JPH0332553B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides 5,6,7,8-tetrahydro-L-
Erythro biopterin (hereinafter abbreviated as BPH ₄ )
The present invention relates to a method for producing L-erythro-biopterin, which is an intermediate for. BPH ₄ is 6-(R) due to the configuration of hydrogen at the 6-position
and the 6-(S) isomer are known [Furrer, HJ et al., Helv.chim.Acta, 62 2577
(1979)]. Above all, 6-(R)BPH ₄ is a coenzyme of phenylalanine hydroxylase, and at the same time,
It is also a coenzyme for other aromatic amino acid hydroxylases. Therefore, its deficiency leads to a deficiency of neurotransmitters such as serotonin, dopamine, noradrenaline, and adrenaline, leading to the development of serious neurological symptoms. In addition, malignant hyperphenylalaninemia, which is a type of congenital metabolic disorder, is an incurable disease that cannot be easily treated with existing drug therapy.
It is known that this occurs because the conversion of phenylalanine to tyrosine is inhibited by a deficiency of (R)BPH ₄ . 6-(R) for the treatment of malignant hyperphenylalaninemia
-Administration of BPH ₄ is considered, but for this purpose, it is desired to develop a method to economically mass-produce this product with high purity. In addition, 6-(R)-BPH ₄ is useful as a reagent in biochemical research. On the other hand, in order to synthesize BPH ₄ , L-erythrobiopterin (hereinafter abbreviated as L-BP) is an important intermediate. Conventionally, methods for producing L-BP include Patterson,
EL et al. [J.Am.Chem.Soc., 78 , 5868 (1956)],
Taylor, EC et al. [Id., 98 , 2301 (1976)],
Andrews, KJM et al. (J.Chem.Soc(C)., 1969 ,
928), Viscontini, M. et al. [Helv.Chim.Acta,
52, 1225 (1969)], but there are many problems with yield, purity, purification method, etc. Also, Matsuura et al. [Brll.Chim.Soc.Jap., 52 , 181
(1979)] obtained L-Bp with a yield of 28%, but many problems such as the complexity of the reaction treatment and the contamination of by-products remain unsolved. The present inventors first conducted extensive research on the production of L-BP and succeeded in obtaining a large amount of L-BP through simple operations. Matsuura et al. oxidized a condensate of 5-deoxy-L-arabinose phenylhydrazone with triaminopyrimidinol using potassium ferricyanide, potassium iodide, hydrogen peroxide, and oxygen.
The product was extracted with aqueous ammonia and purified using a Florisil column. In this case, not only a large amount of water is required for elution, but also pure L-BP because the by-product pretene is eluted immediately after L-BP.
It is difficult to completely remove the BP. The present inventors have discovered that L-BP can be easily obtained as crude crystals by oxidizing the above condensate with iodine, hydrogen peroxide and oxygen and then removing iodine with an organic solvent. The crude crystals were acetylated and subjected to silica gel column chromatography to obtain triacetyl biopretene, which was then hydrolyzed to yield L-BP in high yield. Also, as a method for synthesizing 6-(R or S)-BPH ₄ , Siscontini, M. et al. [Helv.Chim.Acta, 62 2577
(1979)] reduced triacetyl-L-BP and then acetylated it to 2-N-acetyl-5-N-
Acetyl-1',2'-di-O-acetyl-5,6,
7,8-Tetrahydro-L-erythrobiopterin (hereinafter abbreviated as tetraacetyl form) was obtained, and the target product was obtained by optical resolution and hydrolysis.
However, the tetraacetyl compound is extremely sensitive to oxygen, making it difficult to optically resolve it by ordinary recrystallization methods, and it requires great efforts to obtain the target product with high optical purity. The present inventors reduced triacetyl-L-BP and then heated it with acetic anhydride to reduce 2-N.
-acetyl-5,8-di-N-acetyl-1',
2'-di-O-acetyl-5,6,7,8-tetrahydro-L-erythrobiopterin (hereinafter abbreviated as pentaacetyl) was obtained, and this compound was stable and was purified by chromatography using a silica gel column. I learned that optical isomers (R and S) can be easily separated. That is, ()2-N-acetyl-1',2'-
Di-O-acetyl-L-erythrobiopterin [triacetyl-L-BP] was catalytically reduced and then acetylated to give 2-N-acetyl-5,8-di-N-acetyl-1',2'- di-O-acetyl-
5,6,7,8-tetrahydro-L-erythro-
Generates biopterin [pentaacetyl],
Next, this was optically resolved and then hydrolyzed to obtain tetrahydro-L-erythrobiopterin [ _BPH4 ] (2,5,6-triamino-4-pyrimidinol was reacted with hydrazone of 5-deoxyarabinose). Thereafter, iodine, hydrogen peroxide solution and oxygen were reacted to obtain L-erythrobiopterin [L-BP], which was then acetylated to obtain 2-N-acetyl-1',2'-di-O- After catalytic reduction of acetyl-L-erythrobiopterin [triacetyl-L-BP], it was further acetylated to produce 2-N-acetyl-5,8-di-N-acetyl-1',2'-di-O- Acetyl-
producing 5,6,7,8-tetrahydro-L-erythrobiopterin [pentaacetyl],
Next, this is optically resolved and then hydrolyzed to obtain tetrahydro-L-erythrobiopterin [BPH ₄ ]. The reaction steps of the method () of the present invention can be shown as follows. The second half of the reaction step in the above method () is similar to (), but the first half can be expressed as follows. Pentaacetyl is obtained by catalytic reduction and acetylation of the above triacetyl-L-BP. Catalytic reduction can be carried out in a conventional manner in a solvent such as trifluoroacetic acid. Examples of the catalyst include platinum oxide, rhodium, palladium, etc., but platinum oxide is preferred. Next, the obtained reduced product is acetylated. This reaction is the ultimate acetylation, and for this purpose it is best to use an acetylating agent such as acetic anhydride under heat. The thus obtained pentaacetyl is converted into 6-(R)
and 6-(S) body. This division is
For example, it can be carried out by silica gel column chromatography. Each optical isomer is then hydrolyzed. This reaction is preferably carried out under mild conditions in the presence of a mineral acid such as hydrochloric acid. Thus 6-(R)BPH ₄ and 6-(S)
You can get BPH ₄ . According to the present invention, in order to obtain triacetyl L-BP, 2,5,6-triamino-4-pyrimidinol is first reacted with 5-deoxy-L-arabinose hydrazone, and the resulting intermediate is treated with iodine,
Oxidizes with hydrogen peroxide and oxygen. 5-deoxy-L-arabinose hydrazone can be obtained by reacting 5-deoxy-L-arabinose with an aromatic hydrazine such as phenylhydrazine by known methods. The oxidation reaction can be carried out by passing oxygen through a solution of the intermediate to which iodine and hydrogen peroxide are added. The solvent is distilled off from the reaction mixture under reduced pressure, the residue is washed with an organic solvent such as acetone or methanol to remove iodine, extracted with aqueous ammonia, and concentrated to obtain crude L-BP in high yield. Crystals are obtained. This crude crystal can be immediately acetylated without purification. This acetylation can be carried out using, for example, acetic anhydride-acetic acid. The reaction product can then be easily purified by column chromatography using silica gel or the like as a carrier. In this way, triacetyl-L-BP can be produced with good yield. Examples will be shown next, but it goes without saying that the present invention is not limited to these examples. EXAMPLE Preparation of L-erythro-biopterin 13.6 g (0.089 mol) of 5-deoxy-L-arabinose monohydrate are dissolved in 30 ml of ethanol and 3 drops of acetic acid are added. 10.3g (0.107mol)
Dissolve phenylhydrazine in 5 ml of ethanol and add. After a few minutes the reaction solidifies. Reactants are 30
Leave at room temperature for a minute, then add 150 ml of methanol to dissolve the solidified material while stirring. Next 450ml
of water, 11.5 g (0.054 mol) in 300 ml of methanol
The resulting hydrazone solution was added all at once to a solution of 2,5,6-triaminopyrimidinol dihydrochloride, and heated under reflux for 40 minutes under a nitrogen stream.
Upon heating, the solution turns deep red. After heating, cool using ice water. On the other hand, a mixed solution of 22.9g (0.09mol) iodine, 40ml 30% hydrogen peroxide, 15ml 98% formic acid, and 400ml methanol was maintained at 0° to 5°C. The solution of the condensation reaction product was added to the oxidizing agent solution while blowing oxygen gas, and the solution was vigorously stirred while maintaining the temperature at 0° to 5° C. for 30 minutes. The reaction is further carried out at room temperature for 3 hours to complete the reaction. After the reaction, the solid obtained by concentration to dryness under reduced pressure was washed with 300 ml of acetone and 200 ml of methanol to remove iodine, etc., and 1000 ml of 2% ammonia water was used.
Extract BP. When the extract is subsequently concentrated to 1/4, a precipitate forms. Remove the resulting precipitate,
After washing twice with 50 ml of water and three times with 100 ml of methanol and drying, 48 g (37%) of crude crystals are obtained. This crude crystal was 96%L by high performance liquid chromatography ^* .
-Identified as containing BP. (*Manufactured by Whatman) Partisil-
A 10scx column with an elution solvent of 12.5mM PH3.3 acetic acid-ammonia buffer was used. ) Dissolve 4.8 g of the above crude crystals in 750 ml acetic anhydride and 200 ml acetic acid, and heat under reflux for 4 hours. After concentrating to dryness under reduced pressure, it is purified by column chromatography. Column chromatography uses 350 g of silica gel (Merck Kieselgel 60)
Using 5% methanol and methylene chloride as the elution solvent, 6.86 g (0.019 mol) of triacetyl-L-biopterin was obtained as an oil (yield 35%). This triacetyl-L-biopterin corresponds to the physical constants described in the literature. 6.86g of triacetyl-L-biopterin
It is quantitatively hydrolyzed by dissolving it in 550ml of 3N-HCl and heating it at 50°C for 1 hour. After removing the sewage water under reduced pressure, the residue was extracted with 2% aqueous ammonia and recrystallized from 20% acetic acid-water to give 4.40 g (0.019 mol).
Pure crystals were obtained. Yield 34%. The obtained L-erythro biopterin is
NMR spectrum, IR spectrum, UV spectrum, [α] _D , etc. are described in literature (Matsuura et al. Bull.Chim.Soc.
Jap., 52 , 181 (1979). Reference Example 5, Production of 6,7,8-tetrahydro-L-erythrobiopterin 360 mg of platinum oxide is suspended in 20 ml of trifluoroacetic acid and activated under a hydrogen atmosphere. On the other hand, 1.20g
(3.31 mmol) of 2-N-acetyl-1',2'-di-
O-acetylbiopterin (known in literature)
[Furrer, HJ et al. HeLv.Chim.Acta, 62 . 2577
(1979)] in 40 ml of trifluoroacetic acid, and the reduction was carried out under a hydrogen gas atmosphere with vigorous stirring. After the reaction is complete, the catalyst is removed by filtration under reduced pressure under an argon stream, and the solvent is concentrated to dryness. The residue was treated with 2.0 ml of concentrated hydrochloric acid under an argon atmosphere, followed by 24 ml of chilled methanol (oxygen-free),
Addition of 250 ml of chilled oxygen-free ether produces a colorless precipitate. The precipitate is collected by centrifugation and the remaining solvent is removed under reduced pressure. The precipitate was dissolved in 40 ml of acetic anhydride under a stream of argon and heated for 1 hour in an oil bath preheated to 120°C. When the solvent was distilled off under reduced pressure, 1.42g
A residue is obtained. Separation and purification were performed using 140 g of silica gel (manufactured by Merck & Co., Ltd.).
Separate by column chromatography using Kieselgel60). The desired 6-(R) pentaacetyl compound [6-(R)-2-N-acetyl-5,8-dimethyl] was obtained by carefully performing column chromatography using methylene chloride-methanol (95:5) as the elution solvent. -N-acetyl-1',2'-di-O-acetyl-
5,6,7,8-tetrahydro-biopterin] was obtained in an amount of 597 mg (1.32 mmol, yield 40%).
At this time, the optical isomer 6-(S) pentaacetyl body is
329 mg (0.73 mmol, yield 22%) is obtained. Physical constants of 6-(R)-pentaacetyl compound UV spectrum: (λEtOH/max) 237 nm (ξ = 23000), 267 nm (ξ = 8200), 312 nm (ξ = 11000) IR spectrum: (cm ^-1 , in CHCl ₃ ) 1200,
1220, 1615, 1670, 3000 ¹ H-NMR spectrum: (δppm in D ₆ −
DMSO) 1.20 (d, 3H, J=6Hz, 3'-CH ₃ ) 1.85 (s, 3H 2'-OCOCH ₃ ) 2.05 (s, 3H 1'-OCOCH ₃ ) 2.07 (s, 3H 5-NCOCH ₃ ) 2.25 (s, 3H 2-NCOCH ₃ ) 2.26 (s, 3H 8-NCOCH ₃ ) 3.00~3.62 (m, 2H, H-7) 3.95 (m, 1H, H-6) 4.44~5.04 (m, 2H, H-1', H-2') 11.48-11.58 (br, 1H, 3-NH) 12.00 (br, 1H, 2-NH) Mass spectrum: (m/z) 451 (M ⁺ ) 409 (M ⁺ - COCH ₂ ) 367 (M ⁺ -2 (COCH ₂ )) 278, 250, 236, 208, Physical constants of 6-(S)-pentaacetyl compound UV spectrum: (λEtOH/max) 237 nm (ξ = 18200) 267 nm ( ξ=5800) 310nm (ξ=8200) IR spectrum: (cm ^-1 in CHCl ₃ ) 1200, 1220,
1610, 1670, 3000 ¹ H-NMR spectrum: (δppm in D ₆ −
DMSO) 1.16 (d, 3H, J=6Hz 3'-CH ₃ ) 1.89 (s, 3H 2'-OCOCH ₃ ) 1.91 (s, 3H 1'-OCOCH ₃ ) 2.03 (s, 3H 5-NCOCH ₃ ) 2.22 (s, 3H 2-NCOCH ₃ ) 2.25 (s, 3H 8-NCOCH ₃ ) 3.10~3.64 (m, 2H, H-7) 4.02 (m, 1H, 6-H) 4.60~5.27 (m, 2H, H -1', H-2') 11.55 (br, 1H, 3-NH) 11.98 (br, 1H, 2-NH) Mass spectrum: (m/z) 451 (M ⁺ ) 409 (M ⁺ -COCH ₂ ) 367 (M ⁺ -2 (COCH ₂ )) 293, 250, 208, 597 mg of 6-(R)-pentaacetyl compound in 600 ml
The desired 6
-(R)BPH ₄ [6-(R)-5,6,7,8-tetrahydro-L-erythrobiopterin]・2
Hydrochloride is obtained quantitatively. Yield 319mg (1.32m
mole), yield 40%). The obtained 6-(R)-BPH ₄ is described in the literature (Furrer, H.
J. et al. Helv. Chim. Acta, 62 , 2577 (1979)
¹ H-NMR, ¹³ C-NMR, and [α] _D were completely consistent. Similarly, 6-(S)-BPH _4.2 hydrochloride was quantitatively obtained from 6-(R)-pentaacetyl form in a yield of 22%.
(176 mg, 0.73 mol), physical data are literature values, Furrer, HJ et al. Helv.Chim.Acta, 62 , 2577
(1979).

Claims (1)

[Claims] 1. L-erythro, which is characterized in that 2,5,6-triamino-4-pyrimidinol is reacted with hydrazone of 5-deoxyarabinose, and then iodine, hydrogen peroxide and oxygen are reacted. - Method for producing biopterin. 2. The production method according to claim 1, characterized in that the reaction mixture in which iodine, hydrogen peroxide and oxygen are reacted is concentrated to dryness and then washed with an organic solvent to remove iodine. 3. The production method according to claim 2, wherein the organic solvent is acetone and/or methanol.