JPS6210086A - Novel antibiotic lactoquionomycin and production thereof - Google Patents

Abstract

NEW MATERIAL:An antibiotic shown by the formula or its salt or metallic complex. Appearance: orange crystal. Molecular weight: 457. Molecular formula: C24H27NO8. Melting point: 151-159 deg.C (decomposition). Elemental analysis(R): C 61.18, H 6.21, N 3.20, O 27.70. Solubility: soluble in ether, chloroform, ethyl acetate, water, etc., and insoluble in h-hexane. Color reaction: positive in magnesium acetate reaction, Dragendorff's reaction and negative in ninhydrin reaction, Elson-Morgan reaction, etc.,; Specific rotatory power: [alpha]D<28>+316.9 deg. (C=0.2, methanol), etc. USE:An antimicrobial agent and an antitumor agent effective against cancerous cells resistant to adriamycin, etc. PREPARATION:Streptomyces tanashiensis IM8442T strain(FERM P-8327) is cultivated preferably under aerobic condition at 10-40 deg.C at 4-8 pH for 24-144 hours and an antibiotic shown by the formula is obtained from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention has antitumor properties useful in the pharmaceutical field! The present invention relates to a novel naphthoquinone antibiotic, a pharmaceutically acceptable salt thereof, and a method for producing the same.

Many naphthoquinone antitumor antibiotics are known, including anthracycline antitumor antibiotics such as adriamycin and aclarubicin. Adriamycin, aclarpicin, etc. are used clinically and have shown excellent therapeutic effects.

Do existing anticancer drugs such as adriamycin and aclarbine have good effects? However, cancer cells are resistant to these anticancer drugs! This is known to be a problem in treatment.

B) Means for Solving the Problems The present inventor conducted a wide range of searches and conducted extensive research regarding microbial cultures. As a result, they discovered that actinomycetes belonging to the genus Streptomyces, collected and isolated from soil in Hiratsuka City, Kanagawa Prefecture in October 1980, produce naphthoquinone-based substances that have antibiotic and antitumor activities. When we isolated, purified, and determined the structure of this substance, we discovered that it had a new structure that was different from known substances! The substance was named t-lactoquinomycin.

After further investigation, it was found to be resistant to adriamycin and aclarbine! Mouse lymphoma L5178Y with
They discovered that cell growth can be inhibited and completed the present invention.

That is, the present invention uses the novel antibiotic lactokinomycin and its pharmaceutically acceptable salts or metal complexes, as well as the antibiotic lactokinomycin-producing bacteria belonging to the genus Streptomyces, as a nutritional source! Containing medium (: aerobic (biculture,
The present invention relates to lactokinomycin, a salt thereof, or a metal complex thereof, which is a novel antibiotic characterized by producing, accumulating, and collecting lactokinomycin, a salt thereof, or a metal complex thereof.

As an example of the bacteria producing the novel antibiotic lactoquinomycin, its salt, or its metal complex of the present invention, the present inventors cite the newly isolated IM8442T strain.

The mycological properties of the IM8442T strain are as follows.

Mycological characteristics 1 and morphology of the IM8442T strain Under a microscope, the IM8442T strain forms straight aerial hyphae that are relatively longer than the branched basal hyphae, and no whorl formation is observed. A mature spore chain is an almost straight chain of 20 or more spores! The size of the spores was 0.4-0.
6 x 1.0-1.3 micrometers, the surface of the spores is smooth.

2. Growth status on various media (1) Growth on sucrose/nitric acid agar medium is moderate. A small amount of pale yellow powdery aerial mycelium grows on it, and no soluble pigment is observed.

(2) Growth on glucose-asparagine agar medium is slight. A moderate amount of pale yellow powdery aerial mycelium is observed, and no soluble pigment is observed.

(3) Glycerin-asparagine agar medium (ISP
- Medium 5) Growth is moderate. Good epiphyte of gray velvety aerial mycelium, yellowish brown soluble pigment! admit.

(4) Starch/inorganic salt agar medium (ISP-medium) Growth is good. Yellow-brown-gray velvety aerial mycelium is well established, and dark yellow-brown soluble pigment is observed.

(5) Growth was good on tyrosine agar medium (ISP-medium 7). It is covered with abundant brownish-gray hair-like aerial mycelium, and dark brownish-gray soluble pigment is observed.

(6) Growth on nutrient agar medium is slight. Only a few pale yellow powdery aerial mycelia grow on the plant, and a dark brown-gray soluble pigment is observed.

(7) Yeast/malt agar medium (ISP-Medium 2)
Growth is good. Gray velvety aerial mycelia adhere well, and dark brown-gray soluble pigment is observed.

3. Physiological properties (1) Liquefied gelatin glucose peptone gelatin medium (27°C culture)
Liquefaction of gelatin is observed.

(2) Hydrolysis of starch Hydrolysis of starch is observed on starch/inorganic salt agar medium (cultured at 27°C).

(3) Coagulation and peptonization of skimmed milk (skimmed milk, 27°
C culture) No obvious coagulation was observed, and peptonization was observed.

(4) Production of melanin-like pigments Melanin-like pigments are produced in peptone yeast/iron agar medium and tryptone yeast liquid medium.

(5) Utilization of carbon sources (Pridham-Gotlieb agar medium 15P-medium 9.27°C culture) D-glucose, L
-Grows using arabinose, D-xylose, galactose, salicin, but sucrose, D-fructose, raffinose, rhamnose, inositol, D
-Manitol is rarely used.

(6) It grows at a growth temperature of 10-37°C, and the optimal growth temperature is 27-30°C.
C (yeast/malt medium).

It grows at 20-37°C, and the optimum temperature is 20-30°C (oatmeal medium).

Comparing the above characteristics, the IM844.2T strain belongs to the genus Streptomyces.
In addition, L-type diaminopimelic acid (LL-DAP) Y was detected from all the bacterial cells of this strain, the aerial hyphae were straight and slightly curved, and the spore chains were also straight and slightly curved. No whorling is observed and the surface of the spores is smooth. Aerial mycelia are often gray and contain melanin-like pigments! Produces in certain types of media. Regarding protein decomposition ability, coagulation of skim milk is extremely weak, but peptonization of skim milk and liquefaction of gelatin are moderate. D-glucose, L-arabinose, D
-Xylose, galactose, salicin! I use it, but
Sucrose, D-fructose, raffinose, rhamnonis, inositol, and D-manitol are hardly used.

Based on the above characteristics, Streptomyces tanashiensis
-sis), so the inventors determined whether this strain was the same? Streptomyces thanashensis (Slr-ept
omyces tanashiensis ) I M
It was named 8442 T strain.

This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its accession number is No. 8.
No. 327 (FBRMP''-8327).

The IM8442T strain, like many other strains of actinomycetes, is susceptible to changes in its properties, such as exposure to ultraviolet light,
Although it is possible to mutate by artificial mutation means using X-rays, radiation, chemicals, etc., all mutant strains that have the ability to produce the antibiotic lactoquinomycin can be used in the method of the present invention. I can do it.

Next, a method for producing lactoquinomycin, a salt thereof, or a metal complex thereof will be explained.

According to the present invention, lactokinomycin-producing bacteria belonging to the genus Streptomyces are cultured to accumulate lactokinomycin, a salt thereof, or a metal complex thereof, and lactokinomycin, a salt thereof, or a metal complex thereof is obtained from the culture. Lactokinomycin, its salts, or its metal complexes can be produced by collecting lactoquinomycin, its salts, or its metal complexes, but lactoquinomycins, its salts, or its metal complexes can be produced in a pure state.
Alternatively, it is collected in a crude state, in a solution state, or in a solid state.

In carrying out the production of the compounds of the present invention, Streptomyces tanashensis (8 treptomyce
s tanashiensis) I M 8442
A culture containing lactokinomycin is obtained by inoculating the T strain into a nutrient-containing medium and growing it aerobically.

As nutrients, those known as nutritional sources for actinomycetes can be used. For example, commercially available peptone, meat extract, corn
Steep Suba-1 Cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-amine, thawed casein, fish meal, nitrogen sources such as sodium nitrate, ammonium nitrate, and ammonium sulfate, commercially available glycerin, sucrose, and tempura. ,
Carbon sources such as hydrocarbons or fats such as crunine, maltose, molasses, and inorganic salts such as common salt, phosphates, calcium carbonate, magnesium sulfate, etc. can be used.

In addition, metal salts can also be added as needed.

In addition to these, any material can be used as long as it is utilized by the producing bacteria and is useful for producing lactoquinomycin, its salt, or its metal complex.

The culturing method may be the same as a general antibiotic production method, and solid culture or liquid culture may be used. In the case of liquid culture, any of static culture, agitation culture, shaking culture, aerated culture, etc. may be used, but aerated culture is particularly preferred.

Further, the culture temperature is preferably in the range of about 10° C. to 40° C., the pH of the medium is in the range of about 4 to 8, and the culture is carried out for about 8 hours to 168 hours, preferably 24 hours to 144 hours.

Target lactokinomycin from culture! For collection, separation means commonly used to collect metabolites produced by microorganisms from cultures are used. Lactoquinomycin in the culture solution is obtained by adding a P support agent to the culture solution and removing the bacterial cells by filtration or centrifugation.
The operation of extracting the liquid from organic solvent C2, then appropriately changing the pH of the aqueous layer, and dissolving it into the organic solvent layer and the aqueous layer! Repeat or as appropriate, contact with a carrier to remove the active ingredient in liquid or aqueous solution! It can be purified by adsorption and then desorption of the active ingredient with a suitable solvent. High performance liquid chromatography and thin layer chromatography can also be used for extraction and purification.

To explain in more detail, culture liquid 'la0: pH8
After adjusting, lactoquinomycin is dissolved in an organic solvent such as ethyl acetate or butyl acetate, and only the organic solvent layer is separated. After the entire organic solvent layer is extracted with appropriate acidic water, the aqueous layer is separated and adjusted to pH 8. Extraction operation using organic solvents such as ethyl acetate and butyl acetate! Repeatedly, a lactoquinomycin-containing fraction is obtained.

In addition, the aqueous layer containing this lactoquinomycin is freeze-dried, or the organic solvent layer after dissolution is dehydrated and concentrated.
Such as hexane! Upon addition, a powdered salt of lactoquinomycin or its free base can be obtained.

Next, the obtained lactoquinomycin or its salt is dissolved in water and then adsorbed onto a resin such as CI-IP20P. The adsorbed lactoquinomycin is eluted from the resin with a mixed solvent of a solvent such as methanol and acidic water, and lactoquinomycin or its salt can be obtained as a crystalline powder according to the above extraction method. Typical salts include salts with various inorganic or organic acids, such as hydrochlorides, sulfates, acetates, and the like.

Lactokinomycin metal complexes can be easily produced by adding various metal ions to an aqueous solution of lactokinomycin. Typical metal complexes of lactoquinomycin include metal complex Z with iron, copper, cobalt, platinum, zinc, etc. Lactoquinomycin metal complexes can also be obtained from the culture solution. Furthermore, lactoquinomycin or its metal complex can also be produced in the form of a salt.
Typical salts include salts with various inorganic or organic acids, such as hydrochlorides, sulfates, and acetates.

When the antibiotic lactokinomycin of the present invention is used as an antibacterial or anticancer agent, it can be used in the form of a pharmaceutical formulation containing a compatible drug carrier together with the antibiotic lactokinomycin. The carrier is, for example, water, gelatin,
Organic or inorganic inert carriers suitable for oral, subcutaneous or parenteral administration such as gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycols and petroleum sherry. The pharmaceutical formulation may contain therapeutically effective substances other than the antibiotic lactoquinomycin of the invention. Pharmaceutical preparations may be in solid (e.g. tablets, dragees or capsules) or liquid (e.g. tablets, dragees or capsules)
For example, solutions, suspensions or emulsions) C: Can be used. The pharmaceutical formulations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, osmotic agents or buffering salts.

The dosage of the active ingredient varies depending on the method of administration, the age, weight, condition of the patient, and the disease being treated, but typical dosages for adults are oral or parenteral, preferably by intravenous injection. , ranges from 1 to 10 μ per day.

Next, the present invention will be further explained with reference to Examples, but the present invention is not limited only to the Examples, and the present invention (=properties of lactoquinomycin clarified by the present invention) is not limited to the examples. All methods for producing, concentrating, extracting, and purifying lactoquinomycin by applying known means based on the above are also included.

■Example Streptomyces IM8442T strain cultured on agar slant medium was inoculated into a 500-capacity Sakaro flask containing 100-mL liquid medium (pH 7.2) consisting of 2% oatmeal and 0.1% yeast extract for 278,040 hours. Culture on a shaker (150 revolutions per minute) and use the seed culture! Obtained. .

Next, 500- containing liquid medium 100- with the same composition as above
The above seed culture solution 2- was inoculated into 25 Sakaro flasks and cultured at 27°C for 88 hours (agitation: 150 revolutions per minute).

The culture solution (approximately 2/2) was separated into bacterial cells and supernatant using a high-speed centrifuge, the supernatant was adjusted to pH 3 with an aqueous Y 2 N NaOH solution, and then extracted twice with an equal volume of ethyl acetate. A summary of ethyl acetate, 1/10th the amount of 0.01N HeI! Adjust to p)l 8 with an aqueous solution and extract again twice with equal volumes of ethyl acetate. The ethyl acetate layer is dried over anhydrous sodium sulfate and then concentrated to one volume. Add a suitable amount of n-hexitine to this and leave it in a cool place to obtain orange crude crystals.

The crude crystals are dissolved in water and then adsorbed onto a CHP20P (Mitsubishi Kasei) column. After washing the column with water and 40% methanol, 10mM HeI! -Elute with 40% methanol.

Eluate containing active fraction Y I) Methanol under reduced pressure after adjusting to H6! Distill. Adjust the pH to 8 with 2N NaOH aqueous solution and extract again with ethyl acetate. The extract is dried over anhydrous sodium sulfate and concentrated under reduced pressure. After concentration, n-hechitin was added and the mixture was left in a cool place to obtain about 120q of orange crystals of lactokinomycin.

Physical and chemical properties of lactoquinomycin> (a) Molecular weight (based on FAB mass spectrum measurement) 457
(M+H, m/Z 458) (b) Molecular formula % Formula % (dl Elemental analysis HNO as C10H27NOA Experimental value (e) 61.18 6.21 3.20 2
7.70 Theoretical value% 63.01 5.95 3.06
27.98(e) Infrared absorption spectrum The external optical absorption curve of lactoquinomycin measured in chloroform is shown in FIG.

(f) Ultraviolet absorption spectrum and visible absorption spectrum The ultraviolet absorption curve and visible absorption curve of lactoquinomycin are as shown in FIG.

Moreover, the absorption maximum and e value in each solution are as follows.

1) Methanol fi) Methanol containing 0.01 N hydrochloric acid m) o, o
xN sodium hydroxide containing methano.

-L(g)H nuclear magnetic resonance spectra of lactokinomycin measured in deuterated chloroform I)
The nuclear magnetic resonance spectrum (400 MH atoms) is as shown in FIG.

( ), 3 13 C Nuclear Magnetic Resonance Spectrum The 13 C nuclear magnetic resonance spectrum (100 MHz) of lactokinomycin measured in chloroform is shown in FIG.

(it Solubility in solvents Lactoquinomycin is ether, chloroform, ethyl acetate, acetone, methanol, water (= easily soluble, but n
-Almost insoluble in hechitin.

(jl Color reaction The color reaction of lactokinomycin is as followsg Magnesium acetate reaction ・・・Positive Dragendorff reaction ・・・Positive ninhydrin reaction
・・・・・・Negative Elson Morgan reaction ・
... 〃5-troprusidaldehyde reaction...
〃Also, lactoquinomycin is purple in a basic aqueous solution and orange in an acidic aqueous solution! present.

(k) Distinction between basic, acidic, and neutral Lactokinomycin is a basic substance.

(+) Rf value Lactokinomycin was obtained by silica gel thin layer chromatography, developed with a chloroform/ethanol mixture (1:1) to obtain RfO, 27, and methanol to obtain RfO, 30.
Each 'single spot' is shown.

Lactokinomycin was clearly distinguished from known antibiotics based on the above-mentioned properties, and was confirmed to be a new antibiotic.

(1) Antibacterial action The minimum inhibitory concentration of lactoquinomycin against various bacteria on Mueller-Hinton agar medium and against various fungi on yeast extract Siracross agar medium is shown in Table 1-(11
and (2).

Table 1 - (1) Table 1 - (2) (2) Anticancer effect Lactokinomycin is added to RPMI with 10% fetal bovine serum.
In 1640 medium, the growth of human leukemia 562 cells and mouse leukemia P388 cells is inhibited. Also, 10%
Mouse lymphoma L5178Y cells and their adriamycin-resistant strain in RPMI-1640 medium supplemented with horse serum;
Prevents the growth of a'clarubicin-resistant strains and pleomycin-resistant strains.

Table 3 shows the 50% growth inhibitory concentrations for these cancer cells.

(3) The acute toxicity (LDso) of lactoquinomycin in mice is approximately IQIII when administered intravenously.
It was P/Kp.

(→ Effects of the Invention The antibiotic lactokinomycin of the present invention strongly inhibits the growth of mainly Durum-positive bacteria.

It also strongly inhibits the growth of mouse lymphoma L5178Y cells that are resistant to adriamycin, aclapicin, and pleomycin.

Therefore, lactochinmycin can be expected to be used as an antibacterial agent against Durham-positive bacteria and an antitumor agent against drug-resistant cancer cells.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of lactoquinomycin measured in chloroform. Figure 2 shows lactoquinomycin in methanol at 0.0I
Measured in methanol containing N hydrochloric acid and methanol containing 0.01N sodium hydroxide.
The absorption spectrum and visible absorption spectrum are shown. FIG. 3 shows the IH nuclear magnetic resonance spectrum of lactoquinomycin measured in deuterated chloroform. FIG. 4 shows the 13c nuclear magnetic resonance spectrum of lactoquinomycin measured in chloroform.

Claims (1)

[Claims] (1) Antibiotic Lactoquinomycin and its pharmaceutically acceptable salts or metal complexes represented by general formula (I) ▲Mathematical formulas, chemical formulas, tables, etc.▼(I) (2) Antibiotics belonging to the genus Streptomyces Lactokinomycin-producing bacteria are cultured aerobically in a medium containing a nutrient source, producing and accumulating lactokinomycin or a salt thereof, or a metal complex thereof, and collecting this from the culture. A method for producing the antibiotic lactoquinomycin, a salt thereof, or a metal complex thereof.