JPS6035355B2 - Method for producing sodium salt of adenosine derivative - Google Patents

Description

Detailed Description of the Invention The present invention relates to N6,2-0-dibutyryl adenosine-3,5 monocyclic phosphoric acid (hereinafter abbreviated as D-AMP), which has been developed for use as a pharmaceutical and biochemical research reagent. ) The method for producing sodium salts is characterized by the production of adenosine-3',6 monocyclic phosphoric acid (hereinafter c-
In the production process of DBc-AMP from A-Yamisaki (abbreviated as A-Yamisaki), only the D-Tech-AM Enamin salts are selectively extracted and purified from the reaction mixture with a halogenated hydrocarbon, and then this purified D
B-AMP/amine salts are reacted with the sodium salt of an appropriate organic or inorganic acid to exchange the salts and produce highly pure DBc.
- A method for producing AMP sodium salt.

The following three methods are conventionally known as methods for producing the sodium salt of D&-AMP.

[1} Method via barium salt (BiochimB
ioph$. Acte. 148 99 (1967) This method involves adding barium iodide to the DB-AM Yen-TEA salt obtained by reacting the triethylamine (hereinafter abbreviated as TEA) salt of c-AMP with anhydrous n-butyric acid, and ether by sequential addition of DBc
-AMP.barium salt is precipitated and then this barium salt is exchanged with sodium salt, but this method has the drawback that the yield is low and it is extremely difficult to remove the colored substances that have formed.

'2' Method using ion exchange resin (Special Seki Sho 48-527
96) Mr. D-AMP is a method of adsorbing DBc-AMP to a basic anion exchange resin and desorbing it with a suitable desorbent.
・This is a method of treating TEA salt with a sodium-type acidic cation exchange resin, but both methods require a long time to operate, and in order to keep DBc-AMP stable, which is easily decomposed chemically at room temperature, adsorption is required. It is necessary to cool the liquid, ion exchange resin packed column, desorption liquid agent, desorption liquid, etc., and this method is not economically or industrially advantageous.

{3' Neutralization method (Japanese Patent Application Laid-open No. 48-498) Add water to the reaction solution, hydrolyze n-butyric anhydride, neutralize with calcium hydroxide, separate as calcium n-butyrate, and then remove with ion exchange resin. After removing calcium and replacing it with sodium salt with caustic soda, alkali salts were added to form DBc-AMP.
In this method, sodium salts are precipitated, and alkaline salts are removed and purified by repeating moat extraction and extraction with organic solvents.

However, in this method, when replacing the sodium salt with caustic soda, DB-AMP decomposes, and alkali salts are mixed into the sodium salt, and the yield decreases when trying to completely remove the alkali salts. Moreover, it has drawbacks such as complicated operations, and is not necessarily suitable for industrial implementation. Generally, after reacting c-AMP with n-butyric anhydride, water is added to the reaction solution to remove excess n-butyric anhydride.
The reaction mixture, which is hydrolyzed to butyric acid and washed with a suitable organic solvent such as ether or methyl isobutyl ketone to remove n-butyric acid, usually contains 2'- as a by-product in addition to the desired DBc-AM circle. 0-monobutyryl adenosine-3,5 monocyclic phosphoric acid (hereinafter abbreviated as OMc-AM), N6-monobutyryl adenosine-3,6 monocyclic phosphoric acid (hereinafter referred to as NMc-
AMP), c-AMP, etc.

That is, D&-AMP is very easily decomposed in acidic or alkaline conditions, and OMc-AMP is generated in acidic conditions and NMc-AMP is generated in alkaline conditions, so the coexistence of these created products in the reaction mixture is usually unavoidable. Moreover, as by-products, for example, OMc-AMP/TEA salt, NMc-AMP/TEA salt, c-A
DBc-AMP/TEA salt, which coexists with MP/TEA salt, etc., is difficult to purify by recrystallization, which is commonly used industrially.

Further, the present inventors attempted recrystallization after exchanging these TEA salts with sodium salts, but since the physical properties were extremely similar, it was impossible to separate and remove the by-products. As described above, the method for producing purified DB-AMP sodium salt is extremely difficult, and almost no industrially advantageous method has been found.

Therefore, as a result of intensive research into industrial methods for efficiently producing purified DBc-AMP/sodium salt, the present inventors found that DBc-AMP and by-products (OMc-AM, NMc-AMP, c-AMP, etc.) ) When an aqueous solution of TEA salt ( ) is brought into contact with a halogenated hydrocarbon solvent such as chloroform, surprisingly, only DB-AMP/TEA salt selectively migrates to the organic solvent, so this organic layer is separated and the solvent is distilled off. It was discovered that highly purified sodium D-AMP sodium salt could be obtained in good yield by removing and salt-exchanging with sodium n-butyrate, etc., and based on these findings, the present invention was completed.

To describe the present invention in more detail, c-AMP is reacted with n-butyric anhydride, water is added to the reaction solution, excess n-butyric anhydride is hydrolyzed, and the generated n-butyric acid is converted into ether, isopropyl The reaction mixture, which has been washed away with a suitable first organic solvent such as ether or methyl isobutyl ketone, is usually used as a by-product in addition to the desired D-AMP/amine salts.
Contains amine salts such as AMP, NMc-AMP, and c-AMP.

This reaction mixture is concentrated and brought into contact with halogenated hydrocarbons such as methane dichloride, ethane dichloride, chloroform, etc., resulting in the creation of OMc-AMP, NMc-AMP,
Amine salts such as c-AMP remain in the aqueous solution,
Only the DBc-AMP amine salts are selectively extracted into the halogenated hydrocarbon. Examples of amine salts include primary amines such as methylamine, ethylamine, n-propylamine, and cyclohexylamine, secondary amines such as dimethylamine, diethylamine, piperidine, pyrrolidine, piperazine, and morpholine, trimethylamine, triethylamine, tri-n-propylamine, tri- Tertiary amines such as n-butylamine and salts of ammonia have been used. The first organic solvent extracts only n-acid and DB-A
It is preferable to use one that does not extract MP/amine salts. Also,
If the extraction efficiency of DBc-AMP/amine salts is low, dissolve appropriate organic or inorganic salts to extract DBc-AMP.
- The extraction efficiency of amine salts can also be improved.

Furthermore, in the method of the present inventors, OM
DBc-AM salts containing by-products of sodium salts such as c-AMP, NMc-AMm, and c-AMP are also dissolved in water and converted to amine salts by adding equimolar amine mineral salts. After that, by performing the above-mentioned processing, DBc-
AMP can be purified.

That is, purification is also possible when DBC-AMP and by-products are metal salts other than amine salts, such as alkali metal salts such as sodium and potassium, and alkali metal salts such as calcium and barium. Suitable halogenated hydrocarbons used for extraction include methane dichloride, ethane dichloride, and chloroform.

In the case of high boiling point solvents, these solvent fractions are fractionated and then contacted with at least one hydrocarbon-based solvent in the presence of an aqueous solvent to form D-Seichi AMP.
It is also possible to transfer the amine salts into an aqueous solvent.

The solvent of the DBc-AMP/amine salts purified in this manner was distilled off, dissolved in a small amount of water, and a suitable sodium salt of an organic or inorganic acid was added, followed by a hydrophilic organic solvent such as acetone, acetonitrile, or methyl ethyl ketone. By performing the salt exchange, highly pure DR-AM sodium salt can be obtained in high yield. As these sodium salts, for example, sodium acetate, sodium propionate, sodium n-butyrate, sodium iodide, sodium benzenesulfonate, and sodium p-toluenesulfonate are preferably used. This method for producing DB-AMP/sodium salt by salt exchange of sodium salts does not require neutralization with alkali or concentration operations as in known methods, so the decomposition of D-Tech-AMP is significantly suppressed.
It has the advantage of providing highly pure D-technique-AMP sodium salt. As is clear from the above, the method of the present inventors purifies DB-AMP/amine salts by bringing the reaction mixture of D&-AMP and by-product amine salts into contact with an appropriate halogenated hydrocarbon. Then, this purified Mr. D-AMP/amine salt is subjected to salt exchange with a suitable organic acid sodium salt, thereby providing an easy and efficient method for producing highly pure D-AMP/sodium salt. .

According to the present invention, separation and purification of ○
Seiichi AMP sodium salt can be efficiently produced with high purity through simple extraction and salt exchange, and it provides an economically and industrially superior method.

Next, the present invention will be specifically explained with reference to Examples.

Example 1 After reacting c-AMP6.59 with n-butyric anhydride, water was added to the reaction solution to hydrolyze excess n-butyric anhydride, and the n-butyric acid was removed by washing with methyl isobutyl ketone. .

Saiyu residual liquid (analysis value by high performance liquid chromatography:
DBc-AM Yen/TEA salt 93.7%, OMc-AMP
・TEA salt 2.3%, NMc-AMP・TEA salt 3.6
%, c-AMP/TEA salt 0.4%), and the total amount was 4
0 secretion, extracted 3 times with 200ml of chloroform, and when the chloroform was distilled off, the residue was 9.15mm (analytical value, DBc
-AMP/TEA salt 99.7%, OMc-AMP/TE
A salt 0.3%, NMc-AMP/TEA salt 0.0%, c
-AMP/TEA salt 0.0%) is obtained. Water this remaining groove with 1
2. Dissolved in 1.77 g of sodium n-butyrate, dissolved, added 500 g of acetonitrile, left in the freezer overnight, collected the precipitated crystals, and found 6.55 g of sodium n-butyrate (analytical value) :DBc-AMP・Na salt 99.5
%, OMc-AK Naka・Na salt 0.3%, NMc-AMP
・0.1% of Na salt, 0.0% of c-AMP・Na salt) are obtained. Yield 66.3%. This product is a separately synthesized DBc-AM.
The IR is completely identical to the P sodium salt standard. Note that secondary crystals can be obtained from the mother liquor. Example 2 OMc-A Yasaku/Na salt 3.7% as impurities, NMc
-AMP/Na salt 5.4%, c-AMP/Na salt 4.1
% of crude D&-AMP/Na salt (1.88 units as D technique-AMP/Na salt) was dissolved in 10 cups of water, and 0.63 units of TEA/hydrochloride was added and dissolved.

Next, it was extracted 3 times with 50% of chloroform, and when the chloroform was removed, the residual amount was 2.05% (analytical value; DBc-AMP
・TEA salt 99.5%, OMc-AMP・TEA salt 0.
4%, NMc-AMP TEA salt 0.1%, c-AM Yen TEA salt 0.0%). This balance is divided into 2.5 [
Add 0.40 g of sodium n-butyrate and dissolve.
After adding 150 g of acetonitrile and leaving it in the freezer overnight, and removing the precipitated crystals, Mr. D - 1.51 g of AMP Na salt (
Analysis value; D string - AMP/Na salt 99.4%, OMc-A
MP/Na salt 0.4%, NMc-AM yen/Na salt 0.2
%, c-AM yen/Na salt 0.0%).

This product is a separately synthesized sample of Mr. D-AM0・Na salt and IR
matches perfectly. Example 3 DMc-AMP/Na salt 2.3% and NMc as impurities
-AMP/Na salt 3.6%, c-AMP/Na salt 1.8
Crude D & -AMP Na salt containing 2.04% (OR
-Dissolve 1.88 g of AMP/Na salt in 10 g of water, add 0.59 g of TEA/HCI salt and dissolve.

Next, it was extracted three times with 100% dichloromethane, and when the solvent was distilled off, the residue was 1.96 mm (analytical value; DBc-AMP・TE
A salt 99.8%, OMc-AMP/TEA salt 0.2%,
NMc-AMP/TEA salt 0.0%, c-AMm/TE
A salt (0.0%) is obtained. When this residual liquid is crystallized with isopropyl ether, the theoretical value (%): C49.74, day 6.96 as elemental analysis value C24 day ship N608P-ura day 20
, N14.50 analysis value (%): C49.83, day 6.9
2, N14.57 Dissolve this remaining board in 2.5 parts of water, p-
Add 0.67 g of sodium toluene sulfonate and melt, add 170 g of acetone, leave in the freezer overnight, and collect the precipitated crystals to obtain 1.43 g of sodium salt of DBc-AM (analysis value; Yen/Na salt 99.7%, OMc-A
MP/Na salt 0.3%, NMc-AM yen/Na salt 0.0
%, c-AMP.Na salt 0.0%).

This product is a separately synthesized specimen of DBc-AMP/Na salt and I
R matches perfectly. Note that secondary crystals can be obtained from the mother liquor.
Example 4 OMc-AM yen/Na salt 3.7%, NMc as impurities
-AMP/Na salt 5.4%, c-AMP/Na salt 4.1
Crude D&-NMP・Na salt containing 1.96% (DB
-Dissolve 1.70 ml (as AMP.Na salt) in 30 ml of water, add and dissolve 0.92 ml of tri-butylamine hydrochloride.

Next, it was extracted 3 times with 30 volumes of ethane dichloride, and the solvent was distilled off, leaving a residue of 2.09% (analytical value: 98.9% of DBc-AMP/tri-n-butylamine salt, OMc-AMP/tri-n-butylamine salt). 0.9%, NMc-AMP/tri-n-butylamine salt 0.2%, c-AMP/tri-n
-butylamine salt 0.0%). When this afterglow is crystallized with isopropyl ether, the elemental analysis value C3 is 5,
Theoretical value (%) as N608P: C55.03 days 7.8
5N12.84 Analysis value (%): C65.18 days 7.73
N12.65 Dissolve this residue in 2.5 parts of water, add 0.36 parts of sodium n-butyrate, dissolve, and add 1 part of acetonitrile.
After adding 70% of the solution and leaving it in the freezer overnight, and removing the precipitated crystals, Mr. ○ - AMP/Na salt was 1.19% (analytical value; Mr. D - A)
MP・Na salt 98.8%, OMc-AMP・Na salt 0.
9%, NMc-AM yen/Na salt 0.3%, c-AM yen/
Na salt 0.0%) is obtained.

This product is a separately synthesized specimen of D&-AMP/Na salt and IR
matches perfectly. Note that secondary crystals can be obtained from the mother liquor. Example 5 OMc-AM yen/Na salt 3.7%, NMc as impurities
-AMP/Na salt 5.4%, c-AMP/Na salt 4.1
Crude DB-AMP Na salt containing 2.03% (DB
-Dissolve 1.76 ml of AMP/Na salt in 10 portions of water, add 0.53 ml of piverizine hydrochloride, and dissolve.

Next, it was extracted three times with 100% chloroform, and the solvent was distilled off, leaving a residue of 1.69% (analytical value; DBc-AMP/piveridine salt 98.1%, OMc-AMP/piberidine salt 1%).
．． 4%, NMc-AMP/piveridine salt 0.5%, c-
AMP/piveridine salt 0.0%) is obtained. Dissolve this residue in 2.5 parts of water, add 0.34 parts of sodium n-butyrate to dissolve it, add 150 parts of acetonitrile, leave it in the freezer overnight, and collect the precipitated crystals. Salt 1.09% (Analysis value; DB-AMP/Na salt 98.0%
, OMc-AMP・Na salt 1.4%, NMc-AM Yen・
0.6% of Na salt, 0.0% of c-AMm.Na salt).

Example 6 OMc-AM yen/Na salt 2.3%, NMc as impurities
-AMP/Na salt 3.6%, c-AMP/Na salt 1.8
Crude D-AMP Na salt containing 3.21% (DB
AMP.Na salt (2.96 g) was dissolved in 50 g of water, and 1.48 g of tri-n-butylamine hydrochloride was added and dissolved.

Then, the mixture was extracted three times with 50% ethane dichloride, and the solvent was distilled off to obtain a residue of 3.69 g. This residue was dissolved in 4.5 ml of water, 1.1 ml of sodium p-toluenesulfonate was added, dissolved, 300 ml of acetonitrile was added, and the mixture was left in the freezer overnight. When the precipitated crystals were collected, purified DBc-AMP was obtained.・N
a salt 2.34 min (analysis value; DBc-AMP/Na salt 99
．． 5%, OMC-AMP・Na salt 0.4%, NMc-A
MP/Na salt 0.1%, c-AMP/Na salt 0.0%)
get.

Example 7 OMc-AM Yen/Na salt 2.3% as impurities, NMC
-AMP/Na salt 3.6%, c-AMP/Na salt 1.8
% - Dissolve 3.17 days of AMP/Na salt (D base - 2.98 days as AMP/Na salt) on top of water, add 0.91 hours of TEA/HCI salt and dissolve. .

The mixture was then extracted three times with 150 g of methane dichloride, and the solvent was removed to give a residue of 3.08 g. Dissolve this residue in 4.0 parts of water, add 0.529 parts of sodium propionate, dissolve, add 200 parts of methyl ethyl ketone, leave in the freezer overnight, collect the precipitated crystals, and the purified DBc-AM Yen.
Na salt 2.14 hours (analysis value; DB-AMP・Na salt 99
．． 6%, OMC-AMP・Na salt 0.3%, NMc-A
MP/Na salt 0.1%, c-AMP/Na salt 0.0%)
get.

Claims (1)

[Claims] 1 Adenosine-3', 5-cyclic phosphoric acid amine salts, 2'
-0-Monobutyryl adenosine-3', 5'-cyclic phosphoric acid amine salts, N^6-monobutyryl adenosine-3',
5′-N^6・2′- coexisting with cyclic phosphoric acid amine salts, etc.
From an aqueous solution of 0-dibutyryladenosine-3',5'-cyclic phosphoric acid amine salt, N^6,2' is prepared with a halogenated hydrocarbon.
N^ characterized by selectively extracting 0-dibutyryladenosine-3',5'-cyclic phosphoric acid amine salts, and then reacting this with a sodium salt of an organic or inorganic acid to exchange the salt. Process for producing 6,2'-0-dibutyryl adenosine-3',5'-cyclic phosphate sodium salt (here, amines mean linear or cycloaliphatic primary, secondary or tertiary amines and ammonia) ).