JPH0764868B2 - Novel N 6 above, N 6 above, 2'-0-tri-substituted-adenosine-3 ', 5'-cyclic phosphate and process for producing the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention provides a novel N ⁶ , N ⁶ , 2′-O-trisubstituted-adenosine-3 ′, 5′-cyclic phosphate (hereinafter N ⁶ , N ^6, 2'-O-abbreviated as trisubstituted -cAMP) or a salt thereof, and to the new production methods.

Adenosine-3 ', 5'-cyclic phosphate (hereinafter abbreviated as CAMP) and its derivatives have various physiological activities and are expected to have various uses as biochemical reagents and medicines.
This compound is a novel compound, and is expected to have excellent pharmacological actions such as anti-inflammatory action, platelet aggregation inhibitory action, and blood pressure lowering action.

[Conventional technology]

N ^6, N ^6, a method for obtaining 2'-O- trisubstituted -CAMP and this is not known.

[Problems to be solved by the invention]

A method for obtaining N ⁶ , N ⁶ , 2′-O-tri-substituted-CAMP has not been reported yet, and the present inventors have conducted extensive studies on a method for obtaining this, and as a result, have identified CAMP or a salt thereof. Alkali metal hydride such as sodium hydride, potassium hydride, lithium hydride, or potassium tert-
Tertiary alkoxides such as butoxide, alkyllithium compounds such as butyllithium, metal amides such as sodium amide, and the like are reacted with organic halides in the presence of a strong base to give N ⁶ , N ⁶ , 2 ′. -O-
It was found that tri-substituted-CAMP or a salt thereof can be efficiently produced.

[Means for Solving Problems] The present invention has the general formula (In the formula, R represents an alkyl group or an araalkyl group, and
Represents a hydrogen ion, an alkali metal ion, an ammonium ion or an organic ammonium ion)
^{N 6, N 6, 2'-} O- trisubstituted - adenosine-3 ', and 5'-cyclic phosphoric acid or its salt, and the present invention have the general formula (Wherein A represents a hydrogen ion, an alkali metal ion, an ammonium ion or an organic ammonium ion), an adenosine-3 ', 5'-cyclic phosphoric acid or a salt thereof represented by the general formula RX ( III) (wherein R represents an alkyl group or an araalkyl group, X
Is a halogen atom) and is reacted with an organic halide represented by the general formula (Wherein R and A have the above-mentioned meanings)
^{N 6, N 6, 2'-} O- trisubstituted - adenosine-3 ', a process for preparing 5'-cyclic phosphoric acid or its salt.

Hereinafter, the present invention will be described in detail.

CAMP or a salt thereof used as a starting material of the present invention,
For example, it can be manufactured as follows.

Belonging to the genus Microbacterium using a medium containing adenine, xylose or ribose and inorganic phosphate,
A bacterium having the ability to produce CAMP from adenine, xylose or ribose and inorganic phosphate is cultured, and CAMP is collected from the culture (see Japanese Patent No. 651781). When free CAMP is further reacted with an alkali metal or alkaline earth metal hydroxide, carbonate, hydrogen carbonate or the like or an organic amine, preferably a trialkylamine, an alkali metal salt in the phosphoric acid part of CAMP, for example, sodium salt. , Potassium salts and the like, alkaline earth metal salts such as calcium salts and magnesium salts, and organic ammonium salts such as triethylammonium salts.

R in the organic halide represented by the general formula (III) is an alkyl group having 1 to 16 carbon atoms, particularly 2 to 14 carbon atoms,
For example, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, pentyl group, isopentyl group, hexyl group, isohexyl group, heptyl group, octyl group, nonyl group, decyl group, dodecyl group, tetradecyl group, Examples thereof include linear or branched alkyl groups such as hexadecyl group and araalkyl groups such as benzyl group, nitrobenzyl group, chlorobenzyl group, methylbenzyl group, hydroxybenzyl group, phenethyl group and phenylpropyl group.

In carrying out the present invention, the compound of formula (II) is prepared by adding an alkali metal hydride such as sodium hydride, potassium hydride or lithium hydride, or a tertiary alkoxide such as potassium tert-butoxide, or butyl lithium. In the presence of a strong base such as an alkyllithium compound such as or a metal amide such as sodium amide.
Reaction with an organic halide of I) gives a compound of formula (I).

This reaction is carried out in a solvent. As a solvent, water, alcohols such as methanol and ethanol, ethers such as dioxane and tetrahydrofuran, amides such as dimethylformamide and dimethylacetamide, organic carboxylic acid esters such as ethyl acetate, etc. , Or dimethyl sulfoxide and the like, which are appropriately used alone or as a mixture of two or more kinds.

The amount of the organic halide of the formula (III) used with respect to the compound of the formula (II) is usually 3 to 20 times mol, preferably 4 to 10 times mol. Further, the amount of the base used with respect to the compound of the formula (II) is usually 3 to 20 times mol, preferably 4 to 10 times mol. This reaction is generally carried out at a temperature of 10 to 150 ° C., preferably 20 to 70 ° C. for 1 hour or more, preferably 3 hours to 2 days, with standing or stirring.

The target compound of formula (I) can be isolated and purified by, for example, column chromatography using silica gel, alumina, ion exchange resin, activated carbon, etc., recrystallization method, precipitation method by pH adjustment, salting out method using salt, organic method. A purification method such as an extraction method using a solvent can be performed alone or in combination. The free acid of the compound of the formula (I) is reacted with, for example, an alkali metal or alkaline earth metal hydroxide, carbonate, hydrogen carbonate or the like or ammonia or an organic amine, for example, a tertiary amine such as triethylamine or tributylamine. This can lead to the corresponding salt in the cyclic phosphate moiety of the compound of formula (I).

Examples of N ⁶ , N ⁶ , 2'-O-tri-substituted-CAMP of formula (I) and salts thereof obtained by the present invention include the following: N ⁶ , N ⁶ , 2'-O - trimethyl ^{-CAMP, N 6, N 6,} 2'-
O- triethyl ^{-CAMP, N 6, N 6,} 2'-O- tripropylamine ^{-CAMP, N 6, N 6,} 2'-O- triisopropyl -cAMP,
^{N 6, N 6, 2'-} O- tributyl ^{-CAMP, N 6, N 6,} 2'-O-
Triisobutyl ^{-CAMP, N 6, N 6,} 2'-O- tripentyl ^{-CAMP, N 6, N 6,} 2'-O- Toriisopenchiru -cAMP,
^{N 6, N 6, 2'-} O- Torineopenchiru ^{-CAMP, N 6, N 6,} 2 '
-O- trihexyl ^{-CAMP, N 6, N 6,} 2'-O- tri isohexyl ^{-CAMP, N 6, N 6,} 2'-O- triheptyl -CAM
^{P, N 6, N 6,} 2'-O- triisostearate heptyl -CAMP, N ^6, N ^6,
2'-O- trioctyl ^{-CAMP, N 6, N 6,} 2'-O- trinonyl ^{-CAMP, N 6, N 6,} 2'-O- tridecyl -cAMP,
^{N 6, N 6, 2'-} O- Toriunderu ^{-CAMP, N 6, N 6,} 2'-O
- tridodecyl ^{-CAMP, N 6, N 6,} 2'-O- tritriacontanoic decyl ^{-CAMP, N 6, N 6,} 2'-O- tritriacontanoic decyl -cAMP,
^{N 6, N 6, 2'-} O- tri tetradecyl ^{-CAMP, N 6, N 6,} 2 '
-O- tri pentadecyl ^{-CAMP, N 6, N 6,} 2'-O- trihexadecyl ^{-CAMP, N 6, N 6,} 2'-O- tribenzyl -
^{CAMP, N 6, N 6,} 2'-O- tri-nitrobenzyl -cAMP,
^{N 6, N 6, 2'-} O- trichlorobenzyl -CAMP, N ^6, N ^6,
2'-O- tri hydroxy benzyl -CAMP, N ^6, N ^6,
2'-O- trimethylbenzylammonium ^{-CAMP, N 6, N 6,} 2'-O
- tri-2-phenylethyl ^{-CAMP, N 6, N 6,} 2'-O-
Examples include tri-3-phenylpropyl-CAMP and the like, and alkali metal salts, ammonium salts and organic ammonium salts thereof.

In addition, according to the present invention, N ⁶ , N ⁶ , 2'- which is a novel compound
O-tri-substituted-adenosine-3 ', 5'-cyclic phosphoric acid and salts thereof can be efficiently produced.

〔The invention's effect〕

Novel N ^6, N ⁶ of the present invention, 2'-O-trisubstituted - adenosine-3 ', 5'-cyclic phosphoric acid or its salts, for example anti-inflammatory action, platelet aggregation inhibitory activity, such as hypotensive effect An excellent pharmacological action is expected, and the present invention can provide the compound extremely efficiently. The present invention is extremely significant industrially.

Hereinafter, the present invention will be specifically described with reference to examples.

〔Example〕

EXAMPLE ^{1 N 6, N 6, 2'} -O- triethyl -CAMP sodium salt Preparation of tributylamine salt 2.06g of CAMP was dissolved in dimethyl sulfoxide 20 ml, this was added sodium hydride 640 mg, 30 After 20 minutes, 1.20 ml of n-ethyl bromide is added dropwise. Then, 160 mg of sodium hydride is added, and after 30 minutes, 0.3 ml of n-ethyl bromide is added dropwise, and after 8 hours, 0.3 ml of n-ethyl bromide is added dropwise and stirred overnight at room temperature. The solvent of the reaction solution was distilled off under reduced pressure,
The residual oily substance is dissolved in a small amount of water, and 2N-hydrochloric acid is added to adjust the pH to 2, and this is adsorbed on an activated carbon column (2.2 x 28 cm), washed with water, and then ethanol / water / 28% ammonium hydroxide (volume Collect the eluate fraction using the ratio 10: 10: 1) and dry it under reduced pressure. The obtained caramel-like substance was dissolved in a small amount of 1N sodium hydroxide, and this was separated and purified by silica gel thin-layer chromatography (developing solvent; methanol / chloroform, volume ratio; 3: 7) to obtain the UV of the target compound.
The absorption band (Rf value around 0.5) was scraped off, extracted with methanol, and dried under reduced pressure to give N ⁶ , N ⁶ , 2'-O-triethyl-CAMP.
Sodium salt of 960 mg (yield 55.2%) was obtained.

Elemental analysis value: C ₁₆ H ₂₄ N ₅ O ₆ P ・ Na ・ H ₂ O C H N measured value (%) 42.37 5.46 15.36 Calculated value (%) 42.29 5.56 15.45 mp: 158 to 159 ° C. Rf value: 0.30 (developing solvent; methanol / chloroform, volume ratio; 3: 7 or less same) Example 2 Production of N ⁶ , N ⁶ , 2′-O-tripropyl-CAMP: of CAMP 2.0 g of tributylamine salt is dissolved in 30 ml of dimethyl sulfoxide, 468 mg of sodium hydride is added thereto, and after 30 minutes, 0.94 ml of n-propyl bromide is added dropwise. Then, 156 mg of sodium hydride is added, and after 30 minutes, 0.31 ml of n-propyl bromide is added dropwise, and the operation is performed twice, and the mixture is stirred at room temperature overnight. The solvent of the reaction solution was distilled off under reduced pressure, the residual oily substance was dissolved in a small amount of water, and 2N-hydrochloric acid was added to adjust the pH to 2, which was then activated carbon column (2.1 × 31 cm).
After adsorbing to, and washing with water, a section eluting with ethanol / water / 28% ammonium hydroxide (volume ratio; 10: 10: 1) is collected and dried under reduced pressure. The obtained caramel-like substance was dissolved in a small amount of methanol and adjusted to pH 2 by adding 2N-hydrochloric acid, which was separated and purified by silica gel thin layer chromatography (developing solvent; methanol / chloroform, volume ratio; 35:65). , The UV absorption band (Rf value around 0.6) of the target compound was scraped off, extracted with methanol, and dried under reduced pressure to give N ⁶ ,
N ^6, 810 mg of 2'-O- tripropyl -cAMP (yield 45.8
%)Obtained.

Elemental analysis value: C ₁₉ H ₃₀ N ₅ O ₆ P ・ 3 / 4H ₂ O C H N measured value (%) 48.37 6.47 14.70 Calculated value (%) 48.66 6.77 14.93 mp: 123 to 125 ° C. Rf value: 0.37 EXAMPLE ^{3 N 6, N 6, 2'} -O- tributyl -CAMP Preparation of dissolving the tributylamine salt 1.03g of CAMP in dimethyl sulfoxide 10 ml, hydrogen thereto 320 mg of sodium iodide is added, and 30 minutes later, 0.86 ml of n-butyl bromide is added dropwise. Then, 80 mg of sodium hydride is added, 0.22 ml of n-butyl bromide is added dropwise after 30 minutes, and the mixture is stirred overnight at room temperature. After the reaction solution was adjusted to pH 2 with 2N-hydrochloric acid, the solvent was distilled off under reduced pressure. 50 ml of benzene was added to the residual oily substance to dissolve it, and the mixture was extracted 4 times with 150 ml of water. The benzene layer is separated, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The obtained caramel-like substance was dissolved in a small amount of methanol, and this was separated and purified by silica gel thin layer chromatography (developing solvent; methanol / chloroform, volume ratio; 2: 8) to obtain the UV absorption band (Rf value 0.2 of the target compound). scraped around) and extracted with methanol and concentrated to dryness under reduced pressure, N ^6, N ^6,
470 mg of 2'-O-tributyl-CAMP was obtained (yield 47.3%).

Elemental analysis value: C ₂₂ H ₃₆ N ₅ O ₆ P ・ 2 / 3H ₂ O C H N measurement value (%) 52.21 7.19 13.42 Calculated value (%) 51.86 7.38 13.74 mp: 102-104 ° C. Rf value: 0.42 Example 4 Production of N ⁶ , N ⁶ , 2′-O-tripentyl-CAMP: 2.06 g of tributylamine salt of CAMP was dissolved in 20 ml of dimethyl sulfoxide, and hydrogen was added thereto. 480 mg of sodium iodide is added, and 30 minutes later, 1.48 ml of n-amyl bromide is added dropwise. Then, 160 mg of sodium hydride is added, and after 30 minutes, an operation of dropping 0.5 ml of n-amyl bromide is performed three times, and the mixture is stirred at room temperature overnight. After the reaction solution was adjusted to pH 2 with 2N-hydrochloric acid, the solvent was distilled off under reduced pressure. To oily residue
Add 80 ml of benzene to dissolve and extract 4 times with 150 ml of water. The benzene layer is separated, dried over anhydrous sodium sulfate, and then dried under reduced pressure. The obtained caramel-like substance was dissolved in a small amount of methanol, and this was subjected to silica gel thin layer chromatography (developing solvent; methanol / chloroform,
Separation and purification by a volume ratio of 3: 7), scraping off the UV absorption band (Rf value around 0.5) of the target compound, extracting with methanol, and drying to dryness under reduced pressure, N ⁶ , N ⁶ , 2'-O-tripentyl- CAMP 93
7 mg (yield 43.5%) was obtained.

Elemental analysis value: C ₂₅ H ₄₂ N ₅ O ₆ P ・ 2 / 3H ₂ O C H N measurement value (%) 54.43 8.06 12.55 calculated value (%) 54.46 7.92 12.70 mp: 114-116 ° C. Rf value: 0.46 Example 5 Production of N ⁶ , N ⁶ , 2′-O-triheptyl-CAMP: 3.08 g of tributylamine salt of CAMP was dissolved in 100 ml of dimethyl sulfoxide, and potassium was added thereto. 2.69 mg of tert-butoxide is added, 30 minutes later, 3.76 ml of n-heptyl bromide is added dropwise, and the mixture is stirred at room temperature for 2 days. During this period, 673 mg of potassium tert-butoxide is added, and after 30 minutes, 0.94 ml of n-heptyl bromide is added dropwise, which is repeated 4 times. The reaction solution was neutralized with 2N-hydrochloric acid, and the solvent was evaporated under reduced pressure. A small amount of methanol was added to the residual oily substance,
The pH is adjusted to 2 by adding 2N-hydrochloric acid, and a part of the inorganic substances is removed as a precipitate. The filtrate is concentrated, 150 ml of benzene is added to the residue to dissolve it, and the mixture is extracted 4 times with 200 ml of water. Concentrate the benzene layer to a small volume and place it on a 70 g silica gel (packed in chloroform) column. This column was washed with chloroform, and the fraction containing the target compound that was eluted with methanol / chloroform (volume ratio: 3:97 to 1: 9) was collected and dried under reduced pressure to give N ⁶ , N ⁶ , 2, ′ -O-Triheptyl-CAMP
Was obtained in an amount of 1.950 g (52.2%).

Elemental analysis value: C ₃₁ H ₅₄ N ₅ O ₆ P ・ 5 / 4H ₂ O C H N measured value (%) 57.63 8.48 10.76 Calculated value (%) 57.61 8.81 10.84 mp: 166-169 ° C. Rf value: 0.58 Example 6 Production of N ⁶ , N ⁶ , 2′-O-tritetradecyl-CAMP: 3.08 g of tributylamine salt of CAMP was dissolved in 100 ml of dimethyl sulfoxide, 2.69 g of potassium tert-butoxide was added to the mixture, 30 minutes later, 7.14 ml of n-myristyl bromide was added dropwise, and the mixture was stirred at room temperature for 1 day. During this period, 673 mg of potassium tert-butoxide is added, and 30 minutes later, 1.79 ml of n-myristyl bromide is added dropwise. The reaction solution
After adjusting to pH 2 with 2N-hydrochloric acid, the solvent was distilled off under reduced pressure. To the remaining oily substance, 150 ml of ether and 150 ml of water are added and shaken to dissolve the oily substance, and then the ether layer is separated and concentrated to a small volume. This is put on a silica gel 60 g (packed in chloroform) column, and this column is mixed with hexane /
Wash with chloroform (volume ratio; 1: 1 to 1: 9) and elute with methanol / chloroform (volume ratio; 1:99 to 1: 9) to collect the section containing the target compound, and distill this solvent under reduced pressure. When removed, N ⁶ , N ⁶ , 2'-O-tritetradecyl-CAMP was added to 3.08
g (yield 56.0%) was obtained.

Elemental analysis value: C ₅₂ H ₉₆ N ₅ O ₆ P ・ 3 / 2H ₂ O C H N measured value (%) 65.86 10.28 7.41 Calculated value (%) 66.06 10.55 7.41 Rf value: 0.69 Example ^{7 N 6, N 6, 2'} -O- tribenzyl -CAMP Preparation of the tributylamine salt 2.06g of CAMP was dissolved in dimethyl sulfoxide 20 ml, which potassium tert-- butoxide 1.35g 30 minutes later, n-benzyl bromide was added.
Add 1.43 ml dropwise. Then, 449 mg of potassium tert-butoxide is added, and after 30 minutes, 0.48 ml of n-benzyl bromide is added dropwise, and the mixture is stirred at room temperature for 4.5 hours. 2 in the reaction solution
After adjusting the pH to 2 by adding N-hydrochloric acid, the solvent was distilled off under reduced pressure. The remaining oily substance is dissolved by adding 100 ml of benzene, and extraction is performed 4 times with 150 ml of water. The benzene layer is separated, dried over anhydrous sodium sulfate, and then dried under reduced pressure.
The obtained oily substance is dissolved in a small amount of methanol, and this is separated and purified by silica gel thin layer chromatography (developing solvent; methanol / chloroform, volume ratio: 35:65),
The UV absorption band (Rf value around 0.45) of the target compound was scraped off, extracted with methanol, and dried under reduced pressure to give 905 mg (yield 37.8%) of N ⁶ , N ⁶ , 2'-O-tribenzyl-CAMP.

Elemental analysis value: C ₃₁ H ₃₀ N ₅ O ₆ P · 1 / 3H ₂ O C H N measured value (%) 61.70 5.12 11.51 Calculated value (%) 61.48 5.10 11.56 mp: 127-129 ℃ Rf value: 0.42

Claims (2)

[Claims] 1. A general formula (In the formula, R represents an alkyl group or an araalkyl group,
A represents a hydrogen ion, an alkali metal ion, an ammonium ion or an organic ammonium ion)
^{N 6, N 6, 2'-} O- trisubstituted - adenosine-3 ', 5'-cyclic phosphoric acid or its salt. 2. General formula (Wherein A represents hydrogen ion, alkali metal ion, ammonium ion or organic ammonium ion), adenosine-3 ', 5'-cyclic phosphoric acid or a salt thereof is reacted with the compound represented by the general formula RX (III ) (Wherein R represents an alkyl group or an araalkyl group, and X represents a halogen atom), and is reacted with an organic halide represented by the general formula (Wherein R and A have the above-mentioned meanings)
^{N 6, N 6, 2'-} O- trisubstituted - adenosine-3 ', 5'-cyclic phosphate or preparation of a salt thereof.