JPS63122687A - Novel naphthoquinone based compound nqy - Google Patents

Abstract

NEW MATERIAL:A compound expressed by the formula having the following properties; appeArance, yellow powder; melting point, 160-170 deg.C (decomposed); [alpha]D<24>=-96 deg. (C=0.002, CHCl3); molecular formula, C20H2OO7; highly resolving power EI mass spectrum (m/z), 372 and 1224; solubility, soluble in chloroform, methanol and acetone and insoluble in ethyl ether and water, etc. USE:An inhibiting agent of cancer metastasis. PREPARATION:A bacterium strain [e.g. Streptomyces malachiticus No. S-1998 strain (FERM P-8961), etc.] belonging to the genus Streptomyces malachiticus and having NQY-producing ability is cultured preferably by an aerobic liquid submerged cultivation at 25-30 deg.C for 72-96hr.

Description

DETAILED DESCRIPTION OF THE INVENTION [Background of the Invention] Technical Field The present invention relates to a novel naphthoquinone compound that has been shown to have an inhibitory effect on cancer metastasis.

Metastasis is a specific and typical feature of cancer. Although the success rate of removing primary cancer through surgery has increased in recent years, more than half of patients have already metastasized by the time their cancer is diagnosed, and therefore, if a person dies from cancer, it is likely that metastasis is the cause. There are many. As described above, cancer metastasis is an important problem that governs the prognosis of patients, but almost no therapeutic measures have been taken. Currently, after removing the primary tumor through surgical therapy,
A common treatment method is to kill remaining cancer cells with powerful anticancer drugs and suppress metastasis, but as mentioned above, it has not been successful in completely inhibiting metastasis.

Recently, DL-
α-difluoromethylornithine (DFMO) (F
EBS Lett, vol. 150, p. 397, 1982
) and N-chlorosulfonyldicyclohexylamine (
C8D), sulfonyldiamine-N, N'-dicyclohexane (SDD), etc. (Article at the 43rd Annual Meeting of the Japanese Cancer Society, Yang, p. 1150, 319, 1984) have been reported as polyamine synthesis inhibitors; However, problems remain in terms of efficacy, toxicity, and side effects before it can be put into practical use. It is clear that if a cancer metastasis inhibitor with low toxicity and side effects is found, it will greatly contribute to the eradication of cancer, and there is a constant desire for such a drug.

[Summary of the invention]

Abstract The present invention meets the above needs.

That is, the novel naphthoquinone compound NQY according to the present invention is represented by the following formula (I).

CH-COOH and Effect The compound NQY according to the present invention has cancer metastasis suppressing activity. NQY is expected to be developed as a cancer metastasis inhibitor.

[Detailed description of the invention]

Compound NQY 1, Chemical Structure The novel naphthoquinone compound NQY according to the present invention has a chemical structure represented by the above formula (I).

The chemical structure of NQY was determined as described above from the results of instrumental analysis such as ultraviolet-visible absorption spectrum, nuclear magnetic resonance spectrum, high-resolution El mass spectrum, and infrared absorption spectrum.

2. Physicochemical properties 1) Color, properties: yellow powder 2) Melting point: 160-170°C (decomposed) 3) Specific rotation: [α]D-96@ (C0,002, CHCl3) 4) Molecular formula '020H20 °7 5) High resolution El mass spectrum (m/z) real allJ
Value: 372.1224 Calculated value: 372.1209 6) Ultraviolet/visible absorption spectrum Figure 1 λ nm (logε) in CH30H 219 (4,38),
ax 252 (4,08), 420 (3,51) 7) Infrared absorption spectrum Figure 2 CHCl3 1755.1670.1640.910CrT1″″1 8) Solubility in solvent Soluble: Insoluble in chloroform, methanol, acetone: Ethyl ether, water 9) Behavior in thin layer chromatography (using Merck silica gel 60F254 plate) Chloroform:
Methanol-9:I Rf-0,43 to) 'HNMR spectrum Figure 3 400 MHz CD C1all) 1
3CNMR spectrum Figure 4 400 MHz CD Cl a12) Molecular weight: 372 (EIl Mass spectrum Production of compound NQY 1, Overview NQY has currently only been obtained by culturing microorganisms, but synthetic chemistry of related compounds It could be produced by chemical or microbiological modification, or by total synthetic chemistry.

In the case of culturing microorganisms, a strain belonging to the genus Streptomyces and capable of producing NQY is used. Specifically, the present inventors have revealed that the Streptomyces malachiticus No. S-1998 strain isolated by the present inventors produces NQY;
Appropriate strains of other bacteria can also be isolated from nature. There also remains room for increasing NQY production ability by subjecting NQY producing bacteria, including Streptomyces Φ Malachyticus No. S-1998 strain, to irradiation or other treatments. Furthermore, given the advances in genetic engineering, recombinant DNAs containing genes encoding the NQY-producing ability of NQY-producing bacteria, including Streptomyces malachiticus No. S-1998 strain, have been developed.
A method of culturing transformed microorganisms using A is also possible.

2. m5-1998 strain NO,S-1998 strain, which the present inventors discovered as a Streptomyces strain having NQY-producing ability, is
The contents are as follows.

■ Origin and Deposit Number: Strain S-1998 was isolated from soil collected on Kakigashima Kaminaka, and was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on September 5, 1985. It has a number of ``Feikoken Bibori No. 8961.''

(2) Mycological properties and physiological properties The NαS-1998 strain that produces NQY has the following mycological properties.

a) Aerial hyphae extend long from well-branched substratum hyphae, forming spore chains that simply branch from the main axis.

The spore chain has a spiral shape, and there are usually 10 to 50 spores in one spore chain. Occasionally, longer spore chains may form on inorganic salt starch agar. The surface of the spore is spiny, and the size is 0.6-0.
8XO, 9-1.1 μm.

Special forms such as sporangia, flagellated spores, and sclerotia are not observed.

b) Culture properties Properties when cultured at 27°C for 3 weeks on various media are shown in Table 1.

C) Physiological properties As shown in Table 2. From whole bacterial cell hydrolyzate, LL
-diaminopimelic acid (LL-A2p, ) was detected.

Table 2 Production of melanin-like pigments Tyrosine agar medium Negative peptone/yeast extract/iron agar medium Negative tryptone/yeast extract/liquid medium Negative starch hydrolysis Positive gelatin liquefaction Positive (weak) skimmed milk coagulation Negative skimmed milk peptone Reduction of negative nitrate Negative growth temperature range 20-30℃
Optimal temperature 27・C
Assimilation of carbon sources (on Pridham-Gotlieb agar)
D-glucose +D
-Fructose Inositol +D-Mannitol +L-arabinose 10-Beef sirose +L-rhamnose
+ Raffinose
+Sucrose
-Cellulose
From the above characteristics, this strain clearly belongs to the genus Streptomyces. Description of ISP (International Streptomyces Project) (International Journal of φ Systematic Bacteriology, Volume 18, Nos. 69-18
9, pp. 279-392, 1968 and the same magazine, No. 19
Vol., pp. 391-512, 1969 and the same magazine, No. 22
Vol., pp. 265-394, 1972), Bersey's
After referring to the Manual of Determinative Bacteriology (8th edition) and other bacterial species descriptions, we found that Streptomyces is the most closely related species to this strain.
Malachiticus can be given. So N(LS-199
When the 8 strains and the standard Streptomyces malachiticus strain 15P5167 were cultured under the same conditions and compared, slight differences were observed in the color tone of aerial mycelia, ability to assimilate sucrose, and liquefaction of gelatin. However, these were not considered to be two different enough to distinguish between rods. Other properties show very good agreement and therefore S-
The 1998 strain was recognized as the species most closely related to Streptomyces malachiticus, and Streptomyces φ malachiticus (
strcptoa+yccs malachltfcu
s) No. S-1998 to distinguish it from known strains.

3. Culture/Production of NQY NQY can be produced by culturing NQY-producing bacteria belonging to the genus Streptomyces aerobically in an appropriate medium and collecting the target substance from the culture.

The medium can contain any nutrient source that can be utilized by the NQY-producing bacteria. Specifically, for example, glucose, fructose, cellulose, glycerin, etc. can be used as a carbon source, and organic substances such as soybean flour, cornstap liquor, meat extract, peptone, dried yeast, yeast extract, and ammonium can be used as a nitrogen source. Inorganic salts or nitrates such as ammonium sulfate, sodium nitrate, ammonium chloride, etc. can be used. Moreover, inorganic salts such as common salt, potassium chloride, phosphate, and heavy metal salts can be added as necessary. In order to suppress foaming during fermentation, suitable antifoaming agents such as silicones can also be added according to conventional methods. The most suitable culture method is the aerobic liquid deep culture method, which is the same as the commonly used method. The appropriate culture temperature is 20-30℃, but 25-30℃ is suitable.
30°C is preferred. In this method, the production amount of NQY is 48 to 96 hours in shaking culture, and 72 to 9 hours in aerated agitation culture.
Reach i&high in 6 hours.

In the manner described above, a culture in which NQY is accumulated is obtained. Any suitable method can be used to collect NQY from this culture. One method is based on the principle of extraction, and specifically, for example, for NQY in a culture solution, it can be extracted with a water-immiscible solvent such as ethyl acetate, butyl acetate, etc. Extraction with chloroform, butanol, etc. (extraction efficiency is good if the culture solution is neutral or slightly acidic), or the culture itself can be subjected to the above extraction procedure without separating the bacterial cells. You can also.

Another method for collecting NQY from cultures is based on the principle of adsorption, using a liquid containing NQY, such as a culture furnace liquid or an extract obtained by carrying out the extraction operation as described above. , the target NQY can be adsorbed by column chromatography using a suitable adsorbent such as activated carbon, alumina, silica gel, Diaion HP20 (manufactured by Mitsubishi Kasei), etc., and then eluted to obtain NQY. can. The NQY solution thus obtained is concentrated to dryness under reduced pressure to obtain a crude sample of NQY.

In order to further purify the crude sample of NQY, the above-mentioned extraction method and dilution method may be combined as necessary and carried out as many times as necessary. For example, column chromatography using an adsorbent or gel filtration agent such as silica gel or alumina, liquid chromatography using an appropriate solvent, and countercurrent distribution method can be appropriately combined. Specifically, for example, NQY is dissolved in a small amount of chloroform, developed with a suitable solvent using a silica gel column to elute the active ingredient, and the eluate is concentrated under reduced pressure. NQY can be separated as a single substance by applying it to a column (manufactured by Co., Ltd.).

Physiological Activity 1 of Compound NQY, Cancer Metastasis Suppressing Activity NQY is found to have cancer metastasis suppressing activity. An experimental transfer system was used to measure this activity.

B16 suspended in PBS in BDFI 6W male mice
5×10′ melanoma cells were transplanted iv. From day 4 to day 9 after transplantation, a predetermined amount of NQY suspended in PBS was administered ip every day, and 21 days later, the animals were dissected and the number of nodules that had metastasized to the lungs was counted.

Dose Number of lung metastatic nodules n ±SE Control 14.9 ± 3.8 1O
N Q Y O, 4a+g/kg 7.3
±1.56 As shown above, NQY is 0.4mg/k
g, it was observed that lung metastasis of B16 melanoma cells was suppressed by about 50% compared to the control.

2. Anticancer activity When the in vitro anticancer activity of NQY against PBS8 was investigated using a 1p-ip administration system, no anticancer activity was observed in NQY.

3. In vitro cytotoxicity Cytotoxicity to various cells was investigated using conventional methods, and the following results were obtained.

PBS8], 5 Ehrlich 12.5 EL-47,5 8166,3 4, LD5o of acutely toxic NQY administered intraperitoneally to mice was 200 m
g/kg or more.

Usefulness/Applications of Compound NQY As mentioned above, NQY does not exhibit anticancer activity, but
It is a new type of drug that specifically suppresses cancer metastasis and has low toxicity.

NQY as a cancer metastasis inhibitor is suitably administered orally or parenterally to humans and other tumor-bearing animals at doses up to 10 rnz/kg.

Experimental Examples Example 1 (I) 500 ml of 100 ml of a medium containing 1% cultured starch, 1% polypeptone, 1% blackstrap molasses, and 1% meat extract (pH 7,2)
Dispense into Erlenmeyer flasks and add Streptomyces
After inoculating one platinum loop of Malachyticus No. S-1998,
Shaking culture was carried out at 27°C for 2 days.

100 ml of this culture solution was added to 1 liter of the same medium in a 5-liter Erlenmeyer flask, and cultured with shaking at 27° C. for 2 days to obtain a preculture solution.

Glycerin 396, cornstarch liquor 1%, dry yeast 0.3%, Ca CO3 in a 360 liter fermenter
Prepare 200 liters of a medium containing 0-35% and NaC 10.5% (pH 7.2), and add the above preculture solution 3 to this.
After inoculating Little, 27°C/ventilation H0,5vvm/
Stirring rotation speed 1100 rpm / internal pressure 0. It was cultured for 30 days under the condition of 5 kg/cj.

(2) After pre-culture of the crude NQY sample, Celite was added to the culture solution and filtered under pressure to obtain a culture solution. Transfer 400 liters of culture solution to a column (I5φ) of Diaion HP-20J (manufactured by Mitsubishi Kasei).
After washing with 200 liters of water, it was eluted with 100 liters of methanol. The eluate was concentrated under reduced pressure, adjusted to pH 2.0 with hydrochloric acid, and extracted with an equal amount of butyl acetate. After washing the extract with an equal amount of 0.01N hydrochloric acid,
The mixture was concentrated to dryness to obtain 8 g of crude NQY sample.

Example 2 8 g of the crude sample obtained in Example 1 was transferred to a silica gel column (
5 φ x 70 cm) and chloroform-acetone (6
: It was eluted with 1). After concentrating the NQY-containing fraction to dryness,
Dissolve in a small amount of methanol and add [Sephadex L]
Gelton filtration using H-2OJ column (4φ x 60cm) I was developed with methanol. The NQY fraction was concentrated to dryness to obtain 200 rng of NQY purified product.

[Brief explanation of the drawing]

FIG. 1 is a reproduction of the ultraviolet/visible absorption spectrum of compound NQY. FIG. 2 is a reproduction of the infrared absorption spectrum of compound NQY. FIG. 3 is a reproduction of the 'HNMR spectrum of compound NQY. FIG. 4 is a reproduction of the 13C NMR spectrum of compound NQY.

Claims (1)

[Claims] A novel naphthoquinone compound NQ represented by the following formula (I)
Y. ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I)