JPH03216198A - Anthracycline substance, preparation thereof and bacterium producing the same - Google Patents

Abstract

NEW MATERIAL:A compound of formula I (R<1> is H, OH; R<2> is H, lower alkyl; R<3> is H, lower alkanoyl). EXAMPLE:A compound of formula II. USE:An antitumor agent. PREPARATION:A microorganism belonging to the Nocardia strain such as Nocardia sp.-YL-01641P (FERM BP-2737) is cultured preferably at 25-32 deg.C at a pH of 6-8 for 24-120 hours.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to an anthracycline substance which is a compound useful as a medicine, particularly an antitumor drug, a method for producing the substance by fermentation, and a bacterium producing the substance.

(Problem to be Solved by the Invention) The present inventors were conducting research on substances produced by many naturally occurring microorganisms, and found that a microorganism belonging to the genus Nocardia and having the ability to produce anthracycline-based substances. By culturing this substance in a medium, a new anthracycline compound having a strong antitumor effect is produced in the same medium, and the present invention was completed by isolating this substance.

Conventionally, various anthracycline compounds produced by microorganisms include, for example, downmycin and adriamycin.
Aclacinomycin and the like are known, and it is well known that these compounds are clinically very effective drugs. However, during the course of administration of these drugs, target tumor cells almost always acquire resistance to these drugs. As described below, it has been discovered that the novel anthracycline compounds of the present invention are also effective against animal tumor cells that are resistant to such anthracycline compounds. Therefore, the present invention provides anthracycline substances having excellent antitumor effects, and also provides a novel manufacturing method for obtaining antitumor substances. The present invention provides a new bacterial strain that produces the above-mentioned anthracycline substances.

The anthracycline substance (I) provided by the present invention is represented by the following planar chemical structural formula.

(In the formula, R+ means a hydrogen atom or a hydroxyl group, R2 means a hydrogen atom or a lower alkyl group, and R3 means a hydrogen atom or a lower alkanoyl group.) The structural characteristics of the compound (I) of the present invention are that It has sugar chains at the 7th and 10th positions of the cyclin skeleton, and especially the sugar chain at the 10th position is a sugar chain with a dimethylamino group at the 4th position, which has not been previously found in anthracycline antibiotics. That's a certain point.

Furthermore, the production method of the present invention includes culturing a microorganism belonging to the genus Nocardia and having the ability to produce the above-mentioned anthracycline substance in a medium, producing the substance in the culture, and then collecting the substance. This is a method for producing anthracycline-based substances consisting of:

Furthermore, the microorganism of the present invention belongs to the genus Nocardia and has the ability to produce the above-mentioned anthracycline substance (I). The microorganism of the present invention, the production method, and the compound (I) of the present invention obtained by the production method will be described in detail below.

The strain producing the novel antitumor compound of the present invention is a microorganism isolated from soil collected in Indonesia, and has the following mycological properties.

1. The basal hyphae are well developed and branched on various organic and inorganic media.
The width of the hyphae is approximately 04 μm, and fusion of hyphae is observed in the late stage of culture. Due to the medium used, slight rupture of hyphae is observed. Aerial hyphae are long straight or curved, irregularly branched, and form relatively short sporophytes. Culture 5~
From 7 weeks onwards, a chain of 5 to 10 spores is formed near the tip of the sporophyte. Its tip is loosely coiled 2 . On the other hand, long spore chains due to fragmentation may be observed in the main axis of aerial hyphae, and in this case, hyphae may take on bundle-like or loop-like shapes, and a pseudosporangium-like structure may be observed. The shape of the spore is cylindrical, and the size is 1.8-12μm x 05-0.
Its surface is smooth with a thickness of 8 μm. sporangia, sclerotia,
No motile elements or special organs such as sutures are observed.

Properties on various agar media The 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 Research Institute).

(Note) G: Growth degree A; Aerial mycelial epiphytia and its hue R; Hue S on the back side; Soluble pigment bio-embedding properties 1) Growth temperature range Optimum growth temperature 20-40℃ 27-32℃ 2) Gelatin Liquefied simple gelatin (20°C) Glucose-pebutone gelatin (28°C) Negative 3) Coagulation of skimmed milk Positive peptonization of skimmed milk 4) Positive nitrate reducing action 5) Negative starch hydrolysis action 6) Melanin Formation of similar pigment Tributone yeast extract Liquid medium Tyrosine agar medium Pembone yeast iron agar medium Negative Negative (Note) Growth temperature is at each temperature (5, 10, 15, 20,
25, 28, 30, 37, 40. 45. 50
In 'C), the observation results from days 7 to 21 are shown, and the effects on milk are the results from days 3 to 21 at 37°C.Other than that, unless otherwise specified, the results are observed after 2 weeks at 28°C. are shown respectively.

4 Assimilation of carbon sources (Pridm Godleif agar medium,
28°C culture) L-arabinose - Mannose D-Kynlose - Melibiose D-Glucose + Lactose D-Fructose + D-Galactose Sucrose + Maltose inositol + Sarin Rhamnose - Trehalose Raffinose - Glycerin D-Mannitol + Dextrin starch + Xanthine + + + (Note) +: Grows 11: Growth is doubtful: No growth 5 Chemical analysis of bacterial cell components LECHVALIER et al.'s method (LECHVALIE
R, M.P. et al; PP 277-238
in DIETZ, A. et ed. +Acti
nomyceteTaxonomy, SIM Spe
cial publication A6+1980), the acid hydrolyzate and cell wall components of this strain were analyzed, and as shown in Table 4, the cell wall type was rV-A.

Table 4 Total bacterial body reducing sugars arabinose and galactose Main menaquinone species MK-8 Presence of mycolic acid Positive GC content 66% To summarize the above properties, strain YL-01641P has spores on short sporophytes produced on aerial mycelia. Make a chain. Spore chains due to fragmentation are observed in a part of the main axis of aerial hyphae in the late stage of culture. In addition, some of the aerial hyphae are bundle-like, forming a coil-like structure. Pseudosporangium-like structures are also observed. Fractures may also occur in the basal hyphae during the late stage of cultivation. The cell wall type is TV-A, and the main menaquinone is MK-8 (H4, H
2).

When searching various literature for bacterial species with the above properties, this strain was found to be the so-called Sporoactinomycetes.
It falls into the category of Actinobacteria s (genus Streptomyces that forms spore chains).

Among these, the genus Nocardia is a known bacterial genus that matches the chemical classification results and morphological characteristics shown in Table 4. Traditionally, the genus Nocardia was divided into three types depending on the morphology at the initial stage of culture and the presence or absence of aerial hyphae (I+') (Bergey's manual of
determinative bacte-rio1o
gy, 8th edition (1974)), but in particular, many of the bacteria belonging to the ■ and ■ types have been transferred to Rhodococcus, and classification standards are being sought from a completely new perspective. Also Ber
gey's manual of Systemati
c Bacteriology (Volume 2, 1986
Nine species of Nocardia were described in
The L-01641 P strain did not match any of them.

From this point of view, we judged this strain to be a new bacterial species belonging to the genus Nocardia, and identified it as Nocardia S.P.
ardia sp. ) YL O1641P.

This bacterial strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology as FAIKEN Article No. 2737.

In carrying out the method for producing anosthracycline-based substances of the present invention, the production strain of the substance, Nocardia SP strain YL-01641P, is inoculated into a nutrient-containing medium and grown aerobically. A culture containing anthracycline material is obtained. As nutrients, those known as nutritional sources for actinomycetes can be used. For example, commercially available peptone, meat extract, corn steep liquor, cottonseed flour, peanut flour, soybean flour, yeast extract, NZ-
Nitrogen sources such as amines, melted casein, fishmeal, sodium nitrate, ammonium nitrate, commercially available glycerin,
H# starch carbohydrates such as glucose, maltose, and molasses or carbon sources such as fats and inorganic salts such as common salt, phosphate, calcium carbonate, and magnesium sulfate can be used. In addition to these substances, any substance that can be utilized by the producing bacteria and useful for producing the anthracycline substance of the present invention can be used.

The culturing method may be carried out in the same manner as a general antibiotic production method, and the culturing method may be solid culture or liquid culture. For liquid culture, static culture, agitation culture, shaking culture,
Although any method such as aerated culture may be used, aerated agitation culture is particularly preferred. In addition, the culture temperature allows the production bacteria to grow.
The temperature for producing the substance of the invention, i.e. 15°C to 40°C
The pH of the medium can be changed as appropriate within the range of approximately 25°C to 32°C, but is preferably within the range of about 4 to 9, but preferably 6 to 8. The culture time varies depending on various conditions and ranges from lo time to 168 hours, but the target substance accumulated in the culture usually reaches its maximum titer in about 24 hours to 120 hours.

To collect the target compound from the culture, extraction, separation, and
Purification means are utilized as appropriate. The target substance in the culture can be obtained by adding ethyl acetate, chloroform, benzene, toluene, etc. to the culture solution obtained by centrifugation or by adding a furnace aid to the culture and passing it through the door. Extract by adding an immiscible organic solvent. In addition, the target substance can be extracted by bringing culture solution F into contact with a suitable carrier to adsorb the target active ingredient in solution P, and then eluting with an appropriate solvent. To be more specific, for example, Amber Rai}
XAD-2, Diamond ion HP-20, Diamond ion s
The target substance is adsorbed by contacting with a porous adsorption resin such as p-900 or Diaion CHP-20P, or an ion exchange resin such as DOWEX 50W or Amberlite IRC-50.

Then, in the case of the adsorption resin, methanol, ethanol,
A fraction containing the target substance can be obtained by elution with a mixed solvent of an organic solvent such as acetone or acetonitrile and water, or in the case of the ion exchange resin with an aqueous solution such as hydrochloric acid or sulfuric acid. When extracting with organic solvents such as ethyl acetate or butyl acetate, add these solvents to the culture solution, shake well, extract the target substance, and re-extract the resulting extract with acidic water to extract the target substance. Dissolve in water.

Next, by repeating the process of neutralizing the acidic water extract and then extracting it again with the organic solvent, it is possible to obtain a higher proportion of the target substance. Next, the target substance-containing fractions obtained using each of the above procedures are subjected to conventional adsorption treatments such as column chromatography, thin layer chromatography using activated carbon, alumina, silica gel, cellulose, etc. as carriers, and silica gel-based ODS. More pure separation can be achieved by conventional methods such as high performance liquid chromatography using a reverse phase carrier column. Furthermore, the target substances obtained by these methods can be crystallized in a solvent such as methanol or ethyl acetate to obtain even purer substances. The new anthracycline substance thus produced in the culture can be separated in the form of its free base. The free base may also be an acid, i.e. an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, or an inorganic acid such as formic acid, acetic acid,
By reacting with organic acids such as citric acid, tartaric acid, p-toluenesulfonic acid, etc., it can be converted into salts corresponding to the respective acids.

Example 1. (a) (YL-01641P-A, substance manufacturing method)
Dextrin 2.0%, corn steep liquor - 0.5%
, polypeptone 0.5%, yeast extract 0.5%, meat extract 0.3%, plain heart infusion broth 0.52%, calcium carbonate 0.5% (p
H8.0) was prepared, 60 ml of each was dispensed into 500 m7 Erlenmeyer flasks, and sterilized at 120°C for 20 minutes. The hyphae of the strain were scraped and inoculated, and cultured with shaking at 27°C for 48 hours to prepare a seed culture. Next, a medium containing 3.0% potato starch, 1.0% wheat germ, 1.0% copra meal, 0.2% feather meal, and 0.5% calcium carbonate (p}1
7.0), dispensed 60ml each into 500+nt Erlenmeyer flasks, sterilized them at 120"C for 20 minutes, inoculated them with the above culture solution at a ratio of 3.0%, and incubated at 28°C.
The cells were cultured for 96 hours. Culture solution 1 obtained in this way
Radioly} #600 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added to t, stirred, filtered through F, and the culture pH of 650 m7 was adjusted.
I got it. Adjust this r solution to pH 7.0 and add 50% ethyl acetate.
The mixture was divided into two portions of 0 m4 each and stirred and extracted. This extract was sterilized and concentrated to 100 ml, 100 mt of water was added, the aqueous layer was adjusted to pH 2.5 with 1N hydrochloric acid, and stirred thoroughly to transfer the target product from the solvent layer into the water. 50 m4 of ethyl acetate was added to this acidic extract, and the aqueous layer was adjusted to pi{7.5 with 1N caustic soda, stirred well, and re-extracted. Repeat this operation twice to obtain a total of 200 mZ YL.
-0614IP-A, an ethyl acetate extract of the substance was obtained.

After adding Glauber's salt to this extract and thoroughly dehydrating, the Glauber's salt was removed by filtration, and the P solution was concentrated under reduced pressure to 3 ml to obtain crude YL-
01641P-A, a concentrated solution of the substance was obtained. To confirm the substance, use a silica gel plate (Kieselgel 60F254-
20cm x 20cm, manufactured by Merck & Co., Ltd.), and the target YL-01641P A was analyzed using thin layer chromatography 7E developed with a solvent of chloroform:methanol (90:10).
+ substance (Rf = 0.38 > was confirmed. Crude YL-01641P- in the concentrated solvent thus obtained
A. Silica gel plate (Kieselgel 60 F254, 20 cm x 2) to further purify the substance.
Q Cm, manufactured by Merck & Co., Ltd.) were charged in a strip at 2 cm from the bottom, and chloroform:methanol (9
0:10), and after air-drying, a red-orange band (Rr = 0.38) of the YL-01641PA1 substance was confirmed under a UV lamp and visible light, and this part was scraped off and mixed with chloroform:methanol ( 90:10)
After the eluate was concentrated to dryness under reduced pressure, a small amount of methanol was added and dissolved by heating. When this was left in the refrigerator, needle-shaped crystals precipitated, so after removing the solvent, Drying in a desiccator under reduced pressure overnight yielded 2.15 fllg of pure YL-01641P-A1 material.

The above extracted, separated and purified YL-016 4I P-
The free base of the A1 substance has the following physicochemical properties.

(1) Color and shape: Red needle-shaped crystals.

(2) Distinction between acidic, neutral, and basic: Basic.

(3) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, and chloroform, but poorly soluble in water, and almost insoluble in hexane.

(4) Ultraviolet absorption spectrum: YL 01641P
The ultraviolet absorption spectrum of the A+ substance measured in methanol is as shown in FIG.

(5) Mass spectrum (FAB): 790 (
M+ 1) (6) Molecular weight: 789.831 (force Molecular formula: C3.H51NOll! (8) Elemental analysis value: C3o Hs+ NO+.・o,7H20 C (%) H (%) N (%) Actual value 58 ,30 6.57 1.68 Calculated value 58,38 6.58 1.75 (9)
'H-NMR spectrum: YL-01641P
'H-NMR spectrum of A+ substance (500 MHz
, CDCI3) are shown in Figure 2.

GO) 13C-NMR spectrum: YL-016
41P-A, 3C-NMR spectrum of the substance (125
MHz, CDCI,) as shown in Figure 3.

Based on the above physicochemical properties, the YL-01641P A+ substance is represented by the following structural formula.

(b) Preparation method of hydrochloride Purely purified YL-0164 obtained in (a) above
A small amount of water was added to 25.6 mg of 1P-A, and 0.5N hydrochloric acid was carefully added little by little while stirring well to dissolve the YL-01641P A+ substance.

This operation was continued until the YL-01641P-A substance was completely dissolved.

The aqueous solution of YL-01641P-A and hydrochloride thus obtained was freeze-dried, and YL-01641P-A, which is easily soluble in water, was
A1 red powder 26.761! Ig was obtained. The solubility of this substance in water is the raw material YL-0 164I P-
A. Much higher than bases.

Example 2 YL- cultured using the same culture method as that shown in Example 1
Add sulfuric acid to 4 tons of culture solution of 01641P strain to pH 2.0
After adjusting the temperature, stir well. Then Radio Rhy}#6
00 (manufactured by Showa Kagaku Kogyo Co., Ltd.) and stirred again.
passed. When this F solution was adjusted to pH 5.02, unbathable substances precipitated, so much of this was filtered to obtain 2.98 t of clear culture R solution. This F solution was added to Teriyaion HP-20 (
Mitsubishi Kasei Co., Ltd.) column (60 m4, outer diameter 2.3
cm, length 19 cm) to pass through the target object YL-
After adsorbing the 01641P A+ substance, the column was thoroughly washed with 1 liter of distilled water. Next, 30%, 40%, 50%, prepared using 0.01M phosphate buffer.
Elution was carried out stepwise in ascending order of acetone concentration using 120 ml each of 60% and 70% acetone aqueous solutions.

As a result, the substance YL-01641P-A was eluted at both 40% and 50% acetone concentrations. To confirm the YL-01641P A+ substance in this eluate, use a silica gel plate (Kieselgel 60 F'254j 2
0 am x 20 cm, manufactured by Merck & Co., Ltd.), and the target YL-01641P-A was obtained by thin layer chromatography developed with a solvent of chloroform:methanol (90:10).
, substance (Rf-0.38) was confirmed. Next, the elution fraction of the YL-01641P-AI substance obtained in this manner was concentrated under reduced pressure to obtain 10 7. 2■
Obtained. 1 coarse powder of this YL-01641P A+ substance
After dissolving in 00 mZ of ethyl acetate, insoluble matter was removed by P filtration, 100 ml of water was added to the f solution, and while shaking, the aqueous layer was adjusted to pH 2.0 with hydrochloric acid to dissolve YL-0.
The 1641P A+ material was back-extracted into acidic water. Next, after separating this aqueous solution and removing ethyl acetate, 10 On+7 ethyl acetate was newly added, and while shaking, the aqueous layer was adjusted to pH 7.0 with caustic soda, and YL- was added to the ethyl acetate layer. 01641 P A+ material was transferred. Next, this ethyl acetate extract is separated, the aqueous layer is removed, and mirabilite is added. After dehydration, this mirabilite is
Remove by filtration, concentrate under reduced pressure and dry to give crude YL-01 6
4IP-A, red powder of substance (purity 41.9%)
．． 69+11g was obtained. The powder thus obtained has 5
After adding m7 of water and completely dissolving the powder while adding IN hydrochloric acid little by little, the pH was adjusted to 5.0 with caustic soda to prepare a water stone solution of the crude YL-01641P-A1 material. On the other hand, a chromatography tube with an outer diameter of 1.2 cm and a length of 45 cm was filled with 30 ml of Diaion CHP.
A column of -20P (manufactured by Mitsubishi Kasei Corporation) was prepared, and a water bath solution containing the crude YL-01641PA and substance was passed therethrough to adsorb the target substance, -YL-01641P-A. Next, this column was washed with 50 ml of water, and then elution was carried out by a concentration gradient elution method using 600 mt00-80% acetone water in total. The eluate was fractionated into 10 ml portions using a fraction collector. Among the fractions eluted in this way, all seven fractions in which a reddish-orange substance was eluted were placed on a silica gel plate (Kieselgel 60 F254, 20 cm x
20 cm, manufactured by Merck & Co., Ltd.), chloroform:
The target YL-01641P-A, substance (
Rf = 0.38) was confirmed. As a result, the target substance YL-01641P-A was eluted between fractions 46 and 52. Among these, fractions 48 to 50, which were the purest, were collected and concentrated under reduced pressure. After adding an excess of supersaturated sodium bicarbonate water bath solution to bring the pH to 7 or higher, the mixture was extracted twice with 25 ml of ethyl acetate. . After adding Glauber's salt to this extract and thoroughly dehydrating it, it was filtered through P to remove Glauber's salt, and this r solution was concentrated to dryness, and dried overnight in a vacuum desiccator to obtain YL-01641P.
- Red powder of substance A1 (purity 86.2%) was added at 25.3 [
I got 11g.

Example 3. A medium a (pa7) containing 1.0% glucose, 2.0% potato starch, 0.5% polybeptone, 0.5% yeast extract, and 0.4% calcium carbonate was prepared, and this was
Dispense 100mZ into 0rnl Erlenmeyer flasks, 12
Sterilized at 0°C for 20 minutes. Norcadia S. h. grown on Pennett's agar in the medium prepared in this way.
The hyphae of YL-01641P strain were scraped and inoculated, and 27
The culture was cultured with shaking at °C for 96 hours to obtain a seed culture solution (1). Next, prepare 800 mt of medium A and apply it for 500 mt.
Dispense 100mt into each Erlenmeyer flask and incubate at 120℃ for 2 hours.
After sterilization for 0 minutes, the seed culture solution (1) was inoculated at a rate of 2%. This was cultured with shaking at 27°C for 96 hours, and was designated as seed culture i (2). The main culture was carried out in two 30-ton culture tanks as a production medium containing 5% cobra meal, 2.5% wheat germ, 0.15% calcium carbonate, and 0.09% antifoaming agent NKL-5430 (manufactured by NOF Corporation). 20t (p
H70), sterilized in advance at 120°C for 30 minutes, and inoculated with 400 mt each of the seed culture solution (2) that had been cultivated earlier. When cultured at 20 rpm for 3 days, YL-0
1641 P-Al! Bacillus subtilis AT
The antibacterial activity against CC6633 strain was maximum. After adjusting the pH of the thus obtained culture solution to 3.5 with hydrochloric acid, Radioly #600 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added and stirred, it was filtered through P. This F solution was adjusted to pH 7.0 with caustic soda, and precipitated insoluble matter was removed by filtration to obtain 27 tons of culture solution. In order to extract the substance YL-01641P-A from the P solution thus obtained, 181 ethyl acetate was added and the culture P solution layer was adjusted to pH 7.0 using a caustic sorter while shaking.

This extraction operation was repeated twice. YL-0164
The ethyl acetate extract containing the substance 1P-A was removed by separating the culture liquid layer, thoroughly dehydrated by adding Glauber's salt, filtered through F to remove the Glauber's salt, and concentrated under reduced pressure to 2 liters. After adding I-B of water to this, the aqueous layer was adjusted to pH 2.0 with hydrochloric acid while shaking well, and the YL-01641P-A1 substance was extracted by dissolving into the acidic water. Next, this aqueous solution was separated and the ethyl acetate layer was removed. This operation was repeated twice to obtain about 2 volumes of an acidic solution of YL-01641P-A. Add 1 part of ethyl acetate and adjust the aqueous layer to pI{7.0 with caustic soda while shaking. After extracting the YL-01641P-A1 substance to the ethyl acetate layer, separate the layers. The aqueous layer was removed. This operation was repeated twice to obtain an ethyl acetate extract containing about 26 YL-01641P-A substances. Next, after adding Glauber's salt to this ethyl acetate extract and thoroughly dehydrating, Glauber's salt was removed by F filtration, concentrated under reduced pressure, and dried to give crude YL-01641P.
43611@ of red powder of A+ substance was obtained. This coarse YL-
01641P Take 153■ of red powder of At material,
To this, 2 ml of methanol was added and dissolved. Divide this methanol bath solution into 0.4 ml portions, transfer to liquid chromatography YL-01641P-A, and extract the substance;
Ta. Liquid chromatography conditions are YMC-Pack
2cmX of R-ODS-5 (manufactured by Yamamura Kagaku Kenkyusho Co., Ltd.)
0.05M phosphate buffer (pI{4.35):acetonitrile:tetrahydrofuran (75:10:15) was added to a 25cm column as a Ni solution at a rate of 9/min.
1+17 style. 480nm UV
Absorption was used for detection. As a result, YL-01641
Since the P-AI substance was eluted at a retention time of 15 minutes and 48 seconds, this fraction was collected.

This fraction was subjected to liquid chromatography (column: YMC-
PackA 312 (S-5120-ODS)
(Manufactured by Yamamura Kagaku Kenkyusho Co., Ltd.) 6 mm x 150 mm, eluent: 0.05M phosphate buffer (pH 4.35)
: Acetonitrile: Tetrahydrofuran (70: 15
: 15), @output amount: 1ml per minute, retention time =
After confirming that the eluate was pure (1-5 minutes and 51 seconds), it was concentrated under reduced pressure to thoroughly evaporate and remove the organic solvent in the eluate. Excess sodium bicarbonate solution was added to the eluate treated in this way (approximately 120 m7) to adjust the pH to 7.0 or higher, and 100 ml of ethyl acetate was added to perform extraction. This extraction operation was repeated twice to obtain approximately 200 mt of YL-01.
641P-A, a solution of the substance in ethyl acetate was obtained. After adding Glauber's salt to this solution and thoroughly dehydrating, the Glauber's salt was removed by F filtration, concentrated to dryness under reduced pressure, and further dried overnight in a vacuum desiccator to obtain pure YL-01641P-A, a red powder of 83% ■I got it. 40.0111 g of this powder was taken, 1+++7 g of water was added thereto, and 0.5% tartaric acid water bath solution was added dropwise little by little with stirring until the red powder was completely dissolved.

At this time, the pH at the time when the YL-01641P A+ substance was completely dissolved was 4.53. The tartaric acid aqueous solution of the YL-0164 IP-A substance thus prepared was freeze-dried to obtain 42.61I1g of pure YL-01641P-A tartrate crystalline powder. The solubility of this tartrate in water is the raw material YL-01641.
P-A, which is significantly higher than the free base of the substance.

Example 4. (Production method of YL-01641P-A2, B2 and B) [A] Glucose 1.0%, potato starch 2.0%, polypeptone 05%, yeast extract 0
．． Medium A (pH 7) containing 5% calcium carbonate and 0.4% calcium carbonate.
) and put 100ml of this into a 500ml Erlenmeyer flask.
The solution was dispensed into mZ portions and sterilized at 120°C for 20 minutes. The mycelium of Nocardia SP YL-01641P strain grown on Benet agar was scraped and inoculated into the medium thus prepared, and cultured with shaking at 27°C for 96 hours to form a seed culture solution (1). do.

Next, prepare 800 ml of medium A and add it to 500 rnl.
Dispense 100 Int into each Erlenmeyer flask and incubate at 120℃ for 2 hours.
After sterilization for 0 minutes, the seed culture solution (1) was inoculated at a rate of 2%. This was cultured with shaking at 27° C. for 96 hours to obtain a seed culture solution (2). The main culture was carried out in a 30t culture tank with 5% copra meal, 2.5% wheat germ, and 0.15% calcium carbonate.
, Antifoaming agent NKL-5430 (manufactured by NOF Corporation) 0.0
Prepare a production medium 21 (pH 7.0) containing 9%, sterilize it in advance for 30 minutes, and add 400 rnl of the seed culture solution [B] (2) cultured previously to this. Inoculation, aeration rate 30t/min,
Culture was carried out for 2 days at a rotation speed of 200 rpm to obtain 19 L of culture.

After adjusting the pH of 19 tons of the above culture to 3.5 with hydrochloric acid, Radiolite #600 (manufactured by Showa Kagaku Kogyo Co., Ltd.) was added and stirred, the mixture was filtered through f'1 to obtain 15 tons of culture solution f. This f
In order to extract the target substance from the solution, 107 ethyl acetate was added and the culture liquid layer was adjusted to pH 7.
The target substance was extracted into the ethyl acetate layer, and the extraction operation was repeated twice. The culture f liquid layer was separated and removed from the ethyl acetate extract containing the target substance, and after thoroughly dehydrating by adding Glauber's salt, much of the Glauber's salt was removed and concentrated under reduced pressure to 2 tons. 1 ton of water was added to this, and while shaking, the aqueous layer was adjusted to pH 2.0 with hydrochloric acid, and the target substance was extracted by dissolution into acidic water. Next, this aqueous solution was separated and the ethyl acetate layer was removed. This operation was repeated twice to obtain about 2 tons of an acidic aqueous solution of the target substance.

Add another 1 t of ethyl acetate, and while shaking, adjust the aqueous layer to pH 7.0 with caustic soda. After extracting the target substance into the ethyl acetate layer, separate the layers. The aqueous layer was removed. This operation was repeated twice to obtain an ethyl acetate extract containing about 26 target substances. Next,
After adding Glauber's salt to this ethyl acetate extract and thoroughly dehydrating, the sodium sulfate was thoroughly filtered off, concentrated under reduced pressure, and dried to obtain 30,211 g of a crude product containing the crude target substance.

Dissolve 302■ of the above crude product in 5 ml of methanol,
0.5 ml each of this methanol solution was subjected to liquid chromatography to separate three types of target substances based on differences in retention time. The conditions for preparative liquid chromatography are STR-
PREP ODSH (manufactured by Shimadzu Techno Research) 2c
mX2Scm column, 0.02M phosphate buffer (pH 4.2):acetonitrile:tetrihydrofuran:methanol (67:15:8:10) as eluent at 7 min.
ml flow rate, 254 nm UV
Absorption was used for detection.

To confirm the target substance in the preparative fraction, use a silica gel plate (Kieselgel 60F254+ 20cm x 20cm,
Merck & Co., Ltd.) was used, and thin layer chromatography was used, developed with a solvent of chloroform:ethyl acetate:methanol:aqueous ammonia (70:30:10:0.1). Furthermore, each fraction was subjected to liquid chromatography using an analytical column (conditions: analytical column STR-ODS-H).
4.6 mm
:15:10:10), elution volume 0.8ml/min)
It was confirmed that the target substance was pure.

■ Retention time by preparative liquid chromatography: 14 minutes 48 minutes
Target substance obtained from the fraction eluted in seconds: This substance has an Rf value of 0.1 in the thin layer chromatography described above.
7. It is also a single substance whose retention time in liquid chromatography using the analytical ram described above is 5 minutes and 31 seconds. After the organic solvent in the eluate of this fraction was removed by concentration under reduced pressure, sodium hydrogen carbonate solution was added to adjust the pH to 70 or higher, and the mixture was extracted with ethyl acetate. After adding Glauber's salt to the extract and thoroughly dehydrating, Glauber's salt was removed by filtration, concentrated to dryness under reduced pressure, and further dried overnight in a vacuum desiccator. The obtained red powder (8.0 [I1 g) exhibits the following physicochemical properties.

(1) Color and shape: red needle-like crystals.

(2) Distinction between acidic, neutral, and basic: Basic.

(3) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, and chloroform, but poorly soluble in water, and almost insoluble in hexane.

(4) Ultraviolet absorption spectrum: YL-01641P-
The ultraviolet absorption spectrum of the A2 compound measured in methanol is as shown in FIG.

(5) Mass spectrum (FAB): 748 (M+1
)(6) Molecular weight: 747.792 (force Molecular formula: C37H411NO+5(8)'
H-NMR spectrum: 'H-NMR spectrum of YL-01641P-A2 compound (500MHz, CDC
I,) is as shown in FIG.

Based on the above physicochemical properties, this substance is YL-01641P-A2 substance shown by the following structural formula.

■ Retention time by preparative liquid chromatography: 31 minutes 30 minutes
Target substance obtained from the fraction eluted in seconds: This substance has an Rf value of 0 in thin layer chromatography.
．． 15, is a single substance whose retention time in liquid chromatography using the above analytical column was 10 minutes and 14 seconds. This fraction above? The dark red powder (671'rlg) obtained by extraction, concentration, and drying in the same manner as in the method shows the following physicochemical properties.

(1) Color and shape: dark red needle-like crystals.

(2) Distinction between acidic, neutral, and basic: Basic.

(3) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, and chloroform, but poorly soluble in water, and almost insoluble in hexane.

(4) Ultraviolet absorption spectrum: YL-01641P
The ultraviolet absorption spectrum of the B2 compound measured in methanol is shown in FIG.

(5) Mass spectrum (FAB): 734 (M+1
) (6) Molecular weight: 733.765 (7) Molecular formula: C36H, ■NO ,, (81'
H-NMR spectrum: 'H-NMR spectrum of YL-01641P-B2 compound (500MHz, CDC
I,) is as shown in FIG.

Based on the above physicochemical properties, this substance is YL 01641P-B2 substance shown by the following structural formula.

■ Retention time using preparative liquid chromatography: 47 minutes 45 minutes
Target substance obtained from the fraction eluted in seconds: This substance was detected by the Rf
It is a single substance with a value of 0.28 and a retention time of 16 minutes and 08 seconds in liquid chromatography using the above analytical column. This fraction was extracted, concentrated, and
The dark red powder (69.801 g) obtained by drying exhibits the following properties.

(l) Color and shape: dark red needle-like crystals.

(2) Distinction between acidic, neutral, and basic: Basic.

(3) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, and chloroform, but poorly soluble in water, and almost insoluble in hexane.

(4) Ultraviolet absorption spectrum: YL-01641P-
B. The ultraviolet absorption spectrum of the compound measured in methanol is shown in FIG.

(5) Mass spectrum (FAB): 776 (M+1
) (6) Molecular weight: 775.802 (force molecular formula 2 C38 H49 No le (8)
NMR spectrum for compound YL-01641P-B, NMR spectrum for compound (500MHz, CD C
13) as shown in FIG.

(9) 13C-NMR spectrum: YL-016
The 13C-NMR spectrum (125 MHz, CDCI3) of the compound 41P-B is as shown in FIG.

From the above chemical properties, this substance is YL-01641P-B, a substance represented by the following structural formula.

[D] YL-01641P-A2 3[flg, YL-016
41P-B231I1g, YL-01641P-B,
After adding water as needed, 0.5% tartaric acid aqueous solution was added dropwise little by little until the powder was completely dissolved. At this time, the pH at the time when the compound was completely dissolved was from pH 4.5 to pH 4.6.

The tartaric acid aqueous solution of the compound thus prepared was freeze-dried to obtain 3.17111 g of pure YL-0164LP-A2 tartrate, 3.17 g of YL-01641P-82 tartrate, YL-01641P-B, tartrate 31
．． I got 68■. The solubility of the tartrate in water is much higher than that of the free base of the compound as a raw material.

Example 5 (Manufacture of YL-01641P C+ substance) Seed culture solution (following 11, prepare seed culture solution (2)) in the same manner as in Example 3. Separately, prepare a similar seed culture medium in a 306 culture tank, Sterilize it and add 4 seeds of the seed culture solution (2) to it.
After inoculating 00ml, the rotation speed was 2 at an aeration rate of 30 t/min.
00 rpm for 72 hours to prepare a seed culture solution (3).

For the main culture, the same medium as the main culture in Example 3 was used at 230%
t was prepared, sterilized at 120°C for 45 minutes, and 4 t of the above seed culture solution (3) was inoculated into this. After this, 15 per minute
When cultured for 3 days at a rotation speed of 75 rpm with an aeration volume of 0 L, Y
L-016411-C, substance Bacillus subtilis AT
The antibacterial activity against CC6633 strain was maximum.

After adjusting the pH of the culture solution thus obtained to 2.0 with hydrochloric acid, it was added to Radiolite #600 (manufactured by Showa Kagaku Kogyo Co., Ltd.).
was added and stirred. After 1 hour, the culture solution was filtered to remove solid matter, and 1N caustic soda solution was added to the obtained culture solution to adjust the pH to 7.0. At this time, insoluble matter precipitated, so this P solution was filtered again to remove the insoluble matter and obtain 205 tons of clear solution. In order to extract YL-1641P C1 from the culture solution obtained in this way, a 20 t Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) column SV = 2. 5 to adsorb the substance YL01641P-C. Next, after thoroughly washing this column with water, 30% acetone water 41.60% acetone water 601.60% acetone-0
．． 60 t of 05M buffer solution was flowed stepwise to elute the target product YL-01641P. Detection of the target substance was performed using thin layer chromatography (Kieselgel 60F2!, 42
0 x 20 (manufactured by Merck & Co.), developing solvent; chloroform: ethyl acetate: methanol = 28% aqueous ammonia (70
:30:10:0.1), target YL-01641P
C+ORf value: 0.3). As a result, the target product YL-01641P-C was contained in 80t of eluted fractions after 60% acetone water elution. This eluate was concentrated to about 3 OL, and after removing acetone,
20 tons of ethyl acetate was added, and while shaking, the pH of the aqueous layer was adjusted to 7.0 with caustic soda, and the target product, YL-01641p-c, was extracted into the ethyl acetate layer. This extraction operation with ethyl acetate was repeated twice. The ethyl acetate solution obtained in this way was concentrated under reduced pressure to about 3 tons, then 2 tons of water was added, and while shaking well, hydrochloric acid was added until the pH of the aqueous layer became 2.0.
01641PC was transferred and extracted into acidic water. This operation was repeated twice to obtain an acidic aqueous solution containing 4 target substances. 26 ethyl acetate was newly added to this aqueous solution, the pH was adjusted to 7.2 with a caustic nodder while shaking, and the target product YL-01641P-C was re-extracted from the ethyl acetate layer.

This operation was repeated twice to obtain an ethyl acetate extract containing about 4 tons of the target product. The target product was detected using the thin layer chromatography described above at each extraction step. The ethyl acetate solution containing YL01641P-C, the target product extracted in this way, was dehydrated with Glauber's salt, concentrated under reduced pressure at 40°C, dried, and thoroughly dried in a desiccator overnight.
A coarse powder containing g of YL-01641P C+ was obtained. 50 ml of water was added to this crude powder, and hydrochloric acid was added until the powder was completely dissolved to prepare an aqueous solution containing the target product YL-01641P-C. Add this solution in advance to 4crn x 115
A CrrL glass tube was filled with Diaion HP-20 (manufactured by Mitsubishi Kasei Corporation) No. 16, washed with water, and charged into a prepared power ram at a ratio of Sv=o2 for adsorption. After adsorption and thorough washing with water, elution was performed using a concentration gradient elution method using 30-60% acetone water (86% in total).
One fraction of 6 g was fractionated using a fraction collector. The target substance in the eluate was confirmed by the thin layer chromatography described above. As a result, fraction number 341~
Since the target substance was contained in 385, both of these fractions were collected and concentrated under reduced pressure at 40°C to remove acetone, and then the same amount of ethyl acetate as this concentrated solution was added, and the aqueous layer was brought to pH 7. 2
The target product was extracted while adjusting the This operation was repeated twice. This extract was dehydrated with Glauber's salt, concentrated to dryness under reduced pressure at 40°C, dried overnight in a vacuum desiccator, and the target product Y
3.0 g of coarse powder of L-01641P-C was obtained. 2 ml of water was added to 1.0 g of this crude powder, and 1N hydrochloric acid was added little by little to completely dissolve it. 10 of this solution
A 0 μt aliquot was fractionated using liquid chromatography.

The liquid chromatography conditions at this time were STR-
PF! EP ODS-H (manufactured by Shimadzu Techno Research)
Using a 2α x 25 column of
UV detection at 254 nm was used. In this way, the target product YL-0164 was eluted at a retention time of 21 minutes and 6 seconds.
The 1P C+ fraction was collected and concentrated at 40'C under reduced pressure.
After removing the organic solvent, add the same amount of ethyl acetate as this concentrated solution, add sodium hydrogen carbonate solution so that the pH of the aqueous layer becomes 7.5 or higher, and shake well to obtain the target compound YL-
01641P-C, material was extracted into ethyl acetate layer. This extraction operation was repeated twice.

This extract was dehydrated with Glauber's salt, concentrated to dryness at 40'C under reduced pressure, and dried overnight in a vacuum desiccator to obtain the target product YL-0.
1641P-C, was obtained as a crude powder of 78 μm. This powder contained a small amount of components other than the target product, so in order to further increase the purity, Ig water was added and hydrochloride was prepared using the same method as above. An aqueous solution of 50 tons of this aqueous solution was passed through liquid chromatography to obtain the target object YL-01641P C+
The peak was fractionated. The conditions at that time are ST-PREP
ODS-H (manufactured by Shimadzu Techno Research) 2 crr
Using a L x 25 cm column, elute 002M phosphate buffer (pH 4.2):acetonitrile:methanol (55:15:30) as the eluent at a rate of 7ml/min.
UV detection at 254 nm was used. Under these conditions, the target compound YL-016 was eluted at a retention time of 47 minutes and 34 seconds.
Both pure fractions of 41 P-C+ were collected. The target substance in the preparative fractions was confirmed using the above-mentioned thin layer chromatography and liquid chromatography using an analytical column. The liquid chromatography conditions used for analysis were
TR-ODS -H 4.6r+vn X250mm
(manufactured by Shimadzu Techno Research Co., Ltd.) column was used for elution with 002M phosphate buffer (pH 4.2):acetonitrile:methanol (55:15:30) at 0.8 m/min.
254 nm UV detection was used.

The fractions collected in this way were concentrated at 40°C under reduced pressure, and after removing the organic solvent, the same amount of ethyl acetate as the concentrated solution was added, and carbonic acid was added so that the pH of the aqueous layer was 75 or higher. Adjust by adding sodium hydrogen solution and shake well to obtain the target compound Y.
The L O1641P C+ material was extracted into the ethyl acetate layer. This extraction operation was repeated twice. The ethyl acetate extract was then dehydrated with Glauber's salt, concentrated to dryness under reduced pressure at 40°C, and dried overnight in a vacuum desiccator to obtain a pure powder of the target compound YL01641 P-C.
7 lg was obtained.

The target substance obtained from both fractions eluted at a retention time of 47 minutes and 34 seconds in preparative liquid chromatography: This substance has an Rf value of 028 in the thin layer chromatography described above, and a liquid chromatography using the analytical ram. It is a single object with a retention time of 23 minutes and 33 seconds.

(1) Color and shape: orange-yellow crystalline powder.

(2) Distinction between acidic, neutral, and basic: Basic.

(3) Solubility: Soluble in methanol, ethanol, acetone, ethyl acetate, benzene, toluene, and chloroform, slightly soluble in water, and almost insoluble in hexane.

(4) Ultraviolet absorption spectrum: YL-01641P-
The ultraviolet absorption spectrum of compound B measured in methanol is as shown in FIG.

(5) Mass spectrum (FAB) 774 (M+1
) (6) Molecular formula: C39H51 NO+s, molecular weight: 773.830 (force'H-NMR spectrum:
The 'H-NMR spectrum (500 MHz, CDC 13) of the compound YL-01641P-C is as shown in FIG.

Based on the above chemical properties, this substance is YL-01641 P-C, a substance shown by the following structural formula.

(Effect of the invention) The present invention provides novel and useful microorganisms, and at the same time,
The novel anthracycline antitumor antibiotic of the present invention has cytotoxic effects on various tumor cells. These effects are shown below.

(1) In vitro cytotoxicity test method using tumor cells: LL adjusted to 105 cells/ml
210", P388", 4 μt of a solution of tartrate, a new anthracycline compound at each concentration, dissolved in saline was added to 1 ml of each cell suspension, and the cells were cultured for 3 days at 37°C in a carbon dioxide incubator. , and the surviving cells were counted using trypan pool staining. Cytotoxic activity was determined by calculating the cell proliferation inhibition rate at each concentration using the number of cells without drug addition as a control.
It was calculated by plotting it on the graph.

Medium used: *RPMI-1640+10% newborn calf serum.
*”RPMI-1640+ 5% fetal bovine serum+
5μM 2-Hydroxyethyl disulfide Results: (2) Antitumor activity test method in animals using P388: BDFI/SL of P388 tumor cells 5 x 105 cells.
C. The effect of intraperitoneal inoculation of YL-01641P A+ substance tartrate for 5 days after intraperitoneal inoculation into 5-week-old male mice was as follows. The survival rate was calculated from the ratio of median survival days.

Results: When actually administering the compound of the present invention, parenteral administration using an injection or the like is generally used. It can be mixed with a physically acceptable solid or liquid carrier to form a powder,
It can also be formulated into granules, tablets, syrups, etc. and administered orally.

The general administration method for animals is intraperitoneal injection, intravascular injection into a vein or artery, and local administration, and the dosage is determined based on the results of animal studies and various circumstances. It can be administered continuously or intermittently so that the total dose does not exceed a certain amount. However, the dosage depends on the method of administration, the circumstances of the patient or treated animal, such as age, body weight, sex, sensitivity, diet, time of administration, method of administration, etc.
Of course, the administration can be changed as appropriate depending on the concomitant drugs, the patient, or the severity of the disease. When used as an antitumor agent, the usual dosage of the compound of the present invention is 1 dose per day.
0.30] g//kg to 15 mg/kg per serving
It can be in the range of The appropriate dose and frequency of administration under certain conditions must be determined by a specialist in an appropriate dose determination test based on the above guidelines.

These administration methods are also considered for oral administration.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum of the compound YL-01641P-A. Figure 2 shows the 'H of YL-01641P-AI compound.
- Shows the NMR spectrum. Figure 3 shows the 13C of YL-01641P-A, compound
- Shows the NMR spectrum. Figure 4 shows the ultraviolet absorption spectrum of the compound YL-01641P-. Figure 5 shows the IH-
The NMR spectrum is shown. FIG. 6 shows the ultraviolet absorption spectrum of YL-01641P-B2 compound. Figure 7 shows the 'H-
The NMR spectrum is shown. FIG. 8 shows the ultraviolet absorption spectrum of YL-01641P B+ compound. Figure 9 shows YL-01641P-B, 'H-
The NMR spectrum is shown. Figure 10 shows the '3 of YL-01641P B+ compound.
A C-NMR spectrum is shown. Figure 11 is. YL-01641P-C, shows the ultraviolet absorption spectrum of the compound. Figure 12 shows the 'H of YL-01641P-C, compound
- Shows the NMR spectrum. (No 0

Claims (7)

[Claims] (1) Anthracycline substances or acid addition salts thereof represented by the planar chemical structural formula below. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 means a hydrogen atom or a hydroxyl group, R^2 means a hydrogen atom or a lower alkyl group, and R^3 means a hydrogen atom or a lower alkanoyl group. ) (2) The anthracycline substance or its acid addition salt according to claim (1), wherein R^1 is a hydrogen atom or a hydroxyl group, R^2 is a hydrogen atom or a methyl group, and R^3 is a hydrogen atom or an acetyl group. . (3) The anthracycline substance or its acid addition salt according to claim (2), wherein R^1 is a hydroxyl group, R^2 is a methyl group, and R^3 is an acetyl group. (4) Cultivating a microorganism belonging to the genus Nocardia and having the ability to produce the anthracycline substance according to claim (2) in a medium, producing and accumulating the anthracycline substance in the culture; A method for producing an anthracycline substance, which comprises collecting an anthracycline substance produced and accumulated from a culture. (5) The production method according to claim (4), wherein the strain belonging to the genus Nocardia is Nocardia S.P. YL-01641P (Feikoken Joyori No. 2737). (6) A microorganism belonging to the genus Nocardia and capable of producing the anthracycline substance according to claim (1). (7) The strain belonging to the genus Nocardia is Nocardia S.P. YL-01641P (Feikoken Joyori No. 2737)
The microorganism according to claim (6).