JPS604719B2 - Method for producing antibiotic PS-5 with β-lactamase inhibitory activity - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel antibiotic and its derivatives, and more particularly, it has strong antibacterial activity and 8-lactamase inhibitory activity, and is effective against penicillin-type and 8-lactamase-producing bacteria.
The present invention relates to a method for producing antibiotic PS-5, which has the ability to synergistically enhance the antibacterial activity of antibacterial substances such as cephalosporins.

Several antibiotics or 8-lactamase inhibitors having B-lactamase inhibitory activity have been known. For example, MC696-SY2 collected from the culture solution of MC696-SY phantom substance-producing bacteria belonging to the genus Streptomyces.
1A and B (Japanese Patent Publication No. 51-24597), MM4550 or MM1390 collected from a culture of Streptomyces MM4550 or MM13902-producing bacteria
2 substances (Unexamined Japanese Patent Publication No. 135294/1983 and Special Publication No. 1983-
140692), clavaras acid collected from a culture of Streptomyces clavarigelus (Japanese Patent Publication No. 52-1996), etc. have been reported. In addition, chenamycin (thi) is an antibiotic with a penicillin-like skeleton.
cape mycin) has also been reported (Tokubatsu 51-73).
Publication No. 191). This time, the present inventors have developed a novel antibiotic that has physicochemical properties different from the above-mentioned known antibiotics with 3-lactamase inhibitory activity or 3-lactamase inhibitors, and has stronger antibacterial activity and 8-lactamase inhibitory activity. was discovered and named the antibiotic PS-5. Thus, the first objective of the present invention is to provide a novel antibiotic PS-5 with strong antibacterial activity and 3-lactamase inhibitory activity.

A second object of the present invention is to provide derivatives of the antibiotic PS-5, especially trityl derivatives, which have strong antibacterial activity and B-lactamase inhibitory activity.

Still another object of the present invention is to synergistically enhance the antibacterial activity of antibacterial substances such as penicillins and cephalosporins against 6-lactamase-producing resistant bacteria that have strong antibacterial activity and 3-lactamase inhibitory activity. The object of the present invention is to provide a novel antibiotic PS-5 and its trityl derivatives, which have the ability to obtain trityl derivatives.

Another object of the present invention is to provide a method for producing the antibiotic PS-5 by fermentation. Still another object of the present invention is to provide a method for producing the trityl derivative of the antibiotic PS-5. Still another object of the present invention is to use the above antibiotic PS-5 or its derivatives to prevent, treat and/or treat Gram-positive and Gram-negative bacterial infections.

Other objects and advantages of the invention will become apparent from the following description. According to the present invention, a novel antibiotic PS-5 is provided that has a broad spectrum of strong antibacterial activity and 8-lactamase inhibitory activity.

This antibiotic PS-5 is characterized by the physicochemical properties and biological properties shown below. Physicochemical properties of antibiotic PS-5 {1'Soluble antibiotic PS-5 is soluble in water at pH 6-9.

That is, the present antibiotic is not only water with a pH of 7, but also an aqueous medium adjusted to a slightly acidic pH of 6 or higher with, for example, hydrochloric acid, and, for example, sodium bicarbonate,
It is compatible with aqueous media adjusted to a slightly alkaline pH of 9 or less using sodium hydroxide or the like. Furthermore, this antibiotic is substantially insoluble in ethyl acetate and benzene.

4■ Thin layer chromatography (TL
C) When antibiotic PS-5 (sodium salt) is subjected to TLC using the TLC plate and solvent shown below, it shows the Rf value shown below.

Note that the mixing ratio of the mixed solvent used in this specification is a volume ratio unless otherwise specified. {a' Pre-coated glass plate [Adsorbent: Crystalline cellulose (average grain size: 6 to 10 French), thickness: 0.2 to 0.25 side, In this specification, Avicel manufactured by Funakoshi Yakuhin ■ is used as the main plate
Use SF cellulose thin layer plate] n-butanol/
Ethanol/water (7/7/6) Rf = 0.94i-propanol water (7/3) Rf = 0.96'b} Pre-coated glass plate [Adsorbent: silica gel, thickness: 0.25 ribs, book In this specification, as a plate,
Merck & Co., Ltd. DC-Felteitzchplatten-Kiese,
Lugel (DC-Femg-plaUenKiesel degree 1) 6 cape meat 4 used] Ethanol/water (7/3)
Rf=0.82n-propanol/water (7/3)Rf=
0.77 legs Ba-pa chromatography-Antibiotic PS-
5 (sodium salt) is Toyo Paper No. 5 (weight: 140 tano, thickness: 0.25 side, ash content 0.1%, Toyo Paper). 50 (manufactured by Toyo Roshi ■) was used and developed with the following solvent by the descending method, it showed the following Rf value.

n-propanol/water (7/3) Rf=0.68n-
Propanol/i-propanol/water (7/7/6)
Rf=0.70 Acetonitrile/Water (8/2) Rf=0.36 Acetonitrile/Trisno-EDTA (Kono 1) Rf=0.34 Ethanol/Water (7/3) Rf=0.63 [Note 1: Acetonitrile 120 [, 30 Z of 1/10M tris(hydroxymethyl)amide/methane monohydrochloride buffer with pH 7.5 and 1 volume of 1/10M ethylenediaminetetraacetic acid sodium salt aqueous solution with pH 7.5] {41 High pressure Using a high-pressure furnace paper electric device (manufactured by Savant Instruments, high-voltage power supply HV3000A, lacquerware FP180A), use furnace paper [weight: 140 mm / thickness, thickness: 0.25 skin,
The ash content is 0.1%, and Toyoro Paper N is used in this specification as this Roko paper.
o. 50 (made by Toyoro Paper ■) and antibiotics - 5
When (sodium salt) is subjected to electrophoresis, it exhibits the following behavior in a buffer solution with the following pH.

In a buffer solution with a pH of 8.6 consisting of 3000 g of water, 3.3 g of barbital, and 25.5 g of barbital sodium, when electricity is applied for 3 times at 42 V/c to the anode side, at least 5 side, usually 10 ~ Move within the range of 4 things.

{5] Behavior towards 8-lactamases Inactivated by the 8-lactamases of B. vulgaris, C. freundii and Bacillus cereus.

Based on these physicochemical properties, the antibiotic PS-5 of the present invention
is presumed to be an acidic substance having an 8-lactam ring or a similar ring structure.

Furthermore, the antibiotic PS-5 tends to be unstable when isolated, for example, at room temperature;
It is quite stable at low temperatures below 0.000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000 000 Then, the antibiotic P
The antibacterial activity of S is retained at least 50%, usually more than 75%. Biological properties of antibiotic PS-5 '1' Antibacterial spectrum The antibiotic PS-5 of the present invention has a wide range of antibacterial activity,
It shows very strong antibacterial activity against various microorganisms, such as Gram-enteric bacteria belonging to the genera Staphylococcus, Diplococcus, Streptococcus, Sarcina, Bacillus, etc., and also Gram-negative bacteria belonging to the genera Alcaligenes, Comamonas, etc. It also shows very strong antibacterial activity against bacteria.

Furthermore, the antibiotic PS-5 of the present invention exhibits a fairly strong antibacterial activity against Gram-negative bacteria belonging to the genus Escherichia, Klebsiella, Broteus, and the like.

In particular, the antibiotic PS-5 of the present invention is a gram-negative bacterium that is resistant to antibiotics having a B-lactam ring, such as belonging to the genus Citrobacter, Proteus, Enterobacter, Klebsiella, Serratia, etc. It is unique in that it exhibits strong antibacterial activity against humans.

'2} Enhancement of the antibacterial activity of other antibiotics against 8-lactamase producing bacteria The antibiotic M-5 of the present invention is effective against Citrobacter freundii, Proteus. vulgaris, Enterobacter aerogenes, Serratia marcescens, and other 8-lactamase producing bacteria.
In particular, it has the ability to enhance the antibacterial activity of 8-lactam antibiotics such as penicillins and cephalosporins, and in many cases, its ability is synergistic.

When administered to mice infected with '31 active pathogenic Gram-positive bacteria in vivo, a remarkable therapeutic effect was observed.

[4' virulence Approximately 2 of the effective dose to treat infection with pathogenic Gram-positive bacteria]
No significant toxicity was observed when mice were administered 0 and 4 doses of the drug.

According to the present invention, antibiotic PS-5 having the above-mentioned properties can be obtained by culturing antibiotic tower-5 producing bacteria in a nutrient tower, and from the culture, antibiotic PS-5 having 8-lactamase inhibitory activity can be obtained. It can be produced by a method consisting of taking 15.

The antibiotic PS-5 producing bacteria used in the present invention is the antibiotic PS-5 which has the above-mentioned physicochemical properties and biological properties.
Bacteria belonging to any genus can be used as long as they have the ability to produce microorganisms, and can be selected from a wide range of microorganisms.

Therefore, a search for a strain suitable for the purpose of the present invention can be carried out as follows, and a person skilled in the art can:
The antibiotic PS-5 producing bacteria used in the present invention can be easily obtained. That is, a culture solution of soil-isolated bacteria is assayed using a bioassay agar plate in which 8-lactam-sensitive bacteria are used as assay bacteria and a bioassay agar plate in which 8-lactamase is added. and search for soil isolates that give a culture solution with a smaller inhibition circle on the agar plate of the latter than that of the former.

Next, the active ingredients in the culture solution of the isolated bacteria are adsorbed onto activated carbon, and the eluate concentrate is developed using paper chromatography or thin layer chromatography to perform bioautograph analysis using 6-lactam-sensitive bacteria as the test bacteria. - If the antibiotic PS-5 is detected, the bacterium can be said to be an antibiotic PS-5 producing bacterium that can be used in the method of the present invention. This search method will be further explained using a specific example as follows.

A Comamonas assay plate (described later) was used as a bioassay agar plate for 8-lactam-sensitive bacteria, a Comamonas CV assay plate to which 3-lactamase produced by Proteus vulgaris P-5 was added, and a Citrobacter freundii E-9 assay plate. A Comamonas CM assay plate containing the produced 8-lactamase is prepared.

On the other hand, the culture solution of the soil isolate was made into a pulp disk with a diameter of 8 sides, placed on each test plate, and
After culturing for a period of time in the field, soil isolates given a culture furnace solution were given an inhibition circle on the Comamonas assay plate, and the inhibition circle on the Comamonas CV assay plate or the Comamonas CM assay plate was smaller than the inhibition circle on the Comamonas assay plate. elect. Next, special Shirasagi activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.) corresponding to 2% (W/V) of the furnace solution was added to the culture solution of the soil-isolated bacteria.
After stirring for a minute, the precipitate is collected by centrifugation, washed with distilled water of the same volume as the culture solution used, and centrifuged again to collect the precipitate.

To this precipitate was added 50% (V/V) acetone water in an amount equivalent to half the volume of the culture solution used above, and the mixture was mixed with three powders at room temperature and then centrifuged to obtain a top light. This supernatant liquid was concentrated using a rotary evaporator to a concentration of 30 to 360 ml to obtain a concentrated liquid that was 2 times stronger than the culture furnace liquid used above. This concentrated liquid was applied to Toyoro Paper No. 50 (manufactured by Toyoro Paper Co., Ltd.) and 80% acetonitrile/Tris/EDTA [acetonitrile 120, pH 7.5 1/10M Tris(hydroxymethyl)aminomethane-hydrochloric acid buffer 30', pH
7.5 1'loM, a descending method vapor using a solvent consisting of 1'loM of sodium ethylenediaminetetraacetic acid salt aqueous solution]
After the chromatography was carried out for a period of time, bioautography was carried out using Comamonas terrigena B-996 as a test bacterium. Then, soil isolates showing an inhibition zone at the same migration distance (at the Rf value) as the antibiotic PS-5 are selected as antibiotic PS-5 producing bacteria candidates. The candidate bacteria selected in this way are further subjected to paper chromatography and thin layer chromatography to confirm the productivity of antibiotic PS-5.

This allows those skilled in the art to easily search for antibiotic PS-5 producing bacteria that are suitable for the purpose of the present invention.

Typical antibiotic PS-5 producing bacteria searched as above include antibiotic PS-5 producing bacteria belonging to the genus Streptomyces, and a suitable example thereof is:
The strain that the present inventors assigned the number A271 is an actinomycete isolated from Shijo collected near Eiheiji Temple in Yoshida District, Fukui Prefecture.

The mycological properties of this A271 strain are as follows.

1 Morphological characteristics Branching state of spore-forming hyphae: Shape of simple branched spore-forming hyphae: Aerial hyphae tip is hook-shaped
), loops or incomplete spirals, forming section RA (Section Retinac mountain i).
It is considered to belong to apere i).

In particular, this form is koji-matsuri when cultured on oatmeal agar and glycerin-asparagine agar medium. However, on yeast/malt agar medium, it tends to be straight or curved (fl).
The form of ex side US) is often seen. Shape of spores and number of chains: 10 or more spores in circular or cylindrical shape (
Usually 10 to 50 pieces).

Spore size and surface structure: 0.8-1.0 x 1.
0-1.8 micron, smooth surface. Flagellated spores and sporangia are not observed.

The epiphyte location of the sporophyte is on the aerial hyphae.

2 Culture characteristics: The culture characteristics of the A271 strain are summarized in Table 1 below.

In Table 1 below, unless otherwise specified, 28℃
shows the competition results after 2 weeks of culture. Also, the color expression is mainly Japanese. D. Tresner and EJ Butkus (J.D.
Tresner & E. J. ``Systems of Color Wheels for Streptomycete Taxonomy'' (Sys.
r Tax to WheelsforSVeptomyce
The method of ``color standard'' published by the Japan Color Research Institute was also followed.

nine . - 3 Physiological characteristics [1' Growth temperature range: 10-400○, optimal 20-30
00[2} Liquefaction of gelatin (glucose, beptone,
gelatin tower above ground): liquefy (20qo culture) '31
Hydrolysis of starch (starch agar tower above ground): Decomposes {4) Coagulation and veptonization of skim milk: Coagulation is not observed, but veptonization occurs.

■ Production of melanin-like pigments: Tyrosine agar, Beptone
Yeast does not produce melamine-like pigments in iron agar tower ground and tryptone yeast extract broth.

'61 Assimilation of the following carbon sources (on Pridham-Gotlieb agar medium): L-arabinose 10D-xylose 10D-glucose + D-fructose sucrose ± inositol L-rhamnose 10-raffinose D-mannit (10: well assimilated; Soil: Slightly assimilated, difficult to assimilate) As is clear from the above characteristics, the A271 strain is
It is a strain belonging to the genus Streptomyces, the spore-forming hyphae are in the shape of section RA, the color of the surface of the bacterial flora is yellow or red, the spore surface is smooth, and it does not produce melanin or other aqueous pigments. It is a strain belonging to the group.

A bacterial species with the mycological characteristics of the above-mentioned bacterial strain is S. “The Actinomycetes” by A. Waksman (T.
heActjnomycetes Volume 2 (1961), E. B. Shir-ling and D. A paper co-authored by Wtlieb (International Journal of Systematic Bacteriology)
Volume 18, pages 69-189 (196 Mother King), Volume 27
pp. 9-392 (1968 King), Vol. 19, No. 391-5
12 pages (196g hand) and Volume 22, Nos. 265-39
4 pages (i972), as well as Purge's Manual of Determinative Bacteriology.
s N is nual of Detenni cape tiv
eBacteriology) 8th edition (197 French edition), Actinomyces cremeus (Actinomyces cremeus) was found as a similar bacterial species belonging to section RA.
escremeus), Actinomyces flavidovirens (Actinomyces flavidovirens)
ens), Actinomyces alboherbatus ((A
ctinomycesal or helvat), Actinomyces/flabescens ((Actinomyces
navescens), Streptomyces rutgersensis
sis), Streff.

Tomyces chryseus ((Streptomyces)
Streptomyces helvaticus, Streptomyces helvaticus
US) etc. were mentioned. Also, in terms of morphological properties, it is a bacterial species that belongs to section RF, but if you pay attention only to its culture and physiological characteristics, Streptomyces pluricolorescens ((Streptomyces pluricolorescens)
cens) is also mentioned as one of the similar bacterial species. Among these eight cocoon species, except for the last one, Streptomyces pluricolorescens, the aerial hyphae are said to exhibit a straight shape at times and a loop shape at other times, depending on the culture conditions. Therefore, these eight standard strains and A271 according to the present invention
The strains were cultured under the same conditions and compared. As a result, these strains differ in growth on various agar media, color of aerial hyphae, and color of basal hyphae, as well as significant differences in carbon source utilization, and are clearly different from the A271 strain mentioned above. .

Therefore, we identified two bacterial species that are considered to be most similar to the A271 strain, Actinomyces cremeus ISP5147 and Actinomyces flavidvirens ISP5150, and the A271 strain.
Comparative test results with 71 bacterial strains are shown in Tables 2 and 3 below.
and as shown in Table-4. Table 2 Table 3 Table 4 As is clear from the results shown in the table above, when comparing the color of aerial mycelium, Actinomyces flapidovirens is generally white, and even when yellow, the yellow has a green tinge. It is clearly different from the A271 strain of the present invention, and Actinomyces cremes generally has a weaker pale yellow red color than the A271 strain, and a clear difference between it and the A271 strain is observed. It will be done.

Regarding the color of the basal hyphae, the A271 strain generally exhibits a bright yellow color, whereas Actinomyces flavidovirens exhibits a pale yellow color, and many Actinomyces and Cremeus exhibit a bright light yellow color. is clear. Furthermore, if the test results for each tower site are compared in detail, the differences will become even clearer. In addition, as shown in Table 14, the A271 cocoon strain is different from Actinomyces cremeus.
It differs from Actinomyces flavidopyrens in its ability to assimilate -fructose and L-rhamnose, and in its ability to assimilate D-fructose and inositol. As described above, the A271 strain of the present invention is clearly different from the two most similar known bacterial strains. After all, among the known actinobacterial species, there is no bacterial species that exhibits the same properties as the A271 strain.

Therefore, the above A271 strain is recognized as a new bacterial species, and the present inventors identified it as Streptomyces sp. A271 (S
treptmycessp. A271).

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology under the microbial accession number 3984. In the present invention, not only the A271 strain itself, but also its natural mutants or mutants produced by chemical or physical treatments can be used.

The antibiotic PS-5 of the present invention can be obtained from antibiotic PS-5 producing bacteria, such as the above-mentioned Streptomyces Sp. It is produced by inoculating A271 spores or hyphae into a nutrient-containing medium and growing aerobically.

As the nutrient source, those commonly used as nutrient sources for actinomycetes, such as assimilable sources such as carbohydrates, nitrogen sources, and inorganic salts, can be used.

For example, glucose, glycerin, maltose, bran, bran,
Carbon sources such as carbohydrates such as dextrin and starch, oils and fats such as soybean oil, peanut oil, and lard; beptone,
Nitrogen sources such as meat extract, soybean flour, cottonseed flour, dried yeast, corn steep liquor, yeast extract, skim milk, casein, sodium nitrate, ammonium nitrate, ammonium sulfate: inorganic salts such as dicalium phosphate, salt, calcium carbonate, magnesium sulfate can be used, and if necessary, additional metals such as cobalt and manganese can be added.
As a nutrient source, any nutrient source that can be used by antibiotic PS-5-producing bacteria to produce antibiotic PS-5 can be used, and any known culture material for actinomycetes can be used. can. Further, in order to suppress foaming during heat sterilization and culturing, an antifoaming agent such as silicone or vegetable oil may be added. The blending ratio of the nutrient sources as described above is not particularly restricted and can be varied over a wide range, and those skilled in the art will be able to determine the optimal nutrient source composition and blending ratio for the antibiotic PS-5 producing bacteria used. It can be easily determined by simple small-scale experiments.

In addition, the nutrient tower soil can be sterilized prior to cultivation, and the pH of the culture medium can be adjusted to a range of 4 to 9, especially at
It is advantageous to adjust H to a range of 6 to 8.

In principle, the antibiotic PS-5 producing bacteria can be cultured in such a nutrient tower according to the method commonly used in the production of antibiotics using general actinomycetes.

It is usually preferable to culture under aerobic conditions, and it is usually possible to carry out the culture while crossing the ocean and/or while aerating.
In addition, as a culture method, any of static pupil culture, shaking moat culture, and submerged culture with aeration can be used, but submerged culture is advantageous. H. The culture temperature that can be used is not particularly limited as long as the growth of antibiotic PS-5 (sodium salt) is not substantially inhibited and antibiotic PS-5 can be produced, and the culture temperature is not particularly limited, depending on the production strain used. It can be changed depending on the time, but generally 20 to 40 o'clock, preferably 25 o'clock
Temperatures within the range of -390°C are preferred.

Further, in order to carry out the culture suitably, the number of the culture can be adjusted to a range of 4 to 9, particularly 6 to 8, as necessary during the culture. In the case of large-scale mass culture, it is advantageous to perform a seed culture as appropriate, inoculate it into a nutrient tower, and perform liquid culture.

Cultivation can normally be continued until sufficient antibiotic PS-5 has accumulated.

The culture time varies depending on the composition of the medium, the culture temperature, the production strain used, etc., but is usually in the range of 30 to 9 hours. As for the culture conditions to be used, those skilled in the art can easily determine the optimal conditions through simple experiments, depending on the characteristics of the production strain used.

The amount of antibiotic PS-5 accumulated during culture can be quantified by the bioassay method and bioautography described below, thereby making it possible to easily determine the optimum amount of antibiotic PS-5 accumulated. Thus, since the antibiotic PS-5 accumulated in the culture is water-soluble and mainly exists outside the bacterial cells, it is advantageous to use methods known per se, such as incubation, far-D separation, extraction, etc. after t-incubation. The bacterial cells are removed using a separation method and recovered from the furnace liquid, toplight liquid, extract liquid, etc.

Recovery can be carried out by various methods known per se,
In particular, methods often used for recovering carboxylic acid type antibiotics are advantageously applied.

For example, solvent extraction with ethyl acetate, n-butanol, etc. at low pH and transfer from the solvent layer to the high pH aqueous layer:
Lipid quaternary ammonium salts such as penzalkonium chloride, tetrabutyzyl ammonium hydrogen sulfate, or
Solvent extraction with methylene chloride, chloroform, etc. at neutral pH in the presence of crown compounds such as crown ether dicyclohexyl-18-crown-6 and identical 15-crown-5 (manufactured by Nippon Soda) and iodization from the solvent layer. Transfer to neutral water layer containing sodium, potassium iodide, etc.: Activated carbon, Amberlite XAD (Roam & Co., Ltd.)
Adsorption with Diaion HP-20 (manufactured by Mitsubishi Kasei), etc., and elution with methanol water, acetone water, etc.; Dowex 1×2 (manufactured by Dow Chemical),
QAE-Sephadex A-25 (manufactured by Fulmasha)
Adsorption and elution with ion exchange resins such as Sephadex G-10 (manufactured by Pharmasha), Bio-Gel P-2
(manufactured by Bio-Rad), Bio-Beads S1 x 3 (manufactured by Bio-Rad), etc.; cellulose;
Avicel SF (manufactured by American Viscose), DEA
E-cellulose Whatman DE-32 (manufactured by Whatman), DEAE-Sephadex A-25 (manufactured by Pharmacia), column or thin layer chromatography using silica gel, alumina, etc.; hexasol, petroleum ether (boiling point range 30-60 qo) ), etc.; freeze-drying method; and the like may be used alone or in appropriate combinations, and in some cases may be used repeatedly.
The behavior of Antibiotic Kyo-5 during the recovery and purification process can be quantitatively measured by the bioassay method and bioautography described below.

An antibiotic PS-5 is thus obtained which has the properties described above.

As mentioned above, this antibiotic PS-5 is somewhat unstable, so sufficient care must be taken in the recovery and purification process.

Since the salt form of this antibiotic PS-5 is generally more stable than the free form, it can be used for the medicinal purposes described below, or as an intermediate for further conversion into derivatives. Alternatively, when subjecting it to the above-mentioned purification process, it is preferable to treat it in the form of a salt.

Examples of such salts include metal salts such as sodium salts, potassium salts, magnesium salts, aluminum salts, etc.
Ammonium salts; substituted ammonium salts such as trimethylammonium salt; salts with organic bases such as penzathine salt and brocaiso salt; particularly preferred salts are sodium salts and potassium salts.

Furthermore, as described above, the antibiotic PS-5 of the present invention is presumed to be an acidic antibiotic, and therefore, like known acidic antibiotics such as clavalanic acid, various alcohols and mercaptans or their It is presumed that it can change into the form of an ester due to its ester-forming conductor.

Therefore, the scope of the present invention also includes these esters. Further, according to the present invention, when the antibiotic PS-5 is converted into trityl (triphenylmethyl) to introduce a trityl group into the molecular structure, the stability of the antibiotic M-5 is significantly increased. It was discovered that trityl derivatives can be easily isolated and purified, and that their trityl derivatives have strong antibacterial activity and 8-lactamase inhibitory activity.

This also makes it possible to identify and confirm the antibiotic pipe 5 of the present invention. Thus, according to the present invention, there is provided a method for producing an antibiotic PS-5 trityl derivative having B-lactamase inhibitory activity, which comprises reacting said antibiotic Kamo-5 with a reactive derivative of triphenylmethane.

As the reactive derivative of triphenylmethane that can be used in the above method, any functional derivative of triphenylmethane having a reactive group capable of reacting with a functional group such as a carboxyl group can be used, e.g. In the formula, Y
is a cleaving-off atom or group, and compounds of the formula are preferred.

Examples of the leaving atom or group (Y) in the above formula {1) include halogen atoms such as chlorine, bromine, and iodine; hydroxyl groups, thiol groups, etc., with halogen atoms being particularly suitable.

Therefore, the following can be mentioned as representative examples of the compound of the above formula m.

trityl alcohol, trityl mercaptan, trityl chloride, trityl bromide, etc.

The reaction between the antibiotic PS-5 and the above-mentioned reactive converter of triphenylmethane is a reaction known per se that is often used to introduce trityl groups into organic compounds having functional groups such as carboxyl groups, amino groups, hydroxyl groups, etc. This can be done according to the method.

Although the reaction of the antibiotic PS-5 with the reactive converter of triphenylmethane can be carried out in the absence of a medium, it is generally preferred to carry out the reaction in an inert liquid medium, and the inert liquid medium that can be used is For example, hydrocarbons such as benzene, toluene, n-hexane, and cyclohexane; halogenated hydrocarbons such as chloroform and methylene chloride; amides such as dimethylformamide and hexamethylphosphotriamide; dimethyl sulfoxide: jetyl ether, diisopropyl Ethers such as ether, di-n-butyl ether, tetrahydrofuran, and dioxane; Esters such as ethyl acetate and n-butyl acetate;
Examples include ketones such as acetone and methyl ethyl ketone, and these liquid media may be used alone, or two or more types may be mixed as necessary.

The reaction temperature is not critical and can be varied widely depending on the type of the reactive derivative used, the type of liquid medium, etc., and can be arbitrarily selected within a temperature range in which the antibiotic PS-5 does not decompose significantly. However, it is generally advantageous to use a temperature of 6030° C. or lower, preferably in the range of 0 to 40° C., and more preferably in the range of 5° C. to room temperature.

Further, in the above reaction, a reaction accelerator such as trimethylamine, triethylamine, pyridine, dicyclohexylcarbodiimide, etc. may be added as necessary.

Under such conditions, the reaction generally takes place within 1 to 24 hours, usually 3 to 24 hours.
It can be completed in one child's time.

Note that the antibiotic PS-5 to be reacted with the reactive derivative of triphenylmethane does not necessarily need to be isolated; The culture medium after isolation of the body can be used or the crude antibiotic PS-5 purified at least partially according to the isolation and purification method described above.
You can also use

Such partially purified products include, for example, activated carbon adsorption elution concentrate of the culture solution; adsorption and elution concentrate of the culture solution Diaion HP-20 (manufactured by Mitsubishi Chemical Corporation); Extract concentrate with methylene chloride in the presence of penzalkonium chloride; Extract concentrate with chloroform in the presence of crown compound; Examples include butanol extract concentrate at pH 3.5 at low temperature. The antibiotic PS-5 trityl derivative thus obtained can be separated from the reaction mixture or purified by various methods known per se in the antibiotic field.

For example, after the reaction is completed, the reaction solution is first poured into an aqueous medium in order to remove water-soluble impurities such as by-products. At that time p
In order to keep H substantially neutral, it is desirable to use a neutral buffer as the aqueous medium. This mixture is then treated with a non-polar organic solvent that is substantially immiscible with water, such as ethyl acetate, benzene, or chloroform, to form the antibiotic PS.
The -5-trityl derivative is extracted into the organic solvent layer. During this extraction, salts such as common salt and ammonium sulfate may be added to enhance the extraction efficiency due to the salting out effect. After drying the solvent layer with dehydrated moss salt, etc., the solvent layer is dried with a method known per se, for example, Bio-Bead S.
~X3 (manufactured by Bio-Rad), Cephadex LH-2
0 (manufactured by Pharmasha); adsorption chromatography using silica gel, alumina, Florisil (manufactured by Florizil, USA) as a carrier, etc., in combination as appropriate and repeated as necessary, to obtain the trityl derivative. Can be isolated.

Thus, the purified trityl derivative can be further recrystallized from solvents such as benzene, toluene, xylene, ethyl acetate, diethyl ether, methylene chloride, chloroform, hexane, petroleum ether (boiling point range: 30-60 do) alone or in combination. This allows purification to even higher purity.

The antibiotic PS-5 trityl derivative produced as described above is a novel substance that contains a trityl group (triphenylmethyl group) in its molecular structure and has not been described in any literature, and has strong antifungal activity and 8-lactamase inhibition. It is extremely unique in that it has activity.

The physicochemical and biological properties of the trityl derivative are as follows.

Physicochemical properties of the antibiotic PS-5 trityl derivative A Melting point The above trityl derivatives usually exhibit a melting point within the range of 83 to 88°C.

Note that the melting point indicated in this specification is based on a value measured by the Koffler method at a temperature increase rate of 1° C. per minute (measuring device: BY-1 model melting point measuring device manufactured by Yazawa Scientific Instruments Co., Ltd.). B. Infrared absorption spectrum with CH3 state'maX2 315.5 nm, C. Infrared absorption spectrum in chloroform shows the following characteristic maximum absorption wave number, 3430,3100-300
0,2990,2950,1770,1695,166
5, 1445, 1340, 1275 and 1130ka-1
.

D Soluble water, hexane and petroleum ether (boiling range 3
0-6000); benzene, ethyl acetate, chloroform, acetone and dimethyl sulfoxide'
Easy to dissolve.

E Color reaction Ehrlich reagent reaction Positive/positive triphenyltetrazolium chloride reaction Negative ferric chloride reaction
Negative iodine-chloroplatinic acid reaction
Lifted hydroxylamine-ferric chloride reaction Positive chlorine-tolidine reagent reaction Positive ninhydrin reaction Negative F Color of substance G Colorless G Thin layer chromatography (TLC) The above trityl derivative was subjected to TLC using the following TLC plate and solvent. At this time, the following Rf value is shown.

[a - Pre-coated flexible plate [adsorbent: cellulose, thickness: 160〃, fluorescent indicator: lead-manganese activated calcium silicate, herein Eastman
Use "Chromagram Sheet No. 6065" manufactured by Kodak] Upper layer of n-butanol/ethanol/water (4/1/5) Rf = 0.56i - Propanol/water (8/7) Rf = 0.91 n-butanol Rf co1.0i-propanol/
Water (1/4) Rf = 1.0 legs Pre-coated glass plate [Adsorbent: Silica Gale, Thickness: 0.25 side, This plate is referred to herein as DC-
Use Verteizbratten Kieselgel 6 Misaki 254] Benzene/Acetone (2/1) Rf=0.2
Day 9 8- Behavior towards lactamases Proteus vulgaris, Citrobacter frondei and Bacillus vulgaris
It is inactivated by 6-lactamase of S. cereus.

As shown in Example 9 below, the trityl derivative has the following chemical shift NMR658CL3, which is considered to be derived from the trityl group, when tetramethylsilane is used as an internal standard in a 100 MHZ broton nuclear magnetic resonance spectrum in deuterated chloroform: 7.2-7.
6 (multiplet) was observed, which confirmed that the discussion conductor contained a trityl group.

In addition to the above-mentioned physical and chemical properties, the trityl conductor contains carbon, hydrogen, nitrogen, and sulfur atoms in its molecular structure, as is clear from the approximate values of the elemental analysis results below. 62% Hydrogen: about 6% Nitrogen: about 4% Sulfur: about 5% In addition, the above trityl derivative is much more stable than the antibiotic PS-5 itself, and has a pH range from almost neutral to
When heated to 6000 in a weak alkaline aqueous solution of 9 or less,
Within at least 20 minutes, the antibacterial activity of the trityl form is retained by at least 75% and usually more than 90%.

This stability is due to the well-known antibiotic Chenamycin (Japanese Unexamined Patent Publication No.
1-73191)).
Biological properties of antibiotic PS-5 trityl derivative '11
Antibacterial Spectrum The antibiotic PS-5 trityl derivative of the present invention has a broad spectrum of antibacterial activity and is effective against various microorganisms such as Staphylococcus sp.

It exhibits extremely strong antibacterial activity against Gram-positive bacteria belonging to the genus Coccus, Streptococcus, Sarcina, and Bacillus, and is also extremely effective against gram-negative bacteria belonging to the genus Alcaligenes and Comamonas, etc. Shows strong antibacterial activity. Furthermore, the antibiotic PS-5 trityl derivative of the present invention exhibits a fairly strong antibacterial activity against Gram-negative bacteria belonging to the genus Escherichia, Klebsiella, Proteus, and the like.

In particular, the antibiotic PS-5 trityl derivative of the present invention has 6
- resistant to antibiotics with lactam rings;
For example, Citrobacter, Proteus, and Ente.

It is unique in that it exhibits strong antibacterial activity against Gram-negative bacteria such as Bacter, Klebsiella, and Serratia. '2) Enhancement of the antibacterial activity of other antibiotics against 8-lactamase producing bacteria The antibiotic PS-5 trityl derivative of the present invention can be used for Citrobacter freundii, Proteus vulgaris, Enterobacter aeogenes,
It has the ability to enhance the antibacterial activity of other antibiotics, especially 8-lactam antibiotics such as penicillins and cephalosporins, against 8-lactamase-producing bacteria such as Serratia marcescens, and in many cases, these abilities are synergistic. It is true.

'3} Significant therapeutic effects were observed when in vivo active pathogenic Gram-positive bacteria were administered to infected mice.

{4} Toxicity As shown in Example 9 below, approximately four times the amount of CD5o used to treat infection with pathogenic Gram-positive bacteria was administered to mice, but no significant toxicity was observed.

Thus, according to the present invention, by confirming that the antibiotic PS-5 trityl derivative has the above-mentioned physicochemical properties and biological properties, the antibiotic used in the production of the trityl derivative and the target antibiotic can be It can be confirmed that it is PS-5.

As mentioned above, the antibiotic PS-5 and its trityl derivatives have strong antibacterial activity, and have antibacterial activity for the prevention, treatment, and/or treatment of infections caused by gram enteric and gram negative bacteria. As a component, it can be effectively used for the prevention, treatment, treatment, etc. of bacterial infections not only for humans but also for non-human animals such as mammals, livestock, and fish. The antibiotic PS-5 or its trityl derivative of the present invention can be administered orally, topically, or parenterally (intravenously, intramuscularly, intraperitoneally, etc.), and depending on the administration method, usually It can be formulated and used in various pharmaceutical forms as conventionally practiced.

For example, the antibiotic PS-5 or its trityl derivative of the present invention can be used in solid form (e.g., tablets, capsules, powders, jaw granules, coated tablets) together with pharmaceutically acceptable mortar materials, diluents, etc. , troches, powder sprays, suppositories, etc.), semi-solid forms (e.g. cartilages, creams, semi-solid capsules, etc.), or liquid forms (e.g. solutions, emulsions, suspensions, lotions, syrups, injections). , liquid spray, etc.). Antibiotic PS-5 of the present invention
or its trityl derivative, whether liquid, semi-solid or solid, generally costing between $0.1 and $9.
It may contain % by weight of active ingredient, preferably <10 to 6% by weight.

Representative carrier materials, excipients, diluents, and other auxiliary agents that can be used in these formulations, as well as formulation methods, will be further explained as follows.

Tablets and capsules for oral administration may be in unit dosage form, with binders that may be used include:
For example, syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone:
Examples of excipients include lactose, white rice bran, starch, calcium phosphate, sorbitol, or glycine; examples of diluted agents include magnesium stearate, olivine,
Polyethylene glycol, silica, etc. Disintegrants include, for example, starch, starch, etc. Wetting agents include, for example, sodium lauryl sulfate.

In the case of tablets, they can be coated using conventional methods. Oral liquid preparations can be in the form of oil or water suspensions, solutions, emulsions, syrups, etc., or can be provided as a dry product for mixing with water or other suitable carriers for use. can do.

These solutions can usually contain additives such as suspending agents such as sorbitol syrup, methylcellulose, sugar syrup, gelatin, hydroxyethylcellulose, hydroxymethylcellulose, aluminum stearate gel. , hydrogenated edible fats and oils, etc.; emulsifiers, such as lecithin, sorbitan monooleate, or gum arabic; non-aqueous carriers, such as almond oil,
fractionated coconut oil, oil esters, propylene glycol, or ethyl alcohol; preservatives such as p
-Methyl hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid, etc. Suppositories can contain conventional suppository bases such as cocoa butter or other glycerides.

Injectables may be presented in unit dosage form in ampoules or in multi-dose containers containing a preservative. This usually takes the form of a suspension, solution, or emulsion with an oily or aqueous carrier, and may contain a suspending agent stabilizer and/or a solubilizing agent. Alternatively,
The active ingredient may be in the form of a powder that can be formulated at the time of use by dissolving with sterile pyrogen-free water. The antibiotic PS-5 or its trityl derivative can also be formulated into dosage forms suitable for absorption through the mucous membranes of the nose and throat, bronchial tissues, and even as powder sprays, liquid sprays, inhalers, lozenges, and sore throats. It can be made into an appropriate dosage form such as a coating agent.

Formulations for eye and ear administration may be presented as liquid or semisolid capsules, and may also be provided as drops. Topically suitable formulations include ointments, creams, lotions, paints, powders, and the like, which may be formulated with hydrophobic or hydrophilic bases. The above formulation also includes stabilizers, binders, antioxidants,
Components such as preservatives, lubricants, suspending agents, thickening agents, bridging agents, and buffering agents may be included. Furthermore, when the antibiotic PS-5 of the present invention or its trityl derivative is used as a veterinary drug for chickens, cows, sheep, pigs, etc., it can be used, for example, with a long-acting or short-disintegrating base. It can also be formulated as an intramammary preparation, or as a concentrated feed additive according to known methods. The medicament provided by the present invention may contain only the antibiotic PS-5 or its trityl derivative as an active ingredient, or may contain other therapeutically useful active ingredients.

In particular, the antibiotic PS-5 of the present invention or its trityl derivative has the ability to synergistically enhance the antibacterial activity of 8-lactam antibiotics against 3-lactamase-producing bacteria, as described above. It may be used in combination with known 8-lactam antibiotics.

Examples of such 8-lactam antibiotics include penicillin antibiotics such as benzylbenicillin, phenooxymethylbenicillin, urbenicillin, ampicillin, and amoxicillin; cephaloridine, cephalothin, cefazolin, cephalexin, cefoxitin, cefacetryl, and cefamandole. , cephalosporin antibiotics such as cephapirin, cefrazine, and cephaloglycine. When the antibiotic PS-5 of the present invention or its trityl derivative is used in combination with an 8-lactam antibiotic such as those described above, the ratio of the two is not critical and can vary over a wide range, but generally the antibiotic PS-5
Alternatively, the weight ratio of the trityl derivative to the P-lactam antibiotic may range from 20:1 to 1:150, preferably from 10:1 to 1:100.

The dosage of the antibiotic PS-5 or its trityl derivative of the present invention depends on the subject to be treated and its symptoms, the body weight of the host,
Although it can vary widely depending on the type of infection, method of administration, and frequency of administration, it is usually 0.05 to 500 9/1/day.
It is advantageous to administer the k9 body weight, particularly preferably from 0.5 to 200 9/k9 body weight, in one or several doses.

However, depending on the judgment of the specialist, individual differences, severity of symptoms, etc.
It is also possible to administer smaller or larger amounts than the above dosage ranges. The antibiotic PS-5 or its trityl derivative of the present invention can not only be used in the form of a drug as described above, but also be included directly in animal feed or in the form of an additive for animal feed. It can also be used as an active ingredient in disinfectants and preservatives for food preservation.

Next, the present invention will be further explained by examples.

The antibacterial active substances used in the following examples were qualitatively and quantitatively determined by the following method. '1}Bioassay method Comamonas terrigena (comamo ship stemge side) B-9 cultured overnight on nutrient agar
96 cells are suspended in nutrient floss to prepare a seed mother liquor having an absorbance at 61 m of 0.04 derived from the cells.

1% seed mother liquid was inoculated onto a spiky agar base of 0.8% Kyokuto Powder Bouillon (manufactured by Kyokuto Pharmaceutical Industries ■) and 1% Bacto Agar (manufactured by Difco), and this was poured into Dispense into a tri-plate and solidify to prepare a Comamonas assay plate. Staphylococcus aureus (SPphy-loc) cultured overnight in nutrient floss.
Dilute 20 tsubo of the culture solution with nutrient broth to prepare a seed mother liquor.

1% Kyokuto Powder Bouillon (manufactured by Kyokuto Pharmaceutical Industries ■) and 1% Bacto Agar (manufactured by Difco) inoculated with 1% seed mother liquor.
A portion of the agar medium 7' each having a more spiky texture is dispensed into a 9-diameter Petri dish, solidified, and used as a Staphylococcus assay plate. Nkaligenes faeca grown overnight on nutrient agar.
lis) Cells of B-326 are suspended in nutrient floss to prepare a seed mother liquor having an absorbance at 61 m of 0.02 derived from the cells.

1% seed mother liquor was inoculated onto an agar medium made of 0.5% Kyokuto Powder Bouillon (manufactured by Kyokuto Pharmaceutical Industries ■) and 1% Bacto Agar (manufactured by Difco), and this was applied to 7 (9 skin-diameter beads each). Pour into a tray and solidify to make an alkaline assay plate.The test solution is usually made into a pulp disk with an 8-rib diameter.
After placing on furnace paper to remove excess liquid, place on a test plate and culture at 35°C for 2 hours.

Comparing the direct inhibition zone with that of the cephaloridine standard solution,
In this specification, the antibacterial titer that indicates the same inhibition zone diameter as that of cephaloridine 100 r/feather is defined as 100 cephaloridine units/skin. When the solution dissolved in feathers shows an antibacterial potency of 1 cephaloridine unit/', the specific activity of the solid sample is calculated as 1 cephaloridine unit/'.
Let's say Akira 9. However, depending on the test solution, the cephalorhidine units determined by the test bacteria differ, so depending on the type of test bacteria used, the cephalorhidine units for Comamonas (abbreviated as CCU) and Staphylococcus cephalorhidine units (abbreviated as SCU) are used. ), expressed as alcaligenes cephaloridine unit (abbreviated as ACU). In addition, 1 unit of the antibiotic PS-5 trityl derivative indicates 10.8 Comamonas cephaloridine units. {21 Bioautography In the above-mentioned pyoassay method, instead of using a 9-skin Petri dish, use 32 vertical x 24 horizontal pieces of blood and dispense 100 pieces of agar plate inoculated with the test bacteria. Solidify it and make a large test plate.

The paper chromatography furnace paper after the development of the test solution was pasted on the agar surface of the large assay plate prepared above, removed after 15 minutes, and the large assay plate was incubated at 35 qo for 20 hours. The Rf value can be calculated (qualitatively) and semi-quantitatively determined from the size of the inhibition zone.

If a thin layer chromatography plate is used, it is placed on the agar surface with a piece of thin paper so that the component side touches the plate, removed after one plate, and qualitative and semi-quantitative measurements are performed in the same manner as above. Example
A 100% seed culture medium (SE-4) with the following composition was placed in a 1500-year-old Erlenmeyer flask, and by the usual method,
Sterilized at 120qo for 15 minutes.

On the other hand, Streptomyces sp. A271 was grown on an agar slant in vitro.
) Allow the spores of the cocoon strain to adhere sufficiently, and add 0.02% to this test tube.
Tween 80 (manufactured by Atlas, surfactant, Tween
(80) Add 10 minutes of aqueous solution and stir lightly to prepare a spore suspension. This spore suspension 1' was inoculated into the seed culture medium prepared above, and a rotary shaker (2
The cells were cultured in a shaking moat at a relative pm of 0 and an amplitude of 7. This seed culture solution 2' was inoculated into 500 Erlenmeyer flasks containing 100 volumes of production medium with the following composition, and
The cells were cultured in a shaking moat on a rotary shaker for 8 to 9 hours. Composition of seed medium (SE-4) Beef extract (Difco) 0.3% (W/V) Bacto tryptone (Difco) 0.5% (W/V) Glucose 0.1% (W/V) Soluble Starch 2.4% (W/V) yeast extract
0.5% (W/V) calcium carbonate
0.4% (W/V) defatted soybean flour
Composition of 0.5% (W/V) pH 7.5 (before sterilization) production medium (1) AG-1 medium Glucose 1.5% (W/V) Cornstarch 2.5% (W/V) Coarse staple Carr 2.0% (W/V) dry yeast
1.0% (W/V) DL-methionine
0.1% (W/V) cobalt chloride (CoC
12.Skin 20) 0.00013% (W/V) pH 7.2 (before sterilization) '2} AGA-2 medium glucose 1.5% (W/V) potato starch 2.5% (W/V) course Tea liquor 2.0% (W/V) dry yeast
1.0% (W/V) cobalt chloride (CoC
12・Fine 20) 0.00013% (W/v) pH 6.5 (before sterilization) Glue AGB-1 medium Maltose 3.0% (W/V) Coastal liquor 1.0% (W/V) Dry yeast
1.0% (W/V) Cobalt chloride (CoC12/Skin 20) 0.0001% (W/v) pH 6.5 (before sterilization) '4) AGB-41 medium Maltose 5.0% (W/V) Soluble starch 1.0% (W/V) glycerin
0.3% (W/V) dry yeast
2.5% (W no V) salt
0.5% (W/V) dipotassium phosphate
0.05% (W/V) magnesium sulfate (MgS04.
7 days 20) 0.05% (W/V) calcium carbonate
0.3% (W/V) cobalt chloride (CoC12
・Mother LO) 0.00013% (W/V) pH 7.0 (bactericide) (5) ML-1 Vowel Glycerin 4.0% (W/V) Beptone 0.5% (W/V) Glucose
0.2% (W/V) potato starch
0.2% (W/V) defatted soy flour
0.5% (W/V) dry yeast
0.5% (W/V) salt 0.
5% (W/V) Calcium carbonate 0.2%
(WnoV) pH 6.4 (before sterilization) [61AGO-1 medium Soybean oil 3.0% (W/V) Dry yeast 2.0% (W/V) Salt
0.5% (W/V) dipotassium phosphate 0.05% (W/V) magnesium sulfate (M
gS04・7 days 20) 0.05% (W/V) Calcium carbonate 0.3% (W/V) Cobalt chloride (
CoC12・SLO) 0.00013% (W/V) pH 7.0 (before sterilization) The supernatant solution obtained by economical sampling from each culture flask and centrifugation was subjected to antibacterial testing using the disk bioassay method as described above. The titer was measured.

The antibacterial titers for 7 incubations at each site using Comamonas terrigena B-990, Staphylococcus aureus FDA20 bait and Alcaligenes faecalis B-326 as test bacteria were as follows. Table 5 Example 2 100 seeds of the seed culture solution cultured in the same manner as in Example 1 were inoculated into 30 seedling jars containing 15 million pieces of land and 19 residential lots, and incubated at 28°C for 20 minutes. pm fly worship, 7.5
Aerated pseudogroup culture was performed for up to 96 hours with aeration of 1 minute.

Shin-Etsu Silicone KM-75 (manufactured by Shin-Etsu Chemical) as an antifoaming agent
was used at 0.05%. The culture solution was sampled over time, and the antibacterial titer of the centrifuged supernatant solution was measured.
The antibacterial titers at each time were as follows. Table-6 Example 3 Streptomyces sp. A271 (Strep-t
omycesssp. A271) was cultured in the same manner as in Example 1 to prepare a flask seed culture solution, and 100% of this
15 minutes of inoculation into a 30-volume jar fermenter containing SE-4 medium.

At 28o0, 20pm Takahai, 7.5pm/min ventilation, 2
Cultivate for a specific period and add 100 ml of this culture solution to 19 m
The inoculation was carried out in a stainless steel tank fermenter containing 200 ml of culture medium, and cultured at 28° C. with aeration at 10 pm and 50 pm/min for 72 hours.

The antibacterial titer of the centrifuged culture solution was 6 CU/'.

A 5% (W/V) amount of Topcoperlite (manufactured by Toko Perlite ■) was added to the culture solution. 34 was added and filtered through a filter press to obtain a furnace liquid of 80.

Example 4 Preparation of activated carbon adsorption concentrate The culture solution from 10 flasks of ML-19 cultured for 7 hours cultured according to the method of Example 1 was collected, and cultured with Topco Perlite (manufactured by Toko Perlite ■) No. 1. 34 was added at 2% (W/V) and passed through a Buchner funnel through a suction oven to obtain 800 g of culture solution.

After confirming that the pH was 7 to 8, 16% of special Shirasagi activated carbon (manufactured by Takeda Pharmaceutical Co., Ltd.) was added, and after 15 minutes of sedimentation, the precipitate was collected by centrifugation. After washing with 800 °C of distilled water and centrifugation, 400 °C of 50% (V/V) acetone water was added, incubated at room temperature for 30 minutes, and then centrifuged to obtain a top light.

Using a rotary evaporator, 30-35q
Concentrate at 50°C to obtain a 50% concentrated solution. Thus, a concentrate with a titer of 80 CU/barrel was obtained from a culture with a titer of 5 CU/barrel.

Comparative Example 13 - As an antibiotic with lactamase inhibitory activity, Tokukokan 50-1135294 Skin Bi-Tsubaki Kaisho 50-14
In order to prove that MM4550 (composite) proposed in Publication No. 0692 is a substance different from the antibiotic PS-5 of the present invention, the following experiment was conducted. MM455
Strebtomyces olivaceus ATCC31126, which is described in Japanese Patent Application Laid-open No. 50-1135294 and Taroseki No. 50-140692 as a 0 (composite) producing bacterium (
= ATCC21379/M3), ATCC213
80 and the same ATCC 21382, 1.0% soybean flour (Ajinomoto Meat Specialty), 2.0% glucose, 0.2% calcium carbonate, and cobalt chloride (CoC12.
Detail 20) o. 0.001%, (pH 7.0 before sterilization) and inoculated each into sterilized 500' Erlenmeyer flasks with 100ml of medium (pH 7.0 before sterilization). The active ingredients in the culture furnace solution were adsorbed on specially made Shirasagi activated carbon, and the 50% acetone eluate was concentrated, then Toyoro Paper No. 50 (manufactured by Toyo Roshi ■) and 80
% Acetonitrile Notris EDTA (Acetonitrile・
Descending paper chromatography with a solvent consisting of 2 O', Ryo M tris(hydroxymethyl)aminomethane-hydrochloric acid (pH 7.5) buffer, 3 O', Ichi M ethylenediaminetetraacetic acid sodium salt aqueous solution (pH 7.5) 1 After that, bioautography was performed using Comamonas terrigena B-996 as a test bacterium.

In addition, the A271 strain of the present invention was added to glycerin 4.0
%, beptone 0.5%, glucose 0.2%, potato starch 0.2%, defatted soy flour 0.5%, dry yeast 0.5
%, NaCIO. 5% and calcium carbonate 0.2%,
A culture solution cultured in the same manner as above in a medium having a composition of (pH 6.4 before sterilization) was treated in the same manner and bioautography was performed.

The results are shown in FIG. 1 of the accompanying drawings. A in Figure 1 is Streptomyces olivaceus ATCC311
26 (=ATCC21379-M3), B is the same ATC
C21380, C is ATCC21382, and D is a bioautogram of the concentrated eluate of Streptomyces sp. A271 strain of the present invention. As is clear from this figure, component 1, which is the product of D, that is, the antibiotic PS-5 of the present invention, is not found in the products of A, B, and C. Components 0 and A, . It can be seen that component m, which is a product of components B and C, is a different substance that clearly exhibits a different Rf value from component 1. In other words, the antibiotic PS-5, which is a product of the Streptomyces sp. A271 strain, is clearly different from the product of the MM4550 (compound) producing bacteria described in Tokokukan Sho 50-1135294 and No. 50-140692. It is a novel and distinct antibiotic. Example 5 Preparation of Diaion HP-2 skin elution concentrate Example 1
The culture solution from 150 flasks of ML-1$Takuchi7 infant culture cultured by the method described above was collected, 100 ml of disodium ethylenediaminetetraacetate was added, and Tobco Pearlite No. 7 was cultured.
34 was added at 2% (W/V) and filtered by suction using a large Buchner funnel to obtain culture solution 14.1 (pH 7.9).

This is a Diamond Ion HP-20 with a diameter of 7 ga and a height of 50 arc.
It was adsorbed onto a column (manufactured by Mitsubishi Kasei ■), washed with distilled water 7 times, and eluted with 50% (V/V) methanol water. The titers of the culture solution and each elution surface were as follows. Table 7 Collect the port liquids from fraction 8 to fraction 14, and
Concentrate to approximately 100' using an evaporator at 3,000 or less, and freeze-dry the concentrate to obtain a specific activity of 54 CCU.
/ Yellowish brown powder on legs 2.6 times.

It was confirmed by paper chromatography and thin layer chromatography analysis that this powder contained only one type of antibacterial active ingredient. Therefore,
In the following confirmation of the antibacterial active ingredient, that is, antibiotic PS-5, the antibiotic is tracked using the antibacterial activity against Comamonas terrigena B-996 as an indicator, and the antibiotic has the following properties. It was confirmed that 1 Stability: The above-mentioned tan powder containing antibiotic PS-5 was added to 50 CU
Dissolved in distilled water to give /.

Separately, an M/15 aqueous potassium phosphate solution was prepared, and the pH was adjusted by adding phosphoric acid or sodium hydroxide to prepare solutions with pH values ranging from 3 to 9, and one solution was placed in a test tube. Add 1 part of the initially prepared antibiotic PS-5 containing solution to each pH solution, adjust to the required pH with a small amount of phosphoric acid or sodium hydroxide, and then use hot water maintained at 6000. After being immersed in a bath for 30 minutes and cooled with running water, the - was adjusted to 7.0 with a small amount of phosphoric acid or sodium hydroxide. As a control, a pH 7.0 solution soaked in ice water was used, and residual activity was measured by bioassay using Comamonas terrigena B-996 as the test bacterium, and the activity of the control was expressed as 100%. The results were as follows. From this result, antibiotic PS-5
is 3 at 6000 in an aqueous solution with a pH of 6.0 to 9.0.
It can be seen that it is quite stable even after heating for 0 minutes.

The antibiotic PS-5 is also stable to some extent at low temperatures and in a low pH range; for example, it showed at least 30% residual activity after being left at pH 3.0 for 5 minutes at -17°C. 2 Soluble antibiotic PS-5 is soluble in water at pH 6-9.

In other words, this antibiotic is easily soluble not only in water with a pH of 7, but also in an aqueous medium adjusted to a slightly acidic pH of 6 or higher with hydrochloric acid, and an aqueous medium adjusted to a slightly alkaline pH of 9 or less with sodium hydroxide. It is. Also,
The yellowish brown powder of antibiotic PS-5 25 parts 9 was concentrated with 0.5 parts of each of the following solvents at 20 oo for 1 minute, and the insoluble matter was separated and removed. The antibacterial activity in each solvent was The ratios for the antibacterial activity contained in brown powder 25:9 were as shown below. From this result, antibiotic PS-5 is 20q
It is estimated that it is soluble in methanol, slightly soluble in ethanol, and substantially insoluble in ethyl acetate and benzene.

3 Thin Layer Chromatography (TLC) When the yellowish brown powder was subjected to TLC using the TLC plate and solvent described below and detected by the bioassay method described above, the antibiotic PS-5 showed the Rf value shown below.

ta) Avisel■ SF cellulose thin layer plate [manufactured by Funakoshi Yakuhin Co., Ltd.] used n-butanol/ethanol/water (7
/7/6 capacity ratio; same below) Rf=0.9
4i-propanol/water (7/3) Rf=0.96{
b} Silica gel thin layer plate (manufactured by H.Merck; D
C-Verteizhipplattenkieselgel 6 Cape Technique 4)
Used ethanol/water (7/3) Rf=0.82
n-propanol/water (7/3) Rf=0.774
Beichi/G-Chromatography Toyoro Paper Yuzu. When the yellowish brown powder was subjected to paper chromatography using 50 [manufactured by Toyoro Paper ■] and the following solvent, the antibiotic PS-5 showed the following Rf value.

n-propanol/water (7/3) Rf=0.68n-propanol/i-propanol/water (7/7/6)
Rf = 0.70 acetonitrile/water (8/2) Rf = 0.36 acetonitrile notris buffer/EDTA (Kono 1)
Rf=0.34 ethanol/water (7/3)
Rf=0.63 (Speaker 1): Acetonitrile 120
', City of PH7-5.

A mixed solvent consisting of 30 parts of M tris(hydroxymethyl)aminomethane-hydrochloric acid buffer and 1 part of aqueous MI ethylenediaminetetraacetic acid sodium salt solution at pH 7.5.

5 High Pressure Furnace Paper Electrophoresis Laying Using a high pressure furnace paper electrophoresis device (manufactured by Savant Instruments, high voltage power supply HV3000A, electrophoresis tank FP180A), Toyo Furnace Paper No. When the yellowish brown powder was subjected to electric lacquering on a No. 50 (manufactured by Toyo Roshi ■), it was found that it exhibited the following behavior in a buffer solution with the following pH.

[11] When electricity was applied for 30 minutes at 42 V/g at 4°C in a buffer solution with an axis of 8.6 consisting of 3000 g of water, 3.3 g of barbital, and 25.5 g of sodium barbital, 28 skins moved toward the anode side. .

[21 At 7 W/skin at 4°C in a pH 1.9 buffer consisting of 2700 g of water, 240 g of acetic acid, and 60 g of formic acid.
Although electricity was applied for 0 minutes, no movement was confirmed.

6 Antibacterial activity {11 Antibacterial Spectrum Plain Heart Infusion Medium ``Eiken'' (
Antibiotic PS using liquid dilution assay using Eiken Chemical Co., Ltd.
The minimum inhibition unit against 15 different test bacteria was measured.

Antibiotic PS-5 tan powder to 27 CU/top ~5
Antibiotics were dissolved in Plain Heart Infusion Toji "Eiken" (manufactured by Eiken Kagaku Co., Ltd.) with a pH of 7.0 so that the concentration range was above 4 CCU/. A dilution series of substance PS-5 was prepared.

To this dilution series, the seed mother liquor of various test bacteria shown in Table 8 below, obtained by culturing in the same plain heart infusion medium as above at 28℃ for 1 hour, was added to the same plain heart infusion medium as above, and the number of bacteria after inoculation was approximately Inoculated so that 1 x 1 bi/pine [2
The spots were cultured for a period of time, and the presence or absence of growth was then observed, and the lowest concentration unit at which no growth was observed was defined as the minimum growth inhibition unit of the antibiotic PS-5 against the bacteria. The results are in the table below.
8.

Table 1 8 (Note) 2 strains of coolactamase-producing bacteria 3 strains resistant to gentamicin and tobramycin 4 strains resistant to tetracycline, erythromycin, leucomycin, and chloramphenicol KIO Tsutomuma blood added to culture medium [2] Penicillin system for resistant bacteria and enhances the antibacterial activity of cephalosporin antibiotics.
pH 7.0 nutrient agar with /' added
Five chickens were put in and allowed to solidify, and this was used as the bottom layer, and 10M of the same medium inoculated with 8-lactam-resistant bacteria producing 8-lactamase was poured on top of it and allowed to solidify, and this was used as the upper layer.

On this surface, a pulp disk with a diameter of 8 cm was placed, each containing 25 μl of aqueous solution containing the antibiotic PS-5 at various concentrations, and after culturing at 35 q0 for 1 hour, the size of the inhibition circle around the disk was observed. did. As a control, a similar experiment was conducted using the same medium without the addition of either penicillin G or cephaloridine. The results are shown in Table 9 below. Table 19 According to the above results, a clear increase in antibacterial activity was observed when the antibiotic PS-5 was added to penicillin G or cephaloridine at a concentration that did not show antibacterial activity when used alone. Ta.

(B) It was confirmed in the following experiment that this enhancement of antibacterial activity was synergistic.

A strip of furnace paper impregnated with penicillin G or cephaloridine was placed on the surface of nutrient agar that had been inoculated with 8-lactam-resistant 8-lactam-resistant bacteria, Protus vulgaris P-5, into a nutrient medium and hardened. , a strip of furnace paper soaked with antibiotic PS-5 was placed so that the strips of the same antibiotic or two strips of different antibiotics crossed each other, and cultured at 360 for 1 hour. In an appropriate concentration range, an inhibition zone was observed only at the intersection of a strip of antibiotic PS-5 with a strip of penicillin G or cephaloridine, and no inhibition zone was observed at the intersection of strips of the same antibiotic.

From these results, it was concluded that by mixing the above two antibiotics, which do not cause inhibition even when used alone or at two concentrations, by mixing them at one concentration each, they synergistically increase their antibacterial activity and provide an inhibition zone. I can understand.

'3} In vivo activity The in vivo infection treatment activity of the antibiotic PS-5 was determined using mice that were intraperitoneally infected with Staphylococcus aureus Smith strain at 5 x 1 cells per mouse. was measured.

Immediately after inoculation with the infectious agent, a sample containing the substance dissolved in saline was administered subcutaneously, and survival was observed for 72 hours. CD5o when using male ddy mice (Shizuoka) was 810 CU/
It was k9.

7 An injection solution containing the toxic antibiotic PS-5 was subcutaneously administered to male ddy mice (Shizuoka) at 16,200 WCU/k9, and observation was continued for 72 hours, but no deaths were observed.

8 8-Behavior against lactamases Diameter 3 on Comamonas assay plate containing antibiotic PS-5
The solution that gives an inhibition circle of 8 Willow gives an inhibition circle of 21.5 ribs in diameter on the Comamonas assay plate supplemented with 8-lactamase of Proteus vulgaris P-15, and also gives an inhibition circle of 21.5 ribs in diameter on the Comamonas assay plate supplemented with 8-lactamase of Proteus vulgaris P-15. The Comamonas assay plate supplemented with 8-lactamase gave an inhibition circle of 21.5 willow diameter.

From this result, antibiotic PS-5 is effective against Proteus vulgaris P-15 and Citrobacter freundii.
It can be seen that it is inactivated by 8-lactamase produced by E-9. Furthermore, the effect of 8-lactamase on the antibacterial active ingredient (antibiotic PS-5) in the yellowish brown powder was investigated.

The B-lactamase used was a constitutive benicillinase producing bacterium Bacillus licheniformis (Baci-11u).
s licheniformis )749/C(A
TCC25972) and the inducible benicillinase producing bacterium Bacillus cereus (Bacm cereus) 569 (
It was partially purified by adsorption column chromatography from the culture furnace solution of ATCC 27348).

The oxygen titers of these oxygen preparations against penicillin G are as follows: Bacillus licheniformis oxygen 1660 units/kg and Bacillus cereus oxygen 26900 units/kg oxygen
o, pH 6.8, M/10 phosphate buffer, iodometry method).

Note that "1 unit" in this case refers to the oxygen titer that decomposes 1 mole of penicillin G in 60 minutes at 30 qo and 6.8 kg. Bacillus licheniformis oxygen 0.2 [or Bacillus cereus enzyme 0125] (each about 3
．． A Comamonas assay plate containing Benicillinase was prepared by adding 100 pieces of agar plate inoculated with the aforementioned Comamonas terrigena bacteria.

The antibacterial titer on a total of three types of assay plates, including the oxygen-free Comamonas assay plate as a control assay plate, was assayed using an 8-sided paper disk using the conventional method. The size of the inhibition zone was as shown in Tables 10, 11 and 12' below.

In the table, assay plate 1 contains no enzyme, assay plate 0' contains Bacillus licheniformis enzyme, and assay plate skin contains Bacillus cereus enzyme. Table-1
0 Table-11 Table-12 As is clear from the above results, the antibiotic PS-
5 is relatively easily inactivated by Bacillus cereus 569 benicillinase (a type of B-lactamase), and to some extent also by Bacillus licheniformis 749/C benicillinase.

This fact suggests that a 3-lactam ring or a similar ring structure exists in the molecule of the antibiotic PS-5. 9
8-lactamer inhibition activity [Measurement method] 8-lactamase inhibition value, 1 sen R, is the amount necessary to inhibit 50% of the hydrolysis of cephalolidine by P-lactamase for 10 minutes (between 1 and 11 minutes after substrate addition). Represents the amount of a substance.

The rate at which cephaloridine is hydrolyzed can be determined by measuring the decrease in absorbance at 255 nm. As an 8-lactamase, affinity
Citrobacter freundi purified by chromatographic means
i) B-9 or Proteus vulgaris
P-5 8-lactamase is used.

The measurement operation was as follows.

Substrate solution: pH 6.0 containing 0.05 mole/ml of cephaloridine. & 0.1M phosphate buffer (255nm
The absorbance at is approximately 0.75).

Enzyme solution: 255 nm' for 10 minutes without inhibitors
Use a concentration that reduces the absorbance of the sample by approximately 0.1.

Apparatus: Using a Hitachi spectrophotometer UV-VIS13 matrix, measure while reacting in the customer cuvette in optical paths 1 and 3, and keep the cuvette at a constant temperature of 30 oo during the measurement.

Inhibitor dilution: Inhibitor samples are diluted with pH 6.8, 0.1M phosphate buffer or dimethyl sulfoxide (DMSO) as appropriate.

A diluted solution to which no inhibitor is added is used as a control.
Pre-incubation and reaction: Prior to substrate addition, pre-incubate the following composition solution at 3000 °C for 15 minutes.

pH6. & 0.1M phosphate buffer 100 mounds of enzyme
Solution: 0.5 to 2 peaks inhibitor diluted solution
0.5-40 ml Substrate solution after completion of pre-cultivation
Start the reaction by adding 3.0'.

The reaction was carried out at 25 m while measuring the change in absorbance at 3 m
The test is carried out at 0° C. for 10 minutes or longer, and the amount of inhibitor required to inhibit 50% of the hydrolysis of cephaloridine (1 rust RAg) for 10 minutes (between 1 and 11 minutes after addition of the substrate) is measured. [Results] According to the above measurement method, the powder exhibited an R of 1.3 CU/secretion for 8-lactamase produced by Proteus vulgaris P-5. This value means that the active ingredient in the powder inhibits the activity of 3-lactamase of Proteus vulgaris P-5 by 50% at a concentration of 1.38 CCU/'. Example 6 N-Butanol Extraction at Low Temperature In a large test tube, add 20 parts of distilled water, 12 parts of n-butanol,
Then, 7 g of common salt was added, and the mixture was cooled to -17 qo, on the verge of freezing.

Dissolve 9 parts of 200 parts of the mint brown powder obtained in Example 5 in 1 part of distilled water, add the cooled solution to this, and quickly add sulfuric acid while maintaining the temperature below -1 and 0. 2. Carp.
I adjusted it to 7F 3.0 and 3.25 and it performed well. Take each n-butyl/butyl layer and adjust the pH to 6. &0.9M phosphate buffer solution 5 were added, the active ingredient was transferred to the aqueous layer, and the amount of the active ingredient in the aqueous layer was quantified. The extraction rate from the yellowish brown powder used was as follows. pH Extraction rate (%) 2.75 35.0 3.00 32.0 3.25 16.0 Example 7 Preparation of QAE-Sephadex-treated powder Obtained from 100ml of culture solution in the same manner as in Example 5. Yellow-brown freeze-dried powder containing antibiotic PS-5 27.
7g (specific activity 13.CUP guy) at pH 6.8, 25m
M. Dissolve 3 volumes of phosphate buffer, adsorb onto a QAE-Sephadex A-25 (manufactured by Pharmacia) column (3.3 x 25.0) equilibrated with the buffer, and wash with the above buffer. Thereafter, the active fraction was collected by elution with a buffer containing the same salt while linearly changing the salt concentration from 0 to 9M.

The active fraction 30 was cooled to 0°C, adsorbed on activated carbon, washed with water, desalted, eluted with 50% (V/V) acetone water, and the active fraction was freeze-dried to obtain the 727-9 band. A yellowish brown powder of antibiotic PS-5 sodium salt was obtained. The specific activity of this powder was 264 CCU/mg.

Example 8 Preparation of DEAE-Cellulose Treated Powder A yellowish brown freeze-dried powder containing the antibiotic PS-5 obtained in Example 6 was prepared from one 727 female with a skin pH of 6. &Biogel P-2 dissolved in 25hM phosphate buffer and equilibrated with the buffer
(manufactured by Bio-Rad) column (1.5 x 27.0 fox) and developed again with the buffer solution, and active fraction 15' was collected.

This active fraction was passed through a similarly equilibrated jetylaminoethylcellulose-cellulose (DE32) (Watmaso) column (2.5 x 28.0 arc), and the salt concentration was sequentially adjusted linearly from 0 to 0.8M. The active fraction was eluted with a saline-containing buffer while changing temperature at 0°C.
Activated carbon after cooling 4. It was adsorbed to the seaweed, and after washing with water and desalting, it was reduced to 50% (
V/V) Released with acetone water and freeze-dried to 9 of 120
A brownish-white powder of antibiotic PS-5 sodium salt was obtained.

The specific activity of this powder was 60 CU/female. Example
9 Process for producing trityl derivative of antibiotic PS-5 1 A yellow-brown frozen product containing antibiotic PS-5 obtained by treating the 160 culture furnace solution obtained in the same manner as in Example 3 in the same manner as in Example 5 Dry powder 30. Females (specific activity 27 CCU/o) were dissolved in dimethylformamide 100 ml and triethyleneamine 3. Add the females and cool on ice, then trichilk.

9.0 g of Ride was added while controlling the temperature of the solution to not exceed 5°C. When the fly azusa was continued at 5°C, the raw material disappeared in one hour.

After adding 1,000 ml of 0.9M phosphate buffer with pH 6.8, extract the mixture three times with 1,000 ml of ethyl acetate each time, combine the extracts, dry with dehydrated ramie salt, and evaporate the residue under reduced pressure. , dissolved in benzene 20%. This benzene solution is passed through a Biobead S1 x 3 (manufactured by Bio-Rad) column (4.5 x 60.0 arc) that has been swollen with benzene in advance, and developed with benzene. Fractions containing 10% were collected, concentrated, and dried under reduced pressure. benzene 1
Dissolved in silica gel (Kieselgel 60, 70 ~
230 mesh, A-Merck) column (2 bases, 1
．． 5 x 24.0c), developed with benzene monoethyl acetate (10:1), and removed the reagent by welding.
The mixture was developed at a mixing ratio of (1:2), and the active fractions were collected and dried under reduced pressure. This was dissolved in 0.5% of benzene, passed through a neutral alumina (manufactured by Berum Co., Ltd.) column (2 female, 0.9× Shigeru Rene), and mixed with benzene monoacetate (10:1) or a different mixture ratio. The active fractions were collected and concentrated to dryness under reduced pressure. Dissolve this in 0.3' of benzene and use a silica gel column (1 thigh, 0.9
x 22.0, the same type of silica gel as above) was charged, washed with benzene-ethyl acetate (10:1), developed with benzene-ethyl acetate (1:2), and the eluted active fractions were combined. It was concentrated under reduced pressure and dried. Dissolve this in 0.5% of benzene and
．． 0) and developed with benzene, containing the active fraction, which was suitable for drying under reduced pressure. This was dissolved in 0.5 ml of acetone, and a Sephadex LH-20 (manufactured by Pharmacia) column (1.2
．． 0 skin) and developed with acetone, the obtained active fractions were combined and concentrated to dryness under reduced pressure to obtain a white powder of 23-9. This was crystallized from benzene-n-hexane to obtain a colorless crystalline powder of product 9. The colorless crystalline powder obtained above, that is, the antibiotic PS-5 trityl derivative, exhibited the following physical and chemical properties.

1 Color of the substance: Colorless 2 Solubility: Antibiotic PS-5 trityl derivative 5 moths of solvent 0.1'
The solubility was as follows: substantially insoluble in water, n-hexane and petroleum ether (boiling range 30-60 qo): benzene, acetic acid. Easily soluble in ethyl, chloroform, methanol, ethanol, acetone, dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

3. Stability: After dissolving the antibiotic PS-5 trityl derivative in 1 volume of methanol, 9 volumes of distilled water was immediately added to adjust the concentration to 12 bulbs CU/secretion.

Separately, prepare an aqueous M/' 15 dipotassium phosphate solution, add phosphoric acid or sodium hydroxide to it, and adjust the pH.
and prepared solutions of each pH from 3 to 9, and put them one by one into test tubes. Add 1 part of the initially prepared solution of the antibiotic PS-5 trityl derivative to each solution, adjust to the required volume with a small amount of phosphoric acid or sodium hydroxide, and maintain at 60 qo. After immersing the powder in a hot water bath and cooling with running water, the temperature was adjusted to 7.0 with a small amount of phosphoric acid or sodium hydroxide. For the control, residual activity was measured using a pH 7.0 solution soaked in ice water using a bioassay using Comamonas terrigena B-996 as the test bacterium.
Expressed as 0%. The results are shown in the table below. Table-1
3 This result shows that the trityl compound is stable even when heated for 1 hour at 6000 in an aqueous solution with a pH of 6.0 to 9.0.

Furthermore, the activity did not substantially decrease even when heated for 90 minutes at 6,000 ml in the above solution at pH 7.5.

4 Melting point: Melting point when measured at a temperature increase rate of 1°C per minute by the Kofler method using a BY-1 type melting point measuring device manufactured by Yazawa Kagaku Kikai Kogyo ■: mottled. 5-85.50
0.

It gradually turns brown at temperatures above 1400C.

5 Ultraviolet absorption spectrum: The ultraviolet absorption spectrum measured for a solution containing 128 g of the trityl derivative in 3 methanol using a Hitachi self-recording spectrophotometer EPS-Rainbow type is shown in attached Figure 2.

Misakibukuro H = 315-5, E Tegoko = 156 6 Infrared absorption spectrum: Using Hitachi infrared spectrophotometer model 215, chloroform 0
．． The infrared absorption spectrum measured for a solution containing 4.5 females of the 'Nakadantrityl derivative of No.6' is shown in the attached Figure 3.

Moreover, its characteristic maximum absorption wave number is as follows.

3430, 3100-3000 (C of phenyl group per day)
, 2990, 2950, 1770 (C=○), 1695
, 1665, 14451 i 0, 1275 and 1130
-1.

7 Proton nuclear magnetic resonance spectrum: 10 Sengawa z proton nuclear magnetic resonance spectrum measured on a solution containing 4.5 Kite 2 of the trityl derivative in 0.3' deuterated chloroform using JEOL JNAPS-10 top type. It is shown in attached Figure 4.

Its most characteristic signals are a singlet near 1.8 $A and a multiplet originating from the trityl group benzene ring proton at 7.2 to 7.6. 8. Elemental analysis The results of elemental analysis of a specimen prepared by drying the nuclear trityl derivative 3.5 base under a vacuum of 1×10 −2 Hg at room temperature for 4 hours are as follows.

C60.89%, Day 5.78%, N4.48%, S4.
62%9 Color reaction: Ehrlich reagent reaction
Enteric triphenyltetrazolium chloride reaction
Negative ferric chloride reaction Negative ferric chloride-iodide reaction Negative iodomonochloroplatinic acid reaction Positive hydroxyamine-ferric chloride reaction Enteric chlorine-tolidine reagent reaction
Positive I ninhydrin reaction
Negative 10 Thin layer chromatography (TLC) The antibiotic PS-5 trityl derivative has the following Rf value on a thin layer chromatogram using the following solvent.

The movement position was detected by bioautography of Comamonas terrigena 8-996. 'a' The Rf value when using a silica gel TLC plate (DC-Fuerteschbrachttenkiesel gel hexavalent cut, manufactured by Merck & Co., Ltd.) is as follows.

Benzene/acetone (2/1) R 0.29 [b}
The Rf value when using a cellulose TLC plate ("Chromagram Sheet M.6065" manufactured by Eastman Kodak Company) is as follows.

Furthermore, it was confirmed that the antibiotic PS-5 trityl derivative obtained above exhibited the following biological properties.

1 Antibiotic PS- by liquid dilution assay using antibacterial spectrum plain heart infusion medium “Eiken” (manufactured by Eiken Chemical Co., Ltd.)
The minimum inhibitory concentration of the 5-trityl derivative against various pathogenic bacteria including resistant bacteria was measured.

The trityl derivative was dissolved in a small amount of methanol, and this was diluted with Plain Heart Infusion Toji "Eiken" (manufactured by Eiken Kagaku Co., Ltd.) having a pH of 7.0, so that the concentration of the trityl derivative was 40 F. The concentration of methanol was set to be within the range of 1 g/kg to 201 g/kg, and the concentration of methanol was 10% or less.

This was diluted multiple times with the same medium to prepare a dilution series of the trityl derivative. To this dilution series, inoculum of various strains shown in Table 1-14 below, which were obtained by culturing the same plain heart infusion medium as above at 28°C, was added to the same plain heart infusion medium as above, so that the number of bacteria after inoculation was approximately The bacteria were inoculated at a ratio of 1 x 1 '/', statically cultured for 2 hours at 35°C, and then the presence or absence of growth was observed. The minimum inhibitory concentration of the -5 trityl derivative was taken as the minimum inhibitory concentration. As a control, the known antibiotics phan-picillin and cephaloridine were each used at 100 g/g/[
It was dissolved in Plain Heart Infusion medium at pH 7.0 to a concentration of 7.0, diluted multiple times in the same medium to create a dilution series, treated in the same manner, and the minimum inhibitory concentration against the bacteria was measured. The results are shown in Table 14 below. Note that the results for the known antibiotics ampicillin and cephaloIJdine are also listed in the same table for comparison. Table 14 (Note) 2 rice coolamase-producing bacteria 3 bacteria resistant to kanamycin, gentamicin, and tobramycin * 4 bacteria resistant to gentamicin and tobramycin
*Addition of KIO Kyoma blood in the medium From the above results, the antibiotic PS-5 trityl derivative has a broad antibacterial spectrum and is characterized by strong antibacterial activity against 8-lactamase-producing moulactam-resistant bacteria. I know that there is.

2. Enhancement of antibacterial activity of penicillin and cephalosporin antibiotics against resistant bacteria: ■ Put 15% of pH 7.0 nutrient agar supplemented with penicillin G or cephaloridine 50 rg/ml in a 9-diameter petri dish and let it solidify. This was used as the lower layer, and the same medium 10' inoculated with 8-lactam-resistant bacteria capable of producing 8-lactamase was poured onto it and solidified, and this was used as the upper layer.

On this surface, a pulp disk with a diameter of 8 tubes to which 25 tubes of the antibiotic flue-5 trityl derivative solution with the concentration listed in the table below was added was placed, and after incubation for 1 hour at 3yo, the inhibition circle around the disk was I observed that.

As a control, the same experiment was repeated using the same medium without the addition of either penicillin G or cephaloridine. The results were as shown in Table 15 below. Table 15 According to the above results, it was shown that when the trityl derivative at a concentration that does not exhibit antibacterial activity alone is added to penicillin G or cephaloridine at a concentration that does not exhibit antibacterial activity alone, antibacterial activity occurs. According to this, it is recognized that the antibiotic PS-5 trityl derivative clearly enhances the antibacterial activity of penicillin G and cephaloridine.

(B') Next, by using the same liquid dilution assay technique as described above, it was confirmed that the trityl derivative and cephaloridine synergistically enhance antibacterial activity.

First, using Plain Heart Infusion Medium at pH 7.0 as a solvent, solutions containing the antibiotic PS-5 trityl derivative and cephaloridine at five concentration ratios shown in Table 16 below were prepared: Table 16 Various mixed solutions was diluted to 5/10, 4/10, and 3/10 in Plain Heart Infusion Medium, pH 7.0, and each dilution was further diluted multiple times in Plain Heart Infusion Medium, pH 7.0. 15 dilution series were prepared.

The seed mother liquor of Proteus vulgaris P-5 was inoculated into this dilution series so that the number of bacteria after inoculation was approximately 1 x 5 p/cm, and after culturing at 35°C for 20 hours, the presence or absence of growth was determined. Observations were made to determine the respective minimum inhibitory concentrations.

The results were as shown in Table 117 below. Table-17 From this result, both components are Proteus vulgaris P-
It can be concluded that the antibacterial activity against 5 is synergistically enhanced by each other, but in order to make this judgment even easier, the minimum inhibitory concentration in the table above is calculated for each component. The minimum inhibitory concentration when added alone is set as 100 and expressed as an index as shown in Table 18 below.

Table 18 This result shows that by mixing 12.5% of the amount of the trityl derivative added alone and 7.5% of the amount of cephaloridine added alone, the antibacterial effect was the same as when each was administered alone. Since it shows that the force is given, this 8 between the trityl derivative and cephaloridine
-It is clear that there is a strong synergistic effect that mutually enhances the antibacterial activity against Protus vulgaris P-5, which is a lactamase-producing 8-lactam-resistant bacterium.

3 In vivo activity of Staphylococcus aureus Smith strain (Snephy
The in vivo activity of the antibiotic trityl derivative obtained above was measured using mice that were intraperitoneally infected with 5 x 1 cells per mouse with P. lococcusameus Smith.

Immediately after inoculation with the infectious agent, an injection solution containing the trityl derivative was administered subcutaneously. CD50 when using male ddy mice (Shizuoka) is 2.5 females/kg (2754 CU/k9)
Met. 4 8-Lactamase inhibitory activity The antibiotic PS-5 trityl derivative is Example 5, 9)
According to the inhibitory activity measuring method described in , 0.0% against 8-lactamase produced by Proteus vulgaris P-5.
0.060 mountain g/', and 0.80 French g/1 sen R for 8-lactamase produced by Citrobacter freundei E-9.

This value is 0.06 for the antibiotic PS-5 trityl derivative.
Inhibited 50% of the 8-lactamase activity of Broteus vulgaris P-15 at a concentration of 1 g/', and 0.80 mg
This means that the activity of Citrobacter freundii E-9 8-lactamase is inhibited by 50% at a concentration of 50%. 5 Behavior towards 3-lactamase ■ 50g/g of antibiotic PS-5 trityl derivative
The diameter of the inhibition circle when the solution was made into a pulp disk of 8-lactam, placed on a Comamonas assay plate supplemented with 8-lactamase, and cultured overnight was as follows.

Table 19 In addition, the following reaction solution composition was reacted with 8-lactamase produced by Proteus vulgaris P-5 or Citrobacter freundei E-9 for 30 oo for 1 hour in a test tube. Bioassay was performed using a Comamonas assay plate using a pulp disk of 8 willow diameter.

pH68, 0.1M phosphate buffer 0.1
50 upper 8-lactamase (*) or same phosphate buffer 5
0mutrityl PS-5 substance 5.7
(*) In the case of 8-lactamase produced by Proteus vulgaris P-5, 486 units/desktop, and in the case of 8-lactamase produced by Citrobacter freundei E-9, 180 units/ex. In addition, 1 unit is pH
6. Cephaloridine lrmo for 60 minutes at 8,3000
Refers to the enzyme activity that degrades le.

The diameter of the inhibition circle was as follows.

*Table 20 *From this result, the antibiotic PS-5 trityl derivative is effective against Proteus vulgaris P-5 and Citrobacter.
It can be seen that the enzyme was clearly inactivated by 8-lactamase produced by S. freundei E-9.

Tsukuda Furthermore, the behavior of the antibiotic PS-5 trityl derivative against 3-lactamase was investigated in Example 5.
The investigation was conducted under exactly the same conditions and in the same manner as in the case of S-5.

That is, in the Comamonas assay plates and dishes, respectively, containing 3.300 units of Bacillus licheniformis 749/C and Bacillus cereus 569 benicilinase, and in the control assay plate containing no enzyme (assay plate 1). The antibacterial titer is shown below. The measurement results for penicillin G and cephaloridine are as shown in Table 11 and Table 12 above, respectively. Table 21 As is clear from the above results, the antibiotic PS-5 trityl derivative is easily inactivated by Bacillus cereus 569 benicillinase, as is the antibiotic PS-5, and is easily inactivated by Bacillus licheniformis 749/C. It is also inactivated to some extent by Nisilinase.

This fact suggests that an 8-lactam ring or a similar ring structure exists in the molecule of the antibiotic PS-5 trityl derivative.

Example 10 Process for producing trityl derivative of antibiotic PS-5 (b) Brownish yellow lyophilized powder 715 of antibiotic PS-5 obtained by the same method as described in Example 7 (specific activity 23 $CU) 9)
was dissolved in 3.5' of hexamethylphosphotriamide (HMPA), cooled with water, and dissolved in 70' of triethylamine.
Add tritilbe while adjusting so that the dissolution does not exceed 10%.

Mido 250 nail 9 was added. After 7 hours of worship at 1000, the raw material disappeared.

Place it in 50ml of ice water, and after the ice melts, add 1 lb of benzene.
The extract was extracted three times with 5 parts each, and treated in substantially the same manner as in Example 9 to obtain colorless crystalline powder 9.4 part 9 of the trityl derivative of the antibiotic PS-5. Its physicochemical properties were almost the same as those described in Example 9. Example 11 Preparation of antibiotic PS-5 trityl derivative in the presence of crown compound (m) Brownish-white freeze-dried powder of antibiotic PS-5 obtained in the same manner as described in Example 5 10. 1
g was suspended in methylene chloride 200 g, and 15-crown-5 (manufactured by Nippon Soda) was added thereto and dissolved.

Add trityl chloride to this solution while keeping it in ice water so that the temperature does not exceed 5℃. Added acid. After the addition, the mixture was stirred in a room overflowing for 3 hours, and the impurities were removed in an oven. After concentrating the furnace liquid under reduced pressure, 10% of benzene was immediately added, insoluble matter was removed, and a colorless crystalline powder of trityl derivative of antibiotic PS-5 was obtained by purification in the same manner as in Example 9. I got 9. Its physicochemical properties were almost the same as those obtained in Example 9 above. Example 12 Production method of antibiotic PS-5 trityl derivative in the presence of quaternary ammonium salt (N) Culture furnace solution 48 ml (titer: 11.
Extraction was carried out twice with 15 parts of a dichloromethane solution of 0.05% cetyldimethylbenzyl ammonium chloride (24 parts per centimeter on the dichloromethane layer, 5.7 CCUs on top of the aqueous layer).

The extract was dehydrated with anhydrous ramie 40ml and concentrated to 500ml under reduced pressure. The solution obtained by the concentration (45 CU/
After dehydrating the young nitrate again with 1 og of anhydrous E sulfate, the young sulfur was further separated into Molecular Thief 4A (manufactured by Union Carbide Co., Ltd.) 5
Completely dehydrated with g. Cool this solution on ice and add trityl chloride while controlling the temperature to below 5°C. Added spots.

After reacting for 5 hours, dichloromethane was removed at room temperature under reduced pressure, and immediately 15% of benzene was added and purified in the same manner as in Example 10 to obtain anhydrous crystalline powder of trityl derivative of antibiotic PS-5. Ta.

Its physicochemical properties were almost the same as those obtained in Example 9 above. As mentioned above, the antibiotic PS-5 of the present invention containing its trityl derivative can be administered in various unit dosage forms, such as solid or liquid dosage forms that can be taken orally.

The unit dosage form, whether liquid, semi-solid or solid, contains from 0.1 to 99%, preferably from 10 to 6%, as described above.
It can contain 0% active substance. The content of the active ingredient in the drug varies depending on the dosage form and total amount of the drug, but it can usually range from about 9 parts in 10 to 9 parts in 1000, preferably from about 9 parts in 10 to 9 parts in 1000. . The unit dosage form for parenteral administration usually has a purity of 10
Antibiotic PS-5 (sodium salt) of the invention close to 0%
Alternatively, the trityl derivative can be dissolved in sterile water or in the form of a soluble powder that can be easily made into a solution. Next, the antibiotic PS-
5 (sodium salt) or its trityl derivative is illustrated. The above antibiotic and excipient are ground in a mortar and mixed uniformly.

Each capsule contains approximately 200 pieces of 9-ply gelatin, and is packed in a No. 3 hard gelatin capsule with an enteric coating.

Example B: Tablets - Mix antibiotic thoroughly with half of lactose and half of corn starch in the mixing ratio shown.

The mixture is mixed with 10% starch paste solution, granulated, and applied to knots. Add the rest of the corn starch and magnesium stearate to this, mix well, and make a shape with a diameter of 1 arc and a weight of 50.
Form into 0 female tablets. After this is coated with gauze, it is further coated with a positive solvent coating. Example C: Injection The above antibiotic is dissolved in sterile water for injection and filtered to sterilize it.

The solution is dispensed into sterilized ampoules, water is removed aseptically by freeze-drying, and the mouth of the ampule is sealed. When using,
Close and dissolve by adding 2 parts of sterile saline to the contents of the ampoule.

Example D: Tablets Antibiotic PS-5 (sodium salt) and cephaloridine are mixed, formulated and molded in the same manner as in Example B, coated with bran coating and then coated with an enteric coating.

Example E: Tablets Antibiotic PS-5 (sodium salt) and aminobenzyl beniciliso are mixed and formulated in the same manner as in Example B.

Example F: Capsules The above antibiotic conductor and excipients are polished and mixed uniformly.

Each capsule is packed into a No. 3 hard gelatin capsule coated with an enteric coating of approximately 200 ml.

Example G: Tablets The antibiotic derivative is well mixed with half of the lactose corn starch in the above mixing ratio.

The mixture is mixed with 10% starch paste solution, granulated, and applied to knots. Add the remaining corn starch and magnesium stearate to this, mix well, and form into tablets with a diameter of 1 skin and a weight of 500 tablets per tablet. After this is coated with bran coating, it is further coated with enteric coating. Example H: Injection The above antibiotic concessionaire was dissolved in sterile water for injection, filtered in an oven,
Disinfect.

The solution is dispensed into sterilized ampoules, water is removed aseptically by freeze-drying, and the openings of the ampoules are sealed. When using,
Open the package and add two portions of a 70% aqueous solution of sterile N-(8-hydroxylethyl)lactamide to the contents of the ampoule, and after melting,
use. Example 1: Tablet This antibiotic derivative and cephalodysso were mixed, formulated and molded in the same manner as in Example G, coated with sugar, and further coated with an enteric coating.

Example J: Tablet The antibiotic PS-5 trityl derivative and aminobenzylbenicillin are mixed and formulated in the same manner as in [Tablet 1].

[Brief explanation of drawings]

FIG. 1 is a bioautogram of the known MM4550 (composite) substance prepared in Comparative Example 1 and the antibiotic PS-5 of the present invention, and FIG. 2 is a bioautogram of the antibiotic PS-5 of the present invention prepared in Example 9. FIG. 3 is an ultraviolet absorption spectrum diagram of the -5 trityl derivative, FIG. 3 is an infrared edge absorption spectrum diagram thereof, and FIG. 4 is a nuclear magnetic resonance spectrum diagram thereof. Multi-figure map N Moat map M Total size of moat map

Claims (1)

[Claims] 1. Antibiotic PS-5, which is characterized in that an antibiotic PS-5-producing bacterium belonging to the genus Streptomyces is cultured in a nutrient medium, and antibiotic PS-5 having β-lactamase inhibitory activity is collected from the culture. manufacturing method.