JPS6027390A - Fr-900216 substance and its preparation - Google Patents

Abstract

NEW MATERIAL:FR-900216 Substance having the following physical and chemical properties and its salt. Molecular weight: 625(mass spectrometry). Estimated molecular formula: C35H47NO9. Melting point: 130-135 deg.C(decomposition). Specific rotatory power: [alpha]D<20>=+140 deg.(C=1, in CHCl3). Solubility: easily soluble in methanol, acetone, ethyl acetate, soluble in chloroform and benzene, slightly soluble in hexane, ethyl ether, insoluble in water. Color reaction: positive in Dragendorff's reaction and iodine reaction, and negative in ferric chloride reaction, Molish reaction, etc. USE:An antitumor agent. PREPARATION:A fungus [e.g., Rhizopus sp. NO.F-1360(FERM-P 5362), etc.] belonging to the genus Rhizopus, capable of producing FR-900216 substance, is cultivated in a medium and the substance is collected and separated from the culture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel substance FR-900216 having excellent antitumor effects and a method for producing the same.

The physicochemical properties of the FR-900216 substance of this invention are as follows.

■ Molecular weight: 62 s (mass spectrum 7 method) ■
Estimated molecular formula: CHNO 5479 ■ Melting point: 130-135℃ (decomposition) ■ Specific optical rotation °
[α) −+140° (C=1° in chloroform) ■ Ultraviolet absorption spectrum: λCH30H= 232 (E=11.3()O), 23
8 (Sh,).

ax 285 (Sh, ), 296 (ε=55,500)
, 308 (ε=70.300), 324 (ε=51
,900)nm Infrared absorption spectrum: UKBr:3450.3140,2960,2920,
2860°ax 17.30.1715 (Sh,), 1607, 1575
,1455°1445.1435,137711360
,1343,1305°1280.1260,1225
,1190,1170,1140°1107.1077
．． 1045.1030 (Sh, ), 9'80°970.
950,930,915,905,895.87!5゜860.850,835,825,790,780,7
55°705.670.63 nights ■ Nuclear magnetic resonance spectrum of hydrogen: As shown in the figure (solvent: deuterated chloroform Internal standard: tetramethylsilane) ■ Nuclear magnetic resonance spectrum of carbon-13: (in deuterated chloroform) Internal standard: tetra Methylsilane δ (ppm): 9.65, 11.41, 11.71, 1
3.77.14.32゜16.93, 22.57,
29.24, 31.49, 31.85, 34.1
6.

35.86, 36.40, 37.98, 45.3
2, 54.18, 56.12.

63.77.65.10, 76.81, 82.33, 8
9.25, 120.80°123.77, 126.4
4, 129.29, 135.97, 136.21
.

136.57 , 137.67 , 138.51 , 1
39.54, 160.84.

168.01.169.34 ■ Solubility in solvents: Easily soluble: methanol, ethanol, acetone, ethyl acetate Soluble: chloroform, benzene 111 (i: hexane, ethyl ether Insoluble: water ■ Color reaction: Dragendorff reaction, chloride Ferric reaction - Molisch reaction - Ninhydrif reaction - Ehrlich reaction - Iodo reaction FR like production bacteria
It can be produced by culturing -900216 substance-producing bacteria in a medium, and separating and collecting the FR-900216 substance from the resulting culture. FR used in this invention
-900216 substance-producing strains (A, F-13) newly isolated by the inventors from the soil of Uji City, Kyoto Prefecture.
No. 60) has the following mycological properties.

■ Morphological Properties a) Macroscopic Observation 1) Growth on malt extract agar medium is extremely vigorous and fast, and after 3-5 days of culture at 30°C, huge colonies with a diameter of 80 mm or more are formed, covering the vetri dish. I will do my best. The colony surface is flat and thick, and is white at the initial stage of cultivation, but gradually becomes gray or black as spores and sporangia are formed and mature. Spore paste, spores are formed in large numbers. The area around the village is spider web-like and clings to the walls of beet dishes and test tubes and climbs up. The underside is colorless or pale yellow. No diffusible pigments or exudates are produced. Gives off a fragrance. Growth is somewhat slow at 25°C.

ii) Y, on ss agar medium It is roughly the same as the malt extract agar medium.

l11) Potato glucose agar medium is generally similar to malt extract agar medium.

iv) On Czapek agar medium It rarely grows.

b) Microscopic observation The spores are enclosed in a thin membrane, and when they mature, the bag ruptures and the many spores inside come out. Sporangium diameter 30-20
0μ. Spherical. The spores are 7.5 x 15μ''. Oval in shape with a smooth surface and a few wrinkles. The basal hyphae are rhizoids (rhigoi).
d), the conidiophores form stolons (5 tolo
n). Partition wall (5epta) 6″l:
I don't have it.

A columnar axis (columella) is formed at the tip of the spore stalk.

■ Physiological properties a) Growth temperature range 40-48℃ (on potato glucose agar medium) Optimum temperature for spore attachment 14-39℃ (same medium): 33
°C (same medium) b) Pfeffer's solution (pfeffer 5 solution)
n) and does not grow at 10°C. Grows at 25-42°C.

C) Growth pH range pT (2,5-12,0 (on wort and ys agar) s d) Produce organic acids.

Judging comprehensively from the above experimental results, O F-1360
The strain is considered to be a species of the zygomycete Rh1zopus genus. Therefore, this black F-1360 strain was used as Rhizopus sp.
/Ffi, F-1360).

This Rhizopus sp./Ffi, F-1360, has been deposited with the American Type Culture Collection under deposit number ATCCA, 20577.

Also, this Rhizopus sp. A, F-13601d
.

Microbiological Research Institute No. 536 at the Institute of Microbial Technology, Agency of Industrial Science and Technology
It has been deposited as No. 2.

The FR-900216 substance-producing bacteria used in this invention, for example, the FR-900216 substance-producing bacteria belonging to the genus Rhizopus, can be treated with irradiation such as X-rays and ultraviolet rays, such as nitrogen, mustard, azaserine, nitrous acid, 2- FR- The production capacity of 900216 substance can be increased.

FR-90021 is carried out by culturing in a medium a producing bacterium of the FR-900216 substance belonging to the genus Rhizopus.
The production of the six substances basically follows the cultivation method of general microorganisms, but the deep culture method using a liquid medium is usually advantageous.

The medium used for the culture medium is F, which belongs to the genus Rhizopus.
Fortune-telling if the medium contains a nutrient source used by R-900216 substance-producing bacteria. That is, a synthetic medium, a semi-synthetic medium, or a natural medium is used, and the medium composition includes carbon sources such as evaglucose, sucrose, maltose,
Glycerin, starch, liquefied starch, etc. are used, and as nitrogen sources, for example, meat extract, casein hydrolyzate, peptone, gluten meal, corn meal, #ill grain meal, soybean flour, corn steep liquor, dried yeast, yeast extract,
Urea, ammonium phosphate, etc. are used. other than this,
For example, inorganic salts such as disodium hydrogen phosphate, potassium dihydrogen phosphate, magnesium chloride, magnesium sulfate, and calcium carbonate are also added to the medium as necessary.

1. If there is significant foaming during culture, add antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol, and heptano-1, and silicon compounds as appropriate. good.

A suitable culture temperature is around 30°C, and good results are often obtained by carrying out seed culture as appropriate as the culture volume increases. The culture time for the main culture is suitably about 50 to 100 hours, and the culture time may be further extended as the medium becomes more concentrated.

The culture conditions described above are applied by selecting the optimum conditions for each depending on the characteristics of the production strain used.

Next, since the FR-900216 substance produced by culture usually accumulates both inside and outside of the bacterial cells in the culture, it is generally necessary to remove the bacterial cells and the filter by means such as centrifugation or filtration. After separating into a liquid (supernatant), both are separated, purified, and collected by means used in the production of general antibiotics. That is, the target substance within the bacterial cells is usually separated and extracted from the bacterial cells by extraction means such as solvent extraction.

This extract and the above-mentioned filtration 2 (supernatant liquid) are subjected to vacuum concentration, solvent extraction, liquid conversion, treatment with resins such as anion exchange resins, cation exchange resins, and nonionic adsorption resins, such as activated carbon, silica, etc. Separate and purify the target substance, FR-900216 substance, by combining or repeatedly applying methods such as treatment with adsorbents such as acid, silica gel, alumina, and cellulose, crystallization, and recrystallization in any order. can do.

However, the FR-900216 substance thus obtained can be treated in a conventional manner to obtain its pharmaceutically acceptable salt.

Pharmaceutically acceptable salts of such FR-900216 substances include, for example, acid addition salts with inorganic or organic acids (e.g., acetates, lactates, maleates, fumarates, oxalates, citrates). , methanesulfonate, hydrochloride, sulfate, nitrate, phosphate, etc.).

Next, the biological properties of the FR-900216 substance of this invention will be shown.

Test Example 1 Antitumor effect test method for murine leukemia L1210 and P2S5.
The tumor cells were subcultured into B A/2 mice, and ascitic fluid was collected from the peritoneal cavity on the 7th day after transplantation, the tumor cells were separated, and the number of cells was adjusted to prepare a cell suspension. 0.2m of the floating liquid
/ (105 cells for L1210, 106 cells for P2S5) in BDF1 mice (1 body weight for L1210 experiments)
It was transplanted intraperitoneally into a 7-week-old female weighing 7.9-21.79 mm (in the P2S5 experiment, an 8-week-old female weighing 18.4-22.59 mm). The FR-900216 substance was dissolved in methanol and then concentrated, and the concentrate was dissolved in water to obtain an aqueous solution, and a predetermined amount was intraperitoneally administered once a day for 4 days to mice implanted with the above tumor cells. The antitumor activity is expressed as a percentage of the average survival days (1) of each group to the average survival days (C) of the control group (T/CX1
00).

result It is shown in Table 1.

(Margins below) Test Example 2 Test method for antitumor effect on mouse melanoma B16 Mouse melanoma B16 was subcutaneously transplanted into mice, and 14 days after transplantation, the tumor was aseptically collected subcutaneously from the mouth.
1 g of the mixture was mixed with Hank's solution 10 resistant and homogenized. A predetermined amount of the aqueous solution of the substance was administered intraperitoneally once a day for 4 days to 0.5*/f BD F 1 mice (12-week-old females weighing 19.7-24.89 kg). Antitumor activity was calculated in the same manner as in Test Example 1.

result It is shown in Table 2.

(Left below) As described above, the FR-900216 substance of the present invention and its pharmaceutically acceptable salts have antitumor effects and are useful as medicines.

When administering the object compound of this invention, FR-900216 substance or a pharmaceutically acceptable salt thereof, for the purpose of tumor treatment, the FR-900216 substance is used as an active ingredient, and a pharmaceutically acceptable carrier, For example, oral or parenteral organic medicines can be used in the form of preparations with inorganic, solid or liquid excipients. Such preparations include solid preparations such as capsules, tablets, and granules, liquid preparations such as solutions, suspensions, and emulsions, as well as suppositories. Furthermore, if necessary, adjuvants, stabilizers, wetting agents, emulsifiers, buffers, and other frequently used additives can be included in the preparation.

The dosage of the FR-900216 substance or its pharmaceutically acceptable salt varies depending on the patient's age, condition, type of disease, and medical condition, but the average dose of the compound of this invention per dose is about 1001f, 200dah, 400dah. or 1,600
If administered in an amount of #, it is effective in treating tumors. Generally, the FR-900216 substance, or a pharmaceutically acceptable salt thereof, can be administered in an amount of about 200 to 4,000 OOW per day, and in some cases more.

Next, the present invention will be explained based on examples.

Example 1 A medium (pH 7.0 before sterilization) with the composition of 1% corn starch, 0.5% glucose, 1% corn starch meal, 1% dried yeast, and 1% corn starch was grown at 275 m/volume n.
Dispense 80ml each into 6 Erlenmeyer flasks and sterilize by boiling at 120°C for 30 minutes. These include Rhizopus varnish K-S, F-
1 loop of each slant culture of 1360 was inoculated with L;
Rotary shaker at 250 revolutions per minute and 48°C at 30℃
Perform time culture. 2% soluble starch, 0 glycerin
．． 5%, glucose 0.5%, gluten meal 1%, dry yeast 1%, corn starch liquor 1% (
pH7,0) 201! 301! Pour into a jar fermenter and sterilize at 120°C for 30 minutes. Then,
The entire amount of the above culture was inoculated into this, and the aeration amount was IVVM and the stirring speed was 20 per minute.This was used as a seed culture.

Furthermore, a medium (pH 7, 0) with a composition of 2% soluble starch, 0.5% glycerin, 1% dry yeast, 1% cottonseed meal, 0.5% soybean flour, and 1% calcium carbonate 1501f2 0
01! Pour into a jar fermenter and heat at 120℃ for 3 hours.
Sterilize for 0 minutes. Add to this the above seed culture 81! inoculate with
Airflow amount 0. 7 VVM, stirring speed 250 revolutions per minute,
Culture at a culture temperature of 3 ot for 72 hours. After culturing, 5 kg of diatomaceous earth is added to the culture and filtered. Add ethyl acetate to the cake containing the obtained bacterial cells and 801! was added and stirred, and then the ethyl acetate layer was separated. This procedure for extracting the active ingredients from the bacterial cells is repeated again, and the obtained ethyl acetate extract is used as II! Concentrate under reduced pressure in Step 1. Concentrate with 0.5% sodium bicarbonate water
Wash with 00 resistance and separate the ethyl acetate layer. This ethyl acetate mt was dehydrated with anhydrous sodium sulfate to 300 ml.
Concentrate with reeds. This concentrate was subjected to total silica gel chromatography (silica gel: 2500 II/), and after washing the column with chloroform, it was eluted with a mixture of chloroform and methanol (100:1).

Chloroform methanol elution fraction (1 (R
) was concentrated under reduced pressure to about 20 g/hexane.
54' is added, and the resulting precipitate is collected by filtration and dried to obtain 11 pieces of Japanese powder containing the target product. 10 pieces of this coarse powder
The solution was dissolved in 0 g+/ of methanol-7 and subjected to reverse phase chromatography (NS gel: 1500 mm) using NS Gel V (manufactured by Nippon Seikagaku Co., Ltd.), and finely fractionated by elution with methanol. The eluted fraction (800y/) containing the target product was concentrated under reduced pressure to about 10 y/ml and added to hexane Il! and collect the resulting precipitate by filtration to obtain 630 qi of powder.

This powder was subjected to silica gel thin layer chromatography.
Develop with a mixture of chloroform and methanol (20:1), scrape off both parts containing the target substance, and elute with a mixture of chloroform and methanol (10:1). Eluate 8 (1+
Concentrate t under reduced pressure to about 50% hexane.
The precipitate obtained by adding 0 ml was collected by filtration and FR-
300 gjl of white powder of substance 900216 is obtained.

Elemental analysis value (to): C66,30, H7.61,
N2.18 Example 2 Corn starch 1%, glucose 0.5%, gluten meal 1%. Dry fermentation fB t%, cornstarch power
Medium composition of 1% (pH 7.0 before sterilization) i2 7 5
Dispense each bowl into 6 1 ton/Rollenmeyer flasks and sterilize at 120°C for 30 minutes. One platinum loop of Rhizopus sp. AF-1 360 slant culture was inoculated into each of these, and cultured at 30° C. for 48 hours at 250 revolutions per minute in a rotary shaker.

Separately 2% soluble starch, 1% glycerin, 0 glucose
．． 5%, gluten meal 1%, dry fermentation FRt%, corn steeple strength -1% (pH 7.0) 2 (
ll! 301! Inject into Schafer Mentor, 1
Cultivate for 24 hours at 20°C, 200 rotations, and culture temperature of 30°C, and use this as a seed culture.

In addition, 2% soluble starch, 1% glycerin, and 1% dry yeast.
%, cottonseed meal 1%, soybean flour 0.5%, calcium carbonate 1%
A medium with the composition (pH 7.o) xso. /'i2 0 0
1! Pour into a jar fermenter and heat at 120℃ for 30 minutes.
Sterilize for minutes. Add to this the above seed culture 81! was inoculated and cultured for 72 hours at an aeration rate of 0.7 VVM, a stirring speed of 250 revolutions per minute, and a culture temperature of 30''C.

After culturing, ±5 kg of diatoms is added to the culture and filtered. Go7'l of ethyl acetate is added to the resulting cake containing bacterial cells, stirred, and then filtered. This operation was repeated again, and the obtained ethyl acetate extract was added to 1! ! Concentrate under reduced pressure in Step 1. The concentrate was washed with 500w1 of 0.5% sodium bicarbonate solution, and the ethyl acetate layer was separated. This ethyl acetate layer is dehydrated with anhydrous sulfuric acid or thorium and concentrated to a size of 300 x tt. This concentrate was subjected to silica gel column chromatography (silica gel:
After washing the column with chloroform, the column was eluted with a mixture of chloroporum and methanol (100:1). Concentrate the chloroform-methanol elution fraction (10., J) containing the target product under reduced pressure, concentrate at 1 to about 5°C, add 2Jk of hexane, collect the resulting precipitate by filtration, and dry it to contain the target product. 2.59f of coarse powder is obtained. This coarse powder was collected using a high-performance liquid chromatograph 6 manufactured by Nippon Waters Limited, and the column used was Prepack 50.
The active fraction is obtained using a solvent system of chloroform-methanol mixture (100:1.5). The obtained active fraction was concentrated under reduced pressure and added with 100+ liters of hexane.
The resulting precipitate is collected by filtration and dried to obtain powder 1.29. This powder was again subjected to the same treatment using a high-performance liquid chromatograph for mass preparative use under the same conditions to obtain 500 Mg of white powder of the FR-900216 substance.

[Brief explanation of drawings]

The drawing shows the hydrogen nuclear magnetic resonance absorption spectrum of the FR-900216 material obtained by the present invention. Applicant Fujisawa Pharmaceutical Co., Ltd.

Claims (1)

[Claims] (FR-900216 substance having the 11th order immediate chemical properties and its pharmaceutically acceptable salt. ■ Molecular weight: 625 (mass spectrometry) ■ Estimated molecular formula; C35H47NO9 ■ Sub 1 point 130-135 °C (decomposition) ■ Specific optical rotation: (a': J, o = + 140' (C-1. in chloroform) ■ Ultraviolet absorption spectrum: λCH30H-232 (ε = 11,300), 23
8 (Sh,). ax 285 (Sh, ), 296 (ε=55,500),
308 (ε=70.300), 324 (ε=51,90
0) nm ■ Infrared absorption spectrum: Br υmax + 3450.3140, 2960, 292
0,2860゜boiled R乎, 14jb, 1j77.1360
,1343,1305°1280.1260. -122
5.1190,1170,1140°11071107
7.1045,1030 (Sh,), 980°970.
950,930,915,905,895,875゜8
60.850,835,825,790,780,75
5゜1 7 (15,670,6350I! ■ Nuclear magnetic resonance spectrum of hydrogen; as shown in the figure (solvent: hydrogen, internal! quasi: tetramethylsilane) ■ Nuclear magnetic resonance spectrum of the next element 13 l (Solvent weight: 7 rem, internal standard: tetramethylsilane) δ (ppm) = 9.65, 11.41, 11.71, 1
3.77.14.32゜16.93, 22.57,
29.24, 31.49, 31.85, 34.1
(i. 35.86, 36.40, 37.9B, 45.32, 5
4.18, 56.12゜63.77, 65.10,
76.81, 82.33, 89.25. 120.80, 123.77, 126.44, 1
29:29, 135.97. 136.21 , 136.57 , 137.67 , 1
38.51, 139.54. 160.84, 168.01, 169.34 ■ Solubility in solvents Easy soluble: methanol, ethanol, acetone, ethyl acetate Soluble: chloroform, benzene Slightly soluble: hexane, ethyl ether Insoluble: water ■ Color reaction: Dragend! Lef reaction Ferric reaction - Molisch reaction - Ninhydrin reaction - Ko-Risohi reaction - Iodine reaction 10 ■ Distinction between basic, acidic and neutral: Weak basic (2) FR-900216 belonging to the genus Rhizopus FR-900 is obtained from the culture obtained by culturing the substance-producing bacteria in a medium.
FR-9 is characterized by separating and collecting 216 substances.
00216 Method for producing substance.