KR830000619B1 - Preparation of 3-0 (β-D-glucuronopyranosyl) -soyasapogenol B - Google Patents

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Description

Preparation of 3-0 (β-D-glucuronopyranosyl) -soyasapogenol B

Figure 1: Micro Photograph of Starkey Boatless T-791

The present invention has an anti-complementary effect and is useful as an agent for treating autoimmune diseases, collagen diseases, rheumatoid diseases, and 3-0 (β-D-glucuronopyranosyl) -soyasapogenol B of the following structural formula (I) and It relates to a method for producing these salts.

Several compounds similar to the compounds according to the present invention are known and include, for example, glycyrrhizin (see Yasuhiro Ariga, Hiroyuki Sumi, Yumiko Takada and Akikazu Takada, Abridgements of Lecture Programs on Seminar of the Plasmin Research Asscoiation, page 65 (1977); Koretsugu Arimoto, Kaneyuki Mineta, Hiroyuki Sumi, Yumido Takada and Akikazu Takada, Proceedings of the 14th Symposium on Complements, p. 79-82 (1977)] and 3-0 (6-D-glucuronopyranosyl) -soya Sapogenol B (Isao Kitagawa, Masayuki Yoshikawa and Ichiro Yoshioka, Chem. Pharm. Bull., 22, p. 1339 (1974); Ibid., 24, p. 121 (1976)). It has a similar structure to that of steroids, for example, it shows plasmin, urokinase, kallikrein, thrombin and complement inhibitory effects, whereas the pharmacological effects of the latter compounds have not been reported yet. On the contrary, the unity according to the invention And their salts show a very superior anticomplement effect compared to glycyrrhizin, which is 3-0 (6-0-methyl-β-D-glucurono) as shown in the pharmacological test results mentioned so far. It was not expected from pyranosyl) -soyasapogenol B.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for preparing a soya safogenol derivative and a salt thereof having high anti-complement effect.

The compound according to the present invention is used for the treatment of immunological diseases such as rheumatoid arthritis, lupus erythematosus, glomerulonephritis, automatic allergic hemolytic anemia, platelet disease, vasculitis by inhibiting or alleviating the pathological response necessary for the function of complement. do. The compounds according to the invention are non-immune such as night paroxysmal hemochromatosis, hereditary vasculitis, and inflammation induced by the action of bacterial or thiosomal enzymes on appropriate complement components, for example inflammation involving coronary artery occlusion. It can also be used to treat diseases. They are also used to treat graft rejection, and can also be used as blood and transplant media.

Recently, a lot of research has been done on the theoretical analysis of the complement system and its effects on humans and animals. Currently, if a compound has anti-complementary effects, it may have therapeutic effects on the above mentioned syndromes. The fact is widely known. For example, U.S. Patent No. 4,021,544 discloses the following.

"Complement" is a complex of proteins present in body fluids that acts with antibodies or other factors to serve as important intermediates of immune, allergic, immunochemical and / or immunopathological responses. Complementary reactions occur in serum or other body fluids and are therefore thought to be humoral. "

"There are more than 11 proteins in the complement system in human blood, and these complement proteins are represented by the letters C and the letters C1, C2, C3, ... C9. The complement proteins C1 are again Clq, Clr. The numbers attached to the complement proteins refer to the order in which they are activated, but the complement protein C4 reacts after C1 and before C2, so that the numbers attached to the proteins of the complement system before the reaction sequence is fully understood. A more detailed description of the complement system is given in the following references: Bull.World Health Org., 39, 935-938 (1968), Scientific American, 229, (No. 5), 54-66 ( 1973), Medical World News, October 11, 1974, pp. 53-58, 64-66, Harvey Lectures, 66, 75-104 (1972), The New England Journal of Medicine, 287, 489-495, 545-549 , 592-596, 642-646 (1972), The John Hopkins Med. J., 128, 57-74 (1971), and Federation Proceeding, 32, 134-137 (1973). "

"The complement system is thought to consist of three sub-systems: (1) a recognition unit (Clq) capable of binding to an antibody molecule that detects a heterologous invasive agent (2) activation to produce a site on an adjacent membrane. Units (Clr, Cls, C ₂ , C ₄ , C ₃ ) (3) Attack units that make holes in the membrane (C ₅ , C ₆ , C ₇ , C ₈ and C ₉ ) It only destroys the invaders as it is released from adjacent membranes, and in order to reduce damage to the host's cells for the first time, they must limit their active time. It is partially achieved by disruption by destructive enzymes, but because the regulation of complement is not complete, damage to the host cell is a rare occurrence. So immunity is a double war. "

"Activation of the complement system promotes coagulation. This is done by the action of complement from platelets and the secretion of coagulation factors. Some biologically active complements or conjugates participate in a reaction that damages host cells. Toughness eventually leads to immune complex disease, for example, in some nephritis, complement damages the basal membranes of the kidneys, causing them to fall into the urine without reabsorption of proteins in the blood. Symptoms include nephritis, visceral injuries and skin rashes Injecting large amounts of antitoxins to treat diphtheria or tetanus can also lead to serum diseases, one of the immune diseases.

Rheumatoid arthritis is also included in immune diseases. This disease is autoimmune, caused by the pathological effects of the immune system in host tissues, as in the case of sowing erythema. In summary, the immune system is involved in inflammation, coagulation, fibrinolysis, antigen-antibody responses and other metabolic pathways.

In the presence of an antigen-antibody complex, complement proteins undergo a series of irreversible reactions when they are in close proximity to the biofilm, causing membrane damage. This complement, on the one hand, is also part of our body's defense against infection, but at the same time it causes inflammation and damages tissues by immunopathology. The properties of certain types of complement proteins, their binding mechanisms to biological membranes, and the effects of complement causing membrane damage are described in Annual Review in Biochemistry, 38, 389 (1969).

"According to reports, known complement inhibitors, epsilon-aminocapuroic acid, stramine sodium and tranexamic acid are inherently lacking in serum inhibitors or lacking function in the first component of complement. It is effective in treating angiovascular edema that occurs (see The New England Journal of Medicine, 286, 808-812 (1972); Allergol; Et. Immunipath; II, 163-168 (1974); J. Allergy Clin. Immunol) , 53, No. 5, 289-302 (1974); and Annals of Internal Medicine, 84, 580-593 (1976)].

3-0 (β-D-glucuronopyranosyl) -soyasapogenol B prepared according to the present invention exhibits strong anticomplement activity. Therefore, the compound of the present invention has an effect of inhibiting excessive activity of complement in nephritis, rheumatoid disease, systemic lupus erythematosus and the like, which are called "immune-complex diseases" or autoimmune diseases, and also for preventing and treating these diseases. It works.

According to the invention, the compounds of formula (I) can be prepared using the microorganisms newly isolated by the inventors.

This microorganism is described in more detail below.

I. Location

This strain was isolated from soil collected in Tokushima City, Tokushima, Japan.

II. Properties in Processed Culture Medium

The culture characteristics of the strains in various media were visually and microscopically examined (Fig. 1).

A) Visual inspection

1) Malt Extract Agar Medium

Growth is fast and irregular. Clooney's back brown color is tan (3ie) to dark brown (DK Brown, 3pn). Colonies are flat and conidia formation is good. The mycelium color was biscuit (2pc) and yellowish brown (Tan 3ie) with leached droplets. No soluble pigment is produced.

2) Potato Glucose Medium

The growth of bacteria is very fast, and the size of colonies is about 70 mm when incubated at 27 ° C for 30 days. The periphery of the colonies is dendritic and white mycelium is observed. A very large number of droplets are present and sporulation is not observed. The back color of the colonies is light amber (Lt Amber, 3ie) and no soluble pigment is produced.

3) Czapek's Dox Agar Medium

Growth is very low.

4) Synthetic Mucor Agar Medium

Growth is very low.

5) Oatmeal Agar Medium

Growth is so good that the Petri dish is covered in two weeks. Colonies are thin and flat. Mycelia are abundant from the beginning of culture and condydia formation is also fast. The color of colonies changes from white to dark brown (DK Brown, 3pn) over time. Many drops occur on the mycelium, and no soluble pigment is produced.

III. Morphological characteristics

From Figure 1 it can be seen the following. Little vial (Phialophore) mycelium ends in a rod. However, it does not happen as much as in the Stachybotris Echinata 1 F O 7525 and 8825 strains. The mycelial area of this strain is 4.0-4.5μ, slightly wider than pialopore. The piaropore, which extends directly from the plant or mycelium to the scaffold, has two to three diaphragms and has a size of about 40 to 80 × 3.0-3.5 μ. The cell wall of this pyrophore is not epidermal but soft.

Usually three to six pialides are produced from the swollen portion of the pialophore end. Furthermore, cells of size 4.3-5.2 × 3.0-4.2μ with epidermal protrusions form spherical to round pialopores continuously forming at the ends of the pilead, forming chains of 24 to 70 congendia. . The pileads have round bats and range in size from 6.9 to 10.7 × 3.5-4.7μ. The Pyalophore and the Piald have no color, and the Piald is either Coffee (3pn) or Black.

The nomenclature of the strain of the present invention having the above characteristics was cited by reference. [Reference: G.L. Barron, The Genera of Hyphomycets from soil, The Williams & Wilkins Company, Baltimore (1968), J.C. Gilman, A Manual of Soil Fungi, The Iowa State University Press, Ames, Iowa (1971), and J.A. von Arx, The Genera of Fungi Sporelating in Pure Culture, Verag von J. Cremer 3301 Lehre (1970)] According to Saccardo's nomenclature, the strain is Gtaquibotris, Dematiace. And Hyphomycetes. In other words, the strain lacks ascocarps and other reproductive organs, and dark brown pyropores are generated from the pilied, which is collected in a semicircle at the distal end of the pilied. Matches well with the characteristics.

Various characteristics of this strain were confirmed in comparison with the standard strain stored in the Institute for Fermentation (IFO) in Osaka, Japan, with reference to the above-mentioned documents and the following documents. [Reference: G.R. Bisby, Trans. Brit. Mycol. Soc., 26, 133-143 (1943), R. K. Zuck, Mycologia, 38, 69-76 (1946), G.L. Barron, Can. J. Bot., 39, 153-157 (1961)] As a result, the strain belonged to the genus Starchybotris (Memlmoniella). In particular, the strain does not have ascocarps and other reproductive organs, and dark brown pyrophores are produced continuously from the pyride. Long chains of follicles develop. The appearance of this strain T-791 is in good agreement with that of Starkeebotris (Memmonilla).

Several characteristics of this strain were identified through the above-mentioned literature and the following references, and compared with the standard strain stored in the IFO. [Reference: R.K. Zuck, Mycologia, 38, 69-76 (1946) and G. Smith, Trans. Brit. Myco. Soc., 45, 387-394 (1962)] In conclusion, strain T-791, which is a result of the continuous and nourishing of the pilospores from the pilead, was identified as Mmmoniella echinata, which was named by Schenel. You can see that it belongs to). However, the strains of R.K.Juke and G.Smith were considered to be similar strains to Stakibotris ekinata, and thus contrasted with Stakibotris ekinata 7525 and IFO 8856.

Typologically, the specific dendrites of these two strains are not observed in the Pyrophore cell wall of T-791. Not only that, but in standard strains, the ends of the pyropores are expanded two to three times the thickness of the mycelia, but this strain is not seen in this strain. Furthermore, these two standard strains showed excellent growth in various culture media, especially potato agar medium, forming a slightly protruding colony of mycelium. Irregular dendrite with poor hyphae or condydia formation. From the above microscopic differences, it can be seen that the strain is a new species named Stachyboteri species T-791. The above and below color results were performed according to the methods described in the literature.

IV. Physiological features

Starkeebotris species T-791 is an aerobic bacterium and has the following physiological growth.

Staphylococcus spp. T-791, a new strain, is deposited with the accession number FERM-9 No. Deposited under 3803 and ATCC 20513, respectively, to:

Fermentation Research Institute, Agency of Industrial Science and Technology, Japan (at No. 8-1., Nage Higashi 5-chome, Chiba-shi, Chiba, Japan) American Type Culture Collection (12301 Parklawn Drive Rockville, Maryland, USA 20852 )]

Preparation of the compound of the present invention by a strain of the genus Stachybotri is carried out by the following method.

Strains are initially cultured in a medium containing common nutrients and additives. Nitrogen sources generally used as culture substrates include, for example, soy flour, soybean oil, constipate, yeast extract, dry yeast, oatmeal, gravy, hydrolyzed casein, ammonium salt and nitrate. Suitable carbon sources include glucose, glycerol, maltose, starch, lactose, sucrose and molasses. Additives include inorganic salts such as calcium carbonate, sodium chloride, magnesium sulfate and phosphoric acid. If desired, trace amounts of metal salts such as iron, copper, manganese and zinc may be added. Cultivation is carried out by immersion cultivation with surface culturing or aeration and stirring in a conventional aqueous medium containing the substrate. When immersed in culture, it is preferable to give air and stir. As the culture conditions, it is preferable to maintain the pH at 3.5 to 11.5, preferably 4.5 to 9.5 at a temperature of 15 to 38 ° C., preferably 20 to 32 ° C., under normal aeration conditions, preferably at 20 to 32 ° C.

After incubation, the product is recovered from the culture. The recovery method is not particularly limited, and several known methods using the physicochemical properties of the product can be used. For example, the difference in solubility between product and impurities, the difference in adsorption and affinity for conventional adsorbents such as activated carbon XAD-2, silica gel, ion exchange resins, Sephadex, etc. The method using and the above method can be collect | recovered by the method which mixed suitably.

The compound of the present invention thus prepared may form salts with pharmaceutically acceptable basic compounds. Of course, such salts are included in the scope of the present invention.

Suitable basic compounds that can be used to form such salts include, for example, inorganic basic compounds such as sodium hydroxide, potassium hydroxide, aluminum hydroxide, sodium carbonate, potassium carbonate, sodium bicarbonate and piperazine, morpholine, piperidine, Organic basic compounds such as ethylamine, dimethylamine, triethylamine and the like.

The compounds of the present invention may be used as therapeutic agents for nephritis, in which case a pharmaceutical composition may be prepared with conventional pharmaceutically acceptable carriers. Suitable carriers that may be used include fillers, extenders, binders, wetting agents, disintegrants, surfactants and suspending agents which are commonly used to make drugs according to dosage forms.

Several dosage forms that can be used as nephritis medications can be selected depending on the purpose of treatment. Typical dosage forms that can be used include tablets, pills, powders, solutions, suspensions, emulsifiers, granules, capsules, suppositories, and injectable preparations (solutions, emulsifiers, suspensions, etc.).

When preparing a tablet containing a compound of the present invention as an active ingredient, a wide range of carriers known in the art may be used. Examples of suitable carriers include excipients such as lactose, white sugar, sodium chloride, glucose, urine, starch, calcium carbonate, kaolin, crystalline cerulose and siltic acid; Binders such as water, ethanol, propanol, single syrup, glucose, starch solution, gelatin solution, carboxymethylcellulose, shellac, methyl cellulose, potassium phosphate and polyvinylrollidone; Dry starch, sodium alginate, agar powder, laminaria powder, sodium bicarbonate, calcium carbonate, twin, sodium laurylsulfate, disintegrants such as monoglycerides, starch and lactose, white sugar, glyceryl stearate, cacao butter And disintegrant inhibitors such as hydrogenated oil; Absorption accelerators such as quaternary ammonium base and sodium laurylsulfate; Wetting agents such as glycerol and starch; Adsorbents such as starch, lactose, kaolin, bentonite and colloidal silicic acid, and talc such as purified talc, stearate, boric acid powder, macrogol and solid polyethylene glycol.

When the pharmaceutical composition is turned into pills, various carriers conventional in the art can be used. Examples of suitable carriers include excipients such as glucose, tactose, cacao butter, hardened vegetable oil, kaolin and talc; Binders such as gum arabic, tragacanth powder, gelatin and ethanol; There are disintegrants such as laminaria and agar. The tablets can be coated as needed to form sugary tablets, gelatinous tablets, intraocular tablets, thin skin tablets or double or more layers of tablets.

When formulating suppositories, various carriers known in the art can also be used. Suitable carriers include polyethyltenglycol, cacao butter, higher alcohols, esters of higher alcohols, gelatin and semisynthetic glycerides.

When preparing injectable preparations, it is desirable to sterilize the resulting solutions and suspensions and make isotonic solutions for the blood. When preparing a pharmaceutical composition as a solution or a suspension, all diluents commonly used may be used. Suitable diluents include water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters. Sodium chloride, glucose or glycerol can be added to a therapeutic agent such as nephritis in sufficient amounts to make serous fluids. In addition, the therapeutic agent may further contain conventional fusion aids, buffers, pain relief and preservatives, and optionally colorants, flavors, flavors, sweeteners and other agents.

The amount that can contain the compound of the present invention as an active ingredient in a pharmaceutical composition effective as a nephritis therapeutic agent is not particularly limited and can be added in a wide range. Suitable therapeutically effective amounts of the compounds of the invention are from 1 to 70% by weight, preferably from 5 to 50% by weight, based on the total composition.

There are no specific restrictions on how to use it as an anti-complement drug, such as nephritis, and it can be administered by appropriate route for each type of treatment. For example, tablets, pills, suspensions, emulsifiers, granules and capsules can be administered orally. Use formulations are administered alone or intravenously with common adjuvants such as glucose and amino acids. Furthermore, if necessary, intramuscular, subcutaneous, intradermal and intraperitoneal can be administered, and suppositories are administered rectally.

Nephritis dosages are appropriately selected depending on the purpose of use and symptoms. Typically, the dose of a compound of the present invention is 0.5-20 mg / kg body weight per day.

The compound of the present invention has anti-complementary activity and is effective as a therapeutic agent for autoimmune diseases, collagen diseases and rheumatic diseases.

Test results of the pharmacological effects of the compounds of the present invention are as follows.

Pharmacological Test

1. Test Compound

A. 3-0- (β-D-glucuronopyranosyl) soysafogenol B (invention)

B. Glycirizine (Control)

2. Anti-complement activity

Anti-complement activity of the test compound was measured and confirmed according to the method described in the reference. See Meneki Kagaku (Immuno-Chemistry), Yuichi Yamamura et al., Ed. Asakura Shoten, Tokyo, Japan (1973) p. 830-834]

In detail, 1 ml of 5-fold diluted solution of sensitized red blood cells (EA) containing 1 × 10 ⁸ cells / ml in the test tube and Veronal buffer containing isotonic gelatin (this 5-fold diluted solution is GVB ^++). Crude serum) and 0.5 ml of complement serum (complement of guinea pigs) diluted 150-fold with GVB ⁺⁺ are added. The mixture is stored at 37 ° C. for 60 minutes, and then 3 ml of ice cold saline is added and centrifuged. The absorbance of the isolated supernatant is measured by OD413 to determine the degree to which the test compound inhibits the hemolysis of sensitized red blood cells. The hemolytic inhibitory activity (γ / ml) of 50% measured by the above method is shown in Table 1 for each test compound.

3. Acute Toxicity

The acute toxicity level of the test compound (LD ₅₀ mg / kg) is determined by intraperitoneal administration for compound A in mice and intravenous administration for compound B.

The results are shown in Table 1 below.

TABLE 1

As shown in Table 1, in view of the degree of acute toxicity value (LD ₅₀ ), the compound of the present invention is suitable for use as a therapeutic agent.

4. Therapeutic effect on protoxin-type nephritis

Rat endotoxins (denoted "NT") are prepared as follows.

Rat kidney cortex is homogenized with the same amount of saline solution. The homogenized mixture is combined in a 1: 1 volume ratio with furoint's complete adjuvant (manufactured by Difco Company). 2 ml of the resulting mixture is injected intramuscularly into rabbits (3,100 g body weight) to induce immunity. A month and a half later, blood is drawn from the rabbit heart to get serum. The obtained serum is inactivated at 56 ° C. for 30 minutes, salted out with 40% saturated ammonium sulfate solution, and the fractions are separated. Take γ-globurin fraction (IgG) to obtain NT.

The therapeutic effect is repeated three times with each test compound using male Wistar rats weighing 150 to 160 g. Test compounds are administered intraperitoneally every 24 hours for 7 days. NT is administered 1 hour after the test compound is administered on day 3. NT is injected 1 ml intravenously into the tail vein of each rat. Physiological saline is used as a control.

Proteinuria volume (total excreted in urine for 24 hours) is measured by the turbidity method using bovine serum albumin as a control using sulfonsalicylic acid.

The results are shown in Table 2 below.

TABLE 2

The number of days in the table is calculated from the administration of the test compound one hour before the addition of NT.

In healthy rats, the amount of urine protein is 0.5 to 5 mg / day. Nephritis may be considered to occur if the amount of urine protein is above 10 mg / 1 day. As can be seen from the results in Table 2, nephritis occurs in the control group and also with the compound of the present invention, and the amount of urine protein between the time of administration of NT and 10 days after administration is actually the same as that of healthy rats. Therefore, it can be seen that administration of the compound of the present invention inhibits the first and second stage immune responses.

5. Therapeutic Effect on Heymann's Nephritis

Body weights 180-200 g of the asterisk were used in the experiment. The rat kidney cortex is taken and homogenized with the same amount of normal saline. Homogenates are centrifuged at 1500 G for 1 hour. Supernatant is T. s. Eddington's Way

T.S. Edgington et al., Journal of Experimental Medicine, 127, 555 (1968)] and blended with a volume fraction of 0.4: 1 with Puroint's fully supplemented 37Ra (manufactured by DiJco). The resulting product is injected into the rat intraperitoneally by 0.5ml. Every two weeks, the mixture is administered until the amount of urine protein exceeds 100 mg / 1 day (6 to 8 weeks).

Each test compound is administered once a day for 7 days to mice in which Heymann nephritis develops (body weight 300 to 350 g) intraperitoneally to measure the amount of urine protein (mg / day) as described above. Physiological saline was used as a control and the experimental results are shown in Table 3 below.

TABLE 3

Two to three weeks after the start of the experiment, the rats gained weight from 400 to 500 g, and the settled urine protein ranged from 5 to 50 mg / day. As can be seen from the results of Table 3, the compounds of the present invention are effective for Heymann nephritis healing.

In more detail, the preparation of the compound of formula (I) and salts thereof is shown in the following examples, and the preparation of nephritis therapeutic agents containing the structure (I) and salts thereof as an active ingredient is described in the following reference examples. It is.

Example 1

100 ml of the below composition was added to a 500 ml Erlenmeyer flask, followed by addition of Stachybotris species T-791, followed by incubation for 4 days at 25 ° C. and pH 5.5.

Into a 30-l bottle fermenter, 20 l of the above composition and a culture stock solution-containing flask were added thereto, and the mixture was incubated with air at 1 ° per minute per 1 l of medium at a stirring speed of 300 rpm for 5 days at 28 ° C. The obtained culture solution is centrifuged at 8000 rpm to remove the fungal cells, and the pH of the supernatant is adjusted to about 1 with concentrated hydrochloric acid. The resulting precipitate is collected by centrifugation and extracted with methanol. The methanol layer was depressurized and concentrated by evaporation and then adsorbed onto an activated carbon column (500 ml). The column is eluted with an aqueous 30% acetone solution followed by a 50% acetone solution. The 50% acetone solution layer is collected and evaporated to dryness under reduced pressure. The residue was adsorbed onto a silica gel column and chromatographed using a chloroform and acetone mixture (2: 1 and 1: 1 volume ratio) as the eluent.

On the other hand 3-0 (determined by fraction reference containing β-D-glucuronopyranosyl) -soyasapogenol B) (cf. chloroform-methanol-water (volume ratio 65: 35: 8)) The fraction having a Rf value of 0.38 obtained by thin layer chromatography on F ₂₅₄ (trademark of Merck Co.) is collected and concentrated by evaporation under reduced pressure. The concentrated solution is again adsorbed onto the silica gel column, and the chloroform-acetone solution is eluted alternately (2: 1 and 1: 1 volume ratio) to collect the fractions corresponding to the fractions and evaporate to concentrate. The residue is crystallized from a chloroform-acetone (1: 1 volume ratio) solution to give 46 mg of 3-0 (β-D-glucurono pyranosyl) -soyasapogenol B. Melting Point: 231 ~ 232 ℃ (Decomposition)

Reference Example 1

500 mg of sodium salt of the present invention

Glucose 250mg

Add 5 ml of distilled water for injection

A solution prepared by dissolving the sodium salt and glucose of the compound in distilled water for injection is poured into a 5 ml ampoule. Purge air with nitrogen and sterilize the solution at 121 ° C. for 15 minutes to obtain an injectable formulation.

Reference Example 2

Invention cargo 750 mg

Semisynthetic glyceride base (appropriate amount) to 2000 mg total.

The compound of the present invention is added to a semisynthetic glyceride base, mixed and suspended at 50 ° C. The mixture is formulated in a mold and left to cool naturally. The product is separated to obtain suppositories.

Reference Example 3

Compounds of the present invention, Avicel, corn starch and magnesium stearate are mixed and ground to make tablets using a conventional glucose tablet press (R10mm). The obtained tablet was coated with a film-like coating material consisting of TC-5, polyethylene glycol 6000, castor oil and methanol to obtain a film-coated tablet.

Claims (1)

Aerobic culture of Stachybotrys sp. T-791 strain at pH 3.5 to 11.5 and temperature 15 to 38 ° C. in medium containing anabolic nitrogen, carbon, inorganic salts and trace amounts of metal To prepare the following compound of formula (I) and a pharmaceutically acceptable salt thereof.

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