JPH0625164A - Tb toxin - Google Patents

Abstract

PURPOSE:To provide a novel compound useful as a proliferation inhibitor against myeloma cells, fibroblasts or the like and as a biochemical reagent. CONSTITUTION:TB toxin of the formula. This compound is obtained by culturing a fungus, Talaromyces bacillosporus-NMIC 10006 strain in a culture substrate of rice grains, extracting the mildewed rice grains therefrom with a high-polar solvent such as methanol, then transferring the solute into a non-polar solvent such as n-hexane, drying the solution under reduced pressure, subjecting the residue as the essence to column chromatography in a usual manner to remove impurities to give a crude TB toxin, followed by crystallization from a solvent. The properties of TB toxin are as follows. Appearance: pale yellow powder; molecular weight: 435; melting point: 137 to 138 deg.C; coloring reactions: positive to Liebermann-Burchard reaction (red), negative to the ninhydrin reaction; molecular formula: C28H37NO3; solubility: slightly soluble in water, soluble in ethanol, n-hexane, readily soluble in methanol, acetone, chloroform, and benzene.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having cytotoxicity. More specifically, the invention has the formula

[Chemical 2] TB toxin represented by

[0002]

2. Description of the Related Art Cytochalasins, trichothecenes and the like have been reported as fungal cytotoxic metabolites. However, none of the previously reported fungal metabolites having a structure similar to the TB toxin of the present invention is known.

[0003]

An object of the present invention is to provide a novel compound useful not only as a biochemical reagent for clarifying various reactions and biological functions in a living body but also as a medicine.

[0004]

[Means for Solving the Problems] As a result of earnest research, the present inventor has extracted artificial fungus rice obtained by culturing the fungus Tlaromyces bacillosporus NMIC10006 strain using rice as a substrate with an organic solvent. The present invention was completed by discovering that the novel compound obtained by isolation from the extract has cytotoxicity, and named it TB toxin.

[0005] The present microorganism, Talalomyces bacillus polas NMIC10006 strain, is stored in the Chiba Prefectural Central Museum and can be obtained by anyone upon request.

A method for producing TB toxin will be described.
Artificial fungus rice obtained by culturing the fungus Talalomyces bacillus polas NMIC10006 strain using rice as a substrate is extracted with a highly polar solvent, then redissolved in a nonpolar solvent, and the solution is dried under reduced pressure to obtain an extract extract. The extract was subjected to column chromatography according to a conventional method to separate impurities to obtain crude TB.
Toxin is obtained and crystallized from solvent to produce TB toxin.

Here, methanol and acetonitrile are suitable as the polar solvent for extraction, and n-hexane is suitable as the nonpolar solvent for transfer. As column chromatography by a conventional method, a crude TB toxin can be obtained by using a column packed with silica gel and eluting with a chloroform-methanol mixture. Ethanol is suitable as a solvent for crystallization of TB toxin. The physical and chemical properties of TB toxin obtained by the treatment as described above are as follows.

(A) Properties: pale yellow powder (b) Molecular weight: 435 (from M ^{+ of} mass spectrum) (c) Melting point: 137 to 138 ° C. (d) Infrared absorption spectrum (KBr) (cm ^-1 )

1 reference νmax: 2900, 1610, 1550, 1410, 1
120 (e) Nuclear magnetic resonance spectrum (CDCl ₃ ) (ppm) δ: 0.88 (1H), 1.04 (3H), 1.08
(1H), 1.24 (2H), 1.27 (2H), 1.
33 (1H), 1.51 (3H), 1.53 (1H),
1.55 (3H), 1.61 (3H), 1.67 (1
H), 1.72 (2H), 1.81 (1H), 2.08
(2H), 2.15 (2H), 2.19 (1H), 3.
27 (1H), 3.76 (1H), 3.97 (1H),
5.26 (1H), 6.05 (1H), 6.33 (1
H), 7.10 (1H), 7.48 (1H) (f) Color reaction Liebermann-Birchard reaction Positive (red) Ninhydrin reaction Negative (g) Molecular formula: C ₂₈ H ₃₇ NO ₃ (h) Solvent Solubility: Poorly soluble: water, soluble: ethanol, n-hexane Easy soluble: methanol, acetone, chloroform, benzene

The cytotoxicity of the above TB toxin was confirmed by the following experiments. RP containing 10% fetal bovine serum, 10 mM HEPES buffer, 6 mM L-glutamine
Myeloma X63-Ag8-6.5.3 cells or fibroblast Balb3T3 cells were cultured for 2 days in MI1640 medium in the presence of various concentrations of TB toxin, and the proliferation was measured by the MTT method. The 50% inhibitory concentration of growth of these cells was 70 ng / ml in all cases.

[0010]

EXAMPLES The present invention will now be described in detail with reference to examples. Example 1 125 g each of white rice was placed in 10 flasks, sterilized in an autoclave and cooled, and then 40 ml of sterilized water was added, inoculated with the fungus Talalomyces batirosporus NMIC10006 strain, and cultured at 25 ° C. for 20 days to give artificial mold rice. obtain. 360 ml of methanol per 100 g of this artificial mold rice (total 4
(000 ml) while pulverizing in a blender, extracting the extract by filtration and concentrating the solvent under reduced pressure to 800 ml,
-Extract with 1200 ml of hexane and evaporate the n-hexane layer under reduced pressure to obtain 2.78 g of the extract.

This extract was dissolved in a small amount of acetone, adsorbed on 5 g of silica gel (Wakogel-200, manufactured by Wako Pure Chemical Industries), and the solvent was removed by evaporation, followed by silica gel 4
It was layered on a column packed with 00 g and eluted with chloroform. 271.4 mg of an oily substance obtained by distilling off the solvent of the eluate containing TB toxin was dissolved in acetone, and
Silica gel (Wakogel C-200, manufactured by Wako Pure Chemical Industries, Ltd.) 0.5 which was soaked in 1N oxalic acid for 1 minute and then dried at 110 ° C. for 12 hours
g, and the solvent was removed by evaporation, and then layered on a column packed with 30 g of silica gel also treated with oxalic acid,
Elution with chloroform gave 230 mg of crude TB toxin. This was crystallized with ethanol and TB toxin 115
mg was obtained. Its physical and chemical properties were as follows:

(A) Property: pale yellow powder (b) Molecular weight: 435 (from M ^{+ of} mass spectrum) (c) Melting point: 137 to 138 ° C (d) Infrared absorption spectrum (KBr) (cm ^-1 )

1 reference νmax: 2900, 1610, 1550, 1410, 1
120 (e) Nuclear magnetic resonance spectrum (CDCl ₃ ) (ppm) δ: 0.88 (1H), 1.04 (3H), 1.08
(1H), 1.24 (2H), 1.27 (2H), 1.
33 (1H), 1.51 (3H), 1.53 (1H),
1.55 (3H), 1.61 (3H), 1.67 (1
H), 1.72 (2H), 1.81 (1H), 2.08
(2H), 2.15 (2H), 2.19 (1H), 3.
27 (1H), 3.76 (1H), 3.97 (1H),
5.26 (1H), 6.05 (1H), 6.33 (1
H), 7.10 (1H), 7.48 (1H) (f) Color reaction Liebermann-Birchard reaction Positive (red) Ninhydrin reaction Negative (g) Molecular formula: C ₂₈ H ₃₇ NO ₃ (h) Solvent Solubility: Poorly soluble: water, soluble: ethanol, n-hexane Easy soluble: methanol, acetone, chloroform, benzene

[Brief description of drawings]

FIG. 1 is a drawing showing an infrared absorption spectrum (KBr) of TB toxin of the present invention.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Yoshio Ueno 12 Ichigaya, Funagawara-cho, Shinjuku-ku, Tokyo Inside the Faculty of Pharmaceutical Sciences, Tokyo University of Science (72) Kimiko Kobayashi 2-1 Hirosawa, Wako City, Saitama Prefecture (72) Inventor Horie Yoshikazu Chiba Chiba City Aoba 955-2 Chiba Prefecture Central Museum Building

Claims (1)

[Claims] 1. The present invention comprises the following formula: A compound represented by (TB toxin).