factor B has an average elemental composition of 53.97% carbon, 4.75% hydrogen, 5.25% nitrogen, 34.29% oxygen and 1.59% chlorine.

The above elemental composition is particularly in agreement with the preferred empirical formula C98H104N9O47CI 10 (calculated: C = 53.60; H = 4.75; N5.74; O = 34.30; Cl = 1.61). Another preferred empirical formula is C98H103N8O47CI (calculated: C = 54.00; H = 4.75; N = 5.15; O = 34.50; C1 = 1.60).

The ultraviolet absorption spectra of the antibiotic 15 A-35512 factor B show an absorption maximum at 282 nm (epsilon = 15,000) in acidic and neutral methanol and an absorption maximum at 292 nm (epsilon = 16,000) in basic methanol, calculated on a molecular weight of 2000. The ultraviolet spectra of the antibiotic A-35512 Fak-20 tor B also show a final absorption at 225 nm.

The antibiotic A-35512 factor B has the following specific rotation values:

[alphatè5 = -123 ° (c = 1, H2O).

[alphalose = -446 ° (c = 1, H2O).

25 The electrometric titration of the antibiotic A-35512 factor B in 66 percent aqueous dimethylformamide indicates the presence of four titratable groups with pKa values of about 7.15.8.81.10.20 and 12.00 and the possible presence another group with a pKa-30 value of over 13.50.

The apparent molecular weight of the antibiotic A-35512 factor B, determined by titration, is approximately 2143.

The antibiotic A-35512 factor B dihydrochloride is a white crystalline compound (from 50 percent aqueous methanol). The antibiotic A-35512 factor B dihydrochloride is hygroscopic and does not have a well-defined melting point, but a corresponding thermogram shows a weight loss starting at 25 ° C, with a 7.4 percent weight loss at 121 ° C and a further weight loss with decomposition at 135 ° C.

The antibiotic A-35512 factor B dihydrochloride has an average elemental composition of 52.57% carbon, 4.80% hydrogen, 5.66% nitrogen, 32.86% oxygen 45 and 4.51% chlorine. The above elemental composition. exactly matches another possible empirical formula C98H103N9O47CI • 2HC1 (calculated: C = 51.93; H = 4.65; N = 5.57; O = 33.20; Cl = 4.65).

The ultraviolet spectra of the antibiotic A-35512 factor so B dihydrochloride show an absorption maximum at 282 nm (epsilon = 12,000) in acidic and neutral methanol and an absorption maximum at 292 nm (epsilon = 14,000) in basic methanol, calculated using a molecular weight of 2000. The ultraviolet spectrum of the antibiotic 55 k A-35512 factor B dihydrochloride also shows a final absorption even at 225 nm.

The antibiotic A-35512 factor B dihydrochloride has the following specific rotation values:

[alphajo5 = -128 (c = 1, H2O)

60 [alpha] H5 = -475 ° (c = 1, H2O).

The electrometric titration of the antibiotic A-35512 factor B dihydrochloride in 66 percent aqueous dimethylformamide indicates the presence of four titrable groups with pKa values of about 7.15, 8.87.10.30 and 12.10 65 as well as the possible presence another group with a pKa of over 13.1.

The apparent molecular weight of the antibiotic A-35512 factor B dihydrochloride determined by titration is approximately 2027.

5

637 159

The 13C nuclear magnetic resonance spectrum of the antibiotic A-35512 factor B dihydrochloride in D2O has the following characteristics:

Antibiotic A-35512 factor B dihydrochloride recrystallized from methanol-water has the following characteristic powder X-ray diffraction pattern (Cu + + radiation lambda 1.5405, nickel filter, d = interplanar distance in angstroms):

No.

ppm

Height (%)

2nd

173.0

4.1

3rd

171.9

3.7

4th

171.6

3.3

5

171.0

5.8

6

170.8

5.0

7

169.6

3.6

8th

159.0

4.1

9

157.9

4.4

10th

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

154.3

4.2

16

151.7

3.3

17th

144.3

3.1

18th

136.7

3.5

19th

136.2

4.9

20th

135.4

4.0

21st

135.2

4.4

22

133.6

4.2

23

133.3

4.1

24th

129.8

1.7

25th

129.3

3.0

26

128.8

2.6

27th

127.6

1.5

28

126.1

3.9

29

124.2

5.6

30th

122.4

1.4

31

122.0

4.4

32

120.7

3.3

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

d

Relative intensity

17.15

100

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

25 The infrared absorption spectrum of the antibiotic A-35512 factor B dihydrochloride in the form of KBr pressings is shown in FIG. 2 of the drawing. The most significant absorption maxima occur at the following frequencies (cm-1): 3420 (strong), 3300 (shoulder), 2950 (weak), 1752 30 (weak), 1675 (strong), 1630 (shoulder), 1605 (strong), 1520 (strong), 1470 (weak), 1440 (weak), 1410 (weak), 1345 (shoulder), 1312 (medium), 1225 (medium), 1180 (medium), 1135 (weak), 1080 (strong) and 1020 (weak).

Amino acid analysis of the acid hydrolyzed A-35512 35 factor B dihydrochloride shows that the antibiotic A-35512 factor B contains at least 5 amino acid residues, one of which is glycine. The four remaining amino acid residues in antibiotic A-35512 factor B are complex and seem to be identical to those found in antibiotic A-35512 factor A to 40. The structure of one of these amino acid residues is likely to be as follows:

HOOG Mte i r

^ OO

L I

f / ^ COOH

* Dioxane as standard

55 An analysis of his acid-hydrolyzed products shows that the antibiotic A-35512 factor B dihydrochloride contains the following sugars: glucose, fructose, mannose, rhamnose and 3-amino-2,3,6-trideoxy-3-C-methyl-L-xylohexopyranose .

The glucose, fructose, mannose and rhamnose groups are removed by mildly acidic hydrolysis of the antibiotic A-35512 Fak-60 tor B dihydrochloride, and a characteristic aglycone derivative is formed.

Antibiotic A-35512 factor B dihydrochloride has at least one esterifiable hydroxyl group. 65 The antibiotic A-35512 factor B dihydrochloride is soluble in water, partly dissolves in alcohols, such as methanol or ethanol, and is in other less polar organic solvents, such as benzene, chloroform, acetone, diethyl-

637 159

6

ether, ethyl acetate, toluene, hexane, acetonitrile or dioxane, insoluble.

Antibiotic A-35512 factor B dihydrochloride is stable in aqueous solutions with pH values from about 3 to about 10 to 72 hours.

Antibiotic A-35512 factor C.

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

Antibiotic A-35512 factor C has an average elemental composition of 53.93% carbon, 5.15% hydrogen, 5.80% nitrogen, 32.35% oxygen and 1.90% chlorine.

The above elemental composition is in accordance with the preferred empirical formula C84H99N8O38CI. Antibiotic A-35512 factor C has a molecular weight of approximately 1862.

The antibiotic A-35512 factor C dihydrochloride is a white amorphous compound. Antibiotic A-35512 factor C dihydrochloride has a mean elemental composition of 51.76% carbon, 5.07% hydrogen, 5.61% nitrogen, 30.29% oxygen and 4.88% chlorine.

The empirical formula for the antibiotic A-35512 factor C dihydrochloride corresponds approximately to Cgß.gsHsv.ioiNsOsv.sgCb.

The elemental composition is exactly the same as another preferred empirical formula Cs4H97N8038C1.2HCl (calculated: C = 52.2%; H = 5.1%; N = 5.8%; O = 31.5%; Cl = 5.4 %).

The infrared absorption spectrum of the antibiotic A-35512 factor C dihydrochloride in the form of KBr press lines is shown in FIG. 4 of the drawing. The most significant absorption maxima occur at the following frequencies (cm-1): 3370 (strong), 3280 (shoulder); 3040 (shoulder), 2980 (shoulder), 2920 (weak), 1740 (weak), 1658 (strong), 1620 (weak), 1589 (medium), 1503 (strong), 1460 (weak), 1428 (medium), 1385 (weak), 1330 (weak), 1295 (medium), 1210 (strong), 1162 (medium), 1120 (weak), 1060 (strong) and 1005 (medium).

The ultraviolet absorption spectra of the antibiotic A-35512 factor C dihydrochloride show an absorption maximum at 282 nm (epsilon = 14,600) in acidic and neutral methanol and an absorption maximum at 292 nm (epsilon = 16,400) in basic methanol, calculated using a molecular weight of 2000 .

The 13C nuclear magnetic resonance spectrum of the antibiotic A-35512 factor C dihydrochloride in D2O has the following characteristics:

No.

ppm

Height (° / o)

18th

126.5

3.1

19th

124.6

2.1

5 20

129.3

2.7

21st

121.5

2.2

22

120.1

1.2

23

118.0

1.7

24th

116.5

1.2

10 25

107.8

2.7

26

104.5

1.9

27th

101.7

1.9

28

94.5

1.0

29

75.9

3.0

15 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

20.35

67.4

71.7 *

36

64.3

1.3

37

62.2

1.6

38

56.1

1.4

39

55.2

3.5

25 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

30th

* Dioxane as standard

No.

ppm

Height (%)

1

172.9

2.5

2nd

172.2

2.0

3rd

171.5

2.2

4th

171.0

3.9

5

169.6

2.0

6

158.6

1.8

7

157.8

3.0

8th

156.5

2.1

9

156.1

2.8

10th

155.6

4.2

11

154.6

3.9

12

151.1

1.5

13

143.3

1.4

14

136.0

3.2

15

135.4

2.7

16

133.2

3.7

17th

128.7

2.3

The antibiotic A-35512 factor C dihydrochloride has the following specific rotation values:

[alpha] o5 = -161 ° (c = 1.05, H2O)

[alpha] || 5 = -614 ° (c = 1.05, H2O).

The electrometric titration of the antibiotic A-35512 40 factor C dihydrochloride in 66 percent aqueous dimethylformamide indicates the presence of three titrable groups with pKa values of about 7.30.8.92 and 10.99 and the possible presence of two or more Groups with pKa values of over 11.5.

45 The apparent molecular weight of the antibiotic A-35512 factor C dihydrochloride determined by titration is about 1982.

Amino acid analysis of the acid hydrolyzed antibiotic A-35512 factor C dihydrochloride shows that it contains 50 at least 5 amino acid residues, one of which appears to be glycine. The four other amino acid residues have not yet been identified.

The antibiotic A-35512 factor C dihydrochloride is soluble in water, dimethyl sulfoxide and aqueous dimethylformamide 55, partially dissolves in alcohols, such as methanol or ethanol, and is in less polar organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene , Hexane, acetonitrile or dioxane, insoluble.

The antibiotic A-35512 factor C dihydrochloride is stable in 60 aqueous solutions with pH values from about 3 to about 10 to 146 hours.

Antibiotic A-35512 factor E.

The antibiotic A-35512 factor E is a white amor-65 phe basic compound. Antibiotic A-35512 factor E has an elemental composition of about 54.84% carbon, 4.73% hydrogen, 5.26% nitrogen, 32.67% oxygen and 1.72% chlorine.

7

637 159

Antibiotic A-35512 factor E hydrochloride is a white amorphous compound. Antibiotic A-35512 factor E hydrochloride has an elemental composition of approximately 52.67% carbon, 4.59% hydrogen, 5.55% nitrogen, 33.51% oxygen and 3.62% chlorine. 5

The infrared absorption spectrum of the antibiotic A-35512 factor E hydrochloride in the form of KBr pellets is shown in FIG. 5 of the drawing. The most significant absorption maxima occur at the following frequencies (cm-1): 3360 (strong), 3220 (shoulder), 2900 (weak), 1725 io

(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 spectra of the antibiotic A-35512 factor E hydrochloride have the following absorption maxima: in 15 neutral methanol at 270 nm (shoulder) and 359 nm (epsilon = 16 216), in acidic methanol at 286 nm (epsilon = 18 018) and 310 nm (Shoulder), in basic methanol at 270 nm (shoulder), 300 nm (epsilon = 16 216) and 354 nm (epsilon = 17 568), calculated using a molecular weight 20 of 2000.

The antibiotic A-35512 factor E hydrochloride has the following specific rotation:

[alpha] o5 = -108.3 (c = 1, H2O).

(medium), 1118 (weak), 1135 (weak), 1080 (strong) and 1020 (shoulder).

The ultraviolet absorption spectra of the antibiotic A-35512 factor H hydrochloride show an absorption maximum at 282 nm (epsilon = 12,500) in acidic and neutral methanol and an absorption maximum at 292 nm (epsilon = 14,000) in basic methanol, calculated using a molecular weight of 2000. The ultraviolet spectra of the antibiotic A-35512 factor H hydrochloride also show a final absorption at 225 nm.

The 13C nuclear magnetic resonance spectrum of the antibiotic A-35512 factor H hydrochloride in D2O has the following characteristics:

25th

The electrometric titration of the antibiotic A-35512 factor E hydrochloride in 66 percent aqueous dimethylformamide indicates the presence of three titrable groups with pKa values of about 6.30.9.09 and 11.62 and the possible presence of one or two Groups with pKa values of about 12.5 or above (at an initial pH of 5.45).

The apparent molecular weight of the antibiotic A-35512 factor E hydrochloride determined by titration is about 2018.

Amino acid analysis of the acid-hydrolyzed antibiotic A-35512 factor E hydrochloride shows that the antibiotic A-35512 factor E contains six amino acid residues not yet identified.

The antibiotic A-35512 factor E hydrochloride is soluble in water, partially soluble in alcohols, such as methanol or ethanol, in most organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate,

Toluene, hexane, acetonitrile or dioxane, but insoluble.

Antibiotic A-35512 factor H

Antibiotic A-35512 factor H is a white amorphous basic compound. Antibiotic A-35512 factor H has an elemental composition of approximately 53.76% carbon, 5.32% hydrogen, 5.53% nitrogen, 33.48% oxygen and 1.59% chlorine.

The antibiotic A-35512 factor H has the empirical formula C85_87Hio3-io7Ns038_4oClJ, the preferred empirical formula being CsöHkmNsOjsCI, and has a molecular weight of about 1908.

The antibiotic A-35512 factor H hydrochloride is a white amorphous hydroscopic compound. Antibiotic A-35512 factor H hydrochloride has an elemental composition of approximately 53.10% carbon, 5.37% hydrogen, 5.35% nitrogen, 30.12% oxygen and 3.78%

Chlorine.

The infrared absorption spectrum of the antibiotic A-35512 factor H hydrochloride in the form of KBr pellets is shown in FIG. 3 of the drawing. The most significant absorption maxima occur at the following frequencies (cm-1): 3410 (strong), 3240 (shoulder), 2940 (weak), 1670 (strong), 1630 (shoulder), 1605 (strong), 1520 (strong), 1470 (weak), 1442 (weak), 1400 (weak), 1345 (shoulder), 1310 (medium), 1225

30th

35

40

45

50

55

60

65

No.

ppm

Height (%)

2nd

177.2

2.7

3rd

171.6

5.2

4th

170.9

5.8

5

169.6

4.7

6

158.9

3.1

7

157.6

4.3

8th

156.6

3.8

9

155.6

4.1

10th

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

136.0

4.0

16

135.3

1.9

17th

133.5

5.0

18th

129.4

3.7

19th

127.3

1.3

20th

126.1

3.2

21st

124.2

6.9

22

122.6

4.1

23

107.6

2.7

24th

101.8

1.8

25th

76.2

2.8

26

73.5

4.4

27th

72.3

7.4

28

71.0

12.2

29

69.7

4.6

30th

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 as standard

The antibiotic A-35512 factor H hydrochloride has the following specific rotation:

[alpha] o = -123.5 ° (c = 1, H2O).

The electrometric titration of the antibiotic A-35512 factor H hydrochloride in 66 percent aqueous dimethylformamide indicates the presence of five titrable groups with pKa values of about 5.0.7.46.9.80.11.43 and 13.02 (Initial pH 5.93).

637 159

8th

The apparent molecular weight of the antibiotic A-35512 factor H hydrochloride determined by titration is approximately 1660.

Amino acid analysis of the acid hydrolyzed antibiotic A-35512 factor H hydrochloride shows that the antibiotic A-35512 factor H contains at least 5 amino acid residues, one of which is glycine. The other four amino acid residues in antibiotic A-35512 factor H appear to be identical to those found with antibiotic A-35512 factor A and B.

The antibiotic A-35512 factor H hydrochloride is soluble in water, only partially soluble in alcohols, such as methanol or ethanol, but is in most organic solvents, such as benzene, chloroform, acetone, diethyl ether, ethyl acetate, toluene, hexane , Acetonitrile or dioxane, insoluble. J,

Antibiotic A-35512 factor H hydrochloride is stable in aqueous solutions with pH values from about 3 to about 1 hour up to 72 hours.

The separation of the antibiotic mixture À-35512 into the individual factors A, B, C, E and H and the less common factors F and G is carried out by paper chromatography using a solvent system consisting of 1-butanol, pyridine, acetic acid and water (15: 10: 3: 12 ). A preferred method of detection is bioautography using Sarcina lutea. The approximate Rr values of the individual factors of antibiotic A-35512 in this system are shown in Table I below.

Table I

Column size:

3.175x 152.4 mm

Pack:

Polyamide (ZIPAX, DuPont)

Solvent:

KH2P04 (327 g) / HzO (1800 ml)

Flow velocity:

0.5 ml / min

Card speed:

5.1 cm / h

Print:

105 kg / cm2

Table III

A-35512 factor

Dwell time (minutes)

A

9,375

B

13.125

C.

26.25

E

5,625

F

7.5

G

7.5

H

7.5

A-35512 factor

RF value

Factor A-2HC1

0.21

Factor B-2HC1

0.34

Factor C-2HC1

0.46

E-HCl factor

0.64

Factor F

0.81

Factor G.

0.93

H-HCl factor

0.15

In various systems, the antibiotic A-35512 factor H has a chromatographic profile very similar to that of the antibiotic AM374 (US Pat. No. 3,700,768). The antibiotic A-355.12 factor H can be differentiated from the antibiotic AM374 in at least two paper chromatography systems. The Rr values for the antibiotic A-35512 factor H • 2HC1 and AM374 in these two systems are shown in Table II below:

Table II

RF value

Solvent system

A-35512 H

AM374

CH3OH: 0.1N HCl (3: 1)

0.47

0.58

l-propanol: NH40H: H20 (6: 3: 1)

0.11

0.20

Several factors of the antibiotic mixture A-35512 can also be seen by high-performance liquid chromatography (HPLC) using polyamide (ZIPAX, DuPont) as the stationary phase and a 1.33 molar aqueous single-base no. Potassium phosphate solution as the mobile phase and by ultraviolet detection (250 nm ) separate from each other. The following table III shows the retention times for the factors of the antibiotic A-35512 in a typical separation using high-performance liquid chromatography using the following conditions: 5

20 Antibiotic A-35512 factor B aglycone

Antibiotic A-35512 factor B aglycon hydrochloride is a white amorphous compound with an elemental composition of approximately 54.29% carbon, 4.34% hydrogen, 7.40% nitrogen; 5.02% chlorine and 28.95% oxygen 25 (by difference).

The infrared absorption spectrum of the antibiotic A-35512 factor B aglycon hydrochloride in Nujol-Mull is shown in Fig. 6 of the drawing. The most significant absorption maxima occur at the following frequencies (cm-1): 3440 30 (shoulder), 3340 (shoulder), 3215 (strong), 2950 (shoulder), 2910 (strong), 2840 (strong), 2640 (shoulder), 1735 (weak), 1655 (strong), 1590 (medium), 1500 (strong), 1460 (strong), 1378 (medium), 1365 (shoulder), 1298 (medium), 1215 (medium), 1155 (medium), 1120 (shoulder), 1105 (weak), 1060 (weak), 1040 (weak), 35 1008 (medium), 925 (weak), 875 (weak), 765 (shoulder) and 718 (weak).

The electrometric titration of the antibiotic A-35512 factor B aglycon hydrochloride in 66 percent aqueous dimethylformamide indicates the presence of three titrable groups with pKa values of approximately 7.5.9.25 and 11.0 and the possible presence of two further groups with pKa values of over 11.0.

The antibiotic A-35512 factor B aglycon hydrochloride has a molecular weight determined by titration of about 45 1282.

The antibiotic A-35512 factor B aglycon hydrochloride has the following specific rotation values:

[alpha] o = -178 ° (c = 5, CHsOH)

50

[alphaÜs = -716.8 ° (c = 5, CH3OH).

The ultraviolet absorption spectra of the antibiotic A-35512 factor B aglycon hydrochloride show an absorption maximum at 282 nm (E'um = 102.62) in acidic and 55 neutral methanol and an absorption maximum at 302 nm (E '^ m = 182.09) in basic methanol. .

The 13C magnetic nuclear magnetic resonance spectrum of the antibiotic A-35512 factor B aglycone in DMSO-dó at 90 ° C has the following characteristics:

ppm

Height (%)

187.8

172.0 170.7

170.1 169.6

37.2 40.9 45.2 46.9 47.7

9

637 159

No.

ppm

Height (° / o)

Antibiotic A-35512 factor B aglycon hydrochloride

contains at least one esterifiable hydroxyl group.

6

168.4

57.6

Antibiotic A-35512 factor B aglycon hydrochloride

7

166.7

52.5

is soluble in water and methanol, less polar

8th

157.4

49.9

s solvents such as benzene, chloroform, acetone, diethyl

9

156.6

45.5

ether, ethyl acetate, toluene, hexane, acetonitrile or dioxane,

10th

155.7

55.8

however insoluble.

11

155.6

71.5

Antibiotic A-35512 factor B aglycon hydrochloride

12

155.4

56.7

has in a cellulose thin layer chromatography (aluminum

13

154.5

50.5

ion carrier) using a solvent system

14

149.3

43.0

1-butanol, pyridine, acetic acid and water (15: 10: 3: 12) one

15

138.8

38.8

Rr value of around 0.80. The detection is preferably carried out

16

136.9

54.3

by bioautography using Sarcina lutea.

17th

136.3

40.2

Antibiotic A-35512 factor B aglycon hydrochloride

18th

135.2

31.7

15 results in silica gel thin layer chromatography under

19th

134.7

28.4

Use of a solvent system made of methanol, Chlo

20th

133.8

40.9

roform and concentrated ammonium hydroxide (3: 2: 1)

21st

128.2

102.9

Rr value of about 0.26.

22

126.2

77.2

The antibiotic A-35512 factor B aglycone (free base)

23

123.0

57.5

20 is a white amorphous compound with a medium ele

24th

121.3

38.1

mentary composition of about 52.65% carbon, 4.57%

25th

117.7

44.4

Hydrogen, 6.91% nitrogen, 2.94% chlorine, 27.04% oxygen

26

117.0

31.5

and 4.70% ash.

27th

108.6

31.0

The infrared absorption spectrum of the antibiotic

28

106.7

47.9

25 A-35512 factor B aglycone (free base) in the form of KBr press

29

105.7

81.2

lingen has significant absorption maxima in the following

30th

103.2

26.5

Frequencies (cm-1) on: 3360 (strong), 3260 (shoulder), 2940

31

93.6

33.4

(Shoulder), 1735 (shoulder), 1660 (strong), 1598 (medium), 1510

32

74.9

39.8

(strong), 1440 (medium), 1295 (weak), 1215 (medium), 1165 (with-

33

71.8

33.9

3o tel), 1122 (weak), 1070 (weak), 1018 (strong), 940

34

68.9

40.2

(weak) and 920 (weak).

35

63.2

43.1

Electrometric titration of the antibiotic A-35512

36

60.4

33.6

Factor B aglycone (free base) in 66 percent aqueous

37

57.1

57.8

Dimethylformamide indicates the presence of five titrable

38

55.2

33.1

35 ren groups with pKa values of about 6.2, 8.2, 10.1, 11.4 and

39

53.2

30.1

12.4 and the possible presence of one or two others

40

51.8

36.0

Groups with pKa values above 12.5.

41

40.7

43.4 *

The antibiotic A-35512 factor B aglycone (free base)

42

39.9

60.9 *

has the following specific rotation:

43

39.1

43.9 *

40 [alphass5 = -64.5 ° (c = 3, DMSO).

44

23.8

55.7

The ultraviolet absorption spectra of the antibiotic

45

17.1

47.8

A-35512 factor B show aglycone (free base) in neutral and

46

0.0

43.7

acidic methanol an absorption maximum at 282 nm (E '^ n,

= 43.65) and an absorption maximum in basic methanol

* DMSO-dó

45 at 301 nm (E10 / 2m = 67.46).

The antibiotic A-35512 factor B aglycone (free base) The UC magnetic nuclear magnetic resonance spectrum shows that it has the same approximate Rr values as previously for the 3-amino antibiotic A-35512 factor B also in the antibiotic A-35512 factor B aglycone Aglycon hydrochloride have been described.

so In addition to the free base and hydrochloride of the individual factors of the antibiotic mixture A-35512 and the antibiotic A-35512 factor B aglycone, other pharmaceutically acceptable acid addition salts of the antibiotic A-35512 with the factors A, B, C, E and H and 55 of the antibiotic A-35512 factor B aglycone. Pharmaceutically acceptable salts are understood to be salts which, compared to the non-salt form of the compounds, do not result in an overall increase in toxicity to warm-blooded animals. Typical and suitable salts 60 of the antibiotic A-35512 with the factors A, B, C, E and H and of the antibiotic A-35512 factor B aglycone are those which are formed by customary reaction with organic or inorganic acids, for example with sulfuric acid, Phosphoric acid, acetic acid, succinic acid, citric acid 65, lactic acid, maleic acid, fumaric acid, palmitic acid, cholic acid, pamoic acid, mutic acid, D-glutamic acid, d-camphoric acid, glutaric acid, glycolic acid, phthalic acid, tartaric acid, lauric acid, stearic acid, salicylic acid, salicylic acid

2,3,6-trideoxy-3-C-methyl-L-xylohexopyranoseest (namely one of the sugars, which are also present with the antibiotic A-35512 factor B) is contained.

The amino acid analysis of the further acid-hydrolyzed antibiotic A-35512 factor B aglycon hydrochloride shows that the antibiotic A-35512 factor B contains aglycon glycine and at least three complex amino acid residues. The structure of one of these amino acid residues is likely to be as follows:

HOOG RHz i r

YY

1 X

H H COOH

2nd

637 159

10th

15

20th

fonic acid, benzenesulfonic acid, sorbic acid, picric acid, benzoic acid or cinnamic acid.

The new antibiotics are produced according to the invention by cultivating a strain of Streptomyces Candidus NRRL 8156 submerged, which forms the antibiotic A-35512, under aerobic conditions in the culture medium defined above until substantial antibiotic activity is formed. The antibiotics obtained can be obtained using various isolation and purification methods known in fermentation technology.

The new microorganism suitable for the production of antibiotics A-35512 was isolated from a soil sample collected on the Eniwetok Atoll. This microorganism can be identified as a new strain of Streptomyces Candidus (Kressilnikov) Waksman according to E.B. Classify Shirling and D. Gottlieb in “Cooperative Description of Type Cultures of Streptomyces”.

This classification is based on methods as used 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)] may be recommended, along with certain additional studies. The color names are designated according to the ISCC-NBS method (K.L. Kelly and D.B. Judd, "The ISCC-NBS Method of Determining Colors and a Dictionary of Color Names", U.S. Department of Commerce Cir. 553, Washington, D.C., 1955). The numbers in parentheses refer to the color series by Tresner and Backus [H.D. Tresner and S.J. Backus, "System of Color Wheels for Streptomycete Taxonomy", Appi. Microbiol. 11: 335-338 (1963)], with the names of the colored stripes underlined. The color blocks according to Maerz and Paul (A. Maerz and M.R. Paul, "Dictionary of Color", McGraw-Hill, New York, N.Y "1950) are given in brackets. Unless otherwise stated, the cultures are allowed to grow at 30 ° C for 14 days each.

Characterization of the A-35512 producing microorganism

morphology

Long wavy sporophores are formed. The spores, which appear in chains of 10 to 50, are cylindrical and have dimensions of 0.7 to 1.05 jx x 1.4 to 3.5 (x. Sclerotium-like structures arise on several media. Occasionally, broom-shaped or Detect tuft-like hyphae, according to an electron microscopic examination.

medium

Characteristics

Glycerin Asparagine Agar (ISP No. 5)

Emerson agar modified Bennet agar

Czapek solution agar

30th

35

40

Tomato paste oatmeal agar

Breeding behavior on different media

medium

Characteristics

50

Nutrient agar

Glucose-asparagine agar

T rypton yeast extract agar

Tyrosine agar

Yeast extract-malt extract agar (ISP No. 2)

Oatmeal Agar (ISP No. 3)

Inorganic

Salts-Starch Agar (ISP No. 4)

Strong growth, underside greyish yellow (11J5), abundant aerial mycelium and sporulation, white (f) a, no soluble (pigment;

medium growth,

Underside pale yellow-green [10B1], fairly aerial mycelium and sporulation, white (f) 13ba,

no soluble pigment, sclerotium-like bodies can be identified;

good growth, underside amber white [10C1], good aerial mycelium and good sporulation, yellowish gray (GY)

55

Glycerin-glycine agar

60

Calcium malate agar

65

2dc, slightly brown soluble pigment, sclerotium-like bodies can be identified;

good growth, underside pale yellow-green [10B2], good aerial mycelium and good sporulation, white (f) a, no soluble pigment, the mycelium appears to be tufted; good growth, light olive underside [14L6], no aerial mycelium and no sporulation, light brown soluble pigment; abundant growth, underside moderately yellow [11J6], abundant aerial mycelium and abundant sporulation, white (f) a, no soluble pigment; good growth, underside pale yellow-green [10B1], good aerial mycelium and good sporulation, white (w) b, no soluble pigment, the mycelium appears like tufts; abundant growth, greyish yellow underside [1115], abundant aerial mycelium and abundant sporulation, white (f) a, the mycelium rolls off the agar surface, no soluble pigment;

moderate to good growth, underside pale yellow-green [18D2], good aerial mycelium and good sporulation, white (f) a, light brown soluble pigment; good growth, underside pale yellow-green [17E1], good aerial mycelium and good sporulation, white (f) 13ba, no soluble pigment; moderate growth, underside white [10A1],

tolerable aerial mycelium and sporulation, white (f) b, no soluble pigment;

good growth, underside white [10B2], good aerial mycelium and good sporulation, white (f)

a, brown soluble pigment, sclerotium-like pigments can be found;

good growth, underside white [10B2], good aerial mycelium and good sporulation, white (f)

b, light brown soluble pigment;

good growth, underside white [10B1], good aerial mycelium and good sporulation, white (f) a, no soluble pigment, the medium clears up around the zone of the inoculum.

11

637 159

The microorganism is also examined using standard methods for selected physiological properties. This results in the following:

properties

Characteristics

Effect on milk

Nitrate reduction Melanin formation on: Pepton-iron inclined agar T y rosin inclined agar

T rypton yeast extract broth liquefying gelatin

Temperature requirements

Yeast extract-malt extract-inclined agar (ISP Medium No. 2):

Coagulation under a certain clarification after 14

Days;

positive;

negative;

weakly positive (pigment after 7 days)

negative;

completely finished after 14 days;

26-30 ° C, good growth and good sporulation; 37 ° C - sparse growth, no aerial mycelium and no spores;

40 ° C - only slight vegetative growth;

45 ° C - no growth

The results obtained from corresponding studies on the carbon utilization of the microorganism NRRL 8156 can be seen from the following list:

Rating:

+ = good growth, positive recovery (+) = weak to moderate growth (-) = low growth, possibly no recovery - = no growth, no recovery

Carbon source:

None (negative control) D-glucose (positive control) L-arabinose sucrose iso-inositol D-mannitol D-fructose rhamnose raffinose D-xylose

(-) +

(-)

(") +

+ +

(~) (-) (+)

NRRL8156

Published description iso-inositol

+

-

Gelatin liquefaction completely slowly

after 14 days

Effect on milk

Coagulation;

no

certain up

Coagulation;

clarification for good

14 days

Peptinization

Certain properties of the A-35512-forming strain S. Candidus NRRL 8156 differ from the properties of the microorganism described by Shirling and Gottlieb [Elwood, B. Shirling and David Gottlieb, “Cooperative Description of Type Cultures of Streptomyces III. Additio nal Species Descriptions from First and Second Studies »,

Intern. J. Systematic Bacteriol. 18 (2), 69-189 (1968)] and by Waksman [S.A. Waksman, "The Actinomycetes. Classification, Identification and Descriptions of Genera and Species, "Vol. 2, The Williams and Wilkins Co., Baltimore, Md., 1961]. These differences are shown in Table IV below:

Table IV

NRRL 8156

Published description

Carbon utilization

L-arabinose

Rhamnose

+ +

A culture of the microorganism Streptomyces Candidus producing the antibiotic A-35512 is in the permanent collection of the Northern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, Peo-ria, Illinois, 61604, and from there freely available under the filing number NRRL 8156.

As with other organisms, the properties 20 of the culture producing the antibiotic A-35512, namely the Streptomyces Candidus NRRL 8156, are subject to variation. For example, artificial variants and mutants of the strain NRRL 8156 can be formed by treatment with various known mutagenic agents, such as ultraviolet rays, X-rays, high-frequency waves, radioactive radiation or chemicals. All natural and artificial variants and mutants which belong to Streptomyces Candidus and which produce antibiotics A-35512 can be used according to the invention.

30 Any of the many culture media can be used to grow the microorganism Streptomyces Candidus NRRL 8156. However, certain culture media are preferred for reasons of economical production, optimal antibiotic yield and easy isolation of the product. For example, the carbohydrate source preferably used for large-scale fermentation is sucrose, although glucose, tapioca dextrin, fructose, mannitol, maltose, lactose, or the like can be used instead. A preferred nitrogen source is meat peptone, but soybean meal, pig blood meal, amino acids such as glutamic acid, or the like can also be used. As inorganic nutrient salts, customary soluble salts can be added to the culture media, which provide zinc, sodium, magnesium, calcium, ammonium 45, chloride, carbonate, sulfate or nitrate ions.

The trace elements required for the growth and development of the microorganism should also be present in the culture medium. These trace elements normally appear as impurities in the other constituents of the medium in such amounts as are necessary for the growth of the microorganism.

If you have to deal with the problem of foaming of the fermentation media in large-scale fermentation,

then you must preferably add small amounts (namely 55 with 0.2 ml per liter) of an anti-foaming agent, such as propylene glycol.

Submerged aerobic fermentation in tanks or containers is preferred for the formation of substantial amounts of antibiotics A-35512. Small amounts of the antibiotics 60 A-35512 can also be produced by shaking flasks. Because of the time delay that occurs in antibiotic formation when inoculating large tanks or containers with the spores of the microorganism, a vegetative inoculum is preferably used. The vegetative inoculum 65 can be prepared by inoculating a small volume of the culture medium with the spore shape or with mycelium fragments of the microorganism, whereby a fresh, actively growing culture of the organism is obtained. The vegetative

637 159

12

Vaccination culture is then usually transferred to a larger container.

The microorganism producing the antibiotics A-35512 can be grown at temperatures between about 20 and 40 ° C. Optimal formation of the antibiotics 5 A-35512 appears to take place at temperatures of around 30 to 34 ° C.

As usual with submerged aerobic breeding methods,

in this case, too, air is preferably blown through the culture medium. To achieve efficient growth of the microorganism, the volume of air used in tank production should preferably make up more than 0.1 volume of air per volume of culture medium per minute (V / V / M). For a sufficient production of the antibiotics A-35512 one generally works with a tank production with an air volume of approximately 0.25 V / V / M.

The formation of the antibiotics A-35512 can be followed during the fermentation by using appropriate samples of the culture broth or extracts of the mycelium solids with regard to their antibiotic activity against micro-20

Examine organisms whose sensitivity to antibiotics is known. A particularly suitable detection organism for this purpose is Bacillus subtilis ATCC 6633. This bioassay is preferably carried out on paper disks or agar plates which contain a nutrient-limited medium 25.

The antibiotics A-35512 produced under appropriate submerged aerobic fermentation conditions can be obtained from the fermentation medium after their formation by methods customary in fermentation technology. The antibiotic activity which arises during the fermentation of the A-35512 producing microorganism generally occurs in the filtered culture broth. A maximum recovery of the antibiotics A-35512 therefore results if the mycelium mass is first filtered off from the culture broth. The filtered broth obtained in this way can then be purified by any technique to give the antibiotic mixture A-35512. A preferred method of obtaining these antibiotics is to adsorb the filtered broth on a polyamide column and to elute the column with water and aqueous alcohol mixtures. The thereby eluted antibiotic fractions can be combined to form the antibiotic mixture A-35512. Optionally, using this technique, the eluted fractions can also be pooled based on their behavior in thin layer chromatography, resulting in purified antibiotic A-35512 factor B and enriched mixtures of the other A-35512 factors.

The further purification of the individual factors of the antibiotic A-35512 generally consists of an additional 50 adsorption and extraction processes. Used with advantage

30th

35

For this purpose, adsorptive materials, such as aluminum oxide, silica gel, ion exchange resins or the like. The factors of the antibiotic A-35512 occur in the fermentation broth as hydrochloride. The preferred separation method using polyamide gives the antibiotic A-35512 factor B and the balance the mixture of the other factors of the antibiotic A-35512 in the form of the hydrochloride. Each individual factor can be converted into its free base (ionic chlorine-free compound) in a known manner, for example chromatographically, using a weakly basic ion exchange resin.

Optionally, the culture solids including the components of the medium and the mycelium can also be used as a source for the antibiotic A-35512 without extraction or separation, but preferably after removal of the water. For example, after the antibiotic A-35512 has been formed, the culture medium can be dried by lyophilization and the material obtained can then be mixed directly into a feed premix.

The antibiotic A-35512 factor B aglycone is preferably produced by mild acid hydrolysis of the antibiotic A-35512 factor B. Antibiotic A-35512 factor B is most easily available in the form of its dihydrochloride. The antibiotic A-35512 factor B dihydrochloride is therefore preferably used as the starting material for the production of the antibiotic A-35512 factor B aglycone. However, the antibiotic A-35512 factor B or other acid addition salts of the antibiotic A-35512 factor B can also be used for this purpose. The acidic hydrolysis can be carried out in the usual way. Although any acid can be used for this purpose, hydrochloric acid is preferably used to form the antibiotic A-35512 factor B aglycone. When using hydrochloric acid, the antibiotic A-35512 factor B aglycone is obtained as the hydrochloride salt. The hydrolysis is preferably carried out in refluxing water for a period of about 1 to 2 hours. Longer implementation times lead to a breakdown of the Agly-Kons with the formation of less active and later even inactive products. The optimal reaction times for the respective reaction conditions can easily be determined by examining the appropriate partial amounts of the respective reaction mixture with regard to their bioefficacy.

The antibiotic mixture A-35512, the factors A, B, C, E and H of the antibiotic A-35512 and the antibiotic A-35512 factor B aglycone inhibit the growth of certain pathogenic microorganisms, in particular gram-positive bacteria. The minimum inhibitory concentrations (MIC values) at which the factors A, B, C and H of the antibiotic A-35512 inhibit selected bacteria are determined by conventional agar dilution experiments, and the results obtained are shown in Table V below.

Table V

MIC (mcg / ml)

A-35512

A-35512 A-35512

A-35512

Test organism

Factor A -2HC1

Factor B • 2HC1 factor C • 2HC1

H-HCl factor

Staphylococcus aureus 3055

6.25

3.12 6.25

25.0

Staphylococcus aureus 3074

25.0

6.25 6.25

25.0

Streptococcus faecalis

6.25

3.12 3.12

12.5

Of particular interest and importance is the fact that antibiotics A-35512 also inhibit the growth of microorganisms that are resistant to other antibiotics. The effectiveness of the factors obtained in the standard disk-plate test is shown in Table VI below

A, B, C, E and H of the antibiotic A-35512 (at 30 micro-65 grams per disc) against typical microorganisms. The results of these tests are expressed as the diameter (mm) of the zones of inhibition observed.

13

Table VI

637 159

Zone diameter (mm)

A-35512 A-35512 A-35512 A-35512 A-35512

Test organism factorA-2HCl factorB-2HCl factorC-2HCl factorE-HCl factorH-HCl

Staphylococcus aureus 3055

13.4

18.4

0

14.4

11.9

(penicillin-g-susceptible)

Staphylococcus aureus 3074

13.2

16.8

0

14.5

11.6

(penicillin-G resistant)

Staphylococcus aureus 3130

14.4

18.4

0

15.4

12.6

(methicillin resistant)

Streptococcus pyogenes

17.0

19.6

10.5

17.0

16.0

(Group A)

Streptococcus sp. 9960

15.5

19.4

0

16.4

13.8

(Group D)

Diplococcus pneumoniae

-

-

11.0

20.0

-

Park I

Further results from agar dilution experiments are shown in Table VII below for the antibiotic A-35512 factor B dihydrochloride, and this shows the minimal inhibitory concentrations (MIC values) against several microorganisms from group A of streptococci and against Diplococcus pneumonia.

Table VII

25th

MIC (mcg / ml)

Test organism

A-35512 factor B

Streptococcus pyogenes C203

1

Streptococcus pyogenes 10389

1

Streptococcus pyogenes 12344

8th

Streptococcus pyogenes 12961

1

Streptococcus pyogenes 663-72

0.5

Streptococcus pyogenes 664-72

0.5

Streptococcus pyogenes 665-72

1

Streptococcus pyogenes 13234

0.5

Streptococcus pyogenes 19035

0.5

Streptococcus pyogenes M6517

0.5

Streptococcus pyogenes D27781

1

Diplococcus pneumoniae Park I

2nd

Diplococcus pneumoniae type 14

2nd

- The following Table VIII shows further results obtained from the study of the antibiotic A-35512 factor B dihydrochloride in agar dilution tests against several organisms from group D of the streptococci.

45

50

55

Table VIII

Test organism

MIC (mcg / ml) A-35512 factor B

Streptococcus sp. D282 Streptococcus sp. 9901 Streptococcus sp. 9913 Streptococcus sp. 9933 Streptococcus sp. 9960 Streptococcus sp. 12253F Streptococcus sp. Shrigley Streptococcus sp. Mitis

2 2 4 4 4 4 4 4

MIC (mcg / ml)

Test organism

A-35512 factor B

Streptococcus sp. 238

2nd

Streptococcus sp. SS992

4th

30th

Other results obtained from agar dilution experiments using microorganism A-35512 factor B dihydrochloride over other gram positive organisms can be found in Table IX below.

Table IX

Test organism

MIC (mcg / ml) A-35512 factor B

40

Staphylococcus aureus 3123 * Staphylococcus aureus H43 ** Staphylococcus aureus 3124 ** Staphylococcus aureus 3126 ** Staphylococcus aureus 3127 ** Staphylococcus aureus H57 ** Staphylococcus aureus 3074 ** Staphylococcus aureoc 31phus 3131 aureus 3130 *** *** Staphylococcus aureus 3136 *** Staphylococcus aureus 3125 *** Staphylococcus epidermis 3064 ** Staphylococcus epidermis 3078 * Bacillus subtilis ATCC 6633 Sarcina lutea PCI-1001-FDA

4 4 4 2 4 2 4 4 4 2 2 4 2 2 2 1

60

* penicillin-G-susceptible penicilin-G ** -resistant

penicillin-G resistant; methicillin resistant **** penicillin-G resistant; methicillin resistant; Clindamycin resistant i Antibiotics A-35512 also inhibit the growth of certain anaerobic bacteria. The effectiveness of the antibiotic A-35512 factor B dihydrochloride against various anaerobic bacteria, determined according to the standard

637 159

14

dard agar dilution experiment is shown in Table X below.

Table X

Test organism

MIC (mcg / ml) A-35512 factor B

Such (for this purpose 6 mm balls are immersed in solutions which contain 1 milligram of active ingredient per milliliter of solution and are then added to agar plates inoculated with the organisms to be examined in each case) from the following Table XIII.

Table XIII

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 ssp. fragilis 111

64.0

Fusobacterium necrophorum

8.0

Test organism

Inhibition zone (mm)

A-35512 factor B aglycone (free base)

Staphylococcus aureus Bacillus subtilis Sarcina lutea Bacillus subtilis *

19th

13

14 22

The antibiotic A-35512 factor B dihydrochloride has also proven to be antimicrobial in vivo against experimentally induced bacterial infections. For these examinations, two doses of the antibiotic A-35512 factor B are administered subcutaneously to correspondingly infected mice and the resulting effectiveness is determined by measuring the so-called ED 50 values [effective dose in mg / kg to protect 50% of the animals examined, J. Bacte -riol. 81, 233-235 (1961)]. The ED 50 values obtained for the antibiotic A-35512 factor B dihydrochloride are shown in the following table XI.

* with nutritionally limited agar

The following table XIV shows experimental values for the minimal inhibition concentrations (MIC values) with which the antibiotic A-35512 factor B aglycon hydrochloride is effective against certain strains of Staphylococcus aureus. These values have been determined by standard agar dilution tests.

Table XIV

30th

Table XI

Test organism

ED50

infection

Streptococcus pyogenes C 203

1.75

260xLD5o (ip)

Diplococcus pneumoniae Park I

4.3

60.8xLDso (ip)

Staphylococcus aureus 3055

6.9

206xLDso (ip)

40

Test organism

MIC (mgc / ml)

Staphylococcus aureus 3055+

0.5

Staphylococcus aureus H290 +

0.5

Staphylococcus aureus V92 +

0.5

Staphyloccocus aureus V104 +

0.25

Staphyloccocus aureus 3074 ++

0.25

Staphylococcus aureus H43 ++

0.5

Staphylococcus aureus V57 ++

0.5

Staphylococcus aureus H356 ++

1.0

Staphylococcus aureus 3130 +++

0.5

Staphylococcus aureus 3131 ++++

0.5

The antibiotic A-35512 factor B dihydrochloride also provides protection for guinea pigs that have been infected with the anaerobic Clostridium chauvoei (the cause of claw disease in cattle). These investigations are carried out according to the modified USDA method for determining the strength of Clostridium chauvoei-containing products (SAM 200, US Department of Agriculture, Animal and Plant Health Inspection Service, Veterinary Services Laboratories, Ames, Iowa 500 April 7th) 1975, under 9 CFR 50 113.91). The results obtained are shown in Table XII below.

+ penicillin-G susceptible

++ penicillin-G resistant

+++ penicillin-G resistant; methicillin resistant

++++ penicillin-G resistant; methicillin resistant, clindamycin resistant

Further experiments obtained in agar dilution experiments using the antibiotic A-35512 factor B aglycon hydrochloride against group D of the streptococci are shown in the following Table XV.

Table XV

'' Test organism

MIC (mg / ml)

Table XII

infection

Percentage

Treatment dead / total

protection

A-35512 factor B

0/10

100

10 mg / kg *

A-35512 factor B

3/10

70

1 mg / kg *

Infection control

10/10

0

55

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

: intramuscular administration

The efficacy of the antibiotic A-35512 factor B aglycone in a conventional disk thinner The antibiotic A-35512 factor B aglycone 65 also proves to be antibacterial in vivo against bacterial infections caused by experiments. For this purpose, mice with the corresponding infections are administered subcutaneously with two doses of the antibiotic A-35512 factor B aglykonhy-

15

637 159

drochloride and determines the effectiveness observed with A-35512 factor B aglycon hydrochloride ED50 obtained by the so-called ED50 values. The values for the antibiotics are shown in Table XVI below.

Test organism

Streptococcus pyogenes C203 Streptococcus pneumoniae Staphylococcus aureus 3055

Table XVI

ED50 infection

5.8 2570x LDso (ip)

7.0 374xLDso (ip)

1.04 370xLDäo (ip)

Antibiotics A-35512 are relatively non-toxic. For example, the LDo value for the antibiotic A-35512 factor A dihydrochloride when administered subcutaneously to starved female Cox and Harlan ICR mice is below 8000 mg / kg. The LD 50 value for the antibiotic 20 A-35512 Factor B dihydrochloride in the mouse is approximately 1356 mg / kg after intraperitoneal administration and above 1000 and below 1250 mg / kg after intravenous administration. Both the antibiotic A-35512 factor C dihydrochloride and the antibiotic A-35512 factor E hydrochloride have an acute toxicity of more than 300 mg / kg after intraperitoneal administration to mice.

Another important property of the antibiotic mixture A-35512 and the corresponding compounds is their ability to improve the feed conversion in animals. 30 This ability is demonstrated by ruminants with developed rumen function.

As is known, the effectiveness of carbohydrate utilization in ruminants can be increased by treatments which stimulate the rumen flora of the animals to form propionate compounds rather than to form acetate or bityrate compounds. (Church et al. In "Digestive Physiology and Nutrition of Ruminants", volume 2.1971, pages 622 and 625).

The effectiveness for feed utilization can be determined by in-vivo tests using appropriate cattle with an external gastric fistula according to the method described in US Pat. No. 3,794,732 (in particular Example 8). The following table XVII shows the results obtained in such tests using the antibiotic A-35512 factor B dihydrochloride, which represent the percentage propionic acid concentrations in the rumen from an average of 5 analyzes over a 21-day test period.

50

Table XVII

Treatment Number of animals Medium

Mole percentages

mol%

increase

quantity compared to the

Propionic acid

control

Check 5

19.3

A-35512 factor5

26.0

6.7

B 50g / ton

With the antibiotic mixture A-35512 and the corresponding compounds, propionate-increasing and thus feed utilization-improving effects result when these mammals are e.g. administered orally in amounts of about 0.15 to 10.0 mg / kg and day. The best results are obtained with administration amounts of approximately 1.0 to 2.0 mg / kg and day. A preferred method of administering the antibiotic mixture A-35512 or the corresponding compound is by mixing it with the animal feed. However, the present active ingredients can also be administered in other ways, for example in the form of tablets, drinkers, tablets or capsules. The formulation of these various dosage forms can usually be achieved in veterinary medicine. Each individual dosage unit should contain an amount of antibiotic A-35512 or a corresponding compound which corresponds directly to the daily dose suitable for the animal to be treated.

The antibiotic mixture A-35512 and the corresponding compounds are also suitable as growth-promoting agents in animals. The corresponding studies using fried chicken show e.g. With the addition of 20 g of antibiotic A-35512 factor B dihydrochloride per ton of feed, weight gain and improved feed conversion. The results obtained in corresponding investigations with animal batteries with 4 groups of 8 animals each and repetition of the individual experiments are shown in Table XVIII below.

Table XVIII

treatment

Conc. (G / ton)

Weight gain (g)

Percentage improvement over control

Feed conversion

Percentage improvement over control

control

380

1,850

A-35512

20th

394

3.68

1,776

4.00

Factor B

The following table XIX shows the above type in experiments on the results obtained from 12 groups of 80 animals in soil enclosure studies using.

637 159

* p ^ 0.05

16

Table XIX

treatment

Conc. (G / ton)

Weight gain (g)

Percentage improvement over control

Feed conversion

Percentage improvement over control

control

1898

2,250

A-35512

20th

1956 *

3.06

2.177 *

2.81

Factor B

The antibiotic mixture A-35512 and the corresponding compounds have a growth-promoting effect in poultry in particular if they are added to the animal feed in amounts of approximately 1 to 100 g per tonne of feed.

The invention is further illustrated by the following examples.

example 1

A. Shake bottle fermentation of the antibiotic A-35512

A lyophilized cookie from Streptomyces Candidus NRRL 8156 is dissolved in 1 to 2 ml of sterile water. This solution is then used to inoculate a Bacto yeast malt extract slant agar (ISP No. 2, Difco Laboratories, Detroit, Michigan).

The inoculated slant agar is then incubated at a temperature of 30 ° C for about 7 days. The mature slant agar culture is obtained using water (2 ml) and by scraping with a sterile pipette to loosen the spores. A certain amount of this water suspension of the spores (0.1 ml) is used to inoculate another inclined agar from ISP No. 2. The inclined agar inoculated with this is incubated at 30 ° C. for about 7 days. The mature slant agar culture is obtained using water (5 ml) and by scraping with a sterile pipette to loosen the spores. 50 ml of a vegetative medium of the following composition are then inoculated with 2.5 ml of the spore suspension obtained:

Components

Quantity (g / 1)

ZnCk KH2PO4

L-glutamic acid

DL citrulline

CaCCb

Deionized water

0.03 0.1

1.0

0.1 5.0 q.s. 1 liter

Components

amount

Trypticase soy broth

30 g

(Baltimore-Biological

Laboratories, Cockeys

ville, Md.)

Water (deionized)

q.s. 1 liter

The inoculated vegetative medium is then incubated in a 250 ml Erlenmeyer flask at a temperature of 30 ° C over a period of 48 hours on a rotary shaker which is operated under an arc diameter of approximately 5 cm at a rotation speed of 250 revolutions per minute .

50 ml of a production medium of the following composition are then inoculated with the incubated vegetative medium (0.5 ml) obtained in this way:

The inoculated production medium produced in the above manner is incubated in a 250 ml Erlenmeyer flask at a temperature of 32 ° C. for 8 to 10 days on a rotary shaker which is operated at a rotation speed of 250 revolutions per minute under an arc diameter of approximately 5 cm rotates.

30th

B. Tank fermentation to obtain the antibiotic A-35512

To form a larger volume of the vaccine culture, 20 ml of the above-described incubated medium are used to inoculate 400 ml of a vegetative medium of the second stage, which has the same composition as the vegetative medium of the first stage. This inoculated vegetative medium of the second stage is incubated for 24 hours at a temperature of 32 ° C. in a 21-liter flask on a 40 rotary shaker which moves under an arc diameter of about 5 cm at a speed of 250 revolutions per minute.

The incubated vegetative medium of the second stage (800 ml) prepared in this way is used to inoculate 1001 sterile production medium. The inoculated production medium is left to ferment in a 1651 fermentation tank for about 8 to 10 days at 32 ° C. The fermentation medium is aerated by introducing sterile air at a rate of 0.25 V / V / M and stirred with conventional stirrers at a rate of 200 revolutions per minute.

Example 2

The incubated 55 vegetative medium prepared according to Example 1, Section A can optionally also be kept until later use by keeping it in the vapor phase of liquid nitrogen in the following manner.

Into a small (13 x 100 mm) sterilized tube with screw cap, add 2 ml of a suspension. 60 gender composition:

Components

Quantity (g / 1)

Tapioca dextrin

glucose

NH4NO3

KCl

MgSCh

FeCh.4H20

25.0 10.0 2.5 - 1.5 1.1 0.03

Components

amount

Glycerin

20%

Lactose

10%

Water (deionized)

70%

17th

637 159

The suspension thus obtained is then mixed with 2 ml of a vegetative medium prepared in the above manner and incubated over a period of 48 hours. After appropriate mixing, the solution is frozen and saved in the gas phase of a tank with liquid nitrogen.

To use the vegetative medium stored in this way for a shake bottle or tank fermentation, the tube processed in the above manner is thawed in a water bath at a temperature of 43 ° C. A portion of the thawed solution (1 ml) obtained is then inoculated with 50 ml of a 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 for shake bottle fermentation or for the formation of a larger amount of inoculum for tank fermentation.

Example 3

The fermentation process described in Example 1 is repeated using a shake bottle / tank production medium of the following composition:

Ingredients amount (g / 1)

Tapioca dextrin 75.0

Molasses 40.0

soluble meat peptone 15.0

MgSCWHzO 0.5

CaCCb 2.0

Water q.s. 1 liter

Example 4

Separation of the antibiotic mixture A-35512

The entire fermentation broth (9461) described in Example 1 is filtered using a diatomaceous earth filter aid (Hyflo Super-cel, Johns-Manville Products Corp.) at the respective pH of the broth (pH 6.8 to 7.2). The clear filtrate obtained is then passed at a rate of 150 ml per minute through a column containing 10 ml of polymeric adsorbent (Amberlite XAD-4, Rohm and Haas Co.) per 100 ml of broth filtrate. The resulting fractions are monitored for their biological effectiveness using the conventional disc test against Sarcina lutea. The biologically ineffective effluent is discarded. The column is washed with water (Vs of the broth volume) at a rate of 150 ml per minute. The inactive wash water is also discarded.

The column is then eluted using a 50 percent aqueous methanol solution (6001) at a rate of 200 ml per minute. The resulting eluate, which contains the antibiotic mixture A-35512, is then concentrated under vacuum to a volume of 151, giving about 200 g of antibiotic A-35512 per 1.

Example 5

Isolation of the factors of the antibiotic mixture A-35512

The antibiotic mixture A-35512 obtained according to Example 4 (about 3000 g, dissolved in 15 l of methanol) was chromatographed over a polyamide column (Woelm, 100 liters). The column is eluted with deionized water and a flow rate of about 80 to 120 ml per minute.

The individual fractions are monitored by cellulose thin layer chromatography or paper chromatography. graphy using a solvent system of n-butanol, pyridine, acetic acid and water (15: 10: 3: 12) and by bioautography with Sarcina lutea.

The first 100 1 eluate are discarded. Then increase the flow rate to about 160 to 200 ml per minute and collect fractions of 12 1. In this way, a total of 12 fractions are collected.

At this point, the solvent used for elution is changed to a gradient of water and methanol as follows:

A container with 360 1 methanol is lifted into a container with 1201 water. The mixed solution in the water tank is stirred and then applied to the column.

24 fractions (241 each) are collected at a flow rate of 200 to 300 ml per minute.

Based on the results obtained in a bioautography, groups of corresponding fractions are combined and then evaporated to dryness in vacuo, giving the antibiotic A-35512 factor B dihydrochloride and the following enriched mixtures of factors:

Fractions

Volume (liters)

Factors

Weight

1-10

120

A + H

192g

11-24

216

B

269 g

25-31

168

B + C

590 g

32-44

312

C, E, F, G

224 g

Example 6

Purification of the antibiotic B-35512 factor B

The partially purified antibiotic A-35512 factor B dihydrochloride (400 g) obtained according to Example 5 is then dissolved in 1.2 l of 50 percent aqueous methanol and chromatographies, the solution obtained in the following manner over a column filled with aluminum oxide:

Acid aluminum oxide (10 kg, M. Woelm) is stirred into a 50 percent aqueous methanol solution. The mixture is then left to stand, the supernatant solution is decanted and discarded. The aluminum oxide obtained is then stirred with 50 percent aqueous methanol and packed with a column with a diameter of 13.5 cm. Subsequently, the column filled with aluminum oxide is washed with 50 percent aqueous methanol until the column effluent is clear.

The column filled with the above absorbent is eluted using 50 percent aqueous methanol at a flow rate of about 8 to 10 ml per minute to collect fractions with a volume of about 240 to 300 ml. The individual fractions are, as described in Example 5, by thin-layer bioautography supervised. On the basis of the values obtained in this way, purified antibiotic A-35512 factor B dihydrochloride is obtained by combining the following fractions:

Fractions

Weight

17-21

9.6 g

22-29

72.0 g

30-37

117.0g

Each of these fractions is crystallized from a concentrated 50 percent aqueous methanol solution at a temperature of 4 ° C. The purified antibiotic A-35512 factor B dihydrochloride obtained in this way contains about 4.6% chlorine. A solution of the antibiotic A-35512 factor B dihydrochloride in 66 percent aqueous dimethylformamide has a pH of about 6.5.

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

637 159

18th

Example 7

Production of antibiotic A-35512 factor B free of ionic chlorine

Purified antibiotic A-35512 factor B dihydrochloride (1 g) as prepared in Example 6 is dissolved in water (40 ml) and this solution is passed at a flow rate of 0.5 ml per minute through a 2.5 × 18 cm filled with an ion exchanger measuring column (Bio-Rad AG3-4X, in the OH ~ form), which is eluted with deionized water. The first eluate (5 ml) is discarded. The subsequent eluate (50 ml) is evaporated to dryness in vacuo, giving 0.76 g of an antibiotic A-35512 factor B free of ionic chlorine in the form of a white powder which contains about 1.59% chlorine. A solution of this antibiotic A-35512 factor B, which is free of ionic chlorine, in 66 percent aqueous dimethylformamide has a pH of 9.13.

Example 8

Purification of the antibiotic A-35512 factor A

Partially purified antibiotic A-35512 factor A dihydrochloride (1 g) obtained according to Example 5 (fractions 1 to 10) is dissolved in 50 percent aqueous methanol (5 ml). The solution thus obtained is then chromatographed on a column which is filled with acidic aluminum oxide (M. Woelm) and measures 3 × 16 cm and is prepared as described in Example 5. The column is eluted with 50 percent aqueous methanol at a flow rate of 0.5 ml per minute. Fractions with a volume of 5 ml are collected in each case. All fractions are analyzed by thin-layer bioautography using the method described in Example 5. On the basis of these investigations, fractions 7 to 15 are combined, the whole is concentrated to a small volume under reduced pressure and the material obtained is then lyophilized with the formation of 0.3 g of antibiotic A-35512 factor A dihydrochloride (chlorine content = 4.71% ).

Example 9

Production of antibiotic A-35512 factor A free of ionic chlorine

An antibiotic A-35512 factor A dihydrochloride (200 mg) prepared according to Example 8 is dissolved in water (10 ml) and the solution thus obtained is then passed at a flow rate of 0.5 ml per minute through a 1.5 ml filled with ion exchange resin x 10 cm measuring column (Bio-Rad AG3-4X, in the OH form), eluting with deionized water. The eluate coming at the beginning (10 ml) is discarded. The subsequent eluate (20 ml) is concentrated under reduced pressure to a small volume and the whole is then lyophilized to form 115 mg of antibiotic A-35512 factor A free from ionic chlorine.

Example 10

Purification of the antibiotic A-35512 factor C.

A partially purified antibiotic A-35512 factor C dihydrochloride (15 g) obtained according to Example 5 (fractions 25 to 31) is dissolved in deionized water (40 ml). The solution obtained in this way is then poured onto a column filled with polyamide and measuring 4 × 115 cm (MN, with a grain size of less than 0.07 mm from Brinkman Instruments, Inc.), which is prepared in water and thus transferred Night washed. The column is eluted using deionized water at a flow rate of about 3 ml per minute. The first eluate (250 ml) is discarded. Then collect fractions with a volume of 24 ml each.

The individual fractions are monitored by thin-layer chromatographic bioautography. For this, one works with cellulose thin layer chromatography plates (on aluminum foils, E. Merck, Germany), using 5 a solvent system consisting of sec-butanol, pyridine, acetic acid and water (10: 10: 3: 8) and using Bacillus subtilis as a detection organism. On the basis of the results obtained in thin layer chromatography, fractions 1 to 33 are combined, concentrated under vacuum to a volume of 150 ml and then lyophilized.

Using the same conditions, two further quantities of partially purified antibiotic A-35512 factor C dihydrochloride are chromatographed. Here, too, fractions 1 to 33 are combined again, then concentrated under vacuum to a volume of 150 ml and finally the whole is lyophilized. The three lyophilized samples obtained from the three columns are combined to give 12.3 g of partially purified antibiotic A-35512 factor C dihydrochloride.

20 For further chromatographic purification, the antibiotic A-35512 factor C dihydrochloride thus obtained is applied to another polyamide-filled column in the manner described above, but fractions with a volume of 15 ml are collected here at a flow rate of 125 ml per minute . The individual fractions are again monitored by thin-layer chromatography bioautography. Fractions 36 to 58 are combined, concentrated in vacuo to a volume of 150 ml and finally lyophilized, whereby 5.3 g of further purified antibiotic A-35512 factor C dihydrochloride are obtained. For final cleaning, the antibiotic A-35512 factor C dihydrochloride is chromatographed using a column filled with acidic aluminum oxide (Woelm) and measuring 5x41 cm. After appropriate packing, the column is washed with 50 percent aqueous methanol. After the methanol used for washing had become clear, the antibiotic A-35512 factor C (5.3 g in the form of a solution in 30 ml of 50% aqueous methanol) was added to the column. The column is then eluted with 40 50 percent aqueous methanol at a flow rate of 1 ml per minute. Fractions with a volume of 12 ml each are collected and monitored by thin-layer chromatography bioautography as described above. Fractions 22 to 74 are combined, 45 concentrated in vacuo to a volume of 250 ml and then lyophilized, giving 3.86 g of antibiotic A-35512 factor C dihydrochloride.

Example 11

thus production of antibiotic A-35512 factor C free of ionic chlorine

The antibiotic A-35512 factor C dihydrochloride (200 mg) prepared as described in example 10 is chromatographed over a weakly basic ion exchange resin 55 according to the procedure described in example 9, whereby 156 mg of antibiotic A-35512 factor C free of ionic chlorine is obtained. This ionic chlorine-free antibiotic A-35512 factor C contains about 1.90% chlorine.

60 Example 12 Purification of Antibiotic A-35512 Factor E

The partially purified antibiotic A-35512 factor E hydrochloride (8.1 g) obtained according to Example 5 (fractions 32 to 44) is dissolved in deionized water (40 ml). The solution obtained in this way is then applied to a 5 × 110 cm column filled with polyamide (MN, <0.07 mm, Brinkman Instruments, Inc.), this material being prepared in water and washed with water overnight . Connecting

19th

637 159

The column is then eluted with deionized water in an example 15

Flow rate of 20 ml per 15 minutes, producing fractions with a volume of 20 ml in the production of antibiotic free of ionic chlorine. With A-35512 factor H

Fraction 118 changes the elution used. Chromatograph one prepared according to Example 14

Solvent in 50 percent aqueous methanol. The individual antibiotic A-35512 factor H hydrochloride (200 mg) over fractions is monitored by thin-layer chromatography - a weakly basic ion exchange resin according to that described in Beigraphic bioautography as in Example 10. game 9 described, whereby 143 mg of ionic

Fractions 148 to 195 contain the antibiotic Schem chlorine-free antibiotic A-35512 factor H receives.

A-35512 factor E. These fractions are combined under reduced pressure to a volume of 150 ml under this ionic chlorine-free antibiotic A-35512 factor and io H contains about 1.59% chlorine.

then lyophilized, whereby 2.7 g of antibiotic A-35512 factor E hydrochloride are obtained. Example 16

A portion of the partial preparation of antibiotic A-35512 factor B aglycone-purified antibiotic A-35512 factor E hydrochloride prepared in the above manner. Antibiotic prepared according to example 6 is dissolved

(615 mg) is dissolved in 50 percent aqueous methanol (5 15 A-35512 factor B dihydrochloride (5.0 g) in water (200 ml), ml) and the solution thus obtained is then poured onto a 1.5 × 50 cm die The solution obtained in this way is then acidified using a 4-column, which is acidified with acidic aluminum oxide (Woelm) normal hydrochloric acid (14 ml), whereupon it is filled and heated in reflux temperature in 50% aqueous methanol solution for 2 hours. Then prepared and washed with it until the outflow is clear. the solution is cooled and evaporated under vacuum. The column is then eluted with 50 percent aqueous approximately three quarters of its original volume. The solution, which is so popular in methanol, at a flow rate of 1 ml, is then added dropwise per minute, with fractions with a volume of hydrochloric acid (6 normal) until the precipitation collects 10 ml each. The individual fractions are leveled. The precipitate obtained is filtered off and, if necessary, dried again by thin-layer chromatographic bioautogra- phy, which is monitored to give 3.56 g of crude antibiotic. Fractions 5 to 8 are combined and 25 A-35512 factor B aglycon hydrochloride is obtained.

under reduced pressure to a volume of about 10 ml. The filtrate obtained above is concentrated and analyzed. It narrowed. After adding deionized water (approx. 50 ml) lyo- contains glucose, fructose, mannose and rhamnose.

The solution thus obtained is philized, whereby 480 crude antibiotic mg antibiotic A-35512 factor E hydrochloride prepared in the above manner is obtained. A-35512 factor B aglycone is purified chromatographically

30 over aluminum oxide washed with acid (Woelm, type I) Example 13 using a solvent mixture of water

Manufacture of antibiotic free of ionic chlorine and methanol (1: 9). The elution of the column is monitored

A-35512 factor E by cellulose thin layer chromatography. Which the antibiotic

The antibiotic A-35512 factor B aglycone-containing fractions containing A-35512 factor E hydrochloride (200 mg) chromatogra-35 prepared as described in example 12 are combined and evaporated under vacuum, whereby one phases according to the method described in example 9 came to 398 mg of partially purified product. A comparison of a weakly basic ion exchange resin, by means of which these thin-layer chromatographic results confirm 170 mg of ionic chlorine-free antibiotic A-35512 by spraying with ninhydrin to detect that factor E is obtained. This antibiotic free of ionic chlorine is still not bioactive. A-35512 factor E contains about 1.72% chlorine. 40 A subset of this partially purified antibiotic

A-35512 factor B aglycone (100 mg) is therefore purified chroma example 14 topographically over polyamide (4 g, Machery, Nagel & Co. MN-

Antibiotic cleaning A-35512, factor H SC-6, supplier Brinkman Instruments Co.; <0.07 mm) further,

The solution prepared as in Example 5 (fractions 1 to 10) is dissolved, eluting with water. This column, too, monitors partially purified antibiotic A-35512 factor H hydro-45 in the manner already described above using chloride (30 g) in a minimal amount of a mixture of cellulose thin layer chromatography. The antibiotic methanol and water (7: 3). The A-35512 factor B aglycone-containing fractions obtained in this way are

Solution is then adsorbed onto a column combined with acidic aluminum and then lyophilized, whereby a column (3 x 60 cm, Woelm, antibiotic A-35512 factor B aglycon hydrochloride packed in methanol) and filled with 64 mg of purified oxide and with methanol until clear is obtained of the effluent). 50 arrives. (Total yield on the starting material subsequently eluted with methanol under a reversed antibiotic A-35512 factor B = 5.08%). Flow rate of 4 ml per minute. Fractions with a volume of 24 ml each are collected. In example 17

Fraction 59 changes the production of the free base of the antibiotic A-35512 factor used for elution

Solvent in a mixture of methanol and water (1: 1). 55 B Aglykon

The individual fractions are monitored by thin. An antibiotic prepared as in Example 16 is dissolved

Layer chromatography using a solvent with A-35512 factor B aglycon hydrochloride (90 mg) in 30 ml of a system of chloroform, methanol and ammonium hydro-mixture of methanol and water (1: 1). The xid (2: 3: 1) thus obtained and by bioautography with Bacillus subtilis in solution are then neutralized with an ion-exchange alkaline pH. 60 resin [3.5 ml, Bio-Rad AG-3-4X (OH-)]. Then stir

Fractions 51 to 118 are combined and the solution is left at room temperature for 15 minutes, whereupon

Evaporated in vacuo, whereby 6.4 g of purified antibiotic is filtered off the resin. The filtrate is then concentrated under vacuum to give A-35512 factor H hydrochloride. and maintaining a temperature of less than 60 ° C to about half its volume and subsequently lyophilized the whole 65, whereby 68 mg of the free base of the antibiotic A-35512 factor B aglycone are obtained.

G 2 sheet drawings

Claims (10)

637 159 1. Process for the preparation of the antibiotic mixture A-35512 with the factors A, B, C, E, F, G and H, the antibiotic A-35512 factor A, the antibiotic A-35512 factor B, the antibiotic A-35512 factor C, the antibiotic 5 A-35512 factor E, the antibiotic A-35512 factor F, the antibiotic A-35512 factor G and the antibiotic A-35512 factor H and the antibiotic A-35512 factor B Aglycon, characterized in that the microorganism Streptomyces Candidus NRRL 8156 is submerged in a nutrient medium containing assimilable sources of carbohydrates, nitrogen and inorganic salts, under aerobic fermentation conditions, until a substantial amount of the antibiotic is formed and the antibiotic factors obtained optionally converted into pharmaceutically acceptable acid-15 addition salts. 2. The method according to claim 1 for the preparation of the antibiotic mixture A-35512, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 is grown in a nutrient medium and 20 b) the antibiotic mixture A-35512 is separated from the culture medium. 2nd PATENT CLAIMS 3. The method according to claim 1 for the preparation of the antibiotic A-35512 factor A, characterized in that a) the microorganism Streptomyces Candidus NRRL 25 8156 is grown in a nutrient medium, b) the antibiotic mixture A-35512 is separated from the culture medium and c) the antibiotic A-35512 factor A is isolated from the antibiotic mixture A-35512. 30th 4. The method according to claim 1 for the preparation of the antibiotic A-35512 factor B, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 is grown in a nutrient medium, b) separating the antibiotic mixture A-35512 from the culture medium and c) isolating the antibiotic A-35512 factor B from the antibiotic mixture A-35512. 5. The method according to claim 1 for the preparation of the antibiotic A-35512 factor C, characterized in that 40th a) cultivates the microorganism Streptomyces Candidus NRRL 8156 in a nutrient medium, b) the antibiotic mixture A-35512 is separated from the culture medium and c) the antibiotic A-35512 factor C is isolated from the antibiotic mixture A-35512. 6. The method according to claim 1 for the preparation of the antibiotic A-35512 factor E, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 in a culture medium, 50 b) the antibiotic mixture A-35512 is separated from the culture medium and c) the antibiotic A-35512 factor E is isolated from the antibiotic mixture A-35512. 7. The method according to claim 1 for the preparation of the antibiotic 55 T-35512 factor F, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 is grown in a nutrient medium, b) separating the antibiotic mixture A-35512 from the culture medium and 60 c) the antibiotic A-35512 factor F is isolated from the antibiotic mixture A-35512. 8. The method according to claim 1 for the preparation of the antibiotic A-35512 factor G, characterized in that a) the microorganism Streptomyces Candidus NRRL 65 8156 is grown in a nutrient medium, b) the antibiotic mixture A-35512 is separated from the culture medium and c) the antibiotic A-35512 factor G is isolated from the antibiotic mixture A-35512. 9. The method according to claim 1 for the preparation of the antibiotic A-35512 factor H, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 is grown in a nutrient medium, b) the antibiotic mixture A-35512 is separated from the culture medium and c) the antibiotic A-35512 factor H is isolated from the antibiotic mixture A-35512. 10. The method according to claim 1 for the preparation of the antibiotic A-35512 factor B aglycone, characterized in that a) the microorganism Streptomyces Candidus NRRL 8156 is grown in a nutrient medium, b) separating the antibiotic mixture A-35512 from the culture medium, c) isolating the antibiotic A-35512 factor B from the antibiotic mixture A-35512 and d) producing the antibiotic A-35512 factor B aglycone from the antibiotic A-35512 factor B.