JPH07107038B2 - Novel substance FL-657C and its production method - Google Patents

Description

The present invention relates to a novel FL-657C having antitumor activity and Friend leukemia cell differentiation inducing activity, and a method for producing the same.

The present inventors have conducted various searches on various cultures of microorganisms for the purpose of searching for novel antitumor active substances. As a result, actinomycetes belonging to the genus Streptomyces isolated from soil have antitumor activity and Friend leukemia cell differentiation-inducing activity. It was discovered to produce the novel substance FL-657C. This substance was isolated, and the structure of this substance was determined. As a result, this substance was recognized as a novel substance and was designated as FL-657C.

The present invention has been completed based on the above findings.
That is, a novel compound having the following structural formula (A) and a novel substance belonging to the genus Streptomyces The present invention relates to a method for producing a novel substance FL-657C, which comprises culturing a microorganism having the ability to produce FL-657C in a medium to produce a novel substance FL-657C, and collecting this.

Hereinafter, the method for producing the novel substance FL-657C in the present invention will be described.

The microorganism used in the present invention is a microorganism having an ability to produce FL-657C, and specifically, a microorganism isolated from soil is used. Purging this microorganism
When identified according to the Manual of Observed Native Bacteriology 8th edition (1974), it was identified as Streptomyces sioiaensis AJ9451 and was submitted to the Institute of Microbial Science and Technology of the Institute of Industrial Science and Technology, Microbiology Research Institute 7296 (FERM P- 729
6) has been deposited as

As a medium used for producing FL-657C using the present microorganism, a usual liquid medium containing a carbon source, a nitrogen source, inorganic salts and, if necessary, organic trace nutrients is used.

Examples of the carbon source include glucose, fructose, maltose, sucrose, starch, dextrin,
Hydrolyzed starch, carbohydrates such as molasses, citric acid, succinic acid, fumaric acid, acetic acid and the like, organic acids and alcohols such as glycerin are used. Examples of the nitrogen source include ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium nitrate, ammonium salts such as ammonium acetate, nitrates such as sodium nitrate and potassium nitrate, urea, ammonia water, ammonia gas, amino acids, and further peptone, soybean whey, soybean flakes. And proteins such as hydrolysates thereof, rice bran and the like are used. Other inorganic salts include, for example, manganese salts and phosphates, and organic trace nutrients include amino acids, vitamins, peptone containing these, yeast extract, and the like.

Cultivation conditions vary depending on the medium composition and the like, but for example, culture is usually carried out under aerobic conditions such as pH 4.0 to 9.0, temperature 15 to 40 ° C., shaking culture, aeration and stirring culture.

The FL-657C of the present invention is present in the culture solution and the cells obtained by culturing in this manner, and the method for separating and collecting the FL-657C from the culture solution is an organic solvent extraction method such as ethyl acetate, a normal phase. And known separation methods such as reverse-phase silica gel, reverse-phase ODS, adsorption chromatography using an adsorbent such as cellulose, gel permeation method, method utilizing difference in solubility in various solvents, etc.
The purification method is appropriately combined. On the other hand, FL-657C produced in the cells is extracted with an organic solvent such as chloroform, methanol, and acetone ethyl acetate, and then purified and separated according to the above method.

Next, a production example is shown.

A 500 ml dissolution flask into which 100 ml of the medium (pH 7.2) shown in Table 1 was dispensed was sterilized at 120 ° C for 20 minutes, and 1 ml of the culture solution of Streptomyces siaensis FERM P-7296 was inoculated into the flask. Culturing was carried out at 0 ° C for 3 days. On the other hand, 120 seeds of the above-mentioned medium were put into a 200-volume stainless steel jar fermenter and sterilized, and the seed mother 1.3 was inoculated and stirred (320
rpm), aeration (1/2 vvm), and culturing was continued at 27 ° C. for 3 days.
Further, 120 was used as a secondary seed mother, and the medium 2800 having the above composition was placed in a 5,000-volume stainless steel tank and sterilized, inoculated, stirred (180 rpm), aerated (1/1 vvm), and heated at 27 ° C for 2 to 2
Cultured for 3 days. These cultures were performed twice.

Eradication of Table 1 0.2% glucose 1.0% yeast extract _{KH 2 PO 4 0.1% MgSO 4} · 7aq 0.1% Bakutosoiton 0.7% Bacto peptone 0.5% Starch 2.0% Adecanol 0.05% (pH7.2) 5300 culture medium in a filter press 250 kg of mycelium
And the sterilization liquid 5000 was obtained. In order to obtain the section containing FL-657C from the mycelium, the following method may be used.

To 250 kg of this mycelium, a mixed solution 1000 of chloroform: methanol (1: 1) was added, and the mixture was stirred at room temperature for 4 hours, and the mycelium was removed by filtration. The chloroform / methanol mixed solution containing FL-657C was concentrated to obtain an oily substance.

The following method may be used to obtain the category containing FL-657C from the sterilized solution 1000.

Adsorption chromatography column (Mitsubishi Kasei's "Diaion HP-20") was poured with the sterilized solution 1000 to remove substances not adsorbed on the adsorption chromatography column. Then, washed with water, 20% MeOH, 50% MeOH. Pour 100% methanol 1500 into an adsorption chromatography column to 100
A solution 200 containing FL-657C eluted with% methanol was taken. The section was concentrated at room temperature using an evaporator. For the classification of the oily substance obtained by extracting the mycelium with chloroform-methanol and the substance adsorbed to the adsorption chromatography column in the sterilized solution and eluted with 100% methanol, the common FL-657C unit described below is used. A separation and purification step can be used.

Below, FL from the concentrated liquid containing FL-657C obtained from the sterilized liquid was used.
The isolation and purification step of -657C will be described.

Methanol 1 and 2 were added to the above concentrated liquid, and the mixture was vigorously shaken at room temperature for 20 minutes and then allowed to stand to separate methanol-dissolved sections 1 and 2. Next, add 1 to the residual liquid, shake again vigorously for 20 minutes at room temperature and let stand, then dissolve in methanol
2 was collected. After combining these methanol-dissolved sections, the methanol sections were concentrated and dried at room temperature using an evaporator. This concentrated and dried section containing FL-657C was dissolved in 100% methanol 2-3. Next, gel permeation chromatography (“LH-20” manufactured by Pharmacia)
After pouring 100% methanol liquid containing FL-657C into the column, 600% of 100% methanol was newly poured to perform gel filtration, and the liquid was fractionated every 100 ml. A fraction 10 having a growth inhibitory effect on friend leukemia cells was collected from these liquids. Next, this fraction was concentrated and dried at room temperature using an evaporator. The partially purified concentrated and dried product containing FL-657C was dissolved in 200 ml of methanol.

Next, after pouring a 100% methanol solution containing the above FL-657C into a partition chromatography (“LH-20” Pharmacia) column, pour water 100% -methanol 0% to water 0% -methanol 100%, Partition chromatography was performed using a linear gradient, and the eluate was collected in 100 ml portions. A fraction 2.3 having a growth inhibitory effect on Friend leukemia cells was collected from these eluates.

Next, this fraction was concentrated at room temperature using an evaporator.
58.5 ml was obtained.

Next, the partially purified solution containing FL-657C was poured into the partition chromatography (“Protein & Peptide C ₁₈ ” VYDAC Co., Ltd.) column (1), and the concentrated solution containing FL-657C was poured into the column, followed by containing 10 mM EDTA 2Na. Pour a solution of 50% of 10 mM ammonium acetate buffer (pH = 4) and 50% of methanol, and perform partition chromatography to obtain eluates.
Aliquoted every 20 ml. From these solutions, 400 ml of a fraction having a growth inhibitory effect on friend leukemia cells was collected. Next, this fraction was concentrated at room temperature using an evaporator. Next, the partially purified solution containing FL-657C was subjected to adsorption chromatography ("Diaion HP-
20 ”) The concentrate was poured into the column to adsorb the target component in the solution onto the resin. Next, after washing with 50% methanol solution in water, 100% methanol solution was poured into the column to elute the target component, and 20 ml fractions were collected. From these solutions, 150 ml of a fraction having an inhibitory effect on Friend leukemia cells was collected. This fraction was concentrated at room temperature using an evaporator.

Next, high performance liquid chromatography ("Hitachi 635A
Type ”), a reversed-phase column (“ YMC-ODS-D semi-preparative column ”,“ Yamamura Chemical Laboratory ”), mobile phase, acetonitrile: 1 mM EDTA · 2Na, 10 mM acetate buffer (pH 4.0) = 60: Four
0 Concentrated liquid containing the target component under the condition of detection wavelength 254nm 0.1m
l was injected from the injection port, and the section with absorption at 254 nm was fractionated,
I collected it.

From these fractions and fractions, 20 ml of a fraction having the activity of inhibiting the growth of Friend leukemia cells was obtained. Fractionation and fractionation operations were repeated 10 times by this high performance liquid chromatography to obtain 200 ml of a liquid containing FL-657C. This solution was concentrated at room temperature using an evaporator. Then this FL-
The concentrate containing 657C was poured into an adsorption chromatography (“DIAION HP-20” manufactured by Mitsubishi Kasei) column to adsorb the target component in the solution onto the resin. Next, after washing with water and a 50% methanol solution, a 100% methanol solution was poured into the column to elute the target component, and the fraction was collected every 20 ml.

From these solutions, 100 ml of a fraction having an inhibitory effect on Friend leukemia cells was collected. This fraction was concentrated at room temperature with an evaporator and then freeze-dried.

Next, the FL-657C identification results will be described. ^{The 1} H-NMR chemical shift pattern is based on the previously isolated FL-657A.
Although very similar to (trichostatin A) and FL-657B (tricostatin acid), changes in the chemical shift of the 6'and 5'protons were observed. First, the partial structure was estimated from the ¹ H-NMR (400 MHz, JEOL) spectrum assignment of the substance shown in FIG. δ7.87ppm and δ6.73ppm (2 protons, doublet)
Of A ₂ , B ₂ spin system, which are largely separated from each other, and δ3.06ppm
(6 protons, singlet) From the existence of a peak considered to be N-methyl, Was estimated. From δ5.92ppm (1 proton, relatively broad doublet), δ4.54ppm (1 proton, multibread), δ1.27ppm (3 protons, doublet) The existence of the partial structure was estimated. Furthermore, δ 7.15 ppm
The structure of -CH = CH- was deduced from (one proton, doublet) and δ 6.02 ppm (one proton, doublet). And δ 1.94ppm (3 protons, slightly split, Is estimated to be present, and this peak has a long-range coupling with δ5.92 ppm.
Since m and δ 7.15 ppm are also subjected to long range coupling, the following overall structure was estimated. ¹ H-NMR (400
MHz, JEOL) spectrum analysis? ¹³ C in order to confirm the chemical structure of al estimated FL-657C
-NMR analysis was performed. From δ 201.5 ppm of ¹³ C-NMR spectrum shown in Fig. 2. Was estimated. Also from δ171.5ppm Was estimated. FL-657A for ¹³ C-NMR
Analysis was performed with reference to the spectrum of (trichostatin A) FL-657B (trichostatin acid). ^{The 13} C-NMR spectrum shows two pairs of benzene rings (δ132.0p expected from structural formula (A).
pm, δ120.0 ppm), a pair of N-methyl (δ40.1 ppm) approximately double-strength ¹³ C-NMR signal is considered, and the existence of a total of 17 carbon atoms is confirmed. Regarding other attributes, δ201.5ppm is derived from benzoyl group, δ171.5ppm is derived from amide group carbonyl, etc.
It was confirmed to have.

Furthermore, in order to confirm the chemical structure of this substance, FD-MASS spectrum analysis was performed and the results shown in FIG. 3 were obtained. This FD-MA
From the 286M / Z ion peak of the SS spectrum, it was confirmed that this substance has the structural formula (A).

Furthermore, in ¹ H-NMR in CDCl ₃ , a signal corresponding to two protons derived from NH ₂ was observed at 5.5 ppm. Also, when the proton of NH ₂ is irradiated by selective decoupling, it is 171.5.
Carbon from ppm carbonyl reacted.

Next, the elemental analysis results of this substance are shown in Table-2.

The result of this elemental analysis was in agreement with the molecular formula of the structural formula (A) of this substance.

FL-657C has the following physical properties.

a) Molecular weight: 286 (MASS spectrum method) b) Molecular formula: C ₁₇ H ₂₂ N ₂ O ₂ c) Color of substance: White to pale yellow d) Solubility: Soluble in methanol, acetonitrile and chloroform, in water and hexane Dissolution e) ¹ H-NMR spectrum: Fig. 1 f) ¹³ C-NMR spectrum: Fig. 2 g) AD-MASS spectrum: Fig. 3 h) Ultraviolet absorption spectrum: Fig. 4 Structural formula (A) It was investigated whether this substance represented by is a new substance. CAS.ONLINE
When it was examined whether or not the compound represented by Structural Formula (A) was present among the compounds registered in 1965 to the present, no compound corresponding to Structural Formula (A) was found. Next, from among the compounds listed in the formula index of the chemical abstract published from 1947 to the present, the molecular formula of this substance is C ₁₇ H ₂₂ N ₂ O ₂
A compound was selected that gave the same molecular formula as. When it was examined whether or not these compounds correspond to the structural formula (A), the compound given by the structural formula (A) was not found.

From the above results, this substance was identified as a novel compound.

Next, the physiological activity of FL-657C will be described.

Ham ・ SF-12 powdered medium (Gibeo cell culture medium component) 10.4 g and NaHCO ₃ 1.4 g dissolved in 1.0 distilled water, sterile with a pore size 0.22μ Millipore filter,
Fetal bovine serum aseptically prepared (Flow labo)
Was added to prepare a cell culture medium. Pre-cultured Friend leukemia cells (Yoji Ikawa, Metab., 15 , 145 (1978)) were added to this medium to give a cell concentration of 1 × 10 ⁴ / m 2.
l), add this cell suspension to a microtest plate (Nunc
0.1 ml was aseptically dispensed to 96 wells (manufactured by the company) and cultured in a carbon dioxide gas incubator (carbon dioxide gas concentration 5%, temperature 37 ° C.) for 24 hours. FL isolated and purified from the acid generating solution of Streptomyces siaensis FERM P-7296 was added to this culture solution.
The above medium containing a certain amount of -657C was added to 0.1 ml, the culture was continued for 5 days, and the state of cells was observed under an inverted microscope to examine the growth inhibitory effect of FL-657C on Friend leukemia cells. The results are shown in Table 3. In the table, (-) indicates that there is no activity, and +++ indicates that strong activity was present.

Next, Balb 3T transformed with M-MSV virus
Table 4 shows the growth inhibition on 3 cells (see Asronsor and Rowo, Virology. 42 , 9 (1970)). In this experiment, MEM Dulpecco's medium (Gibco) was used as the medium.

As shown in Tables 3 and 4, FL-657C was confirmed to have a markedly strong growth inhibitory activity against the growth inhibitory activity of friend leukemia cells and M-MSV-Balb3T3 cells.

[Brief description of drawings]

FIG. 1 is a ¹ H-NMR spectrum of FL-657C. FIG. 2 is a ¹³ C-NMR spectrum of FL-657C. Fig. 3 shows FL-
It is an FD-MASS spectrum of 657C. FIG. 4 is an ultraviolet absorption spectrum of FL-657C.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 465)

Claims (2)

[Claims] 1. A novel substance FL-657C having the following structural formula: 2. A novel substance FL- which belongs to the genus Streptomyces
Cultivation of microorganisms capable of producing 657C produces novel substances
A process for producing a novel substance FL-657C, which comprises producing FL-657C in a medium and collecting it.