FR2654106A1 - Phosphorated cytosine derivatives, their preparation and their application in therapeutics - Google Patents

Abstract

Phosphorated cytidine derivatives, corresponding to the formula (I) in which C is cytosine R is . an NH4<+>, Et3NH<+> or Na<+> cation . a (CH2)ny radical where n ranges from 1 to 4, Y is an OZ, SZ, , or S-S-Z radical, in which Z is H, (C1-4)alkyl, aryl, CO(C1-4)alkyl or (CH2)nOH, n ranging from 1 to 5. Application in therapeutics.

Description

The present invention relates to phos phore derivatives of cytsin, their preparation and their therapeutic application.
The compounds of the invention correspond to formula (1) given in appendix 1 in which
It is cytosine
R is. a cation N4 +, Et3NH +, Na +
a (CH2) n Y radical where
n goes from 1 to 4,
Y is a CZ, SZ radical,

S 5 - S - Z, in which
Z is H, (C1-4) alkyl, aryl, CO (C1-4) alkyl or
(CH2) nCH, n ranging from 1 to 5.

According to the invention, the compounds (t, according to the reaction schemes given in appendix 2) are prepared from the nuclecid (1) which leads to the intermediate (4) which is reacted with the appropriate reagents to obtain the compounds ( I).

The following examples illustrate the invention.

The thin-layer chromatographies were carried out on Merck 60 F 254 silica plates (art. 55554).

The silica gel column chromatographies were carried out with Merck 60 H militia (art. 7736) or with Merck silanized silica RP2 (art. 7719).

HPLC analyzes were carried out on a column
Waters Radial-Pek (# = # mm, 1 = 100 mm) C16 with a spherical grain size of 10 m. This column is inserted in a module
RCM 100 and protected by a pre-filter and a precolumn
Guard-Pak. The HPLC system is composed of Waters equipment a U6 K injector, two M-6000 A pumps, a programmer
M-720. UV spectra were recorded using a Pye Unicam PU 4021 multi-channel UV detector and a Philips PU 4850 video control center. The elution was resolved with a solution of acetonitrile in 0.1 M ammonium acetate buffer (pH 5.9) at a flow rate of 2.5 ml per minute.

U.V. spectra were recorded on a UVIKON 810 spectrophotometer.

Mass spectra were taken on a device
JEOL JMS DX 300 by the FAB ionization method.

Proton NMR spectra were recorded on
a Varian EM 360 device or on a Brüker AC 250 device.

Chemical shifts are expressed in ppm relative
to the signal of tetramethylsilene (TMS).

The multiplicity and shape of the signals observed by NMR
are indicated by one (or more) lowercase letter (s)
(s): s (singlet), d (doublet), t (triplet), m (multiplet),
l (large), p (pseudo).

Phosphorus NMR spectra were recorded
on a Brüker WP 200 SY device with proton decoupling.

Chemical shifts are expressed in ppm relative
to the H3 PO4 signal taken as an external reference.

Preparation of intermediate (4) 1. O- [N4- (4-methoxy trityl) dideoxy-2'3 'cytidine] -5'
O- (2-chlorophenyl) triethylammonium phosphate (2)
Has a solution of 1.21 g (17.5 mmol) of triazole in 20
ml of anhydrous acetonitrile, added, with stirring, 1.10
ml (6.7 mmol) of 2-chlorophenyl dichlorophosphate and 1.9
ml (13.6 mmol) of anhydrous triethylamine. Stirring is continued for 30 minutes before an on @@@@@@@@@@@@@@@@@@@@@@@@@@@@@@
(2.27 mmolas) of nucleoside 1 dissolved in 20 ml of
anhydrous pyridine.

Phosphorylation is completed in 30 minutes and mixing
0.75 ml of triethylamine and 0.25 ml of water is added.

The reaction mixture is diluted with dichloromethane,
washed with a saturated aqueous solution of NaHCO3 then with
of water and dried over Na2SO4. The solvent is evaporated under
reduced pressure and the resulting oil is co-evaporated
several times with toluene before being precipitated in
hexane to produce 1.43 g (85%) of mononucleotide 2.

This is obtained in the form of triethylammonium salt
with a purity not requiring purifications
additional.

1: Rf = 0.27 (CH2Cl2-MeOH: 8-2)
H NMR (CDCl3): # = 7.6 (bs, 1H exchangeable); 7.3 - 6.6
(m, 19H; mMTr, ClPh, H-6); 5.87 (m, 1H; H-1 '); 5.08 (d,
1H; H-5, J = 7.7Hz); 4.2 (bs, 1H; H-4 '); 4.1 - 3.9 (m,
2H; H-5W, 5 "); 3.73 (s, 3H; CH3O); 2.98 (m, 6H; NH (CH2
CH3) 3); 2.35 (m, 1H; H-2 '); 2.05 - 1.7 (m, 3H; H-2 ", 3 'and 3"); 1.24 (t, 9H t J = 7.3Hz, HN (CH2CH3) 3) ppm.

P NMR (CDCl3): # = 0 4.96 ppm.

2. Di-O- [N4- (4-methoxy trityl) dideoxy-2'3 'cytidine] -5'
O- (2-chlorophenyl) phosphate (3).

A mixture of 1.40 g (1.89 mmol) of nucleotide 2 and
0.870 g (1.80 mmol) of nucleoside 1 dissolved in 25 ml
of anhydrous pyridine is treated with 1.4 g ((4.72 mmol) of
mesitylene sulfonyl-1 $ 3-nitro-1,2,4-triazole. The resotion
is left two hours before being stopped by addition
slow solution of a saturated aqueous solution of NaHCO3.

After extraction with dichloromethane, the organic phase
obtained is washed with water, dried over Na2SO4,
concentrated under reduced pressure and coevaporated with
toluene. Purification by chromatography on a column of
silios gel [elution: MeOH (0 to 4%) in CH2Cl2]
provides 1.48 g (72%) of pure triester 3 dimer.

3: Rf = 0.35 (CH2Cl2 - MeOH: 92-8)
1H NMR (CDCl3): # z 7.6 - 6.7 (m, 34H; 2 mMTr, sClPh, 2
H-6); 5.95 (m, 2H; 2H-1 '); 4.99 (d, 1H; 1H-5, J - 7.6
Hz); 4.98 (d, 1H; 1HS, J = 7.6Hz); 4.25 - 4.0 (m, 6H
2H-4 ', 5' and 5 "); 3.765 and 3.760 (s and s, 3H and 3H; 2 CH3
0); 2.40 (m, 2H; 2H-2 '); 2.05 - 1.8 (m, 2H; 2H-2 ')
1.8 - 1.5 (m, 4H; 2H-3 ', 3 ") ppm.

P NMR (CDCl3): # = - 5.25 ppm.

3. Di-O- [N4- (4-methoxy trityl) dideoxy-2 ', 3' cytidine] -5 '
tetramethylguanidinium phosphate (4).

To a solution of 1.00 g (0.877 mmol) of the triester 3 dimer
in 7.3 ml of dioxane and 3.6 ml of water, 729 mg are added
(4.39 mmol) of 4-nitro benzaldoxime and 0.55 ml (4.38
mmoles) of tgtramethylguanidine (TMG).

After 6 hours of stirring, the reaction mixture is diluted
with dichloromethane, washed with water and dried over Na2SO4.

The solvent is evaporated off under reduced pressure and the oil
obtained is precipitated three times in hexane. A
final purification by gel column chromatography
of silica [elution: MeOH (from 0% to 25%) in CH2Cl2]
followed by filtration of the product dissolved in dichloro
methane, leads, after evaporation, to 910 mg (90%) of
dimer diester 4 as tetramethyl salt
guanidinium.

4: Rf 1 0.22 (CH2Cl2- MeOH: 8 - 2) P NMR (CDCl3): # = 0.97 ppm
Example 1.

Di-O- (dideoxy-2'3 'cytidine) -5 O-methylphosphate.

1.1.Di-O- [N4- (4-methoxy trityl) dideoxy-2 ', 3' cytidine] -5 '
O-methyl phosphate (5)
Has a solution of 211 mg (0.184 mmol) of the dinucleoside
phosphodiester 4 in a mixture of 18 ml of anhydrous DMF,
8.0 ml of lutidine and 7.3 ml (0.180 moles) of methanol
anhydrous 3.5 g (18 mmol) of chloride of
toluene-4 sulfonyl.

After one hour of reaction, 1.5 ml of water are added
and the solvent is evaporated off under reduced pressure. Oil
obtained is taken up with dichloromethane, washed with a
saturated aqueous solution of NaHCO3, then with water and
dried over Na2SO4. After evaporation of the solvent, a purifi
cation by chromatography on silica gel [elution: MeOH
0 to 6% in CH2Cl2] followed by precipitation in hexane yields 116 mg (60%) of the methyl triester
protected 5.

5: RF = 0.30 (CH2Cl2 - MeOH: 92-8)
1H NMR (CDCl3): S = 7.8 (sI; 2 NH); 7.39 (d, 2H; 2H-6, J
- 7.6 Hz); 7.35 - 6.75 (m, 28H; 2 mMTr); 5.94 (m, 2H;
2H-1 '); 5.04 (d, 2H; 2H-5, J = 7.6Hz); 4.2 - 3.95 (m,
6H; 2H-4 ', 5' and 5 "); 3.76 (s, 6H; 2 CH3O); 3.49 (d; 3H
CH3OP, J 11.2Hz); 2.50 - 2.35 (m, 2S; 2H-2 '); 2.65
-1.85 (m, 6H: 2H-2 ', 3' and 3 ") ppm.

P NMR (CDCl3): # = 1.12 ppm.

2.DI-O- (dideoxy-2'3 'cytidine) -5 O-methylphosphate
Protected methyl phosphotriester 5 (110 mg; 0.105
mmoles) is treated with 12.5 ml of a 3 3% acid solution
trifluoroacetic in anhydrous dichloromethane. After 18
hours, the reaction mixture is neutralized by adding
of 10 ml of methanol saturated with ammonia (at 0 C) and is
rapidly evaporated under reduced pressure. The gross obtained is
taken up with water and washed with dichloromethane before being
concentrated.

A first purification on a silanized silica column
RP2 (# = 1.8 cm; h = 26 cm; elution: 0 to 20% methanol
in water) leads to LM 80 (40.2 mg, 76%) containing 1% of
ddC by HPLC (spectrometric percentage).

Three additional purifications on silanized silica RP
2 allow LM 80 to be obtained with a purity of 99.2 * per
HPLC (0.6% contamination by dimer phosphodiester LM
93) before evaporation of the combined fractions. After
filtration (HV-4 filter, Millipore), evaporation under
reduced pressure (at a temperature below 25 C) and
lyophilization in water, 14.9 mg (28 *) of LM are obtained
80 spectroscopic purity of 97.1% by HPLC
(contaminated with 2.9% phosphodiester dimer LM 93).

LM 80: Rf X 0.08 (CH2Cl2- MeOH: 8 - 2)
Retention time: 360 s (12% CH3CN in AcONH4 0.1M aqueous) UV H2O): A max = 271 nm (E = 17200)
# min - 249 nm (s = 11300)
Mass spectrum (FAB positive; matrics: 3-nitro benzyl alcohol) 499 [(M + H) +]), 388 [(M - B) +]); 112 [(SH2) +].

1 H NMR (DMSO): # = 7.64 (d, 2H; 2H-6, J - 7.4Hz) @ 7.1 (bs, 4H; 2NH2); 6.00 (m, 2H; 2H-1 '); 5.70 (d, 2H t 2 HS, J @ 7.4Hz); 4.25 - 4.05 (m, 6H; 2H-4 ', 5' and 5 ") 3.68 (d, 3H; CH3OP, J = 11.2Hz); 3.15 - 2.20 ( m, 2H; 2 H2 '); 2.0 - 1.91 (m, 2H; 2 H-3'); 1.90 - 1.70 (m, 4H; 2
H-2 "and 3") ppm.

31P NMR (DMSO ds): # = 0.762 ppm
Example 2.

Di-O- (dideoxy-2'3 'cytidine) -5 O-ethylphosphate.

2.1. Di-O- [N4- (4-methoxy trityl) dideoxy-2 ', 3' cytidine] -5 '
O-ethyl phosphate (6).

The phosphodiester 4 dimer (200 mg; 0.175 mmol) dissolved in 10.8 ml of anhydrous DMF, 4.8 ml of anhydrous lutidine and 4.4 ml (75 mmol) of ethanol is treated with 2.1 g ( 11 mmol) of 4-toluene sulfonyl chloride.

The reaction is left for 2 hours and 1 ml of water is added. The solvent is evaporated off under reduced pressure to produce an oil. This is taken up with dichloromethane, washed with a saturated aqueous solution of
NaHCO3 and the solvent evaporated.

Purification by chromatography on a silica gel column [elution: MeOH (0 to 4%) in CH2Cl2] allows 85 mg (47 *) of the protected phosphotriester dimer 6 to be isolated.

6: Rf = 0.28 (Ch2Cl2- MeOH; 8 - 2)
H NMR (CDCl3): # = 7.8 (bs, NH); 7.42 (d, 2H; 2H-6, J = 7.5Hz); 7.35 - 6.8 (m, 28H; 2 mMTr), 5.96 (m, 2H; 2H- 1 '); 5.06 (d, 2H; 2H-5, J s 7.6Hz); 3.9 - 4.2 (m, 6H 2H-4X, 5 'and 5 "); 3.92 (td, 2H; CH2CH2OP, JHp - 15.2 Hz; = 7.1 Hz); 3.79 (s, 6H; 2 CH3O); 2.55 - 2.35 (m, 2H; 2 H-2 '); 2.1 (m, 6H; 2 H-2', 3 "and 3"); 1 , 12 (t, 3H; CH3
CH2OP, J - 7.1 Hz) ppm.

2.2.Di-O- (dideoxy-2 ', 3' cytidine) -5 'O-ethyl phosphate
The protected phosphotriester 6 (75 mg, 0.072 mmol) is
treated for 18 hours with an anhydrous solution.

The acid is neutralized by adding 10 ml of methanol
saturated with ammonia and the reaction mixture is
rapidly concentrated under reduced pressure.

The gum formed is taken up with water and washed with
dichloromethane. After evaporation of the aqueous phase, the
residue obtained is chromatographed on a silica column
silanized RP2 (# = 1.5 cm; h = 17 cm; elution: 0 to 20% of
methanol in water). HV-4 filter filtration
Milliore, followed by freeze-drying in water lead
at 23 mg (62%) of LM 91 dimer of spectometric purity
by 87% HPLC.

Further purification on RP2 under conditions
identical to the previous ones allows to obtain LM 91 with
a purity by HPLC of 99.1% after lyophilization (without
contamination by ddC or LM phosphodiester dimer
93).

LM 91: Retention time: 648 s (12% CH3CN in AcONH4
0.1 M aqueous)
UV (H2O): # max: 271 nm (# = 15800)
# min: 249 nm (# = 10700)
Mass spectrum (positive FAB; matrix: glycerol): 513
[(M + H +], 402 [((MB) +], 112 [(BH2) +].

H NMR (DMSO d6): # = 7.62 (d, 2H; 2H-6, J = 7.4Hz);
7.13 (bs, 4H; 2 NH2); 5.99 (dd, 2H; 2H-1 ', J = 3.5 and
6.6 Hz); 5.79 (d, 2H; 2H-5, J = 7.4Hz); 4.25 - 4.1 (m,
Hz, JHH = 7.1 Hz); 2.25 (m, 2H; 2H-2 '); 2.1 - 1.6 (m, 6H
; 2H-2 ', 3' and 3 "); 1.23 (td, 3H; CH3CH2OP, JHP = 0.7Hz,
JHH = 7.1Hz).

-Example 3.

Di-O- (2 ', 3' dideoxy cytidine) -5 'O- (2-hydroxyethyl)
phosphate.

3.1. Di-O- [N4- (4-methoxy trityl) didesoxy-2 ', 3'] - 5 'O - [(4-methoxy
trityl) -2 oxyethyl] -1 phosphate (7).

Has a solution of 230 mg (0.201 mmol) of the phosphodi dimer
ester 4 and 300 mg (0.897 mmol) of the monoether of
ethylene glycol 4-methoxytrityl in 15 ml of
anhydrous pyridine are added 267 mg (0.901 mmol /) of
mesitylene sulfonyl-3 nitro-3 triazole-1, 2, 4 (MSNT). The
reaction is left for 4 hours and 267 mg (0.901 mmol)
additional MSNTs are added.

After 3 hours of stirring, the reaction mixture is
treated with a saturated aqueous solution of NaHCO3 and
extracted with dichloromethane. The organic phase is
washed with water, dried over NaiSO2, evaporated under
reduced pressure and coevaporated with toluene.

The oil obtained is purified by column chromatography
silica gel (elution: MeOH (0 to 6%) in CH2Cl2]
to lead to 170 mg (63%) of the phosphotriester 7 in
foam form.

7: Rf - 0.40 (CH2Cl2 - MeOH: 92 - 8)
H NMR (CDCl3)): s = 7.4 - 5.7 (m, 44H; 3mMTr, 2H-6);
5.8 (m, 2H; 2H-1 '); 5.19 (m, 2H; 2H-5); 4.4 - 3.9 (m,
3H; 2H-4 ', 5', 5 "and OCH2CH2OP); 3.8 - 3.7 (m, 9H; 3 CH3
O); 3.25 (m, 2H; CH3CH2OP); 2.5 - 2.3 (m, 2H; 2H-2 ');
2.25 - 1.6 (m, 6H; 2H-2 ", 3 'and 3") ppm.

3.2.Di-O- (2 ', 3' dideoxy cytidine) -5 'O- (2-hydroxyethyl)
phosphate
The protected dimer 7 (160 mg; 0.119 mmol) is dissolved in
11 ml of a 3% solution of trifluoroecetic acid in the
anhydrous dichloromethane.

After 6 hours, the reaction mixture is treated with 0.5 ml
of methanol saturated (at 0 C) with ammonisc.

Excess ammonia and solvent are quickly removed
under reduced pressure. The residue obtained is taken up with
water, washed with dichdloromethane and the aqueous phase is
concentrated.

The purification is carried out by two chromatographies
successive on a column of silanized silica RP2 [# * 1.5 cm, h = 18 cm; elution: MeOH [from 0 to 5%) in water] to produce, after filtration (HV-4 Millipore filter) and lyophilization in water at 30 mg (48%) of the compound LM 93 of spectrometric purity by 99.5% HPLC.

LM 92: Retention time - 240 s (12% CH3CN dens AcONH4 0.1 M aqueous) UV (H2O): # max = 271 nm (# = 15800)
# min t 248 nm (# = 11600)
Mass speotre (FAB positive; matrix: glycerol): 528 [(M + H) +], 112 [(BH2) +].

H NMR (DMSO d6): # = 7.63 (dd, 2H; 2H-6, J = 7.3 and 1.0
Hz); 7.1 (bs, 4H; 2NH2); 6.01 (dd, 1H, 1H-1, J = 2.4 &4.0Hz); 5.99 (dd, 1H; 1H-1 ', J = 2.3 &4.3Hz); 5.71 (d, 2H; 2H-6, J = 7.4Hz); 4.9 (bs, 1H; OH); 4.25 - 4.1 (m, 6H; 2H-4 ', 5' and 5 "); 3.99 (td, 2H; HOCH2CH2OP); 3.58 (m, 2H; HOCH2CH2OP); 2.3 (m, 2H; 2 H-2 '); 2.0-1.95 (m, 2H; 2 H-3'); 1.90-1.7 (m, 4H; 2 H-2 "and 3 ") ppm.

Example 4.

Di-O- (dideoxy-2 ', 3' cytidine) -5 ammonium phosphate
The protected dimer 4 (190 mg; 0.166 mmol) is dissolved in 10 ml of a 4% solution of trifluoroacetic acid in anhydrous dichloromethane.

After 8 hours, 10 ml of a methanol solution saturated (at 0 ° C.) with ammonia are added.

The excess ammonia and the solvent are driven off under reduced pressure. The residue obtained is taken up in water, washed with dichloromethane and the aqueous phase concentrated under reduced pressure.

The LM 93 compound is isolated pure (45 mg; 54%) after chromatography on a column of silanized silica RP2 (# = 1.5 cm, h = 38 cm; elution: water), filtration on an HV-4 filter.
Millipore and lyophilization in a dioxane water mixture.

LM 93: Retention time: 324 s (elution 5% CH3CN in
AcONH4 0.1 M aqueous).

UV (H2O): # max = 271 nm (e = 17000)
# min = 248 nm (# - 10000)
Mass spectrum (negative FAB; matrix; glycerol): 483 [(MH) +]).

H NMR (DMSO d6): # = 7.92 (d, 2H; 2H-6, J = 7.4Hz); 7.1 (bs, 4H; 2NH2), 5.94 (dd, 2H; 2H-1 ', J = 3.1 and 6.5
Hz); 5.71 (d, 2H; 2H-5, J = 7.4Hz); 4.10 (m, 2H: 2
H-4 '); 3.86 and 3.78 (m and m, 4H p; 2H-5 'and 5 "); 2.27-2.20 (m, 2H, 2H-2'); 1.95-1, 75 (m, 6H; 2H-2 ', 3' and 3 ") ppm.

Example 5.

Di-O- (dideoxy-2 ', 3' cytidine) -5 'O- [S- (2-hydroxy thio-1
ethyl) thio-2 oxy-1 ethyl] -1 phosphate.

5.1.Di-O- [N4- (4-methoxy trityl) dideoxy-2 ', 3' cytidine] -5 '
O- [S- (2-hydroxy-1-thioethyl) 2-oxy-1-ethyl] -1
phosphate (8).

Has a solution of 630 mg (0.553 mmol) of the dimer
phosphotriester 3 are added 5.5 ml (45 mmoles) of
2,2 'dithio diethanol, 5.5 ml of anhydrous dichloromethane and
840 mg (5.5 mmol) of cesium fluoride. The mixture is
plscé under vigorous stirring for 26 hours then diluted with
dichloromethane and washed with water before being
concentrated under reduced pressure. The oil obtained is
chrometographed on silica gel column [elution:
MeOH (0 to 6%) in CH2Cl2] to produce 400 mg (62
%) of compound 8.

8: Rf: 0.21 (CH2Cl2 - MaOH: 92-8) 1 H NMR (CDCl3): # = 8.3 (bs; 2NH); 7.41 (bp, 2H; 2H-6,
J = 7.2Hz); 7.35-6.8 (m, 28H; 2 mMTr); 5.94 (m, 2H; 2
H-1 '); 5.10 (bp, 2H; 2H-5, J = 7.3Hz); 4.3-4.0 (m, 10
H, 2H-4 ', 5' and 5 ", MOCH2CH2SSCH2CH2OP); 3.79 (s, 6H; 2CH3
O); 2.82 (m, 4H; CH2SSCH2); 2.43 (m, 2H; 2H-2 ');
2.15-1.85 (m, 4H; 2H-2 "and 3 '); 1.70 (m, 2H; 2H-3")
ppm.

5.2.Di-O- (2 'dideoxy, 3' cytidine) 5 'O- [S- (2-hydroxy thio-1
ethyl) 2-thio 1-oxyethyl] -1 phosphate
Compound 8 (200 mg; 0.172 mmol) is treated with a
3% trifluoroacetic acid solution in
anhydrous dichloromethane. After 24 hours, 5 ml of methanol
set (at 0 C) of ammonia are added. The solvent and
excess ammonia is immediately evaporated under pressure
scaled down. The residue obtained is taken up in water. wash
with dichloromethane and the concentrated aqueous phase. The
purification is carried out by two chromatographies on
RO2 silanized silica column [# = 1.5 cm, h = 20 cm;
elution: methanol (0 to 100%) in water] to lead
40 mg (38%) of the phosphotriester LM 99 of a purity
99% spectrometric by CLMP (without contamination by
ddC or by phosphodiester dimer LM 93).

LM 99: Retention time = 524 s (15% CH3CN in AcONH4
0.1 M aqueous); 284 s (20% CH3CN in aqueous 0.1 M AcONH4).

UV (H2O): # max = 271 nm (# = 18000)
# min = 249 nm (# = 12000)
Mass spectrum (positive FAB, matrix: glycerol): 621
[((M + / h +], 510 [(MB) +], 112 [(BH2) +].

H NMR (DMSO d6): # = 7.62 (d, 2H; 2H-6, J = 7.4Hz); 7.1 (bs, 4H: 2NH2); 6.01 (dd, 1H; 1H-1 +, J = 2.3 and 4.0
Hz); 5.99 (dd, 1H; 1H-1 +, J = 2.3 &4.3Hz); 5.71 (d, 2H; 2H-5, J - 7.4Hz); 4.87 (t, 1H; OH, J s 5.4 Hz) 4.2-4.1 (m, 8H, 2H-4 ', 5', 5 "and POCH2CH2S): 3.61 (m, 2H CH2 OH); 2.99 (t, 2H; POCH2CH2S, J - 6.3Hz); 2.79 (t, 2H p; SCH2CH2OH, J = 6.4Hz); 2.28 (m, 2H; 2H-2 '); 2.00 (m, 2H: 2H-3'); 1.90-1.75 (m, 4H; 2H-2 "and 3") ppm.

The compounds of the invention have been subjected to pharmacological tests showing their interest in the treatment of viral diseases.

- Evaluation of anti HIV 1 activity on CEM cells
SIV - human immunodeficiency virus
CEM = human T lymphoblastoid cell.

The multiplication of HIV 1 (BRU strain) in CEM cells is evaluated by the number of syncitia formed after 4 days of infection. The compounds tested are added after absorption of the virus into the culture medium at the final concentrations of 10-3M, 10-4M, 10-5M, 10-6M and 10-7M.

The highest concentration used in the analysis of molecules which are poorly soluble in aqueous medium is indicated in brackets.

The results are expressed as the lowest concentration of compound which decreases the formation of syncitia by at least 50%. Confirmation of the inhibition of virus production is sought by assaying reverse transcriptase (RT), the activity of which reflects the presence of virus released into the culture supernatant. (b) indicates that although there is a decrease in the number of syncitia, at the concentration of the compound indicated, the RT activity is identical to that of untreated infected cultures.

The toxic effect on uninfected EMFs is assessed by a colorimetric reaction based on the ability of living cells to reduce 3- (4,5 dimethylthiazol-2-yl) 2,5 diphenyltetrazolium bromide to formazan after four days of incubation. in the presence of different concentrations of the compounds. Results are expressed as the lowest concentration of compounds which causes at least 50% inhibition of formazan formation.

- Evaluation of anti HIV-1 activity in MT4 cells.

MT4 = human To cell transformed by HTLV1
HTLV = human T lymphotropic virus
The multiplication of HIV 1 (strain t.7zLV III B) in MT4 cells (T4 cells transformed with wTLV-1) is followed by the cytopathogenic effect induced by the virus.

The cells are infected with a dose of HIV 1 producing after 4 days a 90% decrease in the number of living cells.

The compounds tested are added, after adsorption of the virus, to the culture medium at different concentrations3.
The highest concentration used is either 10 or that shown in parentheses. The viability of the cells is measured by a colorimetric reaction based on their ability to reduce 3- (4,5 dimethylthiazol-2-yl) 2,5 diphenyl-tetrazolium bromide to formazan, a property due to mitochondrial dehydrogenases. The amount of formazan produced (OD at 540 nm) is proportional to the number of living cells.

The percentage of protection of the cells infected by the treatment with the compounds is calculated by applying the formula proposed by Pauwels et al.

OD 540 of infected cells treated - OD 540 of infected cells
OD 540 of uninfected cells - OD 540 of infected cells.

The concentration of compounds giving 50% protection (ED50) is shown in the table. When the concentrations used do not allow this level to be reached, the percentage of protection is indicated.

The toxic effect of the compounds on uninfected MT4 cells is measured by the same colorimetric reaction. The 50% cytotoxic dose (CD50) is the concentration of compounds causing a reduction by half of the O.D. 540 compared to that of the control cells.

In some cases, the CD50 dose is lower than the lowest concentration of compound tested (<10 21). The possible antiviral effect should be determined at lower concentrations.

The sign - indicates the absence of toxicity.

The results are given in the following table

<tb><SEP> CELLS <SEP> MT4 <SEP> CELLS <SEP> CEM
<tb><SEP> COMPOUND <SEP> CONCENTRATIONS <SEP> GIVING <SEP> A <SEP> EFFECT
<tb><SEP> OF <SEP> EXAMPLE <SEP> ANTIVIRAL <SEP> TOXIC <SEP> ANTIVIRAL <SEP> TOXIC
<tb> 1 <SEP> 4 <SEP> 10-6 <SEP> M <SEP> 6 <SEP> 10-4 <SEP> M <SEP> 5 <SEP> 10-7 <SEP> M <SEP> 6 <SEP> 10-5 <SEP> M
<tb> 2 <SEP> 5 <SEP> 10-5 <SEP> M <SEP> 10-3 <SEP> M <SEP> 5 <SEP> 10-6 <SEP> M <SEP> 10-3 <SEP > M
<tb> 3 <SEP> 4 <SEP> 10-6 <SEP> M <SEP> 8 <SEP> 10-5 <SEP> M <SEP> 10-7 <SEP> M <SEP> 7 <SEP> 10 -5 <SEP> M
<tb> 4 <SEP> 2 <SEP> 10-7 <SEP> M <SEP> 4 <SEP> 10-4 <SEP> M <SEP> 5 <SEP> 10-8 <SEP> M <SEP> 5 <SEP> 10-6 <SEP> M
<tb> 5 <SEP> 4 <SEP> 10-7 <SEP> M <SEP> 2 <SEP> 10-4 <SEP> M <SEP> 5 <SEP> 10-8 <SEP> M <SEP> 6 <SEP> 10-6 <SEP> M
<tb>
The compounds of the invention can be used for the treatment of viral diseases.

They can be presented for this purpose in any suitable form, in combination with one or more excipients.

Annex 1

Annex 2

MTr: 4-methoxy trityl
oClPh; 2-chlorophenyl
Diagram 1

Diagram 2

Schéma 4 Diagram 3
Annex 2 (continued)

Diagram 4

Claims (7)

(CH2) nOH, n ranging from 1 to 5. Z is H, (C1-4) alkyl, aryl, CO (C1-4) alkyl or , S - S - Z, where

Y is a radical OZ, SZ, n goes from 1 to 4, a (CH2) n Y radical where R is. a cation NH4 +, Et3 NH +, Na It is cytosine in which

Claims 1. Phosphorus derivatives of cytidine, corresponding to formula (I) 2. Di-O- (dideoxy-2'3 'cytidine) -5 O-methylphosphate. 3.Di-O- (dideoxy-2'3 'cytidine) -5 O-ethylphosphate. 4.Di-O- (dideoxy-2 ', 3' cytidine) -5 'O- (hydroxy-2 ethyl) phosphate. 5. Ammonium di-O- (2 ', 3' dideoxy) -5 phosphate. 6. Di-O- (2 ′, 3 ′ dideoxy) -5 ′ O-FS- (2-hydroxy thio-1 ethyl) thio-2 oxy-1 ethyl] -1 phosphate. 7. Medicinal product characterized by the fact that it contains a compound as specified in any one of claims I to 6.