JPH04316573A - New compound cf-24, its use and production - Google Patents

Abstract

PURPOSE:To obtain a new substance having antitumor properties. CONSTITUTION:A compound CF-24, expressed by the formula and having the following physico-chemical properties. Appearance; yellow and oily. Solubility; soluble in benzene, chloroform, methanol and ethyl acetate and insoluble in hexane and water. Elementary analysis (%); C, 69.65; H, 10.45; O, 19.90. Fast atom bombardment (FAB)-mass spectrum (m/z); (M+H)<+> 483, etc. The compound expressed by the formula is obtained by aerobically culturing a microorganism [e.g. Saccharothrix sp. 2271-S3 strain (FERM BP-3350)], belonging to the genus Saccharothrix and capable of producing the compound CF-24 in a culture medium and collecting the above-mentioned compound from the resultant culture.

Description

Description: BACKGROUND OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel substance, and more particularly to a novel compound CF-24 having antitumor properties. [0002] Regarding antitumor substances, a large number of them have already been put into practical use as medicines. Generally, the physiological activity of a chemical substance largely depends on its chemical structure;
It can be said that there is a constant desire for new compounds with antitumor properties. [Summary of the Invention] [Problem to be Solved by the Invention] The present invention satisfies the above-mentioned desire. That is, an object of the present invention is to provide a novel compound having antitumor properties. Means for Solving the Problems The present invention has achieved the above object by discovering that a compound represented by the following formula (I) has antitumor properties. That is, the novel compound CF-24 according to the present invention is represented by the following formula (I). embedded image The invention also relates to the use of this substance. That is,
The antitumor agent according to the present invention contains the compound CF-24 represented by the above formula (I) as an active ingredient. The invention furthermore relates to a method for producing this material. That is,
The method for producing CF-24 represented by the above formula (I) according to the present invention involves culturing a strain belonging to the genus Saccharothrix and having the ability to produce CF-24 in an appropriate medium, and producing the compound CF-24 from the culture. It is characterized by obtaining. [Specific description of the invention] New compound CF-2
4 1) Chemical structure The novel compound CF-24 according to the present invention has the above formula (I)
It has the chemical structure shown below. This chemical structure was determined as follows. Analysis of the proton nuclear magnetic resonance spectrum (Figure 1) and carbon-13 nuclear magnetic resonance spectrum (Figure 2) of CF-24 revealed that CF-24 has 1
It was revealed that it consists of a 14-membered β-keto macrolide skeleton having 3 double bonds and an alkyl side chain having 3 methyl groups and 3 hydroxyl groups. A more detailed examination of the spectra revealed the relationship between the substructures, and from the molecular weight determined from the mass spectrum,
The structure of CF-24 was determined as shown in formula (I). 2) Physicochemical properties (1) Appearance: yellow oil (2) Solubility: benzene, chloroform, methanol,
Soluble in ethyl acetate. Insoluble in hexane and water. (3) Rf value (Silica gel 60F254 manufactured by Merck & Co., Ltd.
” used): Chloroform-methanol (19:1) 0.75 Hexane-ethyl acetate (12:3) 0.71
(4) FAB-mass spectrum (m/z): (M+H)
+ 483 (5) Elemental analysis: C 69.65%, H 10.4
5%, O 19.90% (calculated value: C 69.67
%, H 10.44%, O 19.89%; C28
H50O6) (6) Ultraviolet absorption spectrum λmax
nm (ε): Shown in FIG. In methanol: 207 (6370) In 0.01N hydrochloric acid-methanol: 207 (6370)
0.01N sodium hydroxide in methanol: 207
(20000), 276 (5450) (7) Infrared absorption spectrum (solution method in chloroform): Shown in FIG. (8) Proton nuclear magnetic resonance spectrum (500 MHz, in deuterated chloroform): Shown in FIG. (9) Carbon-13 nuclear magnetic resonance spectrum (125 MHz, in deuterated chloroform): Shown in FIG. Production of CF-24 1) Overview Compound CF-24 is currently obtained only by culturing microorganisms, but it can also be produced by synthetic chemical modification of related compounds or by total synthetic chemical modification. You could also do that. It may also be possible to use genetic engineering techniques. That is, it would be possible to introduce a gene involved in the production of compound CF-24 into a suitable microorganism, culture such a transformed microorganism, and obtain the compound from this culture. When culturing a microorganism, for example, a strain belonging to the genus Saccharothrix and capable of producing CF-24 is used. Specifically, the present inventors' isolated Saccharothrix sp. 2271-S3 strain is CF-2
Although the present inventors have shown that antibiotic-producing strains produce antibiotic-producing bacteria, it is possible to isolate suitable strains from nature using conventional methods for isolating antibiotic-producing bacteria. In addition, Saccharothrix sp. 2271
There is also room to increase CF-24 production ability by subjecting CF-24-producing bacteria, including the -S3 strain, to irradiation or other mutation treatments. As mentioned above, genetic engineering techniques are also possible. 2) Strain 2271-S3 The strain 2271-S3, which the present inventors have discovered as a strain of the genus Saccharothrix that has the ability to produce CF-24, is
The contents are as follows. (1) Origin and deposit number Strain 2271-S3 was isolated from soil collected in Yokokawa-cho, Chichibu-gun, Saitama Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on April 10, 1991. As a result, we obtained the number ``FERM BP-3350'' (FERM BP-3350). (2) Mycological properties The mycological properties of strain 2271-S3 are as follows. The "characterization" of this strain was carried out in accordance with the method described in the "Industrial Examination Standards" of the Japan Patent Office. ■Morphological characteristics: The basal hyphae of this strain have a width of 0.3 to 0.8
μm long, branching and elongating, sometimes exhibiting a characteristic zigzag shape, and dividing into short rod shapes. Aerial hyphae elongate their main axis, and a few branches intertwine in a lacy pattern, and all hyphae segment and divide to form spore chains. The spore chain is straight or curved and consists of 10 to 50 or more spores, and when mature, it takes on a slightly zigzag shape. Segmental spores are cylindrical, width 0.3~
0.5 μm, length 1.2-1.7 μm, smooth surface, non-motile. No sporangia, sclerotia, or other special forms were observed. ■Chemical properties: This strain contains meso-diaminopimelic acid as a total bacterial amino acid, galactose, mannose, and rhamnose as a total bacterial sugar pattern, and the cell wall chemical type is III C+rhamnose type. The major menaquinone is MK-9 (H4), and the phospholipid pattern is type PII, including phosphotidylethanolamine. ■Culture properties: The culture properties of this strain are shown in Table 1. The bacterial flora color of the colony was white or gray, the color of the underside was indistinct or dark brown, and no production of melanin-like pigments or other diffusible pigments was observed. ■Physiological properties: The physiological properties of this strain are shown in Table 2. This strain is mesophilic and uses all diagnostic sugars except D-fructose. 3) Based on the results of "characterization" beyond categorical position, Bergey's
Manual of Systematic Bact
eriology, Vol. 4. We searched for the taxonomic position of this strain. To summarize the genus-level characteristics of this strain, it shows a nocardioform morphology, the cell wall chemotype is III C + rhamnose type, and the main menaquinone is M
K-9 (H4), phospholipid pattern is PII type. The genus with these characteristics is Saccharothrix
Only. Therefore, this strain 2271-S3 is Sac
It is placed in the genus Charothrix. Therefore, this strain was classified as Saccharothrix sp. 2271-
It is called the S3 strain. Table 1 Culture properties Medium Bacterial flora color on the surface of the colony Color on the back side of the colony
Sucrose/white series (a)
Pale yellow (2ca~2ea)
nitrate agar

glucose as
Gray series (b to c) Brownish gray (2
ec~2ig) paragine agar

Glycerin As Gray series (b-c)
Brown gray (2ec~2ig) Paragin agar

Later dark brownish gray (2nl) Inorganic salt/starch agar White series (a)
From pale yellow (2ca)

Brownish white (3ca)
Tyrosine agar White series (a) Pale yellow (2ca~3ea
)later
Light yellowish brown (2ic) Nutrient agar
No aerial mycelium
Pale yellow (2ca~2db) Yeast/malt agar Gray series (c~f~10fe
) Pale yellow (2ca) to light yellowish

Orange (3ea), later dark brown Oatmeal agar Gray series (c-b)
Pale yellow (2ca-2ea)
・Production of diffusible pigment into the medium was not observed.・The information in parentheses is the Color Harmony Manual (
Container Corporation of America, 1950) color standard code. Table 2 Physiological properties Growth temperature range
25-37℃
optimal temperature
27-30℃ Melanin-like pigment Tyrosine agar
−
Peptone yeast iron agar
− Tryptone yeast broth − Hydrolysis of starch
+ Liquefaction of gelatin
+
Peptonization of skim milk powder
+ Coagulation of skim milk powder

- reduction of nitrates;
+
Carbon source assimilation D-glucose
+
L-arabinose
+ D-xylose
+
D-fructose
- sucrose
+ L-rhamnose

+ Raffinose
+
i-inositol
+ D-Mannit
+Culture/Production of CF-24 The compound CF-24 is derived from C belonging to the genus Saccharothrix.
It can be produced by culturing F-24 producing bacteria aerobically in an appropriate medium and collecting the target product from the culture. The medium can contain any nutrient source that can be utilized by the CF-24 producing bacteria. Specifically, for example, glucose, sucrose, maltose,
Starches and fats and oils can be used as nitrogen sources, as well as organic substances such as soy flour, cottonseed meal, dried yeast, yeast extract and corn steep liquor, and inorganic substances such as ammonium salts or nitrates, such as ammonium sulfate, sodium nitrate and ammonium chloride. Available. Moreover, inorganic salts such as sodium chloride, potassium chloride, phosphates, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicone oil can also be added according to conventional methods. The most suitable culture method is the aerobic liquid culture method, which is the same as the commonly used antibiotic production method. The appropriate culture temperature is 20-37°C, but 25-37°C is suitable.
0°C is preferred. In this method, the production amount of CF-24 usually reaches its maximum within 3 to 4 days of culture in both shaking culture and aerated agitation culture. In this way, a culture enriched with CF-24 is obtained. In the culture, a part of CF-24 exists in the culture fluid, but most of it exists in the bacterial cells. Any convenient method can be used to harvest CF-24 from such cultures. One method is based on the principle of extraction, and specifically, for CF-24 in the culture fluid, it is extracted with a water-immiscible CF-24 solvent (the physical (See the section on chemical properties), for example, extraction with ethyl acetate, etc.
Alternatively, for CF-24 in bacterial cells, there is a method in which a bacterial mass obtained by filtration, centrifugation, etc. is treated with methanol, ethanol, acetone, etc. and recovered. The culture itself can also be subjected to the above extraction operation without separating the bacterial cells. Countercurrent distribution methods using suitable solvents can also be included in the category of extraction. Another method for collecting CF-24 from a culture is based on the principle of adsorption, in which the CF-24-containing material is already in liquid form, such as the culture broth, or the extraction operation is performed as described above. The target CF-24 is adsorbed using an appropriate adsorbent, such as silica gel, activated carbon, "Diaion HP-20" (manufactured by Mitsubishi Chemical Corporation), etc., on the extract obtained by performing
Thereafter, CF-
24 can be obtained. CF obtained in this way
-24 solution is concentrated to dryness under reduced pressure to obtain a crude sample of CF-24. In order to further purify the crude sample of CF-24 obtained in this way, the extraction method and adsorption method described above may be combined with gel filtration method, high performance liquid chromatography, etc. as necessary, and the procedure may be performed as many times as necessary. Bye. For example, column chromatography using adsorbents such as silica gel, gel filtration agents such as "Sephadex LH-20" (manufactured by Pharmacia), and high-performance liquid chromatography using "YMC Pack" (manufactured by Yamamura Kagaku), etc. It is possible to carry out appropriate combinations of graphography and countercurrent distribution methods. in particular,
For example, a crude sample of CF-24 is
0” column and chloroform-methanol (1:1
) The active fraction was eluted with a mixture and concentrated to dryness, resulting in CF-
24 pure products are obtained. Uses of CF-24 Compound CF-24 according to the present invention is useful in that it has antitumor activity. (1) Biologically active CF-24 showed cytostatic activity against tumor cells. For example, human tumor cells such as KB cells or colchicine-resistant KB cells are prepared in a MEM medium containing 10% heat-inactivated fetal bovine serum to a concentration of 1 x 10 cells/ml,
The IC50 values after 24 hours of incubation at 37°C with various concentrations of CF-24 were 6.0 μg/ml, respectively. Furthermore, when the cell proliferation inhibitory activity of CF-24 in the presence of colchicine against colchicine-resistant KB cells was measured in the same manner as above, the IC50 value was 1.0 μg/ml or less. From this, CF-24 can be used alone as well.
It was revealed that it acts synergistically with colchicine to exhibit stronger inhibition of tumor cell growth. As mentioned above, it was revealed that the CF-24 of the present invention exhibits antitumor activity. Therefore, CF-24 of the present invention can be used as an antitumor agent. (2) Antitumor agent It has thus been revealed that CF-24 according to the present invention exhibits antitumor properties against human tumors, particularly malignant tumors. Therefore, the compounds of the present invention can be used as antitumor agents or tumor therapeutic agents. The compounds of the present invention as antitumor agents can be administered by any convenient route of administration and in a dosage form determined by the route of administration employed. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent. When actually administering the compounds of the present invention as antitumor agents, one typical method is to dissolve them in a pharmaceutically acceptable solvent and inject them. Specifically, methods include intraperitoneal injection, subcutaneous injection, intravascular injection into a vein or artery, and local administration by injection. The dosage of the compound of the present invention is determined in consideration of the results of animal tests and various situations so that the total dosage does not exceed a certain amount when administered continuously or intermittently. The specific dosage will vary depending on the method of administration, the circumstances of the patient or treated animal, such as age, weight, sex, sensitivity, diet, time of administration, concomitant drugs, and the severity of the patient or his or her illness. Needless to say, the appropriate dose and frequency of administration under certain conditions must be determined by a specialist's appropriate dose determination test based on the above guidelines. Specifically, 0.01~ per day for adults
It is about 1g. Effects of the Invention The novel compound CF-24 according to the present invention has the following properties:
It has excellent antitumor effects. It is understood that the property that the compounds of the present invention have such physiological effects was unexpected for those skilled in the art. [Experimental Examples] [Example] The following experimental examples are intended to explain the present invention in more detail, and the present invention is not limited thereby. (1) The culture medium used was one in which the following components were dissolved in 1 liter of water and the pH was adjusted to 6.2. Starch 25g Soy flour 15g Dry yeast
2g calcium carbonate 4
g Prepare 30 liters of the above medium in a 50 liter jar fermenter, sterilize it, and add Saccharothrix s.
p. 2271-S3 strain was inoculated and cultured with aeration at 27°C for 3 days. (2) Collection of CF-24 After culturing under the above conditions, the culture solution (30 liters) was centrifuged, and the bacterial cells were extracted with 5 liters of acetone. The extract was concentrated, extracted three times with 500 ml of ethyl acetate, and concentrated to dryness. This was adsorbed onto a column of silica gel (Wako Gel C200, manufactured by Wako Pure Chemical Industries, Ltd.), and eluted with a chloroform-methanol (150:1) mixture. After concentrating the active fraction to dryness, it was subjected to preparative thin layer chromatography (Silica Gel 60F254 TL manufactured by Merck & Co., Ltd.).
C plate). After developing with a chloroform-methanol (19:1) mixture, the active fraction was scraped off, from which CF-24 was extracted with methanol. The extract was subjected to reverse-phase SEP-PAK (“C18 cartridge” manufactured by Waters Associates), and methanol-
After elution with a mixture of water (87:13), it was concentrated to dryness and the C
5 mg of pure F-24 was obtained.

[Brief explanation of the drawing]

Figure 1: CF-24 at 500% in deuterated chloroform
This is a copy of the megahertz proton nuclear magnetic resonance spectrum.

Figure 2: 125 of CF-24 in deuterated chloroform
This is a reproduction of a megahertz carbon-13 nuclear magnetic resonance spectrum.

[Figure 3] CF-24 in methanol (solid line), 0.01
This is a reproduction of the ultraviolet absorption spectra in normal hydrochloric acid-methanol (solid line) and in 0.01N sodium hydroxide-methanol (dotted line).

FIG. 4 is a reproduction of the infrared absorption spectrum of CF-24 obtained by a solution method in chloroform.

Claims (3)

[Claims] Claims 1: Compound CF-24 represented by the following formula (I). [Chemical formula 1] 2. An antitumor agent comprising the compound CF-24 according to claim 1 as an active ingredient. 3. The method according to claim 1, wherein a strain belonging to the genus Saccharothrix and capable of producing CF-24 is cultured in an appropriate medium, and the compound CF-24 is obtained from the culture. Method for producing compound CF-24.