Phosphoric acid ester of heterorlng compound as medicament

TECHNICAL FIELD

The present invention relates to a new bioactive compound, hereinafter entitled WF11231A substance.

More particularly, it relates to a novel compound, WF11231A substance or a salt thereof which has immunosupressing activity, to a process for preparation thereof, to a pharmaceutical composition comprising the same, which is useful as immunosupressing agents, and to a use thereof as a medicament.

Accordingly, one object of this invention is to provide the novel compound, F11231A substance or a salt thereof which is of use for treating and preventing rejection by transplantation, graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases, and the like.

Another object of this invention is to provide a process for production of the WF11231A substance or a salt thereof by fermentation of a WF11231A substance-producing strain belonging to the genus Cladobotrvum in a nutrient medium.

A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, the WF11231A substance or a salt thereof.

Still further object of this invention is to provide a use of the WF11231A substance or a salt thereof for treating and preventing rejection by transplantation, graft-versus-host diseases by medulla ossium transplantation, autoimmune diseases, and the like.

DISCLOSURE OF INVENTION

The WF11231A substance of the present invention can be represented by the following formula:

With respect to the WF11231 A substance (I) of this invention, it is to be understood that there may be one or more conformer(s) or stereoisomeric pairs such as optical isomer due to asymmetric carbon atoms(s), and such isomers are also included within a scope of this invention.

Suitable salt of the WF11231A substance (I) is conventional pharmaceutically acceptable salt and include a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, etc.), an organic acid salt (e. g. acetate,

trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, etc.), an inorganic acid salt (e. g. hydrochloride, hydrobromide, hydroiodide, sulfate, phosphate, etc.), or a salt with an amino acid (e. g. arginine, aspartic acid, glutamic acid, etc.), and the like.

The WF11231A substance can be produced by fermentation of the WF11231A substance-producing strain belonging to the genus Cladobotryum such as Cladobotrvum sp. No. 11231 in a nutrient medium.

It is to be understood that the production of the WF11231A substance is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF11231A 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-ray, ultra-violet radiation, treatment which N-methyl-N'-nitro-N-nitrosoguanidine, 2- aminopurine, and the like.

Particulars of Cladobotrvum sp. No. 11231 is as follows:

Characteristics of producing Strain No.11231

The fungus strain No.11231 was originally isolated from a litter, collected at Iwaki-shi, Fukushima-ken, Japan. This organism grew very rapidly on various culture media and formed yellow to red colonies. Strain No.11231 formed anamorph, consisting of micronematous conidiophores branching subverticillately with hyaline and septated conidia formed retrogressively on various agar media. The conidiogenesis was holoblastic. This strain did not produce teleomorph structures. On the basis of its morphological characteristics, the strain appears to belong the hyphomycete genus Cladobotrvum Nees ex Steud. 1924^^. Its mycological characteristics were as follows.

Cultural characteristics on various agar media are summarized

in Table 1. Culture on malt extract agar grew spreading broadly, attaining more than 8.5 cm in diameter after two weeks at 25 °C. This colony surface was plane, felty, and reddish yellow. The reverse was brownish yellow. Conidial structures were observed. Colonies on corn meal agar grew spreading broadly, attaining more then 8.5 cm in diameter under the same conditions. The surface was plane, thin, and reddish white. The reverse was reddish white. Conidial structures were formed abundantly.

The morphological characteristics were determined on basis of the cultures on mushroom agar^ ¹⁾ . The conidial structures were micronematous, mononematous, hyaline, smooth, septate. Conidiophores were fragile, hyaline, smooth, often over 1 mm long, and up to 15 μm wide. They branched subverticillately, whorls on main stalks contained 2-3 branches, and ultimate branches bore 2-7 fertile cells verticiUately. Conidiogenous cells were straight, subulate to cylindrical, 20-45 μm long, 3.0-6.0 μm wide above the base, tapering slightly towards the 1.0-3.0 μm wide apex, and formed conidia retrogressively. The conidia were 1-3 septated, straight, smooth, obclavate to cylindrical, and 18.0-28.5 x 7.0-11.0 μm, with rounded apices and acuminate bases.

The vegetative hyphae were smooth, septate, hyaline and branched. The hyphal cells were cylindrical and 2.0-8.0 μm in diameter. Strain No.11231 was able to grow at the temperature range from 4 to 32 °C with the growth optimum at 23 to 26 °C. These temperature data were determined on potato dextrose agar (made by Nissui).

According to the taxonomic criteria of the genus Cladobotrvum^ ¹ ^. strain No.11231 resembled Cladobotryum-state of Hypomyces dactylarioides G. Arnold 1971. But this strain did not produce teleomorph structures, then we named the producing strain Cladobotrvum sp. No.11231. And it was deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) as FERM BP-3665 (deposited date: December 4, 1991).

Table 1. Cultural characteristics of strain No.11231

Medium Cultural characteristics

Malt extract agar G: Spreading broadly, >8.5 cm (Blaskeslee 1915) S: Plane, felty, produced yellow soluble pigment, formed conidial structures, reddish yellow (4B7) R: Brownish yellow (5C8)

Potato dextrose G: Spreading broadly, >8.5 cm agar (Difco 0013) S: Cottony, produced red soluble pigment, formed conidial structures, dark blond

(5D4) R: Brownish violet (11D8)

Czapek's solution G: Spreading broadly, >8.5 cm agar (Raper and S: Irregular, cottony, formed conidial Thorn 1949) structures, yellowish white (3A2), at the center pastel pink (11 A4) R: Yellowish white (3A2), at the center pastel pink (11A4)

Sabouraud dextrose G: Spreading broadly, >8.5 cm agar (Difco 0190) S: Cottony, formed conidial structures, dull red (10B3) R: Brownish yellow (5C7)

Oatmeal agar G: Spreading broadly, >8.5 cm (Difco 0552) S: Felty to cottony, abundantly formed conidial structures, reddish white (7A2) R: Brownish orange (6C5)

Medium Cultural characteristics

Emerson Yp Ss agar G: Spreading broadly, >8.5 cm (Difco 0739) S: Cottony, abundantly formed conidial structures, light brown (7D4), at the margin reddish white (8A2), at the center brownish red (10C6) R: Grayish red (10B5)

Com meal agar G: Spreading broadly, >8.5 cm (Difco 0386) S: Plane, thin, abundantly formed conidial structures, reddish white (8A2) R: Reddish white (8A2)

MY20 agar* G: Spreading broadly, 5.0-5.5 cm

S: Irregular, cottony, abundantly formed conidia, structures, brownish orange (5C3), at the margin pastel yellow (3A4), at center grayish yellow (3C3) R: Pale yellow (3A3), at the center golden brown (5D7)

Abbreviation: G: growth, measuring colony size in diameter S: colony surface R: reverse

* MY20 agar: 5 g peptone, 3 g yeast extract, 3 g malt extract, 200 g glucose and 20 g agar per liter of water

These characteristics were observed after 14 days of incubation at 25 ^* C. The color descriptions were based on the Methuen Handbook of Colour^.

References:

(1) De Hoog, G.S.: Notes on some fungicolous Hyphomycetes and their relatives, Persoonia, 10(1), pp.33-81, 1978.

(2) Komerup, A. and J.H. Wanscher: Methuen Handbook of Colour (3rd ed.), 525p., Methuen, London, 1978.

Production of the WF11231A substance

The WF11231A substance are produced when the WF11231A substance-producing strain belonging the genus Cladobotrvum is grown in a nutrient medium containing sources of assimilable carbon and nitrogen 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, starch, fructose or glycerin, or the like.

The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, com steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like.

The carbon and nitrogen sources, though advantageously employed in combination, need not to 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 slats, copper salts, zinc salts, or salts, or cobalt salts, or the like.

If necessary, especially when the culture medium foams seriously a defaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added.

Agitation and aeration of the culture mixture may be accomplished in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermenter, and the like.

The fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 30°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales.

When the fermentation is completed, the culture broth is then subjected for recovery of the WF11231A substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof.

The salt of the WF11231A substance can be prepared by a conventional manner, during or after the recovery and purification of the WF11231 A substance.

The hydrochloric acid salt of the WF11231A substance as obtained has the following physico-chemical properties:

Appearance:

Colorless needles Molecular formula :

PaftΛPe* ^{• Hα}

Elementary Analysis:

Calcd for ₂₀ H ₃₁ N ₂ O ₆ P ^• HO. ^• 2H ₂ 0:

C 48.14, H 7.27, N 5.61, Cl 7.11, P 6.21 (%) Found:

C 47.65, H 7.31, N 5.75, Cl 6.94, P 6.41 (%) Molecular weight:

FAB-MS: m z 427 (M + H) ⁺ Melting point:

210 - 213 °C (dec.) Specific rotation:

[α] _D (23°Q: -11 ° (c=0.74, in methanol)

Ultraviolet absorption spectrum: ^λ max ( ^metnanol ) ^{: 225} » ²⁷⁵ > 285(shoulder) nm Solubility:

Soluble: methanol

Slightly soluble: water

Insoluble: acetone, ethyl acetate Color reaction:

Positive: eerie sulfate reaction, iodine vapor reaction, ninhydrin reaction

Negative: Molish reaction, Ehrlich reaction Thin layer chromatography (TLC):

. * made by E. Merck

High Performance Liquid Chromatography (HPLC): Condition:

Mobile phase: acetonitrile:water:trifluoroacetic acid (8:92:0.1, v/v)

Column: YMC ODS AM-303** (S-5, 12θA, 4.6mmID x 150mm)

Flow rate: 1.0 ml/minute

Detection: UV 230 nm

Retention time: 7.0 minutes

** trade name: made by Yamamura Chemical Institute Infrared Spectrum: v _m I l _a α _v Λ. (KBr): 3350, 3200, 2960, 2760, 2480, 1660, 1610, 1510,

1460, 1430, 1360, 1320, 1290, 1240, 1180, 1140, 1080, 1040, 980, 950,940 cm ^"1

H Nuclear magnetic resonance spectrum: (400 MHz, CD ₃ OD) δ _R

7.31 (2H d, J=8Hz), 6.88 (2H d, J=8Hz), 4.45 (1H, m), 4.24 - 4.13 (2H, m), 3.84 - 3.76 (2H, m), 3.78 (3H, s), 3.59 (1H, m), 3.28 (1H, m), 3.03 (1H, m), 2.70 (3H, s), 2.60 (1H, m), 2.43 (1H, m), 2.28 - 2.06 (6H, m), 1.89 (1H, m) as shown in Fig. 1

C Nuclear magnetic resonance spectrum: (100 MHz, CD ₃ OD) δ _c

160.4 (s), 131.7 (d) x 2, 128.7 (s), 115.3 (d) x 2, 70.9 (d), 68.2 (s), 64.1 (d), 61.6 (d), 55.8 (q), 54.9 (d), 51.8 (t), 42.9 (d), 41.9 (t), 34.0 (t), 32.3 (q), 28.3 (t), 28.0 (t), 22.4 (t) as shown in Fig. 2.

From the above physical and chemical properties, the WF11231A substance is inferred to have the following plane structural formula.

Biological properties of the WF11231A substance

The WF11231A substance (I) possesses pharmacological activities such as immunosuppressive activity, and the like, and therefore are useful for the treatment and prevention of immune-mediated diseases such as the resistance by transplantation of organs or tissue such as heart, kidney, liver, medulla ossium, skin, cornea, lung, pancreas,

- f -

intestinum tenue, limb, muscle, nervus, 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, and the like.

Further, the WF11231A substance (I) is also useful for the treatment and the prophylaxis of inflammatory and hyperproliferative skin diseases and cutaneous manifestations of immunologically-mediated illnesses, such as, psoriasis, atopical dermatitis, contact dermatitis and further eczematous dermatitises, seborrhoeis dermatitis, Lichen planus, Pemphigus, bullous Pemphigoid, Epidermolysis bullosa, urticaria, angioedemas, vasculitides, erythemas, cutaneous eosinophilias, Lupus erythematosus, acne and Alopecia areata; various eye diseases such as autoimmune diseases and so on (e. g. keratoconjunctivitis, vernal conjunctivitis, uveitis associated with Behcet's diseases, keratitis, herpetic keratitis, conical cornea, dystrophia epithelialis comeae, comeal leukoma, ocular pemphigus, Mooren's ulcer, Scleritis, Graves' ophthalmopathy, Vogt-Koyanagi-Harada syndrome, sarcoidosis, etc.); reversible obstructive airways disease, which includes conditions such as asthma (e. g. bronchial asthma, allergic asthma, intrinsic asthma, extrinsic asthma and dust asthma), particularly chronic or inveterate asthma (e. g. late asthma and airway hyper- responsiveness), bronchitis and the like; inflammation of mucosa and blood vessels such as gastric ulcers, vascular damage caused by ischemic diseases and thrombosis, ischemic bowel disease, inflammatory bowel disease, necrotizing enterocblitis, intestinal lesions associated with thermal bums, leukotriene B^-mediated diseases; intestinal inflammations/allergies such as Coeliac disease, proctitis, eosnophilic gastroenteritis, mastocytosis, Crohn's disease and ulcerative colitis; food related allergic diseases which have symptomatic manifestation remote form the gastro-intestinal tract, for example migraine, rhinitis and eczema; renal diseases such as interstitial nephritis, Goodpasture's syndrome, hemolytic-uremic syndrome and diabetic nephropathy; nervous diseases such as multiple myositis, Guillain-Barrέ syndrome, Meniere's disease, polyneuritis, multiple neuritis, mononeuritis and radiculopathy; endocrine diseases such as hyperthyroidism and Basedow's disease; hematic diseases such as pure red cell aplasia, aplastic anemia,

hypoplastic anemia, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, agranulocytosis, pernicious anemia, megaloblastic anemia and anerythroplasia; bone diseases such as osteoporosis; respiratory diseases such as sarcoidosis, fibroid lung and idiopathic interstitial pneumonia; skin diseases such as dermatomyositis, leukoderma vulgaris, ichthyosis vulgaris, photoallergic sensitivity and cutaneous T cell lymphoma; circulatory diseases such as arteriosclerosis, atherosclerosis, aortitis syndrome, polyarteritis nodosa and myocardosis (e. g. autoimmune myocarditis, vims myocarditis); collagen diseases such as scleroderma, Wegener's granuloma and Sjogren's syndrome; adiposis; eosinophilic fasciitis; periodontal disease such as lesion of gingiva, periodontium, alveolar bone, substantia ossea dentis; nephrotic syndrome such as glomerulonephritis; male pattern alopecia or alopecia senilis; muscular dystrophy; Pyoderma and Sezary's syndrome; Addison's disease; active oxygen-mediated diseases, for example, organ injury such as ischemia-reperfusion injury of organs (e. g. heart, liver, kidney, digestive tract) which occurs on preservation, transplantation or ischemic diseases (e. g. thrombosis, cardiac infarction): intestinal diseases such as endotoxin-shock, pseudomembranous colitis, colitis caused by drug or radiation: renal diseases such as ischemic acute renal insufficiency, chronic renal insufficiency: pulmonary diseases such as toxinosis caused by lung- oxygen or drag (e, g. paracort, bleomycins), lung cancer, pulmonary emphysema: ocular diseases such as cataracta, siderosis, retinitis, pigmentosa, senile macular degeneration, vitreal scarring, comeal alkali bum: dermatitis such as erythema multiforme, linear IgA baUous dermatitis, cement dermatitis: and others such as gingvatis, periodontitis, sepsis, pancreatitis, diseases caused by environmental pollution (e. g. air pollution), aging, carcinogenis, metastasis of carcinoma, hypobaropathy; diseases caused by histamine or leukotriene Λ release; and Behcet's disease such as intestinal-, vasculo-, or neuro-

Behcet's disease, and also the one which affects oral cavity, skin, eye, vulva, articulation, epididymis, lung, kidney and so on; and so on.

And further, the WF11231A substance (I) may have liver regenerating activity and/or activities of stimulating hypertrophy and hyperplasia of hepatocytes. Therefore, they are useful for treatment and prevention of hepatic diseases such as immunogenic diseases (e. g.

chronic autoimmune liver diseases such as the group consisting of autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e. g. necrosis caused by toxins, viral hepatitis, shock or anoxia), B-virus hepatitis, non-A/non-B hepatitis, cirrhosis and hepatic failure such as fulminant hepatic failure, late-onset hepatic failure and "acute-on-chronic" liver failure (acute liver failure on chronic liver diseases).

And further, the WF11231A substance (I) may be useful for various diseases because of its useful pharmaceutical activity such as augmenting activity of chemotherapeutic effect, preventing of treating activity of cytomegalovirus infection, anti-inflammatory activity, and so on.

As examples for showing biological activities of the WF11231A substance, some biological data are explained in the following.

Test 1 Effect of the WF11231A substance on concanavalin A-induced lymphocyte proliferation

Spleens from Balb/c mice were taken under sterile conditions and gently dissociated in RPMI 1640 medium supplemented with penicillin (100 units/ml) and streptomycin (100 μg/ml). Cells were pelleted by centrifugation at 1,000 rpm for 5 minutes. Containing erythrocytes were removed by treating the pellet with ammonium chloride lysing buffer for 2 minutes at room temperature and washed. Washed spleen cells were finally resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum, 50 μM 2-mercaptoethanol, penicillin (100 units/ml) and streptomycin (100 μg/ml). Concanavalin A was added at 10 μg/ml. The cell suspension was immediately distributed into 96 well round-bottomed microculture plates at 50 μl/well. The hydrochloric acid salt of the WF11231A substance, hereinafter entitled WF11231A ^• HC1, of this invention was dissolved in water, further diluted in RPMI 1640 medium and added in triplicate wells at 50 μl/well to give the below-indicated final concentration.

The culture plates were then incubated at 37 °C in a humidified atmosphere of 5% C0 ₂ - 95% air for 72 hours. After the culture period, lymphocyte proliferation was assessed by a MTT [3-(4,5-

dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide] dye reduction assay. MTT was dissolved in phosphate buffer at 5 mg/ml. Ten μl of MTT solution was added to all wells of an assay and plates were incubated at 37 °C for 4 hours. At the end of the incubation, 75 μl of culture medium was removed and acid-isopropanol (100 μl of 0.04N HCI in isopropanol) was added to all wells and mixed thoroughly to dissolve the dark blue crystals. After ten minutes at room temperature to ensure that all crystals were dissolved the plate were read on a microplate photometer, using a test wavelength of 550 nm, a reference wavelength of 630 nm. Mean absorbance of triplicate wells were calculated and the results were expressed as percent inhibition of MTT dye reduction as follows:

% Inhibition = 100 - A B x 100

A: Mean absorbance sample tested B: Mean absorbance control medium

The results are shown in Table 2. The WF11231A ^• HCI suppressed concanavalin A-induced lymphocyte proliferation.

(to be continued)

Table 2 Effect of the WF11231A ^• HCI substance on concanavalin A-induced lymphocyte proliferation

rats

Vental allografts from donor (Lewis) rats were grafted onto the lateral thoracic area of recipient (F344) rats. The dressings were removed 5 days after the skin transplantation. The grafts were inspected daily until rejection which was defined as more than 90% necrosis of the graft epithelium.

The WF11231A ^• HQ was dissolved in saline and administered intraperitoneally either for 14 consecutive days in rats treated at doses of 0, 0.1 and 0.3 mg/kg, for 9 consecutive days in rats treated at 1 mg/kg or for 5 consecutive days in rats treated at 3 mg/kg, beginning at the day of transplantation.

As shown in Table 3, all skin allografts were rejected within 11 days in rats treated intraperitoneally with saline. On the other hand, treatment with the WF11231A ^• HQ at doses of 1 and 3 mg/kg significantly prolonged skin allograft survival. Numbers in the parentheses indicate the days when recipient rats died with active

allograft.

Table 3 Effect of the WF11231A ^• HCI on skin allograft survival

The pharmaceutical composition of this invention can be used in the form of pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains the WF11231A substance or its pharmaceutically acceptable salt, as an active ingredient in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral administrations. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, injections, ointments, liniments, eye drops, lotion, gel, creme, 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, com starch, keratin, colloidal silica, potato starch, urea and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form, and in addition auxiliary, stabilizing, thickening, solubilizing and coloring agents and perfumes may be used.

For applying the composition to human, it is preferable to apply it by intravenous, intramuscular, topical or oral administration. While

the dosage of therapeutically effective amount of the WF11231A substance varies from and also depends upon the age and condition of each individual patient to be treated, in the case of individual patient to be treated, in the case of intravenous administration, a daily dose of 0.01 - 10 mg of the WF11231A substance per kg weight of human being, in the case of intramuscular administration, a daily dose of 0.1 - 10 mg of the WF11231A substance per kg weight of human being, in case of oral administration, a daily dose of 0.5 - 50 mg of the WF11231A substance of human being is generally given for treating.

Following examples are given for the purpose of illustrating the present invention in more detail.

Example 1

(1) Fermentation:

An aqueous seed medium (120 ml) containing sucrose 4%, cotton seed flour 2%, dried yeast 1%, peptone 1%, KH ₂ P0 ₄ 0.2%, CaC0 ₃ 0.2% and Tween 80 0.1% was poured into each of twenty 500 ml-Erlenmyer flasks and sterilized at 121 °C for 30 minutes. A loopful of Cladobotrvum sp. No.11231 was inoculated from a slant culture into each of the flasks. The flasks were shaken on a rotary shaker at 25 °C for 3 days. The resultant seed culture was inoculated to 150 liters of sterile production medium consisting of modified starch 4 %, glucose 1 %, cotton seed flour 2 %, soybean flour 2 %, (NH ₄ ) ₂ P0 ₄ 1%,

Na ₂ HP0 ₄ ^• 12H ₂ 0 0.7 %, ZnS0 ₄ ^• 7H ₂ 0 0.001 %, Adekanol LG-109

(defaming agent, made by Asahi Denka Co.,Ltd.) 0.025% and Silicone KM70 (made by Shinetsu Chemical Co.,Ltd) 0.025 % in a 200 liter-jar fermenter. The fermentation was carried out at 25 °C for 4 days with aeration of 100 liters/minute and agitation of 200 rpm_

The amount of the WF11231A in the fermentation broth was quantified by both its immunosuppressing activity in the in vitro concanavarin A-induced lymphocyte proliferation and HPLC analysis.

(2) Isolation:

The cultured broth (160 liters) was filtered with the aid of diatomaseous earth (1 kg). The filtrate was discarded. 80 liters of methanol was added to the mycelium cake with stirring. The mixture was allowed to stand for 1 hour and was then filtered. The filtrate was concentrated to 20 liters under reduced pressure and the solution was passed through a column (6 liters) of polymeric adsorbent, SEPABEADS SP-207 (Mitsubishi Kasei Co., Ltd.). The column was washed with 18 liters of water, 18 liters of 50 % aqueous methanol and was eluted with 18 liters of 50 % aqueous methanol containing 0.14 % ammonium hydroxide. The eluate was concentrated to 10 liters under reduced pressure. After adjusting the pH to 3.0 with 1 N HCI, the aqueous solution was passed through a column (0.35 liter) of a cation exchange resin, Diaion SK-1B (NH ₄ ⁺ -type, Mitsubishi Kasei

Co.,Ltd.). The column was washed with 3.5 liters of water and then eluted with 0.1 N ammonium hydroxide. After removing the ammonium hydroxide in vacuo, the solution was subjected to a column (0.12 liter) of an anion exchange resin, Dowex 1-X2 (OH- type, Dow Chemical Co.,Ltd.). The column was washed with 3.5 liters of water, 0.6 liter of 0.025N acetic acid and was then eluted with 1 liter of 0.25N acetic acid. The active fraction was evaporated to give crude powder. The resultant crude powder was further purified by preparative HPLC column packed with YMC gel (ODS-AM 120-S50, 1.5 cmID 50 cm, made by Yamamura Chemical Institute), mobile phase, acetonitrile- acid (6:94:0.1, v/v). The eluate was monitored by

UV detector set at 230 nm and appropriate fractions were combined and then lyophilized. The lyophilized powder was dissolved in 5 ml of 97.5 % aqueous acetonitrile and IN HCI solution was added to the solution drop by drop to give colorless crystals of the WF11231A ^• Hα (130 mg).

Brief Description of Drawings

Figure 1. Η - NMR Spectrum of WF11231A ^• HCI Figure 2. ¹³ C - NMR Spectrum of WF11231A • HCI