JPH0463874B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to 1-epidactymicin, a novel dactymicin derivative having antibacterial activity, or a salt thereof. Furthermore, the present invention provides the production of 1-epidactymicin by microbially converting the chemical structure of hotimicin B using a strain of the genus Streptomyces that does not have the ability to produce hotimicin B but has the ability to convert hotimicin B into a dactymicin derivative. It concerns the manufacturing method. 1-Epidactymicin or its acid addition salt provided by the present invention exhibits excellent antibacterial activity and is extremely useful as an antibacterial agent. The following formula Hotimicin B, represented by
of Antibiotics, Vol. 30, pp. 533-570 (197)), but its antibacterial activity is not necessarily satisfactory. During research to produce an antibiotic superior to Hotimicin B, the present inventors found that Hotimicin B was converted to a dactymicin derivative represented by the following formula [] when treated with a certain type of microorganism. did. Dactymicin is a compound described in JP-A-55-64597 under the name of substance SF-2052. Therefore, according to the first invention, the following three-dimensional structural formula 1-epidactymicin or an acid addition salt thereof is provided. 1-epidactymicin corresponds to 1-epi-2''-formimidoylhotymicin A. The compound of the present invention of formula []
It has a chemical structure corresponding to that in which the amino group at position is epitomized and the methylamino group at position 4 is formimidoyl glycylated. The compound of this formula [] (lyophilized product) exhibits the following physical and chemical properties. (1) Basic sulfate of this substance: white powder (2) Solubility: extremely soluble in water, methanol,
Ethanol, acetone, chloroform, benzene, ethyl acetate, butyl acetate, ether, n-
It is insoluble in organic solvents such as hexane. (3) Elemental analysis value (C ₁₈ H ₃₆ N ₆ O ₆・2H ₂ SO ₄・4H ₂ O
): C H N Theoretical value (%) 30.85 6.90 11.99 Experimental value (%) 30.78 7.01 12.00 (4) Melting point: Gradual thermal decomposition (accompanied by browning, foaming, and melting) above 205°C (5) Optical rotation: [α] ²¹ _D +92° (c=0.15%; H ₂ O) (6) Ultraviolet absorption spectrum: Terminal absorption (7) Infrared absorption spectrum (KBr tablet): As shown in FIG. Maximum absorption (cm ^-1 ): 3400, 2940, 2040, 1715,
1630, 1500, 1400, 1360, 1325, 1260,
1110, 1040 (8) Nuclear magnetic resonance spectrum (in heavy water): As shown in Figure 2. Characteristic signal: 1.35ppm (d, 3H, J = 6.5
Hz)...6'-C-methyl, 1.6ppm (m1H)...
4′-methylene, 2.03ppm to 2.15ppm (m3H)
...3',4'-methylene, 3.15ppm (s, 3H)...
4-N-Methyl, 3.41ppm (m, _1HJ6 ', _CH3
= 6.5Hz)...6'-methine, 3.51ppm (s, 3H)
3-0-methyl, 3.60ppm (m, 1H)...2'-
Methine-,. 88ppm (1H)…5′-methine,
3.93ppm (t, 1H.J ₁ ; ₂ = 3.6Hz, J ₁ ; ₆ = 3.6
Hz)...1-methine, 4.06ppm (d, d, 1H,
J ₃ ; ₂ = 3.3Hz, J ₃ ; ₄ = 11Hz) 3-methine,
4.23ppm (t, 1H)...6-methine, 3.38ppm
(d, 1H, J=17.5Hz) and 4.47ppm (d,
1H, J=17.5Hz) 2″-methylene 4.42ppm
(1H)...5 methylene, 4.66ppm (d, d,
1H)...4-methylene, 4.7ppm (t, 1H)...
2-methine, 5.46 ppm (d, 1H, J ₁ ′ _{, 2} ′ = 3.2
Hz)...1′-anomeric proton, 7.98ppm
(s, 1H)...3 (formimidoyl) proton (9) Molecular ion peak in mass spectrum (SIMS): 433 (M+1) (10) Retention time by high performance liquid chromatography: As shown in Table 1 be.

【table】

[Table] (11) Rf value by thin layer chromatography: 2nd
As shown in the table.

【table】

[Table] The antibacterial spectrum of the compound of formula [] according to the present invention is compared with Hotimicin B (abbreviated as FM-B), Hotimicin A (abbreviated as FM-A), and Dactimicin (abbreviated as DAC) as follows. It is shown in Table 3.

【table】

【table】

[Table] As is clear from the above table, the compound of the present invention of formula [] exhibits a wide range of strong antibacterial activity against various Gram-positive and Gram-negative bacteria, and especially compared to the starting material Hotimicin B, The antibacterial activity is enhanced by a factor of two to several tens of times, and it has a stronger antibacterial activity that is comparable to Hotimicin A and Dactimicin. Examples of acid addition salts of compounds of the invention include pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or organic acids such as acetic acid,
There are salts with propionic acid, citric acid, metal sulfonic acid, etc. According to the second aspect of the present invention, Hotimicin B includes one of Hotimicin B that belongs to the genus Streptomyces.
A method for producing 1-epidactymicin of the above formula [] or an acid addition salt thereof, which comprises using a bacterium capable of epitomizing the amino group at the position and introducing a formimidoylglycyl group into the methylamino group at the 4-position. is provided. According to the method of the present invention, the compound [] is produced by contacting Hotimicin B with a bacterial bead belonging to the genus Streptomyces and having the above-mentioned conversion ability or a mutant strain thereof. There are no particular limitations on the strain used in the method of the present invention, as long as it can epiform the amide group at position 1 of the fotamin moiety and formimidoylglysylate the methylamino group at position 4. Examples of fungal beads that can be used in the method of the present invention include:
Streptomyces tenzimariensis, known as istamycin-producing bacterium
tenjimariensis) SS-939 strain (FERM-P4932)
(Refer to Japanese Patent Application Laid-Open No. 55-145697), and Streptomyces tenzimariensis SS1507
can be given. This strain SS-1507 has been deposited with the National Institute of Microbiology, Agency of Industrial Science and Technology, and its accession number is FERM-P. Mutant strains can be obtained from istamycin-producing strains belonging to the genus Streptomyces by using, for example, ultraviolet irradiation, cobalt-60 irradiation, X-ray irradiation, or mutagenic agents such as nitroso compounds, acridine dye compounds, and nucleobase analogues. There are things that can be obtained through artificial mutation. Suitable mutant strains for use in this method include those that do not have the ability to produce istamycin or have an extremely reduced ability to produce istamycin.
Moreover, it has the property of being capable of epitomizing the amino group at the 1-position of Hotimicin B and converting the methylamino group at the 4-position to formimidoylglucylation. A representative example of these mutant strains that can be used in the method of the present invention is Streptomyces tenjimariensis SS-1507U-, which the present inventors derived from Streptomyces tenjimariensis SS-1507. 41
can be given. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, and its accession number is FERM-P7625. These Streptomyces tenjimariensis SS-1507 and Streptomyces tenjimariensis SS-1507U-41
The mycological properties of both are exactly the same and are as shown below. (A) Morphology Streptomyces tenzimariensis growing well on agar medium
strain SS-1507 and Streptomyces tenjimariensis SS-1507U-
Strain 41 has simple branching and extends straight or slightly curved aerial hyphae from well-elongated basal hyphae.
When mature, it forms a chain of 10 to 50 long cylindrical (1 micron wide, 4 to 5 micron long) spores at the tip. No spirals or axle branches are shown. Under an electron microscope, the spore surface is smooth and does not have any spine-like or hair-like structures. It is a typical Streptomyces without flagella or sporangia. (B) Characteristics on various media Seuucrose/nitrate agar medium (cultured at 27°C); white aerial hyphae appear on weak colorless growth, gradually turning blue-green to gray (17ec,
aqua blue, according to the Color Harmony Manual; the same applies hereafter). It does not produce significant diffusible pigments in the medium. Glycerin-asparagine agar medium (27℃
culture); however, aerial mycelia are difficult to attach to. It is almost the same as starch agar medium (cultured at 27℃); however, aerial mycelium grows on it, and the starch around the bacterial growth becomes transparent. The findings are almost the same as those on tyrosine agar medium (cultured at 27°C); melanin-like pigments are produced. Nutrient agar medium (cultured at 27°C): White aerial mycelium grows on colorless growth, and the medium becomes brownish. Yeast/malt agar medium (cultured at 27°C): White aerial mycelium grows on colorless growth, gradually turning blue-greenish gray (19bc aqua gray). The medium appears brownish in color. Oatmeal agar medium (cultured at 27℃); colorless growth with white to blue-gray color (17ec, aqua
Aerial mycelia with a blue color are attached. (C) Physiological properties Can grow at 20-41℃. Hydrolyze the starch on starch agar. Hardly any coagulation or peptonization of skim milk is observed. Melanin-like pigments are produced on tyrosine agar and peptone yeast iron agar. (D) Assimilation of carbon sources (on Prittham-Gotzlieb agar medium) Only glucose and inositol are assimilated, and arabinose, D-xylose, sucrose, rhamnose, raffinose, and D-mannite are not assimilated. Assimilation of D-fructose is questionable. The above properties have already been reported by the present inventors, and istamycin-producing bacterial beads, Streptomyces tenzimariensis SS-939 strain (Japanese Unexamined Patent Application Publication No. 1989-1982)
145697), the ability to form aerial mycelia, the production of melanin-like pigments, the color tone of aerial mycelium, and the assimilation of carbon sources are exactly the same, but Streptomyces tenzimariensis SS-1507U-41 strain It is unique in that it has almost no ability to produce mycin, and is convenient for use in the method of the present invention. To carry out the method of the present invention, in order to convert Ho Chi Minh B into the compound [], Ho Chi Minh B
Generally, the above-mentioned strains of Streptomyces tenzimariensis, which can be used in the method of the present invention, may be cultured in a medium containing . That is, the above-mentioned method is made to work by allowing Hotimicin B to exist in the culture solution in which the bacteria are being cultured. In culturing bacteria in the method of the present invention, conventional culture methods for producing antibiotics are used. Various nutrient sources can be used for culture. Glucose as a carbon source,
Starch dextrin, sucrose, molasses, etc. can be used alone or in combination, and depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, animal oils, vegetable oils, etc. can also be used. Inorganic nitrogen sources and organic nitrogen sources include ammonium chloride, ammonium nitrate, sodium nitrate, soybean flour, defatted soybean flour, cottonseed meal, gluten meal, cornmeal, wheat germ, peptones, meat extract, yeast extract, and drying. Yeast, corn steep liquor, etc. may be used alone or in combination. In addition, amino acids, nucleic acids, vitamins, sodium chloride,
Calcium carbonate, phosphate, magnesium nitrate,
Inorganic salts such as cobalt chloride can also be added. As a culture method, a liquid culture method, particularly a method using a deep stirring method, is suitable. Culture temperature is 20℃~41℃
℃, preferably 25℃ to 32℃, and the pH is preferably around neutral. It goes without saying that culture conditions such as medium composition, liquid properties of the medium, amount of additives, temperature, number of stirrings, amount of aeration, etc. are appropriately selected depending on the bacterial beads used. In order to obtain the compound [], the raw material Hotimicin B may be added at the start of the culture or after the bacteria have grown, but it is preferable to add it within about 96 hours after the start of the culture. The amount of raw materials to be added is approximately 0.1 to 10 g per 1 g of the medium, which may be added all at once, or may be added in portions. Further, Hotimicin B to be added may be in the form of a free base, but may also be in the form of a salt, such as a sulfate or a hydrochloride. The action time of the bacteria and hotimicin B is selected so that the compound [] is accumulated in the largest amount after the addition of the raw materials.
This is for example Bacillus subtilis.
subtilis) PCI219 as a test strain, the amount of the compound [ ] in the culture solution can be monitored using the paper desk method, but it usually takes 3 to 9 days. The method for collecting the compound [], including its isolation and purification from the culture solution, is the method normally used for collecting aminoglycoside antibiotics. Namely, adsorption and desorption methods using cation and anion exchange resins, adsorption and desorption methods using porous polymer resins using counter ions, adsorption and desorption methods using activated carbon,
Methods such as silica gel column chromatography can be used in appropriate combination. Specifically, in order to collect the compound [] from the culture solution, for example, the pH of the culture solution is adjusted to 2 to 3, the bacterial cells are removed by sieving, and the PH is adjusted to 5 to 6 again. Carboxylic acid groups, sulfonic acid groups suitable for adsorption and elution of substances with this chemical structure,
Amberlite IRC-50 (trade name) [Na ⁺ ] as a cation exchange resin with etc., Dowex
Adsorb with 50W (trade name) [Na ⁺ ], etc., and elute with 0.5N sulfuric acid to obtain a crude solution containing compound [T].
In order to further purify this substance, we used sodium 1-pentanesulfonate, sodium 1-hexanesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate, sodium 1-dodecanesulfonate, and sodium 1-dodecanesulfonate as counterion agents. Select an appropriate one from sodium dodecylsulfonate, sodium paratoluenesulfonate, etc., add it and dissolve it, and gradually add about 0.5 to 1N of sodium hydroxide.
After adjusting the pH to 5.0, porous polymer resins Amberlite XAD-2 (trade name), Amberlite XAD-4 (trade name), Diaion HP-20
(product name), Diaion CPH-20P (product name), etc. After adsorbing these and washing the resin thoroughly with water, the metal concentration of the elution solvent was adjusted to
The concentration is gradually increased from 0 stepwise or by concentration gradient method until each substance is eluted. The fractions of each substance eluted in this way were collected and neutralized to pH 6.0 with approximately 0.5N sodium hydroxide, and then mixed with cation exchange resin Amberlite IRC-50 (trade name) [Na ⁺ ]. ,
Adsorb to CG-50 (trade name) [Na ⁺ ], wash well with water, and elute with 0.5N sulfuric acid. This measures the separation from the counter ion agent used.
This eluate contains a large amount of sodium sulfate in addition to the substance of formula [], so it is adsorbed on activated carbon and washed with water to wash away the sodium sulfate, and then eluted with 80% methanol containing 0.05N sulfuric acid. Since the eluate is sulfuric acid, after concentrating it to an appropriate concentration, it is mixed with Amberlite IRA-45, an anion exchange resin.
(Product name) After neutralizing with [OH ^- ] and separating, anion exchange resin IRA-400 (Product name)
The target substance of the formula [] is obtained by passing [SO ₄ ^-- ] through it, concentrating the aqueous solution that has passed through it, and then freeze-drying it. This basic compound [] readily forms non-toxic acid addition salts when reacted with inorganic or organic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid stearic acid, tartaric acid, maleic acid, etc. in a conventional manner. . Next, the present invention will be further explained with reference to Examples. Example 1 Streptomyces grown well by culturing on soluble starch inorganic salt (ISP No. 4) agar medium for 2 weeks.
Streptomyces
tenjimariensis) SS1507U−41 (FERM−P7625)
was inoculated into a sterilized 500 ml flask with 100 ml of a liquid medium (PH 7.0) containing 6.0% cornmeal, 2.0% wheat germ, 0.05% magnesium sulfate, and 0.6% calcium carbonate, and incubated at 27°C for 48 hours. A seed culture solution was obtained by shaking culture for ~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 this medium is 6.5% wheat germ, 0.5% sodium palmitate, 0.05% magnesium sulfate, 0.6% calcium carbonate,
It contains 3.5% soybean oil (PH7.0) and is used after being sterilized at 120°C for 17 minutes. Hochi Sewing Machine B 72 hours after inoculation
(base) was added at 300 mcg per ml of medium. After adding this raw material, shaking culture was performed at 27°C for 168 hours.
By the way, at that time Bacillus subtilis PCI
When the antibacterial activity of the culture solution was measured using the -219 strain by the paper dice method, an inhibition circle of 21.9 mm in diameter was obtained. After adjusting the pH of 22 flasks of the obtained culture solution to 2.0 with 6N sulfuric acid, the bacterial cells were separated, and the bacterial cells were further washed with water, the washing solution was filtered, and the culture solution was mixed with the previously filtered culture solution. Combined. 4.31 of the superculture solution obtained in this way was added to 4N sodium hydroxide to pH 6.0.
Return to Amberlight IRC-50 (product name)
The target compound [ ] was adsorbed by passing through a column packed with 95 ml of [Na ⁺ ]. The column was thoroughly washed with water and eluted with 0.5N sulfuric acid 1.1 to obtain a solution containing the crude target compound [ ]. Add 3.0g of sodium paratoluenesulfonate to this solution and
Gradually add the specified sodium hydroxide to bring the pH to 5.0.
Adjusted to. Add this solution to Diaion CHP-20P.
(trade name) 75 ml column to adsorb the target compound. The column was filled with 200 ml of hydrochloric acid water (PH
After washing with 2.0), concentration gradient column chromatography was performed with 0 to 8% methanol water using hydrochloric and acidic water (PH2.0). Each fraction (10 each
ml) was searched for using high performance liquid chromatography and the compound [] was confirmed. By the way, the operation and conditions of high performance liquid chromatography used at this time were Deveroseal ODS-5 (C-20%, 5μ) on the column.
[Product name] (6mmφ×200mm) was used, and the eluent was 0.2
A solution prepared by adding 4% acetonitrile to a 0.1% acetic acid solution containing 0.02 mol of sodium sulfate and 0.02 mol of sodium 1-pentanesulfonate as a counterion agent is fed at a rate of 1 ml per minute. In addition, ortho-phthalaldehyde reagent (hereinafter abbreviated as OPA) was used to detect this substance.
A solution of 0.6 mg/ml dissolved in 10 boric acid buffer is used. After the sample was injected, the eluent eluted from the column was reacted with the OPA solution in a mixing joint, and the target compound was detected with a UV detector at a wavelength of 344 mμ. According to this operating method, the retention time of the target compound [] is 7 minutes 28 seconds (the first
Table). The compound [] searched in this way was fractionated into fractions 224 to 242, so these were collected and concentrated to about 40 ml, and the pH was adjusted to 6.0 using a 0.4N sodium chloride solution. This fraction of the target compound [] was passed through a 3 ml column of Amberlite IRC-50 (trade name) [Na ⁺ ] to adsorb the target compound []. After thoroughly washing with water, the mixture was eluted with 0.5N sulfuric acid and fractionated into 5ml portions. The target compound [] was assayed by the paper disk method on a plate using Bacillus subtilis PC219 as the test bacterium. Paper disks were neutralized with ammonia gas before assay. Fractions 1 to 8 can also be determined by testing positive for ninhydrin.
After confirming that it was fractionated, the fractions were collected and the pH was adjusted to 6.02 with 0.4N sodium hydroxide solution. The thus obtained fraction of the target compound [] was adsorbed on a column packed with 25 ml of activated carbon, thoroughly washed with 100 ml of water, and then eluted with 50% aqueous methanol containing 0.05N sulfuric acid. Each fraction (5 ml each) was assayed on a Bacillus subtilis PCI219 plate medium using the paper disc method. At this time, the paper disk was neutralized with ammonia gas before the assay. As a result, the target compound [] was eluted from fractions 3 to 40, so it was collected, concentrated, methanol was removed, and then neutralized using Amberlite IRA-45 (trade name) [OH ^- ]. After that, about 1ml
It was further concentrated to This concentrated solution was charged to a 10 ml column of Amberlite IRA-400 (trade name) [SO ₄ ^-- ], eluted with water, and fractionated into 5 ml fractions each. Each fraction was assayed by the paper disc method using Bacillus subtilis PCI219, and after confirming that the target compound was contained in fractions 1 to 5 by testing positive for ninhydrin, the fractions were collected and 0.5 Concentrate to 18.1 ml and lyophilize to 18.1 ml.
mg of the target compound [ ] as a white powder was obtained. The physical and chemical properties of this substance, especially the measurement results of mass spectra, nuclear magnetic resonance spectra, and infrared absorption spectra, as well as Ho Chi Micin B
Based on the fact that it was derived from the formula [-1], it was identified as 1-epi-2''-formimidoylhotimicin A, ie, 1-epi-dactymicin, represented by the above formula [-1].

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of 1-epidactymicin (KBr tablet) according to the present invention;
The figure is a nuclear magnetic resonance spectrum of 1-epidactymicin.

Claims (1)

[Claims] First-order three-dimensional structural formula 1-epidactymicin or an acid addition salt thereof. 2. The following, characterized in that a bacterium belonging to the genus Streptomyces and having the ability to epitomize the amino group at the 1-position of Hotimicin B and introduce a formimidoylglycyl group into the methylamino group at the 4-position is allowed to act on Hotimicin B. 3D structural formula of A method for producing 1-epidactymicin. 3 Streptomyces tenjimariensis in Hochimishin B
The method according to claim 2, in which SS-1507 U-41 (Feikoken Bibori No. 7625) is applied. 4. The method according to claim 2, wherein Hotimicin B is present in the culture medium during which Streptomyces tenzimariensis is being cultured.