JPH021839B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is based on the general formula [] (In the formula, R ₁ represents a hydroxyl group or an amino group, and R ₂ represents a methyl group or a hydrogen atom.) The present invention relates to a novel aminoglycoside compound represented by the following formula and a method for producing the same. In the above compound [], the amino group at the 3'' position of tobramycin is N-methylated, the 4'' position is not or is C-methylated, and in some cases, the amino group at the 2' position is converted to a hydroxyl group. It is a new compound with chemical structural characteristics. The compound [ ] provided by the present invention exhibits excellent antibacterial activity and is promising as an antibacterial agent.
The antibacterial activity (minimum effective inhibitory concentration) of compound [] in comparison with tobramycin (hereinafter abbreviated as TOB) is as follows.

【table】

[Table] As is clear from the table above, the compound [] has strong antibacterial activity against various bacteria, especially the raw material topramycin and other gentamicin, sisomicin,
It has strong antibacterial activity against Proteus inconstans A-2 strain, which is resistant to dibekacin. Furthermore, among the bacteria resistant to kanamycin A and B, it has strong antibacterial activity against Escherichia coli K-12 ML-1629 strain, which inactivates aminoglycosides with a phosphorylating enzyme. On the other hand, compound [] has low toxicity, approximately 1/2 that of TOB. Compound [] is an excellent antibiotic in view of these properties and is extremely useful as a medicine. According to the present invention, the compound [ ] is produced by contacting TOB with a gentamicin-producing strain belonging to the genus Micromonospora or a mutant strain thereof. The strain used in this production method N-methylates the 3″ position of TOB, C-methylates the 4″ position, and in some cases oxidatively deaminates the amino group at the 2′ position to form a hydroxyl group. Yes, there are no restrictions as long as it can be converted. Examples of such strains include Micromonospora expora NRRL 2985 (IFO-
13149), Micromonospora purpurea
In addition to known strains such as NRRL 2953 (IFO-13150), Micromonospora sp.K-6993 strain, which is a new strain of the genus Micromonospora, can be mentioned.
This Micromonospora sp.K-6993 strain was newly isolated by the present inventors from the soil of Ishigaki Island, Okinawa Prefecture, and has been confirmed to produce gentamicin. In addition, mutant strains can be obtained by converting gentamicin-producing strains belonging to the genus Micromonospora to conventional mutagenic agents such as ultraviolet irradiation, cobalt-60 irradiation, X-ray irradiation, and mutagenic agents such as nitroso compounds, acridine dye compounds, and nucleobase analogues. It can be obtained by artificial mutation means. A suitable mutant strain is one that does not have the ability to produce gentamicin, or has an extremely reduced production ability, and has the ability to N-methylate and C-methylate the TOB mentioned above, and convert amino groups to hydroxyl groups. It is something. Representative examples of these mutant strains include Micromonospora sp.K-6993-Y-41 strain and Micromonospora echinospora NRR.2985-N-6 strain, both of which were obtained by the present inventors. Next, Micromonospora sp.K-6993 and its mutant strain Micromonospora sp.K-6993-Y-
41 strains and Micromonospora echinospora
The mycological properties of the NRRL.2985-N-6 strain will be described. These strains have been deposited with the National Institute of Microbiological Technology, No. 4304, No. 4305, and No. 4303, respectively, and have been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology. These strains are also included in the American Type Culture Collection, ATCC Nos. 31348 and 31348, respectively.
Deposited as Nos. 31349 and 31350. Morphological characteristics The morphological characteristics of the above three strains are relatively similar. 3
Both strains do not produce true aerial hyphae, but the diameter is 05~
Forms branched basal hyphae at 1.0μ. Only one spore settles on the tip of a sporophyte that branches from the basal hyphae. The shape of the spores is oblong or oval. All three strains grow well on Zapetsk agar, yeast extract/malt extract agar medium, and show a purple color on egg albumin agar. K-6993 and N-6
The strain is Micromonospora Echinospora
It showed the same morphological characteristics as NRRL・2985 (IFO-13149). Compared to the above two strains, the Y-41 strain does not adhere well to spores, and the color of the growth is slightly lighter overall. Growth status on various media

【table】

[Table] Assimilation of carbon sources Pridham Gottlieb (Pridham)
Gottlieb's medium was used as the basal medium, and 1.0% of each carbon source was added to examine the assimilation ability, and the results are shown in Table 2.

【table】 physiological properties

【table】

[Table] In Table 3, the results for milk and cellulose are shown after one month of culture at 37°C, and the results for gelatin liquefaction, nitrate reduction, and tyrosinase production are shown after two weeks at 29°C. . Strain K-6993 does not form a true aerial fungus system and attaches a single spore to basal hyphae, so it belongs to the genus Micromonospora. The following strains of Micromonospora that produce gentamicin have already been reported. Micromonospora purpurea
NRRL・2953 (Micromonospora purpurea),
Micromonospora Echinospora varietus Echinospora NRRL・2985
(Micromonospora echinospora var.
echinospora), Micromonospora echinospora varietus fuerginia NRRL・2995
(Micromonospora echinospora var.ferru―
ginea), Micromonospora echinospora variesta pallida NRRL・2996
(Micromonospora echinosppora var.
pallida) [Antimicrobial Agents and Chemotherapy 1963, p. 116~
Page 124 (Antimicrobial Agents and
chemotheraphy) and described in the Japanese Patent Publication No. 44-21934] Mikumonospora sagamiensis MK-65
(Micromonospora sagamiensis), Micromonospora sagamiensis varietus nonreducans MK-62 (Micrmonospora sagamiensis)
sagamiensis var.noreducans), Micromonospora sagamiensis varietus flava
Mm-628 (Micr-omonospora sagamiensin)
var.fLlava) [described in Japanese Patent Publication No. 50-39155 and Special Publication No. 51-6755] The taxonomic characteristics of these seven strains are compared with the K-6993 strain, and the differences are shown in Table 4. Post it. As a result, it is considered to be a new strain close to Micromonospora echinospora.

【table】

[Table] Next, to convert TOB to compound [],
Generally, the above-mentioned strains may be cultured in a medium containing TOB. In the culture of the present invention, conventional culture methods for producing antibiotics are used. Various sources of nutrients are used for culturing. As carbon sources, glucose, starch, soluble starch, dextrin, sucrose, molasses, etc. are used singly or in combination, and depending on the assimilation ability of bacteria, hydrocarbons, alcohols, organic acids, animals and plants are used. Oil etc. can also be used. Nitrogen sources include inorganic salts such as ammonium chloride, ammonium sulfate, urea,
Ammonium nitrate, sodium nitrate, etc. are used as natural nitrogen sources, soybean flour, defatted soybean flour, cottonseed meal, etc.
Gluten meal, corn meal, peptone, meat extract, yeast extract, dry yeast, corn steep liquor, etc. may be used alone or in combination. In addition, amino acids, nucleic acids, vitamins, and inorganic salts such as sodium chloride, calcium carbonate, phosphates, magnesium sulfate, and cobalt chloride can also be added as necessary. As for the culture method,
Liquid culture methods, especially methods using deep stirring methods, are suitable. The culture temperature is 25°C to 45°C, preferably
The temperature should be between 28℃ and 32℃, and the pH should be around neutral. In addition, culture composition, liquid properties of the medium, amount of additives, temperature, number of stirring,
It goes without saying that culture conditions such as the amount of aeration must be appropriately selected depending on the bacterial strain used. To obtain compound [], the raw material TOB can be added at the start of culture or after the bacteria have grown, but it is preferable to add it within 72 hours after the start of culture.The amount added is 0.1 It may be added at once to about 10 g/g, or it may be added in portions. Further, TOB may be used as it is, or may be a salt such as a sulfate. The contact time with TObB is selected to be the time when the compound [] accumulates the most after the addition of the raw materials.
It is usually 3 to 7 days after adding the raw materials. The method for collecting the compound [], including its isolation from the culture solution and purification, is the method normally used for collecting aminoglycoside antibiotics. That is, an appropriate combination of adsorption/desorption methods using cation and ion exchange resins, adsorption/desorption methods using activated carbon, adsorption/desorption methods using cellulose column chromatography, silica gel column chromatography, etc. can be used. Specifically, for example, after adjusting the pH of the culture solution to 2 to 3, the bacterial cells are removed by filtration, the pH is neutralized to 6 to 7, and the appropriate carboxone for adsorption and elution of substances with this structure is added. It is adsorbed on resins having groups such as acid and sulfonic acid, such as cation exchange resins Amberlite IRC-50 (trade name) (NH ₄ ⁺ ) and Dowex 50W (trade name) (NH ₄ D ⁺ ),
Elute with 1N aqueous ammonia. Concentrate this eluate under reduced pressure and use Amberlite CG-50 (trade name).
Perform ion exchange chromatography using a concentration gradient (NH ₄ ⁺ ) using aqueous ammonia. To further separate and purify these compounds, for example, use silica gel column chromatography, and if necessary, use Amberlite ccCGG-
50 (NH ₄ ⁺ ) and Dowtux 1×2 (product name)
Column chromatography using (OH ^- ) etc. can also be performed repeatedly. Representative compounds [] obtained by the above method are as follows. 3′-deoxy-4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer [
-T _1a ] 3′-deoxy-4″-C-methyl-3″-N-methylkanamycin or its 4″ epimer [-
T _1b ] 3′-deoxy-3″-N-methylkanamycin B
[-T ₂ ] The basic compound of the present invention [] is an inorganic or organic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid,
Easily forms non-toxic salts with stearic acid, propionic acid, tartaric acid, maleic acid, etc. Next, the manufacturing method of the present invention will be further explained with reference to Examples. Example 1 Target compound obtained from K-6993-Y-41 strain [-
Production of 100 ml of liquid medium ( _PH7.5 ) containing 5% dextrin, 3.5% defatted soybean flour, and 0.0% calcium carbonate.
Dispense into 500ml flasks and sterilize in advance.
Place the flask on Bennett's slant agar at 30°C.
Micromonospora grown sufficiently after being cultured for a week
Sp.K-6993-Y-41 strain was inoculated with one platinum loop and kept at 29℃.
A seed culture was obtained by shaking for 48-72 hours. Separately, prepare 100 ml of main culture medium in a 500 ml flask, and inoculate it with 1 ml of the above seed culture medium. The composition of the main culture medium is 5% dextrin, 3.5% defatted soybean flour (Ssanmeat special grade (trade name)), 0.7% calcium carbonate, 0.000025% cobalt chloride (PH7.5)
It is used after being sterilized in an autoclave at 120°C for 20 minutes. 24 hours after inoculation, 300 ^mcg (titer) of topramycin, which had been sterilized separately, was added per ml of the medium. Culture with shaking at 29℃ for 120 hours after addition.
24 cultures were obtained. After adjusting the pH of this culture solution to 2.0 with 4N hydrochloric acid, the bacterial cells were separated. Adjust the solution to pH 7.0 again with 4N sodium hydroxide and make 500ml of Amberlite IRC-50 (NH ⁺ ).
The target compound [-
T _1a ] was adsorbed. The resin was thoroughly washed with water and eluted with 1.5 N aqueous ammonia, and the eluate was concentrated under reduced pressure and dried to obtain a crude eluate. This crude eluate was adsorbed on a column packed with 900 ml of Amberlite CG-50 (NH ₄ ⁺ ), and after washing the resin thoroughly with water, concentration gradient elution was performed using 4 parts of water and 4 parts of 0.7N ammonia water. , each fraction (each
15 ml) is detected by silica gel thin layer chromatography (Merck Kieselgel 60F ₂₅₄ (trade name) thickness 0.25 mm, developed with a developing solvent (A in Table 5) for 2 hours, ninhydrin color development Rf value 0.21). The target compound [IT _1a ] is eluted before the starting materials tobramycin, [IT _1b ] and [IT ₂ ]. This eluate was concentrated and dried under vacuum to obtain 283 mg of [IT _1a ] as a crude powder. This coarse powder Amberlite CG-50 (N ₄ D ⁺ ) 1cm x 100
Adsorb onto a 1 cm column, perform concentration gradient elution with 1 part of water and 1 part of 0.5N ammonia water, and detect each fraction (each 5.5 ml) using the silica gel thin layer chromatography described above to identify the fraction containing [-T _1a ]. The mixture was collected and concentrated to dryness to obtain 10 mg of pale yellow [-T _1a ] crude powder. This crude powder was applied to a silica gel column (Wako Gel C-200 (trade name) 1 cm x 100 cm) and eluted and fractionated with a developing solvent (A in Table 5). Detected by layer chromatography, the [IT _1a ] fraction was collected and concentrated to dryness under reduced pressure to obtain 73 mg of white coarse powder of [IT _1a ]. This is again Amber Light CG-50
(NH ₄ ⁺ ) adsorbed on a 0.6 cm x 17 cm column and 200 ml of water
Concentration gradient elution was performed with 1 N ammonia water and each fraction (5 ml each) was detected using the silica gel thin layer chromatography described above [II-T _1a ].
The fractions were collected and concentrated under reduced pressure, then lyophilized to 57 mg.
A white powder of [IT _1a ] was obtained. This white powder
Charge 39 mg to a Dowex 1 x 2 (OH) 0.6 cm x 20 cm column and elute with water. The eluted [IT _1a ] fraction was detected by the silica gel thin layer chromatography described above and collected and concentrated under reduced pressure. After that, 31 mg of pure target compound [IT _1a ] was lyophilized.
A white powder was obtained. This [IT _1a ] free base (lyophilized product) exhibits the following physical and chemical properties. Basic white powder (vacuum dried at 80°C for 18 hours) Solubility: Extremely soluble in water, methanol,
Also soluble in ethanol. It is poorly soluble in acetone and insoluble in organic solvents such as chloroform, benzene, ethyl acetate, butyl acetate, ether, and n-hexane. Elemental analysis value (as C ₀ H ₄₀ N ₄ O ₁₀・H ₂ O) C H N Theoretical value (%) 46.68 8.23 10.89 Experimental value (%) 46.68 8.01 10.66 Melting point: 154-156℃ Optical rotation: [α] ²⁵ _D +152.8゜ (C=0.5%, inH ₂ O) Ultraviolet absorption spectrum: Terminal absorption Infrared absorption spectrum (K Br): Figure 1 Absorption maximum (cm ^-1 ) 820, 850, 1030, 1090, 1150, 1250 ,1330,
1360, 1450, 1475, 1595, 2910, 3340 NMR spectrum (in heavy water): Figure 2 Characteristic peaks 1.30ppm...Tertiary 4"-C-methyl (3H, singlet) 2.55ppm...3"-N- Methyl (3H, singlet) 5.13ppm...1″-Anomeric proton (1H, doublet, J=4.1Hz) 5.30ppm...1′-Anomeric proton (1H, doublet, J=3.7Hz) Mass spectrum : Main ion peak (m/
e) 100, 109, 110, 127, 128, 145, 146,
148, 162, 163, 190, 191, 192, 205, 208,
246, 275, 308, 319, 334, 336, 352, 362,
380,497 (M+1) Rf value by thin layer chromatography: 5th
Table (end) The above physical and chemical properties, especially typical fragment ion peaks in mass spectra (e.g. m/e146, 190, 191, 336, 380 and each decomposed ion peak group, as well as m/e497 (M+1)) and NMR In addition to the above results, kanamycin was inactivated by Pseudomonas energinosa strain GN-315, which has an inactivated oxygen that acetylates the amino group at the 6' position, indicating the presence of an amino group at the 6' position. Based on this point, the compound obtained in this example is a 3′-deoxy-
It is recognized to be 4″-C-methyl-3″-N-methylkanamycin A or its 4″ epimer. Example 2 Target compound [I-] obtained by strain K-6993-Y-41
Production of Amberlite shown in Example ₁
^[ _I
-T _1a ] and the target compound [I-T ₂ ] that had been eluted following the raw material tobramycin, etc., was concentrated to obtain a crude fraction of 661 mg of [I-T ₂ ]. This coarse classification is Amber Light CG-50
After adsorbing (NH ₄ ⁺ ) onto a 2 cm x 50 cm column and washing the resin with water, concentration gradient elution was performed with 1 part water and 1 part 0.8N ammonia water, and each fraction (each
6.5 ml) was detected by silica gel thin layer chromatography (Rf value 0.09) as in Example 1 [I-
T ₂ ] fractions were collected and concentrated to dryness to yield 345 mg of [I-
A pale yellow coarse powder of T ₂ ] was obtained. This coarse powder was applied to a silica gel column (Wako Gel C-200, 1 cm x 100
cm), eluted and fractionated with a developing solvent (A in Table 5), and detected each fraction (5 ml each) using the silica gel thin layer chromatography described above [I-T ₂ ].
The fractions were collected and concentrated to dryness under reduced pressure to obtain 293 mg of white coarse powder of [IT ₂ ]. This crude powder was adsorbed on a 1 cm x 50 column of Amberlite CG-50 (NH ₄ ⁺ ), and concentration gradient elution was performed with 1 part water and 0.8N aqueous ammonia, and each fraction (10 ml each) was subjected to the silica gel thin layer chromatography described above. Detected with Graphee [I-
T ₂ ] fractions were collected and concentrated to dryness under reduced pressure to obtain 266 mg of white powder of [IT ₂ ]. This white powder was charged to a Dowex 1 x 2 (OH ^- ) 0.6 cm x 17 cm column and eluted with _water .
The fractions were detected by the silica gel thin layer chromatography described above, collected, concentrated under reduced pressure, and lyophilized to obtain 240 mg of pure white powder of [IT ₂ ]. 20 mg of this [IT ₂ ] free base is dissolved in 0.2 ml of methanol, and a 0.2N methanol solution of sulfuric acid is gradually added to adjust the pH to 3.0, producing a white precipitate.
After taking this and washing it thoroughly with acetone, it was vacuum dried and 36 mg of [IT ₂ ] sulfate (melting point: 224~
A white powder was obtained (227°C, decomposition). This [IT ₂ ] free base (lyophilized product) exhibits the following physical and chemical properties. Basic white powder (vacuum dried at 80℃ for 18 hours) Solubility: Extremely soluble in water and soluble in methanol. Slightly soluble in ethanol, acetone, chloroform, benzene, ethyl acetate,
It is insoluble in organic solvents such as butyl acetate, ether, and n-hexane. Elemental analysis value (as C ₁₉ H ₃₉ N ₅ O ₉・H ₂ O) C H N Theoretical value (%) 45.68 8.27 14.02 Experimental value (%) 45.46 8.45 13.59 Melting point: 160-162℃ Optical rotation: [α] ²⁵ _D +135.3゜ (C=1%, inH ₂ O) Ultraviolet absorption spectrum: Terminal absorption Infrared absorption spectrum (KBr): Figure 3 Absorption maximum (cm ^-1 ) 1030, 1070, 1150, 1235, 1265, 1355, 1460,,
1470, 1595, 1640, 2910, 3340 NMR spectrum (in heavy water): Figure 4 Characteristic peaks 2.5ppm...3"-N-methyl (3H, singlet) 5.07ppm...1"-anomeric proton (1H , doublet, J = 3.6Hz) 5.25ppm...1'-Anomerikpton (1H,
Doublet, J=3.1Hz) Mass spectrum: Main ion peaks (m/
e) 98, 134, 145, 163, 176, 191, 205, 246,
274, 302, 320, 338, 348, 366, 482 (M+1) Rf value by thin layer chromatography: 5th
Table (end) The above physical and chemical properties, especially typical fragment ion peaks in mass spectra (e.g. m/e145, 191, 366 and each decomposition ion peak group, as well as m/e482 (M+1)) and NMR results, Based on the fact that it is derived from topramycin (3'-deoxykanamycin B), the compound obtained in this example was recognized to be 3'-deoxy-3''-N-methylkanamycin B shown by the following formula. It will be done. Example 3 Target compound obtained from K-6993-Y-41 strain [I-
Production of Amberlite shown in Example ₁
In concentration gradient fractionation elution of aqueous ammonia using a column packed with 900 ml of CG-50 [N ₄ D ⁺ ], [I
-T _1a ], the eluted fraction containing the target compound [I-T _1b ] that had been eluted together with tobramycin was concentrated to dryness under reduced pressure to obtain 2.76 g of a crude fraction of [IT _1b ]. This coarse fraction was collected using a silica gel column (Wako Gel C).
-200, 1 cm x 100 cm), elute and fractionate with a developing solvent (A in Table 5), and detect the [I-T _1b ] fraction using silica gel thin layer chromatography in the same manner as in Example 1. The mixture was collected and concentrated to dryness to obtain 417 mg of crude powder of [IT _1b ]. This crude powder still contained tobramycin, so silica gel chromatography was performed again under the same conditions as above.
155 mg of [IT _1b ] crude powder was obtained. This coarse powder is made into Amberlite CG-50 [NH ₄ ⁺ ] 1cm××50cm
A concentration gradient elution operation was performed using 1 part of water and 1 part of 1N ammonia water, and each fraction (10 ml each) was subjected to the same silica gel thin layer chromatography as described above in Example 1 [I-
The fraction containing [IT _1b ] was detected and collected, and concentrated to dryness under reduced pressure to obtain 119 mg of [IT _1b ] as a white powder. Dowex this white powder 1×2 [OH ^- ] 0.6
Charge a cm x 18 cm column, elute with water, detect the eluted [IT _1b ] fraction using silica gel thin layer chromatography as above, collect it, concentrate it under reduced pressure, and freeze-dry it. Pure [I-
109 mg of white powder of T _1b ] was obtained. This [IT _1b ] free base (lyophilized product) exhibits the following physical and chemical properties. Basic white powder (vacuum dried at 80℃ for 18 hours) Solubility: Extremely soluble in water and soluble in methanol. It is poorly soluble in ethanol and insoluble in organic solvents such as acetone, chloroform, benzene, ethyl acetate, butyl acetate, ether, and n-hexane. Elemental analysis value (as C ₂₀ H ₄₁ N ₅ O ₉・2H ₂ O) C H N Theoretical value (%) 45.19 8.53 13.17 Experimental value (%) 45.27 8.45 12.60 Melting point: 162-165℃ Optical rotation: [α] ²⁵ _D +102゜ (in 0.1% H ₂ O) Ultraviolet absorption spectrum: Terminal absorption Infrared absorption spectrum (KBr): Figure 5 Absorption maximum (cm ^-1 ) 1020, 1050, 1260, 1335, 1470,
1585, 2925, 3350 NMR spectrum (in heavy water): Figure 6 Characteristic peaks 1.31ppm...4"-C-methyl (2H, singlet) 2.60ppm...3"-N-methyl (3H, singlet) 5.13ppm ...1''-Anomeric proton (1H, doublet) 5.25ppm...1'-Anomeric proton (1H, doublet) Mass spectrum: Main ion peak (m/
e) 130, 145, 163, 190, 191, 496 (M ⁺¹ ) Rf value by thin layer chromatography: (5th
Table) The above physical and chemical properties, especially typical fragment ion peaks in mass spectra (e.g. resolved ion peaks of m/e 145, 190, 191, m/e 496 (M ⁺¹ ), nuclear magnetic resonance spectra, infrared Based on the absorption spectrum results and derivation from tobramycin, the 3″-N
-Methyl-4''-C-methyl-3'-deoxykanamycin B or its 4'' epimer. Note that the target compound [I-
The Rf values of T _1a ] and [IT ₂ ] measured by thin layer chromatography are shown in comparison with known aminoglycoside antibiotics.

【table】

【table】

【table】 [Brief explanation of drawings]

(1) Figures 1 and 2 show the infrared absorption spectrum and the infrared absorption spectrum of the present compound [IT _1a ], respectively.
The NMR spectrum is shown. (2) Figures 3 and 4 show the infrared absorption spectrum and the infrared absorption spectrum of the present compound [IT ₂ ], respectively.
The NMR spectrum is shown. (3) Figures 5 and ₆ respectively show the infrared absorption spectrum and
The NMR spectrum is shown.

Claims (1)

[Claims] 1. General formula (In the formula, R ₁ represents a hydroxyl group or an amino group, and R ₂ represents a methyl group or a hydrogen atom.) An aminoglycoside compound or an acid addition salt thereof. 2 formulas The aminoglycoside compound or acid addition salt thereof according to claim 1, which is represented by: 3 formulas The aminoglycoside compound or acid addition salt thereof according to claim 1, which is represented by: 4 General formula characterized by contacting tobramycin with a gentamicin-producing strain belonging to the genus Micromonospora or a mutant strain thereof (In the formula, R ₁ represents a hydroxyl group or an amino group, and R ₂ represents a methyl group or a hydrogen atom.) A method for producing a novel aminoglycoside compound or an acid addition salt thereof. 5 Gentamicin-producing strains belonging to the genus Micromonospora or their mutant strains are Micromonospora sp.
K-6993 strain, Micromonospora sp.K-6993-Y-
41 strains, or Micromonospora echinospora
The production method according to claim 4, which is the NRRL 2985-N-6 strain.