JPH0338276B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a pharmacologically active FR-900506 substance or a salt thereof and a method for producing the same. More specifically, this invention provides novel FR-
900506 Substance or its salt and its manufacturing method. That is, one object of the present invention is to provide a novel FR-900506 substance or a salt thereof useful for the treatment and prevention of transplant rejection, graft-versus-host disease caused by bone marrow transplantation, autoimmune diseases, infectious diseases, etc. It is. Another purpose of this invention is to produce
An object of the present invention is to provide a method for producing FR-900506 substance or a salt thereof. The FR-900506 substance of the present invention was isolated for the first time in pure form from a culture broth obtained by fermenting a new bacterial species belonging to the genus Streptomyces. As a result of intensive research, the inventors of this invention discovered that FR-
The chemical structure of substance 900506 was successfully determined. The novel FR-900506 substance of this invention can be represented by the following general formula. [Chemical name: 17-allyl-1,14-hydroxy-12
-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3. 1.0 ^4,9 ] octacos-18-ene-2,3,10,16-tetraone] In the FR-900506 substance of this invention, optical isomers and geometric isomers due to conformers or asymmetric carbon atoms and double bonds There may be one or more pairs of stereoisomers such as conformers or isomers, and such conformers or isomers are also included within the scope of this invention. The method for producing the FR-900506 substance of the present invention will be described in detail below. FR-900506 of this invention is Streptomyces tsukubaensis (Streptomyces tsukubaensis).
It can be produced by fermenting a FR-900506 substance-producing strain belonging to the genus Streptomyces, such as No. 9993 (Tsukubaensis) No. 9993, in a medium. The production of FR-900506 substance by fermentation will be explained below. Microorganisms The bacteria used to produce the FR-900506 substance are FR-900506 producing strains belonging to the genus Streptomyces, of which Streptomyces tsukubaensis No. 9993 was extracted from a soil sample collected in Toyosato Town, Tsukuba District, Ibaraki Prefecture. Newly separated. This newly isolated freeze-dried specimen of Streptomyces tsukubaensis No. 9993 was deposited at the Institute of Microbiology, Agency of Industrial Science and Technology (1-1-3 Higashi, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture) with the deposit number on October 5, 1984. Deposited as Microtechnology Research Institute No. 7886, then in September 1985
On the 19th, the deposit was transferred to the Budapest Treaty route under the new depositary number 927. In the production of the novel FR-900506 substance, the bacteria described in this specification are merely for illustrative purposes and are not intended to be limiting.
This invention also includes natural mutant bacteria, X-ray irradiation, ultraviolet irradiation, N-methyl-N'-nitro-
This includes the use of any mutant strain capable of producing the FR-900506 substance, including artificial mutant strains created by conventional treatment with N-nitrosoguanidine, 2-aminopurine, etc. Streptomyces tsukubaensis No. 9993 has the following morphological properties, culture characteristics, biological and physiological characteristics. [1] Morphological properties Primarily, the method described in Shirling, E. B. and Gottlieb, D.: Methods for Characterization of Streptomyces Species ( International Journal of Systematic Bacteriology (Methods for
Characterization of Streptomyces species.
International Journal of Systematic
Bacteriology) Volume 16, pp. 313-340, 1966
year] was used for taxonomic examination. Morphological observations were made on oatmeal agar, yeast malt extract agar, and inorganic salt
Light and electron microscopy were used on cultures grown on starch agar at 30°C for 14 days. Mature sporophytes formed straight or wavy chains (Rectiflexibiles) with a number of 10 to 50 or more than 50 spores in each spore chain. According to observation with an electron microscope, the spores are oval and cylindrical.
The size is 0.5-0.7×0.7-0.8 μm. The spore surface was smooth. [2] Culture characteristics Culture characteristics are described in the above-mentioned Shearling and Gottlieb and Waksman [Waksman S.A.: The Actinomycetes, Volume 2, Classification, Identification and Description]・Of Generala and Species. The·
Williams & Wilkins Company, Baltimore, 1961 (Waksman, S.
A.: The actinomycetes, Vol.2:
Classification, identification and
description of genera and species.The
Williams and Wilkins Co., Baltimore;
1961)] using 10 types of culture media. Cultured at 30°C for 14 days. The color names used in this study were based on the New Color Name Book (handbook of the Japan Color Research Institute, Tokyo). Oatmeal agar, yeast malt extract agar and inorganic salt
When grown on starch agar, the colonies belonged to the gray color system. Soluble pigments are yeast
It was produced on malt extract agar, but not on other media. The results are shown in Table 1.

【table】

[Table] Cell wall composition analysis was performed using the method of Betzker et al.
R.E. Gordon and H.A.
Receiver Ariel: Rapid Difference Between Nocardia and Streptomyces by Paper Chromatography of Orph Cell Hydrolysates: Applied Microbiology (Becker, B., MPLechvalier, REGordon
and HALechevalier：Rapid differentiation
between Nocardia and Streptomyces by
paper chromatography of whole cell
hydrolysates: Appl.Microbiol.) Volume 12, No.
421-423, 1964] and the method of Yamaguchi (Yamaguchi, T.: Comparison of the Cell Wall Composition of Morphological Distinct Actinomycetes: Journal of Bacteriology ( Yamaguchi, T.: Comparison of the cell.
wall composition of morphologically
distinct actinomycetes: J.Bacteriol.) No. 89
Vol. 444-453, 1965]. Analysis of whole cell hydrolysate of strain No. 9993 showed the presence of LLM diaminopimelic acid. Therefore, the cell wall of this strain is considered to belong to the type. [3] Biological and Physiological Properties The physiological properties of strain No. 9993 were determined by the method described in Shearling and Gottlieb, supra. The results are shown in Table 2. The growth temperature range and optimum growth temperature are
It was measured on malt extract agar using a temperature gradient incubator (manufactured by Toyo Kagaku Sangyo Co., Ltd.). The growth temperature range was 18-35°C, and the optimum growth temperature was 28°C. Milk peptonization and gelatin liquefaction were positive. Production of melamine pigment was negative.

【table】

[Table] The utilization of carbon sources by Pridham and Gottlieb's method
An Aid to Species Determination: Journal of Bacteriology (Pridham, TG and D. Gottlieb: The
utilization of carbon compounds by some
Actinomycetales as an aid for species
determination: J.Bacteriol.) Volume 56, No. 107
~114, 1948]. at 30℃
After culturing for 14 days, the growth status was observed. The carbon source utilization of this strain is summarized in Table 3. Glycerin, maltose and sodium succinate were utilized by strain no.9993. Furthermore, D-glucose, sucrose,
D-mannose and salicin are utilized to some extent.

【table】

[Table] −: Not used Based on the results of microscopic observation and cell wall composition analysis of strain No. 9993, this strain was found to be Waksman, Henrici,
In 1943, it was revealed that it belonged to the genus Streptomyces. In other words, this strain has been evaluated according to its published properties [International Journal of Systematic Bacteriology].
Bacteriology) Volume 18, pp. 69-189, No. 279
~392 pages, 1968 and Volume 19, pp. 301-512,
1969, as well as the Burgess Manual
Bergey's Manual of Determinative Bacteriology
Bacteriology) 8th edition, 1974].
Comparisons were made with various species of the genus Streptomyces. As a result of comparison, strain No. 9993 was found to be Streptomyces aburaviensis Nishimura et.
al.), Streptomyces avuelaneus baldatsusi and grains (Streptomyces
avellaneus Baldacci and Grein) and Streptomyces misakiensis Nakamura. Therefore, we compared the culture characteristics of strain No. 9993 with the corresponding Streptomyces abraviensis.
IFO12830, Streptomyces avuelaneus IFO13451 and Streptomyces misakiensis IFO12891 were compared. The resulting strain
No.9993 is Streptomyces misakiensis
It was most similar to IFO12891. Therefore, strain no.
9993 was further compared in detail with Streptomyces misakiensis IFO12891 shown in Tables 1, 2 and 3 above. From this result, strain No. 9993 is Streptomyces misakiensis IFO12891.
and can be distinguished from each other by the following points, and therefore the strain
No.9993 is thought to be a new species of Streptomyces, and is named after the soil where this microorganism was collected in Tsukuba District, Ibaraki Prefecture.
sp.nov.). Differences from Streptomyces misakiensis IFO12891 The culture characteristics of strain No. 9993 are similar to Streptomyces misakiensis.
misakiensis IFO12891 when cultured on oatmeal agar, yeast malt extract agar, glucose-asparagine agar, Suzapetsk agar and potato dextrose agar. Although starch hydrolysis of strain No. 9993 was negative, Streptomyces misakiensis
In case of IFO12891, it is positive. Gelatin liquefaction is positive for strain No. 9993, but negative for Streptomyces misakiensis IFO12891. In terms of carbon source utilization, strain No. 9993 utilizes glycerin, maltose and sodium succinate, whereas Streptomyces misakiensis
IFO12891 does not use these. In addition, strains
No.9993 does not use D-galactose and inulin, but Streptomyces misakiensis
IFO12891 can use these. Production of FR-900506 substance The novel FR-900506 substance of the present invention is a FR-900506 substance belonging to the genus Streptomyces, such as Streptomyces tsukubaensis No. 9993, (Deposit number: Kaikokenjo Kyori No. 927). It can be produced by culturing the 900506 substance-producing strain in a medium. Generally, FR-900506 material is FR-900506
The substance-producing bacterial strain can be produced by culturing it in an aqueous medium containing assimilable carbon and nitrogen sources, preferably under aerobic conditions, such as shaking culture or submerged culture. Preferred carbon sources for the medium include glucose,
Carbohydrates such as xylose, galactose, glycerin, starch, dextrin, etc. may be mentioned. Other carbon sources include maltose, rhamnose, raffinose, arabinose, mannose,
Examples include salicin and sodium succinate. Preferred nitrogen sources include yeast extract, peptone, gluten meal, cottonseed flour, soy flour, corn staple liquor, dried yeast, wheat germ, feather flour, peanut flour, and, for example, ammonium nitrate, ammonium sulfate, ammonium phosphate, etc. Examples include inorganic or organic nitrogen compounds such as ammonium salts of urea, amino acids, etc. Carbon and nitrogen sources are preferably used in combination, although less pure substances can be used if they contain trace amounts of growth factors and significant amounts of inorganic nutrients and do not necessarily have to be used in pure form. There isn't. If desired, the medium may contain sodium carbonate or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salt,
Inorganic salts such as copper salts and cobalt salts may be added.
Particularly if the culture medium foams significantly, antifoaming agents such as liquid paraffin, fatty oil, vegetable oil, mineral oil or silicone may be added if necessary. The conditions for mass production of FR-900506 substance are as follows:
Deep aerobic culture is preferred. For small-scale production, shaking culture or surface culture is carried out in flasks or bottles. Furthermore, when the growth is carried out in a large tank, it is preferable to inoculate the bacteria into the production tank using a pre-culture of the microorganism in order to avoid growth delays in the production process of the FR-900506 substance. That is, a relatively small amount of cultured ash is inoculated with microbial spores or hyphae, the inoculated ash is cultured to first produce a pre-cultured inoculum of microorganisms, and then the cultured pre-cultured inoculum is aseptically grown to a large size. It is preferable to transfer it to a tank. The medium in which this preculture inoculum is produced may be substantially the same as the medium used to produce the FR-900506 material, or it may be different. Stirring and aeration of the culture mixture can be carried out in various ways, by using a propeller or similar stirring device, by rotating or shaking the fermenter, by using various pump devices, or This can also be done by passing sterile air through the medium. Aeration may be accomplished by passing sterile air through the fermentation mixture. Fermentation is usually carried out at a temperature range of about 20° C. to 40° C., preferably 25° C. to 35° C., for about 50 to 150 hours, but this may be changed as appropriate depending on the fermentation conditions and fermentation scale. The FR-900506 substance produced in this way is
It can be recovered from the culture medium by conventional methods commonly used for recovery of other known biologically active substances. The produced FR-900506 is found in the cultured mycelium and in the filtrate, and therefore the FR-900506 substance can be extracted from the mycelium and filtrate obtained by filtration or centrifugation of the culture broth, vacuum concentration, freeze-drying, Extraction with conventional solvents, pH adjustment, treatment with conventional resins such as anion exchange resins or cation exchange resins, nonionic adsorption resins, conventional adsorbents such as activated carbon, silicic acid, silica gel, cellulose, alumina, etc. It can be separated and purified by conventional methods such as treatment, crystallization, and recrystallization. Physicochemical properties of FR-900506 substance The FR-900506 substance produced by the above fermentation method has the following physicochemical properties. (1) Shape and color: White powder (2) Elemental analysis: C: 64.72%, H: 8.78%, N: 1.59% 64.59%, 8.74%, 1.62% (3) Color reaction: Positive: Cerium sulfate reaction , sulfuric acid reaction, Ehrlichi reaction, Dragendorff reaction, iodine vapor reaction Negative: ferric chloride reaction, ninhydrin reaction, Moritsch reaction (4) Solubility: Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethyl ether , benzene Poorly soluble: hexane, petroleum ether Insoluble: water (5) Melting point: 85-90°C (6) Specific rotation: [α] ²³ _D : -73° (C = 0.8, CHCl ₃ ) (7) Ultraviolet absorption Spectrum: Terminal absorption (8) Infrared absorption spectrum: ν ^CHCl _nax 3: 3680, 3580, 3520, 2930, 2870,
2830, 1745, 1720, 1700, 1645, 1450,
1380, 1350, 1330, 1310, 1285, 1170,
1135, 1090, 1050, 1030, 1000, 990, 960
(sh), 918cm ^-1 (9) ¹³ C nuclear magnetic resonance spectrum: δ (ppm, CDCl ₃ ): {212.59 (s), 212.45 (s)}
,
{196.18(s), 192.87(s)}, {169.07(s),
168.90(s)}, {164.90(s), 166.01(s)},
{138.89(s), 139.67(s)}, {135.73(d),
135.60(d)}, {132.52(s), 131.99(s)},
{130.27(d), 130.21(d)}, {122.87(d),
123.01(d)}, {116.57(t), 116.56(t)},
{97.35(s), 98.76(s)}, 84.41(d), {77.79
(d), 78.22(d)}, {75.54(d), 76.97(d)
},
{73.93(d), 73.09(d)}, {73.72(d), 72.57
(d)}, {70.05(d), 69.15(d)}, 56.75(d
),
{53.03(d), 53.13(d)}, {48.85(t), 48.62
(t)}, {40.33(d), 40.85(d)}, 39.40(t
),
31.58 (t), 30.79 (t), {27.72 (t), 26.34
(t)}, 26.46(d), 24.65(t), {20.45(q)
,
19.73(q)}, {14.06(q), 14.23(q)}, {9.6
9
(q), 9.98(q)} A chart of the above spectrum is shown in FIG. (10) ¹ H nuclear magnetic resonance spectrum: A chart of the above spectrum is shown in Figure 2. (11) Thin layer chromatography:

[Table] (12) Properties of the substance: Neutral substance Regarding the FR-900506 substance, during the measurement of ¹³ C and ¹ H nuclear magnetic resonance spectra, this substance shows one pair of signals each at various chemical shifts. FR-900506 substance with these characteristics is
Furthermore, it has the following properties. () ^13C nuclear magnetic resonance spectra at 25°C and 60°C.
When measured at °C, the intensity ratio of each pair of signals changes. () When thin layer chromatography and high performance liquid chromatography were measured, FR-
Each of the 900506 substances appears as a single spot in thin layer chromatography and as a single peak in high performance liquid chromatography. This white powder of FR-900506 substance changes into the form of crystals when recrystallized from acetonitrile, and the crystals have the following physicochemical properties. (1) Shape and color: Colorless prismatic crystals (2) Elemental analysis: C: 64.30%, H: 8.92%, N: 1.77% 64.20%, 8.86%, 1.72% (3) Melting point: 127-129℃ (4) Specific rotation: [α] ²³ _D : -84.4° (C = 1.02, CHCl ₃ ) (5) ¹³ C nuclear magnetic resonance spectrum: δ (ppm, CDCl ₃ ): {211.98 (s), 211.74 ( s)}
,
{196.28(s), 193.56(s)}, {168.97(s),
168.81 (s)}, {164.85 (s), 165.97 (s)},
{138.76 (s), 139.51 (s)}, {135.73 (d),
135.63(d)}, {132.38(s), 131.90(s)},
{130.39(d), 130.17(d)}, {122.82(d),
122.96(d)}, 116.43(t), {97.19(s),
98.63(s)}, 84.29(d), {77.84(d), 78.21
(d)}, {77.52(d), 76.97(d)}, {69.89
(d), 69.00(d)}, {56.63(d), 54.87(d)
},
{53.97(d), 52.82(d)}, {48.76(t), 48.31
(t)}, {40.21(d), 40.54(d)}, 31.62(t
),
30.72(t), 24.56(t), {21.12(t), 20.86
(t)}, {20.33(q), 19.74(q)}, {16.47
(q), 16.10(q)}, {15.88(q), 15.75(q)
},
{13.89(q), 14.05(q)}, {9.64(q), 9.96
(q)} A chart of the above spectrum is shown in FIG. (6) ¹ H nuclear magnetic resonance spectrum: A chart of the above spectrum is shown in Figure 4. Other physicochemical properties, i.e. FR−
900506 Color reaction of colorless prismatic crystals of substance,
The properties of solubility, ultraviolet absorption spectrum, infrared absorption spectrum, thin layer chromatography and physical properties were the same as those of the white powder. From the above physicochemical properties and X-rotation analysis, FR
-900506 substance was found to have the above chemical structure. The salt in FR-900506 substance is non-toxic,
Conventional pharmaceutically acceptable salts, such as alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, ammonium salts such as doriethylamine salt, N-benzyl-N- Examples include salts with inorganic or organic bases such as amine salts such as methylamine salts. The FR-900506 substance of this invention has immunosuppressive effects,
It has pharmacological effects such as antibacterial effects, and is therefore effective against rejection reactions to organ or tissue transplants such as the heart, kidney, liver, bone marrow, and skin, graft-versus-host reactions caused by bone marrow transplants, and chronic joint damage. It is useful for the treatment and prevention of autoimmune diseases such as rheumatism, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type diabetes, and uveitis, and infectious diseases caused by pathogenic bacteria. In order to show that it has such pharmacological effects,
Pharmacological test data for the FR-900506 substance of the present invention are shown below. Test 1 FR in in vitro mixed lymphocyte reaction (MLR)
Inhibitory effect of -900506 substance C57BL/6 responsive cells (H-2 ^b ) 5x in each well
10 ⁵ pieces and mitomycin C treatment (treated with mitomycin C 25 μg/ml for 30 minutes at 37°C and RPMI1640
5 x 105 BALB/C stimulated cells (H- ^2d ) (washed 3 times with culture medium) were added with 0.2 ^ml of 10% fetal bovine serum.
MLR tests are performed in microtiter plates in RPMI1640 medium [supplemented with 2 mM sodium bicarbonate, penicillin (50 units/ml) and streptomycin (50 μg/ml)], 100% humidity,
In an incubator kept at 5% carbon dioxide and 95% air.
Cells are cultured at 37° C. for 68 hours, pulsed with ³ H-thymidine (0.5 μCi) for 4 hours, and harvested.
Dissolve FR-900506 substance in ethanol,
Dilute in RPMI1640 medium and add to the culture to a final concentration of 0.1 μg/ml or less. The results are shown in Table 4. The FR-900506 substance of this invention suppressed mouse MLR.

[Table] Test 2 Antibacterial activity of FR-900506 substance The antibacterial activity of FR-900506 substance against various fungi was measured in Sabouraud agar by the agar multiple dilution method. After 24 hours of incubation at 30°C, the minimum inhibitory concentration (MIC) is expressed in μg/ml. The FR-900506 substance of this invention is effective against fungi such as Aspergillus fumigatus IF05840 and Fusarium oxysulporum IF05942.
It exhibited antibacterial activity as listed in Tables 5 and 6.

【table】

[Table] Study 3 FR for allograft skin graft survival in rats
Effect of -900506 Substance Abdominal skin grafts from donor (Fisher) rats are transplanted onto the lateral thorax of recipient (WKA) rats. Remove the bandage on the fifth day. Grafts are examined daily until there is rejection, where >90% of the graft epithelium is necrotic. The FR-900506 substance is dissolved in olive oil and administered intramuscularly every day for 14 consecutive days from the day of transplantation. As shown in Table 7, in rats treated intramuscularly with olive oil for 14 consecutive days, all skin allografts were rejected within 8 days, but in rats treated daily with FR-900506, skin allografts survived. has clearly been extended.

[Table] Test 4 Effect of FR-900506 substance on type collagen-induced arthritis in rats Collagen was cooled to a concentration of 2 mg/ml.
Dissolve in 0.01M acetic acid. The solution is emulsified in an equal volume of incomplete Freund's adjuvant. Total volume 0.5ml
This emulsion is injected intradermally into several places on the back and one or two places on the tail of female Lewis rats. FR−
Substance 900506 is dissolved in olive oil and administered orally. Control rats immunized with the same amount of collagen type are orally administered olive oil alone. Observe the incidence of arthritis. The test results are shown in Table 8. Arthritis was induced in all rats treated with olive oil for 14 days starting on the same day as type collagen immunization. When FR-900506 substance is administered daily for 14 days, 3
During the week-long observation period, the induction of arthritis was completely suppressed.

[Table] Quality

Claims (1)

[Claims] 1. General formula: FR-900506 substance shown by or its salt. 2. Cultivate FR-900506 substance-producing bacteria belonging to the genus Streptomyces in a medium, and from the resulting culture
A method for producing an FR-900506 substance or a salt thereof, which comprises separating and collecting the FR-900506 substance and, if necessary, converting it into a salt. 3. The manufacturing method according to claim 2, wherein the FR-900506 substance-producing bacterium is Streptomyces tsukubaensis. 4. The manufacturing method according to claim 3, wherein the FR-900506 substance producing bacterium is Streptomyces tsukubaensis No. 9993.