JPH0641480B2 - Novel glycopeptide antibiotic - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention relates to novel glycopeptide antibiotics useful as antibacterial agents, and more particularly to formula (I): (In the formula, R is hydrogen or Represents a glycopeptidic antibiotic.

Conventional technology and problems Many antibiotics are used for the purpose of prevention and treatment of bacterial diseases, but with the widespread use of such antibiotics, the emergence of viable bacteria has become a big problem. . recent years,
In particular, a resistant bacterium showing resistance to a wide range and antibiotics, especially a methicillin-resistant bacterium has attracted attention. As is well known, methicillin-resistant bacteria are not only methicillin, but also aminoglycoside antibiotics, tetracycline antibiotics,
It is a multidrug-resistant bacterium that is resistant to many antibiotics such as cephem antibiotics, penicillin antibiotics, carbapenam antibiotics and macrolide antibiotics.

The present inventors have previously found that the novel antibiotics PA-42867A and PA-42 which are effective against methicillin-resistant bacteria.
Invented 867B (Japanese Patent Application No. 61-14389).
These compounds are vancomycin derivatives belonging to the glycopeptide antibiotics, Nocardia orientaris (Nocardia orientaris), a strain of the genus Nocardia.
s) produced by PA-42867 (Ministry of Industrial Research, No. 8601). After culturing this bacterium by the method described in detail in the specification of the above patent application, PA-
42867A and PA-42867B are separated and purified,
As a result of detailed examination of their physical properties, they were identified as novel glycopeptide antibiotics. Further, all of them showed excellent antibacterial activity in vitro, and their usefulness as a medicine was proved. However, more is needed to provide effective treatment for individual cases.
Antibiotics with high antibacterial activity are needed. As a means to achieve such purpose, from known antibiotics,
Attempts have been made to obtain derivatives by various methods. However, it is not always easy to efficiently obtain a derivative having antibacterial activity in this way. Therefore, if it is possible to easily and efficiently obtain an antibiotic that is effective against bacteria, especially multidrug-resistant bacteria, it is considered to contribute to the enhancement of medicine.

Means for Solving the Problems As a result of earnest studies for the purpose of developing new antibiotics, the present inventors have found that PA-42867A and / or
Further, it has been found that by hydrolyzing PA-42867B with an acid, a novel glycopeptide antibiotic having excellent antibacterial activity can be selectively obtained in high yield, and the present invention has been completed.

That is, the present invention provides a novel glycopeptide antibiotic represented by formula (I), or a pharmaceutically acceptable salt thereof. More specifically, the present invention provides that R in formula (I) is Dess- (4-Epivancosaminyl) PA-428
Des- (4-epivancosaminyl-O-glucosyl) PA-42867A, wherein 67A and R are hydrogen, and salts thereof.

These compounds are prepared according to the following reaction formulas by using the starting material PA-42867A or PA-4286 represented by the formula (II).
7B or a mixture thereof.

Reaction formula The starting material in the above reaction may be either PA-42867A or PA-42867B, but it is convenient to use the crude product containing both, which is obtained in these production steps.

The starting material PA-42867 (including either A, B, or a mixture) of formula (II) is hydrolyzed with an acid to form two compounds of the invention of formula (I). As is clear from the reaction formula, des- (4-epivancosaminyl) PA-42867A is produced by hydrolyzing only the side chain vancosaminyl group of the compound of formula (II).
On the other hand, des- (4-epivancosaminyl-O-glucosyl) PA-42867A is further hydrolyzed to remove the sugar residue by further hydrolyzing the product obtained by cleaving only this vancosaminyl group. The compound (1) can be obtained by removing these vancosaminyl group and sugar residue at the same time. It is also possible to selectively produce a desired compound by appropriately selecting reaction conditions (acid concentration, temperature, time, etc.). Generally, the reaction is carried out under suitable conditions,
The respective compounds may be isolated by treating the resulting reaction mixture according to a conventional method for separating and purifying antibiotics well known to those skilled in the art.

According to the method for hydrolysis with an acid of the present invention, a novel glycopeptide antibiotic represented by formula (I) can be easily obtained in high yield.

Examples of the acid used in the present invention include inorganic acids such as hydrochloric acid and sulfuric acid, and organic acids such as trifluoroacetic acid. The reaction conditions such as acid concentration, solvent, reaction temperature, reaction time and the like are generally those generally used when hydrolyzing sugar chains of these glycopeptide antibiotics. When the above acid is used, it may be reacted at 0 to 50 ° C. for 10 minutes to 24 hours. The acid concentration varies depending on each acid, but hydrochloric acid is 5 to 36%, sulfuric acid is 2N to 12N, and trifluoroacetic acid is 3%.
It can be 0 to 100%. The reaction is preferably performed under a nitrogen atmosphere.

According to the above reaction, des- (4-epivancosaminyl) PA-42867A represented by the formula (I) and des- (4-
Epivancosaminyl-O-glucosyl) PA-4286
Although a mixture of 7A is obtained, one of these compounds can be selectively produced depending on the reaction conditions. When selectively producing des- (4-epivancosaminyl) PA-42867A, about 20% hydrochloric acid is added to about 0 to 1
C. for about 15-20 hours, 5-10N sulfuric acid for about 1-5 hours at room temperature, about 70-100% trifluoroacetic acid for about 25-3.
It may be carried out under reaction conditions such as 5 ° C. for about 10 minutes to 2 hours. When des- (4-epivancosaminyl-O-glucosyl) PA-42867A is selectively produced, about 80% trifluoroacetic acid is used at about 40-50 ° C for about 2%.
Allow to react for ~ 4 hours. Under the above reaction conditions, one compound is produced in a yield of approximately 70% or more.

Then, the obtained reaction mixture is neutralized with a base such as sodium hydroxide, and then subjected to column chromatography using an adsorbent such as MIC GEL CHP-20P (manufactured by Mitsubishi Kasei Co.), and fractionated with various eluents. The high product fractions are collected by high performance liquid chromatography (HPLC). By repeating the fractionation process as necessary, a solution containing each product in high purity is obtained, and these are individually concentrated to precipitate a desired product by methanol precipitation or the like, and this precipitate is subjected to methanol- Recrystallize from water.
On the other hand, the filtered mother liquor is freeze-dried to obtain a freeze-dried product.

According to the method described above, the compound of formula (I) having an HPLC purity of about 91% or more can be obtained in a high yield of about 50 to 74%, respectively.

The obtained compound of formula (I) can be used as it is for the treatment of humans and animals, but in terms of absorbability in vivo, etc.,
It may be desirable to have the salt form. As a base capable of forming a salt with the compound of the present invention, an alkali metal such as potassium and sodium, an alkaline earth metal such as aluminum and magnesium, and as an acid, an inorganic acid such as hydrochloric acid, sulfuric acid and nitric acid, and acetic acid, fumaric acid and the like. Organic acids are mentioned.

The compound of the present invention or a salt thereof can be orally or parenterally administered to humans or animals as an active ingredient of an antibacterial agent. Can be orally administered as tablets, capsules and powders using commonly used excipients, stabilizers, preservatives, wetting agents, surfactants, etc., and parenterally as injections, coatings and suppositories. It can also be administered locally. The dose varies depending on the purpose of treatment, the age of the patient, symptoms, etc., but when intravenously injected, it is about 0.1 to 10 g per day for an adult.

Effect of the Invention According to the method of the present invention, a highly pure glycopeptide antibiotic derivative, des- (4-epivancosaminyl) PA-42.
867A and Des- (4-epivancosaminyl-O-
Glucosyl) PA-42867A can be efficiently produced.

All of these obtained compounds exhibit strong antibacterial activity against Gram-positive bacteria, especially against methicillin-resistant bacteria, and are useful as medicaments for humans or animals. In addition, since it can be isolated with high purity, as well as an oral agent,
Suitable for use as an injection.

Hereinafter, the present invention will be described in more detail with reference to Examples and Production Examples. However, these examples and manufacturing examples,
The present invention is not limited in any way.

Production Example Preparation of PA-42867A and PA-42867B (a) Fermentation step Soluble starch 0.5%, glucose 0.5%, polypeptone 0.5%, beef extract 0.5%, yeast extract 0.25
%, Salt 0.25%, deionized water (pH 7.0, before sterilization)
In a 2-volume Erlenmeyer flask containing 800 ml of a medium consisting of
No. 601) seed culture slant is inoculated and shake culture is performed at 180 rpm for 48 hours at 28 ° C. This culture fluid 8
00 ml was inoculated into a 30 volume jar fermenter containing the medium 20 having the same components as above, and the aeration rate was 20.
/ Min, internal pressure 0.5 kg / cm ² G, stirring speed 200 rpm,
Incubate at 28 ° C for 24 hours. Then this culture solution 10
Tomato paste 2.4%, dextrin 2.4%,
Dried yeast (Beast, manufactured by Iwaki Pharmaceutical Co., Ltd.) 1.2%, cobalt chloride hexahydrate 0.0006%, antifoaming agent P-2000
(Dainippon Ink) 0.08%, inoculated into a 250 fermentation tank containing a medium 140 consisting of tap water (pH 7.0, before sterilization), aeration rate 150 / min, internal pressure 5 psi, stirring speed 325 rpm, Incubate at 28 ° C for 64 hours.

(b) Separation step The culture solution obtained in the above step was treated with 10% sodium hydroxide.
Adjust the pH to 10.5 and centrifuge the supernatant 145
To get After adjusting this supernatant to pH 4.0, it was poured into a column of 13 Dowex 50 × 2 (Na ⁺ type) (manufactured by Dow Chemical Co.), washed with water 70, and 30% acetone water containing 1% triethylamine. Elute at 40. Fraction 2 showing activity by pulp disk diffusion method using Bacillus subtilis
Collect 2 and adjust to pH 5. This is concentrated under reduced pressure to remove acetone. Next, HP-20 (manufactured by Mitsubishi Kasei) 2
Pour into the column. After washing with water 20, it is eluted with 50% acetone water. Collect the active fraction 6. This is concentrated under reduced pressure and freeze-dried to obtain 35.6 g of a crude powder of PA-42867.

(c) Purification step Using 12 g of the above-mentioned crude powder, a solution of 0.01 N hydrochloric acid in 150 ml is prepared and chromatographed with 100 ml of MIC GEL CHP-20P (manufactured by Mitsubishi Kasei). Elution with 0.01N hydrochloric acid while checking the content by HPLC, PA-428
Fractions containing 67A and PA-42867B are pooled, the solution brought to pH 7.0 and chromatographed again on CHP-20P. After applying the PA-42867A and B-containing solution, it is thoroughly washed with 15% methanol water and eluted with 15% methanol 0.005N hydrochloric acid to obtain a PA-42867A-containing fraction and a PA-42867B-containing fraction. PA-
The 42867B-containing fraction was adjusted to pH 7.0 and then concentrated and lyophilized to obtain 683 mg of a residue. The PA-42867A-containing fraction was adjusted to pH 7.0, concentrated, and then decolorized by CHP.
Chromatography with 10 ml of -20P and elute with dilute hydrochloric acid (pH 5.0) aqueous solution. Collecting fractions containing PA-42867A,
After concentration, lyophilization gives a residue of 571 mg (purity 70%). 571 mg of this residue is dissolved in water, diluted hydrochloric acid is added to adjust the pH to 5.0, and the mixture is applied to a column of 10 ml of CHP-20P and eluted with water. Fractions containing PA-42867A were collected, adjusted to pH 7.0, concentrated and then CHP for desalting.
-20P 10 ml [0.05 M phosphate buffer (pH 7.
Stabilized with 0)] and washed with 0.05 M phosphate buffer (pH 7.0), then water, then 50
Elute with% methanol water and collect the fraction containing PA-42867A. After concentrating the eluate, freeze-dry it, PA-4
256 mg (purity 95%) of 2867A are obtained. PA-42
After dissolving 683 mg of the 867B-containing residue in water, the pH was adjusted to 4.0 by adding dilute hydrochloric acid, and CHP-20P was added for decolorization.
Apply to a 5 ml column and elute with a dilute aqueous hydrochloric acid solution (pH 4.0). Fractions containing PA-42867B were collected and the eluate was
After adjusting to pH 7.0 and concentrating, packed column RQ-
2 (Fuji Gel Sales Co., Ltd.) While checking the purity by HPLC, 7% acetonitrile-0.05M phosphate buffer (pH 4.9), then 8% acetonitrile-
Elute with 0.05M phosphate buffer (pH 4.9), 95
Fractions of% purity or higher are collected, adjusted to pH 7.0, concentrated and applied to a column of 10 ml of CHP-20P [stabilized with 0.05M phosphate buffer (pH 7.0)] for desalting. After washing with water, elution with 50% methanol-water, PA-42867
B-containing fractions were collected, concentrated, freeze-dried, and PA-4286.
102 mg of 7B (purity 98%) are obtained.

Example 1 Crude product obtained in the production example, step (b) (PA-42867A
Containing 53% and PA-42867B 9%) 2.00 g
200 ml of 20% hydrochloric acid (for Wako Pure Chemical Industries, precision analysis)
, And the mixture is cooled in ice under a nitrogen atmosphere (0 to 1 ° C.) and stirred for 16 hours. 6N sodium hydroxide (about 204m)
l) is added to adjust the pH to 9.2. MIC GEL C
HP-20P (200-400 mesh, 100 ml), water (600 ml), 0.01N hydrochloric acid (450 ml),
Water (450 ml), 25% methanol water (450 ml), 5
0% methanol water (400 ml), methanol (400 m
l), 50% methanol-0.005N hydrochloric acid (400m
elute sequentially in l).

HPLC [Nucleosil 300-7C1
8, 4.6φ × 250 mm, 10% acetonitrile-0.
05M PBS (pH3.5) flow rate 1ml / min, 220nmU
Fraction A (0.0
Fraction B (50% methanol, methanol, 50% methanol-0.005N hydrochloric acid eluate) is obtained with 1N hydrochloric acid, water eluate).

After concentrating Fraction B, adjust to pH 3.5 and use MCI GEL
CHP-20P (200-400 mesh, 10 ml), water (adjusted to pH 4.0 with hydrochloric acid water, about 10 ⁻⁴ N hydrochloric acid) 300 ml, 15% methanol-water (pH 4.0) 10
0 ml, 30% methanol-water (pH 4.0) 100 ml, 5
Elution with 100 ml of 0% methanol-water (pH 4.0), 50 ml of methanol and 50 ml of 50% methanol-0.005N hydrochloric acid in this order was carried out to obtain fraction C [water (pH 4.0) elution part] and fraction D.
[50% methanol-water (pH 4.0) elution part] is obtained.

Fractions A and C are combined, concentrated and adjusted to pH 7.0.
Desalt with MCI GEL CHP-20P (200-400 mesh, 10 ml). Water (100 ml), 2
Elution with 5% aqueous methanol (100 ml), 50% aqueous methanol (100 ml), methanol (100 ml), 50% methanol-0.005N hydrochloric acid (50 ml), fraction E
(Undemineralized part) and Fraction F (desalted part) are obtained. Fraction E (undesalted part) is desalted under the same conditions to obtain fraction G (desalted part).

Precipitated portion of the obtained fractions F and G (after concentrating each fraction, adding methanol to form a precipitate) 800 mg of water (39 ml)
After heating to dissolve in, recrystallize by adding methanol (39 ml),
557 mg of crystals are obtained (dried under vacuum at 30 ° C. for 1.5 hours in the presence of phosphorus pentoxide). [Des- (4-epivancosaminyl) PA-42867A, HPLC 93%, yield 46.
0%] Separately, 350 mg of a lyophilized product from a crystal mother liquor and a precipitate-forming mother liquor
[Death- (4-Epivancosaminyl) PA-42867
A, HPLC 91%, yield 28.3%] is obtained.

Fraction D was desalted in the same manner to give 30 mg of freeze-dried product.
[Des- (4-epivancosaminyl-O-glycosyl)
PA-42867A, HPLC 93%, yield 2.8%]
To get

Example 2 PA- with a purity of 90% obtained in the same manner as in Production Example, step (c)
Dissolve 100 mg of 42867A in 10 ml of 20% HCl and stir for 10 minutes while heating under an atmosphere of nitrogen at an oil bath temperature of 40 to 45 ° C. The reaction solution was adjusted to pH 9.2 with 6N NaOH.
To MCI GEL CHP-20P (200-
400 mesh, 10 ml), water (100 ml),
Elute sequentially with 0.01N hydrochloric acid (50 ml), water (50 ml), 25% methanol (50 ml), 50% methanol (50 ml), methanol (50 ml), 50% methanol-0.005N hydrochloric acid (50 ml).

HPLC (Nucleosyl 300-7C18, 10% acetonitrile-0.05M PBS (pH 3.5), 220
Fraction I (0.
Fraction II (50% methanol,
Methanol, 50% methanol-0.005N hydrochloric acid eluate) is obtained.

After concentrating Fraction II, adjust to pH 3.5 and use MCI GEL
CHP-20P (200-400 mesh, 10 ml) was soaked in water (pH 4.0) 50 ml, 15% methanol-water (pH
4.0), 30% methanol-water (pH 4.0), 50%
Methanol-water (pH 4.0), methanol 50 ml, 50
Fractionated by elution with 50% methanol-0.005N hydrochloric acid (50 ml)
III [water (pH 4.0), elution part] and fraction IV [50% methanol-water (pH 4.0), 50% methanol-0.00
5N hydrochloric acid elution part] is obtained.

Fractions I and III were combined, concentrated and adjusted to pH 7.0, and then MCI GEL CHP-20P (200-400).
Desalting using a mesh (5 ml) gives 31.5 mg of des- (4-epivancosaminyl) PA-42867A (yield 38.5%).

Fraction IV was similarly desalted and des- (4-epivancosaminyl-O-glycosyl) PA-42867A 36.3 mg
(50.1%) is obtained.

The physical properties of the compounds of the present invention obtained in the above examples are shown below.

Dess- (4-Epivancosaminyl) PA-42867A Specific Illuminance ▲ [α] ²⁴ _D ▼ °: -91.5 ± 3.1 ° (C = 0.4
22, water) infrared absorption spectrum KBr, cm ⁻¹ : 3380, 2928, 1658, 158
7, 1505, 1422, 1395, 1228, 113
1, 1064, 1029, 1012, 1000 Mass spectrometry (SIMS) MS m / z: 1414 (M + H) ⁺ circular dichroism spectrum (CD spectrum) ▲ λ ^{0.05M PBS (pH3.5)} _max ▼ nm [θ]: 310 (0),
285 (-15400), 277 (sh) (-7300),
260 (-950), 251 (-1140), 249
(0), 228 (+127400), 214 (0) Nuclear magnetic resonance spectrum ( ¹ H NMR) (see FIG. 1) 400 MHz, d ₆ -DMSO + D ₂ O (1 drop), 100
℃, internal standard TMS δppm: 0.889 (3H, d, J = 6.6Hz), 0.916 (3H, d, 6.5), 1.179
(3H, s), 1.179 (3H, d, 6.2), 1.427 (1H, d, d, d, 14.2,7.8,6.
3), 1.529 (1H, d, d, d, 14.2,7.6,5.8), 1.670 (1H, d, d, 13.9
And 4.4), 1.763 (1H, m), 1.942 (1H, d-like, 13.9), 2.178
(1H, d, d, 16.0 and 7.3), 2.314 (3H, s), 2.549 (1H, d, d, 1
6.0,5.2), about 3.3 (2H, m), 3.339 (1H, t, 8.7), 3.439 (1H, d, d,
7.3,8.7), 3.542 (1H, d, d, 11.5 and 4.5), 3.634 (1H, q, d,
6.2 and 9.7), 3.711 (1H, d, d, 11.5 and 2.0), 4.238 (1
H, br.s-), 4.356 (1H, d, d, 7.3 and 5.2), 4.489 (1H, b
rs), 4.519 (1H, s), 4.694 (1H, d-like, 4.4), 4.704 (1H, d, 4.
0), 5.169 (1H, d, -like, 4.0), 5.204 (2H, br.s-like), 5.346
(1H, d, 7.3), 5.659 (1H, br.s), 5.778 (1H, br.s), 6.380 (1H,
d, 2.3), 6.394 (1H, d, 2.3), 6.723 (1H, d, 8.5), 6.806 (1H, d,
d, 8.5 and 2.3), 7.104 (1H, d, d, 8.4 and 2.3), 7.135 (1
H, d, d, 8.4 and 2.4), 7.151 (1H, d, 2.3), 7.228 (1H, br.d,
8.4), 7.294 (1H, d, d, 8.4 and 2.3), 7.338 (1H, d, d, 8.4 and 2.0), 7.602 (1H, d, d, 8.4 and 2.3), 7.864 (1H, d-
, 2.0) Des- (4-epivancosaminyl-O-glycosyl) P
A-42867A Specific light intensity ▲ [α] ²⁴ _D ▼: -62.0 ± 4.9 ° (C = 0.20)
9, water).

Infrared absorption spectrum KBr, cm ⁻¹ : 3392, 2964, 1657, 159
8, 1510, 1429, 1394, 1231, 120
7, 1061, 1012, 1005 mass spectrometry (SIMS) MSm / z: 1252 (M + H) ⁺ circular dichroism spectrum (CD spectrum) ▲ λ ^{0.05M PBS (pH3.5)} _max ▼ nm [θ]: 310 (0) ),
284.5 (-17200), 264 (-2430),
250 (-11600), 246 (0), 225 (+1
39000), 213 (0) Nuclear magnetic resonance spectrum ( ¹ H NMR) (see FIG. 2) 00 MHz, d ₆ -DMSO + D ₂ O (1 drop), 100 ° C.
Internal standard TMS δppm: 0.888 (3H, d, J = 6.6Hz), 0.915 (3H, d, 6.7), 1.142
(3H, s), 1.174 (3H, d, 6.1), 1.425 (1H, d, d, d, 14.0,8.0,6.
5), 1.528 (1H, d, d, d, 14.0,7.6,5.7), 1.626 (1H, d, d, 13.7
And 4.4), 1.763 (1H, m), 1.910 (1H, d-like, 13.7), 2.173
(1H, d, d, 16.0 and 7.5), 2.304 (3H, s), 2.545 (1H, d, d, 1
6.0,5.4), 2.874 (1H, d, 9.7), 3.653 (1H, qd, 6.1 and 9.
7), 4.220 (1H, br.s-like), 4.359 (1H, d, d, 7.5 and 5.0),
4.475 (1H, br.s), 4.513 (1H, s), 4.686 (1H, br.d-like, 4.4),
4.689 (1H, d, 3.9), 5.175 (1H, d-like, 3.9), 5.184 (1H, d, d,
2.3 and 1.1), 5.206 (1H, br.s), 5.598 (1H, br.s), 5.723
(1H, br.s), 6.378 (1H, d, 2.3), 6.401 (1H, d, 2.3), 6.709 (1
H, d, 8.5), 6.794 (1H, d, d, 8.5 and 2.3), 7.074 (1H, d, d,
8.4 and 2.4), 7.119 (1H, d, d, 8.4 and 2.4), 7.130 (1H,
d, 2.3), 7.215 (1H, d, 8.4), 7.288 (1H, d, d, 8.4 and 2.2),
7.342 (1H, d, d, 8.4 and 2.0), 7.601 (1H, d, d-like, 8.4 and 2.0), 7.848 (1H, d, 2.0) Vancosaminyl) PA-42867A and des- (4-epivancosaminyl-O-glycosyl) PA-42867A are considered to have the following stereostructures.

Dess- (4-epivancosaminyl) PA-42867A (as C ₆₆ H ₇₆ O ₂₄ N ₉ Cl, MW 1414.8) R: Des- (4-epivancosaminyl-O-glycosyl) P
A-42867A (as C ₆₀ H ₆₆ O ₁₉ N ₉ Cl, MW 1252.7) R: H Example 3 Purified PA-42867A was treated with hydrochloric acid, sulfuric acid and trifluoroacetic acid in Table 1. Hydrolyzed under the reaction conditions shown in, and des- (4-epivancosaminyl) PA-428
The reaction conditions for selectively producing 67A or des- (4-epivancosaminyl-O-glycosyl) PA-42867A were investigated. The results are shown in Table 1.

As is apparent from Table 1, if appropriate reaction conditions are selected,
One of the compounds can be selectively produced in a yield of 70% or more.

Effect of the Invention The antibacterial activity of the compound of the present invention in vitro and in vivo was evaluated as shown in the following experimental examples.

Experimental Example 1 In Vitro Antibacterial Activity Against Gram-Positive Bacteria Preparation of Antibacterial Suspension 1 platinum loop of test strain on slope medium was inoculated into 1 ml of growth medium [Tryptosoy broth, Eiken Chemical Co., Ltd.] at 37 ° C. 1
Incubate for 8-20 hours. A 100-fold dilution of the culture is used as a cell suspension for inoculation.

Sample solution Weigh accurately 9-10mg of sample and add 2mg / ml to distilled water.
Dissolve so that

Serially dilute the agar plate sample solution with sterile water (2000-
0.25 μg / ml). For each diluted sample solution 0.
51 ml is transferred to a sterilized plastic Petri dish (9 cm in diameter), 9.5 ml of agar medium (susceptibility test medium, Nissui) is poured therein, and the mixture is gently stirred and then solidified.

Measurement of MIC value 1 platinum loop (0.5-1
μl) is transferred to the surface of an agar plate containing various concentrations of the sample prepared by the above method. After culturing at 37 ° C. for 18-20 hours, the growth of bacteria on the agar surface is observed. The minimum concentration at which the growth of bacteria was observed to be completely inhibited was defined as MIC.
Expressed in g / ml.

Addition of horse serum in special medium 0.5% (V / V) horse serum is added to the agar medium for measuring MIC against Streptococcus before injection.

The results are shown in Table 2.

Experimental Example 2 Antibacterial activity of des-4-epivancosaminyl PA-42867A in vivo Test method: Slc-ICR female mice (8 mice per group) were inoculated intraperitoneally with the test bacteria, and 1 hour and 5 hours later. 2 times in total, death-
4-Epivancosaminyl PA-42867A (multiple dilution) is administered subcutaneously.

Results: 50% effective dose (ED ₅₀ ) from the survival rate of mice after 7 days
To calculate.

The results are shown in Table 3.

[Brief description of drawings]

Figure 1 shows Death- (4-Epivancosaminyl) PA-42.
867A nuclear magnetic resonance spectrum, Fig. 2 shows
(4-Epivancosaminyl-O-glycosyl) PA-4
It is the figure which showed the nuclear magnetic resonance spectrum of 2867A.

Claims (3)

[Claims] 1. Formula (I): (In the formula, R is hydrogen or Represents a compound or salt thereof. 2. In the formula (I), R is a group: Dess- (4-epivancosaminyl) PA-42867A according to claim 1. 3. Des- (4-epivancosaminyl-O according to claim 1, wherein R is H in formula (I).
-Glucosyl) PA-42867A.