JPH1045738A - Antibiotic substance epoxyquinomicin c and d, its production and antirheumatic agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound having a new molecular skeleton and exhibiting antirheumatic activity. SOLUTION: The antibiotic substances epoxyquinomicin C and D are expressed by the formula (R is H for epoxyquinomicin C and C1 for epoxyquinomicin D). The epoxyquinomicin C has the following physical and chemical properties; appearance and nature, white powder having weakly acidic nature; melting point, 168-172 deg.C (decomposition); specific rotation, [α]D<25> =+128 deg. (c=1.0, methanol); etc. The compound of the formula can be produced by culturing a microbial strain capable of producing epoxyquinomicin C and D such as Amycolatopsis sp. MK299-95F4 in a nutrient medium at pH6.5-7.5 under aerobic condition.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel compound having an antirheumatic activity, namely, epoxyquinomicin.
omicin) C and epoxyquinomycin D, or salts thereof, and a method for producing epoxyquinomicin C and / or epoxyquinomicin D.
Further, the present invention relates to epoxyquinomicin C and / or epoxyquinomicin D, epoxyquinomicin A
And an antirheumatic agent comprising at least one compound of epoxyquinomicin B or a salt thereof as an active ingredient.

[0002]

2. Description of the Related Art Many various antibacterial substances are known, and various various antitumor substances are known. On the other hand, steroids, acidic anti-inflammatory drugs, immunomodulators, and the like are used in conventional rheumatism treatment.

[0003]

DISCLOSURE OF THE INVENTION In the chemotherapy of bacterial infection, a new compound having a different chemical structure from the conventionally known or used known antibacterial compounds and showing excellent antibacterial activity has been discovered. Or it is always desirable to create. In addition, many antitumor substances generally have strong toxicity, and it is always desired to discover or create an antitumor substance having low toxicity and a novel chemical structure, and research for that purpose has been conducted. I have.

[0004] In addition, steroids and immunomodulators used in the conventional treatment of rheumatism are problematic in that they have various side effects, and acidic anti-inflammatory drugs are truly symptomatic treatments. The emergence of effective rheumatic drugs is desired. Therefore, there is a need to provide a novel antirheumatic agent that is effective in treating or preventing rheumatism and has no or weak side effects. One of the main objects of the present invention is to provide a novel antirheumatic agent.

[0005]

SUMMARY OF THE INVENTION First, the present inventors aimed at providing a novel antibiotic having an antibacterial activity and an antitumor activity to develop and commercialize a more useful antibiotic. Has promoted research. As a result, a strain belonging to the genus Amycolatopsis was successfully isolated as a novel microorganism from a soil sample, and the Amycolatopsis sp.MK 299-95F4 strain named for this strain was obtained from a plurality of strains having a new structural skeleton. He found that he was producing antibiotics. Succeeded in isolating these two new antibiotics,
They were named epoxyquinomicin A and epoxyquinomicin B, respectively. Furthermore, it has been found that these novel antibiotics show antibacterial activity against Gram-positive bacteria including drug-resistant bacteria (such as methicillin-resistant bacteria) and have antitumor activity to suppress the growth of cancer cells (1995). December 4
(See Japanese Patent Application No. 7-315542, filed on Feb. 19, 2009).

[0006] Further, the present inventors proceeded with research, and as a result, as a result, the above-mentioned bacterium belonging to the genus Amycolatopsis produced Epoxyquinomycin A.
And B produced two new compounds having the same chemical structure skeleton but different but different structures. This time, we succeeded in isolating two of these new compounds, one each of epoxyquinomicin C and epoxyquinomicin D.
It was named.

The inventors of the present invention have been diligently researching a substance having anti-rheumatic activity from metabolites of microorganisms. The activity was studied. As a result, it was found that the epoxyquinomicins C and D according to the present invention inhibit collagen-induced arthritis which is an animal experimental model for rheumatoid arthritis. In addition, the present inventors have found that the epoxyquinomicins A and B also have antirheumatic activity. The present invention has been completed based on these findings.

[0008] Epoxyquinomycin C and epoxyquinomycin D, which were newly obtained by the present inventors, showed weak antibacterial activity against specific bacteria, but had an activity of suppressing the growth of various cancer cells. Was found to be quite low.

Therefore, in the first invention, the following general formula (I) (Wherein, R represents a hydrogen atom in epoxyquinomycin C and a chlorine atom in epoxyquinomycin D)
Provided are epoxy quinomycin C and epoxy quinomycin D, or salts thereof.

Epoxyquinomycins C and D are weakly acidic substances. These salts include salts with organic bases such as quaternary ammonium salts, or salts with various metals, for example, alkali metals such as sodium. And these salts also have the above-mentioned antirheumatic activity.

Next, the physicochemical properties of the antibiotics epoxyquinomicins C and D will be described. (1) Physicochemical properties of epoxyquinomicin C A) Appearance and properties: white powder, weakly acidic substance B) Melting point: 168-172 ° C (decomposition) C) Specific rotation: [α] _D ²⁵ + 128 ° ( c) 1.0) Methanol) D) TLC Rf value: 0.31 When measured by thin-layer chromatography on silica gel (Art. 105715, manufactured by Merck) with chloroform-methanol (10: 1) as a developing solvent. spectrum (m / z): 292 ( M + H) + 290 (M-H) - F) high resolution mass spectrum: Found 292.0821 (M ⁺ H) + calcd 292.0804 G) molecular _{formula: C 14 H 13 NO 6 H} ) ultraviolet absorption Spectra: (i) UV absorption spectrum measured in methanol solution is shown by a solid line in FIG. 1 of the accompanying drawings. The main peaks are as follows. λ max nm (ε) 297 (17430) (ii) UV measured in 0.01 N NaOH-methanol solution
The absorption spectrum is shown by a dotted line in FIG. 1 of the accompanying drawings. The main peaks are as follows. λmax nm (ε) 304 (18270), 364 (9750) (iii) The UV absorption spectrum measured in 0.01N HCl-methanol solution is shown by a broken line in FIG. 1 of the accompanying drawings. The main peaks are as follows. λmax nm (ε) 296 (18140) I) Infrared absorption spectrum (KBr tablet method): shown in FIG. 2 of the accompanying drawings. νmax (cm ^-1 ) 3431, 1604, 1537, 1460, 1309, 1232, 1
065, 750 J) ¹³ C-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 3 of the accompanying drawings. K) ¹ H-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 4 of the accompanying drawings. (2) Physicochemical properties of epoxyquinomicin D A) Appearance and properties: yellowish brown powder, weakly acidic substance B) Melting point: 163-168 ° C (decomposition) C) Specific rotation: [α] _D ²⁵ +142 ° (c 1.0, methanol) D) RLC value of TLC: 0.10 When measured by thin-layer chromatography on silica gel (Art. 105715, manufactured by Merck) using chloroform-methanol (10: 1) as a developing solvent. ) mass spectrum (m / z): 326 ( M + H) + 324 (M-H) - F) high resolution mass spectrum: Found 326.0431 (M ⁺ H) + calcd 326.0417 G) molecular _{formula: C 14 H 12 NO 6 Cl} H ) UV absorption spectrum: (i) The UV absorption spectrum measured in a methanol solution is shown by a solid line in FIG. 5 of the accompanying drawings. The main peaks are as follows. λ max nm (ε) 299 (17590) (ii) The absorption spectrum measured in 0.01 N NaOH-methanol solution is shown by a dotted line in FIG. 5 of the accompanying drawings. The main peaks are as follows. λmax nm (ε) 304 (18950), 367 (9230) (iii) The UV spectrum measured in 0.01N HCl-methanol solution is shown by a broken line in FIG. 5 of the accompanying drawings. The main peaks are as follows. λmax nm (ε) 297 (18530) I) Infrared absorption spectrum (KBr tablet method): shown in FIG. 6 of the accompanying drawings. νmax (cm ⁻¹ ) 3438, 1643, 1533, 1281, 1200 J) ¹³ C-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 7 of the accompanying drawings. K) ¹ H-NMR spectrum (CD ₃ OD / TMS): shown in FIG. 8 of the accompanying drawings. In addition, the biological properties of the antibiotics epoxyquinomicins C and D are described below.

A) Inhibitory effect on collagen-induced arthritis The effect on collagen-induced arthritis was evaluated by DBA /
Investigation was performed using 1J male mice. That is, type II collagen was emulsified with an equal volume of Freund's complete adjuvant to prepare a 1 mg / ml administration solution. This was sensitized by inoculating 0.1 ml of the solution into the skin at the ridge of the mouse. Three weeks later, type II collagen emulsified by the same procedure
0.1 ml was intraperitoneally administered to the mice to perform a boost to induce arthritis.

2 mg of epoxyquinomicin A and C
/ Kg or 4 mg / kg, and epoxyquinomicin B
1 mg / kg or 2 mg / kg was administered intraperitoneally three times a week from the day of the first collagen inoculation for a total of 6 weeks. The inhibitory effect of collagen-induced arthritis was evaluated by a score of 0 to 4 according to the degree of redness, swelling and tonicity of the forelimbs and hindlimbs (the highest score of the sum of the four limbs was 16). A score of 0 indicates no symptoms, a score of 1 indicates redness and swelling of only one small joint, such as a finger of a limb, and a score of 2 indicates two or more small joints, or a relatively large wrist or ankle. If the joint shows redness and swelling, a score of 3 indicates redness and swelling of one hand or foot, and a score of 4 indicates that the swelling of one hand or foot has reached the maximum, and the joint has a maximum. The case where it is determined that the tongue is accompanied is shown. Table 1 shows the results.

[0014]

2 mg / kg, 4 mg of epoxyquinomicin A
/ Kg, 1 mg / kg of epoxyquinomicin B, 2 mg / kg,
And 4 mg / kg of Epoxyquinomycin C significantly suppressed the arthritis score.

B) Antibacterial activity Antibiotics epoxyquinomycins C and D according to the invention
Are shown in Table 2 below. This antibacterial spectrum was measured by a multiple dilution method on Muller Hinton agar medium based on the standard method of the Japanese Society of Chemotherapy.

[0017]

C) Cancer Cell Growth Inhibitory Activity The concentration (IC ₅₀ value) of epoxyquinomycin C and epoxyquinomycin D, which inhibits the growth of cancer cells by 50% using various cancer cells, was determined by the MTT method (“Journal of Immunologica”).
l Methods ”, Vol. 65, pp. 55-60 (1983)).
Table 3 shows the results.

[0019]

D) Toxicity A single dose of 100 mg / kg of epoxyquinomycin C and epoxyquinomycin D was intraperitoneally administered to male ICR mice, but there were no dead individuals and no toxic symptoms were observed. In addition, 4 mg / kg / day of epoxyquinomycin C was intraperitoneally administered three times a week for a total of 6 weeks, but no dead individuals or individuals showing toxic symptoms were found. Epoxyquinomycin C has very low toxicity to warm-blooded animals.

As is clear from the results in Table 2, the epoxyquinomicins C and D according to the present invention are useful as antibacterial agents because they have weak antibacterial activity against specific bacteria. However, as is clear from the results in Table 3, epoxyquinomycins C and D inhibited the growth of various cancer cells by 100 μm.
g / ml did not suppress.

According to a second aspect of the present invention, bacteria producing the above-mentioned epoxyquinomycins C and D of the general formula (I) belonging to the genus Amycolatopsis are cultured in a nutrient medium, and the resulting culture is treated with epoxyquinomycin. A method for producing the antibiotics epoxyquinomicin C and / or epoxyquinomicin D is provided, wherein the method comprises collecting the mycin C and / or epoxyquinomicin D.

An example of a bacterium producing epoxyquinomicin C and D which can be used in the second method of the present invention is Amycolatopsis sp. MK299-95F4. This strain is an actinomycete isolated from soil in Sendai City, Miyagi Prefecture at the Institute of Microbial Chemistry in October 1994, and is a microorganism with the strain number of MK299-95F4.

The bacteriological properties of the MK299-95F4 strain are described below. 1. Morphology The basic hypha is well branched and has a zigzag shape. In addition, division is recognized. Aerial hyphae are straight or irregularly curved,
Break into cylindrical to oval fragments or spore-like structures.
Its surface is smooth and its size is about 0.4 ~ 0.6 × 1.1 ~
1.6 microns. Rotating branches, fungal hyphae, sporangia and motile spores are not found.

2. Growing condition in various media About color description Standards shown in [] are the color harmony manual (Container Corporation of America) of Container Corporation of America.
Color harmony manual). (1) Sucrose / nitrate agar medium (cultured at 27 ° C) White aerial hyphae grow slightly on colorless growth, and no soluble pigment is observed. (2) Glucose-asparagine agar medium (cultured at 27 ° C) The growth of light yellow [2ea, Lt Wheat-2gc, Bamboo]
A white aerial mycelium forms and the soluble pigment takes on a yellow tinge. (3) Glycerin-asparagine agar medium (ISP-medium 5, cultured at 27 ° C.) On growth of light yellow tea [3ie, Camel to 3le, Cinnamon], white aerial hyphae are formed, and the soluble pigment is yellow. Takes on tea. (4) Starch / inorganic salt agar medium (ISP-medium 4, 27 ° C
(Culture) White aerial mycelium slightly formed on colorless growth, and no soluble pigment was observed.

(5) Tyrosine agar medium (ISP-medium 7,
27 ° C) Light yellow tea [2lg, Mustard Tan]-grayish yellow tea [3lg, Ad
Obe Brown] develops white aerial hyphae on the development, and the soluble pigment exhibits light yellow tea. (6) Nutrient agar medium (cultured at 27 ° C.) White aerial hyphae grow slightly on the growth of pale yellow [2ea, Lt Wheat], and no soluble pigment is observed. (7) Yeast-malt agar medium (ISP-medium 2, cultivated at 27 ° C) White aerial hyphae grow slightly on the growth of light yellow tea [3ic, Lt Amber], and no soluble pigment is observed. . (8) Oatmeal agar medium (ISP-medium 3, cultivated at 27 ° C) White aerial mycelia slightly formed on the growth of colorless to pale yellow [1 1/2 ca, Cream], and soluble pigment was observed. Absent. (9) Starch agar medium (cultured at 27 ° C) White aerial hyphae grow slightly on colorless growth, and no soluble pigment is observed. (10) Lime malate agar medium (cultured at 27 ° C) White aerial hyphae grow slightly on colorless growth, and no soluble pigment is observed.

3. Physiological properties (1) Growth temperature range Glucose / asparagine agar medium (glucose 1.0
%, L-asparagine 0.05%, dipotassium hydrogen phosphate
0.05%, string agar 3.0%, pH7.0), 10 ℃, 20 ℃
As a result of testing at each temperature of 24 ° C., 24 ° C., 27 ° C., 30 ° C., 37 ° C. and 50 ° C., no growth was observed at 10 ° C. and 50 ° C.
It grew in the range of ° C. The optimal growth temperature seems to be around 27 ° C. (2) Starch hydrolysis (starch / inorganic salt agar,
(ISP-medium 4 and starch agar medium, both cultured at 27 ° C.) After 21 days of culture, the culture medium is negative in any medium. (3) Production of melanin-like pigment (trypton yeast broth, ISP-medium 1: peptone yeast iron agar medium, ISP-medium 6; tyrosine agar medium, ISP-medium 7; all cultured at 27 ° C.) It is also negative in the medium.

(4) Utilization of carbon source (Pridham-Godlieve agar medium, ISP-medium 9; culture at 27 ° C.) D-glucose, D-fructose, inositol, D
-Grown using mannitol, L-arabinose,
Sucrose, rhamnose and raffinose are not used. Whether D-xylose is used is not clear. (5) Dissolution of malate lime (maleic acid lime agar medium, 27 ° C
(Culture) From about 10 days after the culture, lime malate was dissolved,
Its effect is moderate. (6) Nitrate reduction reaction (peptone water containing 0.1% potassium nitrate, ISP-medium 8, cultured at 27 ° C) Negative.

To summarize the above properties, in the MK299-95F4 strain, the basal hypha is well-branched, has a zigzag shape, and is fragmented. Aerial hyphae are straight or irregularly curved and break into cylindrical to oval fragments or spore-like structures. Rotating branches, fungal hyphae, sporangia and motile spores are not found. In various media, white aerial mycelium develops on the development of colorless to light yellow to light yellow tea. In some media, the soluble pigment takes on yellow or yellowish brown. The formation of melanin-like pigments, starch hydrolysis and nitrate reduction were all negative.

The bacterial cell component of the MK299-95F4 strain contained meso-type 2,6-diaminopimelic acid, arabinose and galactose in the cell wall, and exhibited cell wall type IV. The reducing sugar in all cells was type A containing arabinose and galactose. The results of the glycolate test were of the acetyl type. Further, mycolic acid is not contained, the phospholipid PII type (not including phosphatidylcholine and unknown glucosamine-containing phospholipids include phosphatidyl ethanolamine), major menaquinone was MK-9 (H _4). Fatty acids are 16: 0, i-15: 0, 16: 1, i-1
The main components were 6: 0 and 17: 0.

From the above results, the MK299-95F4 strain belongs to the genus Amycolatopsis (Reference: "Internatio
nal Journal of Systematic Bacteriology, Vol. 36, 29-
37, 1986). Searching for a known strain of the genus Amycolatopsis, Amycolatopsis sulphurea (Reference 1: Ibid .; and Reference 2: “International Journal of Systema
tic Bacteriology ”, 37, pp. 292-295, 1987)
It was given as a closely related species. Therefore, the MK299-95F4 strain and Amycolatopsis sulphre strain preserved in our laboratory are being compared on a practical basis. At present, the MK299-95F4 strain was transformed into Amycolatopsis sp.
F4. The MK299-95F4 strain was applied for deposit at the National Institute of Bioscience and Human Technology, and received the deposit number FERM P-15243 on October 17, 1995.

In carrying out the second method of the present invention, bacteria producing epoxyquinomycins C and D belonging to the genus Amycolatopsis are inoculated into a nutrient medium and cultured in this medium. The nutrient medium used here contains a carbon source and a nitrogen source that can be assimilated by the above-mentioned production bacteria as nutrient components.

As the nutrient, those usually used as nutrients for microorganisms, for example, assimilable nutrients such as carbon source, nitrogen source and inorganic salts can be used. For example, carbohydrates such as glucose, maltose, molasses, dextrin, glycerin and starch, and carbon sources such as soybean oil and oils such as peanut oil, and peptone, meat extract, cottonseed powder, soybean powder, yeast extract, casein, corn・ Nitrogen sources such as steep liquor, NZ-amine, ammonium sulfate, ammonium nitrate, and ammonium chloride; and inorganic salts such as dipotassium phosphate, sodium phosphate, salt, calcium carbonate, magnesium sulfate, and manganese chloride can be used. , Iron and the like can be added. As a nutrient, any known nutrient can be used as long as the bacteria used can produce the antibiotics epoxyquinomicin C and D.

The mixing ratio of the above-mentioned nutrients in the medium is not particularly limited, and can be varied over a wide range.
Depending on the producing bacteria, the optimal composition and composition of the nutrient source is
Those skilled in the art can easily determine by a simple small-scale experiment. Further, the nutrient medium consisting of the above-mentioned nutrients can be sterilized prior to cultivation, and before or after this sterilization, the pH of the medium is in the range of 6-8, especially pH 6.5-.
It is advantageous to adjust it to a range of 7.5.

The cultivation of the epoxyquinomycin C and D producing bacteria in such a nutrient medium can be carried out according to a method generally used in the production of antibiotics by general actinomycetes. Usually, the culture is preferably performed under aerobic conditions, and can be performed with stirring and / or aeration. As the culture method, any of stationary culture, shaking culture, and submerged culture with aeration and stirring can be used, but liquid culture is suitable for mass production of epoxyquinomicins C and D.

The cultivation temperature that can be used is not particularly limited as long as the growth of the epoxyquinomycin C and D producing bacteria is not substantially inhibited and the antibiotic can be produced. It can be selected appropriately according to the bacteria,
Particularly preferred are temperatures in the range of 25-30 ° C. The cultivation can usually be continued until the epoxyquinomicins C and D have sufficiently accumulated. The culturing time varies depending on the composition of the medium, the culturing temperature, the working temperature, the used bacterial strain, and the like, but usually the desired antibiotic can be obtained by culturing for 72 to 120 hours. The amount of accumulated epoxyquinomycins C and D in the medium during the culturing can be determined by using a Staphylococcus aureus smith by a cylindrical plate method used for the usual determination of antibiotics.

The epoxyquinomicins C and D thus accumulated in the culture are taken from the culture.
After the culture, if necessary, filtration, after removing the cells from the culture by a known separation method such as centrifugation,
The culture filtrate is adjusted to acidic (pH 2-4) and extracted with an organic solvent, especially ethyl acetate, chromatography using adsorption or ion exchange, gel filtration, chromatography using countercurrent distribution. Can be used alone or in combination to isolate and purify the desired antibiotic. Activated carbon, silica gel, porous polystyrene-divinylbenzene resin, or various ion exchange resins can be used as a chromatography carrier having adsorption or ion exchange ability. From the separated cells, the desired antibiotic can be extracted from the cells by a solvent extraction method using an appropriate organic solvent or an elution method by crushing the cells, and can be isolated and purified in the same manner as described above. Thus, new compounds epoxyquinomycins C and D having the above-mentioned properties are obtained.

Further, in the third invention, the above-mentioned general formula (I) (Wherein, R represents a hydrogen atom in epoxyquinomycin C and a chlorine atom in epoxyquinomycin D)
The antibiotics epoxyquinomycin C and epoxyquinomycin D which are compounds represented by the following general formula (II): (Wherein, R represents a chlorine atom in epoxyquinomicin A, and a hydrogen atom in epoxyquinomycin B)
An antirheumatic agent comprising as an active ingredient at least one compound selected from the antibiotics epoxyquinomycin A and epoxyquinomycin B, which are compounds represented by the formula: or salts thereof.

In the third antirheumatic agent according to the present invention, the epoxyquinomycins or pharmaceutically acceptable salts thereof as active ingredients are pharmaceutically acceptable conventional solid or liquid carriers such as ethanol, water and the like. , Starch and the like.

Epoxyquinomycin A and epoxyquinomycin B used as active ingredients in the third antirheumatic drug according to the present invention are novel substances, and their physicochemical properties are described in detail in Japanese Patent Application No. Hei 7-315542. MK 299-95F4 is described in the specification, and the method for producing them by culturing the above-mentioned Amycolatopsis sp. Strain MK 299-95F4 is also described.

The main physicochemical properties of epoxyquinomicins A and B are described below. (1) Physicochemical properties of epoxyquinomicin A A) Appearance and properties: pale yellow powder, weakly acidic substance B) Melting point: 168-173 ° C (decomposition) C) Specific rotation: [α] _D ²⁵ +44.6 ° (c 0.51, methanol) D) RLC value of TLC: 0.28 When measured by thin-layer chromatography on silica gel (Art. 105715, manufactured by Merck) using chloroform-methanol (10: 1) as a developing solvent.

E) Molecular formula: C ₁₄ H ₁₀ NO ₆ Cl F) Ultraviolet absorption spectrum: The main peaks of the UV absorption spectrum measured in a methanol solution are as follows. λmax nm (ε) 236 (sh. 8900), 255 (sh. 5900), 325 (80
00), 370 (sh, 2700) G) Infrared absorption spectrum (KBr tablet method) νmax (cm ^-1 ) 3450, 1710, 1670, 1600, 1520, 1460, 1
340, 1230

(2) Physicochemical properties of epoxyquinomicin B A) Appearance and properties: pale yellow powder, weakly acidic substance B) Melting point: 178-184 ° C (decomposition) C) Specific rotation: [α] _D ²⁵ + 32.2 ° (c 0.51, methanol) D) Rf value of TLC: 0.52 Measured by developing with thin-layer chromatography on silica gel (Art. 105715, Merck) with chloroform-methanol (10: 1) as a developing solvent. if you did this.

E) Molecular formula: C ₁₄ H ₁₁ NO ₆ F) Ultraviolet absorption spectrum: The main peaks of the UV absorption spectrum measured in a methanol solution are as follows. λmax nm (ε) 237 (6100), 253 (sh, 5400), 326 (6300) G) Infrared absorption spectrum (KBr tablet method) νmax (cm ⁻¹ ) 3430, 1710, 1660, 1610, 1530, 1340, 1
230

Epoxyquinomycins C and D and Epoxyquinomycins A and B used as active ingredients in the third antirheumatic agent according to the present invention, as described above, inhibit collagen-induced arthritis which is an animal experimental model of rheumatoid arthritis. Has inhibitory activity. Epoxyquinomycins C and D and epoxyquinomycins A and B, especially when used as antirheumatic agents, vary in their dosage depending on the condition, but generally do not exceed the adult daily dose of 10%.
The dose is 20002000 mg, preferably 20-600 mg, and it may be administered in 1 to 3 divided doses as necessary according to the symptoms. The administration method can be in a form suitable for administration, and oral administration or intravenous administration is particularly desirable.

Epoxyquinomycins A to D have an inhibitory effect on collagen-induced arthritis as described above. Therefore, they can be used not only for rheumatoid arthritis but also as autoimmune reducing or inhibiting agents for systemic lupus erythematosus, systemic sclerosis, It can be expected to be effectively applied to the prevention or treatment of autoimmune diseases such as periarteritis nodosa, ulcerative colitis and juvenile diabetes.

[0047]

Next, the present invention will be described in more detail by way of examples, but the present invention is not limited to the following examples.

Example 1 Preparation of Antibiotic Epoxyquinomicins C and D and Epoxyquinomicins A and B (A) Glycerin 0.5%, sucrose 2%, soybean flour 1%, dried yeast 1%, corn steep liquor
Dispense 110 ml of a liquid medium (adjusted to pH 7.0) containing 0.5% and cobalt chloride 0.001% into an Erlenmeyer flask (500 ml volume),
The solution was sterilized at 120 ° C for 20 minutes by a conventional method. In these media, Amycolatopsis sp. MK299-95F cultured on an agar slant medium
Four strains (FERM P-15243) were inoculated, followed by rotary shaking culture at 30 ° C. for 5 days. Thus, a seed culture was obtained.

Glycerin 2%, dextrin 2%,
A liquid medium (adjusted to pH 7.4) containing 1% Bacto-Soyton, 0.3% powdered yeast extract, 0.2% ammonium sulfate, 0.2% calcium carbonate and 1 drop of silicone oil was dispensed into Erlenmeyer flasks (500ml volume) in 110ml increments. 120 ℃ by standard method
For 20 minutes. Thereafter, 2 ml of each of the above seed cultures was inoculated into each of these culture media, and cultured with rotation and shaking at 27 ° C. for 4 days.

The culture thus obtained was centrifuged to remove the cells. 1.8 liters (L) of the culture filtrate
After adjusting the pH to 2 with 6N-HCl, the mixture was extracted with 1.8 L of butyl acetate, and the butyl acetate layer was dried over anhydrous sodium sulfate. The butyl acetate layer was concentrated to dryness under reduced pressure, and the residue was dissolved in 50 ml of methanol and washed twice with 50 ml of hexane. The methanol layer was concentrated to dryness under reduced pressure to give a brown oil (980
mg) was obtained. This oily substance was added to a silica gel column (Merc
k, Kieselgel 60, 120 ml) and sequentially eluted with a toluene-acetone system (10: 1, 5: 1, 3: 1) to obtain 18 mg of epoxyquinomycin A and 19 mg of epoxyquinomycin B.
mg, 170mg of a mixture of epoxyquinomicin C and D
Obtained. 51 mg of this mixture was applied to silica gel TLC (Merc
k, Art.105715, chloroform-10% aqueous methanol = 1
(3 developments at 0: 1) to give 13 mg of epoxyquinomycin C as a white solid, and 23 mg of epoxyquinomycin D as a yellowish brown powder. That is, epoxyquinomycin C was obtained as a white powder having a melting point of 168 to 172 ° C (decomposition) in a yield of 13 mg, and epoxyquinomycin D was obtained as a yellowish brown powder having a melting point of 163 to 168 ° C (decomposition).
Obtained in a yield of 23 mg. (B) The culture solution obtained in the same manner as in the above (A) was filtered to separate the cells. After 2.55 liters (L) of the culture filtrate was adjusted to pH 2 with 6N-HCl, butyl acetate was added.
The mixture was extracted with 2.55 L, and the butyl acetate layer was dried over anhydrous sodium sulfate. The butyl acetate layer was concentrated to dryness under reduced pressure, the residue was dissolved in 50 ml of methanol, washed twice with 50 ml of hexane, and the methanol layer was concentrated to dryness under reduced pressure. The obtained residue was subjected to chloroform-methanol-water (50: 10: 40,100m
l) and the lower layer was concentrated to dryness under reduced pressure to give a brown oil (0.515 g). This oil was subjected to silica gel column chromatography (Kieselgel 60, manufactured by Merck, 50 ml), and a toluene-acetone mixed solvent (10:10) was used.
1,7: 1,5: 1,3: 1,2: 1). The obtained active fraction was subjected to silica gel column chromatography under the same conditions, and eluted sequentially with a toluene-acetone mixed solvent (50: 1, 20: 1, 10: 1, 7: 1).
124 mg of a mixture of epoxyquinomicins A and B was obtained. 35 mg of this mixture was separated and purified by silica gel TLC (developing solvent: chloroform-methanol, 20: 1). Epoxyquinomicin A has a melting point of 168-173 ° C
Epoxyquinomycin B was obtained in a yield of 20 mg as a pale yellow powder (decomposed) and as a pale yellow powder having a melting point of 178-184 ° C. (decomposed).

[Brief description of the drawings]

FIG. 1. In a methanol solution of epoxyquinomycin C,
It is each ultraviolet-ray absorption spectrum in a 0.01N NaOH-methanol solution and a 0.01N HCl-methanol solution.

FIG. 2 is an infrared absorption spectrum of epoxyquinomycin C measured by a KBr tablet method.

FIG. 3 is a proton nuclear magnetic resonance spectrum measured with a heavy methanol solution of epoxyquinomicin C (internal standard: trimethylsilane).

FIG. 4 is a 13 C nuclear magnetic resonance spectrum measured with a heavy methanol solution of epoxyquinomicin C (internal standard: trimethylsilane).

FIG. 5: In a methanol solution of epoxyquinomicin D,
It is each ultraviolet-ray absorption spectrum in a 0.01N NaOH-methanol solution and a 0.01N HCl-methanol solution.

FIG. 6 is an infrared absorption spectrum of epoxyquinomicin D measured by a KBr tablet method.

FIG. 7 is a proton nuclear magnetic resonance spectrum measured with a heavy methanol solution of epoxyquinomicin D (internal standard: trimethylsilane).

FIG. 8 is a 13 C nuclear magnetic resonance spectrum measured with a heavy methanol solution of epoxyquinomycin D (internal standard: trimethylsilane).

 ──────────────────────────────────────────────────続 き Continuation of the front page (72) Inventor Hironobu Iinuma 4-61-17, Hakusan, Midori-ku, Yokohama-shi, Kanagawa Prefecture (72) Inventor Riki Sawa 4-6-7-1, Ayanishi, Ayase-shi, Kanagawa Prefecture (72) Inventor Hiroshi Naganawa 3-17, Denonchofuhonmachi, Ota-ku, Tokyo (72) Inventor Masaru Hamada 1-26 Naitocho, Shinjuku-ku, Tokyo Shuwa Residence 405 (72) Inventor Shinichi Hirano 5--8, Honmura, Chigasaki-shi, Kanagawa 1-207 (72) Inventor Naoki Matsumoto 11-3, Sachigaoka, Asahi-ku, Yokohama-shi, Kanagawa Prefecture 102 1st Green Corp. 102 (72) Inventor Masaaki Ishizuka 6-5 Nishiwakacho, Mishima-shi, Shizuoka Pastoral Heim Ichibankan 411

Claims (3)

[Claims] 1. The following general formula (I) (Wherein, R represents a hydrogen atom in epoxyquinomycin C and a chlorine atom in epoxyquinomycin D)
The antibiotics epoxyquinomycin C and epoxyquinomycin D, which are compounds represented by the formula: or salts thereof. 2. The method according to claim 1, which belongs to the genus Amycolatopsis.
The antibiotics epoxyquinomicin C and / or D are obtained by culturing the bacteria producing epoxyquinomicin C and D described in 1) in a nutrient medium, and collecting epoxyquinomicin C and / or D from the culture. ) Method for producing epoxyquinomicin D. 3. The following general formula (I) (Wherein, R represents a hydrogen atom in epoxyquinomycin C and a chlorine atom in epoxyquinomycin D)
The antibiotics epoxyquinomycin C and epoxyquinomycin D which are compounds represented by the following general formula (II): (Wherein, R represents a chlorine atom in epoxyquinomicin A, and a hydrogen atom in epoxyquinomycin B)
A rheumatic agent comprising, as an active ingredient, at least one compound selected from the antibiotics epoxyquinomycin A and epoxyquinomycin B, which are compounds represented by the formula: