JPS5830036B2 - Antibiotic SU-2 and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antibiotic 5U-2 and its non-toxic acid addition salt, and a method for producing the same.

Non-toxic addition salts of antibiotic 5U-2 refer to mono-, di-, tri-, and tetra-salts produced by the reaction of one molecule of 5U-2 with 1 to 4 equivalents of a pharmacologically acceptable non-toxic acid. .

Examples of the non-toxic acids include inorganic salts such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, and nitric acid, acetic acid, fumaric acid, malic acid, citric acid, mandelic acid, ascorbic acid, and tartaric acid. , organic acids such as succinic acid.

The present inventors studied the productivity of antibiotics using various microorganisms.

As a result, we found that in cultures of certain microorganisms belonging to the genus Micromonospora, there were antibiotics that behaved differently in chromatography from known antibiotics.

The present inventors purified and isolated the substance from the culture and investigated its physicochemical properties. As a result, the present inventors discovered that the substance was a new antibiotic and named it antibiotic 5U-2. .

Hereinafter, the present antibiotic 5U-2 and its manufacturing method will be explained in detail.

The physicochemical properties of the free base of antibiotic 5U-2 related to the present invention are as follows.

(1) Basic white powder (2) Elemental analysis values C48, 38% H8, 72% N 11.83% (3) Molecular weight: 450 (4) Molecular formula: C1, H38N408 (5) Melting point: 102-112° C (6) Ultraviolet Absorption Spectrum The ultraviolet absorption spectrum of an aqueous solution of the two substances does not show any characteristic absorption maximum between 220 and 360 nm, but only shows terminal absorption.

(7) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr tablet method is shown in FIG.

The wave number (crn, -1) showing the absorption maximum is as follows.

3350.2920.1570.1480.

1345.1105, 1050, 1025 (8) This substance is extremely soluble in water, soluble in methanol, slightly soluble in ethanol and acetone, chloroform, benzene, ethyl acetate, butyl acetate, ether, butanol,
Insoluble in organic solvents such as petroleum ether and n-hexane (9) Color reaction: Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Elson-Morgan reaction: Negative Biuret reaction: Negative (10) Mass spectrum: Book The mass spectrum of the substance gives the following M+H ion button and fragment ions.

However, the composition formula obtained by accurate mass measurement is shown in parentheses.

m/e 451 M+H (C1, H3, N408).

433 (C19H3, N308), 351 (C14H2
7N208).

323 (C13H2□N20?), 305 (C10
H25N206)t290(C13H24NO6),
192 (C7H, 4NO,).

160 (C7H14NO8), 129 (C6H13N
O2) From this, the molecular weight of this substance is 450, and the molecular formula is C19.
It was determined to be H38N408.

Also, the theoretical elemental analysis values of this substance (-hydrate) obtained from this molecular formula are C48, 71%, H8, 61% N1
It becomes 1.96%.

0υ The Rf value of paper chromatography button and thin layer chromatography using various developing agents for this substance is
~As shown in Table 3.

For comparison with known antibiotics, select those that are considered to be closely related and record their Rf values.

As described above, the antibiotic S tJ 2 has extremely strong antibacterial activity against a wide range of Durham-positive and -negative bacteria.

Among its wide-ranging and powerful antibacterial properties, it is remarkable in that it is extremely effective against bacteria of the genus Proteus and Puneudomonas, for which there have been few effective antibiotics.

Antibiotic 5U-2, which has extremely excellent antibacterial properties as described above, is medicinally useful as an antibacterial agent.

Furthermore, antibiotic 5U-2 can also be used in cleaning agents for hygienic purposes, such as cleaning laboratory glassware and equipment.

Next, we compared this substance with known antibiotics and found that dientamycin Corrplex (M, J) is a water-soluble, basic, and broad-spectrum antibiotic produced by Micromonospora.

Weinstein b Antimicrob, Ag
,Chermther,.

1963.1 and J-Cooper et al. J-I
nfect.

Dis, 119, 342, 1969, JA-We
Itz et al. Antimicrob-Ag, Chemo
ther,,2,464.

1972), Antibiotic 4460 (Tokuko Sho 4)
6-16153), cinmycin (M.

J0%! Instein et al., J-Antibiot
ics, 23, 551.

555.559, 1970), berdamycin (M-J
, Weinstein et al., Antimicrob
,Ag-Chemother, ,E,246,1
975), antibiotic G-52 (J-A, M
Arquez et al., J. Antibiotics, 24
, 483, 1976, h and P-J-L, Dan
iels et al. 1. J. Antibiotics, 24
.

488.1976),
No. 9155), Hortimycin A (Special Publication No. 1987-
No. 45675), Hortimycin C (%
-18888 Publication), Hortimycin D (Japanese Patent Application No. 1888)
1-128837), XK-62-3 (
(described in Japanese Patent Application No. 52-57827), XK-62
-4 (described in Japanese Patent Application No. 52-83038).

However, as shown in Tables 1, 2 and 3,
Antibiotic 5U-2 differs from both of the above in its behavior on paper chromatography and thin layer chromatography.

In addition, water-soluble, basic, and broad-spectrum antibacterial antibiotics produced by actinobacteria other than microorganisms belonging to the genus Micromonospora include streptomycin, lipostamycin, lividomycin, neomycin, tsunamycin, paromomycin, and neomycin. However, as shown in Table 3, the antibiotic 5U-2
It is distinguished from these antibiotics based on its Rf value.

Furthermore, from mass spectrum data, antibiotic 5U-
2 is clearly distinguished from known antibiotics.

Based on the above, antibiotic 5U-2 is considered to be a new antibiotic.

Next, the method for producing antibiotic 5U-2 according to the present invention will be explained.

Antibiotic 5IJ-2 is produced by culturing an antibiotic 5U-2-producing strain belonging to the genus Micromonospora in a nutrient medium, producing and accumulating antibiotic 5U-2 in the culture, and removing the antibiotic from the culture. It can be obtained by collecting.

The microorganisms used in the present invention include antibiotic 5U-2-producing bacteria belonging to the genus Micromonospora [for example, Micromonospora sagamiensis 5U-2 (Feikoken Bacteria No. 4230), (NRRLI 1. 182)).

The mycological properties of the Kori fungus species are described in Japanese Patent Publication No. 50-39155.

Culture for producing antibiotic 5U-2 is carried out by the following method.

That is, in culturing the microorganisms used in the present invention, the usual culturing method for actinobacteria is generally used.

Various sources of nutrients can be used for culture.

As the carbon source, glucose, starch, dextrin, mannose, fructose, sucrose, molasses, etc. are used alone or in combination.

Inorganic and organic nitrogen sources include ammonium chloride, ammonium sulfate, urea, ammonium nitrate, sodium nitrate, etc. Natural nitrogen sources include peptone, meat extract, yeast extract, dried yeast, corn steep liquor,
Soybean flour, casamino acids, soluble vegetable protein, cottonseed waste, etc. are used alone or in combination.

In addition, inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates can be added as appropriate, as well as organic and inorganic substances that promote the growth of the bacteria used and the production of antibiotic 5U-2. .

As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable.

It is desirable to culture at a culture temperature of 25° to 40°C and a pH around neutral.

When culture is carried out in liquid culture for usually 1 B to 12 days, antibiotic 5U-2 is accumulated in the culture solution.

When the amount produced in the culture solution reaches the maximum, the culture is stopped and the target product is purified and isolated from the culture solution.

Purification and isolation of antibiotic 5U-2 from the culture solution utilizes separation and purification methods commonly used to isolate microbial metabolites from the culture solution.

Since antibiotic 5U-2 is a water-soluble, basic substance as described above, it can be purified by a method commonly used for purifying so-called water-soluble/basic antibiotics.

Namely, adsorption/desorption method using cation exchange resin, cellulose column chromatography, Sephadex LH-20
This can be carried out by appropriately combining methods such as adsorption/desorption using a column, silica gel chromatography, and carbon chromatography.

Furthermore, the free base of this substance is soluble in acetone, but the sulfate salt is difficult to dissolve in the same solvent, so this point can be utilized to isolate and purify the free base or sulfate of this substance.

Next, an example of purification and isolation of antibiotic 5U-2 from a culture solution will be shown.

After culturing, solid matter is removed from the culture solution, and the resulting cultured solution is adjusted to be slightly alkaline. ) (Nni type) to adsorb the active substance, and after washing with water, the active substance is eluted with dilute ammonia water.

After several trace components have been eluted, the active fraction containing the antibiotic SU2 is eluted.

The active fraction containing antibiotic 5U-2 is collected and concentrated to dryness under reduced pressure to obtain antibiotic 5U-2 crude powder.

Next, column chromatography is performed using activated carbon (Shirasagi, manufactured by Takeda Pharmaceutical Co., Ltd.).

Dilute hydrochloric acid is used as the developing solvent.

The crude powder is dissolved in a solvent, introduced into a column packed with activated carbon, and eluted with the same solvent.

After the first few components are eluted, antibiotic 5U-2 is eluted.

After collecting this fraction and adjusting it to slightly alkaline, it was passed through a cation exchange resin Amberlite RC-50 (NH4+ type) to adsorb antibiotic 5U-2. After washing with water, it was eluted with IN-ammonia water, and the antibiotic Purified antibiotic 5U-2 can be obtained by collecting the 5U-2 fractions, concentrating them under reduced pressure, dissolving the residue in a small amount of water, and performing lyophilization.

The trends of the active fraction during the above refining process are as follows: Toyoji Paper/16
Check by ascending paper chromatography using No. 51.

As a developing solvent, chloroform, methanol, 17%
Using the lower layer of ammonia water (volume ratio (2:1:1),
Develop at room temperature for 6-16 hours.

The method for producing the antibiotic SU2 of the present invention will be specifically explained below with reference to Examples, but these are merely illustrative and do not limit the present invention in any way.

[Example 1] A. Culture of Micromonospora sagamiensis 5U-2. Micromonospora sagamiensis (Mi
cromonospora sagamiensis
) SU-2 (Microtechnical Research Institute No. 4230 NRRLIL
182) was used.

The first type medium includes Stabilose K [manufactured by Matsutani Kagaku ■] 2% (W/V) (hereinafter referred to), glucose 0.5. %
, peptone 0.5%, yeast extract 0.5%, meat extract 0
．． 3%, calcium carbonate 0.2% (pH 8.0 before sterilization)
A medium was used.

A platinum loopful of inoculum 1 is inoculated into 10 of the above-mentioned medium in a large test tube, and cultured with shaking at 30°C for 3 days.

This seed culture solution 10- is inoculated into a second type medium of 350- in a Kierlenmeyer flask with 21 volumes.

The composition of the second type medium is the same as that of the first type medium.

The second type culture is cultured with shaking at 30°C for 2 days.

350 ml of this seed culture was inoculated into 3.5 mL of the third type medium in a 51-volume Jar Fermentor, and the mixture was kept at 34°C for 24 hours with aeration and stirring (rotation speed: 400 rp11, aeration rate: 3.5 ml).
1Ailt) 2. Perform the culture.

The composition of the third type medium is the same as that of the first type medium.

Finally, 1.5 liters of this third type culture solution is inoculated into fermentation medium 157 in a 301-volume Jar Fermentor.

The fermentation medium composition was 5% Stabilose, 1% Soybeanmeal, and Pharmamedia (Traders 01).
1M1l company U, S, A, ) 2%
% Corn meal 1%, Soybean casein 0.5%, Dipotassium phosphate 0.025%, Magnesium sulfate 0.05%, Calcium phytin 0.2%,
Corn oil 0.1%, ferrous sulfate 0.015%, L-glutamine 0.0001%, L
- Cystine 0.005%, β-alanine 0.005%, nicotinic acid 0.0005%, calcium pantothenate 0.
0005%, zinc sulfate (heptahydrate) 0.0005%, potassium alum (tetrahydrate) 0.001%, ammonium molybdate (tetrahydrate) 0.025% (pH before sterilization 8.0
) was used.

This fermentation is carried out at 30℃ for 3 days using aeration stirring culture method r rotation speed 3
00rpHl, ventilation rate 151/mm).

B. Purification and isolation of antibiotic 5U-2 After the completion of the above culture, the pH of the culture solution was adjusted to pH 2 with 12N sulfuric acid.
0 and heat and stir at 80°C for 10 minutes.

Thereafter, 500 g of Radiolite #600 (manufactured by Showa Kagaku Kogyo ■) was added as a filter aid and the bacterial cells were separated by furnace.

This filtrate was mixed with Diaion HPK-25 (manufactured by Mitsubishi Kasei) (
Pass it through a column packed with NH7 type) and discard the running water.

After washing the resin with water, it is eluted with 2N aqueous ammonia, and both fractions containing the active substance are concentrated under reduced pressure to 500 rrll.

After adjusting the concentrated solution to pH 7.5 with 6N hydrochloric acid, it was passed through a column packed with Amberlite CG-50 Type I (NH"; type) 300-.
After washing with water, elute with 0, IN aqueous ammonia containing 0.2 M ammonium chloride.

After a few trace components were eluted at the beginning, the antibiotic SU
Active substances containing 2 are eluted.

The fractions were collected, adjusted to pH 7.5 with 6N hydrochloric acid, passed through a column packed with Amberlite RC-50 (NHt type) 100-, washed with water, and eluted with IN aqueous ammonia.

Concentrate and dry the fraction containing the active substance - this in a small amount of 0.5N
Dissolved in hydrochloric acid, activated carbon (Shirasagi, Takeda Pharmaceutical Co., Ltd.) 157727! is introduced into the top of the column packed with

After that, elution was performed with 0.5N hydrochloric acid and the eluted fraction was collected at 3fn! ,
Separate each portion.

The active fraction is detected by the method described above, and the fraction corresponding to antibiotic 5U-2 is collected.

Both fractions were adjusted to pH 7.5 with 6N ammonia water, passed through a column packed with 10ml of Amberlite RC-50 (NH type), washed with water, eluted with 2N ammonia, collected the active fraction, and reduced the pressure. Concentrate.

The solid obtained by concentration was dissolved in a small amount of water and lyophilized to obtain a purified sample of 5.8 m9 of antibiotic 5U-2 free base.

Example 2 5 μ of antibiotic 5 U-2 free base obtained in the same manner as in Example 1 was dissolved in 2- of water, and 0.9 of 0.05 N sulfuric acid was added.
Add rrll.

By freeze-drying this solution, antibiotic 5U-2.
7.2 μ of sulfate powder was obtained.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of antibiotic 5U-2.

Claims (1)

[Scope of Claims] 1. An antibiotic 5U-2 button and its non-toxic acid addition salt having the following physical and chemical properties. ■ Basic white powder ■ Elemental analysis: C48, 38%, H8, 72% N11
．． 83% ■ Molecular weight: 450 ■ Molecular formula: C19H38N408 ■ Melting point: 102 to 112°C ■ Ultraviolet absorption spectrum: The ultraviolet absorption spectrum of an aqueous solution of this substance shows no characteristic absorption maximum between 220 and 360 nm, with terminal absorption. It only shows. (2) Infrared absorption spectrum: The infrared absorption spectrum measured by the KBr tablet method is as shown in FIG. ■ Extremely soluble in water, soluble in methanol, slightly soluble in ethanol and acetone, but chloroform, benzene, ethyl acetate, butyl acetate, ether, butanol,
It is insoluble in organic solvents such as petroleum ether and n-hexane. ■ Color reaction Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Elson-Morgan reaction: Negative Biuret reaction: Negative [phase] Mass spectrum: The mass spectrum of this substance gives the following M+H ions and fragment ions. However, the composition formula obtained by accurate mass measurement is shown in parentheses. m/e 451M+H (C19H3, N408). 433 (C1, H35N308), 351 (C14H2
□N208) 2323 (C13H27N207), 30
5 (C13H2, N206). 290 (C13H24NO6), 192 (C7H14
NO, ). 160 (C7H14NO3), 129 (C6H13NO
2) 2 Antibiotic SU2-producing bacteria belonging to the genus Micromonospora were cultured in a nutrient medium, and antibiotic 5U-2 was added to the culture.
A method for producing antibiotic 5U-2, which comprises producing the antibiotic 5U-2 and collecting the antibiotic from the culture.