JPH0627103B2 - New substance UCN-1028C - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel substance UCN-1028C.

UCN-1028C has antitumor activity, protein kinase C inhibitory activity, etc., and is useful as an antitumor agent, a carcinogenesis inhibitor, etc.

Conventional material UCN-1028C is similar in structure to Cladospoi frey, which causes leaf spot disease on grass.
Freichrome isolated as a phytotoxin from rium phlei is known [Agricultural & Biological Chemistry (Agricultural & Biological
Chemistry) 39, 1683 (1975) , day grass Journal, 28 (4), 426 (198
3)]. The similar substance UCN-1028A having antitumor activity and protein kinase C inhibitory activity has been disclosed by the applicant in Japanese Patent Application No. 62-72378.

Problems to be Solved by the Invention Freychrome has weak antitumor activity and protein kinase C inhibitory activity.

According to the present invention, by culturing a microorganism belonging to the genus Cladosporium in a medium, a compound having excellent antitumor activity and protein kinase C inhibitory activity can be obtained. UCN-1028C represented by is provided.

The physicochemical properties, physiological activity and antitumor activity of UCN-1028C are shown below.

(A) Physicochemical properties (1) Molecular formula: C ₄₄ H ₃₈ O ₁₄ (2) Molecular weight: 790 (3) Mass spectrometry: EI mass spectrum method, shown in FIG. 1.

(4) Ultraviolet absorption spectrum: shown in FIG.

(Measured in MeOH) (5) Red external absorption spectrum: shown in FIG.

(Measured in CHCl ₃ ) (6) Solubility: Insoluble in water Soluble in alkaline water, chloroform, acetone, ethyl acetate Soluble in methanol, DMSO (7) ₁ H-NMR spectrum (400 MHz, in CD ₃ OD Measurement, internal standard TMS) δ (ppm) 1.10 (d, 3H, J = 6.3Hz), 1.22 (d, 3H, J = 6.3Hz), 3.00
(dd, 1H, J = 9.8,13.5Hz), 3.11 (dd, 1H, J = 10.1,13.5Hz), 3.6
0 (dd, 1H, J = 2.5,13.5Hz), 3.62 (dd, 1H, J = 1.8,13.5Hz), 3.6
4 (s, 3H), 3.82 (s, 3H), 4.18 (s, 3H), 4.19 (s, 3H), 4.7 (m, 1
H), 5.0 (m, 1H), 5.87 (d, 2H, J = 8.9Hz), 6.30 (s, 1H), 6.31 (s,
1H), 6.36 (d, 2H, J = 8.9Hz), 6.74 (dd, 2H, J = 8.1,1.2Hz), 6.8
6 (t, 2H, J = 8.1Hz), 7.22 (dt, 1H, J = 8.1,1.2Hz) (8) ¹³ C-NMR spectrum (400 MHz, measured in CDCl ₃ , internal standard TMS) δ (ppm ) As shown in FIG.

(9) Thin Layer Chromatography Silica gel TLC (Art 5715 E. Merck) with chloroform: methanol (97: 3 v / v) Rf of 0.3.

(10) CD spectrum: shown in FIG. (Measured in MeOH) (B) Physiological activity (1) Protein kinase C inhibitory activity The measurement of protein kinase C inhibitory activity was performed by the method of Yoshikawa et al. [Journal of Biological Chemistry 257 , 13341 ( 198
2)].

Specifically, 2.5 μmole magnesium acetate, 50 μg histone type IIS (manufactured by Sigma), 20 μg phosphatidylserine, 0.8 μg diolein, 25 n mole CaC
l ₂ , 5 μg crude enzyme (partially purified from rat brain by the method of Yoshikawa et al.) 5 μmole Tris-HCl buffer (pH 7.
5μ) in a solution of 250μ containing 10μ of sample (UCN-1028C
Or Freychrome) solution and add 3 minutes at 30 ℃,
Incubated. Next, 1.25n mole [γ- ₃₂ P] A
After adding TP (5-10 × 10 ₃ cpm / n mole) and phosphorylation at 30 ℃ for 3 minutes, 25% trichloroacetic acid (TCA)
Was added to stop the reaction. The reaction solution was filtered through a cellulose acetate membrane (pore size 0.45 μm) (manufactured by Toyo Roshi Kaisha, Ltd.) to obtain 5
After washing 4 times with% TCA, the radioactivity remaining on the membrane was measured. On the other hand, the radioactivity was measured in the same manner as above without adding the sample, and used as a control. The concentration of the sample showing 50% inhibition relative to the control was defined as IC ₅₀ .

The results are shown in Table 1.

(2) Measurement of protein kinase A inhibitory activity Protein kinase A inhibitory activity Kuo (Kuo)'s method [Biochemistry (Biochemistry) 64, 1349
(1969)]. Specifically, 5 μmole Tris-HCl buffer (pH 6.8), 2.5 μmole magnesium acetate, 100 μm
Histone type IIS (manufactured by Sigma), 0.25nmole cAM
P, 200 μg of a crude enzyme [partially purified from calf heart by the method of Kuo et al.] Was added to a solution of 250 μm and a sample solution of 10 μm was added. The IC ₅₀ was determined in the same manner. The results are shown in Table 1.

UCN-1028C selectively inhibits protein kinase C over flarechrome.

(C) Antitumor activity (1) MCF7 cells 96-well microtiter plate with RPMI-1640 medium (Gib
MCF7 cells prepared at 4.5 × 10 ₄ cells / ml in a medium (hereinafter referred to as medium A) containing 10% fetal bovine serum, 10 μg / ml insulin and 10 ₋₈ M estradiol.
0.1 ml was dispensed into each well of the plate. After culturing the plate in a carbon dioxide gas incubator at 37 ° C. for 20 hours,
To this, 0.05 ml of a sample appropriately diluted with the medium A was added. Then, after culturing at 37 ° C. for 1 hour in a carbon dioxide gas incubator, the culture supernatant was removed. The residue is ProS (composition: Na
Cl 8g /, KCl 0.2g /, Na ₂ HPO ₄ 1.5g / and KH ₂ PO ₄
After washing once with 0.2 g /) 0.1 ml of fresh medium A
Each of them was added and cultured in a carbon dioxide gas incubator at 37 ° C. for 72 hours. After removing the culture supernatant, the medium A and 0.02 were added to the residue.
Add 0.1 ml of medium consisting of% Neutral Red,
The cells were stained by culturing in a carbon dioxide gas incubator at 37 ° C for 1 hour. After removing the culture supernatant, the residue was washed once with physiological saline. Then, after extracting the dye with 0.001 N hydrochloric acid / 30% ethanol, 55 using a microplate reader.
Absorbance at 0 nm was measured. The sample concentration (IC ₅₀ ) at which cell growth was inhibited by 50% was calculated by comparing the absorbances of untreated cells and cells treated with a sample of known concentration. The results are shown in Table 2.

(2) HelaS ₃ cells 3 × 10 ₄ cells / well in 96-well microtiter plate with MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and 2 mM glutamine.
0.1 ml of HelaS ₃ cells prepared in ml was dispensed into each well of the plate. Then, in the same manner as for the MCF7 cells, IC
₅₀ was calculated. The results are shown in Table 2.

Next, a method for manufacturing UCN-1028C will be described. UCN-1028C can be obtained by culturing a UCN-1028C-producing strain belonging to the genus Cladosporium in a medium and collecting UCN-1028C from the culture. As the UCN-1028C producing strain, any strain can be used as long as it belongs to the genus Cladosporium and has a UCN-1028C producing ability. A representative strain is the KAC-2215 strain isolated by the present inventors from an outdoor wall block wall in Osaka.

The mycological properties of the KAC-2215 strain are as follows.

When cultured at 20 ° C using malt extract agar medium, 3
The diameter of the village reaches 5.5 to 6 cm in a week. The center of the surface of the village is grayish green, and the surrounding area is gray. The back surface of the village is dark green and black. Mycelia have septa and grow in and on the medium. The hypha has a diameter of 1.5 to 5 μm and is well branched. The conidia peduncle is brown or dark brown, rises from hyphae, and has septa. Conidia are necrotized and concatenated on the conidia peduncle or its branches. The mode of ontogeny of conidia is budding, with the youngest conidia located at the tip of the conidial chain. Branched cells at the tip of conidia stalk consist of 1-2 cells with a width of 3-4 μm, and single-cell conidia have an oval, lemon-shaped, or elliptical shape, and are pale brown with a length of 4-6 μm and a width of 3 μm. ~ 4 μm.

As a result of the above observations, this bacterium was identified as Gladosporium cladosporioides. The mycological properties of Cladosporium cladosporioides are described by Ellis, "Dematiaceous hyphomyce.
tes) [1971] ”.

This strain is "Kladsporium cladsporioides KA.
C-2215 ”, and is designated as Micro Engineering Research Article No. 1285, Showa 6
It was deposited at the Institute of Microbial Technology of the Institute of Industrial Technology on February 10, 2nd.

In culturing the bacterium used in the present invention, an ordinary filamentous bacterium culturing method is generally used. As the culture medium, either a natural medium or a synthetic medium can be used as long as it is a medium containing a carbon source, a nitrogen source, an inorganic substance and the like. As the carbon source, glucose, starch, glycerol, mannol, fructose, sucrose, molasses, hydrocarbon, alcohol (methanol, ethanol, etc.), organic acid (succinic acid, acetic acid, etc.) are used. As the nitrogen source, ammonium chloride, ammonium sulfate, ammonium nitrate, sodium nitrate, urea, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, soybean powder, casamino acid and the like are used. As the inorganic substance, sodium chloride, potassium chloride, ferrous sulfate, zinc sulfate, manganese sulfate, copper sulfate, calcium carbonate, phosphate and the like are used. Further, a substance that promotes the production of UCN-1028C, such as biotin or vitamin, may be added to the medium.

The culturing method may be either a liquid culturing method or a solid culturing method, but a liquid culturing method, particularly a deep agitation culturing method is usually used. Culture temperature is 20 to 35 ° C, especially 23 to 28 ° C
Is preferred, and the pH of the medium is preferably in the range of 4 to 10, particularly 5 to 7 by adding ammonia water, ammonium carbonate solution and the like. When liquid culture is normally performed for 1 to 7 days, UC
N-1028C is produced and accumulated in the culture medium. When the amount of production in the culture reaches the maximum, the culture is stopped and UCN-
Purify and isolate 1028C.

For the isolation and purification of UCN-1028C from the culture broth, the separation and purification methods usually used for isolating microbial metabolites from the culture broth are used. For example, the culture product is first separated into a culture solution and cells by filtration, centrifugation, or the like. Cells are chloroform, acetone, etc. UCN-1028C
Extract with a soluble solvent, concentrate the extract under reduced pressure to remove the solvent, and then dissolve in water to form an aqueous solution. The liquid obtained by removing the above-mentioned bacterial cells and the liquid obtained by treating the bacterial cells are treated with a nonionic porous resin such as HP-20 (manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) to adsorb the active ingredient onto the resin. After that, elute with methanol. After concentrating the eluate, combine with extraction with a solvent such as ethyl acetate, column chromatography using a carrier such as gel filtration agent, silica gel, etc. to make UCN-1028C
Can be isolated.

Examples will be shown below.

Example 1 As inoculum, Cladosporium cladsporioides
KAC-2215 strain was used. 1 platinum loop of the strain was inoculated into 50 ml of a seed medium of the following composition in a 300 ml Erlenmeyer flask,
The culture was carried out at 25 ° C. for 72 hours with shaking.

Seed medium composition: Peptone 5 g /, Ebios (Ebios Pharmaceutical Co., Ltd.) 5 g /, glucose 10 g /, vegetable juice (V-8 vegetable juice, Campbell) 200
ml /, calcium carbonate 3g / (pH 6.0, NaO before sterilization
(Adjusted with H) The seed culture solution was transferred to a fermentation medium 18 having the following composition in a 30-volume jar fermenter at a rate of 5% (volume) 2
Aeration stirring at 5 ° C (rotation speed 350 rpm, aeration rate 18 / mi
Culture was performed according to n).

Fermentation medium composition: Soluble starch 50 g /, dry yeast 2
0 g /, KH ₂ PO ₄ 0.5 g /, MgSO ₄ .7H ₂ O 0.5 g /,
Calcium carbonate 5 g / (pH 7.0, adjusted with NaOH before sterilization), and culturing for 80 hours without adjusting pH during culturing.

The culture solution was filtered to obtain bacterial cells. Acetone 30 was added to the cells and stirred to extract the UCN-1028C substance. After deionized water 90 was added to the acetone extract, the solution was passed through a column packed with 2 nonionic porous resins (HP-20; manufactured by Mitsubishi Kasei Co., Ltd.) to adsorb UCN-1028C. . Then 5
After elution of impurities with 0% methanol solution, UCN-1028
C was eluted with methanol. After concentrating the eluate, adjust the pH to 1.7.
The mixture was adjusted to, and extracted with ethyl acetate. The ethyl acetate layer was concentrated, applied to a silica gel column (Wakogel C-200; Wako Pure Chemical Industries, Ltd., 1), and developed with chloroform: methanol (97: 3 v / v). The fraction containing UCN-1028C was concentrated, and the concentrated liquid was added to 300 ml of a nonionic porous resin (HP-2
0SS; manufactured by Mitsubishi Kasei Kogyo Co., Ltd.) was passed through the column.
After washing with 50% methanol solution, 70% methanol,
Elution was performed with 90% methanol and 100% methanol (1 each) to obtain a fraction containing UCN-1028C. After the lysate containing the components was concentrated, the concentrate was subjected to Sephadex LH-20 column chromatography and eluted with methanol.
Fractions containing the active substance were concentrated to give UCN-1028C (647 mg).

The trend of UCN-1028C during the culture and purification procedure was determined by the inhibitory activity of protein kinase C and thin layer chromatography.
The UCN-1028C was traced using the red-orange spot as a guide.

Effect of the Invention The present invention provides UCN-1028C having excellent antitumor activity and protein kinase C inhibitory activity.

[Brief description of drawings]

Figure 1 shows the EI mass spectrum of UCN-1028C. Figure 2 shows the UV absorption spectrum of UCN-1028C. Figure 3 shows the infrared absorption spectrum of UCN-1028C. FIG. 4 shows the ¹³ C-NMR spectrum of UCN-1028C. FIG. 5 shows the CD spectrum of UCN-1028C.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location (C12N 1/14 C12R 1: 645) (C12P 15/00 C12R 1: 645)

Claims (1)

[Claims] 1. A formula UCN-1028C, a novel substance represented by.