CH668598A5 - ANTITUMOR ANTIBIOTICS. - Google Patents

Description

DESCRIPTION

The present invention relates to new antitumor antibiotics and their manufacture and isolation.

The compounds according to the invention have not yet been structurally elucidated. Given their uniform physical, chemical and biological properties, however, it must be assumed that BBM-1675C and BBM-1675D are new compounds.

In GB-A-2141425, which was published on December 19, 1984, the fermentation of Actinomadura verrucosopora strain H964-92 (ATCC 39334) or Actinomadura verrucoso-spora strain A1327Y (ATCC 39638) is used to produce a new anti-tumor complex called BBM -1675, apparently. Two major biologically active components of the BBM-1675 complex were designated BBM-1675A1 and BBM-1675A2. The structures of these antibiotics, which are also known as Esperamicin Ai and Esperamicin A2, have also not yet been elucidated, but both components show excellent antimicrobial and antitumor activities.

US-A-4,530,835, issued Wed. 23, 1985 to Bunge et al., Discloses the fermentation of an unidentified Actinomy cetes isolate WP-444 (ATCC 39363) to produce the anti-tumor antibiotics, which are known as CL-1577A and CL-1577B were designated. The structures of the CL-1577 antibiotics have not yet been elucidated, but have distinctive characteristics that show that CL-1577A and CL-1577B are structurally similar to the BBM-1675 antibiotics, particularly the BBM-1675Ai and A2 described in GB-A-2141425.

R.H. Bunge et al., In J. Antibiotics, 37 (12), 1566-1571 (1984) describes the fermentation of Actinomadura sp. (ATCC 39363) for the preparation of a bioactive complex with the two main components PD114759 and PD 115 028, which were isolated. J.H. Wilton et al., In J. Chem. Soc. Chem. Commun., 919-920 (1985) describes the partial structure elucidation of the antibiotics PD 114759 and PD 115 028. The production, isolation and characterization of the PD 114 759 and PD 115 028 antibiotics seems to be identical to that of the above-mentioned CL-1577A and CL-1577B be.

EP-A-95154, which was published on November 30, 1983, discloses the fermentation of Actinomadura pulver-aceus sp. nov No. 6049 (ATCC 39100) for the preparation of the anti-tumor antibiotics designated WS 6049-B and WS 6049-B. The structures of the WX 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics show that WS 6049-A and WS 6049-B structurally correspond to the BBM-1675 antibiotics of GB-A-2141425 and are related to the CL-1577 antibiotics according to US-A-4,530,835. However, the spectral data show that neither WS 6049-A nor WS 6049-B is identical to any of the BBM-1675 components. Furthermore, the producing microorganisms described in EP-A-95154 are clearly different from Actinomadura verrucosospora used in GB-A-2141485, namely in the color of their aerial medium in ISP medium No. 2,3 and 4, in their positive milk peptonization and positive use of D-fructose, D-mannitol, trehalose and cellulose.

The present invention relates to the new antitumor antibiotics, which are referred to here as BBM-1675C and BBM-1675D and which are also known as BMY-27305 and BMY-27307. These substances are produced by selective chemical hydrolysis of the bioactive components BBM-1675AX (Esperamicin Aj) or BBM-1675 A2 (Esperamicin A2), which are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-1675C and

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BBM-1675D can be separated and purified by conventional chromatographic methods and the two substances show excellent antimicrobial and anti-tumor properties.

Description of the drawings:

Fig. 1 shows the ultraviolet absorption spectrum of BBM-1675C;

Figure 2 shows the ultraviolet absorption spectrum of BBM-1675D;

Figure 3 shows the infrared absorption spectrum of BBM-1675C (KBr, film);

Figure 4 shows the infrared absorption spectrum of BBM-1675D (KBr, film);

Figure 5 shows the relative frequency mass spectrum of BBM-1675C;

Figure 6 shows the relative frequency mass spectrum of BBM-1675D;

Figure 7 shows the proton nuclear magnetic resonance spectrum of BBM-1675C in CDC13 (360 MHz);

Figure 8 shows the proton nuclear magnetic resonance spectrum of BBM-1675D in CDCI3 + 10% CD3OD (360 MHz);

Figure 9 shows the 13C nuclear magnetic resonance spectrum of BBM-1675C in CDCI3 (90.6 MHz);

Figure 10A shows the 13C nuclear magnetic resonance spectrum (110-220 ppm) of BBM-1675D in CDEC13 + 10% CD3OD (90.6 MHz);

Figure 10B shows the 13C nuclear magnetic resonance spectrum (0-110 ppm) of BBM-1675D in CDC13 + 10% CD3OD (90.6 MHz);

IIA shows the proton resonance spectrum of compound 3A (a-anomer) in CDC13 (360 MHz);

IIB shows the proton resonance spectrum of compound 3B (β-anomer) in CDC13 (360 MHz).

The anti-tumor-active substances according to the invention with the names BBM-1675C and BBM-1675D, which are also known as BMY-27305 and BMY-27307, can be obtained by selective chemical hydrolysis of the biologically active components BBM-1675A! (Esperamicin Ai) or BBM-1675A2 (Esperamicin A2), the latter being cultivated by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora, preferably Actinomadura verrucosospora strain H964-92 (ATCC 39 334) or Actinomadura ver12cos7por (strain A3 39 638) or a mutant thereof.

Another object of the present invention is also a method for producing the substance BBM-1675C by selective hydrolysis of the bioactive components BBM-1675Ai or BBM-1675A2. Another object of the present invention is a method for producing the substance BBM-1675D by selective hydrolysis of the substance BBM-1675C, preferably from the bioactive components BBM-1675A! or BBM-1675A2. The isolation and purification of BBM-1675 C and BBM-1675D from the reaction mixture can be carried out by conventional chromatographic methods. The bioactive substances BBM-1675C and BBM-1675D show an antimicrobial activity against a broad spectrum of microorganisms and also show an inhibitory effect against various tumor systems in mice, such as P-388 leukemia and B16 melanoma. The compounds according to the invention can accordingly be used as antimicrobial agents or as antitumor agents for the inhibition of tumors in mammals.

During decomposition studies to elucidate the structure of the anti-tumor antibiotics BBM-1675Aj (Esperamicin A [) and BBM-1675A2 (EsperamicinA2), a mixture of the components was produced which lead to the isolation and identification of two inactive fragments, inventions of the formulas 1 and 2 respectively However, it was surprisingly found that the chemical decomposition for the gradual release of

3rd

5

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20th

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30th

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40

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50

55

60

65

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4th

two bioactive fragments, namely BBM-1675C and BBM-D, which had a small molecular weight (approximately 70 and 1675D, respectively). It was even more surprising that the two 55% of the molecular weight of the BBM-1675A! and A2 the same bioak-1675Ai and A2) are even more effective than BBM-1675A2 and formed with active fragments, as shown in Scheme 1. the BBM-1675A! as an antitumor and antimicrobial

Even more surprising is that fragments BBM-1675C and 5 are comparable.

Scheme 1

BBM-16 75 A] (Esperamicin A ^)

BBM-16 75C ■

"7 ^

BBM-16 75D

BBM-16 75A, (Esperamicin A ^)

The substances BBM-1675C and BBM-1675D can be obtained by selective chemical hydrolysis of the antibiotic BBM-1675Aj, as shown in Scheme 2.

Scheme 2

BBM-16 75A,

(KG 1248)

ch3oh

BBM-1675C (MG 85 5)

V

BBM-16 75D (MG 695)

och

och

Compound l (MG 425) (mixture of the et and β anoruers)

h® / ch3oh och.

Compound 3 (MW 192) (mixture of the a and β anomers)

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The starting compound BBM-1675AJ is prepared according to the procedure described in GB-A-2141425 (published December 19, 1984). The adjusted component BBM-1675A! is hydrolyzed with a mineral acid or an organic acid such as hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid or the like in an organic solvent or in a mixture of an inert aqueous / organic solvent at a temperature from 0 ° C. to the reflux temperature of the solvent until a essential part of the desired BBM-1675C or BBM-1675D has been formed. The hydrolysis is preferably carried out in an alcohol having 1-6 C atoms and the alcoholysis is particularly preferably carried out in methanol. The reaction temperature is not critical, but it is preferred that the reaction be carried out at about room temperature to 60 ° C and preferably at 40 ° C.

The selective hydrolysis of BBM-1675Ax is carried out in stages, whereby the antibiotic BBM-1675C and the inactive fragment of formula 1 are obtained first. Subsequently or with continued treatment under hydrolyzing conditions, a mixture of the a and β anomers of the thio sugar of the formula 3 is released and the antibiotic BBM-1675D is obtained. Those skilled in the art will recognize that changing reaction conditions such as time, temperature and acid concentration will change the proportions of the antibiotics BBM-1675C and D producing accordingly. However, it is desirable to monitor the progress of the reaction by thin layer chromatography as described in the examples.

If the antibiotic BBM-1675D is now to be produced, the selective hydrolysis is preferably carried out in an organic acid such as p-toluenesulfonic acid, as described here, with a quantitative yield of BBM-1675D being obtained.

The substances BBM-1675C and BBM-1675D can also be obtained by selective chemical hydrolysis of the antibiotic BBM-1675A2, as shown in Scheme 3.

15

Scheme 3

bbm-1675a

ch

(mg 1248)

2 CH3OH

-> bbm-1675c (mg 85 5)

ch3Ov ch3o

O

3 / ^ O oh

Oh no,

och

O- / ^ 2

och

Compound 2 (MW 425) (mixture of the et and β anomers)

I ^ / ch3OH

V

bbm-1675d (MG 695)

och

Compound 3, (MG 192) ^ Mixture of et and ß-orders) '

668 598

6

The starting material BBM-1675 A2 is produced according to the method as described in GB-A-2141425 (published on December 19, 1984). Selective hydrolysis of the purified BBM-1675A2 also takes place gradually, first obtaining the antibiotic BBM-1675C and the inactive fragment of Formula 2. Continued treatment under hydrolyzing conditions results in the release of a mixture of the α or β anomers of the thio sugar of Formula 3 to produce the antibiotic BBM-1675D.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A2 are essentially the same as those for the hydrolysis of BBM-1675Ax as described above. In a similar manner to the production of BBM-1675D from BBM-1675Ai, if only production of BBM-1675D is desired, the hydrolysis of BBM-1675A2 is continued until essentially all of BBM-1675A2 and BBM-1675CinBBM -1675D is converted. The hydrolysis is preferably carried out with an organic acid, such as p-toluenesulfonic acid.

Another finding is that the same antibiotics BBM-1675C and D are made from two different antibiotics BBM-1675Ax and BBM-1675 A2 with the same loss of two inactive fragments of formula 1 and 2 and the thio sugar of formula 3 Advantage of the present invention. Accordingly, another subject of the invention is a selective hydrolysis process of a mixture of BBM-1675Ai and A2 to produce BBM-1675C and D, as shown in Scheme 4.

Scheme 4

BBM-1675Aj + BBM-1675A2 -> BBM-1675C BBM-1675D

This advantage becomes apparent when you consider the relative proportions of BBM-1675Ax and A2, which can be different. The manufacture of BBM-1675C and D is therefore independent of the relative proportions of BBM-1675 Ax and A2, which are used as starting material in the process according to the invention.

As described, hydrolysis of BBM-1675Ai, A2 and C leads to the release of an inactive thiosugar fragment. This thiosugar was isolated in order to obtain further information about the chemical structure of BBM-1675C and consequently also for BBM-1675Ai and A2. The compound of formula 3 was determined as a mixture of the α and β anomers of thio sugar, the structure of which is explained in Schemes 2 and 3. A further characterization became possible after the alcoholysis of the a and β anomers was separated. The nuclear magnetic resonance spectra (360 MHz) of compound 3A (a-anomer) and compound 3B (β-anomer) are shown in FIGS. IIA and IIB, respectively. From an analysis of the spectral data, the thio sugar methyl glycosides of the formula 3 were provisionally ascribed the stereochemistry of the following formula: OH

ch och

Currently, absolute stereochemistry, i.e. H. D orL Not determined yet. Based on the interpretation of the spectral data, it is concluded that the thiosugar of formula 3 (less the CH3 group of the anomeric methoxy that is introduced during methanolysis) is a component of the structure of the antibiotic BBM-1675C and thus also a component of the Output antibiotics are BBM-1675Ai and A2.

Physicochemical properties of BBM-1675C

Description: amorphous solid

Ultraviolet absorption spectrum: compare FIG. 1

Instrument: Hewlett-Packard 8458

Solvent: methanol

Concentration: 0.0155 g / 1

Xma ^ nm) absorptivities

210 21,770

274 9.340

313 sh (shoulder) 4.190

No significant change was observed with acid or base.

Infrared absorption spectrum: compare FIG. 3 instrument: Nicolet 5DX FT-IR

Main absorption bands (KBr, film): 540,740,955,990,1017,

1065, 1080, 1118, 1150, 1250, 1305, 1325, 1340, 1370, 1385, 1440, 1690, 1705, 1735, 299, 2920, 2930, 2970, 3450 cm "1.

Mass spectrum: compare FIG. 5

Instrument: Finigan 4500 TSQ

Procedure: rapid fire

Atoms (FAB) ionization

Matrix m / z

Molecular ion

Relative frequency

Glycerin

856

[M + H] +

10%

Glycerin + NaCl

878

[M + Na] +

100%

Dithiotreitol:

Dithioerythritol

(3 + 1) (w.w)

856

[M + H] +

100%

Instrument: Kratos MS-50

High resolution FAB (m / z): [M + H] + = 856.3362 Molecular Weight: Apparent MG = 855 (based on previous mass spectra)

Elemental composition: C36H61B3014S3 (based on the high-resolution data above) Proton resonance spectrum: compare FIG. 7 Instrument: WM 360 Bruker

Solvent: CDC13

! HNMR 360 MHz Ô (ppm): 6.54 (1H, dd, J = 7,7,7,0); 6.21

(1H, brs); 5.87 (1H, d, J = 9.6); 5.78 (1H, dd, J = 89.6, 1.5); 5.66 (1H, brd, J = 2.9); 4.94 (1H, dd, J = 10,3,1,8); 4.61 (1H, d, J = 7.7); 4.25 (1H, s); 4.09 (1H, q, J = 2.6); 3.97 (1H, t, J = 9.6); 3.92-3.53 (10H), 3.45 (1H, dt J = 10,3,2,6); 2.48 (3H, s); 2.30 (2H, m); 2.13 (1H, m); 2.09 (3H, s), 1.50 (2H, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3); 1.08 (6H).

5

10th

15

20th

25th

30th

35

40

45

50

55

60

65

7

668 598

13C nuclear magnetic resonance spectrum:

Instrument:

Solvent:

13C NMR 90.6 MHz ô (ppm):

see Fig. 9 WM 360 Bruker CDCI3

13.7, 17.5, 19.8, 22.3, 22.7,

23.5.34.2.35.2.39.5.47.7,

52.7.55.8.56.1.57.7.62.4,

64.7.67.4.69.3.69.8.71.9,

76.1.77.1.77.7.79.7.83.2,

88,4,97,3,99,7,123,4,124,6,

130,1,193.1.

Physicochemical properties of BBM-1675D

Description:

Ultraviolet-

Absorption spectrum:

Instrument:

Solvent:

Concentration:

amorphous solid see FIG. 2 "Hewlett-Packard 8458 methanol 0.01 g / 1

Xmax (nm)

Absorptivities

214 274 325

27,000 12,800 5,400

No noticeable changes are observed with acid and base.

Infrared absorption spectrum: instrument:

Main absorption bands (KBr, film):

Mass spectrum:

Instrument:

Method:

Matrix:

Molecular ion (m / z):

Relative frequency: Instrument:

High-resolution FAB (m / z): molecular weight:

Elemental composition:

Proton resonance spectrum:

Instrument:

Solvent:

see Fig. 8 WM 360 Bruker CDCI3 + 10% CD3OD

13 Nuclear Resonance Spectrum:

Instrument:

Solvent:

compare Figures 10A and 10B WM 360 Bruker CDCI3 + 10% CD3OD

10th

13CNMR 90.6MHzô (ppm): 17.5.21.6.22.2.23.0.33.4.39.2, 46.4.52.3.55.8 62.1.67.8 , 69,8,70,1,71,3,75,8,77,1,78,1,82,4, 83,3,88,2,97,4,99,6,122,6,124,8,130,1,130 , 8,134,3,148,7, 192,8.

see Fig. 4

Nicolet 5DX FT-IR

735,755,910,960,1000,1020,

1085, 1150, 1195, 1250, 1310,

1335.1365.1385.1445.1510,

1685.1720.1735.2880.2930,

2960, 3400 cm "1.

see Fig. 6

Finigan 4500 TSQ

Rapid fire

Atoms (FAB) ionization

Thioglycerin

[M + H] + = 696

100%

Kratos MS-50 [M + H] + = 696.2794 Apparent MG = 695 (based on the mass spectral data above)

C ^ HäO ^ Sj (based on the high-resolution data above)

Biological properties of the substances BBM-1675 The antimicrobial activity of the substances BBM-1675 was determined in a variety of gram-positive and gram-negative 15 microorganisms. Table I below presents the data in the form of results from an antimicrobial screening process that includes the original component BBM-1675Ai and substances BBM-1675C and BBM-1675D of the present invention. In the screening procedure, each test compound was in the form of a solution with a uniform concentration of 10 [ig / ml] for impregnating it on a paper strip which was placed in the growth culture and the antibiotic activity was measured by determining the growth inhibition zone next to the paper strip. As shown in Table I, the substances BBM-1675C and D showed a broad spectrum of antimicrobial activity and proved to be at least as effective as the component BBM-1675Aj; substances BBM-1675C and D in particular were more effective than inhibitors of gram-negative organisms.

25th

30th

35

Table I

Antimicrobial activity of the BBM-1675 substances

The correlation of [M + H] + and [(M + H) +2] + of the relative frequencies with the corresponding calculated values confirms the elementary composition from the measurements of the high-resolution FAB.

XH NMR 360 MHz (ppm): 6.43 (1H, dd, J = 4,4,10,3); 6.13 (1H, s); 5.81 (1H, d, J = 8.8); 5.70 (1H, d, J = 8.8); 5.48 (1H, 6 brs); 4.48 (IH, d, J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3.80; 3.77 (1H, t, J = 9.0); 3.70-3.50 (11H, brm); 3.35 (1H, m); 3.28 (3H, s); 3.22 (3H, brs); 2.66-2.55 (2H, m); 2.38 (3H, s); 2.23-2.12 (2H, m); 1.42 (1H, brdt); 1.22 (3H, d, J = 5.9); 0.94 (3H, d, J = 6.6); 0.87 (3H, d, J = 5.9).

Inhibition zone in mm

Test microorganism

BBM-

BBM-

BBM-

1675A,

1675D

1675D

40

Escherichia coli AS 19

22

52

51

Escherichia coli K12

13

36

35

Escherichia coli P1373

12

34

33

Escherichia coli R Azaserine

14

35

34

45 Escherichia coli R netropsin

11

32

32

Escherichia coli R Mitomycin C

12

35

34

Escherichia coli R bleomycin

16

38

36

Escherichia coli R daunomycin

19th

45

44

Escherichia coli R neomycin

24th

53

52

50 Escherichia coli R Sibiromycin

14

32

30th

Escherichia coli R Hedamydin

14

30th

25th

Escherichia coli R aclacinomycin 15

41

40

Bacillus subtilis ATCC 6633

34

43

41

Klebsiella pneumoniae

17th

35

35

55 Staphylococcus 209 P.

32

47

44

Staphylococcus R actinoleukin

33

35

35

Staphylococcus R streptonigrin

37

50

48

Staphylococcus faecalis P1377

30th

39

38

Streptococcus aereus Smith P

36

47

45

60 Staphylococcus aureus Smith R

40

55

53

Actinomycin D

Staphyolcoccus aureus Smith R

17th

32

31

Aureolinic acid

Acinetobacter

16

33

32

65 Micrococcus luteus

35

57

55

Saccharomyces cerevisiae petite

22

42

43

R = resistant to the named antibiotic

668 598 p

Activity against leukemia P-388 Tables II and III contain the results of laboratory tests with CDFr mice which were implanted intraperitoneally with a tumor rinoculum of 106 ascites cells of leukemia P-388 and which were in different doses of BBM-5 1675A! C or D were treated. The substances were administered by intraperitoneal injection. For each dose amount, a group of six mice was used, which was treated with a single dose of the substance the day after the inoculation. A group of 10 control mice treated with saline io were also included in each series of experiments. The group treated with BBM-1675Ai was included in Table III as a direct comparison. A 30-day protocol was run, with the mean survival in days for each group of 15 mice and the number of survivors at the end of the 5-day period. The mice were weighed before treatment and on day four. The change in weight served as a measure of the toxicity of the medication. Mice weighing 20 g were used and weight loss to about 2 g was not considered excessive. The control animals treated with the vehicle normally died within 9 days. The results were from% T / C

determined, the ratio of the mean survival time of the treated group to the mean survival time of the control group treated with the vehicle times 100. An effect in the form of% T / C equal to or greater than 125 showed that a noticeable antitumor effect was achieved. The results of the screening in Table II show the originally unexpected level of the anti-tumor activity of the substance BBM-1675C. Table III shows the results of a direct comparison of BBM-1675Aj (Esperamicin Aj) and BBM-1675C and BBM-1675D. The data show that BBM-1675C is comparable to BBM-1675Aj in terms of potency and anti-tumor efficacy and that it is not plan-dependent, whereas BBM-1675D is only slightly less effective.

In addition, it is described in the present invention that the substances BBM-1675C and BBM-1675D can also be obtained as BBM-1675A2 (Esperamicin A2). In comparison to the data described here for BBM-1675C and BBM-1675D and the data described in GB-A-2141425 for BBM-1675A2, it was surprisingly found that the substances BBM-1675C and D as anti-tumor agents are more effective than the parent compound BBM-1675A2 from which they were derived.

Table II

Effect of BBM-1675C on P-388 leukemia (day 1 treatment)

Dose, IP

MST

MST effect

AWC

Survivors

Compound mg / kg / inj.

Days

% T / C

g day 4

Day 5

BBM-1675C

3.2

TOX

TOX

0/6

0.8

TOX

TOX

-

0/6

- 0.2

TOX

TOX

-

0/6

0.05

TOX

TOX

-1.8

1/6

0.0125

11.0

122

-2.5

5/6

0.003125

13.5

150

-2.5

6/6

Vehicle

T

9.0

100

0.4

10/10

Tumor inoculum: host:

Evaluation: Effect: Criterion:

AWC:

^ Jo6 "Ascites cells implanted i.p.

Male mice CDFt

MST = mean survival time

% T / C = (MST treated / BST control) x 100

% T / C> 125 is considered significant anti-tumor activity

Mean weight change (treated control) in g (on day 4)

Table III

Effect of BBM-1675 substances on P-388 leukemia

11

Connection BBM-1675A!

J

Dose, IP

MST

MST effect

AWC g

Survivors

. Treatment plan mg / kg / inj.

Days

% T / C

Day 4

Day 5

d.l

0.0512

TOX

TOX

0/6

0.0256

TOX

TOX

-

0/6

0.0128

TOX

TOX

-1.8

3/6

0.0064

15.5

172

-0.3

6/6

0.0032

15.0

167

-0.6

6/6

0.0016

15.5

172

0.6

6/6

0.0008

12.5

139

0.3

6/6

0.0004

12.0

133

1.4

6/6

0.0002

11.0

122

0.8

6/6

0.0001

11.5

128

1.4

6/6

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Table III

Effect of BBM-1675 substances on P-388 leukemia

Dose, IP

MST

MST effect

AWC g

Survivors

connection

Treatment plan rag / kg / in j.

Days

% T / C

Day 4

Day 5

BBM-1675C

d.l

0.0256

TOX

TOX

0/6

0.0128

TOX

TOX

-0.8

3/6

0.0064

11.5

128

-0.3

6/6

0.0032

"14.5

161

-0.1

6/6

0.0016

10.5

117

0.0

6/6

0.0008

12.0

133

0.3

6/6

0.0004

11.5

128

0.8

6/6

0.0002

11.0.

122

1.4

6/6

0.0001

11.0

122

0.8

6/6

0.00005

10.5

117

1.3

6/6

BBM-1675D

d.l

0.0256

9.0

100

0.1

6/6

0.0128

11.5

128

0.3

6/6

0.0064

12.5

139

0.3

6/6

0.0032

12.0

133

0.1

6/6

0.0016

11.5

128

0.8

6/6

0.0008

10.0

111

0.2

6/6

0.0004

10.0

111

0.5

6/6

0.0002

9.5

106

1.7

6/6

0.0001

9.5

106

1.7

6/6

0.00005

9.0

100

2.0

6/6

BBM-1675C

d.l- »5

0.0032

16.0

178

-1.3

6/6

0.0016

13.5

150

-1.0

6/6

0.0008

13.5

150

-0.3

6/6

0.0004

12.0

133

-0.4

6/6

0.0002

12.0

133

-0.4

6/6

0.0001

11.0

122

-0.4

5/6

0.00005

11.0

122

0.9

6/6

0.000025

8.5

94

2.2

6/6

0.0000125

8.0

89

2.4

6/6

0.00000625

8.0

89

2.4

6/6

Vehicle

-

9.0

100

2.4

10/10

Tumor inoculum: 106 ascites cells implanted i.p.

Host: CDFi female mice

Evaluation: MST = mean survival time

Effect:% T / C = (MST treated / MST control) x 100

Criterion:% T / C> 125 was considered significant anti-tumor activity

AWC: mean weight change (treated control) in g (on day 4)

Activity against B16 melanoma

Table IV contains the results of the anti-tumor tests using a B16 melanoma that grew in mice. BDFr mice were used and subcutaneously inoculated with a tumor implant. A 60 day protocol was used. Groups of 10 mice were tested for each dose unit and mean survival was determined for each group. The control animals were inoculated in the same way as the test animals and treated with the injection vehicle, whereby they had an average survival time of 22.5 days without active ingredient. For each dose level, a test animal was treated with the test compound on days 1, 5 and 9 by an intraperi-50 tonal injection. An effect expressed in% T / C equal to or greater than 125 indicates that a significant anti-tumor effect has been achieved. The results in Table IV show that in a direct comparison, BBM-1675C was also effective for the treatment of mice that had 55 B16 melanoma and was approximately comparable in potency to BBM-1675Ai.

Dose, IP MST

Compound mg / kg / inj. Days

BBM-1675A! 0.0032 37.5

0.0016 37.5

0.0008 38.5

0.0004 37.0

0.0002 34.5

0.0001 32.0

Effect AWC survivors

MST g day 10

% T / C day 12

167 0.3 10/10

167 0.3 10/10

171 1.4 10/10

164 1.8 10/10

153 2.0 10/10

142 1.9 10/10

668 598

10th

effect

AWC

Survivors

Dose, IP

MST

MST

G

Day 10

Compound mg / kg / inj.

Days

% T / C

Day 12

BBM-1675C

0.0008

31.5

140

0.6

10/10

0.0004

37.0

164

1.2

10/10

0.0002

31.0

138

0.6

10/10

0.0001

31.5

140

1.0

10/10

0.00005

27.5

122

0.8

10/10

0.000025

25.0

111

0.5

10/10

Vehicle

-

22.5

100

0.3

10/10

Tumor inoculum: 0.5 ml of a 10% porridge, IP

Host: BDFi female mice

Evaluation: MST = mean survival time

Effect:% T / C = (MST treated / MST control) x 100

Criterion:% T / C>: 125 is considered significant antitumor activity

AWC: mean weight change (treated control) in g (on day 12)

As can be seen from the data regarding the antimicrobial activity, using conventional dose determinations

and the effect on mouse tumor can be seen, BBM-1675C mung test are

and BBM-1675D can thus be determined as antibiotics useful for therapy.

see Treatment of Infectious Diseases in Mammals The examples below serve to illustrate the and other animals and as anti-tumor agents for the therapeutic invention.

see inhibition of mammalian tumor.

The present invention also includes pharmaceutical “, T ^

see compositions containing as active component BBM- Chemical manufacture and isolation of BBM-1675C and 1675C or BBM-1675D. The compositions BBM-1675D generally contain an antimicrobial or anti-tumor 30 Example 1 effective amount of the active compounds according to the invention in combination. A sample of BBM-1675Aj (50 mg) was in 2.5 ml nation with an inert. pharmaceutically acceptable carrier methanol dissolved and with 2.5 ml 0.1 molar hydrogen chloride or diluent !. Such compositions can be treated in methanol solution. The reaction was allowed to proceed at another active antimicrobial or antitumor agent temperature of approximately 50 ° C. and the disappearance of the contained and, depending on the administration form, in 35 starting materials (approximately after 30 min), every 5 to any corresponding pharmaceutical administration 10 min by thin layer chromatography (TLC) can be processed on silica form. Examples of such compositions gel plates (Analtech, 250 micron, GF) with toluene / acetone (3: 2, include solid compositions for oral administration / v) tested as eluents. After consumption of the digest such as tablets, capsules, pills, powders and granules, raw material, the reaction mixture was neutralized with a saturated liquid composition for oral administration, such as 40th NaHC03 solution in methanol, then under redu solutions, suspensions, Syrups or elixirs and prepared pressure evaporated, whereby a dry residue gene for parenteral administration such as sterile, aqueous or was obtained, which contained the bioactive fragments. The non-aqueous solutions, suspensions or emulsions. The BBM-1675C substance would also be flash chromatographed from the residue from sterile, solid compositions by column chromatography, placing the column, which was then placed in sterile water, physiological 45 cm by mass. X10 cm and was packed with WoelmSilica gel saline or other sterile injectable media 32-63 micron particle size. The pillar was dissolved with just before consumption. Toluene / acetone (3: 2 v / v) eluted, with 3 ml fractions in total

BBM has been melted for use as an antimicrobial. Each fraction was analyzed by TLC (silica

1675C or BBM-1675D or a pharmaceutical combination with toluene / acetone, 3: 2, v / v, as eluent) and the TLC-

The concentration was administered in such a way that the 50 areas were with a UV light source with a wavelength tion of the active ingredient greater than the minimum inhibition of 254 nm and a cerium sulfate spray (1% cerium sulfate and 2.5%

concentration for the organism to be treated. Made visible for molybdic acid in 10% sulfuric acid). The

Use as an anti-tumor agent can range the optimal dose fraction 6-12 (Rr value of BBM-1675C is 0.28) and dosage plans for BBM-1675C or BBM-1675D were pooled and evaporated to dryness, taking 12 mg (35% ),

for a particular mammalian host, substantially pure BBM-1675C was obtained.

be communicated. It is pointed out, however, that the actual- The physico-chemical properties of BM-1675C the dose of BBM-1675C or BBM-1675D depending on the are given in the description and the ultraviolet, infrared, formulated composition, the administration -, Mass, H NMR and 13-CNMR spectra of the type of connection and the particular site, host and gene are shown in FIGS. 1, 3,5, 7 and 9 respectively.

treating suffering varies. Many factors that affect the effect of the active ingredient must be considered, including age, weight, gender, diet, example 2

time of administration, route of administration, excretion rate, when extending the reaction time of the method in addition

The patient's condition, drug combinations, reac- tion 1, the percentage of BBM-1675C decreases and two new product sensitivity and seriousness of suffering. The ver 65 appear that from compounds 3 (Rf = 0.65) and BBM-

Discharge can be designated continuously or periodically within the 1675D (RF remains at baseline) (TLC:

maximum tolerated dose. The opti-silicon dioxide, toluene / acetone, 3: 2 v / v) and, over time, become predominant for a given set of conditions.

11

668 598

Compound BBM-1675D, which usually accompanies the production of BBM-1675C, was isolated from the chromatography columns as described in Example 1 by eluting the column with chloroform / methanol (5: 1, v / v). The appropriate fractions were pooled and evaporated to dryness to give 18 mg, essentially pure BBM-1675D, of the reaction described in Example 1.

Substance BBM-1675D shows a major spot at Rf = 0.37 in reverse phase thin layer chromatography (Whatman MKQgF, 200 micron) using 30% water, methanol as eluent and at Rf = 0.22 in normal phase silica gel Thin layer chromatography using chloroform / methanol as eluent (5: 0.5, v / v).

Example 3

A significant improvement in the yield of BBM-1675D can be achieved by using p-toluenesulfonic acid instead of hydrogen chloride for the chemical hydrolysis of BBM-1675 A2 or BBM-1675Ai, as explained in the methods of Examples 3 and 5, respectively.

A sample of BBM-1675A2 (15.2 mg) was hydrolyzed with a 0.03 molar solution of p-toluenesulfonic acid in methanol (1 ml) at a temperature of about 63 ° C for about 1 hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30 ° C. The substance BBM-1675D was isolated from the dry residue by flash column chromatography on a column using Woelm silica gel (32-63 micron particle size). The column was eluted with chloroform / methanol (5: 0.5, v / v) and the collected fractions were analyzed by TLC (silica gel with chloroform / methanol, 5: 0.5, v / v as eluent). The chromatographic conditions used allowed the separation of the mixture of inactive components 2 and 3 (7 mg) from the bioactive substance BBM-1675D, which has an Rr value of 0.22. The appropriate fractions were pooled and evaporated to dryness to give 8 mg of essentially pure BBM-1675D in almost quantitative yield.

The physicochemical properties of BBM-1675D are given in the description and the ultraviolet, infrared, mass, * H NMR and 13C NMR spectra of the compound are shown in Figures 2,4,6,8 and 10A, respectively and 10B combined.

Example 4

A sample of BBM-1675A2 (40 mg) was treated with 5 ml of a 0.5 molar solution of hydrogen chloride in methanol at about 50 ° C for about 2 hours according to the general procedure and isolation method described in Example 1. After neutralization with NaHC03 and evaporation to dryness, the substance BBM-1675C was isolated from the residue by flash chromatography on a column containing Woelm silica gel (particle size 32-36 microns) using toluene / acetone (3: 2, v / v) as eluent. The appropriate fractions were cleaned and dried

evaporated to give 8.4 mg of substantially pure BBM-1675C which was identical to the product isolated in Example 1.

The above chromatography column was then eluted with chloro-5 form / methanol (5: 0.25, v / v) and the collected fractions were pooled and evaporated to dryness to give BBM-1675D. The BBM-1675D substance was further purified by an additional flash chromatography column with silica gel using chloroform / methanol (5: 0.5, 10 v / v) as the eluent. The appropriate fractions were combined and evaporated to dryness to give 6.3 mg of substantially pure BBM-1675D, which was identical to the product isolated in Example 3.

15

Example 5

A sample of BBM-1675Ai (49.3 mg) was hydrolyzed with a 0.037 M solution of p-toluenesulfonic acid in methanol (1.5 ml) at a temperature of about 60 ° C for about 1.5 hours. The reaction mixture was evaporated to dryness under reduced pressure at about 30 ° C to give a residue containing BBM-1675D and inactive compounds 1 and 3. The bioactive substance BBM-1675D was isolated by flash chromatography on a column packed with Woelm silica gel (32-63 micron particle size) using chloroform / methanol (5: 0.25, v / v) as Eluent. The appropriate fractions were combined and evaporated to dryness to give 27 mg of substantially pure BBM-1675D which was with the product

30th

was identical according to Example 3.

Example 6

A sample of BBM-1675C (5.1 mg) was hydrolyzed with 0.5 molar solution of hydrogen chloride in methanol (1 ml) at 40 to 50 ° C overnight. After neutralization with NaHC03 and evaporation to dryness, the bioactive substance BBM-1675D was isolated from the residue by flash chromatography, on a column packed with Woelm silica gel (32-63 micron 40 particle size) and using chloroform / methanol (5: 0.25, v / v) as eluent. The suitable fractions gave essentially pure BBM-1675D, which was identical to the product according to Example 3.

45 Example 7

When the procedures according to Examples 1 and 2 were repeated, except that the starting material BBM-1675Âi was replaced by an equimolar amount of a mixture of BBM-1675 At and BBM-1675A2, the sub-50 punch BBM-1675C was obtained and BBM-1675D.

Example 8

When repeating the general procedure according to 55 Example 5, but with the exception that the starting material BBM-1675Ai by an equimolar amount of a mixture, the BBM-1675A! and BBM-1675 A2 was replaced, the substance BBM-1675D was obtained.

M

13 sheets of drawings

Claims (10)

668 598 2nd PATENT CLAIMS 1. Anti-tumor antibiotic BBM-1675C in a substantially pure form, which a) appears as an amorphous solid; b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; c) in silica gel thin-layer chromatography in the toluene / acetone solvent system in a volume ratio of 3: 2 has an Rr value of 0.28; d) has an apparent molecular weight of 855 as determined by high resolution FAB mass spectroscopy; e) has an ultraviolet absorption spectrum in methanol solution, as is shown essentially in FIG. 1, and the absorption maxima and absorptivities at 210 nm (a = 21.770), 274 nm (a = 9.340) and 313 nm (shoulder) (a = 4,190), with no significant changes, when an acid or base is added; f) has an infrared absorption spectrum (KBr, film), as is essentially represented by FIG. 3 and has the main absorption values at the following values in cm'1: 540,740,955,990,1017,1065,1080,1118,1150,1250,1305, 1323,1340,1370,1385,1440,1690,1705,1735,2900,2920,2930, 2970 and 3450; g) has a low resolution mass spectrum as essentially shown in Figure 5 and has a molecular ion [M + H] + of 856; h) has a 360 MHz proton nuclear resonance spectrum in CDC13, which essentially has the following signals shown in FIG. 7: 6.54 (1H, dd, J = 7,7,7,0); 6.21 (1H, brs); 5.87 (1H, d, J = 9.6); 5.78 (1H, dd, J = 9.6, 1.5); 5.66 (1H, brd, J = 2.9); 4.94 (1H, dd, J = 10,3,1,8); 4.61 (1H, d, J = 7.7); 4.25 (1H, s); 4.09 (1H, q, J = 2.6); 3.97 (1H, t, J = 9.6); 3.92-3.53 (10H); 3.45 (1H, dt, J = 10,3,4,0); 3.37 (3H, s); 2.77 (1H, m); 2.69 (1H, dt, J = 9.9, 5.2); 2.49 (1H, dd, J = 10,3,2,6); 2.48 (3H, s); 2.30 (2H, m); 2.13 (1H, m); 2.09 (3H, s); 1.50 (2H, m); 1.37 (3H, d, J = 5.9); 1.32 (3H, d, J = 6.3); and 1.03 (6H) parts per million of tetramethyl silane down; i) has a 90.6 MHz carbon 13 nuclear magnetic resonance spectrum in CDC13, essentially as shown in FIG. 9 and has the following signals: 13.7.17.5.19.8.22.3.22.7.23.5.34.2.35.2.39.5.47.7.52.7, 55.8.56, 1.57.7.62.4.64.7.67.4.69.3.69.8.71.9.76.1.77.1, 77.7.79.9.83.2, 88.4.97.3.99.7, 123.4, 124.6, 130.1 and 193.1 parts per million tetramethylsilane downwards. 2. Anti-tumor antibiotic BBM-1675D, which is essentially in pure form: a) is present as an amorphous solid; b) is soluble in methanol, ethanol, acetone and tetrahydrofuran and is slightly soluble in chloroform; c) with thin-layer chromatography on silica gel has an Rr value of 0.22, with the solvent system chloroform / methanol in a volume ratio of 5: 0.5 and in reverse phase silica gel thin-layer chromatography has an Rr value of 0.37, in the solvent system Methanol / water in a volume ratio of 70:30; d) has an apparent molecular weight of 695 as determined by high resolution FAB mass spectroscopy; e) has an ultraviolet absorption spectrum in methanol solution, essentially as shown in FIG. 2 and the ultraviolet absorption maxima and absorption values at 214 nm (a = 27,000), 274 nm (a = 12,800) and 325 nm (a = 5,400) has no significant change in acids or base addition; f) has an infrared absorption spectrum with (KBr, film), essentially as shown in FIG. 4 and has the following main peaks: 735,755,910,960,1000,1020,1085,1150,1195,1250,1310, 1335,1365,1385,1445,1510,1685,1720,1735,2880,2930,2960 and 3400 cm "1; g) has a low resolution mass spectrum, substantially as shown in Figure 6, and has a molecular ion [M + H] + of 696; h) has a 360 MHz proton resonance spectrum in CDCl3 + 10% CD3OD, which is essentially shown in FIG. 8 and has the following signals: 6.43 (1H, dd, J = 4,4,10,3); 6.13 (1H, s); 5.81 (1H, d, J = 8.8); 5.70 (1H, d, J = 8.8); 5.48 (1H, 6brs); 4.48 (1H, d, J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3.80; 3.77 (1H, t, J = 9.0); 3.70-3.40 (11H, brm); 3.34 (1H, m); 3.28 (3H, s); 3.22 (3H, brs); 2.66-2.55 (2H, m), 2.38 (3H, s); 2.23-2.12 (3H, d, J = 6.6); and 0.87 (3H, d, J = 5.9) parts per million of tetramethylsilane; i) has a 90.6 MHz carbon 13 nuclear magnetic resonance spectrum in CDCI3 + 10% CD3OD, which is essentially shown in FIG. 10, and has the following signals: 17.5.21.6.22.2.23.0.33.4.39.2.46.4.52.3.55.8.62.1.67.8, 69.8.70, 1.71.3.75.8.77.1.78.1.82.4.83.3.88.2.97.4.99.6, 122.6.124.8.130.1.130.8.134.1.148, 7 and 192.8 parts per million downward from tetramethylsilane. 3. A method for producing the anti-tumor antibiotic BBM-1675C according to claim 1, characterized in that BBM-1675A! or BBM-1675A2 is hydrolyzed with a mineral acid or an organic acid and the BBM-1675C formed is recovered from the reaction medium. 4. A method for producing the anti-tumor antibiotic BBM-1675D according to claim 2, characterized in that BBM-1675A! or BBM-1675 A2 is hydrolyzed with a mineral acid or an organic acid and the BBM-1675D formed is recovered from the reaction medium. 5. A method for producing the anti-tumor antibiotic BBM-1675D according to claim 2, characterized in that BBM-1675C is hydrolyzed with a mineral acid or an organic acid and the BBM-1675D formed is obtained from the reaction medium. 6. The method for producing the anti-tumor antibiotic BBM-1675C according to claim 1, characterized in that a mixture of BBM-1675Ai and BBM-1675A2 is hydrolyzed with a mineral acid or an organic acid and the BBM-1675C formed is obtained from the reaction medium . 7. The method for producing the anti-tumor antibiotic BBM-1675D according to claim 2, characterized in that a mixture of BBM-1675AJ and BBM-1675A2 is hydrolyzed with a mineral acid or an organic acid and the BBM-1675D formed is obtained from the reaction medium . 8. Pharmaceutical composition, characterized in that it contains BBM-1675C according to claim 1 or BBM-1675D according to claim 2. 9. Pharmaceutical composition according to claim 8, characterized in that it contains an antimicrobially effective amount of BBM-1675C or BBM-1675D in combination with a pharmaceutical carrier or diluent. 10. Pharmaceutical composition according to claim 8, characterized in that it contains a tumor-inhibiting amount BBM-1675C or BBM-1675D in combination with a pharmaceutical carrier or diluent. 5 10th 15 20th 25th 30th 35 40 45 50 55 60 65