JPH01207299A - Compound tan-1054 and production thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula (R is isobutyl or secondary butyl). Physical properties; appearance; white powder. Specific rotatory power; [alpha]21+101+ or -20 deg. (C=0.5, H2O). Molecular weight; m/z 258(M+H)<+>. Molecular formula; C12H23N3O3.2HCl. Color reaction; positive to ninhydrin, Greig.Leaback, iodine and potassium permanganate reactions and negative to the Ehrlich, Sakaguchi and Dragendorff reactions. Solubility; soluble in water and methanol and sparingly soluble in acetone. USE:A remedy for mycosis. EXAMPLE:A microorganism (FERM P-9844), belonging to the genus Serratia and having the ability to produce the aimed compound TAN-1054 is cultivated on a culture medium under conditions of 20-300 deg.C and pH6-8 for 15-80hr.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a novel compound TAN useful as a therapeutic agent for fungal diseases.
-1054 and its manufacturing method.

Conventional technology TAN-1054 is a dipeptide containing novel amino acids based on its physicochemical data, and no other antifungal compound has been reported yet.

Problems to be Solved by the Invention Chemotherapy by administering antifungal agents is the main treatment for infections caused by fungi. Bacterial replacement or the emergence of resistant bacteria (resistance phenomenon) due to large doses are currently a major problem in the field of chemotherapy for fungal infections. To overcome this problem, the field is constantly looking for compounds with new skeletons and exhibiting new biological activities, or intermediate raw materials for synthesizing them.

Means for Solving the Problems The present inventors isolated a large number of microorganisms from natural samples for the purpose of searching for new compounds, and conducted a separate search for the compounds they produced. that the microorganism belongs to the genus Serratia;
By cultivating the microorganism in an appropriate medium, we learned that a compound that inhibits the growth of fungi accumulates in the medium, isolated this compound, and determined that the compound suppresses the growth of the fungus based on its physicochemical and biological properties. was confirmed to be a new compound, and this was designated as compound TAN-1054. Moreover, TAN-1054 was composed of two components, which were named TAN-1054A and B, respectively.

The present inventors completed the present invention as a result of further research based on these findings.

That is, the present invention has the general formula, NH,R 112NCIIyCH2CII= CHCHCONHC
Compound TAN-1054 or a salt thereof represented by IICOtH [wherein R represents isobutyl or secondary butyl], and compound TAN-105 belonging to the genus Serratia
The present invention provides a method for producing the compound TAN-1054, which comprises culturing a microorganism capable of producing TAN-1054 in a medium, producing and accumulating the compound TAN-1054 in the culture, and collecting the compound.

In addition, in this specification, the above-mentioned TAN-105
4A and B may be collectively referred to as compound TAN-1054 or simply TAN-1054.

The microorganism that produces the compound TAN-1054 used in the method of the present invention may be any microorganism that belongs to the genus Serratia and has the ability to produce the compound TAN-1054'.

Examples include Serratia sp.
ratia sp,). A specific example thereof is Serratia sp. U-001 strain (hereinafter sometimes abbreviated as rU-001 strain) isolated from a sample collected in Hiroshima City, Hiroshima Prefecture.

The mycological properties of the U-001 strain are listed below.

(a) Morphology Cells on the gravy agar slant are rod-shaped with the order of 0.2 to 0.3 micrometers x 1.0 to 1.3 micrometers, and motility due to periciliary hairs is observed. Does not form spores. In addition, Durham staining was negative and showed no acid-fast properties.

(b) Growth status on various media The cells were cultured at 30°C for 24 hours and observed.

(1) Broth agar plate culture: Good growth, colony size is 2-3 mm in diameter, S-shaped, and translucent to slightly milky white. No diffusible dye is produced.

(2) Meat juice agar slant culture: Same as on meat juice agar plate.

(3) Meat juice liquid culture - Grows evenly throughout, but the upper layer is slightly better.

(4) Meat juice gelatin puncture culture: Liquefaction is observed after culturing at 30'C for 18 hours.

(5) Lidmus milk: Lidmus turns slightly red and curdles. The upper layer is liquefied.

(c) Physiological properties (1) Nitrate reduction: + (2) Denitrification reaction knee (3) MR test knee (4) VP (Voges Proscalaer) test + (5) Indole formation knee (6) Hydrogen sulfide Formation of (lead acetate paper) (7) Hydrolysis of starch: (8) Utilization of citric acid (Kozel, Christensen and Simmons media): + (9) Inorganic nitrogen source nitrate (ii) Ammonium salts: + (10) Pigment production (11) Urease: + (12) Oxidase (13) Catalase: + (14) Growth range 1) pH: Grows at pH 4.0 to 9.0, but optimum p)
(6.0-8.0 ii) Temperature: Grows at 8-42℃, but the optimum temperature is 25-42℃
35°C (15) Attitude towards oxygen: Facultative anaerobic (16) O-F
(oxidative fermentative) test [HughLeifson method]: fermentative type (17) type of production of acids and gases from sugars with and without sugars
Acid gas L-arabinose -- D-xylose -- D-glucose 10 -D-mannose + -D-fructose
-D-galactose 10 -maltose 10 -sucrose
10 - Lactose -- Trehalose 10 - D- Sorbitol + - D- Mannitol
+ - Inosit + - Glycerin + - Starch - - +: Positive, -: Negative (18) Utilization of malonic acid: Not utilized (19) No decomposition or bicomposition of alkynine (20) Decarboxylation reaction of lysine: + ( 21) Decarboxylation reaction of ornithine: + The U-001 strain having the above mycological properties was purified according to Bergey's Manual of Systeraic Bacteriology.
Stematic Bacteriology Volume 1 and Identification of Medical Bacteria
As a result of searching with the 2nd edition of ``edical Bacteria'', it was found that strain U-001 most appropriately belongs to the genus Serratia, and that it is most appropriate for strain U-001 to belong to the genus Serratia.
However, considering the fact that there are some differences in the properties, such as not producing pigment, and that it produces a new substance, TAN-1054, this strain has been classified as a new strain. Serradea SP U-OO1 (Serrat
ia sp, U-001).

This Serratia sp. U-001 strain was born in 1986.
On the 25th of May, the Fermentation Research Institute (IFO) received the accession number I.
This strain was deposited as FO14721 and was developed in 1932.
On January 28, 2013, the Ministry of Commerce and Industry, Agency of Industrial Science and Technology, Microbial Research Institute (FRI) received accession number FERM P-9844.
It has also been deposited as

The Serratia bacteria used in the method of the present invention are generally susceptible to changes in their properties, such as exposure to ultraviolet rays, X-rays, chemicals (
Although it can be easily mutated by artificial mutagenesis methods using, for example, nitrosoguanidine, ethyl methanesulfonic acid, etc., any mutant strain has the ability to produce TAN-1054, which is the subject of the present invention. All can be used in the method of the present invention.

When culturing the bacteria that produces TAN-1054, carbon sources such as glucose, glycerin, starch, maltose, lactose, blackstrap molasses, oils and fats (e.g., soybean oil, olive oil, etc.), organic acids (e.g., citric acid), etc. , succinic acid, gluconic acid, etc.) that can be assimilated by bacteria are used as appropriate. Nitrogen sources include organic nitrogen compounds such as soy flour, meat extract, cottonseed flour, corn steep liquor, dried yeast, yeast extract, meat extract, peptone, urea, ammonium sulfate, ammonium nitrate, ammonium chloride, ammonium phosphate, etc. and inorganic nitrogen compounds can be used. In addition, as the inorganic salt, for example, inorganic salts normally required for culturing bacteria such as sodium chloride, potassium chloride, calcium carbonate, magnesium sulfate, potassium phosphate, and disodium phosphate are used alone or in appropriate combinations. .

In addition, sulfur compounds that can be assimilated by the bacteria that produce TAN-1054, such as sulfates (e.g., ammonium sulfate, etc.)
, inorganic sulfur compounds such as thiosulfates (e.g., ammonium thiosulfate, etc.), sulfites (e.g., ammonium sulfite, etc.), sulfur-containing amino acids (e.g., cystine, cysteine, L-
When organic sulfur compounds such as thiazoline-4-carboxylic acid), hypotaurine, sulfur-containing peptides (eg, glutathione), or mixtures thereof are added to the culture medium, the production amount of the target product may be increased.

In addition, heavy metals such as ferrous sulfate and copper sulfate, and vitamin B
1. Vitamins such as biotin are also added as necessary. Furthermore, antifoaming agents and surfactants such as silicone oil and polyalkylene glycol ether may be added to the medium. In addition, TAN-10 helps the growth of bacteria.
Organic or inorganic substances that promote the production of 54 may be added as appropriate.

The culturing method may be the same as a general antibiotic production method, and solid culture or liquid culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aeration culture, etc. may be carried out, but aeration agitation culture is particularly preferred. Further, the culture temperature is preferably in the range of about 17°C to 35°C, more preferably about 20°C to 30°C.
℃, the pH of the medium is in the range of about 4 to 9, more preferably about 6 to 8, and the culture is carried out for about 10 to 90 hours, preferably about 15 to 80 hours.

In order to collect the target TAN-1054 from the culture, separation means that are normally used to collect metabolites produced by microorganisms from the culture of the microorganisms are appropriately used. For example, TAN-1054 exhibits the properties of a water-soluble basic substance and is mainly contained in the culture filtrate. Therefore, first, a filter aid is added to the culture solution and the bacterial cells are removed by filtration or centrifugation. Advantageously, a method is used in which the obtained culture filtrate is brought into contact with an appropriate carrier to adsorb the active ingredient in the filtrate, and then the effective substance is desorbed with an appropriate solvent and then fractionated and collected. As a carrier for chromatography, use the differences in the adsorption properties of compounds such as activated carbon, powdered cellulose, and adsorption resins, or use the differences in the functional groups of compounds such as cation exchange resins, cation exchange cellulose, and cation exchange Sephadex. Alternatively, those that utilize the difference in molecular weight of compounds such as Sephadex are advantageously used. In order to elute the target compound from these carriers, the combination differs depending on the type and nature of the carrier, but for example, aqueous solutions of water-soluble organic solvents, such as aqueous acetone, aqueous alcohols, acids, alkalis, buffer solutions, etc. Aqueous solutions containing inorganic or organic salts are used in appropriate combinations.

In addition, a purified product may be obtained by subjecting a composite material containing the compound obtained by these chromatography to preparative high performance liquid chromatography.

More specifically, when a cation exchange resin such as Amberlite IR-120, I RC-50 or CG-50 (manufactured by Rohm and Haas, USA) is used as a carrier, antibacterial substances in the filtrate can be adsorbed. It is eluted with an aqueous solution or buffer solution containing salts or acids.

Alternatively, a cation exchange molecular sieving resin such as CM-
The compound is adsorbed onto a carrier such as Sephadex (manufactured by Pharmacia Fine Chemicals, Sweden),
Elution can be performed with an aqueous solution or buffer solution containing salts or acids. In order to remove salts and colored substances from the eluate, activated carbon for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.) or an adsorbent resin such as Diamond Iont IP-20 or 5P-207 can be used.
(manufactured by Mitsubishi Chemical Industries, Ltd.), Amberlight XAD-
tI (manufactured by Rohm and Haas, USA) and the like are advantageously used. The fractionated eluted fraction is powdered through processes such as concentration and freeze-drying. If the purity of the powder thus obtained is low, high performance liquid chromatography (HPLC) is advantageously used for further purification.

The carrier used is, for example, YMC gel (manufactured by Yamamura Kagaku Kenkyushin), and the mobile phase is a buffer alone or a mixture of methanol, acetonitrile, etc., and an aqueous solution containing an inorganic salt or a buffer solution. . Note that TAN-1054 is isolated as a salt of mineral acids such as hydrochloric acid, sulfuric acid, phosphoric acid, etc. or organic acids such as formic acid, acetic acid, oxalic acid, etc.

Thus, TAN-1054 isolated in salt form
The salt can be converted into the free form of TAN-1054 by conventional means, and the free form of the compound can also be converted into the same salt form by conventional means.

The physicochemical properties of TAN-1054A dihydrochloride and TAN-1054B dihydrochloride obtained in Example 2 below are as shown in the figure.

[1] TAN-1054A dihydrochloride (1) Appearance: White powder (2) Specific rotation = [α]Ul+101°±20° (cm
0.5, HtO) (3) Measured molecular weight: m/z 25g (M+H)+(
(by SI-MS method) (M represents the molecular weight of the educt.

) (4) Molecular formula: C1t H2a N 303・2HC
Q (5) Elemental analysis value: The sample was dried under reduced pressure at 40°C for 6 hours over phosphorus pentoxide (calculated on the assumption that it contains 1.5 moles of water).
(%) Actual value Calculated value C, 40,66C, 40,34H,7,63H,7,9O N, 11.98N, 11.76CL19.82
CC,19,85(6) UV absorption spectrum:
Terminal Absorption in Water (7) IR Absorption Spectrum - See Figure 1 Showing high absorption (wave number) 3450, 2970.1730.1680.1600.
1550.1439°1390, 1240.1150.
940.850.790.730 (Rho 1) (g) +3c nuclear magnetic resonance (NMR) spectrum.

At 75λlHz, δppm, and heavy water, the following signals are observed.

181.6(s), 170.2(s), 137.8
(d), 125.6(d).

57.0(d), 53.0(d), 43.0(t)
, 41.1. (t), 28.1(t), 27.2
(d), 25.0 (q), 23.6 (Q) (S: singlet, d: doublet, L nitriblet (tripl)
et), representing quartet)
(9) High performance liquid chromatography (HPLC): Column: YMC pack A312 (manufactured by Yamamura Kagaku Kenkyushin), mobile phase: 0.0! M phosphoric acid solution (pi-r3), flow rate: 2
mff/min, Rt=4.2 (min) (10) Color reaction.

Positive, Ninhydrin, Brave Ryback, Iodine,
Potassium permanganate reaction Negative: Ehrlich, Itame, Dragendorff reaction (11) Solubility: Soluble; Water, dimethyl sulfoxide, methanol Poorly soluble: Acetone, ethyl acetate, diethyl ether (12) Acidic, medium Distinction between basicity and basicity: Free form is basic (13) Amino acid analysis value: TAN-1054A is 6N
Leucine (1 mole) was detected in the sample heated at 1108C for 15 hours in the T-I CC.

(14) Structural formula (TAN-1054A educt) (in the formula,
(L) indicates the configuration at the asymmetric center. ) [2] TAN-1054B dihydrochloride (1) Appearance/White powder (2) Specific rotation: [α]2'+109°±20" (
cm0.5, H2O) (3) Molecular weight measurement: m/z 25g (M+ t-t
) 4 (S[-by MS method) (4) Molecular formula: C, 21-1! 3 N 303・2
HC ((5) Elemental analysis value: Same conditions as A (calculated assuming that it contains 1 mol of water) (%) Actual value Calculated value 0.41.27 C, 41,39 [(, 7,49
I-I, 7.81 N, 12.48 N, 12.07 CLI9.82
CC,20,36(6) UV absorption spectrum:
Terminal absorption in water (7) IR absorption spectrum, see Figure 2 3
430, 3240.2970, 1690.1600.1
560.1500゜1400, 1350.1280.1
240.1150.1080. +010°940,7
90.750.580 (cm-') (8) 13CNM
R spectrum.

The following signals are observed at 75 MHz, δppm, and in heavy water.

179.4(s), 170.3(s), 136.7
(d), 125.6(d).

62.4(d), 53.1(d), 41.1(t)
, 39J(d), 28.2(t), 27.4(t
) (9) HPLC: Same conditions as A Rt = 3, 7 (min) (10) Color reaction: Positive: Ninhydrin, Brave Reback, Iodine,
Potassium permanganate reaction negative; Ehrlich, Itame, Dragendorff reaction (11) Solubility.

Soluble, water, dimethyl sulfoxide, methanol Poorly soluble: acetone, ethyl acetate, diethyl ether (12) Distinction between acidic, neutral, and basic: The free form is basic (13) Amino acid analysis value: TAN-1054B 6NH
Isoleucine (1 mol) was detected in the sample heated at 110° C. for 15 hours in CC.

(14) Structural formula (TAN-1054B educt) (in the formula,
(L) indicates the configuration at the asymmetric center. ) Next, we will discuss the biological properties of TAN-1054.

First, Tables 1 and 2 show the antibacterial spectrum of TAN-1054 (mixture of A and B) obtained in Example 2 against various microorganisms. 1000 μ9 of antibiotic TAN-1054
/l1I (filter paper discs (manufactured by Toyo Seisaku Shin, diameter 811 m) immersed in an aqueous solution of various fungi and bacteria are pasted on agar plates for phagocytic fungi and bacteria, and after culturing at a predetermined concentration for a predetermined time, the growth that has occurred around the filter paper discs is measured. The diameter of the suppression circle is shown in Table 1.
0” indicates that no growth inhibition circle was observed, and “+
” indicates that very weak growth inhibition was observed. The medium used was as follows.

A. Yeast Nitrogen Base Agar Medium, manufactured by Difco, USA. Yeast Nitrogen Base with 2% glucose and 1.5% agar added. B: Tributicase Soy Agar (Becton Dickinson, USA) Furthermore, The acute toxicity of TAN-1054 in mice is shown in Table 2.

Table 2 As is clear from these data, TAN-1054
has low toxicity to mammals. Therefore, T.A.
N-1054 can be used in the treatment of infectious diseases in mammals (eg, mice, rats, rabbits, dogs, humans), especially as an antifungal agent against fungi.

To use TAN-1054 or a salt thereof as a therapeutic agent for, for example, a fungal infection, TAN-1054 or a salt thereof is mixed with a pharmacologically acceptable carrier, excipient, diluent, etc. or a salt thereof is administered parenterally to the mammal subcutaneously or intramuscularly as an injection at a dose of about 1 to 50 m9/kg/day, preferably about 5 m9/kg/day.
Administer between 20m and 97kg/day. Orally, T
AN-1054 or its salt is used as a capsule, and TAN
-1054 for about 1 to 100197 days, preferably about 5 to 50 m9/97 days.

Moreover, TAN-1054 or its salt can be used as a disinfectant. For example, TAN-1054 or a salt thereof is about 0.0% as TAN-1054. Ol or O,
Solution dissolved in distilled water at a concentration of Iw/v%, or 1 g
Approximately 0.2 to 20 rxg per TAN-1054
By applying it as an ointment containing preferably about 1 to 10 R9 to the hands, feet, ears, etc. of the mammal, it can be used for sterilization and disinfection of these parts.

Next, the present invention will be explained in more detail by giving examples.

Example 1 Glycerin 209/Q, Peptone 20910. , dipotassium hydrogen phosphate 2, 59/Q, sodium chloride 59/
A medium containing ρ (hereinafter referred to as A medium) 2 rs (l) was dispensed into test tubes, sterilized in an autoclave, and U-0 grown on a trypdicase soy agar slant medium was added.
A loopful of the 01 strain was inoculated and cultured with shaking at 30°C for 24 hours.

50 mQ of medium A was dispensed into a shaking flask (300 x Q capacity), sterilized, inoculated with 2 mQ of the above-mentioned total culture solution, and cultured at 30°C for 124 hours in a reciprocating shaking incubator.

Next, A medium IQ was dispensed into a shaking flask (3 f2 capacity), sterilized, inoculated with the above-mentioned whole culture solution 507Q, and cultured at 30° C. for 172 hours in a reciprocating shaker.

Example 2 Example! Hyfloth-Purcel (manufactured by Johns Manville Products, USA) was added to the culture solution (5Q) obtained in , and the mixture was filtered and washed with water to obtain a filtrate (5Q). pH the filtrate
7, the filtrate was subjected to activated carbon chromatography (75
It was assigned to 0+0. After washing the column with water, the active fraction was eluted with 8% isobutanol water (3Q). The eluate was concentrated and the concentrated solution was passed through a column packed with I RC-50 (H+ form, 250 tttQ). After washing the column with water,
% saline (750 mQ). Transfer the eluate to activated carbon (
350 mQ) column, water (1 g) and 8
The active fraction was eluted with % isobutanol in water (1.5 f2). The eluate was concentrated, lyophilized, and TAN-1054A
, 1.039 of a crude material containing B was obtained. 1.79 g of the crude material obtained in the same manner was dissolved in water and passed through a column packed with CM Sephadex C-25 (Na+ form, 0.212). The active fraction was eluted and fractionated using 0.003 to 0.05 M saline solution (4). After confirming the active fraction by HPLC, it was desalted by activated carbon chromatography, and the eluate was concentrated and lyophilized to obtain TAN-1054A.

0.8 g of mixture B was obtained.

The mixture (0.8 g) was subjected to a preparative reverse-phase HPLC [
Carrier: YMC pack S-363 (manufactured by Yamamura Kagaku Kenkyu Shin)
, mobile phase: 0.01M phosphoric acid solution (pH 3)],
Two active fractions were obtained based on the difference in retention time. These fractions were each subjected to activated carbon chromatography to obtain a purified solution fraction. Both of these fractions were concentrated, freeze-dried, White powders of TAN-1054A dihydrochloride (230 mg) and TAN-1054B dihydrochloride (95z9) were obtained.

Effects of the Invention TAN-1054 of the present invention is a novel compound produced by Serratia bacteria, and particularly inhibits the growth of various fungi, so it can be administered orally, parenterally, or externally to humans and other animals. By administering it, it can be advantageously used for the treatment of these fungal infections.

[Brief explanation of the drawing]

FIG. 1 shows the IR spectrum of TAN-1054A dihydrochloride, and FIG. 2 shows the IR spectrum of TAN-1054B dihydrochloride. Patent applicant Ueno Pharmaceutical Application Research Institute Co., Ltd. and 1 other person

Claims (2)

[Claims] (1) Compound TAN-1054 or its salt represented by the general formula ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R represents isobutyl or secondary butyl]. (2) A microorganism belonging to the genus Serratia and capable of producing compound TAN-1054 is cultured in a medium, and compound TAN-1054 is present in the culture.
A method for producing a compound according to claim 1, which comprises producing and accumulating AN-1054 and collecting the same.