Bola-amphiphilic compounds and their uses for biomedical applications

Abstract

The invention relates to bola-amphiphilic compounds and their uses for biomedical application. The invention particularly relates to the use of bola-amphiphilic compounds for providing low molecular weight gels (LMWG), useful, in particular, as culture media for animal or human cells, or as biocompatible material for biomedical applications.

Claims

1. A compound of formula (I)
R.sub.1—X-A-X—R.sub.2  (I) in which X is oxygen, —NH—C(O)—NH— or —C(O)—NH— A is a C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated or unsaturated, which is unsubstituted or substituted by one or more C.sub.1-C.sub.12 linear or branched alkyl groups, or A represents a C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated or unsaturated, which is partially or completely halogenated; R.sub.1 and R.sub.2, identical or different, represent —(CH.sub.2).sub.n—R.sub.3—(CH.sub.2).sub.m—R.sub.4-(CH.sub.2).sub.p—R.sub.5-R.sub.6 in which n, m and p, identical or different, are 0 to 10; R.sub.3 represents a heteroaryl group comprising 1 to 4 oxygen or nitrogen atom(s); R.sub.4 represents a nucleosidyl group; R.sub.5 represents a heteroaryl group comprising 1 to 4 heteroatom(s); R.sub.6 represents a residue of a cyclic carbohydrate or a derivative of the said carbohydrate.

2. A compound of formula (I) according to claim 1, in which the carbohydrate derivative of R.sub.6 is selected from an oligosaccharide-type glycan deriving from a cyclic carbohydrate, a N-acyl derivative of said cyclic carbohydrate and a protected derivative of said cyclic carbohydrate

3. A compound of formula (I) according to claim 1, in which at least one of the following conditions is fulfilled: n=m=p=1 R.sub.3 and R.sub.5, identical or different, represent a heteroaryl group containing 1 to 4 nitrogen atoms selected from the group consisting of pyrazole, triazole, tetrazole and imidazole; R.sub.4 represents a nucleosidyl group selected from adenosine, deoxyadenosine, guanosine, deoxyguanosine, thymidine, deoxythymidine, uridine, deoxyuridine, cytidine and deoxycytidine. R.sub.6 represents a residue of a cyclic carbohydrate selected from D-glucopyranose, D-galactopyranose, D-mannopyranose, D-fructopyranose D-ribofuranose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylmannosamine and sialic acid or a protected derivative thereof.

4. A compound of formula (I) according to claim 1, in which: A represents a C.sub.12 saturated hydrocarbon chain; R.sub.1 is hydrogen, or —(CH.sub.2).sub.n—R.sub.3—(CH.sub.2).sub.m—R.sub.4-(CH.sub.2).sub.p—R.sub.5-R.sub.6; R.sub.2 is —(CH.sub.2).sub.n—R.sub.3—(CH.sub.2).sub.m—R.sub.4-(CH.sub.2).sub.p—R.sub.5-R.sub.6; n=m=p=1; R.sub.3 is a triazole group; R.sub.4 is thymidine; R.sub.5 is a triazole group, and R.sub.6 is D-glucopyranosyl or 2,3,4,6 tetra-O-protected-glucopyranosyl.

5. A compound of formula (I) according to claim 1, in which: A represents a C.sub.10 hydrocarbon chain which is partially fluorinated; R.sub.1 and R.sub.2 are —(CH.sub.2).sub.n—R.sub.3—(CH.sub.2).sub.m—R.sub.4-(CH.sub.2).sub.p—R.sub.5-R.sub.6; n=m=p=1; R.sub.3 is a triazole group; R.sub.4 is thymidine; R.sub.5 is a triazole group, and R.sub.6 is D-glucopyranosyl or 2,3,4,6 tetra-O-protected-glucopyranosyl.

6. A compound of formula (I) according to claim 1, in which R.sub.5 is covalently linked to R.sub.4 via —(CH.sub.2)p- to the carbon atom at position 5′ of the ribose or deoxyribose ring of the nucleosidyl group, or, alternatively, R.sub.5 is covalently linked to R.sub.4 via —(CH.sub.2)p- to the oxygen atom at position 3′ of the ribose or deoxyribose ring of the nucleosidyl group.

7. A compound of formula (I) according to claim 1, which is selected from the group consisting of: 5′-[4-(12-hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-N3-(1-((β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl thymidine, 1,12-bis-dodecanyl-5′-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine, 1,12-bis-dodecanyl-5′-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-3′-O-1-((b-D-glucopyrano side)-1-H-1,2,3-triazol-4-yl)methyl thymidine, 1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5′-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-N-3-[1-((b-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl]-5′-deoxythymidine, 12-bis-dodecanyl-5′-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxythymidine, 1,12-bis-dodecanyl-5′-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine, 11,14-bis-tetradecanyl-5′-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine, and 1,14-bis-tetradecanyl-5′-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine.

8. A process for preparing a compound of formula (I) according to claim 1 in which X is oxygen, comprising the following steps: reacting an alkane-diol or a fluoroalkane-diol of formula R.sub.1—O-A-O—R.sub.2 at both ends with an alkylating agent of formula R—(CH.sub.2).sub.q—C≡CH, where q is 1 or 2 and R is an halide, in particular chloride or bromide, reacting the resulting compound with the 5′-azido derivative of R.sub.4, reacting the purine or pyrimidine base or universal base moiety of R.sub.4 in the resulting compound with an alkylating agent of formula R—(CH.sub.2).sub.q—C≡CH, where q and R is as defined above, in order to obtain the N-alkylated derivative of the purine or pyrimidine base or universal base of R.sub.4, and reacting the resulting compound with the 1-azido derivative of R.sub.6 (click-reaction).

9. A process for preparing a compound of formula (I) according to claim 1 in which X is —NH—C(O)—NH—, comprising the following steps: reacting a diisocyanatoalkane of formula OCN-A-NCO with NH.sub.2—CH.sub.2—C≡CH (propargylamine) or any primary amine containing a terminal alkyne group in order to obtain a compound of formula (II)
CH≡C—CH.sub.2—NH—C(O)—NH-A-NH—C(O)—NH—CH.sub.2—C≡CH  (II), reacting a purine or pyrimidine base or a universal base with an alkylating agent of formula R—(CH.sub.2).sub.q—C≡CH, where q is 1 or 2 and R is an halide, preferably bromide or chlorine, in order to obtain the N-alkylated derivative of the purine or pyrimidine base or universal base of R.sub.4, reacting the resulting compound with the 1-azido derivative of R.sub.6 (click-reaction), converting the 5′-OH group on the ribose or deoxyribose moiety of R.sub.4 into an azide, and subjecting the resulting compound to a second click-reaction with a compound of formula (II).

10. A compound of formula (IV)
R.sub.7—X-A-X—(CH.sub.2).sub.n—R.sub.3—(CH.sub.2).sub.m—R.sub.4-R.sub.7  (IV) in which X is —NH—C(O)—NH—; A is a C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated or unsaturated, which is unsubstituted or substituted by one or more C.sub.1-C.sub.12 linear or branched alkyl groups, or A represents a C.sub.4-C.sub.30 hydrocarbon chain, linear or branched, saturated or unsaturated, which is partially or completely halogenated; R.sub.3 represents a heteroaryl group comprising 1 to 4 oxygen or nitrogen atom(s); R.sub.4 represents a nucleosidyl group; R.sub.7 is the residue of an alkylating agent of formula R—(CH.sub.2)q-C≡CH where q is 1 or 2 and R is an halide, which is linked by a covalent bond with a nitrogen atom of the purine, pyrimidine or universal base moiety of R.sub.4; n and m, identical or different, are 0 to 10.

11. A compound of formula (V)
R.sub.4—(CH.sub.2).sub.p—R.sub.5-R.sub.6  (V) in which R.sub.4 represents a nucleosidyl group; R.sub.5 represents a heteroaryl group comprising 1 to 4 heteroatom(s), which is linked to R.sub.4 by a covalent bond to a nitrogen atom of the pyrimidine base or of the purine base of the nucleosidyl group R.sub.6 represents a residue of a cyclic carbohydrate or a derivative of the said carbohydrate, optionally substituted; p is 0 to 10.

12. A compound of formula (V) according to claim 11, which is selected from 5′-deoxy-N3-(1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl) thymidine, and 5′-Azido-N3-(1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl) thymidine.

13. Hydrogel formed from one or more compound(s) of formula (I) according to claim 1.

14. Hydrogel according to claim 13, characterized in that they are formed from an aqueous solution of compounds of formula (I) containing 0.1 to 10 wt % of compound of formula (I), in particular 1 to 5 wt %.

15. Hydrogel according to claim 13, characterized in that comprise one or more compound(s) of formula (I), or a mixture of one or more compound(s) of formula (I) and one or more glycosyl-nucleoside(s)-lipid(s) (GNL) and/or glycosyl-nucleoside(s)-fluorolipid(s) (GNF), or else a mixture of one or more compound(s) of formula (I) with one or more protein(s) or glycoprotein(s), such as for example collagen or hyaluronic acid, where said mixture contains, optionally, one or more GNL or GNF.

16. In a cell culture media containing a hydrogel, the improvement wherein said hydrogel is one according to claim 13.

17. A cell culture media according to claim 16, where the cells are eukaryotic cells.

18. A cell culture media according to claim 16, where the cells are stem cells.

19. A hydrogel according to claim 13 for use as a biocompatible material for tissue engineering, cellular therapy or prevention of adhesion formation after abdomino-pelvic surgery.

20. A hydrogel according to claim 13 for use as a biocompatible material for controlled-release delivery of an active ingredient which is entrapped into the gel.

Description

[0171] The examples below, entitled “Preparation” describe the preparation of synthesis intermediates used for preparing the compounds of formula (I). The preparation of the compounds of formula (I) and their applications are then described as “Examples”. When present, the number that accompanies the title compound in the “Preparation” or in the “Example” refers to that shown in the schemes of FIGS. 1, 2 and 3.

[0172] FIG. 1 shows the synthetic scheme used in the Preparations and Examples for compounds of formula (I) in which X is oxygen.

[0173] FIG. 2 shows the synthetic scheme used in the Preparations and Examples for compounds of formula (I) in which X is NH—C(O)—NH—.

[0174] FIG. 3 shows the synthetic scheme used in the Preparations and Examples for compounds of formula (I) in which X is —NH—C(O)—.

[0175] FIG. 4 shows the frequency sweep results for hydrogels obtained from compounds 5 and 6.

[0176] FIG. 5 shows the step-strain measurement of the hydrogel obtained from compound 6.

[0177] FIG. 6 shows the determination of the cytotoxicity of compound 6 by MTT test on human mesenchymal stem cells.

[0178] FIG. 7 shows the cytocompatibility of compound 6 by monitoring alamar blue metabolism in rat osteoblastic cells (A) or human stem cells ASCs (B) grown in compound 6-based gels.

[0179] FIG. 8 shows the cytotoxycity of compound GNL of G. Godeau et al., Chem. Comm., 2009, 34, 5127-5130_by MTT test in human hepatic carcinoma cells HuH-7 (comparative example).

PREPARATION 1

1,12-Dipropargyloxydecane (2)

[0180] To a cooled (0° C.) solution of compound 1 (commercially available) (5 g, 24.7 mmol) in anhydrous DMF (60 mL) was added sodium hydride in small portions (55% in mineral oil, 6.5 g, 148.2 mmol) under stirring. Propargyl bromide (80% w/w in toluene, 11 g, 74.1 mmol) was added followed by TBAI (0.91 g, 2.4 mmol), and stirring was continued for 15 hours at room temperature. The reaction was quenched with methanol (10 mL) and stirred a further 30 min. After addition of DCM (200 mL) the solution was washed with water (3×50 mL) then brine (50 mL), the organic layer dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. Product 2 was isolated after purification on silica gel (hexane/ethyl acetate 100/0 then 97/3) as a colorless liquid. Yield: 4.48 g (65%).

[0181] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.22-1.43 (m, 16H, CH.sub.2), 1.52-1.66 (m, 4H, CH.sub.2CH.sub.2O), 2.43 (t, J=2.4 Hz, 2H, CH propargyl), 3.52 (t, J=6.6 Hz, 4H, CH.sub.2CH.sub.2O), 4.15 (d, J=2.4 Hz, 4H, CH.sub.2 propargyl).

[0182] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 26.08, 29.42, 29.50, 29.55 (CH.sub.2, CH.sub.2CH.sub.2O), 57.97 (CH.sub.2 propargyl), 70.31 (CH.sub.2O), 74.01 (CH propargyl).

PREPARATION 2

12-Propargyloxydodecan-1-ol (7)

[0183] To a cooled (0° C.) solution of compound 1 (commercially available) (4 g, 20 mmol) in anhydrous DMF (100 mL) was added sodium hydride in small portions (55% in mineral oil, 2.5 g, 109 mmol) under stirring. Propargyl bromide (80% w/w in toluene, 9 g, 60 mmol) was added dropwise, and stirring was continued for 12 hours at low temperature. The reaction mixture was concentrated under reduced pressure. The resulting mixture was poured into water and extracted with DCM. The extracts were combined, washed with water (3×50 mL) then brine (50 mL). The organic layer was dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. Product 7 was isolated after purification on silica gel (hexane/ethyl acetate 6/4) as a brown solid. Yield: 2.24 g (46%).

[0184] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.19-1.30 (m, 16H, CH.sub.2), 1.46-1.59 (m, 4H, CH.sub.2CH.sub.2O), 2.37 (large s, 1H, OH), 2.40 (t, J=2.4 Hz, 1H, CH propargyl), 3.46 (t, J=6.6 Hz, 2H, CH.sub.2O), 3.56 (t, J=6.7 Hz, 2H, CH.sub.2O), 4.08 (d, J=2.4 Hz, CH.sub.2 propargyl).

[0185] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 25.74, 26.03 (CH.sub.2), 29.39, 29.43, 29.53, 29.58 (CH.sub.2+CH.sub.2CH.sub.2O), 32.70 (CH.sub.2CH.sub.2O), 57.92 (CH.sub.2 propargyl), 62.72, 70.22 (CH.sub.2O), 74.13 (CH propargyl).

PREPARATION 3

1,12-bis-dodecanyl-5′-[(4-oxymethyl)-1H-1,2,3-triazol-1-yl)]-5′-deoxy thymidine (3)

[0186] To a solution of 7 obtained in Preparation 2 (1.50 g, 5.39 mmol) and 5′-azido-5′-deoxythymidine (3.02 g, 11.30 mmol) in 140 mL of THF/H.sub.2O (1:1) was added copper sulfate (172 mg, 1.07 mmol) followed by sodium ascorbate (427 mg, 2.15 mmol). The mixture was stirred at 60° C. for 20 hours. After cooling to room temperature, the solvents were removed under reduced pressure. The resulting solid was washed with water until the washings were colorless, and then with ethanol. After drying under high vacuum, the resulting white solid was used in the next step without further purification. Yield: 4.00 g (91%).

[0187] .sup.1H NMR (300 MHz, in DMSO-d.sub.6) δ 1.22 (s, 16H, 8CH.sub.2), 1.41-1.52 (m, 4H, CH.sub.2CH.sub.2O), 1.79 (s, 6H, CH.sub.3 thymine), 2.08-2.15 (m, 4H, H-2′), 3.35-3.41 (m, 4H, CH.sub.2O), 4.06-4.10 (m, 2H, H-4′), 4.24-4.29 (m, 2H, H-3′), 4.27 (s, 4H, OCH.sub.2-triazole), 4.57-4.74 (m, 4H, H-5′), 5.52 large s, 2H, OH thymine), 6.16 (t, J=7.5 Hz, 2H, H-1′), 7.33 (s, 2H, H-6 thymine), 8.06 (s, 2H, CH triazole), 11.34 (s, 2H, NH thymine).

[0188] .sup.13C NMR (75 MHz, in DMSO-d.sub.6) δ 12.53 (CH.sub.3 thymine), 6.11, 29.33, 29.48, 29.55 (CH.sub.2), 38.31 (C-2′), 51.51 (C-5′), 63.68 (CH.sub.2O-triazole), 70.01 (CH.sub.2O), 71.16 (C-3′), 84.40 (C1′ and C-4′), 125.00 (CH triazole), 136.45 (C-6 thymine).

PREPARATION 4

1,12-bis-dodecanyl-5′-[(4-oxymethyl)-1H-1,2,3-triazol-1-yl)]-5′-deoxy-N-3-propargyl thymidine (4)

[0189] To as solution of compound 3 obtained in Preparation 3 (3.50 g, 4.30 mmol) in anhydrous DMF (120 mL) was added sodium carbonate (1.7 g, 12.92 mmol) followed by propargyl bromide (80% w/w in toluene, 1.92 g, 12.92 mmol) and TBAI (0.16 g, 043 mmol). The mixture was stirred at room temperature for 20 hours, then poured into DCM (200 mL) and washed with water (3×50 mL) and brine (50 mL). The organic extract was dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The product was purified by column chromatography on silica gel eluting with DCM/MeOH (100:0 to 96:4) and obtained as a white solid foam. Yield: 3.07 g (77%).

[0190] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.27 (s, 16H, 8 CH.sub.2), 1.53-1.60 (m, 4H, 2 CH.sub.2CH.sub.2O), 1.94 (s, 6H, 2 CH.sub.3 thymine), 2.22 (t, 2H, J=2.3 Hz, propargylic CH), 2.31-2.41 (m, 4H, 2H-2′), 3.52 (t, 4H, J=6.5 Hz, 2 CH.sub.2O), 4.23-4.27 (m, 2H, 2H-4′), 4.53-4.59 (m, 2H, 2H-3′), 4.61 (s, 4H, 2 CH.sub.2O triazole), 4.71 (s, 4H, 2 propargylic CH.sub.2), 4.70-4.77 (m, 4H, 2H-5′), 6.23 (t, 2H, J=6.6 Hz, 2H-1′), 6.81 (s, 2H, 2H-6 thymine), 7.68 (s, 2H, 2H triazole).

[0191] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 13.16 (CH.sub.3 thymine), 29.22, 29.31, 29.34, 29.47 (CH.sub.2), 30.50 (propargyl CH.sub.2), 38.61 (C-2′), 51.11 (C-5′), 63.93 (OCH.sub.2 triazole), 71.14 (CH.sub.2O), 71.23 (C-3′), 77.25 (propargyl CH), 83.79 (C-4′), 86.85 (C-1′), 124.60 (CH triazole), 134.72 (C-6 thymine).

PREPARATION 5

5′-[4-((12-Hydroxydodecanyloxy)methyl)1H-1,2,3-triazol-1-yl]-5′-deoxy thymidine (8)

[0192] To a degassed solution of compound 7 obtained in Preparation 1 (2.23 g, 9.30 mmol) in 80 mL THF/H.sub.2O (1:1) was added 5′-azido-5′-deoxy thymidine (2.48 g, 9.30 mmol), copper sulfate (0.15 g, 0.93 mmol) and sodium ascorbate (0.37 g, 1.86 mmol). The mixture was stirred at 60° C. for 3 hours. After removal of THF in vacuo, the aqueous mixture was filtered, the resulting yellow solid was washed with water, then purified by column chromatography on silica gel eluting with DCM/MeOH (9:1) to elute the product as a white solid.

[0193] Yield: 3.88 g (82%).

[0194] .sup.1H NMR (300 MHz, in MeOD @ 313K) δ 1.30-1.39 (m, 16H, CH.sub.2), 1.52-1.60 (m, 4H, CH.sub.2CH.sub.2O), 1.89 (d, J=1.2 Hz, 3H, CH.sub.3 thymine), 2.23-2.29 (m, 2H, H-2′), 3.49-3.57 (m, 4H chain CH.sub.2O+CH.sub.2OH), 4.16-4.21 (m, 1H, H-4′), 4.39-4.43 (m, 1H, H-3′), 4.72-4.81 (m, 2H, H-5′), 6.20 (t, J=6.7 Hz, H-1′), 7.20 (d, 1H, H-6 thymine), 7.95 (s, 1H, CH triazole).

[0195] .sup.13C NMR (75 MHz, in MeOD @ 313K) δ 10.95 (CH.sub.3 thymine), 25.51, 25.74, 29.06, 29.13, 29.22, 29.27, 32.25 (CH.sub.2), 38.26 (C-2′), 51.09 (C-5′), 61.64 (CH.sub.2OH or CH.sub.2CH.sub.2O), 63.27 (CH.sub.2O triazole), 70.38 (CH.sub.2CH.sub.2O or CH.sub.2OH), 70.98 (C-3′), 84.06 (C-4′), 85.46 (C-1′), 110.10 (C-5 thymine), 124.61 (CH triazole), 136.55 (C-6 thymine), 150.30 & 164.31 (C═O).

[0196] HRMS: (M+Na) 530.2943 (calculated 530.2949)

PREPARATION 6

5′-[4-((12-Hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5′deoxy-N-3-propargylthymidine (9)

[0197] To a solution of compound 8 obtained in Preparation 5 (1.27 g, 25.0 mmol) in anhydrous DMF (60 mL) was added potassium carbonate (0.69 g, 50.0 mmol) and TBAI (0.09 g, 2.5 mmol), followed by propargyl bromide (80% w/w in toluene, 1.19 g, 50.0 mmol). The mixture was stirred at room temperature for 24 hours, then filtered, and the filtrate was concentrated under reduced pressure. The resulting yellow oil was poured into water (50 mL) and extracted with DCM (3×50 mL). The combined organic extracts were washed with water (50 mL), then brine (50 mL), dried (Na.sub.2SO.sub.4), and the solvent removed in vacuo. The crude product was purified by column chromatography on silica gel eluting with DCM/MeOH (95:5) to elute the title compound as a viscous yellow oil which crystalizes on standing (white solid). Yield: 1.20 g (87%).

[0198] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.26-1.34 (m, 16H, CH.sub.2), 1.51-1.62 (m, 4H, CH.sub.2CH.sub.2OH+CH.sub.2CH.sub.2O), 1.93 (d, J=1.1 Hz, 3H, CH.sub.3 thymine), 2.20 (t, J=2.4 Hz, 1H, propargylic CH), 2.22-2.29 (m, 1H, H-2′a), 2.35-2.44 (m, 1H, H-2′b), 3.48-3.56 (m, 2H CH.sub.2O), 3.65 (t, J=6.6 Hz, 2H, CH.sub.2OH), 4.18-4.23 (m, 1H, H-4′), 4.45-4.51 (m, 1H, H-3′), 4.59 (s, 2H triazole CH.sub.2O), 4.70-4.74 (m, 4H, H-5′+propargylic CH.sub.2), 6.25 (t, J=6.6 Hz, H-1′), 6.74 (d, 1H, H-6 thymine), 7.68 (s, 1H, CH triazole).

[0199] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 12.93 (CH.sub.3 thymine), 25.68, 25.91, 29.30, 29.33, 29.41, 29.47 (CH.sub.2+CH.sub.2CH.sub.2OH or CH.sub.2CH.sub.2O), 30.36 (propargylic CH.sub.2), 32.55 (CH.sub.2CH.sub.2O or CH.sub.2CH.sub.2OH), 38.58 (C-2′), 51.00 (C-5′), 62.49 (CH.sub.2OH), 63.85 (CH.sub.2 triazole), 70.65 (C-3′), 70.85 (propargylic CH), 70.99 (triazole CH.sub.2O), 83.77 (C-4′), 86.30 (C-1′), 124.48 (CH triazole), 134.83 (C-6 thymine).

[0200] HRMS: (M+Na) 568.3098 (calculated 568.3105)

PREPARATION 7

5′-[4-((12-Propargyloxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-N-3-propargylthymidine (10)

[0201] To a cold (0° C.) solution of compound 8 obtained in Preparation 5 (2 g, 3.66 mmol) in anhydrous DMF (100 mL) was added sodium hydride in one portion (55% in mineral oil, 0.90 g, 22 mmol). After 15 min, TBAI (0.135 g, 0.36 mmol) was added followed by propargyl bromide in small portions (80% w/w in toluene, 1.63 g, 11 mmol), and stirring continued for 5 hours at 0° then at room temperature overnight. The reaction was quenched with water (10 mL) with stirring and cooling on ice, and the solution was concentrated under reduced pressure. The brown residue was dissolved in water (200 mL) and extracted with DCM (3×100 mL) The combined organic extracts were washed with brine (3×100 mL) and aqueous 10% KCl solution (100 mL), dried (Na.sub.2SO.sub.4) and the solvent removed in vacuo. The crude product was purified by chromatography on silica gel eluting with ethyl acetate and obtained as a yellow oil. Yield: 0.87 g (40%).

[0202] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.23-1.27 (m, 16H, CH.sub.2), 1.48-1.56 (m, 4H, CH.sub.2CH.sub.2O), 1.89 (s, 3H, CH.sub.3 thymine), 2.08-2.17 (m, 1H, H-2′a), 2.18 (m, 1H thymine propargylic CH), 2.35-2.43 (m, 1H, H-2′b), 2.51 (m, 1H, chain propargylic CH), 3.48 (t, J=6.7 Hz, 2H CH.sub.2OCH.sub.2-triazole), 3.59 (t, J=6.6 Hz, 2H, chain CH.sub.2O-propargyl), 4.17-4.20 (m, 2H, propargylic CH.sub.2O), 4.24-4.30 (m, 1H, H-4′), 4.35-4.40 (m, 1H, H-3′), 4.57 (s, 2H CH.sub.2 triazole), 4.57-4.70 (m, 4H, H-5′+propargylic CH.sub.2N), 6.14 (t, J=6.9 Hz, H-1′), 6.73 (s, 1H, H-6 thymine), 7.62 (s, 1H, CH triazole).

[0203] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 13.06 (CH.sub.3 thymine), 25.73, 26.03, 29.49, 29.54 (CH.sub.2), 29.39 (CH.sub.2CH.sub.2O), 30.42 (thymine propargylic CH.sub.2), 32.76 (CH.sub.2CH.sub.2O propargyl), 36.23 (C-2′), 50.92 (C-5′), 57.41 (propargylic CH.sub.2O), 62.84 (chain CH.sub.2O-propargyl), 64.16 (CH.sub.2 triazole), 70.80 (thymine propargylic CH), 71.08 (chain CH.sub.2OCH.sub.2 triazole), 75.70 (chain propargylic CH), 78.26 (C-3′), 81.76 (C-4′), 86.69 (C-1′), 124.18 (CH triazole), 134.30 (C-6 thymine).

[0204] HRMS: (M+Na) 606.3258 (calculated 606.3262)

PREPARATION 8

1,1′-(dodecane-1,12-diyl)bis(3-[propargyl]urea) (12)

[0205] To a solution of 1,12-diisocyanatododecane (0.53 mL, 2 mmol) in anhydrous DCM (20 mL) was added slowly propargylamine (0.30 mL, 4.8 mmol). The mixture was stirred for 16 hours at room temperature. The precipitate was filtered and washed abundantly with DCM to remove the excess of propargylamine. After drying, the product was obtained as a white solid.

[0206] Yield: (99%).

[0207] .sup.1H NMR (300 MHz, in DMSO-d.sub.6 @ 350 K) δ (ppm) 1.50-1.63 (m, 20H), 3.15 (t, J=2.6 Hz, 2H), 3.20-3.26 (q, 4H), 4.03 (m, J=2.6 Hz, 4H), 6.08-6.25 (large s, 4H).

[0208] .sup.13C NMR (75 MHz, DMSO-d.sub.6, 350 K) δ (ppm) 26.82, 27.91, 28.05, 29.26, 29.32, 29.69, 30.34, 31.0, 31.09, 72.58, 83.05, 158.02.

PREPARATION 9

N3-propargylthymidine (13)

[0209] To a solution of thymidine (3 g, 12.39 mmol) in anhydrous DMF (20 mL) was added potassium carbonate (2.58 g, 18.58 mmol), propargyl bromide (80% w/w in toluene, 2.07 mL, 18.58 mmol). The reaction was stirred for 2 days at room temperature. After removal of DMF in vacuo, the residue was dissolved in ethyl acetate (100 mL) and then washed with water (3×30 mL). The organic extract was dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The resulting oil was used in the next step without further purification. Yield: 3 g (86%).

[0210] .sup.1H NMR (300 MHz, in DMSO d.sub.6) δ 1.85 (s, 3H), 2.10-2.18 (m, 2H), 3.10 (s, 1H), 3.59 (m, 2H), 3.79 (s, 1H), 4.25 (m, 1H), 4.53 (s, 2H), 5.04 (t, J=6 Hz, 1H), 5.27 (s, 1H), 6.21 (t, J=6 Hz, 1H), 7.82 (s, 1H).

[0211] .sup.13C NMR (75 MHz, in DMSO d.sub.6) δ 13.0, 30.3, 40.3, 62.0, 70.9, 71.1, 78.2, 86; 0, 87.1, 116.9, 135.2, 150; 2, 162.7.

[0212] HRMS: (M+H) 281, 1135 (calculated 281.1137)

PREPARATION 10

[0213] Compound (14)

##STR00001##

5′-deoxy-N3-(1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl) thymidine (14)

[0214] To a solution of 13 obtained in Preparation 9 (2.80 g, 10 mmol) and 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (3.73 g, 10 mmol) in 80 mL of tert-butanol/H.sub.2O (1:1) was added copper sulfate (159 mg, 1 mmol) and sodium ascorbate (396 mg, 2 mmol). The mixture was stirred at 60° C. for 20 hours. The solvent was removed under reduced pressure and the residual solid was dissolved in ethyl acetate (150 mL) and then washed with water (3×50 mL). The organic extract was dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel eluting with ethyl acetate and obtained as a white solid. Yield: 5.33 g (82%).

[0215] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.84-2.08 (m, 15H), 2.35 (m, 2H), 3.82-3.92 (m, 2H), 4.00 (m, 2H), 4.11-4.15 (dd, 1H), 4.25-4.31 (dd, 1H), 4.58 (m, 1H), 5.19-5.31 (m, 3H), 5.36-5.48 (m, 2H), 5.85 (d, J=8.8 Hz, 1H), 6.23 (t, J=6.4 Hz, 1H), 7.52 (s, 1H), 7.88 (s, 1H).

[0216] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 12.47, 19.93, 20.32, 20.47, 35.18, 39.60, 61.00, 61.22, 67.03, 69.58, 70.15, 72.03, 74.14, 84.72, 85.45, 86.62, 99.37, 109.15, 122.30, 134.58, 142.94, 149.97, 162.53, 168.23, 168.77, 169.22, 169.99.

PREPARATION 11

[0217] Compound (15)

##STR00002##

5′-Azido-N3-(1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl) thymidine (15)

[0218] To a solution of 14 obtained in Preparation 10 (1 g, 1.53 mmol) in anhydrous DMF (6 mL) was successively added PPh.sub.3 (0.48 g, 1.84 mmol), NaN.sub.3 (0.50 g, 7.65 mmol) and CBr.sub.4 (0.61 g, 1.84 mmol). After stirring for 24 hours, the reaction was quenched with methanol (1.5 mL) and stirred a further 30 min. The solvent was removed under reduced pressure and the product was isolated after purification on silica gel (DCM/MeOH 100/0 then 97/3) as a white solid. Yield: 1.31 g (77%).

[0219] .sup.1H NMR (300 MHz, in CDCl.sub.3) δ 1.72-1.97 (m, 15H), 2.10-2.17 (m, 1H), 2.30-2.33 (m, 1H), 3.49-3.65 (m, 2H), 4.00-4.22 (m, 3H), 4.25-4.31 (dd, 1H), 4.38 (m, 1H), 5.05-5.22 (m, 3H), 5.35-5.45 (m, 2H), 5.85 (d, J=8.8 Hz, 1H), 6.23 (t, J=6.4 Hz, 1H), 7.33 (s, 1H), 7.85 (s, 1H).

[0220] .sup.13C NMR (75 MHz, in CDCl.sub.3) δ 12.47, 19.98-20.53, 35.76, 39.97, 52.09, 61.50, 67.56, 70.06, 71.07, 72.58, 74.72, 84.44, 85.32, 110.17, 122.68, 134.04, 143.49, 150.44, 162.87, 168.68, 170.50.

PREPARATION 12

1,1′-(tetradecane-1,14-diyl)bis(3-[propargyl]amide) (18)

[0221] Thionyl chloride (9.12 mmol, 2.1 eq) and triethylamine (13.02 mmol, 3 eq) was added dropwise to a suspension of hexadecanedioic acid (4.34 mmol) in dry DCM. The mixture was vigorously stirred for 1 hour at room temperature. Then, propargylamine (9.55 mmol, 2.2 eq) was added slowly. After stirring for 16 hours, the mixture was filtrated and the crude product was washed abundantly with water and ethanol. Yield: 36%.

[0222] .sup.1H NMR (300 MHz, DMSO-d.sub.6, 340 K) δ 1.25-1.49 (m, 24H), 2.08 (t, 4H,), 2.93 (t, 2H), 3.83 (m, 4H), 7.99 (large s, 4H).

[0223] .sup.13C NMR (75 MHz, DMSO-d.sub.6, 340 K) δ 24.6, 27.3, 28.2, 28.3, 28.4, 28.5, 34.7, 71.9, 81.0, 171.5.

Example 1

5′-[4-(12-hydroxydodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-N3-(1-((β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl thymidine (5)

[0224] To a degassed solution of compound 9 obtained in Preparation 6 (1.182 g, 2.0 mmol) and 1-azido-β-D-glucopyranose (0.534 g, 2.0 mmol) in 20 mL of THF/H.sub.2O (1:1) was added copper sulfate (32 mg, 0.2 mmol) followed by sodium ascorbate (80 mg, 0.4 mmol). The mixture was stirred at 60° C. for 20 hours. After cooling to room temperature, the solvents were removed under reduced pressure. The resulting green solid was dissolved in methanol and filtered through celite, the resulting solution was concentrated under reduced pressure and applied to a column of silica gel. The product was eluted with DCM/MeOH (9:1 to 8:2). Yield: 0.84 g (55%).

[0225] .sup.1H NMR (300 MHz, in MeOD) δ 1.28-1.35 (s, 18H, CH.sub.2), 1.51-1.60 (m, 4H, CH.sub.2CH.sub.2O), 1.94 (s, 3H, CH.sub.3 thymine), 2.28-2.32 (dd, J=6.0 Hz, 2H, H-2′), 3.45-3.60 (m, 5H, H-3, H-4, H-5, CH.sub.2O), 3.66-3.72 (dd, J=12.0, 5.2 Hz, 2H, H-6a), 3.84-3.91 (m, 2H, H-2, H-6b), 4.19-4.21 (m, 1H, H-4′), 4.42-4.45 (m, 1H, H-3′), 4.56 (s, 2H, CH.sub.2 triazole), 4.75-4.78 (m, 2H, H-5′), 5.22 (s, 2H, CH.sub.2—N triazole), 5.58 (d, J=9.2 Hz, 1H, H-1), 6.24 (dd, J=6.0 Hz, 1H, H-1′), 7.31 (s, 1H, H-6 thymine), 8.00 (s, 1H, H triazole), 8.11 (s, 1H, H triazole).

[0226] .sup.13C NMR (75 MHz, in MeOD) δ 11.80 (CH.sub.3 thymine), 25.56, 25.80, 29.23, 29.26, 32.27 (CH.sub.2 and CH.sub.2CH.sub.2O), 35.62 (CH.sub.2N triazole), 38.21 (C-2′), 51.21 (C-5′), 60.92 (C-6), 61.60 (CH.sub.2O), 63.14 (OCH.sub.2 triazole), 70.33 (CH.sub.2O), 69.41, 76.93, 79.67 (C-3 or C-4 or C-5), 71.00 (C-3′), 72.50 (C-2), 84.27 (C-4′), 86.53 (C-1′), 88.15 (C-1), 109.82 (C-5 thymine), 123.03, 124.82 (CH triazole), 135.47 (C-6 thymine), 143.12, 144.72 (C quat. triazole), 150.59, 163.37 (C═O thymine).

[0227] HRMS: (M+Na) 773.3795 (calculated 773.3804)

Example 2

1,12-bis-dodecanyl-5′-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine (6)

[0228] To a solution of 4 obtained in preparation 4 (200 mg, 0.225 mmol) and 1-azido-β-D-glucopyranose (111 mg, 0.540 mmol) in 12 mL of THF/H.sub.2O (1:1) was added copper sulfate (14 mg, 0.09 mmol) followed by sodium ascorbate (53 mg, 0.27 mmol). The mixture was irradiated under stirring in a microwave reactor (open vessel equipped with a reflux condenser) at 200 W and 75° C. for 5 min. The crude resulting mixture was concentrated under reduced pressure and purified by column chromatography on silica gel eluting with DCM/MeOH (8:2 to 7:3) to give 5 then 6. Yields: 5, 16 mg (9.5%); 6, 177 mg (58%).

[0229] .sup.1H NMR (300 MHz, in MeOD) δ 1.28-1.35 (s, 16H, CH.sub.2), 1.52-1.61 (m, 4H, CH.sub.2CH.sub.2O), 1.92 (s, 6H, CH.sub.3 thymine), 2.27-2.31 (dd, J=6.0 Hz, 4H, H-2′), 3.48-3.60 (m, 10H, H-3, H-4, H-5, CH.sub.2O), 3.67-3.73 (dd, J=12.0, 5.2 Hz, 2H, H-6a), 3.85-3.93 (m, 4H, H-2, H-6b), 4.17-4.22 (m, 2H, H-4′), 4.41-4.46 (m, 2H, H-3′), 4.56 (s, 4H, triazole CH.sub.2O), 4.74-4.77 (m, 4H, H-5′), 5.21 (s, 4H, triazole CH.sub.2N), 5.59 (d, J=9.2 Hz, 2H, H-1), 6.23 (dd, J=6.0 Hz, 2H, H-1′), 7.28 (s, 2H, H-6 thymine), 7.99 (s, 2H, H triazole), 8.13 (s, 2H, H triazole).

[0230] .sup.13C NMR (75 MHz, in MeOD) δ 11.80 (CH.sub.3 thymine), 25.76 (CH.sub.2), 29.23 (CH.sub.2CH.sub.2O), 35.67 (CH.sub.2N triazole), 38.21 (C-2′), 48.45 (C-5′), 60.95 (C-6), 63.16 (OCH.sub.2 triazole), 69.42, 76.98, 79.69 (C-3, C-4, C-5), 70.32 (CH.sub.2O), 70.98 (C-3′), 72.51 (C-2), 84.23 (C-4′), 86.63 (C-1′), 88.17 (C-1), 122.91, 124.79 (CH triazole), 135.44 (C-6 thymine).

[0231] HRMS: (M+Na) 1321.5802 (calculated 1321.5783)

Example 3

5′-[4-((β-D-Glucopyranosyloxy)dodecanyloxy)methyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl]thymidine (11)

[0232] To a degassed solution of compound 10 obtained in Preparation 7 (0.87 g, 1.49 mmol) in 24 mL THF/H.sub.2O (6:4) was added 1-azido-β-D-glucopyranose (0.62 g, 3.00 mmol), copper sulfate (120.00 mg, 0.60 mmol) and sodium ascorbate (47.80 mg, 0.30 mmol). The mixture was stirred at 60° C. for 3 hours. After removal of the solvents in vacuo, the resulting green solid was dissolved in methanol and filtered through celite, the resulting solution was concentrated under reduced pressure and applied to a column of silica gel. The product was eluted with DCM/MeOH (8:2). Yield: 0.81 g (54%).

[0233] .sup.1H NMR (300 MHz, in MeOD) δ 1.30-1.33 (m, 16H, CH.sub.2), 1.50-1.62 (m, 4H, chain-CH.sub.2CH.sub.2O), 1.93 (d, J=1.0 Hz, 3H, CH.sub.3 thymine), 2.26-2.47 (m, 1H, H-2′), 3.49-3.60 (m, 10H, 2 chain-CH.sub.2O, H-3A, H-3B, H-4A, H-4B, H-5A, H-5B), 3.67-3.76 (2 dd, 2H, J=11.6, 5.2 Hz, H-6A, a and H-6B, a), 3.85-3.97 (m, 4H, H-6A, b, H-6B, a and H-2A, H-2B), 4.33-4.37 (m, 2H, H-3′ and H-4′), 4.56 (s, 2H, triazole-CH.sub.2O), 4.68-4.70 (d, J=4.7 Hz, 2H, triazole-CH.sub.2O), 4.74-4.76 (m, 2H, H-5′), 5.22 (s, 2H, triazole-CH.sub.2N), 5.58 (d, J=9.2 Hz, 1H, H-1A or H-1B), 5.65 (d, J=9.2 Hz, 1H, H-1B or H-1A), 6.19 (t, J=7.0 Hz, H-1′), 7.30 (d, 1H, H-6 thymine), 7.98, 8.13 and 8.25 (3s, 1H, CH triazole).

[0234] .sup.13C NMR (75 MHz, in MeOD) δ 11.75 (CH.sub.3 thymine), 25.55, 25.80, 29.18, 29.22, 29.26, 29.33 (CH.sub.2 and CH.sub.2CH.sub.2O), 32.26 (CH.sub.2CH.sub.2O), 32.50 (triazole-CH.sub.2-thymine), 51.07 (C-5′), 60.94 (C-6A, C-6B), 61.58 (chain-CH.sub.2O), 61.93 and 63.11 (triazole-CH.sub.2O), 69.40, 69.44 (C-3, C-4 or C-5 A and B), 70.36 (chain-CH.sub.2O), 72.49, 72.61 (C-2A, C-2B), 76.98, 77.01 (C-3, C-4 or C-5 A and B), 78.49, 82.12 (C-3′, C-4′), 79.69, 79.74 (C-3, C-4 or C-5 A and B), 87.04 (C-1′), 88.17, 88.25 (C-1A, C-1B), 122.93, 123.18, 124.79 (CH triazole), 135.53 (C-6 thymine).

[0235] HRMS: (M+Na) 1016.4648 (calculated 1016.4859).

Example 4

1,12-bis-dodecanyl-5′-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-5′-deoxy-3′-O-1-((β-D-glucopyranoside)-1-H-1,2,3-triazol-4-yl)methyl thymidine

[0236] ##STR00003##

[0237] To a degassed solution of 7 obtained in preparation 1 (139.0 mg, 0.5 mmol) in 30 mL THF/H.sub.2O (2:1) was added 5′-azido-5′-deoxy-3′-O-1-((β-D-glucopyranoside tetraacetate)-1H-1,2,3-triazol-4-yl)methyl thymidine [L. Latxague, A. Patwa, E. Amigues, P. Barthélémy, Molecules 18 (2013) 12241-63], copper sulfate (15.9 mg, 0.1 mmol) and sodium ascorbate (39.6 mg, 0.2 mmol). The mixture was stirred at 65° C. for 6 hours then overnight at rt. After removal of the solvents in vacuo, the remaining crude material was applied to a column of silica gel. The product was eluted with DCM/MeOH (95:5) then subjected to a Zemplen deacetylation. Yield: 301 mg (46%).

[0238] .sup.1H NMR (300 MHz, in MeOD @ 323K) δ 1.30 (s, 16H, CH.sub.2), 1.55-1.63 (m, 4H, CH.sub.2CH.sub.2O), 1.91 (s, 6H, CH.sub.3 thymine), 2.22-2.31 (m, 2H, H-2′a), 2.39-2.47 (m, 2H, H-2′b), 3.39 (t, J=6.5 Hz, 4H, CH.sub.2CH.sub.2O), 3.54-3.66 (m, 6H, H-3, 4, 5), 3.74-3.81 (m, 2H, H-6a), 3.91-4.00 (m, 4H, H-2, 6b), 4.34-4.39 (m, 4H, H-3′, 4′), 4.60 (s, 4H, OCH.sub.2 triazole), 4.71-4.76 (m, 8H, H-5′ and OCH.sub.2 triazole), 5.66 (d, J=9.2 Hz, 2H, H-1), 6.18 (dd, J=6.7 Hz, 2H, H-1′), 7.22 (s, 2H, H-6 thymine), 7.96 (s, 2H, H triazole), 8.21 (s, 2H, H triazole).

[0239] .sup.13C NMR (75 MHz, in MeOD @ 323K) δ 9.75 (CH.sub.3 thymine), 24.49 (CH.sub.2), 27.76, 27.89, 27.97 (CH.sub.2 and CH.sub.2CH.sub.2O), 34.39 (C-2′), 49.87 (C-5′), 59.91 (C-6), 60.97 and 62.07 (CH.sub.2 triazole), 68.47, 75.95, 78.55 (C, 3, 4, 5), 69.20 (CH.sub.2CH.sub.2O), 71.49 (C-2), 77.42, 80.77 (C-3′, 4′), 84.60 (C-1′), 87.08 (C-1), 109.44 (C-5 thymine), 121.88, 123.43 (CH triazole), 135.32 (C-6 thymine), 142.91, 143.87 (C-4 triazole), 149.51, 163.46 (C═O thymine).

Example 5

1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5′-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazol-4-yl)methyl]-5′-deoxythymidine

[0240] ##STR00004##

[0241] To a degassed solution of 1,10-bis-2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexafluorodecanyl-5′-[4-(oxymethyl)-1H-1,2,3-triazol-1-yl]-N-3-propargyl-5′-deoxythymidine obtained from commercial 2,2,3,3,4,4,5,5,6,6,7,7,8,8,9,9-hexadecafluoro-1,10-decanediol subjected to a similar set of reactions as shown in FIG. 1, namely propargylation/azidothymidine coupling/nucleobase propargylation) (782.00 mg, 0.68 mmol) in 15 mL THF/H.sub.2O (2:1) was added 1-azido-1-deoxyglucopyranose, copper sulfate (26.90 mg, 0.14 mmol) and sodium ascorbate (43.40 mg, 0.27 mmol). The mixture was stirred at 60° C. overnight. After removal of the solvents in vacuo, the remaining crude material was washed with water and the filtrate was concentrated under reduced pressure. The oily residue was applied to a column of silica gel. The product was eluted with DCM/MeOH (8:2 then 7:3). Yield: 514.00 mg (48%).

[0242] .sup.1H NMR (300 MHz, in MeOD) d 1.91 (d, J=1.1 Hz, 6H, CH.sub.3 thymine), 2.28-2.32 (t, J=6.2 Hz, 4H, H-2′), 3.47-3.58 (m, 6H, H-2,3,5), 3.66-3.71 (dd, J=5.1, 12.1 Hz, 2H, H-6a), 3.83-3.90 (m, 4H, H-4, 6b), 4.09-4.18 (t, J=14.1 Hz, 4H, CH.sub.2CF.sub.2), 4.17-4.22 (m, 2H, H-4′), 4.40-4.45 (dd, J=5.2 Hz 2H, H-3′), 4.74-4.79 (m, 8H, H-5′ and OCH.sub.2 triazole), 5.20 (s, 4H, OCH.sub.2 triazole), 6.56 (d, J=9.2 Hz, 2H, H-1), 6.20 (dd, J=6.7 Hz, 2H, H-1′), 7.29 (d, 2H, H-6 thymine), 8.05 (s, 2H, H triazole), 8.10 (s, 2H, H-triazole).

[0243] .sup.13C NMR (75 MHz, in MeOD) d 10.14 (CH.sub.3 thymine), 34.15 (OCH.sub.2 triazole), 36.72 (C-2′), 49.76 (C-5′), 59.47 (C-6), 63.11 (OCH.sub.2 triazole), 64.97 (t, .sup.2J.sub.C-F=26.0 Hz, CH.sub.2CF.sub.2), 67.98, 75.54, 78.22 (C2, 3 or 5), 69.60 (C-3′), 71.05 (C-4), 82.80 (C-4′), 85.44 (C-1′), 86.72 (C-1), 108.26 (C-5 thymine), 121.36, 123.75 (CH triazole), 134.00 (C-6 thymine), 141.91 (C-4 triazole), 149.05 and 161.87 (C═O thymine).

Example 6

1,12-bis-dodecanyl-5′-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxythymidine (16)

[0244] To a solution of 15 obtained in Preparation 11 (1 g, 1.47 mmol) in 36 mL of tert-butanol/H.sub.2O (1:1) was added compound 12 obtained in Preparation 8 (243 mg, 0.67 mmol), copper sulfate (33 mg, 0.13 mmol) and sodium ascorbate (53 mg, 0.27 mmol). The mixture was stirred at 75° C. for 20 hours. The solvent was removed under reduced pressure and the residual solid was dissolved in DCM (150 mL) and then washed with water (3×50 mL). The organic extract was dried (Na.sub.2SO.sub.4) and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel eluting with ethyl acetate/MeOH (10:0 to 90:10) and obtained as a white solid. Yield: 0.90 g (78%).

[0245] .sup.1H NMR (300 MHz, in DMSO-d.sub.6) δ 1.23-1.32 (m, 20H, chain CH.sub.2), 1.76-2.00 (m, 15H), 2.17 (m, 2H), 2.95 (q, 4H), 4.08 (m, 6H), 4.20-4.36 (m, 8H), 4.57-4.73 (m, 4H), 4.97-5.11 (q, 4H), 5.16 (t, 2H), 5.54 (m, 2H), 5.66 (t, 2H), 6.21 (t, 2H), 6.31 (d, 2H), 7.50 (s, 2H), 7.85 (s, 2H), 8.26 (s, 2H).

[0246] .sup.13C NMR (75 MHz, in DMSO-d.sub.6) δ 13.12, 20.32-20.96, 35.41, 36.36, 38.54, 51.53, 62.27, 67.97, 70.38, 71.18, 72.68, 72.85, 73.73, 84.20, 84.74, 85.72, 109.18, 123.07, 135.55, 143.81, 150.61, 158.32, 162.69, 168.68, 169.82, 170.03, 170.51.

Example 7

1,12-bis-dodecanyl-5′-[(4-methylurea)-1H-1,2,3-triazol-1-yl]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine (17)

[0247] A solution of sodium methoxide (1M in methanol, 0.2 mL) was added dropwise to a solution of compound 16 prepared in example 6 (0.49 g, 0.28 mmol) in 20 mL of methanol anhydrous. After heating for 1 h minutes at 60° C., amberlite IRC-50 was added to convert from Na.sup.+ to H.sup.+ ions. After 20 minutes at the same temperature, the resin was removed by filtration and washed with methanol. The filtrate was concentrated and the product was purified by column chromatography on silica gel eluting with MeOH:CH.sub.2Cl.sub.2 (30:70 to 50:50) to afford as a white solid (Yield: 79%, 0.31 g).

[0248] .sup.1H NMR (300 MHz, in MeOD) δ 1.26-1.41 (m, 20H), 1.89 (s, 3H), 2.24-2.29 (m, 4H), 3.03-3.07 (t, 4H), 3.40-3.54 (m, 6H), 3.62-3.68 (dd, 2H), 3.81-3.86 (m, 4H), 4.06-4.16 (m, 2H), (4.12 (s, 4H), 4.32-4.39 (m, 2H), 4.60-4.74 (m, 4H), 4.02 (s, 6, 160.89, 164.79.

Example 8

1,14-bis-tetradecanyl-5′-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine (19)

[0249] To a solution of compound 18 (0.24 g, 0.67 mmol) and compound 15 (1 g, 1.47 mmol, 2.2 eq.) in 36 mL of tert-Butanol/H2O (1:1) was added sodium ascorbate (53 mg, 0.27 mmol, 0.4 eq.) followed by copper sulfate pentahydrate (33 mg, 0.13 mmol, 0.2 eq.). The mixture was stirred at 75° C. for 24 hours.

[0250] After cooling to room temperature, the solvents were removed under reduced pressure. The resulting solid was dissolved in DCM and the solution was washed with water. The organic layer was dried over Na.sub.2SO.sub.4, filtered and concentrated. The product was purified by column chromatography on silica gel eluting with ethyl acetate/MeOH (100:0 to 80:20) to afford as a white solid (Yield: 76%, 0.87 g).

[0251] .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ 1.21 (s, 20H), 1.43-1.49 (m, 4H), 1.76-2.03 (m, 24H), 1.87 (s, 6H), 2.04-2.09 (t, J=7.5 Hz, 4H), 2.12-2.23 (m, 4H), 4.03-4.14 (m, 6H), 4.26-4.37 (m, 8H), 4.58-4.73 (m, 4H), 4.97-5.11 (m, 4H), 5.14-5.20 (t, J=9.8 Hz, 2H), 5.49-5.54 (t, J=9.6 Hz, 2H), 5.49-5.54 (d, 2H), 5.63-5.69 (t, J=9.4 Hz, 2H), 6.20-6.24 (t, J=6.6 Hz, 2H), 6.28-6.32 (d, J=9.2 Hz, 2H), 7.49 (s, 2H), 7.90 (s, 2H), 8.25 (s, 2H), 8.25-8.29 (m, 2H).

[0252] .sup.13C NMR (75 MHz, DMSO-d.sub.6) δ 12.68, 20.33, 20.70, 20.84, 20.97, 25.65, 29.16, 29.26, 29.41, 29.53, 34.07, 35.21, 35.92, 38.12, 51.11, 61.83, 67.52, 70.00, 70.72, 72.22, 73.29, 83.74, 84.27, 85.24, 109.05, 122.62, 123.48, 135.11, 143.33, 145.14, 150.17, 162.23, 168.42, 169.36, 169.57, 170.04, 172.10.

Example 9

1,14-bis-tetradecanyl-5′-[(4-methylamide)-1H-1,2,3-triazol-1-yl]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine (20)

[0253] A solution of sodium methoxide (1M in methanol, 0.2 mL) was added dropwise to a solution of compound 19 prepared in example 8 (0.64 g, 0.37 mmol) in 20 mL of methanol anhydrous. After heating for 30 minutes at 45° C., Amberlite IRC-50 was added to convert from Na.sup.+ to H.sup.+ ions. After 20 minutes at the same temperature, the resin was removed by filtration and washed with methanol. The filtrate was concentrated and the product was purified by column chromatography on silica gel eluting with MeOH:CH.sub.2Cl.sub.2 (30:70 to 50:50) to afford as a white solid (Yield: 84%, 0.46 g).

[0254] .sup.1H NMR (300 MHz, MeOD) δ 1.22 (s, 20H), 1.48-1.57 (m, 4H), 1.87 (s, 6H), 2.11-2.16 (t, 4H), 2.22-2.27 (m, 4H), 3.41-3.51 (m, 6H), 3.64-3.70 (dd, 2H), 3.79-3.85 (m, 4H), 4.13-4.18 (m, 2H), 4.33-4.39 (m, 6H), 4.63-4.77 (m, 4H), 5.16 (s, 4H), 5.51-5.54 (d, 2H), 6.13-6.17 (t, 2H), 7.28 (s, 2H), 7.82 (s, 2H), 8.06 (s, 2H).

[0255] .sup.13C NMR (75 MHz, MeOD) δ 13.15, 26.89, 30.25, 30.38, 30.56, 30.66, 30.69, 35.57, 36.92, 37.06, 39.59, 52.73, 62.37, 70.87, 72.56, 73.94, 78.42, 81.13, 85.82, 88.31, 89.61, 111.15, 124.29, 125.34, 137.07, 144.58, 146.39, 151.95, 164.77, 176.27.

Example 10: Gelation Test

[0256] The aqueous dispersions (Milli-Q water, 18 MΩ.Math.cm.sup.−1) containing 1.5 wt % of compounds 5 and 6 obtained in examples 1 and 2 were heated until dissolution (at 45° C.) and gradually allowed to cool to room temperature unless otherwise stated. The resulting solid aggregate mass was stable to inversion of the container when the test tube was turned upside down, which shows that the compounds 5 and 6 form gels.

Example 11: Rheology

[0257] Rheological measurements were carried out on a Malvern Kinexus Pro+ rheometer with steel plate-plate geometry (20 mm diameter). The lower plate is equipped with a Peltier temperature control system, and all samples were studied at 25±0.01° C. unless indicated otherwise. A solvent trap was used to ensure homogeneous temperature and to prevent water evaporation. A gap distance of 0.3 mm was maintained between the plates. The gels based on compounds 5 (example 1) and 6 (example 2) (obtained in example were heated at 55° C. and the liquid resulting was placed into the rheometer and subjected to sinusoidal oscillations. All the measurements were carried out within the linear viscoelastic regime (LVR). For this purpose the experimental conditions to achieve a linear viscoelastic regime were determined by performing an amplitude strain sweep from 0.01 to 10% at an angular frequency of 1 Hz (6.283 rad.s.sup.−1).

[0258] FIG. 4 shows the frequency sweep results for hydrogels obtained from compound 5 and 6 (at 23.10 mM) at a constant strain of 0.03%.

[0259] FIG. 5 shows the step-strain measurement of the hydrogel obtained from compound 6 at 1% (w/v) at a fixed angular frequency of 6.283 rad.s.sup.−1. This experiment was repeated at least three times to verify its reproducibility.

[0260] These results show the thixotropic behaviour of the hydrogel according to the invention. The mechanical energy which is provided destabilizes the gel, which is able to auto-regenerate when the mechanical stress stops, without causing aging of the sample (several stress/restauration of G′ and G″ modulus were observed).

[0261] Sol-Gel Transition Temperature (T.sub.gel)

[0262] The determination of gel-sol transition temperature can also be determinated by rheology. The hydrogel (3% product 6 of example 2, w/v) was heated progressively from 25 to 60° C. (3° C./min). The oscillatory stress applied was set to 5 Pa at a constant frequency (1 Hz). The sol-gel transition point was taken as the temperature at which the gel became liquid. This melting temperature is reached at the intersection of the viscoelastic moduli (G′ and G″). This value is of 46° C. which is compatible with the cell culture at the gel state (37° C.).

Example 12: Cytotoxicity and Cytocompatibility

[0263] The experiments were performed using compound 6 of example 2, namely the 1,12-bis-dodecanyl-5′-[(4-oxymethyl)-1H-1,2,3-triazole-1-yl)]-N-3-[1-((β-D-glucopyranoside)-1H-1,2,3-triazole-4-yl)methyl]-5′-deoxy thymidine.

[0264] a) Cell Culture—Isolation and Culture of hASCs

[0265] Mesenchymal stem cells were isolated from human adipose tissue. Human subcutaneous fat was obtained from healthy patients aged 20 to 80 years old who underwent hip surgery in Bordeaux Pellegrin CHU (Bordeaux, France). Fat mass was separated from other tissues, washed with sterilized PBS, finely cut and incubated with 0.1% (w/v) collagenase type I (Worthington, Lakewood, N.J., USA) at 37° C. with gentle agitation for 1 h30. After filtration and centrifugation, the top liquid layer was removed and the remaining Stromal Vascular Fraction (SVF) was treated for 10 min with ELB (Erythocyte Lysis Buffer; 155 mM NH.sub.4Cl (Sigma-Aldrich, St. Louis, Mo., USA), 5.7 mM K.sub.2HPO.sub.4, 7.4 mM K.sub.2HPO.sub.4-3H.sub.2O, 0.1 mm EDTA (all Sigma-Aldrich)), and then centrifuged. The pellet was resuspended in DMEM F12 medium (Invitrogen/Life Technologies, Sergy Pontoise, France) supplemented with 10% (v/v) Foetal Bovine Serum (FBS) (Lonza, Basel, Switzerland) and sequentially filtered through 100, 70 and 40 μm cell strainer (BD Falcon, Franklin Lakes, N.J., USA). Cells were plated and cultivated at 37° C. in 5% CO.sub.2. Culture medium was replaced every two days.

[0266] b) Cell Culture—D1-ORL-UVA

[0267] D1-ORL-UVA (ATCC® CRL-12424) were cultured in DMEM (Invitrogen/Life Technologies) containing 10% FBS, and 1% Penicillin-Streptomycin (Invitrogen/Life Technologies). Culture medium was replaced every two days.

[0268] c) Preparation of 3D Scaffold with hASCs or D1 Seeding

[0269] For all in vitro assays, the hydrogel obtained from compound 6 was prepared at a concentration of 3% (w/v). The compound 6 powder was solubilized in PBS 1× under agitation (900 rpm) at 25° C. during 45 minutes. Then, when the gel was formed, it was left at room temperature, without agitation, during 3 to 4 hours. To allow cell seeding, the gel was heated during 30 minutes at 55° C. Once the liquid solution was obtained, it was rapidly cooled down to 37° C. and thoroughly mixed with 10 μL cell suspension at a concentration of 1 million cells per mL of gel. Finally, the cellularized hydrogel was maintained under agitation (600 rpm), at 25° C. during 45 minutes. After this period, medium was been added (250 μL/tube) and tubes were incubated at controlled atmosphere (5% CO.sub.2, 37° C.)

[0270] d) Evaluation of Compound 6 Cytotoxicity and of BOLA Gel Cytocompatibility

[0271] The cytotoxicity of compound 6 was evaluated by the measurement of the cell metabolic activity (MTT assay). This tests uses tetrazolium bromide MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) which is reduced into formazan by the mitochondrial enzyme succinate dehydrogenase of living cells. This reduction results in a change of colour from yellow to violet-blue. The colour intensity is proportional to the amount of living cells.

[0272] Cells (hASCs or D1) were seeded in a 96 wells plate (Nunc) at a density of 10 000 cells/cm.sup.2. Product 6 was solubilized at concentrations from 5 μM to 5 mM in culture media.

[0273] MTT assay was performed after 72 h of incubation. As a positive control, the culture medium without GNBA was used. As a negative control, Triton X100 was added in the culture medium. Results were expressed as a percentage of the metabolic activity of the positive control. Absorbance was measured at a wavelength of 570 nm with background subtraction at 630 nm.

[0274] The cytotoxicity of compound 6 was determined by MTT test to measure the metabolic activity of human mesenchymal stem cells (ASCs) 48 hours after incubation with increasing concentrations of compound 6. Results are shown as percentage of positive control consisting in BOLA-free cell culture medium. **: p<0.01 (Mann-Whitney U-Test).

[0275] The results, represented on FIG. 6, show that compound 6 is non toxic at concentrations up to approximately 1 mM.

[0276] Cell viability was also evaluated by using the Alamar Blue metabolic activity assay. Resazurin sodium salt solution (Sigma-Aldrich), suspended at a concentration of 0.1 mg/ml in PBS 1×), was diluted at 1/10 (v/v) in the culture media. The solution was added to each well (250 μL) and incubated during 3 h at 37° C. After this time, the fluorescence was measured at wavelengths excitation 530 nm and emission 590 nm. Triplicate samples were analyzed for each experiment. The results are shown on FIG. 7A.

[0277] Cytocompatibility was determined by monitoring alamar blue metabolism in rat osteoblastic cells (A) or human ASCs (B) grown in compound 6-based gels. Results are shown as percentage of the value obtained at day 1. *: p<0.05; **: p<0.01 (Mann-Whitney U-Test).

[0278] The results are shown on FIG. 7B.

[0279] Viability was assessed after 1, 4 and 7 days by using Live/Dead staining with calcein-AM and ethidium homodimer (Molecular Probes, Invitrogen, USA). Cells seeded within gels were incubated for 1 h at 37° C. in Hank's medium supplemented with 1.25 μL calcein-AM and 5 μL EthD-1. Samples were observed with a Confocal microscope (Leica TCS SPE).

[0280] Live/dead staining at 2 weeks showed a vast majority of green (living) cells.

[0281] The results show that compound 6 is not cytotoxic and has a god cytocompatibility when used as cell culture medium.

Example 13 (Comparative Example) Cytotoxycity of Compound GNL of G. Godeau et al., Chem. Comm., 2009, 34, 5127-5130

[0282] The toxicity of the compound GNL disclosed in G. Godeau et al., on page 5127, right column, was evaluated in human hepatic carcinoma cells HuH-7 by MTT assay, in order to measure cellular mortality.

[0283] Huh-7 cells were grown in DMEM medium supplemented with 10% foetal calf serum, 2 mM L-glutamine and 1% non-essential amino acids, at 37° C. in a 5% CO2 atmosphere. All culture reagents were purchased from Invitrogen. 2000 Huh-7 cells per well were seeded into a 96-well plate and incubated the following day with increasing concentrations of compound GNL in complete growth medium. After 5 days in the presence of the compounds, the living cells were quantified by the colorimetric CellTiter Aqueous One Solution Cell Proliferation Assay (Promega), as recommended.

[0284] The viability results are shown on FIG. 8.

[0285] The results show that the GNL compound does not have a significant toxicity for concentrations which are equal to or lower than 100 μM. For higher concentration, toxicity quickly becomes important and 100% of the cells die at a concentration of 200 μM.