WO1993014111A1

WO1993014111A1 - Novel compound ustiloxin a or b, or derivative thereof

Info

Publication number: WO1993014111A1
Application number: PCT/JP1993/000018
Authority: WO
Inventors: Shigeo Iwasaki; Kuniko Koiso; Tomowo Kobayashi
Original assignee: Sankyo Company, Limited
Priority date: 1992-01-08
Filing date: 1993-01-08
Publication date: 1993-07-22
Also published as: AU3266593A

Abstract

A novel compound ustiloxin A or B, represented by general formula (I), having a potent antitumor activity, derivatives thereof and salts thereof, wherein R1 represents hydrogen or C¿1?-C5 alkyl, R?2¿ represents hydrogen or C¿2?-C6 aliphatic acyl, and R?3¿ represents methyl or propyl.

Description

Ming ίϋ m

Novel compounds styroxine A or usthyroxine B or their derivatives ^ [Technical Field]

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 method for producing them.

[Background technology]

The rice koji disease fungus (Ustyraginoidea 'Virens Cooke') belongs to imperfect fungi, but a complete generation formed on the sclerotium has been observed, and Claviceps virens Sakurai and (Yadao Harada, Nissho Disease Report, Volume 3, p. 387 (1984)). Ustilagino idinn A, B and C (Tadaharu Yabuta, Yusuke Sumiki, Journal of the Japanese Society of Agricultural Chemistry, Vol. 9, p. 47S (1933)) and Ustilaginoidin D, E, F, G, I1, I and J (K. Koama and S. Natori, Chem. Pharm. Bull., Vol. 36, p. 146, (1988)) are known. However, these compounds and the ustyroxins of the present invention are clearly different in physicochemical properties and structure.

Examples of the 13-membered ring compound having one ether bond and two peptide bonds in 璟 include Phomopsisin A (Phomopsin A) produced from Phomopsis leptostromiformis (CLAUDE CJ CULVENOR et al., Tetrahedron, Vol. 45, No. 8, p. 2351 (1989)) is known. However, the structure of the side chain is completely different from usthyroxines.

[Disclosure of the Invention]

The present inventors have found that the novel compounds ustiloxin A and ゥ styroxine B, which are novel compounds isolated from rice koji, which naturally occurred in rice ears or artificially generated in rice ears, caused a strong antitumor effect. The present inventors have found that the inducer has a strong antitumor effect, and have completed the present invention. The present invention General formula

(Where

R ¹ is In addition hydrogen atom an alkyl group having 1 to 5 carbon atoms, R ² is an aliphatic Ashiru group having 2 to 6 water桌原resonator or carbon atoms, R ³ is methyl group or propyl group

Is shown. )

With respect to the compound represented by or a salt thereof,

More preferably,

In the above general formula (I),

General formula

(Where

R ¹ is a water atom or a alkyl group having 1 to 5 carbon atoms,

R ² is water, an atom or an aliphatic acyl group having 2 to 6 carbon atoms,

n R ³ is a methyl group or a pulp ί

Is shown. )

Or a salt thereof.

When R ¹ is an alkyl group having 1 to 5 carbon atoms, examples thereof include straight-chain or branched-chain alkyl groups such as methyl, ethyl, propyl, butyl, t-butyl, and pentyl. 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 acetyl group having 2 to 6 carbon atoms, examples thereof include a linear or branched aliphatic acetyl group such as acetyl, propionyl, butyryl, valeryl, and hexanoyl. Preferably, it is an aliphatic acetyl group having 2 to 4 carbon atoms in a direct sale form or a branched sale form, and 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, dimethylamine, and dipyramine And salts of aliphatic primary to tertiary amines such as trimethylamine; salts of amino acids such as lysine and arginine; and ammonium salts. Preferably, it is a salt of an alkaline 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, hydroiodic acid; nitrates; perchlorates; sulfates; Or an alkanesulfonic acid salt of a lower alkanesulfonic acid such as methanesulfonic acid, trifluoromethanesulfonic acid or ethanesulfonic acid; a salt of an arylarylsulfonic acid such as benzenesulfonic acid or di-toluenesulfonic acid; glutamic acid or asparagine Organic acid salts such as salts of amino acids such as acids; 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 optimal one is a pharmacologically acceptable i. ' The compound having the above general formula (I) has ^ isomerism. In the compound having the above general formula (I '), all of these isomers are represented by the formula ^5- . Therefore, the present invention includes all of these isomers and mixtures of these isomers.

Further, in the compound having the general formula (I) of the present invention, a so-called “prodrug compound” which is derived into a compound having the general formula (I) in vivo

All are included.

Compound having the general formula (I):

Preferably,

R ¹ represents a hydrogen atom or an alkyl group having 1 to 3 carbon atoms,

R ² represents a hydrogen atom or an aliphatic acyl group having 2 to 4 carbon atoms,

R represents a methyl group or a propyl group,

A compound or a salt thereof.

a¾ properly t,

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,

A compound or a salt thereof.

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

【table 1】

Compound number R ¹ RR

1 H H M e One H A c M e

3 H Prn M e 4 H B y r M e 5 H C O (CH CH M e 6 e H M e 7 M e A c M e 8 M e Prn M e 9 M e B yr M e 0 E t H M e

E t Ac Me

1 2 P r H M e 1 3 P r A c M e 14 B u H M e 15 B u A c M e 16 H H P r

H = hydrogen atom, Me = methyl, Et = ethyl,

Ρ r = propyl, Bu = butyl, A c = acyl,

Prn = propionyl, Byr = butyryl

Represents a group.

In the above compound ^, Preferably, they are exemplified compound numbers 2, 3, G. 7.10.11, 112, 1.1, I6 or a salt thereof.

Optimally, the compound No. 1-2.6.7.10, 16 or a salt thereof. According to the present invention, the thyroxine belonging to the genus Ustyraginoidea or the thyroxine B 'production ability A method for producing us thyroxine A or us thyroxine B, comprising producing rice koji in rice using a rice koji disease fungus having In particular, rice cypress A, which has the ability to produce ustyroxin A or usty sigma xin に belonging to the genus Styraginoidea, is available from Ustilaginoidea virens (Cooke) Takahashi SAN 15391 strain. (1) Among the compounds of the present invention having the general formula (I),

R ¹ represents a hydrogen atom,

R ² represents a hydrogen atom,

R ³ represents a methyl group

A compound (ie, ustyroxin A), or

R ¹ represents a hydrogen atom,

R ² represents a hydrogen atom,

R ³ represents a propyl group,

Compound (ie, usthyroxine B)

Is obtained as follows.

That is, the rice koji (koji grain) generated in the rice spike due to the infection of the rice koji disease fungus (Ustilaginoidea virens Cooke) in the rice field is collected. The koji grains can be picked up by hand from the 'spreading ears' in the piloerection, air-dried indoors for about one week, and pulverized with a mill. In addition, after crushing, those stored in a refrigerator at around 5 ° C can also be used.

By extracting water or alcohols such as methanol, ethanol and the like, organic acids such as ethyl acetate and ters, and ketones such as acetone and methyl ethyl ketone alone or in combination, extraction is performed. An extract containing thyroxine A or usthyroxine B can be obtained. This can be achieved by isolating and purifying Usuloxin A or Ustiloxin B from the product: 2. Utilizing its physicochemical properties. For example, it can be removed using an adsorbent. Examples of the adsorbent used include activated carbon or adsorbent resin Amberlite XAI3-2, XAD-4, XAD-7, etc. (ROHM-AND ■ '\-S), Diaion HP 10 , HP20, IIP50, CHP20P (manufactured by Mitsubishi Kasei), Polyamide gel (manufactured by Elm) and the like are used. The extract containing usthyroxine A or usthyroxin B is passed through the above adsorbent layer to adsorb and remove impurities contained in the solution containing ustyroxin A or ゥ thyroxine B, or ustyroxine A or usthyroxine. It is obtained by adsorbing B and eluting with a suitable solvent. In order to further purify the thus obtained usthyroxine A or usthyroxine B, cellulose such as Avicel (Asahi Kasei Kogyo Co., Ltd.) or Sephadex LH-20 (manufactured by Pharmacia) is used. Effective methods include column chromatography, extraction using the difference in the partition ratio of solvents mixed with usthyroxine A or b, and the counter-current partitioning method.

Ustiloxin 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, a spore suspension of the rice koji disease strain Ustilaginoidea virens (Cooke) Takahashi SANK 15391 isolated by the present inventors was sprayed onto rice ears growing in a field, and rice koji generated after a certain period of time was sprayed. Ustyroxin A or Ustyroxine B can be obtained in the same manner as described above.

In this case, the rice two to six weeks before heading can be inoculated from above with conidia or chlamydospores of SANK 15391 to generate rice koji.

The conidiospore suspension can be obtained by transplanting the mycelium pieces into an Erlenmeyer flask containing a potato decoction medium for adding sucrose, and performing liquid culture under dark conditions at 26 ° C. for 12 to 20 days. A surfactant 20, such as Tween This suspension was added 0.05% equivalent amount, field of rice, a method of approximately IL / ra ² sprayed and inoculated from the top of the body is preferred.

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

Chlamydospores can be obtained by the following method. If ^ 搪 if] Using a potato decoction medium at step 26 26: 菡してして培養!!!! 濁濁濁濁濁濁濁濁.

Culture in 2 dark cows.

Conidiospores are formed in the first week or so, then !? Membrane spores are formed. After culturing for 1 to 3 months under these conditions, leave it for 10 days with the lid closed and leave it in a 22 C 內 outside room10 for 7 days. Thus, desired chlamydospores can be obtained.

The chaos of the chlamydospores directly above the rice plant in the field can produce the koji. 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.

There are no particular restrictions on the test varieties, but late-growing varieties such as No. 1 and Norin 21 are preferred.

Rice koji strain Ustilaginoidea virens (Cooke) Takahashi SAN K 15391 which makes rice koji can be isolated, stored and used as needed as follows. Immediately, spores (chlamydospores) are collected from rice panicles that show diseased and dark green symptoms, and are placed in a culture medium such as a potato-glu-use 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 a suitable agar medium, for example, potato dextrose agar medium, and cultured at 23 to 28 ° C, and the well-grown one is stored. Growth on potato dextrose agar is slow, with initially white fluffy hyphae growing, but then dark greenish brown. It also produces a dark green soluble pigment in the medium. The mycelium is colored and has partition walls. Its diameter is 2-0 to 5.0 μιη, the wall is very thick, dark green-brown, and often forms hyphal mass. Conidia occur in the symposiform type, and are 1 cell, colorless, 5.0-8.0 Χ 3.0-4.5 m. Chlamydospores found in rice koji are not observed.

These properties are described in detail in Takaoka et al.'S report (Saitama Prefectural Agricultural Experiment Station Research Report No. 2, pp. 1-20, published in 351).

Rice koji fungus isolated by the present inventors Ust i lagino idea virens iCooke) Takahashi SA K15391 has been deposited internationally with the Research Institute of Microbial Industry and Technology of the Ministry of International Trade and Industry (Deposit No., Deposit No. 36! H ^ i FERM BP-36! 31): Deposit date December 24, 1991 Says).

2) a compound of the present invention having the general formula (I),

R ¹ represents an alkyl group,

The compound is obtained as follows.

That is, it can be attained by carrying out an esterification reaction with ustiloxin Α or ustiloxin Β and an alcohol such as methanol or ethanol with an acid. The acid used in the reaction is not particularly limited. For example, mineral acids such as sulfuric acid, hydrochloric acid, chloric acid, and perchloric acid; and organic acids such as formic acid and trifluoroacetic acid are preferred. The reaction temperature and reaction time vary depending on the reagents and solvents used, but are usually 0-100. C can be converted to another alkyl group by transesterification in 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. If an acid is used in excess of usthyroxine A or usthyroxine B as the 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 also be achieved by reacting with diazoal compounds such as diazome. 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. Ethers such as getyl ether, tetrahydrofuran, dioxane, and dimethoxetane; esters such as ethyl acetate; dimethylform Amides such as amides; sulfoxides such as dimethyl sulfoxide; are preferred. The reaction temperature and the reaction time vary depending on the reagents, solvents and the like to be used.

Alternatively, it can be obtained by alkylating a compound in which R ¹ represents a hydrogen atom. The reaction is carried out in the presence of an alkali metal carbonate such as potassium carbonate or sodium carbonate, with an alkyl halide compound such as methyl iodide or butyl iodide. 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. Tetrahydrofuran, dioxane, dioxane Ethers such as methoxenone: dimethylpho; remamides; sulphoxides such as dimethylsulfoxide; are preferred, and the reaction temperature and reaction time are ffl. Although different, usually 0 to 0 ° C \ L to time is suitable.

After completion of the reaction, the solvent of the target compound is distilled off from the reaction mixture, and water and an organic solvent such as nitrile acetate are added to the residue to carry out liquid separation. It is obtained by concentrating the organic layer and purifying the residue by recrystallization or ordinary column chromatography.

3) Among the compounds of the present invention having the general formula fl),

R ² represents an aliphatic acyl group,

The compound is obtained as follows.

That is, it can be 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; and 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. For example, water, alcohols such as methanol and ethanol, or a mixed solvent thereof are preferable. The reaction is performed in the presence of a base. Examples of the base used include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal carbonates such as sodium carbonate and potassium carbonate; sodium hydrogen carbonate and hydrogen carbonate Metal bicarbonates such as potassium sulfide; aromatic amines such as pyridine; heterocyclic amines such as proline; aliphatic amines such as triethylamine and getylamine. The reaction temperature varies depending on the type of the acylating agent, etc. Usually, 0 to 50 ° C is preferable. The reaction time varies depending on the reaction conditions, but is usually 1 to 5 hours.

The target compound thus obtained may be subjected, if necessary, to adsorption column chromatography using a carrier such as silica gel or florisil; reverse-phase partition chromatography using silica gel having octadecyl or octyl groups; Distribution column chromatography using frullose or SEPHADE "NOX Lii-20 [manufactured by FULL MACIA CORPORATION] or the like; Use a difference in the distribution ratio of the pure substance to the solvent to make the purification process easier.

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

Compounds in which R ¹ is a hydrogen atom include compounds in which R ¹ is an alkyl compound, such as sodium hydroxide, hydroxide hydroxide, and other alkali metal hydroxides; and sodium carbonate, potassium carbonate, and other alkali metals. It can also be obtained by hydrolyzing in the presence of a metal carbonate.

The compound having the above general formula (I) thus obtained is further exemplified by an adsorption column chromatograph using a carrier such as silica gel or Florisil, and Fadex LH-20 (manufactured by Pharmacia). It can be purified by partition column chromatography used, high-performance liquid chromatography using normal phase or reverse phase columns, and the like.

[Best Mode for Carrying Out the Invention]

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

Example 1. Styroxine A

Rice ears caused by the infection of rice koji fungus (Ustilaginoidea virens Cooke) :; spikelets that were spontaneously generated were collected. After drying, 100 _g of the rice koji were pulverized in a mixer with 10 times the volume of water, and extracted by shaking at room temperature for i hours. 100 () ml of the extract was filtered, concentrated to one tenth, and applied to a 50 ml iDODS column (ODS-T. Senshiyu Kagaku (Sekiyu)). The eluate was developed and eluted with 2.5 liters of water, 2.5 liters of a mixture of methanol: water = 2: 8, and 1.5 liters of 100% methanol while monitoring at 254 nm of ultraviolet absorption. 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 0DS column. The mixture was developed and eluted with 0.5 liter of a mixture of methanol and water at a ratio of 2:98, and 200 ml of the eluted fraction between 200 and 400 ml was collected to obtain a fraction containing ustiloxin A. When these fractions were concentrated, crude crystals of about 20 nig were obtained. When this crude crystal is recrystallized with water, the melting point

14 mg of pure target compound having a temperature of 195 ° C (decomposition) were obtained as crystals. Ustyroxine A obtained in this example exhibited the following physical properties.

1) Structural formula

NH-CH ₂ -C00H

H00C

) Substance properties: water-soluble, white needle-like crystals

) Molecular formula: C ₂₃ Η ,, 0, _Ξ Ν ₅ : ·;

) Molecule-: 673

) Mass spectrometry: FAB-MS; m / z 674 (M + H !:

HR FAB-S; m / 2 674.2730 (M + H! (Theoretical 674.2718) Elemental analysis: C ₂₈ II., 30 _12. S 2.5 il ₂ () as (%)

Measured: 46.69; H &92;, :). 64 S, 4.23 Theoretical: 46.80; H 6.82 ;, 9.75 S, 4.45

) UV absorption spectrum: max nm (ε)

The ultraviolet absorption spectrum measured in water is as follows.

207 (21300), 249 (6000). 287 (3200).

) Infrared absorption spectrum:: y max cm " ¹

Infrared absorption spectrum measured by the bromide power (KBr) tablet method is as shown below.

3700 ~ 2400 (broad), 1650, 1640, 1540, 1500, 1465, 1400, 1295, 1225, 1155, 1130, 1090. 1030, 900.

) — Nuclear magnetic resonance spectrum: (δ; ρρπι)

The nuclear magnetic resonance spectrum (500 MHz) measured in heavy water at 50 ° C using tetramethylsilane as an external standard is as shown below.

0.80 (311, d, J = 7.0 Hz), 0.88 (3Η, d, J = 7.0 Hz), 1.09 (3H, t, J = 7.2 Hz), 1.73 (lH, dd, J = 7.2 and 14.2 Hz), 1.77 (3H, s),

1.532 (111, dd, J = 7.0 and 10.0 Hz),

2.12 (lH, ddd, J = 3.0,8.0 and 15.0 Hz),

2.22 (111, ddd, J = 8.0, 10.0 and 15.0 Hz),

2.24 (lll, dd, J = 7.2 and 14.2 Hz), 2.77 (3H, s),

3.04 (lll, dd, J = 3.0 and 13. & Hz),

3.33 (lH, dd, J = 10.0 and 1: 5.6 Hz),

3.73 (211, s), 4.01 (1H.dd, J = 4.0 and 8ί) Hz), ' 4. I3i "lH, d. J = ll). () IIz 4.23 (Hi, J, J = H). Π Hz!

4.33 1 [, tt.i = 3.0 and 10.0 Hz) l.S 11i [.si.

4.96il (l, d, .J = ll) .0 Hz), 7.LI (lILs) 7.61 (lH, s)

) ¹³ C nuclear magnetic resonance spectrum: S _; ppm I

Nuclear magnetic spectrum (00 MHz) measured in heavy water using tetra-ethylsilane as an external standard is as follows.

7.8 (q 17.9 (q) 13.3 (q)> 2i.1 iq) 28.7 (d)

22.1 ( _(} 2), 36.7it), 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) .13G.4 (s)

146.0 (s), 152.2 (s), 16B.4 (s) .170.3 (s), m.o (s),

174.4 (s), 176.3 (s) ₀

) Solubility:

Easily soluble in water, soluble in aqueous methanol.

) Color reaction:

Positive for ninhydrin.

) Thin layer chromatography:

Rf value; 0.16

Adsorbent; silica gel plate Art. 5715 (Merck)

Developing solvent: π-butanol: acetic acid: water = 3: 1: 1

Detection: UV 254 nra and ninhydrin coloring.

) Amino acid analysis:

After hydrolysis with 6 N hydrochloric acid for 11 hours at 20 ° C., the solvent was distilled off from the reaction mixture. The residue was analyzed with an amino acid analyzer to detect palin and glycine. Analysis of valine by high performance liquid chromatography> L-valine.

Column: Enantio L1 (4.6 mm ID X250, manufactured by Tosoh Corporation) Solvent: 0.8 mM CuSOi

am 'ϊ Flow ill; 1 ml / '→

Inspection; infrared 25ϋ nm-.

Example 2. 'Styroxine A

Koji grains were generated in rice plants by artificial inoculation, and Soxin A was isolated from O and purified.

Rice was raised by the following method.

The seedlings sown on April 14 were transplanted to Honda on May 20. The cultivation density was: i. 75 plants per 3 m ² , i. Fertilizer: nitrogen (N) as the basal dressing, phosphate (P ₂ 0,) and using the per 10a 8 Kg potassium to (K ₂ 0) as the component amount, the nitrogen (N) as a top dressing on July 14 2 kg was applied per llhi. Other methods followed the customary law.

Rice koji disease SANK 15391 strain was cultured, and chlamydospores formed after standing were obtained by the following method.

The mycelial suspension obtained by liquid culture at 26 ° C for 7 days using a potato decoction medium for sucrose was poured onto a potato decoction agar medium for sucrose so that it spreads thinly.

The cells were cultured in the dark at 25 ° C.

Conidia formed in the first week, but chlamydospores formed thereafter. After culturing for 1 to 3 months under these conditions, leave the lid of the Petri dish in the room inside and outside for 7 days with the lid on, and pulverize the dried product together with the medium with a mixer to obtain thick film spores. Was.

The rice spores were generated by inoculating the chlamydospores directly on the rice plant in the field. In other words, rice spikelets were generated when sprayed on rice ears growing in the field and left for a certain period of time (about 2 months). These rice koji were collected and uthyroxine A was isolated and purified in the same manner as described in Example 1. 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 the same as those of ustyroxin A obtained in Example 1.

Example 3. Ustyroxine A dimethyl ester 'dihydrochloride

5 mg of usthyroxine A obtained in Example 1 was dissolved in 0.5 ml of methanol containing 12% hydrochloric acid and released overnight at 4 ° (: overnight. The solution was concentrated under reduced E; After distilling off the agent, the desired product-m.m¾ was obtained.

■■ V Physical properties of Roxin A dimers and dihydrochloride

1) Structural formula

2) W—nuclear magnetic resonance spectrum: (5; ppm)

The nuclear magnetic resonance spectrum (500 MHz) measured at 50 using tetramethylsilane as an external standard in deuterated dimethyl sulfoxide is as follows.

$> O o

0.77 (3H, d), 0.83 (3H, d), 1.02 (3H, dd), 1.43 (3H, s) 1.87 (lH, m), 1.98 (2H, m), 2.08 (lH, dd), 2.43 ( 3H, s), 2.79 (lH, dd),

3.04 (III, dd), 3.61 (3H, s), 3.74 (3H, s) \ 3.82 (3H, m) .4.07 (lH, m), 4.22 (lH, dd), 4.47 (1H, ddK 4-83 (lH, d). 4.93 (lH, dd),

6.51 (lH, s), 7.07 (1H, s)

3) Thin layer chromatography:

Rf ϋ; 0.33

Adsorption 剂; Silica gel plate Art. 5715 (Merck)

Developing solvent: n-butanol: methanol: water = 3: 1: 1.

Example 4. '-Acetylus thyroxine A

Dissolve 5.4 mg of ustyroxine A obtained in Example 1 in 0.5 ml of a 2% aqueous sodium hydrogen carbonate solution, add an excess of anhydrous II, and stir at 0 for 2 hours. . The reaction mixture bicarbonate - after neutralization with Totori ¹ ^ anhydrous solution, Guiyaio (manufactured by Mitsubishi Kasei Co.) Secondary HP 2 forces the ram was adsorbed to 2 ml. Elution was performed using methanol as a developing solvent, and 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 the target compounds 4.6. .,

The physical properties of 4'-N-acetylus thyroxine A. dinatrium salt are as follows3.

1) Structural formula

NaOOC

2) Color reaction:

Negative for ninhydrin.

3) Thin layer chromatography:

Rf ί [β; 0.39

Adsorption 剂; Silica gel plate Art. 5715 (Merck)

Developing solvent: n-butanol: methanol: water = 3: 1: 1.

Example 5 Ustyroxine B

Koji grains were generated in rice by artificial inoculation, and ustyroxin B was isolated and purified from this. The artificial inoculation was performed using the rice koji fungus SANK 15391 strain, and the inoculation was performed according to the method described in Example 2.

The rice koji generated in the rice ears was collected, dried, and then 10D _g was pulverized with a mixer together with 10 times the amount of water, and extracted by shaking at room temperature for 1 hour. After filtering 1000 ml of the extract, it is concentrated to one-tenth, and a 50 ml ODS column (ODS-SS-1 () 2ϋ 、, Senshuyu Kagaku Co., Ltd. Made). UV absorption at 254 am, while mixing: 2.5 liters of water, 1 liter of water, and 2.5% of a mixture of water = 2: 8. 'L-, ίθί)% Ά. It was eluted with δ solution. The fraction eluted with the g solution of methanol: water = 2: 8 was collected and concentrated. After dissolving this concentrate in water, re-inject into a 30 ml 0DS column? ,did. The eluate was developed and eluted with D-5 liters of a mixture of Knorr: water = 2: 98 to obtain a fraction containing Ustyroxine B, which flows out before Ustyroxine A. This fraction was again applied to the ODS column; Then, the mixture was developed and eluted with a mixture of methanol and water at a ratio of 1:33 to obtain a powder of genus thyroxine. This powder was injected into a 0DS column: this was eluted and developed with a mixture of methanol: water = 25: 75 containing 0.02% trifluoroacetic acid to obtain a purified fraction of ustiloxin II. About 3 mg of ustyroxin B was obtained per 100 g of rice koji.

Ustyroxine B obtained in this example exhibited the following physical properties.

1) Structural formula

2) Property of substance: water-soluble, white solid

) Molecular formula: C _2S H ₃₉ N _S 012 S

) Molecular weight: G 45

) Mass spec: FAB-MS: m / z 646 (M + H)

) Optical rotation: +14.1 ° (c = 0.55, H ₂ 0)

) UV absorption spectrum:; Lmax nm (ε)

The UV absorption spectrum measured in water is as shown below; ! 13:) 1) ()), 252 ίηΟΟΠ '. 290! 2501) 1

8 Infrared absorption spectrum: ν cm " ¹

The infrared absorption spectrum measured with calcium fluoride (CaF) ^ film (film) is as follows.

37 (M) -24 () 0 (broad), 1670, 1B00, 1550, 151) 0, 1465. 1430, 1380, 1290, 1200, 1140

9) 'Η- nuclear magnetic resonance spectrum: (δ; ρρπι)

Nuclear magnetic resonance spectra (500) measured in 5 () using 2,2-dimethyl-2-shiventan-5-sulfonic acid sodium (DSS) as an external standard in heavy water. MHz) is as shown below.

() .92 (3H, d (i, J = 7.2, 7.2Hz), 1.14 (311, d,, J = 7.0Hz), .63 (1H, dq, J = 14.0, 7.2Hz), l.BS (3H, s), 2.01 (1H, ddd, J = 3. (), 15.0, 8.0 Hz), 2.0δ (1Η, dq, J = 14.0, 7.2 Hz), 2.12 (1H, ddd,,) = 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 = 17.0Hz, 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 (11m , J = 2.8, 10.0, 3.0, 10.0Hz), 4.37 (1H, q, J = 7.OHz), 4.65 (1H, s), 4.80 (111, d, J = 10.0Hz), 7.28 (1H, s), 7.47 (1H, s)

1 0) ¹³ H-nuclear magnetic resonance spectrum (δ; ρρπι)

The nuclear magnetic resonance spectrum (100 ΜΗ 測定) measured in heavy water using DSS as the external standard is as follows.

8.36 (q), 1B.72 (q), 22.24 (q), 31.68 (t), 33.00 (q), 37.0S (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), 84.48 (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), 172.72 (s), 174.82 (s), 176.92 (s)

1 1) Solubility:

Soluble in water, soluble in water methanol.

1 2) Color reaction: positive for ninhydrin.

1 3) Thin layer chromatography:

Rf cruise: D.14 Adsorbent-Shiri gel plate 5715

Expanded solution ¾; n-bukunol: m: water = 3 _: I: i

Greetings: UV light 254 nm. Ninihydrin% color

1 4) Amino acid analysis:

After hydrolysis with 6 N hydrochloric acid at 110 ° C for 20 hours, the solvent was distilled off from the reaction mixture. The resulting residue was separated using an amino acid analyzer. Alanine and -lysine were detected. Analysis of alanine by HPI was consistent with L-alanine:

Column; Enantio L1 (4.6 ID Χ2δί) mm, E ,, 'single ¾ made': solvent; 0.8 mM CuS0 ₄

? Ίτπ / ■ ϊ 2 /

Flow rate; 1 ml / min ''

Detection: Infrared 250 rim UV absorption spectrum of usthyroxine B, optical rotation is similar to usthyroxin A, and its molecular weight is 645, which is 28 (corresponding to C ₂ H ₄ ) smaller than usthyroxine A. This indicates that the chromophore contained in the structure and the absolute configuration of the steric structure are consistent with ustyroxin A.

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

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

Example 6

Ustyroxine Α and ustyroxine の of the present invention and their induction have an antitumor effect.

The following shows the antitumor activity of styroxine A and X "^ oral toxin B.

0 The result is i.

Investigation of antitumor activity of ustyroxine / λ¾ubistyroxine B according to the method of Array (Michael C. Alley) et al. (Canc''Research, 48, 589-f; 01 (1988)) did.,

That was suspended various cancer cells 9Β well culture Blanket Ixlt) ^three each Z100 μ ΐ medium (RPMI-1 40 + 10% FCS) .., however, RERF- IX- ΜΑ the ^two 4Χ 10, WiDr , SW- 480, KU-2 is 5X 10 ^2, Ζίί- 75- 30 is 3Χ 1 (1 ³ pieces, SBC-5 is 2. suspended ^two [delta] X 10. these suspensions 5% C0 ₂ under 24 hours incubation at 37, added medium of 100 M l containing diluted sample, an additional 48-hr cultured. after incubation under the same conditions, washed 3 times with medium of 200 ll, removing agent was. then, under 5% C0 _2, after 96 hours at 37 ° C, 50 / ig / 50μ while handling Μπ of 1, and cultured further for 4 hours under the same conditions. remove the medium, 150 μ ΐ of added and stirred DMS0, it was calculated cell growth rate from the absorbance at 540 nm the concentration of test compound required to inhibit cell growth by 50%; shows the (IC _S / zg Zml.) Table 1.. .

[Table 2] Inhibitory effect of ustiloxin A and usthyroxine B on human Bl «« vesicle »cells. (Ue

Ustylokidine A S-FU (vs. s compound)

MKN-1 Ι9β gland] Squamous epithelium S 2.46 2.S5 5.73

Yang-7 Xiao as differentiated gland as 3.75 5.04 3.34

-74 Well differentiated tubular gland s 3.9 & 11 1.40

RERF-LC-HA Lung s Kosode 4.27 β.8 0.314

SBC-5 Lung s Small sleeve vesicle S 0.254

MCF-7 breast s gland S 0.656 1.38 0.915

ZR-75-30 Solid gland duct 35 2.93> 1D

WiDr connection 3.57 5.S 1.05

SW-480 Result MS gland as 4..G4 7.3 3.44

KU-2 3.3S 2.55 2.79

2. As in FIJI, Ustyroxine A and Ustyroxine B are various types of human Excellent growth inhibitory effect on tumor cells

[Industrial potential]

Prior to the present invention, the compound having the s-general formula (I) is a novel compound which has not been described in the literature, and has a strong growth inhibitory effect on various human cancer cells. Before the invention; E—The compound having the general formula (r) is used as a medicament as a medicament, in a funeral hall, or in accordance with a conventional method, by itself or an appropriate pharmaceutically acceptable carrier, excipient, or diluent. They can be mixed and safely administered orally or parenterally in the form of powders, condyles, tablets, capsules, injections and the like. The dose varies depending on the target disease, the administration route and the number of administrations. For example, the effective daily dose for adults is 1

It is preferable to administer 0.01 mg to 80 mg per kg, or 0.5 mg to 12 G mg for oral administration, once or in several divided doses according to the symptoms.

Claims

Range of claim General formula

(Where

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,

R ³ is a methyl or propyl group

Is shown. )

Or a salt thereof.

2.

In the general formula (I) described in claim 1,

General formula

R! OOC /

(Where

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,

R ³ is a methyl or propyl group

Is shown. )

Or a salt thereof.

3-Generating a rice koji in a rice plant using a rice koji fungus capable of producing ustyroxine A or ustyroxine B belonging to the genus Ustyraginoidea, and isolating ustyroxine A or ustyroxine B from the rice koji. A method for producing ustiloxin A or ustiloxin B, which is characterized in that: ·

4. Aspergillus oryzae having the ability to produce ustyroxine A or ustyroxine B belonging to the genus Ustyraginoidea was produced by Ustilaginoidea virens (Cooke) Takahashi SAN K15391 strain (Microe Kenjiro No. 3691: BP-3691). The method of claim 3.

5. A pharmaceutical composition comprising a therapeutically effective amount of a compound as defined in claim 1 or 2, or a salt thereof, together with a pharmaceutically acceptable carrier or excipient.

6. An anti-cancer agent comprising a therapeutically effective amount of the compound defined in claim 1 or 2 or a salt thereof together with a pharmaceutically acceptable carrier or excipient.

7. A method for treating or preventing cancer, comprising administering to a cancer patient a therapeutically effective amount of the compound defined in claim 1 or 2 or a salt thereof.