CY1676A

CY1676A - Antibiotics

Info

Publication number: CY1676A
Application number: CY1676A
Authority: CY
Original assignee: Bristol Myers Squibb Co
Priority date: 1985-08-27
Filing date: 1993-10-10
Publication date: 1993-10-10
Also published as: IL79519A0; GB2179649A; KR920010226B1; CH668598A5; KR870002269A; ZA865796B; JPS62116589A; SG109692G; DE3629052C2; FR2586686A1; IT8621527A0; IT1229176B; AU604464B2; HUT44035A; FI863405A0; LU86562A1; SE9200428D0; FI863405L; GB8620118D0; DK170671B1

Description

1

GB 2 179 649 A

1

SPECIFICATION Antibiotics

5 This invention relates to new antitumor antibiotic substances and to their production and isolation.

Th antitumor compounds of the present invention have not yet been identified in terms of structure. In view of their unique physical, chemical and biological properties, however, applicant believes that the BBM-1675C and BBM-1675D antibiotics are novel substances.

United Kingdom Patent Application No. 2,141,425, published December 19,1984, discloses fermentation of 10 Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) to produce a new antitumor antibiotic complex designated as BBM-1675. Two major bioactive components of the BBM-1675 complex described therein were designated as BBM-1675A, and BBM-1675A2. The structures of the BBM-1675A, and BBM-16752 antibiotics, also known as esperamicin A, and esperamicin A2, respectively, have not yet been elucidated, but both components exhibit excellent antimicrobial and 15 antitumor activity.

United States Patent No. 4,530,835, issued July 23,1985 to Bunge et a!., discloses fermentation of an unidentified Actinomycete isolate WP-444 (ATCC 39363) to produce antitumor antibiotics designated CL-1577A and CL-1577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that CL-1577A and CL-1577B are similar in structure 20 to the BBM-1675 antibiotics, and especially BBM-1675A, and A2 mentioned above in United Kingdom Patent Application No. 2,141,425.

There is disclosed by R.H. Bunge et a/., in J. Antibiotics, 37 (12), 1566-1571 (1984) the fermentation of Actinomadura sp. (ATCC 39363) to produce a bioactive complex from which two major components, PD 114,759 and PD 115,028, were isolated. In J. Chem. Soc. Chem. Commun., 919-920 (1985), J. H. Wilton et ai. 25 described the partial structural elucidation of the antibiotics PD 114,759 and PD 115,028. The production, isolation and characterization of the PD 114,759 and PD 115,028 antibiotics appear to be identical to the above-mentioned CL-1577A and CL-1577B antibiotics, respectively.

European Patent Application No. 95,154, published November 30,1983, discloses fermentation of Actinomadura puiveraceus sp. nov. No. 6049 (ATCC 39100) to produce antitumor antibiotics designated WS 30 6049-A and WS 6049-B. The structures of the WS 6049 antibiotics have not yet been elucidated, but the characterizing properties given for the antibiotics indicate that WS 6049-A and WS 6049-B are related in structure to the BBM-1675 antibiotics of United Kingdom Patent Application No. 2,141,425 and to the CL-1577 antibiotics of United States Patent No. 4,530,835. Spectral data show, however, that neither WS 6049-A nor WS 6049-B is identical to any of the BBM-1675 components. Moreover, the producing organism described in 35 European Patent Application No. 95,154 may be clearly differentiated from Actinomadura verrucosospora employed in United Kingdom Patent Application No. 2,141,425 in the color of its aerial mycelium on ISP Medium Nos. 2, 3 and 4, in its positive milk peptonization and in its positive utilization of D-fructose, D-mannitol, trehalose and cellulose.

There is provided by the present invention new autitumor antibiotic substances designated herein as 40 BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, said substances being produced by selective chemical hydrolysis of the bioactive components BBM-1675A, (esperamicin A,) or BBM-1675A2 (esperamicin A2), which are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura verrucosospora. The bioactive substances BBM-1675C and BBM-1675D may be separated and purified by conventional chromatographic procedures, and both substances exhibit excellent antimicrobial and 45 antitumor activity.

Description of the drawings

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

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

50 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 abundance mass spectrum of BBM-1675C.

Figure 6 shows the relative abundance mass spectrum of BBM-1675D.

Figure 7 shows the proton magnetic resonance spectrum of BBM-1675C in CDCI3 (360 MHz).

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

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

Figure 10A shows the 13C magnetic resonance spectrum (110-200 ppm) of BBM-1675D in CDCI3 + 10% CD3OD (90.6 MHz).

60 Figure 10B shows the 13C magnetic resonance spectrum (0-110 ppm) of BBM-1675D in CDCI3 + 10% CD3OD (90.6 MHz).

Figure 11A shows the proton magnetic resonance spectrum of compound 3A (a-anomer) in CDCI3 (360 MHz).

Figure 11B shows the proton magnetic resonance spectrum of compound 3B (P-anomer) in CDCI3 65 (360MHz).

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2

GB 2179 649 A

2

Detailed description of the invention

This invention relates to two novel antitumor antibiotic substances designated herein as BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, said substances being produced by selective chemical hydrolysis of the bioactive components BBM-1675A, (esperamicin A,) or BBM-1675A2 5 (esperamicin A2), which are themselves produced by cultivating a BBM-1675-producing strain of Actinomadura 5 verrucosospora, most preferably Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638), or a mutant thereof. In another aspect, the present invention provides a process for producing the BBM-1675C substance by selective hydrolysis of the bioactive components BBM-1675A, or BBM-1675A2. In a further aspect, the present invention provides a process for the preparation 10 of BBM-1675D by selective hydrolysis of the BBM-1675C substance or, more preferably, from the bioactive 10 components BBM-16752 or BBM-1675A2. The isolation and purification of BBM-1675C and BBM-1675D from the reaction mixture may be accomplished by conventional chromatographic procedures.

The bioactive substances BBM-1675C and BBM-1675D exhibit antimicrobial activity against a broad spectrum of microorganisms and have also been shown to exhibit inhibitory activity against various mouse tumor 15 systems, such as P-388 leukemia and B16 melanoma. The newly described substances of the present invention, 15 therefore, may be used as antimicrobial agents or as antitumor agents for inhibiting mammalian tumors.

During the course of degradation studies to elucidate the structure of the antitumor antibiotics BBM-1675A, (esperamicin A,) and BBM-1675A2 (esperamicin A2), a mixture of components were produced which lead to the isolation and identification of two inactive fragments, compounds of the Formulas 1 and 2, respectively. 20 However, it was surprisingly found that the chemical degradation lead to the stepwise liberation of two bioactive 20 fragments BBM-1675C and BBM-1675D. Even more surprising, it was found that the two different antibiotics BBM-1675A-, and A2 produced the same bioactive fragments as illustrated in Scheme 1. Still more surprising, the smaller molecular weight fragments BBM-1675C and D (having approximately 70% and 55% of the molecular weight of the parent antibiotics BBM-1675A, and A2, respectively) were found to be more effective than 25 BBM-1675A2 and comparable to BBM-1675A, as antitumor and antimicrobial agents. 25

Scheme 1

BBM-1675A^ 30 (esperamicin

30

35

BBM-16 75C

■^>BBM-1675D 35

40

BBM-1675A2^ (esperamicin A2)

45

The BBM-1675C and BBM-1675D substances may be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A5 as outlined in Scheme 2.

BNSDOCID: <GB 217964SA_I_>

3

GB 2 179 649 A

3

Scheme 2

10 BBK-16 75A,—-(m.w. 1248) J

15

->BBM-16 75C + (m.w. 855)

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OCH.

Compound 1 (m.w. 425) (A mixture of a and B anoners)

I^/CH3OH

V

BBM-16 75D (m.w. 695)

OCH.

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Compound 3 (m.w. 192) (A mixture of a and 6 anoroers)

The starting BBM-1675A-, compound is prepared according to the procedure described in United Kingdom Patent Application No. 2,141,425, published December 19,1984. The purified BBM-1675A, component is hydrolyzed with a mineral or organic acid such as hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, 60 benzenesulfonic acid or the like, in an organic or mixed aqueous-organic inert solvent at a temperature of about O'C to the refluxing temperature of the solvent until a substantial amount of the desired BBM-1675C or BBM-1675D is produced. Preferably, the hydrolysis is carried out in (VC6 alcohol solvents, and most preferably, the alcoholysis is carried out in methanol. The temperature of the reaction is not critical, but it is preferred to conduct the reaction at about ambient temperature to 60°C, and most preferably from about 40° to 60°C. 65 The selective hydrolysis of BBM-1675A, proceeds in a stepwise manner with the initial production of the

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BNSDQCID: <GB 2179649A_L>

GB 2179 649 A

10

BBM-1675C antibiotic and the inactive fragment of Formula 1. Subsequent or continued treatment under hydroiyzing conditions leads to the liberation of a mixture of a and P anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1675D. It should be appreciated by those skilled in the art that altering the reaction conditions such as time, temperature and concentration of acid will produce varying relative amounts of the antibiotics BBM-1675C and D. Thus, it is desirable to monitor the progress of reaction by thin layer chromatography as described in the examples herein.

When it is desired to prepare only the BBM-1675D antibiotic, the selective hydrolysis is preferably carried out with an organic acid such as p-toluenesulfonic acid as described herein to yield a quantitative amount of BBM-1675D.

The BBM-1675C and BBM-1675D substances may also be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A2 as outlined in Scheme 3.

10

Scheme 3

15

BBM-16 75A.

->BBM-16 75C +

2 CH3OH (m.w. 1248} (m.w. 855)

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55

O'"" 2

OCH.

CH, 3

Compound 2 (m.w. 425) (A mixture of a and 0 anomers)

H®/ch3OH

BBM-16 75D (m.w. 695)

+ ch3S

£>

OCH-

315

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50

55

OH

60

65

Compound 3 (m.w. 192) (A mixture of a and 6 anomers)

60

65

BNSDOCID: <GB 2179649A_J_>

5

GB 2179 649 A

5

The starting BBM-1675A2 compound is prepared according to the procedure described in United Kingdom Patent Application No. 2,141,425, published December 19,1984. The selective hydrolysis of purified BBM-1675A2 likewise proceeds in a stepwise manner with the initial production of the BBM-1675C antibiotic and the inactive fragment of Formula 2. Continued treatment under hydroiyzing conditions leads to the liberation 5 of a mixture of a and p anomers of the thiosugar of Formula 3 and the production of the antibiotic BBM-1675D. 5 The reaction conditions utilized for the selective chemical hydrolysis of 8BM-1675A2 are substantially the same as those utilized for the hydrolysis of BBM-1675A, described above. In a manner similar to the production of BBM-1675D from BBM-1675A,, when it is preferred to produce only the BBM-1675D antibiotic, the hydrolysis of BBM-1675A2 is carried out until substantially all of BBM-1675A2 and BBM-1675C is converted to 10 BBM-1675D. Most preferably, the hydrolysis is carried out with an organic acid such asp-toluenesulfonic acid. 10 The discovery, as described herein, that the same BBM-1675C and D antibiotics are produced from two different antibiotics BBM-1675A, and BBM-1675A2 with the concurrent loss of two inactive fragments of Formulas 1 and 2, respectively, and the thiosugar of Formula 3, provides an additional advantage for the present invention. Accordingly, in a further aspect of the present invention, there is provided a process for the selective 15 hydrolysis of a mixture of BBM-1 675At and A2 to produce BBM-1675C and D as illustrated in Scheme 4. 15

Scheme 4

BBM-1675A, + BBM-1675A2 ► BBM-1675C > BBM-1675D

20 20

This advantage becomes apparent when one considers that the relative amounts of BBM-1675A, and A2 produced in the fermentation process is subject to variability. The production of BBM-1675C and D is therefore independent of the relative amounts of BBM-1675A, andA2 utilized as starting material in the present invention. As described herein, the hydrolysis of the BBM-1675A1f A2 and C antibiotics results in the release of an 25 inactive thiosugar fragment. The said thiosugar was isolated to provide further information into the chemical 25

structure of the BBM-1675C antibiotic and hence, for the BBM-1675A, and A2 antibiotics. The compound of Formula 3 was identified as a mixture of a and /? anomers of a thiosugar which has the structure illustrated in Schemes 2 and 3. Further characterization was made possible when the products of the alcoholysis, the a and p anomers, were separated. The proton magnetic resonance spectra (360 MHz) of the compound 3A (a-anomer) 30 and compound 3B (P-anomer) are shown in Figures 11A and 11 B, respectively. From an analysis of the spectral 30 data, the thiosugar methyl glycosides of Formula 3 were tentatively assigned the relative stereochemistry of the formula

35

OCH.

40

45

At the present time, the absolute stereochemistry, i.e. D or L, has not yet been determined. Accordingly, based on the present interpretation of the spectral data, it is concluded that the thiosugar of Formula 3 (less the CH3 group from the anomeric methoxy which is incorporated during the methanolysis) is a component in the structure of the antibiotic BBM-1675C and furthermore, is a component in the structure of the starting BBM-1675A1 and A? antibiotics.

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Physico-chemical Properties of B8M-1675C

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Description: amorphous solid

Ultraviolet absorption spectrum: See Figure 1

Instrument: Hewlett-Packard 8458

Solvent: methanol

Concentration: 0.0155g/l

X (nm) -max-1—-

210 274

313 sh (shoulder)

absorptivities

21,770 9,340 4,190

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55

60

No significant change is observed with acid or base.

Infrared absorption spectrum: See Figure 3 65 Instrument Nicolet 5DX FT-IR

65

BNSDOCID: <GB 2179649A_I_>

6

GB 2 179 649 A

6

Major absorption bands (KBr, film):

540, 740, 955, 990,1017,1065,1080,1118,1150,1250,1305,1325,1340,1370,1385,1440,1690,1705, 1735, 2900, 2920, 2930, 2970, 3450 cm"1.

Mass spectrum: See Figure 5 Instrument: Finigan 4500 TSQ

Method: fast atom bombardment (FAB) ionization

Molecular

Relative

10 Matrix mlz

Ion

Abundance glycerol

856

[M + H] +

100%

glycerol + NaCI

876

[M + Na] +

100%

dithiothreitol:dithioerythritol

856

[M + H] +

100%

15 (3:1) (w:w)

Instrument:

Kratos MS-50

High resolution FAB (m/z):

[M + H]+ =

856.3362

20 Molecular weight: apparent MW = 855

(based on above-described high resolution data)

25

Elemental composition: C30H61N3014S3 (based on above-described high resolution data)

See Figure 7 WM 360 Bruker CDCI3

Proton Magnetic Resonance Spectrum:

Instrument:

Solvent:

'H NMR 360 MHz 8 (ppm):

30 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 (1 H, d, J=7.7); 4.25 (1H, s); 4.09 (1H, q, J = 2.6); 3.97 (1 H, 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); 1.08 (6H).

35

40

13C Magnetic Resonance Spectrum: See Figure 9

Instrument: WM 360 Bruker Solvent: CDCI3 ,3C NMR 90.6 MHz 5 (ppm):

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.

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Physico-chemical Properties of BBM-1675D Description: amorphous solid

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Ultraviolet absorption spectrum: See Figure 2

Instrument: Hewlett-Packard 8458

Solvent: methanol

Concentration: 0.01 g/i

50

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X (nm) -max- -

214 274 325

absorptivities

27,000 12,800 5,400

55

No significant change is observed with acid or base.

60 infrared absorption spectrum: See Figure 4

Instrument: Nicolet 5DX FT-IR Major absorption bands (KBr, film):

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.

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BNSDOCID: <GB 2179649A_I_>

7

GB 2 1 79 649 A

7

Mass spectrum: See Figure 6

Instrument: Finigan 4500 TSQ

Method: fast atom bombardment (FAB) ionization Matrix: thioglycerol Molecular ion (m/z): [M + H]+ = 696 Relative abundance: 100%

Instrument: Kratos MS-50

10 High resolution FAB (m/z): [M + H]+ = 696.2794 10

Molecular weight: apparent MW = 695 (based on above-described high resolution data)

15 Elemental composition: C29H49N3012S2 15

(based on above-described high resolution data)

Correlation of [M+ H]+ and [(M + H) + 2]+ relative abundances to their calculated values confirms the elemental composition derived from high resolution-FAB measurements. 20 20

Proton Magnetic Resonance Spectrum: See Figure 8

Instrument: WM 360 Bruker Solvent: CDCI3 + 10% CD3OD 1H NMR 360 MHz 5 (ppm):

25 6.43 (1H, dd, J = 4.4, 10.3); 6.13(1 H,s); 5.81 (1H, d, J = 8.8); 5.70 (1H, d, J = 8.8); 5.48 (1H, 6 brs); 4.48 25 (1H,d, J = 8.1); 4.02 (1H, d, J =2.0); 3.95-3.80 (solvent background); 3.77 (1 H,t, J =9.0); 3.70-3.40 (11H, brm); 3.35 (1 H, 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).

30 13C Magnetic resonance spectrum: See Figures 10A and 10B 30

Instrument: WM 360 Bruker Solvent: CDC(S + 10% CD3OD ,3C NMR 90.6 MHz 5 (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, 35 88.2, 97.4, 99.6,122.6,124.8,130.1,130.8,134.3,148.7,192.8. 35

Biological properties of BBM-1675 substances Antimicrobial activity of the BBM-1675 substances was determined for a variety of gram-positive and 40 gram-negative microorganisms. Table I below provides data in the form of results of an antimicrobial screening 40 procedure involving the parent BBM-1675A, component and the BBM-1675C and BBM-1675D substances of the present invention. In the screening procedure, each test compound at a uniform concentration of 10 ng/ml of solution impregnated on a paper strip was placed on the growth culture, and the measure of antibiotic activity is the resulting zone of inhibition from the paper strip. As shown in Table I, the BBM-1675C 45 and D substances showed a broad spectrum of antimicrobial activity which were at least as effective as the 45 BBM-1675A-, component; and in particular, the BBM-1675C and D substances were more effective as inhibitors of gram-negative organisms.

TABLE I

50 50

Antimicrobial activity of BBM-1675 substances

Zone of inhibition, mm

Test Microorganism

BBM-1675A,

BBM-1675C

BBM-1675D

55 Escherichia coli AS 19

22

52

51

Escherichia coli K 12

13

36

35

Escherichia coli P 1373

12

34

33

Escherichia coli R Azaserine

14

35

34

Escherichia coli R Netropsin

11

.32

32

60 Escherichia coli R Mitomycin C

12

35

34

Escherichia coli R Bleomycin

16

38

36

Escherichia coli R Daunomycin

19

45

44

Escherichia coli R Neomycin

24

53

52

Escherichia coli R Sibiromycin

14

32

30

65 Escherichia coli R Hedamycin

14

30

25

BNSDOCID: <GB 2179648A_J_>

8

GB 2 179 649 A

8

Zone of inhibition, mm

Test Microorganism

BBM-1675A,

BBM-1675C

BBM-1675D

Escherichia co/i R Aclacinomycin

15

41

40

Bacillus subtilis ATCC 6633

34

43

41

Klebsiella pneumoniae

17

35

Staphylococcus 209 P

32

47

44

Staphylococcus R Actinoleukin

33

35

33

Staphylococcus R Streptonigrin

37

50

48

Staphylococcus faecafis P1377

30

39

38

Streptococcus aureus Smith P

36

47

45

Staphylococcus aureus Smith R

40

55

53

Actinomvcin D

Staphylococcus aureus Smith R

17

32

31

Aureolic acid

Acinetobacter

16

33

32

Micrococcus luteus

35

57

55

Saccharomyces cerevisiae petite

22

42

43

R = resistant to named antibiotic

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Activity against P-388 Leukemia

Tables II and ill contain the results of laboratory tests with CDF, mice implanted intraperitoneally with a tumor inoculum of 10® ascites cells of P-388 leukemia and treated with various doses of BBM-1675A,, C or D. The substances were administered by intraperitoneal injection. Groups of six mice were used for each dosage 25 amount, and they were treated with a single dose of the substance on the day after inoculation. A group of ten saline treated control mice was included in each series of experiments. The BBM-1675A, treated group in Table III was included as a direct comparison. A 30-day protocol was employed with the mean survival time in days being determined for each group of mice and the number of survivors at the end of the 5-day period being noted. The mice were weighed before treatment and again on day four. The change in weight was taken as a 30 measure of drug toxicity. Mice weighing 20 grams each were employed, and a loss in weight of up to approximately 2 grams was not considered excessive. The vehicle treated control animals usually died within nine days. The results were determined in terms of a % T/C which is the ratio of the mean survival time of the treated group to the mean survival time of the vehicle treated control group times 100. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor effect was achieved. The screening results 35 in Table II show the initially unexpected level of antitumor activity of the BBM-1675C substance. In Table III, the results of a direct comparison of BBM-1675A1 (esjj0ramicin Ai) and the BBM-1675C and BBM-1675D substances are reported. The data suggest that BBM-1675C is about comparable to BBM-1675A, in potency and antitumor effectiveness and that it is not schedule dependent, while BBM-1675D is only slightly less effective.

40 Additionally, it is reported in the present invention that the same substances BBM-1675C and BBM-1675D can also be obtained from the BBM-1675AZ (esperamicin Az) component. In comparison of the data reported herein for BBM-1675C and BBM-1675D and the data reported in published U.K. Patent Application No. 2,141,425 for the BBM-1675A2 component, it is surprisingly found that the substances BBM-1675C and D are mora effective as antitumor agents than the parent BBM-1675A2 component from which they were derived.

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TABLE II

Effect of BBM-1675C on P-388 Leukemia (Day 1 Treatment)

Effect

AWC

50

Dose, IP

MST

gm

Survivors

Compound mg/kg/inj.

days

% T/C

Day 4

Day 5

BBM-1675C

3.2

TOX

—

0/6

0.8

TOX

—

0/6

0.2

TOX

—

0/6

55

0.05

TOX

-1.8

1/6

0.0125

11.0

122

-2.5

5/6

0.003125

13.5

150

-2.5

6/6

20

25

30

35

40

45

50

55

Vehicle 9.0 100 0.4 10/10

60 60

Tumor inoculum: 10s ascites cells implanted i.p.

Host: CDF, male mice Evaluation: MST = median survival time Effect: % T/C = (MST treated/MST control) x 100 65 Criteria: % T/C > 125 considered significant antitumor activity 65

BNSDOCID: <GB 2179649A__L>

9

GB 2 179 649 A 9

AWC: average weight change (treated-control) in grams (on day 4)

TABLE III

Effect of BBM-1675 substances on P-388 Leukemia

5

Effect

AWC

Treatment

Dose, IP

MST

gm

Sun/ivors

Compound

Schedule mg/kg/inj.

days

% T/C

Day 4

Day 5

BBM-1675A,

d. 1

0.0512

TOX

—

0/6

0.0256

TOX

—

0/6

10

0.0128

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

15

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

BBM-1675C

d. 1

0.0256

TOX

—

0.6

20

0.0128

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

25

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

30 BBM-1675D

d. 1

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

35

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

40

BBM-1675C

d. 1-+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

45

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

50

0.00000625

8.0

89

2.4

6/6

Vehicle

—

9.0

100

2.4

10/10

Tumor inoculum: 10® ascites cells implanted i.p.

55 Host: CDF, female mice

Evaluation: MST = median survival time

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

Criteria: % T/C > 125 considered significant antitumor activity

AWC: average weight change (treated-control) in grams (on day 4)

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Activity against B16 Melanoma

Table IV contains results of antitumor tests using the B16 melanoma grown in mice. BDF, mice were employed and inoculated subcutaneously with the tumor implant A 60-day protocol was used. Groups of ten mice were used for each dosage amount tested, and the mean survival time for each group was determined. 65 Control animals inoculated in the same way as the test animals and treated with the injection vehicle and no

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drug exhibited a mean survival time of 22.5 days. For each dosage level, the test animals were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. An effect in terms of % T/C equal to or greater than 125 indicates that a significant antitumor effect was achieved. The results in Table IV show that in a direct comparison BBM-1675C was also effective in treatment of mice bearing B16 melanoma and was about 5 comparable to BBM-1675A, in potency.

TABLE iV

Effect of BBM-1675 substances on B16 melanoma (Day 1,5 and 9 Treatments)

10

Effect

AWC

Dose, IP

MST

gm

Survivors

Compound mg/kg/inj.

days

% TIC

Day 12

Day 10

BBM-1675A,

0.0032

37.5

167

0.3

10/10

0.0016

37.5

167

0.3

10/10

15

0.0008

38.5

171

1.4

10/10

0.0004

37.0

164

1.8

10/10

0.0002

34.5

153

2.0

10/10

0.0001

32.0

142

1.9

10/10

20 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

25

0.000025

25.0

111

0.5

10/10

Vehicle

—

22.5

100

0.3

10/10

Tumor inoculum: 0.5ml of a 10% brei, IP 30 Host: BDF, female mice

Evaluation: MST = median survival time

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

Criteria: % T/C > 125 considered significant antitumor activity

AWC: average weight change (treated-control) in grams (on day 12)

35

As indicated by the antimicrobial and mouse tumor data provided above, BBM-1675C and BBM-1675D are thus useful as antibiotics in the therapeutic treatment of mammals and other animals for infectious diseases and also as antitumor agents for therapeutically inhibiting the growth of mammalian tumors.

The present invention, therefore, provides a method for therapeutically treating an animal host affected by a 40 microbial infection or by a malignant tumor which comprises administering to said host an effective antimicrobial or tumor-inhibiting dose of BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof.

The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of BBM-1675C or BBM-1675D in combination with an inert pharmaceutically acceptable carrier or diluent. Such compositions may also contain other active antimicrobial or antitumor agents 45 and may be made up in any pharmaceutical form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenteral administration such as sterile aqueous or non-aqueous solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in 50 sterile water, physiological saline or some other sterile injectable medium immediately before use.

For use as an antimicrobial agent, the BBM-1675C or BBM-1675D, or a pharmaceutical composition thereof is administered so that the concentration of active ingredient is greater than the minimum inhibitory concentration for the particular organism being treated. For use as an antitumor agent, optimal dosages and regimens of BBM-1675C or BBM-1675D for a given mammalian host can be readily ascertained by those skilled 55 in the art. It will, of course, be appreciated that the actual dose of BBM-1675C or BBM-1675D used will vary according to the particular composition formulated, the mode of application and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease. Administration can be carried out continuously or 60 periodically within the maximum tolerated dose. Optimal application rates for a given set of conditions can be ascertained by those skilled in the art using conventional dosage determination tests in view of the above guidelines.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

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Chemical Preparation and Isolation of BBM-1675C and BBM-1675D Example 1

Asample of BBM-1675A, (50mg) was dissolved in 2.5ml of methanol and treated with 2.5ml of 0.1 molar 5 solution of hydrogen chloride in methanol. The reaction was allowed to proceed at a temperature of about 50°C, and the disappearance of the starting material (approximately 30 minutes) was monitored every 5 to 10 minutes by thin layer chromatography (TLC) on silica gel plates (Analtech, 250 micron, GF) with toluene:acetone (3:2, v/v) as the eluting solvent. After the starting material has been consumed, the reaction mixture was neutralized with a saturated solution of NaHC03 in methanol, then evaporated under reduced pressure to yield a dry residue 10 containing the bioactive fragments. The BBM-1675C substance was isolated from the residue by flash column chromatography on a 2 cm i.d. x 10 cm column packed with Woelm silica gel (32-63 micron particle size). The column was eluted with toluene:acetone (3:2, v/v) collecting 3 ml fractions. Each fraction was analyzed by TLC (silica gel with toluene:acetone (3:2, v/v) as eluent), and the TLC spots were visualized with a UV 254 nm light source and a eerie sulfate spray (1 % eerie sulfate and 2.5% molybolic acid in 10% sulfuric acid). Fractions 6-12 15 (R, value for BBM-1675C is 0.28) were pooled and evaporated to dryness to yield 12 mg (35%) of substantially pure BBM-1675C.

The physico-chemical properties of BBM-1675C appear in the specification and the ultraviolet, infrared, mass, 'H NMR and 13C NMR spectra of the compound appear as Figures 1, 3, 5,7 and 9, respectively.

20 Example 2

When the reaction time of the procedure in Example 1 is extended, the amount of BBM-1675C decreases, and two new products denoted as compound 3 (Rf = 0.65) and BBM-1675D (R, remains at baseline) [TLC: silica, toluene:acetone (3:2, v/v)] appear and become more prominent with time.

Compound BBM-1675D which usually accompanies the production of BBM-1675C was isolated from the 25 chromatographic column 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 yield 18 mg of substantially pure BBM-1675D from the reaction described in Example 1.

The BBM-1675D substance exhibits one major spot at Rf = 0.37 in reverse phase TLC (Whatman MKC18F, 20C micron) using 30% water in methanol as the eluent and Rf = 0.22 in normal phase silica gel TLC using 30 chloroform:methanol (5:0.5, v/v) as the eluent.

Example 3

Substantial improvement in the yield of BBM-1675D can be achieved by using p-toluenesulfonic acid in place of hydrogen chloride in the chemical hydrolysis of BBM-1675AZ or BBM-1675A, as illustrated by the 35 procedures of Examples 3 and 5, respectively.

A sample of BBM-1675A2 (15.2 mg) was hydrolyzed with 0.03 molar solution of p-toluenesulfonic acid in methanol (1 ml) at a temperature of about 63°C for about one hour. The reaction mixture was then evaporated to dryness under reduced pressure at about 30°C. The BBM-1675D substance was isolated from the dry residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size). The 40 column was eluted with chloroform:methanol (5:0.5, v/v), and the collected fractions were analyzed by TLC [silica gel with chloroforrrrmethanol (5:0.5, v/v) as eluent]. The applied chromatography conditions permitted the separation of the mixture of inactive compounds 2 and3 (7 mg) from the bioactive BBM-1675D substance which has an R, value of 0.22. The appropriate fractions were pooled and evaporated to dryness to yield 8 mg of substantially pure BBM-1675D in near quantitative yield.

45 The physico-chemical properties of BBM-1675D appear in the specification and the ultraviolet, infrared, mass, 1H NMR and 13C NMR spectra of the compound appear as Figures 2,4, 6,8 and combined lOAand 10B, respectively.

Example 4

50 A sample of BBM-1675A2 (40 mg) was treated with 5 ml of an 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 BBM-1675C substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) using toluene.acetone (3:2, v/v) as the eluent. The appropriate fractions were combined 55 and evaporated to dryness to yield 8.4 mg of substantially pure BBM-1675C which is identical to the product isolated in Example 1.

The chromatographic column of above was then eluted with chloroform:methanol (5:0.25, v/v) and the fractions collected were pooled and evaporated to dryness to yield BBM-1675D. The BBM-1675D substance was further purified by an additional flash chromatography column with silica gel utilizing chloroform:methanol 60 (5:0.5, v/v) as the eluent. The appropriate fractions were combined and evaporated to dryness to yield 6.3 mg of substantially pure BBM-1675D which is identical to the product isolated in Example 3.

Example 5

A sample of BBM-1675A, (48.3 mg) was hydrolyzed with 0.037 M solution of p-toluenesulfonic acid in 65 methanol (1.5 ml) at a temperature of about 60°C for about 1.5 hours. The reaction mixture was evaporated to

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dryness under reduced pressure at about 30°C to give a residue which contains BBM-1675D and the inactive compounds 1 and 3. The BBM-1675D bioactive substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) utilizing chloroform:methanol (5:0.25, v/v) as the eluent. The appropriate fractions were combined and evaporated to 5 dryness to yield 27mg of substantially pure BBM-1675D which is identical to the product isolated in 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 about 40-50°C overnight. After neutralization with NaHC03 and evaporation to dryness, the 10 BBM-1675D bioactive substance was isolated from the residue by flash column chromatography on a column packed with Woelm silica gel (32-63 micron particle size) utilizing chloroform:methanol (5:0.25, v/v) as the eluent. The appropriate fractions yielded substantially pure BBM-1675D which is identical to the product isolated in Example 3.

15 Example 7

When the general procedure of Examples 1 and 2 are repeated, except that the starting material BBM-1675A, is replaced by an equimolar amount of a mixture containing BBM-1675A, and BBM-1675A2, there is thereby produced the BBM-1675C and BBM-1675D subtances.

20 Example 8

When the general procedure of Example 5 is repeated, except that the starting material BBM-1675A! is replaced by an equimolar amount of a mixture containing BBM-1675A, and BBM-1675A2, there is thereby produced the BBM-1675D substance.

Claims

25 CLAIMS

1. The antitumor antibiotic BBM-1675C which in substantially pure form:

(a) appears as an amorphous solid;

(b) is soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform; 30 (c) exhibits in silica gel thin layer chromatography an Rf value of 0.28 with the solvent system toluene:acetone (3:2, v/v);

(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 substantially as shown in Figure 1 exhibiting ultraviolet absorption maxima and absorptivities at 210nm (a = 21,770), 274 nm (a = 9,340)

35 and 313 nm (shoulder) (a = 4,190) with no significant change upon addition of acid or base;

(f) has an infrared absorption spectrum (KBr, film) substantially as shown in Figure 3 exhibiting principal absorption peaks at

540, 740, 955, 990,1017,1065,1080,1118,

1150,1250,1305,1325,1340,1370,1385,

40 1440,1690,1705,1735, 2900, 2920, 2930,

2970, and 3450 reciprocal centimeters;

(g) has a low resolution mass spectrum substantially as shown in Figure 5 exhibiting a molecular ion [M+ H]+ of 856;

(h) has a 360 MHz proton magnetic resonance spectrum in CDCI3 substantially as shown in Figure 7 45 exhibiting signals at

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 (1 H, q, J=2.6); 3.97 (1H, t, J = 9.6); 3.92-3.53 50 (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 (1 H, 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 1.08 (6H) parts per million downfield from tetramethylsilane;

55 (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCI3 substantially as shown in Figure 9 exhibiting signals at

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,

60 1 24.6,130.1, and 193.1 parts per million downfield from tetramethylsilane.

2. The antitumor antibiotic BBM-1675D which in substantially pure form:

(a) appears as an amorphous solid;

(b) is soluble in methanol, ethanol, acetone and tetrahydrofuran, and slightly soluble in chloroform;

65 (c) exhibits in silica gel thin-layer chromatography an R( value of 0.22 with the solvent system chloroform:

<GB 217964SA__I_>

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methanol (5:0.5, v/v) and exhibits in reverse phase silica gel thin layer chromatography an Rf value of 0.37 with the solvent system methanohwater (70:30, v/v);

(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 substantially as shown in Figure 2 exhibiting

5 ultraviolet absorption maxima and absorptivities at 214 nm (a = 27,000), 274 nm (a = 12,800), and 325 nm (a = 5,400) with no significant change upon addition of acid or base;

(f) has an infrared absorption spectrum (KBr, film) substantially as shown in Figure 4 exhibiting principal absorption peaks at

735, 755, 910, 960,1000,1020,1085,1150,1195,

10 1250,1310,1335,1365,1385, 1445,1510, 1685,

1720,1735, 2880, 2930, 2960, and 3400 reciprocal centimeters;

(g) has a low resolution mass spectrum substantially as shown in Figure 6 exhibiting a molecular ion [M + H] + of 696;

15 (h) has a 360 MHz proton magnetic resonance spectrum in CDCI3 + 10% CD30D substantially as shown in Figure 8 exhibiting signals at

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 (1H, d,

J =8.1); 4.02 (1H, d, J =2.0); 3.95-3.80 (solvent background);

20 3.77 (1 H, t, J = 9.0); 3.70-3.40 (11H, brm); 3.35(1 H, 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); and 0.87 (3H, d, J=5.9) parts per million downfield from tetramethylsilane;

25 (i) has a 90.6 MHz carbon-13 magnetic resonance spectrum in CDCI3 + 10% CD30D substantially as shown in Figure 10 (Figure 10A + 10B) exhibiting signals at

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, and 30 192.8 parts per million downfield from tetramethylsilane.

3. The process for the production of the antitumor antibiotic BBM-1675C, which comprises hydrolyzing BBM-1675A, or BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675C is produced and then recovering BBM-1675C from the reaction medium.

4. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing 35 BBM-1675A, or BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675D is produced and then recovering BBM-1675D from the reaction medium.

5. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing BBM-1675C with a mineral or organic acid until a substantial amount of BBM-1675D is produced and then recovering BBM-1675D from the reaction medium.

40 6. The process for the production of the antitumor antibiotic BBM-1675C, which comprises hydrolyzing a mixture of BBM-1675A, and BBM-1675A2 with a mineral or organic acid until a substantial amount of BBM-1675C is produced and then recovering BBM-1675C from the reaction medium.

7. The process for the production of the antitumor antibiotic BBM-1675D, which comprises hydrolyzing a mixture of BBM-1675A, and BBM-1675A2 with a mineral or organic acid until a substantial amount of

45 BBM-1675D is produced and then recovering BBM-1675D from the reaction medium.

8. The antitumor antibiotic BBM-1675C, substantially as hereinbefore specifically described with particular reference to the examples.

9. The antitumor antibiotic BBM-1679D, substantially as hereinbefore specifically described with particular reference to the examples.

50 10. A process for the production of BBM-1675C, substantially as hereinbefore specifically described with particular reference to the examples.

11. A process for the production of BBM-1675D, substantially as hereinbefore specifically described with particular reference to the examples.

12. A pharmaceutical composition comprising an effective antimicrobial amount of BBM-1675C or 55 BBM-1675D in combination with a pharmaceutical carrier or diluent.

13. A pharmaceutical compostion comprising an effective tumor-inhibitng amount of BBM-1675C or BBM-1675D in combination with a pharmaceutical carrier or diluent.

14. A pharmaceutical compound as defined in claim 1 for use in a method of treatment of the human or animal body.

60 15. A pharmaceutical compound as defined in claim 2 for use in a method of treatment of the human or animal body.

16. A compound according to claims 1 or 2 for use as an antitumor antibiotic.

17. A compound according to either of claims 1 or 2 substantially as hereinbefore specifically described in each of the examples for the use hereinbefore specifically described.

65 18. A pharmaceutical composition according to either of claims 12 or 13 substantially as hereinbefore

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specifically described in examples.

19. A compound when produced by a process as claimed in any one of claims 3 to 7,10 or 11.

20. The use of a substance according to either of claims 1 or 2 for the manufacture of a medicament which is antitumor antibiotic.

5

Printed for Her Majesty's Stationery Office by Croydon Printing Company (UK) Ltd 1 /87. D8817356. Published by The Patent Office, 25 Southampton Buildings, London, WC2A1 AY, from which copies may be obtained.

BNSDOCID: <GB 2179649A_l_>