JPH034080B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel chemical method for the production of Bredinin.

Bredeinin (4-carbamoyl-1-β-D
-ribofuranosyl-imidazolium-5-oleate) has the formula Eupenicillium
It was first produced by a fermentation method using Bredeinin-producing bacteria belonging to the genus [J. Antibiotics, Vol. 27,
No. 10, 775-782 (1974), Special Publication No. 49-12720], and has not only strong immunosuppressive activity, but also anticandida activity, antiviral activity, and antitumor activity [J. Antibiotics, Vol. 28, No. 10, 798-803
(1975) Chem.Pharm.Bull., 23(1), 245-246
(1975), Cancer Research, 35, 1643-1648
(1975), Special Publication No. 49-12720].

Other methods for producing bredeinin other than the above include 4.
-Chemical N-glycosidation method to carbamoyl-imidazolium-5-oleate and β-D-ribofuranose [Special Publication No. 47196/1986, Japanese Patent Publication No. 1986-1999
−52038, Chem.Pharm.Bull., 23(1), 245-246
(1975)], 4-carbamoyl-imidazolium-
A method for biochemically salvage synthesizing 5-oleate using microorganisms (Japanese Patent Publication No. 54-36678) is mentioned.

As a result of various studies on the chemical production method of bredeinin, the present inventors discovered that AICA riboside (5-
The present invention was completed by discovering a new method for converting amino-imidazole-4-carboxamide riboside (amino-imidazole-4-carboxamide riboside) into bredeinine.

The present invention is based on the formula (In the formula, R ₁ and R ₂ each represent a hydrogen atom or a hydroxyl group-protecting group, and R ₃ represents a hydrogen atom or a hydroxyl group-protecting group.) AICA riboside represented by the formula (wherein R ₁ , R ₂ and R ₃ have the same meanings as above) was obtained, and the compound [3]
is ring-closed with imidazole, and when the hydroxyl groups at the 2′, 3′, and/or 5′ positions are protected,
The first objective of the invention is to provide a novel manufacturing method for chemically synthesizing bredeinin from AICA riboside, which is characterized by removing the protective group. The second objective is to provide a new method for synthesizing bredeinin at a lower cost than conventional chemical methods.

AICA riboside [2], which is the starting material in the present invention, is AICA riboside or 2'-position, 3'-position and
AICA with the 5′-hydroxyl group protected with an appropriate protecting group
Ribosides are used.

As the above-mentioned protecting group, a known hydroxyl group-protecting group used in the fields of nucleic acid chemistry or sugar chemistry is used. Examples of protecting groups for hydroxyl groups at the 2' and 3' positions include acyl groups such as formyl, acetyl, methoxyacetyl, benzoyl, p-chlorobenzyloxyacetyl, t-butyl, benzyl, α-ethoxyethyl, α- Examples include methoxyisopropyl, tetrahydropyranyl, methoxytetrahydropyranyl, o-nitrobenzyl, and t-butyldiphenylsilyl groups. Furthermore, the 2'- and 3'-position hydroxyl groups are protected in such a way that they form a cyclic acetal together with the adjacent oxygen atom. Examples of such protecting groups include isopropylidene, methoxymethylene, methoxyethylidene, ethoxymethylene, ethoxyethylidene, benzylidene, and cycloalkylidene groups. Examples of protecting groups for the hydroxyl group at the 5' position include acyl groups such as formyl, acetyl, chloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, pivaloyl, benzoyl, β-benzoylpropionyl, phenoxyacetyl, and trityloxyacetyl. , trityl groups such as trityl, monomethoxytrityl, dimethoxytrityl, and trimethoxytrityl, and methoxymethyl groups.

The above-mentioned protecting groups can be introduced using known methods, but it is important to select a protecting group that can be removed later in an efficient manner and that can be removed in one step. preferable.

In the present invention, first, AICA riboside [2]
Intermediate compound [3] is produced by a photochemical reaction caused by irradiation with light under acidic conditions. The above acidic conditions may be within a PH range where AICA riboside [2] can be protonated, but the acidic conditions are usually PH 0.1 to 4. To achieve such acidic conditions, a solution prepared by appropriately diluting an inorganic acid such as hydrochloric acid, sulfuric acid, nitric acid, or phosphoric acid or an organic acid such as acetic acid or trifluoroacetic acid may be used.

The above photochemical reaction can be performed even under cooling, but
Usually done at room temperature. The reaction time mainly depends on the type of starting material [2] and its concentration; the lower the concentration, the faster the reaction, and the higher the concentration, the longer the reaction takes, but the end point of the reaction depends on the use of an appropriate carrier. It can be appropriately determined by tracking the starting material [2] and the produced intermediate compound [3] by thin layer chromatography or high performance liquid chromatography. Normally
It takes about 30 minutes to 24 hours. The light irradiation method is carried out by irradiation with an ultraviolet light bulb, for example, a mercury lamp. During the reaction, if oxygen is present in the reaction solution, it may be converted to ozone, which may adversely affect the reaction, so do not use it under a flow of inert gas, such as argon gas or nitrogen gas. It is advantageous to carry out the reaction in terms of preventing side reactions.

The intermediate compound [3] thus obtained is
After being neutralized, it is optionally concentrated under reduced pressure and extracted with a non-hydrophilic organic solvent such as chloroform or dichloromethane. If further purification is required, it can be purified by chromatography using a carrier such as silica gel, activated alumina, or adsorption resin.

Next, the intermediate compound [3] is subjected to imidazole ring closure in an appropriate organic solvent such as dimethyl sulfoxide, dimethyl formamide, dimethyl acetamide, hexamethylene phosphorylamide, etc., using formic acid, orthoformate, form This can be carried out by heating with iminoether, diethoxymethyl acetate or N-formylmorpholine, or by reacting with a pyridine solution of carbon disulfide, an alkali dithioformate or thiourea, and then subjecting it to a desulfurization reaction.

In this way, bredeinin or bredeinin in which the hydroxyl groups at the 2′, 3′, and 5′ positions are protected with protecting groups is obtained, but in order to isolate and purify these reaction products, conventional known methods are used. Just use the means. For example, a solution containing a reaction product is concentrated, a solvent is added to the resulting residue for extraction, the resulting extract is concentrated to obtain a crude reaction product, and in order to further purify this, silica gel, It may be purified by chromatography using a carrier such as activated alumina or adsorption resin.

When removing a protecting group, it is carried out by a known removal method used in nucleic acid chemistry or sugar chemistry. For example, the 2', 3', 5'-tri-O-acetyl group can be treated in ammonia-saturated methanol at room temperature or under heating, or in an alcoholic solution of an alkali metal alcoholate or an aqueous solution of an alkali metal hydroxide. It's okay. In order to obtain bredeinin, which is the main product, from the reaction solution, the reaction solution is concentrated and the residue is crystallized with an appropriate alcoholic solvent, or if necessary, chromatography using a carrier such as silica gel, activated alumina, or adsorption resin is performed. It can be purified by E.

Next, the present invention will be specifically explained with reference to Examples and Reference Examples, but the present invention is not limited thereto.

In the thin layer chromatography (TLC) in Examples and Reference Examples, the following carriers and developing solvents were used unless otherwise specified. In addition, Examples 1, 3, 6
The NMR assignment of the title compound and 7 was performed based on the position number described in formula [3].

Support: Silica gel (Merck Art5715) Developing solvent: a: Butyl acetate-acetic acid-acetone-water (10:6:
3:4) b: Chloroform-methanol (10:1) c: Chloroform-methanol (5:1) Reference example 1 2-Amino-N-β-D-ribofuranosylmalonamide AICA riboside 1548mg (6mM) at 0.02 N-hydrochloric acid
The solution was dissolved in 500 ml and irradiated with a high-pressure mercury lamp (400 W) for 15 hours under an argon gas flow. Add ion exchange resin Dowex/(OH ^- type) to the reaction solution to neutralize it.
I also added Dowex/(OH ^- type).
The liquid was dried under reduced pressure to obtain 2-amino-N-β-D-ribofuranosylmalonamide as a yellow amorphous solid.

NMR (DMSO-d ₆ -D ₂ O) δ ^TMS _ppn ; 3.5-3.9 (m.,
6H), 5.20 (d., 1H, H-1') IR; ν ^KBr _CO 1710cm ^-1 TLC; Rfa=0.13 Example 1 Production of Bredeinin 2-Amino-N-β-D- obtained in Example 1 After drying ribofuranosylmalonamide under vacuum at 40°C for 5 hours, it was heated and stirred at 133°C for 7 minutes with 20ml of dimethylformamide and 0.4ml of ethyl orthoformate. Ion conversion resin IRA-411 (OH ^- type) for the reaction solution
The column was charged to a column (2 x 15 cm), washed with 500 ml of water, and eluted with 200 ml of 2% aqueous acetic acid. Each fraction was followed by TLC, and fractions around Rfa=0.30 were collected and concentrated under reduced pressure. Develop the residue with butyl acetate-acetic acid-acetone-water (10:6:3:4) on preparative silica gel (Merck, Art5717, 20 x 20
cm) Thin layer chromatography was performed. Rfa=
A portion having a spot around 0.30 was collected and eluted with chloroform-methanol-acetic acid (6:12:1). The eluate was concentrated under reduced pressure to obtain a viscous oil. Dissolve this in a small amount of water and use Dowex50W.
(H ⁺ type) column (2 x 15 cm), eluted with water, and fractions containing bredeinin were collected and dried under reduced pressure. The residue was crystallized from water-isopropanol and then dried under vacuum at 90°C to obtain 174 mg of bredeinin (yield 11.2%).

NMR (DMSO−d ₆ ) δ ^DSS _ppn ; 3.4 to 3.7 (m., 2H,
H-5'), 3.8-4.0 (m., 1H, H-4'), 4.10 (t.,
1H, H-3'), 4.39 (t., 1H, H-2'), 4.4-6.2
(br., 3H, OH), 6.76, 7.02 (each br., 2H,
CONH ₂ ) 8.30 (s., 1H, H-2) UV: λ ^H2O _nax 277nm, 244nm Biological activity and other instrumental analysis data were completely consistent with natural bredeinin.

Reference example 2 2',3',5'-tri-O-acetyl AICA riboside 2.58g (10mM) of AICA riboside was added to 50% of pyridine.
After adding 5.0 ml of acetic anhydride under ice-cooling, the mixture was stirred at room temperature for 2 hours. The reaction solution was poured into ice water and extracted with chloroform. The chloroform layer was dried over anhydrous magnesium sulfate and then concentrated under reduced pressure. The residue was charged to a column of silica gel (Wako Gel C-200, manufactured by Wako Pure Chemical Industries, Ltd.), and chromatography was performed by eluting with methanol-chloroform (1:20). Fractions around Rfb = 0.5 were collected and dried under reduced pressure to give 3.21 g of candy-like 2',3',5'-tri-O-acetyl AICA riboside (yield 85%).
I got it.

NMR (CDCl ₃ , D ₂ O) δ ^TMS _ppn ; 2.13 (9H,
3XCH ₃ CO), 4.38 (m., 3H, H-4', H-5'),
5.32 (m., 1H, H-3'), 5.48 (d, d., 1H, H-
2'), 5.67 (d, 1H, H-1'), 7.12 (s., 1H, H-
2) Reference Example 3 2-Amino-N-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)malonamide 2.6 g of 2',3',5'-tri-O-acetyl AICA riboside It was dissolved in 500ml of 0.02N hydrochloric acid and irradiated with a high-pressure mercury lamp (400W) for 15 hours under an argon stream.
After the reaction, Dowex1 (OH ^- type) was added to neutralize it,
After filtering, the liquid was concentrated under reduced pressure. The residue was extracted with chloroform, the extract was passed through Watmann 1-PS filter paper, and then concentrated under reduced pressure. The residue was charged to a column (3 x 15 cm) of silica gel (Art9385 manufactured by Merck & Co.), and chloroform-methanol (17:
Flash chromatography was performed using 1). Collect the fraction around Rfb=0.33,
The mixture was dried under reduced pressure to obtain 2-amino-N-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)malonamide as an amorphous solid. Yield 802mg
(Yield 32%) This product was confirmed to consist of two types of optical isomers by NMR, and one epimer was obtained as a crystal by treatment with methanol. Yield 459 mg Melting point: 122-124℃ [α] ²⁴ _D −39.8 (C = 0.5, chloroform) NMR (CDCl ₃ C−D ₂ O) δ ^TMS _ppn ; 2.09 (s., 6H,
2XCH ₃ CO), 2.15 (s., 3H, CH ₃ CO), 4.06 (s.,
1H, H-3), 4.24 (m., 3H, H-4', H-5'),
5.36-5.12 (m., 2H, H-2', H-3'), 5.67 (d.,
1H, H-1', J ₁ ', ₂ ' = 5Hz) Elemental analysis [as C ₁₄ H ₂₁ N ₃ O ₉ ] C% H% N% Calculated value 44.80 5.64 11.20 Actual value 44.77 5.68 11.14 The above crystal mother liquor Concentration under reduced pressure yielded an amorphous solid mainly composed of other epimers.

NMR (CDCl ₃ −D ₂ O) δ ^TMS _ppn ; 2.09 (s., 6H,
2XCH ₃ CO), 2.14 (s., 3H, CH ₃ CO), 4.24 (m.,
4H, H-3, H-4', H-5'), 5.28 (m., 2H,
H-2', H-3'), 5.62 (d 1H, H-1', J ₁ ' ₂ '
=
3Hz) Example 2 2',3',5'-tri-O-acetylbredeinin 2-amino-N-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)malonamide (2 960 mg (mixture of seed epimers) was dissolved in 25 ml of DMF, 0.554 ml (1.3 equivalents) of ethyl orthoformate was added thereto, and the mixture was stirred at 110°C for 20 minutes. After the reaction,
DMF was distilled off from the reaction solution under reduced pressure, and the residue was purified using a silica gel (Merck Art9385) column (3 x 15
cm) and flash chromatography was performed using chloroform-methanol (10:1) as elution. Fractions around Rfc=0.28 were collected and dried under reduced pressure. The residue was crystallized by treatment with methanol to obtain 736 mg (yield 74.1%) of 2',3',5'-tri-O-acetylbredeinine.

NMR (CDCl ₃ − CD ₃ OD) δ ^TMS _ppn ; 2.13 (s., 9H,
3XCH ₃ CO), 4.38 (m., 3H H-4', H-5'),
5.44 (dd, 1H, H-3'), 5.64 (dd, 1H, H-
2'), 5.92 (d., 1H, H-1'), 7.90 (s., 1H, H-
2) UV: λ ^MeOH _nax 244nm, 282nm λ ^MeOH,H+ _nax 243nm, 286nm λ ^MeOH,OH- _nax 276nm MS (CI); 386 (MH ⁺ ) Elemental analysis [as C ₁₅ H ₁₉ N ₃ O ₉ ] C% H% N% Calculated value 46.76 4.97 10.90 Actual value 47.05 5.09 10.90 Example 3 Production of bredeinin 510 mg of 2',3',5'-tri-O-acetylbredeinin obtained in Example 4 was suspended in 20 ml of methanol. ,
Add dry ammonia gas while stirring while cooling with a cryogen.
It lasted 20 minutes. Then, the reaction mixture was sealed and stirred at room temperature for 5 and a half hours, and then the reaction solution was dried under reduced pressure. When the residue was dissolved in hot methanol, propanol was added, and the mixture was concentrated under reduced pressure, bredeinin precipitated as crystals, which were collected and vacuum-dried at 90°C to obtain 283 mg of bredeinin (yield: 83%). .

This was the same as bredeinin obtained in Example 2.

Reference Example 4 2-Amino-N-(1-β-D-ribofuranosyl)malonamide 2-Amino-N-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)malonamide (two types of epimers) mixture) 375 mg methanol
The mixture was dissolved in 10 ml and saturated with dry ammonia gas while stirring under ice cooling. The mixture was then tightly stoppered and stirred at room temperature overnight. The reaction solution was dried under reduced pressure, the residue was dissolved in a small amount of water, this was charged to Dowex 1 (H ⁺ type), and then washed thoroughly with water. Next, it was eluted with 0.1N aqueous ammonia, and the fractions around Rfa = 0.13 were collected and dried under reduced pressure to obtain the amorphous solid 2-amino-N.
-β-D-ribofuranosylmalonamide 208mg
(yield 84%).

NMR (DMSO-d ₆ -D ₂ O) δ ^TMS _ppn ; 3.5-3.9 (m.,
6H), 5.20 (d., 1H, H-1′) IR; ν ^KBr1 _CO 1710cm ^-1 TLC; Rfa=0.13 Reference example 5 2-Amino-N-(2,3-O-isopropylidene-β-D -ribofuranosyl)malonamide 2',3'-O-isopropylidene-AICA riboside (298 mg) was dissolved in 500 ml of 0.05N acetic acid and irradiated with a high-pressure mercury lamp (400 W, equipped with a Pyrex filter) under an argon atmosphere for 20 hours. The reaction solution was neutralized with 1N aqueous sodium hydroxide solution and concentrated under reduced pressure. Dissolve the residue in as small a volume as possible of 50% aqueous methanol, and add silica gel (Wacordel C-200) 6 to this.
g was added, mixed, dried under reduced pressure, and then packed into a column. Chloroform-methanol (20:1~
Column chromatography was performed using an elution ratio of 15:1). Collect the fraction around Rfc=0.33,
The mixture was dried under reduced pressure to obtain a candy-like target product.

Yield; 33 mg (yield 11.4%) TLC; Rfc = 0.33 Mass (CI, isobutane); 290 (MH ⁺ ) NMR (CDCl ₃ ) δppm; 3.75 (br, 2H, H-5'),
4.09, 412 (each s., 1H, H-3), 4.28 (br.s., 1H,
H-4'), 4.65 (d., 1H, H-3'), 5.71 (br.s.,
1H, H-1′), 7.75 (br, 2H, NH ₂ ), 8.40 (br.,
2H, _NH2 ), 8.7 (br., 1H, NH).

Claims (1)

[Claims] 1 formula (In the formula, R ₁ and R ₂ each represent a hydrogen atom or a hydroxyl group-protecting group, and R ₃ represents a hydrogen atom or a hydroxyl group-protecting group.) AICA riboside represented by the formula (wherein R ₁ , R ₂ and R ₃ have the same meanings as above), the compound is ring-closed with imidazole, and the 2'-position, 3'-position and/or
A method for producing bredeinine, which comprises removing the protecting group when the 5'-position hydroxyl group is protected with a protecting group. 2. The production method according to claim 1, wherein the protecting group is an acetyl or benzoyl group. 3. The manufacturing method according to claim 1, wherein imidazole ring closure is carried out by heating with formic acid or orthoformate in an aprotic polar solvent.