JPS6025999A - Anthracyclin compound ag-2 and its use - Google Patents

Abstract

NEW MATERIAL:Anthracyclin AG-2 shown by the formula. USE:An antitumor agent. Usually, it is dissolved in distilled water for injection or isotonic sodium chloride solution, and administered as an injection. PREPARATION:For example, argomycin of an anthracyclin compound is hydrolyzed to give a compound shown by the formula. Argomycin of an anthracyclin compound as a raw material is usually prepared from a culture of a strain [e.g., Streptomyces violochromogenes 1089-AV2(S92) strain(FERM-P 450)] belonging to the genus Streptomyces, capable of producing argomycin.

Description

DETAILED DESCRIPTION OF THE INVENTION Background of the Invention The present invention relates to a novel anthracycline compound AG-2 and uses thereof.

Anthracycline compounds as anticancer antibiotics occupy an important position as medicines, and various compounds have already been proposed. In general, the physiological activity of a chemical compound 'IIf largely depends on its chemical structure or physicochemical properties, so anthracycline compounds are also different from existing ones in the type of sugar or substituent in the aglycon moiety and sugar chain moiety. It can be said that there is a constant desire for compounds that have physical and chemical properties different from those of existing compounds.

The essence of invention The present invention meets the above needs.

That is, the anthracycline compound AG according to the present invention
-2 is represented by formula (1).

The anti-inflammatory agent according to the present invention contains an anthracycline compound AG-2 represented by formula (I) as an active ingredient. 1 Of course AG, 2 Chemical structure AG-2 which is an anthracycline compound according to the present invention
has the chemical formula (I) l-f\ structure.

This chemical structure was determined as follows.

So, AG-, 2 is 40% ant 1! After that, nogalalol (No2Xalol
), but also three types of Fi-constituent sugars as sugar moieties, obtained) t
reactor. The aglycone was found to be nogalalol by analysis of :vJ-Papa chromatographic behavior, H-NMR, and 5IJt4S spectrum bamboo analysis. .

OHOOHOH Nogalalol Also, regarding the constituent sugars, from the comparison of chromatographic FjII with the standard sample, '11-NMR spectrum and its coupling knot, 2-de, = +
-deoxy-7 course (2-deoxy-fu, cos
e), diginose and desio
= o -th (decilonitrose).

117 in the S engineering L4S spectrum of this compound
From the observation of the parent 1 ion peak (M0H) in 8 and the analysis of the 1H-NMR 0-NIQ spectrum, it was found that this compound contains nogalalol as an aglycone, two tuginoses, and 2-deoxy-fucose. It was found that it has a total of 4 sugars, 1 sugar and 1 sugar nitrose.

The literature values for nogalalol (J, Am, C!hem.

soc, dish, 542, (1977)) and literature values for desylonitrose (Journal of Antib
iotics ; 46.454 (198 phantom), it was found that sugars were bound to the 41st and 7th positions of the aglycone and the 4th position of the desylonitrose. In addition, since diginose was obtained by catalytic reduction of the present compound with 5% pa-BaSO4, it was found that (.magiginose) was bound to the 7th position.

When this compound is further hydrolyzed with a 50% acetic acid aqueous solution at 50° C. for 10 minutes, a highly polar decomposition product is obtained. 'H-NMR and 130-NI of this decomposition product
From the fragment ions of the ylR spectrum, this minute)
It was revealed that the +ir product has nogalalol as an aglycone, and sugars are bound only at the 41st position in the order of 2-deoxy-fucose and tesironitrose. 'H-
From the NME spectrum, it was revealed that only the glycosidic bond of desylonitrose was β, and the others were α.

Combining the above results, the incisal edge of AG-2 was determined as shown in formula (I) in the previous section.

Physical and chemical properties (1) Appearance Red powder (2) Melting point 201-203°C (3) Specific degree of rotation [α]”, knee +369° (C!-
(4) Molecular weight (EIIMS) 1177 (5) Elemental analysis value C' HN et al. 5 6 5 2 3 (6) Ultraviolet and visible absorption spectra (Figure 1) λma
X ('ツC!H3O1(207(205), 235(406)
), 259 (1!12), 293 (163), 4
7B (117) OH30H+HO1205 (194)
, 234(419), 256(183), 293(
67), 466 (119) OHa OH+N a OH207 (350), 24
0 (490), 295 (67), 54 et al. (111
) (7) Infrared absorption spectrum (C1Ω) (Figure 2) 3d30, 2+370.1725.1620.15
45, 1450, 1410.

1385, 1300.1230, 1205.117
0.1095.1070.1010.990.972 (8) Solubility in bath agents Chloroform, methanol, DMSO, pyridine, acetone, ethyl acetate, butanol, ethanol, soluble in alkaline water Hexane, insoluble in water (9) Thin I. Cochromatography result TIfIl (Merck & Co., Ltd. "Silica Gel 60F (254) J Braid Calyx (") Rf value Chloroform: Methanol - 9: 1 0.40 Toluene: Acetone = 2: 3 0.55Qo) 'H-
Nl spectrum (Figure 3) (n) 13cm 1q
yH Spectrum (No. 41￥1) Use of AG-2 The anthracycline compound AG-2 according to the present invention is a useful compound in that it has antipulmonary activity. .

Physiological activity (1) Antitumor AG-2 exhibits antitumor activity against leukemia in experimental animals. For example, a suspension of P388 leukemia cells (1 x 10 cells/mouse) was intraperitoneally transplanted into a CDF1 mouse, and AG-2 was intraperitoneally administered at the 1st and 51st eyes after transplantation, and physiological saline was administered intraperitoneally. The effect was expressed as a survival rate (%) based on the number of days the mice in the administered control group survived as 100, and was as follows.

10 156 20 150 (2) Acute toxicity LD5 by intraperitoneal injection of AG-2 of the present invention in mice
The o value is 80 mg/kg.

Antitumor agents Thus, the anthracycline compound AG-
2 is the ratio of animals including humans! It was revealed that it exhibits anti-IBiθ activity against 14 malignant tumors.

Therefore, AG-2 of the present invention can be used as an anti-inflammatory agent or a therapeutic agent for lung cancer.

AG-2 as an anti-tumor agent can be administered by any convenient route of administration and in a dosage form determined by the route of administration adopted. The drug is usually in a diluted form with a pharmaceutically acceptable carrier or diluent. for example,
The AG-2 of the present invention can be used alone or as a mixture with a carrier such as maltose, lactose, or a non-toxic
'J: complex, for example, deoxyliponucleus (niZ
It can be administered as a complex with The deoxyribonucleic acid of the sling is, for example, calf l1
Extracts from animals such as Fyl gland, F. fyl gland, F. f. j., and microbial cells can be used.

When AG-2 of the present invention is actually administered, one typical method is to dissolve it in distilled water for injection or physiological saline and inject it. Specifically, 1) the platform for lifting the object is intraperitoneal injection, subcutaneous injection, static injection or p
y, J) Intravascular injection into the pulse and administration, but in humans, there are injection methods such as intravenous injection into the vein or artery, or local administration. Administration: Taking into account the results of animal tests and the serious circumstances, the total dose is determined to exceed a certain level when administered continuously or intermittently. The specific administration: r1;'' is determined depending on the membrane force method, the situation of the patient or treated animal, such as age, body weight, sex, sensitivity, diet, administration time, concomitant drugs, and the degree of the patient or his/her disease. It goes without saying that the appropriate dose and frequency of administration under certain conditions will be determined by a specialist's appropriate dose determination test based on the above guidelines. The anthracycline compound AG-2 is currently the anthracycline compound albomycin (*rgomyc
Although it has only been obtained by hydrolysis of in),
It could be produced by synthetic chemical or microbiological modification of other related compounds, or by total synthetic chemistry.

The anthracycline compound albomycin is produced by a strain of Streptomyces that has the ability to produce albomycin, specifically Streptomyces piolochromogenes 1.
It is obtained from a culture of 089-AV2 (892) strain (Feikoken Bacterium No. 6865).

892 strain anthracycline compound albomycin producing bacterium 139
The two stocks have the following contents.

(1) Origin and deposit number Strain S92 was isolated from soil collected from a paddy field in Kiyama-cho, Kanzaki-gun, Saga Prefecture, and was deposited at the Institute of Microbial Technology, Agency of Industrial Science and Technology on December 28, 1981. , the number of "Chokoken Bibori No. 6865" is C).

(2) Mycological properties and physiological properties (a) Morphology Aerial hyphae have a long main axis (elongate), irregularly multi-branched branches (branched in clusters and uniaxial branches), and form spore chains at their tips. The spore chain usually consists of 10-25 spores and presents a dense closed spiral (diameter 2-3μ, 1-4 turns).The spore surface is a flat steel with a diameter of 0.7-0. 9μ, 0.5-0.6μ cylindrical or oblong shape.Depending on the culture medium (nutrient agar medium), bent short branches are branched on the basal hyphae in single or tassel-like shapes, and the tip has an indentation. Regularly bent spore chains (2 to 15 spores) may be formed. No other special morphology is observed.

LI4J diaminopimelic acid is included in whole bacterial cell hydrolysis.

(b) Growth status in various media
Observations were made according to the ods of Manual 1941). The observation results are summarized in Table 1.

(c) Physiological properties and assimilation of the carbon source The physiological properties and assimilation of the carbon source are shown in Table 2 and Table 3, respectively.

Discussion Since this strain 1089-AV2 contains L-type diaminopimelic acid as a cell wall component and forms spore chains consisting of 10 or more spores, it is considered to be a member of Streptomyces (Streptomyces).
tomycee), p%, and is characterized as follows: a) the spore chain is tightly closed coiled, b) the spore chain surface is smooth, and C) the flora color is Gray and red series, d) The negative side color of the prefecture ranges from charcoal orange to light yellow to dark orange, e) Production of melanin-like pigment is No. 1.

As a result of comparing the above five characteristics with known bacterial species, this strain was found to be a member of Actinomyces (Streptomyces).
Violochromogenes' (Actinomyces (S)
treptomycee) violochromog
enes). When comparing this strain with Violochromogenes A., only the ability to utilize rhamnose and O-raffinose differs, and this is not considered to be a significant difference in performance. (The same species is included in the genus.) Therefore, this strain is considered to be Streptomyces violochromogenes (Streptomyces violochromogenes).
ces violo-chromogenes) 10
It is called 89-AY2.

Table 2 Physiological Properties Growth Temperature Range -20-42℃ Optimum Growth Temperature 27-37℃ Liquefaction of gelatin Hydrolysis of starch Coagulation of skimmed milk - Peptonization of skimmed milk Formation of decamelanin-like pigment Tyrosine agar Tenpeftone Yeast/iron agar Ten tryptone/yeast liquid medium →- Note) +: Positive m: Negative Table 3 Assimilability of carbon sources L-arabinose + D-xylose + D-glucose Shiri-fructose + sucrose + inositol + L- Rhamnose - Lan-inose Soil D- Mannide-)'L-Juprid Ham-Gotzlieb agar medium was used Pillar) +: clustered growth ±: slight growth - no growth (3) Cultivation/Production of albomycin Anthracycline compound Albo Mycin can be produced by culturing argomycin-producing bacteria belonging to Streptomyces coffin aerobically in an appropriate medium and collecting the target product from the culture.

The medium can contain any nutrients available to the albomycin-producing bacteria. Specifically, for example,
Glucose, sucrose, maltose, starch, and fats and oils can be used as carbon sources, and soybean flour, cottonseed meal, meat extract, peptone, dried yeast, and nitrogen sources can be used.
Organics such as yeast extract and corn steep liquor as well as inorganics such as ammonium salts or nitrates such as ammonium salt, sodium nitrate, and ammonium chloride can be used. In addition, salts such as common salt, potassium chloride, phosphate, heavy metal salt, etc. may be added depending on safety. In order to suppress foaming during fermentation, suitable blowing 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 for producing antibiotics. The appropriate culture temperature is 20°C to 42°C, but 27°C
C to 37C is preferred. With this method, the production amount of albomycin reaches its maximum in 3 to 6 days in both shaking culture and agitation culture.

In this way a culture enriched with albomycin is obtained. In the culture, most of albomycin exists in the culture solution, but a portion of it exists in the bacterial cells.

To obtain albomycin from such cultures,
Any suitable method can be used.

One method is based on the principle of extraction, and specifically, for example, for lubomycin in culture: For albomycin in bacterial cells, 111 bacterial cells obtained by extraction with butyl, chloroform, butanol, etc., or by centrifugation, etc., were extracted with chloroform, vinegar rs7 ethyl, butanol, methanol, ethanol, acetone, methyl ethyl ketone, etc. It can be recovered by treatment with hydrochloric acid f6 W'l- or acetic acid d solution.

It is also possible to subject the culture as is to the above-mentioned extraction operation without subjecting the bacterial cells to 1 part (IL). Countercurrent distribution methods using an appropriate solvent can also be included in the scope of extraction.

Another method for harvesting albomycin from cultures is based on the principle of adsorption, using albomycin-containing substances already in liquid form, such as medium 4'f:i solution or as described above. A suitable adsorbent, such as activated carbon, alumina, silica gel, [Diaion HP2
Albomycin can be obtained by adsorbing the target albomycin by column chromatography, liquid chromatography, etc. using 01 (manufactured by Mitsubishi Kasei), etc., and then subjecting it to M Filtration. By concentrating the albomycin solution to dryness under reduced pressure, a crude sample of albomycin can be obtained (1).

In order to further purify the crude preparation of albomycin thus obtained, the above-mentioned extraction method and adsorption method may be combined as necessary and carried out as many times as necessary. for example,
Column chromatography using silica gel, an adsorbent such as "Diaion HP20J" or a gel filtration agent,
Liquid chromatography using a suitable solvent and countercurrent distribution method can be carried out in combination as appropriate. Specifically, for example, a crude sample of albomycin is dissolved in a small amount of chloroform, developed with an appropriate solvent using an acidic silica column to remove the active ingredient, and the eluate is concentrated under reduced pressure. Furthermore, when eluted with a Sephadex LH20J (Pharmacia trademark) column, albomycin is separated as a single substance, so if this is concentrated to dryness, albomycin can be obtained.

The physicochemical properties of albomycin thus obtained are as follows.

(1) Color, properties: Orange powder (z) M'lt point: 207-213℃ (decomposition) (3)
Specific optical rotation: [α] Fu + 112° (c = o, i,
Chloroform: methanol = 9:1) (4) Elemental analysis value C: 56.2 section (experimental value) H: 6.9 section 0: 35.1% N: 1.8% (5) Ultraviolet visible region The absorption spectrum is shown in Figure 5.

0H30Hλn1aX (”+IF) 235 (363), z5a (xb7), 292 (
61) , 476 (104) cH30H + HO1 235 (387) , 258 (159) , 292
(61), 468(110)OHs OH+IJ
aOH 239 (302), 294 (41), 543 (8
8) (6) Infrared absorption spectrum (measured by potassium bromide method) as shown in Figure 6.

(7) Solubility in solvents Easily soluble in chloroform-methanol mixture, dimethyl sulfoxide, pyridine and basic water.

Soluble in chloroform, methanol, ethyl acetate, methyl ethyl ketone, butanol, butyl acetate, ethanol, acetone and acidic water.

(8) Thin layer chromatography (using Merck [silica gel 60F254] plate)
] Solvent Rf Tsukuda dichloroform methanol O+20 28:1 Chloroform:methanol/I/:29t! /) Ammonia water 0.25 = 8:1:0.1 Chloroform: Methanol: Vinegar! 'i'2 0. '30
=8:1:0.1 chloroform:benzene:methanol l), 2G=7
:2:2 (9) Nuclear magnetic resonance spectrum (400 MHz, in small chloroform-1μr methanol) 10 as shown in FIG.

(4) Albomycin hydrolysis 1i'F/AG-2 #
:'fi Jin1! .

Suitable acidic water, such as acetic acid water, salt: t'! Dissolve albomycin in water, formic acid water, etc. and heat it at 50 to 90℃.
Process for 10 to 60 minutes. The hydrolyzed product of albomycin that has been removed is subjected to vacuum eχi<iL, and then subjected to column chromatography using an adsorbent such as silica gel or acidic silica, or gel I13 filter, or liquid chromatography using an appropriate solvent. , scraping by silica gel thin layer chromatography, countercurrent distribution method, etc., as appropriate, can be combined to obtain AG-2.

Experimental example In the following, "related" means "l-W/V%".

Example 1 (1) Preparation of Seed Mother The medium used was prepared by dissolving the following components in 1 liter of water and adjusting the pH to 7.0.

Glucose 4th grade Malt Extract 10% Yeast Extract 4% Vitamin Complex 1o +n1/1 In addition, Vitamin Complex consists of the following ingredients:
1 of distilled water.

Thiamine hydrochloride 0.5 mg Riboflavin 0.5 mg Niamine 0.5 mg Pyridone Schiff hydrochloride 0.5 mg Inositol o, 5 mg Calcium pantothenate 0.5 mg Para-aminobenshii 2-quaside o, s mg Biotin 0.25 mg 15 ml of the above medium was prepared in a large sample # Dispense Strephtomyces piolochromogenes tosc+-Av2 into 2 tubes.
(S92) was seeded with one platinum loop from a slant and placed in a test tube shaker (23 Orpm) at 27° C. for 72 hours to use as a seed mother.

(2) The medium used for culturing was prepared by dissolving the ingredients in the following set (1) in 1 liter of water and adjusting the pH to 7.0.

Sludge 4 minutes 2.5 Bean flour 1°5 Dried yeast 0.2% Calcium carbonate (sedimentation) 0.4% Dispense 1 liter of the above medium into 500 ml Erlenmeyer flasks and sterilize them. 2 ml of the above seed mother was added and cultured at 27° C. for 4 days using a rotary shaker (230 rprn).

(3) Collection of albomycin After culturing, the culture solution is filtered, and 1 liter of the obtained culture solution is adjusted to pJ (2.0), then adsorbed to Diaio%P20, and then mixed with water and then Wash with 100'% methanol and elute with 100'% methanol.

This is concentrated under reduced pressure and then extracted with chloroform.

This chloroform layer is dried under reduced pressure to obtain 1 g of red powder (albomycin crude powder).

Example 2 1 g of the crude albomycin powder obtained in Example 1 was added to Y8Wi in a solution of chloroform:methanol=20:1, and 500 g of acidic silica was added to a solution of chloroform:methanol=20:1.
The column was loaded onto a 6 x 5Q cm column equilibrated with 20:1 chloroform:metallic acid.

After drying the obtained active fraction under reduced pressure, it was further dissolved in a bath of chloroform:methanol=1=1 to give chloroform:
"Sephadex L" which was normalized with methanol = l:1
Place on H204 column, chloroform:methanol=1
: 1 and get out of the bath. 31) The obtained active substance 4: Dry the fraction under reduced pressure I2, orange albomycin powder Zoo mg
I got it.

Example 3 200 mg of albomycin was added to a 50% acetic acid aqueous solution (
2ml) and hydrolyzed at 85°C for 20 minutes.

The hydrolyzate was removed under reduced pressure and subjected to chloroform:methanol=9=10P) JFt chromatography (Merck's "Silica Gel 601i'254").
(Use J plate) K and unfold. Rf value obtained at this time. , 4. Scrape off the alcohol-colored area near I=J and drain it with chloroborum: methanol = 1:1 sewage, d;
A crude sample of <i L-c AG-2 is obtained.

When this crude sample of AG-2 was placed on a column (Toyopearl UVI4O'FJ (bundle) manufactured by Kakudasha Co., Ltd.) flattened with methanol and eluted with methanol, two colored particles (
The trough comes out, but the one that elutes at the 2'flr is AG-2.
This is g's i'ni dried to obtain A(J -2
(1o mg)l')6.

[Brief explanation of drawings]

FIG. 1 is a reproduction of the ultraviolet and visible absorption spectra of AG-2. 1 in methanol 2 in methanol + NOH 3 in methanol + NaOH Figure 2 is a reproduction of the infrared absorption spectrum of AG-2. Fig. 3 is a partial copy of the 'I(-Nll/IR spectrum) of AG-2. Fig. 4 is a copy of the 130-NMR spectrum of AG-2. Fig. 5 is a reproduction of the ultraviolet/visible absorption spectrum of albomycin. 1 in methanol 2 in methanol 10 HO'l 3 in methanol A/ + NaOH Figure 6 is a reproduction of the infrared absorption spectrum of albomycin Figure 7 is a reproduction of the 'H-NMR spectrum of albomycin. s Correction Agency Director Kazuto Wakasugi Ij1-1 Indication of Matter A1 1982 Particularly Application No. 130916 2 Title of Invention Anthracycline Compound ΔG-2J: i, J, Use of Bizo 3 Matters to be amended f1 Which relationship 1.° J revision applicant shave (one side &, c song 4:1, ceremony meeting 1 generation)
Person 7 Subject of Supplement F , -,, -''i Note 1 to the specification, "Detailed description of the invention" column 8 Contents of amendment (1) Specification 1q, page 4, line 9 [Nogalarol is , 1 is corrected as [a compound represented by the following formula <A)". (2) On page 4, line 12, ``Nogalalol'' is amended to read ``a compound represented by the following formula (△)''. (4) Amend "Nogaralol" in the second line from the bottom of page 4 to r(A)J. (5) Change "Nogalalol" in the 6th line from the bottom of the same page 5 to [a compound represented by the formula (Air)]. It is corrected as "data corresponding to Aglycon's S.1." (7) 6th page 12-13 t-j “Spectrum”
is corrected as [spectrum and S[lIS spectrum]. (8) "Nogalalol" on page 6, lines 13-14, is corrected by compound J represented by formula (Δ). (9) - Line 9 from the bottom of page 9, “Anthracycline,” means “Anthracycline.”
There is a lot of correction. (10) Same page 11 line 10 JArgomycin J [Arugomycin J]
Correct as n J. (11) The same page 11, lines 17-18 [Feiko Kenboku No. 6865] has been amended to read ``Feiko Kenbi No. 450.'' (12) Riru Sodeyama, page 12, line 6, ``Microbiosis (ill fungus No. 6865) [micro]2ωI article odd no. 450.'' (13) Page 24, line 13, `` 4%" becomes "0.4%" (+li i [E Rir. (14) The 24th true line 14th line 110%" becomes "1.0%" and t+Ii in Lee. (15) The same "14%" on page 24, line 15 is corrected to "084%". (16) "Niamin" on page 25, line 1 is corrected to "niacin". (17) From right below line 21 on page 25 Line 2 ~ 22nd line 2nd line “(
4) ~1.8%'', as shown below. (4) Elemental analysis value (%): C1-1ON (actual training value)
5G, '26,935.11.8 (Theory II (i)
! i6.7 G, 7344] 1.7 j (18) l
Add the following between "i'il, page 23, line 15 and 16t". r(10) molecule Frl (S I MS ) m/z 1
694 (m+It) -1 Notification of change in accession number April 77, 1980 Director of the Patent Office Kazuo Wakasugi 1 Indication of the case 1981 Pre-patent No. 130916 2 Name of the invention Anthrazycline compound AG-2 and its Application 3
Relationship with the person who filed the procedure Seki's name Same as the name of the old depository institution 8 Kami accession number Micro ■ Kenjoki No. 450 9 List of attached documents (1) Kawamori (copy) certifying the new accession number 6

Claims (1)

[Claims] 1. Anthracycline compound AG2 represented by the following formula,
An antitumor agent containing an anthracycline compound AG-2 represented by the following formula as an active ingredient.