JPS5924998B2 - Antibiotic SUM-3 and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a new antimicrobial substance SUM-3, its nontoxic acid addition salt, and a method for producing the same.

The non-toxic acid addition salt of the antibiotic substance SUM-3 is SUM-3,1
It refers to the mono-, di-, tri-, and tetra-salts produced by the reaction of the molecule with 1 to 4 equivalents of a pharmaceutically acceptable non-toxic acid. Examples of the non-toxic acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroioccic acid, sulfuric acid, phosphoric acid, carbonic acid, and nitric acid; acetic acid;
Examples include organic acids such as fumaric acid, malic acid, citric acid, mandelic acid, ascorbic acid, tartaric acid, and succinic acid.
The present inventors studied the productivity of antibiotic substances using various microorganisms.

As a result, we found that in cultures of certain microorganisms belonging to the genus Micromonospora, there were antibiotic substances that behaved differently in chromatography from known antibiotic substances. The present inventors purified and isolated the substance from the culture and examined its physicochemical properties. As a result, the substance has the formula CH2NHCH3NHCH3.

. . □Second. N-.

It was discovered that this substance has the structure shown by N□OH and is a new antibiotic.
It was named M-3.

Hereinafter, the present antibiotic SUM-3 and its manufacturing method will be explained in detail.

The physicochemical properties of the free base of the antibiotic SUM-3 related to the present invention are as follows.

5 (1) Basic white powder (2) Elemental analysis value C: 49.51% The ultraviolet absorption spectrum of an aqueous solution of this substance is 220360
It does not show a characteristic absorption maximum in the nm range, but only shows terminal absorption.

(6) Specific rotation (7) This substance is extremely soluble in water, soluble in methanol, and slightly soluble in ethanol and acetone;
It is insoluble in organic solvents such as butanol, petroleum ether, and n-hexane.

(8) Color reaction: Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Elson-Morgan reaction: Negative Biuret reaction: Negative (9) Mass vector reactivity The mass spectrum of this substance gives the following molecular ions and fragment ions. .

m/E464 (M+), 434, 402-, 351, 3
47,334,323,306,290,289,27
2,192,160,1430 (10) The proton magnetic resonance spectrum (heavy water) (PDll.3) of this substance shows the following values.

δ1.20 (3H, s), 1.20 to 1.90 (5H,
m), 1.90-2.21 (1H, m), 2.31 (3
H, s), 2.50 (3H, s), 2.58 (1H, d
J=10.8Hz), 2.60(2H, dJ=6.6H
z), 2.64-3.10 (2H, m), 3.30 (1
H, dJ=12.3Hz), 3.31~3.62 (3H
, m), 3.75 (1H, d, dJ=3.9, 10.8
Hz), 4.05 (1H, dJ2 12.2Hz), 3.
62-4.11 (2H, m), 5.15 (1H, dJ=
3.7Hz), 5.27 (1 H, dJ=3.9Hz)
(11) The carbon nuclear magnetic resonance spectrum of this substance shows the following value (δPpm).

(12) The Rf values of this substance in paper chromatography and thin layer chromatography using various developing agents are shown in Tables 1 to 3.

In addition, for comparison with known antibiotics, we select those that are considered to be closely related, and also record their Rf values. Rising paper chromatography 1 (Toyofuchi paper 5 as paper)
1 and developed at 28°C) of the antibiotic SUM-3.
f value silica gel thin layer chromatography [E. Me-R
KieselGel6O manufactured by ck (carried out at room temperature).

Rf value at 3 hours development time)*Developing agent: 10% ammonium acetate, methanol (1:1) (volume ratio)Developing agent: chloroform, methanol, concentrated aqueous ammonia (1:1)
1:1) (capacity ratio) lower layer ※※ JP-A-54-592
Antibiotics listed in Publication No. 02 ※※※ Distance of the substance from the origin RfCl value = 1 Distance of dientamicin C1 from the origin ※※※※
Antibiotics described in JP-A-53-56640 [Table 3] Rising type using lower layer of chloroform, methanol, and 17% ammonia water (2:1:1) (volume ratio) as developing agents Paper chromatography (perform at room temperature).

Rf value at 12 hours deployment time* JP-A-53-566
Antibiotics described in Publication No. 40※※ JP-A-53-9
Antibiotics described in Publication No. 0241 ※※※ JP-A-5
Antibiotics listed in Publication No. 3-144547※※※※
Antibiotics described in JP-A-54-19939 ※※※※※ Antibiotics described in JP-A-54-59202 Next, the antibacterial spectrum of antibiotic SUM-3 (
MICγ/a) is shown in Table 4.

The measurement is performed using a medium with a pH of 7.2 and by the agar dilution method.
The medium is Hard Infusion Pross (DifcO
) was used. * Produces dientamycin acetyltransferase type I * * Produces dientamycin nutaleotidyl transferase * * * Produces kanamycin acetyltransferase In this way, the antibiotic SUM-3 is effective against a wide range of Gram-positive bacteria and It has extremely strong antibacterial activity against negative bacteria.

Among its wide-ranging and powerful antibacterial properties, a remarkable feature is that it is extremely effective against bacteria of the genus Proteus and Busheudomonas, for which it has been thought that there are few effective antibiotics. It also has strong antibacterial activity against Escherichia coli, which is resistant to various known antibiotics.
Antibiotic SUM-3, which has extremely excellent antibacterial properties as described above, is medicinally useful as an antibacterial property.

Furthermore, the antibiotic SUM-3 can also be used in cleaning agents for hygienic purposes, such as cleaning laboratory glassware and equipment. Next, let's compare this substance with known antibiotics.

Dientamycin Complex (M.J. Weinst) is a water-soluble, basic and broad-spectrum antibiotic produced by Myuromonospora.
ein et al. AntimicrOb. Ag. ChemOth
er. , l963, l and D. J. COOper et al. J
・Infect. Disll9,342,l969,J
．． A. Weitz et al. AntimicrOb. Ag. Ch
emOther. , 2,464, 1972), Anti-Biochicken 460 (Special Publication No. 16253/1983)
, sisomicin (M.J. Weinstein et al., J.
AntibiOtics, 23, 55l, 555, 55
9,197O), perdamycin (M.J. Weins
Teln et al., AntimicOrb. Ag. ChemO
ther. , 7, 246, 1975), antibiotic G-52 (J. A. Marquez et al., J. Ant
iblOtics, 24,483, l976, and P
．． J. L. Daniels et al., J. AntibOtic
s, 24,488, l976), XK-162-2 (Japanese Patent Publication No. 50-39155), Horteimycin A (Japanese Patent Publication No. 51-45675), Horteimycin C (
JP-A-52-18888), Horteimycin D
(described in Japanese Patent Application Laid-open No. 53-56640), XK-62
-3 (described in JP-A-53-144547), XK
-62-4 (described in the specification of Japanese Patent Application No. 52-83038)
, SU-2 (described in Japanese Unexamined Patent Publication No. 54-59202). However, as shown in Tables 1, 2 and 3, the behavior of antibiotic SUM-3 in paper chromatography and thin layer chromatography
is different from any of the above. In addition, streptomycin, ribostamycin, lividomycin, neomycin, kanamycin, paromomycin, nebulomycin, which are water-soluble, basic, and have a broad antibacterial spectrum, are produced by actinobacteria other than microorganisms belonging to the genus Micromonospora. Examples include mycin, but the antibiotic SU
As is clear from Table 3, M-3 is distinguished from these antibiotics by its Rf value. Furthermore, PMR and C
A substance with the same structure as the chemical structure of the antibiotic SUM-3 (above) revealed from MRl mass spectrum data cannot be found among known substances, and from the above, the antibiotic SUM-3 is It is considered to be a new anti-inflammatory substance.

Next, the method for producing the antibiotic SUM-3 in the present invention will be explained.

Antibiotic SUM-3 is produced by culturing an antibiotic SUM-3-producing strain belonging to the genus Micromonospora in a nutrient medium, producing and accumulating the antibiotic SUM-3 in the culture, and extracting the antibiotic from the culture. It can be obtained by collecting.

The microorganisms used in the present invention include antibiotic SUM-3 producing bacteria belonging to the genus Micromonospora [e.g.
Micromonosvora sagamiensis SU-2 (Fiber Science and Technology Research Institute No. 4230), (NRRLll, l82) (Unexamined Japanese Patent Publication No. 5
4-59202)].

The mycological properties of this species are described in Japanese Patent Publication No. 39155/1983. Culture for producing the antibiotic SUM-3 is carried out by the following method. That is, in culturing the microorganisms used in the present invention, a conventional method for culturing actiobacteria is generally used.

Various sources of nutrients can be used for culture. As the carbon source, glucose, starch, dextrin, mannose, flagose, 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 factory scratches, yeast factory scratches, dried yeast, corn, steep liquor, soy flour, Casamino acids, solid vegetable proteins, cottonseed gas, 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 the antibiotic SUM-3. . 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 400 and a pH near neutral. Liquid culture usually produces 1E1. When the culture is carried out for 12 days, the antibiotic SUM-3 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. The purification and isolation of the antibiotic SUM-3 from the culture fluid allows for the isolation of microbial metabolites.
The separation method commonly used to isolate from the culture fluid
Purification methods are utilized.

Since the antibiotic SUM-3 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. In addition, the free base of this substance is soluble in acetone, but the sulfate salt is poorly soluble in the same solvent.
Utilizing this point, the free base or sulfate of this substance can be isolated and purified. Next, antibiotic SU was added from the culture solution.
An example of purification/isolation of M-3 is shown.

After completion of the culture, the solids were removed from the culture solution, and the resulting culture solution was adjusted to be slightly alkaline, and then treated with cation exchange resin Amberlite RC-50 (manufactured by Hum & Haas, U.S.). .A.) (NH4 type 10) to adsorb the active substance, and after washing with water, the active substance is eluted with 2N aqueous ammonia.

The active fraction was collected and concentrated under reduced pressure, adjusted to be slightly alkaline, and passed through an Amberlite CG-50 Type 1 (manufactured by Rohm and Haas, USA) (NH4+) type to adsorb the active substance. urge 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 SUM-3 is eluted.

The active fraction containing antibiotic SUM-3 is collected and concentrated to dryness under reduced pressure to obtain a white powder of crude antibiotic SUM-3. Next, this is dissolved in water and adjusted to be slightly alkaline, passed through Biorex 70 (manufactured by Biorat Lab., USA) to be adsorbed, and after washing with water, the active fraction is eluted with dilute ammonia water. . After several trace components are eluted, the antibiotic SUM-3 is eluted. The purified antibiotic SUM-3 can be obtained by collecting and concentrating the fractions under reduced pressure, dissolving the residue in a small amount of water, and freeze-drying. Activity segment during the above purification process (antibiotic SUM-3
) trends are checked by ascending paper chromatography using Toyo P Kamini 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 16 hours. The compound of the present invention, antibiotic SUM-3, can also be chemically synthesized by the steps shown in Example 3 using antibiotic XK-62-2 (described in Sagamisin Japanese Patent Publication No. 50-139155) as a raw material.

That is, after protecting the amino groups other than the amino group at position 1 of antibiotic -3 can be synthesized. The method for producing the antibiotic SUM3 of the present invention will be specifically explained below with reference to Examples, but these are merely examples and do not limit the present invention in any way.

[Example 1] A. Cultivation of Micromonospora sagamiensis SU-2: Micromonospora sagamiensis (Mi
crOmOnOspOrasagamiensis)S
U-2 (Microtechnical Research Institute No. 4230NRRLll, l82
) was used.

As the first type medium, Stabilose K [Matsuya Chemical Co., Ltd.]
] 2% (W/) (same below), glucose 0.5%,
Peptone 0.5%, yeast scratches 0.5%, meat scratches 0.
A medium containing 3% calcium carbonate and 0.2% calcium carbonate (pH 8.0 before sterilization) was used.

A platinum loopful of seed culture 1 is inoculated into 10 d of the above medium in a large test tube, and cultured with shaking at 30° C. for 3 days. 10m of this seed culture solution
1 was inoculated into 350 ml of a second type medium placed in a 21-tube Erlenmeyer flask. The composition of the second type medium is the same as that of the first type medium. Type 2 culture is 3
Culture with shaking at 0°C for 2 days. 1.51 of this seed culture (for 5 flasks) was inoculated into 151 of the third type culture medium in a 301-capacity jar fermenter, and kept at 34°C for 24 hours using an aeration stirring method (rotation speed 250 rpm, aeration volume). 151/Mln)
Culture is performed by The composition of the third type medium is the same as that of the first type medium. Finally, add this third type culture solution 152 to 30
Fermentation medium 1501 in a 01 volume tank is inoculated. The fermentation medium composition is Stabilose K4%, soybean meal (
SOybeanmeal) 1%, Pharmamedia (P
(harmamedia) (TradersOilMil
Company made U. S. A. ) 2%, soybean casein 0
．． 5%, dipotassium phosphate 0.025%, magnesium sulfate 0.05%, calcium phytate 0.2%, corn oil (COrnOil) 0.1%, ferrous sulfate 0.01
5%, L-glutamine 0.0001%, L-cystine 0
．． 005%, β-alanine 0.005%, nicotinic acid 0
．． 0005%, calcium pantothenate 0,0005%
A medium containing 0.0005% zinc sulfate (7 hydrate), 0.001% potassium alum (24 hydrate), and 0.025% ammonium molybdate (4 hydrate) (pH 8.0 before sterilization) was used. . This fermentation was carried out at 30℃ for 3 days using an aerated agitation culture method (
The rotation speed is 180 rpm and the ventilation rate is 1501/min). B. Purification and isolation of antibiotic SUM-3 After the completion of the above culture, the pH of the culture solution was adjusted to PH2 with 12N monosulfuric acid.
The mixture was heated and stirred at 80°C for 10 minutes.

Thereafter, 2K7 of Radiolite+600 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added as a filtering agent, and the bacterial cells were separated. This deep liquid was used as Diaion HRK-25 (manufactured by Mitsubishi Kasei) (NH).
Pass it through a column packed with magazine type) 101, and discard the running water. After washing the resin with water, it is eluted with 2N aqueous ammonia, and the fraction containing the active substance is concentrated under reduced pressure to 301 ml. The concentrated solution was adjusted to pH 8. with 6N hydrochloric acid. After adjusting to O, it is passed through a column packed with Amberlite IRC-50 (NH4+ type) 41, washed with water, eluted with 2N aqueous ammonia, and the active fraction is dried under reduced pressure. Dissolve the residue in water, pH 8. After adjusting to O, Amberlight CG-50 Type I (NH4l
Pass through a column packed with type) 200Tn1, and after washing with water, 0.
Elute with 0.1N ammonia water containing 2M ammonium monochloride. First, several trace components are eluted, and then an active fraction containing this substance is eluted.
The fractions were collected and adjusted to pH 7.8 with 6N hydrochloric acid, passed through a column packed with 50 ml of Amberlite RC5O (NHj type), washed with water, and eluted with 1N aqueous ammonia.
The fraction containing the active substance was concentrated and lyophilized to give a crude powder of 56.3W1f. Dissolve this in a small amount of water and adjust the pH to 7.8.
Pass through a column packed with 10 ml of + type), and after washing with water, 0.
Elute with 0.06N aqueous ammonia containing 1M ammonium acetate. The eluted fractions are separated into 5a portions, the active fraction is detected by the method described above, and the fractions corresponding to antibiotic SUM-3 are collected. This fraction was adjusted to pH 7.7 with 6N hydrochloric acid,
Amberlight CG-50 Type I (NH4+ type) 10
A purified sample of the free base of the antibiotic SUM-3 is passed through a column packed with 5.5 mL of SUM-3, washed with water, eluted with 1-N' aqueous ammonia, and the active fraction is collected, concentrated under reduced pressure, and lyophilized. Goods were obtained.

Example 2 Antibiotic SUM-3 obtained in the same manner as Example 1
Dissolve 5 liters of free base in 2 liters of water and add 0.05N sulfuric acid.
．． Add 9T11.

By freeze-drying this solution, the antibiotic SUM-3
707 nv of sulphate powder was obtained. Example 3 Synthesis of antibiotic SUM-3: A. Preparation of l-N-t-butoxycarbonyl-2',6'-di-1N-benzyloxycarbonyl XK-62-2:
27,6'-di-N-benzyloxycarbonyl XK
-62-2 (described in JP-A-50-101336)
1.49 was dissolved in 20 ml of tetrahydrofuran and t
-Butyl-S-4,6-dimethylpyrimidin-2-ylthiocarbonate 480% was added and left at room temperature for 18 hours.

The reaction mixture was concentrated, 10 ml of chloroform was added to the residue to dissolve it, and silica gel (Kiese-1gel
60E. The same silica gel manufactured by Talc was used. ) Inner diameter 2,5 filled with 809? column. Elution is chloroform-methanol (10:1 volume ratio)
I went there. Fractions 64 to 94 were collected into fractions of 20 d each, and the solvent was distilled off to obtain a white powder of 960 η.

This powder exhibited the following properties and was identified as 1-N-t-butoxycarbonyl-2',6'-di-N-benzyloxycarbonyl XK-62-2. Yield 60.3%00・
Rf value of silica gel thin layer chromatography (hereinafter abbreviated as TLC) (Developing agent: chloroformrimethanol: 14%
Lower layer of asmonia water 4:1:1) 0.66○ Nuclear magnetic resonance absorption spectrum (methanol D4) δ (Ppm) 1.10 (3H, s), 1.43 (9H,
s), 2.53 (3H, s) 2.93 (3H, s), 5
．． 01 (4H, s), 7.27 (5H, s), 7.33
(5H,s)B. 3,2',6',3'L tetra-N-
Preparation of benzyloxycarbonyl XK-62-2: 1-N-t-butoxycarbonyl 21,6'-di-N-benzyloxycarbonyl-XK-62-2900mf obtained in the above step 680W9 (2.5 times mole) of N dissolved in tetrahydrofuran at room temperature
- Add benzyloxycarbonyloxysuccinimide and react for 8 hours with stirring.

The reaction mixture was concentrated to dryness, and the resulting pale yellow powder was
Dissolve LI in chloroform, add trifluoroacetic acid 5a, and leave at room temperature for 30 minutes. Concentrate the reaction mixture;
Add 100 ml of ethyl acetate to the residue, add 100 ml of saturated aqueous sodium hydrogen carbonate solution twice, and twice with 100 d of water.
After washing, the ethyl acetate layer is separated and the solvent is distilled off. Thereafter, the product was purified by silica gel column chromatography in the same manner as in Section A above, and fractions 12 to 50 were combined and the solvent was distilled off to obtain 840 THi white powder. This powder showed the following physical properties and was identified as 3,2',6',3''-tetraN-benzyloxycarbonyl XK-62-2.Yield 77.6% Rimethanol = 9:1) 0.37○
Nuclear magnetic resonance absorption spectrum (methanol D4) δ (Ppm) 1.02 (3H, s), 2,83 (3H,
s), 3.06 (3H, s), 5.07 (6H, s) 5
．． 13 (2H, s), 7.34 (20H, s)C. Synthesis of antibiotic SUM-3: 3 obtained by the above step B
, 2', 6', 3'' tetra-N-benzyloxycarbonyl XK62-2600 ~ in 50% acetic acid aqueous solution 1571
L1! and tetrahydrofuran 15d, and while cooling on ice, 30% sodium nitrite aqueous solution 3a was added dropwise, and stirring was continued at the same temperature for 1 hour.

The reaction mixture is poured into 1007nl of water and extracted twice with 100d of ethyl acetate. The ethyl acetate layer was washed with 100 ml of a saturated aqueous sodium bicarbonate solution and 100 ml of water and separated, and the solvent was distilled off. The residue is dissolved in 50W11 methanol, 0.5TfL1 of 2N hydrochloric acid and 100T11f of palladium carbonate catalyst are added, and reductive hydrogenolysis is carried out for 3 hours at room temperature and pressure while bubbling hydrogen. The solution obtained by filtering the catalyst was concentrated, and 50ml of water was added to the residue to give a 1N
Adjust the pH to 6 with sodium hydroxide. This aqueous solution is passed through a column with an inner diameter of 2.5 cm filled with Amberlite CG-50 (NH4+ type) 100a. Column with water 500ml
The fraction was washed with ml of water, then eluted with 0.2N ammonia water, and collected into fractions of 10Tn1 each. When fractions 20 to 46 are combined and ammonia water is distilled off, the result is 63W.
! A white powder of f was obtained. This powder completely matched the physical properties and biological activity of the antibiotic SUM-3 obtained by fermentation, and was identified as the antibiotic SUM-3.

(Yield 22.6%) In the above treatment, when fractions 50 to 72 were combined and the solvent was distilled off, a white powder of 105 mf was obtained, and this powder showed the following physical properties. -XK62-2 (1-epi-SUM-3).

(Yield 37.7%) (1) Silica gel TLC (7) Rf
Value developing agent: Chloroform, methanol, concentrated ammonia water (
1:1:1) (capacity ratio) lower layer RfC2:0,88 (2) Light intensity [α] Blue = +196.5 ((C=0.2, H20) (3
) Nuclear magnetic resonance absorption spectrum (heavy water) δ (Ppm) 1.
20 (3H, s) 1.3-1.8 (6H, m) 2,32
(3H, s) 2.50 (3H, s) 5,04 (1H, d
) 5.15 (1H, d) (4) C-13 nuclear magnetic resonance absorption spectrum (heavy water) (5) Mass spectrum (m/e
)464(M+),435,402,351,347,
334, 323, 306, 290, 289, 272, 1
92,160,143(6) Elemental analysis values, molecular weight, and molecular formula are the same as antibiotic SUM-3.

From the above, it was revealed that 1-epi mountain SUM-3 has the same planar structure as SUM 3.

1-Epi-SUM-3 antibacterial spectrum (MIC, γ/d).

Claims (1)

[Claims] 1 Antibiotic SUM-3 and its nontoxic acid addition salt represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2 Antibiotic SUM-3-producing bacteria belonging to the genus Micromonospora were cultured in a nutrient medium, and the antibiotic SUM-3 was added to the culture solution.
A method for producing antibiotic SUM-3, which comprises producing SUM-3 and collecting the antibiotic from the culture.