JPH04282395A - New physiologically active substance ta-3,037a and production thereof - Google Patents

Abstract

PURPOSE:To obtain the title substance useful for an agent of conquering drug resistance of cancer, having inhibitory action on glutathione transferase by culturing a bacterium belonging to the genus Streptomyces, capable of producing new physiologically active substance TA-3,037A and collecting the product from the culture mixture. CONSTITUTION:A bacterium [e.g. Streptomyces TA-3,037A strain (FERM P-11,991)] belonging to the genus Streptomyces, capable of producing new physiologically active substance TA-3,037A is cultured and the product is collected from the culture mixture to give physiologically active substance TA-3,037 (salt) shown by the formula, having 281 molecular weight, molecular formula C16H11O4N1, 248-250 deg.C melting point (decomposition), specific rotatory power [alpha]<23>D=0 deg. (C 0.5, DMF) and ultraviolet light absorption spectrum of lambdamax nm(epsilon) 204(26,816) 242(15,674), 282(9385), 383(14,561), (EtOH); 205(21,997), 241(18,307), 282(9,043), 350sh(8615), 382(14,345), (01NHCl 90% EtOH); 324 (0.1N NaOH 90% EtOH).

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention provides a novel physiologically active substance TA-3 having glutathione transferase inhibitory activity.
037A, its production method and drugs containing it.

0002

BACKGROUND OF THE INVENTION In cancer chemotherapy using anticancer drugs, the emergence of anticancer drug-resistant cells has become a major therapeutic barrier.

In particular, in cancer cells that have become resistant to cisplatin and alkylating agents, increased intracellular glutatine concentration and enhanced glutathione transferase activity have been observed [Cancer Chemother. Ph
armacol. , Vol. 17, pp. 223-226 (
1986); Biochem. Pharmacol. ，
Volume 34, pages 2583-2586 (1985)].

Conventionally, attempts have been made to increase the intracellular concentration of anticancer drugs using calcium antagonists and to inhibit the inactivation mechanism of anticancer drugs using glutathione biosynthesis inhibitors, but these efforts have had side effects and effects. This was not satisfactory in that respect, and there was no means to defeat the anticancer drug resistance mechanism of cancer.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an inhibitor of glutathione transferase involved in the inactivation of anticancer drugs, a method for producing the same, and a drug containing the same.

[0006]

[Means for Solving the Problems] To summarize the present invention, the first invention relates to a novel physiologically active substance TA-3037A (hereinafter referred to as TA-3037A substance) having the following physicochemical properties and a salt thereof. It is something. (1) Molecular weight 281
(2) Molecular formula C16
H11O4 N1 (3) Melting point
248-250℃ (decomposition) (4) Optical rotation [α]
D23=0° (C0.5, DMF)

(5) Ultraviolet absorption spectrum λmax nm
(ε) 204 (26816), 242 (15674
), 282 (9385), 383 (14561), (E
tOH)

[0008]λmax nm(ε) 205(219
97), 241 (18307), 282 (9043),
350sh (8615), 382 (14345) (0.
1N HCl 90% EtOH) λmax n
m(ε) 324 (9152) (0.1N NaO
H 90% EtOH) (6) Infrared absorption spectrum (KBr Disk) (cm-1) 3440 (br), 2930, 1752, 1663, 1
275,1258,758

(7) 1H-NMR spectrum (heavy DM
in SO, 400 MHz) (ppm, multiplicity) 6.65 (
1H, s), 6.90 (1H, t), 7.32 (1H,
dd), 7.34 (1H, t), 7.46 (2H, t)
,7.60(2H,d),7.66(1H,dd),1
1.1 (1H, s)

(8) 13C-NMR spectrum (heavy DM
in SO, 100MHz) (ppm, multiplicity) 109.4
d, 112.0s, 118.7d, 120.4d, 12
3.3s, 126.7d, 127.8d, 128.0d
×2,129.0d×2,129.9s,134.0s
, 140.3s, 157.3s, 169.3s

The second invention of the present invention relates to a method for producing TA-3037A substance, in which a TA-3037A substance-producing bacterium belonging to the genus Streptomyces is cultured, and the TA-3037A substance is collected from the culture. The present invention relates to a method for producing a TA-3037A substance characterized by the following. The third invention of the present invention relates to a glutathione transferase inhibitor containing TA-3037A substance or a salt thereof as an active ingredient.

The present inventors have extensively searched nature for glutathione transferase inhibitors, and have completed the present invention by obtaining substance TA-3037A, a substance that exhibits significant glutathione transferase inhibitory activity in adriamycin-resistant murine leukemia cells.

The TA-3037A substance-producing bacteria used in the present invention belongs to the genus Streptomyces, and is not particularly limited as long as it produces the TA-3037A substance. Streptomyces isolated from soil collected at Chakidaira, Nikko City, Prefecture.
strain TA-3037, which belongs to the genus P. tomyces. Its mycological characteristics are as follows. This strain was deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology on February 7, 1991, and was given the accession number FERM P-11991. There is.

TA-3037A substance producing bacteria TA-303
The 7 strains are actinomycetes with a hyphae width of around 1 micron, and under an optical microscope, the spore chains on the aerial hyphae of actinomycetes are Rectusf.
lexibilis. Rod-shaped spores are observed under an electron microscope, and the spore surface is smooth. L,L-diaminopimelic acid was detected as a component of the cell wall, and no spores or other characteristic structures were observed even under an electron microscope. From the above, this bacterium belongs to the genus Streptomyces.

The culture properties, physiological properties, and sugar assimilation ability of this bacterium are shown in the table below. Regarding the description of colors in various media, the standards shown in parentheses are the Color Harmony Manual of Container Corporation of America. All property tests were determined after culturing for 2 weeks at 27°C.

[0016]

[Table 1]

[0017]

[Table 2]

[0018]

[Table 3]

The above properties indicate that this bacterium belongs to the genus Streptomyces. Therefore, the TA used in the present invention
-3037A-producing bacteria include actinomycetes of the genus Streptomyces that are equivalent to or related to strain TA-3037.

The TA-3037A substance is characterized by the following physicochemical and biological properties. (A) Physical and chemical properties of TA-3037A substance (1) Molecular weight 281 (2) Molecular formula C16H11O4
N1 (3) Melting point
248-250℃ (decomposition) (4) Optical rotation [α]
D23=0° (C0.5, DMF)

(5) Ultraviolet absorption spectrum λmax nm
(ε) 204 (26816), 242 (15674
), 282 (9385), 383 (14561) (Et
OH)

λmax nm(ε) 205(219
97), 241 (18307), 282 (9043),
350sh (8615), 382 (14345) (0.
1N HCl 90% EtOH) λmax n
m(ε) 324 (9152) (0.1N NaO
H90% EtOH)

(6) Infrared absorption spectrum (KBr Disk) (cm-1) 3440 (br), 2930, 1752, 1663, 1
275,1258,758

(7) 1H-NMR spectrum (heavy DM
in SO, 400 MHz) (ppm, multiplicity) 6.65 (
1H, s), 6.90 (1H, t), 7.32 (1H,
dd), 7.34 (1H, t), 7.46 (2H, t)
,7.60(2H,d),7.66(1H,dd),1
1.1 (1H, s)

(8) 13C-NMR spectrum (heavy DM
in SO, 100MHz) (ppm, multiplicity) 109.4
d, 112.0s, 118.7d, 120.4d, 12
3.3s, 126.7d, 127.8d, 128.0d
×2,129.0d×2,129.9s,134.0s
, 140.3s, 157.3s, 169.3s (9) Appearance yellow needle-like crystals.

(B) Biological properties of TA-3037A substance (1) Antibacterial activity In the agar dilution method, TA-3037A substance did not inhibit the growth of various bacteria and molds at 50 μg/ml. .

(2) Acute toxicity As a result of testing the acute toxicity of the substance TA-3037A to mice, it showed no toxicity when administered intraperitoneally to mice at 100 mg/kg.

(3) The glutathione transferase inhibitory activity of the physiologically active TA-3037A substance was measured by the method of Habig et al. [J. Biol
．． Chem. , Volume 249, Pages 7130-7139 (
1974)]. That is, 100mM
To a reaction solution containing 1mM glutathione and 1mM chlorodinitrobenzene in potassium phosphate buffer (pH 6.5),
340n at 30℃ when adding sample and enzyme solution
The increase in absorbance of m was measured. As the enzyme solution, a cell lysate of adriamycin-resistant mouse leukemia cells was used. As a result, the 50% inhibitory concentration of the TA-3037A substance against glutathione transferase of adriamycin-resistant mouse leukemia cells was 2.5 μg/ml.

As described above, TA-3037A of the present invention
The substance inhibits glutathione transferase in adriamycin-resistant murine leukemia cells at low concentrations, and has extremely low antibacterial activity and toxicity, so it is expected to be used as an agent to overcome drug resistance in cancer. As the salt of TA-3037A substance,
Examples include commonly used pharmacologically acceptable salts such as acid addition salts with commonly used acids, and salts with alkali metals and alkaline earth metals.

The TA-3037A substance of the present invention is TA-3
It can be produced by culturing 037A substance-producing bacteria and collecting from the culture. The medium used when culturing the TA-3037A substance-producing bacteria is TA-303
Any nutrient source that can be used by the 7A substance-producing bacteria may be used. For example, as a carbon source, glycerin, glucose, maltose, sucrose, dextrin,
Starch, fats and oils, etc. can be used. As nitrogen sources, organic substances such as soybean flour, cottonseed meal, meat extract, peptone, dried yeast, yeast extract, and corn steep liquor, as well as inorganic substances such as ammonium nitrate, sodium nitrate, calcium carbonate, and ammonium chloride can be used.

[0031] If necessary, salt, potassium chloride,
Inorganic salts such as malates, heavy metal salts, etc. can also be added. In order to suppress foaming during fermentation, an appropriate antifoaming agent such as silicone, soybean oil, etc. may be added according to a conventional method.

As the culture method, an aerobic liquid deep culture method is most suitable, similar to the commonly used method for producing antibiotics. The culture temperature that can be used is TA-3037A.
The growth of substance-producing bacteria is not substantially inhibited, and TA-303
Although there are no particular restrictions as long as the temperature is within a range where the 7A substance can be produced, a particularly preferred temperature range is 25 to 30°C.

[0033] The incubation time can generally be continued until sufficient accumulation of the TA-3037A substance, and the culture medium composition,
Although it varies depending on the culture temperature, the desired TA-3037A substance can usually be obtained by culturing for 48 to 96 hours.

From the culture thus obtained, TA-30
Any convenient method can be used to isolate the 37A material. For example, the culture is filtered, the bacterial cells are removed by a separation method known per se, such as centrifugation, and the filtrate supernatant is extracted using a solvent extraction method using an appropriate organic solvent, or by using adsorption or ion exchange ability. It can be isolated and purified using chromatography alone or in combination.

The TA-3037A substance of the present invention has the effect of strongly inhibiting glutathione transferase. Therefore, the present invention provides a glutathione transferase inhibitor containing the substance TA-3037A or a salt thereof as an active ingredient. Glutathione transferase inhibitors are useful as reagents such as enzyme inhibitors or medicines such as agents for overcoming cancer drug resistance. In order to use the glutathione transferase inhibitor of the present invention to overcome drug resistance in cancer, an effective amount of the TA-3037A substance may be administered to cancer-bearing warm-blooded animals including humans. Administration methods include injection, oral administration, and parenteral administration other than injection. The dosage varies depending on the subject to be administered and the route of administration, but is usually 0.1 mg to 100 mg/kg/day.
Preferably it is about 0.5 to 50 mg/kg/day.

There are no particular restrictions on the formulation for administration, and the TA-3037A substance can be used in liquid formulations, solid formulations (capsules, tablets, lyophilized formulations, etc.) together with commonly used pharmaceutical additives such as carriers and adjuvants. ) etc. and are administered. Hereinafter, TA-3 of the present invention will be explained based on examples.
The present invention will be further explained in detail by showing production examples of the 037A substance, but the present invention is not limited to these examples.

[0037]

[Example] Actinomycetes (Feikoken Bibori No. 11991) cultured on agar slant medium were treated with 2.5% glycerol and meat extract (
Far East) 0.5%, polypeptone (Nippon Pharmaceutical) 0.5%,
A liquid medium (adjusted to pH 7.4) containing 1.0% yeast extract (Difco), 0.05% magnesium sulfate, and 0.32% calcium carbonate was added to an Erlenmeyer flask (500 ml volume).
Dispense 100 ml each into
The cells were inoculated into cells that had been previously sterilized for minutes, and cultured with rotary shaking (180 revolutions per minute) at 27°C for 72 hours. This seed culture solution was similarly dispensed into Erlenmeyer flasks, 2% inoculated into 10 liters of the above sterilized medium, and cultured with rotational shaking at 27° C. for 72 hours.

The culture solution thus obtained was centrifuged to separate the bacterial cells, and 9 liters of the supernatant was adjusted to pH 2 and extracted with an equal amount of butyl acetate. This extract was concentrated under reduced pressure, and 6.6 g of the resulting residue was added to Sephadex LH-
20 columns and developed with methanol. The active fractions were collected and concentrated under reduced pressure, and 206 mg of the resulting crude powder was applied to a silica gel column and eluted and developed with chloroform-methanol (50:1). The active fractions were collected and concentrated under reduced pressure, heated to dissolve in methanol:chloroform=1:1, and allowed to stand to crystallize. In this way, T.A.
125 mg of yellow crystals of -3037A substance were obtained.

[0039]

Effects of the Invention The TA-3037A substance of the present invention strongly inhibits glutathione transferase of adriamycin-resistant murine leukemia cells, has almost no antibacterial activity, and is expected to be an excellent agent for cancer treatment as a low-toxic anticancer drug resistance overcoming agent. It is a new substance. Furthermore, the TA-
Substance 3037A also has the possibility of being used as a therapeutic agent for allergic diseases that inhibits leukotriene biosynthesis through its glutathione transferase inhibitory activity. The present invention is an extremely useful invention that provides a new substance having these useful properties.

Claims (3)

[Claims] Claim 1: A novel physiologically active substance TA-3037A and its salts, characterized by having the following physicochemical properties:
(1) Molecular weight 281
(2) Molecular formula C16
H11O4 N1 (3) Melting point
248-250℃ (decomposition) (4) Optical rotation [α]
D 23 = 0° (C0.5, DMF) (5) Ultraviolet absorption spectrum λmax nm (ε) 204 (26816), 24
2 (15674), 282 (9385), 383 (14
561), (EtOH) λmax nm(ε) 205 (21997), 24
1 (18307), 282 (9043), 350sh (
8615), 382 (14345) (0.1N HCl 90% EtOH) λma
x nm (ε) 324 (9152) (0.1N
NaOH 90% EtOH). 2. Production of a novel physiologically active substance TA-3037A, which comprises culturing a novel physiologically active substance TA-3037A-producing bacterium belonging to the genus Streptomyces and collecting the novel physiologically active substance TA-3037A from the culture. Law. 3. A glutathione transferase inhibitor comprising a novel physiologically active substance TA-3037A or a salt thereof as an active ingredient.