JPS5934356B2 - New substance D-53 and its manufacturing method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel substance D-53 and its acid addition salts and processes for their production.

The features of the present invention reside in the novel substance D-53, which has strong antibacterial activity against a wide range of Gram-positive and -negative bacteria, and its production method using microorganisms. The present inventors conducted various studies with the aim of developing new useful substances.

As a result, the present inventors discovered that in the culture of Micromonospora sagamiensis KG-52, a substance that behaves differently in various chromatographic assays than known antibiotics accumulates. The material was then purified and isolated from the culture and named D-53. As a result of analysis, D-53 was found to have the following physical and chemical properties. (1) Basic white powder (2) Elemental analysis value (%) C5O. l5H 8.82Nl4. 88 (3) Molecular weight 447 (4) Molecular formula Cl7H37N,O7 (51 Ultraviolet absorption Svectori) The ultraviolet absorption spectrum of an aqueous solution of this substance does not show a characteristic absorption maximum between 230 and 360 nm and only shows terminal absorption. be.

(6) 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.

(7) Color reaction Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Elson-Morrigan reaction: Negative Biuret reaction: Negative (8) Mass vector reactivity The mass spectrum of this substance gives the following M+{on and fragment ions. .

However, the composition formula obtained by accurate mass measurement is shown in parentheses. RrL/E447M+ (Cl, H37N5O7), 4
30(Cl9H34N4O7)348(Cl4H28N
4O6)9336(Cl3H26N3O7)318(C
l3H24N3O6), 308(Cl2H26N3O6
)290(Cl2H24N3O,),203(C8Hl
7N3O3) 191(C7Hl5N2O3), 163(
C6Hl5N2O3)146(C6H,2NO3),1
45(C6Hl3N2O2) 141(C7Hl3N2O
) From this, the molecular weight of this substance is 447, and the molecular formula is Cl,H.
It was determined to be 37N5O7.

In addition, the elemental analysis values of this substance (monohydrate) obtained from this molecular formula (theoretical values of (2) are C: 49.02, H: 8.4
4,N: 15,04. (9) PM of PMR Svectory substance in heavy water solution (hydrochloric acid acidic POO.8)
The R spectrum gives the following chemical shift values.

(Unit is fermentation) δ 1.29 (3H, s), 1.60-2.70 (4H
, m) 2.74 (3H, s), 3.30-430 (12
H, m) 5.11 (1H, d, J = 3.5), 5.31
(1H, t, J=4.0), 5.70 (1H, d, J=
1.8) Based on the above physical and chemical properties, this substance is considered to have the structure shown by the formula.

(3) The Rf value of paper chromatography and thin layer chromatography using various developing agents for this substance is as follows:
~As shown in Table 3. For comparison with known substances, we select substances that are considered to be closely related, and also record their Rf values. [Table 1] R of D-53 in ascending paper chromatography (using Toyo Sake Paper 51 as paper and developing at 28°C)
f value [Table 2] Silica gel thin layer chromatography using a lower layer of chloroform, methanol, and concentrated ammonia water (1:1:1) (volume ratio) as developing agents [Kies manufactured by E. Merck]
elGel6O (performed at room temperature).

RfCl value 8 at a developing time of 3 hours (Table 3) Rising paper chromatography using a lower layer of chloroform, methanol and 17% aqueous ammonia (2:1:1) (volume ratio) as developing agents. 1 (Carry out at room temperature.

Rf value at a development time of 12 hours) This substance exhibits extremely strong antibacterial activity against a wide range of Gram-positive and Gram-negative bacteria.

Among its wide-ranging and powerful antibacterial properties, the most distinctive feature is
This substance is extremely effective against bacteria of the genus Proteus and Pseudomonas, for which there have been few effective antibiotics, and it is effective against these bacteria at concentrations of 0.1 μ7/ml to 10.0 μ7/ml shows antibacterial activity. Examples of bacterial species that are susceptible to this substance include Bacillus Staphylococcus aureus 209p, Bacillus subtilis ATCC
6633 Escherichia coli NIHJC-2, Klebscilla pneumonia 8045, Pseudomonas aeruginosa +1, Proteus mirabilis 1287, Serratia marsetuscens T-55, and the like. Furthermore, this substance is effective against Staphylocotcus aureus and Pseudomonas aeruginosa bacteria that are resistant to various known antibiotics, such as Staphylococcus aureus and Pseudomonas aeruginosa, which produce aminoglycoside nucleotidyltransferase [ANT (4●)]. Irokotsucus aureus KY897O
and aminoglycoside acetyltransferase [AA
It has strong antibacterial activity against Pseudomonas aerugisosa KY851O, which produces C(6')]. As mentioned above, D-53, which has excellent antibacterial properties,
It is medicinally useful as an antibacterial agent. Additionally, D-53 can be used as a disinfectant for hygienic purposes, such as sterilizing laboratory glassware and equipment. A comparison of this substance having the above-mentioned physical and chemical properties with other substances is as follows.

An example of an antibiotic produced by a microorganism belonging to the genus Micromonospora that is water-soluble, basic, and has a wide range of antibacterial vectors is dientamycin Complex (
M Weinstein et al., Antimicr-0b.
Ag. Chen]0ther. 1963, 1 and D.
J. COOperi/)J. nfect. Dis. U, 342, 1969, J-A-Weitz et al. Antim
lcrOb●Ag. ChemOther. 2,464,
1972) Anti-biochicken boiled 460 (Special Publication 1972-
16153), Sisomicin (M.J. Wein
Stein et al., J. AntibiOtics4th, 55th
1,555,559, 1970), berdamycin (M
．． J. Weinstein et al., AntimicrOb-
Ag-ChemOther, 7, 246, 1975),
Antibiotic G-52 (J.A. Marquez
et al., J. AntiblOtics,? 4,483,19
76 and P. J. L. Daniels et al., J. Ant
iblOtlcs, 24, 488, l976), XK-
62-2 (Japanese Patent Publication No. 50-39155), Horteimycin A (Japanese Patent Publication No. 51-45675), Horteimycin C (Japanese Unexamined Patent Publication No. 18888/1988), Horteimycin D (Patent Application No. 18888/1988) 51-128838), XK-62-3 (described in Japanese Patent Application No. 57827, 1983),
8) SU-2 (Japanese Patent Application No. 52-12403)
There are antibiotics such as (described in specification No. 0).

However, as shown in Tables 1, 2, and 3, D-53 differs from any of the above in terms of paper chromatography and thin layer chromatography behavior.

Also, recently reported 31-N-demethylsisomicin (
T. L. Nagabhus Han et al., J. Antib
lOtics, 3l, 43, l978 and M. Kug
Elman et al. J. AntiblOtics, 3l, 64
3, 1978) can be clearly distinguished from the mass spectrum, PMR spectrum, and elemental analysis data. In addition, water-soluble, basic, and broad-spectrum antibacterial antibiotics produced by actinomycetes other than microorganisms belonging to the genus Micromonosvora include streptomycin, ribostamicin, ribidomycin, neomycin, kanamycin, paromomycin, and nebula. Examples include mycin, but as is clear from Table 3, D-53 has Rf
It is distinguished from these antibiotics by its value. Furthermore, from mass spectrum and PMR spectrum data, D-
53 is clearly distinguished from other antibiotics. Based on the above, D-53 is considered to be a novel antibiotic.

Next, the method for producing D-53 in the present invention will be explained.

D-53 is obtained by culturing D-53-producing microorganisms belonging to the genus Micromonospora in a nutrient medium, producing and accumulating D-53 in the culture, and collecting the substance from the culture. be able to.

As the microorganism used in the present invention, any strain that belongs to the genus Micromonospora and has the ability to produce D-53 can be used, and specific examples include Micromonospora.
sagamiensis KG-52 (Feikoken Bokuyori No. 4456) (
NRRL-11290). The mycological properties of this species are described in Japanese Patent Publication No. 39155/1983. Culture for producing D-53 is carried out by the following method. That is, in culturing the microorganism in the present invention, a normal method for culturing actinomycetes is generally used.

Various sources of nutrients can be used for culture. Carbon sources include glucose, starch, dextrin,
Mannose, flagose, shakerose, 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 protein, cottonseed gas, etc. are used alone or in combination. In addition, in addition to appropriately adding inorganic salts such as table salt, potassium chloride, calcium carbonate, and phosphates as necessary,
Organic or inorganic substances that promote the production of -53 can be appropriately added. As a culture method, a liquid culture method, particularly a deep aeration agitation culture method, is suitable. The culture temperature is
It is desirable to culture at 25° C. to 40° C. and pH around neutral. D-53 is accumulated in the culture solution when the culture is carried out in liquid culture for usually 1 to 12 days. In the culture solution, D-5
When the accumulation of No. 3 is confirmed, preferably when the accumulated amount reaches a large amount, the culture is stopped and the target product is purified and isolated from the culture solution. For the purification and isolation of D-53 from the culture solution, separation and purification methods commonly used for isolating microbial metabolites from the culture solution are utilized. Since D-53G is a water-soluble basic substance as mentioned above, it can be purified by a method commonly used for purifying water-soluble basic substances.

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 has the ability to dissolve in acetone (sulfate is difficult to dissolve in the same solvent, so
Utilizing this point, the free base or sulfate of this substance can be isolated and purified. Next, an example of purification and isolation of D-53 from a culture solution will be shown.

After the completion of the culture, the solid matter was removed from the culture solution, and the obtained culture solution was adjusted to be slightly alkaline, and then treated with cation exchange resin Amberlite IRC-50 (manufactured by Kuchim & Haas, USA). ) (NH; type) to adsorb the active substance, and after washing with water, the active substance is eluted with 2N aqueous ammonia.

The active fraction was collected, concentrated under reduced pressure, and adjusted to slightly alkaline.
This is passed through Amberlite CG-50 Type I (manufactured by Rohm and Haas, USA) (NH') to adsorb the active substance. After washing with water, the active substance is eluted with dilute ammonia water. After several trace components were eluted, D-53
is eluted. The active fraction containing D-53 is collected and concentrated to dryness under reduced pressure to obtain a pale yellow powder of D-53. Next, dissolve this in a small amount of water and use silica gel (Wakogel C-
200 (manufactured by Wako Pure Chemical Industries, Ltd.) to adsorb it through a column filled with chloroform, iso-propanol,
The active substance is eluted using a lower layer of concentrated aqueous ammonia (volume ratio 2:1:1). After several trace components were eluted, D-
53 is eluted. The active fraction containing D-53 is collected and concentrated to dryness under reduced pressure to obtain a white powder of D-53. Next, this is dissolved in water, adjusted to be slightly alkaline, passed through Biorex 70 (manufactured by Biorat Lab., USA), and adsorbed. After washing with water, the active substance is eluted with dilute ammonia water. do. After several trace components are eluted, D-53 is eluted. Purified D-53 can be obtained by collecting and concentrating the fractions under reduced pressure, dissolving the residue in a small amount of water, and freeze-drying. The trend of the active substance (D-53) during the above purification process is checked by ascending paper chromatography using Toyofuchi Paper Ni 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. D-53 thus obtained
produces mono-, tri-, tetra-, and penta-salts by reacting D-53-1 molecule with 1 to 5 equivalents of acid,
These acid addition salts of D-53 are also target substances in the present invention.

In this case, the acid addition salts include hydrochloride, hydrobromide,
Inorganic acid salts such as hydroiodide sulfate, phosphate, carbonate, nitrate, acetate, fumarate, malic acid citrate, mandelate, ascorbate, tartrate, succinate Examples include organic acid salts such as D
A specific example of the method for producing the acid addition salt of -53 will be described in Examples below. The following examples show that D of the present invention
The method for producing -53 and its acid addition salt will be specifically explained.

Example 1 A. Cultivation of Micromonosvora sagamiensis KG-52: Micromonosvora sagamiensis (M
IcrOmOnOspOrasagamjensis)
K to 52 (Microtechnical Research Institute No. 4456) (NRRL-1112
90) is used.

As the first type medium, Stabilose K [Matsuya Chemical Co., Ltd.]
[manufactured] 27/Dt, glucose 0.57/Dt peptone 0
．． 57/Dt, yeast scratches 0.57/Dt meat scratches 0.
39/dl, calcium carbonate 0.2L x u (PH before sterilization
8. Use the medium of O).

Inoculum 1 platinum loop was inoculated into 10 mt of the above medium in a large test tube, and cultured with shaking at 3'C for 3 days. This seed culture solution 10
mf. was inoculated into 350 ml of a second type medium having the same composition as the first type medium, which was placed in an Erlenmeyer flask with a 2-t buffer. The second type culture is cultured with shaking at 30°C for 2 days. 30 tons of this seed culture solution (1.5 tons (5 flasks))
The cells are inoculated into 15 t of a third type medium having the same composition as the first type medium in a Jia Huamenta, and cultured at 34° C. for 24 hours using an aeration stirring method (rotation speed: 250 rpm, aeration rate: 15 t/Mm).

Finally, 15 t of this third type culture solution is inoculated into 150 t of fermentation medium in a 300 t capacity tank. The fermentation medium composition was shakerose 57/Dtl soy bean meal (SOyb
-Eanmeal) 17/Dt, Pharmamedia (P
harmamedia) (cottonseed meal) (TradersO
Made by ilmil Company. S. A) 37/Dt,
Soybean casein 0.57/d of dipotassium phosphate 0.002
5'Ii/Dt. Magnesium sulfate (heptahydrate) 0.05
7/Dt, Calcium Fitch 0.27/DL Corn Oil (COrnOil) O, 1V/D4 Ferrous Sulfate (7
water salt) 0.0157/D4L-glutamine 0.0001
7/D4L-cystine 0.0057/Dt, β-alanine 0.0057/Dt. Nicotinic acid 0.00057/D
Zinc 4-sulfate (heptahydrate) 0.0005y/Dt. A culture medium of potassium alum (24 Water Co., Ltd.) 0.0017/Dt. Ammonium molybdate (tetrahydrate) 0.025y/Dt (PH8O before sterilization) is used. This fermentation is carried out at 30°C for 6 days using an aerated agitation culture method. (Rotation speed 180rpII1, ventilation amount 1
50t/Mm). B. Purification and isolation of D-53 After the above culture, the pH of the culture solution was adjusted to 2 with 12N monosulfuric acid.
The mixture was heated and stirred at 80°C for 10 minutes.

After that, as a filter aid, Radiolight≠600 [Showa Chemical Industry? Add 2k9 of [Made in Japan] and separate the bacterial cells. This liquid is passed through a column packed with 10 tons of Diaion HPK-25 (strong acid meter cation exchange resin) (Mitsubishi Kasei? (Nl-[: type)], and the effluent is discarded. After washing the resin with water, 2N-
Elute with aqueous ammonia, collect the fraction containing the active substance, and concentrate under reduced pressure to 30t. The concentrated solution was adjusted to pH 8 with 6N hydrochloric acid.
．． After adjusting to O, Amberlight IRC-50 (N
Pass through a column packed with HZ type) 4t, wash with water, elute the active substance with 2N ammonia water, collect the active substance fraction, and dry under reduced pressure. Dissolve the residue in water, pH 8. After adjusting to O and 2, Amberlight CG-50 type (NH4 type)
Pass through a column packed with 500 ml of water, wash with water, and elute with 0.1N ammonium water containing 0.2M ammonium monochloride.

After a few trace components are eluted at the beginning, the elution volume is 5.
D-53 is eluted in the section from 0t to 9.5t.
Fractions containing D-53 were collected and adjusted to pH 7.8 with 6N monohydrochloric acid.
After adjustment, Amberlight IRC-50 (NHZ type) 1
The column was passed through a column packed with 00 ml of water, washed with water, and then eluted with 2N aqueous ammonia. The fraction containing the active substance is concentrated and freeze-dried to obtain a powder 1047. After dissolving this in a small amount of water and adsorbing it through a column packed with silica gel 5007, the active substance is eluted using the lower layer of chloro[]form, isopropanol, and concentrated aqueous ammonia (volume ratio 2:1:1). . After several trace components are eluted, D-53 is eluted in the elution volume range of 6.5 t to 9.0 t. The active fraction containing D-53 is collected and concentrated to dryness under reduced pressure. The residue was dissolved in water and adjusted to pH 8.0, passed through a column packed with 50 ml of Amberlite IRC-50 (NH+4 type), washed with water, and eluted with 2N aqueous ammonia. The fraction containing the active substance is concentrated and freeze-dried to give a pale yellow powder. Dissolve this in a small amount of water and adjust the pH to 7.6, then Biorex 70 (weakly acidic cation exchange resin) (BioRat, USA) (NH?
Pass through a column packed with 20ml of 0.1M
Elute with 0.06N aqueous ammonia containing ammonium acetate.

The eluted fraction is collected in 10 ml portions, the active fraction is detected by the method described above, and the fractions containing D-53 are collected. D-53 elutes in the elution volume category of 1.8t to 24t. This fraction was adjusted to pH 7.8 with 6N monohydrochloric acid, passed through a column packed with 10ml of Amberlite CG-50 Type I (NHZ type), washed with water, eluted with 2N aqueous ammonia, and the active fraction was collected and vacuumed. After concentration and lyophilization, D
A purified preparation of the free base of -53 is obtained. Example 2 D-53 free base 5 obtained in the same manner as Example 1
Dissolve the solution in 2 ml of water and add 0.6 ml of 0.05N monosulfuric acid.

Claims (1)

[Claims] 1. D-53 and its acid addition salt having the following physical and chemical properties (1) Basic white powder (2) Elemental analysis value (%): C50.15 H8.82 N14.88 ( 3) Molecular weight: 447 (4) Molecular formula: C_1_9H_3_7N_5O_7(5
) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum of an aqueous solution of this substance does not show a characteristic absorption maximum between 220 and 360 nm, but only shows terminal absorption. (6) Color reaction Ninhydrin reaction: Positive Potassium permanganate reaction: Positive Elson-Morgan reaction: Negative Biuret reaction: Negative (7) Mass spectrum: The mass spectrum of this substance shows the following M^+ ions and fragment ions. give. However, the composition formula obtained by accurate mass measurement is shown in parentheses. m/e447M^+(C_1_9H_3_7N_5O
_7), 430 (C_1_9H_3_4N_4O_7)
348 (C_1_4H_2_8N_4O_6), 336
(C_1_3H_2_6N_3O_7) 318 (C_1
_3H_2_4N_3O_6), 308(C_1_2H
_2_6N_3O_6), 290(C_1_2H_2_
4N_3O_5), 203(C_8H_1_7N_3O
_5) 191 (C_7H_1_5N_2O_3), 16
3(C_6H_1_5N_2O_3) 146(C_6H
_1_2NO_3) 145(C_6H_1_3N_2O
_2) 141 (C_7H_1_3N_2O) (8) PM
R spectrum: heavy aqueous solution of this substance (hydrochloric acid PD0.8)
The PMR spectrum within gives the following chemical shift values. (Units are ppm) δ1.29 (3H, s), 1.60 to 270 (4H, m
), 2.74 (3H, s), 3.30-4.30 (12
H, m), 5.11 (1H, d, J=3.5), 5.3
1 (1H, t, J = 4.0), 5.70 (1H, d, J
= 1.8) 2 A D-53-producing microorganism belonging to the genus Micromonospora is cultured in a nutrient medium, D-53 is produced and accumulated in the culture solution, and the substance is collected from the culture. A method for producing D-53.