JPH0227328B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to an antitumor agent containing an ansatrienin compound as an active ingredient. As a result of research into the fermentation products of Streptomyces liciliensis strain T-23 [FERMBP-243], a new strain belonging to Streptomyces liciliensis, the present inventors found that One of the products, T-23-substance, has promising anti-tumor activity, and such T-23-substance is also one of the above-mentioned fermentation products, under oxidizing conditions. It has been found that it can be easily obtained chemically by subjecting it to. T-23-- substance which is the object of the present invention and T-23- which is the starting material for chemically producing it
The physicochemical properties of each substance are as follows. T-23-Physicochemical properties of the substance (1) Appearance properties Amorphous yellow powder (2) Structural formula (3) Specific rotation [α] _D = +91.8 (CHCl ₃ , C = 1.0%) (4) Melting point 117℃ (decomposition) (5) Elemental analysis (as C ₃₆ H ₄₈ N ₂ O ₈ ) C% H% N% O% Theoretical value: 67.92 7.55 4.40 20.13 Actual value: 67.72 7.68 4.28 19.74 (6) Ultraviolet absorption λ _nax molecular extinction coefficient 262nm ε=38500 272nm ε=49600 282nm ε=38800 383nm ε=3 400 (7) Infrared absorption (see Figure 1) 3330cm ^-1 1410cm ^-1 1100cm ^-1 1730cm -1 1376cm ^-1 998cm ^-1 1650cm ^-1 1300cm ^-1 849cm ^-1 1535cm ^-1 1202cm ^{-1 720cm -1 (} 8) 13C− NMR (in CDCl ₃ )

[Formula] 188.2, 182.5

[Formula] 176.6, 169.4

[Formula] 172.9 C= 145.4, 137.9, 129.3 CH= 139.9, 133.7, 133.6, 133.2, 133.1, 131.3, 129.5, 122.5, 114.5 CH−O 79.2, 75.2, 68.0 −OCH ₃ 56.6

[Formula] 48.5

[Formula] 44.9

[Formula] 44.8 CH− 39.9 −CH ₂ − 33.0, 29.4, 29.4, 29.3, 25.6, 25.6, 25.5, 25.5 −CH ₃ 20.5, 17.4, 9.6 (9) PMR (in CDCl ₃ ) CH− CH ₃ 0.90 3H, d1.41 3H, d

[Formula] 1.80 3H, s -OCH ₃ 3.28 3H, s -OCH 4.02 1H, dt 4.75 1H, bs 4.96 1H, dd

[Formula] 4.36 1H, dq Aromatic H 6.50 1H, d 7.51 1H, d CONH 8.18 1H, s (10) Color reaction Ninhydrin (-) Billet (-) Anthrone (-) Fehring (-) (11) Dissolution in solvent Properties Easily soluble in methanol, ethanol, acetone, chloroform and ethyl acetate Slightly soluble in benzene and ether Insoluble in water, petroleum ether and hexane Physical and chemical properties of the T-23 substance (1) Appearance White amorphous powder (2) Structural formula (3) Specific rotation [α] _D = +288 (CH ₃ OH, C = 1.0
%) (4) Melting point 151℃ (decomposition) (5) Elemental analysis (as C ₃₆ H ₅₀ N ₂ O ₈ ) C% H% N% O% Theoretical value: 67.72 7.68 4.28 20.32 Actual value: 67.92 7.55 4.40 20.13 ( 6) Ultraviolet absorption λ _nax molecular extinction coefficient 260 ε=40800 270 ε=52300 280 ε=40500 310 ε=5900 (7) Infrared absorption (see Figure 2) 3340 (NH, OH), 1730, 1200 (ester ),
1650, 1535 (amide) (8) ¹³ C−NMR (in CDCl ₃ )

[Formula] 176.9, 173.3

[Formula] 179.7 C= 149.2, 141.1, 137.8, 132.7, 125.5 CH= 134.9, 134.4, 133.9, 129.6, 129.5, 129.1, 124.3, 115.8, 107.5 CH−O 79.6, 75.8, 68.7 −OCH ₃ 56 .6

[Formula] 48.7

[Formula] 45.1

[Formula] 43.1 CH− 39.0 −CH ₂ − 33.7, 31.7, 29.5, 29.4, 26.7, 26.6, 25.7, 25.6, −CH ₃ 20.3, 17.7, 9.7 (9) PMR (in pyridine) CH− CH ₃ 0.85 3H, d 1.57 3H, d

[Formula] 1.98 3H, s -OCH ₃ 3.27 3H, s -O-CH 4.49 1H, dt 5.28 1H, bs 5.36 1H, dd

[Formula] 4.80 1H, dq

[Formula] 5.53 1H, m 5.70 1H, dd 6.06 1H, m 6.23 1H, dd 6.37 1H, dd 6.53 1H, dd 6.64 1H, dd Aromatic H 7.12 2H, s CONH 8.78 1H, bs 9.01 1H, d (10) Color reaction Ninhydrin (-) Biuret (-) Anthrone (-) Fehring (-) (11) Solubility in solvents Easily soluble in chloroform, methanol, ethanol, acetone and ethyl acetate Slightly soluble in benzene, ether Water, petroleum ether and hexane The T-23-substance, which is a new substance of the present invention, exhibits the following biological activities and is particularly promising as an antitumor agent. T-23-Biological activity of substances (1) Antibacterial action against yeast and mold

[Table] All test bacterial strains were cultured at 30°C for 72 hours in a medium containing 1.0% glucose and 0.2% malt extract, and the presence or absence of bacterial growth was observed. (2) Growth inhibitory effect on L-5178Y tumor cells (in vitro)

[Table] L-5178y cells were grown in Eagle MEM medium (manufactured by Nitsusui) with horse serum 10.0% and asparagine.
The cells were cultured for 120 hours at 37°C in a culture solution supplemented at a rate of 100 mg/ml, and the presence or absence of cell growth was determined. The T-23-substance was dissolved in ethanol and added to the assay system. (3) Inhibitory effect on mouse P-388 leukemia tumor Table 3 Dose survival rate T/C (%) 24 mg/Kg/day x 2 doses 56.0 12 mg/Kg/day x 2 doses 123.3 6 mg/Kg/day × 2 doses 120.2 3 mg/Kg/day × 2 doses 105.7 1.5 mg/Kg/day × 2 doses 107.3 0.75 mg/Kg/day × 2 doses 105.7 0 mg/Kg/day × 2 doses 100 CDF ₁ mouse (20g±1g〓) 6 fish per group,
¹⁰⁶ P-388 ascites tumor cells were cultured using CDF ₁ mice and were inoculated intraperitoneally, and 24 hours later, the first
A second drug administration was given 120 hours later. The drug is suspended in gum arabic.
0.2 ml was administered. Life extension rate T/ in Table 3 above
C (%) was determined by the following formula. Number of days of survival of the administration group/number of days of survival of the control group x 100 Note that the control group is the case where only the gum arabic suspension was administered. (4) Acute toxicity LD ₅₀ (mouse, ip) 55.9 mg/Kg The Streptomyces liciliensis T-23 strain referred to herein is FERM BP-243. It has been deposited with the National Institute of Fine Technology, and its mycological properties are as follows. (a) Mycological morphology Vegetative hyphae extend long into the medium while branching without dividing or segmenting. Aerial hyphae elongate their main axis, branch out from spore-forming hyphae (spore stalks), and have loop-shaped, curved, or spiral-shaped (coiled diameter 15-20 μm, 2
~6 rotations) and settle on long chains of 10 to 50 or more spores. The surface structure of the spore is smooth (some rough surfaces can be seen depending on the medium), and the diameter is 0.5 to 0.7μ.
It has an oval shape with a length of 1 to 1.5 μm. Special forms such as sclerotia, flagellated spores, and sporangia are not observed. (b) Growth status on various media The following observation of growth status on various media:
According to "Methods Manual 1941" edited by International Streptomyces Projects.
The observation results are summarized in the table below.

[Table] (c) Physiological properties Growth temperature range 10-37℃ Optimum growth temperature 20-30℃ Liquefaction of gelatin + (positive) Hydrolysis of starch + coagulation of skim milk - (negative) Peptonization of skim milk + melanin Tyrosine agar + peptone yeast iron agar + tryptone yeast liquid medium + (d) Assimilation of carbon sources (Pridham-Gottlieb agar medium) L-arabinos (good growth) D-xylose D-glucose D-fructose + (weak growth) Sucrose Inositol L-Rhamnose Raffinose D-Mannite - (No growth) As a result of observation, strain T-23 is an actinomycete included in the genus Streptomyces and is characterized as follows.
In other words, (1) the spore chain morphology is spirally curved and loop-shaped, (2) the surface structure of the spore chain is smooth, (3) the color of the bacterial flora is gray, and (4) the color of the underside of the colony is indistinct. , (5) melanin-like pigment production is positive, and (6) does not assimilate D-mannite as a carbon source. These 6
Based on the characteristics, “Bergey’s Manual of
Determinative Bacteriology” 8th edition (1974)
and “J.Ferment.Technol.” Volume 52, No. 2
As a result of searching from pages 78 to 92 (1974), the characteristics of this strain are Streptomyces liciliensis (S
rishiriensis). Therefore, this strain was identified as a strain included in Streptomyces rishiriensis, and was named Streptomyces rishiriensis strain T-23. A normal actinomycete culture method is applied to culture this strain, and carbon sugars such as glucose, lactose, and fructose, starch, or starch hydrolysates can be used alone or in a mixed form as carbon sources. In particular, a mixture of starch and glucose is most suitable. Meat extract, polypeptone, soy flour, corn steep liquor and various inorganic nitrogen sources are used as nitrogen sources. In order to carry out fermentation production more appropriately, trace amounts of additives such as dry yeast, yeast extract, malt extract, various plant seed extracts, vitamins, and various inorganic salts may be added.
Antifoaming agents such as silicone and vegetable oil are added as necessary. The strain can be cultured using a medium obtained by distributing appropriate amounts of the nutrients shown above using a Kolben or Jar type fermenter or a larger fermentation tank. Culture temperature is 20-35℃, preferably 25℃
The temperature is ~30°C, and a deep culture method is used. Fermentation time may be 17 to 96 hours, and T-23 is usually heated around 24 hours.
Material production reaches its peak. T-23-substances and active substances containing T-23-substances are mainly contained in bacterial cells in the fermentation solids,
Some amount is also present in liquids. After culturing, the culture solution is immediately cooled and separated into bacterial cells and supernatant by centrifugation or filtration. 60~ than the bacterial body
Extract the active substance-containing fraction with 70% acetone water.
On the other hand, the supernatant liquid is passed through a nonionic exchange resin to adsorb some active substances contained therein, and then treated with an organic solvent such as acetone, lower alcohol, etc.
The active substances are eluted with aqueous solutions containing these organic solvents. Alternatively, without passing through a nonionic exchange resin,
It is also possible to directly extract the supernatant with the organic solvent. Next, the liquid obtained from the bacterial cells containing the active substance and the liquid obtained from the supernatant liquid are combined, and the pH is adjusted.
The organic solvent is distilled off under reduced pressure. From the aqueous solution containing the residual active substance, extract it as is, or in order to increase the extraction rate, use a water-immiscible solvent such as chloroform, ethyl acetate, or isopropyl alcohol in the presence of an industrially available inorganic salt such as common salt. Extract active substances. Glauber's salt is added to the obtained extract in a conventional manner, and after being allowed to stand for a while, water contained in the solvent is removed, and then concentrated under reduced pressure. Thereafter, petroleum ether, hexane, etc. are added to obtain a precipitate containing the active substance. In this,
Contains T-23-substance and T-23-substance,
This is further purified by chromatography using silica gel, nonionic exchange resin, Cefdex LH-20, etc. As an example, high purity T-23- using silica gel chromatography
, and/or T-23-substances are described below. Fill a column with silica gel using benzene and charge the T-23-substance and the precipitate containing the T-23-substance. Exclude the fraction eluted by passing benzene through the column first. Then, the active fraction is eluted with a mixed solvent of benzene/acetone (=4:1). The resulting active fraction is concentrated under reduced pressure and precipitated using a non-polar solvent such as petroleum ether, hexane, etc. The obtained precipitate is further purified by chromatography or recrystallized, if necessary, to obtain a highly pure T-23-substance.
Then, in the same column, benzene/acetone (=
7:3), the active fraction containing the T-23 substance is eluted. High purity T-23- can be obtained by the same post-treatment. For reference, production examples of the ansatrienin compound of the present invention are shown below. Production example 1 Soluble starch 1.0%, yeast extract 0.2% and agar
Take one platinum loopful from Streptomyces T-23 strain that has been subcultured in a test tube slant medium with a composition of 1.5% and add 1.0% soluble starch and 1.0% waste molasses.
%, meat extract 1.0% and polypeptone 1.0% (PH
Inoculate a Sakaguchi flask containing 100 ml of a seed medium with the composition of 7.0). Culture with shaking at 30°C for 48 hours, use the resulting culture as a seed, and use the same medium.
A 100 ml Sakaguchi flask was inoculated with 0.5 ml each. Shaking culture was carried out at 30°C for 24 hours, and the culture was used as the inoculum for main culture in a jar fermenter. The main culture contains 1.0% glucose, 1.5% soluble starch,
Soy flour 1.5%, dry yeast 0.2%, ammonium sulfate 0.2%,
The experiment was carried out using six 30-volume stainless steel jar-type fermenters containing a medium (PH 7.0) of 15.0 consisting of 0.5% NaCl, 0.4% precipitated calcium carbonate, and 0.33% antifoaming agent (Toshiba Silicon YMA6509). That is, the above-mentioned inoculum was inoculated at a rate of 4.0%, and 30
Aeration stirring culture (aeration rate 15.0/min, stirring rotation speed 200 rpm) was carried out at ℃ for 24 hours. Immediately after culturing, adjust the culture solution to pH5.8 with 4.0N sulfuric acid.
Adjusted to. After separating the bacterial cells using a large continuous centrifuge, the bacterial cells were immersed in 20% of a 60% acetone aqueous solution, stirred for a while, and then left for 3 hours. Then, the bacterial cells were filtered. The same treatment was repeated twice and the obtained filtrates were combined to make 40 extracts. Next, acetone was distilled off from this extract under reduced pressure to obtain aqueous solution 18. The resulting aqueous solution 18.0
6.5 kg of ordinary salt (Japan Monopoly Corporation's salt brand) was added and dissolved, and extracted twice with 9.0 ml of ethyl acetate. 1.0 kg of Glauber's salt was added to the obtained ethyl acetate solution, allowed to stand for a while, dehydrated and concentrated under reduced pressure, and then hexane was added to obtain a precipitate containing the active substance. Furthermore, after washing with hexane and drying, T-23
- material, T-23 - 42 g of a crude mixture of material was obtained. The resulting crude mixture was dissolved in 100 ml of chloroform and adsorbed on a silica gel column (packed with 400 g of silica gel), first passing benzene through it.
Then, T-23- was added with benzene/acetone (4:1).
The material was eluted and then the T-23- material was eluted with benzene/acetone (7:3). Each of the obtained fractions was concentrated under reduced pressure, and hexane was added to precipitate crystals to obtain 1.5 g of T-23-substance powder and 11.7 g of T-23-substance powder. Production Example 2 As shown in Production Example 1, Streptomyces T.
-23 strains were cultured, and 10.0% of the supernatant was collected from the resulting culture using a centrifuge using nonionic exchange resin HP.
-20 and after washing with water, the fraction containing the active substance was extracted with 50% acetone water 2.0.
Acetone was distilled off under reduced pressure to form an aqueous solution, which was extracted twice with ethyl acetate. The extract was dehydrated with Glauber's salt, and ethyl acetate was distilled off under reduced pressure to obtain 2.0 g of a dark brown oil.
I got it. The resulting oil was dissolved in 10 ml of chloroform and adsorbed onto a silica gel column, passing first benzene and then eluting the T-23-substance first with benzene/acetone (4:1), then The T-23- material was eluted with benzene/acetone (7:3). Each of the obtained fractions was concentrated under reduced pressure, hexane was added to each, and the precipitate was collected, washed with hexane, and dried under reduced pressure to obtain 48 mg of T-23-substance powder and 0.63 g of T-23-substance powder. Ta. Production example 3 1.25g of T-23-substance is pre-mixed with 0.1g of ferric chloride
was added to 100 ml of methanol in which it had been dissolved, stirred and dissolved under ice cooling, and allowed to stand for 15 minutes. Next, methanol was distilled off under reduced pressure, 100 ml of ethyl acetate was added to the resulting dry solid, the resulting solution was filtered through a glass filter, the liquid was collected, and ethyl acetate was distilled off again under reduced pressure. 1.05 g of yellow T-23 material powder was thus obtained. The pharmaceutical composition of the present invention can be administered orally or parenterally, usually in the form of a pharmaceutical preparation. Forms of pharmaceutical preparations include tablets, capsules, granules, powders, injections, suspensions, and the like. It can also be made into double-layer tablets or multi-layer tablets together with other drugs. Furthermore, the tablets can be made into tablets with conventional coatings, such as sugar-coated tablets, enteric-coated tablets, or film-coated tablets, if necessary. When preparing a solid preparation, additives such as lactose, sucrose, crystalline cellulose, corn starch, calcium phosphate, sorbitol, glycine, carboxymethylcellulose, gum arabic, polyvinylpyrrolidone, hydroxypropylcellulose, glycerin, polyethylene glycol, stearic acid, stearin are used. magnesium acid,
Talc etc. are used. When preparing a liquid preparation, additives such as sodium chloride, sorbitol, glycerin, olive oil, almond oil, propylene glycol, and ethyl alcohol are used. The amount of active ingredient in these preparations ranges from 0.01 to 50 of the preparation
% by weight, suitably from 0.1 to 20% by weight in the case of formulations for oral administration and from 0.05 to 10% by weight in the case of formulations for injection. The administration method and dosage of the antiulcer agent of the present invention are not particularly limited, and are appropriately selected depending on various formulation forms, administration routes, patient age, sex, degree of disease, etc. Dosage is 0.01
~100mg. Formulation Example 1 Injection T-23-Compound 1 mg Sodium chloride 10 mg Distilled water Appropriate amount Total volume 1.0 ml Dissolve sodium chloride and the active ingredient by adding distilled water to make a total volume of 1.0 ml. Formulation Example 2 Tablet (1 tablet) T-23-compound 5 mg Lactose 72 mg Crystalline cellulose 15 mg Corn starch 7 mg Magnesium stearate 1 mg 100 mg Each component is mixed uniformly to form a powder for direct injection. This is molded into tablets with a diameter of 6 mm and a weight of 100 mg using a rotary tablet press. Formulation Example 3 Granules (1 sachet) T-23-Compound Lactose Corn Starch Crystalline Cellulose 5mg 85mg 50mg 50mgA Hydroxypropyl Cellulose Ethanol 10mg 90mgB After uniformly mixing the components of A, add the solution of B and knead. The particles are sized using an extrusion granulation method, and then dried in a dryer at 50°C. Particle size of dried granules is 297μm
The granules are sieved to ~1460 μm.
The amount of one sachet is 200mg.

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are infrared absorption spectra of T-23-substance and T-23-substance, respectively.

Claims (1)

[Scope of Claims] 1. An antitumor agent containing an ansatrienin compound having the following structural formula as an active ingredient.