NO834433L - PROCEDURE FOR THE PREPARATION OF 3`, 5` DIARABINO SIDES - Google Patents

Description

The present invention relates to a process for the production of monophosphate dinucleosides with the formula Ara A-p-Ara C, Ara C-p-Ara A and Ara A-p-Ara-A

in which

Ara A is 9-3-D-arabinofuranosyl-adenine,

Ara C is 1-3-D-arabinofuranosyl-cytosine, and

p is the monophosphate group and

the characteristic feature of the invention is that

1) one prepares the synthons adenine and cytosine which carry a free OH group in the 3' position, 2) one prepares the synthons adenine and cytosine which carry a free OH group at the 5' position, 3) one carries out the synthesis of monophosphate the dinucleosides by - phosphorylation of the synthon bearing a free OH group in the 3' position by means of a bifunctional phosphorylating agent,

- condensation of the diester obtained with it

selected nuclide by means of a condensing agent, and - deprotection of the obtained compounds to obtain the desired diirers in the form of the triethylammonium salts.

These and other features of the invention appear in the patent claims.

The nucleosides of arabinose or the arabinosides, which have analogous structures to the ribosides, have interesting antiviral and antitumoral properties that allow their use in the therapy of certain compounds, such as 9-3-D-arabinofuranosyl-adenine (adenine-arabinoside) and 1-3-D -arabinofuranosyl-cytosine (cytosine-arabinoside).

A disadvantage that occurs in the use of therapeutic forms of these substances is due to their poor solubility (about 1 part per 1000 parts at ordinary temperature) which necessitates the use of a solvent such as DMSO in topical preparations or the perfusion of considerable volumes of liquid when proceeding in the usual way .

Attempts have been made to remedy this difficulty by using, instead of the nucleosides, their 5'-monophosphates, which are highly soluble. These molecules, however, carry two negative electrical charges that only penetrate the cells with difficulty and their antiviral and antitumoral effects are therefore very low. In contrast, the dinucleotides of general formula arabinoside-3',5'-phosphate-arabinoside have only a single electric charge for two nucleoside molecules and experience shows that their biological activity is similar to the nucleoside constituents. Their solubility is satisfactory.

It has previously been shown (Brivat de Garilhe and De Rudder Progr. Antimicr. & Anticancer Chemother., Vol. II,

pages 180-184, Tokyo 1970) that between adenine-arabinoside and cytcsine-arabinoside there exists a synergistic activity shown especially against ascitic Ehrlich tumor in mice.

The purpose of the present invention is, based on the previous findings, to produce molecules of 3',5'-diarabinosides where the solubility is satisfactory and which exhibit a very good activity.

The procedure for preparing the compounds appears from the following reaction schemes: In the first step, the two starting synthons are prepared which carry a free OH group in the 3'-position (fig. 1 and 2).

In another step, the two starting synthons are prepared which carry a free OH group in the 5' position (fig. 3 and 4).

And finally, according to the invention, the monophosphate dinucleotides Ara A-p-Ara C, Ara C-p-Ara A are produced

and Ara A-p-Ara A (reaction schemes 5, 6, 7) by the phosphoric acid triester method which requires two steps: the first step consists in a phosphorylation of the synthon bearing a free OH group in the 3' position

(fig. 5)

the second step consists in a condensation of the obtained diester and the nucleoside with the help of a condensation agent such as e.g. 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT) (Fig. 6) and then the finally obtained dimers are deprotected in the form of the triethylammonium salt (Fig. 7).

The phosphorylation of the synthon which carries a free OH group in the 3' position can be carried out with the help of a bifunctional phosphorylating agent such as o-chlorophenyl phosphorus dichloridate.

The reaction of the nucleoside derivatives protected in the 2'-position and 5'-position (6) and (12) with an excess of O-chlorophenyl-phosphordi-(L,2,4-triazolide) (19) prepared in situ in pyridine leads after treatment with triethylamine to phosphoric acid diesters (20a) and (20b). The latter are isolated in the form of the triethylammonium salt after extraction with chloroform and precipitation in petroleum ether (Fig. 5).

In the second step, the condensation agent chosen is 1-mesitylene-sulfonyl-3-nitro-1,2,4-triazole (MSNT) (13).

A solution in pyridine of the phosphodiester nucleosides (20a) or (20b) and the synthons (15) or (18) is treated with 2.5 to 3 equivalents of MSNT for 45 min. at normal temperature. The crude reaction mixture is then poured into an aqueous saturated solution of sodium bicarbonate and extracted with chloroform (fig. 6).

The three monophosphate dinucleosides (21a), (21b) and (21c) are obtained after purification over a silica gel column and precipitation from petroleum ether. The deprotection of compounds (21a), (21b) and (21c) is carried out as follows: the o-chlorophenyl groups are deblocked with a solution of 4-nitro-benzaldoxime (0.3M) and triethylamine (0.3M) in a mixture of dioxane /water (1/1, volume/volume) for 24 hours at 35°C.

the benzoyl groups are removed using ammonia at 50°C for 24 hours.

The monomethoxytrityl group is hydrolysed with 80% acetic acid for 1 hour 15 minutes.

After extraction, the dimers (22a), (22b) and (22c) are purified

on a column of DEAE-Sephadex A2 5 and then purified again by means of semi-preparative HPLC on column C18 in reverse phase with a water/acetonitrile gradient (Fig. 7).

The compounds are obtained in the form of their triethylammonium salts.

The reaction forms are indicated in the following.

The following examples illustrate the invention.

Analyzes and spectra UV, IR and RMN confirm the structure of the compounds.

Example 1:

Synton Ara-adenosine bearing a free OH group in the 3' position.

1.1 N 3-benzoyl-D-9-arabinofuranosyl-adenine (2)

To a suspension of 1 g (3.74 mmol) of arabinosyl-adenine

(1) 18 ml (150 mmol) of benzoyl chloride are added dropwise to 30 ml of anhydrous pyridine while cooling. After 36 hours of stirring at normal temperature, 100 ml of water mixed with ice is added. The reaction mixture is extracted with chloroform (4 x 60 ml).

The organic phase is washed with aqueous sodium bicarbonate solution (5 x 70 ml) and then with water (5 x 50 ml), dried over sodium sulfate, filtered,

evaporated and then evaporated together with toluene (2 x 50 ml).

After purification on a silica gel column (eluent CHCl3), the obtained nucleosides are dissolved in 25 ml of pyridine. This solution is added to 40 ml of NaOH (2N) and then stirred for 20 min. 80 ml resin "Dowex 50 - X2(pyridinium)

(pH = 6-7) is then added. The resin is filtered off, washed with water (3 x 50 ml). The aqueous phase is extracted with ether (6 x 100 ml), evaporated, the precipitate formed is filtered and then crystallized from water. Compound (2) is obtained.

Temp. = 145 - 147°C (water)

1.2 3',5'-TIPDSi-N<6>3-benzoyl-D-9-arabinofuranosyl-adenine (3)

To 1.98 g (6.71 mmol) TIPDSiCl2, add 1.34 g

(19.66 mmol) of imidazole, 1.66 g (4.47 mmol) of compound (2) and 50 ml of anhydrous DMF.

After stirring for 1 hour at normal temperature, add

50 ml of water. The mixture is evaporated under reduced pressure. The residue is redissolved in 100 ml of water and then extracted with chloroform (3 x 80 ml). The organic phase is dried over magnesium sulphate, filtered and evaporated to dryness. After purification on a silica gel column (eluent CHC^/AcOEt, 50/50, volume/volume) the compound (3) is obtained in the form of a white foam.

1.3 3' ,5'-TIPDSi-2',N 3-dibenzoyl-D-9-arabinofuranosyl-adenine (4)

A solution containing 2.1 g (3.42 mmol) of compound (3) is stirred for 3 hours at room temperature

0.851 g (3.76 mmol) of benzoic anhydride and 0.138 g (1.14 mmol) of 4-dimethylaminopyridine in 15 ml of anhydrous pyridine.

After evaporation under reduced pressure of the pyridine

dissolve the residue in 100 ml of chloroform. The resulting solution is washed sequentially with a saturated sodium bicarbonate solution and with water (2 x 100 mL). The organic phase is dried over sodium sulphate, filtered and evaporated under reduced pressure.

After chromatography on a silica gel column (eluent: CHCl3/ether, 98/2, 95/5, 90/10, volume/volume) the compound (4) is obtained in the form of a white foam.

1.4 2',N 3-di-benzoyl-D-9-arabinofuranosyl-adenine (5)

100 mg (0.139 mmol) of the compound (4) is dissolved in

6 ml of a solution of (0.5M) tetrabutylammonium fluoride in tetrahydrofuran. After 45 min. stirring at ordinary temperature, the solution is evaporated under reduced pressure. The residue is chromatographed on a silica gel column (eluent: CHCl3/MeOH, 99/1, 98/2, volume/volume). The compound (5) is obtained after precipitation from chloroform.

Temp. = 124 - 126°C (CHC13) 1.5 5'-monomethoxytrityl-2',N^3-dibenzoyl-D-9-arabinofuranosyl-adenine (6)

A solution containing 0.900 g (1.89 mmol) of compound (5) and 1.10 g (3.78 mmol) of monomethoxytrityl chloride in 20 ml of anhydrous pyridine is stirred for 22 hours at room temperature in the absence of moisture and light. After adding 150 ml of water mixed with ice, the reaction mixture is extracted with chloroform (3 x 70 ml). The organic phase is dried over sodium sulfate, filtered, evaporated to dryness, then evaporated together with toluene (2 x 30 ml). The residue is chromatographed on a silica gel column (eluent: CHCl3/Et3N 100/0.1, volume/volume). The product (6) is obtained after precipitation from petroleum ether.

Example 2:

Synton-Ara-cytidine bearing a free OG group in the 3-position.

2.1 N 4 3- benzoyl- Q- 1- arabinofuranosyl- cytosine (8)

To a suspension of 500 mg (1.79 mmol) of the hydrochloride of Ara-C in 5 ml of anhydrous methanol is added 10 ml (1.79 mmol of sodium) of sodium methylate.

After 15 min. stirring at ordinary temperature, the residue is evaporated, it is evaporated together with ethanol 100 and then with anhydrous pyridine. To the white precipitate obtained, 10 ml of pyridine and 8 ml of benzoyl chloride (at 0°C) are added. After stirring overnight, the mixture is poured into 50 ml of ice-cold water and extracted with chloroform (3 x 50 ml).

The organic phase is washed with water (3 x 30 ml), with sodium bicarbonate solution, filtered and evaporated with toluene (2 x 10 ml). The reaction mixture is purified on a silica gel column (eluent: CHCI3)• The resulting foam is dissolved in 22 ml of pyridine and then 11 ml

NaOH (2N).

After 20 min. stirring, the solution is neutralized with resin Dowex-50 W X2 (pyridinium). The resin is filtered off, washed with water (3 x 80 ml) and the aqueous phase is extracted with ether (5 x 100 ml) and then evaporated under reduced pressure. The product (8) is crystallized from methanol.

Temp. = 186 - 190°C (methanol)

2.2 3',5'-TIPDSi-N<4>3-benzoyl-D-1-arabinofuranosyl-

cytosine (9)

200 mg (0.82 mmol) of the compound (8) is dissolved in 4 ml of pyridine and to this solution is added 0.3 ml (0.97 mmol) TIPDSiCl,,. After stirring for 1 hour at room temperature, the mixture is evaporated to dryness. The residue obtained is redissolved in 10 ml of chloroform, washed with 20 ml of water and extracted with (3 x 20 ml) chloroform. The organic phases are collected, dried over magnesium sulphate, filtered and evaporated under reduced pressure. The resulting oil is fractionated on a silica gel column (eluent: CHCl^,

CHCl3/MeOH, 99/1, v/v). The compound (9) is obtained.

2.3 3',5'-TIPDSi-2',N 43-dibenzoyl-D-1-arabinofuranosyl-cytosine (10)

A solution of 250 mg (0.42 mmol) of compound (9),

105 mg (0.46 mmol) of benzoic anhydride and 17 mg (0.14 mmol) of 4-dimethylaminopyridine in 2 ml of anhydrous pyridine are stirred at ordinary temperature for 2 hours. The reaction mixture is poured over 10 ml of water and extracted with chloroform (3 x 10 ml). The organic phase is washed with water (3 x 10 ml), dried over magnesium sulphate, filtered, evaporated and then evaporated with toluene (2 x 5 ml). The residue is fractionated on a silica gel column (eluent: CHCl3/MeOH, 99.5/0.5, volume/volume.

The compound (10) is obtained in the form of a white foam.

2.4 2',N 43-dibenzoyl-D-1-arabinofuranosyl-cytosine (11)

245 mg (0.35 mmol) of the compound (10) is dissolved in

6.5 ml of a solution (0.5M) of tetrabutylammonium fluoride in anhydrous tetrahydrofuran. After 45 min. stirring at normal temperature, the solution is incorporated under reduced pressure. The residue is chromatographed on a silica gel column (eluent: CHCl3/MeOH, 98/2, volume/volume). The compound (11) is obtained.

Temp. = 219 - 222°C (chloroform).

2.5 5'-monomethoxytrityl-2',N 43-dibenzoyl-D-1-arabinofuranosyl-cytosine (12)

A solution of 140 mg (0.31 mmol) of the anhydrous compound (11) and 184 mg (0.62 mmol) of monomethoxytrityl chloride in 2 ml of anhydrous pyridine is stirred for 22 hours at room temperature under exclusion of moisture and light.

After adding 10 ml of water mixed with ice, the reaction mixture is extracted with chloroform (3 x 10 ml). The organic phase is dried over magnesium sulphate, filtered, evaporated and co-evaporated with toluene (2 x 5 ml). The obtained oil is chromatographed on a silica gel column (eluent: CHCl3/MeOH/Et3N/, 99/1/0.1, volume/volume/volume. Compound (12) is obtained.

Example 3:

Synton Ara-adenosine bearing a free OH group in the 5' position.

3.1 5'-monomethoxytrityl-N<6>0-benzoyl-D-arabinofuranosyl-adenine (13)

A solution of 400 mg (1.08 mmol) of compound (12) and 650 mg (2.16 mmol) of monomethoxytrityl chloride in 7 ml of anhydrous pyridine is stirred at room temperature for 22 hours under exclusion of moisture and light. After adding 25 ml of water mixed with ice, the reaction mixture is extracted with chloroform (3 x 15 ml). The organic phase is dried over sodium sulphate, filtered, evaporated and co-evaporated with toluene (2 x 10 ml).

The residue is chromatographed on a silica gel column (eluent: CHCl3/MeOH/Et3N, 98/1/1, volume/volume/volume). The compound (13) is obtained.

3.2 5'-monomethoxytrityl-2',3',3-tribenzoyl-D-9- arabinofuranosyl- adenine (14)

A solution of 200 mg (0.31 mmol) of the compound (13), 146 mg (0.69 mmol) of benzoic anhydride and 13 mg (0.10 mmol) of 4-dimethylaminopyridine in 2 ml of anhydrous pyridine is stirred at ordinary temperature for 1 hour and 15 minutes. After adding 30 ml of aqueous sodium bicarbonate solution, the solution is extracted with chloroform (3 x 20 ml). The organic phase is washed with water (3 x 15 ml), dried over sodium sulfate, filtered, evaporated, coevaporated with toluene (2 x 10 ml). The residue is purified on a silica gel column (eluent: CHCl3/hexane/Et3N, 80/19/1, volume/volume/volume.

The compound (14) is obtained after precipitation from hexane.

3.3 2', 3' ,N6(3-tribenzoyl-D-9-arabinofuranosyl-adenine (15)

Dissolve 200 mg (0.24 mmol) of the compound (14) in

9 ml of a mixture of chloroform/methanol (7/3, volume/volume) containing 2% benzenesulfonic acid. The reaction mixture is stirred for 20 min. at 0°C. 10 ml of aqueous sodium bicarbonate solution is added. The mixture is extracted with chloroform (3 x 8 ml). The organic phase is washed with 10 ml of anhydrous hydrogen carbonate solution and then with water (3 x 10 ml), dried over sodium sulphate, filtered and evaporated under reduced pressure. The residue is purified on a silica gel column (eluent: CHCl3/MeOH, 99/1, volume/volume).

The product (15) is obtained after crystallization from ethanol.

M.p.=135 - 137°C (ethanol).

Example 4:

Synton-Ara-cytidine bearing a free OH group in the 5' position. 4.1 5'3-monomethoxytrityl-D-1-arabinofuranosyl-cytosine (16) The mixture containing 100 mg (0.41 mmol) of Ara-C(7) and 186 mg (0.61 mmol) of monomethoxytrityl chloride in 3 ml of anhydrous pyridine is stirred at normal temperature for 10 hours under the exclusion of moisture and light. After the addition of

The reaction mixture is extracted with 10 ml of ice-cold water

(3 x 10 ml) chloroform. The organic phase is dried over magnesium sulphate, filtered, evaporated, co-evaporated with toluene (2 x 10 ml). The residue is chromatographed on a silica gel column (eluent: CHCl3/MeOH/Et3N, 95/5/0.1, volume/volume/volume) which gives the compound (16).

4.2 5'-monomethoxytrityl-2',3',N 43-tribenzoyl-D-1- arabinofuranosyl-cytosine (17)

A solution containing 415 mg (0.82 mmol) of compound (16), 615 mg (2.72 mmol) benzoic anhydride, 34 mg (0.27 mmol) 4-dimethylamino-pyridine in 3 ml pyridine is stirred overnight at room temperature . 20 ml of water is added. The resulting solution is extracted with chloroform (3 x 20 mL). The organic phase is dried over magnesium sulphate, filtered, evaporated and co-evaporated with toluene. The residue is chromatographed on a silica gel column (eluent: CHCl3/hexane/Et3N, 79/20/1, volume/volume/volume). The compound (17) is obtained.

4.3 2',3',N 43-tribenzoyl-D-1-arabinofuranosyl-cytosine (18)

Dissolve 672 mg (0.82 mmol) of the compound (17) in

20 ml of a mixture of chloroform/methanol (7/3, volume/volume) containing 2% benzenesulfonic acid, the reaction mixture is stirred for 20 min. at 0°C. 20 ml of aqueous sodium hydrogen carbonate solution is added, the mixture is extracted with chloroform (3 x 20 ml). The organic phase is dried over magnesium sulfate, filtered, evaporated under reduced pressure. The residue is fractionated on a silica gel column (eluent: CHCl3/MeOH, 98.5/1.9, volume/volume). The product (18) is obtained after crystallization from ethanol 95%.

Temp. = 169 - 171°C (ethanol 95).

Example 5: triethylammonium-3'-o-chlorophenylphosphate-(5'-monomethoxytrityl-2',N6-dibenzoyl-Ara A) (20 a).

A solution of o-chloro-phenyl-phosphordi(1,2,4-triazolide) (12) in 1 ml of anhydrous pyridine is prepared in situ by starting from 100 mg (0.408 mmol) of o-chlorophenyl phosphorodichloridate and 162 mg (2.347 mmol) 1,2,4-triazole. After 20 min. stirring at normal temperature, 80 mg (0.107 mmol) of the compound (6) are added. Stirring is continued for 20 min. A solution composed of 0.75 ml of triethylamine, 0.5 ml of water and 0.5 ml of pyridine is then added. The reaction is stopped for 10 min. then with the addition of 8 ml of aqueous sodium hydrogen carbonate solution. The mixture is extracted with chloroform

(3 x 8 ml) .

The organic phase is dried over magnesium sulphate, filtered, evaporated, co-evaporated with toluene (2 x 5 ml).

After purification on a silica gel column (eluent: CHCl3/MeOH/Et3N, 98/2/0.1 volume/volume/volume) the compound (20 a) is obtained after precipitation from petroleum ether.

Rf = 0.11 (eluent CHCl3/MeOH, 90/10, v/v)

U.V.: m^ks (ethanol 95): 278 mm (e = 20,700) 232 nm

(e = 33,500)

Fully protected dimer- Ara- Ap- Ara C( 21 a)

To a solution of 601 mg (0.578 mmol) of the phosphodiester (20 a) and 305 mg (0.549 mmol) of 2', 3', N4-tribenzoyl-Ara-

C (18) in 2.9 ml of pyridine is added 513 mg (1.735 mmol) of mesitylene-1-sulfonyl-3-nitro-1,2,4-triazole. The reaction is stopped after 45 min. stirring at ordinary temperature by adding 0.8 ml of sodium bicarbonate solution. After 10 min. the obtained solution is poured into 20 ml of aqueous sodium bicarbonate solution and then extracted with chloroform (4 x 20 ml). The organic phase is dried over magnesium sulfate, filtered, evaporated and coevaporated with toluene (2 x 10 ml). The residue is fractionated on a silica gel column (eluent: CHCl3/Et3N, 100/1, volume/volume). The dimer (21 a) is obtained after precipitation of petroleum ether.

R f = 0.20; 0.34 two diastereoisomers (eluent:

CHCl 3 /MeOH; 97/3; volume/volume).

U.V.: m^ks (ethanol 95): 269 nm (e=11.00) 234 nm (e-20.000).

Example 6: triethylammonium-3'-o-chlorophenylphosphate-(5'-monomethoxytrityl-2',N<4>-dibenzoyl-Ara C) (20 b).

A solution of o-chlorophenyl phosphorodi-(1,2,4-triazolide) (12) in 2 ml of anhydrous pyridine is prepared in situ from 272 mg (1.11 mmol) of o-chlorophenyl phosphorodichloridate,

377 mg (5.54 mmol) of 1,2,4-triazole. After 20 min. stirring at ordinary temperature, 160 mg (0.22 mmol) of the compound (12) are added. Stirring is then continued for 20 min. A solution consisting of 0.5 ml of triethylamine, 0.3 ml of water and 0.3 ml of pyridine is then added. The reaction is stopped after 10 min. The reaction mixture is treated in the same way as for the synthesis of (20 a). The residue is chromatographed on a silica gel column (eluent: CHCl3/MeOH/Et3N, 97/3/0.1, volume/volume/volume).

The compound (20 b) is obtained after precipitation from petroleum ether.

Rf = 0.2 3 (eluent: CHCl3/MeOH, 90/10, v/v)

UV :m_^s(ethanol 95): 303 nm (e = 12.300)

261 nm (e = 31,700)

232 nm (e = 54,100)

Fully protected dimer- Ara C- p- Ara A ( 21 b)

To a solution of 200 mg (0.197 mmol) phosphodiester

(20 b) and 110 mg (0.191 mmol) of 2',3'-N<6->tribenzoyl-Ara-A (15) in 1 ml of pyridine are added to 175 mg (0.591 mmol) of mesitylene-1-sulfonyl-3-nitro -1,2,4-triazole. The reaction is stopped after 45 min. stirring at ordinary temperature and treated as previously indicated. The residue obtained is purified on a silica gel column (eluent benzene/MeOH/Et3N, 99/0.9, 0.1, volume/volume/volume). The dimer (21 b) is obtained after precipitation from petroleum ether.

Rf = 0.20. Two diastereoisomers (eluent:

CHCl3/MeOH, 9 7/3, v/v)

U.V.: ^ (ethanol 95): 269 nm (e = 9,700)

max

232 nm (e =17,600)

Example 7: Triethylammonium-3'-o-chlorophenylphosphate-(5'-monomethoxytrityl-2',N-dibenzoyl-Ara-A) (20 c).

A solution of o-chlorophenyl-phosphorusdi-(1,2,4-triazolide) (12) in 1 ml of anhydrous pyridine is prepared in situ by starting from 100 mg (0.408 mmol) of o-chlorophenylphosphorus dichloridate and 162 mg (2.347 mmol ) 1,2,4-triazole. After 20 min. stirring at normal temperature, 80 mg (0.107 mmol) of the compound (6) are added. Stirring is continued for 20 min. A solution composed of 0.75 ml of triethylamine, 0.5 ml of water and 0.5 ml of pyridine is then added. The reaction is stopped after 10 min. by adding 8 ml of aqueous sodium bicarbonate solution. The mixture is extracted with chloroform (3 x 8 ml).

The organic phase is dried over magnesium sulphate, filtered, evaporated, co-evaporated with toluene (2x5 ml).

After purification on a silica gel column (eluent: CHCl3/MeOH/Et3N, 98/2/0.1, volume/volume/volume) the compound (20 a) is obtained after precipitation from petroleum ether.

Rf = 0.11 (eluent CHCl3/MeOH, 90/10, v/v)

U.V.: m^ks (ethanol 95): 278 nm (e= 20,700)

232 nm (e= 33,500)

Fully protected dimer- Ara- A- p- Ara- A ( 21 a)

To a solution of 106 mg (0.103 mmol) of the phosphodiester (20a) and 58 mg (0.100 mmol) of 2',3',N6-tribenzoyl-Ara-A (15)

91 mg (0.307 mmol) of mesitylene-1-sulfonyl-3-nitro-1,2,4-triazole is added to 0.5 ml of pyridine. The reaction is stopped after 45 min. stirring at ordinary temperature by adding 0.2 ml of sodium bicarbonate solution. After 10 min. the resulting solution is poured into 12 ml of aqueous sodium bicarbonate solution and then extracted with chloroform (4 x 10 ml). The organic phase is dried over magnesium sulphate, filtered, evaporated, co-evaporated with toluene (2x5 ml). The residue is fractionated on a silica gel column (eluent: CHCl3/MeOH/pyridine, 99/1/0.1, volume/volume/volume). The dimer (21 a) is obtained after precipitation from petroleum ether.

Rf = 0.57 - two diastereoisomers (eluent: CHCl^/MeOH

95/5, volume/volume).

Example 8:

Deprotection and purification of dimers Ara-A-p-Ara C (21 a)

and Ara C-p-Ara-A (21b).

a) Deprotection.

A solution of 0.020 mmol of fully protected oligonucleotide (21 a) or (21 b) of 0.3 M 4-nitro-syn-benzaldoxime and 0.3 M triethylamine, in a mixture of dioxane - water (4 ml, 1 /1, volume/volume) is stirred for 24 hours at 30°C. After evaporation at reduced pressure, 5.5 ml of concentrated ammonia (20%) is added to the obtained soft rubber. This solution is heated for 24 hours at 50°C in a sealed tube.

The crude reaction mixture is neutralized with resin

Dowex 50 W-X2 (pyridinium form). The resin is filtered off, washed with water (3 x 20 ml) and EtOH (3 x 20 ml). The water - ethanol phase is evaporated to dryness. The remainder is dissolved in 40 ml of water. The aqueous phase is washed with ether (10 x 30 ml) and evaporated under reduced pressure. 4 ml of aqueous 80% acetic acid is added to the residue obtained. The obtained solution is stirred at ordinary temperature for 1 hour and 15 minutes. and evaporated, coevaporated with water to neutral pH and washed successively with chloroform (15 x 30 ml) and ethyl ether (5 x 20 ml).

The aqueous phase is evaporated under reduced pressure,

b) Purification.

The residue is purified on a column with DEAE Sephadex A-2 5 (linear

_3

gradient 10 M to 0.3 M, aqueous triethylammonium hydrogen carbonate buffer, pH = 7.5).

The fractions containing the dimer (21) are combined again and evaporated. The product obtained is purified again using semi-preparative HPLC. This is carried out in column C"18 with a water gradient, CH^CN (5%) during 13 min.

The fractions containing the dimer (21) are evaporated to dryness, coevaporated with water and freeze-dried.

The triethylammonium salts (22 a) and (22 b) of the dimers Ara-A-p-Ara-C and Ara-C-p-Ara-A are then obtained.

Example 9:

Deprotection and purification of the dimer Ara-A-p-Ara-A (21 c).

1 g of the mixture (dimer 21 c + impurity) is coevaporated with anhydrous pyridine (3 x 20 ml) and dissolved in 5 ml of pyridine. 2 ml of benzoyl chloride are added. After stirring for 24 hours, 100 ml of water is added, the mixture is extracted with chloroform (5 x 20 ml). The organic phase is washed with water (5 x 10 ml), with aqueous sodium bicarbonate solution (5 x 10 ml), with water (5 x 10 ml), dried over magnesium sulfate, filtered, evaporated, coevaporated with toluene, and with anhydrous methanol . The residue is dissolved in 15 ml of methanol and 6 ml (0.92 M) of sodium methylate is added. After stirring for 4 hours, neutralize with resin Dowex 50 W-X2 (pyridinium form), filter off the resin, wash with water

(3 x 50 ml). The solution is evaporated under reduced pressure. The soft gum obtained is dissolved in 20 ml of water, washed with chloroform (15 x 20 ml) and with ether (10 x 20 ml).

After purification on DEAE-Sephadex A-25 with linear gradient

_3

(10 M to 0.3 M) with triethylammonium hydrogen carbonate (pH = 7.5), the fractions containing the dimer (21 a) are purified

again by means of semi-preparative HPLC on C^g column with a linear gradient (CH^CN 4% to 20%, 15 min., amount 10 ml/min.), evap. co-evaporated with water, and freeze-dried. The triethylammonium salt (22c) of the dimer Ara-A-p-Ara A is then obtained.

The compounds that can be prepared by the invention were subjected to pharmacological tests which show that they are interesting in the antitumoral field. The effect of the compounds on the development of Erlich's ascites tumor in mice was observed.

The tests were carried out with groups of 5 or 6 Swiss mice

weighing 12 to 15 g. Each animal received 2 x 10 malignant cells by peritoneal administration. The treatment began 16 hours after the inoculation and consisted of two injections per day of 0.75 ml of a solution of the dinucleotide of 2 mg/ml in the peritoneum, i.e. 3 mg per animals per day or 200 mg/kg. The duration of the treatment is 4 days.

The dinucleotides produced by the invention were tested in the manner indicated and the results are given in the following tables.

The compounds that can be produced by the invention have antitumoral properties and can be used for the treatment of various disorders such as certain viral affections caused by ADV virus: . Herpes in all localizations, herpes 1 and herpes 2,

. Varicella

. Zone

. Cytomegaly

. Infectious mononucleosis, Burkitt tumor (Epstein-Barr virus)

. Hepatitis B

. Diseases in the Variola vaccine group

. Adenovirus

. Retroviruses

certain malignant affections sensitive to Ara A (chronic myeloid leukemias with an acute course) or Ara C (acute myeloblastic leukosis).

Pharmaceutical preparations with the compounds can be liquid or solid and are available in the form of solutions, ointments, dispersions, pomades.

The active principle can be incorporated into the preparations and additives can consist of common solvents, stabilizers, softeners, vaselines, lanolins, polyethylene glycols, fatty acid esters of sorbitan which are optionally polyhydroxyethylated.

The concentration of active principle in pharmaceutical preparations is varied according to the case and the method of administration and can e.g. be between 0.1% and 5%.

Claims (4)

1. Process for the production of therapeutically active monophosphate dinucleosides of formula Ara A-p-Ara C, Ara C-p-Ara A and Ara A-p-Ara A in which Ara A is 9-3-D-arabinofuranosyl-adenine Ara C is 1-3-D-arabinofuranosyl-cytosine, and p is the monophosphate group, characterized by 1) the synthons adenine and cytosine are produced which carry a free OH group in the 3' position, 2) the synthons adenine and cytosine are produced which carry a free OH group in the 5' position, 3) the synthesis of the monophosphate dinucleosides is carried out by - phosphocrylation of the synthon which carries a free OH group in the 3' position using a bifunctional phosphorylating agent, - the resulting diester is condensed with the chosen nucleoside using a condensing agent, and - the obtained compounds are deprotected to arrive at the desired dimers in the form of the triethylammonium salts. 2. Method as stated in claim 1, characterized in that o-chloro-phenyl-phosphorus-dichloridate is used as bifunctional phosphorylating agent. 3. Method as stated in claim 1, characterized in that mesitylene-1-sulfonyl-3-nitro-1,2,4-triazole (MSNT) is used as condensation agent. 4. Method as stated in claim 1, characterized in that the o-chloro-phenyl groups are deprotected with a solution of 4-nitrobenzaldoxime and triethylamine, the benzoyl groups are removed with ammonia, and the monomethoxytrityl group is hydrolysed using acetic acid.