NO176523B - Process for preparing the tricyclo compound FR-900523 - Google Patents

Description

The present invention relates to a method for the production of a hitherto unknown tricyclo compound with pharmacological activity.

More specifically, the invention relates to a method for producing a hitherto unknown tricyclo compound which has pharmacological activity, such as immunosuppressive activity, antimicrobial activity and the like.

One object of the present invention is therefore to produce the hitherto unknown tricyclo compound which is useful for the treatment and prevention of resistance in transplantation, graft-versus-host diseases in bone marrow transplantation, autoimmune diseases, infectious diseases and the like.

With regard to the present invention, it should be noted that it originates from and is based on the recognition of certain hitherto unknown specific compounds, the substances FR-900506 and FR-900523. More specifically, the substances FR-900506 and FR-900523 were first isolated in pure form from culture fluids that were produced by fermentation of hitherto unknown species belonging to the genus Streptomyces.

As a result of extensive investigations to elucidate the chemical structures of the substances FR-900506 and FR-900523, the present inventors have succeeded in determining their chemical structure and in preparing the tricyclo compound of formula I.

The hitherto unknown tricyclo compound produced according to the invention has the following formula I

According to the invention, the tricyclo compound with formula I can be prepared by the following method:

I) Fermentation processes:

Streptomyces Fermentation \ FR-900523 hygroscopicus subsp.

yakushimaensis, FERM BP-928

or mutants thereof with those

same essential morphological and

biochemical properties.

The process according to the invention for producing the tricyclo compound of formula I is explained in more detail below.

I) Fermentation process

The substance FR-900523 is produced by fermenting Streptomyces hygroscopicus subsp. in a nutrient medium. yakushimaensis number 7238.

The microorganism

Further information on the microorganism used to produce the substance FR-900523 is given below.

The microorganism that can be used for the production of the substance FR-900523 is the strain Streptomyces hygroscopicus subsp. yakushimaensis number 7238 which has been recently isolated from a soil sample collected in Yakushima, Kagoshima Prefecture, Japan.

A lyophilized sample of the newly isolated Streptomyces hygroscopicus subsp. yakushimaensis number 7238 has been deposited in 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 FERM P-8043 (deposit date 12 . January 1985) and has then on 19 October 1985 been converted to deposit in accordance with the Budapest Treaty with the same depositing authority under the new deposit number FERM BP-928.

It is clear that the production of the hitherto unknown substance FR-900523 is not limited to the use of the particular organism described herein and which is only given as an example. The invention also relates to the use of all mutants with the same essential morphological and biochemical characteristics that are capable of producing the substance FR-900523, including both natural mutants and artificial mutants that can be produced from the described organism by conventional methods such as X-ray irradiation, ultraviolet irradiation, treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 2-amino-purine and the like.

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

1) Morphological characteristics:

For this taxonomic investigation, the methods described by Shirling and Gottlieb (E.B. Shirling and D. Gottlieb, "Methods for characterization of Streptomyces species", International Journal of Systematic Bacteriology 16, 1966, pp. 313-340) were mainly used.

Morphological observations were made with light and electron microscopes on cultures grown at 30°C for 14 days on oat flour agar, yeast/malt extract agar and inorganic salts/starch agar. The mature sporophores were moderately short and formed Retinaculiaperti and spirals with approx. 20 spurs in each chain. Hygroscopic spore mass is sown in the aerial mycelium on oatmeal agar and inorganic salts/starch agar. Surface irregularities on the tracks were a cross between very short, thick spikes and warts.

2) Cultural identity:

Culture characteristics were observed on ten media as described by Shirling and Gottlieb (loe. eit.) and by Waksmann (S.A. Waksman, The Actinomycetes, volume 2, "Classification, identification and description of genera and species", The Williams and Wilkins Co. Baltimore , 1961).

Incubation was carried out at 3 0"C for 14 days. The color names used in this study are based on the 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. No soluble pigment was formed in the investigated media. The results are shown in table 1.

The cell wall analysis was carried out according to the methods described by Becker et al., as well as Yamaguchi (B. Becker, M.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, 1964, pp. 421-423, and T. Yamaguchi, "Comparison of the cell wall composition of morphologically distinct Actinomycetes",

J. Bacteriol. 89, 1965, pp. 444-453). Analysis of whole cell hydrolysates of strain No. 7238 showed the presence of LL-diaminopimelic acid. Consequently, the cell wall of this strain is assumed to be of type I.

3) Biological and physiological characteristics.

The physiological characteristics of strain No. 7238 were determined according to the methods described by Shirling and Gottlieb 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 (manufactured by Toyo Kagaku Sangyo Co., Ltd.). The temperature range for growth was 18-36°C with an optimum temperature of 28°C. Starch hydrolysis and gelatin liquefaction were positive. No melanoid pigment was formed.

The utilization of carbon sources was investigated according to

the methods described by Pridham and Gottlieb (T.G. Pridham and D. Gottlieb, "The utilization of carbon compounds by some Actinomycetales as an aid for species determination",

J. Bacteriol. 56, 1948, pp. 107-114). Growth was observed after 14 days of incubation at 30°C.

The carbon source utilization of this strain is shown in summary in table 6. D-glucose, sucrose, lactose, maltose, D-trehalose, inositol, inulin and salicin could be utilized by strain no. 7238.

Symbols:

+ = utilization

= questionable exploitation

- = no utilization

Microscope examinations and analysis of the cell wall composition in strain no. 7238 show that this strain belongs to the genus Streptomyces Waksman and Henrici 1943.

Accordingly, a comparison of this strain with various Streptomyces species was made in the light of the published descriptions (International Journal of Systematic Bacteriology 18, 1968, pp. 69-189 and 279-392, and 19, 1969 pp. 391- 512 as well as Bergey<1>'s Manual of Determinative Bacteriology,

8th edition 19 74) .

As a result of the comparison, strain No. 7238 is considered to be similar to Streptomyces antimycoticus Waksman 19 57 and Streptomyces hygroscopicus subsp. glebosus Ohmori et al., 1962. The culture characteristics of strain no. 7238 were therefore further compared with the corresponding strains Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13 786 as shown in Tables 1, 2 and 3 above. Based on further comparisons, strain no. 7238 could be distinguished from Streptomyces antimycoticus IFO 12839 and Streptomyces hygroscopicus subsp. glebosus IFO 13786 on the following points.

i) Difference from Streptomyces antimycoticus IFO 12839

The culture characteristics of strain #7238 differ from Streptomyces antimycoticus IFO 12839 on yeast/malt extract agar, glucose/asparagine agar, glycerol/asparagine agar, potato/dextrose agar and tyrosine agar.

With respect to carbon source utilization, strain No. 7238 can utilize maltose, which Streptomyces antimycoticus IFO 12839 cannot. Moreover, strain No. 7238 cannot utilize glycerol, D-fructose, rhamnose, raffinose, D-galactose, D-mannose, mannitol and sodium succinate, while Streptomyces antimycoticus IFO 128 39 can utilize them.

ii) Difference from Streptomyces hygroscopicus subsp. glebosus IFO 13786

The culture characteristics of strain No. 7238 differ from Streptomyces hygroscopicus subsp. glebosus IFO 13 786 on yeast/malt extract agar, potato/dextrose agar and tyrosine agar.

Milk peptonization is negative for strain no. 7238 but is positive for Streptomyces hygroscopicus subsp. glebosus IFO 13786. Strain No. 7238 can grow in the presence of 7% NaCl, whereas Streptomyces hygroscopicus subsp. glebosus IFO 1378 6 cannot grow under the same conditions.

With respect to carbon source utilization, strain No. 7238 can utilize lactose, inulin and salicin, while Streptomyces hygroscopicus subsp. glebosus IFO 13786 cannot utilize them.

Strain no. 7238 cannot utilize glycerol, D-xylose, D-fructose, raffinose, D-galactose, D-mannose, mannitol and sodium succinate, but Streptomyces hygroscopicus subsp. glebosus IFO 13786 can utilize them.

However, strain No. 7238 forms hygroscopic spore mass

in the aerial mycelium on oat flour agar and inorganic salts/starch agar, and furthermore the morphological and cultural characteristics of strain no. 7238 are very similar to the characteristics of Streptomyces hygroscopicus subsp. glebosus IFO 1378 6. Therefore, strain No. 7238 is considered to belong to Streptomyces hygroscopicus, but strain No. 7238 is different from Streptomyces hygroscopicus subsp. glebosus IFO 13786, although this known strain is the one most similar to strain No. 7238 of the Streptomyces hygroscopicus subspecies. Based on the above facts, strain no. 7238 is considered to be a new species of Streptomyces hygroscopicus and has been given the designation Streptomyces hygroscopicus subsp. yakushimaensis subsp. Nov. referring to the soil collected in Yakushima from which the organism was isolated.

Preparation of the substance FR-900523.

The hitherto unknown substance FR-900523 is produced by

grow the strain Streptomyces hygroscopicus subsp. yakushimaensis No. 7238 FERM BP-928 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 carbon sources in the nutrient medium are carbohydrates such as glucose, sucrose, lactose, glycerol, starch, dextrin and the like. Other sources that can be incorporated are maltose, D-trehalose, inositol, inulin, salicin and the like.

The preferred nitrogen sources are yeast extract, peptone, gluten meal, cottonseed meal, soybean meal, corn casting liquid, dry yeast, wheat germ, feather meal ("feather meal"), peanut powder, etc. as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulfate , ammonium phosphate, etc), urea, amino acids and the like.

The carbon and nitrogen sources, even if they are advantageously used in combination, do not need to be used in their pure form, as less pure materials containing traces of growth factors and significant amounts of mineral nutrients are also suitable for use. If desired, 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 can be added to the medium. If necessary, especially if the culture medium foams a lot, an antifoam agent such as liquid paraffin, a fatty oil, vegetable oil, mineral oil or silicone can be added.

Submerged aerobic culture conditions are preferred as conditions for the production of the substances FR-900520 and FR-900523 in large quantities. For production in small quantities, a shaking or surface culture in a flask or bottle is used. When cultivation is carried out in large tanks, it is further preferred to use the vegetative form of the organism for inoculation in the production tanks to avoid delayed growth during the production of the substance FR-900523. It is therefore desirable to first produce a vegetative inoculum of the organism by inoculating a relatively small amount of culture medium with spores or mycelia of the organism, and culture this inoculated medium, and then aseptically transfer the cultured vegetative inoculum to large tanks. The medium in which the vegetative inoculum is produced is essentially the same as, or is different from, the medium used to produce the substance FR-900523.

Agitation and aeration of the culture mixture can be done in several different ways. Agitation can be done with a stirrer or similar mechanical agitation equipment, by turning or shaking the fermentation vessel, with various pumping equipment or by passing sterile air through the medium. Aeration can be performed by passing sterile air through the fermentation mixture.

The fermentation is usually carried out at a temperature of approx. 20-40°C, preferably 25-35°C, for approx. 50-150 hours, which can be varied depending on the fermentation conditions and measuring stick.

The thus produced substance FR-90052 3 can be isolated from the culture medium by conventional methods which are usually used for the isolation of other known biologically active substances. The manufactured substance FR-900523 is mainly found in the cultured mycelium, therefore the substance FR-900523 can be isolated and purified from the mycelium, which is obtained after filtering or centrifuging the culture liquid, by a common method such as concentration under reduced pressure, lyophilization, extraction with a common solvent, pH adjustment, treatment with a common resin (e.g. anion or cation exchange resin, non-ionic adsorption resin, etc), treatment with a common adsorbent (e.g. activated carbon, silicic acid, silica gel , cellulose, aluminum oxide, etc), crystallization, recrystallization and the like.

The substance FR-900523 can be separated in particular by dissolving the materials containing both products produced by fermentation in a suitable solvent such as ethyl acetate, n-hexane and the like, after which this solution is subjected to chromatography, e.g. on silica gel in a column with an appropriate organic solvent, such as ethyl acetate and n-hexane or a mixture thereof. The thus-separated substance FR-900523 can be further purified by a conventional method, e.g. recrystallization, re-chromatography, high pressure liquid chromatography and the like.

The substance FR-900523

1) Shape and colour:

Colorless needles

2) Elementary analysis:

C, 64.57; H, 8.24; N, 1.81

3) Color reaction:

Positive: cerium sulfate reaction, sulfuric acid reaction,

Ehrlich reaction, Dragendorff reaction and iodine vapor reaction.

Negative: ferric chloride reaction and ninhydrin reaction.

4) solubility:

Soluble: methanol, ethanol, acetone, ethyl acetate,

chloroform, diethyl ether and benzene.

Sparingly soluble: n-hexane and petroleum ether

Insoluble: water.

5) Melting point: 152-154"C.

6) Specific rotatability: [oc]q<3>= -73.0° (c = 0.65 in CHCl-j) .

7) Ultraviolet absorption spectrum: Total absorption.

8) Infrared absorption spectrum (CHCl^): vmax <=>

3670, 3580, 3510, 2930, 2875, 2825, 1745, 1722, 1700, 1647, 1450, 1380, 1350, 1330, 1307, 1285, 1170, 1135, 1090, 1050, 1090, 909, 909, 1030, 1096 930, 915, 888, 870 and 850 cm"<1>.

9) <i>3C-NMR spectrum (CDCl3): (ppm) =

213.82 (s) + 213.32 (s), 196.31 (s) + 193.34 (s), 168.96 (s) + 168.85 (s), 164.84 (s) + 165 .98 (s), 137.80 (s) + 138.41 (s), 132.89 (s) + 131.96 (s), 129.62 (d) + 130.03 (d) , 124, 51 (d) + 124 .84 (d) , 97.13 (s) + 98.67 (s) , 84.38 (d), 76.69 (d) + 78.06 (d), 75.45 (d) + 76.91 (d) , 73, 89 (d) + 73.70 (d) , 73.70 (d) , 73.09 (d) + 72.84 (d) , 70.40 ( d) + 69.24 (d) , 56.75 (d) + 52.89 (d) , 56.93 (g) + 57.43 (q), 56.61 (q) + 56.56 (q ) , 56.24 (q) + 55.94 (q) , 48.58 (t) + 48.32 (t) , 47.14 (d) + 47.38 (d) , 40.23 (d) + 40.65 (d) , 27.85 (t) + 26.32 (t) , 26.48 (d) + 26.64 (d) , 24.68 (t), 21.33 (t) + 20.83 (t), 20.63 (q) + 19.77 (q), 16.24 (q) + 16.34 (q) , 15.70 (q) + 15.96 (q) , 15 .51 (q) + 15.96 (q), 14.31 (q) + 14.18 (q), 9.64 (q) + 10.04 (q),

which spectrum is shown in fig. 1.

10) <1>H-NMR spectrum: is shown in fig. 2. 11) Thin layer chromatography:

12) Property of the substance: neutral substance.

With regard to the substance FR-900523, it should be noted that with regard to measurements of 13C and ^-H NMR spectra, the substance shows signal pairs at different chemical shift values, and that when measured by thin-layer chromatography and high-pressure liquid chromatography it showed respectively a single spot by thin-layer chromatography and a single peak by high-pressure liquid chromatography.

Based on the above physical and chemical properties and the previous successful determination of the chemical structure of the substance FR-900506, it could be determined that the substance FR-900523 has the following chemical structure:

1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-2 3,25-dimethyloxy-13,19,17,21,27-pentamethyl-1,28-dioxa -4-azatricyclo[22.3.1.04'9]octacos-18-ene-2,3,10,16-tetraone.

The tricyclo compound of formula I has pharmacological activity such as immunosuppressive activity, antimicrobial activity and the like and is therefore useful for the treatment and prevention of resistance in transplantation of organs or tissues, such as hearts, kidneys, liver, spinal cord, skin, etc., "graft -versus-host" diseases in spinal cord transplantation autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type I diabetes, uveitis, etc, infectious diseases caused by pathogenic microorganisms and the like.

As examples for demonstrating such pharmacological activities, some pharmacological test data for the tricyclo compound are given in the following.

Test 1

Suppression caused by the tricyclo compounds I in the in vitro mixed lymphocyte reaction (MLR)

The MLR test was performed on microtiter plates where each well contains 5 x IO<5> C57BL/6 responder cells (H-2<b>), 5 x IO<5 >mitomycin C-treated (25 µg/ml mitomycin C at 37°C for 30 minutes and washed 3 times with RPMI 1640 medium) BALB/C stimulator cells (H-2<d>) in 0.2 ml RPMI 1640 medium supplemented with 10% fetal calf serum, 2 mM sodium bicarbonate, 50 units/ml penicillin and 50 µg/ml streptomycin. The cells were incubated at 37°C in a humid atmosphere with 5% carbon dioxide and 95% air for 68 hours and radiolabeled with 0.5 iiCi <3>H-thymidine 4 hours before isolation of the cells. The subject compound prepared according to the invention was dissolved in ethanol and further diluted in RPMI 164 0 medium and added to the cultures, giving final concentrations of 0.1 µg/ml or less.

The results are shown in Table 4. The tricyclo compounds prepared according to the invention suppressed mouse MLR.

Test 2

Antimicrobial activity of the tricyclo compound I

The antimicrobial activity of the tricyclo compound I against various fungi was determined by an agar serial dilution method in a Sabouraud agar. Minimal inhibitory concentrations (MIC) were expressed as nq/ml after incubation at 30°C for 24 hours.

The tricyclo compound prepared according to the present invention exhibited antimicrobial activity against fungi, e.g. Aspergillus fumigatus IFO 5840 and Fusarium oxysporum IFO 5942, which is shown in table 5 below.

A pharmaceutical preparation containing the tricyclo compound (I) produced according to the invention can e.g. be in solid, semi-solid or liquid form, containing the tricyclo compound I prepared according to the present invention, as active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral use. The active ingredient can e.g. be mixed with common non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions and any other form suitable for use. The carriers that can be used are water, glucose, lactose, acacia gum, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silicon dioxide, potato starch, urea and other carriers that are suitable for use in the preparation of preparations in solid , semi-solid or liquid form, and auxiliaries, stabilizers, thickeners, dyes and perfumes can also be used. The active compound is incorporated into the pharmaceutical preparation in an amount sufficient to induce the desired effect on the course of the disease or the patient's condition.

For administration of this preparation to humans, it is preferred to administer it parenterally or enterally. Although the dose of the therapeutically effective amount of tricyclo compound I varies with, and also depends on, the age and condition of each individual patient to be treated, generally a daily dose of about 0.01 - 1000 mg, preferably 0.1 - 500 mg and especially 0.5 - 100 mg, of the active ingredient for the treatment of diseases, and an average single dose of approx. 0.5 mg, 1 mg,

5 mg, 10 mg, 50 mg, 100 mg, 250 mg and 500 mg.

The invention is explained in more detail by the references and examples below.

Reference 1

Isolation of Streptomyces tsukubaensis No. 9993.

Streptomyces tsukubaensis No. 9993 was isolated using dilution plate techniques as described below.

About. 1 g of soil, which was collected in Toyosato-cho, Tsukuba Gun, Ibaraki Prefecture, Japan, was added to a sterile test tube and the volume was made up to 5 ml with sterile water. The mixture was then thoroughly mixed for 10 seconds with a tubular vibrator and mixing continued for 10 minutes. The supernatant was successively diluted 100 times with sterile water. The diluted solution (0.1 ml) was plated on Czapek agar enriched with thiamine hydrochloride (30 g sucrose, 3 g sodium nitrate, 1 g dicalcium phosphate, 0.5 g magnesium sulfate, 0.5 g potassium chloride, 0.01 g ferrous sulfate , 0.1 g thiamine hydrochloride, 20 g agar, 1000 ml tap water; pH value 7.2) in a Petri dish. The growing colonies that developed on the plates after 21 days of incubation at 30°C were transferred to slant agar [yeast/malt extract agar (ISP medium 2)] and were grown for 10 days at 30°C. Among the isolated colonies, Streptomyces tsukubaensis no. 9993 could be found.

Streptomyces tsukubaensis No. 9993 has the following morphological, cultural, biological and physiological characteristics: 1) Morphological characteristics

The methods described by Shirling and Gottlieb (E.B. Shirling and D. Gottlieb "Methods for characterization of Streptomyces species", International Journal of Systematic Bacteriology 16, 1966, pp. 313-340) were mainly used for this taxonomic investigation.

Morphological observations were made with light and electron microscopes on cultures grown at 30°C for 14 days on oat flour agar, yeast/malt extract agar and inorganic salts/starch agar. The mature sporophores formed Rectiflexi-biles with 10-50 or over 50 spores in each chain. The spores were oblong or cylindrical, 0.5-0.7 x 0.7-0.8 µm in size, as determined by electron microscopic observation. The spore surfaces were smooth.

2) Cultural characteristics

Culture characteristics were observed on ten kinds of media as described by Shirling and Gottlieb mentioned above, as well as by Waksman (S.A. Waksman, The Actinomycetes, volume 2, "Classification identification and description of genera and species", The Williams and Wilkins Co., Baltimore, 1961 ).

Incubation was carried out at 30°C for 14 days. The color names used in this survey are based on the Guide to Color

Standard (manual published by Nippon Shikisai Kenkyusho, Tokyo). The colonies belonged to the gray color series when grown on oatmeal agar, yeast/malt extract agar and inorganic salts/starch agar. Soluble pigment was formed in yeast/malt extract agar but not in other media. The results are listed in table 6.

Cell wall analysis was performed according to those of Becker

et al., as well as methods described by Yamaguchi (B. Becker, M.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, 1964, pp. 421- 423, and T. Yamaguchi, "Comparison of the cell wall composition of morphologically distinct Actinomycetes", J. Bacteriol. 89, 1965, pp. 444-453). Analysis of whole cell hydrolysates of strain #9993 showed the presence of LL-diaminopimelic acid. Consequently, the cell wall of this strain is assumed to be of type I.

3) Biological and physiological characteristics

The physiological characteristics of strain No. 9993 were determined

according to the methods described by Shirling and Gottlieb mentioned above. The results are shown in Table 7. Temperature range and optimum temperature for growth were determined on yeast/malt extract agar using a temperature gradient incubator (manufactured by Toyo Kagaku Sangyo Co., Ltd).

The temperature range for growth was 18-35°C with an optimum temperature of 28°C. Milk peptonization and gelatin-fluidization were positive. Melanoid pigment formation was negative.

The utilization of carbon sources was examined according to the methods described by Pridham and Gottlieb (T.G. Pridham and D. Gottlieb, "The utilization of carbon compounds by some Actinomycetales as an aid for species determination", J. Bacteriol. 5_6, 1948, 2. 107-1 14). The growth was observed after

14 days incubation at 30°C.

The carbon source utilization of this strain is shown in summary in table 8. Glycerol, maltose and sodium succinate could be utilized by strain no. 9993. Furthermore, questionable utilization of D-glucose, sucrose, D-mannose and salicin was also observed.

Microscope examinations and analysis of the cell wall composition of strain no. 999 3 show that this strain belongs to the genus Streptomyces Waksman and Henrici 1943.

Accordingly, a comparison of this strain with various Streptomyces species was made in the light of the published descriptions (International Journal of Systematic Bacteriology 18, 1968, pp. 69-189 and 279-392, and 19, 1969, p. 391 -512, as well as Bergey's Manual of Determinative Bacteriology, 8th edition 1974).

As a result of the comparison, strain No. 9993 is considered to be similar to Streptomyces aburaviensis Nishimura et al., Streptomyces misakiensis Nakamura. The culture characteristics of strain number 9993 were therefore compared with the corresponding strains Streptomyces aburaviensis IFO 12830, Streptomyces avellaneus IFO 13451 and Streptomyces misakiensis IFO 12891. As a result, strain number 999 3 resembled Streptomyces misakiensis IFO 12891 the most. Strain number 9993 was therefore further compared with Streptomyces misakiensis IFO 12891 as shown in Tables 6,7 and 8 above. Based on further comparisons, strain number 9993 could be distinguished from Streptomyces misakiensis IFO 12891 on the following points, which is why strain number 9993 is considered to be a hitherto unknown Streptomyces species that has been given the designation Streptomyces tsukubaensis sp. nov., referring to the soil collected in Tsukuba-gun, from which the organism was isolated.

Difference from Streptomyces misakiensis IFO 12891.

Culture characteristics of strain number 9993 differ from Streptomyces misakiensis IFO 12891 on oat flour agar, yeast/malt extract agar, glucose/asparagine agar, Czapek agar and potato/dextrose agar.

Starch hydrolysis caused by strain number 9993 is negative, but is positive for Streptomyces misakiensis IFO 12891.

Gelatin liquefaction caused by strain No. 9993 is positive but is negative for Streptomyces misakiensis IFO 12891.

In terms of carbon source utilization, strain number 9993 can utilize glycerol, maltose and sodium succinate, but Streptomyces misakiensis IFO 12891 cannot utilize them. Moreover, strain no. 9993 cannot utilize D-galactose and inulin, whereas Streptomyces misakiensis IFO 12891, on the other hand, can utilize them.

Fermentation

A 160-ml culture medium containing 1% glycerol, 1% soluble starch, 0.5% glucose, 0.5% cottonseed meal, 0.5% dry yeast, 0.5% corn molasses and 0.2% calcium carbonate (adjusted to pH 6, 5) was poured into each of 20,500 ml Erlen- . meyer flask and sterilized at 120°C for 30 minutes. An inoculum full slant agar culture of Streptomyces tsukubaensis number 9993, FERM BP-927 was inoculated into each of the media and grown at 30°C for 4 days on a rotary shaker. The resulting culture was inoculated into a 150 L medium containing 4.5% soluble starch, 1% corn molasses, 1% dry yeast, 0.1% calcium carbonate and 0.1% Adekanol™ (antifoam agent manufactured by Asahi Denka Co) in a 200 liter jar fermenter that had been previously sterilized at 120°C for 20 minutes, and the culture was grown at 30°C for 4 days under aeration at 150 l/min and shaking at 250 rpm.

Isolation and purification

The culture liquid thus obtained was filtered using 5 kg of diatomaceous earth. The mycelial cake was extracted with 50 1 of methanol, which gave 50 1 of extract. The methanol extract from the mycelium and the filtrate were combined and passed through a 10 liter column of a nonionic adsorption resin (Diaion™ HP-20, manufactured by Mitsubishi Chemical Industries Ltd.). After washing with 30 1 of water and 30 1 of aqueous methanol, elution with methanol was carried out. The eluate was evaporated under reduced pressure, giving 2 1 of residual water. This residue was extracted with 2 l of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to an oily residue. The oily residue was mixed with twice its weight of acidic silica gel (special silica gel, grade 12, manufactured by Fuji Devison Co.), and this mixture was slurried in ethyl acetate. After evaporation of the solvent, the resulting dry powder was subjected to column chromatography on 800 ml of the same acidic silica gel packed with n-hexane. The column was eluted with 3 L of n-hexane, a mixture of n-hexane and ethyl acetate (9:1 vol/vol, 3 L and 4:1 vol/vol, 3 L) and 3 L of ethyl acetate. The fractions containing the desired compound were collected and concentrated under reduced pressure to an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1:1 vol/vol, 30 ml) and subjected to column chromatography on 500 ml silica gel (manufactured by Merck & Co., Ltd., 0.063-0.037 mm) packed with the same solvent system.

Elution was carried out with a mixture of n-hexane and ethyl acetate (1:1 vol/vol, 2 1 and 1:2 vol/vol, 1.5 1). Fractions containing the first subject compound were collected and concentrated under reduced pressure to a yellowish oil. The oily residue was mixed with twice its weight of acidic silica gel and this mixture was slurried in ethyl acetate. After evaporation of the solvent, the resulting dry powder was chromatographed on acid silica gel which was packed and eluted with n-hexane. Fractions containing the desired compound were collected and concentrated under reduced pressure to give 1054 mg of crude FR-900506 as a white powder.

100 mg of this crude product was subjected to high pressure liquid chromatography. Elution was carried out using a column (diameter 8 x 500 mm) with Lichrosorb™ SI 60 (manufactured by Merck & CO.) as carrier. This chromatography was monitored with a UV detector at 230 nm and the mobile phase was a mixture of methylene chloride and dioxane (85:15 vol/vol) at a flow rate of 5 ml/min. The active fractions were collected and evaporated. High pressure liquid chromatography was repeated and this gave 14 mg of the purified FR-900506 substance as a white powder.

Physiological and chemical properties of substance FR-900506.

The substance FR-900506 produced by the above method has the following physical and chemical properties: FR-900506

1) Shape and colour:

White powder

2) Elementary analysis

Calculated: C, 64.72; H, 8.78; N, 1.59

Found: C, 64.59; H, 8.74; N, 1.62

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: hexane, petroleum ether.

Insoluble: water

5) Melting point: 85-90°C

6) Specific rotatability: [a]^<3>= -73° (c = 0.8 in CHC13).

7) Ultraviolet absorption spectrum: Total absorption

8) Infrared absorption spectrum (CHCl3): <v>max<=><3>680, 3580, 3520, 2930, 2870, 2830, 1745, 1720, 1700, 1645, 1450, 1380, 1350, 1330, 1310, 1285, 1170, 1135, 1090, 1050, 1030, 1000, 990, 960 (shoulder) and 918 cm"<1>.

<9*><13>C-NMR spectrum (CDCl3): δ (ppm)= ;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), 9.69 (q) + 9.98 (q), which spectrum is shown in fig. 3. ;1<0>* <1>H-NMR spectrum is shown in fig. 4.

11) Thin layer chromatography:

12) Property of the substance: neutral substance.

With regard to the substance FR-9 00506, it should be noted that in terms of measurements of 13 C and 1 H NMR spectra, the substance showed signal pairs at different chemical shift values.

The thus characterized substance FR-9 00506 has the following additional properties: i) Measurements of <13>C-NMR spectra at 25°C and 60°C showed that the intensity of each pair of the different signals therein was changed.

ii) Measurements by thin-layer chromatography and high-pressure liquid chromatography showed that the substance FR-9 00506 occurs respectively as a single spot by thin-layer chromatography and a single peak by high-pressure liquid chromatography.

This white powder of the substance FR-900506 could be converted into crystal form by recrystallization from acetonitrile, which crystals have the following physical and chemical properties:

1) Shape and colour:

Colorless prisms.

2) Elementary analysis:

Calculated: C, 64.30; H, 8.92; N, 1.77

Found: C, 64.20; H, 8.86; N, 1.72.

3) Melting point: 127-129°C.

4) Specific rotatability: [a]^<3>= -84.4° (c = 1.02 in CHCl3).

5) <13>C-NMR spectrum (CDCl3) 5 (ppm)=

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) , 52.97 (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), 16.17 (q) + 16.10 (q), 15.88 (q) + 15.75 ( q), 13.89 (q) + 14.05 (q), 9.64 (q) + 9.96 (q),

which spectrum is shown in fig. 5.

6) -1H-NMR spectrum: is shown in fig. 6.

Other physical and chemical properties, i.e., color reaction, solubility, UV spectrum, IR spectrum, thin layer chromatography and properties of the colorless prisms of the substance FR-900506, were the same as those of the white powder of the substance under identical conditions.

Based on the above physical and chemical properties and X-ray diffraction analysis, it could be determined that the substance FR-900506 has the following chemical structure:

17-allyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxy-cyclohexyl)-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.

Example 1

Isolation of Streptomyces hygroscopicus subsp. yakushimaensis number 72 38

Streptomyces hygroscopicus subsp. yakushimaensis number 72 38 was isolated using dilution plate techniques as described below.

About. lg of soil, which was collected in Yakushima, Kagoshima, Prefecture, Japan, was added to a sterile test tube and the volume was brought up to 5 ml with sterile water. The mixture was then thoroughly mixed for 10 seconds with a tubular vibrator and mixing was continued for 10 minutes. The supernatant was successively diluted 100 times with sterile water. The diluted solution (0.1 ml) was plated on Czapek agar supplemented with thiamine hydrochloride (30 g sucrose, 3 g sodium nitrate, 1 g dipotassium phosphate, 0.5 g magnesium sulfate,

0.5 g of potassium chloride, 0.01 g of ferrous sulfate, 0.1 g of thiamine hydrochloride, 20 g of agar, 1000 ml of tap water; pH value 7.2) in a Petri dish. The growing colonies that developed on the plates after 21 days of incubation at 30°C were transferred to slant agar [yeast/malt extract agar (ISP medium 2)] and were grown for 10 days at 30°C. Among the isolated colonies, Streptomyces hygroscopicus subsp. yakushimaensis No. 7238.

Fermentation

A 160-ml culture medium containing 1% glycerol, 1% soluble starch, 0.5% glucose, 0.5% cottonseed meal, 0.5% dry yeast, 0.5% corn molasses and 0.2% calcium carbonate (adjusted to pH 6, 5) was poured into each of 20,500 ml Erlenmeyer flasks and sterilized at 12CC for 30 minutes. An inoculum full slant agar culture of Streptomyces hygroscopicus subsp. yakushimaensis No. 7238, FERM BP-928 was inoculated into each of the media and grown at 30°C for 4 days on a rotary shaker. The resulting culture was inoculated into a 150 L medium containing 4.5% glucose, 1% corn slurry, 1% dry yeast, 1% gluten meal, 0.5% wheat germ, 0.1% calcium carbonate and 0.1% Adekanol™ (antifoam agent manufactured by Asahi Denka Co) in a 200 liter jar fermenter previously sterilized at 120°C for 20 minutes, and the culture was grown at 30°C for 4 days under aeration at 150 L/minute and shaking at 250 rpm.

Isolation and purification

The culture liquid thus recovered was filtered by

■with the help of 5 kg of diatomaceous earth. The mycelial cake was extracted with 50 L of acetone, yielding 50 L of extract. The acetone extract from the mycelium and the filtrate (135 L) were combined and passed through a 10 liter column of a nonionic adsorption resin (Diaion™ HP-20, manufactured by Mitsubishi Chemical Industries Ltd.). After washing with 30 1 of water and 30 1 of aqueous acetone, elution with acetone was carried out. The eluate was evaporated under reduced pressure, giving 2 1 of residual water. This residue was extracted with 4 l of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to an oily residue. The oily residue was mixed with twice its weight of acidic silica gel (special silica gel, grade 12, manufactured by Fuji Devison Co.), and this mixture was slurried in an ethyl acetate. After evaporation of the solvent, the resulting dry powder was subjected to column chromatography on 800 ml of the same acidic silica gel, which was packed with n-hexane. The column was eluted with 3 L of n-hexane, a mixture of n-hexane and ethyl acetate (4:1 vol/vol, 3 L) and 3 L of ethyl acetate. The fractions containing the substances FR-900520 and FR-900523 were collected and concentrated under reduced pressure to an oily residue. The oily residue was dissolved in a mixture of n-hexane and ethyl acetate (1:1 vol/vol, 50 ml) and subjected to column chromatography on 1000 ml silica gel (manufactured by Merck & Co., Ltd., 0.210-0.063 mm) packed with same solvent system. Elution was carried out with a mixture of n-hexane and ethyl acetate (1:1 vol/vol, 3 1 and 1:2 vol/vol, 3 1) and 3 1 ethyl acetate. Fractions containing the desired compounds were collected and concentrated under reduced pressure to give 4.5 g of a yellowish powder. This

powder was dissolved in 20 ml of methanol and mixed with 10 ml of water. The mixture was chromatographed on 500 ml of reverse-phase silica gel YMC™ (0.250-0.074 mm, manufactured by Yamamura Chemical Institute), which was packed and eluted with a mixture of methanol and water (4:1 vol/vol).

Chromatography was performed on reverse-phase silica gel, and the resulting fractions containing FR-900523 were collected and concentrated under reduced pressure, yielding 0.51 g of crude FR-900523 as a pale yellow powder. This crude product was dissolved in 3 ml of acetonitrile and subjected to 70 ml of reverse phase silica gel YMC™ which was packed and eluted with a mixture of acetonitrile, tetrahydrofuran and 50 mM phosphate buffer solution (pH 2.0) (3:2.5 vol/vol) . Fractions containing the desired compound were collected and extracted with ethyl acetate. This extract was concentrated under reduced pressure to give 190 mg of yellowish-white powder. The yellow-white powder was again chromatographed on reverse-phase silica gel YMC™, yielding 80 mg of a white powder. This white powder was dissolved in a small amount of diethyl ether and allowed to stand overnight at room temperature yielding 56 mg of crystals. Recrystallization from diethyl ether gave 34 mg of FR-900523 material in the form of colorless needles.

Claims (1)

Method for preparing a compound FR-900523 with formula I characterized in that Streptomyces hygroscopicus subsp. yåku shimaensis, FERM BP-928 or mutants thereof with the same essential morphological and biochemical characteristics are cultivated in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under aerobic conditions, and the substance FR-900523 is extracted in a conventional manner.