JPH0733393B2 - Novel BBM-1675C antitumor antibiotic - Google Patents

Description

Description: BACKGROUND OF THE INVENTION (1) Field of the Invention The present invention relates to novel antitumor antibiotics and their production and isolation.

(2) Disclosure Statement The antitumor compound of the present invention has not been confirmed in terms of structure. However, considering their unique physical, chemical and biological properties, the BBM-1675C and BBM-1675D antibiotics appear to be novel.

British Patent Application No. 2,141,425, published December 19, 1984
In the issue, Actinomadura verrucosospora strain H964-92 (ATCC39, ATCC39, which produces a novel antitumor antibiotic complex designated as BBM-1675.
334) or Actinomazura Bercosospora (Actinom
Fermentation of adura verrucosospora) strain A1327Y (ATCC 39,638) is disclosed. The two major bioactive components of the BBM-1675 complex described therein are BBM-1675A ₁ and BBM-16.
Shown as 75A ₂ . BBM-1675A ₁ and BBM-1675A ₂ antibiotics, or esperamicin (esperamici
n) The structures of A ₁ and known as esperamicin A ₂ have not yet been elucidated, but both components show excellent antibacterial and antitumor activity.

US Pat. No. 4,530,835 to Bunge et al., Issued July 23, 1985, contains CL-1577A and CL-
Actinomycete isolate WP-444 (ATCC3, which produces an antitumor antibiotic designated as 1577B
9,363) fermentation is disclosed. The structure of the CL-1577 antibiotic has not yet been elucidated, but the characteristic properties conferred on the antibiotic are that the CL-1577A and CL-1577B were described in British Patent Application No. 2,141,425 BBM-1675 antibiotics. , In particular structurally similar to BBM-1675A ₁ and A ₂ .

Journal of Antibiotics, 37 (12), 1 by RHBunge et al.
566 ~ 1571 (1984) 2 main ingredients PD114,759 and PD115,
Fermentation of Actinomadura sp. (ATCC 39,363) producing a bioactive complex with 028 isolated is disclosed. Journal of the Chemical Society, Chemical Communications (J.Chem.S
oc., Chem.Commun.), 919-920 (1985) Wilton (JH Wilton) et al. antibiotics PD114,759 and PD115,0
The elucidation of 28 partial structures was disclosed. PD114,759 and PD11
The production, separation and confirmation of 5,028 antibiotics are described in the above C respectively.
It appears to be the same as the L-1577A and CL-1577B antibiotics.

European Patent Application No. 95,154 published on November 30, 1983
Actinomadura purveraceus (Ac that produces antitumor antibiotics designated as WS6049-A and WS6049-B)
tinomadura pulveraceus) sp.nov.No.6049 (ATCC39,10
0) fermentation is disclosed. The structure of the WS6049 antibiotic has not yet been elucidated, but the characteristic properties given for elucidation are that WS6049-A and WS6049-B are BBM-1675 antibiotics of British Patent Application No. 2,141,425 and US Pat. No. 4,530,835. No. CL-1577, which is related to the structure to the antibiotic. However, the spectral data is WS-6049
Neither -A nor WS-6049-B is identical to any of the BBM-1675 components. Furthermore, the producing strain described in European Patent Application No. 95154 was used in British Patent Application No. 2,141,425 and Actinomadura vercosospora (Actinomadura v
errucosospora) is clear in terms of the color of aerial hyphae on ISP medium Nos. 2, 3 and 4, their possible milk peptonization and their possible availability of D-fructose, D-mannitol, trehalose and cellulose. Can be different.

SUMMARY OF THE INVENTIONThe present invention provides novel antitumor antibiotics, designated BBM-1675C and BBM-1675D, also known as BMY-27305 and BMY-27307, respectively, which comprises:
Actinomadura verru
cosospora) produced by culturing a BBM-1675 producing strain of BBM-1675A ₁ (esperamycin A ₁ ) or BBM-1675A ₂ (esperamycin A ₂ ) produced by selective chemical hydrolysis . The bioactive substances BBM-1675C and BBM-1675D can be separated and purified by conventional chromatographic procedures, both substances exhibiting excellent antibacterial and antitumor activity.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to two novel antitumor antibiotics designated BBM-1675C and BBM-1675D and known as BMY-27305 and BMY-27307, respectively, which actinomadura bercosospora (Actinomadura verrucososp
ora) BBM-1675 producing strain, most preferably Actinomadura verrucosospora
Strain H964-92 (ATCC39,334) or Actinomadura verrucosospora strain A1327Y
Selective chemical hydrolysis of BBM-1675A ₁ (esperamycin A ₁ ) and BBM-1675A ₂ (esperamycin A ₂ ) produced by culturing (ATCC39,368) or mutants thereof Manufactured by. The present invention in another aspect, the bioactive ingredient BBM-1675A ₁ or BBM-1675
To provide a method for producing the BBM-1675C substance by selective hydrolysis of A _2. In another aspect, the invention features BBM-1675C.
By selective hydrolysis of the substance, or more preferably from the bioactive ingredient BBM-1675A ₁ or BBM-1675A ₂ ,
A method of manufacturing BBM-1675D is provided. Separation and purification of BBM-1675C and BBM-1675D from the reaction mixture can be carried out by conventional chromatographic procedures.

The bioactive substances BBM-1675C and BBM-1675D have been shown to exhibit antibacterial activity against a broad spectrum of microorganisms and suppressive activity against various mouse tumor lines such as P-388 leukemia and B-16 melanoma. . Thus, the newly described substances of the invention can be used as antibacterial agents or as antitumor agents to suppress mammalian tumors.

Antitumor antibiotic BBM-1675A ₁ (esperamycin A ₁ )
And a mixture of components leading to the separation and confirmation of two inactive fragments during the course of a degradation study to elucidate the structure of BBM-1675A ₂ (esperamycin A ₂ ) respectively a compound of formula 1 and a compound of formula 2. Occurred. Surprisingly, however, the chemical degradation resulted in two biologically active fragments, BBM-1675C.
And BBM-1675D was observed to produce a gradual release. More surprisingly, two different antibiotics BBM-1675A ₁
And A ₂ were found to produce the same bioactive fragment as shown in Scheme 1. Even more surprisingly, the small molecular weight fragments BBM-1675C and D (approximately 70% and 55% of the parent antibiotics BBM-1675A ₁ and A ₂ , respectively).
% BB as an antineoplastic and antibacterial agent
It was found to be more effective than M-1675A ₂ and comparable to BBM-1675A ₁ .

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

BBM-1675A ₁ compounds as the starting materials are prepared by the procedure described in British Patent Application No. 2,141,425, published on December 19, 1984. The purified BBM-1675A ₁ component is treated 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 or mixed aqueous-organic inert solvent at about 0 ° C. Hydrolyze at the reflux temperature until a substantial amount of the desired BBM-1675C or BBM-1675D is formed. Preferably the hydrolysis is C ₁
Place at -C ₆ alcoholic solvent, most preferably alcoholysis is carried out in methanol. The temperature of the reaction is not critical, but the reaction is carried out at room temperature to 60 ° C, most preferably at about 40-60 ° C.

Selective hydrolysis of BBM-1675A ₁ proceeded stepwise, and BBM-
The 1675C antibiotic and the inactive fragment of Formula 1 are initially generated. Subsequent or subsequent treatment under hydrolytic conditions results in the liberation of a mixture of α and β anomers of the thiosugar of formula 3 and formation of the antibiotic BBM-1675D. When the reaction conditions such as time, temperature and acid concentration are changed, the antibiotic BB
It should be appreciated by those skilled in the art that M-1675C and D occur in various relative amounts. Therefore, it is desirable to monitor the progress of the reaction by thin layer chromatography as described in the examples.

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

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

The starting BBM-1675A ₂ compound is prepared according to the procedure described in British Patent Application No. 2,141,425, published on December 19, 1984. Selective hydrolysis of purified BBM-1675A ₂ likewise proceeds stepwise, initially producing BBM-1675 (antibiotic and inactive fragment of formula 2. When treatment under hydrolysis conditions is continued. Release of the mixture of α and β anomers of the thiosugar of formula 3 and formation of the antibiotic BBM-1675D occurs.

The reaction conditions used for the selective chemical hydrolysis of BBM-1675A ₂ are substantially the same as those used for the hydrolysis of BBM-1675A ₁ . Hydrolysis of BBM-1675A ₂ results in virtually all BBM when it is preferred to produce only BBM-1675D antibiotic as well as the production of BBM-1675D from BBM-1675A _1.
-1675A ₂ and BBM-1675C until converted to BBM-1675D. Most preferably the hydrolysis is with an organic acid such as p
-Toluenesulfonic acid.

As noted, the same BBM-1675C and D antibiotics are 2
It has been found that it is produced from two different antibiotics, BBM-1675A ₁ and BBM-1675A ₂ , at the same time without the _two inactive fragments of formulas 1 and 2 and the thiosugar of formula 3 respectively. Give the advantage of. Therefore, in another aspect of the present invention, as shown in Scheme 4, the BBM
-1675 Selectively hydrolyze a mixture of A ₁ and A ₂ to produce BBM
A method of making -1675C and D is provided.

Scheme 4 BBM-1675A ₁ + BBM-1675A ₂ → BBM-1675C → BBM-1675D This advantage is due to the fact that BBM-1675A ₁ and A ₂ produced in the fermentation process.
It becomes clear considering that the relative amount of fluctuates easily. BB
The formation of M-1675C and D is therefore independent of the relative amounts of BBM-1675A ₁ and A ₂ used as starting materials in the present invention.

As described, hydrolysis of BBM-1675A ₁ , A ₂ and C antibiotics results in the release of inactive thiosugar fragments.
The thiosugar was isolated to isolate the BBM-1675C antibiotic, and thus the BB
Further information was given to the chemical structures of the M-1675 A ₁ and A ₂ antibiotics. The compound of Formula 3 was identified as a mixture of α and β anomers of the thiosugar having the structures shown in Schemes 2 and 3. Furthermore, confirmation was possible when the products of alcoholysis, the α and β anomers, were separated. The proton magnetic resonance spectra (360 MHz) of compounds 3A (α-anomer) and 3B (β-anomer) were 11A, respectively.
And shown in Figure 11B. From the analysis of the spectral data, the thiosugar methyl glycoside of formula 3 has the formula Is tentatively assigned to the relative stereochemistry of.

The absolute stereochemistry, D or L, has not yet been determined at this time. Therefore, based on this interpretation of the spectral data, the thiosugar of formula 3 (which lacks the CH ₃ group from the anomeric methoxy incorporated during methanolysis) is the antibiotic BBM-16.
It is concluded that it is a component in the structure of 75C and also a component in the structure of the starting BBM-1675A ₁ and A ₂ antibiotics.

Physicochemical properties of BBM-1675C Description: Amorphous solid UV absorption spectrum: See Fig. 1 Device: Hewlett-Packard
8458 Solvent: Methanol Concentration: 0.0155g / λmax (nm) Absorptivity 210 21,770 274 9,340 313sh (shoulder) 4,190 No significant change was observed with acid or base.

Infrared absorption spectrum: See Fig. 3 Device: Nicolet 5DX FT-IR main absorption band (KBr, film) 540,740,955,990,1017,
1065,1080,1118,1150,1250,1305,1325,1340,1370,1385,
1440,1690,1705,1735,2900,2920,2930,2970,3450cm ^-1 . Mass spectrum: See Fig. 5 Device: Finigan 4500TSQ Method: Fast atom bombardment (FAB) ionization Apparatus: Kratos MS-50 High resolution FAB (m / z): [M + H] ⁺ = 856.3362 Molecular weight: Apparent MW = 855 (based on the above mass spectrum data) Elemental composition: C ₃₆ H ₆₁ N ₃ O ₁₄ S ₃ (Based on the above high resolution data) Proton magnetic resonance spectrum: See Fig. 7 Device: WM360 Bruker Solvent: CDCl ₃ ¹ H-NMR, 360MHz, 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, d
d, 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 (10
H); 3.45 (1H, dt, J = 10.3,4.0); 3.37 (3H, s); 2.77 (1
H, 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); 2.0
9 (3H, s); 1.50 (2H, m); 1.37 (3H, d, J = 5.9); 1.32 (3
H, d, J = 6.3); 1.08 (6H). ¹³ C magnetic resonance spectrum: see FIG. 9 Device: WM360 Bruker Solvent: CDCl ₃ ¹³ C-NMR, 90.6 MHz, δ (ppm): 13.7,17.5,19.8,22.3,2
2.7,23.5,34.2,35.2,39.5,47.7,52.7,55.8,56.1,57.7,6
2.4,64.7,67.4,69.3,69.8,71.9,76.1,77.1,77.7,79.7,8
3.2,88.4,97.3,99.7,123.4,124.6,130.1,193.1. Physicochemical properties of BBM-1675D Description: Amorphous solid UV absorption spectrum: See Fig. 2 Device: Hewlett-Packard
8458 Solvent: Methanol Concentration: 0.01g / λmax (nm) Absorptivity 214 27,000 274 12,800 325 5,400 No significant change with acid or base.

Infrared absorption spectrum: See Fig. 4 Device: Nicolet 5DX FT-IR main absorption band (KBr, film) 735,755,910,960,1000,
1020,1085,1150,1195,1250,1310,1335,1365,1385,1445,
1510,1685,1720,1735,2880,2930,2960,3400cm ^-1 . Mass spectrum: See Fig. 6 Device: Finigan 4500TSQ Method: Fast atom bombardment (FAB) ionization Matrix: Thioglycerin Molecular ion (m / m) z): [M + H] ⁺ = 696 Relative abundance: 100% Device: Kratos MS-50 High resolution FAB (m / z): [M + H] ⁺ = 696.2794 Molecular weight: Apparent MW = 695 (Mass spectrum data above Elemental composition: C ₂₉ H ₄₉ N ₃ O ₁₂ S ₂ (based on the high resolution data above) [M + H] ⁺ and [(M + H) +2] ⁺ Correlation of their relative abundances with their calculated values is high resolution FAB Support the elemental composition obtained from the measurements.

Proton magnetic resonance spectrum: see Fig. 8 Device: WM360 Bruker Solvent: CDCl ₃ + 10% CD ₃ OD ¹ H-NMR, 360MHz, 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, 6brs); 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); 3.3
5 (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); 0.8
7 (3H, d, J = 5.9). ¹³ C magnetic resonance spectrum: see Figures 10A and 10B Device: WM360 Bruker Solvent: CDCl ₃ + 10% CD ₃ OD ¹³ C-NMR, 90.6MHz, δ (ppm): 17.5, 21.6, 22.2, 23.0, 3
3.4,39.2,46.4,52.3,55.8,62.1,67.8,69.8,70.1,71.3,7
5.8,77.1,78.1,82.4,83.3,88.2,97.4,99.6,122.6,124.
8,130.1,130.8,134.3,148.7,192.8. Biological properties of BBM-1675 substance The antibacterial activity of BBM-1675 substance was measured against various Gram-positive and Gram-negative microorganisms. Table 1 shows parent BBM-167
Data are provided in the form of results of antimicrobial screening procedures involving the 5A ₁ component and the BBM-1675C and BBM-1675D agents of the present invention. In the screening procedure, each test compound was placed on the growth medium in a uniform concentration solution of 10 μg / ml impregnated on a piece of filter paper and a measure of antibiotic activity is the inhibition circle generated from the piece of filter paper. As shown in Table 1, BBM-167
5C and D exhibit broad spectrum antibacterial activity, which is at least as effective as the BBM-1675A ₁ component, especially B
BM-1675C and D substances were more effective as inhibitors of Gram-negative bacteria.

Activity against P-388 Leukemia Tables II and III show CDF ₁ mice intraperitoneally implanted with a tumor inoculum of 10 ⁶ ascites cells of P-388 leukemia and treated with various doses of BBM-1675A ₁ , C or D. Includes laboratory test results. The substance was administered by intraperitoneal injection. Groups of 6 mice were used for each dose and they were dosed with a single dose the day after inoculation. Groups of 10 saline treated control mice were included in each series of studies. BBM-1675A _{1 in} Table III
Treatment groups were included as a direct comparison. Mean survival time (days) was measured for each group of mice using a 30 day protocol and the number of survivors at the end of 5 days was recorded. Mice were weighed before treatment and on day 4. The change in body weight was used as a measure of drug toxicity. Mice weighing 20 g each were used and weight loss to about 2 g was not considered excessive. Vehicle treated control animals usually died within 9 days. The results were determined in terms of% T / C, which is 100 times the ratio of the mean survival time of the treated group to that of the vehicle treated control group. 125% or more T / C
The effect for indicates that a significant antitumor effect was achieved. The screening results in Table II show initially unexpected levels of antitumor activity of BBM-1675C substance. Table III shows BBM
-1675A ₁ (esperamycin A ₁ ) and BBM-1675C and BB
Results of a direct comparison with the M-1675D material are shown. The data show that BBM-1675C is BBM in terms of potency and antitumor efficacy.
It is almost comparable to -1675A ₁ and although they are not dose schedule dependent, BBM-1675D suggests that it is only slightly less effective.

In addition the same substances BBM-1675C and BBM-1675D are also BBM
It has been reported in the present invention that it can be obtained from the -1675A ₂ (esperamycin A ₂ ) component. BBM-1675C and
In comparison of the data reported here for BBM-1675D with the data reported for the BBM-1675A ₂ component in published British Patent Application No. 2,141,425 surprisingly the substance BBM-1
It is observed that 675C and D are more effective as antineoplastic agents than the parent BBM-1675A ₂ component that induced them.

Activity Against B16 Melanoma Table IV contains the results of anti-tumor studies using B16 melanoma grown in mice. Tumor grafts were inoculated subcutaneously using BDF ₁ mice. A 60 day protocol was used. Ten mice in each group were used for each test dose and the mean survival time for each group was measured. Control animals are inoculated by the same route as test animals,
The vehicle was injected and treated and showed a mean survival of 22.5 days without drug. For each dose level, test animals were treated with the test compound by intraperitoneal injection on days 1, 5 and 9. Effects on% T / C of 125 and above indicate that a significant antitumor effect was achieved. The results in Table IV are effective in treating mice with BBM-1675C Kamata B16 melanoma in a direct comparison, indicating that the roughly equal to BBM-1675A ₁ in titer.

As shown by the antibacterial and mouse tumor data, BBM-1675C and BBM-1675D thus serve as antibiotics in the therapeutic treatment of mammals and other animals against infectious diseases, and therapeutically in the growth of mammalian tumors. It is useful as an antitumor drug.

Accordingly, the present invention provides a therapeutic treatment of an animal host affected by a microbial infection and by a malignant tumor, comprising administering an effective antibacterial or tumor suppressive amount of BBM-1675C or BBM-1675D or a pharmaceutical composition thereof. Provide a way to do.

The present invention includes within its scope a pharmaceutical composition containing an effective antibacterial or tumor-inhibiting amount of BBM-1675C or BBM-1675D in combination with an inert formulation acceptable carrier or diluent.
Such compositions may also contain other active antibacterial or antitumor agents and may be prepared in any formulation suitable 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 are included. They can also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, saline, or some sterile injectable medium immediately before use.

For use as an antibacterial agent, BBM-1675C or BBM-1675D
Alternatively, the pharmaceutical compositions are administered such that the concentration of active ingredient is greater than the minimum inhibitory concentration for the individual bacteria to be treated. Optimal doses and regulations of BBM-1675C or BBM-1675D for a given mammalian host for use as anti-tumor agents can be readily ascertained by one of skill in the art. It will, of course, be appreciated that the actual dose of BBM-1675C or BBM-1675D used will depend on the particular composition formulated, the mode of administration, and the particular location to be treated, host and disease. Many factors that alter the action of drugs are considered, including age, weight, sex, diet, time of administration, route of administration, excretion rate, patient condition, drug combination, reaction susceptibility and disease condition. It Administration can be carried out continuously or periodically within the maximum tolerated dose. Optimal dose rates for a given set of conditions can be ascertained by one of ordinary skill in the art using routine dosing studies in view of the above guidelines.

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

Chemical production and separation of BBM-1675C and BBM-1675D, Example ₁ A sample of BBM-1675A ₁ (50 mg) was dissolved in 2.5 ml of methanol and 2.5 ml of a 0.1 molar solution of hydrogen chloride in methanol was added. The reaction is allowed to proceed at a temperature of about 50 ° C and the disappearance of the starting material (about 30 minutes) is monitored by silica gel plates [Analte
ch), 250μ, GF], toluene: acetone (3: 2, v /
Monitored every 5-10 minutes by thin layer chromatography (GLC) with v) as the eluting solvent. After consumption of the starting material, the reaction mixture was neutralized with saturated NaHCO ₃ solution in methanol and then evaporated under reduced pressure to give a dry residue containing bioactive fragments. The BBM-1675C material was separated from the residue by flash column chromatography on a 2 cm id x 10 cm column packed with Woelm silica gel (particle size 32-63μ). The column was eluted with toluene: acetone (3: 2, v / v) and 3 ml fractions were collected. Each fraction was analyzed by TLC [silica gel, eluent toluene: acetone (3: 2, v / v)], and TLC spots were analyzed by UV2.
Visualization with a 54 nm light source and cerium sulfate spray (1% cerium sulfate and 2.5% molybdic acid in 10% sulfuric acid). Fractions 6 to 12 (Rf value for BBM-1675C is 0.28
Is pure BBM when pooled and evaporated to dryness
-1675C 12 mg (35%) was obtained.

The physicochemical properties of BBM-1675C are shown in the specification, and the UV, infrared, mass, ¹ H-NMR, and ¹³ H-NMR spectra of the compound are shown in FIGS. 1, 3, and 5, respectively. , FIGS. 7 and 9.

Example 2 Prolonging the reaction time of the procedure of Example 1 reduces the amount of BBM-1675C, resulting in compound 3 (Rf = 0.65) and BBM-1675D.
Two new products, denoted as (TLf stays at baseline) [TLC: silica, toluene: acetone (3: 2, v / v)], become more prominent with time.

Compound BBM-1675D, which is usually associated with the formation of BBM-1675C, was separated from the chromatographic column described in Example 1 by eluting the column with chloroform: methanol (5: 1, v / v) to give the appropriate fraction. Were pooled and evaporated to dryness to give 18 mg of substantially pure BBM-1675D from the reaction described in Example 1.

The BBM-1675D material had Rf = 0.37 on a reverse phase TLC [Whatman MKC ₁₈ F, 200 micron] using 30% water in methanol as eluent and chloroform: methanol (5: 0.5, 5: 0.5, eluent). One major spot is shown at Rf = 0.22 in normal phase silica gel TLC with v / v).

Example 3 A substantial improvement in the yield of BBM-1675D was achieved in Examples 3 and 5
BBM-1675A ₂
Alternatively, it can be achieved by substituting p-toluenesulfonic acid for hydrogen chloride in the chemical hydrolysis of BBM-1675A ₁ .

A sample of BBM-1675A ₁ (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 BBM-1675D material was separated from the dry residue by flash column chromatography on a column packed with warm silica gel (particle size 32-63μ). Replace the column with chloroform: methanol (5:
0.5, v / v), and the collected fractions were TLC [silica gel, chloroform: methanol (5: 0.5, v
/ v)]. The chromatographic conditions applied allowed the separation of a mixture (7 mg) of inactive compounds 2 and 3 from the bioactive BBM-1675D material with an Rf value of 0.22. Appropriate fractions were pooled and evaporated to dryness to give 8 mg of substantially pure BBM-1675D in almost quantitative yield.

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

Example 4 A sample (40 mg) of BBM-1675A ₂ was prepared according to the general procedure and separation method described in Example 1 with 0.
It was treated with 5 ml of a 5 molar solution at about 50 ° C. for about 2 hours. After neutralizing with NaHCO ₃ and evaporating to dryness, toluene: acetone (3: 2, v / v) was applied to the residue by flash column chromatography on a column packed with warm silica gel (particle size 32-63μ). The BBM-1675C material was separated using as eluent. When the appropriate fractions were combined and evaporated to dryness, Example 1
Substantially pure BBM-1675C consistent with the product isolated in
8.4 mg was obtained.

The chromatographic column was then eluted with chloroform: methanol (5: 0.25, v / v) and the collected fractions were pooled and evaporated to dryness to give BBM-1675D. BBM-167
The 5D material was further chromatographed on silica gel with an additional flash chromatographic column chloroform: methanol (5: 0.
5, v / v) as eluent. Appropriate fractions were combined and evaporated to dryness, yielding 6.3 mg of substantially pure BBM-1675D, consistent with the product isolated in 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. to give a residue containing BBM-1675D and inert compounds 1 and 3. From the residue, BBM-1675D bioactive substance was obtained by flash column chromatography on a column packed with warm silica gel (particle size 32-63μ) using chloroform: methanol (5: 0.25, v / v) as eluent. separated. The appropriate images were combined and evaporated to dryness, yielding 27 mg of substantially pure BBM-1675D, consistent with the product isolated in Example 3.

Example 6 A sample of BBM-1675C (5.1 mg) in hydrogen chloride methanol
It was hydrolyzed with 0.5 molar solution (1 ml) at about 40-50 ° C. overnight. After neutralizing with NAHCO ₃ and evaporating to dryness, the residue was subjected to flash column chromatography on a column filled with warm silica gel (particle size 32-63μ) with chloroform: methanol (5: 0.25, v / v). Was used as the eluent to separate the BBM-1675D bioactive material. From the appropriate fractions, substantially pure BBM-1675D was obtained, which corresponds to the product isolated in Example 3.

Instead of Example 7 starting material BBM-1675A ₁ BBM-1675A ₁ and BBM-1675A
The general procedure of Examples 1 and 2 was repeated using equimolar amounts of the mixture containing ₂ and thereby resulting in BBM-1675C and BB
M-1675D material is produced.

Example 8 Instead of the starting materials BBM-1675A ₁ , BBM-1675A ₁ and BBM-1675A
Repeating the general procedure of Example 5 with an equimolar amount of a mixture containing ₂ and this yields the BBM-1675D material.

[Brief description of drawings]

Fig. 1 shows the UV absorption spectrum of BBM-1675C, Fig. 2 shows the UV absorption spectrum of BBM-1675D, Fig. 3 shows the infrared absorption spectrum (KBr, film) of BBM-1675C, and Fig. 4 The figure shows the infrared absorption spectrum (KBr, film) of BBM-1675D, FIG. 5 shows the relative abundance mass spectrum of BBM-1675C, and FIG. 6 shows the relative abundance mass spectrum of BBM-1675D. Fig. 7 shows the proton nuclear magnetic resonance spectrum of BBM-1675C in CDCl ₃ (360 MHz), and Fig. 8 shows the proton nuclear magnetic resonance spectrum of BBM-1675D of CDCl ₃ + 10% CD ₃ OD (360 MHz), Figure 9 shows the ¹³ C nuclear magnetic resonance spectrum of BBM-1675C in CDCl ₃ (90.6 MHz). Figure 10 consists of Figures 10A and 10B. Figure 10A shows CDCl ₃ +10 of BBM-1675D. % In CD ₃ OD (90.6MHz)
¹³ C magnetic resonance spectrum (110-200 ppm) of is shown in Fig. 10B in CDCl ₃ + 10% CD ₃ OD of BBM-1675D (90.6 MHz).
¹³ C magnetic resonance spectrum (0 to 110 ppm) of Compound 3A (α-anomer) in CDCl ₃ (360 MHz
z) shows the proton magnetic resonance spectrum, and Fig. 11B shows the compound 3B (β-anomer) in CDCl ₃ (360 MH).
2 shows the proton magnetic resonance spectrum of z).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display area // (C12P 1/06 C12R 1:03)

Claims (5)

[Claims] 1. A substantially pure form, (a) occurring as an amorphous solid, (b) soluble in methanol, ethanol, ethyl acetate, acetone, tetrahydrofuran and chloroform, (c) silica gel thin layer chromatography. In solvent system toluene: acetone (3: 2, v / v), has an Rf value of 0.28, (d) has an apparent molecular weight of 855 as measured by high resolution FAB mass spectroscopy, and (e) 210 nm ( a = 21,770), 274 nm (a = 9,340) and 313 nm (shoulder) (a = 4,190), which show the UV absorption maximum and absorptance without any significant change by the addition of acid or base. (F) 540, 740, 955, 990, 1017, 1065, 1080, 111
8, 1150, 1250, 1305, 1325, 1340, 1370, 1385, 144
It has an infrared absorption spectrum (KBr, film) showing main absorption peaks at ⁰ , 1690, 1705, 1735, 2900, 2920, 2930, 2970, and 3450 cm ⁻¹ , and (g) 856 molecular ion (M + H) ⁺ (H) from tetramethylsilane in a low magnetic field 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 (2
H, 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 (6
H) having a 360 MHz proton magnetic resonance spectrum in CDCl ₃ showing a signal at ppm, and (i) low-field 13.7,17.5,19.8,22.3,23.5,23.5,34.2,35.2,39.5,47.7, from tetramethylsilane.
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 a 90.6MHz carbon-13 nuclear magnetic resonance spectrum in CDCl ₃ showing the signal to 193.1Ppm, antitumor antibiotics BBM-1675C. 2. Antitumor activity comprising hydrolyzing BBM-1675A ₁ or BBM-1675A ₂ with a mineral or organic acid until a substantial amount of BBM-1675C is formed, and then recovering BBM-1675C from the reaction medium. Method for producing the antibiotic BBM-1675C. 3. Hydrolyzing a mixture of BBM-1675A ₁ and BBM-1675A ₂ with a mineral or organic acid until a substantial amount of BBM-1675C is formed and then recovering BBM-1675C from the reaction medium. A method for producing the antitumor antibiotic BBM-1675C. 4. A pharmaceutical composition for treating infectious diseases, which comprises an effective antibacterial amount of BBM-1675C in combination with a pharmaceutical carrier or diluent. 5. A pharmaceutical composition for treating malignant tumor, which comprises an effective tumor-suppressing amount of BBM-1675C in combination with a pharmaceutical carrier or diluent.