JPH06279481A - Substance ws64826 - Google Patents

Abstract

PURPOSE:To obtain a new substance, having antimicrobial and antitumor activities and useful as an antitumor agent. CONSTITUTION:A substance, WS64826, or its salt. appearance: red powder; molecular formula: C39H47NO16; molecular weight: 785; solubility: soluble in methanol, acetone, etc., slightly soluble in water and insoluble in n-hexane; color reaction: positive to the cerium acetate reaction, Molisch reaction, etc., and negative to the ninhydrin reaction and Dragendorff reaction and essential properties: amphoteric substance. This substance is obtained by culturing a strain, belonging to the genus Streptomyces and capable of producing the substance WS64826 [e.g. Streptomyces sp. No 64826 (FERM BP-3876) which is a new microorganism] in a nutrient culture medium usually under aerobic conditions and recovering the substance from the culture solution. Furthermore, the culturing is preferably carried out at 20-30 deg.C for 50-150hr.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel compound having biological activity and is used in the field of medicine.

[0002]

The present invention relates to novel compounds having biological activity, more specifically, WS64826 substance. More specifically, the present invention relates to a biologically active novel WS64826 substance having antibacterial and antitumor activities, a method for producing the same, and an antitumor agent containing the same. Accordingly, one object of this invention is WS6 having antitumor activity.
4826 substance or a salt thereof. Another object of the present invention is Streptomyces sp.
No. It is intended to provide a method for producing a WS64826 substance or a salt thereof by fermenting a WS64826 substance-producing strain such as 64826 in a nutrient medium. Another object of the present invention is to provide an antitumor agent containing the substance WS64826 or a salt thereof as an active ingredient.
WS64826 substance is Streptomyces sp. It can be produced by fermentation of a WS64826 substance-producing strain such as 64826 in a nutrient medium. WS64826
It should be appreciated that the production of the substance is not limited to the use of the particular microorganisms described herein for purposes of illustration only. This invention applies X-ray irradiation, ultraviolet irradiation, N-methyl-
It also covers the use of any WS64826 substance-producing mutant strain, including artificial mutants that can be produced by conventional means such as treatment with N'-nitro-N-nitrosoguanidine, 2-aminopurine and the like. Streptomyces SP No. Details of 64826 are as follows.

No. Characteristics of 64826 strain: No. 648
The 26 strains were originally isolated from soil samples collected at Mt. Himuro in Tochigi Prefecture, Japan. Newly isolated Streptomyces sp. The freeze-dried sample of 64826 was deposited at the Institute of Microbial Science and Technology, Institute of Industrial Science and Technology (currently Institute of Biotechnology, Institute of Biotechnology; 1-3, Higashi 1-3, Tsukuba, Ibaraki, Japan) under the deposit number FERM BP-3876 (deposit date). : May 27, 1992). N
o. For taxonomic studies of the 64826 strain, Shirring and Gottliep were used.
Method ¹⁾ described by b) and Waxman (Wak
The method ²⁾ described by Sman) was adopted. After culturing at 30 ° C. for 21 days, observation was performed. The morphological observation is
Cultures grown on yeast extract-malt extract agar and oatmeal agar were performed using light and scanning electron microscopy. The color names used in this study are the Methuen Handbook
Taken from Of Color ³⁾ . The growth temperature range was determined for yeast extract-malt extract agar. Preparation of cells and detection of diaminopimelic acid isomerism was performed by Becke
It was carried out by the method ⁴⁾ of r) et al.

(1) Morphological characteristics The basal mycelium was well developed and irregularly branched. This strain produced aerial mycelia composed of a branched aerial mycelium network structure, and the aerial mycelium segmented into spores. Short club-shaped side branches are formed on the triangular spores,
Or 2-3 spores were produced. The spores have an oval or columnar shape, and sometimes have a triangular shape (0.6 to 0.8 × 0.
8 to 1.0 μm). Their surface was smooth. No sclerotium granules, sporangia and spores were found. (2) Characteristics in culture and biological characteristics Tables 1 and 2 show the results of examining the characteristics in culture and the physiological characteristics, respectively. Growth was moderate to good on most of the media used in the test, but poor on glucose-asparagine agar and sucrose-nitrate agar. The color of the aerial body was bluish white in most of the media used in the test. The rear growth color is
Oatmeal agar was yellowish white, inorganic salts-starch agar was pale yellow, and glycerol-asparagine agar was reddish brown. Melanoid pigments were produced in tryptone-yeast extract broth, peptone-yeast extract-iron agar and tyrosine agar. These mycelial pigments and soluble pigments were not pH sensitive. (3) Chemotaxonomic characteristics LL-diaminopimelic acid was detected in the whole cell hydrolyzate of this strain. (4) Classification Based on the above morphological and chemical characteristics, No. 64
The 826 strain is considered to belong to the Streptomyces genus ^5,6) and the triangular sporulation group ^7,8) . Therefore, this strain was designated Streptomyces sp. 64826
(Streptomyces sp. No. 6482
6).

Table 1 No. Culture characteristics of strain 64826

[Table 2] Abbreviations: G: Growth A: Aerial mycelium R: Color on back side S: Soluble pigment

Table 2 No. Physiological characteristics of strain 64826

[Table 3] +: Positive ±: Weak positive −: Negative

References: 1) E. B. Shirring and D. Gottlieb: A method for characterizing Streptomyces species. International ·
Journal of Systematic Bacteriology Vol. 16, pp. 313-340, 1966 2) S.M. A. Waxman: Radioactive fungus (The ac
Tinomycetes) Volume 2: Classification of genera and species,
Identification and description. The Williams and Wilkins, Baltimore, 1961 3) A. Kornerup and J.
H. Wanscher: Mechuen
Handbook of Color, Methuen, London, 1978 4) B. Becker, M.A. P. Lechevalier, R.A. E. Gordon and H.M. A. Le Chevalier (L
(Echevalier): Rapid identification of whole cell hydrolysates of Nocardia and Streptomyces by paper chromatography: Applied: Microbiology 12: 421-423, 1964 5) R. E. Buchanan and N.M.
E. Gibbons: Vergi's Manual of Definite Bacteriology,
Eighth Edition: The Williams and Wilkins,
Baltimore, 1974 6) S.S. T. Williams: The Burmese Manual of Systematic Bacteriology Volume 4 2
599-2601: Williams and Wilkins, Baltimore, 1989 7) C.I. E. Higgens and R.H. E.
Kastner: Streptomyces
Crabrigels, a new β-lactam antibiotic-producing bacterium. International Journal of Systematic Bacteriology Vol. 21, 326-331,
1971 8) M.M. Arai, A. Toricata, R.K. Enokita, H.K.
Fukkatsu, R.K. Nakayama and K.K. Yoshida: Folipomycin, a new member of phospholipid antibiotics. Journal of Antibiotics Vol. 30, 1049-10
54 pages, 1977

Production of WS64826 substance W in a nutrient medium containing assimilable carbon and nitrogen sources
When the S64826 substance-producing bacterium is grown under aerobic conditions (eg, shaking culture, aerobic submerged culture, etc.), WS6
4826 substances are produced. Preferred carbon sources in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose, glycerin. Preferred nitrogen sources are yeast extract, peptone, gluten meal, cottonseed flour,
Soybean flour, corn steep liquor, dry yeast, malt, etc.,
And ammonium and inorganic nitrogen compounds such as ammonium nitrate, ammonium sulfate, ammonium phosphate, urea, amino acids and the like.
The carbon source and the nitrogen source are advantageously used in combination, but need not be used in pure form. This is because relatively low-purity substances containing trace amounts of growth factors and a considerable amount of inorganic nutrients are also suitable for use. If desired, the medium may be supplemented with inorganic salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts and cobalt salts. If necessary, liquid paraffin, fatty oils, vegetable oils, mineral oils,
An antifoaming agent such as silicone may be added. Agitation and aeration of the culture mixture can be carried out by various methods such as agitation with a propeller or similar agitation device, agitation by rotation or shaking of the fermentor. Fermentation is usually carried out at a temperature of between about 10 ° C and 40 ° C, preferably between 20 ° C and 30 ° C and a temperature of between about 50 ° C and about 50 ° C.
Perform for 150 hours. This time may be changed depending on the fermentation conditions and scale. Upon completion of fermentation, the culture solution is subjected to various operations commonly used for collecting and purifying biologically active substances, for example, extraction with a suitable solvent or solvent mixture, chromatography, recrystallization from a suitable solvent or solvent mixture. Then, WS64826 substance is recovered. Since the WS64826 substance is an amphoteric substance, it may be converted into its salt. The salt of the WS64826 substance can be prepared by a conventional method during or after the collection and purification of the WS64826 substance. Suitable salts of the substance WS64826 are conventional pharmaceutically acceptable salts, such as alkali metal salts (eg sodium salt, potassium salt etc.), alkaline earth metal salts (eg calcium salt, magnesium salt etc.) and the like. , Ammonium salts, organic base salts (eg trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N′-dibenzylethylenediamine salt, etc.), organic acid addition salts (eg acetate salt, trifluoroacetate salt) , Maleate, tartrate, methanesulfonate, benzenesulfonate, formate, toluenesulfonate, etc., inorganic acid addition salts (eg hydrochloride, hydrobromide, hydroiodide, sulfuric acid) Salts, phosphates, etc., amino acids (eg arginine, aspartic acid, glutamic acid) Etc.) and the like.

The WS64826 substance has the following physicochemical properties: Appearance: Red powder Molecular formula: C ₃₉ H ₄₇ NO ₁₆ Molecular weight: 785 High resolution FAB-MS: m / z Calculated value as C ₃₉ H ₄₈ NO ₁₆ : 786.2973 (M + H) ⁺ measured value: 786.2991 (M + H) ⁺ ultraviolet absorption spectrum: λmax (methanol) (ε): 232 (40820), 250 (2983
0), 285 (9420), 388 (3140), 470 (13345), 492 (1334
5), 530 (sh, 7850), 570 (2355) nm λmax (0.01N HCl, in methanol) (ε): 232 (40820),
250 (29830), 285 (9420), 388 (3140), 470 (13345),
492 (13345), 530 (sh, 7850), 570 (2355) nm λmax (0.01N NaOH, in methanol) (ε): 232 (3297
0), 247 (33755), 290 (7850), 350 (4710), 550 (1256
0), 593 (10990) nm Solubility: Soluble: Methanol, ethanol, acetone, ethyl acetate, chloroform Slightly soluble: Water Insoluble: n-hexane Color reaction: Positive: Cerium sulfate reaction, Maurish reaction, iodine Vapor reaction Negative: Ninhydrin reaction, Dragendorff reaction

Thin layer chromatography:

[Table 4] High performance liquid chromatography (HPLC): Conditions: Mobile phase: 0.2% NH ₄ H ₂ PO ₄ containing acetonitrile:
Methanol: water = 5: 1: 5 Column: YMC-AM-303 ^** (S-5, 120ÅO
DS, 4.6 mm ID x 250 mm) Flow rate: 1.0 ml / min Detection: UV 235 nm Retention time: 12.0 min ^** Product name: YMC Infrared absorption spectrum: νmax (KBr): 3477, 2971, 2935, 2872, 1716, 1618,
1579, 1447, 1415, 1380, 1354, 1287, 1232, 1210, 111
8, 1067, 1035, 991,945 cm ^-1

¹ H nuclear magnetic resonance spectrum: (400 MHz, CD ₃ OD) δH 7.45 (1H, m), 7.37 (1H, m), 7.15 (1H, d, J = 8Hz), 5.47
(1H, s), 5.00 (2H, m), 4.59 (1H, d, J = 8Hz), 4.26 (1H,
m), 4.24 (1H, s), 3.98 (1H, brs), 3.88 (1H, m), 3.78
(3H, s), 3.78-3.55 (5H, m), 3.52 (1H, brs), 3.42 (1H,
m), 2.83 (1H, d, J = 18Hz), 2.65 (1H, d, J = 18Hz), 2.42
(1H, m), 2.38 (3H, s), 2.25 (1H, d, J = 14Hz), 2.00 (1H, b
rd, J = 14Hz), 1.73 (2H, m), 1.67 (1H, brd, J = 11Hz), 1.2
5 (3H, d, J = 6Hz), 1.19 (3H, d, J = 6Hz), 1.18 (3H, d, J = 6H)
z) (See Fig. 1) ¹³ C nuclear magnetic resonance spectrum: (100MHz, CD ₃ OD) δC 213.4 (s), 186.9 (s), 186.8 (s), 161.9 (s), 156.9
(s), 155.8 (s), 136.9 (d), 135.7 (s), 135.6 (s), 13
5.3 (s), 120.8 (s), 120.2 (d), 119.8 (d), 112.0
(s), 111.8 (s), 106.9 (d), 105.9 (s), 102.0 (d), 9
5.0 (d), 87.6 (d), 83.3 (d), 82.0 (d), 77.7 (s), 7
7.0 (d), 75.1 (t), 70.0 (d), 69.9 (d), 65.4 (d), 6
4.2 (t), 56.9 (q), 50.9 (d), 47.4 (t), 36.4 (t), 3
3.8 (t), 30.6 (t), 24.8 (q), 24.2 (q), 18.5 (q), 1
7.6 (q) (See FIG. 2) Essentiality: Amphoteric substance In order to show the usefulness of the WS64826 substance, test data on antitumor and antibacterial activity are shown below.

Biological Properties of WS64826 Substance As an example showing the biological activity of the WS64826 substance, some biological data will be described below. In Vitro Inhibition of Tumor Cell Growth by WS64826 Substance A cytotoxicity test was performed using a microtiter plate. Tumor cells, ie mouse leukemia P, in each well
3 × 10 ³ 388 cells or lymphoma EL-4 cells, 10% fetal bovine serum, penicillin (50 units / m 2
1) and streptomycin (50 μg / ml) in 100 μl of Dulbecco's minimum essential medium added. The cells were cultured at 37 ° C for 3 days. Cell proliferation was measured by the MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, Sigma] assay. Colorimetric M
The TT assay was carried out according to the method described by Mosann (Journal of Immunological Methods Vol. 65, pages 55-63). MT
T was dissolved in phosphate buffer (PBS) at a concentration of 5 mg / ml, filtered through a Millipore filter (pore size 0.45 μm) for sterilization, and at the same time, a small amount of insoluble residue was removed. After culturing the human tumor cells, this MTT solution (10 μl /
100 μl medium) to all assay wells,
The plate was further incubated at 37 ° C for 4 hours.
Acid-isopropanol (0.04N H
Cl isopropanol solution (100 μl) was added and mixed well to dissolve dark blue crystals. After all the crystals were dissolved, the MTT concentration in each well was measured using a two-wavelength microplate photometer (MTP-120; Corona Electric Co., Katsuta City) at 550 nm with 660 nm as a reference wavelength. The WS64826 substance was dissolved in methanol, diluted with Dulbecco's minimum essential medium, and added to the culture at a final concentration of 1 μg / ml or less. The results are shown in Table 3.

Table 3 Inhibition of tumor cell growth by substance WS64826

[Table 5] Antibacterial activity of WS64826 substance The antibacterial effect of WS64826 substance was determined by the multiple broth dilution method using normal broth for Gram-positive and Gram-negative bacteria and Sabouraud medium for fungi. Inoculation amount is 5 × 10 ⁵ cfu / ml for bacteria
In the case of fungi, it was adjusted to 1 × 10 ⁶ cfu / ml.
18 hours at 37 ° C for bacteria, 28 for fungi
The minimum inhibitory concentration (MIC) was determined in μg / ml after incubation at 48 ° C. for 48-72 hours. WS6
4826 substances are Staphylococcus aureus 20
9P (MIC 0.16 μg / ml) and Bacillus subtilis ATCC 6633 (MIC 0.08 μg / m)
It had an antibacterial action against l). However, for the following microorganisms, WS64826 substance is 100 μg /
Not showing antibacterial action in ml: Escherichia coli N
IHJ JC-2, Pseudomonas aeruginosa A
TCC14970 and Candida albicans FP6
33. Antitumor effect of WS64826 substance on P388 mouse leukemia The antitumor effect of WS64826 substance in a mouse tumor system was determined. P388 in BDF1 mice (female, 6 weeks old)
Leukemia cells (1 × 10 ⁶ ) were transplanted intraperitoneally. 24 hours after tumor cell inoculation, the mice were intraperitoneally administered with WS64826 substance. Intraperitoneal administration of the WS64826 substance was continued once daily for 3 days. WS64826 substance is HCO
-60 (Nikko Chemicals Co., Ltd.) 10%.
It was dissolved in 9% saline. Control mice have 10% HC
0.9% saline containing O-60 was intraperitoneally administered.

The results are shown in Table 4. Anti-tumor activity was measured as T / C% (1
00 × treatment group / control group). Table 4 WS64826 against P388 mouse leukemia
Antitumor effect of substances

[Table 6] Acute toxicity of WS64826 substance The acute toxicity of the WS64826 substance when administered intraperitoneally to ICR mice was 3 mg / kg. For therapeutic administration, a mixture of WS64826 substance or a salt thereof with a pharmaceutically acceptable carrier such as an organic or inorganic solid or liquid excipient suitable for oral administration, parenteral administration and external application (topical administration). In the form of a conventional pharmaceutical preparation containing the substance as an active ingredient. The pharmaceutical preparation includes solid forms such as tablets, granules, powders, capsules and suppositories, liquids,
It may take the form of a suspension, syrup, emulsion, limonade, lotion, ointment, gel and the like. If necessary,
Auxiliary substances, stabilizers, humectants, and other conventional additives such as lactose, stearic acid, magnesium stearate, clay, sucrose, corn starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cocoa butter. , Ethylene glycol, tartaric acid, citric acid, fumaric acid, etc. may be added. WS6482
The doses of the 6 substances will depend on the patient's age, condition, type of disease, etc., and may depend on them.
Generally, from about 0.1 mg to about 10 per patient per day
Amounts between 00 mg or higher, preferably about 1 mg
Amounts between about 500 mg and about 500 mg may be administered. When treating cancer or infectious disease, the average single dose is about 0.1 mg, 1 mg,
10mg, 20mg, 30mg, 50mg, 100m
g, 200 mg, 250 mg of WS64826 substance may be used. The following examples are given for the purpose of illustrating the invention in more detail.

Example 1 (1) Fermentation Glucose (0.5%), glycerin (1%), corn starch (1%), cottonseed flour (1%), dry yeast (1.5)
%), Corn steep liquor (0.5%) and CaC
Seed culture medium containing O ₃ (0.5%) (160 ml)
(Adjust the pH to 8.0 with 6N NaOH)
Inject each into a 00 ml Erlenmeyer flask and store at 30
Sterilize over a period of minutes. Streptomyces SP No. was added to each medium. Strain culture 1 platinum loop of 64826 was inoculated and orbital shaker (220 rpm, stroke 5.1 cm)
Incubate at 25 ° C for 3 days. Large-scale fermentation process
Perform in a 200 l jar fermentor. Glucose (1%), soluble starch (3%), soybean flour (2%),
Potato protein (1%), CaCO ₃ (2%), Fe
_{SO 4 · 7H 2 O (0.04} %), Adekanol LG-1
09 (antifoaming agent, Asahi Denka, 0.05%) and silicone KM-70 (antifoaming agent, Shin-Etsu Chemical, 0.05%) were added to the jar and the medium was injected into the jar at 30 ° C. at 30 ° C. Sterilize for minutes. The above-obtained seed culture is inoculated into the above medium and cultivated at 25 ° C. for 5 days under aeration of 160 l / min and stirring at 200 rpm. The production of active compound in the fermentation broth is monitored by HPLC analysis.

(2) Isolation and Purification The culture solution (150 l) is added to acetone (150 l), and the broth-acetone mixture is stirred overnight. This mixture
Filter with diatomaceous earth (6 kg) as an aid. Filtrate (300
1) to water (300 l) and pH 6 with 6N HCl.
Adjust to 0. Filtrate (finally 25% hydrous acetone)
Is passed through a column of Diaion HP-20 (Mitsubishi Kasei) (15 l), which is washed with water (100 l) and eluted with methanol (100 l). 20 l of water are added to the eluate during the vacuum evaporation of methanol. The resulting aqueous solution (10
l) is extracted with 10 l of ethyl acetate. The extract is concentrated under reduced pressure. The residue is dissolved in dichloromethane (2 l), subjected to silica gel (500 g) column chromatography, eluting with a dichloromethane-methanol (25: 1) mixture (30 l). The active fractions are combined (61) and concentrated under reduced pressure. The residue was treated with 60% aqueous methanol solution (1
Dissolve in l). The solution was mixed with reverse phase silica gel (500
g) (ODS-AM120-S-50, YMC) column chromatography, eluting with acetonitrile-methanol-water mixture (4: 1: 5) containing 0.2% ammonium dihydrogen phosphate. Combine the active fractions (1 l),
Concentrate under reduced pressure to a 300 ml aqueous solution. The solution is extracted with 500 ml of ethyl acetate. The extract is concentrated under reduced pressure to give a pure red powder (80 mg) of material WS64826.

[Brief description of drawings]

FIG. 1 ¹ H nuclear magnetic resonance spectrum of WS64826 substance

FIG. 2 ¹³ C nuclear magnetic resonance spectrum of WS64826 substance

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12R 1: 465) (C12P 1/06 C12R 1: 465) (72) Inventor Hiro Terano 2600 Manabe, Tsuchiura City, Ibaraki Prefecture -3 (72) Inventor Masakuni Okuhara 2-14-10 Umezono, Tsukuba City, Ibaraki Prefecture

Claims (5)

[Claims] 1. A WS648 having the following physicochemical properties:
26 substances or salts thereof: Appearance: Red powder Molecular formula: C ₃₉ H ₄₇ NO ₁₆ Molecular weight: 785 High resolution FAB-MS: m / z Calculated value as C ₃₉ H ₄₈ NO ₁₆ : 786.2973 (M + H) ⁺ measured value : 786.2991 (M + H) ⁺ UV absorption spectrum: λmax (methanol) (ε): 232 (40820), 250 (29830),
285 (9420), 388 (3140), 470 (13345), 492 (13345), 5
30 (sh, 7850), 570 (2355) nm λmax (0.01N HCl, in methanol) (ε): 232 (40820),
250 (29830), 285 (9420), 388 (3140), 470 (13345), 49
2 (13345), 530 (sh, 7850), 570 (2355) nm λmax (0.01N NaOH, in methanol) (ε): 232 (32970),
247 (33755), 290 (7850), 350 (4710), 550 (12560), 5
93 (10990) nm Solubility: Soluble: Methanol, ethanol, acetone, ethyl acetate, chloroform Slightly soluble: Water Insoluble: n-hexane Color reaction: Positive: Cerium sulfate reaction, Maurish reaction, iodine vapor reaction Negative : Ninhydrin reaction, Dragendorff reaction Thin layer chromatography: [Table 1] High performance liquid chromatography (HPLC): Conditions: Mobile phase: 0.2% NH ₄ H ₂ PO ₄ containing acetonitrile:
Methanol: water = 5: 1: 5 Column: YMC-AM-303 ^** (S-5, 120ÅO
DS, 4.6 mm ID × 250 mm) Flow rate: 1.0 ml / min Detection: UV235 nm Retention time: 12.0 min ^** Product name: YMC Infrared absorption spectrum: νmax (KBr): 3477, 2971, 2935, 2872 , 1716, 1618,
1579, 1447, 1415, 1380, 1354, 1287, 1232, 1210, 111
8, 1067,1035, 991, 945 cm ^{-1 1} H Nuclear magnetic resonance spectrum: (400MHz, CD ₃ OD) δH 7.45 (1H, m), 7.37 (1H, m), 7.15 (1H, d, J = 8Hz ), 5.47
(1H, s), 5.00 (2H, m), 4.59 (1H, d, J = 8Hz), 4.26 (1H,
m), 4.24 (1H, s), 3.98 (1H, brs), 3.88 (1H, m), 3.78
(3H, s), 3.78-3.55 (5H, m), 3.52 (1H, brs), 3.42 (1H,
m), 2.83 (1H, d, J = 18Hz), 2.65 (1H, d, J = 18Hz), 2.42
(1H, m), 2.38 (3H, s), 2.25 (1H, d, J = 14Hz), 2.00 (1H,
brd, J = 14Hz), 1.73 (2H, m), 1.67 (1H, brd, J = 11Hz), 1.2
5 (3H, d, J = 6Hz), 1.19 (3H, d, J = 6Hz), 1.18 (3H, d, J = 6H)
z) ¹³ C nuclear magnetic resonance spectrum: (100MHz, CD ₃ OD) δC 213.4 (s), 186.9 (s), 186.8 (s), 161.9 (s), 156.9
(s), 155.8 (s), 136.9 (d), 135.7 (s), 135.6 (s), 13
5.3 (s), 120.8 (s), 120.2 (d), 119.8 (d), 112.0
(s), 111.8 (s), 106.9 (d), 105.9 (s), 102.0 (d), 9
5.0 (d), 87.6 (d), 83.3 (d), 82.0 (d), 77.7 (s), 7
7.0 (d), 75.1 (t), 70.0 (d), 69.9 (d), 65.4 (d), 6
4.2 (t), 56.9 (q), 50.9 (d), 47.4 (t), 36.4 (t), 3
3.8 (t), 30.6 (t), 24.8 (q), 24.2 (q), 18.5 (q), 1
7.6 (q) Essence: Amphoteric substance 2. WS64 belonging to the genus Streptomyces
A method for producing a WS64826 substance or a salt thereof, which comprises culturing an 826 substance-producing strain in a nutrient medium and recovering the substance. 3. WS64 belonging to the genus Streptomyces
826 substance producing strain is Streptomyces sp.
No. The method according to claim 2, which is 64826 (FERM BP-3876). 4. Streptomyces SP No.
A biologically pure strain of 64826 (FERM BP-3876). 5. An antitumor agent containing the substance WS64826 as an active ingredient.