CS202046B2 - Process for preparing functionalized derivatives of adenine - Google Patents

Description

The present invention provides a process for the preparation of adenine derivatives having reactive functions and the products thus obtained. More particularly, the present invention relates to a process for preparing adenine derivatives with reactive functions having an omega-carboxylic acid side chain at position 8, wherein the adenine derivatives have a reactive function ^. it starts from compounds that contain an adenine nucleus with a halogen atom in this position. Starting materials for this process can be obtained by conventional procedures, by halogenating compounds containing an adenine cycle such as nicotinic amide, adenine dinucleotide (NAD +), nicotinoylamidadenine dinucleotide phosphate (NADP +), adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate ( AMP), adenosine.

Most of these compounds are important in biochemistry and their functionalization extends their application field.

For example, in the case of NAD ⁺ , but note that these terminals are also well suited for other members, its functionalized derivatives can be used after coupling by covalent bonding with macromolecules that are either water soluble or insoluble, such as non-diffusing coenzymes or affinity chromatography. When bound to water-soluble macromolecular substances, the resulting products can be used as non-diffusible water-soluble macromolecularized coenzymes. These substances extend the possibilities of using known enzyme systems in which the enzyme is physically incorporated in insoluble porous structures such as fibers, polyacrylamide gel, microcapsules and the like that are impermeable to macromolecules. As a result of the physical incorporation of a water-soluble macromolecularized coenzyme into an enzyme or polyenzymatic system, in fact, both the enzyme and the coenzyme remain in intimate contact and the dispersion of the coenzyme out of the closed structure is prevented. This phenomenon is not possible with natural coenzyme due to its low molecular weight. In the case of binding to water-soluble macromolecular compounds, the products can be used in affinity chromatography or for enzymatic reactions in heterogeneous phase, whereby coenzyme can be regenerated.

SUMMARY OF THE INVENTION The present invention provides a process for the preparation of a functionalized adenine derivative comprising a compound selected from the group consisting of nicotinoylamidadenine dinucleotide, nicotinoylamidadenine dinucleotide phosphate, adenosine monophosphate, cyclic adenosine monophosphate, adenosine triphosphate, adenosine and adenine. of a polar aprotic solvent at 20 to 60 ° C to react with a compound of formula

M + -S- (CH 2) _n -COO-M ⁺ , where

M is an alkali metal ion and n is an integer of 2 to 4.

As aprotic polar solvent, for example, hexamethylphosphoric triamide, dimethylsulfoxide and dimethylformamide can be used. The reaction is carried out under anhydrous conditions and preferably at room temperature.

The reaction substitutes the halogen at the 8-position of the adenine nucleus according to the following scheme:

HIM

R where M ⁺ have the same meaning as above,

X is halogen and

R is a non-adenine residue of the compound.

The salt of the carboxyl derivative thus obtained is then converted to the corresponding free acid during processing.

The method is quite general, but the following describes the reaction of the disodium salt of 3-mercaptopropionic acid with NAD ⁺ and NADP + derivatives, which have accumulated on the carbon at the 8-position of the adenine nucleus of adenosine, in order to illuminate the methods required of said procedure.

However, it will be readily understood by one of ordinary skill in the art that the skilled person can obtain the functionalized adenine derivatives described above starting from any halogen-substituted adenine backbone at the 8-position by simply adapting the operating conditions to the nature of the parent compound and

Example 1

Preparation of 8- (2-carboxyethylthio) adenosine

To a solution of 380 mg (1.1 mmol) of 8-bromoadenosine in 8 mL of anhydrous hexamethylphosphoric triamide was added under stirring at room temperature and an anhydrous atmosphere (nitrogen) 633 mg (4.2 mmol) of 3-mercaptopropionic acid disodium salt (prepared from mercapto acids by adding a stoichiometric amount of sodium hydride at room temperature in anhydrous tetrahydrofuran and removing the solvent under reduced pressure).

After stirring at room temperature for 16 hours, the mixture is filtered and the filtrate is made up to 15 ml with water and extracted several times with chloroform until it is freed from hexamethyltriophthiamide.

The aqueous solution is adjusted by adding dilute hydrochloric acid to pH 8.5 and chromatographed on a Dowex-1 (HCOO ^-), eluting with a gradient of formic acid in water. The fractions with an A _{max of} 282 mm were combined, freeze-dried to give 327 mg of 8- (2-carboxyethylthio) -adenosine.

Thin layer analysis (silica gel with fluorescent indicator; eluent isopropanol-water-32% ammonia 7: 2: 1; visualization of the spot with UF source at 254 nm; Rf = 0.56) showed the product to be pure and also in high-voltage electrophoresis (Whatman paper 3MM 11 x 57 cm; electrolyte 0.02 M ammonium acetate pH 5.0, potential 5000 V in 40 minutes, visualization of the spot with UF source at 254 · nm; movement of the product towards the anode coincides with the presence carboxyl groups while adenosine migrates towards the cathode).

The UV spectrum in 0.1 M NaOH had peak absorption at 282 nm while 8-bromadenosine had it at 263 nm.

^{The 1} H NMR in NaO ² H shows, outside the adenosine-excluded signals from the 8-position proton, those attributed to the side chain protons: 2.88 (2H, t; CH 2 COO) and 3.86 (2H, t; CH 2 S) ).

Also, the mass spectrum confirms the structure attributed to the product (m / e 239, 221, 192, 167).

Example 1 a d 2

Preparation of Nicotinoylamide-8- (2-carboxyethylthio) -adenosine dinucleotide.

To 118 mg (160 mmol) of nicotinoylamide-8-bromadenine dinucleotide dissolved in 2 ml of anhydrous dimethylsulfoxide was added, under stirring at room temperature under an anhydrous atmosphere (nitrogen), 98.4 mg (656 mmol) of 3-mercapto202046 propionic acid disodium salt. Example 1).

After stirring at room temperature for 16 hours, the mixture was filtered and ten times its volume of acetone was added to the filtrate. The resulting precipitate collected by centrifugation and washed with acetone was dried under reduced pressure and dissolved in 15 ml of 0.1 M HCl. The solution was adjusted to pH 7.5 with dilute sodium and chromatographed on DOWEX-1 (HCOO) eluting with a gradient of formic acid in water.

The chromatographic fractions with an A _{max of} 276.5 were combined and freeze-dried to give 90 mg of nicotinoylamide-8- (2-carboxyethylthio) -adenine dinucleotide. Thin layer chromatography on silica gel with a fluorescent indicator showed the product to be pure; eluent: isobutyric acid-water-32% ammonia in a 66: 33: 1.7 by volume ratio; visualization of the spot with a UF source at 254 nm; Rf 0.31 (and in high-voltage electrophoresis with 3MM Whatman paper 11 x 57 cm; electrolyte: 0.02 M ammonium acetate, pH 5.0, potential 5000 V in 30 minutes; visualization of the spot with a UF source at 254 nm; mobility toward anode than NAD + in agreement with the presence of a carboxyl group).

The ultraviolet spectrum in the pyrophosphate buffer solution at pH 8.7 has a maximum at 276.5 nm, which on enzymatic reduction by yeast alcohol dehydrogenase changes to 282 nm to form a new peak at 340 nm, characteristic of the reduced nicotinoylamide nucleus.

NMR in ² H showed, outside the NAD ²⁺ related signals excluding proton from carbon at position 8 of the adenine nucleus, those attributable to side chain protons: delta 2.88 (2H, t;

CH 2 COO) and 3.86 (2H, t; CH 2 S). Also, the mass spectrum confirms the structure attributed to the product (m / e 221, 192, 167). Example 3

Preparation of Nicotinoylamide-8- (2-carboxyethylthio) adenine dinucleotide phosphate.

To 50 milligrams (57.6 micromoles of nicotinoylamide-8-bromadenine dinucleotide phosphate dissolved in 1 ml of anhydrous dimethylsulfoxide) was added 36 milligrams (240 micromoles of 3-mercaptopropionic acid disodium salt (prepared as was described in Example 1).

After stirring at room temperature for 16 hours, the mixture was filtered and 10 volumes of acetone were added to the filtrate. The precipitate obtained is collected by centrifugation, washed with acetone, dried under reduced pressure and dissolved in 10 ml of 0.1 M HCl.

The solution is adjusted to pH with dilute soda

7.5 and chromatographed on DOWEX-1 (Cl ^- ) eluting with a gradient of CaCl 2 in acidified water to pH 3 by addition of HCl. Chromatographic reactions containing the expected product are combined, concentrated to a small volume and freed from salts by gel filtration on Sephadex G-10 eluting the product with water.

The characterization of the nicotinoylamide-8- (2-carboxyethylthio) -adenine dinucleotide phosphate was performed similarly as described in Example 2 for the corresponding NAD ⁺ derivative (glucose-6-phosphate dehydrogenase was used for enzymatic reduction).

Claims (1)

SUBJECT Process for the preparation of functionalized adenine derivatives, characterized in that a compound selected from the group of the nicotinoylamidadenine dinucleotide, the nicotinoylamidadenine dinucleotide phosphate, adenosine monophosphate, cyclic adenosine monophosphate, adenosine triphosphate, adenosine and adenine. temperature 20 to ynAlez 60 ° C to react with a compound of formula M ^{+ -} S ^- (CH 2) _n ^- CO ^- M +, where M is an alkali metal ion and n is an integer of 2 to 4.