JPH0360836B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to novel compounds useful as pharmaceuticals.

Prior Art The compound of the present invention is a novel compound that has not been described in any literature.

Problems to be Solved by the Invention The purpose of the present invention is to provide a new substance having anticancer and antibacterial effects.

Means for Solving the Problems The present invention is based on the general formula [In the formula, R ₁ and R ₂ both represent hydrogen atoms,
Or R ₁ is a methyl group and R ₂ is a hydrogen atom, base or Indicates the group. ] 301 substances and their salts are provided.

The 301 substance of the present invention is an _A1 substance in which R ₁ is a methyl group and R ₂ is a hydrogen atom in the above general formula (),
A ₂ substance in which R ₁ and R ₂ are both hydrogen atoms, R ₁ is a methyl group and R ₂ is Substance B is a group, and R ₁ is a methyl group and R ₂ is It includes substance C which is a group.

The physical and chemical properties of each substance A ₁ , A ₂ , B, and C are as follows.

(1) 301−A ₁ substance (hydrochloride) (i) Empirical formula: C ₃₅ H ₄₄ N ₂ O ₁₂ (high resolution first atom
bombardment mass spectrometry).

(ii) Molecular weight: 684 (mass spectrometry).

(iii) Solubility: Easily soluble in water and methanol. Poorly soluble in chloroform. Insoluble in diethyl ether and hexane.

(iv) Ultraviolet absorption spectrum, λ ^MeOH _nax , nm (ε): 530 (4620), 495 (8000), 480 (7200), 292 (5420), 254 (17240), 235 (21900).

The chart is shown in Figure 1.

(v) Infrared absorption spectrum, ν ^KBr _nax , cm ^-1 : 995, 1030, 1202, 1425, 1578, 1620, 2980, 3400.

The chart is shown in Figure 2.

(vi) Properties of the substance: Red powder basic substance.

(vii) Melting point: 245-250°C (decomposition).

(2) 301−A ₂ substances (hydrochloride) (i) Empirical formula: C ₃₄ H ₄₂ N ₂ O ₁₂ (high resolution first atom
bombardment mass spectrometry).

(ii) Molecular weight: 670 (mass spectrometry).

(iii) Solubility: Easily soluble in water and methanol. Poorly soluble in chloroform. Insoluble in diethyl ether and hexane.

(iv) Ultraviolet absorption spectrum, λ ^MeOH _nax , nm (ε): 542 (4490), 497 (7310), 478 (7040), 292 (5090), 253 (16200), 236 (18200).

The chart is shown in Figure 3.

(v) Infrared absorption spectrum, ν ^KBr _nax , cm ^-1 : 990, 1030, 1202, 1250, 1420, 1570, 1620, 2970, 3400.

The chart is shown in Figure 4.

(vi) Properties of the substance: Red powder basic substance.

(vii) Melting point: 240-245°C (decomposed).

(3) 301-B substance (hydrochloride) (i) Empirical formula: C ₅₉ H ₈₀ N ₄ O ₂₆ (high resolution first atom
bombardment mass spectrometry).

(ii) Molecular weight: 1260 (mass spectrometry).

(iii) Solubility: Easily soluble in water and methanol. Poorly soluble in chloroform. Insoluble in diethyl ether and hexane.

(iv) Ultraviolet absorption spectrum, λ ^MeOH _nax , nm (ε), 542 (6120), 497 (10200), 480 (9920), 296 (7140), 253 (22700), 236 (26900).

The chart is shown in Figure 5.

(v) Infrared absorption spectrum, ν ^KBr _nax , cm ^-1 : 960, 1000, 1030, 1060, 1200, 1258, 1405, 1540, 1570, 1610, 1725, 2990, 3400.

The chart is shown in Figure 6.

(vi) Properties of substance: Red powder amphoteric substance.

(vii) Melting point: 224-227°C (decomposed).

(4) 301-C substance (hydrochloride) (1) Empirical formula: C ₆₀ H ₈₂ N ₄ O ₂₇ (high resolution first atom
bombardment mass spectrometry).

(ii) Molecular weight: 1290 (mass spectrometry).

(iii) Solubility: Easily soluble in water and methanol. Poorly soluble in chloroform. Insoluble in diethyl ether and hexane.

(iv) Ultraviolet absorption spectrum, λ ^MeOH _nax , nm (ε): 543 (4770), 498 (9540), 485 (9220), 292 (6830), 253 (22600), 236 (28200).

The chart is shown in Figure 7.

(v) Infrared absorption spectrum, ν ^KBr _nax , cm ^-1 : 960, 1000, 1030, 1060, 1200, 1380, 1540, 1578, 1620, 1720, 2980, 3400.

The chart is shown in Figure 8.

(vi) Properties of substance: Red powder amphoteric substance.

(vii) Melting point: 260-265°C (decomposed).

Substance 301 of the present invention can be produced by culturing Substance 301-producing bacteria belonging to the genus Streptomyces in a medium, and separating and collecting Substance 301 from the resulting culture.

An example of a 301 substance-producing bacterium is a strain belonging to the genus Streptomyces that was newly isolated by the inventors from the soil of Nanping, Fujian Province, People's Republic of China. This 301 substance-producing bacterium was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, with accession number 789.
No. (FERM BP-789).

The mycological properties of the above-mentioned 301 substance-producing bacteria are as follows.

(a) Morphological properties (1) Branching method of spore-forming hyphae Shows simple branching, no whorled branches.

(2) Morphology Forms hook-shaped (Retinaculum Apartum type) or spiral-shaped aerial hyphae.

(3) Spore stalk epiphyte position It grows on aerial hyphae.

(4) Number of spores A chain of about 10 to 60 spores is observed.

(5) Spore surface structure and size The spore surface is smooth, and the spore size is
It is approximately 0.9 to 1.2 x 2.0 to 2.8 microns.

(6) Others Flagellated spores, sporangia, and sclerotia formation are not observed.

(b) Growth status on various media (1) Seuculose/nitrate agar medium Bright purplish gray aerial mycelium grows on pale yellow-brown growth, and no soluble pigments are observed.

(2) Glucose-asparagine agar medium White to grayish-brown aerial mycelium grows on the yellowish growth, and no production of soluble pigments is observed.

(3) Glycerin-asparagine agar medium (ISP medium-5) Bright purplish gray aerial mycelium grows on the yellowish growth, and no production of soluble pigment is observed.

(4) Starch/inorganic salt agar medium (ISP medium-4) Light gray aerial mycelium grows on the growth of light yellow brown, and no production of soluble pigment is observed.

(5) Tyrosine agar medium (ISP medium-7) Gray brown aerial mycelium grows on the growth of light yellow brown, and the soluble pigment has a slight brown tinge.

(6) Nutrient agar medium Growth is light yellowish brown, no aerial mycelia are attached, and no production of soluble pigment is observed.

(7) Yeast/malt agar medium (ISP medium-2) Gray brown aerial mycelium grows on the yellow brown growth, and the soluble pigment has a slight brownish tinge.

(8) Oat meal agar medium (ISP medium-3) Gray brown aerial mycelium grows on the brownish growth, and no production of soluble pigment is observed.

(9) Krasilnikov No. 1 synthetic agar medium Bright purplish gray aerial mycelium grows on the pale yellow-orange growth, and the soluble pigment has a slight pinkish tinge.

(10) Glass No. 1 synthetic agar medium Aerial mycelium of gray tea grows on the growing tea ash, and no production of soluble pigment is observed.

(c) Physiological properties (1) Growth temperature range It grows at any temperature between 20 and 37°C.

(2) Production of melanin-like pigments (tyrosine agar, ISP
Medium-7), reduction of nitrate, production of hydrogen sulfide,
Liquefaction of gelatin, coagulation and peptonization of milk,
Cellulose decomposition and starch hydrolysis are both positive.

(3) Utilization of carbon sources (Pridham-Gotlieb agar medium, ISP medium-9) D-glucose, D-fructose, L-rhamnose, D-mannite, L-arabinose, D-xylose, and sucrose may be used. grown, inositol, raffinose,
It is determined that galactose is probably utilized.

(4) Analysis of cell wall components L,L-diaminopimelic acid and glycine were detected in the whole bacterial cell hydrolyzate. In addition, galactose, D-glucose, and L-arabinose were detected as major sugar components of the whole bacterial cells.

If you search for known bacterial species based on these properties, Book 301
Streptomyces virgiinia is the species most similar to substance-producing bacteria.
Virginiae) [S. A. Waxman (SA)
Waksman), The Actinomycetes (The
Actinomycetes), 2 volumes (1961), The Journal of Antibiotics (The
Journal of Antibiotics, Volume 36, pp. 451-453 (1983), International Journal of Systematic Bacteriology.
bacteriology), vol. 18, p. 178 (1968)]. When compared with this strain, it was found to be very similar to Streptomyces bilgiinia.
There is only a slight difference in the availability of sucrose and D-mannite. Therefore, the present 301 substance-producing bacterium is Streptomyces Virginiae Var.
It was identified as nanpingenesis. Next, 301 of the present invention
A case will be described in which a substance is produced by culturing, for example, the above-mentioned 301 substance-producing bacteria belonging to the genus Streptomyces in a medium.

The culture method is basically similar to that of general microorganisms, but a stirring aeration culture method using a liquid medium is usually advantageous. The medium used for culture may be any medium containing a nutrient source that can be used by the 301 substance-producing bacteria belonging to the genus Streptomyces.

That is, a synthetic medium, a semi-synthetic medium, or a natural medium can be used. The composition of the medium includes carbon sources such as glucose, mannose, glycerin, sucrose, molasses, starch, and liquefied starch, and nitrogen sources such as meat extract, casamino acids, peptone, gluten meal, corn meal, cottonseed oil, Soy bean meal, corn steep liquor, dried yeast, ammonium phosphate, ammonium sulfate, urea, etc. are used. Inorganic salts such as metal phosphates, carbonates, and chlorides, such as disodium hydrogen phosphate, potassium dihydrogen phosphate, calcium carbonate, and magnesium chloride, may also be added as necessary. In addition, if foaming is significant during culturing, antifoaming agents such as vegetable oils such as soybean oil and linseed oil, higher alcohols such as octadecanol, tetradecanol, and heptadecanol, and various silicon compounds may be added as appropriate. good.

301 substance-producing bacteria usually grow at temperatures of about 20 to 37 degrees Celsius. It is preferable to produce the 301 substance at a temperature of about 25 to 37°C. The culture time for the main culture is usually about 50 to 100 hours, and the culture time may be further extended as the medium becomes more concentrated. During cultivation, it is preferable to blow sterile air into the medium, as is commonly done in agitated aerated cultivation. For efficient growth of microorganisms, the amount of air used for tank culture should be 0.3 to 0.6 per 1 volume part of nutrient medium per minute.
It is best to use about 1 part by volume and stir at about 200 to 400 revolutions per minute. From the culture conditions described above, optimal conditions can be appropriately selected and applied depending on the characteristics of the bacterial strain used.

The 301 substances accumulated in the culture in this way are mainly contained in the cultured bacterial cells, so the bacterial cells are separated from the culture by centrifugation or filtration.
It can be isolated and purified from bacterial cells by conventional means used in the production of general antibiotics. That is, vacuum concentration, freeze drying, solvent extraction with acetone, methanol, ethanol, chloroform, etc., liquid exchange, treatment with resins such as cation exchange resins, anion exchange resins, nonionic adsorption resins, activated carbon, silicic acid, etc. The 301 substance can be separated, purified, and collected by treatment with an adsorbent such as silica gel or alumina, or by using methods such as crystallization, recrystallization, etc. singly or in combination in any order, or repeatedly.

The salts used for 301 substances refer to salts commonly used in medicine, such as alkali metal salts such as sodium, potassium, and lithium, calcium,
Alkaline earth metal salts such as magnesium, organic amine salts such as trimethylamine, triethylamine, etc., and acids used include inorganic acid salts such as hydrochloric acid, nitric acid, and sulfuric acid, or formic acid, acetic acid, oxalic acid, maleic acid, and succinic acid. , tartaric acid, para-toluenesulfonic acid, and other organic acid salts.

Substance 301 of the present invention thus obtained has anticancer and antibacterial effects and is useful as a medicine.

Example Next, a more detailed explanation will be given with reference to examples.

Example 1 Glucose 2.0%, Starch 1.0%, Soy Bean Meal 0.5%, "Soiton" [DIFCO]
Fermentation medium (PH 7.0 to 7.5) 1 having a composition of 0.2% (manufactured by J.D. Co., Ltd.), 0.3% sodium chloride, 0.3% sodium nitrate, and 0.3% calcium carbonate was dispensed into 10 500 ml Erlenmeyer flasks (100 ml each). 121℃, 20
301 substance producing bacteria grown on starch agar medium after sterilization in an autoclave for minutes (Feikoken Joyori No. 789
No.) 0.5 ^cm2 of conidia and mycelia were inoculated into each flask, and cultured at 27℃ for 2 days with rotary shaking (220 m/min).
Rotation, 10 cm) was performed. The obtained preculture solution was
Inoculate 300 ml each into two 10-volume fermenters containing fermentation medium 5 with the same composition as above, and culture with aeration at 27°C for 3 days (aeration rate 0.3 to 0.6 V/V/min,
250 revolutions per minute). "Silicon KM70" (manufactured by Shin-Etsu Chemical Co., Ltd.) was used as an antifoaming agent in the culture solution.

After completion of the culture, centrifugation is performed, and the bacterial cells are separated from Staphylococcus aureus (Staphylococcus aureus).
aureus) 209P, the following separation and purification operations were performed using the antibacterial activity against 209P as an indicator.

Approximately 1.2 kg of wet bacterial cells were extracted with 80% acetone (6), and the extract was concentrated using a rotary evaporator under reduced pressure. The resulting red aqueous solution was adjusted to pH 8.0 to 8.5 and extracted with chloroform in Step 6. The chloroform layer was concentrated using a rotary evaporator under reduced pressure to obtain 5 g of an active oil.

Dissolve 1 g of the red oil in 3 ml of chloroform-methanol (9:1) mixture, add octadecylsilyl (ODS) resin (manufactured by Yamamura Kagaku Kenkyujo, 30μ)
m) column (15 x 500 mm) for adsorption.
Gradient elution with a mixture of 0.05% trifluoroacetic acid solution and acetonitrile (acetonitrile, 30→60
%) to elute the active ingredient, and in the order of elution A, B,
It was divided into three fractions designated as C and collected.

These three fractions were concentrated using a rotary evaporator and then freeze-dried to obtain active crude powders A 20 mg, B 25 mg, and C 40 mg.

The active substances were isolated from these crude powders by preparative purification using high performance liquid chromatography as described below.

a Purification of coarse powder A Dissolve coarse powder A in water to a concentration of 10 mg/ml, and
200μ was injected into the high performance liquid chromatograph shown below.

Column: “Micro Bonder Pack (μ
Bondapak) C18” (7.8 x 300mm, manufactured by Waters).

Mobile phase: 25% acetonitrile/0.05% trifluoroacetic acid aqueous solution.

Flow rate: 2.0ml/min.

Detection: UV-254nm.

Peaks with retention times of about 7.8 to 8.2 minutes and about 8.4 to 9.0 minutes were fractionated, and this operation was repeated to collect two fractions. This fractionated solution was concentrated using a rotary evaporator, then adjusted to pH 8.0 to 8.5 with a 5% sodium carbonate solution, and extracted with 5 times the amount of chloroform. The chloroform layer was concentrated to dryness using a rotary evaporator, and then dissolved in a 0.001N hydrochloric acid solution and freeze-dried to obtain 2 mg of hydrochloride 301-A ₁ and 1.5 mg of 301-A ₂ as active substances.

b Purification of coarse powder B Dissolve coarse powder B in water to a concentration of 10 mg/ml, and
200μ was injected into the high performance liquid chromatograph described below.

Column: “Micro Bonder Pack (μ
Bondapak) C18” (7.8 x 300mm, manufactured by Waters).

Mobile phase: 33% acetonitrile/0.05% trifluoroacetic acid aqueous solution.

Flow rate: 2.0ml/min.

Detection: UV-254nm.

A peak with a retention time of about 11.0 to 12.0 minutes was fractionated, and this operation was repeated to collect this fraction. The obtained fractionated solution was concentrated using a rotary evaporator, then adjusted to pH 8.0 to 8.5 with a 5% sodium carbonate solution, and extracted with 5 times the amount of chloroform. The chloroform layer was concentrated to dryness using a rotary evaporator, then dissolved in 0.001N hydrochloric acid solution and freeze-dried to obtain 5 mg of the active substance hydrochloride 301-B.

c Purification of crude powder C Dissolve crude powder C in water to a concentration of 10 mg/ml, and
200μ was injected into the high performance liquid chromatograph shown below.

Column: “Micro Bonder Pack (μ
Bondapak) C18” (7.8 x 300mm, manufactured by Waters).

Mobile phase: 50% acetonitrile/0.05% trifluoroacetic acid aqueous solution.

Flow rate: 2.0ml/min.

Detection: UV-254nm.

A peak with a retention time of about 8.0 to 9.0 minutes was fractionated, and this operation was repeated to collect this fraction. The obtained fractionated solution was concentrated using a rotary evaporator, then adjusted to pH 8.0 to 8.5 with a 5% sodium carbonate solution, and extracted with 5 times the amount of chloroform. After concentrating the chloroform layer to dryness using a rotary evaporator, it was dissolved in a 0.001N hydrochloric acid solution and freeze-dried to obtain 2 mg of the active substance hydrochloride 301-C.
I got it.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the 301-A ₁ substance (hydrochloride) of the present invention, and FIG. 2 shows its infrared absorption spectrum (KBr method). Figure 3 shows the ultraviolet absorption spectrum of the 301-A ₂ substance (hydrochloride), and Figure 4 shows its infrared absorption spectrum (KBr method). Fifth
The figure shows the ultraviolet absorption spectrum of substance 301-B (hydrochloride), and Figure 6 shows its infrared absorption spectrum (KBr
law). FIG. 7 shows the ultraviolet absorption spectrum of the 301-C substance (hydrochloride), and FIG. 8 shows its infrared absorption spectrum (KBr method).

Claims (1)

[Claims] 1. General formula [In the formula, R ₁ and R ₂ both represent hydrogen atoms,
Or R ₁ is a methyl group and R ₂ is a hydrogen atom, base or Indicates the group. ] 301 substances and their salts.