DE3629052A1 - ANTITUMOR ANTIBIOTICS - Google Patents

Description

The invention relates to new anti-tumor antibiotic substances as well their manufacture and isolation.

The structure of the anti-tumor compounds according to the invention is up to not yet cleared up. Because of their unique physical, chemical and biological properties antibiotics BBM-1675C and BBM-1675D for new substances.

British patent application 21 41 425, published on December 19, 1984, describes the fermentation of Actinomadura verrucosospora strain H964-92 (ATCC 39334) or Actinomadura verrucosospora strain A1327Y (ATCC 39638) for the production of a new antitumor-antibiotic complex called BBM -1675. Two mainly bioactive components of the BBM-1675 complex described there are referred to as BBM-1675A ₁ and BBM-1675A ₂ . The structures of the BBM-1675A ₁ and BBM-1675A ₂ antibiotics, which are also referred to as Esperamicin A ₁ and Esperamicin A ₂ , have not yet been elucidated, but both components show excellent antimicrobial and antitumor activity.

U.S. Patent 4,530,835, issued July 23, 1985 (Bunge et al.) Discloses the fermentation of an unidentified Actinomycetes isolate WP-444 (ATCC 39363) for the manufacture of anti-tumor antibiotics called CL-1577A and CL- 1577B. The structures of the CL-1577 antibiotics have not yet been elucidated, but characteristic properties of these antibiotics show that CL-1577A and CL-1577B in the structure of the BBM-1675 antibiotics and in particular BBM-1675A ₁ and A _{2 of} the British patent application 21 41 425 are similar.

In Journal Antibiotics, 37 (12), 1566-1571 (1984), R. H. Bunge et al. the fermentation of Actinomadura sp. (ATCC 19363) to get a bioactive complex that consists of two main components, PD 114.759 and PD 115.028 were isolated. In J. Chem. Soc. Chem. Commun., 919-920 (1985) describe J.H. Wilton et al. the partial structure elucidation of antibiotics PD 1 14 759 and PD 1 15 028. The manufacture, isolation and characterization of antibiotics PD 1 14 759 and PD 1 15 028 appear to be the same as those above mentioned antibiotics CL-1577A and CL-1577B.

European patent application 95 154, published November 30 1983, describes the fermentation of Actinomadura pulveraceus sp. nov No. 6049 (ATCC 39100) for the production of anti-tumor antibiotics with the designation WS 6049-A and WS 6049-B. The structure the antibiotics WS 6049 has not yet been elucidated, the characterizing ones However, properties of these antibiotics show that WS 6049-A and WS-6049B in the structure of the antibiotics BBM-1675  British patent application 21 41 425 and antibiotics CL-1577 of U.S. Patent 4,530,835. However, spectral data show that neither WS 6049-A nor WS 6049-B are identical to a BBM-1675 component is. The one described in European patent application 95 154 producing organism can clearly differentiate are used by Actinomadura verrucosospora, the process according to the British Patent application 21 41 425 in relation to the color his aerial mycelium on ISP medium No. 2, 3 and 4 and his positive Milk peptonization and its positive utilization on D-fructose, D-mannitol, trehalose and cellulose.

The present invention creates new anti-tumor antibiotics called BBM-1675C and BBM-1675D (also referred to as BMY-27305 and BMY-27307, respectively). These substances can be produced by selective chemical hydrolysis of the bioactive constituents BBM-1675A ₁ (Esperamicin A ₁ ) or BBM-1675A ₂ (Esperamicin A ₂ ). These components can in turn be made by cultivating a strain of Actinomadura verrucosospora producing BBM-1675. The BMM-1675C and BBM-1675D bioactive substances can be isolated and purified by conventional chromatographic methods. Both substances show excellent antimicrobial activity and antitumor activity.

Further features and details of the invention emerge from the following description in connection with the drawing and the subclaims.

Show in the drawing

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

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

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

Fig. 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

Fig. 7 shows the proton magnetic resonance spectrum of BBM-1675C in _CDCl3 (360 MHz)

Fig. 8 is the proton magnetic resonance spectrum of BBM-1675D in _CDCl3 + 10% _CD3OD (360 MHz)

Fig. 9 shows the ¹³ C magnetic resonance spectrum of BBM1675C in _CDCl3 (90.6 MHz)

FIG. 10A, the ¹³ C magnetic resonance spectrum (110-200 ppm) of BBM-1675D in _CDCl3 + 10% _CD3OD (90.6 MHz)

FIG. 10B, ¹³ C magnetic resonance spectrum (0-110 ppm) of BBM-1675D in _CDCl3 + 10% _CD3OD (90.6 MHz)

FIG. 11A, the proton magnetic resonance spectrum of compound 3A (α-anomer) in _CDCl3 (360 MHz) and

FIG. 11B, the proton magnetic resonance spectrum of compound 3B (β-anomer) in _CDCl3 (360 MHz).

As mentioned above, the invention relates to two new anti-tumor antibiotics called BBM-1675C and BBM-1675D (also referred to as BMY-27305 and BMY-27307, respectively). These substances can be produced by selective chemical hydrolysis of the bioactive components BBM-1675A ₁ (Esperamicin A ₁ ) or BBM-1675A ₂ (Esperamicin A ₂ ). These components can in turn be produced by culturing a BBM-1675 producing strain of Aktinomadura verrucosospora, in particular Aktinomadura verrucosospora strain H964-92 (ATCC 39334) or Aktinomadura verrucosospora strain A1327Y (ATCC 39638), a mutant thereof. The invention further relates to a process for the production of BBM-1675C by selective hydrolysis of the bioactive constituents BBM-1675A ₁ or BBM-1675A ₂ , furthermore the production of BBM-1675D by selective hydrolysis of BBM-1675C or in particular the bioactive components BBM-1675A ₁ or BBM-1675A ₂ . Isolation and purification of BBM-1675C and BBM-1675D from the reaction mixture can be accomplished by conventional chromatographic methods.

The bioactive substances BBM-1675C and BBM-1675D show antimicrobial Activity against a wide range of microorganisms and have also shown that they have inhibitory activity against have different mouse tumor systems, such as P-388 and leukemia B16 melanoma. The new substances according to the invention described here can therefore be used both as antimicrobial agents and as Antitumor agents used to inhibit tumors in mammals will.

During the course of degradation studies to clarify the structure of the anti-tumor antibiotics BBM-1675A ₁ (Esperamicin A ₁ ) and BBM-1675A ₂ (Esperamicin A ₂ ), a mixture of components was produced which led to the isolation and identification of two inactive fragments , namely compound of formula 1 and formula 2. Surprisingly, it was found that chemical degradation led to the gradual release of two bioactive fragments BBM-1675C and BBM-1675D. It was even more surprising that the two different antibiotics BBM-1675A ₁ and A _{2 formed the} same bioactive fragments as shown in Scheme 1. It is particularly surprising that the lower molecular weight fragments BBM-1675C and BBM-1675D (they have about 70% and 55% of the molecular weight of the starting antibiotics BBM-1675A ₁ and A _2, respectively) are more effective than BBM-1675A as anti-tumor and anti-microbial agents ₂ and comparable to BBM-1675A ₁ .

Scheme 1

The substances BBM-1675C and BBM-1675D can be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A ₁ , as shown in Scheme 2.

Scheme 2

The starting compound BBM-1675A ₁ is prepared according to the method described in British patent application 21 41 425. The purified BBM-1675A ₁ is hydrolyzed with a mineral acid or organic acid such as hydrogen chloride, sulfuric acid, p-toluenesulfonic acid, benzenesulfonic acid and the like in an organic or mixed aqueous / organic inert solvent at a temperature of about 0 ° C. to the reflux temperature of the solvent until a substantial amount of the desired BBM-1675c or BBM-1675D is produced. The hydrolysis is preferably carried out in C ₁ -C ₆ alcohol solvents, with alcoholysis in methanol being particularly preferred. The temperature of the reaction is not critical, but the reaction is preferably carried out at about ambient temperature to 60 ° C, especially between about 40 and 60 ° C.

The selective hydrolysis of BBM-1675A ₁ progresses gradually with the initial production of the BBM-1675C antibiotic and the inactive fragment of Formula 1. Subsequent or continuous treatment under hydrolysis conditions leads to the release of a mixture of α and β anomers of thio sugar according to formula 3 and the formation of the antibiotic BBM-1675D. It will be understood by those skilled in the art that changing reaction conditions such as time, temperature and acid concentration will result in a variation in the relative amounts of the antibiotics BBM-1675C and BBM-1675D. It is therefore desirable to monitor the progress of the reaction by thin layer chromatography as described in the examples below.

If it is desired to make only the BBM-1675D antibiotic, then the selective hydrolysis is preferably carried out with an organic one Acid such as p-toluenesulfonic acid, as described here, to get a quantitative amount of BBM-1675D.  

The substances BBM-1675C and BBM-1675D can also be prepared by selective chemical hydrolysis of the antibiotic BBM-1675A ₂ , as shown in Scheme 3.

Scheme 3

The starting compound BBM-1675A ₂ is prepared according to the procedure described in British patent application 21 41 425. The selective hydrolysis of the purified BBM-1675A ₂ also progresses gradually with the initial production of the antibiotic BBM-1675C and the inactive fragment of the formula 2. Continued treatment under hydrolysis conditions leads to the release of a mixture of α and β anomers of the thio sugar according to formula 3 and formation of the antibiotic BBM-1675D.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A ₂ are essentially the same as those used for the hydrolysis of BBM-1675A ₁ described above.

Similar to the production of BBM-1675D from BBM-1675A ₁ , the hydrolysis of BBM-1675A _{2 is} carried out until essentially all of the BBM-1675A ₂ and BBM-1675C are converted to BBM-1675D, if preferred is to make only the BBM-1675D antibiotic. The hydrolysis is particularly preferably carried out with an organic acid such as p-toluenesulfonic acid.

Since it was found that the same antibiotics BBM-1675C and BBM-1675D can be produced from two different antibiotics BBM-1675A ₁ and BBM-1675A ₂ with the simultaneous loss of two inactive fragments of the formulas 1 and 2 and the thio sugar of the formula 3 , there is an additional advantage for the invention. Accordingly, according to a further embodiment of the invention, the process for producing BBM-1675C and BBM-1675D as illustrated in Scheme 4 can also be carried out by selective hydrolysis of a mixture of BBM-1675A ₁ and A ₂ .

Scheme 4

BBM-1675A ₁ + BBM-1675A ₂ → BBM-1675C → BBM-1675D

This advantage is evident when one considers that the relative amounts of BBM-1675A ₁ and A ₂ that are formed in the fermentation process are subject to variation. In contrast, the production of BBM-1675C and BBM-1675D is independent of the relative amounts of BBM-1675A ₁ and A ₂ used as the starting material according to the invention.

As described above, hydrolysis of the antibiotics BBM-1675A ₁ , BBM-1675A ₂ and BBM-1675C 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 thus the antibiotics BBM-1675A ₁ and A ₂ . The compound of Formula 3 was identified as a mixture of α and β anomers of a thio sugar having the structure described in Schemes 2 and 3. A further characterization was possible after isolation of the products of alcoholysis, the and anomers. Proton magnetic resonance spectra (360 MHz) of compound 3A (α-anomer) and compound 3B (β-anomer) are shown in Figures 11A and 11B, respectively. From an analysis of the spectral data, the thiosugar methylglycosides according to Formula 3 were tentatively assigned to the relative stereochemistry of the following formula

Absolute stereochemistry, ie D or L, has not yet been determined. Accordingly, based on the current interpretation of the spectral data, it is concluded that the thiosugar according to formula 3 (reduced by the CH ₃ group of the anomeric methoxy which was introduced during the methanolysis) is a component in the structure of the antibiotic BBM-1675C and also a component Component in the structure of the parent antibiotics BBM-1675A ₁ and A ₂ .

The physiochemical properties of BBM-1675C were examined:
Description: amorphous solid
Ultraviolet absorption spectrum: Fig. 1
Instrument: Hewlett-Packard 8458
Solvent: methanol
Concentration: 0.0155 g / l
λ _max (nm) Absorbance 21021770 2749340 313 sh (shoulder) 4190 No significant change was observed with acid or base.

Infrared absorption spectrum: see 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, 2900, 2920, 2930,
2970, 3450 cm ^-1

Mass spectrum: see Fig. 5
Instrument: Finigan 4500 TSQ
Method: rapid atomic bombing (FAB) ionization

Instrument: Kratos MS-50
High resolution FAB (m / z): [M + H] ⁺ = 856.3362
Molecular weight: apparent molecular weight = 855
(based on the mass spectral data described above)
Elemental composition: C ₃₆ H ₆₁ N ₃ O ₁₄ S ₃
(based on the high-resolution data described above)
Proton magnetic resonance spectrum: see Fig. 7
Instrument: WM 360 Bruker
Solvent: CDCl ₃

¹ H NMR 360 MHz δ (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 = 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 (1OH), 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). [Downfield from tetramethylsilane]

¹³ C Magnetic resonance spectrum: see Fig. 9
Instrument: WM 360 Bruker
Solvent: CDCl ₃
¹³ C NMR 90.6 MHz δ (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. [Downfield from tetramethylsilane]

The physiochemical properties of BBM-1675D were examined:
Description: amorphous solid
Ultraviolet absorption spectrum: see Fig. 2
Instrument: Hewlett-Packard 8458
Solvent: methanol
Concentration: 0.01 g / l
λ _max (nm) Absorbance 21427000 27412800 3255400 No significant change was observed with acid or base.

Infrared absorption spectrum: see Fig. 4
Instrument: Nicolet 5DX FT-IR
Main absorption bands (Kr, 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
Mass spectrum: see Fig. 6
Instrument: Finigan 4500 TSQ
Method: rapid atomic bombardment (FAB ionization
Matrix: thioglycerin
Molecular ion (m / z): [M + H] ⁺ = 696
Relative abundance: 100%
Instrument: Kratos MS-50
High resolution FAB (m / z): [M + H] ⁺ = 696.2794

Molecular weight: apparent molecular weight = 695 (based on the mass spectral data described above)

Elemental composition: C ₂₉ H ₄₉ N ₃ O ₁₂ S ₂ (based on the high-resolution data described above)

The correlation of [M + H] ⁺ and [(M + H) + 2] ⁺ relative abundances to their calculated values confirmed by the high-resolution FAB measurements derived elementary compositions.

Proton magnetic resonance spectrum: see Fig. 8
Instrument: WM 360 Bruker
Solvent: CDCl ₃ + 10% CD ₃ OD

¹ H NMR 360 MHz δ (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 (1H, d, J = 8.1); 4.02 (1H, d, J = 2.0); 3.95-3.80 (solvent background); 3.77 (1H, t, J = 9.0); 3.70-3.40 (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). [Downfield from tetramethylsilane]

¹³ C magnetic resonance spectrum: see Figs. 10A and 10B
Instrument: WM 360 Bruker
Solvent: CDCl ₃ + 10% CD ₃ OD

¹³ C NMR 90.6 MHz δ (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. [Downfield from tetramethylsilane]

Below are the biological properties of the BBM-1675 substances described.

The antimicrobial activity of the BBM-1675 substances was determined against a number of gram-positive and gram-negative microorganisms. Table 1 below shows data in the form of the results of an antimicrobial screening process which comprised the parent substance BBM-1675A ₁ and the substances BBM-1675C and BBM-1675D according to the invention. In the screening process, each test substance was placed on the growth culture in the form of a paper strip impregnated with a uniform concentration of 10 µg / ml solution. The measure of antibiotic activity is the zone of inhibition resulting from the paper strip. As shown in Table 1, the substances BBM-1675C and BBM-1675D showed a broad spectrum of antimicrobial activity which was at least as great as that of BBM-1675A ₁ . The substances BBM-1675C and BBM-1675D were in particular more active than inhibitors against gram-negative organisms.

Table 1

Antimicrobial activity of BBM-1675 substances

Activity against P-388 leukemia was examined:

Tables II and III contain the results of laboratory tests with CDF ₁ mice, in which a tumor inoculum of 10 ⁶ ascites cells of P-388 leukemia was implanted intraperitoneally and which were treated with different doses of BBM-1675A ₁ , C or D. The substances were administered by intraperitoneal injection. Groups of 6 mice were used for each dose amount. They were treated with a single dose of the substance one day after the inoculation. A group of 10 control mice treated with saline (vehicle) were included in each series of experiments. The group treated with BBM-1675A ₁ in Table III was included in the direct comparison. A 30-day log was kept. The average survival time in days determined for each group of mice and the number of survivors at the end of the 5-day period were noted. The mice were weighed before treatment and again on day 4. The change in weight was taken as a measure of the toxicity of the sample. Mice weighing 20 g each were used, and weight loss up to approximately 2 g was not considered excessive. The control animals treated with the vehicle usually died within 9 days. The results were calculated in terms of% T / C, which is the ratio of the mean survival time of the treated group to the mean lifetime of the control group treated with the vehicle × 100. An effect expressed in% T / C = 125 shows that a significant Antitumor effect was achieved. The screening results in Table II show the initially unexpected level of anti-tumor activity of the BBM-1675C substance. Table III shows the results of a direct comparison of BBM-1675A ₁ (esperamicin A ₁ ) and the substances BBM-1675C and BBM-1675D. The data indicate that BBM-1675C is roughly comparable to BBM-1675A ₁ in terms of strength and anti-tumor activity and that it is not dependent on administration, while BBM-1675D is only slightly less effective. Furthermore, it has already been pointed out that according to. the invention, the same substances BBM-1675C and BBM-1675D can also be obtained from component BBM-1675A ₂ (Esperamicin A ₂ ). In comparison to the reported data for BBM-1675C and BBM-1675D and the data given in British patent application 21 41 425 for BBM-1675A ₂ , it was surprisingly found that the substances BBM-1675C and BBM-1675D as an anti-tumor Are more effective than the parent substance BBM-1675A ₂ from which they are derived.

Table II

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

Tumor inoculum: 10 ⁶

Ascites cells implanted ip
Host: CDF ₁

male mice
Evaluation: MST = mean survival time
Effect: §T / C = (MST treated / MST control × 100
Criteria:% T / C 125 is considered significant anti-tumor activity
AWC: mean weight change (treated control) in grams (on day 4)

Table III

Effect of BBM-1675 substances on P-388 leukemia

Tumor inoculum: 10 ⁶

Ascites cells implanted ip
Host: CDF ₁

female mice
Evaluation: MST = mean survival time
Effect:% T / C = (MST treated / MST control) × 100
Criteria:% T / C 125 is considered significant anti-tumor activity
AWC: mean weight change (treated - control) in grams (on day 4)

The activity against B16 melanoma was examined:

Table IV contains the results of anti-tumor tests using B16 melanoma grown in mice. The tumor implant was subcutaneously inoculated in BDF ₁ mice. A 60 day protocol was used. Groups of 10 mice were tested for each dose level. The mean survival for each group was determined. Control animals that were inoculated in the same manner as the test animals and treated with the injection vehicle without the drug showed an average survival time of 22.5 days. For each dose level, the test animals were treated with the test compound on days 1, 5 and 9 by intraperitoneal injection. An effect expressed in% T / C 125 indicates a significant anti-tumor effect. The results in Table 4 show that in a direct comparison BBM-1675C is also effective in the treatment of B16 melanoma-bearing mice and that the potency is approximately comparable to that of BBM-1675A ₁ .

Table IV

Effect of BBM-1675 substances on B16 melanoma

(Treatments on day 1, 5 and 9)

Tumor inoculum: 0.5 ml of a 10% pulp
Host: BDF ₁

female mice
Evaluation: MST = mean survival time
Effect:% T / C = (MST treated / MST control) × 100
Criterion:% T / C 125 is considered significant anti-tumor activity
AWC: mean weight change (treated - control) in grams (on day 12)

As can be seen from the above antimicrobial and mouse tumor data, BBM-1675C and BBM-1675D are valuable as antibiotics in therapeutic Treatment of mammals and other animals in infectious Diseases and also as anti-tumor agents for therapeutic Inhibition of mammalian tumor growth.

The present invention therefore also offers a method for therapeutic Treatment of an animal host by a microbial Infection or malignant tumor is compromised the host an effective antimicrobial or tumor inhibiting Dose of BBM-1675C or BBM-1675D or a pharmaceutical composition this is administered.

The invention also relates to pharmaceutical compositions, an effective antimicrobial or tumor inhibiting amount BBM-1675C or BBM-1674D in combination with a pharmaceutical acceptable carrier or diluent. Such compositions other active antimicrobial or anti-tumor Agents contain and can be in any pharmaceutical form be prepared, suitable for the desired form of administration is. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, Pills, powders and granules, liquid compositions for the 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 can also be in the form of sterile solid compositions, which in sterile water, physiological saline or a other sterile injection medium just before use will be made.  

For use as an antimicrobial agent, the BBM-1675C or BBM-1675D or a pharmaceutical composition thereof administered that the concentration of the active ingredient is greater is the minimum inhibitory concentration for each organism to be treated. For use as an anti-tumor agent the optimal dosage and handling of BBM-1675C or BBM- 1675D easily assured by those skilled in the art for a given mammalian host will. It is understandable that the actually used Dose of BBM-1675C or BBM-1675D depending on the respective formulated composition, the mode of administration and the respective site, host and the disease to be treated varies. Other factors that affect the effectiveness of the agent may be considered, such as age, weight, gender, Diet, administration time, route of administration, excretion rate, condition of the patient, drug combinations, reactive sensitivities and severity of the disease. Administration can be continuous or periodically within the maximum tolerated dose. The optimal administration rates for given conditions can Expert using conventional tests to determine the dose related ensure with the guidelines above.

The following examples serve to explain preferred ones Embodiments for making BBM-1675C and BBM-1675D.

example 1

A sample of BBM-1675A ₁ (50 mg) was dissolved in 2.5 ml of methanol and treated with 2.5 ml of a 0.1 molar 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 was monitored (approximately 30 min.) By washing every 5-10 min. performed a thin layer chromatography (TLC) on silica gel plates (Analtech, 250 micron, GF) with toluene to acetone (3: 2, vol / vol) as eluent. After the starting material was consumed, the reaction mixture was neutralized with a saturated solution of NaHCO ₃ in methanol and then evaporated under reduced pressure to obtain a dry residue containing the bioactive fragments. The BBM-1675C substance was isolated from the residue by rapid column chromatography on a 2 cm (diameter) by 10 cm column packed with Woelm silica gel (32-63 micrometer particle size). The column was eluted with toluene: acetone (3: 2, vol / vol), collecting 3 ml fractions. Each fraction was analyzed by thin layer chromatography (TLC) [silica gel with toluene: acetone (3: 2, vol / vol) as eluent] and the spots of thin layer chromatography were analyzed with a 254 µm UV light source and a cerosulfate spray (1% Cerium sulfate and 2.5% molybdic acid in 10% sulfuric acid). Fractions 6-12 (R _f value for BBM-1675C is 0.28) were pooled and evaporated to dryness to give 12 mg (35%) essentially of a BBM-1675C.

The physicochemical properties of BBM-1675C are listed in the foregoing description, and the ultraviolet, infrared, mass, ¹ H NMR and ¹³ C NMR spectra of the compound are shown in Figures 1, 3, 5, 7 and 9 shown.

Example 2

If the reaction time of the process according to Example 1 is extended, the amount of BBM-1675C decreases and two new products, designated compound 3 (R _f = 0.65) and BBM-1675D (R _f remains at the baseline) [TLC: silica , Toluene: acetone (3: 2, vol / vol)], appear and increase in quantity over time.

BBM-1675D, which is normally used to make BBM- Accompanied by 1675C, was from the chromatography column described in Example 1 isolated by column with chloroform: methanol (5: 1, vol / vol) was eluted. The right fractions were pooled and evaporated to dryness, 18 mg essentially pure BBM-1675D were obtained by the method described in Example.

The substance BBM-1675D shows a main spot at R _f = 0.37 in reverse phase thin layer chromatography (Whatman MKT ₁₈ F, 200 micrometers) using 30% water in methanol as eluent and at R _f = 0.22 in normal phase thin layer chromatography (TLC) using chloroform: methanol (5: 0.5, vol / vol) as eluent.

Example 3

A significant improvement in the yield of BBM-1675D can be achieved using p-toluenesulfonic acid instead of hydrogen chloride in the chemical hydrolysis of BBM-1675A ₂ or BBM-1675A ₁ , as explained in the procedures of Examples 3 and 5, respectively.

A sample of BBM-1675A ₂ (15.2 mg) was hydrolyzed with a 0.03 molar solution of p-toluenesulfonic acid in methanol (1 ml) at a temperature of approx. 360 ° C. for approx. 1 hour. The reaction mixture was then evaporated to dryness at about 30 ° C. under reduced pressure. The BBM-1675D substance was isolated from the dry residue by rapid column chromatography on a Woelm silica gel (32-63 micron particle size). The column was eluted with chloroform: methanol (5: 0.5, vol / vol) and the collected fractions were analyzed by thin layer chromatography [(silica gel with chloroform: methanol (5: 0.5, vol / vol) as the eluent). Chromatography conditions used allowed the mixture of inactive compounds 2 and 3 (7 mg) to be separated from the bioactive substance BBM-1675D, which had an R _f value of 0.22, and the correct fractions were pooled and evaporated to dryness, with 8 mg of essentially pure BBM -1675D were obtained in almost quantitative yield.

The physicochemical properties of BBM-1675D are mentioned in the description, and the ultraviolet, infrared, mass, ¹ H NMR and ¹³ C NMR spectra of the compound are shown in Figs. 2, 4, 6, 8 and 10A and 10B.

Example 4

A sample of BBM-1675A ₂ (40 mg) was treated with 5 ml of a 0.5 molar solution of hydrogen chloride in methanol at approx. 50 ° C. for approx. 2 hours according to the general procedure and isolation method according to Example 1. After neutralization with NaHCO ₃ and evaporation to dryness, the BBM-1675C substance was removed from the residue by rapid column chromatography on a column packed with Woelm silica gel (32-63 micrometer particle size) using toluene: acetone (3: 2 , vol / vol) isolated as eluent. The correct fractions were combined and evaporated to dryness to give 8.4 mg of substantially pure BBM-1675C, which is identical to the product obtained in Example 1.

The above chromatography column was then run with chloroform: methanol (5: 0.25, vol / vol) eluted and the fractions collected combined and evaporated to dryness to give BBM-1675D. The BBM-1675D substance was further purified by an additional one Rapid chromatography column with silica gel using of chloroform: methanol (5: 0.5, vol / vol) as eluent. The  correct fractions were pooled and evaporated to dryness, whereby 6.3 mg of substantially pure BBM-1675D was obtained which is identical to the product according to Example 3.

Example 5

A sample of BBM-1675A ₁ (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, whereby a residue was obtained which contained BBM-1675D and the inactive compounds 1 and 3. The bioactive substance BBM-1675D was removed from the residue by rapid column chromatography on a column packed with Woelm silica gel (32-63 micrometer particle size) using chloroform: methanol (5: 0.25, vol / vol) as the eluent isolated. The correct fractions were pooled and evaporated to dryness to give 27 mg 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 a 0.5 molar solution of hydrogen chloride in methanol (1 ml) at approx. 40-50 ° C overnight. After neutralization with NaHCO ₃ and evaporation to dryness, the bioactive substance BBM-1675D was removed from the residue by rapid column chromatography on a column packed with Woelm silica gel (32-63 micrometer particle size) using chloroform: methanol (5: 0.5 , vol / vol) isolated as eluent. The correct fractions gave essentially pure BBM-1675D, which is identical to the product isolated in Example 3.

Example 7

If the general procedure of Examples 1 and 2 is repeated with the exception that the starting material BBM-1675A _{1 is} replaced by an equimolar amount of a mixture of BBM-1675A ₁ and BBM-1675A ₂ , the substances BBM-1675C and BBM- 1675D also formed.

Example 8

If the general procedure of Example 5 is repeated, except that the starting material BBM-1675A _{1 is} replaced by an equimolar amount of a mixture of BBM-1675A ₁ and BBM-1675A ₂ , then the substance BBM-1675D is also formed.

Claims (29)

1. Antitumor antibiotic BBM-1675C in essentially pure form with the following characteristics: (a) it is an amorphous solid; (b) it is soluble in methanol, ethanol, ethyl acetate, acetone, Tetrahydrofuran and chloroform; (c) it shows an R _f value of 0.28 in silica gel thin layer chromatography with the solvent system toluene: acetone (3: 2, vol / vol); (d) after high-resolution FAB mass spectroscopy, a Measured molecular weight of 855;   (e) It has an ultraviolet absorption spectrum in methanol solution substantially as shown in Fig. 1, and shows ultraviolet absorption maxima and absorbency at 210 nm (a = 21770), 274 nm (a = 9340) and 313 nm (Shoulder) (a = 4190) without significant change when acid or base is added; (f) it has an infrared absorption spectrum (KBr, film) substantially as shown in Fig. 3 and shows major absorption peaks
540, 740, 955, 990, 1017, 1065, 1080, 1118,
1150, 1250, 1305, 1325, 1340, 1370, 1385,
1440, 1690, 1705, 1735, 2900, 2920, 2930,
2970 and 3450 reciprocal centimeters; (g) has a low resolution mass spectrum essentially as in FIG. 5 with a molecular ion [M + H] ⁺ of 856; (h) has a 360 MHz proton magnetic resonance spectrum in CDDl ₃ essentially as in Fig. 7 and shows 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 (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 = 20.3, 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 from tetramethylsilane; (i) It has a 90.6 MHz carbon 13 nuclear magnetic resonance spectrum in CDCl ₃ essentially as in FIG. 9 and shows 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, 124.6, 130.1, and 193.1 parts per million from tetramethylsilane. 2. Anti-tumor antibiotic BBM-1675D in essentially pure form with the following characteristics: (a) it is an amorphous solid; (b) it is soluble in methanol, ethanol, acetone and tetrahydrofuran and slightly soluble in chloroform; (c) it shows an R _f value of 0.22 in silica gel thin layer chromatography with the chloroform: methanol solvent system (5: 0.5, vol / vol) and shows an R _f value in reverse phase silica gel thin layer chromatography from 0.37 with the solvent system methanol: water (70:30, vol / vol); (d) it has a molecular weight of 695 when determined by high-resolution FAB mass spectroscopy; (e) It has an ultraviolet absorption spectrum in methanolic solution substantially as in Fig. 2 and shows ultraviolet absorption maxima and absorbency at 214 nm (a = 27,000), 274 nm (a = 12,800), and 325 nm (a = 5,400) without significant change when adding acid or base; (f) It has an infrared absorption spectrum (Kbr, film) substantially as in Fig. 4 and shows major absorption 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 reciprocal centimeters (g) it has a low resolution mass spectrum essentially as in Fig. 6 and shows a molecular ion M + H⁺ of 696; (h) it has a 316 MHz proton magnetic resonance spectrum in CDCl ₃ + 10% DC ₃ OD essentially as in Fig. 8 and shows 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); 3.77 (1H, t, J = 9.0); 3.70-3.40 (11H, brm); 3.35 (1H, m); 3.28 (3H, s); 3.22 (3H, brs); 2.66-255 (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 from tetramethylsilane; (i) It has a 90.6 MHz carbon 13 nuclear magnetic resonance spectrum in CDCl ₃ + 10% CD ₃ OD essentially like Fig. 10 ( Fig. 10A + 10B) and shows 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 192.8 parts per million from tetramethylsilane. 3. A process for the preparation of the anti-tumor antibiotic BBM-1675C, characterized in that BBM-1675A ₁ or BBM-1675A _{2 is} hydrolyzed with a mineral acid or organic acid until a substantial amount of BBM-1675C is produced and that BBM-1675C is then isolated from the reaction medium. 4. A process for the preparation of the anti-tumor antibiotic BBM-1675D, characterized in that BBM-1675A ₁ or BBM-1675A _{2 is} hydrolyzed with a mineral acid or an organic acid until a substantial amount of BBM-1675D is produced and that the BBM -1675D is then isolated from the reaction medium. 5. Process for the preparation of the anti-tumor antibiotic BBM-1675D, characterized in that BBM-1675C with a mineral acid or an organic acid is hydrolyzed until substantial Amount of BBM-1675D is manufactured and that then the BBM-1675D is obtained from the reaction medium. 6. A process for the preparation of the anti-tumor antibiotic BBM-1675C, characterized in that a mixture of BBM-1675A ₁ and BBM- 1675A _{2 is} hydrolyzed with a mineral acid or an organic acid until a substantial amount of BBM-1675C is produced and the BBM-1675C is then isolated from the reaction medium. 7. A process for the preparation of the anti-tumor antibiotic BBM-1675D, characterized in that a mixture of BBM-1675A ₁ and BBM-1675A _{2 is} hydrolyzed with a mineral acid or organic acid until a substantial amount of BBM-1675D is produced and then the BBM-1675D is isolated from the reaction medium. 8. Pharmaceutical composition containing an effective antimicrobial Amount of BBM-1675C or BBM-1675D in combination with a pharmaceutical carrier or diluent. 9. Pharmaceutical composition containing an effective tumor inhibiting Amount of BBM-1675C or BBM 1675D in combination with a pharmaceutical carrier or diluent. 10. Use of BBM-1675C or BBM-1675D for pharmaceutical Treatment of someone infected with a microbial infection animal host. 11. Use of BBM-1675C or BBM-1675D for therapeutic Treatment of an animal host by one BBM-1675C or 1675D affected sensitive malignant tumor is.