Process for producing an 11,28-dioxa-4-azatricyclo-(22.3.1.049)octacos-18-ene-2,3,10,16-tetraone derivative and a pharmaceutical composition containing the same

^PROCESS FOR PRODUCING AN?ll,2a-DIOXA-4-AZATRICYtl0{22.3.1.0⁴.⁹] 0CTAC0S-18-ENE2,^,10,16-TETRAONE DERIVATIVE^ AND A PHARMACEUTICAL COMPOSITION; CONTAINING THE! S/IME׳

מיולי ר לייצור תולךח.'0ל 28,11-דיאוקםא-4-אזאטריציקן*ו-; 22.3.1.0) ן אוקםאקום-18האיו-2;3_״16,10-טטראוו ותכמיר רוקחות חמכיל אותוו

.׳ץ. .׳;.ג^ י >׳ . י־.^,־^, י ί

The present specification is divided from Israel Specification

77222 filed 3.12.85, and relates to a process for production of

FR-900520 substance and to an immunosuppressive pharmaceutical composition containing the same.

More particularly, it relates to a process for production of the FR-900520 substance which has pharmacological activities such as immunosuppressive activity, and the like, and to an immunosuppressive pharmaceutical composition containing the same.

Accordingly, one object of this invention is to provide a process for production of the FR-900520 substance which is useful for treatment and prevention of resistance by transplantation, graftversus-host diseases by medulla ossium transplantation, autoimmune diseases, and the like.

A further object of this invention is to provide an immunosuppressive pharmaceutical composition containing, as active ingredients, the FR-900520 substance.

In the present invention, the FR-900520 substance is represented by the following formula:

<img file="IL92345A_D0001.tif" />

With respect to the FR-900520 substance of this invention, it is to be understood that there may be one or more conformer(s) or stereoisomeric pairs such as optical and geometrical isomers due to asymmetric carbon atom(s) and double bond(s), and such isomers are also included within a scope of this invention.

2a

Can. J. Chem. 58, 579 (1980) discloses that the antifungal antibiotic rapamycin of the following formula:

<img file="IL92345A_D0002.tif" />

was isolated from cultures of

Streptomyces hygroscopicus.

US patent No.4,212,881 discloses that the antituberclosis antibiotic Streptovaricin C ” of the following formula:

<img file="IL92345A_D0003.tif" />

was produced by culturing the strain of Streptomyces 101 and its derivatives of the following formula:

2b

<img file="IL92345A_D0004.tif" />

have antiviral activity.

US patent No.3,244,592 and J.Antibiotics, 15, 231 (1962) discloses that the antifungal ” ascomycin ״ the chemical structure of which was not described, was obtained by cultivating a strain of Streptomyces hygroscopicus var. ascomycetius. f

Though all of the three compounds mentioned above are macrocyclic compounds, they are quite different from the compounds of the present invention in chemical structure. Further, it is not known at all that these known compounds possess immunosuppressive activity.

c The process for production of ascomycin by fermentation of Streptomyces hygroscopicus var, ascomyceticus (ATCC 14891) and its antifungal activity were shown in USP 3,244,592 and J. Antibiotics 15, 231-232 (1962). And it was ascertained that ascomycin was the same compound as the FR-9OO52O substance by our researcher's further profound investigations. However, the chemical structure of ascomycin and its immunosuppressive activity had never been shown in any documents.

And further, the process of its production by fermentation of the specific microorganisms, i.e. Streptomyces tsukubaensis No. 9993, Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 had never been shown in any documents, either.

The process for production of the FR-900520 substance of this invention is explained in detail in the following.

Fermentation Process:

The FR-900520 substance of this invention can be produced by fermentation of Streptomyces tsukubaensis No. 9993 and Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 in a nutrient medium.

Particulars of microorganisms used for the production of the FR-900520 substance are explained in the following.

[A] The FR-900520 substance of this invention can be produced by fermentation of Streptomyces tsukubaensis No. 9993 in a nutrient medium,

THE MICROORGANISM

The microorganism which can be used for the production of the FR-900520 substance is Streptomyces tsukubaensis No, 9993, which has been newly isolated from a soil sample collected at Toyosato-cho, Tsukuba-gun, Ibaraki Prefecture, Japan.

A lyophilized sample of the newly isolated Streptomyces tsukubaensis No. 9993 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (No.

1-3, Higashi 1-chome, Yatabemachi Tsukuba-gun, Ibaraki Prefecture, Japan) under the deposit number of FERM P-7886 (deposited date: October 5th, 1984), and then converted to Budapest Treaty route of the same depository on October 19, 1985 under the new deposit number of FERM BP-927.

This invention also includes the use of any mutants which are capable of producing the FR-900520 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of ־ 5 X-rays, ultra-violet radiation, treatment with

N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.

The Streptomyces tsukubaensis No. 9993 has the following morphological, cultural, biological and physiological characteristics.

(1] Morphological Characteristics:

The methods described by Shirling and Gottlieb (Shirling, E. B. and D. Gottlieb: Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology, 16, 313 - 340, 1966) were employed principally for this taxonomic study.

Morphological observations were made with light and electron microscopes on cultures grown at 30 °C for 14 days on oatmeal agar, yeast-malt extract agar and inorganic salts-starch agar. The mature sporophores formed Rectiflexibiles with 10 to 50 or more than 50 spores in each chain. The spores were oblong or cylindrical, 0.5 - 0.7 x 0.7-0.8 pm in size by electron microscopic observation. Spore surfaces were smooth.

(2] Cultural Characteristics:

Cultural characteristics were observed on ten kinds of media described by Shirling and Gottlieb as mentioned above, and by Waksman (Waksman, S. A.: The actinomycetes, vol. 2: Classification, identification and description of genera and species. The Williams and Wilkins Co., Baltimore, 1961).

The incubation was made at 30°C for 14 days. The color names used in this study were based on Guide to Color Standard (manual published by Nippon Shikisai Kenkyusho, Tokyo). Colonies belonged to the gray color series when grown on oatmeal agar, yeast-malt extract agar and inorganic salts-starch agar. Soluble pigment was produced in yeast-malt extract agar but not in other media. The results are shown in Table 1.

(continued to the next page)

Table 1 Cultural Characteristics of Strain Mo, 9993 and Streptomyces misakiensls IFO 12891

<td colspan="2" rowspan="2"> Medium</td><td colspan="2"> Cultural characteristics</td>

<td> No. 9993</td><td> IFO 12891</td>

<td> Oatmeal Agar</td><td> G</td><td> Moderate</td><td> ♦ Moderate</td>

<td></td><td> Λ</td><td> Gray</td><td> Grayish White</td>

<td></td><td> R</td><td> Pale Pink</td><td> Colorless</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td> Yeast-Malt</td><td> G</td><td> Moderate</td><td> Moderate</td>

<td> Extract Agar</td><td> Λ</td><td> Light Gray</td><td> Grayish White</td>

<td></td><td> R</td><td> Dull Reddish Orange</td><td> Light Brown</td>

<td></td><td> S</td><td> Dull Reddish Orange.</td><td> None</td>

<td> Inorganic Salts-</td><td> G</td><td> Moderate</td><td> Moderate</td>

<td> Starch Agar</td><td> A</td><td> Pale Yellow Orange to</td><td> Grayish White</td>

<td></td><td></td><td> Light Gray</td><td></td>

<td></td><td> R</td><td> Dark Orange</td><td> Pale Yellowish Brown</td>

<td></td><td> S</td><td> None</td><td> None</td>

in

ס

מ

ויי

<td colspan="2"> Medium</td><td> No. 9993</td><td> IFO 12091</td>

<td> Glucose-</td><td> G</td><td> Poor</td><td> Moderate</td>

<td> Asparagine</td><td> Λ</td><td> White</td><td> Grayish White</td>

<td> Agar</td><td> R</td><td> Pale Drown</td><td> Pale Yellowish Brown</td>

<td></td><td> S</td><td> None</td><td> Pale Brown</td>

<td> Glycerin-</td><td> G</td><td> Moderate</td><td> Moderate</td>

<td> Asparagine</td><td> A</td><td> Pale Pink tq White</td><td> Grayish White</td>

<td> Agar</td><td> R</td><td> Pale Pink</td><td> Pale Yellowish Brown</td>

<td></td><td> S</td><td> None</td><td> Pale Brown</td>

<td> Czapek Agar</td><td> G</td><td> Poor</td><td> 1 Abundant</td>

<td></td><td> A</td><td> None</td><td> Grayish White</td>

<td></td><td> R</td><td> Pale Pink</td><td> Dark Orange to Dark Brown</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td> Nutrient Agar</td><td> G</td><td> Poor</td><td> Poor</td>

<td></td><td> A</td><td> White, Poor</td><td> White</td>

<td></td><td> R</td><td> Colorless</td><td> Colorless</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td colspan="2"> Medium</td><td> No. 9993</td><td> IFO 12091</td>

<td> Potato-Dextrose .</td><td> G</td><td> Poor</td><td> Moderate</td>

<td> Agar</td><td> A</td><td> None</td><td> Yellowish Gray</td>

<td></td><td> R</td><td> Pale Pink</td><td> Brown</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td> Tyrosine Agar</td><td> G</td><td> Moderate</td><td> Moderate</td>

<td></td><td> A</td><td> White</td><td> Grayish White to Light Gray</td>

<td></td><td> R</td><td> Dull Reddish Orange</td><td> Dark Orange to Black</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td> Peptone-Yeast</td><td> G</td><td> Poor</td><td> Poor</td>

<td> Extract-Iron</td><td> A</td><td> None</td><td> None</td>

<td> Agar</td><td> R</td><td> Colorless</td><td> Colorless</td>

<td></td><td> S</td><td> None</td><td> None</td>

<td> Abbreviation !</td><td> ! G ~ Growth, R = Reverse Side Color,</td><td> Λ = Aerial Hass Color, S = Soluble Pigment,</td>

<td> in</td><td> S ή ^u ו-י</td><td> S'» s</td>

סו

The cell.wall analysis was performed by the methods of Becker et al. (Becker, Β., Μ. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between Nocardia and Streptomyces by paper chromatography of whole cell hydrolysates: Appl. Microbiol., 12, 421-423, 1964) and Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacterial., 89, 444-453, 1965). Analysis of whole cell hydrolysates of the strain No. 9993 showed the presence of LL-diaminopimelic acid. Accordingly, the cell wall of this strain is believed to be of type I.

[3] Biological and Physiological Properties:

Physiological properties of the strain No. 9993 were determined according to the methods described by Shirling f and Gottlieb as mentioned above. The results are shown in Table 2. Temperature range and optimum temperature for growth were determined on yeast-malt extract agar using a temperature gradient incubator (made by Toyo Kagaku Sangyo Co., Ltd.). Temperature range for growth was from 18 to 35°C with optimum temperature at 28°C. Milk peptonization and gelatin liquefaction were positive. Melanoid pigment production was negative.

(continued to the next page)

Table 2 Physiological Properties of Strain No, 9993 and

Streptomyces mlsakiensis IFO 12091

<td> Physiological properties</td><td> No. 9993</td><td> IFO 12891</td>

<td> Temperature Range for Growth</td><td> 18 .C 35 ־ ’C</td><td> 12 .C - 35 ^,C</td>

<td> Optimum Temperature</td><td> 20 .C</td><td> 28 ’C</td>

<td> Nitrate Reduction</td><td> Negative</td><td> Negative</td>

<td> Starch Hydrolysis</td><td> Negative</td><td> Positive</td>

<td> Milk Coagulation</td><td> Negative</td><td> Negative</td>

<td> Milk Peptonization</td><td> Positive</td><td> Weakly Positive</td>

<td> Melanin Production</td><td> Negative</td><td> Negative</td>

<td> Gelatin Liquefaction</td><td> Positive</td><td> Negative</td>

<td> II^S Production</td><td> Negative</td><td> Negative</td>

<td> NaCl Tolerance 1%)</td><td> £3 »</td><td> 3»&lt;₍&lt;5t '</td>

ו Utilization of carbon sources was examined according to the methods of Pridham and Gottlieb (Pridham, T. G. and D. Gottlieb: The utilization of carbon compounds bv some Actinomycetales as an aid for species determination: J. Bacteriol., 56, 107-114, 1948). The growth was observed after 14 days incubation at 30°C.

Summarized carbon sources utilization of this strain is shown in Table 3. Glycerin, maltose and sodium succinate could be utilized by the strain No. 9993. Further, doubtful utilization of D-glucose, sucrose, D-mannose and salicin was also observed.

(continued to the next page) ו Table 3 Carbon Sources Utilization of Strain No. 9993 and

Streptomvces misakiensis IFO 12891

<td colspan="2"> Carbon</td><td rowspan="2"> No. 1</td><td rowspan="2"> 9993 IFO 12891</td>

<td> 5</td><td> Sources</td>

<td></td><td> D-Glucose</td><td> 4*</td><td> —</td>

<td></td><td> Sucrose</td><td> “ז-</td><td> —</td>

<td></td><td> Glycerin</td><td> -ד</td><td> —</td>

<td> 10</td><td> D-Xylose</td><td> —</td><td> —</td>

<td></td><td> D-Fructose</td><td> —</td><td> —</td>

<td></td><td> Lactose</td><td> —</td><td> —</td>

<td></td><td> Maltose</td><td> +</td><td></td>

<td></td><td> Rhamnose</td><td> —</td><td> —</td>

<td> 15</td><td> Raffinose</td><td> —</td><td> —</td>

<td></td><td> D-Galactose</td><td> *.</td><td> 4</td>

<td></td><td> L-Arabinose</td><td> —</td><td> —</td>

<td></td><td> D-Mannose</td><td> +</td><td> —</td>

<td></td><td> D-Trehalose</td><td> .*</td><td> —</td>

<td> 20</td><td> Inositol</td><td> —</td><td> —</td>

<td></td><td> D-Mannitol</td><td> —</td><td> —</td>

<td></td><td> Inulin</td><td> —</td><td> .ו-</td>

<td></td><td> Cellulose</td><td> —</td><td> «.</td>

<td></td><td> Salicin</td><td> +</td><td> —</td>

<td> 25</td><td> Chitin</td><td></td><td> +</td>

<td></td><td> Sodium Citrate</td><td> —</td><td></td>

<td></td><td> Sodium Succinate</td><td> +</td><td> —</td>

<td></td><td> Sodium Acetate</td><td> M</td><td></td>

<td> 30</td><td> Symbols:</td><td> + :</td><td> utilization</td>

<td></td><td></td><td> + :</td><td> doubtful utilization</td>

<td></td><td></td><td> — .</td><td> no utilization</td>

Microscopic studies and cell wall composition analysis of the strain No. 9993 indicate that this strain belongs to the genus Streptomyces Waksman and Henrici 1943.

Accordingly, a comparison of this strain was made with various Streptomyces species in the light of the published descriptions [International Journal of Systematic Bacteriology, 18, 69 to 189, 279 to 392 (1968) and 19, 391 to 512 (1969) , and Bergy's Manual of Determinative

Bacteriology 8th Edition (1974)].

As a result of the comparison, the strain No. 9993 is considered to resemble Streptomyces aburaviensis Nishimura et. al., Streptomyces avellaneus Baldacci and Grein and 15 Streptomyces misakiensis Nakamura. Therefore, the cultural characteristics of the strain No. 9993 were compared with the corresponding Streptomyces aburaviensis IFO 12830, Streptomyces avellaneus IFO 13451 and Streptomyces misakiensis IFO 12891. As a result, the strain No. 9993 was 20 the most similar to Streptomyces misakiensis IFO 12891.

Therefore, the strain No. 9993 was further compared with Streptomyces misakiensis IFO 12891 as shown in the above Tables 1, 2 and 3. From further comparison, the strain No. 9993 could be differentiated from Streptomyces misakiensis 25 IFO 12891 in the following points, and therefore the strain

No. 9993 is considered to be a new species of Streptomyces and has been designated as Streptomyces tsukubaensis sp. nov., referring to the soil collected at Tsukuba-gun, from which the organism was isolated.

Difference from Streptomyces misakiensis IFO 12891

Cultural characteristics of the strain No. 9993 are different from the Streptomyces misakiensis IFO 12891 on oatmeal agar, yeast-malt extract agar, glucose-asparagine agar, Czapek agar and potato-dextrose agar.

Starch hydrolysis of the strain No. 9993 is negative, but that of the Streptomyces misakiensis IFO 12891 is positive.

Gelatin liquefaction of the strain No. 9993 is positive, but that of the Streptomyces misakiensis IFO 12891 is negative.

In carbon sources utilization, the strain No. 9993 can utilize glycerin, maltose and sodium succinate, but the Streptomyces misakiensis IFO 12891 can not utilize them. And, the strain No. 9993 can not utilize D-galactose and inulin, but the Streptomyces misakiensis IFO 12891 can utilize them.

PRODUCTION OF FR-900520 SUBSTANCE

The FR-900520 substance of this invention can be produced by culturing Streptomyces tsukubaensis No. 9993 (FERM BP-927) in a nutrient medium.

In general, the FR-900520 substance can be produced by culturing Streptomyces tsukubaensis No. 9993 in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under aerobic conditions (e.g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, xylose, galactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, rhamnose, raffinose, arabinose, mannose, salicin, sodium succinate, and the like.

The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cottonseed meal, soybean meal, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, amonium sulfate, ammonium phosphate, etc.), urea, amino acid, and the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salt and the like. If necessary, especially when

92345/2 the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone may be added.

As the conditions for the production of the FR-900520 substance in massive amounts, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of the FR-900520 substance. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism and culturing said inoculated medium, and then to transfer the cultured vegetative inoculum aseptically to large tanks. The medium, in which the vegetative inoculum is produced, is substantially the same as or different from the medium utilized for the production of the FR-900520 substance.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.

92345/2

The fermentation is usually conducted at a temperature between about 20°C and 40°C, preferably 25-35°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.

Thus produced FR—900520 substance can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances, the FR-900520 substance produced is found in the cultured mycelium and filtrate, and accordingly, the FR-900520 substance can be isolated and purified from the mycelium and the filtrate, which are obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc.), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.

[B] The FR-900520 substance of this invention can be produced by fermentaion of Streptomyces hygroscopicus subsp. yakushimaensis No, '7238 in a nutrient medium.

92345/2

THE MICROORGANISM

The microorganism which can be used for the production of the FR-900520 substance is Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 which has been newly isolated from a soil sample collected at Yakushima, Kagoshima Prefecture, Japan.

A lyophilized sample of the newly isolated Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 has been deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology (No. 1-3, Higashi 1-chome, Yatabemachi, Tsukuba-gun, Ibaraki Prefecture, Japan) under the number of FERM P-8043 (deposited date: January 12th, 1985), and then converted to Budapest Treaty route of the same depository on October 19, 1985 under the new deposit number of FERM BP-928.

This invention also includes the use of any mutants which are capable of producing the FR-900520 substance including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-rays, ultra-violet radiation, treatment with

N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine, and the like.

The Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 has the following morphological, cultural, biological and physiological characteristics.

[1] Morphological Characteristics:

The methods described by Shirling and Gottlieb (Shirling, E. B. and D. Gottlieb: Methods for characterization of Streptomyces species.. International Journal of Systematic Bacteriology, 16, 313 - 340, 1966) were -employed principally for this taxonomic study.

Morphological observations were made with light and electron microscopes on cultures grown at 30°C for 14 days on oatmeal agar, yeast-malt extract agar and inorganic salts-starch agar. The mature sporophores were moderately short and formed Retinaculiaperti and Spirales with about 20 spores in each chain. Hygroscopic spore mass were seen in the aerial mycelia on oatmeal agar and inorganic salts-starch agar. Surface irregularities on spores were intermediate between very short, thick spines and warts.

(2] Cultural Characteristics:

Cultural characteristics were observed on ten kinds of media described by Shirling and Gottlieb as mentioned above, and by Waksman (Waksman, S. A.: The actinomycetes, vol. 2: Classification, identification and description of genera and species. The Williams and Wilkins Co., Baltimore, 1961).

The incubation was made at 30°C for 14 days. The color names used in this study were based on Guide to Color Standard (manual published by Nippon Shikisai Kenkyusho, Tokyo). Colonies belonged to the gray color series when grown on oatmeal agar, yeast-malt extract agar and inorganic salts-starch agar. Soluble pigment was not produced in the examined media. The results are shown in Table 4.

(continued to the next page)

Table 4 Cultural Characteristics of Strain No. 7238, Streptomyces antimycoticus IFO 12839 and Streptomyces hyqroscopicus subsp. glebosus IFO 1378¢

Cultural Characteristics

<td> Medium</td><td></td><td> No. 7238</td><td> IFO 12839</td><td> IFO 13786</td>

<td> Oatmeal Agar</td><td> G</td><td> Poor</td><td> Poor</td><td> Poor</td>

<td></td><td> A</td><td> Grayish ;Yellow Brown</td><td> Grayish Yellow Brown</td><td> Grayish Yellow Brown</td>

<td></td><td> R</td><td> Pale Yellow</td><td> Pale Yellow</td><td> Pale Yellow</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

<td> Yeast-Malt</td><td> G</td><td> Moderate</td><td> Abundant</td><td> Moderate</td>

<td> Extract Agar</td><td> A</td><td> Grayish White</td><td> Gray</td><td> Gray</td>

<td></td><td> R</td><td> Pale Yellowish Brown</td><td> Pale Yellowish Brown</td><td> Dark Orange</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

<td> Inorganic Salts-</td><td> G</td><td> Moderate</td><td> Moderate</td><td> Moderate</td>

<td> Starch Agar</td><td> A</td><td> Gray to Black</td><td> Gray</td><td> Light Gray</td>

<td></td><td> R</td><td> Pale Yellow Orange</td><td> Yellowish Gray</td><td> Pale Yellow Orange</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

<td> in</td><td></td><td> 2 2</td><td> 20 25</td><td> ςε οε</td>

<td> Medium</td><td colspan="2"> No. 7238</td><td> IFO 12839</td><td> IFO 13786</td>

<td> Glucose-</td><td> G</td><td> Moderate</td><td> Moderate</td><td> Moderate</td>

<td> Asparagine</td><td> A</td><td> Grayish White</td><td> Gray</td><td> White</td>

<td> Agar</td><td> R</td><td> Pale Yellow Orange</td><td> Pale Yellow Orange</td><td> Pale Yellow Orange</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

<td> Glycerin-</td><td> G</td><td> Moderate</td><td> Moderate</td><td> Moderate</td>

<td> Asparagine</td><td> A</td><td> White</td><td> Gray</td><td> Light Gray</td>

<td> Agar</td><td> R</td><td> Yellowish Gray</td><td> Yellowish Gray</td><td> Grayish Yellow Brown</td>

<td></td><td> S</td><td> None.</td><td> None</td><td> None</td>

<td> Czapek Agar</td><td> G</td><td> Moderate</td><td> Moderate</td><td> Moderate</td>

<td></td><td> A</td><td> Grayish White</td><td> Grayish White</td><td> White</td>

<td></td><td> R</td><td> Pale Yellowish Brown</td><td> Pale Yellowish Brown</td><td> Pale Yellowish Brown</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

<td> Nutrient Agar</td><td> G</td><td> Moderate . ’</td><td> Moderate</td><td> Moderate</td>

<td></td><td> A</td><td> Grayish White</td><td> Grayish White</td><td> None</td>

<td></td><td> R</td><td> Pale Yellow</td><td> Pale Yellow</td><td> Pale Yellow</td>

<td></td><td> S</td><td> None</td><td> None</td><td> None</td>

1Λ '0 mom ν η (ף

<td> Medium</td><td> No, 7238</td><td> IFO 12839</td><td> IFO 13786</td>

<td> Potato-Dextrose</td><td> G Moderate</td><td> Moderate</td><td> Moderate</td>

<td> Agar</td><td> A White, Poor</td><td> Pale Reddish Brown</td><td> Pale Pink to White</td>

<td></td><td> R Pale Yellow Orange</td><td> Pale Yellow Orange</td><td> Pale Yellowish Brown</td>

<td></td><td> S None</td><td> None</td><td> None</td>

<td> Tyrosine Agar</td><td> G Moderate</td><td> Moderate</td><td> Moderate</td>

<td></td><td> A White</td><td> Grayish White</td><td> Gray to Black</td>

<td></td><td> R Pale Yellowish Brown</td><td> Brown</td><td> Pale Yellowish Brown</td>

<td></td><td> S None</td><td> Brown -</td><td> None</td>

<td> Peptone-Yeast</td><td> G Moderate</td><td> Moderate</td><td> Moderate</td>

<td> Extract-Iron</td><td> A None</td><td> Grayish White</td><td> None</td>

<td> Agar</td><td> R Pale Yellow</td><td> Pale Yellow</td><td> Colorless</td>

<td></td><td> S None</td><td> None</td><td> None</td>

<td> Abbreviation :</td><td> G = Growth, R: = Reverse Side Color,</td><td> A = Aerial Mass Color, S = Soluble Pigment,</td><td></td>

<td></td><td> 0 to</td><td> ו/ו 0 CM CM</td><td> 0 m רה ף&gt;</td>

The cell wall analysis was performed by the methods of

Becker et al. (Becker, Β., Μ. P. Lechevalier, R. E. Gordon and H. A. Lechevalier: Rapid differentiation between

Nocardia and Streptomyces by paper chromatography of whole cell hydrolysates: Appl. Microbiol., 12, 421-423, 1964) and

Yamaguchi (Yamaguchi, T.: Comparison of the cell wall composition of morphologically distinct actinomycetes: J. Bacterial., 89, 444-453, 1965). Analysis of whole cell hydrolysates of the strain No. 7238 showed the presence of 10 LL-diaminopimelic acid. Accordingly, the cell wall of this strain is believed to be of type I.

[3] Biological and Physiological Properties:

Physiological properties of the strain No. 7238 were determined according to the methods described by Shirling and Gottli'eb as mentioned above. The results are shown in Table 5. Temperature range and optimum temperature for growth were determined on yeast-malt extract agar using a 20 temperature gradient incubator (made by Toyo Kagaku Sangyo

Co., Ltd.). Temperature range for growth was from. 18 to 36°C with optimum temperature at 28°C. Starch hydrolysis .and gelatin liquefaction were positive. No melanoid pigment was produced.

(continued to the next page)

Table 5 Physiological Properties of Strain No. 7238, Streptomyces antimycoticus IFO 12839 and

Streptomyces hygroscopicus subsp. glebosus IFO 13786

I

ט

CM

I

<td> Physiological properties</td><td> No. 7238</td><td> IFO 12839</td><td> IFO 13786</td>

<td> Temperature Range for Growth</td><td> 18 °C - 36 °C</td><td> 18 ’C - 38 .C</td><td> 16°C - 35°C</td>

<td> Optimum Temperature</td><td> 28 °C</td><td> 28 .C</td><td> 27°C</td>

<td> Nitrate Reduction</td><td> Negative</td><td> Negative</td><td> \ Negative</td>

<td> Starch Hydrolysis</td><td> Positive</td><td> Positive</td><td> Positive</td>

<td> Milk Coagulation</td><td> Negative</td><td> Negative</td><td> Negative</td>

<td> Milk Peptonization</td><td> Negative</td><td> Negative</td><td> Positive</td>

<td> Melanin Production</td><td> Negative</td><td> Negative</td><td> Negative</td>

<td> ׳ Gelatin Liquefaction</td><td> Positive</td><td> Positive</td><td> Positive</td>

<td> H₂S Production</td><td> Negative *</td><td> Negative</td><td> Negative</td>

<td> Urease Activity</td><td> Negative</td><td> Negative</td><td> Negative ׳</td>

<td> NaCl Tolerance (%)</td><td> 7% , 10%</td><td> n , 10»</td><td> 5% , 7%</td>

q in 0 m ף, ח ni n nd

Utilization of carbon sources was examined according to the methods of Pridham and Gottlieb (Pridham, T. G. and D. Gottlieb: The utilization of carbon compounds by some Actinomycetales as an aid for species determination: J. Bacteriol., 56, 107-114, 1948). The growth was observed after 14 days incubation at 30°C.

Summarized carbon sources utilization of this strain is shown in Table 6. D-Glucose, sucrose, lactose., maltose, D-trehalose, inositol, inulin and salicin could be utilized by the strain No. 7238.

(continued to the next page)

Table 6 Carbon Sources Utilization of Strain No. 7238,

<td colspan="5"> Streptomyces antimycoticus IFO 12839 and</td>

<td rowspan="2"> 5</td><td colspan="4"> Streptomyces hygroscopicus subsp. glebosus IFO 13786</td>

<td> Carbon Sources</td><td> No. 7238</td><td> IFO 12839</td><td> IFO 13786</td>

<td></td><td> D-Glucose</td><td> +</td><td> +</td><td> +</td>

<td> 10</td><td> Sucrose</td><td> +</td><td> +</td><td> +</td>

<td></td><td> Glycerin</td><td> -</td><td> +</td><td> +</td>

<td></td><td> D-Xylose</td><td> -</td><td> +</td><td> +</td>

<td></td><td> D-Fructose</td><td> -</td><td> +</td><td> .ז-</td>

<td></td><td> Lactose</td><td> +</td><td> +</td><td> —</td>

<td> 15</td><td> Maltose</td><td> +</td><td> —</td><td></td>

<td></td><td> Rhamnose</td><td> -</td><td> +</td><td> —</td>

<td></td><td> Raffinose ־'</td><td> —</td><td> +</td><td> +</td>

<td></td><td> D-Galactose</td><td> —</td><td> +</td><td> 4.</td>

<td></td><td> L-Arabinose</td><td> —</td><td> +</td><td> +</td>

<td> 20</td><td> D-Mannose</td><td> —</td><td> +</td><td> +</td>

<td></td><td> D-Trehalose</td><td> +</td><td> +</td><td> +</td>

<td></td><td> Inositol</td><td> +</td><td> +</td><td> +</td>

<td></td><td> D-Mannitol</td><td> -</td><td> +</td><td> +</td>

<td></td><td> Inulin</td><td> +</td><td> +</td><td> —</td>

<td> 25</td><td> Cellulose</td><td> +</td><td> —</td><td></td>

<td></td><td> Salicin</td><td> +</td><td> +</td><td> —</td>

<td></td><td> Chitin</td><td> +</td><td> -</td><td> —</td>

<td></td><td> Sodium Citrate</td><td> -</td><td> —</td><td> +</td>

<td></td><td> Sodium Succinate</td><td> -</td><td> +</td><td> +</td>

<td> 30</td><td> Sodium Acetate</td><td> -</td><td> -</td><td> «Μ</td>

<td></td><td> Symbols:</td><td> + :</td><td> utilization</td><td></td>

+ : doubtful utilization : no utilization

־

Microscopic studies and cell wall composition analysis of the strain No. 7238 indicate that this strain belongs to the genus Streptomyces Waksman and Henrici 1943.

Accordingly, a comparison of this strain was made with various Streptomyces species in the light of the published descriptions (International Journal of Systematic Bacteriology, 18, 69 to 189, 279 to 392 (1968) and 19, 391 to 512 (1969), and Bergy's Manual of Determinative Bacteriology 8th Edition (1974)].

As a result of the comparison, the strain No. 7238 is considered to resemble Streptomyces antimycoticus Waksman 1957 and Streptomyces hygroscopicus.subsp. glebosus Ohmori, ct. al. 1962. Therefore, the cultural characteristics of the strain No. 7238 were further compared with the corresponding Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13786 as shown in the above Tables 4, 5 and 6. From further comparison, the strain No. 7238 could be differentiated from Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13786 in the following points.

(i) Difference from Streptomyces antimycoticus IFO 12839

Cultural characteristics of the strain No. 7238 are different from the Streptomyces antimycoticus IFO 12839 on yeast-malt extract agar, glucose-asparagine agar, glycerinasparagine agar, potato-dextrose agar and tyrosine agar.

In carbon sources utilization, the strain No. 7238 can utilize maltose, but the Streptomyces antimycoticus IFO 12839 can not utilize it. And, the strain No. 7238 can not utilize glycerin, D-fructose, rhamnose, raffinose,

35.

D-galactose, D-mannose, mannitol and sodium succinate, but the Streptomyces antimycoticus IFO 12839 can utilize them.

(ii) Difference from Streptomyces hygroscopicus subsp. glebosus IFO 13786

Cultural characteristics of the strain No. 7238 are different from the Streptomyces hygroscopicus subsp. glebosus IFO 13786 on yeast-malt extract agar, potato-dextrose agar and tyrosine agar.

Milk peptonization of the strain No. 7238 is negative, but that of the Streptomyces hygroscopicus subsp. glebosus IFO 13786 is positive. The strain No. 7238 can grow in the presence of 7% NaCl, but the Streptomyces hygroscopicus subsp. glebosus IFO 13786 can not grow under the same condition׳.

In carbon sources utilization, the strain No. 7238 can utilize lactose, inulin and salicin, but the Streptomyces hygroscopicus subsp. glebosus IFO 13786 can not utilize them. And, the strain No. 7238 can not utilize glycerin, D-xylose, D-fructose, raffinose, D-galactose, D-mannose, mannitol and sodium succinate, but the Streptomyces hygroscopicus subsp. glebosus IFO 13786 can utilize them.

However, the strain No. 7238 forms hygroscopic spore mass in the aerial mycelia on oatmeal agar and inorganic salts-starch agar, and further morphological and cultural characteristics of the strain No. 7238 are similar to the Streptomyces hygroscopicus subsp. glebosus IFO 13786. Therefore, the strain No. 7238 is considered to belong to Streptomyces hygroscopicus, but the strain No. 7238 is different from the Streptomyces hygroscopicus subsp. glebosus IFO 13786, though this known strain is the most similar to the strain No. 7238 Streptomyces hyqroscopicus subspecies. From the above facts, the strain No. 7238 is considered to be a new species of Streptomyces hyqroscopicus and has been designated as Streptomyces hygroscopicus subsp. yakushimaensis subsp. nov., referring to the soil collected at Yakushima, from which the organism was isolated.

PRODUCTION OF FR-900520 SUBSTANCE

The FR-900520 substance can be produced by culturing Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 (FERM BP-928) in a nutrient medium containing sources of assimilable carbon and nitrogen, preferably under aerobic conditions (e.g. shaking culture, submerged culture, etc.).

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, lactose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, D-trehalose, inositol, inulin, salicin, and the like.

The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cottonseed meal, soybean meal, corn steep liquor, dried yeast, wheat germ, feather meal, peanut powder, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, amonium sulfate, ammonium phosphate, etc.), urea, amino acid, and the like.

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The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form, because less pure materials which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, cobalt salt and the like. If necessary, especially when the culture medium foams seriously, a defoaming agent, such as liquid paraffin, fatty oil, plant oil, 'mineral oil or silicone may be added.

As the conditions for the production of the FR-900520 substance in massive amounts, submerged aerobic cultural conditions are preferred therefor. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of the FR-900520 substance. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism and culturing said inoculated medium, and then to transfer the cultured vegetative inoculum aseptically to large tanks. The medium, in which the vegetative inoculum is produced, is substantially the same as or different from the medium utilized for the production of the FR-900520 substance.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermentor, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C and 40°C, preferably 25-35°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.

Thus produced FR-900520 substance can be recovered from the culture medium by conventional means which are commonly used for the recovery of other known biologically active substances. The FR-900520 substance produced is mainly found in the cultured mycelium, and accordingly, the FR-900520 substance can be isolated and purified from the mycelium, which is obtained by filtering or centrifuging the cultured broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, pH adjustment, treatment with a conventional resin (e.g. anion or 'cation exchange resin, non-ionic adsorption resin, etc.)» treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina, etc.), crystallization, recrystallization, and the like.

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Particularly the FR-900520 substance can be separated by dissolvIng the materials containing the product produced by fermentation in acetate an appropriate solvent such as ethyl n-hexane, and the like, and then by subjecting said solution to chromatography, for example, on silica gel in a column with an appropriate organic solvent such as ethyl acetate and n-hexane, or a mixture thereof. And the FR-900520 substance thus separated can be further purified by a conventional method, for example, recrystallization, re-chromatography, high performance liquid chromatography, and the like.

־)

PHYSIOLOGICAL AND CHEMICAL PROPERTIES OF FR-900520 SUBSTANCE (1) Form and Color:

colorless plates (2) Elemental Analysis:

C: 64.817, H: 8.827: N: 1.557 (3) Color Reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction, Ehrlich reaction, Dragendorff reaction and iodine vapor reaction

Negative: ferric chloride reaction, ninhydrin reaction and

Molish reaction (4) Solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate, chloroform, diethyl ether and benzene

Sparingly Soluble: n-hexane, petroleum ether

Insoluble: water (5) Melting Point:

163 - 165 °C (6) Specific Rotation:

[α]θ : 84.1°־ (c 1.0 ־־, CHC1₃) (7) Ultraviolet Absorption Spectrum:

end absorption (8) Infrared Absorption Spectrum:

<td> CHC1., ״_on^vmax ^{3 : 3680}'</td><td> 3575,</td><td> 3520,</td><td> 2940, 2875, 2825,</td>

<td> 1745,</td><td> 1725,</td><td> 1700,</td><td> 1647, 1610(sh), 1452,</td>

<td> 1380,</td><td> 1350,</td><td> 1330,</td><td> 1285, 1170, 1135,</td>

<td> 1090,</td><td> 1030,</td><td> 1005,</td><td> 990, 980(sh),</td>

960(sh), 913, 908(sh) cm¹ (9) C Nuclear Magnetic Resonance Spectrum:

δ(ppm, CDC1₃I: 213.04 (s),j196.21 (s) (169.07 (s) hs3.23 (s),’168.85 (s) ,

1164.92 (s) &lt;138.67 (s) &lt;132.46 (s) '165.97 (31,1139.53 (s) , 1131.98 (s) , (130.20 (d) &lt;123.42 (d) &lt;97.28 (s) 1130.08 (d), 1123.59 (d), 198.75 (s), 84.37 (d), (77.80 (d) (75.53 (d)

178.24 (d) , 176.98 (d), 73.92 (d)73.69 (d), &lt;73.11 (d)

172.72 (d) , &lt;70.11 (d) 57.02 (q), &lt;56.60 (q) '69.21 (d), ‘57.43 (q), &lt;56.23 (q) &lt;56.72 (d) &lt;55.10 (d)

155.98 (q) , (52.91 (d) , 154.90 (d) , (48.90 (t) (40.19 (d) (27.67 (t)

148.57 (t) , '40.63 (d) , 126.32(t) , (26.51 (d) 24.60 (t), &lt;21.19(t)

126.44 (d), (20.86(t), (20.47 (q) &lt;16.21 (q) &lt;15.83(q)

119.75 (q) , 115.97 (q) , (!5.94(q) , (14.04 (q) 11.68 (q) , &lt;9.64,(q) (14.16 (q), !9.93(q), the chart of which being shown in Figure 1, (10) ¹H Nuclear Magnetic Resonance Spectrum: ׳׳ the chart of which being shown in Figure 2, (11) Thin Layer Chromatography:

Deve loping

Stationary Phase Solvent Rf Values chloroform silica gel plate : methanol (20:1, v/v) 0.38 ethyl acetate 0.51 (12) Property of the Substance:

neutral substance

With regard to the FR-900520 substance, it is to be noted that in case of measurements of C and H nuclear magnetic resonance spectra, this substance shows pairs of the signals in various chemical shifts, however, in case of measurements of the thin layer chromatography and the high performance liquid chromatography, the FR-9OO52O substance showed a single spot in the thin layer chromatography and a single peak in the high performance liquid chromatography, respectively.

From the above physical and chemical properties, the FR-900520 substance could be determined to have the following chemical structure.

<img file="IL92345A_D0005.tif" />

och₃ och₃

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17-Ethy 1 -1,14-dו hydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl )-1 methylvוnyl]-23,25-dimethoxy-13,19,21,27-tetramethyl-l1,28-di oxa-4azatri cyclo-[22.3.1.0^’^]octacos-18-ene-2,3,10,16-tetraone,

The FR-900520 substance used in the present invention possesses pharmacological activities such as immunosuppressive activity, and therefore are useful for the treatment and prevention of the resistance by transplantation of organs or tissues such as heart, kidney, liver, medulla ossium, skin, etc., graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, etc., and the like.

As examples for showing such pharmacological activities, some pharmacological test data of the FR-900520 substance are illustrated in the following.

TEST I

Suppression of the FR-900520 substance in in vitro Mixed

Lymphocyte Reaction (MLR)

The MLR test was performed in microtiter plates, with each well containing 5 x 10^ C57BL/6 responder cells (H-2^), 5 x 10^ mitomycin C ²⁵ ^0 ריזז/סλ treated (2-5ug-AftL mitomycin C at 37 C for 30 minutes and washed three times with RPMI 1640 medium) BALB/C stimulator cells (Η-2^) in 0.2 ml

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RPM1 1540 medium supplemented with 10^ fetal calf serum, 2mM sodium hydrogen carbonate, penicillin (50 unit/ml) and streptomycin (50 /ug/ml). The cells were incubated at 37°C in humidified atmosphere of 5% carbon dioxide and 95X of air for 68 hours and pulsed with ^Η-thymidine (0.5 jiCi) 4 hours before the cells were collected. The FR-900520 substance was dissolved in ethanol and further diluted in RPMI 1640 medium ^and^ Ridded to the cultures to give final 1 concentrations of ApI-or less.

The result is shown in Table 7. The FR-900520 substance of the present invention suppressed mouse MLR.

(continued on following page)

Table 7: Effect of FR-900520 Substance on MLR

<td> FR-900520</td><td> Radioactivities</td><td> Suppression</td><td>^IC50</td>

<td> concentration</td><td> (mean C.P.M. + S.E.)</td><td> (%)</td><td> (ng/tnl)</td>

<td> 100</td><td> 175 + 16</td><td> 99.2</td><td></td>

<td> 10</td><td> 515 + 55</td><td> 97.8</td><td></td>

<td> 1</td><td> 2744 + 527</td><td> 88.1</td><td> 0.38</td>

<td> 0.500</td><td> 9434 + 1546</td><td> 59.2</td><td></td>

<td> 0.25</td><td> 14987 + 1786</td><td> 35.1</td><td></td>

<td> 0</td><td> 23106 + 1652</td><td> 0</td><td></td>

Test 2

Acute toxicity of the FR-900520 substance

Test on acute toxicity of the FR-900520 substance in ddY mice by a intraperitoneal injection was conducted, and -the-־dead at/dose of 100 סח dead mice mg/kg/could .net be observed.

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The pharmaceutical composition of this invention can be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the FR-900520 substance of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The carriers which can be used are water, glucose, lactose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, dr liquid form, and in addition auxiliary, stabilizing, thickening and coloring agents and perfumes may be used. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of diseases.

For applying this composition to human, it is preferable to apply it by parenteral or enteral administration. While the dosage of therapeutically effective -amount of the FR-900520 substance varies and also depends upon the age and condition of each individual patient to be treated, a daily dose of about 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100 mg, of the active ingredient is generally given for treating diseases, and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg is generally administered.

The following examples are given for the purpose of illustrating the present invention.

Example 1

Isolation of Streptomyces tsukubaensis No. 9993 .

Streptomyces tsukubaensis No. 9993 was isolated by using dilution plate techniques as shown in the following.

About one gram soil which was collected at Toyosato-cho, Tsukuba Gun, Ibaraki Prefecture, Japan, was added to a sterile test tube and the volume made up to 5 ml with sterile water. The mixture was then blended for 10 second by a tube buzzer and kept on •10 minutes. The supernatant was sequentially diluted by 100 fold with sterile water. The diluted solution (0.1 ml) was spread on Czapek agar supplemented with thiamine hydrochloride (saccharose 30 g, sodium nitrate 3 g, dipotassium phosphate 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, ferrous sulfate 0.01 g, thiamine hydrochloride 0.1 g, agar 20 g, tap water 1000 ml; pH 7.2) in a Petri dish. The growing colonies developed on the plates after 21 days incubation at 30°C were transferred to slants [yeast-malt extract agar (ISP-medium 2)], and cultured for 10 days at 30°C. Among of the colonies isolated, the Streptomyces tsukubaensis No. 9993 could be found.

Fermentation

A culture medium (160 ml) containing glycerin (1%), soluble starch (1 %), glucose (0.5%), cottonseed meal (0.5%), dried yeast (0.5%), corn steep liquor (0.5%) and calcium carbonate (0.2%) (adjusted to pH 6.5) was poured into each of twenty 500 ml-Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of slant culture of Streptomyces tsukubaensis No.9993, FERM BP-927 was inoculated to each of the media and cultured at 30“C for 4 days on a rotary shaker. The resultant culture was inoculated to a medium containing soluble starch (4.5%), corn steep liquor (1%), dried yeast (1%), calcium carbonate (0.1%) and Adekanol (defoaming agent. Trade Mark, maker; Asahi Denka Co.) (0.1%) (150 liters) in a 200-liter jar-fermentor, which had been sterilized at 120°C for 20 minutes ϊή advance, and cultured at 30^QC for 4 days under aeration of 150 liters/minutes and agitation of 250 rpm.

Isolation and Purification

The cultured broth thus obtained was filtered with an c aid of diatomaceous earth (5 kg). The mycelial cake was extracted with methanol (50 liters), yielding 50 liters of the extract. The methanol extract from mycelium and the filtrate were combined and passed through a column of a non-ionic adsorption resin Diaion HP-20 (Trade Mark, maker Mitsubishi Chemical Industries Ltd.)( 10 liters). After washing with water (30 liters) and aqueous methanol (30 liters), elution was carried out with methanol.

The eluate was evaporated under reduced pressure to give residual water (2 liters). This residue was extracted with ethyl acetate (2 liters). The ethyl acetate extract was concentrated under reduced pressure to give an oily residue. The oily residue was mixed with twice weight of acidic

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3;12.85 silica gel (special silica gel grade 12, maker Fuji Division Co.), and this mixture was slurred in ethyl acetate. After evaporating the solvent, the resultant dry powder was subjected to column chromatography on the same acid silica gel (800 ml) which was packed with n-hexane. The column was developed with n-hexane (3 liters), a mixture of n-hexane and ethyl acetate (9:1 v/v, 3 liters and 4:1 v/v, 3 liters) and ethyl acetate (3 liters). The fractions containing the object compound were collected and concentrated under reduced pressure to give an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1:1 v/v, 30 ml) and subjected to column chromatography of silica gel (maker Merck Co., Ltd. 230-400 mesh) (500 ml) packed with the same solvents system.

Elution was carried out with a mixture of n-hexane and ethyl acetate (1:1 v/v, 2 liters and 1:2 v/v, 1.5 liters). Fractions containing the FR-900520 substance were collected and concentrated under reduced pressure to give a yellowish oil. The oily residue was mixed with twice the weight of acidic silica gel and this mixture was slurried in ethyl acetate. After evaporating the solvent, the resultant dry powder was chromatographed on acidic silica gel packed and developed with n-hexane. Fractions containing the object compound were collected and concentrated under reduced pressure to give crude product (1054 mg) in the form of white powder.

The white powder of crude produce (1 g) was dissolved in acetonitrile (5 ml) and subjected to high performance liquid chromatography (HPLC) using Shimazu LC4A (Trade Mark, made by Shimazu Seisaku-sho). Steel column (25

3.12.85 mm inside diameter, 250 mm length) packed with YMC-S343 (ODS) (Trade Mark, made by Shimakyu Co., Ltd.) was used at a flow rate of 12 ml/min. Mobile phase was an aqueous mixture of 28% acetonitrile, 10% n-butancl, 0.075% phosphoric acid, 3.75 mM sodium dodecyl sulfate (SDS) and detection was carried out using Hitachi UV-recorder at 210 nm. One hundred ul of the sample was injected each time and the HPLC was repeated 50 times so that all the sample could be subjected to the column. Each eluate with a retention time of 85 min. to 90 min. was collected and extracted with an equal volume of ethyl acetate (3.6 liters). The ethyl acetate layer was separated and washed with an aqueous sodium hydrogen carbonate (1%, 2 liters) and concentrated in vacuo to a small amount. SDS crystallized on concentration was removed by filtration. Crude powder obtained was dissolved in acetonitrile at a concentration of 100 ing/ml and applied again to HPLC. Mobile phase was an aqueous mixture of 12.5% acetonitrile, 9.75% n-butanol, 0.075% phosphoric acid, 3.75 mM SDS. The column was eluted at a flow rate of 10 ml/min. The eluates with a retention time of 131 min. to 143 min. were collected and extracted with equal volume of ethyl acetate. The solvent layer was separated and washed with 1% aqueous sodium hydrogen carbonate and concentrated-in vacuo to a small volume. SDS crystallized on concentration was removed by filtration.

Crude powder thus obtained was dissolved in a small amount of ethyl acetate and subjected to column chromatography using silica gel (10 ml) (Kiesel gel, 230-400 mesh, maker: Merck Co., Ltd.). The column was washed with a mixture of n-hexane and ethyl acetate (30 ml) (1:1 v/v) and a mixture of n-hexane and ethyl acetate (60 ml) (1:2 v/v). elution was carried out using ethyl acetate and fractionated (each fraction : 3 ml). Fractions 18 to 24 were collected and concentrated in vacuo to dryness to give FR-900520 substance (24 mg).

Example

Isolation of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238

Streptomyces hygroscopicus subsp. yakushimaensis No.

7238 was isolated by using dilution plate techniques as shown in the following.

About one gram soil which was collected at Yakushima, Kagoshima Prefecture, Japan, was added to a sterile test tube and the volume made up to 5 ml with sterile water. The mixture was then blended for 10 seconds by a tube buzzer and kept on 10 minutes. The supernatant was sequentially diluted by 100 fold with sterile water. The diluted solution (0.1 ml) was spread on Czapek agar supplemented with thiamine hydrochloride (saccharose 30 g, sodium nitrate 3 g, dipotassium phosphate. 1 g, magnesium sulfate 0.5 g, potassium chloride 0.5 g, ferrous sulfate 0.01 g, thiamine hydrochloride 0.1 g, agar 20 g, tap water 1000 ml; pH 7.2) in a Petri dish. The growing colonies developed on the plates after 21 days incubation at 30^eC were transferred to slants (yeast-malt extract agar (ISP-medium 2)1, and cultured for 10 days at 30°C. Among of the colonies isolated, the Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 could be found.

Fermentation

A culture medium (160 ml) containing glycerin (3¾) soluble starch (1 %) , glucose (0.5%), cottonseed meal (0.5%), dried yeast (0.5%), corn steep liquor (0.5%) and calcium carbonate (0.2%) (adjusted to pH 6.5) was poured into each of twenty 500 ml-Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of slant culture of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238, FERM BP-928 was inoculated to each of the media and cultured at 30°C for 4 days on a rotary shaker. The resultant culture was inoculated to a medium containing glucose (4.5%), corn steep liquor (1%), dried yeast (1%), gluten meal (1%), wheat germ (0.5%), calcium carbonate (0.1%) and Adekanol (defoaming agent, Trade Mark, maker Asahi Denka Co.) (0.1%) (150 liters) in a 200-liter jar-fermentor, which had been sterilized at 120°C for 20 minutes in advance, and cultured at 30®C for 4 days under aeration of 150 liters/minutes and agitation of 250 rpm.

Isolation and Purification

The cultured broth thus obtained was filtered with an c aid of diatomaceous earth (5 kg) . The mycelial cake was extracted with acetone (50 liters), yielding 50 liters of the extract. The acetone extract from mycelium and the filtrate (135 liters) were combined and passed through a column of a non-ionic adsorption resin Diaion HP-20 (Trade Mark, maker Mitsubishi Chemical Industries Ltd.)( 10 liters). After washing with water (30 liters) and aqueous acetone ('30 liters) , elution was carried out with acetone. The eluate was evaporated under reduced pressure to give residual water (2 liters). This residue was extracted with ethyl acetate (4 liters). The ethyl acetate extract was concentrated under reduced pressure to give an oily residue. The oily residue was mixed with twice weight of acidic silica gel (special silica gel grade 12, maker Fuji Devison Co.), and this mixture was slurried in ethyl acetate. After evaporating the solvent, the resultant dry powder was subjected to column chromatography on the same acid silica gel (800 ml) which was packed with n-hexane. The column was developed with n-hexane (3 liters), a mixture of n-hexane and ethyl acetate (4:1 v/v, 3 liters) and ethyl acetate (3 liters). The fractions containing the FR-900520 substance were collected and concentrated under reduced pressure to give an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1:1 v/v, 50 ml) and subjected to column chromatography silica gel (maker Merck Co., Ltd. 70 - 230 mesh) (1000.ml ) packed withthe same solvents system. Elution was carried out with a mixture of n-hexane and ethyl acetate (1:1 v/v, 3 liters and 1:2 v/v, 3 liters) and ethyl acetate (3 liters). Fractions containing the object compounds were collected and concentrated under reduced pressure to give a yellowish powder (4.5 g). This powder was dissolved in methanol (20ml) and mixed with water (10ml). The mixture was chromatographed on a reverse phase silica gel YMC (60-200 mesh) (500ml) (Trade Mark, maker Yamamura Chemical Institute) packed and developed with a mixture of methanol and water (4:1 v/v).

Fractions containing the FR-900520 substance were collected and concentrated under reduced pressure to give crude product of the FR-900520 substance (1.8 g) in the form of pale yellowish powder. This powder was dissolved in a small amount of diethyl ether. After standing overnight, the precipitated crystals were collected by filtration, washed with diethyl ether and then dried under reduced pressure. Recrystallization from diethyl ether gave 600 mg of the purified FR-900520 substance in the form of colorless plates.