JPH0372493A - Production of 2'-o-substituted-adenosine-3',5'-cyclic phosphoric acid or its salt - Google Patents

Abstract

PURPOSE:To obtain the title compound excellent in anti-inflammatory effect, platelet agglutination-inhibitive effect, vasodepressing activity, etc., by the reaction between adenosine-cyclic phosphoric acid (salt) and an organic halide under alkaline condition in the presence of crown ether. CONSTITUTION:The objective compound of formula II can be obtained by the reaction of adenosine-3',5'-cyclic phosphoric acid (salt) of formula I [A<+> is H<+>, alkali (alkaline earth) metal ion, ammonium ion or organic ammonium ion] with an organic halide of formula RX (R is alkyl or aralkyl; X is halogen) under alkaline condition in the presence of crown ether. The amounts of the organic halide and crown ether to be used are pref. each 1-10 molar times based on the compound of the formula I, and the amount of the alkali to be used is pref. 5-10 molar times based on the compound of the formula I.

Description

Detailed Description of the Invention [Industrial Application Field] The present invention provides 2'-〇-substituted-7deno/-3',5L cyclic 7m (hereinafter referred to as 2'-〇-substituted-cAMP).
or a new method for producing the salt thereof.

Adenosine-3', 5'-cyclic phosphate (hereinafter cAMP)
) and its derivatives have various physiological activities and are expected to have a variety of uses as biochemical reagents and medicines. This compound is considered to be useful as having excellent pharmacological effects such as anti-inflammatory effect, platelet aggregation inhibiting effect, and blood pressure lowering effect.

[Conventional technology]

The method for producing 2'-O-methyl-cAMP is to use 2'-0-methyladenosine as a raw material, react with oxygen/chloride to form 2'-0-methyladenosine-57-phosphate, and then convert 4-morpholine into 2'-0-methyladenosine-57-phosphate. -N,N'-zinc-hexylcarboxamidine (Biochemistry, Vol. 12, pp. 1010-1016 (1973)); A method of reacting cAMP with methyl iodide in an aqueous Flukali solution. (Biochemistry
11, pp. 4931-4937 (1972)).

[Problems to be solved by the present invention]

In all of the above methods, the operations are complicated, and the 2'-OH group cannot be substituted with a medium-chain or long-chain alkyl group.

[Means to solve the problem]

As a result of intensive research aimed at solving these drawbacks, the present inventors found that by reacting CAMP or a salt thereof with an organic halide in the presence of an alkali and a crown ether, cAMP can be obtained even with medium-chain and long-chain alkyl groups. or its salt can be easily and efficiently substituted, and based on this knowledge, the present invention was completed.

That is, the present invention is characterized by reacting with a general formula halide (in the formula, A represents a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion, or an organic ammonium ion). cAMP or a salt thereof represented by the following formula is added under alkaline conditions and in the presence of crown ether: (in the formula R and AG
) has the above meaning) is a method for producing 2'-0-substituted-cAMP or a salt thereof.

The present invention will be explained in detail below.

cAMP or a salt thereof used as a starting material in the present invention is produced, for example, as follows.

belonging to the genus Microbacterium using a medium containing adenine, xylose or lipose and inorganic phosphate,
Patent No. 65178, which involves culturing a bacterium that has the ability to produce cAMP from adenine, xylose, halibose, and free phosphate, and producing cAMP from the culture.
(See specification No. 1).

Furthermore, cAMP or its salt can also be obtained by synthetic methods.

Alternatively, when free cAMP is treated with an alkali metal or alkaline earth metal hydroxide, carbonate, hydrogen carbonate, or an organic amine, preferably a trialkylamine, an alkali metal salt in the phosphate moiety of cAMP is formed. ,
For example, sodium salts, potassium salts, alkaline earth metal salts, such as calcium salts, magnesium salts, etc., organic ammonium salts, such as evatriethylammonium salts, ammonium salts, etc. can be obtained.

As R of the organic halide represented by the general formula (1), an alkyl group having 1 to 16 carbon atoms, preferably 3 to 14 carbon atoms, such as a methyl group, an ethyl group, a propyl group, a butyl group,
Straight chain or branched alkyl groups such as pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undenol group, dodecyl group, tritecyl group, tetradecyl group, pentadecyl group, hexadecyl group, aralkyl groups, such as benzyl/l&i, nitrobenzyl group, polybenzyl group, methylbenzyl group, hydroxybenzyl group, aminobenzyl group, phenethyl group,
Examples include phenylpropyl group.

The reaction between cAMP represented by the general formula ([I) or its salt and the organic halide represented by the general formula (1)]
The reaction is carried out under alkaline conditions and in the presence of crown ether.

Examples of alkalis include alkali metals or alkaline earths such as sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium carbonate, potassium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, calcium hydrogen carbonate, sodium hydride, and potassium hydride. Metal hydroxides, carbonates, hydrogen carbonates, hydrides, or alkoxides such as sodium methoxide are used alone or in combination.

Further, as the crown ether, for example, 18-crown-6, 15-crown-5, etc. are used.

This reaction takes place in a solvent. As a solvent, water, alcohols such as methanol, ethanol, oxane,
Ethers such as tetrahydrofuran, amides such as dimethylformamide and dimethylacetamide, esters of organic carboxylic acids such as ethyl acetate, dimethyl sulfoxide, and the like may be used individually or as a mixture of two or more as appropriate.

The amount of the organic halide represented by the general formula (厘) to be used is usually 1 to 20 times the molar amount of cAMP represented by the general formula (Q) or its salt, preferably 1 to 10@molar. In addition, cAMP represented by -monana ([l)
Or the amount of alkali used for the salt is usually 2 to 2.
The amount of crown ether used is usually 0.5 to 20 times, preferably t-to@solu.

This reaction is usually carried out at a temperature below the boiling point of the solvent used, generally from 10 to 150'C, preferably from 40 to 100'C.
At a temperature of ℃, for 1 hour or more while standing or stirring,
It is preferably carried out for 1 hour to 4 days.

In order to isolate and purify the target compound 2'-O-substituted-cAMP represented by general formula (1) or a salt thereof, for example, column chromatography using silica gel, alumina, io-74[1 fat, activated carbon, etc. Purification methods such as a crystallization method, a precipitation method by pH adjustment, a salting-out method using common salt, and an extraction method using an organic solvent may be used alone or in appropriate combinations.

In addition to the target compound, Ncl, 2LO-disubstituted-cA
In some cases, MP powder may be produced, and as a separation and purification method in this case, for example, a silica gel column or thin layer chromatography (developing solvent: methanol/chloroform, etc.) may be used. Furthermore, when using silica gel thin layer chromatography, the Rf value is 2'-〇-substituted-cA
Compared to MP, N',2'-0-disubstituted-cAMP
becomes large.

- The free acid of cAMP represented by the pattern (1), for example +i alkali metal or alkaline earth metal hydroxide,
The cyclic phosphoric acid moiety can be converted to the corresponding salt by the action of a carbonate, a hydrogen carbonate, or a tertiary amine such as ammonia or a ta-amine, such as triethylamine or tributylamine.

2'- represented by general formula (I) obtained by the present invention
Examples of 〇-substituted-cAMP and its salts include the following.

2'-〇-methyl-cAMP 2'-0-zthyl-
cAMP2'-0-propyl-cAMP2'
O-butyl-cAMP 2'-Q-pentyl-c
AMP2'-〇-hexyl-cAMP2'-0
-hebutyl-cAMP 2' -0-octyl-c
AMP2'-0-nonyl-cAMP2'-0-
Decyl-cAMP2'-〇-undecyl-cAMP2'
-0-dodenru-cAMP2'-0-tridecyl-cA
MP2'-〇-tetradecyl-cAMP2'-0-pentadecyl-cAMP 2'-0-hexadenyl-CAMP% or branched versions of these alkyl groups, 2'-〇-benzyl-cAMP 2'-0
-nido pbenzyl-cAMP 2' -Q-chlorobenzyl CAMP % 2' Q i thylbenzyl-cAMP2'-0-hydroxybenzyl-cAMP
2'-O-aminobe/dyl-cAMP 2
'-〇 -2-phenylethyl-cAMP, 2'
-0-3~Phenylpvir-CAMP% and their alkali metal salts, alkaline earth metal salts, ammonium salts, organic ammonium salts, etc.

According to the present invention, in the conventional method, the 2'-
Even medium-chain and long-chain alkyl groups that cannot be introduced into OH groups can be introduced very easily.

〔Effect of the invention〕

The present invention enables extremely efficient production of useful 2'-0-substituted-cAMP or its salts, which are expected to have excellent pharmacological effects such as anti-inflammatory effects, platelet aggregation inhibiting effects, and antihypertensive effects. It is possible to do so, and is of great industrial significance.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples.
3.299 cAMP was dissolved, and dioxane 150- in which 7.93 g of 18-crown-6 was dissolved was added. Then, n-pyl bromide 5.45-
and stirred in a water bath at 50° C. for 6 hours. 2 of the reaction solution
After neutralizing with N-hydrochloric acid, the solvent was distilled off under reduced pressure. Dissolve the remaining oily substance in a small amount of water and adjust the pH by adding 2N-hydrochloric acid.
2 to obtain crude crystals. A small amount of methanol was added to the obtained crude crystals, 2N-sodium hydroxide was added and dissolved, and this was subjected to silica gel thin layer chromatography (Development: methanol/chloroform, volume ratio: 1:1)
The target compound is separated and purified by UV absorption band (Rf
(value around 0.57) was extracted with methanol and dried under reduced pressure to obtain 1.38 g (yield: 37.5%) of the sodium salt of 2'-0-propyl-cAMP.

A portion of this was dissolved in water, then adjusted to pH 2 with 2N hydrochloric acid, and recrystallized.

Elemental analysis value: C'+ 3H+ sN 50 aP・
Measured value as 5/4H20 (%> Calculated value (%) 39.92 39.65 5.16 5.25 17.53 17.78 UV: λ 'Q2':: "0H (g)
258 (13200) nmRf [: 0.
42 (Silica gel thin layer μmography (developing solvent; 0.IN heptammonium chloride:methanol:acetonitrile, volume ratio: L:l:5), the same applies to the following examples) Example 2 2'-〇-Butyl Preparation of sodium salt of cAMP 10 Jl of an aqueous solution of potassium hydroxide 1.01.9
Dissolve cAMP 1.009 in / and add 18-
Dioxane 50 in which 2.014 g of Crown-6 was dissolved
− is added. Then n-butyl bromide 1.30
+w/ and stir for 2 days at 50°C in a water bath. After neutralizing the reaction solution with 2N hydrochloric acid, the solvent was distilled off under reduced pressure.

The remaining oily substance is dissolved in a small amount of water, and the pH is adjusted to 2 by adding 2N hydrochloric acid to obtain crude crystals. A small amount of methanol was added to the obtained crude crystals, 2N-sodium hydroxide was added and dissolved, and this was subjected to silica gel thin layer chromatography (developing solvent: methanol/couloform, volume ratio;
4:6), scrape off the UV absorption band (Rf value around 0.5) of the target compound, extract the oil with methanol, and dry under reduced pressure to obtain the sodium salt of 2'-〇-butyl-cAMP at 580 s+v ( Yield: 49.5%).

A portion of this was dissolved in water, then adjusted to pH 2 with 2N hydrochloric acid, and recrystallized.

Elemental analysis value: C+ aH2ON so 6P・5/
4 Measured value (%) as H20 Calculated value (%) 41.29 41.23 N 5.29 16.90 5.56 L7. L7 UV: λ Q2iN N・0H(t)
258 (14100) nmRf value: 0.44 Example 3 Production of 2'-〇-benzene cAMP To 30 ml of an aqueous solution in which 5.28 y of potassium hydroxide was dissolved, cAMP was added.
3.29 g was dissolved, and dioxane 150- in which 18-crown-6 was dissolved at 7.93 F was added thereto.

7.41- of n7 milbromide is then added and stirred for 2 days at 50<0>C in a water bath. After neutralizing the reaction solution with 2N-hydrochloric acid, the solvent was distilled off under reduced pressure.The remaining oily substance was dissolved in a small amount of water, the pH was adjusted to 2 by adding 2N-hydrochloric acid, and this was transferred to an activated carbon column (2, 2x After washing with water, the fraction eluted with methanol/water/28% ammonium hydroxide (volume ratio: 10: to: l) is collected and 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: 35:65), and the absorption band of the target compound (Rf value 0) was separated and purified. .3) was scraped off, extracted with methanol, and dried under reduced pressure. Dissolve this in water and then adjust the pH to 2 with 2N hydrochloric acid, resulting in 2L
1.13 g of O-benzene-cAMP crystals (yield: 2
8.8%) was obtained.

Elemental analysis value: Cl5H2□N 50 ap-1/2 H
HN as 20 Measured value (%) 44.07 5.69 17.26 Calculated value (%) 44.12 5.68 17.15 Uv
: λ'=4P: ”H(t) 258
(14500) nmRf [: 0.46 Example 4 Preparation of sodium salt of 2'-〇-heptyl-cAMP 1.65 p of cAMP was dissolved in an aqueous solution 15 in which 2.64 g of potassium hydroxide was dissolved. Add 3.97 g of 18-crown-6 and 75 g of dissolved dioxane. Then n-hebutyl bromide 4.7
Add 0 Jl/ and stir for 1 day at 60°C in a water bath.

After neutralizing the reaction solution with 2N hydrochloric acid, the solvent was distilled off under reduced pressure. The remaining oily substance is dissolved in a small amount of water, and the pH is adjusted to 2 by adding 2N hydrochloric acid to obtain crude crystals. A small amount of methanol was added to the obtained crude crystals, and 2N-sodium hydroxide was added to dissolve the crystals, which was then subjected to 7L gel thin layer chromatography (developing solvent: methanol/liroform, volume ratio: 35:65). After separating and purifying the target compound, scraping off the four absorption bands (Rf value around 0.4), extracting with methanol and drying under reduced pressure, 2'-0-hebutyl-cAMP is obtained.
1.34 g (yield 62.6%) of the sodium salt of was obtained.

A portion of this was dissolved in water, then adjusted to pH 2 with 2N hydrochloric acid, and recrystallized.

Elemental analysis value: HN as C1□H26N 50 oP-H20 Measured value (%) 46.12 6.35 15.37 Calculated value (%) 45.84 6.34 15.72 Uv
: λ';:” ”0H(g) 258 (13
500) nmRf value: 0.49 Example 5 Production of triethylamine salt of 2'-O-tetraden-cAMP 30 ml of an aqueous solution in which 5.28.5+ potassium hydroxide was dissolved
Dissolve e-cAMP 3.291/, and dissolve this di-18-
Dioxa 7150 dissolved in 7.93 g of Ri5un-6
Add ml. Next, n-mi! Add 17.85 ml of J Stirpmite and stir at 50°C in a water bath. Sarani n-myristyl bromide after 3 days 5.9
5m/added. After further stirring the reaction solution at 50°C for 2 days, the reaction solution was neutralized with 2N-hydrochloric acid, and the solvent was distilled off under reduced pressure. The remaining oily substance was dissolved in a small amount of water, and the pH was adjusted to 2 by adding 2N hydrochloric acid to obtain crude crystals. This was treated with dichloromethane 70:11/w, triethylamine was added and dissolved, and the mixture was extracted twice with 200 d of water. Dichloromethane is concentrated to a small volume and loaded onto a 30 g silica gel (packed in chloroform) column. Wash this column with chloroform and methanol/chloroform (volume JI ratio;
1:9 to 4:6) was used to collect the fraction containing the target compound, which was dried under reduced pressure.
5 g (yield 22%) was obtained.

A portion of this was dissolved in water, then adjusted to pH 2 with 2N hydrochloric acid, and recrystallized.

Elemental analysis value: C24H4゜N sOaP・3/4
Measured value (%) Calculated value (%) as H20 53.76 53.47 7.70 7.76 12.76 12.99 UV: λ 〇22': ``0H(t) 2
58 (12000) nmRf value: 0.55 Example 6 Production of 2'-〇-benzy-cAMP cAMP was added to 60 yr of an aqueous solution in which 1.32 g of potassium hydroxide was dissolved.
Dissolve 3.29 g and add 18-5 rough
-6 Add 150 #Il of total 2.6417 dissolved dioxane. Next, 1.19 g of n-benzyl bromide
/ and stir at room temperature for 1 day. After neutralizing the reaction solution with 2N hydrochloric acid, the solvent was distilled off under reduced pressure. The remaining oily substance is dissolved in a small amount of water, and the pH is adjusted to 2 by adding 2N hydrochloric acid to obtain crude crystals. A small amount of methanol was added to the obtained crude crystals, 2N sodium hydroxide was added to dissolve the crystals, and this was separated and purified using a thin layer of silica gel rhomadography (developing solvent: methanol/chloroform, volume ratio: 4:6). Then, the image absorption band of the target compound (Rf value around 0.25) is scraped off, extracted with methanol, and dried under reduced pressure. When this was dissolved in water and the pH was adjusted to 2 with 2N hydrochloric acid, 2'-0-benzyl cAMP crystals were obtained.
g (yield 50.7%) was obtained.

Elemental analysis value: C17H18N so oP・1/2
H as H20 Measured value (%) 47.61 4.23 16.20 Calculated value (%) 47.67 4.47 16.34 UV: λ 0 (ε) 58 (14700) f value: 0.47

Claims (1)

[Claims] (1) General formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (II) (In the formula, A^■ represents a hydrogen ion, an alkali metal ion, an alkaline earth metal ion, an ammonium ion, or an organic ammonium ion) adenosine-3
',5'-cyclic phosphoric acid or its salt under alkaline conditions.
and in the presence of a crown ether, the general formula is reacted with an organic halide represented by the general formula RX (III) (wherein R represents an alkyl group or an aralkyl group, and X represents a halogen atom). ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) 2'-O-substituted-adenosine-3',5'-cyclic phosphorus represented by (in the formula, R and A^■ have the above meanings) Process for producing acids or their salts.