JP2000226391A - Macrolide compound and evaluation of immunosuppressive agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new compound having an excellent immunosuppressive activity and an excellent antifungal activity and useful as a medicine, especially an immunosuppressive agent, or the like, for improving symptoms such as rejection or autoimmune diseases especially at the time of organ transplantation. SOLUTION: This compound is represented by the formula. The compound represented by the formula is obtained by culturing a microorganism capable of producing the compound represented by the formula [preferably Streptomyces sp. SANK 66797 (FERM BP-6235)] preferably at pH 5.0-8.0 and preferably at 26-28 deg.C according to an aerobic culture method such as a usually used aeration spinner culture method and then recovering the compound represented by the formula from the resultant cultured product by extraction, or the like, with acetone. The compound represented by the formula preferably has physicochemical properties such as a yellow powder and soluble in dimethyl sulfoxixe, methanol and ethanol and insoluble in water.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel substance useful as an immunosuppressant or an antifungal agent and a method for producing the same,
And a novel test method for immunosuppressants.

[0002]

2. Description of the Related Art Immunosuppressant FK50 used for suppressing rejection during organ transplantation and treating autoimmune diseases.
6 acts at the early stage of T cell activation and inhibits transcription and expression of interleukin 2 gene,
It exerts a strong immunosuppressive effect. Immunosuppressants such as FK506 and cyclosporin A form a complex with a binding protein (FK506-binding protein (FKBP), cyclophilin, etc.) in T cells, and this complex is a kind of protein phosphatase, calcineurin ( Inhibits its activity by binding to calcium-calmodulin-dependent protein phosphatase, also called protein phosphatase 2B (hereinafter referred to as "CN") (Kunz, J. and Hall, MN (1993) Trend. Bio)
chem. Sci. 18, 334-338).

As a test method for the above immunosuppressants, a method utilizing a mixed lymphocyte culture reaction (Adler, W. et al.
H. (1970) J. Immunol. 105, 984) is common,
This method requires a great deal of labor when testing many specimens at once, for example, in the primary screening of secondary metabolites of microorganisms. Therefore, a simpler and more suitable test method for multi-sample processing has been demanded.

[0004] By the way, yeast also has the same enzyme as CN, and a specific binding protein (yFKBP) for immunosuppressants.
-12) is known to exist (Foor, F., et.
al., (1992) Nature 360, 682-684 and Liu, J. et a
l., (1991) Cell 66, 807-815). CN is not essential for normal growth of yeast, but is involved in the maintenance of intracellular ion homeostasis and is required for growth under stress conditions involving certain ions. For example, when yeast is cultured in a medium containing a large amount of sodium chloride, the intracellular sodium ion concentration increases, and the growth stops due to a decrease in potassium ion concentration. Needs CN to resume growth (Naka
mura, T., et al., EMBO J., 12, 4063-4071, 1993).
When the immunosuppressant FK506 coexists in this system, FK5
06-binding protein-dependent inhibition of yeast growth resumption (Miyagawa et al., (1994) Proteins, nucleic acids, enzymes, 39, 420). That is, the process of activating mammalian T cells is similar to the process by which yeast resumes growth through the CN. However, it was unclear whether yeast could be used in place of mammalian lymphocytes in the search for mammalian immunosuppressants, as both processes differed in the initial stimulus to initiate signal transduction.

On the other hand, as a compound having a similar structure or activity to the compound of the present invention, a compound represented by the following formula (II) described in JP-A-9-29980:

[0006]

Embedded image And a compound represented by the following formula (III) described in JP-A-9-100290:

[0007]

Embedded image However, all have different structures from the compound of the present invention.

[0008]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel compound having excellent immunosuppressive activity and antifungal activity, and a method for producing the same. Another object of the present invention is to provide a novel method for evaluating compounds having immunosuppressive activity.

[0009]

Means for Solving the Problems The present invention provides (1) the following formula (I):

[0010]

Embedded image Or a salt thereof, (2) a compound having the following physicochemical properties or a salt thereof: 1) Property: yellow powder; 2) Solubility: soluble in dimethyl sulfoxide, methanol, ethanol, and insoluble in water 3) molecular formula: C ₇₅ H ₁₁₄ O ₂₆ S; 4) molecular weight: 1462; 5) FAB mass spectrum: m / z amu positive mode (matrix: m-NBA);
(M + Na) ⁺ negative mode (matrix: m-NBA); 1462
(M) ⁻ ; 6) High-resolution FAB mass spectrum: m / z amu positive mode (matrix: m-NBA) measured value: 1485.72111 (M + Na) ⁺ calculated value as C ₇₅ H ₁₁₄ O ₂₆ S + Na; 7
217; 7) Ultraviolet absorption spectrum (ethanol): λ _max nm
(Ε) 214 (19328), 275 (12281), 333 (4825), 358 (5088); 8) Infrared absorption spectrum (KBr): νmax cm ⁻¹ 3419, 2921, 2852, 1718, 1665, 1595, 1379, 1256, 11
69, 1064; 9) ¹ H-nuclear magnetic resonance spectrum: δ ppm (integration, multiplicity, coupling constant) In a heavy dimethyl sulfoxide solution, 293 K based on tetramethylsilane (hereinafter referred to as “TMS”) as an internal standard.
The nuclear magnetic resonance spectrum (600 MHz) measured in step is as follows: 8.02 (1H, d, 8.03), 7.89 (1H, s), 7.75
(1H, d, 8.03), 6.82 (1H, m), 6.72 (1H, s), 6.64 (1H,
s), 5.89 (1H, d, 15.39), 5.56 (1H, m), 5.39 (1H, dd, 1
5.29, 6.54), 5.38 (1H, m), 5.30 (1H, s), 5.27 (1H,
m), 5.01 (1H, m), 4.82 (1H, br d), 4.74 (1H, m), 4.74
(1H, m), 4.67 (1H, d, 3.95), 4.62 (1H, br d), 4.53 (1
H, m), 4.53 (1H, m), 4.50 (1H, m), 4.36 (1H, d, 4.28),
4.33 (1H, m), 4.27 (1H, d, 5.62), 4.19 (1H, d, 5.88),
4.16 (1H, m), 4.15 (1H, m), 4.12 (1H, d, 3.34), 3.97
(1H, m), 3.86 (1H, m), 3.85 (1H, m), 3.79 (1H, m), 3.
70 (1H, m), 3.68 (1H, m), 3.62 (1H, m), 3.61 (1H, m),
3.53 (1H, m), 3.47 (1H, m), 3.29 (1H, m), 3.23 (1H, m),
3.16 (1H, m), 2.67 (1H, m), 2.67 (1H, m), 2.60 (1H,
m), 2.44 (3H, s), 2.37 (1H, m), 2.26 (1H, m), 2.00 (2
H, m), 1.99 (3H, s), 1.98 (1H, m), 1.86 (3H, s), 1.85
(1H, m), 1.84 (1H, m), 1.82 (2H, m), 1.80 (1H, m), 1.7
7 (1H, m), 1.63 (1H, m), 1.62 (1H, m), 1.61 (2H, m), 1.
61 (2H, m), 1.58 (1H, m), 1.58 (1H, m), 1.55 (2H, m),
1.55 (1H, m), 1.52 (1H, m), 1.48 (1H, m), 1.45 (1H, m
m), 1.43 (1H, m), 1.34 (1H, m), 1.27 (1H, m), 1.25 (2H,
m), 1.12 (1H, m), 1.12 (1H, m), 1.12 (3H, d, 5.58),
1.11 (1H, m), 1.02 (3H, d, 6.15), 1.01 (3H, m), 0.97 (3
H, m), 0.97 (3H, m), 0.80 (3H, m), 0.77 (3H, m), 0.76
(3H, m), 0.70 (3H, d, 6.00); 10) ¹³ C-nuclear magnetic resonance spectrum: δ ppm (multiplicity) in heavy dimethyl sulfoxide solution (TMS internal standard), 2
Nuclear magnetic resonance spectrum measured at 93 K (125 MH
z) is as follows: 181.15 (s), 180.70 (s), 173.64
(s), 170.14 (s), 165.44 (s), 155.58 (s), 146.76 (d), 14
3.41 (s), 143.01 (s), 133.56 (d), 133.56 (d), 129.94
(d), 129.16 (s), 126.98 (d), 126.84 (d), 126.72 (s), 12
6.05 (d), 125.72 (d), 122.75 (d), 102.84 (d), 98.26 (s),
94.51 (d), 78.91 (d), 77.01 (d), 75.57 (d), 75.17 (d),
75.17 (d), 72.97 (d), 72.29 (d), 72.22 (d), 71.42 (d),
69.37 (d), 69.07 (d), 69.07 (d), 67.19 (d), 66.87 (d), 6
5.58 (d), 64.29 (d), 64.07 (d), 63.22 (d), 62.89 (d), 6
2.89 (d), 56.88 (d), 46.06 (t), 44.65 (t), 43.55 (d), 4
2.76 (t), 40.61 (t), 40.31 (t), 40.31 (t), 39.63 (t), 3
9.63 (t), 38.53 (d), 37.93 (d), 37.92 (d), 36.70 (d), 3
4.49 (d), 31.86 (t), 30.00 (t), 29.17 (t), 27.15 (t), 2
4.46 (t), 24.33 (t), 21.03 (q), 17.93 (q), 16.84 (q), 1
4.96 (q), 14.52 (q), 14.33 (q), 14.28 (q), 13.02 (q), 1
2.70 (q), 9.46 (q), 9.14 (q), 4.87 (q); 11) High performance liquid chromatography: Separation column: Symmetric ODS (φ4.6 × 150)
mobile phase: 65% acetonitrile-water; flow rate: 1.0 ml / min; detection wavelength: 210 nm; temperature: 25 ° C; retention time: 6.4 minutes; (3) belonging to the genus Streptomyces A bacterium that produces the compound described in (1) or (2) is cultured under conditions that allow the production of the compound described in (1) or (2), and then the culture described in (1) or (2) is cultured from the culture. (1) The method for producing a compound according to (1) or (2), wherein the compound belongs to the genus Streptomyces (1).
Alternatively, the bacterium producing the compound according to (2) is Streptomyces sp. (Streptomyces sp.) SANK 66797.
(FERM BP-6235), a method according to (3), a microorganism belonging to the genus Streptomyces, which produces the compound according to (1) or (2), (6) Streptomyces sp. SANK 66797 (FE
RM BP-6235), the microorganism according to (5),
(7) A drug containing the compound according to (1) or (2) or a pharmacologically acceptable salt thereof as an active ingredient, (8) a compound according to (1) or (2), or a compound thereof. An immunosuppressant containing a pharmacologically acceptable salt as an active ingredient; (9) an anti-drug containing the compound according to (1) or (2) or a pharmacologically acceptable salt thereof as an active ingredient. A method for evaluating the immunosuppressive activity of a fungal agent, (10) substance, comprising culturing yeast in the presence of 0.1 to 1.5 M sodium chloride, potassium chloride or lithium chloride, and / or a test substance. And then measuring the yeast growth inhibitory activity of the test substance. (11) Saccharo as yeast
myces cerevisiae SANK 50182 (FERM
(BP-4530). (10)
The method described.

The present inventors have conducted intensive studies on an evaluation method for an immunosuppressant more suitable for the purpose of primary screening of secondary metabolites of microorganisms, and as a result, have developed a novel method using yeast instead of lymphocytes. did. And it discovered that the substance which has an immunosuppressive activity and an antifungal activity was produced in the microbial secondary metabolite using this method. We further isolated and purified this material,
The structure and physicochemical properties were determined, and the present invention was completed.

The compound A of the present invention represented by the above formula (I)
-77951 has some asymmetric carbon atoms and some double bonds. Therefore, there are various optical and geometric isomers. In the present invention, all of these isomers are represented by a single formula, but the present invention includes all of these isomers including racemates and mixtures of these isomers. When a stereospecific synthesis method is used, or when an optically active compound is used as a starting compound, individual isomers may be directly produced, while a mixture of isomers may be produced. The individual isomers may be obtained by a conventional method.

[0013] Compound A-77951 of the present invention can be salted using methods well known to those skilled in the art. The present invention also includes such a salt of A-77951. A-779
The salt of 51 is not particularly limited as long as it is used medically and is pharmacologically acceptable. A-779
When the salt of 51 is used for purposes other than medicine, for example, when used as an intermediate, there is no limitation. Such salts are preferably sodium salts, potassium salts,
Alkali metal salts such as lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; metal salts such as aluminum salts, iron salts, zinc salts, copper salts, nickel salts, cobalt salts; Inorganic salts; t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N , N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt,
Amine salts such as organic salts such as piperazine salt, tetramethylammonium salt and tris (hydroxymethyl) aminomethane salt; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt and asparagine salt. Can be. More preferably, those preferably used as pharmacologically acceptable salts, that is, sodium salt, potassium salt, lithium salt, calcium salt, magnesium salt and the like can be mentioned.

The compound of the present invention or a salt thereof may be a solvate. For example, by leaving it in the air or by recrystallizing, it absorbs moisture,
The solvate may be adsorbed water or form a hydrate, and such a solvate is also included in the present invention.

The present invention further includes all compounds which are metabolized in the living body and converted to A-77951, so-called prodrugs.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION The method for evaluating an immunosuppressant of the present invention comprises the steps of: culturing yeast in a medium having a high salt concentration; and determining whether a test sample added to the medium inhibits the growth of yeast. Can be implemented by examining However, when the test sample inhibits the growth of yeast irrespective of the salt concentration in the medium, the sample is determined to be negative as showing antifungal activity irrespective of CN.

As the yeast used herein, commercially available baker's yeast is preferably used, but Saccharomyces cerevisi is used.
ae SANK 50182 strain is more preferred. In addition,
Saccharomyces cerevisiae SANK 50182 strain was internationally deposited with the National Institute of Bioscience and Human Technology on January 11, 1994, under the accession number FERMB.
P-4530 is attached.

As a medium used for culturing yeast, any of general-purpose media can be used.
Medium (1% yeast extract, 2% bactopeptone, 2% glucose) is preferred. Further, the temperature condition during yeast culture is preferably 20 to 37 ° C, and 28 ° C.
Is most preferred.

As the salt to be added to the medium, sodium chloride, potassium chloride or lithium chloride can be used. The concentration range of these salts in the medium is preferably 0.1 to 1.5M. More preferably, it is used at a concentration of 1.0 M for sodium chloride and potassium chloride, and 0.1 M for lithium chloride, respectively. In some cases, these salts may be used in combination. In addition, as a test sample, various microbial secondary metabolites and low molecular compound libraries, etc., a sample or a mixture thereof in which a substance having immunosuppressive activity is to be found,
Use it after diluting it appropriately.

Under the above-described conditions, for example, a certain amount of a seed yeast, a test sample, and a salt are simultaneously added to a fresh medium and cultured for about 8 to 24 hours. Thereafter, the growth rate of the yeast is quantified by the absorbance at 595 nm or the like, and it is determined whether the growth of the yeast is inhibited by the presence or absence of the test sample.

According to the above method, not only the compound of the present invention described in the above (1) but also the known immunosuppressant FK5
06 and cyclosporin A are also determined to be positive. Further, in order to confirm the activity as an immunosuppressant for a sample determined to be positive in this way, the most common mixed lymphocyte culture reaction test as a conventional method for evaluating an immunosuppressant was carried out. It is confirmed that the result of the determination according to the above method matches the result of the determination by the mixed lymphocyte culture reaction test. That is, the method of the present invention is useful as a novel method for evaluating immunosuppressants. Thereafter, the above method is performed on each of the fractions generated in the process of isolating and purifying the test sample, and the operation of further purifying the positively determined fractions is repeated, thereby suppressing immunosuppression from secondary metabolites of microorganisms and the like. It is possible to screen for substances having activity.

Next, the compound according to the above (1) is cultured in a bacterium which produces the compound according to the above (1) belonging to the genus Streptomyces, and then the compound according to the above (1) is separated and purified from the culture. Can be obtained.

As the strain belonging to the genus Streptomyces used in the production of the compound of the present invention, for example, Streptomyces isolated from plant leaves of the genus Carex sp. Collected in Iwai City, Ibaraki Prefecture.・ SP (Streptomyces sp.) SANK 6
6797 strains can be mentioned. SANK 6679
The mycological characteristics of the seven strains are as follows.

1. Morphological characteristics SANK 66797 strain was obtained from ISP [International Streptomyces Project (International
Streptomyces Project)] on a defined agar medium at 28 ° C, 1
After culturing for 4 days, SANK 6679 was observed under a microscope.
The basic mycelia of the seven strains elongate and diverge favorably, exhibiting a light yellowish brown to dull yellowish orange color, but no hypha rupture or zigzag elongation like Nocardia strains is observed. Aerial hyphae are simply branched. 10 to 50 or more spore chains are formed at the tip of the aerial hyphae, and the spore chain forms a spiral. Observation with a scanning electron microscope shows that the surface structure of the spores is spiny. The spores are elliptical and their size is 0.6-0.9 × 0.9-1.3 μm. In addition, no special organs such as axle branching of aerial hyphae, sclerotium and sporangia are observed.

2. Various properties on various culture media SANK 66 after cultivation at 28 ° C for 14 days on various culture media
The properties of the 797 strain are as shown in Table 1. The color tone indicates the color chip number of the Munsell method "Standard color chart" of the Japan Color Research Institute.

[0026]

[Table 1] Type of medium Item ^{* 1} Properties of SANK 66797 strain Sucrose G Not very good, flat, light yellowish tea (2.5Y 8/3) ^{* 2} Nitrate agar AM Not so good, velvety, brownish white (5YR 6/1) R Brown ash (2.5Y 6/2) SP No glucose and G good, flat, pale yellowish tea (2.5Y 8/6) Asparagine agar AM Originally traced, white R Light yellowish tea (2.5Y 8/6 ) SP No glycerin / G good, flat, light yellowish tea (2.5Y 8/4) Asparagine agar AM Rich, velvety, brownish ash (2.5Y 6/1) (ISP5) R brownish ash or yellowish brown (2.5Y 8/1) Y 6/2 ～ 10YR 7/6) Starch / inorganic salt agar without SP production G Very good, flat, dull yellowish orange (10YR 7/8) (ISP4) AM rich, velvety, light brown ash (10YR 6/1) R Brown ash or yellowish tea (10YR 6/2 to 10YR 7/6) SP Tyrosine agar without producing G Very good, flat, yellowish tea (10YR 7/6) (ISP7) AM good, velvety, moist, bright brown ash (7.5YR 7/1) R Brown ash or yellowish brown (2.5Y 6 / 1-10YR 7/6) SP No production, nutrient agar G Good, flat, light yellowish tea (2.5Y 8/4) (Difco) AM Good, velvety, brownish white (5YR 6/1) R Light yellowish tea (10YR 6/4) SP Yeast extract G without production Good, flat, light yellowish tea (10YR 6/4) Malt extract agar AM Rich, velvety, brownish ash (5YR 5/1) (ISP2) R Bright brown (7.5YR 6/6) SP Yellowish tea (10YR 6 / 6) Oatmeal agar G Not very good, flat, light yellowish orange (10YR 8/3) (ISP3) AM good, velvety, brownish ash (7.5YR 5/1) R Grayish brown (7.5YR 6/3) SP Light brown (7.5YR 7/3) Water agar G Not good, flat, light brown (2.5Y 8/2) AM Slightly formed, light brown ash (7.5YR 7/1) R Light brown (2.5Y 8/2) SP Potato extract not produced , G not good, flat, light yellow tea (2.5Y 8/3) Ginseng extract agar AM well formed, velvety, ashy tea (7.5YR 6/2) R tea ash (10YR 6/2) SP No production * 1 G: growth, AM: ki Mycelium, R: back surface, SP: soluble dye * 2 The parentheses in the column of properties indicate color tone display by the Munsell method.

3. Physiological properties SANK observed 2 to 21 days after incubation at 28 ° C
Physiological properties of strain 66797 are shown in Table 2.

[0028]

[Table 2] * Medium 1: Tryptone yeast extract broth (IS
P1) 2: Peptone yeast extract / iron agar (ISP6) 3: tyrosine agar (ISP7) 4: yeast extract / malt extract agar (ISP2).

Table 3 shows the assimilation of the carbon source of the SANK 66797 strain, which was observed after cultivation at 28 ° C. for 14 days using a Prideham Gottlieb agar medium (ISP9).

[0030]

[Table 3] Carbon source assimilation D-glucose + D-fructose ± D-arabinose + L-rhamnose + D-xylose ± sucrose-inositol + raffinose-D-mannitol +Control − +: Use, ±: Use weakly,-: Do not use.

4. About the cell components The cell wall of the SANK 66797 strain was prepared according to the method of Hasegawa et al. [T.
Hasegawa et al., J. Gen. Appl. Microbiol., Vol. 29,
319-322, (1983)]. As a result, LL-diaminopimelic acid was detected.
Was confirmed. Also, SANK 66797
As a result of examining the sugar components in all cells of the strain according to the method of Hasegawa et al., No characteristic pattern was observed.

ISP [International Streptomyces Project
roject)], by Waxman, SA Waksman, The Actinomycetes, Vol. 2, Buchanan and Gibbons, Barges Manual (RE
Buchanan and NE Gibbons, Bergey's Manual of Det
erminative Bacteriology, 8th edition (1974), Bergey's Manual of Systematic Ba
cteriology), Vol. 4 (1989), and recent literature on Streptomyces actinomycetes, and it was determined that this strain belongs to the genus Streptomyces among actinomycetes. Therefore,
This strain was transformed into Streptomyces sp.
p.) It was named SANK 66797 strain. This strain was deposited on January 21, 1998 with the National Institute of Bioscience and Human-Technology, National Institute of Advanced Industrial Science and Technology, and the deposit number was FERM.
BP-6235 was attached.

Although the SANK 66797 strain has been described above, it is well known that the properties of actinomycetes are not constant, and mutations can be easily caused naturally and artificially. Strains that can be used in the present invention include all such mutants. That is, the present invention includes all strains belonging to the genus Streptomyces and producing A-77951.

In culturing the bacterium producing the compound of the present invention, an ordinary culturing method for actinomycetes is generally used.

In order to obtain the compound of the present invention, the cultivation of the bacterium is carried out in a medium conventionally used for culturing fungal or actinomycete strains. As such a medium, any synthetic or natural medium can be used as long as the medium appropriately contains one selected from a carbon source, a nitrogen source, an inorganic ion, an organic nutrient source, and the like, which can be assimilated by the microorganism.

Specifically, glucose, fructose, maltose, sucrose, mannitol, glycerol, dextrin, oats, rye, corn starch, potato, corn powder,
Soybean powder, cottonseed oil, starch syrup, molasses, soybean oil, citric acid, tartaric acid and the like can be used alone or in combination. The content of the carbon source in the medium is generally 1 to the amount of the medium.
The amount varies at 10% by weight, but is not limited to this range.

As the nitrogen source, generally, a substance containing a protein or a hydrolyzate thereof can be used. Suitable nitrogen sources include, for example, soybean flour, bran, peanut flour, cottonseed flour, casein hydrolyzate, pharmamine,
Fishmeal, corn steep liquor, peptone, meat extract, raw yeast, dried yeast, yeast extract, malt extract, potato, sodium nitrate, ammonium nitrate,
Ammonium sulfate or the like may be used. These nitrogen sources are preferably used alone or in combination in the range of 0.2 to 10% by weight of the medium volume.

Further, as nutrient inorganic salts, sodium,
Usable salts that can obtain ions such as ammonium, calcium, phosphate, sulfate, chloride, carbonate and the like can be used. Also, trace metals such as potassium, calcium, cobalt, manganese, iron, magnesium and the like can be used.

In the liquid culture, silicone oil, vegetable oil, surfactant and the like can be used as an antifoaming agent.

The pH of the medium for culturing the SANK 66797 strain to produce the compound of the present invention is preferably 5.0 to 8.0.

The SANK 66797 strain was grown from 14 ° C. to 35
It grows in the temperature range of 21 ° C, especially in the range of 21 ° C to 30 ° C. Further, for the production of the compound of the present invention, it is preferable to culture at 22 ° C. to 28 ° C.
It is preferable to culture at a temperature of from 28 ° C to 28 ° C. The compound of the present invention can be obtained by culturing aerobically, and aerobic culturing methods usually used, for example, a solid culturing method, a shaking culturing method, an aerated stirring culturing method and the like are used.

In small-scale culture, it is preferable to carry out shaking culture at 28 ° C. for several days. The culture is initiated by one or more stages of seed development in an Erlenmeyer flask. The seed flask is shaken in a constant temperature incubator for several days, or until fully grown. The grown seed is used to inoculate part or all of the seed in the next stage seed or production medium. The flask containing the inoculated production medium is shaken at a constant temperature for several days, and after completion of the culture, the contents of the flask are separated by centrifugation or filtration. In the case of mass culture, it is preferable to culture in a suitable tank equipped with a stirrer and aeration device. According to this method, a nutrient medium can be prepared in a tank. The nutrient medium is sterilized by heating to 121 ° C. and, after cooling, the sterile medium is inoculated with seeds that have been grown beforehand. Culture is performed at 28 ° C. with aeration and stirring. This method is suitable for obtaining large amounts of compounds.

To know the change over time of the amount of the compound of the present invention produced as the culture proceeds, for example, an acetone extract obtained by adding an equal amount of acetone to the culture solution, stirring, and filtering is used.
It measures by performing the above-mentioned yeast growth inhibition test. The production of the compounds according to the invention usually reaches a maximum after 48 to 140 hours of culture.

After completion of the culture, the cells and the filtrate are separated by a filtration operation using diatomaceous earth as a filter aid or by centrifugation. The compound of the present invention is present in the cells or the precipitate layer, and can be obtained by extraction and purification utilizing its physicochemical properties.

For example, a solution containing the compound of the present invention obtained by extracting the cells with acetone and distilling off acetone is added to an organic solvent immiscible with water, for example, n-butanol, ethyl acetate, or the like.
The compound of the present invention can be obtained by extraction with an organic solvent such as methylene chloride alone or in combination thereof.

Further, activated carbon or adsorption resins such as Amberlite XAD-2 and XAD-4 (manufactured by Rohm and Haas), Diaion HP-10 and HPION
-20, HP-50, and CHP20P (manufactured by Mitsubishi Chemical Corporation) or the like, and the liquid containing the compound of the present invention is passed through the adsorbent layer to adsorb and remove impurities, or After adsorbing the compound of the present invention, the compound of the present invention can also be recovered by elution using a mixed solvent of water and an organic solvent such as methanol water and acetone water.

The compound of the present invention thus obtained is further subjected to adsorption column chromatography using a carrier such as silica gel or florisil, Avicel (manufactured by Asahi Kasei Kogyo Co., Ltd.), Sephadex LH-20 (Pharmacia) Purification can be carried out by partition column chromatography using, for example) and high-performance liquid chromatography using normal-phase and reverse-phase columns.

The compound A-77951 of the present invention can be separated and purified by using the above-mentioned separation and purification means alone or in an appropriate combination, and in some cases, repeatedly using them. The location of the compound of the present invention in the purification process can be detected by any of the following methods in addition to the method for examining the growth inhibitory activity of yeast under a high salt concentration.

Mixed lymphocyte culture reaction: A lymphocyte fraction was separated from peripheral blood of a human or mouse, etc., and a group stimulated with mitomycin C or irradiated with X-rays was used as a stimulator cell, an untreated group was used as a reaction cell, and both were mixed and cultured. When (the ratio of the responding cells 1 to 2 with respect to the stimulating cells 1), the responding cells start to grow and start proliferating. When a test sample is added to this culture system, the growth inhibitory activity of the reaction cells is examined. The proliferation of the reaction cells can be examined using a known cell proliferation assay such as the incorporation of ³ H-thymidine.

High performance liquid chromatography (HPLC)
Method using: HPL for test sample under the following conditions
Perform C: Separation column: Symmetric ODS (φ4.6 × 150
mobile phase: 65% acetonitrile-water; flow rate: 1.0 ml / min; detection wavelength: 210 nm; temperature: 25 ° C.

Under the above conditions, the compound of the present invention is detected as a single peak having a retention time of 6.4 minutes.

The compound of the present invention obtained as described above is a novel compound which has not been published in the literature, and its immunosuppressive activity is examined by a known immunosuppressive activity measuring method such as the above-mentioned mixed lymphocyte culture reaction. be able to. Further, since the compound of the present invention inhibits the growth of yeast under high salt concentration conditions, it can be used as an antifungal agent, for example, in combination with sodium chloride.

The compound of the present invention or a pharmaceutically acceptable salt thereof is administered in various forms. As the administration form, for example, oral administration by tablets, capsules, granules, powders, syrups, etc., or parenteral administration by injections (intravenous, intramuscular, subcutaneous), drops, eye drops, suppositories, etc. Can be mentioned. These various preparations are commonly used in the field of pharmaceutical preparation technology such as excipients, binders, disintegrants, lubricants, flavoring agents, solubilizers, dissolution aids, suspensions, coatings, etc., in accordance with the usual methods for the main drug. It can be formulated using known adjuvants.

In molding into tablets, those conventionally known in the art can be widely used as carriers, for example, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and the like. Excipients such as water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc .; dried starch, sodium alginate, agar powder , Laminaran end,
Disintegrating agents such as sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, lactose;
Disintegration inhibitors such as sucrose, stearin, cocoa butter, and hydrogenated oil; absorption promoters such as quaternary ammonium bases and sodium lauryl sulfate; humectants such as glycerin and starch; starch, lactose, kaolin, bentonite and colloidal silica An adsorbent such as an acid; and a lubricant such as purified talc, stearates, powdered boric acid, and polyethylene glycol can be exemplified. Further, the tablet can be made into a tablet coated with a usual coating, if necessary, such as a sugar-coated tablet, a gelatin-encapsulated tablet, an enteric-coated tablet, a film-coated tablet or a double tablet or a multilayer tablet.

In shaping into pill form, carriers conventionally known in the art can be widely used, such as glucose, lactose, cocoa butter, starch, hydrogenated vegetable oil, and the like.
Excipients such as kaolin and talc; binders such as gum arabic powder, tragacanth powder, gelatin, and ethanol; disintegrating agents such as laminaran agar;

For shaping in the form of suppositories, those conventionally known in the art can be widely used as carriers, for example, polyethylene glycol, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides and the like. Can be mentioned.

When prepared as an injection, the solution and suspension are preferably sterilized and isotonic with blood. When formed into these solutions, emulsions and suspensions, they are diluted. All the agents commonly used in this field can be used as the agent, and examples thereof include water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. In this case, a sufficient amount of salt, glucose, or glycerin to prepare an isotonic solution may be included in the pharmaceutical preparation, and ordinary solubilizers, buffers, soothing agents, etc. May be added.

If necessary, coloring agents, preservatives, fragrances, flavors, sweeteners and other pharmaceuticals may be added.

The amount of the active ingredient compound contained in the above-mentioned pharmaceutical preparation is not particularly limited and may be appropriately selected in a wide range, but is usually 1 to 70% by weight, preferably 1 to 30% by weight of the whole composition.
It is appropriate that the amount is contained by weight%.

The method of administering the above pharmaceutical preparation is not particularly limited, and is determined according to various preparation forms, age, weight, sex and other conditions of the patient, degree of disease, and the like. For example, tablets, pills, solutions, suspensions, emulsions, granules and capsules are orally administered. In the case of injections, they are administered intravenously either alone or mixed with normal replacement fluids such as glucose and amino acids.
It is administered intradermally, subcutaneously or intraperitoneally. In the case of suppositories, they are administered rectally.

The amount used varies depending on symptoms, age, body weight, administration method, dosage form, etc.
200mg per day (preferably 100m
g) The lower limit of 0.1 mg (preferably 1 mg) can be administered once or several times depending on the symptoms.

[0062]

EXAMPLES The present invention will be described more specifically with reference to examples and test examples below, but the present invention is not limited to these.

Embodiment 1 Screening using yeast growth
Selection of A-77951 Producing Bacteria by the Saccharomyces cerevisiae SANK 50182 (F
ERM BP-4530) strain in 1 × YPD medium (1%
Yeast extract, 2% bactopeptone, 2
(% Glucose) One loopful was inoculated into a 100 ml Erlenmeyer flask containing 10 ml, and cultured overnight at 28 ° C. and 210 rpm as a seed yeast. 96-well plate (MS-8096F: manufactured by Sumitomo Bakelite Co., Ltd.)
In each well, 150 μl of 2 × YPD medium, 60 μl of 5 M sodium chloride, 5 μl of a test sample, and 5 μl of seed yeast were added, and the volume of each well was adjusted to 300 μl with sterile water. At the same time, a plate was prepared under exactly the same conditions except that 5M sodium chloride was not added. After culturing them at 28 ° C. for 16 hours, each well was suspended and the absorbance at 595 nm was measured using a microplate reader (Model 450: manufactured by Bio-Rad). Based on the absorbance of the test sample-free group, the rate of decrease in absorbance of the test sample was defined as the yeast growth inhibition rate. Only the sodium chloride-added group showed the yeast growth inhibitory activity and the wells whose growth inhibition rate was 50% or more were evaluated as positive.

As a result of screening of various secondary metabolites of microorganisms using the above method, Streptomyces sp. Isolated from fresh leaves of the plant of the genus Sedge of the family Cyperaceae collected in Iwai City, Ibaraki Prefecture. S
The bacterial cell extract of ANK 66797 strain was evaluated as positive.

Further, a known immunosuppressant, FK506,
As a result of performing the same test for cyclosporin A and cyclosporin A, all were determined to be positive.

Embodiment 2 FIG . Production of A-77951 (A) Culture 500 ml of a medium having the following composition was placed in four Erlenmeyer flasks having a capacity of 2 liters and sterilized by heating at 121 ° C. for 30 minutes. 28
After cooling to ° C, Streptomyces sp.
ptomyces sp.) SANK 66797 strain (FERM B)
P-6235) is homogenized and inoculated.
Incubation was performed at 28 ° C, 210 rpm in a rotary shaker for 168 hours to obtain a seed culture solution.

Medium composition: Glucose 5.0% Soy flour (Soya flour: manufactured by Nisshin Flour Milling Co., Ltd.) 1.0% Meat extract (Ehrlich meat extract: manufactured by Kyokuto Pharmaceutical Co., Ltd.) 0.4% Polypeptone (Manufactured by Nippon Pharmaceutical Co., Ltd.) 0.4% yeast extract (manufactured by Difco) 0.1% calcium carbonate 0.5% sodium chloride 0.25% antifoaming agent (CB-422 10% acetone solution: Japan) (Manufactured by Yushi Co., Ltd.) 0.01% pH 7.2 before sterilization.

Further, 15 liters of a medium having the above composition was added to 3
Four jar fermenters having a capacity of 0 liter were charged and sterilized by heating at 121 ° C. for 30 minutes. After cooling this to 28 ° C., 450 ml of a seed culture solution was inoculated, and 15 liters of air per minute was aerated, and the dissolved oxygen content was 5.0 ppm.
The rotation speed was adjusted within the range of 100 to 230 rpm so as to maintain the temperature, and culturing was performed with stirring at 28 ° C. for 72 hours.

(B) Isolation To 55 liters of the culture solution obtained in the above (A), 2.4 kg of Celite 545 (manufactured by Johns Manville Product Corporation) was added as a filter aid, followed by filtration. .2 kg were obtained. 40 for this cake
One liter of 80% acetone-water was added, and the mixture was stirred and extracted for 1 hour, and then filtered to obtain an acetone extract. The acetone extract (40 liters) was concentrated under reduced pressure to obtain a concentrate (12 liters). The pH of this concentrated solution was adjusted to 7.0, and extraction was performed three times with 10 liters of ethyl acetate, and the organic layer was recovered. The organic layers from the three extractions with ethyl acetate were combined, washed with saturated saline, dried over anhydrous sodium sulfate, filtered through filter paper, and concentrated to dryness under reduced pressure to obtain 26.8 g of an extract containing A-77951. Obtained.

Next, 26.8 g of this extract was dissolved in 500 ml of ethyl acetate, and then dissolved in Cosmosil C18-OP.
N (manufactured by Nacalai Tesque, Inc.) was coated on a column packed with 1 liter of Cosmosil C18-OPN previously equilibrated with 50% acetonitrile-water. The column was washed with 2.5 liters of 50% acetonitrile-water, and eluted with 1.9 liters of 50% acetonitrile-water and 1.6 liters of 60% acetonitrile-water. The eluted fractions were each adjusted to pH 7 and extracted with ethyl acetate, and the organic layer was collected, washed with brine, dried over anhydrous sodium sulfate, filtered through filter paper, and concentrated to dryness under reduced pressure to give an oil. 14 g (fraction eluted with 50% acetonitrile) and 0.96 g (fraction eluted with 60% acetonitrile) were obtained.

The resulting oils (4.14 g and 0.96 g) were separated by high performance liquid chromatography (HPLC) to purify A-77951. H in advance
PLC column (YMC-Pack S-1050-20)
-SR ODS (15/30) φ100 × 500mm:
Equilibrated with 70% acetonitrile-water, 0.96 g of an oily substance was dissolved in 15 ml of methanol, and the whole was injected as a sample solution.
The development was carried out at a flow rate of 240 ml / min using water as a mobile phase. Monitoring was performed at a detection wavelength of 210 nm, and the retention time was 48 minutes to 5 minutes.
480 ml of the 0 minute eluate was collected. 4.1 remaining oil
HPLC separation of 4 g was carried out in the same manner, and a retention time of 4 g was obtained.
960 ml of eluate from 8 minutes to 52 minutes was collected. These eluates were concentrated to dryness under reduced pressure to obtain 48.2 mg of a yellow powder.

Further, an HPLC column (Sensepak.RTM.) Previously equilibrated with 70% acetonitrile-water was used.
After injecting half of the sample solution obtained by dissolving the entire amount of the powder with 0.8 ml of methanol into Pegasil ODS (φ20 × 250 mm, manufactured by Senshu Kagaku KK), the flow rate is 6.0 ml / min using 70% acetonitrile-water as a mobile phase. Unfolded.
Monitoring at a detection wavelength of 210 nm, 6 ml of eluate was collected for a retention time of 34 to 35 minutes. The same operation was performed on the remaining sample solutions to collect the eluate. These eluates were concentrated to dryness under reduced pressure to obtain 9.4 mg of A-77951 yellow powder.

A portion of this yellow powder was analyzed by high performance liquid chromatography (HPLC), and the following results were obtained.

Separation column: Symmetry ODS (φ
Mobile phase: 65% acetonitrile-water; Flow rate: 1.0 ml / min; Detection wavelength: 210 nm; Temperature: 25 ° C; Retention time: 6.4 minutes.

Test Example The immunosuppressive activity of a sample that was active in the yeast growth inhibition test was confirmed using a mixed lymphocyte culture (hereinafter, referred to as "MLC") reaction.

Preparation of lymphocytes: Human peripheral blood (40 ml) was collected, and Hanks' Balanced Salt S
olution: Hereinafter referred to as “HBSS”. After diluting twice with Gibco BRL, and laminating on Ficoll-Pac Plus (Pharmacia), 1700 rpm
Centrifuged at 20 ° C. for 30 minutes. Recover the middle layer and add HBSS
And then centrifuged at 4 ° C and 1000 rpm for 10 minutes.
Precipitate 10% fetal bovine serum (hereinafter referred to as “FBS”)
Was resuspended in RPMI1640 medium (manufactured by Gibco BRL) containing This was used as floating cells of human peripheral blood lymphocytes. After counting the cells, the cells were adjusted to 2 × 10 ⁶ cells / ml in RPMI1640 medium containing 10% FBS and used as “reacting cells” in the MLC reaction.

Preparation of stimulator cells: The above-mentioned suspension cells of human peripheral blood lymphocytes were transformed into RPMI 1640 containing 5% FBS.
It was adjusted to 1 × 10 ⁵ cells / ml with a medium, placed in a cell culture flask, and cultured at 37 ° C. for 5 hours in 5% carbon dioxide. After collecting the supernatant, the cells were adjusted to 1 × 10 ⁶ cells / ml with RPMI1640 medium containing 5% FBS, and mitomycin C was removed.
(Kyowa Hakko Co., Ltd.) was added at a concentration of 50 μg / ml, and the mixture was kept at 37 ° C. for 60 minutes. RPMI 1640
After washing the cells three times with medium, RPM containing 10% FBS
The cells adjusted to 2 × 10 ⁶ cells / ml with I1640 medium were used as “stimulator cells” for the MLC reaction.

MLC reaction: 50 μl of the reaction cell solution, 50 μl of the stimulating cell solution and 100 μl of the test sample solution prepared as described above were placed in a 96-well plate, and 5% carbon dioxide gas was added.
The cells were cultured at 7 ° C for 7 days. Test sample is 0.5%
R containing dimethylsulfoxide and 10% FBS
It was adjusted to 200 μg / ml with a PMI1640 medium, and this was used as a stock sample after being serially diluted 10-fold.

After the cell culture for 7 days, ³ H-thymidine was added at 18.5 Kbq / well, and after further culturing for 18 hours, the cells in each well were collected using a cell harvester and taken up into the cells. The radioactivity was measured with a liquid scintillation counter. Based on the radioactivity taken up by the cells of the group to which the test sample was not added, the suppression rate of the radioactivity taken up by the cells of the group to which the test sample was added was calculated. Table 4 shows the results.

[0080]

[Table 4] A-77951 ³ H-thymidine Inhibition rate Addition concentration Uptake amount (%) (μg / ml) (cpm) Only reaction cells 0 486 − Only stimulation cells 0 32 − Reaction cells + stimulator cells 0 57080 0 0.00001 18455 67.7 0.0001 14161 75.2 0.001 25653 55.1 0.01 13988 75.5 0.1 24686 56.8 1 7605 86.7 A-77951 showed immunosuppressive activity at low concentrations.

Cytotoxicity: Human-derived cell line U937 (A
TCC No. CRL-1593) was adjusted to 1 × 10 ⁴ cells / ml with RPMI1640 medium containing 10% FBS, and 100 μl / well was added to a 96-well plate. At the same time, 10 μL of the same test sample used in the above MLC reaction was added.
0 μl was added. This was cultured for 4 days under conditions of 5% carbon dioxide and 37 ° C., and the supernatant of each well was removed.
MTT reagent (3- (4,4) diluted to 5 mg / ml with BS
5-dimethyl-2-thiazoyl) -2,5-diphenyl-2H-tetrazolium bromide: Wako Pure Chemical Industries, Ltd.
10 μl / well was added. Further at 37 ° C 4
After culturing for 1 hour, the supernatant was removed and dimethyl sulfoxide was added for 1 hour.
After adding 50 μl / well and stirring, the absorbance at 550 nm was measured with a microplate reader. The cytotoxicity was evaluated by calculating the suppression rate of the absorbance of the group to which the test sample was added, based on the absorbance of the group to which the test sample was not added. Table 5 shows the results.

[0082]

[Table 5] A-77951 addition concentration OD550nm Inhibition rate (μg / ml) (%) 0 1.48 0 0.00001 1.37 7.4 0.0001 1.37 7.4 0.001 1.50 1.4 0.01 1.33 10.1 0.1 1.67 -12.8 A-77951 has a concentration of 0.1 μg / ml or less.
It had little effect on the growth of U937.

Formulation Examples Capsules: Compound obtained in Example 2 100 mg Lactose 100 mg Corn starch 148.8 mg Magnesium stearate 1.2 mg Total amount 350 mg The powder of the above formulation was mixed and passed through a 60 mesh sieve. The powder is placed in a gelatin capsule to form a capsule.

[0084]

According to the present invention, a novel method for evaluating a compound having immunosuppressive activity is provided. The present invention also provided a novel compound having excellent immunosuppressive activity, a bacterium producing the compound, and a method for producing the compound. The compound of the present invention is useful as a drug, particularly as an immunosuppressant for improving symptoms such as rejection at the time of organ transplantation and autoimmune diseases. In addition, the compounds of the present invention are useful as antifungal agents.

Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (Reference) C12P 17/18 C12P 17/18 D C12Q 1/04 C12Q 1/04 G01N 33/15 G01N 33/15 Z 33/50 33 / 50 T // (C12P 17/18 C12R 1: 465) (72) Matsuo Nakajima 1-58, Hiromachi, Shinagawa-ku, Tokyo Sankyo Co., Ltd. (72) Tomoko Nishizaki, Inventor Tomoko Shinagawa-ku, Tokyo 1-258 Hiromachi Sankyo Co., Ltd. (72) Inventor Masaaki Kitzuka 33 Miyukigaoka, Tsukuba, Ibaraki Prefecture Sankyo Co., Ltd.

Claims (11)

[Claims] (1) The following formula (I): Or a salt thereof. 2. A compound having the following physicochemical properties or a salt thereof: 1) Property: yellow powder; 2) Solubility: soluble in dimethylsulfoxide, methanol, ethanol, insoluble in water; 3) Molecular formula: C ₇₅ H ₁₁₄ O ₂₆ S; 4) molecular weight: 1462; 5) FAB mass spectrum: m / z amu positive mode (matrix: m-NBA); 1485
(M + Na) ⁺ negative mode (matrix: m-NBA); 1462
(M) ⁻ ; 6) High-resolution FAB mass spectrum: m / z amu positive mode (matrix: m-NBA) measured value: 1485.72111 (M + Na) ⁺ calculated value as C ₇₅ H ₁₁₄ O ₂₆ S + Na; 7
217; 7) Ultraviolet absorption spectrum (ethanol): λ _max nm
(Ε) 214 (19328), 275 (12281), 333 (4825), 358 (5088); 8) Infrared absorption spectrum (KBr): νmax cm ⁻¹ 3419, 2921, 2852, 1718, 1665, 1595, 1379, 1256, 11
69, 1064; 9) ¹ H-nuclear magnetic resonance spectrum: δ ppm (integration, multiplicity, coupling constant) In a heavy dimethyl sulfoxide solution, 293 K based on tetramethylsilane (hereinafter referred to as “TMS”) as an internal standard.
The nuclear magnetic resonance spectrum (600 MHz) measured in step is as follows: 8.02 (1H, d, 8.03), 7.89 (1H, s), 7.75
(1H, d, 8.03), 6.82 (1H, m), 6.72 (1H, s), 6.64 (1H,
s), 5.89 (1H, d, 15.39), 5.56 (1H, m), 5.39 (1H, dd, 1
5.29, 6.54), 5.38 (1H, m), 5.30 (1H, s), 5.27 (1H,
m), 5.01 (1H, m), 4.82 (1H, br d), 4.74 (1H, m), 4.74
(1H, m), 4.67 (1H, d, 3.95), 4.62 (1H, br d), 4.53 (1
H, m), 4.53 (1H, m), 4.50 (1H, m), 4.36 (1H, d, 4.28),
4.33 (1H, m), 4.27 (1H, d, 5.62), 4.19 (1H, d, 5.88),
4.16 (1H, m), 4.15 (1H, m), 4.12 (1H, d, 3.34), 3.97
(1H, m), 3.86 (1H, m), 3.85 (1H, m), 3.79 (1H, m), 3.
70 (1H, m), 3.68 (1H, m), 3.62 (1H, m), 3.61 (1H, m),
3.53 (1H, m), 3.47 (1H, m), 3.29 (1H, m), 3.23 (1H, m),
3.16 (1H, m), 2.67 (1H, m), 2.67 (1H, m), 2.60 (1H,
m), 2.44 (3H, s), 2.37 (1H, m), 2.26 (1H, m), 2.00 (2
H, m), 1.99 (3H, s), 1.98 (1H, m), 1.86 (3H, s), 1.85
(1H, m), 1.84 (1H, m), 1.82 (2H, m), 1.80 (1H, m), 1.7
7 (1H, m), 1.63 (1H, m), 1.62 (1H, m), 1.61 (2H, m), 1.
61 (2H, m), 1.58 (1H, m), 1.58 (1H, m), 1.55 (2H, m),
1.55 (1H, m), 1.52 (1H, m), 1.48 (1H, m), 1.45 (1H, m
m), 1.43 (1H, m), 1.34 (1H, m), 1.27 (1H, m), 1.25 (2H,
m), 1.12 (1H, m), 1.12 (1H, m), 1.12 (3H, d, 5.58),
1.11 (1H, m), 1.02 (3H, d, 6.15), 1.01 (3H, m), 0.97 (3
H, m), 0.97 (3H, m), 0.80 (3H, m), 0.77 (3H, m), 0.76
(3H, m), 0.70 (3H, d, 6.00); 10) ¹³ C-nuclear magnetic resonance spectrum: δ ppm (multiplicity) in heavy dimethyl sulfoxide solution (TMS internal standard), 2
Nuclear magnetic resonance spectrum measured at 93 K (125 MH
z) is as follows: 181.15 (s), 180.70 (s), 173.64
(s), 170.14 (s), 165.44 (s), 155.58 (s), 146.76 (d), 14
3.41 (s), 143.01 (s), 133.56 (d), 133.56 (d), 129.94
(d), 129.16 (s), 126.98 (d), 126.84 (d), 126.72 (s), 12
6.05 (d), 125.72 (d), 122.75 (d), 102.84 (d), 98.26 (s),
94.51 (d), 78.91 (d), 77.01 (d), 75.57 (d), 75.17 (d),
75.17 (d), 72.97 (d), 72.29 (d), 72.22 (d), 71.42 (d),
69.37 (d), 69.07 (d), 69.07 (d), 67.19 (d), 66.87 (d), 6
5.58 (d), 64.29 (d), 64.07 (d), 63.22 (d), 62.89 (d), 6
2.89 (d), 56.88 (d), 46.06 (t), 44.65 (t), 43.55 (d), 4
2.76 (t), 40.61 (t), 40.31 (t), 40.31 (t), 39.63 (t), 3
9.63 (t), 38.53 (d), 37.93 (d), 37.92 (d), 36.70 (d), 3
4.49 (d), 31.86 (t), 30.00 (t), 29.17 (t), 27.15 (t), 2
4.46 (t), 24.33 (t), 21.03 (q), 17.93 (q), 16.84 (q), 1
4.96 (q), 14.52 (q), 14.33 (q), 14.28 (q), 13.02 (q), 1
2.70 (q), 9.46 (q), 9.14 (q), 4.87 (q); 11) High performance liquid chromatography: Separation column: Symmetric ODS (φ4.6 × 150)
mobile phase: 65% acetonitrile-water; flow rate: 1.0 ml / min; detection wavelength: 210 nm; temperature: 25 ° C; retention time: 6.4 minutes. 3. A bacterium that produces the compound according to claim 1 or 2 belonging to the genus Streptomyces, is cultured under conditions that allow the production of the compound according to claim 1 or 2, and then from the culture. The method for producing a compound according to claim 1 or 2, wherein the compound according to (1) or (2) is recovered. 4. The bacterium according to claim 1 or 2, which belongs to the genus Streptomyces, wherein the bacterium is Streptomyces sp. SANK 66797 (FE).
RM BP-6235). 5. A microorganism belonging to the genus Streptomyces and producing the compound according to claim 1 or 2. 6. Streptomyces sp.
yces sp.) SANK66797 (FERM BP-62)
The microorganism according to claim 5, which is 35). 7. A medicament comprising the compound according to claim 1 or 2 or a pharmacologically acceptable salt thereof as an active ingredient. 8. An immunosuppressant comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient. 9. An antifungal agent comprising the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient. 10. A method for evaluating the immunosuppressive activity of a substance, comprising the steps of:
A method comprising culturing in the presence of potassium chloride or lithium chloride and / or a test substance, and then measuring the yeast growth inhibitory activity of the test substance. 11. Saccharomyces cerevisiae as a yeast
SANK 50182 (FERM BP-4530)
The method according to claim 10, characterized in that: