JPS6232174B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula (In the formula, R represents C ₁₀ H ₂₁ , C ₁₁ H ₂₃ or C ₁₂ H _25. ) The present invention relates to a cell normalizing antitumor agent having an acyl group represented by the following formula. The present inventors previously discovered that a peptide having an acyl group represented by the above structural formula could be produced by a microbial method, and at the same time, cyclic adenosine-3',
We discovered that this substance has an inhibitory effect on 5'-monophosphate phosphodiesterase, and as a result of intensive research into the pharmacological effects of this substance, we found that it has sufficient pharmacological effects as an antitumor agent.
This has led to the completion of the present invention. That is, the general formula (In the formula, R represents C ₁₀ H ₂₁ , C ₁₁ H ₂₃ or C ₁₂ H _25. ) The present invention relates to a cell normalizing antitumor agent having an acyl group represented by the following formula. The method for producing the substance of the present invention is carried out by producing Bacillus bacteria newly isolated from soil by the present inventors in a culture filtrate through conventional aerobic culture, and recovering the bacterium. An example of the method for producing and separating and purifying the cell normalizing antitumor agent of the present invention is as follows. Glucose, peptone, yeast extract, sodium chloride, magnesium sulfate, dipotassium phosphate (PH
6.8) Bacillus subtilis C-756 in a medium consisting of
(Feikoken Bibori No. 6785) is cultured at 30° C. for 2 to 3 days and then sterilized, and a cell-normalizing antitumor agent can be obtained from the obtained culture filtrate. Recovery from the culture filtrate is usually done by adding concentrated hydrochloric acid to the culture filtrate to a pH of 2, or adding copper sulfate to a final concentration of 0.6% to precipitate the cell-normalizing antitumor agent. can be collected and extracted several times with ethyl acetate. The ethyl acetate extracted fraction is washed with aqueous sodium bicarbonate, dehydrated with anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude extract. Next, this can be sequentially applied to a Sephadex G-50 column and a silica gel column to obtain an active fraction. Further purification is performed sequentially using a Sephadex LH-20 column and a silica gel column to obtain a colorless amorphous powder. This shows a single spot on a thin layer using various solvents, but it can be further separated and purified by high performance liquid chromatography equipped with an octadecylated silica gel column. The substance of the present invention produced in the culture filtrate is a peptide having an acyl group represented by the above general formula, where the R-corresponding moiety is C ₁₀ H ₂₁ , C ₁₁ H ₂₃ or
Three new peptides with C ₁₂ H ₂₅ attached can be obtained. The physicochemical properties of these substances of the present invention are as follows.

[Table] The sequence order of the constituent amino acids from the molecular ion fragments appearing in the mass spectrometry spectrum of permethylated compounds is Glu, Leu, Leu, Val, Asp, Leu, Leu from the N-terminus for all peptides. was recognized. In addition, chemical analysis confirmed that the presence of the lactone ring shown in the infrared absorption spectrum is a bond between the β-hydroxylated fatty acid and the C-terminal amino acid Leu, and the overall structure of the substance of the present invention is as described above. It was recognized that Next, as a microorganism that produces the substance of the present invention, for example, a Bacillus bacterium newly isolated by the present inventors can be exemplified, and the mycological properties of this strain are as follows. Mycological properties Gram-positive aerobic bacillus that is motile with periflagella and forms spores. The growth state on the broth agar medium is medium, butter-like pale yellow-brown colonies are formed, the colony surface is wrinkled and dull glossy, and no change in the medium is observed. Growth test 7% sodium chloride medium + Sabouraud dextrose medium + Azide medium - Anaerobic culture medium - Growth temperature 52-15℃ Production/decomposition test Pigment production (glucose, tyrosine) - Acetoin production + Acid production (glucose, arabinose) , xylose, mannitol) + starch degradation + citrate assimilation + propionate assimilation - lysozyme resistance + indole formation - nitrate reduction + casein degradation + milk hydrolysis + gelatin liquefaction ＋ Degradation of tyrosine − Degradation of hippurate ＋ Egg yolk reaction − Catalase test ＋ As a result of the above morphological and physiological properties, this strain was identified as Bergey's Manual of Determinative
Bacteriology (8th edition) and The Genus Bacillus
As a result of the search, it seems most appropriate to identify it as Bacillus subtilis, although there is no agreement in that it decomposes hippurate. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as Bacillus subtilis C-756 (Fiber Science and Technology Research Institute No. 6785). Examples are shown below. Example 1 An example of the production method and separation and purification of the cell normalizing antitumor agent of the present invention will be described. Glucose 1%, peptone 1%, yeast extract 0.3
%, sodium chloride 0.3%, magnesium sulfate 0.1
%, dipotassium phosphate 0.1% (PH6.8).
No. 6785) at 30° C. for 2 to 3 days, bacteria were removed, and a cell-normalizing antitumor agent was obtained from the resulting culture filtrate. If you add concentrated hydrochloric acid to the culture filtrate 40 to make the pH 2, or add copper sulfate to the final concentration of 0.6%, the cell normalizing antitumor agent will precipitate.
This is collected and extracted several times with ethyl acetate. The ethyl acetate extracted fraction was washed with aqueous sodium bicarbonate, dehydrated over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain about 14 g of crude extract. Next, this was sequentially applied to a Sephadex G-50 column (emitted with 80mM Tris-HCl buffer (PH7.5)) and a silica gel column (eluted with chloroform:methanol = 8:1), yielding about 8 g as an active fraction. It was done. Furthermore, security
It was sequentially purified using an LH-20 column (emitted with acetone) and a silica gel column (emitted with chloroform:methanol=5:1) to obtain about 3 g of colorless amorphous powder. This shows a single spot on a thin layer using various solvents, but it is further separated and purified using high performance liquid chromatography equipped with an octadecylated silica gel column, and the three types of cell-normalizing antitumor agents of the present invention are separated and recovered. did. Example 2 The following chemical analysis experiment was conducted to determine the β-hydroxylated fatty acid and amino acid forming the lactone ring of the present invention. After reducing the cell normalizing antitumor agent with lithium borohydride by three methods, hydrochloric acid hydrolysis,
The loss of amino acids was investigated. Lithium borohydride can reduce esters and lactones, but cannot reduce carboxylic acids, so (1) it directly reduces them without pretreatment. (2) After methylating the cell-normalizing antitumor agent, it is reduced. (3) After opening the lactone ring of the inhibitor with an alkali, it is reduced. After the above three types of reduction, the amino acid composition is (1) Glu:Asp:Val:Leu=1:1:1:3 (2) Glu:Asp:Val:Leu=0:0:1:3 ( 3) Glu:Asp:Val:Leu=1:1:1:4, and this result proved that it was the C-terminal Leu that was bonded to the β-hydroxylated fatty acid. Example 3 After hydrochloric acid hydrolysis of 2 to 3 mg of each of the three cell-normalizing antitumor agents isolated by the method of Example 1, the water-soluble peptide part was used for amino acid composition analysis, and the oily part was analyzed using gas chromatography. This was confirmed to be a β-hydroxylated fatty acid using tograph-mass spectrometry (GC-MS). That is, when the oily substance produced from the hydrochloric acid hydrolyzate was extracted with ether, methylated with diazomethane, and then subjected to gas chromatography-mass spectrometry (GC-MS),
Each cell normalizing antitumor agent is m/z244,
It was found to have fatty acids with molecular weights of 258 and 272. In addition, other physicochemical properties are as follows.

[Table] Based on the above properties, three types of cell normalizing antitumor drugs are
C ₁₀ H ₂₁ binding peptide, C ₁₁ H ₂₃ binding peptide,
It was confirmed to be a C ₁₂ H ₂₅ binding peptide. Example 4 Converting this substance into normal cells in tumor-type Chinese hamster ovary cells The method of Puck et al. (TTPuck et al. Proc. Nat. Acad.
Sci.USA, vol. 68, p. 358, 1971). Specifically, Chinese hamster ovary cell (ATCC CCL-61) strain was cultured in Ham's F-12 medium containing 10% fetal bovine serum at 37°C in a 5% CO ₂ incubator, and pharmacological effects were determined. investigated. After dibutyryl cyclic AMP is taken into cells, it is degraded and converted into cyclic
It has been reported that it normalizes tumor cells by converting into AMP and increasing intracellular cyclic AMP concentration. When 1mM of dibutyryl cyclic AMP is added to tumor-type Chinese hamster ovary cells, they recover to the differentiated morphology before tumorigenesis, and at the same time, their contact inhibition ability is restored and monolayer proliferation occurs (TTPuck). etProc.Nat.Acad.Sci.US
A, vol. 68, p. 358, 1971, I. Pastan et al., Proc. Nat.
Acad.Sci.USA, vol. 68, p. 425, 1971). The C ₁₀ H _21- binding peptide, C ₁₁ H _23- binding peptide, and C ₁₂ H ₂₅ -binding peptide obtained by the method of Example 1 were added to cultured cells at 0.1 mM each, and after 2 days, the results showed that 1 mM Djibouti In the case of a C ₁₀ H ₂₁ -binding peptide like Rilcylic AMP, the tumor-type cells in Figure 1 (Photo 1) differentiated into monolayer-proliferating fibroblasts with the orientation shown in Figure 2 (Photo 2). It was confirmed that there was a change in Similarly, it was confirmed that the C ₁₁ H ₂₃ -binding peptide and the C ₁₂ H ₂₅ -binding peptide changed to the fibroblast-differentiated form shown in FIG. 3 (Photo 3) and FIG. 4 (Photo 4), respectively. Example 5 Growth inhibition effect on mouse tumor cells When mouse tumor cell (Sarcoma 180) strain was cultured in MEM Earl medium containing 10% calf serum,
1 ml of Sarcoma 180 (ATCC CCL-8) strain
1-2 x ¹⁰⁵ cells per 7-day culture
Multiply by 15 times. When 1mM of each of the substances obtained according to the present invention was added, no proliferation was observed even after 7 days had passed since the addition.

[Brief explanation of the drawing]

Figure 1 shows a photograph of tumor-type cells of Chinese hamster ovary, Figure 2 shows a photograph of fibroblasts after addition of _{C10H21 -} binding peptide, and Figure 3 shows a photograph of tumor-type cells of Chinese hamster ovary with _C11 Figure 4 shows a photograph of fibroblasts after adding the _H23- binding peptide, Figure 4 shows a photograph of fibroblasts after adding the _{C12H25 -} binding peptide to the tumor-type cells in Figure 1, and Figure 5 Infrared absorption spectrum measured in potassium bromide tablets of inhibitor,
The vertical axis shows absorbance and the horizontal axis shows wavelength.

Claims (1)

[Claims] 1. General formula (Wherein, R represents C ₁₀ H ₂₁ , C ₁₁ H ₂₃ or C ₁₂ H _25. ) A cell normalizing anti-tumor agent having an acyl group represented by the formula: