JPH0339517B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to 2-
The present invention relates to amino-N-β-D-ribofuranosylmalonamide derivatives.

[Prior art] 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), Japanese Patent Publication No. 49-12720], not only has strong immunosuppressive activity,
It has anticandidal 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. 12720].

Other methods for producing bredeinin other than the above include 4.
- Method for chemically converting carbamoyl-imidazolium-5-oleate and β-D-ribofuranose into N-glycosidation [Special Publication No. 47196/1986;
−52038 Publication, Chem.Pharm.Bull., 23(1), 245
~246 (1975)], a method for the biochemical salvage synthesis of 4-carbamoyl-imidazolium-5-oleate using microorganisms [Japanese Patent Publication No. 1983-36678]
can be mentioned.

[Problem that the invention seeks to solve]

It is important for the production of pharmaceuticals to find a new method for producing Bredeinin other than the conventional method.

[Means to solve the problem]

As a result of various studies on the chemical production method of bredeinin, the present inventors discovered that AICA riboside (5-
A novel production method for converting aminoimidazole-4-carboxamide riboside) to bredeinine, i.e., 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, R and R have the same meanings as above), the compound [3] is ring-closed with imidazole, and the 2'-position, 3'-position and/or
When the 5'-position hydroxyl group is protected, the inventors discovered a method for advantageously producing bredeinine by removing the protecting group, and completed the present invention.

The present invention is a compound represented by the above formula [3] or a salt thereof, and its object is to
The object of the present invention is to provide a useful intermediate for the chemical production of bredeinin.

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-protecting group used in the field of oxidation chemistry or sugar chemistry can be 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 formyl, acetyl,
Chloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, pivaloyl,
Examples include acyl groups such as benzoyl, β-benzoylpropionyl, phenoxyacetyl, and trityloxyacetyl, trityl groups such as trityl, monomethoxytrityl, dimethoxytrityl, and trimethoxytrityl, and methoxymethyl groups.

The above-mentioned protecting group can be introduced by a known method, but it is preferable to select a protecting group that can be efficiently removed later, but can be removed in one step.

In the present invention, first, AICA riboside [2]
Intermediate compound [3] is produced by photochemical reaction 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 intermediate compound [3] by thin layer chromatography or high performance liquid chromatography. Usually 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 have an adverse effect on the reaction, so do not use it under a flow of inert gas, such as aregon gas or nitrogen gas. Carrying out the reaction is advantageous 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.

The desired bredeinine can be produced by subsequently ring-closing this intermediate compound [3] with imidazole. For imidazole ring closure, formic acid,
It is carried out by a method of heating with orthoformic acid ester, formimino ether, diethoxymethyl acetate or N-formylmorpholine, etc., a method of 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 the reaction product is concentrated, the resulting residue is extracted with a solvent, and the resulting extract is concentrated to obtain a crude reaction product, which can be further purified using 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. You may. To obtain bredeinin produced 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

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.

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) In addition, the NMR assignment of the title compounds of Examples 1 to 4 is based on the position number described in formula [3]. I went based on this.

Example 1 2-Amino-N-β-D-ribofuranosylmalonamide AICA riboside 1548mg (6mM) was added to 0.02N-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. Ion exchange resin Dowex 1 (OH ^- type) was added to the reaction solution to neutralize it, and Dowex 1 (OH ^- type) was further added and filtered. The filtrate was dried under reduced pressure to give a yellow amorphous solid of 2-amino-
N-β-D-ribofuranosylmalonamide was obtained.

NMR (DMSO- _d6 - _D2O , TMS) δppm; 3.5~
3.9 (m., 6H), 5.20 (d., 1H, H-1') IR (KBr method); 1710 cm ^-1 (CO) TLC; Rfa = 0.13 Reference example 1 Production of Bredeinin 2 obtained in Example 1 -Amino-N-β-D-ribofuranosylmalonamide was vacuum-dried at 40°C for 5 hours, and then heated and stirred with 20ml of dimethylformamide and 0.4ml of ethyl orthoformate at 133°C for 7 minutes. The reaction solution was mixed with ion exchange resin IRA-411 (OH ^- type)
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. The residue was developed with butyl acetate-acetic acid-acetone-water (10:6:3:4). Preparative silica gel (Merck, Art5717, 20
×20cm) 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 , DSS) δppm; 3.4-3.7 (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; λmax 277, 244 nm (in water) 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-water 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 and chloroform (1:20). The fractions around Rfb = 0.5 were collected and dried under reduced pressure to form candy-like 2',3',5'-tri-O.
-Acetyl AICA riboside (yield 85%) was obtained.

NMR (CDCI ₃ , D ₂ O, TMS) δppm; 2.13 (9H,
3×CH ₃ CO), 4.38 (m., 3H, H−4′, H−
5'), 5.32 (m., 1H, H-3', 5.48 (dd1H, H
−2′), 5.67(d., 1H, H−1′), 7.12(s., 1H,
H-2) Example 2 2-Amino-N-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)malonamide 2',3',5'-tri-O-acetyl AICA riboside 2.6 g was dissolved in 500 ml of 0.02 N calculation, and irradiated with a high-pressure mercury lamp (400 W) for 15 hours under an argon gas flow. Ion exchange resin Dowexl (OH ^- type) in the reaction solution
The mixture was neutralized by adding and filtered, and the filtrate was concentrated under reduced pressure. The residue was extracted with chloroform, the extract was passed through Watmann 1PS filter paper, and then concentrated under reduced pressure. The residue was charged to a column (3 x 15 cm) of silica gel (Merck & Co., Art 9385), and flash chromatography was performed by eluting with chloroform-methanol (17:1). The fraction around Rfb=0.33 was collected and dried under reduced pressure to 2-amino-N-(2,
3,5-Tri-O-acetyl-β-D-ribofuranosyl)malonamide was obtained as an amorphous solid.
Yield 802 mg (yield 32%).

It was confirmed by NMR that this product consisted of two types of optical isomers, and one epimer was obtained as a crystal by treatment with methanol. Yield 459mg.

Melting point; 122-124°C [α] ²⁴ _D −39.8 (c = 0.5, chloroform) NMR (CDCl ₃ , D ₂ O, TMS) δppm; 2.09 (6H,
2 x CH 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 to 5.12 (m., 1H, 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 crystalline mother liquor was concentrated under reduced pressure to obtain an amorphous solid mainly composed of other epimers.

NMR (CDCl ₃ , D ₂ O, TMS) δppm; 2.09 (6H,
2 x CH ₃ CO), 2.15 (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) Reference example 3 2′,3′,5′-tri-O-acetylbredeinine 2-amino-N-(2,3,5-tri-O-acetyl-β- 960 mg of D-ribofuranosyl) malonamide (mixture of two epimers) was dissolved in 25 ml of DMF, and 0.554 ml (1.3 equivalents) of ethyl orthoformate was added thereto, followed by stirring at 110°C for 20 minutes. After the reaction, DMF was distilled off from the reaction solution under reduced pressure, and the residue was transferred to a column (3 x 15 cm) of silica gel (Merck Art9385).
and chloroform-methanol (10:
Flash chromatography was performed using 1). Collect the fraction around Rfc=0.28,
It was dried under reduced pressure. The residue was crystallized by treatment with methanol to obtain 736 ml of 2',3',5'-tri-O-acetylbredeinine (yield 741%).

NMR ( _CDCI3 , _CD3OD , TMS) δppm; 2.13
(9H, 3×CH 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; λmax244, 282nm (in methanol) λmax243, 286nm (in methanol, H ⁺ ) λmax276nm (in methanol, OH ^- ) MS (Cl); 386 (MH ⁺ ) Elemental analysis [C As ₁₅ H ₁₉ N ₃ O ₉ ] C% H% N% Calculated value 44.76 4.97 10.90 Actual value 47.05 5.09 10.94 Reference example 4 Production of bredeinin 2', 3', 5'-tri-O- obtained in Reference example 3 Suspend 510ml of acetylbredeinin in 20ml 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, bropanol was added, and the mixture was concentrated under reduced pressure, bredeinin precipitated as crystals, which were collected by filtration and vacuum dried at 90°C to obtain 283 mg of bredeinin (yield: 83%). Ta.

This product was the same as bredeinin obtained in Reference Example 1.

Example 3 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 to dryness 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- _d6 - _D2O , TMS) δppm; 3.5~
3.9 (m., 6H), 5.20 (d., 1H., H-1') IR (KBr method); 1710 cm ^-1 (CO) TLC; Rfa = 0.13 Example 4 2-Amino-N-(2, 3-O-isopropylidene-β-D-ribofuranosyl) malonamide 298 mg of 2',3'-O-isopropylidene-AICA riboside was dissolved in 500 ml of 0.05N acetic acid and heated under a high-pressure mercury lamp (400 W, with Pyrex filter) under an argon gas stream. was irradiated 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 amount as possible of 50% aqueous methanol, and add silica gel (Wako Gel C-200) to this.
6 g was added, mixed, dried under reduced pressure, and then packed into a column. Chloroform-methanol (20:1
Column chromatography was performed eluting with ~15:1). Collect the fraction around Rfc=0.33,
The product was dried under reduced pressure to obtain a candy-like target product. Yield: 33mg
(Yield 11.4%).

TLC; Rfc = 0.03 Mass (Cl, isobutane); 290 (MH ⁺ ) NMR (CDCl ₃ ) δppm; 3.75 (br, 2H, H-5'),
4.09, 4.12 (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, (wherein 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) or a salt thereof. 2. The compound or salt thereof according to claim 1, wherein the protecting group is an acetyl or benzoyl group. 3 2-Amino-N-β-D-ribofuranosylmalonamide or 2-amino-N-(2,3,5
-tri-O-acetyl-β-D-ribofuranosyl)malonamide or a salt thereof according to claim 1.