JPH01272593A - 17-membered ring lactone compound t-2636k - Google Patents

Abstract

NEW MATERIAL:A 17-membered ring lactone compound T-2636K, having following properties. Shape: colorless crystal. Melting point; 180 deg.C+ or -10 deg.C. Specific rotation; [alpha] -48+ or -10 deg. (C=0.5, ethanol). Thin layer chromatography; benzene : acetone : acetic acid (50:50:2), Rf=0.42. Color reaction; blue violet with conc. sulfuric acid. Solubility; readily soluble in acetone and alcohols, a little sparingly soluble in chloroform and ethyl acetate and insoluble in water. USE:A preventive and remedy for pig's dysentery or a growth promoter for the pig. PREPARATION:For example, Streptomyces.rochei.var. volubilis (FERM 6155) is cultured at 37 deg.C for 30-72hr, and the resultant culture filtrate is extracted and separated to afford the aimed T-2636K.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a 17-membered ring lactone compound T-
2636 and salts thereof, the present compounds are useful in preventing, treating swine dysentery, and promoting growth in pigs.

Japanese Patent Publication No. 45-6071 describes the antibiotic T-
Manufacturing method of 2636A, B, D, Japanese Patent Publication 1984-20
Publication No. 959 describes the method for producing antibiotic T-26360, and Japanese Patent Publication No. 1987-7355 describes the method for producing antibiotic T-26.
The manufacturing method of 36E and F is described.

Furthermore, Japanese Patent Publication No. 1987-74 has T-26
Japanese Patent Publication No. 47-46915 discloses a method for producing T-2636A using T-2636A as a raw material and enzymatic reaction.
Each method for producing 36C is described.

- It is known that all of the above antibiotics T-2636, except for T-2636B, are 17-membered ring lactone compounds (Japanese Patent Publication No. 10317/1983).
.

Under these circumstances, the present inventors isolated microorganisms from a large number of soils for the purpose of searching for new antibiotics, and as a result of separating and searching for the antibiotics produced by them, certain types of microorganisms were found to be novel. that the microorganism belongs to the genus Streptomyces; that by culturing the microorganism in an appropriate medium, a large amount of the new antibiotic can be accumulated in the culture; and that the antibiotic is physicochemical. I learned that scales can be collected to any purity by conveniently using their properties.

And, the antibiotic is the above-mentioned antibiotic T-2636A,
While B, C, D, E, and F are neutral fat-soluble substances,
It has been found that two types of novel 17-membered ring lactone compounds are acidic and fat-soluble and can be mutually converted under specific conditions. The inventors have designated these compounds as T-2636 and L. The present invention was completed based on this knowledge.

That is, the present invention provides a 17-membered ring lactone compound T-263
6K or its salt.

T-2636 can be produced by using a bacterium that belongs to the genus Streptomyces and produces a 17-membered ring lactone compound T-2636K or a small amount of L.

For example, a strain called No. T-2636, which the present inventors isolated from soil in the Nukata region of Osaka Prefecture, is an example of a strain most advantageously used in the method of the present invention. No, T-
The various properties of the 2636 strain on the medium are as follows, for example.

(1) Morphology Spore-forming hyphae are simply branched or pseudowhorled (Pseud
overticillus), the tip of which is looped or spiral-shaped, and the spores are attached in a chain. The spores are oval or oval in shape, and the size is 0.1-1.0μ x 0.9-
1.5μ and its surface is smooth.

(2) Culture characteristics Regarding the culture findings, - Numerically, the basal hyphae are colorless;
Aerial hyphae are brown to brownish-gray, do not produce soluble pigments in almost all media, and are non-chromogenic. Moreover, this strain grows well when the pH of the medium is 5 to 9 and the culture temperature is 20'C to 45C.

The symbols marked with Rdg in the descriptions showing the properties of honkan in various media are those in the standard color name table written by Ridgiway.

Growth on Zapek agar medium Growth 2 Spread thin, colorless Aerial mycelia: Poor, powdery, white to light drab (light grayish brown) Rdg-XL, 17"-b) Background: Colorless Soluble pigment: None 2 Dextrose Zapek Growth on agar medium: thinly spread, colorless aerial hyphae: poor, white or light drab (light gray color
Rdg-xt, L t7”-b) Background: Colorless soluble pigment: None 3 Glycerol Czapek agar medium Growth: Thin spread, colorless aerial mycelia: Poor, powdery, white to light cinnamon-trap (Rdg-XLI) , 13″-b) or lie l-
-F Love (Rdg-'XLIt, 17"-b) Back
Surface: Colorless Soluble pigment: None 4 Glucose asparagine agar medium Growth: Spreading and showing fairly good growth, colorless aerial mycelia:
Quite abundant, powdery, light drab (Rdg, LM,
17°-b) to drab (Rdg・XLM, 1
7) or Drab Gray (Rdg/XLW, 17”
-d) may show white color.

Back side: colorless to pale yellow Soluble pigment: None 5 Nutrient agar medium Growth: Spreading and showing fairly good growth, colorless to pale yellow Aerial mycelium: Poor, white, slightly light drab (1?d)
g-XL stomach, 17”-b) section.

Back side: Colorless to light brown Soluble pigment: None 6 Glucose nutrient agar growth: Spreading and wrinkled abundant growth, colorless to light yellow Aerial mycelium: Poor, white to drab gray (Rdg/XL)
17°-d) Back side: Light brown soluble pigment: None or may produce light brown pigment.

7 Glycerol nutrient agar medium Growth: abundant growth with wrinkles, colorless or pale yellow Aerial mycelium: shows considerable growth and is white Back: Light brown or dark brown in places.

No soluble pigment or pale yellow 8 Juicy Growth: Quite abundant, colorless to pale yellow ring-shaped growth, with growth also visible on the bottom of the vessel.

Aerial hyphae: None or slightly grown, white soluble pigment: None 9 Peptone agar medium Growth: Poor, thinly spread growth, colorless Aerial hyphae: Poor, white Back side: Colorless soluble pigment: None IO starch agar medium Growth: Poor, colorless aerial hyphae: None Back side: Colorless soluble pigment: None 11 Yeast extract agar medium Growth: Abundant wrinkled growth, colorless to light brown Aerial hyphae: Quite abundant, white to drab gray (Rdg
-XL stomach, 17′ζd) or Mouse Gray (Rdg
-LI, 15”) Reverse side: Yellowish brown Soluble pigment: None 1z Whole egg medium (37℃) Growth: Spreads and shows abundant growth, becoming colorless and later spreading with moist black spots appearing from above the slope. I'm coming.

Aerial mycelium: Abundant, white to veil olive gray (R
dg-Ll, 23”-f) or Mouse Gray (R
dg-LI, Is”) or Drab Gray (R
dg-XLW, 17”-d) Soluble pigment: None 13 Potato section medium Growth: Abundant, spreading, wrinkled growth, colorless aerial hyphae; Abundant, white to drab gray (Rdg・XL
l, 17”-d) or Mouse Gray (Rdg-L
Soluble pigment: None14 Carrot section medium Growth: Abundant, spreading, showing wrinkled growth; Colorless aerial mycelia: Abundant, powdery, white to light drab (Rdg
-XL'A, 17"-b) to drab (Rdg ・X
17”) Soluble pigment: None 15 Lidomus milk (37℃) Growth: Shows ring-shaped growth, colorless to pale yellow Aerial mycelium: Poor, white Soluble pigment: None Does not coagulate and peptonizes.pH is Almost no change.

16 Leffler's serum medium (37°C) Growth: Spread and wrinkled growth, achromatic aerial hyphae: absent or slightly grown, drab gray (Rdg-XLW,
17''-d) Soluble dye: None Almost does not liquefy, but shows weak liquefaction.

17 Gelatin puncture (21 months at 24°C) Growth: poor, showing ring-shaped growth, colorless aerial hyphae: poor, white or veil drab gray (Rdg-XLII, 1
7” Soluble dye: None Almost never liquefies or shows weak liquefaction.

18 Growth on cellulose medium: May not grow or may show slight colorless growth.

Aerial mycelium: no growth or slightly growing, drab gray (Rdg4LII, 17”-d) 19 Bennett's medium Growth: abundant and spread, colorless Aerial mycelium: abundant, powdery, drab (Rdg-XLII, 1
7”) Reverse side: Brown-gray Soluble pigment: None 20 Calcium malate medium Growth: Abundant, spreading and colorless, or may show poor and pale growth.

Aerial mycelium: Shows considerable growth, powdery, white to light.
Drab (Rdg-XLW, !7”-b) Poor white backside: Colorless to pale yellow Soluble pigment: None 21 Growth on tyrosine agar medium: Considerable growth, spreading, colorless!J
-Mu Huff (Rdg -xxx, t9'-d)
Aerial mycelium: Poor, white Back side: Colorless to pale yellow Soluble pigment: None 22 It shows almost no reducing property in nitrate-reducing Czapek liquid medium, but
Peptone water shows quite strong reducing properties.

23 Starch hydrolysis Growing area near 10am Enzyme area: 26-3026 -3O carbon source availability The method of Pridham and Gottlieb (J.

Bacteriol vol. 59 p. 107 1948)
I investigated according to the following.

Erythritol + Adonitor ± D-Sorbitol + Inositol + D-Mannitol Fence Dolcitol + D-Pheasant Rose + L-arabinose Shi-Tsuru Pose + D-galactose Mother D-glucose D-fructose Rhamnose + Melibiose + Maltose Mother Sucrose　　　　　10～ Ukutose + Raffinose + Trehalose Fence Surisin Fence Varnished cow - Lin + Inulin + Dextran　　　　　　　　 Sodium acetate Sodium succinate Sodium citrate Citrus D-mannose + Starch 　 　 　 　 　 　 　 Glycerin Contrast 　　　　10 Fence: Grows very well.

++: Grows well.

+: Slight growth.

±: Very little growth.

The above properties and Nis A. Waxman's The Actinomycetes, Volume 2, The Williams & Wilkins Company (1961), Bird's Manual of Determinative Batchyliology, 7th Edition, The... Williams & Wilkins Company (1957), L. Ettrinker et al.
8), it is considered to be similar to Streptomyces rotchei.

Streptomyces rotii has spore-forming hyphae that are linear or spiral, and it liquefies gelatin quickly and produces a pale yellow soluble pigment. The potato sections turn reddish brown.

While the growth on starch medium turns brown, No. T-26
The 36 spore-forming bacteria are rarely found in a straight line, but generally in a loop or spiral shape. Slightly liquefies gelatin and produces no soluble dyes. Potato sections are hardly colored and growth on starch medium is colorless.

However, since other culture properties are very similar, No. T-2636 is considered to be a mutant of Streptomyces rotchei. So no.

T-2636 is Streptomyces rochei barr 0 volubilis (Streptoa+yces roch
ei varvolubilis). This strain has been deposited with the Institute of Microbiology, Agency of Industrial Science and Technology under application accession number 6155, and with the Fermentation Research Institute under IFo 12507.

Neutral macrolide antibiotics and other T-26
The following bacteria have been reported as producing antibiotics similar to No. 36, but No. T-2636 is different from any of them.

Similar antibiotic-producing bacteria 1, Streptomyces flagae (megacidin-producing bacteria) [Ettlinger et al., Monachefte et al. Streptomyces violazeoniger (lankamycin and lancascidin producing bacterium) [Ebormann et al., Japanese Patent Publication No. 37-16700] 3, Streptomyces bikiniensis (calcomycin producing bacterium) [Radia
Philip Frohart et al., Special Publication No. 36-22650] 4. Streptomyces albogriseolus (
cominomycin-producing bacteria) [German Patent No. 1110820 by Egoimann et al.] 5. Streptomyces and labentenillae mutant strains (artugamycin-producing bacteria) [M. Peekenstmann et al. Antimicrobial Agents and Chemotherapy, p. 87 1964) 6, Streptomyces lavendulae mutant strain (Arjamycin strain antibiotic producing bacterium) [Toru Haseyo et al. Ota Research Institute Annual Report Vol. 25, p. 15 (1)
966)] 7. Streptomyces goshikiensis (pandamycin-producing bacterium) [Nobu Kondo et al., Special Publication No. 38-26948] 8. Streptomyces rimosus (unitathramycin-producing bacterium) [Dewielfmin et al. Antimicrobial Agents and Chemotherapy, p. 41 (1963)] 9. Streptomyces griseofnux (bandolin-producing bacterium) [G.M., G., Sakamoto et al., Journal of Antibiotics, Vol. 15, A 98
(1962)) Of course, it is well known that the properties of microorganisms belonging to the genus Streptomyces, especially Streptomyces, vary naturally or artificially, and Streptomyces φ rotchei var volubilis is also an exception. isn't it. That is, Streptomyces rotchei var. volpilis may naturally or artificially mutate, resulting in a different appearance of M on the culture medium.

However, it goes without saying that even these strains can be used for the purpose of the present invention as long as they do not lose the ability to produce a small amount of either T-2636K or L.

In the method of the present invention, for example, Streptomyces rotchei var. volubilis and its variants or mutant strains thereof (hereinafter referred to as T-2636 producing bacteria) are used.
are cultured on the medium. The medium may be either liquid or solid, but it is more convenient to use a liquid medium. The medium contains assimilable carbon sources such as glucose, soluble starch, and digestible nitrogen sources such as cornstape liquor and polypeptone for the T-2636 producing bacteria.

Raw soybean flour, inorganic salts, heavy metal salts, antifoaming agents, etc. are contained.

To culture T-2636-producing bacteria in a medium containing these nutrients, an aerated agitation culture method using a liquid medium is advantageous, but shaking culture may also be used in some cases.

For example, strain No. T~2636 contains 5% glucose, 0.5% glycerin, 1.0% polypeptone, 0.5% meat extract, and 1.0% raw soybean flour.

Magnesium sulfate 0.1%, calcium carbonate 0.5% (
pH 7,0) 30-7 in a medium containing 0.3% soybean oil
When cultured for 2 hours, T-2636 and L are accumulated in the culture solution. In addition, the culture solution contains T-2636A, B, and C.
, D, E, and F are produced, but these can be easily removed by the separation method described below. In order to collect the target T-2636K from the culture, separation means commonly used for collecting metabolites produced by microorganisms from the culture of the microorganisms can be used as appropriate. For example T-26
36 and L are acidic substances, and T-2636A, B, C, D, E, and F, which are co-produced at the same time, are neutral substances, so they can be collected by means that take advantage of this property. I can do it.

On the other hand, T-2636 is an acetyl form of T-2636L, and can be separated by changing the ratio of both in the culture solution using the action of enzymes contained in the culture solution.

That is, when the culture solution is extracted with a non-aqueous solvent that does not contain an acetyl donor, such as methyl isobutyl ketone or n-butanol, an extract that mainly contains T-2636L is obtained, whereas a non-aqueous solvent that does not contain an acetyl donor is obtained. When extracted with a water-soluble solvent such as ethyl acetate, mainly T
An extract containing -2636K is obtained.

When the extract thus obtained is extracted with a dilute alkaline solution, such as a cooled sodium hydrogen oxide solution, the target substance moves to the aqueous layer. Next, these aqueous layers are adjusted to pH 2 to 4 with a dilute acid, such as dilute hydrochloric acid, and extracted with a non-aqueous solvent to obtain the target extract containing T-2636K as the main component. During these operations, neutral fat-soluble substances T-2636A, B,
C,D.

E, F, etc. are efficiently removed. When the obtained extract is washed with water and concentrated, a crude substance of T-2636, which is the compound of the present invention, is obtained. When these crude substances are subjected to column chromatography using an adsorbent carrier such as silica gel or alumina or a high porous resin, purified T.
-2636K is obtained as a crystal.

As mentioned above, T-2636 is an acetyl form of T-2636L, and the target compound can be produced by converting T-2636L into K using an enzymatic reaction.

This conversion-based production method usually involves combining an aqueous solution of a culture of Streptomyces bacteria or its processed product with an organic solvent that is hardly miscible with water and containing an acetyl donor (e.g.
T-2636K can be produced by combining a solution of T-2636L dissolved in (chloroform, ethyl acetate, methyl isobutyl ketone, butanol) and stirring at room temperature.

Here, the acetyl donor refers to a compound that supplies an acetyl group to an enzyme in an acetyltransferase reaction, and includes, for example, ethyl acetate, monoacetin, diacetin, triacetin, and the like.

In addition, in the above, the culture solution is the T-2636K mentioned above.
or (and) the whole culture solution itself when culturing L-producing bacteria. In addition, the treated culture solution refers to the culture solution that has been subjected to centrifugation, filtration, washing, drying, grinding, extraction, insolubilization, etc. Live bacterial cells, dried bacterial cells, ground bacterial cells, crude or purified enzymes, insolubilized enzymes, etc. obtained by appropriate treatment T-2636
Contains an enzyme system involved in the reaction that converts L to crab.

T-2636 obtained in Example 1 and Reference Example 1 described below
The physicochemical properties of K or L are as follows.

T-2636K (1) Shape: Colorless crystal (ethyl acetate-acetone) (2)
Melting point = 180℃±lO℃ (decomposition point) (3) Specific optical rotation: [
α] 48°±10° (C=0.5. Ethanol) (4) Molecular formula: Estimated molecular formula CshH4l-41N Or
+-tt(5) Elemental analysis value (%) (sample vacuum dried over phosphorus pentoxide at 60°C for 8 hours) C, 61,96±1. O H, 6,32±0.5 N, 2.37±0.5 O, 28.71±1.0 (6) Ultraviolet absorption spectrum: The spectrum measured in ethanol is as shown in Figure 1. , its maximum value is λ 229±2nm (E P =727±50)t
O11 n+ax (7) Infrared absorption spectrum: The spectrum measured as a potassium bromide tablet is as shown in Figure 2, and the main peaks (wavelengths) are as follows.

336O, 2950.1?55.1710. 1660
．． 1550.1460°1375, 1310.12B0
．． 1190. 1150. Ion, 1050°10
2O, 970. 930. 890. 810. 7
50. 715° (8) 8-layer chromatography [Silica gel F□4 (manufactured by Merck & Co.)] Solvent system R1 value methyl ethyl ketone: ethyl acetate (2:8) o,
33 Benzene: Acetone: Acetic acid (50:50:2)
o, 42(9) Color reaction: Colors blue-purple with concentrated sulfuric acid.

(10) Solubility Ml for acetone and alcohols.

Slightly soluble in chloroform, ethyl acetate, etc.

Insoluble in water.

T-2636L (1) Shape: Colorless crystals (methanol-ethyl acetate) (2
) Melting point: 176°c+io°C (decomposition point) (3) Specific optical rotation: [α] 38°±lO9 (C=0.5. Ethanol) (4) Molecular formula: Estimated molecular formula Cs t H4IN Or.

,.

(5) Elemental analysis value (%) (Phosphorous pentoxide, vacuum dried at 60°C for 8 hours) C, 61,80±1.0 H, 6,58±0.5 N, 2.12±0. 5 O, 28.39 ± 1.0 (6) Ultraviolet absorption spectrum: The spectrum measured in ethanol is as shown in Figure 3, and its maximum value is λ 227 ± 2 nm (E”% = 813 ± 50) tO
11 wax la m (7) Infrared absorption spectrum: The spectrum measured as a potassium bromide tablet is as shown in FIG. 4, and the main peaks (wavelengths) are as follows.

340O, 2950.1755.1?20. 1665
．． 1520.1450°1: (80,1250,11
65,1140,1070,1010,970°88O
, 705. 620. 550 (8) Thin layer chromatography [Silica gel FtS4 (manufactured by Merck & Co.)] Solvent system Rf value Benzene: Acetone: Acetic acid (50:50:2)
o, 22(9) Color reaction: Colors blue-purple with concentrated sulfuric acid.

(10) Easily soluble in soluble alcohols, acetone, ethyl acetate, etc.
Poorly soluble, insoluble in water.

Next, the biological activities of T-2636 and L will be described. That is, bouillon agar is used as a test medium for general bacteria, glycerin bouillon agar for acid-fast bacteria, and glucose bouillon agar for mold and yeast. The antibacterial spectrum is as follows.

Antibacterial spectrum Escherichia coli >too
>to.

Staphylococcus aureus 20 10
0 Bacillus subtilis >100
>100 Zarcina Lutea 52
0 Micrococcus flavus 50
>100 Mycobacterium avium >100
>100 Candeida albicans
>100 >100 Next, the present invention will be explained in more detail with reference to Examples. In the following, percentages indicate weight ffi/% by volume.

Example 1 Glucose 5%, Glycerin 0.5%, Polypeptone 1
．． 0%, meat extract 0.5%, raw soybean flour 1.0%, magnesium sulfate 0.1%, calcium carbonate 0.5% (pH 7
, 0) was dispensed into a 2-g slope flask, and Streptomyces rochei burr volubilis (IFO12507, IFO12507, FIKEN application accession number FIKEN Bacillus No. 6155 strain) was poured onto the slope. One platinum lug was seeded from the culture and cultured on a reciprocating shaker at 37°C for 40 hours.
5, in which 3012 of the same medium was injected using the culture solution of
0<) Transplanted into a stainless steel tank. Soybean oil was used as an antifoaming agent, and 30 g was used at the time of preparation. Culture with ventilation 10
0% temperature, 37°C, stirring at 180 rpm for 20 hours,
This culture solution 102 is transplanted as a seed to the next tank, and 3
The cells were cultured at 7°C for 143 hours. That is, the same medium 100
In a 200Q stainless steel tank injected with 12, 100 g of soybean oil was used as an antifoaming agent during culture medium preparation and 500 g during culture.
Cultured with 100% aeration and 20 Orpm agitation. High Flow Supercel (2%) was added to 1ffl 100 of the obtained culture, filtered, and the pH of the fermentation liquid 80R was adjusted to 7.
Dityl acetate 5012 was added and stirred for 2 hours. The upper ethyl acetate layer was separated, washed with water, and then the antibacterial fraction was dissolved in the aqueous layer using 2% sodium bicarbonate solution (1012×2). The pH of the dissolved aqueous layer was adjusted to 2 to 4 with dilute hydrochloric acid, and ethyl acetate (
512X2). The extract was washed with water and then concentrated, and the concentrated solution was subjected to column chromatography on silica gel (0,812, manufactured by Merck & Co., Ltd.) and toluene:ethyl acetate (2:
Antibacterial activity was eluted at 8.41 ml. The active fraction was concentrated, and the concentrate was crystallized from a mixed solvent of acetone and ethyl acetate. T-2636K (10.3 g) was obtained as colorless crystals.

Reference Example 1 The culture solution (75i2) obtained as in Example 1 was adjusted to pH 7 and extracted with n-butanol (50!2). After washing the extract with water, add 2% sodium hydrogen carbonate solution (101
The antibacterial fraction was transferred to the aqueous layer using 2×2). p of the dissolved water layer
Adjust H to 2 to 4 with dilute hydrochloric acid, and add n-butanol (5QX
2). The extract was washed with water and concentrated, and the concentrated solution was subjected to column chromatography on silica gel (0,5i2), and the active fraction was eluted with ethyl acetate (5Q). The active fraction was concentrated, and the concentrate was crystallized from acetone. T-26
36L (8.6 g) was obtained as colorless crystals.

Reference Example 2 T-2636K (10g) obtained as in Example 1
) was dissolved in methanol (300-), and the culture solution of the T-2636 producing bacteria was obtained by J. Antibiotics.

It was added to the crude enzyme solution (le) obtained by the method described in Lambda, 23-28 (1971), and stirred at 25°C for 2.5 hours. Concentrate the reaction solution and adjust the concentrated solution (112) to pH 2.5.
After adjustment, extraction was performed with the same amount of n-butanol. The extract was washed with water and concentrated, and the concentrated residue was subjected to column chromatography on silica gel (40 g).

Antibiotics were eluted and fractionated with a mixture of ethyl acetate and acetone. The effective fraction was concentrated and the concentrated residue was crystallized from methanol/ethyl acetate to give colorless crystals of T-2636L (6,
38g) was obtained.

[Brief explanation of the drawing]

Figures 1 and 2 show the ultraviolet absorption spectrum and infrared absorption spectrum of the 17-membered ring lactone compound T-2636 obtained in Example 1, and Figures 3 and 4 show the spectrum obtained in Reference Example 1. 17-membered ring lactone compound T-263
The ultraviolet absorption spectrum and infrared absorption spectrum of 6L are shown, respectively. Agent Patent Attorney Iwa 1) Hiroi Nawatebakugeji 9-? V castle

Claims (1)

[Claims] 17-membered ring lactone compound T-263 having the following properties
6K or its salts (1) Shape: Colorless crystal (2) Melting point: 180°C ± 10°C (decomposition point) (3) Specific rotation: ▲ Numerical formula, chemical formula, table, etc. ▼ (C = 0.5, ethanol ) (4) Ultraviolet absorption spectrum: ▲Mathematical formulas, chemical formulas, tables, etc.▼ (727±50) (5) Infrared absorption spectrum (KBr), main peak (
cm^-^1): 3360, 2950, 1755, 1710, 1660,
1550, 1460, 1375, 1310, 1260,
1190, 1150, 1080, 1050, 1020,
970, 930, 890, 810, 750, 715, 6
20. (6) Thin layer chromatography [silica gel F_2_5
_4 (manufactured by Merck & Co.)]: Benzene: Acetone: Acetic acid (50:50:2), Rf=
0.42 (7) Color reaction: Colors blue-purple with concentrated sulfuric acid. (8) Solubility: Easily soluble in acetone and alcohols, slightly soluble in chloroform and ethyl acetate, insoluble in water. (9) Elemental analysis values (%) (materials vacuum dried over phosphorus pentoxide at 60°C for 8 hours): C, 61.96 ± 1.0 H, 6.32 ± 0.5 N, 2.37 ± 0 .5 O, 28.71±1.0