JPH0272884A - Q-1043 substances and production thereof - Google Patents

Abstract

NEW MATERIAL:The Q-1043A, A1, B and B1 substances having the following physical and chemical properties. Q-1043A substance: elemental analysis (%) (as C49H60O19.H2O), C 60.33, H 6.09; melting point, 245-250 deg.C, molecular weight, 952. Q-1043A1 substance: elemental analysis (%) (as C49H58O18.1.5H2O), C 60.98, H 6.11; melting point, 206-208 deg.C, molecular weight, 934; etc. USE:An antitumor agent, a preventive and remedy for phlebo-thrombosis, pulmonary embolism, cerebral thrombosis, etc., and antibacterial agent. PREPARATION:A microbial strain belonging to genus Streptomyces and capable of producing Q-1043A, A1, B and B1 substances [preferably Streptomyces sp. Q-1043 (FERM P-10125), etc.] is cultured preferably under aerobic condition at 28-30 deg.C and pH 5.5-8.5 for 1-5 days.

Description

Detailed Description of the Invention (Industrial Field of Application) The present invention provides novel Q-1043A, A, B, which are useful as medicines, and more specifically, have cytotoxic effects, platelet aggregation inhibitory effects, antibacterial effects, etc. B.

This invention relates to substances (hereinafter these substances are referred to as Q-1043 substances) and methods for producing the same.

(Means for Solving the Invention) The Q-1043 substances of the present invention are new substances having the following physical and chemical properties.

(Il Q-1043A material elemental analysis value (as CnHeoo+o ・H2O)C(
Transfer H (@Theoretical value 60,61 6.44 Experimental value 60,33 6.09 Melting point 245-250°C Molecular weight 952 (FAB-Mass spectrum) Ultraviolet absorption spectrum (in methanol) 470 nm, 445
nm, 420nm, 310nm, 260nm.

20nm infrared absorption spectrum (potassium bromide tablet) 1730cm-
t, 1680cm-1,1620cm-1,1100
~1000100O Specific rotation (in chloroform, concentration = 0.5%) [α]2
o. Knee 22° thin layer chromatography (Kieselgel 60, Merck & Co.) Solvent (■): Benzene/Chloroform/Methanol (
1:1:0.1) Solvation: Ethyl acetate (same below) Solvent (Il Solvent circle 0.34 0.38 Rf value Q -1043A, substance ■ Elemental analysis value (C4QH58018・1.5H,, O
) C('n H(@ Theoretical value 61.17 6.39 Experimental value 60,98 6.11 ■ Melting point 206-208℃ ■ Molecule ft 934 (FAB-Mass spectrum) ■ Ultraviolet absorption spectrum (in methanol )49
0nm, 310nm, 290nm, 262nm,
250nm ■ Infrared absorption spectrum (potassium bromide tablet) 1735cm-1,1620cm-t, 1100~
1000 cm-1■ Specific optical rotation (in chloroform, concentration = 05%) [α] temporary = + 6.0゜■ Thin layer chromatography (Kieselgel 60, Merck & Co.) Solvent (Il Solvation Rf value 0.48 0. 51 (〕→ Q -1043B substance ■ Elemental analysis value (C55H70021" as H2O) , 1066 (FAB-mass spectrum) ■ Ultraviolet absorption spectrum (in methanol) 4
70nm, 445nm, 420nm, 310nm
, 260nm.

20nm ■ Infrared absorption spectrum (potassium bromide tablet) 1730c
m-1,1680cr+rt, 1100~1000c
m-1 ■ Specific optical rotation (in chloroform, concentration = 05%)
[α] knee 12゜■ Thin layer chromatography (Kieselgel 60, Merck, Inc.) Solvent (I) Solvent (II) Rf value 0.211 0.30) Q-1043B, substance■ Elemental analysis (Cs5H6sOn・= (as IH20)
C(□□□ H(Model value 58,91 6.83 Experimental value 58,88 6.52 ■ Melting point 252-256°C ■ Molecular weight 1048 (FAB-mass spectrum) ■ ■ ■ ■ Ultraviolet absorption spectrum ( in methanol) 490 nm,
310nm, 290nm, 262nm, 250n
m infrared absorption spectrum (potassium bromide tablet) 1735cm
-+, 1610cm-1,1100~1000cm-
1 specific optical rotation (in chloroform, (HI=0.5%) [α
]202+42゜Thin layer chromatography (Kieselgel 60, Merck & Co.) Solvent (Il Solvent (11) Rf O,340,41 (Effects of the invention) The Q-1043 substances of the present invention are strong against various tumor cells. Q-1043 of the present invention has a cytotoxic effect, and also has a platelet aggregation inhibiting effect and an antibacterial effect.
Substances include antitumor agents, agents for preventing and treating diseases such as venous blood vessels, pulmonary embolism, and cerebral blood clots resulting from thrombus formation.

The test methods and results for the cytotoxic effect, platelet aggregation inhibitory effect, and antibacterial effect of Q-1043 substances are shown below.

(1) Cytotoxicity ■ Cytotoxicity using He1a cells Cytotoxicity is as follows:
The number of cells was measured microscopically after culturing with carbon dioxide gas at each concentration for 72 hours.

Q-1043A, Q-10438゜Q by this method
Table 1 shows the toxicity of -1043B to He1a cells.
Shown below.

Table 1 ■ Cytotoxicity test method using L1210 and P388 cells: Q-1043A at various concentrations dissolved in dimethyl sulfoxide to tL1 ml of each L1210 P2S5 cell adjusted to I X 10' Ce1ls/ml.
, or add 4μt of Q-1043B substance, 37°
After culturing for 3 days in a carbon dioxide incubator, surviving cells were counted by trypan blue staining. Cytotoxic activity is based on the number of cells without drug addition.

The cell growth inhibition rate at each concentration was calculated and plotted on a graph.

Table 2 (2) Test method for inhibiting platelet aggregation; 9 volumes of blood were collected from the ear artery of a male Japanese white rabbit weighing approximately 3 kg into a plastic syringe containing 1 volume of 38% sodium citrate aqueous solution. 270 blood
Centrifuge at room temperature for 10 minutes at
Centrifuge at 0X for 15 minutes to extract platelet-poor plasma (hereinafter referred to as PPP).
) was obtained.

After diluting PRP with PPP and adjusting the platelet count to 500,000/μt, various platelet aggregating substances (PAF; t) were added.

nM, ADP; 3 μM, ColCo11a;
10μg/1m4
d; Platelet aggregation with 100 μM) and cross [
G., V., R.Born and K.J., Cro.
ss, Journal of Physiology
y.

168.178-195. (1963)]. That is, PR with two different flocculating substances using NBS hematotracer (binary bioscience).
Changes in light transmittance of P were measured. The test sample was added 2 minutes before the addition of the aggregating substance, and the effect on aggregation was determined from the inhibition rate against the maximum light transmittance in the solvent control.

(2) Antibacterial effect Table 4 shows the minimum inhibitory concentration (MIC) of substances Q-1043A and B against various bacteria.

(Manufacturing method) Q-1043 substances belong to the genus Streptomyces.
1043 substances producing bacteria are cultured and Q-10 is obtained from the culture.
It can be manufactured by collecting 43 substances.

An example of Q-1043 substance-producing bacteria used in the production method of the present invention is Streptomyces sp.
strain (Feikoken Bibori No. 10125). The mycological properties of this strain are described below.

1. Morphology The basal hyphae are well developed on various organic and inorganic media, and the width of the hyphae is approximately 0.4-0.8 μm. The epiphytion of aerial hyphae is good on l5P-2 and Benenoto agar media.・It adheres well on other media, but it grows poorly or does not adhere at all on other media.Aerial hyphae are long straight or curved and simply branched.

Mature aerial hyphae form a long spore chain of 20 or more. The surface of the spore is smooth and cylindrical in shape.

Prespore, motile spores.

No sclerotia, two-ring silk, seedling bundle thread, etc. were observed (・.

2. Properties on various agar media Properties on various agar media are as shown below. Unless otherwise specified, the cells were cultured at 28° C. for 21 days and observed according to conventional methods. The description of color tone was based on the color standard (Japan Color Collection Institute).

Table 5 Assimilation of carbon sources (Bridham-Godelive obturation medium, 2
(8°C culture) 2) Liquefied simple gelatin (20°C) Negative 4) Nitrate reducing action 5) Starch hydrolyzing action 6) Production of melanin-like pigment Negative (no growth) Negative 7) In a salt-containing medium It grows at 3% growth rate, but not at 6% growth rate.

37, 40, 45. Observation results from 7 to 21 days at 50°C.

The effects on milk are the results observed after 3 to 21 days at 37°C.Other than that, unless otherwise specified, the results are shown after 2 weeks at 28°C.

Q-1043 strain L-arabinose D-xylose D-glucose L-rhamnose D-fructose D-galactose raffinose D-mannitol inositol mannose trehalose sucrose glycerin starch salicin xanthine + + 5, chemical analysis of bacterial cell components Method of LECHVLIER et al. LECHVALIER,
M.P.

et al; PP277-2381nDIETZ, A
et al ed,,Act-inomycete T
axonomy, S IM 5special pub
As a result of analyzing the acid hydrolyzate of this strain (whole cells and cell walls) according to lication No. 6.1980, LL-diaminopimelic acid was detected.

To summarize the above properties, strain Q-1043 creates a long chain of spores on the aerial mycelium, and the width of the hyphae is approximately 0.4-
It is 0.8 μm. The hue of the aerial hyphae was red/leeds, and the aerial hyphae growth was good on the l5P-2 and bean-nod occlusion medium.

On other media, the seedlings are poorly established or not adhered to at all. In addition, it produces five different reddish dashi and reddish-brown soluble pigments on the agar medium. The surface of the spore is smooth and cylindrical in shape.

No sporangia, motile spores, 9 rings of sclerotia, 7 fascicle threads, etc. were observed, and according to the chemical classification method, diaminopimelic acid in the whole bacterial body or cell wall was classified as LL type.

No characteristic sugar components were detected.

Based on the above characteristics, known bacterial strains similar to the nine strains were listed in Bergey's Manual of Determinative Bacteriology.
eterminative Bacteriology
) 8th edition, 1974”, [International Journal of Systematic Bacteriology
(International Journal of
Systematic Bacteriology) Volume 18, No. 2.

pp. 69-189, No. 4, pp. 279-392 (196
8).

Volume 19, No. 4, pp. 391-512 (1969),
Vol. 22, No. 4, pp. 265-394 (1972) J
As a result of searching for and other literature, 9 strains were found.

It was recognized as a species belonging to the genus Streptomyces. Furthermore, Strepto
As a result of a search for known bacterial species belonging to the genus myces, Q
- As a bacterial species similar to strain 1,043, (1) Str
eptomyces phae.

purpureus, (21 Streptomyce
s roseosporus, (3) Strepto
myces 5ubrut mountain S is mentioned. This strain has a hyphal shape.

Although the species (2) and (3) are similar to the above-mentioned species in terms of hue and general physiological properties, they are different from the species (2) and (3) in that they are capable of assimilating aerial mycelial dust and carbon sources in l5P3°4 and 5 media. They are distinguished by gender, etc. Furthermore, it differs from (1) in the assimilation ability of the carbon source. Table 8 summarizes the differences between these strains.

Based on the above results, nine strains were identified as Streptomyces (Streptomyces).
It was recognized as a new bacterial species belonging to the genus Streptomyces sp.
tomycessp, Q-1043). This strain has been deposited with the National Institute of Microbial Technology, Agency of Industrial Science and Technology as Microbiological Research Institute No. 10125.

The strain used in the present invention, as seen in other actinobacteria, tends to undergo artificial and natural mutations.
Alternatively, it also includes strains that have been artificially mutated using ultraviolet rays, xi, chemical agents, etc., and natural mutant strains thereof.

The microorganisms belonging to the new bacterial species of the present invention were obtained by separating and miscellaneous from natural soil, but they can be easily obtained by restoring the freeze-dried product of the strain deposited with the Microbial Technology Research Institute. Be able to do it. In the present invention, the Q-1043 substance-producing bacteria belonging to the genus Sprebutomyces is cultured according to the culture method of general microorganisms, but the deep culture method using a liquid medium is usually advantageous. The medium used for culturing may be any medium as long as it contains a nutrient source used by the producing bacteria. That is, a synthetic medium, a semi-synthetic medium or a natural medium is used, and the composition of the medium is, for example, carbon sources such as D-galactose, glucose, D-fructose, L-rhamnose, mannitol, trehalose, glycerin, dextrin, and starch.

Vegetable oil, etc. Nitrogen sources include meat extract, peptone, gluten meal, cottonseed meal, soybean flour, fallen raw flour, fish meal, corn steep liquor, and dried yeast.

Yeast extract, ammonium sulfate, ammonium nitrate, urea and other organic or inorganic nitrogen sources are used. Also, Na, K, Mg, Ca as metal salts.

Sulfates, nitrates, chlorides, carbon salts of Zn, Fe, etc.

Phosphates and the like are added as necessary. Further, if necessary, antibiotic production promoting substances or antifoaming agents such as methionine, cysteine, cystine, methyl oleate, lard oil, silicone oil, and surfactants are appropriately used.

It is generally advantageous to culture under aerobic conditions, and the culture temperature is preferably in the range of about 20 to 33°C, preferably around 28 to 30°C, and the pH of the medium is is preferably kept in the range of about 5.5 to 8.5 to obtain good results. The culture period varies depending on the composition of the medium, temperature, etc., but is generally about 1 to 5 days.

At the end of the culture, the target substance is selectively accumulated.

In order to collect the target Q-1043 substances from the culture, extraction, separation and purification means commonly used from the culture of metabolites produced by microorganisms are appropriately used. Since the Q-1043 substances in the culture are contained in the culture solution, after removing the bacterial cells by centrifugation or filtration, the effective substances are extracted from the filtrate. In other words, general methods utilize differences in solubility and solubility in appropriate solvents, differences in precipitability from solutions and precipitation rates, differences in adsorption affinity for various adsorbents, and differences in distribution between two liquid phases. It is separated, collected, and purified by the same means used in the production of antibiotics. These methods can be used alone, combined or repeated in any order as required.

(Example) The two present inventions have been described in detail above, and will be further explained below with reference to Examples.

Example 1 Medium A (pH 7.0) containing 1.0% glucose, 2.0% hotel starch, 0.5% malt extract, 0.5% polypeptone, and 0.4% calcium carbonate was prepared.

Dispense this into 500mZ Erlenmeyer flasks in amounts of 60+nZ.

Streptomyces sp. Q-104 grown on Benenoto agar medium after sterilization at 120°C for 20 minutes.
Scrape the mycelia of 3 strains and inoculate them.

The culture was cultured with shaking at 27°C for 72 hours to prepare a seed culture.

Next, culture medium A5Z (pH 7,
0) was dispensed into 500 ml Erlenmeyer flasks at 6 On + Z, sterilized at 120°C for 20 minutes, inoculated with the seed culture solution at a rate of 4%, and cultured with shaking at 27°C for 296 hours in a rotary shaker at 220 revolutions per minute. Go on.

Adjust 5 L of culture solution of Q-1043 strain to pH 7 with diluted hydrochloric acid, and centrifuge at 3000 rpm for 10 minutes.

The supernatant was extracted with 6 L of ethyl acetate, and the extract was washed with water and concentrated under reduced pressure to obtain 2.34 g of a solid. The resulting material was subjected to column chromatography using 300 ml of silica gel (Kieselgel 60, Merli). Elution was performed with chloroform-methanol (75:1), and fractions 27 to 39 containing Q-1043A substance and Q-1043AI substance were collected and concentrated.

220 mg of a reddish-brown substance are obtained. Similarly, Q-1043
Fraction 40 containing substance B, Q-1043B, substance
~54 was collected and concentrated to yield 900 ml of reddish brown material. The fraction 27-39 concentrate was further subjected to thin layer silica gel chromatography (Kiesel get 60).
and develop with ethyl acetate. Fractions containing Q-1043A substance and Q-1043A substance were separated.

After extraction with ethyl acetate, the mixture was concentrated to dryness under reduced pressure. Q-104
3A substance orange powder 13■ and Ql 043
A. Obtain 22.5171 g of substance Azuki powder. On the other hand, fractions 40 to 54 concentrates were similarly subjected to chromatography and developed with ethyl acetate.

Fractions containing Q-1043B substance and Q-1043B substance are separated, extracted with ethyl acetate, and the extracts are concentrated to dryness under reduced pressure. 120 mg of orange powder of substance Q-1043B, and 86 μg of russet powder of substance Q-1043B are obtained.

The above extraction 9 separation, purified Q-1043A, and B1
The substance is considered to be a substance having the following planar structural formula from the above-mentioned physical and chemical properties.

Q-1043A, substance; Q-1043B, substance; (In the formula, one of R1 and R2 is a hydrogen atom and means the rest.) Also, Q-1043A substance is Q-1043A, substance has Q-
The 1043B substance is considered to be Q-1043B, a substance in which each substance has one molecule of water in its two-molecular formula.

Patent applicant Yamanouchi Pharmaceutical Co., Ltd.

Claims (3)

[Claims] (1) Q-1043A, A_ with the following physical and chemical properties
1, B and B_1 substance (a) Q-1043A substance [1] Elemental analysis value (C_4_9H_6_0O_1_9・
(as H_2O) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ [2] Melting point 245-250℃ [3] Molecular weight 952 [4] Ultraviolet absorption spectrum (in methanol) 470n
m, 445nm, 420nm, 310nm, 260nm
, exhibits absorption at 220 nm. [5] Infrared absorption spectrum (potassium bromide tablet) 1730
cm^-^1, 1680cm^-^1, 1620cm^
-^1, shows absorption at 1100 to 1000 cm^-^1. (b) Q-1043A_1 substance [1] Elemental analysis value (C_4_9H_5_8O_1_8・
1.5H_2O) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ [2] Melting point 206-208℃ [3] Molecular weight 934 [4] Ultraviolet absorption spectrum (in methanol) 490n
m, 310nm, 290nm, 262nm, 250nm
shows absorption. [5] Infrared absorption spectrum (potassium bromide tablet) 1735
cm^-^1, 1620cm^-^1, 1100~10
It shows absorption at 00cm^-^1. (c) Q-1043B substance [1] Elemental analysis value (C_5_5H_7_0O_2_1・
(as H_2O) ▲ Numerical formulas, chemical formulas, tables, etc. are available ▼ [2] Melting point 261-263℃ [3] Molecular weight 1066 [4] Ultraviolet absorption spectrum (in methanol) 470n
m, 445nm, 420nm, 310nm, 260nm
, exhibits absorption at 220 nm. [5] Infrared absorption spectrum (potassium bromide tablet) 1730
cm^-^1, 1680cm^-^1, 1100~10
It shows absorption at 00cm^-^1. (d) Q-1043B_1 substance [1] Elemental analysis value (C_5_5H_6_8O_2_0・
(as 4H_2O) ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [2] Melting point 252-256℃ [3] Molecular weight 1048 [4] Ultraviolet absorption spectrum (in methanol) 490n
m, 310nm, 290nm, 262nm, 250nm
shows absorption. [5] Infrared absorption spectrum (potassium bromide tablet) 1735
cm^-^1, 1610cm^-^1, 1100~10
It shows absorption at 00cm^-^1. (2) Q-1043A, A belonging to the genus Streptomyces
A method for producing Q-1043A, A_1, and B_1 substances, characterized by culturing _1, B, and B_1 substance-producing bacteria, and collecting Q-1043A, A_1, B, and B_1 substances from the obtained culture. (3) The bacteria belonging to the genus Streptomyces is Streptomyces sp.Q-1
043 (Feikoken Bibori No. 10125)
) manufacturing method described