JPH0443912B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention has symbols CL1565-A, CL1565-B and
The present invention relates to a novel phosphorus-containing antibiotic designated as CL1565-T, an antitumor agent, and related substances, as well as pharmaceutically acceptable salts of these substances, methods for their production, and uses of these substances.
More specifically, the method for producing this substance is based on the isolated bacterial strain for producing the CL1565 composite of the present invention, which has been deposited under deposit no.
This invention relates to a fermentation method using a Streptomyces sp. strain having ATCC31906. Three types of CL1565 antibiotic components: CL1565-A,
The chemical structures of CL1565-B and CL1565-T are shown in formulas 1, 2a and 2b, respectively. 1 CL1565-A 2a R ₁ = R ₂ = H; CL1565-B 2b R ₁ = R ₂ = OH; CL1565-T CL1565-A, CL1565- as formulas 1, 2a and 2b
The chemical structures given for B and CL1565-T, respectively, are the best representations of the structure of the materials known to the inventors up to the date of filing of this application. The structure is CL1565−
It was obtained by comparing the physical and spectroscopic properties of A, B and T and certain of their derivatives.
Results obtained from other physical measurements, such as those based on X-ray crystallography, may lead to future modifications of the structures shown in equations 1, 2a and 2b. However, any chemical structural formula that may be modified in the future is included within the scope of the present invention. The present invention further relates to pharmaceutical compositions containing various salts of the above-mentioned compounds of the present invention, alone or in combination with other anti-tumor agents, in the treatment of tumor diseases. According to the present invention, a Streptomyces strain that produces the CL1565 complex and has deposit number ATCC 31906 as an isolated strain is selected, and a substantial amount of the CL1565 complex is produced.
CL1565 compound or compounds (especially CL1565-A,
CL1565-B and CL1565-T) are produced under artificial conditions in a suitable nutrient medium and by isolating one or more of the compounds in the free acid or salt form. Compounds of CL1565 are produced. A Streptomyces strain suitable for the purposes of the present invention was found in a soil sample taken in São Paulo, Brazil. The microorganisms were isolated from the soil samples by using a suitable agar plate. An example of this medium is a medium containing salts such as potassium phosphate, magnesium sulfate and ferrous sulfate and carbon compounds such as glycerin and asparagine. Before a soil sample is placed on an agar medium, it is pretreated with lime carbonate and kept constant at a suitable temperature, particularly 24°C, to allow the growth of soil microorganisms. The isolated CL1565 complex-producing bacterium is an unidentified strain of the genus Streptomyces and belongs to the American Type Culture Collection.
Culture Collection, Rockville;
Maryland 20852) and is kept in its permanent deposit as ATCC 31906. CL1565
-A and its congeners are produced by the Warner-Lambert Culture Laboratory (Warner-Lambert/
Freeze tubes at Parke-Davis Culture Laboratory)
The isolate was stored as a dormant culture in cold bottles and clay tubes and labeled as isolate WP-426. Antitumor compounds CL1565-A and “CL1565-B”
and closely related congeners including CL-1565-T”
is produced by aerated fermentation of isolated WP-426 under controlled conditions. The fermentation medium consists of a carbon source, a nitrogen source, minerals and growth factors. Examples of carbon sources are various simple sugars such as cerelose,
Mannose, fructose, xylose, ribose and glycerin or other carbohydrate-containing substances such as dextrin, starch, corn flour and whey. The usual amount of carbon source can vary from 0.1 to 10% by weight. The nitrogen source can be an organic, inorganic or mixed organic-inorganic source. Examples of such compounds are cottonseed flour, soybean flour, corn germ flour, corn soaking liquid (corn
steep llquor), distillers solubles, groundnut flour, peptonized milk and various ammonium salts. The usual amount added is
Higher amounts are also acceptable, although they can vary from 0.1 to 5%. Addition of minerals and growth factors to the fermentation medium
It also promotes the production of CL1565-A and its congeners. Examples of media components that provide minerals are potassium phosphate, sodium chloride, ferrous sulfur, calcium carbonate, cobalt chloride, and zinc sulfate. Sources of growth factors include various yeasts and dairy products. A preferred method for producing CL1565-A and congeners is submerged culture fermentation. According to one embodiment of the present invention, various components for fermentation are made into a solution, which is sterilized by heating under pressure or heating with steam. The pH of the aqueous medium is preferably 4 to 8. This fermentation medium is cooled to an appropriate temperature, that is, 16 to 45°C, and an appropriate culture is inoculated. When fermented under aeration and agitation, maximum production of CL1565-A and its congeners is usually achieved in 2 to 10 days. CL1565-A
Solid state fermentation for the production of and congeners may also be used. In the deep culture method, fermentation is carried out in shake flasks or static fermenters. In the case of a shake flask, aeration is achieved by shaking the flask to mix the medium and air. In the case of static fermenters, stirring is carried out using impellers in the form of disk turbines, bladed discs, open turbines or marine propellers. Aeration can also be carried out by blowing air or oxygen into the fermentation mixture. The following examples are illustrative of preferred methods of manufacturing products, namely CL1565-A and congeners. Example 1 Inoculum growth and shake flask fermentation The dormant culture is transferred to a CIM-23 slanted agar medium and kept at 28° C. for 7 to 14 days. A portion of the slant culture was added to 5 ml of ARM1550 seed medium.
Inoculate into a ×150mm seed tube. This seed tube
Shake at 24°C for 3-4 days. CIM23 slanted agar medium Amidex (trade name) (Amidex) corn starch 10g N-Z amine (type A) 2g Beef extract [Difco] 1g Yeast extract [Difco] 1g Cobaltous chloride hexahydrate 0.020g Agar 20g Distilled water 1000ml ARM1550 base % Bacto (trade name) - Yeast extract [Bacto-
Yeast Extract (Difco) 0.5 Glucose-hydrate 0.1 Soluble starch (Difco) 2.4 Bacto-Tryptone
(Difco) 0.5 Bacto-Beef Extract
(Difco)] 0.3 CaCO ₃ 0.2 Note: Adjust the pH to 7.5 with NaOH before adding CaCO ₃ Take a portion (1 ml) of the grown microorganisms from the seed tube and
Transfer this to a 300 ml Erlenmeyer shake flask with a baffle containing 50 ml of SM64 manufacturing medium. After inoculation, the flask was shaken for 24 hours using a rotary shaker (2″ throw) set at 180 RPM.
Incubate at ℃ for 5 days. After 5 days of fermentation, the fermented liquid has a yellow-brown color, the mycelium is granular, and the pH of the fermented liquid is approximately 5.5. Milk whey for SM64 production [Kroger Dairy]
35.0% by volume Dextrin [Amidex B411] American corn 1.5% by weight Pharmamedia (Traders
Protein) 431307〕 1.5% by weight Distilled water Note: Adjust the pH to 6.5 with NaOH. A 1:100 dilution of fermentation broth containing CL1565-A and congeners is tested in vitro on L1210 murine leukemia cells. 0-50% growth of cells indicates activity when compared to L1210 cell controls, with 0% being most active. The cytotoxicity in the two experimental shake flask fermentations was as shown in the table below.

[Table] The above fermentation liquid was tested against several types of microorganisms using an agar circular medium. The crude fermented liquid is made from Neisseria catarrhalis.
03569), Staphylococcus aureus02482, Bacillus subtilis04555, Crechera
Brevis (Kloeckera brevis M1378), Rhodotorula glutinls
M1384), Saccharomyces cerevisiae01525 and Penicillum avellaneum
M2988). Example 2 Fermentation in a 200 Gallon (757) Fermenter (A) Inoculum Growth A cold bottle containing approximately 1 ml of culture suspension is used as the inoculum. The contents of the cryobottle were thawed and transferred aseptically to a 2-volume baffled Erlenmeyer flask containing 500 ml of SD-05 inoculum medium. 130RPM flask after inoculation
The cells were incubated at 24 °C for 46-48 h on a rotary shaker at a speed of . SD-05 Inoculum medium % Amberex [Amberex×1003 (Amber
Labs)] 0.5 Glucose-hydrate [Cerelose]
0.1 Dextrin-amidex (trade name)
[Dextrin−Amidex B411 (Corn Products)]
2.4 N-ZCase (Humko
Spray Dried Meat 0.5 Spray Dried Meat
Solubles (Daylln Labs)] 0.3 CaCO ₃ 0.2 Distilled water After 48 hours of constant temperature maintenance, the contents of the inoculum flask were
Aseptically transfer to a 30 volume stainless steel fermenter containing SD-05 seed culture medium. After inoculation, the fermenter is incubated at 24° C. for 18-24 hours with agitation at 300 RPM and air flow at a rate of 1 VVM. This grown microorganism is used to inoculate a 200 gallon (757) production fermentor. (B) Fermenter production with 160 gallons (606) of SM64 medium
Steam sterilize a 200 gallon (757) fermenter at 121°C for 40 minutes. The medium is cooled to 24° C. and then inoculated with approximately 16 grown microorganisms from a 30-volume starter fermenter. Stir the inoculated medium at
Ferment at 24°C for 5-7 days at 190RPM and 1VVM ventilation. Dow Corning C as antifoaming agent
(Dow Corning "C") and polyglycol (polyglycol P-2000) are used to control foaming. CL1565-A and at least two related anti-tumor antibiotics throughout the fermentation cycle, viz.
The production status of CL1565-B and CL1565-T was monitored by recording fermentation parameters such as PH and percentage of deposits or growths, and by performing in vitro tests on L1210 murine leukemia cells.
Then, high pressure liquid chromatography treatment (see below) is performed. An example of the fermentation situation in a 200 gallon (757) capacity fermenter is shown in the table below.

【table】

[Table] The yield (volume) from the fermenter after 142 hours of fermentation is
It was 140 gallons (530). Example 3 Isolation of CL1565-A The fermented liquor obtained by the above fermentation was mixed with 34 kg of Cerite 545 (trade name) and passed through a filter press with plate and frame.
Liquid (473) to 120 resin HP-20 (trade name)
[HP−20resln(Gillies International, Inc., La
The percolate was passed through a 30.5 cm [OD] column with a 30.5 cm [OD] column. Next, mix this resin with water (605) and water:methanol (90:10).
Washed with mixed solution (170). Most of the CL1565-A was then eluted from the resin with a water:methanol (80:20) root solution. High pressure liquid chromatography (HPLC) analysis (see below) of the eluate showed the elution state shown in the table below.

TABLE Elutions #2 and #3 were individually concentrated and lyophilized to give 90.2 g and 78.7 g of dark brown solid, respectively. The products were combined and dissolved in 3 parts of water and the resulting solution was added to 2 parts of methanol with stirring. After standing at 5° C. overnight, the mixture was stirred and the precipitate was washed with 5 portions of methanol.
The liquid and washings were combined, concentrated in vacuo, and lyophilized to yield 104.5 g of solid. Add 1.5 of water to a portion of this product (95 g) and mix it with 1-
Add slowly to propanol with stirring. The resulting mixture was filtered after 1 hour to remove the precipitate.
Washed with propanol. The liquid and washings were combined, concentrated, and lyophilized to give 57 g of solid, which was
When subjected to HPLC analysis, this material weighs approximately 15g.
It contained CL1565-A. This product was made into a 7.6 cm [O.
1.2 40 μm C18− packed in a column of D.
Silica gel (trade name) (Analytichem)
International, Inc., Harbor City, California)
Chromatographed above. The eluent used is PH
0.005M ammonium acetate buffer at 4.5 and then 0.005M ammonium acetate at PH containing 5% acetonitrile. Each fraction was collected and tested by HPLC. Collect fractions containing CL1565-A,
Concentrate and lyophilize. A portion (570 mg) of the obtained product was transferred to a chromatography device [Prep LC/
System500apparatus fitted with a prep−
Pak-500/C ₁₈ column (Waters Instruments,
Inc., Milford, Massachusetts) at pH 6.5 containing 10% acetonitrile as the eluent.
Chromatographed again using 0.1M phosphate buffer. The main fraction containing about 375 mg of CL1565-A was collected and concentrated in vacuo. 200ml of HP-20 aqueous solution
Passed through a column with resin (filled in water). Wash this resin with 1400ml of water and leave it on the column.
CL1565-A was eluted with 350 ml of 50% methanol. Concentrate the eluate in vacuo and transfer to 35 ml of resin (trade name
Dowex-50×2) (Na ⁺ ) was passed through the column. The effluent (PH5.5) and the resin washing solution were combined and lyophilized to isolate the sodium salt.
Yielded 180 mg of purified CL1565-A. Analysis of this product showed that it contained approximately 1.3 moles of sodium per 1.0 mole of parent CL1565-A free acid. Since the free acids (CL1565-A, CL1565-B, and CL1565-T) are unstable, the CL1565 free acid compound is prepared in the form of a salt, such as a sodium salt, preferably at about 1.0 to about 2.0 moles of sodium per 1.0 mole of free acid. It is preferable to isolate the compound as a salt containing the compound. Example 4 Using the filtrate of the fermented liquor produced in the same manner as above,
It was passed through 31 resin (Dowex-1×2) (chloride type) packed in a 30.5 cm [OD] column. The effluent and subsequent flushing water are
It did not contain any CL1565 components. 0.1M as a solvent system for all fractions listed here.
Phosphate buffer (Na ⁺ )-acetonitrile (88:
12) was monitored by the HPLC method (see below). The DOWEX-1™ resin was eluted with a 1M sodium chloride-methanol (1:1) mixture and the eluate was collected in two 10 fractions and six 15 fractions. CL1565-A, CL1565-B,
The majority of CL1565-T and additional amounts of CL1565 components were present in the second to sixth eluates. These fractions were combined and diluted with 246 ml of acetone.
The resulting mixture was kept at a final temperature of 5°C. The clear supernatant was removed and concentrated under vacuum to a concentration of 16.
Lyophilization of the concentrate gave 800 g of solid.
This product (740 g) was added to 552 g of similarly obtained product and the combined solid was dissolved in 20 g of water. Obtained (PH6.0) 15cm [OD]
Chromatographed on 50 HP-20 resin packed in a column. HP-20 resin column
Elution with 175 water separated most of the CL1565-T and all of the minor polar CL1565 components. Most of the CL1565-A component was eluted with a 100% methanol-water (15:85) mixture;
CL1565-B and the remaining amount of CL1565-A were eluted with 83 methanol-water (50:50) mixture.
The CL1565-A enriched eluates were combined, concentrated, and lyophilized to give 123 g of solid, which contained approximately 110 g of CL1565-A by HPLC analysis. 75g of this product at pH 6.8 of 2
52 (25 Kg) of silica gel [100 μm] dissolved in 0.05 M phosphate buffer and filled with 0.05 M phosphate buffer (Na ⁺ ) of pH 6.8 in a 15 cm [OD] column.
C ₁₈ reverse phase silicagel (Analytlchem
International, Inc., Harbor Clty,
Further purification was achieved by chromatography on a commercially available (California). The columns were developed using 0.05M phosphate buffer containing increasing amounts (4.0-6.5%) of acetonitrile. The early fractions contained primarily CL1565-T and a small additional amount of polar CL1565 component(s). Most of the CL1565-A component was eluted in later fractions. The CL1565-A containing fractions were the only UV-absorbing component and were pooled and concentrated under vacuum to a volume of 20. This concentrate was stored at 5° C. to remove precipitated inorganic salts. Then 15 cm [O.] with 28 HP-20 resin.
D.] The liquid was loaded onto the column. water (66)
Wash the resin with 42 methanol-water (50:
50) CL1565-A was eluted with the mixed solution. CL1565-A
Combine the eluate(s) containing the majority of the
), concentrated and lyophilized to give 34 g of CL1565-A containing some inorganic impurities. Dissolve the above product (50-100 mg/ml) in methanol,
Since the insoluble substances are removed and the liquid is concentrated under vacuum, the above-mentioned inorganic impurities can be removed by frequently adding water to the liquid to make it an aqueous solution. The resulting aqueous concentrate was HP
CL1565-A is finally purified by chromatography on -20 resin. Isolation of Additional Amounts of CL1565 Component(s) The concentrate obtained from the CL1565-containing fermentation broth was immersed in silica gel (trade name C18-Silica Gel) or HP-20.
By careful chromatography on a resin, fractions containing CL1565 components other than CL1565-A are obtained. In this way, six or more compounds can be detected by HPLC, based on the fact that they have similar ultraviolet absorption spectra.
It is a CL1565-A related substance. Two of these components, CL1565-B and CL1565-T, were isolated as essentially pure compounds. CL1565 components A, B, and T were prepared using a silica gel column [μBondapak C ₁₈ -silica gel column (3.9 mm I.
D. × 30cm)] easily distinguished by HPLC on
Detected by ultraviolet absorption at 254 nm. above
Typical retention times (minutes) for CL1565-A, B, and T using HPLC conditions are shown in the table below.

[Table] The crude fermentation liquid can be tested in the same manner as above, except that the solvent used is a 0.1M phosphate buffer-acetonitrile (88:12) mixture with a pH of 6.8. In this case, at a flow rate of 2 ml/min, the retention times for CL1565-T, CL1565-A and CL1565-B are approximately 2.7, 5.0 and >12 minutes, respectively. The elution rate from HP-20 resin and from reverse phase silica gel is
CL1565-T is faster than CL1565-A. HP
It can be isolated from the initial fraction of the _C18 -silica gel column described in Example 4 using -20 resin. The elution rate from HP-20 resin and from reverse phase silica gel is slower for CL1565-B than for CL1565-A. CL1565-B is the crude Dowex-1 described in Example 4
During HP-20 chromatography of the product by
It is eluted using 50% methanol. This component can most conveniently be isolated by rechromatography on HP-20 followed by chromatography on 40 μm C ₁₈ -silica gel using essentially the same procedure for the CL1565-A purification described above. Properties of CL1565-A sodium salt: Ultraviolet absorption spectrum in methanol (see _Figure ¹ ) Infrared absorption spectrum in KBr (see Figure 2) Mainly Absorption position: 3400, 1710, 1630, 1420, 1387,
1260, 1155, 1090, 1060, 975, 920, 820, and
775cm ^-1 Optical rotation [α] ²³ _D +28.2° (1.0% in 0.1MPH7 phosphate buffer) Elemental analysis Calculated value: (as C ₁₉ H _27.7 O ₁₀ Na _1.3 P) %C %H %Na % P 47.84 5.86 6.27 6.49 Experimental value: 48.01 5.88 6.05 6.3 Mass spectrum (vla fast atom
bombardment) Calculated value: [C ₁₉ H ₂₅ Na ₂ O ₉ P+H] ⁺ as m/z 475 [C ₁₉ H ₂₆ Na O ₉ P+H] ⁺ as m/z 453 Experimental value: m/z 475, 453 360MHz proton magnetic resonance spectrum ( (during D ₂ O) (see Figure 3) Main signal: (s=single line, d=double line, t=triple line, m=multiple line) 1.29s (3H), 1.58t (1H), 17.0m ( 1H), 2.49−
2.58m (2H), 4.13−4.18m (3H), 4.86t (1H),
5.09m (1H), 5.53t (1H), 5.9−6.0m (4H), 6.14t
(1H), 6.32t (1H), 6.55t (1H), 6.75dd (1H), and 7.09m (1H) ppm downfield (based on DSS) [parts per milion down−field from
sodium 2,2-dimethyl-2-silapentane-
5-suifonate (DSS)] 13C-nuclear magnetic resonance spectrum (in D ₂ O) (see Figure 4) Main signal: Maximum number Maximum number 1 168.4 12 79.5 2 149.8 13 79.0 3 138.1 14 75.6 4 135.0 15 64.4 5 134.4 16 62.7 6 131.3 17 39.4 7 127.4 18 29.7 8 126.7 19 23.5 9 124.9 10 124.8 11 120.1 ppm downfield (based on TMS)
[parts permililon downfilld
fromtetramethylsilane (TMS)] 31P-nuclear magnetic resonance spectrum (in D ₂ O) shows a doublet [a doublet (J=10Hz) at 0.504ppm
downfield from 85% phosphoric acid]. High pressure liquid chromatography column: μBondapak C ₁₈ silica gel (3.9mm1.D.
×30cm) Solvent: 0.005MPH7.3 sodium phosphate buffer - acetonitrile (90:10) Flow rate: 2ml/min Detection: Ultraviolet absorption retention time at 254nm: 2.8min Antibacterial activity Aqueous solution containing 500μg CL1565-A per ml A soaked paper circle (12.7 mm in diameter) was placed on the agar layer inoculated with the following microorganisms. After keeping the temperature at 28°C overnight, the following inhibition circle was observed. Microbial inhibition circle diameter Saccharomyces cerevisiae 25mm Saccharomyces italicus 17mm Saccharomycoides ludwigii 25mm In vitro activity of CL1565-A against L1210 leukemia cells ID ₅₀ = 0.078 μg/ ml Mouse P388 CL1565 for lymphocytic leukemia
- In vivo antitumor activity of A.

[Table] Properties of CL1565-T sodium salt Ultraviolet absorption spectrum in methanol Almost the same as shown in Figure 1 except that there are refraction points at λmax 268 nm (a ¹ ₁ = 774) 260 and 278 nm. External absorption spectrum main absorption positions: 3400, 1715, 1630, 1380, 1260,
1090, 970, 830 and 770 cm ^-1 mass spectra (via fast atom
bombardment) Calculated value: [C ₁₉ H ₂₅ Na ₂ O ₁₀ P+H] ⁺ as m/z 491 Experimental value: m/z 491 360MHz proton magnetic resonance spectrum (in D ₂ O) ¹ H-NMR spectrum of CL1565-T is CL1565
-It is strikingly similar to the ¹ H ^-NMR spectrum of T, except that in the former spectrum,
At 4.34 ppm it shows a characteristic single proton signal where the doublets appear as one doublet, and in the 2.2-3.2 ppm downfield (based on DSS) it shows no signal. Main signal of CL1565-T sodium salt: (s = single line, d = doublet, t = triplet, m = multiplet) 1.30s (3H), 1.55-164m (1H), 1.73
(1H), 4.13−4.20m (1H), 4.16d (2H), 4.34dd
(1H), 4.94t (1H), 5.09dd (1H), 5.55t (1t),
5.89−6.06m (3H), 6.16m (2H), 6.36t (1H),
6.56t (1H), 6.76dd (1H), 7.14dd (1H) ppm Downfield (based on DSS) 90.4MHz13c - Nuclear Magnetic Resonance Spectrum (in _D2O ): Maximum Number Chemical Shift ^* 1 24.10 2 41.60 3 64.68 4 64.90 5 66.67 6 78.28 7 79.81 8 84.33 9 124.40 10 126.21 11 126.91 12 127.18 13 128.99 14 133.36 15 136.87 16 137.2 3 17 142.27 18 149.46 19 169.66 Note: *ppm downfield (based on TMS) Mouse P388 lymphocytic CL1565 against leukemia
- In vivo antitumor activity of T.

[Table] Properties of CL1565-B sodium salt Ultraviolet absorption spectrum in methanol λmax 267nm (a ¹ ₁ = 805) Refraction points at 259 and 277nm Infrared absorption spectrum in KBr Main absorption positions: 1720, 1640, 1385, 1200 , 1060, 970,
and 820 cm ^-1 360 MHz proton magnetic resonance spectrum (in D ₂ O) Main signal: (s = single line, d = doublet, t = triplet, m = multiplet) 1.32s (3H), 1.58t (1H) , 1.75t (1H),
1.79d (3H), 2.45-2,68m (2H), 4.15t (1H),
4.89t (1H), 5.10m (1H), 5.49t (1H), 5.83−
6.21m (6H), 6.50−6.64m (2H), 7.06−7.13m
(1H) ppm downfield (based on DSS) 90.4MHz13c - Nuclear Magnetic Resonance Spectrum (in _D20 ) Maximum Number Chemical Shift ^* 1 20.70 2 25.06 3 31.91 4 41.85 5 66.85 6 77.87 7 80.87 8 81.64 9 122.41 10 124.45 11 127.24 12 129.47 13 129.90 14 134.66 15 135.94 16 136.67 17 140.42 18 152.01 19 170.56 Note *ppm downfield (based on TMS)
CL1565 against P388 lymphocytic leukemia in mice
- In vivo antitumor activity of B.

[Table] Antibiotic CL1565-A and its congeners are classified into various metal salts such as sodium, potassium, calcium or magnesium salts and analogs or other salts such as ammonium salts or appropriate organic salts based on their antimicrobial and antitumor activities. It can be used in the form of pharmaceutical compositions containing any of the salts formed from amines. The pharmaceutical composition is used in conjunction with a pharmaceutically acceptable co-carrier. The composition may also contain other active antimicrobial and/or antitumor agents. The composition may be made into any pharmaceutical form suitable for the intended route of administration.
Examples of such compositions are solid compositions for oral administration such as tablets, capsules, pills, powders and granules, and liquid compositions for topical or oral administration such as solutions, suspensions, syrups and Includes elixirs as well as products for parenteral administration such as sterile solutions, suspensions or emulsions. For use as an antimicrobial agent, the composition is administered at a concentration of active ingredient that is greater than the minimum inhibitory concentration for the particular microorganism of interest. The following examples describe one or more products for the treatment of diseases, particularly neoplastic diseases: CL1565-A;
1 is an illustration of a suitable pharmaceutical composition containing CL1565-B and CL1565-T. Example A. 75 mg of CL1565 in each ampoule for parenteral use.
A sterile, lyophilized product containing the -A sodium salt was prepared from the compound (in water for injection). Example B CL1565-A sodium salt 75mg Sodium ascorbate 33mg Place the above ingredients in a sterile bottle. An injection solution is prepared by dissolving the above mixture in physiological saline using a conventional method. A buffer may be added if necessary. Example C Dissolve CL1565-A sodium salt (1000 mg) and sodium ascorbate (440 mg) in 100 ml of water. This solution is passed through a sterile strainer,
This solution is aseptically filled into a previously sterilized bottle and freeze-dried. The bottle is sealed in a nitrogen atmosphere with a previously sterilized lid and stored at a temperature of 5° C. or below. CL1565-B and CL1565-B instead of CL1565-A
Other suitable formulations may be formulated using CL1565-T as described in Examples A, B and C. CL1565− for use as an antitumor agent for mammals
The suggested doses for each of A, CL1565-B and CL1565-T are 1.0 to 100 mg per square meter per intravenous injection per day.

[Brief explanation of drawings]

Figure 1 of the accompanying drawings shows the ultraviolet absorption spectrum of CL1565-A sodium salt in methanol; Figure 2 shows the infrared absorption spectrum of CL1565-A sodium salt in KBr; Figure 3 shows the infrared absorption spectrum of CL1565-A sodium salt in KBr;
360MHz proton magnetic resonance spectrum in _D2O of CL1565-A sodium salt;
The figure shows 13C− of CL1565-A sodium salt in D ₂ O.
represents the magnetic resonance spectrum.

Claims (1)

[Claims] 1. The following formula (In the formula, 1 R ₁ = H and R ₂ = OH is CL1565-A; 2a R ₁ = R ₂ = H is CL1565-B; 2b R ₁ = R ₂ = OH (CL1565-T is CL1565-T) and pharmaceutically acceptable salts thereof. 2 In claim 1, in the formula, R ₁ is H and R ₂ is
CL1565-A which is OH and pharmaceutically acceptable salts thereof. 3. CL1565-B according to claim 1, wherein R ₁ and R ₂ are each H, and a pharmaceutically acceptable salt thereof. 4. CL1565-T and pharmaceutically acceptable salts thereof according to claim 1, wherein R ₁ and R ₂ are each OH. 5 The following formula (In the formula, 1 R ₁ = H and R ₂ = OH is CL1565-A; 2a R ₁ = R ₂ = H is CL1565-B; 2b R ₁ = R ₂ = OH CL1565-T is a method for producing CL1565 compounds, which comprises using a strain of Streptomyces with accession number ATCC 31906 that produces CL1565 compounds,
CL1565 compound or compounds are cultured in a suitable medium until a substantial amount is obtained, and the compound or compounds are isolated in the form of free acid or salt. 6 A pharmaceutically acceptable carrier and the following formula as an active ingredient: (In the formula, 1 R ₁ = H and R ₂ = OH is CL1565-A; 2a R ₁ = R ₂ = H is CL1565-B; 2b R ₁ = R ₂ = OH (CL1565-T is CL1565-T.) An antitumor agent characterized by containing the CL1565 compounds shown in the formula (CL1565-T) or their pharmaceutically acceptable salts. 7. Claim 6, wherein the CL1565 compound is CL1565-A.
The antitumor agent described in. 8. Claim 6, wherein the CL1565 compound is CL1565-B.
The antitumor agent described in. 9. Claim 6, wherein the CL1565 compound is CL1565-T.
The antitumor agent described in.