JPS6053033B2 - New anticancer antibiotic mazethramycin and its production method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a novel anticancer antibiotic, Mazethramycin A., which is obtained by culturing microorganisms belonging to the genus Streptomyces and collecting the cultured product. ．．
B. ．． C and anhydromazethramycin (hereinafter, these new compounds will be collectively referred to as mazethramycin compounds or simply mazethramycin).

The present invention also relates to a method for producing these mazethramycin compounds. According to the inventors, Showa 4
In October, an actinomycete isolated from soil in Niijima, Tokyo, was identified as Streptomyces thiorteus.
! When the 4E561-14 strain is cultured, mazethramycin is produced and accumulated in the culture, and by collecting mazethramycin from the culture, new anticancer antibiotics mazethlamycin A, . B. ．． It has been found that C and/or anhydromazethramycin can be produced.

Mazethramycin A according to the invention, . B. sC and anhydromazethlysin are recognized as compounds having the chemical structure shown below, and in a solution dissolved in an appropriate solvent, they react with a solvent compound as shown in the reaction formula below, thereby causing mutual interaction. It is a compound that is easily converted into

That is, when mazethlamycin A is refluxed in a nonpolar solvent, it is dehydrated to become anhydromazethlamycin.

Further, when anhydromazethramycin reacts with water in a water-containing solvent, it is easily converted to mazethlamycin A. The unstable mazethlamycin A or anhydromazethramycin easily becomes stable mazethlamycin B when reacted with methanol in a methanol-containing solution, and stable mazethlamycin C when reacted with ethanol in an ethanol-containing solution. becomes. Therefore, mazethramycin compounds are mostly present in aqueous culture medium! It is thought that mazethlamycin exists as mazethlamycin A, but if an alcoholic solvent is used during extraction or purification to collect the mazethlamycin compound, it can be collected as stable mazethlamycin B or mazethlamycin C. preferable. Mazethramycin AlB. ．． C and anhydromazethramycin both exhibit antibacterial activity against bacteria and fungi, and especially against murine leukemia L-1.
It is an anticancer antibiotic that strongly suppresses the growth of 21E cells and/or cancer cells, and there is no essential difference in their effects. Therefore, each suitable mazethramycin compound can be used as an anticancer agent as well. Therefore, according to the first aspect of the present invention, the following general formula [wherein... A... is a group -].

An anticancer antibiotic mazethramycin compound is provided, which is a compound represented by the following formula: monoene or a group -N=CH, where R represents a hydrogen atom or a lower alkyl group. In the compound of general formula (1)... A... is a group K, L
When IU is a hydrogen atom and R is a hydrogen atom, this compound is mazethlamycin A.

In the compound of general formula (1)...A... is a group,
,, above,, and when R is a methyl group, in the compound of general formula (1)... A... is a group -,,
When R is ethyl and R is an ethyl group, the compound is mazethlamycin C. Furthermore, in the compound of general formula (1), when A... is a group -N=CH-, this is anhydromazethlamycin, which is an anhydro form and is represented by the following formula (■). .

The physiochemical properties of mazethramycin, a new anticancer antibiotic according to the present invention, are as shown below.

(1) Mazethramycin A is a pale yellow powder with a melting point of 181~
19.3 SuC (disassembly), [α] Eup 1+7301 (CO.
(O62, dimethylformamide), the ultraviolet absorption spectrum curve is as shown in FIG.

λ recognition ξCNTrL, P (ε): 320 (shoulder 34600)
, 335 (39400).

The infrared grade yield spectrum curve measured with potassium bromide tablets is the second
As shown in the figure. Elemental analysis is experimental value: C62.3
5%, H5.72%, Nl2.82%, 018.99%
, theoretical value (Cl7Hl9N3O4): C6l. 99%,
The H content was 5.82%, the Nl content was 2.76%, and the content was 19.43%. No molecular peak was observed in the high-resolution mass spectrum, but a dehydration peak was observed. The nuclear magnetic resonance spectrum measured with a deuterated dimethyl sulfoxide solution is compared with that of Mazethramycin B described below: -0CH3 signal (δ 3.44 ppm)) disappears, δ 5.09 ppm (singlet) and δ 4.83 ppm (tablet) A new signal (1H) was observed.

This means that the -0CH3 group in Mazethramycin B is -
Converted to 0H group to indicate the presence of epimer (approximately 50%).
(Ii) Mazethramycin B is yellow needle-like crystals that do not show clear dots and decomposes around 245 to 270 tons. The specific rotation is [
A value of α]Ba3+9000 (CO.2, dimethylformamide) was obtained. Elemental analysis is experimental value: C63.38%, H
6. l8%, Nl2.4O%, 018.19%, theoretical value (Cl8H2lN3O4): C62.96%, H6. l
6%, Nl 2.24%, and 018.64%.

It is soluble in methanol, ethanol, butanol, acetone, ethyl acetate, acetonitrile, and chloroform, but sparingly soluble in butyl acetate, benzene, and ethyl ether. The color reaction is a fast blue B reaction, which results in a brick-red color. Ehrrich reaction, Sakaguchi reaction, and Landon-Smith reaction were negative. When left on a thin layer of silica gel for about one hour, it develops a brown color. Chloroform-methanol (1
0:1), Rf is 0.21.
The ultraviolet absorption spectrum curve (5 μHmL) is as shown in FIG. 3, and a shift toward longer wavelengths is observed in the alkaline solution. The maximum absorption is 2 in 1% methanol solution.
15rrL,μ(ε25600), 235mμ(ε22
200) and 3347TLμ (E461OO). In 1% methanol solution containing 0.1N sodium hydroxide, 2587nμ (shoulder ε17200) and 351T
rL,μ(ε43400).

The infrared absorption spectrum curve of Mazethramycin B measured with potassium bromide tablets is as shown in Figure 4, and 3350
, 3120, 2950, 1660, 1565 to 16301161, 1515s146 飄 1410, 1370, 1
11 to 345, 1315s1250, 1220, 117
It has main absorption bands at 45, 1070, 1025, 990s955.694 to 910, wings 0, 85\82 to 7600-1. The nuclear magnetic resonance spectrum measured with a heavy dimethyl sulfoxide solution is shown in FIG. Mazethramycin B was determined from its ultraviolet absorption spectrum, infrared absorption spectrum, and nuclear magnetic resonance spectrum as anthramycin (Journal of American Chemical
Society, Vol. 87, pp. 5791-5795, 196 details). The double resonance method in nuclear magnetic resonance spectra revealed that the acrylamide side chain of anthramycin ● methyl ether is N-methyl (
It is estimated that this is a compound with an E of 2.66 ppm). Furthermore, the demethanol peak characteristically seen in the mass spectrum of anthramycin methyl ether was also observed in the high-resolution mass spectrum of mazethramycin B, and the acid hydrolyzate of mazethramycin B (1N hydrochloric acid, heated under reflux for 2 hours) Mazethramycin A, . It was confirmed that B and C each have the following structures. Mazethramycin A
:R=H Mazethlamycin B: R=CH3 Mazethlamycin C: R=-CH2CH3l (Ii) Mazethlamycin C is a pale yellow crystalline powder with a melting point of 216-2
23 MaC (disassembly), [α] Bas 1 + 4500 (CO.O6
7, dimethylformamide).

The ultraviolet absorption spectrum curve is as shown in FIG.
λ? ^ICNwl. μ( )E: 217 (25700)
, 2352 (shoulder 19300), 333 (43600). The infrared absorption spectrum curve measured with the potassium bromide tablet is as shown in FIG. Elemental analysis is experimental value C6
3.25%, H6.53, 12.25%, 015.83
%, theoretical value (Cl9H23N3O4): C63.85%
, H6.48%, 2N 11.76%, and 017.91%.

The nuclear magnetic resonance spectrum measured with a deuterated dimethyl sulfoxide solution showed a methyl group signal (-0CH2-, δ3.1-3.6ppm) compared to that of mazethlamycin B.
:-CH3, δ1.15 ppm) was observed. (I
v) Anhydromazethramycin is a pale yellow crystal, melting point 252-267C (decomposed), [α]f+19405 (
C.O. O5, dimethylformamide).

The ultraviolet absorption spectrum curve is as shown in FIG. λ Cape FNWl, μ (ε): 229 (16100), 2
35 (shoulder 15800), 298 (shoulder 19300), 31
5 (21800) and 352 (21100). The infrared absorption curve measured with the potassium bromide tablet is as shown in FIG. The experimental value for elemental analysis is C65. O4%, H6. l
O%, Nl3. O4%, 016.38%, theoretical value (Cl
7Hl7N3O3): C65.58%, H5.5O%,
The Nl content was 3.5% and 0.15%. A molecular peak (experimental value: 311.12, calculated value: 311.126) was observed in the high-resolution mass spectrum.

The nuclear magnetic resonance spectrum measured with deuterated dimethyl sulfoxide solution was compared with that of mazethramycin B, and -0CH
The signal of 3 (δ3.44 ppm) disappeared, and the signal of azomethine (δ8.15 ppm) was observed. It was confirmed that anhydromazethramycin is a dehydrated form of mazethlamycin A having the above structure. Furthermore, when anhydromazethramycin was dissolved in a lower alcohol such as methanol, ethanol, propyl alcohol, butanol, it was confirmed from the change in the ultraviolet absorption spectrum that it became an alcohol adduct.

However, alcohol adducts other than mazethramycin B and C, which are methanol and ethanol adducts, are unstable, and when dried by heating (approximately 50° C.) under reduced pressure, it was found that they reverted to anhydromazethramycin. Mazethramycin A. ．． B. ．． The minimum inhibitory concentrations for various test bacteria on nutrient agar for each of C and anhydromazethramycin are shown in Table 1.

Mazethramycin A. ．． In order to examine the therapeutic effect on leukemia in mice B and C, 1 CP/kg was injected into the peritoneal cavity of the mice.
After transplantation of L-1210 cells at the rate of mice, mazethramycin A. ．． B. ．． When each of C was administered by intraperitoneal injection at the doses shown in Table 2 for 10 consecutive days, the survival effects shown in Table 2 were shown.

However, the life extension rate was calculated using the following formula.

mazethramycin A,. The acute toxicity of BNC and anhydromazethramycin was determined by the LI when administered intraperitoneally to mice as a 10% aqueous methanol solution.
50 value is 0.8m91k9.

Mazethramycin A according to the invention, . B. ．． It is recognized that the biological properties of C. C. and anhydromazethramycin do not show any essential differences. According to the second gist of the present invention, a mazethramycin compound-producing bacterium belonging to the genus Streptomyces is aerobically cultured in a medium containing a nutrient source, and the culture has the following general formula (1). [In the formula,...A... is a group ,, on u.

or a group -N=CH-, where R represents a hydrogen atom or a lower alkyl group], and the mazethlamycin compound is collected from the culture, according to the general formula (1). Provided is a method for producing an antibiotic mazethramycin compound represented by: An example of the mazethramycin compound-producing bacterium used in the second invention is Streptomyces thiorteus ME56l-14 strain.

The mycological properties of this ME56l-14 strain are as follows. Under the microscope, the ME56l-14 strain of the first form elongates aerial hyphae with whorled branches rather than a branched basal fungal system, but no spiral formation is observed.

A mature spore chain consists of one or more spores, the size of the spores is about 1.0 to 1.2 x 0.4 to 0.5 microns, and the surface of the spores is smooth. 2 Regarding the description of growth status colors in various media [ ] The standard shown in brackets was the Color Harmony Manual of Container Corporation of America.

(1) Sucrose/nitrate agar medium (27°C culture)
White aerial mycelium grows on the colorless growth, and no soluble pigment is observed.

(2) Glucose-asparagine agar medium (2rC culture) Colorless to pale yellow to dark yellow [1 volume E, . Antique
On the growth of white to yellowish gray [1Cb1Parc]
hment-2cb. , Iv0ryTint], the soluble pigment is slightly yellowish.

(3) Glycerin/asparagine agar medium (ISP-1 medium 5.2 rC culture) Light yellow to light yellow brown [3 ng. ．． Y
e110wMapIe] ~ Yellow tea [3pi,. G0Idn
Br0wn~4pi0akBr0wT1],
White to yellowish gray [1ba1Ye110wTint-Additional, Pe
It grows on argy aerial mycelium, and the soluble pigment is brownish.

(4) Starch/inorganic salt agar medium (ISP-1 medium 4.2
rc culture) colorless to light yellow tea 0mg, YellowMap
le], white to yellowish gray [2cb, . Iv0ry
Tint] aerial mycelia were attached, and the soluble pigment was 15% after culture.
It takes on a slight yellowish tinge from the first day onwards.

(5) Tyrosine agar medium (ISP-1 medium 7, 2rC culture) Light yellow tea to yellow tea [2pi-2r1i, . Musta
rdBr0wn]~Dark yellow brown [3pi1DeepBr0
On the growth of white to yellowish gray [1ba, . Ye110
wTint~2ba,. Pear 1) aerial mycelia are attached, and the soluble pigment is yellowish to brownish.

(6) Nutrient agar medium (2TC culture) Light yellow to light yellow tea [3 ng, . Ye110wMap1
e], white aerial mycelium grows on it, and the soluble pigment is yellowish.

(7) Yeast/malt agar medium (ISP-1 medium 2.27
°C culture) Light yellow tea to yellow tea [3ni. ．． C10veBr
0wn], white to yellowish gray [1cb. sParc
hmet~2cb. ．． Aerial mycelia of Iv0ryTint] are attached, and the soluble pigment is slightly brownish.

(8) Oatmil agar medium (ISP one medium 3.2TC culture) On the growth of light yellow to light yellow brown, white to yellowish gray [2 cb
．． Aerial mycelia of Iv0ryTint] are attached, and the soluble pigment is yellowish.

(9) Glycerin/nitrate agar medium (2rc culture) On the colorless to pale yellow growth, white to yellowish gray aerial mycelia grow loosely, and no soluble pigment is observed.

[Phase] Starch agar culture (culture at 27°C) colorless to light yellowish brown [3r1g. ．． White to yellowish gray [2cb, . Aerial mycelia of Iv0ryTint] were attached, and the soluble pigment became slightly yellowish from about 15 days after culturing.

(11) Malate lime agar medium (2TC culture) White to yellowish gray [1ba1Ye110wTint
Aerial hyphae (like Pearl) are attached, and no soluble pigment is observed.

(12) Simple gelatin puncture culture (20C culture) Growth is light yellow to light yellow brown, aerial mycelia are attached from around 14 days after culture,
It has a yellowish gray color.

The soluble pigment becomes slightly yellowish from around the 14th day after culture. (13) Glyose/peptone/gelatin puncture culture (2rC culture) Aerial mycelia of yellowish ash grow in layers on the growth of nibu-yellow to light yellow-brown, and the soluble pigment has a yellowish tinge.

(10 Skimmed milk (3rC culture) White to yellowish gray aerial mycelium grows on the pale yellow to dark yellow growth, and the soluble pigment has a yellowish color.

(15) Cellulose (2rc culture) Growth is colorless, no aerial mycelia are attached, and no soluble pigments are observed.

3 Physiological properties (1) Growth temperature range Starch yeast agar (1.0% soluble starch, 0.2% yeast scratches, 3.0% string agar, pH 7.0) was used at 20℃, 24℃. C127℃, 30℃, 3rc150
As a result of tests at various temperatures of .degree. C., it grows at all temperatures except 50.degree. C., but the optimum growth temperature seems to be around 2rC to 30.degree.

(2) Liquefaction of gelatin (15% simple gelatin, 20'C
Culture: glucose, pebtone, gelatin, 27°C culture)
In the case of simple gelatin, liquefaction of gelatin is observed from about the fifth day after culturing, but the effect is moderate to weak.

In the glucose-peptone-gelatin medium, no liquefaction was observed even after 3 weeks of culture. (3) Hydrolysis of starch (starch/inorganic salt agar and starch agar, both cultured at 2rC) Water decomposition is observed from about 10 to 14 days after culture, but its effect is extremely weak.

(4) Coagulation and peptonization of skimmed milk (skimmed milk, 37℃
Culture) Coagulation is completed on the 3rd day after culture, and post-peptonization begins, and peptonization is almost completed on the 10th day after culture.

Both coagulation and peptonization have strong effects. (5
) Production of melanin-like pigments (tryptone, yeast, prosthesis, ISP-1 medium 1; peptone, yeast, iron, agar,
(ISP-1 medium 6; tyrosine agar, ISP-1 medium 7; both cultured at 2rC) No production of melanin-like pigment was observed in tryptone yeast prosthesis, and in the case of peptone yeast iron agar and tyrosine agar, no melanin-like pigment was observed. It shows only a faint brown soluble pigment and is probably negative.

(6) Utilization of carbon sources (Pridham-Gotlieb agar,
ISP-1 medium 9, 2rC culture) Growth utilizes glucose, and it was determined that inositol is probably utilized, L-arabinose, D-xylose, D-fructose, sucrose, L-rhamnose, raffinose, D
-Do not use mannitol.

(7) Dissolution of malate lime (malate lime agar, 2rC
Culture) No dissolution of lime malate was observed.

(8) Nitrate reduction reaction (peptone water containing 1% sodium nitrate, ISP-1 medium 8, 2rC culture) Negative.

To summarize the above characteristics, strain ME56l-14 belongs to the genus Streptomyces, the hyphae have whorled branches, no spiral formation is observed, and the surface of the spores is smooth.

Growth on various media is light yellow to light yellowish brown to yellowish brown, aerial mycelia are generally yellowish gray, and soluble pigments are colorless to yellowish to brownish. Melanin-like pigments are negative, proteolysis is moderate to strong, and starch water decomposition is extremely weak. When searching for known bacterial species based on these properties and the fact that this strain produces aureothricin, ME56l was found.
The species most closely related to strain -14 is Streptomyces thioltheus [StreptOmycesthiOlute].
us document 11ntemati0na1J0ur'Rla
lOfSystematicBacteriOlOgy
Volume l22, page 362 (1972): and Reference 2:'Nl
eJapaneseMedicaIJOwnal, 1
Vol., p. 512, (1948)]. Next, we actually obtained Streptomyces thiortaeus ISP5O2 and directly compared it with the ME564-14 strain. A summary of the comparative results is shown in Table 3 below. As mentioned above, Streptomyces Thiorteus ISP5O2
The strain ME56l-14 does not form aerial hyphae, and its morphological characteristics were unknown, but according to the literature, it forms white or yellowish white aerial hyphae with whorled branches, which is similar to strain ME56l-14.

On the other hand, the ME56l-14 strain is different from the Streptomyces thiorteus Isp5On strain in terms of the reduction reaction of glucose, peptone, gelatin, and nitrate, but they are in good agreement in other respects.

Therefore, the ME56l-14 strain was transformed into Streptomyces thioluteus (StreptOmycesthiOluteus).
us) ME56l-14.

This ME56l-14 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on November 27, 1976, under the name Takko Streptomyces ME56l-14, and has since been deposited as Microbiological Research Institute No. 3825. It has been done.

Actinobacteria tend to undergo mutations both artificially and naturally, but Streptomyces thiortheus ME5 as referred to in the present invention
The 6l-14 strain includes all of these mutant bacteria.

The mazethramycin compound-producing bacterial species referred to in the present invention includes all strains that produce mazethlamycin compounds and cannot be clearly distinguished from the present strain ME561-14 and its mutants. In carrying out the method according to the second aspect of the present invention, mazethlamycin compounds, particularly mazethlamycin A Obtain a culture medium containing. As a nutrient source for the culture medium, any known nutrient source used as a nutrient source for actinomycetes can be used.
For example, glucose, maltose, dextrin, starch,
Lactose, sucrose, galactose, glycerin, soybean oil, etc. can be used as carbon sources. Examples are shown in Table 4. Peptone 0.75%, meat scratches 0.75%, N
aClO. 3%, CaCO3O. 32%, MgSO4・
7H200.1%, CUSO4I5H2OO. OOO5
6%, FesO4●7H200.00008%, MnC
l24H2OO. OOO64%, ZnSO4・7H20
A medium containing 0.00016% was used as a basal medium, and 125 ml of the medium to which the above carbon source was added at the concentration shown in Table 4 below was dispensed into a 500 ml volume Sakaro flask,
Autoclave sterilize at 120'C for 20 minutes and cool.
When inoculated with spores and hyphae from a culture of Streptomyces strain ME56l-14 and cultured aerobically with shaking at 2rC, culture 3
The production amount of mazethramycin compound on day 4 is as shown in Table 4. As mentioned above, any carbon source can be used to produce these compounds, but glycerin and glycol are particularly preferred carbon sources.

As a nitrogen source, all known nitrogen sources used as a nutrient source for actinomycetes can be used for the production of mazethramycin compounds.

For example, peptone, meat scratches, yeast scratches, soy flour, soybean meal, cornstarch liquor, cottonseed flour, fish meal, casamino acids, NZ-amines, etc. can be used.
Shown in the table. Glucose 1%, starch 1% NaC as above
lO. 3%, CaCO3O. 32%, MgSO, 7H2
OO. l, CUSO4・5H200.00056%, F
esO4・7H200.00008%, MnCl24H
2OO. OOO64%, ZnsO4●7H200.00
Using a medium containing 0.016% as a basal medium, add a nitrogen source to the concentration shown in Table 5 below to sterilize the medium, and add the spores or hyphae grown in the liquid medium to this. Table 5 shows the production amount of the mazethramycin compound when the cells were inoculated and cultured with shaking for 3 or 4 days. As mentioned above, any nitrogen source can be used, but meat scratches and peptone are particularly preferred nitrogen sources.

Trace amounts of inorganic salts, metal salts, and heavy metal salts are added if necessary to produce the mazethramycin compound. Also, when defoaming is required during culturing, silicone resin, soybean oil,
Antifoaming agents such as Adekanol can be added and used. Liquid culture is preferred for mass production of mazethlamycin compounds, and the culture temperature can be within a temperature range at which producing bacteria grow and produce mazethlamycin compounds, but 25 to 35°C is particularly preferred.

Cultivation is usually continued until sufficient mazethramycin compound has accumulated. For example, 1.5% glycerin, 1% cottonseed powder.
5%, NaClO. 3. Adjust the pH of a medium containing 0.2% L-asparagine to 7.4, and add Streptomyces ME56l-1 to this.
When spores and mycelia were inoculated from the slant cultures of the four strains and aerobically stirred culture was carried out at 2rc, the highest accumulation of the target antibiotic mazethramycin was observed on the 2nd to 4th day of culture. Quantitation of mazethramycin compounds is carried out using Bacillus subtilis PCI2l9 etc. as a test bacterium and by the usual cylindrical plate method used for the quantification of antibiotics.
Pure mazethramycin B obtained according to the present invention is used as a standard substance.

When other antibiotics such as thiolthine and aureothricin are simultaneously produced in the culture solution, extract the culture solution with a solvent such as ethyl acetate, and measure the remaining aqueous layer using the cylindrical plate method described above. By doing so, the mazethramycin compound can be quantified. In this case, the mazethlamycin compound also partially migrates into a solvent such as ethyl acetate, so the same procedure can be performed using mazethlamycin as a control to prepare a standard curve, which can be used for quantification. The antibiotic can be extracted from the culture of the bacteria producing the mazethlamycin compound by solvent extraction methods using water-immiscible organic solvents such as butanol and adsorption methods using activated carbon or porous resins as adsorbents. It is carried out at the same time.

The partition coefficient of mazethramycin B in butano-water is 101) in the pH range of 6 to 8). Show above. Therefore,
The mazethramycin compound can be extracted from the culture within this pH range. In addition, when extracting the mazethramycin compound from the culture solution, it is possible to use activated carbon, nonionic porous adsorption resin, etc. as an adsorbent.
It is valid. In particular, it is preferable to perform column chromatography using Amperlite XAD-2 (manufactured by Rohm and Haas, USA), a polystyrene resin crosslinked with divinylbenzene. It is eluted with water and concentrated by vacuum distillation. Mazethramycin compounds in solid matter such as bacterial cells are extracted with commonly used organic solvents such as methanol, ethanol, acetone, butanol, etc., and concentrated by vacuum distillation. It is also possible to extract the mazethlamycin compound in the liquid portion and the bacterial cell portion into a solvent in which the mazethlamycin compound is well soluble, such as butanol, without removing the bacterial cells from the culture solution containing the bacterial cells. When the extracted dry product obtained as described above is treated with ethyl ether, n-hexane, etc., the mazethramycin compound remains in the insoluble portion. Furthermore, when this insoluble part is extracted with methanol, the mazethramycin compound is extracted into the solvent layer.
The residue is removed as an impurity. By collecting the powder containing the mazethlamycin compound in this way and then extracting this powder with methanol or a mixed solvent containing methanol, an extract mainly containing mazethlamycin B can be collected. Mazethramycin can be purified by appropriately combining or repeating these extraction methods.
It is purified by conventional column chromatography using cellulose and silica gel. The known antibiotics thiolthine and aureothricin, which often coexist in culture, can be easily separated from the mazethramycin compound by the above-mentioned treatment with ethyl ether, n-hexane, etc. or by silica gel column chromatography. The mazesulamycin compound can be purified by the above-described purification treatment by extraction alone or in any combination as necessary.

Mazethramycin A. ．． B.. Anhydromazethramycin is produced by dehydrating or dealcoholizing C by heating under reflux in a nonpolar organic solvent. Examples of non-polar organic solvents used here include acetone, acetonitrile, ethyl acetate, and chloroform. When anhydromazethramycin is dissolved in water or a water-containing non-alcoholic organic solvent, water is added to obtain mazethlamycin A. When mazethramycin A or anhydromazethramycin is dissolved in methanol, the methanol reacts and can be converted to relatively stable mazethlamycin B. Similarly, when mazethramycin A or anhydromazethramycin is dissolved in ethanol, the ethanol reacts to yield relatively stable mazethlamycin C. Therefore, the gist of the third invention is to prepare mazethramycin B or C, which consists of reacting mazethramycin A with methanol or ethanol.
It is in the manufacturing method.

Furthermore, the fourth aspect of the present invention is to prepare anhydromazethramycin by heating mazethlamycin A, mazethlamycin B, or mazethlamycin C in a nonpolar organic solvent to dehydrate, demethanol, or deethanol, respectively. It's in the manufacturing method.

Furthermore, the fifth aspect of the present invention is a method for producing mazethramycin A, mazethramycin B, or mazethramycin C, which comprises reacting anhydromazethramycin with water and methanol with ethanol.

An example of an effective combination of the above extraction and purification methods in order to collect mazethlamycin from a culture solution of mazethlamycin-producing bacteria is as follows.

After adjusting the pH of the culture solution to 8, the antibiotic is extracted with butanol, water is added, and the mixture is concentrated under reduced pressure at 40° C. or lower to completely remove butanol and form an aqueous solution. This is PH7
．． 5 and passed through a column of Amperlite XAD-2 to adsorb it, and after thorough washing with water, it was eluted with 50% acetone water. This is concentrated to dryness under reduced pressure at below 40°C to obtain a crude powder. Dissolve this in a small amount of methanol, remove impurities that are insoluble in methanol by centrifugation or filtration, add silica gel, mix uniformly, dry it, and place it on top of a column filled with silica gel suspended in a developing solvent. and then diluted with chloroform-methanol (10:5 volume ratio). After concentrating the eluted active fraction, the residue is dissolved in methanol and left in a refrigerator to obtain mazethlamycin B as yellow needle-like crystals. When there are many impurities and it is difficult to precipitate crystals, perform rechromatography on silica gel using ethyl acetate as the developing solvent, concentrate the eluted active fraction, dissolve it in methanol, and leave it in the refrigerator to form Mazethlamycin B crystals. Obtainable.

Examples of methods for producing mazethramycin compounds are shown below. B
、． Since the properties of C and anhydromazethramycin have been clarified, various methods for producing mazethramycin compounds can be devised based on these properties.

Therefore, the present invention is not limited to the examples, and may be modified by means of modification of the examples, as well as by applying known concentration, extraction, and purification means based on the properties of the mazethramycin compound revealed by the present invention. Mazethramycin A. ．． B. ．． It covers all methods of producing C and anhydromazethramycin.

Example 1 Actinomycetes ME56l-1 cultured on agar slant medium
4 strains (Feikoken Bacteria No. 3825) were added to glycerin 1.5
%, cottonseed flour 1.5%, L-asparagine 0.2%, and salt 0.3% was inoculated into a liquid medium containing 1.5% cottonseed powder, 0.2% L-asparagine, and 0.3% salt, and cultured with shaking at 27°C for an hour to obtain a primary seed culture.

Next, 125 ml of the liquid medium 5e having the above composition was dispensed into a 500 ml capacity Sakalo flask, and 1 of the primary seed culture solution was added.
ml each was inoculated and cultured with shaking at 2rc for 4 days. P.H.
4740 ml of culture p solution of 7.6 was obtained. The liquid is 46μY
1 ml (total volume 216 m9) of the Mazethlamycin compound. 215g of bacterial cells separated by filtration weighs 6
Contained 0 mg of mazethlamycin compound. 4,740 ml of the above culture solution was adjusted to pH 8 with sodium hydroxide. Adjust to O, add 5000ml of butanol, stir and extract.
It was concentrated under reduced pressure and dissolved in 1600 ml of purified water. 191TfLg, which is 89% of the total amount of mazethramycin compounds.
was obtained by butanol extraction, and the pH of the aqueous solution was 4.
．． It was 5. Adjust the pH to 7 with sodium hydroxide and use Amperlite
m) column. 3000m of purified water in the column
Wash by passing through 50% acetone water 2
The mazethlamycin compound was eluted using 000 mt and concentrated to dryness under reduced pressure to obtain a 1.4y brown powder (mazethlamycin A crude powder). This brown powder containing 184 m9 of mazethramycin compound (mainly mazethlamycin A) was dissolved in a small amount of methanol, and silica gel (
After adding Wakogel C-200) 4y and mixing uniformly,
Dry under reduced pressure.

This was placed on top of a column (inner diameter 20 m) filled with 50 y of silica gel suspended in chloroform. Next, 250 mt of chloroform/methanol (50:1 volume ratio) was passed through, and then the mixture was expanded with chloroform/methanol (20:1 volume ratio), and fractions of 15 y were collected. Fractions 32-45
Mazethramycin B was eluted. This fraction was concentrated under reduced pressure to obtain 118m9 of an ocher powder containing Mazethramycin B7lm9. The yield was 33%. Example 2 The ocher powder 118Tn9 obtained in Example 1 was added to 6
After dissolving in 50 ml of methanol at 0° C. and cooling, 46 ml of needle-like crystals of Mazethramycin B were obtained.

The crystallization yield was 65%. Example 3 115 Tng of dry powder obtained in the same manner as in Example 1 is dissolved in 1 ml of methanol, silica gel 1f is added thereto, mixed uniformly, and then dried under reduced pressure.

This was placed on top of a column (inner diameter 14Wr!R) filled with silica gel 11y suspended in ethyl acetate. Next, expand with 600 mL of ethyl acetate and collect 7y fractions. Mazethramycin B was eluted in fractions 23-39. This fraction was concentrated under reduced pressure to obtain 61 mg of Mazethlamycin B as a pure dry powder. This was dissolved in 6 ml of methanol while heating, and then cooled to obtain 40 mg of Mazethramycin B crystals. Example 4 Streptomyces strain ME56l-14 (Feikoken Bibori No. 3825) cultured on agar slant medium was treated with 2.5% glycerin, 0.5% beef scratches, 0.5% polypeptone, and yeast. Scratches 1.0%, salt 0.2%
, MgSO4.7H200.05%, K2HPO4O
．． A liquid medium 5e containing 5% OO and 0.32% precipitated calcium carbonate was rotary cultured at 27° C. for 4 days in 110 ml portions of a 500 mL Erlenmeyer flask with a waffle.

3530 mt of culture broth with pH 5.6 and 1160 bacterial cells
I got y. The bacterial cells were extracted with stirring by adding 72,500 rfL1 of methanol, and the extract was concentrated under reduced pressure, dissolved in 500 ml of water, and combined with the culture p solution. Thereafter, butanol extraction, amperlite X and AD-2 adsorption, and elution treatments were performed in the same manner as in Example 1 to obtain a 2.2y crude powder. This crude powder was subjected to silica gel column chromatography on a scale twice that of Example 1, and fractions containing mazethlamycin B were collected and concentrated under reduced pressure to obtain 150 m9 of pure mazethlamycin B powder. This was dissolved by adding 2 ml of dimethylformamide, and 35 ml of methanol was added and cooled to obtain 68 ml of needle-shaped crystals of Mazethramycin B.
Example 5 Mazethramycin B crystal 124Tn9 was dissolved in 100 ml of acetonitrile, a very small amount of Amperlite CG-50 was added, and the mixture was refluxed for 1 hour.

When Amperlite CG-50 was removed by filtration with a glass filter and acetonitrile was removed by vacuum concentration, needle-like crystals were precipitated. This was recrystallized from acetonitrile to obtain 80 mg of anhydromazethramycin crystalline powder. In addition, Mazethramycin C crystal 60
m9 was dissolved in 50 ml of acetonitrile and treated as above to obtain 38 mg of anhydromazethramycin crystalline powder.

Example-6 50% of anhydromazethramycin obtained in Example 5
m9 was dissolved in 50 mt of 50% acetone water and concentrated under reduced pressure to obtain Mazethramycin A.

Example 7 50% of Mazethramycin A obtained in Example 6
mg was dissolved in 15 ml of methanol and concentrated under reduced pressure to obtain 48 ml of Mazethramycin B crystals.

Example 8 50ml of Mazethramycin A was dissolved in 15ml of ethanol and concentrated under reduced pressure to form Mazethlamycin C.
Crystals of 45Tn9 were obtained.

Example 9 50% of anhydromazethramycin obtained in Example 5
mg was dissolved in 15 mt of methanol and concentrated under reduced pressure to obtain crystals of Mazethramycin B 327n9.

Example 10 21 mg of anhydromazethramycin obtained in Example 5 was dissolved in 30 ml of ethanol and concentrated under reduced pressure to obtain 24 ml of crystalline powder of Mazethramycin C.

[Brief explanation of drawings]

FIG. 1 shows the ultraviolet absorption spectrum curve of mazethramycin A in a 4.14 μYlml acetonitrile solution.

Claims (1)

[Claims] 1 The following general formula (I) ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula,
... A... is a group ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ or a group -N=CH-, where R is a hydrogen atom or a lower alkyl group] An anticancer antibiotic that is a compound represented by a group Mazethramycin compounds. 2. In the compound of general formula (I), A... is a group ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and R is mazethramycin A represented by a hydrogen atom, as stated in claim 1 compound. 3 In the compound of general formula (I), A... is a group ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and R is mazethramycin B represented by a methyl group, as stated in claim 1 compound. 4 In the compound of general formula (I), A... is a group ▲There are mathematical formulas, chemical formulas, tables, etc.▼, and R is mazethramycin C represented by an ethyl group, as stated in claim 1 compound. 5 In the compound of general formula (I)...A... is an anhydro compound in which the group -N=CH-, and the compound has the following formula (II)
▲There are mathematical formulas, chemical formulas, tables, etc.▼The first claim, which is anhydromazethramycin represented by (II)
Compounds described in Section. 6 A mazethramycin compound-producing bacterium belonging to the genus Streptomyces is cultured aerobically in a medium containing a nutrient source, and the culture contains the following general formula (I)▲mathematical formula, chemical formula, table, etc.▼ (I) [In the formula, ... A ... is a group ▲ There is a mathematical formula, chemical formula, table, etc. ▼ or a group -N=CH
-, where R represents a hydrogen atom or a lower alkyl group] is produced, and the mazethlamycin compound is collected from the culture. Method of manufacturing compounds. 7 Streptomyces thiorteus ME561-14
strain (Feikoken Bacteria No. 3825) in a nutrient-containing medium for 2 hours.
7. The method according to claim 6, wherein the mazethramycin compound of general formula (I) is produced in the culture by culturing aerobically in a temperature range of 5 to 35°C. 8. The method according to claim 6, wherein the mazethlamycin compound of general formula (I) is extracted from a culture of mazethlamycin compound-producing bacteria by extraction with a water-immiscible organic solvent. 9. The method according to claim 6, wherein the mazethlamycin compound of general formula (I) is collected from the culture filtrate of the mazethlamycin compound-producing bacteria by adsorption onto an adsorbent. 10. A process for producing mazethramycin B or C, which comprises reacting mazethramycin A with methanol or ethanol. 11 A method for producing anhydromazethramycin, which comprises heating mazethlamycin A, mazethlamycin B, or mazethlamycin C in a nonpolar organic solvent to dehydrate, remove methanol, or remove ethanol, respectively. 12. A process for producing mazethramycin A, mazethramycin B or mazethlamycin C, which comprises reacting anhydromazethramycin with water, methanol or ethanol.