JP3257605B2 - New physiologically active substances Mer-W8020A and Mer-W8020B and methods for producing them - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel physiologically active substance having an antirheumatic effect and a method for producing the same.

[0002]

2. Description of the Related Art Conventionally, steroid hormones, gold preparations, D-penicillamine, acidic anti-inflammatory drugs and the like have been used as therapeutic drugs for autoimmune diseases (for example, rheumatoid arthritis). Occasionally serious side effects such as adrenal insufficiency, infectious disease, renal disorder, hematopoietic disorder, or gastrointestinal disorder are caused, which is a great limitation in use. In addition, there is a limit in reducing toxicity and enhancing efficacy by chemical modification of existing drugs, and there is a need for the development of new drugs.

[0003]

SUMMARY OF THE INVENTION The present invention provides a novel physiologically active substance which has excellent antirheumatic activity, is low toxic and can be used safely, and a process for producing the same.

[0004]

Means for Solving the Problems The present inventors have isolated strains from various soils for the purpose of searching for a novel substance having an anti-rheumatic effect, and have repeatedly studied metabolites produced by the strains. Separated Streptomyces (Strep
It was found that a strain belonging to the genus tomyces) produced a substance that suppresses adjuvant arthritis in a rat, which is a model of rheumatoid arthritis, in a culture solution. When the active substance was separated and purified from the culture solution and its physicochemical properties were examined, the active substance was a novel substance not described in the conventional literature,
The present inventors have found that they have an excellent antirheumatic effect and completed the present invention.

That is, the present invention provides a physiologically active substance Mer-W8020A having the following physicochemical properties. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1472.8760 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1472 (M + H) ⁺ (4) Melting point 178-181 ° C (5) Optical rotation [α] ²⁴ _D -150.5 ゜ (c 0.4, methanol)

(6) Ultraviolet absorption spectrum The result of measurement in methanol is as shown in FIG. (7) Infrared absorption spectrum The results measured in the potassium bromide tablet are as shown in FIG. (8) ¹ H-NMR spectrum (400 MHz) The result measured in deuterated methanol is as shown in FIG. (9) ¹³ C-NMR spectrum (100 MHz) The result measured in deuterated methanol is as shown in FIG. (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water.

(11) Color reaction A color is formed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.30 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1)

[0008] The present invention further provides a physiologically active substance Mer-W8020B having the following physicochemical properties. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1458.8956 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1458 (M + H) ⁺ (4) Melting point 174-177 ° C (5) Optical rotation [α] ²⁴ _D -145 ゜ (c 0.4, methanol)

(6) Ultraviolet absorption spectrum The result measured in methanol is as shown in FIG. (7) Infrared absorption spectrum The result of measurement in a potassium bromide tablet is as shown in FIG. (8) ¹ H-NMR spectrum (400 MHz) The result measured in deuterated methanol is as shown in FIG. (9) ¹³ C-NMR spectrum (100 MHz) The result of measurement in deuterated methanol is as shown in FIG. (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water.

(11) Color reaction A color is formed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.26 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1)

The present invention further relates to Streptomyces (Streptomyces).
tomyces) belonging to the genus, bioactive substances Mer-W8020A and Mer-
An object of the present invention is to provide a method for producing the novel bioactive substance, which comprises culturing a microorganism capable of producing W8020B and collecting the bioactive substance from the culture.

The microorganism used in the present invention belongs to the genus Streptomyces, and is a physiologically active substance Mer-W802.
Any strain may be used as long as it has the ability to produce 0A and Mer-W8020B. For example, the present inventors include Mer-W8020 strain isolated from soil on the shore of the Hikichi River, Fujisawa City, Kanagawa Prefecture, but not limited to this strain, Streptomyces (Strepto
myces) belonging to the genus, bioactive substances Mer-W8020A and Mer-W8
Any strain having the ability to produce 020B, including those mutants, can be used in the present invention.

Hereinafter, the mycological properties of the Mer-W8020 strain will be described. (1) Morphological properties Spiral aerial hyphae elongate from well-branched basic hyphae. A spore chain consisting of 10 to 50 elliptic to cylindrical spores is formed at the tip of a mature aerial hypha. Spore sac is not observed. The size of the spores is about 0.4-0.5 × 0.8-1.0 micron, and the surface of the spores is smooth and no flagella are observed.

(2) Growth state in various media All cultures were performed at 28 ° C. The description of the color tone is a container
Corporation of America
tion of America) Color Harmony Manual (C
(oror Harmony Manual). (2-1) Yeast / malt agar medium The growth is good, and the aerial mycelia develop well. The color of the aerial hyphae is creamy, but yields red-grey (5dc) spores thereon. The back of the culture is colorless or pale olive. Produces large amounts of black-violet or black-brown soluble pigments.

(2-2) Starch / inorganic salt agar medium The growth is good, many aerial mycelia and spores are formed, and the color of the colony surface and the back color are the same as those in the yeast / malt agar medium. The amount of soluble dye is small. (2-3) Tyrosine agar medium Growth is moderate. It develops aerial hyphae and spores. Produces soluble dye in the medium. Their colors are similar to those on yeast-malt agar medium.

(3) Assimilation of Various Carbon Sources Various carbon sources were added to a pred ham Gottlieb agar medium, and growth was observed. (3-1) L-arabinose + (3-2) D-xylose + (3-3) D-glucose + (3-4) D-fructose + (3-5) sucrose-(3-6) inositol -(3-7) L-rhamnose + (3-8) raffinose-(3-9) D-mannitol ++ assimilates,-does not assimilate

(4) Properties of Cell Wall Component When the cells hydrolyzed were analyzed by thin-layer chromatography of cellulose, the isoform of diamino pimeric acid of the cell wall component of the bacterium was LL-type. The sugar component had no characteristic.

From the above mycological properties, it is clear that this bacterium is a bacterium belonging to the genus Streptomyces, and is described in International Journal of Systematic Bacteriology, Vol. 18, No. 2, 1968 (International Jou
(rnal of Systematic Bacteriology, vol. 18, No. 2, 1968)
In, the Streptomyces project (International Streptomyces Project) was approved by the Streptomyces (Streptomyces) and compared with the described properties of the genus strain, Streptomyces glyceoviridis (Streptomyces griseoviridis) described, the bacterium is dissolved The results were almost the same except that a large amount of sex pigment was produced and the color of the back of the culture was almost nonexistent.

Therefore, the present inventors have proposed that this strain be Streptomyces griseoviridis (Streptomyces griseoviridis).
dis) It was named Mer-W8020 and deposited on January 19, 1993 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology under the number FERM-P13374.

The physiologically active substances Mer-W8020A and Me of the present invention
r-W8020B is produced by inoculating the above strain into a nutrient source-containing medium and aerobically culturing. The method for culturing the above microorganisms is in principle according to the method for culturing general microorganisms,
Usually, it is preferably carried out under aerobic conditions such as shaking culture by liquid culture and aeration and agitation culture.

The medium used for the culture may be any medium containing a nutrient source that can be used by microorganisms belonging to the genus Streptomyces, and any of various synthetic, semi-synthetic media, natural media, etc. can be used. is there. As a medium composition, glucose as a carbon source, sucrose, fructose, glycerin, dextrin, starch,
Molasses can be used alone or in combination.

Pharmaceutical media, peptone, meat extract, soy flour, casein, amino acids, yeast extract, organic nitrogen sources such as urea, and inorganic nitrogen sources such as sodium nitrate and ammonium sulfate may be used alone or in combination as the nitrogen source. it can. In addition, salts such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, sodium phosphate, potassium phosphate, and cobalt chloride, salts of heavy metals, vitamins such as vitamin B and biotin can be added and used as necessary. . In the case where foaming during the culturing is remarkable, various known antifoaming agents can be appropriately added to the medium. When adding an antifoaming agent, the concentration must be such that it does not adversely affect the production of the target substance. For example, the use concentration is desirably 0.05% or less.

Culture conditions can be appropriately selected within a range where the strain can grow well and produce the above-mentioned substances. For example, the pH of the medium is preferably about 5 to 9, usually around neutrality. The culture temperature is generally maintained at 20 to 40 ° C, preferably at 28 to 35 ° C. The culturing days are about 2 to 8 days, usually 3 to 5 days.
The various culture conditions described above are based on the type and characteristics of the microorganism used,
Needless to say, it can be appropriately changed according to the external conditions and the like, and the optimum conditions can be selected.

The physiologically active substance Mer-W8 accumulated in the culture solution
020A and Mer-W8020B can be recovered by separating cells by a known ordinary solid-liquid separation means such as filtration and centrifugation, and extracting from the cells.

Physiologically active substances Mer-W8020A and Mer-W8020B
Can be separated and purified by selecting and combining various known methods. For example, methanol, ethyl acetate,
Solvent extraction using n-butanol, etc., activated carbon, Amberlite XAD (manufactured by Rohm and Haas), Diaion H
Adsorption to P-20 (manufactured by Mitsubishi Kasei) and elution with aqueous alcohol, aqueous acetone, etc., Sephadex LH-20 (Pharmacia), gel with Bio-Gel P-2 (Bio-Rad) Filtration, column method using silica gel, alumina, etc., thin-layer chromatography, preparative high-performance liquid chromatography using normal-phase or reverse-phase column (preparative HPLC), etc., alone or in appropriate combination, and repeated use in some cases And can be purified.

The physiologically active substance Me obtained as described above
r-W8020A and Mer-W8020B have the following physicochemical properties. The physicochemical properties of the physiologically active substance Mer-W8020A of the present invention are as follows. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1472.8760 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1472 (M + H) ⁺ (4) Melting point 178-181 ° C (5) Optical rotation [α] ²⁴ _D -150.5 ゜ (c 0.4, methanol)

(6) Ultraviolet absorption spectrum The result measured in methanol is as shown in FIG. 1, and the maximum absorption is as shown below. λmax nm (ε): 218 (50500), 269 (5700), 281 (4900), 292 (4
700) (7) Infrared absorption spectrum The result of measurement in a potassium bromide tablet is as shown in FIG. 2, and the characteristic absorption is as shown below. νmax cm ^-1 : 3426,3354,2965,1730,1640,1510 (8) ¹ H-NMR spectrum (400 MHz) The result measured in deuterated methanol is as shown in FIG. As shown. (ppm, multiplicity, coupling constant) 7.23 (5H, m), 6.95 (2H, m), 6.70 (1H, s), 6.44 (1H, d, J = 7.3H
z), 5.79 (1H, m), 5.36 (1H, s), 5.21 (1H, d, J = 2.2 Hz), 5.01 (1
(H, d, J = 4.0Hz), 3.83 (3H, s), 3.12 (3H, s), 2.32 (3H, s), 0.33
(3H, d, J = 6.6Hz), 0.13 (3H, d, J = 6.6Hz)

(9) ¹³ C-NMR spectrum (100 MHz) The result of measurement in deuterated methanol is as shown in FIG. 4, and the clear absorption is as shown below. (ppm, multiplicity) 174.9s, 174.2s, 174.0s, 172.0s, 171.7s, 171.3s, 171.0s, 1
55.4s, 142.7s, 140.0s, 129.5d, 128.5d, 127.2d, 124.0d, 12
3.5d, 118.5s, 112.6s, 106.1d, 99.9d, 73.1d, 71.5d, 71.2d,
70.4d, 66.9d, 63.1d, 59.6d, 59.3d, 58.6d, 56.9d, 55.8q, 5
3.4d, 41.0q, 40.5q, 39.5t, 38.0d, 27.1t, 26.0t, 19.8q, 19.
6q, 19.3q, 16.8q, 15.9q, 11.1q (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water.

(11) Color reaction A color is formed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.30 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1)

The physicochemical properties of the physiologically active substance Mer-W8020B of the present invention are as follows. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1458.8956 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1458 (M + H) ⁺ (4) Melting point 174-177 ° C (5) Optical rotation [α] ²⁴ _D -145 ゜ (c 0.4, methanol)

(6) Ultraviolet absorption spectrum The result of measurement in methanol is as shown in FIG. 5, and the maximum absorption is as shown below. λmax nm (ε): 218 (48200), 268 (5400), 282 (4600), 292 (4
500) (7) Infrared absorption spectrum The result of measurement in a potassium bromide tablet is as shown in FIG. 6, and the characteristic absorption is as shown below. νmax cm ^-1 : 3324,3356,2965,1730,1638,1510 (8) ¹ H-NMR spectrum (400 MHz) The result measured in deuterated methanol is as shown in FIG. As shown. (ppm, multiplicity, coupling constant) 7.23 (5H, m), 6.95 (2H, m), 6.70 (1H, s), 6.44 (1H, d, J = 7.3H
z), 5.79 (1H, m), 5.36 (1H, s), 5.21 (1H, d, J = 2.2 Hz), 5.01 (1
(H, d, J = 4.4Hz), 3.83 (3H, s), 3.60 (3H, s), 3.12 (3H, s), 2.34
(3H, s), 0.33 (3H, d, J = 6.6Hz), 0.13 (3H, d, J = 6.6Hz)

(9) ¹³ C-NMR spectrum (100 MHz) The result of measurement in deuterated methanol is as shown in FIG. 8, and the clear absorption is as shown below. (ppm, multiplicity) 174.9s, 174.2s, 174.0s, 172.1s, 171.7s, 171.3s, 171.0s, 1
55.4s, 142.7s, 140.1s, 129.5d, 128.5d, 127.2d, 124.0d, 1
23.5d, 118.5s, 112.6s, 106.1d, 100.0d, 73.1d, 71.5d, 71.2
d, 70.4d, 66.9d, 63.1d, 59.7d, 59.6d, 59.3d, 56.8d, 55.8q,
53.6d, 41.0q, 40.5q, 39.5t, 27.1t, 25.8d, 20.1q, 19.9q, 1
9.6q, 16.8q (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water.

(11) Color reaction A color is formed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.26 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1)

The physiologically active substances Mer-W8020A and Me of the present invention
r-W8020B shows the inhibitory effect of adjuvant arthritis in rats, which is a pathological model of rheumatoid arthritis, as shown below, rheumatoid arthritis, systemic lupus erythematosus (SLE)
It is expected to be applicable to autoimmune diseases such as. In addition, it has almost no toxicity and can be used effectively and safely.

(1) Adjuvant Arthritis Inhibitory Action Six to eight female Lewis rats (10 weeks old) per group were anesthetized with ether and suspended in liquid paraffin with 0.1 m of killed tuberculosis (6 mg / ml).
l was inoculated intradermally into the right hind footpad to induce adjuvant arthritis. Mer-W8020A was intraperitoneally administered at 1 and 10 mg / Kg, each once a day for 20 days from the day of sensitization with adjuvant. Adjuvant arthritis is suppressed using digital calipers
Evaluation was made by periodically measuring the thickness of the footpads of the hind limb of the sensitized site and the non-sensitized site. Table 1 shows the results.

[0036]

[Table 1]

The Mer-W8020A of the present invention, by intraperitoneal administration of 1 and 10 mg / Kg, significantly suppresses swelling of hind limbs of sensitized and non-sensitized sites of adjuvant arthritis and has an antirheumatic effect. Admitted.

(2) Acute toxicity Three groups of ICR mice were administered intraperitoneally with 50 and 100 mg / kg of Mer-W8020A of the present invention, respectively. No animals died during 20 days of observation, and no toxic symptoms were observed.

[0039]

As described above, the physiologically active substance of the present invention
Mer-W8020A and Mer-W8020B are expected to be applied to autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus (SLE). Further, since it has very low toxicity, it can be used effectively and safely for a long period of time.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. In the examples, percentages (%) indicate weight / volume percentages, unless otherwise specified.

【Example】

From the slant medium (ISP-2) of the strain Mer-W8020, one platinum loop was added to 50 ml of a seed culture medium (glycerin 2%, glucose 2%,
Essan Meat 2%, Yeast Extract 0.5%, Sodium Chloride
0.25%, calcium carbonate 0.32%, copper sulfate 0.0005%, manganese chloride 0.0005%, zinc sulfate 0.0005%, defoamer 0.05%,
pH 7.4) and inoculated into a 500 ml Erlenmeyer flask.
The seed culture was obtained by culturing on a rotary shaker for days.

Each 100 ml of this seed culture was used as a main culture medium.
(2.5% glycerin, 0.5% meat extract, 0.5 polypeptone)
%, Yeast extract 1%, sodium chloride 0.2%, magnesium sulfate 0.05%, monopotassium phosphate 0.05%, calcium carbonate
0.32%, defoamer 0.05%, pH 7.4) Inoculate 4 10-liter jar fermenters containing 5 liters
Aeration and agitation culture for a period of time (aeration rate 5 L / min, agitation 300 r.
pm).

After completion of the culture, 15 liters of methanol was added to about 4 kg of cells recovered by suction filtration, and the methanol layer was concentrated to 2 liters under reduced pressure. Add 3 liters of water to this and add 2.5 liters of butanol.
Extracted times. The extract was concentrated under reduced pressure, and the dried product was dissolved in 50 ml of methanol, washed with 50 ml of n-hexane, and concentrated under reduced pressure to obtain 12 g of a dried product.

This was dissolved in 40 ml of warm methanol, applied to a Sephadex LH-20 column (4.5 × 45 cm, manufactured by Pharmacia) pre-filled with methanol, and developed with methanol. Each fraction was chloroform / methanol /
The mixture was subjected to silica gel thin-layer chromatography (Merck, Art. 5715) using a water mixture (80: 10: 1) as a developing solvent, and fractions containing the same Rf substance were collected and concentrated according to the color of the Erich reagent. The rats were allowed to dry and in each case induced adjuvant arthritis, and administered intraperitoneally for 2 weeks to examine the preventive effect.

As a result, it was clarified that the fraction containing a substance having an Rf of 0.26 to 0.30 showed an inhibitory effect on rat adjuvant arthritis in the above-mentioned silica gel thin-layer chromatography. Was detected using silica gel thin-layer chromatography (manufactured by Merck, Art. 5715, color development by Erich reagent) using a chloroform / methanol / water mixture (80: 10: 1) as a developing solvent as an index. .

Rf 0.30 (Mer-W8020A) and Rf 0.26 (Mer-W
From the fraction showing 8020B), 970 mg of a dried product was obtained.
This was dissolved in 10 ml of a mixed solution of toluene / methanol (20: 1) and subjected to silica gel column chromatography.
That is, the mixture was applied to a silica gel column (3φ × 50 cm, manufactured by Merck) previously filled with a toluene / methanol mixture (20: 1), and developed with a toluene / methanol concentration gradient (20: 1 to 2: 1). The fraction was subjected to silica gel thin-layer chromatography, detected with Erich reagent, and Mer-W8020A 4
30 mg and 344 mg of Mer-W8020B were obtained. 0.5m each
The mixture was dissolved in 1 l of methanol, 50 ml of toluene was added while heating to about 40 ° C., and the mixture was allowed to cool to room temperature, to obtain 308 mg of Mer-W8020A and 265 mg of Mer-W8020B as white crystals, respectively.

[0047]

[Brief description of the drawings]

FIG. 1 shows an ultraviolet absorption spectrum of a physiologically active substance Mer-W8020A in methanol.

FIG. 2 shows the infrared absorption spectrum of the physiologically active substance Mer-W8020A by the KBr method.

FIG. 3 shows the physiologically active substance Mer-W8020A in heavy methanol.
3 shows a 400 MHz ¹ H-NMR spectrum.

FIG. 4 shows the physiologically active substance Mer-W8020A in deuterated methanol.
3 shows a 100 MHz ¹³ C-NMR spectrum.

FIG. 5 shows an ultraviolet absorption spectrum of a physiologically active substance Mer-W8020B in methanol.

FIG. 6 shows an infrared absorption spectrum of a physiologically active substance Mer-W8020B by the KBr method.

FIG. 7 shows the physiologically active substance Mer-W8020B in deuterated methanol.
3 shows a 400 MHz ¹ H-NMR spectrum.

FIG. 8 shows the bioactive substance Mer-W8020B in heavy methanol.
3 shows a 100 MHz ¹³ C-NMR spectrum.

────────────────────────────────────────────────── ─── Continuation of the front page Examiner Minako Mitsumoto (56) References JP-A-59-154992 (JP, A) JP-A-2-16995 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C07G 17/00 C12P 1/06 BIOSIS (DIALOG) WPI (DIALOG) JICST file (JOIS)

Claims (4)

(57) [Claims] 1. A physiologically active substance Mer-W8020A having the following physicochemical properties. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1472.8760 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1472 (M + H) ⁺ (4) Melting point 178-181 ° C (5) Optical rotation [α] ²⁴ _D -150.5 ゜ (c 0.4, methanol) (6) Ultraviolet absorption spectrum Maximum absorption of ultraviolet absorption spectrum measured in methanol
The yield is as shown below. λmax nm (ε): 218 (50500), 269 (5700), 281 (4900), 292 (4
700) (7) Infrared absorption spectrum of infrared absorption spectrum measured in potassium bromide tablet
Characteristic absorption is as shown below. νmax cm ^-1 : 3426,3354,2965,1730,1640,1510 (8) ¹ H-NMR spectrum (400 MHz) Clear absorption of ¹ H-NMR spectrum measured in deuterated methanol
The yield is as shown below. (ppm, multiplicity, coupling constant) 7.23 (5H, m), 6.95 (2H, m), 6.70 (1H, s), 6.44 (1H, d, J = 7.3H
z), 5.79 (1H, m), 5.36 (1H, s), 5.21 (1H, d, J = 2.2 Hz), 5.01 (1
(H, d, J = 4.0Hz), 3.83 (3H, s), 3.12 (3H, s), 2.32 (3H, s), 0.33
(3H, d, J = 6.6 Hz), 0.13 (3H, d, J = 6.6 Hz) (9) ¹³ C-NMR spectrum (100 MHz) Clear ¹³ C-NMR spectrum measured in deuterated methanol
The absorption is as shown below. (ppm, multiplicity) 174.9s, 174.2s, 174.0s, 172.0s, 171.7s, 171.3s, 171.0s, 1
55.4s, 142.7s, 140.0s, 129.5d, 128.5d, 127.2d, 124.0d, 12
3.5d, 118.5s, 112.6s, 106.1d, 99.9d, 73.1d, 71.5d, 71.2d,
70.4d, 66.9d, 63.1d, 59.6d, 59.3d, 58.6d, 56.9d, 55.8q, 5
3.4d, 41.0q, 40.5q, 39.5t, 38.0d, 27.1t, 26.0t, 19.8q, 19.
6q, 19.3q, 16.8q, 15.9q, 11.1q (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water. (11) Color reaction A color is developed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.30 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1) 2. A physiologically active substance Mer-W8020B having the following physicochemical properties. (1) Color and shape: white crystal (2) High-resolution FAB mass spectrum m / z: 1458.8956 (M + H) ⁺ (3) FAB mass spectrum (matrix; m-nitrobenzyl alcohol) m / z: 1458 (M + H) ⁺ (4) Melting point 174-177 ° C (5) Optical rotation [α] ²⁴ _D -145 ゜ (c 0.4, methanol) (6) Ultraviolet absorption spectrum Maximum absorption of ultraviolet absorption spectrum measured in methanol
The yield is as shown below. λmax nm (ε): 218 (48200), 268 (5400), 282 (4600), 292 (4
500) (7) Infrared absorption spectrum of infrared absorption spectrum measured in potassium bromide tablet
Characteristic absorption is as shown below. νmax cm ^-1 : 3324,3356,2965,1730,1638,1510 (8) ¹ H-NMR spectrum (400 MHz) Clear absorption of ¹ H-NMR spectrum measured in deuterated methanol
The yield is as shown below. (ppm, multiplicity, coupling constant) 7.23 (5H, m), 6.95 (2H, m), 6.70 (1H, s), 6.44 (1H, d, J = 7.3H
z), 5.79 (1H, m), 5.36 (1H, s), 5.21 (1H, d, J = 2.2 Hz), 5.01 (1
(H, d, J = 4.4Hz), 3.83 (3H, s), 3.60 (3H, s), 3.12 (3H, s), 2.34
(3H, s), 0.33 (3H, d, J = 6.6 Hz), 0.13 (3H, d, J = 6.6 Hz) (9) ¹³ C-NMR spectrum (100 MHz) ¹³ C-NMR measured in deuterated methanol Spectrum clear
The absorption is as shown below. (ppm, multiplicity) 174.9s, 174.2s, 174.0s, 172.1s, 171.7s, 171.3s, 171.0s, 1
55.4s, 142.7s, 140.1s, 129.5d, 128.5d, 127.2d, 124.0d, 12
3.5d, 118.5s, 112.6s, 106.1d, 100.0d, 73.1d, 71.5d, 71.2
d, 70.4d, 66.9d, 63.1d, 59.7d, 59.6d, 59.3d, 56.8d, 55.8q,
53.6d, 41.0q, 40.5q, 39.5t, 27.1t, 25.8d, 20.1q, 19.9q, 1
9.6q, 16.8q (10) Solubility Soluble in chloroform, acetone, methanol and ethyl acetate, insoluble in hexane and water. (11) Color reaction A color is developed with a Lydon-Smith reagent, a phosphomolybdic acid reagent, and an Erich reagent. No coloration with Dragendorff's reagent or ninhydrin reagent. (12) Rf value of thin layer chromatography 0.26 on silica gel plate F-254 (manufactured by Merck) (developing solvent is chloroform: methanol: water = 80: 10:
1) 3. The method according to claim 1, wherein a physiologically active substance Mer-W8020A-producing bacterium belonging to the genus Streptomyces is cultured, and the physiologically active substance Mer-W8020A is collected from the culture . New bioactive substance Mer-W8020A
Manufacturing method. 4. The method according to claim 2, wherein a physiologically active substance Mer-W8020B-producing bacterium belonging to the genus Streptomyces is cultured, and the physiologically active substance Mer-W8020B is collected from the culture . New bioactive substance Mer-W8020B
Manufacturing method.