JPS5914039B2 - Houteimycin KO and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Horteimycin KO and its non-toxic acid addition salts and methods for producing the same.

Horteimycin KO is a compound with strong antibacterial activity against a wide range of Gram-positive bacteria5 and Gram-negative bacteria.
This is a new compound that is expected to be used as an antibiotic.

As for hortimycin compounds, hortimycin A (Japanese Unexamined Patent Publication No. 50-29789), hortimycin B (Japanese Unexamined Patent Publication No. 49-126892), and hortimycin C (Japanese Unexamined Patent Publication No. 52-18888) are available. It is known to be produced by microorganisms belonging to the genus Monospora.

Also, hortimycin D (patent application 128837/1983)
and 15 and hortimycin KE (patent application 1986-233)
8) is currently being filed by the present applicant. The present inventors discovered that microorganisms belonging to the genus Micromonospora further
The present invention was completed by discovering the production of a new compound named O, which has antibacterial activity. The present invention will be explained in detail below.

Horteimycin KO (free base) of the present invention has the following physicochemical properties.

(1) Properties: Basic white powder 25 (2) High resolution mass spectrum + 406.27↓4 (M+1) (C17H3606N5
), 235.1295 (C9H19O5N2), 207
．． 1364 (C8H1904N2), 200.1408
(C9H1802N3) 30189.1226 (C8H
1703N2)O This gives Horteimycin KO free base a molecular weight of 405 and a molecular formula of C17H35O6N5.

(3) Melting point: 130-132°C 35 (4) Ultraviolet absorption spectrum: 220-130°C for aqueous solution
It does not show a characteristic absorption maximum at 360 nm, but only shows terminal absorption.

(5) Specific rotation [α]=+93 H2O) (6) Infrared absorption spectrum: The results measured by the KBr tablet method are shown in FIG.

The wave number showing the absorption maximum (?-ri is 3480, 3380, 3
300, 1672, 1548, 1085, 1040, 1
010,909.

(7) Carbon nuclear nuclear magnetic resonance spectrum: heavy aqueous solution (PD-
The results measured in 10.6) are shown in Figure 2. Signal assignments are as follows: 175.7 (C=0), 99.5 (C-1′), 82.
8 (C-6), 82.4 (C-3), 74.8 (C-5
'), 73.3 (C-5), 72.4 (C2), 68.
4 (dioxane, internal standard substance), 60.4 (C-4)
, 57.7 (0CH3), 56.3 (C-1), 50.
6 (C-6'), 50.0 (C2'), 44.7 (glycine part CH2), 38.4 (NCH3), 26.8 (C
-4○, 23.6 (C3'), 18.2 (6'-CH3
) (8) Color reactions: Ninhydrin reaction, positive potassium permanganate reaction, positive Elson-Morgan reaction, negative Biuretz reaction, negative (9) From the above physicochemical properties, the three-dimensional structural formula of Horteimycin KO is estimated as follows.

Solubility in solvents: Extremely soluble in water, methanol, and slightly soluble in enonol and acetone, but chloroform, benzene, ethyl acetate, butyl acetate, ether, butiool, petroleum ether, n-hexane It is insoluble in organic solvents such as.

C Whatmanf. l(W&RBals
R at room temperature, development time 12 hours]
The f values are shown in Table 1.

Rf of an antibiotic considered to be close to Horteimycin KO
Values are also listed for comparison. (Own) Silica gel thin layer chromatography 1 [Merck & CO. Table 2 shows the Rf values of the products manufactured by the company (the same applies hereinafter) at room temperature and development time of 3 hours]. Other hortimycins are also listed for comparison. Next, Table 3 shows the antibacterial spectrum of Horteimycin KO against various microorganisms.

In Table 3, MIC is PH8. It was measured by the agar dilution method using O agar medium. In this way, Horteimycin K
O has strong antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria.

Among its wide-ranging and powerful antibacterial properties, a particularly distinctive feature is that it is effective against certain types of Escherichia coli that are resistant to kanamycin, diemmemycin, tobramycin, and the like. It has also been found that the present hortimycin KO exhibits extremely excellent therapeutic effects against various infectious diseases (in humans and animals) caused by the various bacteria mentioned above. The extremely excellent antibacterial properties of Horteimycin KO indicate that this substance can be particularly applied for medical purposes as an antibacterial antibiotic. Next, let's compare this substance with known antibiotics.

Dienmemycin Complex (M.J. weinstei) is a water-soluble basic antibacterial substance with a broad antibacterial spectrum produced by Micromonospora.
n et al.: Antimicrobial Agents and
ChemOtherapy, 1963, 1 and D. J.
COOper et al., J. nfect. Dis. ll9,3
42, 1969, J. A. Waitz et al., Antimi
crOb. Ag. ChemOth. 2, 464, 197
2), Antibiotic Black 460 (Special Publication 1976-16)
No. 153), Sisomicin (M.J. Weinst
ein et al., J. AntiblOtics23, 551,
555, 559, 1970),
-126892), Horteimycin A (JP-A-1983-126892)
29789), Horteimycin C (JP-A-52-18)
888), hortimycin D (patent application 1988-1288)
37), Horteimycin KE (patent application 1982-2338)
) and other antibiotics. First, when comparing each component of diemmemycin, as shown in Table 1, diemmemycin A, B. cla. The Rf values of c2 and C1 are 0.00, 0.00, 0.18, 0.38, 0.5, respectively.
9, whereas the Horteimycin KOf)Rf value was 0.48, which is clearly different from these. In addition, Antibiotic Black 460 and Sisomicin have Rf values of 0.01 and 0.18, which are clearly different from Horteimycin KO. In addition, each component of Horteimycin A. B.. C,. D and KEf) Rf values are 0.37, 0.65, 0.18, 0.18, 0.
59, and these also have different Rf values from Horteimycin KO. However, the Rf value of XK-622 was 0.49, which was similar to the Rf value of Horteimycin KO, 0.48, but as a result of silica gel thin layer chromatography shown in Table 2, the developer 1''The Rf value of XK-62-2 is 05
On the other hand, Horteimycin KO is a 0.501 developing agent, and the Rf value of XK62-2 is 0.10, whereas Horteimycin KO has a different Rf value of 0.30, and Horteimycin KO has a different Rf value. It is clear that it is a different substance from XK-62-2. Furthermore, streptomycin, a water-soluble basic antibiotic with a broad antibacterial spectrum produced by actinomycetes other than Micromonospora,
Examples include ribosmemycin, lividomycin, spectinomycin, kasugamycin, neomycin, kanamycin, nebramycin, paromomycin, etc., but horteimycin KO is different from any of these antibiotics in terms of physicochemical properties and the list shown in Table 1. It is obvious that the paper chromatographs have different Rf values.

From the above, Horteimycin KO is considered to be a new antibiotic. Next, an example of a method for producing the compound hortimycin KO of the present invention will be shown.

A microorganism belonging to the genus Micromonospora and having the ability to produce Horteimycin KO is cultured in a medium, Horteimycin KO is accumulated in the culture, and Horteimycin KO is collected from the culture.

As the microorganism used, any strain can be used as long as it belongs to the genus Micromonospora and can produce and accumulate Horteimycin KO in the medium.
A specific preferred example is Micromonospora oliboasterospora MK7O (Feikoken Bibori No. 1560) (A
TCC2l8l9), Micromonospora oliboasterospora MK8O (Feikoken Bibori No. 2192) (ATC
C3lOlO), Micromonospora oliboasterospora Mm744 (Feikoken Bibori No. 2193) (ATCC
3lOO9). The mycological properties of these strains are specified in JP-A-50-29789.

As the medium, a medium commonly used for culturing actinomycetes is used.

As the carbon source, glucose, starch, mannose, flagose, shakerose, molasses, etc. are used alone or in combination.

Furthermore, depending on the assimilation ability of the bacteria, hydrocarbons, alcohols, organic acids, etc. may also be used. 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, etc.
Steep liquor, soybean flour, casamino acids, soluble
Vegetable protein and the like are used alone or in combination. In addition to adding inorganic salts such as common salt, potassium chloride, calcium carbonate, and phosphates as necessary, organic or inorganic substances that promote the growth of the strain used and the production of Horteimycin KO can be added as appropriate. As a culture method, a liquid culture method, especially a deep agitation culture method, is suitable.

It is desirable that the culture be carried out at a culture temperature of 25 to 40°C and a pH near neutral. When culture is carried out in liquid culture for usually 2 to 15 days, horteimycin KO is produced and accumulated in the culture solution. When the production amount of the target substance in the culture solution reaches the maximum, the culture is stopped, and the target substance is purified and isolated from the culture solution. Purification and isolation of Horteimycin KO is carried out as follows.

For the purification and isolation of Horteimycin KO from the culture fluid, separation and purification methods commonly used for isolating microbial metabolic products from the culture fluid are utilized.

Since Horteimycin KO is a basic substance that is well soluble in water but not well soluble in general organic solvents, it is purified by a method that is often used for the purification of so-called water-soluble basic antibiotics.

Namely, adsorption/desorption method using cation exchange resin, column chromatography of cellulose, Sephadex LH
Adsorption/desorption using a 2O column, silica gel column chromatography, and other methods can be used in appropriate combination. Next, one example of a specific purification method for Horteimycin KO will be shown.

That is, after adjusting the culture broth to pH 7.5, cation exchange resin Amberlite IRC-50 (ammonia type) (
ROllm&HaasCO. (manufactured by) and after washing with water,
Elute with 0.5N aqueous ammonia. After collecting the active fractions and concentrating them under reduced pressure, the concentrated liquid was treated with cation exchange resin Amberlite C.
G-50 (ammonia type) (ROhnl & HaasCO
．． After washing with water, it is eluted with 0.1N ammonia water containing 0.1M NH4C1. At this time, Horteimycin KO is first eluted, and other components Horteimycin A, . BlC. ．． D. ．． KE elutes later.
Fractions containing Horteimycin KO were collected, neutralized with hydrochloric acid, adsorbed on Amberlite IRC-50 (ammonia type), washed with water, eluted with 0.5N ammonia water, and the active fractions were collected and concentrated to dryness. Then, a coarse powder of Horteimycin KO is obtained. This crude powder was placed on a silica gel column, and the column was developed and eluted with a lower layer of chloroform, menol, and aqueous ammonia (2:1:1). In this case, the mixed hortimycin B is eluted first, and then the hortimycin K is eluted.
O is eluted. The white free base of Horteimycin KO can be obtained by collecting the fractions containing Horteimycin KO, concentrating under reduced pressure, and freeze-drying. In this way, it is possible to obtain fairly clean Horteimycin KO using silica gel column chromatography, but since it contains some impurities, it is necessary to obtain BioRex 70 (ammonium type) (BiO-RadLabOrat).
After washing with water, it is eluted with 0.04N ammonia water containing 0.04M ammonium acetate.
Fractions containing Horteimycin KO are collected, neutralized, adsorbed on Amberlite CG-50 (ammonia type), washed with water, eluted with 0.5N ammonia water, active fractions are collected, concentrated, and freeze-dried. By doing so, Horteimycin KO
A pure preparation (free base) can be obtained.

Column chromatography using carboxymethylcellulose is also effective for removing mixed ninhydrin-positive substances. That is, in this case, a solution of coarse powder is passed through a column filled with carboxymethyl cellulose (ammonia type) to adsorb the active substance. Thereafter, it is thoroughly washed with water to elute most of the dye and inorganic salt, and then the active substance is eluted by elution with 0.2M citric acid/phosphate buffer (PH3.O). Purified Horteimycin KO can be obtained by collecting fractions containing Horteimycin KO and freeze-drying them. The trend of the active fraction of Horteimycin KO during the above purification process was checked by silica gel thin layer chromatography.

As developing solvents, the lower layer of chloroform-memenol-aqueous ammonia (2:1:1) and the lower layer of 10% aqueous ammonium acetate solution memenol-aqueous ammonia (50:50:1) were used.
), and detection was performed using the ninhydrin color method and Bacillus subtilis X). The test was carried out using a bioautograph method using 10707 as the test bacterium. The Rf values of this silica gel thin layer chromatography are shown in Table 2. The non-toxic acid addition salt of Horteimycin KO is Horteimycin K.
Mono-, di-, tri-, tetra-,
Meaning penta-salt.

Examples of acids include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, carbonic acid, nitric acid, acetic acid, fumaric acid, malic acid, citric acid, mandelic acid, ascorbic acid, tartaric acid, succinic acid, etc. of organic acids.

Hereinafter, the production of the compounds of the present invention will be shown in Examples, but these are merely illustrative examples and do not limit the present invention in any way.

Example 1 A. Micromonospora oliboasterospora (
MicrOmOnOspOraOllvOasterO
spOra) MK-70 (ATCC2l8l9) (Feikoken Bacteria No. 1560), glucose 2t/dl, peptone 0.5y/dl, yeast engineering scratches 0 as the medium
．． 57/Dll calcium carbonate 0.17/dl (pH 7.5 before sterilization) medium was used.

A loopful of the inoculum is inoculated into 10 ml of the above medium in one 50 ml large test tube, and cultured at 30° C. for 5 days. 10 ml of this seed culture solution is inoculated into 30 ml of second seed medium placed in a 250 ml Erlenmeyer flask. The composition of the second type medium is the same as that of the first type medium. Type 2 culture is 30
Incubate with shaking at ℃ for 2 days. 21 ml of this seed culture
300 in an Erlenmeyer flask with a battleship
Inoculate the tertiary medium of m1. The composition of the third type medium is the same as that of the first type medium. The third type culture is cultured with shaking at 30°C for 2 days. This third type medium 1.51 (flask 5
151 of the fourth type medium in a 301-capacity stainless steel jar. The composition of the fourth type medium is the same as the composition of the first type medium.

This culture was incubated at 37°C for 2 days.
Aeration stirring method (rotation speed 350 rpm aeration amount 151/MO
This is done by This fourth type culture solution 151 was mixed with 3001 volumes of Fertan Shioichi medium fermentation medium 150. Inoculate e.
The fermentation medium composition was 47/dl soluble starch, 27/dl soy bean meal, 17/dl cornstarch.
．． K2HPO4O. O57/DjlMgSO4・7H2
00.05f7/Dl. ．． KClO. O37/Dl. C
aCO3O. A medium of 17/dl (pH 7.5 before sterilization) was used. This fermentation culture was carried out at 37°C for 4 days with aeration and stirring (rotation speed: 150 rpm, aeration volume: 80f!/Mi!).
L). S. After isolation and purification of Horteimycin KO, the pH of the culture solution was adjusted to pH by adding concentrated sulfuric acid.
Adjust to H25 and stir for 30 minutes.

Thereafter, about 7 kg of Radiolite #600 (manufactured by Showa Kagaku Kogyo) was added as a filter aid to remove the bacterial cells. Add 6N sodium monohydroxide to the slurry to adjust the pH to 7.5. Use this sap with cation exchange resin Amberlite IR.
Pass through a column packed with approximately 201 g of C-50 (ammonia type) and discard the effluent. Since the active substance is adsorbed on the resin, after washing the resin with water, the adsorbed active substance is eluted with 0.5N aqueous ammonia. The activity of the eluate is measured by the paper disk method using an agar plate of Bacillus subtilis black 10707. The active fractions are combined and concentrated under reduced pressure to ca. This concentrated solution was adjusted to pH 7 with concentrated hydrochloric acid, and then passed through a column of Amberlite CG-50 (ammonia type) 31.

The active substance is adsorbed, so after washing with water, add 0.1M NH4C.
Elute with 0.1N NH40H containing 1. 50% of the eluate
The components in each fraction were analyzed for antibacterial activity against Bacillus subtilis black 10707 (KY4273) and silica gel thin layer chromatography. Underlayer,
It was investigated using ninhydrin coloring.

Horteimycin KO is eluted at about 101 points. Fractions containing Horteimycin KO were collected, concentrated under reduced pressure to remove ammonia, neutralized, passed through a column containing 500ml of Amberlite RC-50 (ammonium type), washed with water, and the active substance was removed with 0.5N ammonia. It was eluted with water, concentrated under reduced pressure, and lyophilized to obtain a white powder of about 1y. The activity was 920 units/η. (Activity is defined as 1000 units of pure product 1η). C. Preparation of pure Horteimycin KO The crude Horteimycin KO preparation 17 obtained in the above step B was placed on 11 silica gel columns (silica gel, manufactured by Wako Pure Chemical Industries, Ltd., column diameter approximately 3 CTrL), and chloroform-menonol-ammonia was added. Develop and elute with the lower layer of water (2:1:1).

The eluate was fractionated into 20ml portions, the components in each fraction were examined using silica gel thin layer chromatography, and the fractions containing Horteimycin KO were collected, concentrated under reduced pressure, and lyophilized.
Approximately 500 mg of white powder was obtained. The activity was 980 units/η. The obtained hortimycin KO5OO was dissolved in water to adjust the pH to 7, adsorbed on 50 ml of Biolex 70 (ammonium type), washed with water, and eluted with 0.04N ammonia water containing 0.04M ammonium acetate.

The eluate was fractionated into 10ml portions, and each fraction was subjected to silica gel thin layer chromatography.
Fractions containing only O were collected, ammonia was removed by vacuum concentration, the pH was adjusted to 7, and the Amberlite IRC
-50 (ammonia type) is adsorbed onto 100 ml of water, washed with water, and eluted with 0.5N ammonia water. Collect the active fraction,
After vacuum concentration and final lyophilization, 380 η of pure Horteimycin KO free base was obtained as a white powder. Example 2 A culture solution obtained by culturing in the same manner as in Section A of Example 1 is treated in the same manner as in Section B of Example 1.

The obtained coarse powder 500W19 was dissolved in 5WL1 of water and about 200m of carboxymethyl cellulose (ammonia type) was dissolved.
Pass through a column packed with 1. When approximately 1000 ml of water is subsequently flushed, the active ingredient is adsorbed onto the carboxymethylcellulose, and most of the dyes and inorganic salts that are not adsorbed onto the carboxymethylcellulose are washed out. Then 0.
Elute the column with 2M citric acid/phosphate buffer (PH3.O) (flow rate approximately 50 ml/hour). The eluted solution is collected in fractions of 10 ml each, and the activity of each fraction is assayed by the paper disc method. Perform silica gel thin layer chromatography on the active fraction,
Fractions containing components corresponding to Horteimycin KO are collected. This fraction is passed through a 50 ml column of Amberlite IRC-50 (ammonium type) to adsorb the active ingredient. After washing with water, elute with 0.5N ammonia water,
By lyophilizing the active fraction, approximately 300 η of Horteimycin KO free base can be obtained. The activity was 980 units/η. Example 3 Micromonospora oliboasterosporha Tsukuda 744, KYllO67 strain (Feikoken Bibori No. 2193,
ATCC31OO9) was used as the first type medium, glucose 2y/dl, peptone 0.57/dl, yeast scratches 0.37/dl, calcium carbonate 0.1y/dl (
A medium with a pH of 7.2) before sterilization was used.

Seed culture (first type culture ~
Type 4 culture) was performed. The compositions of the second type medium to the fourth type medium are all the same as the composition of the tenth type medium. The fourth type culture solution 151 thus obtained was heated to 300. The main fermentation medium 1501 in an e-capacity stainless steel fermenter is inoculated. Main fermentation medium composition: soluble starch 27/dl, soybean meal 0.5y/dll, glucose 2t/dl
l Corn stapler 1st floor/Dll yeast engineering scratches 17
/Dl,. K2HPO4O. O57/Dl,. MgSO
47H2OO. O5t/Dl. KClO. O3t/Dl
．． ．． caCO3O. A medium of 17/dl (pH 7.0 before sterilization) is used. This main fermentation culture was carried out at 30° C. for 4 days using an aeration stirring method (rotation speed: 150 rpm, aeration rate: 801/Mm). The main fermentation liquid thus obtained was used in Example 1 and B.
When horteimycin KO crude powder was isolated according to the method shown in section 1, about 800 m9 of crude horteimycin KO having an activity of about 900 units/watt was obtained. This crude powder was purified in the same manner as shown in Example 1, Section C, to yield ~250~ of Horteimycin KO free base with an activity of ~980 units/~. Example 4 Micromonospora oliboathrospora MK as seed fungus
8O,. KYllO55 strain (Feikoken Bibori No. 2192,
ATCC 3lOlO) was used as the first type medium, glucose 1y/dl, soluble starch 17/Dll yeast scratch 0.5y/Dll peptone 0.57/Dll calcium carbonate 0.17/dl (PH7.O before sterilization) A medium was used.

Seed culture (first type culture ~
Type 4 culture) was performed. The compositions of the second type medium to the fourth type medium were all the same as the composition of the first type medium of this example. The thus obtained type 4 culture solution was subjected to main fermentation culture in the same manner as shown in Example 3. The main fermentation medium composition was the same as the main fermentation medium shown in Example 3. The main fermentation culture solution thus obtained was used in Example 1 and B.
When the crude hortimycin KO powder was isolated by the same method as described in section 1, crude hortimycin KO powder with an activity of about 910 units/η was obtained.

When this crude horteimycin KO powder was purified in the same manner as shown in Example 1, Section C, a purified sample containing about 300 η of horteimycin KO free base was obtained. The activity of this purified Horteimycin KO was about 990 units/~. Example 5 Horteimycin KO free base 1V was dissolved in a small amount of water and after adjusting the pH to 4.5 using 6N monosulfuric acid, the
By adding 0 times the amount of acetone, Horteimycin KO sulfate precipitates.

The precipitate was collected in a centrifuge and dried to obtain a white powder of about 1.57 Horteimycin KO sulfate. Activity is 6
It was 20 units/M9.

[Brief explanation of the drawing]

Figure 1 shows the infrared absorption spectrum of Horteimycin KO, and Figure 2 shows the carbon nuclear magnetic resonance spectrum of Horteimycin KO.

Claims (1)

[Claims] 1 Horteimycin KO and its non-toxic acid addition salt represented by the formula ▲ Numerical formula, chemical formula, table, etc. ▼. 2 Belongs to the genus Micromonospora, Horteimycin KO
A method for producing horteimycin KO, which comprises culturing a microorganism capable of producing it in a medium, accumulating horteimycin KO in the culture, and collecting horteimycin KO from the culture.