JPH0565260A - Polyene compound and its production - Google Patents

Abstract

PURPOSE:To obtain a new compound useful for multiple drug resistant cancer treatment, having conquering action on anticancer drug resistance of cancer cell having acquired anticancer drug resistance. CONSTITUTION:A polyene compound shown by the formula. Properties: molecular weight: 492. Solubility: soluble in methanol, carbon tetrachloride and chloroform and slightly soluble in water. Shape: yellow oily substance at normal temperature. The compound is obtained by culturing a fungus (e.g. Muxococcus steibitus AJ12, 587) belonging to the genus Muxococcus a common culture method, isolating and purifying.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a polyene compound derived from a microorganism which is expected to be useful for treating multidrug-resistant cancer which is a problem in cancer chemotherapy and a method for producing the same.

[0002]

2. Description of the Related Art In chemotherapy for cancer, it is often recognized that the anticancer drug used gradually loses its efficacy. This resistance phenomenon often shows "multidrug resistance" in which resistance to a plurality of drugs is simultaneously exhibited, and has become a clinically important problem. The mechanism of action of this multidrug resistance is considered to be activation of the anticancer drug excretion mechanism in resistance-acquired cells.
Since the compound (glycoprotein on the cell membrane) that has a pumping action that excretes this anticancer drug has low selectivity for drugs, it is considered that many drugs are simultaneously excreted and resistant (Tsuruo Takashi, Oncologia, vol-22, No.4, 5
~ 31 (1989)).

Due to this multidrug resistance, administration of a large amount of an anticancer drug is not an effective therapeutic method due to the toxicity of the anticancer drug. Therefore, it is considered effective to use various calcium antagonists such as verapamil and calmodulin inhibitors in combination with anticancer agents.

[0004]

However, the use amount of the calcium antagonist effective in this method has drawbacks such that side effects such as antihypertensive action are large and toxicity is strong.
The problem to be solved by the present invention is to provide a new anticancer drug resistance overactive substance for the purpose of developing a multidrug resistance overcoming agent having a higher therapeutic effect and lower toxicity.

[0005]

Means for Solving the Problems As a result of intensive studies to solve the above problems, the present inventors have developed a novel horiene compound 102C produced by a myxobacteria belonging to the genus Myxococcus represented by the following chemical formula: The inventors have found that 102T has a remarkable anticancer drug resistance overcoming activity, and completed the present invention.

[0006]

[Chemical 3]

[0007]

[Chemical 4]

That is, the present invention provides polyene compounds 102C and 1
02T, an anticancer drug resistance overactivator containing the polyene compound 102C or 102T, and a microorganism belonging to the genus Myxococcus are cultured, and the polyene compound 102C or 102T produced and accumulated in the culture is collected. It relates to a manufacturing method of.

The chemical structures of the polyene compounds 102C and 102T of the present invention are as shown in the above formulas, both of which are cis-type and trans-type with a double bond between the carbon atom at the 6-position and the carbon atom at the 7-position as the center. There is a type isomer relationship. The physicochemical properties of both compounds are shown below. (1) Molecular weight The molecular weight measured by the mass spectrum (FABMS method) is 1
Both 02C and 102T were 492. The mass spectrum of 102C is shown in FIG. 1, and the mass spectrum of 102T is shown in FIG.
Are shown respectively. (2) Ultraviolet absorption spectrum The absorption peak in methanol is shown below (nm (ε)). 102C: 206.5 (9820), 263.5 (6730), 358.5 (3150
0) 102T: 206.5 (9670), 263.5 (4490), 358.5 (4550
0) (3) Infrared absorption spectrum 102C: See FIG. 3 102T: See FIG. 4 (4) Solubility in solvent Both 102C and 102T are soluble in methanol, carbon tetrachloride, and chloroform, and hardly soluble in water. (5) Color and shape of material Both 102C and 102T are yellow oily materials at room temperature. (6) NMR spectrum The ¹ H-NMR spectrum of 102C in heavy acetone is shown in FIG.
FIG. 6 shows the ¹ H-NMR spectrum of ¹⁰ C in heavy acetone, ¹³ C-NMR spectrum of the 102 C in heavy acetone in FIG. 7, and 102
The ¹³ C-NMR spectra of T in deuterated acetone are shown in FIG. 8, respectively. (7) Chromatography Rf values 102C and 102T were subjected to silica gel 60 F254 adsorption chromatography using a mixed solvent of hexane: ethyl acetate (2: 3) as a developing solvent. give. (8) Anticancer drug resistance overcoming action Both 102C and 102T have anticancer drug resistance overcoming action against anticancer drug resistant cancer cells.

The polyene compounds 102C and 102T of the present invention belong to the genus Myxococcus, and the polyene compounds 102C and 102T.
It can be produced by culturing myxobacteria having the ability to produce T in a culture medium. As an example of a microorganism having such an ability, there can be mentioned Myxococcus stavitatus AJ12587 (FERM P-11976).

As the medium for producing the polyene compound 102C or 102T in the culture medium by the bacterium belonging to the genus Myxococcus, an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and the like may be used. That is, as the carbon source, glucose, fructose, maltose, raffinose,
Rhamnose, glycerol, xylose, lactose,
Sugars such as sucrose and starch, alcohols such as ethanol and sorbitol, and organic acids such as acetic acid can be used. Nitrogen source is ammonium ion in addition to ammonium ion, amino acid, protein and yeast extract containing these,
Casein hydrolyzate and the like can be used. If necessary, potassium ion, phosphate ion, calcium ion,
Inorganic ions such as magnesium ion, copper ion, zinc ion, manganese ion and iron ion may be added.

The culture is preferably carried out under aerobic conditions. Of medium
The pH is not particularly limited, but it is usually a suitable pH within the range of 4 to 9.
The culture may be performed while adjusting to. Culture temperature is 25 ℃ to 38 ℃
The range of is desirable. Other than this, it may be carried out based on a usual culture method.

102C and 102T are mainly accumulated in the cells, but some of them are also present in the medium. When obtained from bacterial cells, the bacterial cells may be destroyed in advance by a known method, if necessary. The method for isolating and purifying 102C and 102T is a solvent extraction method using an organic solvent such as butanol or ethyl acetate that is immiscible with water from the culture solution, or an organic solvent such as methanol, ethanol or acetone from the cells collected by centrifugation from the culture solution. Extraction method, silica gel, Diaion HP-
Adsorption chromatography such as 20 or the like, Toyopearl HW-40 chromatography, reverse phase partition chromatography such as Microbonder pack C18, Liclosolve Rp-8 or the like, and other methods that can be used for separation and purification of usual antibiotics can be applied.

When the polyene compound of the present invention is used as an agent for overcoming anticancer drug resistance, it may be diluted with a buffer, physiological saline or the like, or may be absorbed in a suitable carrier.

[0015]

[Example] 2.0% Kaziton (product of Difco, casein
Decomposition product) and 0.2% MgSO_Four・ 7H ₂PH 7.2 consisting of O
Add 100 ml of each medium to five 500 ml shoulder flasks and
I sterilized each. Mixococcus stippi on each medium
Inoculation with Tatsusu FERM P-11976 (AJ 12587) at 28 ℃ for 3 days
Interculture was performed to obtain seed mother liquor. On the other hand, the same medium as above for each 0.9
1 was placed in 5 5 l flasks for sterilization. Seed mother liquor
0.1 l was inoculated and cultured at 28 ° C for 4 days. 5 l of obtained
The culture solution was centrifuged to obtain 35 g of bacterial cells.

500 ml of acetone was added to the obtained cells, and the mixture was extracted at room temperature for 2 hours with stirring. The acetone extract was concentrated under reduced pressure, and the active substance was extracted from a small amount of the water-soluble residue with ethyl acetate (100 ml × 2 times). The ethyl acetate layer was dehydrated by adding anhydrous sodium sulfate and then concentrated to dryness under reduced pressure to obtain a dark brown oily substance. This is subjected to silica gel column chromatography (Wakogel C-200, 5x
10 cm). After washing with chloroform, chloroform:
Elution with methanol (49: 1). The active fraction was concentrated to dryness under reduced pressure and then subjected to preparative thin layer chromatography (Merck, 574).
4) (Developing solvent; chloroform: methanol (2
1: 1)). At this time, compounds 102C and 102T each have an Rf value of 0.2.
Bands were given at 8 and 0.24. After collecting the respective active bands, they were again subjected to preparative thin layer chromatography (developing solvent; hexane: ethyl acetate (2:
3)), compound 102C gave a single band at Rf of 0.37 and 102T at 0.28.

The action of compounds 102C and 102T to overcome colchicine resistance against colchicine resistant cancer cells (KB cells) was evaluated. Table 1 shows the IC ₅₀ (concentration of a drug required to cause 50% growth inhibition) in the presence (1.5 μg / ml) of colchicine and in the absence thereof.

As shown in Table 1, 102C and 102T
Remarkably suppresses the growth of KB cells in the presence of colchicine.

[0019]

[Table 1]

[0020]

From the above results, the polyene compounds 102C and 102T of the present invention have an action to overcome resistance to anti-cancer drugs, and are extremely useful in cancer chemotherapy.

[Brief description of drawings]

FIG. 1 shows a FABMS spectrum of compound 102C.

FIG. 2 is a diagram showing a FABMS spectrum of compound 102T.

FIG. 3 is a diagram showing an infrared absorption spectrum of Compound 102C.

FIG. 4 is a diagram showing an infrared absorption spectrum of compound 102T.

FIG. 5 is a chart showing ¹ H-NMR spectrum of a compound 102C.

FIG. 6 is a chart showing ¹ H-NMR spectrum of a compound 102T.

FIG. 7 is a chart showing ¹³ C-NMR spectrum of a compound 102C.

FIG. 8 is a chart showing ¹³ C-NMR spectrum of a compound 102T.

Claims (4)

[Claims] 1. A polyene compound 102 represented by the following formula:
C [Chemical formula 1] 2. A polyene compound 102 represented by the following formula:
T [Chemical formula 2] 3. The polyene compound 102C according to claim 1.
Or an anticancer drug resistance overcoming activator comprising the polyene compound 102T according to claim 2. 4. A polyene compound 102C or 102T according to claim 1 or 2, wherein a microorganism belonging to the genus Myxococcus is cultured and the polyene compound 102C or 102T produced and accumulated in the culture is collected. Manufacturing method