JP2873341B2 - Antibiotic TAN-950, its production and use - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a novel antibiotic TAN-950 and a salt thereof, which are useful as a therapeutic agent for fungal infections, a method for producing them, and uses thereof.

2. Description of the Related Art The novel antibiotic TAN-950 of the present invention is a novel amino acid antibiotic based on its chemical structure and biological data, and such an antifungal antibiotic has not yet been reported.

SUMMARY OF THE INVENTION Diseases caused by fungi have been considerably overcome by the development of therapeutics with antibiotics. However, many of the conventional antibiotics have high toxicity (side effects), and changes in the causative bacteria (bacteria replacement phenomenon) or emergence of resistant bacteria (phenotyping phenomenon) due to long-term or large dose administration are currently being considered. The fungal infection has become a major problem in the medical treatment field. In order to overcome these problems, there is a need in the art for antibiotics that have always weak toxicity (side effects), have a new skeleton and exhibit new biological activity, or intermediate materials for synthesizing them.

Means for Solving the Problems The present inventors separated a large number of microorganisms from soil for the purpose of searching for a novel antibiotic and separated and searched for the antibiotic produced by the microorganism. Producing a substance, that the microorganism is a species belonging to the genus Streptomyces, that an antibiotic exhibiting antibacterial activity against fungi can be accumulated in the medium by culturing the microorganism in an appropriate medium, etc. Knowing and isolating this antibiotic,
Based on its physicochemical and biological properties, it was confirmed that the antibiotic was a new antibiotic, and this was designated as antibiotic TAN-950. TAN-950 consists of five components, which were named TAN-950A, B, C, D and E, respectively. Further, subsequent studies revealed that the structure of each component of TAN-950 was represented by the following formula. The interconversion of these compounds will be described later.

Furthermore, the present inventors used TAN-950A as a raw material to synthesize a derivative by introducing a protecting group to stabilize the derivative, and also used L-glutamic acid as a raw material to prepare an intermediate raw material for synthesizing each component of TAN-950. Obtained.

The present inventors have further studied based on these findings, and as a result, completed the present invention.

That is, the present invention provides: (1) the following formula (I) or (II) [Wherein -COOR ¹ represents a carboxyl group which may be esterified, R ² and R ³ represent hydrogen or an N-protecting group, and R ⁴
Represents NOH or O, and in the formula (I), -COO
When R ¹ is carboxyl and R ² is hydrogen, R ³ represents an N-protecting group. Or a salt thereof, (2) a member of the genus Streptomyces, wherein
COOR ¹ is carboxyl, R ² is hydrogen, and R ⁴ is NOH.A microorganism capable of producing a compound capable of producing a compound is cultured in a medium, the compound is produced and accumulated in the culture, and this is collected. (3) Formula (V): [Wherein, -COOR ¹ represents a carboxyl group which may be esterified, and R ² represents hydrogen or an N-protecting group. A compound of formula (I-1) [a compound of the formula I wherein R ³ is hydrogen] or a salt thereof, (4) a method of formula (III) or (IV) [Wherein -COOR ¹ 'represents an esterified carboxyl group, and R ² ' represents an N-protecting group. A compound represented by the formula (VI), wherein the compound represented by the formula [Wherein —COOR ¹ ′, R ² ′ and R ⁴ have the same meaning as described above. Methods for preparation of the compounds represented by, and (5) R ² is hydrogen -COOR ¹ is a carboxyl in formula (I), the compound wherein R ³ is hydrogen, is -COOR ¹ in formula (II) carboxyl Wherein R ² is hydrogen and R ⁴ is NOH.

In the above formula, the carboxyl group and esterified in esterified carboxyl group which may be represented by -COOR ^1, as the esterified carboxyl group represented by -COOR ¹ ', for example, methoxycarbonyl, ethoxycarbonyl Carbonyl, t-butoxycarbonyl, isopropoxycarbonyl, 2-cyanoethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, benzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p
-Methoxybenzyloxycarbonyl, diphenylmethyloxycarbonyl, methoxymethyloxycarbonyl, acetylmethyloxycarbonyl, isobornyloxycarbonyl, phenyloxycarbonyl and the like, among which methoxycarbonyl and p-nitrobenzyloxycarbonyl are preferable.

R ^2, as the N- protecting group represented by R ² 'and R ^3, for example, methyl, ethyl, propyl, isopropyl, butyl, alkyl isobutyl, phthaloyl, p- nitrobenzoyl, p-tert-butyl-benzoyl aromatic acyl groups such as, p-tert-butylbenzenesulfonyl, benzenesulfonyl, toluenesulfonyl, for example, formyl,
Acetyl, propionyl, monochloroacetyl, dichloroacetyl, trichloroacetyl, methanesulfonyl,
Ethanesulfonyl, trifluoroacetyl, malonyl,
An aliphatic acyl group such as succinyl, a carboxyl group esterified as described above, for example, a methylene group such as (hexahydro-1H-azepin-1-yl) methylene, a sulfonyl group such as 2-amino-2-carboxyethylsulfonyl,
Further, for example, N-protecting groups other than acyl groups such as trityl, 2-nitrophenylthio, benzylidene, p-nitrobenzylidene, di- or trialkylsilyl, benzyl and p-nitrobenzyl may be mentioned. , Benzoyl, t-butoxycarbonyl, p-nitrobenzyl, p-methoxybenzyloxycarbonyl and the like are preferred.

The compound represented by the formula (I) may be represented by an enol type as described below.

The conversion to the enol form as described above is also included in the concept of the present invention.

In the present specification, “antibiotic TAN-950” may be simply referred to as “TAN-950”.

Bacteria that produce the antibiotic TAN-950 include the antibiotic TAN-9.
Any microorganism can be used as long as it has an ability to produce 50. For example, the microorganism can be isolated by the present inventors and used in Streptomyces platensis A-136 (Streptomyces platensi).
The strain named sA-136) or a strain related thereto is one example that is most effectively used. After that, this bacterium was changed to A
It may be abbreviated as -136 bacteria.

The morphological characteristics of the bacterium and the culture findings on the classification medium are as follows, for example.

In this bacterium, aerial hyphae are formed on an ordinary classified medium, they show simple branching, and the spore-forming hyphae has a spiral shape. Ten or more spores are linked, the surface is smooth, and the size is 0.8 to 1.2 μm × 0.9 to 1.3 μm.
No sporangia, flagellated spores, sclerotia, etc. are formed on the normal classification medium.

Table 1 shows the growth state of this bacterium on the classification medium. Culture findings observed at 28 ° C for 21 days unless otherwise noted.
The names in parentheses in the description are the names of colors according to the Color Harmony Manual, 4th edition (Container Corporation of America, 1958).

The physiological properties of the A-136 bacterium are as follows.

(1) Growth temperature range: grows at 10-36 ° C, but shows better growth at 28-32 ° C.

(2) Liquefaction of gelatin: positive (3) Starch hydrolysis: positive (4) Coagulation of skim milk: peptone formation: negative (5) Melanin-like pigment formation: negative (peptone yeast iron agar medium and tyrosine agar medium) (6) Nitrate reduction: negative (International Streptomyces project No. 8 medium) (7) Utilization of carbon source (Pridham Gottlieb agar medium) Commonly used carbon sources Inositol, D-mannitol, D-xylose , D-glucose, D-fructose, raffinose Moderately used carbon source L-arabinose Unused carbon source rhamnose, sucrose Was. This suggests that the bacterium belongs to the genus Streptomyces. As a result of an attempt to compare with the existing bacterial species based on the morphological characteristics, culture findings, and physiological properties of the A-136 bacterium, the bacterium was identified as Streptomyces platensis, and the Streptomyces platensis was identified.
(Streptomyces platensis A-136). Streptomyces platensis A- used in the present invention
136 is from April 23, 1987, Fermentation Research Institute (IFO)
Deposit No. as IFO14603 and April 1987
The deposit was deposited with the Research Institute of Microbial Industry and Technology (FRI) of the Ministry of International Trade and Industry under the accession number FERM P-9358 from the 30th. Is stored in

As a general property of Streptomyces, mycological properties are extremely susceptible to variation, and Streptomyces platensis A-136 is no exception. Therefore, the properties of the bacterium are not constant as described above, and various mutants can be easily obtained. However, even these mutants can be used in the method of the present invention as long as they do not lose the property of producing the antibiotic TAN-950. Of course, the mutation may be caused by a natural cause or artificially performed by using various mutagenic agents (for example, ultraviolet ray, X-ray, radiation, nitrosoguanidine, etc.).

Known strains of the genus Streptomyces other than the above-mentioned A-136 strain, for example, Streptomyces hygroscopicus A-300 (see Japanese Patent Publication No. 1312, JP-B-54-43600), Streptomyces hygroscopicus Mrs. hygroscopicus subspecies angst mysetics (Streptomyces hygroscopicus subs
p. angustmyceticus) IFO 3934, IFO 3935, Streptomyces hygroscopicus subsp. hygroscopicus (Streptomyces hygroscopicus subsp. hy)
(groscopicus) IFO 14012 and the like can also be used in the present invention.

In culturing in the method of the present invention, a medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, inorganic salts, and the like is generally used. If necessary, a trace amount of an effective substance such as a micronutrient, a growth promoting substance, or a precursor may be added to the medium. In general, glucose, sucrose, molasses, starch,
There are dextrin and glycerin, and the digestible nitrogen sources include meat extract, soy flour, corn steep liquor, peptone, casein, cottonseed meal, and inorganic nitrogen compounds such as nitrates and ammonium compounds. Both are used effectively. The culture may be performed by a surface culture method, but is usually performed by a submerged aeration culture method. In the case of the deep aeration culture method, the properties of the culture medium are preferably near neutral, and the temperature during the culture is preferably maintained at about 20 to 36 ° C, preferably 24 to 30 ° C. However, the culture conditions such as the culture composition, the liquid properties of the culture medium, the culture temperature, and the number of agitation may be appropriately adjusted and selected so as to obtain favorable results according to the type of the strain used and external conditions. Needless to say.

In order to collect the desired antibiotic TAN-950 from the culture, a separation means usually used for collecting a metabolite produced by the microorganism from the culture of the microorganism is appropriately used. For example, the antibiotic TAN-950 exhibits the properties of a water-soluble amphoteric substance and is mainly contained in the culture filtrate, so that the culture medium is first added with a filter aid to remove cells by filtration or centrifugation. A means for appropriately contacting the culture filtrate with a carrier to adsorb the active ingredient in the filtrate, desorbing the active substance with an appropriate solvent, and separating and collecting the fraction is advantageously used. As a carrier for chromatography, activated carbon, silica gel, powdered cellulose, use the difference in the adsorptivity of the compound such as adsorbent resin, or ion exchange resin, ion exchange cellulose, use the difference in the functional group of the compound such as ion exchange Sephadex, or Those utilizing the difference in molecular weight of compounds, such as molecular sieve carriers, are advantageously used. In order to elute the target compound from these carriers, the combination differs depending on the type and properties of the carrier. For example, an aqueous solution of a water-soluble organic solvent, that is, aqueous acetone, aqueous alcohols, etc. An aqueous solution containing an inorganic or organic salt or the like is used in appropriate combination.

More specifically, as a carrier, a cation exchange resin such as Amberlite IR-120 (manufactured by Rohm and Haas, USA), Dowex 50W (manufactured by Dow Chemical,
US), Diaion SK1A (Mitsubishi Kasei Co., Japan) or an anion exchange resin such as Amberlite IRA-402, I
Use RA-68 (manufactured by Rohm and Haas, USA), Dowex 1 (manufactured by Dow Chemical, USA), Diaion SA10B, PA-404, WA-30 (manufactured by Mitsubishi Kasei, Japan) The antibiotic in the filtrate is adsorbed and eluted with a salt, alkali or acid-containing aqueous solution or buffer. Further, the present antibiotic can be adsorbed on a carrier such as an ion exchange molecular sieve resin such as QAE or CM-Sephadex (Pharmacia, Sweden) and eluted with an aqueous solution or buffer containing salts, alkalis or acids. I can do it. Activated carbon for chromatography (Takeda Pharmaceutical Co., Japan) and adsorbent resins such as Diaion HP-20 and SP-207 (Mitsubishi Kasei Co., Japan) to remove salts, coloring substances, etc. in these eluates. , Amberlite XAD-II (Rohm and Haas, USA), molecular sieve resin Sephadex (Pharmacia, Sweden) or crystalline cellulose (Asahi Kasei, Japan) is advantageously used. In order to remove fat-soluble substances in the filtrate or eluate, pass through a column such as activated carbon or an adsorptive resin, or use an organic solvent immiscible with water, such as dichloromethane, ethyl acetate, methyl isobutyl ketone, etc. And the like are appropriately combined.

Furthermore, when the compound is finally purified, a preparative high performance liquid chromatography (HPLC) method is also advantageously used.
When this method is applied, a reversed-phase resin such as YMC gel (Yamamura Chemical Laboratory, Japan) or TSK gel (Toyo Soda Kogyo, Japan) is used as a carrier, and a buffer solution is used as a mobile phase. A solvent system to which an ion-paired reagent such as tetrabutylammonium hydride is added is used.

The elution fraction purified and fractionated as above is concentrated,
TAN-950A can be powdered or crystallized through a process such as lyophilization or crystallization.

TAN-950A was isolated as a monosodium salt,
A free form of this compound is prepared as follows. That is, TAN-950A monosodium salt is dissolved in water, 1 equivalent of hydrochloric acid is added thereto, and the solution is subjected to column chromatography of crystalline cellulose.
Elution with aqueous propanol or the like gives TAN-950A (free form). Further, a method in which the antibiotic-containing solution is subjected to column chromatography such as Amberlite IR-120 (H ⁺ type) and eluted with ammonia water or the like is also used as appropriate.

Preparation of a pharmacologically acceptable salt (eg, potassium salt, calcium salt, etc.) from the free form is carried out by a method known per se.

Next, the relationship between TAN-950A and TAN-950B, C, D and E will be described. TAN-950A and T obtained in Example 2 described later
Each of the AN-950A to E-E mixtures was subjected to analytical HPLC under the following conditions to obtain elution patterns shown in FIG.

Carrier: YMC-Pack AQ-312 (Yamamura Chemical Laboratory) Moving layer: 0.0025 M tetrabutylammonium hydride / 0.02 M phosphate buffer (pH 6.0) Detection method: UV absorption (214 nm) Flow rate: 2 ml / min The mixture showed four peaks in addition to TAN-950A,
The t value and the area percentage are as follows.

HPLC separation of the TAN-950A-E mixture to isolate each component
Each component was isolated as an almost single peak compound, but the aliquot was heated to 60 ° C., and after one hour by HPLC again, it was found that it had been converted to other components. (See FIG. 2). Table 2 summarizes this data.

Almost the same results are obtained in water or a phosphate buffer.

As is apparent from Table 2, TAN-950B and D are TAN-950B.
It is converted to TAN-950A faster than 950C and E,
AN-950A is mainly converted to TAN-950B and D, and TAN-95
Small amounts to OC and E. The data mentioned above is TAN
-950A, B, C, D and E clearly indicate that they are present as an equilibrium mixture in the aqueous solution. It is difficult to convert TA
N-950C and E were also rapidly converted to TAN-950A when left in a dilute aqueous alkaline solution at room temperature. When the same mixture is converted to t-butoxycarbonyl (Boc), only the N-Boc form of TAN-950A is obtained. When the mixture of the BE components is converted to p-nitrobenzyl (PNB), four types of PNB are obtained. A mixture of ester bodies was obtained. When this ester derivative was subjected to chromatography, one component ester was isolated as a pure product, but the remaining three types could not be isolated.
Various physicochemical data of the pure product supported the following formula. Considering the above data comprehensively, the relationship between TAN-950A-E is as follows.

The physicochemical properties of TAN-950A (monosodium salt) obtained in Example 2 described below are shown below.

1) Appearance: white solid 2) Specific rotation: ▲ [α] ²³ _D ▼ -70 ° ± 10 ° (c = 0.5
24, in water) 3) Elemental analysis value (%) Measured value Calculated value ^* C, 33.64 C, 33.97 H, 4.31 H, 4.28 N, 12.72 N, 13.21 O, 37.82 Na, 11.0 Na, 10.66 ( ^* 1 mole of water 4) Measured molecular weight: m / z 195 (M + H) ⁺ , 217 (M + Na) ⁺ , 239 (M + 2Na) ⁺ 5) Molecular formula: C ₆ H ₇ N ₂ O ₄ Na 6) UV spectrum: in water (see Fig. 3), 7) IR spectrum: In KBr tablet (see Fig. 4), main wave numbers (cm ^-1 ) 3430,1640,1500,1410,1350,1180,1070,930,870,810,75
0,610,530 8) ¹ H-NMR spectrum: 400 MHz, in heavy water (see Fig. 5) The following signals are observed (δ _ppm J _(Hz) ) 2.70 (1H, dd, J = 7.3, 15.6), 2.80 (1H, dd, J = 4.4, 15.6), 3.86 (1H, dd, J = 4.4, 7.3), 7.98 (1H, s) (however, dd: double doublet, s: singlet) 9) ¹³ C-NMR spectrum: 100 MHz, In heavy water (see Fig. 6), the following signals are observed (δ _ppm ): 180.6 (s), 177.1 (s), 158.5 (d), 82.9 (s), 58.
5 (d), 26.5 (t) (however, s: singlet, d: doublet, t: triplet) 10) Solubility: Soluble: water, dimethyl sulfoxide, methanol Poorly soluble: ethyl acetate, acetone, chloroform 11) Color reaction: Positive: Ninhydrin, Ehrlich (acidic), phosphomolybdic acid, dimethylaminobenzaldehyde reaction Negative: Greig-Liebach, Dragendorf reaction 12) Thin-layer chromatography (TLC): 13) High performance liquid chromatography (HPLC): carrier; YMC-
Pack AM-324, manufactured by Yamamura Chemical Laboratory, mobile phase; 0.005 M tetrabutylammonium hydride / 0.02 M phosphate buffer (pH 6.0), flow rate: 2 ml / min .; Rt = 4.8 (min.) 14) Substance Category: Amphoteric substance 15) Amino acid analysis: TAN-950A in 6N HCl, 16 hours, 110 ° C
After heating in, amino acid analysis revealed that glutamic acid was detected.

The compound numbers, names,
1 shows a chemical structure.

The most effective way to convert compound 6 to compound 1 is to subject them to an alkali reaction. That is, the compound is dissolved in a 0.1 to 1.0N, preferably 0.2 to 0.5N aqueous sodium hydroxide solution at a concentration of 5 to 50 mg / ml, preferably 15 to 30 mg / ml, and the reaction temperature is 20 ° C to 80 ° C, preferably 40 ° C to 70 ° C. At 15 ° C for 15 minutes to 2 days, preferably
The reaction may be performed for 30 minutes to 4 hours.

A common method for introducing a protecting group is TW
“Protective Groups in Organic Synthesis” by Greene
ic Synthesis), page 218 (1981), John Wiley & Sons. As a typical example of the introduction of an N-protecting group, for example, in the case of t-butoxycarbonylation, the sample is treated with a polar solvent such as 50%. Dissolved in dioxane-water, etc.
About 1 to 4 equivalents of triethylamine and about 1 to 3 equivalents of 2- (t-butoxycarbonyloxyimino) -2-phenylacetonitrile (hereinafter sometimes abbreviated as BOC-ON) are added, and the mixture is added at room temperature for 0.5 to 0.5 equivalents. The reaction is completed under stirring for 24 hours, preferably 3 to 10 hours.

The same is true for p-methoxybenzyloxycarbonylation. In the case of benzoylation or acetylation, dissolve the sample in dilute aqueous sodium bicarbonate, add about 1 to 3 equivalents of benzoyl chloride or acetic anhydride, and react at room temperature with stirring for about 1 to 16 hours. Done by

Further, as an example of introducing a protecting group into an acidic group, for example, the following method is used.

1) The compound is reacted with a diazoalkane (eg, diazomethane) in a solvent (eg, ethyl ether, tetrahydrofuran, dioxane, methanol, etc.) at about 0 ° C. to reflux temperature for about 2 minutes to 10 hours.

2) reacting the compound with an activated alkyl halide (eg, methyl iodide, p-nitrobenzyl bromide, etc.); As a suitable reaction condition, a solvent (eg, dimethylformamide, dimethylacetamide, etc.) is used, and the reaction is carried out at about 0 ° C. to 60 ° C. for about 2 minutes to 20 hours.
Even if an alkali metal salt solution (eg, sodium hydroxide, potassium hydroxide), ammonia, triethylamine, or the like is co-present in this reaction solution, the reaction does not hinder the progress of the reaction.

3) Reacting the compound with an alcohol such as methanol. In this reaction, a mineral acid such as hydrochloric acid, sulfuric acid or p-toluenesulfonic acid is used as a condensing agent.
The reaction is carried out at about 0 ° C. to reflux temperature for 15 minutes to 20 minutes. As the solvent, a corresponding alcohol or a mixture thereof with chloroform, dichloromethane, dioxane or the like is also used.

4) The compound is reacted with an acid anhydride or an acid halide such as acetic anhydride, acetyl chloride, di-t-butyl dicarbonate or the like in an organic solvent such as chloroform or dichloroethane at 0 ° C. to reflux temperature for 15 minutes to 15 hours. The solvent may contain a basic reaction catalyst such as triethylamine and pyridine.

The method for removing the introduced protecting group can be appropriately selected from a method using an acid or a base depending on the type of the protecting group. Here, in the case of the method using an acid, although it depends on the type of the protecting group and other conditions, the acid may be an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or the like, or an organic acid such as formic acid, acetic acid, trifluoroacetic acid, or propionic acid. In addition, an acidic ion exchange resin or the like is used. In the case of the method using a base, the base varies depending on the kind of the protecting group and other conditions, but the base may be an inorganic base such as an alkali metal such as sodium or potassium or a hydroxide or carbonate of an alkaline earth metal such as calcium or magnesium. In addition to organic bases such as metal alkoxides, organic amines and quaternary ammonium salts, basic ion exchange resins and the like are used. When a solvent is used in the method using an acid or a base, a hydrophilic organic solvent, water or a mixed solvent is often used. In the method using a metal and an acid, water, acetone and the like are frequently used. However, when the acid is a liquid, the acid itself can be used as a solvent. In the method using an acid and a base, the reaction is usually carried out under cooling or about warming, and the reaction time is 30 minutes to 20 hours.

 Next, the synthesis method of TAN-950A will be described.

Starting materials (III or IV, known compounds) can be obtained from glutamic acid by a known method.

First, an aldehyde group is introduced into these compounds by activating the methylene adjacent to the carbonyl group and reacting with a reagent having an aldehyde group. That is, when a compound (III-1 or IV-1) of the formula III or IV in which —COOR ^{1 *} is methoxycarbonyl and R ² ′ is BOC is used as the starting material, an organic solvent such as tetrahydrofuran, ethyl ether, Dissolved in n-hexane and mixtures thereof, at a temperature between -100 ° C and 40 ° C
C., preferably at -78.degree. C. to 0.degree. C., adding an activating agent such as lithium diisopropylamide or lithium bistrimethylsilylamide, preferably for 5 minutes to 2 hours.
Hold for 10 minutes to 1 hour. Then, an aldehyde introduction reagent such as ethyl formate, isopropyl formate,
Isobutyl formate or t-butyl formate is added, and the reaction temperature is
The reaction time is 0.5 to 10 hours, preferably 1 to 4 hours at 78 ° C to room temperature, preferably -40 ° C to 0 ° C.
React in time. Compound 17 is obtained by the reaction.

Compound 18 is obtained by subjecting compound 17 to the following oximation reaction. That is, compound 17 is dissolved in a water-containing solvent such as dioxane water, tetrahydrofuran water and ethanol, and 1 to 3 equivalents, preferably 1.2 to 2.0 equivalents of hydroxylamine or a salt thereof is added, and the reaction temperature is 0 ° C to reflux temperature, preferably 20 ° C to 20 ° C. The reaction is carried out at 40 ° C. for a reaction time of 10 minutes to 5 hours, preferably 30 minutes to 2 hours.

The obtained compound 18 is dissolved in alcohols such as methanol, and about 1 equivalent of an alkali solution is added. The reaction temperature is -20 ° C to 40 ° C, preferably 0 ° C to room temperature, and the reaction time is 10 minutes to 4 hours, preferably The reaction is carried out with stirring for 30 minutes to 2 hours. This reaction cleaves the amide group of compound 18 and reacts with the oxime group to give compound 19. Further, Compound 19 is dissolved in about 2 to 4 equivalents, preferably about 3 equivalents of an aqueous alkali solution,
The reaction is carried out at preferably 20 ° C. to 40 ° C. for 15 minutes to 6 hours, preferably 30 minutes to 2 hours to obtain compound 9. The compound was identical in physicochemical data with a compound derived from naturally occurring TAN-950A.

 Next, the biological properties of TAN-950 will be described.

A filter paper disc (8 mm in diameter, manufactured by Toyo Seisakusho) immersed in a 1000 μg / ml aqueous solution of each of Compounds 1 and 6 is attached to a fungal- and bacteria-containing agar plate and cultured at a predetermined concentration for a predetermined time. Table 3 shows the diameters of the growth-suppressing circles generated in Table 1. In the table, "0" indicates that no growth suppression circle was observed. The medium used was as follows.

A: Addition of 2% glucose and 1.5% agar to yeast nitrogen-based agar medium (Difco, USA) B: Trypticase soy agar (Becton Dickinson, USA) As shown in Table 3, the antibiotic TAN- 950 shows antibacterial activity against certain fungi.

Next, the therapeutic effects of Compound 1 on experimental mouse infections are as shown in Table 4.

Furthermore, no acute toxicity was observed when Compound 1 was administered intravenously, subcutaneously, intraperitoneally or orally to mice at a dose of 4000 mg / kg.

As apparent from these data, it can be said that TAN-950 is an antibiotic that exhibits antibacterial activity against fungi and does not exhibit toxicity in mammals and the like. Therefore, TAN-950 can be used for treating fungal infections in humans, livestock, poultry, and the like.

For this treatment, TAN-950 can be formulated and used in various dosage forms of pharmaceutical compositions according to known formulation techniques.

To normally use TAN-950 as a therapeutic agent for, for example, Candida infection, for example, TAN-950 is dissolved in physiological saline and injected parenterally as an intravenous, subcutaneous, or intramuscular usually at 1 to 50 mg / mg. kg / day, preferably 5-20 mg / kg / day. As an oral preparation, the antibiotic TAN-950 is mixed with lactose to form a capsule, or a sugar-coated tablet produced by a known method, usually 1 to 100 mg / kg as TAN-950.
/ Day, preferably 5 to 50 mg / kg / day.

Further, TAN-950 obtained by the present invention can be used as an external germicide. For example, TAN-950 is usually
0.1 to 10 W / V%, preferably 0.5 to 5 W / V%, dissolved in distilled water or the like, or Vaseline or lanolin as a base, and TAN-950 is usually 0.2 to 100 mg, preferably 1 to 1 g per gram. As an ointment containing 20 mg, it can be used for sterilization and disinfection of human and animal skin or mucous membranes.

The antibiotic TAN-950 is also a promising compound as an intermediate for the synthesis of new pharmaceuticals, and TAN-950B-E are TAN-950A
It can also be a raw material for the synthesis of

As is clear from the chemical and biological properties described above, TAN-950A, B, C, D and E are novel antibiotics.

EXAMPLES Next, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto. Percentages refer to weight / volume% unless otherwise noted. The conditions described above were employed as the HPLC conditions in the examples.

Example 1 A medium (pH 7) consisting of 2.0% glucose, 3.0% soluble starch, 1.0% corn steep liquor, 1.0% defatted soy flour, 0.5% peptone, 0.3% sodium chloride, 0.5% calcium carbonate in a 3 liter Sakaguchi corben. Adjustment) 500 ml after injection, sterilize, and add Streptomyces platensis
A-136 (IFO 14603, FERM BP-1786)
After inoculating a platinum loop, 28 ° C on a reciprocating shaker at 120 reciprocations / minute
For 48 hours. Actol (defoamer, Takeda Pharmaceutical Co., Ltd.
(Japan) 30 l of medium supplemented with 0.05% was prepared, sterilized, and inoculated with 500 ml of the whole culture of Sakaguchi Kolben, which had been previously cultivated, and submerged at 28 ° C for 48 hours at aeration rate of 30 l / min and stirring at 280 rpm at 280 rpm. A seed culture was obtained.

0.5% glucose, 5% dextrin, 3.5% defatted soy flour, 0.7% calcium carbonate in a 200l stainless steel tank
% Of a medium (pH 7.0) prepared and sterilized, inoculated with 6 l of the seed culture solution, aeration rate 120 l / min, stirring speed 200 rpm
At 28 ° C for 90 hours.

Example 2 After adjusting the culture solution (100 l) obtained in Example 1 to pH 8.0, Hyflo supercell (manufactured by Johns Manville; USA) was added, and the mixture was filtered to obtain a filtrate (85 l). After the filtrate was adjusted to pH 3, ethyl acetate (40 l) was added and the mixture was stirred, and the ethyl acetate layer was removed. The obtained aqueous layer is concentrated, and the concentrated solution (50
l) was adjusted to pH 5 and then subjected to column chromatography with Amberlite IR-120 (H ⁺ type, 8 l). The antibiotic was eluted with 2% aqueous ammonia, and the eluate (40 l) was concentrated. The concentrate (18 l) was passed through a column of Diaion HP-20 (8 l) and washed with water (12 l). The combined filtrate and washings were concentrated, and the concentrate (3.3 l) was subjected to column chromatography on Diaion SP-207 (2 l) to elute and fractionate with water. The fractions containing the antibiotic (4l), QAE-Sephadex A-25 (Cl ^≠ type, 2.5 l) and subjected to column chromatography, eluted with 0.02~0.04M saline and fractionated. The active fractions were collected (5 l), concentrated, and the concentrated liquid (0.4 l) was subjected to chromatography on crystalline cellulose (manufactured by Asahi Kasei Corporation, Japan). Elution and fractionation with a solvent system of acetonitrile: water (9: 1 to 4: 1). The fraction showing a single peak by HPLC was collected and concentrated to obtain Compound 1 (monosodium salt, 2.59 g) as a white powder.

The antimicrobial activity fractions other than a single peak were collected by chromatography on QAE-Sephadex, and Amberlite IR-12 was collected.
After elution with 0% (H ⁺ type, 0.8 l) chromatography and elution with 2% aqueous ammonia, the eluate was concentrated, lyophilized, and treated with TAN-9.
Compound 6 (4.1 g) containing 50A, B, C, D and E was obtained as a white powder.

Example 3 In a 200-ml Erlenmeyer flask, 2.0% glucose, 3.0% soluble starch, corn steep,
Liquor 1.0%, defatted soy flour 1.0%, peptone 0.5%, sodium chloride 0.3%, calcium carbonate 0.5%, a medium (pH 7 adjustment) was injected in 40 ml each, sterilized, and then streptomyces hygroscopicus A-300. One platinum loop was inoculated from the slant culture (Microtechnical Laboratories No. 1312), and cultured at 28 ° C. for 48 hours on a rotary shaker at 200 rpm, and this was combined into a seed culture solution.

On the other hand, glycerin 5%, profuro (absorbent cotton seed powder, manufactured by Traders Oil Mill Company, USA) 3.5%
5% of medium (pH7) consisting of 0.7% sedimentable calcium carbonate and 0.7%
Was prepared, and 40 ml was poured into each 200 ml Erlenmeyer flask and sterilized. After cooling, 2 ml of the above-described seed culture solution was inoculated per flask and cultured at 28 ° C. for 5 days on a rotary shaker at 200 rpm.

The obtained culture was filtered to obtain a filtrate (4.3 l). After adjusting the filtrate to pH 3, ethyl acetate (2 l) was added, and the mixture was stirred for 30 minutes. The aqueous layer separated after filtration was further subjected to ethyl acetate (1.5
Washing in l) gave an aqueous filtrate (4.2 l). The aqueous layer filtrate was subjected to column chromatography using Amberlite IR-120 (H ⁺ type, 1), and the active component was eluted with 2% aqueous ammonia. After concentrating the eluate (5 l), the concentrated solution is
The column was subjected to SP-207 (0.3 l) column chromatography.
Eluted with water. The antibacterial activity section (350 ml) was subjected to column chromatography on QAE-Sephadex (C1 ^- type, 100 ml), and fractionated by elution with 0.02-0.03 M saline. Active fraction (1
00 ml) was subjected to column chromatography on crystalline cellulose (70 ml), and acetonitrile (85-70): water (15-3
The antimicrobial substance was eluted and fractionated with the solvent system of 0). Then H for analysis
Fractions containing only compound 1 were collected by PLC, concentrated and lyophilized. The obtained powder (180 mg) had the same physicochemical properties as the preparation of compound 1 (sodium salt).

In addition, Streptomyces hygroscopicus subspecies hygroscopicus (Streptomyces hygroscopicus)
(microscopicus subsp. hygroscopicus) IFO 14012 was cultured under the same conditions as above for 4 days. The culture filtrate (2.8 l) was purified in the same manner as described above, and Compound 1 (sodium salt) (302 m
g) was obtained.

Example 4 59 l of the culture solution (105 l) obtained in the same manner as in Example 1 was filtered using Hyflo Super Cell. The obtained filtrate (7.6 l) was passed through a column of Diaion SP-207 (2 l). Mixture of passing liquid and washing liquid (8.1
) Of Amberlite IR-120 (H ⁺ type, 700m)
The active substance was eluted with 2% aqueous ammonia (1.2 l) by chromatography in l). The eluted fraction is directly used (pH
10) and column chromatography with Amberlite IR-402 (C1 ^- type, 100 ml), and fractionated by elution with 0.2 M saline (1.3 l). After concentrating the active fraction, dioxane (150 ml) was added to the concentrate (150 ml, containing compound 1.84 g in HPLC analysis), and the pH was adjusted to 9.3 by adding triethylamine. BOC-ON (4.5 g, 1.8 equivalent) was added to the solution, and the solution was added at room temperature for 3 hours.
Stirred for hours. The reaction mixture was concentrated and evaporated to dryness.
0 ml). The obtained aqueous solution was diluted with ethyl acetate (100 ml
After washing with × 3), sodium chloride was added to the aqueous layer, and the pH was adjusted to 2.7.
Extracted with ethyl acetate (100 ml × 3). Concentrate the extract,
A mixture of ether and hexane was added to the concentrated solution to obtain a powder of compound 9 (2.8 g). This powder was analyzed by HPLC (assuming that the sample obtained in Example 8 was 100%) and had a purity of 65%.
%showed that.

Example 5 Compound 6 (240 mg) was added to a 0.3 N aqueous sodium hydroxide solution (1
2 ml) and stirred at 60 ° C. for 1 hour. After adjusting the obtained reaction solution to pH 6.7, it was concentrated, and microcrystalline cellulose (200 m
The mixture was subjected to column chromatography in 1), and eluted and fractionated with a solvent system of acetonitrile: water (9: 1 to 7: 3). The fraction showing a single peak by HPLC was collected, concentrated and freeze-dried to obtain a white powder (134 mg) of compound 1 (monosodium salt).

Example 6 Compound 1 (517 mg of monosodium salt) was dissolved in a 2% aqueous sodium hydrogen carbonate solution (30 ml), benzoyl chloride (350 μl) was added, and the mixture was stirred at room temperature for 1 hour. further,
Benzoyl chloride (300 μl) was added, and the mixture was stirred at room temperature for 3 hours while maintaining the pH at 8.0 to 8.5. The obtained reaction solution was washed with ethyl acetate, adjusted to pH 3.0 with 2N hydrochloric acid, and washed with ether. After adding sodium chloride to the aqueous layer, the mixture was extracted with ethyl acetate (50 ml). After dehydrating the obtained organic layer with sodium sulfate, concentrated to dryness,
The residue was treated with ether to give compound 7 as a white powder (460 m
g) was obtained. The obtained powder (100 mg) was crystallized with a mixed solvent of methanol and ether to obtain a crystal of Compound 7 (70 mg).

Melting point 147-148.5 ° C (decomposition) Optical rotation ▲ [α] ²² _D ▼ -28.1 ° (c = 0.50, methanol) IR (KBr): 3080,1745,1650,1580,1540 (cm ^-1 ) ¹ H-NMR
_{(DMSO-d 6) 12.66 (} 1H, br.), 8.71 (1H, d, J = 7.8Hz),
8.32 (1H, s), 7.85 (2H, m), 7.50 (3H, m), 4.52 (1H, dd
d, J = 5.2,7.8,9.3Hz), 2.76 (1H, dd, J = 5.2,14.8Hz),
2.67 (1H, dd, J = 9.3,14.8Hz) FD-MS m / z 277 (M + H) + Elemental analysis _{(C 13 H 12 N 2 O} 5) Calculated: C, 56.52, H, 4.38 , N, 10.14 Found: C, 56.50, H, 4.47 , N, 10.02 13 C NMR (DMSO-d 6) 172.66 (s), 171.71 (s), 166.2
5 (s), 151.94 (d), 133.77 (s), 131.33 (d), 12
8.20 (d), 127.22 (d), 93.36 (s), 52.21 (d), 2
3.64 (t) Example 7 Compound 1 (500 mg of monosodium salt) was dissolved in a 2% aqueous sodium hydrogen carbonate solution (50 ml), and acetic anhydride (270 μm) was dissolved.
l) was added and the mixture was stirred at room temperature for 30 minutes. Further, sodium hydrogen carbonate (200 mg) and acetic anhydride (100 μl) were added,
Stirred at room temperature for 30 minutes. After concentrating the reaction solution, Bio-Gel P-2 (100-200 mesh, 500 m
It was subjected to column chromatography in l). Eluted with water,
After fractionating and concentrating the effective fraction, freeze-drying and coarse powder (570m
g) was obtained. The obtained coarse powder is mixed with microcrystalline cellulose (200 m
The mixture was subjected to column chromatography in l), and eluted and fractionated with a solvent system of acetonitrile: water (85:15 to 70:30). HP
The fractions showing a single LC peak were collected, concentrated, lyophilized,
A white powder (245 mg) of compound 8 (disodium salt) was obtained.

^{IR (KBr): 3440,1640,1520cm -1 1} H-NMR (D 2 O) 7.98 (1H, s), 4.26 (1H, dd, J = 4.6,8.3H
z), 2.68 (1H, dd, J = 4.6,15.0Hz), 2.51 (1H, dd, J = 8.
3,15.0Hz), 2.01 (3H, s ) Elemental analysis _{(C 8 H 8 N 2 O} 5 Na 2 · 1.5H 2 O) Calcd: C, 33.70, H, 3.89 , N, 9.82, Na, 16.12 Found Value: C, 33.99, H, 3.63, N, 9.91, Na, 16.00 Example 8 Compound 1 (6.36 g of monosodium salt) was dissolved in a 50% aqueous dioxane solution (150 ml), and triethylamine (6.3 m
l), 2- (t-Butoxycarbonyloxyimino) -2
Phenylacetonitrile (11.4 g of BOC-ON)
Stir at room temperature for 3 hours. After concentrating the reaction solution to about 50 ml,
Water (300 ml) was added, and the mixture was washed three times with ethyl acetate (150 ml). After adjusting the aqueous layer to pH 2.7, sodium chloride was added and ethyl acetate (150
ml) three times. The obtained organic layer was washed with a saturated saline solution, dehydrated with sodium sulfate and concentrated to dryness. The residue was treated with n-hexane-ether to give compound 9 as a white powder (6.32 g).
I got

Optical rotation ▲ [α] ¹⁹ _D ▼ -29.3 ° (c = 0.54, methanol) IR (KBr): (. 1H , br) 3110,2990,1675,1580,1510cm -1 1 H-NMR (DMSO-d 6) 12.50, 8.23 (1H, s), 7.
06 (1H, d, J = 8.2Hz), 4.02 (1H, ddd, J = 5.1,8.2,9.4H
z), 2.57 (1H, dd, J = 5.1, 14.6 Hz), 2.43 (1H, dd, J = 9.
4,14.6Hz), 1.36 (9H, s ) SI-MS m / z 273 (M + H) + Elemental analysis _{(C 11 H 16 N 2 O} 6) Calculated: C, 48.53, H, 5.92 , N, 10.29 Found Value: C, 48.51, H, 6.05, N, 10.23 Example 9 Compound 9 (148 mg) was dissolved in trifluoroacetic acid (1 ml) and left at room temperature for 20 minutes. The reaction solution was concentrated to dryness, and the obtained residue was treated with ether to obtain a crude powder (136 mg). The coarse powder is dissolved in water (2 ml), adjusted to pH 7.0 by adding sodium hydrogen carbonate, and then microcrystalline cellulose (100 ml)
Column chromatography. After elution and fractionation with a solvent system of acetonitrile: water (85:15 to 80:20), the obtained active fraction was concentrated, and then Sephadex LH-20 (500 m
It was subjected to column chromatography in l). The fraction was eluted and fractionated with 20% aqueous methanol, fractions showing a single peak by HPLC were collected, concentrated, and lyophilized to obtain a white powder (56 mg) of compound 1 (monosodium salt).

Example 10 Compound 9 (1.0 g) was dissolved in dimethylformamide (10 ml), sodium carbonate (214 mg) and methyl iodide (0.69 ml) were added, and the mixture was stirred at room temperature for 2 hours. After concentrating the reaction solution, a 0.05 M phosphate buffer (pH 7.0, 100 ml) was added, and sodium hydrogen carbonate was further added to adjust the pH to 8.0. After washing the obtained aqueous solution twice with ethyl acetate (50 ml), the pH was adjusted to 2.8.
And extracted three times with ethyl acetate (50 ml). The obtained organic layer was washed with a saturated saline solution, dried over sodium sulfate and concentrated to dryness to obtain an N'-methyl form of Compound 2 (996 mg) as a colorless oil.

The obtained compound (996 mg) was treated with trifluoroacetic acid (3 ml)
And left at room temperature for 30 minutes. The reaction solution was concentrated to dryness, and the obtained residue was treated with n-hexane-ether to obtain a crude powder (982 mg). The coarse powder was dissolved in water (6 ml), adjusted to pH 6.9 by adding sodium hydrogen carbonate, and then subjected to column chromatography on microcrystalline cellulose (400 ml). Elution and fractionation with a solvent system of acetonitrile: water (85:15 to 80:20), concentration of the effective fraction,
-2 (100-200 mesh, 500 ml) column chromatography. The fraction was eluted with water and fractionated, and a fraction showing a single peak by HPLC was collected. The obtained aqueous solution was concentrated and freeze-dried to obtain a white powder of Compound 10 (367 mg).

Optical rotation ▲ [α] ²³ _D ▼ -42.8 ° (c = 0.58, water) ^{IR (KBr): 3430,3050,1720,1630,1600,1495cm -1 1} H-NMR (D 2 O) 8.13 (1H, s), 3.92 (1H, t, J = 5.8Hz),
3.58 (3H, s), 2.83 (2H, d, J = 5.8Hz) SI-MS m / z 187 (M + H) + Elemental analysis _{(C 7 H 10 N 2 O} 4 · 0.5H 2 O) Calculated: C, , 43.08, H, 5.68, N, 14.35 Found: C, 42.50, H, 5.79, N, 13.88 Example 11 Compound 2 (800 mg) was dissolved in pyridine (4 ml), and acetic anhydride (2 ml) was added. For 20 minutes. After adding 0.5N hydrochloric acid (80 ml) to the obtained reaction solution, the pH was adjusted to 2.3 and extracted with ethyl acetate (100 ml). The organic layer was washed with saturated saline, dehydrated with sodium sulfate, and concentrated to dryness. The residue was treated with ether to give Compound 11 as a white powder (875 mg).

IR (KBr): (. 1H , br) 3370,1770,1735,1685,1615,1520cm -1 1 H-NMR (DMSO-d 6) 12.70, 8.74 (1H, s), 7.
14 (1H, d, J = 7.0Hz), 4.13 (1H, m), 2.50-2.74 (2H,
m), 2.35 (3H, s ), 1.36 (9H, s) Elemental analysis _{(C 13 H 18 N 2 O} 7) Calculated: C, 49.68, H, 5.77 , N, 8.91 Found: C, 49.41, H , 5.70, N, 8.65 Example 12 Compound 11 (157 mg) was dissolved in methanol (3 ml).
A 5N aqueous sodium hydroxide solution (3 ml) was added, and the mixture was allowed to stand at room temperature for 30 minutes. The resulting reaction solution was adjusted to pH 6.4 with N hydrochloric acid, concentrated, and the mixture was added with saturated saline, adjusted to pH 2.5 with N hydrochloric acid, and extracted with ethyl acetate. The organic layer was washed with saturated saline, dehydrated with sodium sulfate and concentrated to dryness. The residue was n-hexane-
Treatment with ether provided compound 9 as a white powder (113 mg).

Example 13 Compound 1 (monosodium salt, 3.0 g, purity 92%) was suspended in hydrochloric acid-methanol (50 ml) and allowed to stand at room temperature for 20 hours. The reaction solution was filtered, the filtrate was concentrated, and the pH was adjusted to 6.2 by adding a saturated aqueous solution of sodium hydrogen carbonate. After the obtained solution was concentrated, it was subjected to column chromatography on microcrystalline cellulose (500 ml). Acetonitrile: water (90:10
~ 85: 15) elution and fractionation with the solvent system of approx.
After concentration, the mixture was subjected to column chromatography on microcrystalline cellulose (300 ml). The fraction was eluted with acetonitrile: water (9: 1), fractionated, and fractions showing a single peak by HPLC were collected.
The obtained solution was concentrated and freeze-dried to obtain a white powder of Compound 12 (722 mg).

^{IR (KBr): 3420,2970,1750,1630,1500cm -1 1} H-NMR (D 2 O) 7.97 (1H, s), 4.27 (1H, t, J = 5.7Hz),
3.84 (3H, s), 2.85 (2H, d, J = 5.7Hz) SI-MS m / z 187 (M + H) + Elemental analysis _{(C 7 H 10 N 2 O} 4 · 0.5H 2 O) Calculated: C, , 43.08, H, 5.68, H, 14.35 Found: C, 42.91, H, 5.20, H, 14.15 Example 14 Compound 7 (270 mg) was dissolved in methanol (10 ml).
An ether solution of diazomethane was added until the reaction turned yellow. After concentrating the reaction mixture to dryness, silica gel (15 g)
It was subjected to column chromatography. Elution with a solvent system of n-hexane: ethyl acetate (1: 1), fractionation, concentration to dryness,
A white powder of compound 13 (138 mg) was obtained. Further, elution with a solvent system of n-hexane: ethyl acetate (1: 4), fractionation, and concentration gave a colorless oily compound 14 (142 mg).

Compound 13: Optical rotation ▲ [α] ¹⁹ _D ▼ -38.5 ° (c = 0.49, methanol) ^{IR (KBr): 3340,1760,1640,1515cm -1 1} H-NMR (CDCl 3) 7.92 (1H, s), 7.75-7.80 (2H, m), 7.
40−7.54 (3H, m), 6.77 (1H, d, J = 6.7Hz), 4.93 (1H, dd
d, J = 4.9,5.3,6.7Hz), 4.01 (3H, s), 3.79 (3H, s), 3.0
4 (1H, dd, J = 5.3,15.0Hz), 2.87 (1H, dd, J = 4.9,15.0H
z), SI-MS m / z 305 (M + H) + Elemental analysis _{(C 15 H 16 N 2 O} 5) Calculated: C, 59.21, H, 5.30 , N, 9.21 Found: C, 59.16, H, 5.32 , N, 9.16 Compound 14: Optical rotation ▲ [α] ¹⁹ _D ▼ -26.5 ° (c = 0.54, methanol) ^{IR (KBr): 3350,1740,1665,1605,1535cm -1 1} H-NMR (CDCl 3) 7.88-7.93 (2H, m), 7.83 (1H, d, J =
7.2Hz), 7.63 (1H, s), 7.41-7.55 (3H, m), 4.90 (1H, d
t, J = 7.2,5.6Hz), 3.75 (3H, s), 3.37 (3H, s), 2.91 (2
H, d, J = 5.6Hz) SI-MS m / z 305 (M + H) + Elemental analysis _{(C 15 H 16 N 2 O} 5) Calculated: C, 59.21, H, 5.30 , N, 9.21 Found: C , 59.59, H, 5.22, N, 8.77 Example 15 Compound 6 (4.8 g) was dissolved in dimethylformamide (50 ml), p-nitrobenzylbromide (5.0 g) was added, and the mixture was stirred at room temperature for 1.5 hours. Further, p-nitrobenzyl bromide (5.0 g) was added, and the mixture was stirred at room temperature for 6 hours. After the reaction solution was concentrated, a saturated saline solution was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated saline, dehydrated with sodium sulfate, and concentrated to dryness.
The residue was treated with n-hexane to obtain 10.2 g of a crude powder. The resulting crude powder was subjected to silica gel (250 g) column chromatography, and n-hexane: ethyl acetate (25:75).
After washing with n-hexane: ethyl acetate (20: 80-0: 100)
Elution, fractionation and concentration with a solvent system of
0 mg) and a crude powder (2.4 g) of the p-nitrobenzyl column ester of Compounds 2 to 5. Both are subjected to silica gel chromatography (solvent system: chloroform-
Purified with ethyl acetate), powder of compound 15 (390 mg), powder of any one of p-nitrobenzyl esters of compounds 2 to 5 (compound 16,155 mg), mixture of three isomers of compound 16 (880 mg) And a mixture of compound 16 and three isomers (220 mg) was obtained.

Compound ^{15 IR (KBr) 3450,3090,2930,2860,1740,1605,1515cm -1 1} H-NMR (DMSO-d 6) 8.50 (1H, s), 8.22,8.18,8.15 ( each
2H, d, J = 8.6Hz), 7.63,7.60,7.56 (2H, d, J = 8.6Hz each),
5.24 (2H, ABq, J = 14.0Hz), 4.99 (2H, s), 3.93 (1H, br.
d, J = 15.0), 3.78 (1H, br.d, J = 15.0Hz), 3.51 (1H,
. m), 2.94 (1H, br), 2.54 (2H, m) Elemental analysis _{(C 27 H 23 N 5 O} 10) Calculated: C, 56.15, H, 4.01 , N, 12.13 Found: C, 56.18, H, 3.91, N, 12.03 compound 16 optical rotation _{^{▲ [α] 19 D ▼ +}} 44.9 ° (c = 0.52, ^{methanol) IR (KBr) 3250,1755,1705,1610,1520cm -1 1} H-NMR (DMSO _{-d 6) 10.83 (1H, s} ), 8.38 (1H, br.), 8.
25 (2H, d, J = 8.6Hz), 7.68 (2H, d, J = 8.6Hz), 7.29 (1
H, d, J = 6.2Hz), 5.32 (2H, s), 4.32 (1H, d, J = 8.3Hz),
3.24 (1H, dt, J = 6.2,9.2Hz), 2.52 (1H, m), 2.34 (1H,
m) SI-MS m / z 308 (M + H) + Elemental analysis _{(C 13 H 13 N 3 O} 6) Calculated: C, 50.82, H, 4.26 , N, 13.68 Found: C, 50.82, H, 3.98 , N, 13.62 compound 16 isomers three mixtures: optical rotation _{^{▲ [α] 19 D ▼ +}} 19.1 ° (c = 0.47, ^{methanol) IR (KBr) 3230,1755,1715,1610,1520cm -1 1} H _{-NMR (DMSO-d 6) 11.11} (0.5H, s), 11.80 (0.5H,
s), 8.37 (1H, br), 8.25 (2H, d, J = 8.5 Hz), 7.67 (2H, br)
d, J = 8.5Hz), 7.26 (0.5H, d, J = 6.2Hz), 6.70 (0.2H, d,
J = 6.2Hz), 6.67 (0.3H, d, J = 6.2Hz), 5.32 (2H, s), 4.
35 (1H, m), 3.73 (0.5H, m), 3.30 (0.5H, m), 2.82-1.8
4 (2H, m) Elemental analysis _{(C 13 H 13 N 3 O} 6) Calculated: C, 50.82, H, 4.26 , N, 13.68 Found: C, 50.99, H, 4.12 , N, 13.69 Example 16 Diisopropyl A solution of the amine (1.99 ml) in anhydrous tetrahydrofuran (20 ml) was added at -78 ° C under an argon atmosphere.
1.6 Mn-butyl lithium hexane solution (7.2 ml) was added,
Stirred at 0 ° C. for 10 minutes. After the solution was cooled again to -78 ° C, N-BOCL-glutamic acid dimethyl ester (1.32 g) anhydrous tetrahydrofuran (2
5 ml) solution was added dropwise through a stainless steel tube.
Ethyl formate (0.84 ml) was added to the solution, and the temperature of the reaction solution was returned to 0 ° C. over 1.5 hours. After further stirring at 0 ° C. for 1 hour, ethanol (5.0 ml) was added, followed by ether (40 ml). The obtained mixture was poured into a mixture composed of 2N hydrochloric acid (12 ml) and a saturated aqueous solution of ammonium chloride (30 ml). The ether layer was extracted with a 1% aqueous sodium carbonate solution (80 ml), and the extracted aqueous layer was washed twice with a mixture of ethyl acetate-hexane (1: 1) (50 ml), adjusted to pH 2.5-3, and extracted with ethyl acetate. Extracted. The obtained ethyl acetate layer is
The extract was washed twice with water (100 ml) and then with saturated saline (80 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a white powder of compound 17 (0.53 g). Powder was crystallized from ether-hexane Compound 17: Melting point 106-108.5 ° C IR (KBr) 3290,3000,1780,1760,1670,1440,1370,1300,1
260,1210,1150,1030,960,850,800,780 ¹ H-NMR (CDCl ₃ ) δ 9.90 (s), 9.70 (s), 6.99 (br.
s), 4.65 (dd, J = 10.5, 3.6Hz), 4.61 (dd, J = 10, 2.6H
z), 3.80 (s), 3.78 (s), 3.75 (s), 3.58 (dd, J = 1
0,4Hz), 2.99 (ddd, J = 15.5, 10.5, 2.2Hz), 2.72 (dt.J
= 14,9.5Hz), 2.62 (ddd, J = 14,3.7,3.2Hz), 2.59 (dd
d, J = 15.5,3.6,1.8Hz), 2.40 (dt.J = 14,9.5Hz), 2.11
(Ddd, J = 14,9.4,2.5Hz), 1.51 (s), 1.51 (s), 1.50
(S) Elemental analysis (C ₁₂ H ₁₇ NO ₆₎ Calculated: C, 53.13, H, 6.32 , N, 5.16 Found: C, 53.18, H, 6.26 , N, 5.06 EXAMPLE 17 N-BOC L-glutamic acid Dimethyl ester (112mg)
To a solution of anhydrous tetrahydrofuran (3.0 ml) was added a 1 M lithium bistrimethylsilylamide hexane solution (0.55 ml) at -78 ° C under an argon atmosphere, followed by stirring at -40 ° C for 20 minutes. The reaction solution was cooled again to -78 ° C, isopropyl formate (69 µl) was added, the temperature of the reaction solution was raised to -40 ° C over 1 hour, and the mixture was further stirred at -40 ° C for 3 hours. I
-Propanol (0.5 ml) was added, then diluted with ethyl ether (10 ml) and poured into a mixture of 1N HCl (0.5 ml) and water (10 ml). To this was added a saturated aqueous sodium hydrogen carbonate solution (2.5 ml), and the ether layer was partitioned and removed. The obtained aqueous layer was washed twice with ether (10 ml), and then washed with 2N hydrochloric acid to pH 2.0.
Adjusted to 8 and further saturated with saline. This was extracted three times with ethyl acetate (10 ml), and these were combined, washed twice with saturated saline (10 ml), dried over anhydrous sodium sulfate, and then dried.
After concentration under reduced pressure, compound 17 (57 mg) was obtained as a white powder.

Compound 17: SIMS (measured with a mixture of methanol and diethanolamine) M + H + DEA = 377 (DEA: diethanolamine, molecular weight 10
5) M + K = 310 Example 18 10% water-dioxane solution of compound 17 (378 mg) (10 m
To l) was added hydroxylamine hydrochloride (107 mg), and the mixture was stirred at room temperature for 1 hour. The reaction solution was diluted with ethyl acetate (50 ml), washed three times with water (25 ml), then with saturated saline (20 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain compound 18 (360 mg). .

At diethanolamine mixture - C, 50.68, H, 6.46 , N, 9.24 SIMS ( methanol: Compound 18 Elemental analysis _{(C 12 H 18 N 2 O} 6) Calculated: C, 50.35, H, 6.34 , N, 9.79 Found M + H + DEA = 392 (DEA: diethanolamine, molecular weight 10)
5) M + K = 325 The obtained compound 18 (76 mg) was dissolved in methanol (2.5 ml), 2N sodium hydroxide (0.10 ml) was added, and the mixture was stirred at room temperature for 2 hours. Dilute the reaction with ether (10 ml)
Poured into water (10 ml) and the aqueous layer was washed with diethyl ether (10 ml). The aqueous layer was adjusted to pH 3 with N hydrochloric acid, and then ethyl acetate (1
0 mL), washed twice with 1/2 saturated saline (10 ml), then with saturated saline (10 ml), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound 19 ( 57 mg) as a white powder.

Elemental analysis _{(C 12 H 18 N 2 O} 6) Calculated: C, 50.35, H, 6.34 , N, 9.79 Found: C, 50.11, H, 6.42 , N, 8.96 1 H-NMR (300MHz, CDCl 3) δ1.43 (9H, s), 2.71 (1H, dd,
J = 14.5,6.2Hz), 2.80 (1H, dd, J = 14.5,5.3Hz), 3.76
(3H, s), 4.43 (1H, m), 5.62 (1H, m), 7.94 (1H, br.s) IR (KBr) 3350,3100,2990,1710,1600,1520,1440,1400,1
370,1260,1220,1160,1050,1025,860,760,640cm ^-1 ▲ [α] ²⁰ _D ▼ -19.2 ° (c = 0.50, MeOH) Example 19 2N was added to a methanol solution (1.5 ml) of compound 19 (57 mg).
An aqueous sodium hydroxide solution (0.5 ml) was added, and the mixture was stirred at room temperature for 2 hours. The reaction solution was diluted with water (10 ml), washed twice with ether (10 ml), adjusted to pH 2.5, and extracted three times with ethyl acetate (10 ml). The obtained ethyl acetate layers were combined, and twice with a 1/2 saturated saline solution (10 ml).
Each was washed with l), dried using anhydrous sodium sulfate, and concentrated under reduced pressure to obtain Compound 9 (46 mg) as a white powder.

Example 20 Compound 9 (136 mg) was suspended in chloroform (5 ml), and triethylamine (146 μl), di-t-butyl dicarbonate (120 μl) and 4-dimethylaminopyridine (1.5 g) were added, and the mixture was stirred at room temperature for 1 hour. Stirred. Water was added to the obtained reaction solution to adjust the pH to 2.5, followed by extraction with chloroform. The obtained organic layer was washed with a saturated saline solution, dehydrated with sodium sulfate and concentrated to dryness. The obtained residue was washed with n-hexane to obtain a white powder of compound 20 (183 mg).

^{IR (KBr): 3380,3120,2990,1745,1620,1515cm -1 1} H-NMR (CDCl 3) 8.20 (1H, s), 5.58 (1H, d, J = 6.0H
z), 4.60 (1H, br.), 4.48 (1H, m), 2.93 (1H, dd, J = 5.
0,14.9Hz), 2.30 (1H, m ), 1.58 (9H, s), 1.44 (9H, s) Elemental analysis _{(C 16 H 24 N 2 O} 8) Calculated: C, 51.61, H, 6.50 , N , 7.52 Found: C, 51.71, H, 6.50, N, 7.70 Example 21 Compound 20 (15 mg) was dissolved in trifluoroacetic acid (100 μl) and allowed to stand at room temperature for 15 minutes. The reaction solution was concentrated to dryness,
When 0.1 M phosphate buffer (pH 7.0, 8.5 ml) was added to the residue and subjected to HPLC analysis, 7.8 mg of compound 1 was detected.

Example 22 Compound 20 (16 mg) was suspended in a 1% aqueous sodium hydrogen carbonate solution (3 ml) and stirred at room temperature for 30 minutes. When the obtained solution was subjected to HPLC analysis, 10.6 mg of compound 9 was detected.

EFFECT OF THE INVENTION The novel antibiotic TAN-950 of the present invention and salts thereof exhibit antibacterial activity against fungi, and these fungicidal infections can be caused by administering orally, parenterally or topically to humans and other animals. Can be used advantageously for the treatment of

[Brief description of the drawings]

Fig. 1 shows the HPLC pattern of each component of TAN-950, and Fig. 2 shows the TAN-950.
-950 HPLC pattern showing conversion of each component in aqueous solution. FIG. 3 shows antibiotic TAN-950A obtained in Example 2.
UV absorption spectrum of monosodium salt (in water), FIG. 4 shows infrared absorption spectrum (KBr method), FIG.
The figure shows the ¹ H nuclear magnetic resonance spectrum (400 MHz, in heavy water), No. 6.
The figure shows the ¹³ C nuclear magnetic resonance spectrum (100 MHz, in heavy water).

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12P 17/14 C12P 17/14 // (C12P 17/10 C12R 1: 465) (C12P 17/14 C12R 1: 465) (58 6) Fields investigated (Int. Cl. ⁶ , DB name) C12P 17/00-17/18 C07D 207/28 C07D 261/12 A61K 31/40 A61K 31/42 BIOSIS (DIALOG) WPI (DIALOG) CA (STN) REGISTRY (STN)

Claims (6)

(57) [Claims] (1) The following formula (I) or (II) [Wherein -COOR ¹ represents a carboxyl group which may be esterified, R ² and R ³ represent hydrogen or an N-protecting group, and R ⁴
Represents NOH or O, respectively. However, when -COOR ¹ is carboxyl and R ² is hydrogen in the formula (I), R ³ is N
-Represents a protecting group. Or a salt thereof. 2. The compound or a salt thereof according to claim 1, wherein -COOR ¹ is a carboxyl group, R ² is hydrogen and R ⁴ is NOH. (3) The present invention belongs to the genus Streptomyces, and (2).
A method for producing the compound or a salt thereof, comprising culturing a microorganism having the ability to produce the compound described in a medium, producing and accumulating the compound in a culture, and collecting the compound. (4) [Wherein, -COOR ¹ represents a carboxyl group which may be esterified, and R ² represents hydrogen or an N-protecting group. A compound represented by the formula: [Wherein -COOR ¹ represents a carboxyl group which may be esterified, R ² represents hydrogen or an N-protecting group, and R ³ represents hydrogen. ] Or a salt thereof. 5. A compound of the following formula (III) or (IV) [In the formula, -COOR ¹ 'represents an esterified carboxyl group, and R ² ' represents an N-protecting group. Carbonylation of a compound represented by the formula [Wherein -COOR ¹ ′ and R ² ′ have the same meaning as described above;
⁴ represents NOH or O. ] The manufacturing method of the compound represented by this. (6) Wherein -COOR ¹ is a carboxyl group, R ² is hydrogen, R ³
Represents hydrogen. A compound represented by the formula: Wherein -COOR ¹ is a carboxyl group, R ² is hydrogen, R ⁴
Indicates NOH, respectively. Or a salt thereof, for treating a fungal infection.