JPS58224698A - Preparation of 2-hydroxyaclacinomycin a and n - Google Patents

Abstract

PURPOSE:To prepare 2-hydroxyaclacinomycin A and N which are anthracycline- type antibiotic substances having antitumor activity, by culturing a microbial strain belonging to Streptomyces genus. CONSTITUTION:A microbial strain belonging to Streptomyces genus and capable of producing 2-hydroxyaclacinomycin A or N, e.g. Streptomyces galilaeus A-862 (FERM-BP No.45), is innoculated in a nutrient medium, and cultured under aerobic condition at 20-40 deg.C, preferably 28 deg.C, and 5-9pH, preferably 7.4pH for 1-5 days, and the objective 2-hydroxyaclacinomycin A and N are separated from the culture liquid and the microbial cells.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides anti-+11I! Jll-related anthracycline antibiotics 2-hydroxyaclacinomycin A and N
Concerning the manufacturing method.

Streptomyces galilaeus MA144-M1 (A
Aclacinomycin A, an antibiotic produced by 'I'CC31133), is an excellent antitumor antibiotic that is highly effective against white plum disease and solid cancer (see US Pat. No. 3,988,315). On the other hand, it was discovered that 2-hydroxyaclacinomycin A, which is one of its related compounds and is represented by the following formula, exhibits an anti-am activity greater than that of aclacinomycin A, and its 6-toxic effect is also MM. The present inventors filed an application earlier (see specification of Japanese Patent Application No. 55-49400).

According to the specification, 2-hydroxyaclacinomycin A is produced by Streptomyces galilaeus MA144-M.
Streptomyces galilaeus M.
A 144-M is added to the culture solution of a mutant 1 strain that is non-antibiotic producing and has glycosidation ability, and is obtained by conversion using this strain (as described in the above-mentioned application specification). Reference) 0 1 The present inventors, in order to further improve the method for producing 2-hydroxyaclacinomycin A using the above two-step fermentation method,
After searching for microorganisms that can be used in the direct fermentation method, we found that Mayuki 2 - A human roxyaclavinone-producing mutant strain and an antibiotic non-producing mutant strain were crossed by protoplast fusion, and from their progeny, 2. - A strain capable of producing hydroxyaclacinomycin A was successfully constructed. The present inventors further discovered that by culturing the strain, 2-hydroxyaclacinomycin A and a reduced compound of the antibiotic, 2-hydroxyaclacinomycin N<tn 106522
We discovered that 2-hydroxyaclacinomycin B represented by the following formula, which has not yet been published in the literature, can be produced, and that this new compound has strong antitumor properties. (Buttocks M 1976-
125826 Particulars No. 8).

The present invention is a two-step fermentation method for 2-hydroxyaclacinomycin A and 2-hydroxyaclacinomycin N, which have antitumor properties and can be expected to be used as an anticancer agent for dairy animals including humans. This is a method to directly manufacture these without relying on

According to the invention, 2-hydroxyaclacinomycin A
Microorganisms belonging to any genus can be used for the production of N and N, as long as they have the ability to produce the compound, but microorganisms belonging to the genus Streptomyces are generally used, especially by the protoplast fusion method described above. Among the strains obtained, for example, Streptomyces galilaeuy,
A-862 may be used to advantage.

Creation of recombinants using the protoplast fusion method of microorganisms is already known in the microbial world, and has been reported for antibiotic-producing actinomycetes, but it has not been reported for Streptomyces galilaeus. There are no examples of the creation of recombinant strains that are applied to crossbreeding between strains with characteristics regarding substance production, that have the seasons of the parent strains, and that produce antibiotics different from the parent strains. This is something that is clarified for the first time by this application.

To construct the 2-hydroxyaclacinomycin A-poor and N-producing bacteria of the present invention by the protoplast fusion method,
Streptomyces galilaeus MA144-M,
Weird result! %-2-hydroxyacrabinone-accumulating fAI strains, such as Streptomyces galilaeus l
0U-29361: ANR58 strain (isolated by the same method as the ANR58 strain described in JP-A-56-49341 (No. 5081)) and an antibiotic non-producing @converting mutant strain, e.g. Streb) Mrs. Galilaeus 11U-1
11 strains [KE- described in JP-A-56-15299]
303 (Imitation Koken Chinyo No. 4808) by the same method as υ
[separated] are crossed, and the recombinants are single-selected.

In order to facilitate the selection of recombinants, we genetically mark the strain with a trophic marker, and select the recombinants that can grow on minimal medium. Select as auxotrophic colony.

Among the non-auxotrophic recombinants thus obtained, strains that produce the desired 2-hydroxyaclacinomycin A and N are selected.

Below, 2-hydroxya9cladinomycin A and N
Detailed Description of Method for Construction and Isolation of Production Strains Two strains having the following characteristics, isolated by mutation treatment from Streptomyces galilaeus MA144-M, were subjected to mating experiments using protoplast fusion.

+10U-2936 strain: ACM, AKN,
2HO-AKN.

Glyc + adc 11U-1111 strain: ACM, AKN,
2) io-AKN.

10-Glyc, ura The above genetic knowledge of one strain is indicated by symbols for convenience, and each represents the following content.

ACM-niacladinomycin non-productive AKN: akravinone non-productive + 2KO-AKN: 2-hydroxyaclavinone non-productive 2HO-AKN"-: 2-hydroxyaclavinone non-productive Glycl": externally added Aclavinone or 2
- Ability to bind sugar to hydroxyaclavinone to produce aclacinomycin or 2-hydroxyaclavinone Glyc: Externally added aclavinone or 2-hydroxyaclavinone
Deficient in the ability to bind sugar to hydroxyaclavinone to produce aclacinomycin or 2-hydroxyaclacinomycin ade -: Requirement for adenine ura -: Requirement for uradil Super cultured platinum loops and seeds, separate sterilized Y8 yeast medium (0.3g yeast extract,
1 Chronic Ti3 Common Starch, pl (7,2,4-/test tube)
and cultured overnight at 28°C. This 0.3- is 30
mt Okanishi et al.'s MSG medium CJ, Gen, Microb
iol, 80:389 (1974)) was inoculated into a 250rne'8 Erlenmeyer flask and incubated at 28°C on a rotary shaker (rotation speed 220/min).
Culture by shaking for 0 hours.

Both culture fluids and υ20- were collected, the bacterial cells were collected by centrifugation, and 1 () ml of P medium (J, Gen.

Microbiol, 80:389t, 19
74)] After washing once with P medium 2Orig containing lysozyme at a concentration of 1.
The disease relapses inside. The reaction was carried out at 28°C for 1 hour, and then a p-applied glass tube (φ15 x 2
The F solution that had passed through 01 mm) was centrifuged at low temperature (upper oooxr, lo sorted), and the collected protoplasts and l1til were suspended in 1.5-〇P medium, and the turbidity (0, D 61JO) is i.

Dilute with P medium as much as possible to prepare a protoplast cell solution for fusion. These two-stall alveolar suspensions were subjected to analysis using 1 colony-forming unit (4.4 x 10 /d for IU-2936ff1 strain Yumai and one derived from IIU-111).

That is, 0.2 - of the mixed protogelatin cell solution was mixed with 1.
8-40% polyethylene glycol (hereinafter referred to as [PEG
(abbreviated as J) was added to P medium containing 4000, mixed and stirred, and then cultured at 28°C for 5 minutes. Then, the mixture was diluted with P medium and plated on a plate consisting of the following regenerated minimal medium and regenerated complete medium supplemented with 100 μf/ml each of adenine and uracil, and cultured at 28° C. for 10 days.

Regeneration minimal medium composition Sucrose 110 f Polyethylene glycol 1 ooo so PK2
So, 0.25
5'Trace element 5solution
2 rM Future KH2PO40,05F MgCt2-61 (,04,06f Glucose 102 L-Asparagine 3f O,1M TES (pH7,4) 100- Make the total amount to 1 t with distilled water.

lk ZnC22”4H2040119, FeC42・
6H20200mf.

CuC62*2H2010my, MnCA254H
2010my.

Na2B40,610) 120 10 1Y l
(N1(4mCMO,O□4)e4H2010～1
Dissolve the solution in distilled water and sterilize it separately.

Approximately 500 colonies generated on the minimal plates are isolated and picked on the following minimal agar slants. After culturing at 28°C for 5 days, add one more line to the same sphere.

Minimum Division Ten Glaze Surface I @ Geological Composition Glucose 102 L-Asparagine 3f KNO31y K, HPo, 0.5 fMg
SO, 7H200, 5f CaC62 2H200, 5f O, IM Trls-HC4 (pal 7.2)
Make the total volume 1 ton with 100 ml distilled water.

From this, one platinum loopful was inoculated into YS liquid medium (described above), and -
Culture with shaking at 28°C overnight. Fermentation medium Fermentation medium composition Soluble starch 1.5 Tyglucose
1. θ % Essan Meat (soybean flour, manufactured by Ajinomoto Co., Ltd.) 3.0% yeast extract
0.2% to 2HP0.
0.1 1MgSO4・7H20θ, 1% NaC6O, 3T mineral 0.125T Cu80
4'5H202,8f, FeSO4*71 (200
,4t +MnC62s4H403,2f , ZnS
The entire amount of 200.8 tons of O4.7H was inoculated into a 250-mL Erlenmeyer flask containing 30-mL dissolved in 500 mg of distilled water, and cultured with shaking on a rotary shaker (described above) for 2 days. To test the product, culture II? l[Collect 5m,
Add chloroform/methanol mixture (3/2) to 5-
Starved, stirred with a mixer, centrifuged to separate and harvest the chloroform extract layer? I# Condensate to dry. Dissolve this in a small amount of chloroform, spot it 1.5σ below the bottom edge of a thin layer of silica gel F (manufactured by Merck & Co., Ltd.), and place
Develop with the solvent system of 110.3), evaluate 2-human xiaclacinomycin A and 2-hydroxyaclavinone as controls, and search for and isolate 2-hydroxyaclacinomycin-producing bacteria. Of the 500 strains tested, 420 strains were non-antibiotic-producing strains, 4 billion strains were 2-hydroxyaclavinone-producing strains, and 301 strains were 2-hydroxyaclavinone-producing strains.

Among the 2-hydroxyaclacinomycin-producing strains thus obtained, Streptomyces galilaeus A-862
One strain, named , is advantageously used for the production of the antibiotic. This strain has been internationally deposited with the Agency of Industrial Science and Technology's Institute of Microbiology and Industrial Technology t5Fe as Copyright Research Article No. 45 (FERMBP-45).

The main pathogenic properties of this strain are those of its sibling Streptomyces galilaeus FvlA144-M (%
There is almost no difference from the above (see Japanese Publication No. 51-34916).

The orchidological properties of strain A-862 are described below.

All of the υ-shaped Akumoto strains elongate aerial hyphae with clearly branched, basal, straight, hook-shaped to spiral formations. Especially on the starch inorganic salt agar medium, the aerial mycelium was observed to have a concentric to spiral shape.

Whorled branches are not allowed.

A mature spore chain is a chain of 10 or more spores, the size of the spores is 0.4 to 1.6 mm, and the surface of the spores is smooth. No aerial mycelium is formed on many media, only yeast, malt agar and starch.
Although a spore system was formed on an inorganic salt agar medium, no settlement of mature spores was observed.

■ Regarding the description of growth status colors in various media, the symbols in [ ] are based on the Color Harmony Manual of Container Corporation 6 Op.
Co1ar harmo on of America
nymanual), and the color standards of the Japan Color Research Institute were also referenced and described.

(1) Sea rose/nitrate agar medium (cultured at 27°C) Growth is colorless to pale yellow (2 db), no aerial mycelia are attached, and no water-soluble pigments are observed.

(9) Glucose and aspartic acid growth (27°C culture)
Growth is bright orange-yellow (3ea) to light brown [4ie]
Almost no aerial mycelium was observed, and a slight brownish water-soluble pigment was observed.

(3) Glycerin φ asparagine medium (ISP-
Culture medium 5.27°C) Growth is pale yellow (2 db) to yellowish brown, almost no aerial mycelia are attached, and no water-soluble pigments are observed.

(4) Starch/inorganic salt agar medium (IEliP-medium 4
゜27℃ culture) Pale yellow (2 db) to bright gray on the development of yellow tea [d]
Aerial mycelium gradually appears, and no water-soluble pigments are observed.

(5) Tyrosine agar medium (l5P-medium 7.27℃ culture) growth fi bright olive tea C2ge) ~ Hojo [4t
g) The a-type does not adhere and the water-soluble pigment exhibits a brown color.

(6) Nutrient agar medium (cultured at 27°C) Yellowish gray (2 da) to light gray (
Aerial cocoon filaments (d) are deposited, and a slight brown water-soluble pigment is observed.

(7) Yeast Mugami Kanten Soji (Is)? -Medium 2,2
7℃ culture) Bright orange-yellow (3ea) to bright brown [: 4ie]
On the growth, yellowish gray (2 dc) aerial mycelium was produced.
Water-soluble dyes exhibit a red color.

(8) Oatmeal agar medium (15P-medium 3.27℃
Cultivation) The appearance is yellowish brown to pale yellow (3ec), almost no air 1d filaments are attached, and the bathing pigment is slightly brown.

3) Physiological properties There are no differences between the parent strain and this strain in terms of physiological properties such as gelatin decomposition, melanin pigment production, starch decomposition ability, peptonization of skim milk, and carbohydrate utilization. It is as follows.

+l) Growth temperature dry curve: Maltose/yeast extract agar (maltose 1, oqb,
Yeast extract (manufactured by Oriental Yeast Co., Ltd.) o, 4%
, agar 2.0%, pH 6.0) at 24°C.
As a result of examining the growth at each temperature of .degree. C., 27.degree. C., 30.degree. C., 37.degree. C. and 50.degree. C., it grew at all mi except 50.degree. C., but the optimum temperature was around 27.degree. C. to m.degree.

(2) Liquefaction of gelatin (glucose, peptone, gelatin, cultured at 20°C): Liquefy.

(3) Hydrolysis of starch (starch/inorganic salt hemorrhoids, 2
7°C culture 51): Decompose.

(4) Coagulation and peptonization of skim milk (cultured at 27°C): Peptonization occurs, but no coagulation is observed.

(5) Production of melanin-like pigments (tryptone, yeast,
Broth + l5P-medium 1; peptone, yeast, iron agar, l5P-medium 6; tyrosine agar, l5P-medium 7. (Culture at 27°C): Production of melanin-like pigments is observed in all media.

(6) Availability of carbon sources (Pridham-Gottlieb agar, l5P-medium 9.27°C culture): L-75 binose, D-xylose, glucose, D-7 lactose, sucrose, inositol, L-rhamnose, raffinose Take advantage of. D-Manni) - #c Do not use.

In order to obtain 2-hydroxyaclacinomycin B by the method of the present invention, the 2-hydroxyaclacinomycin B bioma strain is first grown in a medium containing known nutritional sources that can be utilized by a healing imitator. Cultivate using a known method. For example, carbon sources that can be used include glucose, glycerin, #sugar, starch, maltose, animal and vegetable oils, and nitrogen sources that can be used, such as soybean flour, meat extract, yeast extract, peptone, cornstap liquor, and cottonseed meal. Inorganic nitrogen such as ammonium sulfate, ammonium chloride, ammonium nitrate, and ammonium phosphate can be used. In addition to cooling inorganic salts such as salt, potassium chloride, phosphates, other heavy metal salts, and vitamins as necessary, silicone (manufactured by Shin-Etsu Chemical, Inc., An antifoaming agent such as KM75 (trademark) can be added as appropriate.

Fermentation conditions such as aeration agitation and fermentation time are selected such that the fermentation used accumulates the maximum amount of the compound. For example, fermentation conditions such as 20-40
℃, preferably 28℃ and a pH of 5 to 9, preferably 7.4, for 1 to 5 days, preferably 3 days.'
is advantageous.

2-Hydroxyaclacinomycin A and N can be produced from the fermentation culture by conventional means in the art for the production of anthracycline antibiotics, such as by centrifugation, or by using suitable quartz clay. Like! After the culture solution is filtered in the presence of a filter aid and separated into bacterial cells and liquid f, acetone is extracted from the bacterial cells.

Extraction can be performed using an organic solvent that is miscible with water, such as methanol, ethanol, or butanol, and rp* can be extracted using an organic solvent such as chloroform, ethyl acetate, etc., but these solvents can be selected as appropriate. To do...
Therefore, it is also possible to directly extract the bacterial cells from the nutrient solution without pre-fractionating them. In the thus extracted organic solvent, one active ingredient is concentrated and dried under reduced pressure, and then the desired chemical compound is added. In order to isolate substances, adsorbents such as silica gel, activated carbon, alumina, acidic or weakly basic ion exchange resin, Sephadex LH-20 are used.
(manufactured by Pharmacia: i[l) or other gel filtration agents such as force chromatography, preparative silica gel thin layer chromatography, liquid chromatography, countercurrent distribution, etc. alone or in combination as appropriate. This can be done to great advantage.

The compounds 2-hydroxyaclacinomycin A and N of the present invention thus obtained can form addition salts with various types of 2-hydroxyaclacinomycin A and N.

That is, acid addition salts of 2-hydroxyaclacinomycin A and N can be obtained by a known method used for salt formation of free bases. For example, it can be obtained as an addition salt of hydrochloric acid, sulfuric acid, citric acid, succinic acid, tartaric acid, zumaru, glutamic acid, pantothenic acid, laurylsulfonic acid, benzenesulfonic acid, naphthalenesulfonic acid, and the like. - A convergent method for forming salts is a freeze-drying method in which the free bases of 2-hydroxyaclacinomycin A and N are reacted with the above-mentioned acids in a suitable solvent, or
The compounds 2-hydroxyaclacinomycin A and N of the present invention can be used for the growth of murine leukemia cultured cells (L1210) and for nucleic acid recovery. Significantly inhibits synthesis. 91'' was inoculated with L1210 cells at 5 x 10 cells/me into RI) MI 1640 medium containing 20% calf serum (Rothwell Burke Laboratory 164 (1)), and at the same time, the compound of the present invention was inoculated at 0.01 to 0. It was added at a concentration of .25 μt/me and cultured at 37°C in a carbon dioxide gas incubator to determine the 50% growth inhibitory concentration relative to the control.The above cultured L1210 cells were further cultured in RPMI 1640 crucible containing 10% calf serum. 5 x 10 to the ground?
After 1 to 2 hours of culture in a vertical cough incubator at 37°C, the compound of the present invention was added at various concentrations, and 15 minutes later, 140-uridine was added. (0
,05μCi/d) and 14C-thymidine (0,05μCi/d)
μct hawk) was added and cultured at 37°C for 60 minutes. Add 10%) IJ chloroacetic acid solution to the reaction solution to stop the reaction, and at the same time precipitate the acid-insoluble matter. After washing three more times with 10-5 dichloroacetic acid, dissolve in formic acid and remove the acid-insoluble matter. The effect on experimental tumor animals was determined by measuring the radioactivity in the cells and calculating the degree of 50% uptake inhibition from the radioactivity uptake rate in the control group. 24 hours after transplantation, 2-human p-xiaclacinomycin A and N were intraperitoneally administered for 10 days, and the survival rate calculated compared to the control group (physiological saline administration) and the anti-L1210 leukemia + that of 2-hydroxyac2dinomycin A and N with respect to tumor efficacy and toxicity and the known antitumor properties.
$75! Table 1 also shows that of L-aclacinomycin A.

From the results shown in Table 1, 2-hydroxyaclacinomycin A and N of the present invention kill the proliferation of mouse leukemia L1210 cells at low concentrations, and exhibit an excellent survival effect on leukemia cell-transplanted mice. In addition, it has much lower toxicity than known anthracycline antibiotics such as adriamycin or daunomycin, and is the least toxic of the anthracycline antibiotics. Aclacinomycin was developed through research as an anti-pulmonary agent with low cardiotoxicity. It has lower toxicity than Aclacinomycin A, and has the same or better antitumor effect, and the dark wrinkle area showing antitumor effect is approximately twice that of Aclacinomycin A. It has excellent properties as an anticancer drug.

Furthermore, with respect to nucleic acid synthesis, which is considered to be the direct point of action of anthracycline antibiotics, both of these substances are effective against RN.
It has the characteristic of highly specific inhibition of A synthesis, and its inhibitory action is similar to known Aclacinomycin Z and rhodomycin group antibiotics.

The present invention will be explained in more detail below using examples.

Example 1 Soluble starch 1.0%, Glucose 1.0%, Nissanmeat (soybean flour, manufactured by Ajinomoto Co., Ltd.) 0.1%, vi Mother Kiss 01%, K, HPo, 0.1%, MgSO4
・Dispense 100-ml of seed medium consisting of 7H200.1% and 0.3% NaCA into 15 killed 500-capacity flasks and add Streptomyces galilaeus A-86.
2 (@Koken Joyori No. 45) was inoculated one platinum loop at a time, and cultured with shaking at 28°C for 48 hours to obtain 14 seeds.
1! I did it.

2 t glycerol, 3% nissanmeat, 0 yeast extract
．． 2%, K2HPO40,1% + MgSO4”7
H200, 1%, NhCtO, 3% + CuSO4
'5H,10o,ooo7%, FeSO4・7H,O
O,0001%.

MnC62.4H, 00,0008% and ZnSO4
- 15 tons of fermentation medium consisting of 200,0002% 7H (pH 7.4) was dispensed and collected into five 301-volume jars and 7 amentors, each of which was mixed and inoculated with the amount of the above-mentioned 3 flasks. Culture was carried out for 3 days with aeration Ik1/2 m and rotation speed 300/min. The culture solution was mixed and separated into bacterial cells and supernatant by centrifugation.

The supernatant was extracted with stirring by adding 20 t of chloroform, and the chloroform layer was separated. On the other hand, the bacterial cell components were extracted with 30 tons of acetone, concentrated to dryness under reduced pressure to reduce the acetone layer to about 1/3 of its volume, further extracted with 5 tons of chloroform, mixed with the chloroform extract layer from the supernatant, and concentrated to dryness. An oily crude extract containing 2-hydroxyaclacinomycin A and N was obtained.

Example 2゜The crude extract obtained in Example 1 was mixed with ammoniacal methanol (4
After dissolving the insoluble matter in methanol (adding 0.4 mm of concentrated ammonia water) and removing insoluble matter by centrifugation, it was further quenched.
Sephadex LH divided into 4 doses of 100-100 l-1 time
-20 column (φ4.OX 35cnI) and eluted with the above ammoniacal methanol. The first eluted red pigment fraction was collected and condensed to dryness to obtain a crude component containing 2-hydroxyaclacinomycin A, B and N.

Next, this crude component was dissolved in a small amount of chloroform, applied to a thin layer of preparative silica gel 60 PFxsa (manufactured by Merck & Co., Ltd.) in powder form, and mixed with chloroform/methanol (10/t).
). 2-hydroxyaclacinomycin components A, B and N are approximately 0.5 and 0, respectively.
．． The Rf values were around 7 and 0.2. Scrape off each and add chlorotetraform/methanol/concentrated ammonia water (4
0/to/o, t) mixture was eluted and dried.

Next, for A, B and N components, using the same preparative silica gel 60 PF254 thin layer, chloroform/
Rechromatography was performed by developing with a solvent system of methanol/acetic acid (i5o/15/1). Classes A, B, and N were extracted with persimmons and chloroform/methanol (5/1) mixture, and each was dried into mm. 0.2M vinegar for each dry item! The solution was dissolved in a 20-mL solution of 13.5-mL E-Run solution (p), and the remaining insoluble matter was removed by centrifugation, and then washed by shaking and mixing with a 10-mL toluene solution. Water layer 4
The mixture was neutralized with N sodium hydroxide solution and extracted with chloroform. The chloroform extract was mixed with 0.01 M EDTA (
After washing at pH 7.2>, washing with water and drying with Glauber's salt, the mixture was vacuum-treated using a III-sieve filter. An excess of n-hexane was added to the concentrated solution to form a yellowish brown precipitate, which was collected by filtration and vacuum dried to give 2-hydroxyaclacinomycin A and N of 61 to 24N, respectively! The physicochemical properties of the obtained 2-hydroxyaclacinomycin A and N are shown below. Analysis C4211 (C as 58N 01g) I
NO Calculated value (%i 60.93 6.45 1.69 3
0.93Experiment I01"60.276.201.64-Specific optical rotation (a)D' + 42-3°(c O,
04, MeOH) UV-visible absorption spectrum 295 (207), 450 (110) external absorption spectrum (KBr tablet) ctn3450.2975.
2940.1735.1675.1620.

1610, 1450, 1400, 1380, 1
300, 1255.

1230, 1170, 1120, 1010 boutons nuclear magnetic resonance spectrum (100MHz, CDCl2 CDaOD) δ,
PF11 2.20 (6)1. ! 1. N(CHs)i )3
．． 65 (3H,s, C00C)11 )6.20
(IH, d, J=2Hz, ArH)6.70 (I
H, d, J=2Hz, ArH)7.30 (IH,
s, ArH) 2-hydroxyaclacinomycin N proton nuclear magnetic resonance spectrum (100 MHz, eDct, -CD30D) δ,
pP11 2.20 (6H,s, N(CHg)2) 3.67
(3M, g, C00CI(s)6.56 ([4,
d, J=2Hz+ ArH)7.19 (H1+ d
, 2Hz, ArH)7.57 (IH,s, Ar
H)

Claims (1)

[Scope of Claims] 1. A microorganism having the ability to produce 2-hydroxyaclacinomycin A represented by the formula and 2-hydroxyaclacinomycin N represented by the formula belonging to the genus Streptomyces is cultivated in a nutrient medium. A method for producing 2-hydroxyaclacinomycin A and N, which comprises recovering the compound from a culture of kernels. 2. The microorganism belonging to the genus Streptomyces is Streptomyces galilaeus.