JPH083188A - New compound chymostatinols and its production - Google Patents

Abstract

PURPOSE:To obtain new chymostatinols having strong action to inhibit cathepsin L and weak inhibiting action on cathepsin B and useful for the treatment of osteoporosis, etc., by culturing a microbial strain belonging to the genus Streptomyces and capable of producing chymostatinols A-C. CONSTITUTION:The new chymostatinols effective to specifically and strongly inhibiting cathepsin L and weakly inhibiting cathepsin B, having high safety and useful as an agent for the treatment of osteoporosis is expressed by formula I (R<1> is isopropyl, see-butyl or isobutyl; R<2> is H or a 1-5C alkyl) and composed of chymostatinol A of formula II, chymostatinol B of formula III and chymostatinol C of formula IV. These compounds are produced by culturing microorganisms belonging to the genus Streptomyces and capable of producing chymostatinol A, chymostatinol B and/or chymostatinol C and collecting the products from the cultured material.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to three novel compounds having a cathepsin L inhibitory activity, chymostatinol A, chymostatinol B and chymostatinol C or salts thereof, and a process for producing them.

[0002]

2. Description of the Related Art Senile osteoporosis is a major social problem in today's rapidly aging society. Bone is turned over and maintains a constant bone mass by maintaining balance between bone resorption and bone formation. When such an equilibrium state breaks down and bone resorption exceeds bone formation, the bone mass gradually decreases and causes osteoporosis. Older people are often forced to be bedridden due to bone fracture due to osteoporosis, which is an important social issue.

One of the fundamental causes of osteoporosis is abnormally increased bone resorption by activated osteoclasts. Bone resorption by osteoclasts has been reported to be triggered by the command of osteoblasts. That is, active vitamin D3,
The receptors for most bone resorption promoters such as parathyroid hormone (PTH) and interleukins are present on osteoblasts, and the osteoblasts that receive this command are activated, and the information is somehow transmitted to osteoclasts. Be transmitted to. The osteoclasts to which these information is transmitted are activated and, together with the secretion of H ⁺ , decompose the collagen, which is a protein supporting tissue of bone, to form a resorption space between the osteoclast and the bone. It is known that cathepsin L in lysosome plays the most important role in the degradation of bone collagen, and that bone resorption can be suppressed by inhibiting cathepsin L (Nobuhiko Katsunuma: intracellular protein degradation: Tokyo Kagaku Dojin, (1992): Agricultural B
iological Chemistry 42 : 523, (1978)).

As an inhibitor of cathepsin L, E-64 which significantly reduces blood calcium level in an animal model
(Agricultural Biological Chemistry 42 : 523, (197
8)), a molecular weight of 10 which is an endogenous inhibitor of the cathepsin group,
Of 000 peptides (Nobuhiko Katsunuma: Intracellular proteolysis: Tokyo Kagaku Dojin, (1992), 73-8
8), a specific inhibitor of cathepsin L, cathelin (FEBS-
Lett. (1989); 255 (2): 211-214) and the like are known. These substances could be promising drugs,
It has not been put to practical use yet.

Compounds having a partial structure common to the chymostatinol compounds of the present invention include chymostantines (J. Antibiot., 23 , 425-427, (1970)) and M.
er-N5075A (J. Antibiot., 46 , 1622-1624, (1993))
Etc. are known.

[0006]

DISCLOSURE OF THE INVENTION The present inventors strongly inhibit cathepsin L in a culture of a newly isolated microorganism Streptomyces nashvillensis belonging to the genus Streptomyces. And found that three compounds with novel structures, chymostatinol A, chymostatinol B and chymostatinol C, were isolated and purified,
Further, those ester derivatives were prepared to complete the present invention.

[0007]

The present invention provides (1) general formula (I):

[0008]

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(Wherein R ¹ represents an isopropyl group, a secondary butyl group or an isobutyl group, and R ² represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms). Or a salt thereof, (2) the following formula (II):

[0010]

[Chemical 6]

Chymostatinol A or a salt thereof represented by: (3) the following formula (III):

[0012]

[Chemical 7]

Chymostatinol B represented by: or a salt thereof, (4) the following formula (IV):

[0014]

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The chymostatinol C or salt thereof represented by (5) chymostatinol A, chymostatinol B and / or chymostatinol C producing bacterium belonging to the genus Streptomyces is cultured, and chymosin is obtained from the culture. A method for producing chymostatinol A, chymostatinol B and / or chymostatinol C or a salt thereof, comprising collecting tatinol A, chymostatinol B and / or chymostatinol C, (6 ) A method for producing chymostatin A or a salt thereof, comprising culturing a chymostatin A producing bacterium belonging to the genus Streptomyces and collecting chymostatin A from the culture, (7)
A method for producing chymostatinol B or a salt thereof, comprising culturing a chymostatinol B-producing bacterium belonging to the genus Streptomyces and collecting chymostatinol B from the culture, (8) Streptomyces genus (9) Streptomyces nashbilensis SANK63093, characterized by culturing a chymostatinol-C-producing bacterium belonging to the category: and collecting chymostatinol-C from the culture. Stocks.

When R ^{2 of the} compound represented by the general formula (I) of the present invention is an alkyl group having 1 to 5 carbon atoms, for example, methyl, ethyl, propyl, butyl, t-
Examples thereof include linear or branched alkyl groups such as butyl and pentyl. It is preferably a linear or branched alkyl group having 1 to 3 carbon atoms, and most preferably a methyl group.

The compound represented by the general formula (I) of the present invention can be converted into a salt according to a conventional method.

When R ¹ is a hydrogen atom, such salts include salts of alkali metals such as lithium, sodium and potassium; salts of alkaline earth metals such as magnesium, calcium and barium; methylamine, ethylamine and dimethyl. Examples thereof include salts of aliphatic primary to tertiary amines such as amine, dipropylamine and trimethylamine; salts of amino acids such as lysine and arginine; ammonium salts. Preferred are alkali metal salts such as sodium and potassium.

Of these salts, the most suitable one is a pharmacologically acceptable salt.

The compound represented by the general formula (I) has various isomers. In the compound having the general formula (I), these isomers are all represented by a single formula. Therefore, in the present invention, all of these isomers and a mixture of these isomers are also included.

Further, the compound having the general formula (I) of the present invention includes all so-called "prodrug compounds" which are derived from the compound having the general formula (I) in vivo. .

In the compound having the general formula (I), R ¹ is preferably an isopropyl group, a secondary butyl group or an isobutyl group, and R ² is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. It is the compound or its salt shown.

Most preferably, R ¹ represents an isopropyl group, a secondary butyl group or an isobutyl group, and R ² represents a hydrogen atom, or a salt thereof.

Specific examples of the compound having the general formula (I) of the present invention include the compounds shown in Table 1 below.

[0025]

[Table 1] ──────────────────────────────────── Compound number R ¹ R ² ──── ──────────────────────────────── 1 i-Pr H 2 sec-Bu H 3 i-Bu H 4 i- Pr Me 5 sec-Bu Me 6 i-Bu Me 7 i-Pr Et 8 sec-Bu Et 9 i-Bu Et 10 i-Pr Pr 11 sec-Bu Pr 12 i-Bu Pr ───────── ──────────────────────────── However, in the table, H = hydrogen atom, Me = methyl,
Et = ethyl, i-Pr = isopropyl, Pr = propyl, i-Bu = isobutyl, sec-Bu = secondary butyl, a group is shown.

In the above compound table, Exemplified Compound Nos. 1, 2, 3 and salts thereof are preferable.

The compound of Exemplified Compound No. 1 is chymostatinol A, the compound of Exemplified Compound No. 2 is chymostatinol B, and the compound of Exemplified Compound 3 is chymostatinol C.

(I) Of the compounds of the present invention represented by the general formula (I), chymostatinol A, chymostatinol B and chymostatinol C are obtained by the following method.

Chymostatinol A, chymostatinol B and chymostatinol C isolated by the present inventors
The morphological characteristics, physiological properties, and chemotaxonomic properties of the actinomycete SANK 63093 strain producing L. are shown below.

1. Morphological characteristics SANK63093 strain is ISP "International
Streptomyces Project (International St
The following morphological characteristics were exhibited by culturing at 28 ° C. for 14 days on a medium defined by “reptomyces Project”). Substrate mycelia is good elongation SANK sixty-three thousand and ninety-three the observation with an optical microscope shows a dark brown to no branched Chami white torn or zigzag extension of Nocardia (Nocardia) sp strain like is not observed. The aerial hyphae simply branch. The spore linkage morphology is linear or curved and is approximately 10 to 5
Form a chain of zero or more spores. Observation with a scanning electron microscope shows that the spores have an elliptical shape and the surface structure is smooth. Spore size is 0.7-0.8
x 0.7 to 1.7 μm. Spores are formed only on aerial hyphae. No special organs such as sporangia, aerial mycelial axle branching, aerial hyphae rupture, and sclerotia were not observed.

2. Culture properties on various culture media Table 2 shows the culture properties of the SANK 63093 strain on an agar medium after culturing at 28 ° C for 14 days.

[0032]

[Table 2] Type of medium Item ^{* 1} Properties of SANK63093 strain ───────────────────────────────────── Yeast extract G: Very good, flat, light yellowish green tea (2.5Y 8/3) ^{* 2} Malt extract agar AM: Richly formed, velvety, light brown taste ash (2.5Y 8/1) (ISP 2) R: Dark brown (10YR 3/2) SP: Dark brown (10YR 4/4) Oatmeal agar G: Very good, flat, light yellowish orange (2.5Y 9/2) (ISP 3) AM: Richly formed, velvet Shape, bright tea taste ash (2.5Y 8/1) R: Gray taste yellow tea or dark tea taste ash (2.5Y 8/3 ~ 2.5Y 3/2) SP: Yellow tea (2.5Y 6/4) Starch / inorganic salt agar G: Very good, flat, yellowish gray (5Y 8/2) (ISP 4) AM: Richly formed, velvety, light brownish gray (2.5Y 7/1) R: Grayish yellow tea (2.5Y 6) / 3) SP: Not produced Glycerin / G: Very good, flat, yellowish ash (7.5Y 9/2) Asparagine agar AM: Richly formed, velvety, bright tea taste ash (2.5Y 7/1) (ISP 5) R: Tea taste ash (2.5Y 5/2) SP: Not produced Peptone yeast G: Good, flat, tea taste ash (2.5Y 6/2) Extract / Iron Agar AM: Not formed (ISP 6) R: Tea taste ash (2.5Y 6/2) SP: Dark yellow tea (7.5YR 2/2) Tyrosine agar G: Very good, Flat, dark brown (10YR 3/2) (ISP 7) AM: Richly formed, velvety, light brown Taste ash (2.5Y 7/1) R: Dark brown (10YR 2/2) SP: Dark brown (10YR 3 / 2) Sucrose G: Not good, flat, brownish white (2.5Y 9/1) Nitrate agar AM: Good, velvety, bright brownish ash (2.5Y 8/1) R: Light brown (2.5Y 8/2) ) SP: Not produced Glucose / G: Not so good, flat, brownish white (2.5Y 9/1) Asparagine agar AM: Slightly formed, white R: Light yellowish orange (2.5Y 9/2) SP: Produced Without Nutrient agar G: Not very good, flat, light yellowish orange (2.5Y 9/2) (DIFCO) AM: Not very good, velvety, white R: Yellowish tea (2.5Y 7/4) SP: Yellowish tea (10YR 6/6) Potato extract / G: Poor, flat, brownish white (2.5Y 9/1) Carrot extract Agar AM: Good, velvety, light brown ash (2.5Y 7/1) R: Light brown ash (10YR 8/1) SP: Not produced Water agar G: Poor, flat, brownish white (2.5Y 9 / 1) AM: Not so good, velvety, brownish white (10YR 9/2) R: Brownish white (10YR 9/2) SP: Not produced ──────────────── ──────────────────── * 1: G: Growth, AM: Aerial mycelium, R: Back side, SP: Soluble pigment * 2: Color tone based on Munsell method Display 3. Physiological properties SANK observed for 2 to 21 days after culturing at 28 ° C
The physiological properties of strain 63093 are shown in Table 3.

[0033]

[Table 3] In addition, SANK63 was observed after culturing at 28 ° C for 14 days using Pridham Gortribe agar medium (ISP 9).
The carbon source assimilation of the 093 strain is shown in Table 4.

[0034]

[Table 4] ─────────────────────── D-glucose + L-arabinose + D-xylose ± inositol-D-mannitol-D-fructose-L − Rhamnose − Sucrose ± Raffinose − Contrast − ─────────────────────── +: Utilization, ±: Weak utilization, −: Utilization Not 4. Chemotaxonomic properties The cell wall of the SANK63093 strain was analyzed by the method of Hasegawa et al., “Hasegawa et al., The Journal of General and Applied Microbiology.
l and Applied Microbiology) 29, 319-32.
2 page, 1983 ”, LL-diaminopimelic acid was detected. Also, SANK 6309
The major sugar components in the whole cells of the three strains were analyzed by MP Rechevalier, “MP Lechevalier, Journal of Laboratory and Clinical Medicine (Journa
l of Laboratory and Clinical Medicine) 71, 9
34-944, 1968 ”, no characteristic component was detected. Regarding the acyl group of cell wall peptidoglycan, the method of Uchida et al., “The Journal of General and Applied Microbiology (The Journal of General and Applied
Microbiology) 25, 169-183, 1979.
As a result of examination according to “year”, it was acetyl type.

From the above mycological properties, SANK 6309
The three strains are Streptomyces among Streptomyces.
yces ). Description of ISP strains by shirring and gotrieb [(EB Shirling
and D. Gottlieb), International Journal of Systematic Bacteriology (Intern)
ational Journal of Systematic Bacteriology), Vol. 18, 68-189 (1968), Vol. 18, 2
79-392 (1968), Volume 19, 391-5.
12 (1969), Vol. 22, 265-394 (1972)], Waxman, SA Waksman; The Actinomycetes, Vol. 2, Buchanan and Gibbons, Vergiz Manual (RE Bu).
chanan and N. E. Gibbons; Bergey's Manual of Determ
8th edition (1974), Williams, Sharp and Holt, edited by Bergey's Manual of Systematic Bacteriology
Volume 4 (1989), and Streptomyces ( St
When compared with the bacterial species described in the recent literature on actinomycetes of the genus reptomyces ), it was found to be extremely closely related to Streptomyces nashvillensis . However, Streptomyces nashv
illensis ) was positive in milk coagulation and peptonization . The SANK 63093 strain having such mycological characteristics is apparently Streptomyces nashvillens.
It is believed that the strain is a new strain different from Streptomyces nashville ( Streptomyces nashville).
nsis ). SANK 63093 strain was sent to the Institute of Biotechnology, Institute of Biotechnology, AIST on April 4, 1994.
Deposited as ERM BP-4627.

The physiologically active substances chymostatinol A, chymostatinol B and / or chymostatinol C of the present invention are, for example, cultivated chymostatinol compound producing bacteria belonging to the genus Streptomyces. It is produced by collecting chymostatinol A, chymostatinol B, and / or chymostatinol C from the culture.

Chymostatinol A used in the present invention,
Chymostatinol B and / or chymostatinol C
Examples of the producing bacterium include the aforementioned Streptomyces genus. Strains belonging to the genus Streptomyces and having the ability to produce chymostatinol A, chymostatinol B and / or chymostatinol C are Its properties tend to change, as seen in the case of general microorganism strains,
For example, it can be easily mutated by artificial mutagenesis means such as ultraviolet rays, high frequencies, radiation and chemical mutagens. Even with such mutant strains, all strains having the desired activity can be used in the method of the present invention. That is, in the present invention, a chymostatinol compound is produced, and SANK 63093 is produced.
All strains that are not clearly distinguished from the strain and its mutants are included in the SANK 63093 strain.

In the production method of the present invention, the culture can be carried out under the conditions generally used for culturing actinomycetes. As a medium containing nutrients that can be used by microorganisms, a known medium used for general microorganism culture can be used, and a carbon source that can be assimilated by microorganisms (usually 1 to 10% by weight of the amount of medium) , Nitrogen source (usually 0.2
Any of a natural medium and a synthetic medium can be used as long as the medium appropriately contains an inorganic substance and the like.

The carbon source is not particularly limited as long as it can be utilized by the bacterium as a carbon source, and examples thereof include glucose, fructose, maltose, lactose, sucrose, starch, mannitol,
Carbohydrates such as dextrin, glycerin, starch syrup, molasses, molasses, oat flour, rye flour, corn starch, potato, corn flour, soy flour, malt extract; soybean oil, cottonseed oil, olive oil, cod liver oil, fats such as lard. Kinds; citric acid, sodium ascorbate, malic acid, acetic acid, fumaric acid, tartaric acid, succinic acid,
Organic acids such as gluconic acid; methanol, ethanol
, N-propanol, isopropanol, n-butanol
Alcohols such as alcohol, isobutanol and t-butanol; and amino acids such as glutamic acid can be used alone or in combination.

The nitrogen source is not particularly limited as long as it can be assimilated by the fungus as a nitrogen source. For example, bran,
Peanut flour, casein hydrolyzate, fish meal, malt extract,
Soybean flour, soybean casein, casamino acid, pharmamedia, cottonseed flour, wheat germ, meat extract, peptone, corn steep liquor, self-digesting yeast, dried yeast, yeast extract, organic nitrogen compounds such as urea; aspartic acid, glutamine. , Amino acids such as cystine, alanine; ammonium sulfate, ammonium nitrate, ammonium chloride,
Inorganic nitrogen compounds such as ammonium salts such as ammonium phosphate and nitrates such as sodium nitrate and potassium nitrate can be used alone or in combination.

The inorganic salts taken into the medium are ordinary salts capable of obtaining ions such as sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride and carbonate, and molybdenum, boron, copper and cobalt. It also contains trace amounts of metals such as manganese and iron. Specifically, for example, sodium chloride, manganese chloride, cobalt chloride, potassium chloride, calcium chloride, calcium carbonate, potassium aluminum sulfate, manganese sulfate, copper sulfate, cobalt sulfate, sulfuric acid. Examples thereof include zinc, ferrous sulfate, magnesium sulfate, monopotassium phosphate, dipotassium phosphate, disodium phosphate, and ammonium molybdate, which can be used in appropriate combination.

When a sulfur compound that can be assimilated by bacteria is added to the medium, the amount of the desired product produced may increase. For example, zinc sulfate, copper sulfate, ferrous sulfate, sulfates such as ammonium sulfate, thiosulfates such as ammonium thiosulfate, inorganic sulfur compounds such as sulfites such as ammonium sulfite; cystine, cystine, L-thiazolidine- Organic sulfur compounds such as sulfur-containing amino acids such as 4-carboxylic acid, sulfur-containing peptides such as hypotaurine and glutathione, ferrous sulfate, heavy metals such as copper sulfate, vitamin B1, vitamins such as biotin, and thiamine. Such bacterial cell growth promoting substance and the like are also added as necessary.

Further, a defoaming agent such as silicone oil, polyalkylene glycol ether, vegetable oil, animal oil, or surfactant may be added to the medium (especially suitable for liquid culture).

As a culture method suitable for growing the producing bacterium,
There is no particular limitation, and any culturing method generally used for microorganisms can be used, and for example, a solid culturing method, a static culturing method, a stirring culturing method, a shaking culturing method, and an aeration culturing method can be used. Preferably, a stirring culture method that is an aerobic liquid culture method, a shaking culture method, or an aeration culture method,
The shaking culture method is more preferable. Incidentally, the aeration stirring culture method is industrially suitable.

The pH of the medium is usually 5.0 to 8.0, preferably 7.0.

The culture temperature suitable for the growth of the bacterium is usually 15
C. to 30.degree. C., preferably 28.degree.

Chemostatinol A, Chemostatinol B
And / or chymostatinol C production usually peaks in 1-3 days in shake culture.

The production amount of the chymostatinol compounds of the present invention can be quantified by measuring the cathepsin L inhibitory activity.

The inhibitory activity was measured by the following method.

Cathepsin L activity was measured using 82 mM NaOAc, 15
mM CH ₃ COOH (pH 5.5), 1 mM EDTA, 0.05% Brij 35, 2
The enzyme preparation and the test sample were added to mM DTT, and the total amount was 1 ml. Pre-incubate for 5 minutes at 30 ° C, then
Substrate 10 mM Z-Phe-Arg-MCA was added and incubated at 30 ° C for 20 minutes. The reaction was stopped with 10 mM leupeptin, the mixture was centrifuged at 10,000 rpm for 5 minutes, and the fluorescence intensity (Ex 370 nm, Em 460 nm) of the supernatant was measured.

The product after the culture is present in the culture filtrate and the cells. The bacterial cells and other solid parts in the culture can be removed by centrifugation or a filtering operation using diatomaceous earth as a filter aid. The chymostatinol A, chymostatinol B and / or chymostatinol C of the present invention is present in the supernatant fraction or filtrate obtained here.

Chymostatinol A, chymostatinol B and / or chymostatinol C in the supernatant fraction or the filtrate can be extracted and purified by utilizing its physicochemical properties.

After subjecting the supernatant fraction or the filtrate to acidic conditions,
Extraction operation is performed with an organic solvent immiscible with water, such as butanol, and the extract is concentrated to give chymostatin A, chymostatin B and / or
Alternatively, chymostatinol C is obtained.

An organic solvent miscible with water (for example, methanol, so that the final concentration is 50 to 90%).
Alcohols such as ethanol; Ketones such as acetone; Nitriles such as acetonitrile and isobutyronitrile; Formamide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methyl- Two
-Pyrrolidone, N-methylpyrrolidinone, and hexamethylphosphorotriamide) are added to the cells to perform an extraction operation to remove the solvent, and then the same treatment as that of the supernatant fraction or the filtrate is carried out. Chymostatinol A, chymostatinol B and / or chymostatinol C can be obtained in the same manner as the clear fraction or the filtrate.

Further, the compound is prepared by a method commonly used for separation and purification of organic compounds, for example, Sephadex LH-20 (Pharmacia), Amberlite XA.
A method such as partition column chromatography using a synthetic adsorbent such as D-11 (made by Rohm and Haas) or Diaion HP-20 (made by Mitsubishi Kasei), or normal phase using silica gel or alkylated silica gel. The target compound can be separated and purified by appropriately combining reverse phase column chromatography methods (preferably high performance liquid chromatography) and eluting with a suitable eluent.

The compound can also be purified by an adsorption column chromatography method using a carrier such as alumina and magnesium-silica gel florisil.

The solvent used is preferably aliphatic hydrocarbons; aromatic hydrocarbons such as benzene, toluene and xylene; methylene chloride, chloroform,
Halogenated hydrocarbons such as carbon tetrachloride and dichloroethane; Esters such as butyl acetate and diethyl carbonate;
Tetrahydrofuran, dioxane, dimethoxyethane,
There may be mentioned ethers such as diethylene glycol dimethyl ether.

Furthermore, the compound of the present invention can be efficiently purified by column chromatography using the following carriers. Cation exchange resins such as Dowex 50W (manufactured by Dow Chemical Co.), Amberlite IRC-50 (manufactured by Rohm and Haas Co.), Diaion WX series, SK series (manufactured by Mitsubishi Kasei) and SP
The target compound is prepared by adsorbing the compound on a cation exchange Sephadex such as Sephadex or CM Sephadex (manufactured by Pharmacia) and adjusting the salt concentration and pH appropriately using various acids, bases, inorganic salts and organic salts. Can be eluted.

The elution of the target compound can be assayed by HPLC analysis using a reverse phase column or by measuring the above-mentioned cathepsin L inhibitory activity.

(II) Of the compounds of the present invention represented by the general formula (I), the compounds in which R ² represents an alkyl group can be obtained as follows.

That is, it is achieved by carrying out an esterification reaction with an acid using chymostatinol A, chymostatinol B and / or chymostatinol C and an alcohol such as methanol or ethanol. The acid used in the reaction is not particularly limited, for example, sulfuric acid,
Mineral acids such as hydrochloric acid, chloric acid and perchloric acid; organic acids such as formic acid and trifluoroacetic acid are preferred. The reaction temperature and reaction time will vary depending on the reagents and solvents used, etc., but are usually 0-10
It can be converted to another alkyl group by a transesterification reaction at 0 ° C for 1 to 24 hours.

After completion of the reaction, the desired compound is obtained by distilling the solvent from the reaction mixture and drying the residue. The target compound thus obtained can be further purified by recrystallization or ordinary column chromatography.

Alternatively, it can be achieved by reacting with a diazoalkane such as diazomethane. The reaction is carried out in the presence of solvent. The solvent to be used is not particularly limited as long as it does not affect the reaction, ethers such as diethyl ether, tetrahydrofuran, dioxane and dimethoxyethane; esters such as ethyl acetate; amides such as dimethylformamide. Kind;
Sulfoxides such as dimethyl sulfoxide; are preferred. The reaction temperature and reaction time will differ depending on the reagents and solvents used, but they are generally preferably 0 ° C to room temperature, and 1 to 24 hours.

Alternatively, a compound in which R ² represents a hydrogen atom is
Also by reacting with an alkyl halide compound such as methyl iodide or ethyl iodide in the presence of an alkali metal carbonate such as potassium carbonate or sodium carbonate,
A compound in which R ² represents an alkyl group can be obtained. The reaction is carried out in the presence of solvent. The solvent used is not particularly limited as long as it does not affect the reaction,
Ethers such as tetrahydrofuran, dioxane, dimethoxyethane; amides such as dimethylformamide; sulfoxides such as dimethylsulfoxide; are preferred. The reaction temperature and reaction time depend on the reagents used,
Although it varies depending on the solvent and the like, it is usually suitable at 0 to 100 ° C. for 1 to 72 hours.

After the completion of the reaction, the solvent of the target compound is distilled off from the reaction mixture, and water and an organic solvent such as ethyl acetate are added to the residue for partitioning. It is obtained by concentrating the organic layer and purifying the residue by recrystallization or usual column chromatography.

The physicochemical properties of the chymostatinol compound obtained as described above are as follows.

1 chymostatinol A 1) Properties of substance White powder 2) Molecular weight (determined by FAB-MASS method) 595HR
FABMS [M + H] ⁺ measured value: 596.3198 calculated value: 596.3197 (C ₃₀ H ₄₂ O ₆ N
₇ ) 3) Molecular formula C ₃₀ H ₄₁ N ₇ O ₆ 4) Infrared absorption spectrum (ν max c in KBr tablets
m ^-1 ) 3329,2963,2934,1676,1639,1550,1386,1246,107
7,1046,702 5) ¹ H-nuclear magnetic resonance spectrum The δ value (multiplicity) when measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent is as follows.

0.73 (3H, d), 0.73 (3H, d), 1.87 (1H, m), 4.05
(1H, dd), 7.89 (1H, brd), 3.25 (2H, m), 3.87 (1H, m), 2.6 (1H,
dd), 2.76 (1H, dd), 7.89 (1H, brd), 7.02 (1H), 4.0 (1H, m), 2.
82 (1H, m), 2.95 (1H, m), 7.08 (1H), 6.15 (1H), 4.4 (1H, dd),
3.5 (1H, m), 1.6 (2H, m), 2.84 (1H, m), 3.0 (1H, m), 6.8 (1H),
7.2 (10H, m) 6) ¹³ C-nuclear magnetic resonance spectrum The δ value (multiplicity) when measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent is as follows.

175.3 (s), 169.5 (s), 170.3 (s), 157.1 (s), 15
4.4 (s), 125.5 (s), 125.8 (s), 139 (d) x2,127.6 (d), 128 (d),
129 (d), 129.5 (d), 38.5 (t), 21.2 (t), 38.4 (t), 36.3 (t), 6
2.2 (t), 50.6 (d), 54.9 (d), 125.5 (d), 56 (d), 125.8 (d), 52.
4 (d), 57.9 (d), 30.6 (d), 17.8 (q), 19.1 (q), 7) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184
) Waters mobile phase: acetonitrile / triethylamine-phosphate buffer pH 3.2 (25:75) Flow rate: 2.0 ml / min Detection: UV 210 nm retention time: 7.2 minutes 8) Amino acid analysis 100 ° C in sealed tube Detect valine and phenylalanine by hydrolysis with 6N hydrochloric acid for 18 hours 2 Chemostatinol B 1) Properties of substance White powder 2) Molecular weight (determined by FAB-MASS method) 609HR
FABMS [M + H] ⁺ Actual value: 610.3350 Calculated value: 610.3353 (C ₃₁ H ₄₄ N ₇ O
₆ ) 3) Molecular formula C ₃₁ H ₄₃ N ₇ O ₆ 4) Infrared absorption spectrum (ν max c in KBr tablets
m-1) 3324,2964,2935,1640,1551,1498,1454,1386,126
0,701 5) ¹ H-nuclear magnetic resonance spectrum The δ value (multiplicity) when measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent is as follows.

0.73 (3H, d), 0.73 (3H, d), 1.87 (1H, m), 4.05
(1H, dd), 7.89 (1H, brd), 3.25 (2H, m), 3.87 (1H, m), 2.6 (1H,
dd), 2.76 (1H, dd), 7.89 (1H, brd), 7.02 (1H), 4.0 (1H, m), 2.
82 (1H, m), 2.95 (1H, m), 7.08 (1H), 6.15 (1H), 4.4 (1H, dd),
3.5 (1H, m), 1.6 (2H, m), 2.84 (1H, m), 3.0 (1H, m), 6.8 (1H),
7.2 (10H, m) 6) ¹³ C-nuclear magnetic resonance spectrum The δ value (multiplicity) when measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent is as follows.

175.0 (s), 169.3 (s), 170.4 (s), 157.2 (s), 15
4.3 (s), 138.9 (d) x2,127.6 (d), 128 (d), 129 (d), 129.4 (d),
125.6 (s), 125.8 (s), 38.4 (t), 21.1 (t), 50.7 (d), 54.8 (d),
125.6 (d), 38.5 (t), 58.8 (d), 125.8 (d), 36.3 (t), 52.4 (d),
62.2 (t), 57.2 (d), 45.4 (d), 36.7 (t), 15.2 (q), 11.0q), 7) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184
) Waters mobile phase: acetonitrile / triethylamine-phosphate buffer pH 3.2 (25:75) Flow rate: 2.0 ml / min Detection: UV 210 nm Retention time: 12.9 minutes 8) Amino acid analysis 100 ° C in sealed tube, Isoleucine and phenylalanine were detected by hydrolysis with 6N hydrochloric acid for 18 hours 3 Chemostatinol C 1) Property of substance White powder 2) Molecular weight (determined by FAB-MASS method) 609HR
FABMS [M + H] ⁺ Actual value: 610.3350 Calculated value: 610.3353 (C ₃₁ H ₄₄ N ₇ O
₆ ) 3) Molecular formula C ₃₁ H ₄₃ N ₇ O ₆ 4) Infrared absorption spectrum (ν max c in KBr tablets
m-1) 3325,3085,3030,2961,2875,1642,1551,1497 5) ¹ H-nuclear magnetic resonance spectrum When measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent. The δ value (multiplicity) is as follows.

0.78 (3H, d), 0.8 (3H, d), 1.35 (2H, m), 1.5 (1
H, m), 4.21 (1H, dd), 8.05 (1H, brd), 3.25 (2H, m), 3.8 (1H,
m), 2.6 (1H, dd), 2.75 (1H, dd), 7.85 (1H, brd), 7.02 (1H), 3.
98 (1H, m), 2.92 (1H, m), 2.75 (1H, m), 6.15 (1H), 6.12 (1H),
4.31 (1H, dd), 3.5 (1H, m), 1.62 (2H, m), 3.01 (1H, m), 3.12 (1
H, m), 6.75 (1H), 7.1 (10H, m) 6) ¹³ C-nuclear magnetic resonance spectrum δ value when measured with tetramethylsilane as an internal standard in a heavy dimethyl sulfoxide solvent ( The multiplicity) is as follows.

175.4 (s), 169.2 (s), 171.5 (s), 157.0 (s), 15
4.3 (s), 138.9 (d), 139.1 (d), 127.6 (d), 128 (d), 129.1 (d),
129.4 (d), 125.6 (s), 125.8 (s), 38.0 (t), 21.3 (t), 50.8
(d), 54.9 (d), 125.5 (d), 38.8 (t), 58.8 (d), 125.8 (d), 36.3
(t), 52.4 (d), 62.0 (t), 57.0 (d), 21.6,22.9,24.1 (q), 11.2
q), 7) High Performance Liquid Chromatography Column: Radial Pack Cartridge (8NVC184
) Waters mobile phase: acetonitrile / triethylamine-phosphate buffer pH 3.2 (25:75) Flow rate: 2.0 ml / min Detection: UV 210 nm Retention time: 15.7 minutes 8) Amino acid analysis 100 ° C in sealed tube Leucine and phenylalanine are detected by hydrolyzing 6N hydrochloric acid for 18 hours. When chymostatinol A, chymostatinol B and / or chymostatinol C and / or salts thereof of the present invention are used as therapeutic agents for osteoporosis, various Administered in a form. Examples of the dosage form include oral administration such as tablets, capsules, powders, granules and syrups, and injections (intravenous, intramuscular, subcutaneous) and the like. These various preparations are prepared by using known auxiliary agents such as an excipient, a binder, a disintegrating agent, a lubricant, a solubilizer, a flavoring agent, a coating agent, etc., which are commonly used in the field of known pharmaceutical preparations, as the main ingredient according to a conventional method. It can be formulated. The amount used varies depending on symptoms, age, weight, administration method, dosage form, etc., but is usually 10 mg to 1 per day for adults.
000 mg can be administered.

[0074]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited thereto.

Example-1 A) Culture For seed culture, a seed medium having the following composition was used.

Medium composition (seed medium) Glucose 1% Glycerin 1% Oatmeal 0.5% Sucrose 1% Nissin soyaflower 2% Raw yeast 1% Casamino acid 0.5% Calcium carbonate 0.1% Antifoam 0 Dissolve in 0.05% tap water and adjust to pH 7.

A seed medium having the above composition was used for seed culture.
Add 100 ml to a 00 ml Erlenmeyer flask with baffles and
Sterilized for 40 minutes at 1 ° C. For seed culture, two Erlenmeyer flasks containing this medium were placed in Streptomyces nashvillensis SAN.
Inoculate one platinum loop each from K63093 slant,
Rotary shaker (200rpm, 5cm thr for 3 days at 28 ℃)
ow). Add 30 L of seed medium to a 60 L jar fermenter for the second seed culture at 121 ° C.
It was sterilized for 40 minutes, and the whole amount of the seed culture solution was transferred. 28
Culturing was carried out at 0 ° C for 2 days.

For production, 300 L of a medium having the following composition was put in a 600 L fermenter and sterilized at 121 ° C. for 40 minutes.

Glucose 0.5% Glycerin 2.5% Corn steep liquor 1% Nisshin soya flower 1% Raw yeast 1% Calcium carbonate 0.3% 1 potassium phosphate 1% Trace metal salt solution (Note) 0.1 % Antifoaming agent 0.05% Dissolve in deionized water to adjust the pH to 7.2.

(Note) Minor bacterial salt: FeSO ₄ .7H ₂ O 0.1 g, M
A solution obtained by dissolving 0.1 g of nCl ₂ .4H ₂ O and 0.1 g of ZnSO ₄ 7H ₂ O in 100 ml of distilled water.

The entire amount of the above-mentioned 60 L jar fermenter seed culture solution was put into a production medium, and the mixture was subjected to aeration and stirring culture at 28 ° C. for 24 hours.

B) Isolation 300 L of the culture broth was separated by filtration through Celite into a filtrate and cells.
After 300 L of the filtrate was adjusted to pH 6.3 with hydrochloric acid, it was adsorbed on a column filled with 50 L of HP-20 resin (manufactured by Mitsubishi Kasei). After washing with 50 L of water and 100 L of 15% acetone / water, the active substance was eluted with 200 L of 60% acetone / water. Acetone was distilled off under reduced pressure to make 120 L.
After adjusting the pH to 3.5 with hydrochloric acid, the mixture was washed twice with 100 L of ethyl acetate, the aqueous layers were collected, and ethyl acetate was distilled off under reduced pressure.

This liquid was added to 7 L of Dowex-50W X4.
Donate to a column packed with cation exchange resin (H ⁺ type),
Adsorbed. After washing with 30 L of water, it was eluted with 0.5N aqueous ammonia. 30 L of active fractions were obtained, concentrated under reduced pressure to remove ammonia, then 2.5 L of HP
Adsorbed on a column filled with -20 resin. Wash with 5 L of water and elute with 60% acetone / water. The active fraction was concentrated and freeze-dried to obtain 12 g of a coarse powder.

The crude powder was dissolved in 50 ml of water and the pH was adjusted to 7.
It was applied to a column packed with 500 ml of CM-Sephadex (Pharmacia) swollen and equilibrated with a phosphate buffer of 0. The active fraction obtained by developing with distilled water was freeze-dried to give a powder, and the CM-Sephadex column treatment was repeated again under the same conditions. 800 mg of active lyophilized powder were obtained.

The obtained powder was subjected to HPLC fractionation under the fractionation conditions shown below for further purification.

Column: Senshu ODS H-5251 (20mm x 2
Mobile phase: Acetonitrile / 0.5% triethylamine-phosphate buffer pH 3.2 (28:72) Flow rate: 6 ml / min Detection: Differential refractometer 8 ml of oil dissolved in mobile phase 100 mg was injected once. Retention time 2 in this HPLC
The peaks of chymostatinol A for 5 minutes, chymostatinol B for 42 minutes, and chymostatinol C for 48 minutes were separately collected. After repeating this 8 times, the fractionated liquids of each of the 3 types of fractions were combined, and acetonitrile was distilled off. Concentrate each fraction with 10 ml of HP-
It was adsorbed on a column packed with 20 resins. After washing with 50 ml of water, elution with 60% acetone / water, concentration, and freeze-drying, 12 mg of pure chymostatinol A, 10 mg of chymostatinol B, and 9 mg of chymostatinol C were obtained.

[0087] After mixing the powders of the above formulation and passing through a 30 mesh sieve, 330 mg of this powder is put into a gelatin capsule,
It was used as a capsule.

[0088]

The effects of the present invention will be described with reference to test examples.

Test Example-1 1-1 Materials, Reagents Z-Phe-Arg-MCA and Z-Arg-Arg-MCA were purchased from Peptide Institute Co., Ltd. Cathepsin B and α-chymotrypsin were purchased from Sigma. The rat liver was collected from a 5-week-old male SD rat (Japan SLC).

1-2 Purification of Cathepsin L The liver collected from a rat was washed with PBS and then finely chopped to obtain a double amount.
(v / w) homogenate buffer (1% NaCl, 0.1% ED
TA, 2% Butanol) and homogenized with Polytron. The homogenate fraction was centrifuged at 3600 rpm for 30 minutes,
The supernatant was adjusted to pH 4.2 with 2M HCl. The supernatant was autolyzed at 37 ° C. for 4 hours and further at 4 ° C. overnight.

After self-decomposition, centrifugation was performed at 5000 rpm for 30 minutes,
The supernatant was subjected to 20-80% saturated ammonium sulfate fractionation. 1 ml of Buffer A (20 mM NaOAc, 1 mM
EDTA) and stir at 4 ° C for 1 hour, then at 25,000 rpm for 25 hours.
Ultracentrifuged for minutes. The supernatant was dialyzed against buffer A.
The dialysis solution was applied to a CM-Sephadex column equilibrated with buffer solution A, washed with buffer solution A, and then washed with 0 M to 1 M NaC.
Linear gradient elution was carried out with buffer A containing 1 l. Cathepsin L and cathepsin B activities were measured for each fraction, and fractions having only cathepsin L activity were collected.
It was dispensed and stored at -80 ° C.

1-3 Cathepsin L activity assay method Cathepsin L activity assay was 82 mM NaOAc, 15 mM CH ₃ COOH.
(pH 5.5), 1 mM EDTA, 0.05% Brij 35, 2 mM DTT, the enzyme preparation and the test sample were added, and the total amount was 1 ml. 3
After preincubation at 0 ℃ for 5 minutes, 10 mM Z-Phe-Arg-MCA, a substrate of cathepsin L, was added and the temperature was increased to 30 ℃.
Incubate for 20 minutes. The reaction was stopped with 10 mM leupeptin, centrifugation was performed at 10,000 rpm for 5 minutes, and the fluorescence intensity (Ex 370 nm, Em 460 nm) of the supernatant was measured.

1-4 Cathepsin B activity assay method Cathepsin B activity assay was performed according to the cathepsin L activity assay method, and Z-Arg-Arg-MCA was used as a substrate.

1-5 Method for measuring α-chymotrypsin activity α-chymotrypsin activity was measured according to the method of Umezawa et al. That is, in 2% casein 500 ml dissolved in 0.025 M borate buffer (pH 7.4), 0.1 M CaCl ₂ 25 ml and 0.025 M as a test sample or a negative control.
425 ml of M borate buffer (pH 7.4) was added and preincubated at 37 ° C for 3 minutes. 0.025M borate buffer
The reaction was started by adding 3 mg of α-chymotrypsin dissolved in (pH 7.4), and incubated at 37 ° C for 30 minutes. The reaction was stopped with 1 ml of 1.7 M perchloric acid, left at room temperature for 1 hour, and then centrifuged at 10,000 rpm for 5 minutes to measure the absorbance at 280 nm of the supernatant.

Results The 50% inhibitory concentration (IC50) of this substance for cathepsin L in the above assay method is summarized in Table 5 below.

[0096]

Table 5 Cathepsin L Cathepsin B Chymotrypsin (ng / ml) (μg / ml) (μg / ml) Chymostatin A 46 12 8.4 Chemostatinol B 32 4 1.6 Chymostatin C 44 14 5. As described above, not only the chymostatinol A, chymostatinol B, chymostatinol C, and cathepsin L of the physiologically active substances of the present invention are specifically and strongly inhibited, but also the inhibitory activity against cathepsin B is weak and safe. Because it is highly
It is useful as a therapeutic drug for osteoporosis.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location (C12N 1/20 C12R 1: 465) (C12P 17/10 C12R 1: 465) (72) Inventor Ryuzo Enokida 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd. (72) Inventor Nao Okazaki 33 Miyukigaoka, Tsukuba City, Ibaraki Sankyo Co., Ltd. (72) Inventor Takeshi Kinoshita 1-2 chome, Hiromachi, Shinagawa-ku, Tokyo No. 58 Sankyo Co., Ltd. (72) Inventor Yasuyuki Takamatsu 389-4 Oigata, Shimokawa, Izumi-cho, Iwaki-shi, Fukushima Sankyo Co., Ltd.

Claims (9)

[Claims] 1. General formula (I): (Wherein R ¹ represents an isopropyl group, a secondary butyl group or an isobutyl group, and R ² represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms) or a salt thereof. . 2. The following formula (II): The chymostatinol A or its salt shown by these. 3. The following formula (III): Of chymostatinol B or a salt thereof. 4. The following formula (IV): The chymostatinol C or its salt shown by these. 5. A chymostatinol A, chymostatinol B and / or chymostatinol C-producing bacterium belonging to the genus Streptomyces is cultured, and chymostatinol A, chymostatinol B and / or chymos is produced from the culture. A method for producing chymostatinol A, chymostatinol B and / or chymostatinol C or a salt thereof, which comprises collecting tatinol C. 6. A chymostatinol A, which is obtained by culturing a chymostatinol A-producing bacterium belonging to the genus Streptomyces and collecting chymostatinol A from the culture.
Or the manufacturing method of the salt. 7. A chymostatinol B-producing microorganism belonging to the genus Streptomyces is cultured, and chymostatinol B is collected from the culture.
Or the manufacturing method of the salt. 8. A chymostatinol C producing a chymostatinol C-producing bacterium belonging to the genus Streptomyces and collecting chymostatinol C from the culture.
Or the manufacturing method of the salt. 9. Streptomyces nashbilensis SANK63093 strain.