JP3276187B2 - Novel Compound Ustiloxin A or Ustiloxin B or Derivatives Thereof - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel compound having an antitumor effect, ustyroxin A or ustyroxin B, a derivative thereof or a salt thereof (hereinafter referred to as "us thyroxines"), and a process for producing them.

[0002]

2. Description of the Prior Art Ustilaginoidea virens cooke belongs to imperfect fungi, but a complete generation formed on the sclerotium has been recognized and is called Claviceps virens Sakurai. (Yukio Harada, Nihon Keisho, Vol. 3, p. 387 (1984
Year)). Ustilaginoidi as a metabolite of rice koji fungus
n A, B, C (Tadajiro Yabuta, Yusuke Sumiki, Journal of the Japanese Society of Agricultural Chemistry, Vol. 9, p. 478 (1933)) and Ustilaginoi
din D, E, F, G, H, I, J (K. Koyama and SN
atori, Chem. Pharm. Bull., 36, 146, (1988
Year)) is known. However, these compounds and the usthyroxines of the present invention are clearly different in physicochemical properties and structure.

In addition, one ether bond and 2
As a 13-membered ring compound having two peptide bonds, for example, Phomopsin A (Phomopsin A) produced from Phomopsis leptostromiformis (CLAUDE C.
J. CULVENOR et al., Tetrahedron, Volume 45, Issue 8, 235
1 (1989)) is known. However, the structure of its side chain is completely different from usthyroxines.

[0004]

DISCLOSURE OF THE INVENTION The present inventors have developed a novel compound, ustyroxin A and a novel compound, which are isolated from rice koji, which have been spontaneously generated in rice ears or artificially generated in rice ears by rice koji disease fungi. The inventors have found that thyroxine B has a strong antitumor effect, and have also found that derivatives obtained by chemical modification thereof have a strong antitumor effect, thereby completing the present invention.

[0005]

According to the present invention, there is provided a compound of the general formula

[0006]

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Wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R ² is a hydrogen atom or an aliphatic acyl group having 2 to 6 carbon atoms, and R ³ is a methyl group or propyl Or a salt thereof, more preferably, in the above general formula (I),

[0008]

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Wherein R ¹ is a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R ² is a hydrogen atom or an aliphatic acyl group having 2 to 6 carbon atoms, and R ³ is a methyl group or propyl Or a salt thereof.

Here, when R ¹ is an alkyl group having 1 to 5 carbon atoms, for example, a linear or branched alkyl group such as methyl, ethyl, propyl, butyl, t-butyl and pentyl Is raised. It is preferably a linear or branched alkyl group having 1 to 3 carbon atoms, and most preferably a methyl group.

When R ² is an aliphatic acyl group having 2 to 6 carbon atoms, examples thereof include linear or branched aliphatic acyl groups such as acetyl, propionyl, butyryl, valeryl and hexanoyl. . It is preferably a linear or branched aliphatic acyl group having 2 to 4 carbon atoms, most preferably an acetyl group.

The compound represented by the above general formula (I) of the present invention can be converted into a salt according to a conventional method.

When R ¹ is a hydrogen atom, for example, salts of alkali metals such as lithium, sodium and potassium; salts of alkaline earth metals such as magnesium, calcium and barium; methylamine, ethylamine and dimethyl Salts of aliphatic primary to tertiary amines such as amine, dipropylamine and trimethylamine; salts of amino acids such as lysine and arginine; ammonium salts; It is preferably a salt of an alkali metal such as sodium or potassium.

When R ² is a hydrogen atom, salts of hydrohalic acids such as hydrofluoric acid, hydrochloric acid, hydrobromic acid and hydroiodic acid; nitrates; perchlorates; sulfates; Inorganic salts such as salts; salts of lower alkanesulfonic acids such as methanesulfonic acid, trifluoromethanesulfonic acid and ethanesulfonic acid; salts of arylsulfonic acids such as benzenesulfonic acid and p-toluenesulfonic acid; glutamic acid; Organic acid salts such as salts of amino acids such as aspartic acid; salts of carboxylic acids such as fumaric acid, succinic acid, citric acid, tartaric acid, oxalic acid and maleic acid; Preferably it is a hydrochloride.

Of these salts, the most suitable are pharmacologically acceptable salts.

The compound having the general formula (I) has various isomers. In the compound having the general formula (I), all of these isomers are represented by a single formula. Therefore, in the present invention, all of these isomers and a mixture of these isomers are also included.

Further, the compound having the general formula (I) of the present invention includes all so-called "prodrug compounds" derived in vivo into the compound having the general formula (I). .

In the compound having the general formula (I), preferably, R ¹ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and R ² represents a hydrogen atom or 2 to 4 carbon atoms. A compound or salt thereof, wherein the compound has an aliphatic acyl group and R ³ represents a methyl group or a propyl group.

[0019] Optimally, R ¹ represents a hydrogen atom or a methyl group, R ² represents a hydrogen atom or an acetyl group, R ³
Represents a methyl group or a propyl group, or a salt thereof.

Specific examples of the compound having the general formula (I) of the present invention include the compounds shown in the following table.

[0021]

[Table 1] ------------------------------------------------------------------------- Compound number R ¹ R ² R ³ --- ------------------------ ---------------------------------------------------------------------- 1 H H Me 2 H Ac Me 3 H Prn Me 4 H Byr Me 5 H _{CO (CH 2) 3 CH 3} Me 6 Me H Me 7 Me Ac Me 8 Me Prn Me 9 Me Byr Me 10 Et H Me 11 Et Ac Me 12 Pr H Me 13 Pr Ac Me 14 Bu H Me 15 Bu Ac Me 16 H H Pr ----------------------------------.

However, in the table, H = hydrogen atom, Me = methyl, E
t = ethyl, Pr = propyl, Bu = butyl,
Ac = acyl, Prn = propionyl, B
yr represents a butyryl group.

In the above-mentioned compound table, preferably, exemplified compound numbers 1, 2, 3, 6, 7, 10, 11, 12, 14, 16
Or a salt thereof.

Most preferably, Exemplified Compound Nos. 1, 2, 6,
7, 10, 16 or a salt thereof. The present invention also provides
A method of producing rice koji in a rice plant using a rice koji disease fungus having an ability to produce ustyroxine A or ustyroxine B belonging to the genus Ustyraginoidea, and isolating ustyroxine A or ustyroxine B from the rice koji. With respect to the method for producing ustiloxin A or ustiloxin B, preferably, a rice koji disease bacterium belonging to the genus Ustyraginoidea and having the ability to produce ustiloxin A or ustiloxin B is used as Ustilaginoidea virens (Cooke) Tak
ahashi SANK 15391 shares (Microtechnical Laboratories No. 3691: BP-3691
).

1) Among the compounds having the general formula (I) of the present invention, a compound wherein R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, and R ³ represents a methyl group (ie, usthyroxine B ), or, R ¹ represents a hydrogen atom, R ² represents a hydrogen atom, R ³ represents an isopropyl group, a compound (i.e.,
Ustyroxine A ) is obtained as follows.

In other words, the rice koji fungus (Ustila
ginoidea virens Cooke). Koji grains can be picked up by hand from diseased ears in piloerection, air-dried indoors for about one week, and pulverized by a pulverizer before use. In addition, after pulverization, those stored in a refrigerator at about 5 ° C. can be used.

The thyroxine A or thyroxine A or thyroxine A is extracted by extracting water, alcohols such as methanol and ethanol, organic acid esters such as ethyl acetate, and ketones such as acetone and methyl ethyl ketone singly or in combination. An extract containing thyroxine B can be obtained. Isolation and purification of ustiloxin A or ustiloxin B from the extract thus obtained is achieved by utilizing its physicochemical properties. For example, it can be collected using an adsorbent. As the adsorbent to be used, for example, activated carbon, or Amberlite XAD-2, X
AD-4, XAD-7 etc. (manufactured by Rohm and Haas),
Diaion HP 10, HP 20, HP 50, CHP 20P (manufactured by Mitsubishi Kasei Co., Ltd.), polyamide gel (manufactured by Welm) and the like are used. Ustyroxine A or Ustyroxine B
Is passed through the above adsorbent layer to adsorb and remove impurities contained in the liquid containing ustiloxin A or ustiloxin B, or
Alternatively, it can be obtained by adsorbing ustiloxin B and eluting with a suitable solvent. In order to further purify the thus obtained usthyroxine A or usthyroxin B, cellulose such as Avicel (Asahi Kasei Kogyo Co., Ltd.) or Sephadex LH-20 (manufactured by Pharmacia) is used.
An effective method is a partition column chromatography using, for example, an extraction method utilizing the difference in the partition ratio of the solvent mixed with usthyroxine A or usthyroxine B to the solvent, or a countercurrent partitioning method.

Usthyroxin A or usthyroxin B can be separated and purified by repeatedly using the above separation and purification means alone or in an appropriate combination.

Alternatively, the rice koji disease strain Ustilaginoidea virens (Cooke) Takaha isolated by the present inventors.
The spore suspension of shi SANK 15391 is sprayed on rice ears growing in a field, and using a rice koji that has been generated after a certain period of time, ustyroxin A or ustyroxin B can be obtained in the same manner as described above.

[0030] In this case, the rice 2-6 weeks before heading has SANK 1
5391 conidia or chlamydospores can be spray inoculated from above to generate rice koji.

The suspension of conidia was transferred to an Erlenmeyer flask containing a potato decoction medium for adding sucrose.
It can be obtained by liquid culturing for 12 to 20 days under dark conditions at ℃. Add 0.05% surfactant such as Tween 20 to this suspension
A preferred method is to add a considerable amount and spray inoculate about 1 L / m ² from above the rice plant in the field.

The time of the spray inoculation is preferably around 19:00.

Chlamydospores can be obtained by the following method.

Using a potato decoction medium for adding sucrose, 26
The mycelial suspension obtained by liquid culture at 7 ° C. for 7 days is poured onto a potato decoction agar medium for sucrose addition so as to spread thinly, and cultured under dark conditions at about 25 ° C.

In the first week or so, conidiospores are formed.
Thereafter, chlamydospores are formed. After culturing under these conditions for 1 to 3 months, the petri dish was capped with 22
The mixture was allowed to stand for 7 days in a room at or below ° C and dried, and the whole was crushed by a mixer together with the medium to obtain a desired thick spore.

By inoculating the chlamydospores directly on the rice plant in the field, rice koji can be generated. The inoculation is preferably used a small dusting device, also, as the intake of 30 to 40 sheets per m ² petri dish chlamydospores are preferred.

The inoculation time is preferably performed during the day.

The test variety is not particularly limited, but a late variety such as Tori No. 1 or Norin 21 is preferred.

Rice koji strain Ustilaginoi which makes rice koji
dea virens (Cooke) Takahashi SANK 15391 can be isolated, stored and used as needed as follows. That is, spores (chlamydospores) are collected from rice panicles that show diseased and dark green symptoms and are placed in a culture solution such as a potato glucose medium and cultured at 23 to 28 ° C. for 1 to 2 days. A part of this culture solution is taken, observed under a microscope, and spores that have begun to germinate are subjected to monospore separation using a manipulator. This is placed on an appropriate agar medium, for example, a potato dextrose agar medium, and cultured at 23 to 28 ° C., and a well-grown one is stored. Growth on potato dextrose agar is slow, with white fluffy hyphaes growing initially, but eventually becoming dark greenish brown. It also produces a dark green soluble pigment in the medium. The hypha is colored and has a partition,
The diameter is 2.0 to 5.0 μm, and the wall is very thick, dark greenish brown, and often forms a hyphal mass. Conidia occur in a sympgio form, 1 cell, colorless, 5.0-8.0 ×
3.0 to 4.5 μm. Chlamydospores found in rice koji are not observed.

These properties are reported by Hashioka et al. (Saitama Prefectural Agricultural Experiment Station Research Report No. 2, pp. 1-20, 1951
Year publication).

Rice koji fungus isolated by the present inventors
Ustilaginoidea virens (Cooke) Takahashi SANK1539
1 has been internationally deposited with the Research Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry (Deposit No.
M BP-3691): Deposit date December 24, 1991).

2) Among the compounds having the general formula (I) of the present invention, the compound wherein R ¹ represents an alkyl group is obtained as follows.

That is, the esterification is achieved by carrying out an esterification reaction with an acid using usthyroxin A or usthyroxin B and an alcohol such as methanol or ethanol. The acid used in the reaction is not particularly limited, and is preferably, for example, a mineral acid such as sulfuric acid, hydrochloric acid, chloric acid, or perchloric acid; or an organic acid such as formic acid or trifluoroacetic acid. The reaction temperature and reaction time vary depending on the reagents and solvents used, but can be usually converted into another alkyl group by transesterification at 0 to 100 ° C for 1 to 24 hours.

After completion of the reaction, the target compound is obtained by distilling off the solvent from the reaction mixture and drying the residue. When an acid is used in excess of usthyroxine A or usthyroxine B as a starting compound, the target compound is obtained as a salt of the acid. The target compound thus obtained can be further purified by recrystallization or ordinary column chromatography.

Alternatively, it can be achieved by reacting with a diazoalkane such as diazomethane. The reaction is performed in the presence of a solvent. The solvent used is not particularly limited as long as it does not affect the reaction, and ethers such as diethyl ether, tetrahydrofuran, dioxane and dimethoxyethane; esters such as ethyl acetate; amides such as dimethylformamide Kind;
Sulfoxides such as dimethyl sulfoxide; The reaction temperature and reaction time vary depending on the reagents, solvents and the like used, but are usually preferably room temperature to 50 ° C and 1 to 24 hours.

Alternatively, it can be obtained by alkylating a compound in which R ¹ represents a hydrogen atom. The reaction is carried out with an alkyl halide compound such as methyl iodide or ethyl iodide in the presence of an alkali metal carbonate such as potassium carbonate or sodium carbonate. The reaction is performed in the presence of a solvent. The solvent to be used is not particularly limited as long as it does not affect the reaction, and ethers such as tetrahydrofuran, dioxane and dimethoxyethane; amides such as dimethylformamide; sulfoxides such as dimethyl sulfoxide. Is preferred. The reaction temperature and reaction time vary depending on the reagents, solvents and the like to be used, but usually 0 to 100 ° C and 1 to 72 hours are preferred.

After completion of the reaction, the solvent for the target compound is distilled off from the reaction mixture, and the residue is separated by adding water and an organic solvent such as ethyl acetate. It is obtained by concentrating the organic layer and purifying the residue by recrystallization or ordinary column chromatography.

3) Among the compounds having the general formula (I) of the present invention, the compound wherein R ² represents an aliphatic acyl group can be obtained as follows.

That is, the compound is obtained by selectively acylating a primary amino group of a compound in which R ² represents a hydrogen atom in the presence of a base. As the acylating agent used in the reaction, for example, acid anhydrides such as acetic anhydride and propionic anhydride; acid halides such as acetyl chloride and acetyl bromide are preferable. The reaction is suitably performed in the presence of a solvent. The solvent used is not particularly limited as long as it does not affect the reaction, and is preferably, for example, water, an alcohol such as methanol or ethanol, or a mixed solvent thereof. The reaction is performed in the presence of a base. The base used is, for example, an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide; an alkali metal carbonate such as sodium carbonate or potassium carbonate; an alkali metal bicarbonate such as sodium hydrogen carbonate or potassium hydrogen carbonate. salt;
Preferred are aromatic amines such as pyridine; heterocyclic amines such as proline; aliphatic amines such as triethylamine and diethylamine. The reaction temperature varies depending on the type of the acylating agent and the like, but is usually preferably 0 to 50 ° C. The reaction time varies depending on the reaction conditions, but is usually 1 to 5 hours.

The target compound thus obtained is optionally subjected to adsorption column chromatography using a carrier such as silica gel or florisil; reverse phase partition chromatography using silica gel having an octadecyl group, an octyl group or the like; Partition column chromatography using cellulose such as Avicel (made by Asahi Kasei Kogyo Co., Ltd.) or Sephadex LH-20 (manufactured by Pharmacia); or an extraction method utilizing the difference in the partition ratio of a mixed impurity with a solvent to a solvent. And the like.

4) The compound having the general formula (I) of the present invention can be obtained by appropriately combining the above reactions 1) to 3).

Compounds in which R ¹ is a hydrogen atom are represented by R
^It can also be obtained by hydrolyzing a compound in which ¹ is alkyl in the presence of an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide; an alkali metal carbonate such as sodium carbonate or potassium carbonate.

The thus obtained general formula (I)
Compounds having the following are used: adsorption column chromatography using a carrier such as silica gel and florisil, distribution column chromatography using Sephadex LH-20 (manufactured by Pharmacia), and normal and reverse phase columns. It can be purified by high performance liquid chromatography or the like.

[0054]

The compound having the general formula (I) of the present invention is
It is a novel compound that has not been published in the literature, exhibits a strong growth inhibitory effect on various human cancer cells, and is useful as an antitumor agent.

When the compound having the general formula (I) of the present invention is used as a medicament, it is mixed with itself or an appropriate pharmaceutically acceptable carrier, excipient or diluent according to a conventional method to obtain a powder or granule. It can be safely administered orally or parenterally in the form of tablets, capsules, injections and the like. The dose varies depending on the target disease, the route of administration and the number of administrations. For example, the effective daily dose for adults is 0.01 mg to 80 mg per kg of patient body weight when injected.
In the case of oral administration, it is preferable to administer 0.5 mg to 120 mg once or in several divided doses according to the symptoms.

[0056]

EXAMPLES Next, the present invention will be described more specifically with reference to examples and test examples, but the present invention is not limited to these examples.

Embodiment 1 Ustyroxine A The rice koji naturally occurring in the rice ear due to the infection of the rice koji disease fungus (Ustilaginoidea virens Cooke) is collected and dried.
Combine 100 g with 10 times the volume of water in a mixer and mix at room temperature.
Extracted by shaking for 1 hour. After filtering 1000 ml of extract,
Concentrate to one tenth and use a 50 ml ODS column (ODS-SS-1020
T, manufactured by Senshu Kagaku Co., Ltd.). UV absorption 25
While monitoring at 4 nm, elution was performed with 2.5 liters of water, 2.5 liters of a mixture of methanol: water = 2: 8, and 1.5 liters of 100% methanol. The fraction eluted with a mixture of methanol: water = 2: 8 was collected and concentrated. After dissolving this concentrate in water, it was injected again into a 30 ml ODS column. The eluate was developed and eluted with 0.5 liter of a mixture of methanol: water = 2: 98, and 200 ml of the eluted fraction between 200 ml and 400 ml was collected to obtain a fraction containing uthyroxin A. When these fractions were concentrated, about 20 mg of crude crystals were obtained. When this crude crystal is recrystallized with water, the melting point is 195 ° C.
14 mg of a pure product of the target compound having (decomposition) was obtained as crystals.

Ustyroxine A obtained in this example was
The following physical property values are shown. 1) Structural formula

[0059]

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2) Properties of substance: water-soluble, white needle-like crystals 3) Molecular formula: C ₂₈ H ₄₃ O ₁₂ N ₅ S 4) Molecular weight: 673 5) Mass spectrometry: FAB-MS; m / z 674 (M + H): HR FAB-MS; m / z 674.2730 (M + H) (theoretical 674.2718, C
_{28 H 44 O 12 N 5 S} ) 6) Elemental analysis: C ₂₈ H ₄₃ O ₁₂ N as _{5 S · 2.5 H 2 O (} %) Found: C, 46.69; H, 6.92 ; N, 9.64;
S, 4.28 theoretical: C, 46.80; H, 6.82; N, 9.75;
S, 4.45 7) Ultraviolet absorption spectrum: λ _max nm (ε) The ultraviolet absorption spectrum measured in water is as follows. 207 (21300), 249 (6000), 287 (3200).

8) Infrared absorption spectrum: ν _max cm ^-1 potassium bromide (KBr) The infrared absorption spectrum measured by the tablet method is as follows. 3700-2400 (broad), 1650, 1640, 1540, 1500, 1465,
1400, 1295, 1225, 1155, 1130, 1090, 1030, 900.

9) ¹ H-nuclear magnetic resonance spectrum: (δ; pp
m) 50% in heavy water using tetramethylsilane as external standard
The nuclear magnetic resonance spectrum (500 MHz) measured at ° C. is as follows. 0.80 (3H, d, J = 7.0 Hz), 0.88 (3H, d, J = 7.0 Hz), 1.09
(3H, t, J = 7.2 Hz), 1.73 (1H, dd, J = 7.2 and 14.2 Hz)
, 1.77 (3H, s), 1.92 (1H, dd, J = 7.0 and 10.0 Hz)
, 2.12 (1H, ddd, J = 3.0,8.0 and 15.0 Hz), 2.22 (1
H, ddd, J = 8.0,10.0 and 15.0 Hz), 2.24 (1H, dd, J = 7.2
And 14.2 Hz), 2.77 (3H, s), 3.04 (1H, dd, J = 3.0 and 13.6 Hz), 3.33 (1H, dd, J = 10.0 and 13.6 Hz)
, 3.79 (2H, s), 4.01 (1H, dd, J = 4.0 and 8.0 Hz),
4.13 (1H, d, J = 10.0 Hz), 4.28 (1H, d, J = 10.0 Hz), 4.39
(1H, tt, J = 3.0 and 10.0 Hz), 4.83 (1H, s), 4.96 (1
H, d, J = 10.0 Hz), 7.11 (1H, s), 7.61 (1H, s).

10) ¹³ C nuclear magnetic resonance spectrum:
(Δ; ppm) The nuclear magnetic resonance spectrum (100 MHz) measured in heavy water using tetramethylsilane as an external standard is as follows.

7.8 (q), 17.9 (q), 18.3 (q), 21.1
(q), 28.7 (d), 32.1 (q × 2), 36.7 (t), 43.8
(t), 52.7 (d), 59.6 (d), 60.1 (d), 63.8 (d)
, 64.8 (t), 66.7 (d), 73.9 (d), 87.2 (s),
114.0 (d), 124.2 (d), 128.0 (s), 136.4 (s), 14
6.0 (s), 152.2 (s), 166.4 (s), 170.3 (s), 171.
0 (s), 174.4 (s), 176.3 (s).

11) Solubility: easily soluble in water, soluble in aqueous methanol. 12) Color reaction: positive for ninhydrin. 13) Thin-layer chromatography: Rf value; 0.16 adsorbent; silica gel plate Art. 5715 (manufactured by Merck) Developing solvent; n-butanol: acetic acid: water = 3: 1: 1 detection; UV at 254 nm and ninhydrin coloring.

14) Amino acid analysis: 110 with 6 N hydrochloric acid
After hydrolysis at ℃ for 20 hours, the solvent was distilled off from the reaction mixture. The obtained residue was analyzed with an amino acid analyzer to detect valine and glycine. Analysis of valine by high performance liquid chromatography indicated that it was consistent with L-valine. Column; Enantio Ll (4.6 mm ID × 250 mm, manufactured by Tosoh Corporation) Solvent: 0.8 mM CuSO ₄ Temperature; 27 ° C. Flow rate; 1 ml / min Detection;

Embodiment 2 FIG. Ustyroxine A Koji grains were generated in rice by artificial inoculation, and ustyroxin A was isolated and purified from this.

The rice was grown by the following method.

The seedlings sown on April 14 were transplanted to Honda on May 20. The cultivation density was set at 75 plants per 3.3 m ² , and one plant per line. Fertilizer is nitrogen (N), phosphoric acid (P ₂ O ₅ ) as base fertilizer
And potassium (K ₂ O) as an ingredient amount of 8 kg each for 10 a, and on July 14, nitrogen (N) was added as top fertilizer at 2 kg for 10 a.
Applied. Other methods followed the customary method.

The rice koji disease fungus SANK 15391 was cultured and chlamydospores formed after standing were obtained by the following method.

Using a potato decoction medium for adding sucrose, 26
The mycelial suspension obtained by liquid culture at 7 ° C. for 7 days was poured onto a potato decoction agar medium for sucrose addition so as to spread thinly, and cultured under dark conditions of about 25 ° C.

Although conidiospores were formed in the first week or so,
Thereafter, chlamydospores formed. After culturing under these conditions for 1 to 3 months, the petri dish was capped with 22
The mixture was allowed to stand for 7 days in a room at or below ° C and dried, and the medium was pulverized with a mixer to obtain thick-film spores.

A rice koji was generated by inoculating the chlamydospores directly on a rice plant in a field. That is,
Spraying rice ears growing in the field and leaving them for a certain period (about 2 months) produced rice koji. These rice koji were collected. Ustyroxin A was isolated and purified in the same manner as described. About 1 mg of the target compound was isolated from 10 g of rice koji. The physical property values of usthyroxine A obtained in this example were as shown in Example 1. Were the same as the physical properties of the ustyroxin A obtained in the above.

Embodiment 3 FIG. Usthyroxine A dimethyl ester dihydrochloride 5 mg of usthyroxine A obtained in Example 1 was added to 12%
It was dissolved in 0.5 ml of methanol containing hydrochloric acid and left at 4 ° C overnight. The solution was concentrated under reduced pressure, and the solvent was distilled off to obtain 5.9 mg of the desired compound.

The physical properties of ustiloxin A dimethyl ester dihydrochloride are as follows. 1) Structural formula

[0076]

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2) ¹ H-nuclear magnetic resonance spectrum: (δ; pp
m) A nuclear magnetic resonance spectrum (500 MHz) measured at 50 ° C. using tetramethylsilane as an external standard in deuterated dimethyl sulfoxide is as follows. 0.77 (3H, d), 0.83 (3H, d), 1.02 (3H, dd), 1.43 (3H,
s), 1.87 (1H, m), 1.98 (2H, m), 2.08 (1H, dd), 2.43 (3
H, s), 2.79 (1H, dd), 3.04 (1H, dd), 3.61 (3H, s)
, 3.74 (3H, s), 3.82 (3H, m), 4.07 (1H, m), 4.22 (1H, d
d), 4.47 (1H, dd), 4.83 (1H, d), 4.93 (1H, dd), 6.51
(1H, s), 7.07 (1H, s) 3) Thin layer chromatography: Rf value; 0.33 adsorbent; silica gel plate Art. 5715 (Merck) Developing solvent; n-butanol: methanol: water = 3: 1 :
1.

Embodiment 4 FIG. 4′-N-acetylusthyroxine A disodium salt 5.4 mg of usthyroxine A obtained in Example 1 was added to 2%
It was dissolved in 0.5 ml of an aqueous solution of sodium hydrogen carbonate, an excess amount of acetic anhydride was added, and the mixture was stirred at 0 ° C for 2 hours. The reaction mixture was neutralized with an aqueous solution of sodium hydrogen carbonate and adsorbed on a Diaion HP 20 column (manufactured by Mitsubishi Kasei Corporation) (2 ml). Eluting with matanol as the developing solvent,
Fractions containing the target compound were collected. This fraction was concentrated under reduced pressure, the solvent was distilled off, and the residue was dried to obtain 4.6 mg of the target compound.

The physical properties of 4'-N-acetylusthyroxine A. disodium salt are as follows. 1) Structural formula

[0080]

Embedded image

2) Color reaction: Ninhydrin negative. 3) Thin layer chromatography: Rf value; 0.39 adsorbent; silica gel plate Art. 5715 (Merck) Developing solvent; n-butanol: methanol: water = 3: 1:
1.

Embodiment 5 FIG. Ustyroxine B Koji grains were generated in rice by artificial inoculation, and uthyroxine B was isolated and purified from this. The artificial inoculation used the rice koji disease fungus SANK 15391 strain, and the inoculation was performed according to the method described in Example 2.

The rice koji generated on the rice ear was collected and dried, and then 100 g of the rice koji was pulverized with a 10-fold amount of water by a mixer and extracted by shaking at room temperature for 1 hour. After filtering 1000 ml of the extract, it is concentrated to one-tenth and concentrated to a 50 ml ODS column (ODS-SS-1).
020 T, manufactured by Senshu Kagaku Co., Ltd.). 2.5 liters of water, 2.5 liters of a mixture of methanol: water = 2.5 liters, 100% while monitoring with ultraviolet absorption at 254 nm
The mixture was eluted with 1.5 liters of methanol. The fraction eluted with a mixture of methanol: water = 2: 8 was collected and concentrated. After dissolving this concentrate in water, it was injected again into a 30 ml ODS column. Methanol: water = 2:98 mixture
A fraction containing eustyroxin B that eluted with 0.5 l and eluted before eustyroxin A was obtained. This fraction was again injected into the ODS column, and methanol: water = 1: 99
The mixture was developed and eluted with a mixture of the above to obtain crude ustiloxin B powder. This crude powder was injected into an ODS column and eluted and developed with a 25:75 mixture of methanol and water containing 0.02% trifluoroacetic acid to obtain a purified fraction of ustyroxin B. About 3 mg of ustyroxin B was obtained per 100 g of rice koji.

Ustyroxine B obtained in this example was
The following physical property values are shown.

1) Structural formula

[0086]

Embedded image

2) Properties of substance: water-soluble, white solid 3) Molecular formula: C ₂₆ H ₃₉ N ₅ O ₁₂ S 4) Molecular weight: 645 5) Mass spectrometry: FAB-MS: m / z 646
(M + H) 6) Optical rotation: + 14.1 ° (c = 0.55, H ₂ O) 7) Ultraviolet absorption spectrum: λmax nm (ε) The ultraviolet absorption spectrum measured in water is as shown below.

213 (13900), 252 (5000), 290 (2500) 8) Infrared absorption spectrum: ν _max cm ^-1 calcium fluoride (CaF) The infrared absorption spectrum measured by the thin film method is shown below. It is on the street.

3700-2400 (broad), 1670, 1600, 1550, 1
500, 1465, 1430, 1380, 1290, 1200, 1140 9) ¹ H-nuclear magnetic resonance spectrum: (δ; ppm) In heavy water, 2,2-dimethyl-2-silapentane-5 as an external standard
-The nuclear magnetic resonance spectrum (500 MHz) measured at 50 ° C. using sodium sulfonate (DSS) is as follows:

0.92 (3H, dd, J = 7.2, 7.2 Hz), 1.14 (3H, d,
J = 7.0Hz), 1.63 (1H, dq, J = 14.0,7.2Hz), 1.68 (3H,
s), 2.01 (1H, ddd, J = 3.0, 15.0, 8.0 Hz), 2.05 (1H, d
q, J = 14.0, 7.2 Hz), 2.12 (1H, ddd, J = 10.0, 15.0, 4.
0Hz), 2.60 (3H, s), 2.96 (1H, dd, J = 13.4, 2.8Hz),
3.30 (1H, dd, J = 13.4, 10.0Hz), 3.70 (1H, d, J = 17.0H
z), 3.76 (1H, d, J = 17.0Hz), 3.90 (1H, d, J = 10.0Hz),
3.90 (1H, dd, J = 8.0, 4.0Hz), 4.29 (1H, m, J = 2.8, 10.
0, 3.0, 10.0Hz), 4.37 (1H, q, J = 7.0Hz), 4.65 (1H, s),
4.80 (1H, d, J = 10.0Hz), 7.28 (1H, s), 7.47 (1H, s) 10) ¹³ H-nuclear magnetic resonance spectrum (δ; ppm) In heavy water, using DSS as an external standard The measured nuclear magnetic resonance spectrum (100 MHz) is as follows.

8.36 (q), 15.72 (q), 22.24 (q), 31.68 (t),
33.00 (q), 37.08 (t), 44.24 (t), 50.00 (d), 53.16 (d),
60.26 (d), 64.22 (d), 65.11 (t), 68.15 (d), 74.70 (d), 8
4.48 (s), 114.42 (d), 124.62 (d), 128.96 (s), 137.14
(s), 146.34 (s), 153.24 (s), 169.40 (s), 170.59 (s), 17
2.72 (s), 174.82 (s), 176.92 (s) 11) Solubility: easily soluble in water, soluble in aqueous methanol.

12) Color reaction: positive for ninhydrin.

13) Thin-layer chromatography: Rf value; 0.14 adsorbent; silica gel plate Art. 5715 Developing solvent; n-butanol: acetic acid: water = 3: 1: 1 detection; ultraviolet ray 254 nm, ninhydrin coloring 14) amino acid analysis : After hydrolyzing with 6 N hydrochloric acid at 110 ° C for 20 hours, the solvent was distilled off from the reaction mixture. The obtained residue was analyzed with an amino acid analyzer, and alanine and glycine were detected. Alanine was analyzed by HPLC and found to be consistent with L-alanine.

Column: Enantio Ll (4.6 mm ID × 250 mm
Solvent; 0.8 mM CuSO ₄ temperature; 27 ° C. flow rate; 1 ml / min detection; infrared 250 nm.

The ultraviolet absorption spectrum and optical rotation of ustiloxin B agree well with that of ustiloxin A, and the molecular weight is 645 smaller than that of ustiloxin A (corresponding to C ₂ H ₄ ). This indicates that the chromophore contained in the structure and the absolute configuration of the steric structure are consistent with usthyroxin A.

In the amino acid analysis, L-alanine was detected instead of L-valine detected in usthyroxine A. ^{In 1} H-NMR, two methyl signals at δ 0.80 and 0.88 ppm and a methyl signal at 0.92 ppm derived from the valine residue, which were shown in the spectrum of ustiloxin A, were not observed. A methyl signal appears in ppm. Similarly, ¹³ C-NMR also showed that the number of carbon signals was two less and that an alanine residue was present instead of a valine residue.

From the above data, it was determined that the structure of usthyroxine B was such that L-valine in usthyroxine A was replaced by L-alanine.

Embodiment 6 FIG. Anti-tumor activity Ustyroxine A and U-thyroxine B and derivatives thereof of the present invention have anti-tumor activity.

The results of measuring the antitumor activity of usthyroxine A and usthyroxine B are described below.

The method of Array (Michael C. Alley) et al.
The antitumor effect of ustiloxin A and ustiloxin B was examined according to ncer Research, 48, 589-601 (1988). That is, various cancer cells were placed in a 96-well culture plate at 1 × 10 ³ cells / 100 μl medium (RPMI-1640 +
10% FCS). However, RERF-LC-MA is 4
× 10 ² pieces, WiDr, SW−480, KU−2 5 × 10 ² pieces, ZR−
3 × 10 ^{3 of} 75-30 and 2.5 × 10 ^{2 of} SBC-5 were suspended. After culturing these suspensions at 37 ° C. for 24 hours under 5% CO ₂ , 100 μl of the medium containing the diluted sample was added, and the cells were further cultured under the same conditions for 48 hours. After culturing, 20
Washed three times with 0 μl of medium to remove drug.
Then, after culturing at 37 ° C under 5% CO ₂ for 96 hours, 50μg
/ 50 µl of MTT was added, and the cells were further cultured under the same conditions for 4 hours. The culture solution was removed, 150 μl of DMSO was added, the mixture was stirred, and the cell growth rate was calculated from the absorbance at 540 nm. The concentration of the test compound required to inhibit cell proliferation by 50% (IC ₅₀ ; μg / ml) is shown in Table 2.

[0101]

[Table 2]

As can be seen from Table 2, Ustyroxine A
And Ustyroxine B against various human tumor cells,
It showed an excellent growth inhibitory effect.

[0103]

The thyroxine A and the thyroxine B of the present invention and their derivatives show an excellent growth inhibitory effect on tumor cells and are useful as antitumor agents.

──────────────────────────────────────────────────続 き Continued on the front page (58) Field surveyed (Int. Cl. ⁷ , DB name) C07K 5/103 C12P 21/00-21/06 BIOSIS (DIALOG) WPI (DIALOG) REGISTRY (STN) CA (STN) )

Claims (4)

(57) [Claims] 1. A compound of the general formula (Wherein, R ¹ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R ² represents a hydrogen atom or an aliphatic acyl group having 2 to 6 carbon atoms, and R ³ represents a methyl group or a propyl group. Or a salt thereof. 2. The compound represented by the general formula (I) according to claim 1, wherein (Wherein, R ¹ represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms, R ² represents a hydrogen atom or an aliphatic acyl group having 2 to 6 carbon atoms, and R ³ represents a methyl group or a propyl group. Or a salt thereof. 3. A general formula belonging to the genus Ustyraginoidea
(II) ## STR3 ## (Wherein R ¹ and R ² are hydrogen atoms, R ³ is a methyl group or
Shows a propyl group. Using a rice koji fungus capable of producing the compound represented by
From the rice koji, general formula (II) (wherein R ¹ and R ² are water
An element atom and R ³ represent a methyl group or an isopropyl group. )
A compound represented by the general formula:
(II) (wherein R ¹ and R ² are hydrogen atoms, and R ³ is a methyl group
Or an isopropyl group. )) . 4. A general formula belonging to the genus Ustyraginoidea
(II) ## STR4 ## (Wherein R ¹ and R ² are hydrogen atoms, R ³ is a methyl group or
Shows a propyl group. The rice koji fungus having the ability to produce the compound represented by the following formula: Ustilaginoidea virens (Cooke)
Takahashi SANK 15391 strain (FERM BP-3691: FERM
BP-3691).