WO1993012131A1

WO1993012131A1 - 2',3'-dideoxy-3'-aminothymidine derivatives, method for preparing same, and therapeutical uses thereof

Info

Publication number: WO1993012131A1
Application number: PCT/FR1992/001173
Authority: WO
Inventors: Jean-Louis Imbach; Gilles Gosselin
Original assignee: Centre National De La Recherche Scientifique (Cnrs)
Priority date: 1991-12-12
Filing date: 1992-12-11
Publication date: 1993-06-24
Also published as: FR2684996A1

Abstract

2',3'-dideoxy-3'-amino-thymidine derivatives of formula (I), wherein R is NH4+ or HO(CH¿2?)2-S-S-(CH2)2; and therapeutical uses thereof.

Description

The present invention relates to derivatives of 2 ', 3'-dideoxy-3'-aminothymidine (or amino-dT), their preparation and their application in therapy.

The compounds of the invention correspond to the formula given in Annex 1 in which

R is NH ₄ ⁺ or HO (CH ₂ ) ₂ -SS- (CH ₂ ) ₂ -.

The preparation of the compounds of the invention is indicated below.

The thin layer chromatographies were carried out on Merck 60F 254 silica plates (art.5554). The chromatographies on a silica gel column were carried out with Merck 60 H silica (art. 7736) or with RP2 Merck silanized silica (art. 7719).

The HPLC analyzes were carried out on a Waters Radial-Pak column (diam .: 8 mm, 1: 100 mm) C ₁₈ with a spherical particle size of 10 μm. This column is protected by a Guard-Pak precolumn. The HPLC system is composed of a U ₆ K injector, two M-6000 A pumps, an M-720 programmer

(Waters), a Pye Unicam PU 4021 multichannel UV detector and a PU 4850 video control center (Philipps). The elution was carried out with a solution of acetonitrile in a buffer of 0.1 M ammonium acetate (pH 5.9) at a flow rate of 2 ml per minute (TR retention time).

The HPLC purifications were carried out on a SFCC Nucleosil column (diam .: 19 mm, 1: 150 mm) with a spherical particle size of 10 μm. The HPLC system is composed of a U ₆ K injector, two M-510 EF pumps, an M-720 programmer, a M-481 UV detector and a Data Module 746 recorder (Waters) . Elution is carried out with a solution of acetonitrile in water at a flow rate of 6.25 ml per minute. Before analysis, HPLC purification or lyophilization, the solutions were filtered through a Millex HV-4 filter (Millipore).

UV spectra were recorded on a spectrophotometer

UVIKON 810.

Mass spectra were taken on a JEOL JMS device DX 300 by the FAB ionization method in a matrix of glycerol (G), glycerol / thioglycerol (GT) or 3-nitrobenzyl alcohol (NBA).

The proton NMR spectra were recorded on a Varian EM 360 device or on a Brϋker AC 250 device.

The chemical shifts are expressed in ppm relative to the tetramethylsilane (TMS) signal.

The multiplicity and the pace of the signals observed by NMR are indicated by one (or more) letter (s): s (singlet), d (doublet), t (triplet), m (multiplet), 1 (wide).

The phosphorus NMR spectra were recorded on a Brϋker WP 200 SY device with decoupling of the proton. The chemical shifts are expressed in ppm relative to the H ₃ P0 ₄ signal taken as an external reference.

5'-O-Tertiobutyldimethylsilyl 3'-amino 2 ', 3'-dideoxythymidin 2.

To a solution of 8.35 g (34.6 mol.) Of 3'-amino-2 ', 3'-di-deoxythymidine 1_ in 250 ml of pyridine are added 6.26 (41.5 mol.) Of tert-butyldimetylsilyl chloride. The reaction is left for 24 h. The mixture is diluted with CH _g Cl _g , washed with an aqueous solution of ΝaHC0 ₃ then with water. The organic phase is dried over Na ₂ S0 ₄ and concentrated. The oil obtained is chromatographed on silica gel.

(eluent MeOH (0-10%) in CH ₂ C1 ₂ ) to yield 11.8 g (96%) of 2 in the form of foam.

2UV (EtOH): max 266 nm (ε 10600) min 233 nm (ε 1700)

MS (FAB positive, GT): 711 (2M + H) ⁺ , 356 (M + H) ⁺ , 127 (BH ₂ ) ⁺ NMRΗ (DMSO-? _E ): δ = 0.07 (s; 6H, (CH ₃ ) ₂ If); 0.88 (s, 9H, (CH ₃ ) ₃ CSi); 1.77 (s, 3H, CH ₃ ); 2.03 (m, 2H, H-2 ', 2' ¹ ); 3.38 (m, 1H, H-3 *); 3.58 (m, 1H, H-4 '); 3.72 (dd, 1H, H-5 ¹ , J = 3.9 and 10.4 Hz); 3.83 (dd, 1H, H-5 '*, J = 2.8 and 10.4 Hz); 6.11 (t, 1H, H-1 ', J = 6.2 Hz), 7.48 (s, -1H, H-6) ppm.

5'— O- ertiobutyldimethylsilyl 3 '- (__— (4-methoxytrityl) —amino) 2 *, 3'— dideoxythymidine 3.

Compound 2 (11.7 g; 32.9 mmol.) Is treated with 15.2 g (49.2 mmol.) Of 4-methoxytrityl chloride in 295 ml of pyridine for 20 h. The mixture is taken up with CH ₂ C1 ₂ (1 And ₃ N), washed with an aqueous solution of NaHCO ₃ and then with water. The organic phase is dried over Na ₂ S0 ₄ , evaporated and coevap rated with toluene. Purification on a column of silica gel (eluent: MeOH (0-3%), and ₃ N (1%) in CH ₂ C1 ₂ ) gives 17.5 g (85%) of 3 in the form of foam.

3UV (EtOH): λ max 266 nm (ε 11300) λ min 254 nm (ε 10200) λ inflex 232 nm (ε 15800)

SM (FAB positive, GT): 628 (M + H) ⁺ , 127 (BH ₂ ) ⁺

RM ^ H (OMSO-d ₆ ): δ = -0.08 and -0.04 (s and s, 3H and 3H, (CH ₃ ) ₂ Si); 0.79 (s, 9H, (CH ₃ ) ₃ CSi); 1.10-1.37 (m, 2H, H-2 ', 2''); 1.69 (s, 3H, CH ₃ ); 3.17 (m, 1H, H-3 '); 3.50 (m, 2H, H-4 ', NH); 3.70 (m, 1H, H-5 '); 3.71 (s, 3H, CH ₃ OTr); 3.85 (m, 1H, H-5 ''); 6.09 (t, 1H, H-1 '; J = 6.9 Hz); 6.82-7.47 (m, 14H, Tr), 7.20 (s, 1H, H-6) ppm.

3 '- (N- (4-Methoxytrityl) -amino) 2 *, 3'-dideoxythymidine 4.

Treatment of 17.4 g (27.7 mmol.) Of 3 with 83 ml (91 mmol.) Of a 1.1 M solution of tetrabutylammoniu fluoride in THF results in 10.2 g (72%) of after evaporation of the solvent and 4 chromatographies on a silica gel column (eluent: MeOH (0-15%), And ₃ N (0.5%) in CH ₂ C1 ₂ ) to remove the butylaπunonium salts.

4UV (EtOH): λ max 265 nm (ε 9900) X min 255 nm (ε 8600) λ inflex 231 nm (ε 14000)

SM (FAB positive, GT): 514 (M + H) ⁺ , 127 (BH ₂ ) ⁺ R ^ H (DMSO-d ₅ ): δ = 1.02-1.19 (m, 1H, H-2 '); 1.19-1.37 (m, 1H, H-2 ''); 1.68 (s, 3H, CH ₃ ); 2.8 (si, 1H, NH); 3.19 (m, 1H, H-3 '); 3.44 (m, 1H, H-5 '); 3.64 (m, 1H, H-5 "); 3.71 (s, 3H, CH ₃ 0); 3.72 (m, 1H, H-4 '); 4.90 (t, 1H, OH , J = 4, Hz); 5.97 (t, 1H, H-1 ', J = 6.5 Hz); 6.81-7.55 (m, 14H, Tr); 7.55 (s, 1H, H-6); 1 1, 2 (si, 1 H, NHCO) ppm.

O- (3 '- (N- (4-methoxy rityl) -amino) 2', 3'-dideoxy-thymidin-5'-y1) -hydrogenophosphonate 5.

A solution of 6.65 g (97.7 mmol.) Of imidazole in 69 ml of acetonitrile is treated at 0 ° C. with 2.60 ml (29.8 mmol.) Of phosphorus trichloride and 15.3 ml (110 mmol.) Of triethyl anine for 30 '. This mixture is added to 5.12 g (9.99 mmol.) Of 4 in 69 ml of acetonitrile. The phosphorylation is left for 7 h and then 5 ml of water are added. The solution is then concentrated under reduced pressure, taken up with a solution of triethylammonium bicarbonate and extracted with CH ₂ Cl2 _* The organic phase is washed with water, dried over sodium sulfate and evaporated. The crude is purified on a column of silica gel (eluent: MeOH (0-20%), And ₃ N (0.05%) in CH ₂ Cl2) to yield 5.1 g (75%) of 5.

J5UV (EtOH): λ max 265 nm (ε 8700) λ min 254 nm (ε 7600) λ inflex 231 nm (ε 12300)

SM (FOB negative, GT): 576 (M) ^"

¹ H NMR (DMS0-d): δ = 1.00-1.50 (m, 2H, H-2 ', 2''), 1.18 (t,

9H, (CH ₃ CH ₂ ) ₃ N, J = 7.3 Hz); 1.75 (s, 3H, CH ₃ ); 3.04 (quadruplet, 6H, (CH ₃ CH ₂ ) ₃ N, J = 7.3 Hz), 3.21 (m, 1H, H-3 ');

3.60-3.95 (m, 3H, H-4 ', 5', 5 ''); 3.72 (s, 3H, CH ₃ OTr); 6.00

(t, 1H, H-V, J = 6.3 Hz); 6.61 (d, 1H, HP, J = 585 Hz);

6.80-7.55 (m, 14H, Tr); 7.66 (s, 1H, H-6); 10.5 (if, 1H,

NH); 10.8 (si, 1H, NH) ppm ³¹ P NMR (DMS0-d _$ ): δ = 2.055 ppm.

0-0'-bis (3 * -N- 4-methoxy ri yl) -amino) 2 ', 3 * dideoxy-thymidin-5'-yl) -phosphate 6. To the mixture of 2.19 g (3.23 mmol.) of hydrogenophosphonate 5 and 1.49 g (2.90 mmol.) of nucleoside A in 42 ml of pyridine are added 1.20 ml (9.74 mmol.) of pivaloyl chloride. After 2 h of reaction, the medium is diluted with CH ₂ Cl2, washed with an aqueous NaHCO ₃ solution and then with water. The organic phase is dried over Na2SO4, concentrated and co-evaporated with toluene. The crude is taken up with 82 ml of a 2% iodine solution in the pyridine, water mixture (98: 2). After 30 ', the reaction medium is diluted with CH ₂ Cl2 containing 1% of Et ₃ N and with an aqueous solution of NaHC0 ₃ , then is treated with an aqueous solution of sodium thiosulfate until the coloring due to iodine. The organic phase is separated, washed with water, dried over Na ₂ S0 ₄ , concentrated and coevaporated with toluene. Diester 6 (2.27 g, 66%) is obtained after chromatography on a silica gel column (eluent: MeOH (0-10%), And ₃ N (0.5%) in CH ₂ C1 ₂ ).

6UV (EtOH): λ max 265 nm (ε 12300) λ min 254 nm (ε 9200) λ inflex 231 nm (ε 15300)

SM (FAB negative, GT): 1088 (M) ^" , 816 (MH-MTr) ^"

¹ H NMR (DMSO-d ₆ ): δ = 1.08-1.50 (m, 2H, H-2 ', 2''); 1.19 (t,

9H, (CH ₃ CH ₂ ) ₃ N, J = 7.3 Hz); 1.75 (s, 3H, CH ₃ ); 3.05 (quadruplet, 6H, (CH ₃ CH ₂ ) ₃ N, J = 7.3 Hz); 3.20 (m, 1H, H-3 '); ≈ 3.4 (m, H-4 'masked by water); 3.70 (s, 3H, CH ₃ OTr); 3.78 (m, 2H, H-5 ', 5''); 6.04 (t, 1H, H-1 ', J = 6.1 Hz); 6.80-7.52 (m, 14H, Tr); 10.3 (si, 1H, NH); 10.9 (si, 1H, NH) ppm ³¹ P NMR (DMSO-d ₅ ): δ = -1.126 ppm.

0,0 ^, -bis (3 ^, -amino-2 ', 3'-dideoxy hymidin-5'-yl) phosphate (ammonium salt) 7, (compound 1). Compound 6 (300 mg, 0.252 mmol.) Is deprotected by reaction with 7.6 ml of a 2% solution of benzene sulfonic acid in methanol for 2 h. The acid is neutralized by adding an aqueous solution of triethylammonium bicarbonate. The aqueous phase is extracted with CH ₂ Cl and then concentrated under reduced pressure. The residue obtained is chromatographed. 2 times on a thin layer of silica gel (eluent: ammonia (10%), water (10%) in isopropanol) to give, after filtration on a Millipore filter and lyophilization in water, 41 mg (31%) of 1_ diester form of ammonium salt.

7CLHP: TR: 510 S (99.4%) (5% CH ₃ CH / Ac ONH ₄ 0.1M) UV (H ₂ 0): λ max 266 nm (ε 15000) λ min 235 nm (ε 4000)

MS (FAB negative, GT): 543 (M) ^" ; (FAB positive, GT): 589 (M + 2Na) ⁺ , 567 (MH + Na) ⁺ , 545 (M + 2H) ⁺

¹ H NMR (DMSO-d ₅ ): δ = 1.77 (s, 6H, 2CH ₃ ); 2.00-2.29 (m, 4H, 2H-2 ', 2 "); 3.46 (m, 2H, 2H-3 *); 3.70 (m, 2H, 2H-4'); 3 , 9 (m, 4H, 2H-5 *, 5 ''); 6.07 (t, 2H, 2H-1 ', J = 5.4 Hz); 7.64 (s, 2H, 2H-6) ppm ³¹ P NMR (DMS0-d ₅ ): δ = -0.627 ppm.

0.0'-bis (3'-amino-2 *, 3 * -dideoxy hymidin-5 * -yl) 0- (S- (2-hydr xyethylsulfidyl) 2-thioethyl) phosphate 8 (compound 2). A mixture of 300 mg (0.252 mmol) of diester 6, 35 mg (2.52 mmol) of 4-methoxypyridine N-oxide and 1.07 g (2.51 mmol.) Of 0-mono- (4-methoxytrityl) dithiodiethanol (prepared by reacting a large excess of dithiodiethanol with 4-methoxytrityl chloride) dissolved in 63 ml of CH ₂ C1 is treated with 373 mg (1.26 mmol.) Of 1- ( 2-mesitylene-sulfonyl) 3-nitro 1,2,4-triazole. After 3 h of reaction, the reaction medium is diluted with CH ₂ Cl2. washed with an aqueous solution of NaHC0 ₃ then with water. The organic phase is dried over Na ₂ S0 ₄ , concentrated and coevaporated with toluene. The crude is directly deprotected by reaction with 7.5 ml of a 2% solution of benzene sulfonic acid in MeOH in 2 h. The acid is neutralized by the addition of a 1 M solution of triethylammonium bicarbonate. The aqueous phase is washed with CH ₂ Cl2 and concentrated under reduced pressure. The crude product obtained is purified by semi-preparative HPLC (nucleosil column C ₁ 5, eluent: 12% CH ₃ CN in a 0.05 M solution of triethylammonium acetate). The appropriate fractions are evaporated and lyophilized 4 times in water. After filtration, on a Millipore filter, a final lyophilization gives 50 mg (25%) of triester 8_ associated with two molecules of acetic acid. 8CLHP: TR 320s (94%; 7: 4%) (15% CH ₃ CN / AcONH ₄ 0.1 M) UV (H ₂ 0): λ max 265 nm (ε 15700) λ min 234 nm (ε 4100)

SM (FAB positive, GT): 681 (M + H) ⁺ , 545 (M- (OCH ₂ CH ₂ S) ₂ + H) ⁺ ¹ H NMR (DMS0-d ₅ ): δ = 1.78 (s, 6H, 2CH ₃ ); 1.89 (s, 6H, 2CH ₃ COO ^" ); 1.95-2.19 (m, 4H, 2H-2 ', 2"); 2.77 (t, 2H, CH ₂ CH ₂ OH, J = 6.4 Hz); 2.96 (t, 2H, (CH ₂ CH ₂ OP, J = 6.5 Hz); 3.40 (m, 2H, 2H-3 '); 3.59 (t, 2H, (CH ₂ CH ₂ ) OH, J = 6.4 Hz); 3.71 (m, 2H, 2H-4 '); 4.10-4.22 (m, 6H, 2H-5', 5 '\ CH ₂ CH ₂ OP ) 6.14 (dd, 2H, 2H-1 *, J = 5.7 and 6.7 Hz); 7.47 and 7.46 (ses, 2H, 2H-6) ppm ³¹ P NMR (DMSO-d ₆ , D ₂ 0): δ = -0.504 ppm.

BOARD

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

- Evaluation of anti HIV 1 activity in MT4 cells MT4 = human T cell transformed by HTLV1 HTLV = human T ly photropic virus.

The multiplication of HIV-1 (strain HTLV IIIB) in MT4 cells is followed by the cytopathic effect induced by the virus.

The cells are infected with a dose of HIV-1 produisan after 5 days a 90% decrease in the number of living cells.

The compounds tested are added, after adsorption of the. virus, in the culture medium at different concentrations. The highest concentration used is 10 M. The viability of the cells is measured by a colorimetric reaction based on their ability to reduce the bromide of 3- (4,5 dimethylthiazol-2-yl) -2,5 diphenyl-tetrazolium to formazan , property due to mitochondrial dehydrogenases. The amount of formazan produced is assessed by measuring the D.O. at 540 nm: this D.O. is proportional to the number of living cells. The percentage of protection of infected cells by treatment with the compounds is calculated by applying the formula proposed by Pauwels et al.

D.O. 540 of cells D.O. 540 of infected infected cells treated untreated

D.O. 540 of cells D.O. 540 of untreated infected uninfected cells

The compounds of the invention exhibit anti-HIV activity,

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 compound causing a halving of the OD 540 by _ * - compared to that of the control cells.

ANNEX 1

-O-aminodT

Claims

claims

1. 2 ', 3'-dideoxy-3'-amino-thymidine derivatives corresponding to the formula

in which

R is NH ₄ ⁺ or H0 (CH ₂ ) 2-SS- (CH ₂ ) 2- -

2. A drug containing a derivative according to claim 1.

3. Pharmaceutical composition containing a derivative according to claim 1 in association with any acceptable pharmaceutical excipient.