JPH04108787A - New 18-membered macrolide compound - Google Patents

Abstract

NEW MATERIAL:A compound shown by the formula (R is H or methyl), wherein R is methyl has the following physical and chemical properties. Appearance: colorless needle-like crystal. Melting point: 117-120 deg.C. Mass spectrometry value: cation FABMS spectrum m/z 729(M+Na)<+>, m/Z 745(M+K)<+>; Elemental analysis (%): 68.13 H 9.24, O 22.63. Molecular formula: C40H66O10:[alpha] : +108 deg. (C=0.05, methanol). Solubility: readily soluble in methanol, chloroform, acetone, ethyl acetate and benzene, sparingly soluble in n-hexane. Color reaction: positive in sulfuric acid and iodine and negative in ninhydrin and FeCl3. Having neutrality, etc. USE:An antitumor agent. PREPARATION:Streptomyces graminofaciens A-8,800 (FERM P-11,504) is cultured.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a novel physiologically active substance having antitumor activity.

[Prior art] The compound of the present invention has a molecular weight of 7062 and a molecular formula of C4°H660.
11! , and molecular weight 692/molecular formula 〇39H1340,
, (7) It is a new substance, and the existence of a substance with the same physicochemical properties and physiological activity is unknown.

[Problem M to be Solved by the Invention] An object of the present invention is to provide a novel physiologically active substance having an antitumor effect.

[Means for Solving the Problems] As a result of repeated exploratory research in order to obtain new substances with anticancer activity from soil-isolated bacteria, the present inventors discovered that a specific microorganism that The present invention was completed by the discovery of the production of a new physiologically active substance that has a growth-inhibitory effect on cultured cancer cells.

An object of the present invention is to provide a compound represented by formula (1) (in which R represents a hydrogen atom or a methyl group) (hereinafter, a compound in which R is a methyl group will be referred to as FD-893). , FD is a compound in which R is a hydrogen atom
-894. ).

The strain producing the new physiologically active substances FD-893 and FD-894 was newly isolated by the present inventors from soil collected in Nirasaki City, Yamanashi Prefecture, and the microorganism name is strep.
tomyces #gram:nofaciens r
A-8890J and microbial deposit number FERM ρ-11504 J, which has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and the present invention obtained by culturing this strain. The physiologically active substances were named physiologically active substances FD-893 and FD-894.

The mycological properties are shown below.

■ Morphology The vegetative filaments of this strain develop well on synthetic agar media and branch irregularly. Also, no bulkheads are allowed. Spores are well formed on the tips of aerial hyphae extending from vegetative hyphae on starch/inorganic salt agar media and oatmeal agar media. When observed under a microscope, the branching method of spore-forming hyphae is simple, and spores are usually formed in a spiral shape at the tips of aerial hyphae.

Ten or more spores are chained together, and the surface is layered.

Spore formation is cylindrical and its size is 1.3-1.7
X is 0.7 to 0.8μ. No sclerotia, sporangia, or flagellated spores are observed.

■ Growth status on culture media Table 1 shows the results of macroscopic observation when cultured on various media at 28°C for 14 days.

(Margins below this page) Table 1 Growth status ■ Physiological and ecological properties 1) Growth temperature range Grows well in yeast/malt extract medium in the range of 20 to 33°C. It does not grow at temperatures below 14°C and above 39°C.

2) Biochemical properties a) Distinction between aerobic and anaerobic; aerobic b) Liquefaction of gelatin; positive C) Coagulation of skim milk; IIJI d) Peptonization of skim milk; positive e) Starch hydrolysis: positive f) Production of melanin-like pigment; negative 9) Type of cell wall 3) Utilization of carbon sources (on Bridham-Godelive agar) Use: L-arabinose, D-glucose, D-xylose, D-fructose.

From the properties of sucrose, inositol, rhamnose, raffinose, and D-mannite, it was revealed that this strain belongs to the genus Streptomyces among actinomycetes.
``The International Streptomycetes Project'', Bersey's Manual of Systematic Bacteriology, Volume 4, (1989) and ``The Actinomycetes'' by Waxman, Volume 2 (1989)
As a result of comparison with many known strains reported in 1981, this strain was found to be more closely related to Streptomyces graminofascens.
cien).

Based on the above results, it was determined that this strain is the same species as Streptomyces graminofaciens, and this strain was classified as Streptomyces graminofaciens A-8890.
It was named.

FD-893, a physiologically active substance produced in this culture solution,
FD-894 can be isolated according to a general method for collecting fermentation products.

That is, after completion of the culture, the culture solution is extracted with acetone and further extracted with ethyl acetate. The extracted fraction of physiologically active substances FD-893 and FD-894 is concentrated to form a syrup. This syrup was subjected to column chromatography using silica gel and gel filtration using Sephadex LH-20 (product name: Pharmacia) to remove the physiologically active substance F.
D-893, 894 can be purified and isolated. ・Physiologically active substance FD-893, obtained by the above purification method.
The physical and chemical properties of FD-894 are shown below.

(Physicochemical properties of physiologically active substance FD-893) (1) Appearance: Colorless needle crystals (2) Melting point: 117-120°C (3) Mass spectrometry value: Cation FABMS spectrum m/z 729 (M+Na)”m /z 7
45 (M+K)” (4) Elemental analysis values: Actual values: C, 68,13%, H, 9,24%, O
;'12.63% Theoretical value: C, 67,99%, H:
Calculated as 9.35%, O; 22.66% Ca5HeeO+e (5) Molecular formula: CaeHeso Il! (6) [a
] p: 10 + 08' (c = 0.05. Methanol solution) (7) UV absorption spectrum: Measured with methanol solution, λ,, □ = 201 nm (ε = 9 + 78) 242
nm (ε=38 1 95) 282 nm (ε=17368) (8) IR absorption spectrum: The spectrum measured in the KBr tablet is shown in FIG.

(9) IH-NMR spectrum: The spectrum measured at 400 MHz in deuterated chloroform is shown in FIG.

(1o) 13C-NMR spectrum: The spectrum measured at +OOMHz in deuterated chloroform is shown in FIG.

(11) Solubility: Easily soluble in methanol, chloroform, acetone, acetic acid ethyl ester, and benzene.

n-Hexane, poorly soluble in water.

(12) Color reaction: Positive, sulfuric acid, iodine negative: Ninhydrin, FeC13 (13) Basic, acidic, neutral Distinction: Neutral In addition, the physiologically active substance FD-893 has its elemental analysis value, molecular weight, and ultraviolet spectrum. , infrared spectrum, 'H-NMR spectrum, and 13C-NMR spectrum, its structural formula was determined as follows.

(Structural formula of physiologically active substance FD-893) Next, the physicochemical properties of physiologically active substance FD-894 are shown below.

(Physicochemical properties of physiologically active substance FD-894) (1) Appearance: White powder (2) Melting point: 110-117°C (3) Mass spectrometry value: Cation FABMS spectrum m/z 715 (M+Na)" m/Z 73 1 (M+K)” (4) Elemental analysis value: Actual value: C; 55.91%, ) 1.9.27%,
O; 23.82% Theoretical value: C; 57.53%, )1
．． 9.25%, 0.23. +2%039 days 84018
(7) UV absorption spectrum: Measured with methanol solution, λ,, x = 243% m (ε = 26607) 282%
m(ε=12456) (8) IR absorption spectrum The spectrum measured in the KBr tablet is shown in FIG.

(9) IH-NMR spectrum: The spectrum measured at 400 MHz in deuterated chloroform is shown in FIG.

(10) 13C-NMR spectrum: The spectrum measured at 100 MHz in deuterated chloroform is shown in FIG.

(11) Solubility: Easily soluble in methanol, chloroform, acetone, acetic acid ethyl ester, and benzene.

n-hexane, poorly soluble in water.

(12) Color reaction: Positive (sulfuric acid, negative iodine, ninhydrin, Fe CI 3) (13) Distinction between basic, acidic, and neutral/neutral and physiologically active substance FD-8
94ζ years old, its elemental analysis value 1 molecular weight, ultraviolet spectrum, infrared spectrum, 'H-NMR spectrum,'
The structural formula was determined as follows by analysis of 3C-NMR spectrum.

(Structural formula of physiologically active substance FD-894) [Effects of the invention] The physiologically active substance of the present invention has a growth-inhibiting effect on cultured cancer cells and is therefore useful as a medicine.

[Example] Hereinafter, the present invention will be specifically explained with reference to Examples and Test Examples.

Example 1 (Isolation and purification of FD-893 and FD-894) (1) Per iooml: 2 g of oatmeal, 29 glucose, 0.39 sodium chloride, 0.39 meat extract
, ferric sulfate o, o4g, manganese chloride (tetrahydrate) o
strain A113890 was inoculated into a sterile liquid medium containing 039, 04 g, and calcium carbonate, and cultured with shaking at 28° C. for 96 hours. Next, use 2 mini jars with a capacity of 5 oz and use sterile medium 3 with the same composition as the seed medium! 100 ml of the above medium was inoculated into the culture medium, and cultured with aeration at 28°C for 96 hours.

(2) After culturing, add 2 culture solutions 61 to acetone 6
! Extracted with. After removing acetone, extraction was performed with ethyl acetate 61, and after dehydration with anhydrous sodium sulfate,
Concentration yielded a yellow syrupy substance 29.

(3) Silica gel (trade name: Silica gel 6) prepared with chloroform by dissolving the syrup-like substance in chloroform
0. Methanol was adsorbed onto a 500 m column packed with methanol (manufactured by Merck & Co.), and the concentration of methanol was gradually increased (from 0 to 10
0%) methanol/chloroform solution. The active fractions were combined and concentrated to dryness to give a pale yellow syrupy substance 7.
00 mg was obtained.

(4) 700m9 of the pale yellow syrupy substance from the previous section,
Gel filtration was performed with methanol using a 200ml O column packed with Sephadex LH=20 (trade name, manufactured by Pharmacia), which was dissolved in 3rnl of methanol and prepared with methanol.The active fraction was collected and a pale yellow syrup was obtained. 500 mg of a similar substance was obtained.

(5) 500mg of the pale yellow syrupy substance from the previous section,
Silica gel dissolved in chloroform imml and treated with n-hexane (trade name: Silica Gel 60, manufactured by Merck & Co.)
was adsorbed onto a 25ml column packed with FD-893 and FD-894, and eluted with a mixed solvent of n-hexane-acetone (7:3) to separate into two active fractions. 20mg, 90m9 was obtained.

(6) 20ml of the white powder containing FD-893 from the previous section was dissolved in 0.5ml of chloroform and adsorbed onto a 20ml column filled with silica gel (trade name: Silica Gel 60, manufactured by Merck & Co., Ltd.) prepared with n-hexane. , elute with a mixed solvent of n-hexane-acetone (173), collect the active fractions, and convert TPCI-893 into colorless needle-like crystals.
, 8 mg was obtained.

(7) 90mg of white powder containing FD-894 from the previous item
was dissolved in 0.5 ml of chloroform and prepared with n-hexane.
Adsorbed in a 20ml column packed with Merck & Co., Ltd.)
Elution was performed with a mixed solvent of n-hexane-acetone (4:1), and active fractions were collected to obtain 71 mg of a white powder.

Test Example 1 (Proliferation inhibitory effect on various cultured cells) (Sample) Dissolve 5 mg each of FD-893 and FD-894 obtained in Example 1 in methanol and dilute with sterile physiological saline to reach the target concentration. I used the one I made.

(Test cells) ■ 238 days mouse leukemia ■ L-1240 mouse leukemia ■ HL-60 human leukemia (culture medium used) ■ RPMI-1640 medium (test method) Using the above culture medium, various cancer cells were cultured at 2X+O'~ lX1
05/ml, and put 2ml into a 6-hole shear dish with a diameter of 35mm.
Dispensed in portions. Then, 50 μl of the specimen previously diluted to the desired concentration was added at the same time as the start of the culture.

The test cells were cultured for 3 to 4 days at 37°C in a 5% carbon dioxide incubator, then the living cells were measured and the IC5,a value (for 50% inhibition) was determined from the sample concentration and inhibition rate. concentration) was determined.

(result) The results are shown in Table 2.

Table 2

[Brief explanation of drawings]

FIG. 1 shows the IR spectrum of FD-893 measured in KBr tablets. Figure 2 shows F measured at 400MHz in deuterated chloroform.
The 'H-NMR spectrum of D-893 is shown. Third
The figure shows FD- measured at ioOMHz in deuterated chloroform.
Figure 4 shows the IR spectrum of FD-894 measured in KBr tablets. Figure 5 shows the C-NMR spectrum of FD-894 measured at ζ-400MHz in deuterated chloroform. The H-NMR spectrum is shown. 6th
The figure shows FD measured at +OOMHZ in deuterated chloroform.
13C-NMR spectrum of -894 is shown.

Claims (1)

[Claims] 1) A compound represented by the general formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R represents a hydrogen atom or a methyl group.)