JPH02218676A - Estrogen substance be-14348b - Google Patents

Abstract

NEW MATERIAL:The BE-14348B expressed by formula. USE:A drug. An active component of estrogen preparation. It has strong antagonistic action against the bonding of estradiol to a receptor and exhibits against activity, however, it has no antagonistic action against the bonding of hormones such as progesterone to a receptor and is useful for the remedy of gynecologic diseases, osteoporosis, prostatic cancer, prostatomegaly, etc., caused by the deficiency of estrogen. PREPARATION:A microbial strain belonging to genus Streptomyces and capable of producing BE-14348B such as Streptomyces sp. BA-14348B (FERM P-10491) or its mutant is made to contact with a medium containing nutrient source for actinomycetes, aerobically cultured at 12-37 deg.C and a medium pH of 4-8 for 48-144hr to produce and accumulate BE-14348B in the cultured material, the cells are separated from filtrate by filtration, etc., and the objective compound of formula is separated and purified by high-performance liquid chromatography.

Description

[Detailed description of the invention] 1! The present invention is directed to the use of BE-1, a novel substance that exerts estrogenic effects by specifically binding to estrogen receptors.
4348B, its production process and its use as an estrogenic agent. Furthermore, the present invention relates to a microorganism belonging to the genus Streptomyces that produces BE-14348B, a substance exhibiting estrogenic effects.

Estrogen is a type of sex hormone that is also known as estrous hormone, female hormone-like substance, or follicular hormone, and is a general term for hormones that exhibit estrous effects. Known steroidal estrogens include, for example, estradiol, estrone, estriol, echilin, homoestrone, and ethinylestradiol.

For example, diethylstilbestrol phosphate and hexestrol are known as steroidal estrogen agents.

These estrogen drugs are used to treat gynecological diseases such as amenorrhea, uterine hypoplasia, 1-year ovarian deficiency syndrome, and vaginitis caused by a decrease in estrogen, as well as diseases such as prostate cancer and benign prostatic hyperplasia. Osamu#! Used as a medicine.

The problem that we are trying to solve Because the estrogen drugs already in use have strong physiological effects, the use of estrogen drugs in clinical settings requires extreme caution to prevent side effects. Side effects include hypercalcemia, sodium and fluid retention when steroidal estrogens are administered intramuscularly or subcutaneously.

Uterine withdrawal bleeding and hypersensitivity are known, and when steroid estrogens are administered orally, in addition to side effects when administered intramuscularly or subcutaneously, they may also cause nausea, vomiting, loss of appetite, diarrhea, and abdominal pain. Presents with gastrointestinal disorder. Furthermore, serious side effects such as blood clots, myocardial infarction, heart failure, electrocardiogram abnormalities, and cerebrovascular disorders have been reported with large doses of non-steroidal estrogens.

Therefore, although conventional estrogen agents have excellent medicinal efficacy, they also suffer from many side effects, and it is desired to reduce or eliminate these side effects.

Means for Accomplishing the Problem The present inventors searched for various substances in microorganism cultures that antagonize estradiol in its binding to estrogen receptors, and determined the estrogenic activity (agonist activity) of the obtained estrogen antagonists. ) was further investigated. As a result, we found that a microorganism belonging to the genus Streptomyces isolated from the soil of Tanna City, Hyogo Prefecture, produced a substance with this activity.We isolated and purified one substance, determined its structure, and found that it was a known substance. The present invention was completed based on the discovery that the compound is a novel compound different from that of the present invention.

(The following is a blank space) That is, the present invention relates to BE-14348B represented by the formula, its production method, and its use as an estrogen agent.

In addition, the present invention is directed to the use of BE-14, a substance exhibiting estrogenic action.
The present invention relates to a microorganism belonging to the genus Streptomyces that produces 348B.

The microorganism used in the production of BE-14348B of the present invention may be any microorganism as long as it produces BE-14348B; for example, BA-143 having the following mycological properties:
I can list 48 stocks.

1. Morphology When observed under a microscope, strain BA-14348 grows aerial hyphae, which are relatively shorter than the substratum hyphae. Many of the tips of these hyphae are loop-shaped, sometimes wavy or straight, and contain more than 10 spores. A chain of six rings is observed, and no fragmentation of hyphae is observed.Spore size is 0.8-1.2 x 0.8-1.6
It has a cylindrical shape on the order of μm in size, and its surface is smooth or rough, and special organs such as sporangia, flagellated spores, and sclerotia are not observed.

2. Culture properties on various agar media (28°C, 14 days culture) (1) Growth on yeast/malt agar medium (ISP-2 medium) was very good, with substratum hyphae being light brown.

Forms fine powdery white aerial mycelium. Production of yellow soluble pigment is observed.

(2) Oatmeal agar medium (ISP-3 medium) Growth is good, and the substratum hyphae are pale yellow-orange. Formation of aerial hyphae was minimal, and no production of soluble pigment was observed.

(3) Starch/inorganic salt agar medium (ISP-4 medium) Growth is good, substratum hyphae are bright reddish-yellow, and powdery gray-white aerial hyphae are well formed. No production of soluble pigment is observed.

(4) Glycerin-asparagine agar medium (ISP-5
Medium) Growth is very good, and the substratum hyphae are pale yellowish-orange. Formation of aerial hyphae was minimal, and no production of soluble pigment was observed.

(5) Peptone yeast iron agar medium (I 5P-6
Medium) Growth is good, and the subsoil hyphae are pale yellow-orange. No aerial mycelia were formed and no production of soluble pigment was observed.

(6) Tyrosine agar medium (ISP-7 medium) Growth is good, substratum hyphae are brown, and powdery light gray to dark yellow aerial hyphae are well formed.

No production of soluble pigment is observed.

(7) Growth on nutrient agar medium is good, substratum hyphae are pale yellow-orange, and formation of powdery, light gray aerial hyphae is poor.

No production of soluble pigment is observed.

(8) Growth on sucrose/nitrate agar medium is very good, and the substratum hyphae are pale yellowish orange. Formation of aerial hyphae was minimal, and no production of soluble pigment was observed.

(9) Glucose-asparagine agar medium growth is good;
Substrate hyphae are colorless. Formation of aerial hyphae is minimal;
No production of soluble pigment is observed.

3. Physiological properties (1) Liquefaction of gelatin Liquefaction of gelatin is observed in glucose-peptone-gelatin medium (cultured at 28°C).

(2) Hydrolysis of starch Hydrolysis of starch is observed on starch/inorganic salt agar medium (cultured at 28°C).

[3) Coagulation of skim milk powder No coagulation of skim milk was observed in skim milk medium (cultured at 28°C).

(4) Peptonization of skim milk powder Peptonization of skim milk is observed in skim milk medium (cultured at 28°C).

(5) Generation of melanin-like pigment No melanin-like pigment formation was observed.

(6) Utilization of carbon sources (cultured on Bridham-Gotlieb agar medium at 28°C for 14 days) D-glucose, D-xylose, L-arabinose.

Utilizes L-rhamnose, D-fructose, D-galactose, raffinose, D-mannitol, inositol and sucrose. The use of salicin is questionable.

(7) Growth temperature using Bennett agar medium: 12°C, 20°C, 28°C, 3
As a result of culturing at each temperature of 7℃ and 45℃, it grew at all temperatures except 45℃, but the optimal temperature was 20 to 28℃.
It seems to be nearby.

(8) As a result of culturing (28° C.) using a salt-tolerant yeast/malt agar medium to which salt was added, growth occurred with a salt content of 2% or less.

4. Chemical properties of cells LL-diaminopimelic acid and glycine were detected from the cell wall hydrolyzate.

The above mycological properties revealed that strain BA-14348 belonged to the genus Streptomyces.

Therefore, the present inventors used the BA-14348 strain as Streptomyces SB BA-14348 (Strept.
onyces sp.

BA-14348).

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its FERN accession number is FERN P-10491.

The mutant strain of the microorganism that produces BE-14348B used in the present invention can be treated, for example, by irradiation treatment with X-rays or ultraviolet rays, etc.
For example, nitrogen mustard, azaserine, nitrous acid, 2-aminopurine or N-methyl-No-nitro-N-nitrosoguanidine (NTG'). Treatment with 4 inducers, phage contact, and transformation.

This is a microorganism obtained by mutating BE-14348B-producing bacteria using commonly used bacterial species conversion treatment methods such as transduction or conjugation.

In manufacturing BE-14348B of the present invention, BI
By inoculating the E-14348B production strain BA-14348 strain into a nutrient-containing medium and growing it aerobically,
A culture containing BE-14348B is obtained. As the nutrient source, those known as nutrient sources for actinomycetes can be used.

For example, as the carbon source, commercially available glucose, glycerin, maltose, starch, sucrose, molasses, dextrin, etc. may be used alone or in a mixture. As the nitrogen source, commercially available soybean flour, corn staple liquor, corn gluten meal, meat extract, yeast extract, cottonseed flour, peptone, wheat germ, fish meal, inorganic ammonium salt, and the like can be used alone or as a mixture. As the inorganic salt, commercially available calcium chloride, sodium chloride, potassium chloride, magnesium sulfate, various phosphates, etc. can be used. Other iron, as required.
Trace amounts of heavy metal salts such as manganese, zinc, copper, and cobalt can also be added. In addition, when foaming is significant, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, and various silicon compounds may be added as appropriate. Any material that is available and useful for the production of BE-14348B can be used. The culturing method may be the same as a general method for producing microbial metabolites, and solid culture or liquid culture may be used.

The platform for liquid culture may be used for static culture, agitation culture, shaking culture, aeration culture, etc., but shaking culture or deep aeration agitation culture is particularly preferred.

The appropriate culture temperature is between 12°C and 37°C, but between 20°C and 20°C.
The temperature is preferably 8°C, and the culture time is 48 hours to 144 hours, preferably 72 hours to 1 hour, when the pH of the medium is in the range of 4 to 8.
It is 20 hours.

In order to collect the target BE-14348B from the culture, separation means that are normally used for collecting metabolites produced by microorganisms from the culture may be appropriately used.

Since BE-14348B of the present invention is contained in the bacterial cells and the culture filtrate, after separating the culture liquid into the bacterial cells and the culture filtrate by centrifugation or filtration, the bacterial cells and the culture filtrate are separated by ordinary separation means. For example, separation and purification are carried out by appropriately combining solvent extraction, adsorption resin, adsorption or distribution column chromatography, high performance liquid chromatography, and the like.

Examples of preferred separation and purification include the following method. The culture solution is separated into bacterial cells and culture filtrate by centrifugation or filtration. The active ingredient is extracted from the bacterial cells using methanol, acetone, etc.

The culture filtrate is treated with a suitable adsorbent resin 1 such as Diaion H.
After passing through a P20G (manufactured by Mitsubishi Chemical Industries, Ltd.) column to adsorb the active ingredient, it is eluted using water-containing methanol, water-containing acetone, or the like. The eluate and the bacterial cell extract are combined, the solvent is removed under reduced pressure, and then extracted using an organic solvent that is hardly miscible with water, such as ethyl acetate or methyl ethyl ketone. After concentrating the extract to dryness, it is purified using silica gel column chromatography using chloroform-ethyl acetate as an eluent and reverse-phase high performance liquid chromatography using aqueous acetonitrile as a mobile phase.

The physicochemical properties of BE-143488 obtained in the Examples described below are as follows.

(1) Color of substance Pale yellow solid (2) Molecular formula % Formula % (3) Elemental analysis value Actual value C: 67.19%, H: 4.91% Theoretical value C:
67.13%, H: 4.93% (4) Molecular weight FAB-MS method: II/Z 287 (N+H)"HR
FAB-MS method: i/z 287.0948 (N+H
)"(5) Optical rotation [α]-"x-115,7' (C=0.95.Me
OH) (6) Ultraviolet absorption spectrum (Figure 1) 0, IN
Measurement in ICI-MeOH (shown by solid line in Figure 1) λwax 227Hm (E' *936) 1cm 288Hm (E'' ・690) aa 330Hm (shoulder E'' 5132)a
1 Measurement in 0.1N Na0H-NeOfl (indicated by the broken line in Figure 1) λmat 245Hm (E ” *725) 1 mouth 323□Iris (El・1105) IC!II (7) Infrared absorption spectrum ( KBr tablet method) See Figure 2 (8)' H-NNR spectrum (acetone-d,
300MIIz) 3rd r! ! J reference (9)” C-NMR spectrum (acetone-d, 7
5MHz) See Figure 4 (10) Solubility in solvents Soluble in methanol, ethanol, acetone, ethyl acetate, insoluble in water and n-hexane (11) Color reaction Potassium permanganate reaction...・Positive (1
2) Distinguish between basic, acidic, and neutral neutral substances (13) Rf value (manufactured by Merck & Co., Ltd., Kieselgel 60F,
, , ) (J! Open solvent) (Rf value)
CBCI, :Ac0Et(4:1) 0.
23CHCI, :MeOH (10:1)
0.41 (14) High performance liquid chromatography carrier: tnertsil 005 (manufactured by Gascro Industries), inner diameter 4.6 am, length 250 mm Mobile phase: C1'l, CN:H, O: Ac0H (450
:550:2) Detection method: [IV(2a8nffi) Retention time (Rt): 12.7m1nBE-1434
8B was clearly distinguished from known substances based on the above-mentioned properties, and was confirmed to be a new substance.

Next, the biological activity of BE-14348B of the present invention will be described.

[Estradiol antagonistic activity] Pig uterus was ground and 38. The estrogen receptor (estrogen binding protein) in the cytoplasmic soluble fraction obtained by ultracentrifugation at OOOrpm for 1 hour was used for testing. T
ED buffer (10 cod Tris-hydrochloric acid buffer pH 7,4°1
A reaction solution containing 504.10 nM (I) estradiol (30,000 cps) containing 204 mM ethylenecinmintetraacetic acid, 11 M dithiothreitol, 25 components of the above cell-soluble fraction, and 5 parts of the sample solution was incubated at 4°C for 2 hours. After keeping warm, it is treated with dextran-coated activated carbon to remove free [”
'I) Removal of estradiol and binding to estrogen receptors (''I) Removal of estradiol and measurement of the amount of estradiol bound to estrogen receptors (''I) The estrogen activity in the sample was determined.

As a result, BE-143488 was found to be at a concentration (IC,
) was 9.4Hg/m (32.9n?).In contrast, the IC of tamoxifen, an anti-estrogen agent frequently used in hormone therapy for estrogen-dependent cancers such as breast cancer and uterine cancer, , is 33.8Hg/d (91
, OnM), BE-1434 of the present invention
It can be said that 8B is a compound having excellent estrogen antagonistic activity.

[Estrogen activity and cytotoxicity]

The estrogenic activity of BE-14348B was tested using CF-7 in a human breast cancer-derived cell line whose proliferation is promoted by the presence of estrogen. Estrogen was removed with activated charcoal, and 10,000 CF-7 cells were plated in a modified Dulbecco culture medium containing 10% fetal serum and 10,000 CF-7 cells were plated in each format of a 96-well microtest plate. 1434
8B was added at each concentration shown in Table 1, and cultured at 37°C for 4 days. Diethylstilbestrol, an estrogen agent, was used as a control. After culturing, the estrogenic effect of BE-14348B was determined by counting the number of cells, with the ability to promote Jl cell growth being #1s. Experiments were also conducted under conditions in which estradiol 1n was added to the medium. the result,
As shown in Table 1, BE-143488 is at least 1 to
A concentration of 10.000 nH promoted the proliferation of MCF-7 cells in the absence of estradiol. However, although not shown in the table, this growth-promoting effect was not observed in culture under conditions in which estradiol was added.

On the other hand, diethylstilbestrol is also 1-1,000n
Cell proliferation was promoted at a concentration of H, but proliferation was suppressed at a concentration of 10 μH or higher.

(Margin below) Table 1 Next, the effect of BE-14348B on human uterine cancer-derived HeLa cells, whose proliferation does not depend on estrogen, was tested. The test method was the same as that for MCF-7 cells, but untreated fetal bovine serum was used and the culture period was 3 days. CF-7 cells were also tested under the same conditions. As a result, as shown in Table 2, BE-14
3488 at a concentration that promotes the proliferation of MCF-7 cells.
It did not promote proliferation of eLa cells. Also, HeLa,
The concentration that inhibits the proliferation of CF-7 cells by 50% is BE
-14348B was 26μg/m (91μ?ri and 40μg/m)
μg/it (140 μM), and diethylstilbestrol is 2.0 μg/it (5,3 μM) and 15 μg/it (140 μM).
．． ccg/me (39 μM) inhibited the proliferation of each cell by 50%.

As described above, BE-14348B exhibited estrogenic effects over a wide concentration range, and its NM1-toxicity was weak, being less than one-tenth that of diethylstilbestrol (IIeLa cells).

Table 2: Comparison with other steroid hormones The effect of BE-14348B on the binding of dexamethasone and promegestone to Noritase was tested. For dexamethasone glue septa, a soluble fraction of liver cytoplasm obtained from a rat that had undergone adrenalectomy in advance was used. For promegestone, the #r cytosolic soluble fraction of pig uterus used for estradiol glue septa binding was used. The experimental method was similar to that for estrogen. As a result, BE-1
4348B is a 10-
No binding inhibition activity was shown at a concentration of 10,000 ng/ml.

[Acute toxicity]

CDF, female mice were injected intraperitoneally with respective amounts of BE-143488 suspended at a concentration of 50 mg/m9 in saline containing 5% DMSO and 0.3% Tween. As a result, all of the treated mice survived up to the dose of 500 mg/kg, and no abnormalities were observed in their behavior during the 7-day observation period or in their external appearance at the subsequent autopsy.

As is clear from the above test examples, BE-14348B
is a compound with estrogenic activity, characterized by very low toxicity and weak cytotoxicity, and is therefore useful in the pharmaceutical field. Therefore, gynecological diseases caused by estrogen deficiency.

BE-14348B can be used alone as a therapeutic agent for osteoporosis, prostate cancer, and benign prostatic hyperplasia.

Alternatively, it may be used in combination with therapeutically effective substances other than BE-14348B.

The compounds of the invention may be mixed with pharmacologically acceptable solid or liquid carriers, excipients, diluents, etc., and used in the form of pharmaceutical preparations suitable for parenteral, oral or external administration. Can be done. Pharmaceutical preparations include, for example, injections,
Examples include liquid preparations such as syrups or emulsions, solid preparations such as tablets, capsules or granules, and external preparations such as ointments or suppositories. These preparations may also contain commonly used additives such as auxiliaries, stabilizers, wetting agents, emulsifiers, absorption enhancers, or surfactants, as necessary. Distilled water, Ringer's solution, glucose, sucrose syrup, gelatin, edible oil5
Examples include cocoa butter, ethylene glycol, sucrose, corn starch, magnesium stearate, and talc.

The dosage of BE-14348B of the present invention varies depending on the target disease, age, weight, and condition of the patient, but the dosage per adult per day is for oral, intramuscular, or intravenous administration. 0.1 to 251 g for gynecology and 0.1 to 500 mg for prostate cancer and benign prostatic hyperplasia. The number of administrations varies depending on the administration method and symptoms, but 1
Once or several times a day.

The three inventions that will be specifically explained by giving examples below are:
BE-143 disclosed by the present invention is not limited to the examples, and may include modification means of the examples.
Based on the properties of 48B, using known means, BE-1
It covers all methods for producing, concentrating, extracting, and purifying 43488.

Streptomyces SB BA-14348 strain cultured on an agar slant medium was mixed with 0.1% glucose, 2.0% dextrin (MS-3600), 1.0% corn gluten meal, and 0.5% fishmeal. , yeast extract 0.1%, sodium chloride 0.1%, magnesium sulfate 0.05%, calcium chloride 0.05%, 3-(N-morpholino)propanesulfonic acid 0.5%, ferrous sulfate 0. 0002%,
Cuprous chloride 0.00004%, manganese chloride 0.000
04%, cobalt chloride 0.00004%, zinc sulfate o,
A medium consisting of ooooos%, sodium borate o, ooooa%, ammonium molybdate 0.00024% (pH corrected to 6.8, however, the percentage in the medium is weight/volume%)
) 110m! ! The seeds were inoculated into three Erlenmeyer flasks with a capacity of 500 and cultured on a rotary shaker (180 revolutions per minute) at 28°C for 72 hours to obtain a seed culture. 2M of the obtained seed culture solution was added to a medium containing 1.1Ω of the same composition as above.
Transplanted into 9 Q volume Erlenmeyer flasks and incubated at 28°C for 96 hours.
Cultured on a rotary shaker. The culture solution was filtered to obtain bacterial cells and a culture filtrate (8,812). The obtained culture filtrate was directly passed through a column made of Diaion HP20 1 Q to adsorb the active ingredient, and after washing with water and 50% acetone water, the active substance was eluted with 80% acetone water (3 g). . The active substance was extracted from the bacterial cells with methanol (5Q), combined with the eluate obtained from the culture filtrate, and concentrated under reduced pressure. From this concentrate, n-hexane (6oom
) to remove impurities, and the aqueous layer (500 mg) was extracted with tt ethyl acetate. The obtained ethyl acetate layer (850+d
) was washed with pJ (2) and water at pH 8, and the ethyl acetate layer was concentrated to dryness under reduced pressure. This was subjected to silica gel column chromatography (inner diameter 2.2 cm, length 42 cm).
and eluted with chloroform:ethyl acetate (4:1).

Both parts containing BE~14348B were concentrated to dryness under reduced pressure,
Dissolve in a small amount of methanol and add an ODS column (InertsLl-ODS, manufactured by Gascro Industries Co., Ltd., inner diameter 7.6 mm).
, length 250 im) and preparative high performance liquid chromatography using acetonitrile:water:acetic acid (450:550:2) as a mobile phase was performed to collect a fraction containing BE-14348B. This fraction was concentrated to dryness under reduced pressure, and B
E-14348B 28.5a+g was obtained.

BE-143488 of the present invention strongly antagonizes the binding of estradiol to the receptor and exhibits agonist activity.
BE-14348B, a compound of the present invention, does not antagonize the binding of hormones such as progesterone and dexamethasone to their receptors. It can be used as a therapeutic agent for cancer and benign prostatic hyperplasia.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of BE-14348B, Figure 2 shows the infrared absorption spectrum of BE-143488 by the KBr tablet method, and Figure 3 shows the BE-14348B infrared absorption spectrum.
-14348B at 300MHz in heavy acetone
'H-nuclear magnetic resonance vector diagram, Figure 4 is 75MH
Figures of z ”C-NHR nuclear magnetic resonance spectra are shown respectively. Figure 1

Claims (6)

[Claims] (1) Formula ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) BE-14348B represented by (2) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) In the manufacturing method of BE-14348B, BE-
A production method comprising culturing a microorganism belonging to the genus Streptomyces or a mutant strain thereof having the ability to produce 14348B in a nutrient medium, producing and accumulating BE-14348B in the culture, and collecting the same. (3) A microorganism belonging to the genus Streptomyces that has the ability to produce BE-14348B is Streptomyces sb.
The production method according to claim 2, characterized in that the strain is BA-14348. (4) A microorganism belonging to the genus Streptomyces or a mutant strain thereof having the ability to produce BE-14348B. (5) The microorganism belonging to the genus Streptomyces that has the ability to produce BE-14348B is Streptomyces sb.
The microorganism according to claim 4, which is strain BA-14348. (6) Formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (I) An estrogenic agent containing BE-14348B as an active ingredient.