Lipopolythioureas: A New Non-Cationic System for Gene Transfer

Inserm, U640, Paris, F-75006, CNRS, UMR8151, Paris, F-75006, Ecole Nationale Supérieure de Chimie de Paris, Paris, F-75005, Unité de Pharmacologie ...
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Bioconjugate Chem. 2007, 18, 484−493

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Lipopolythioureas: A New Non-Cationic System for Gene Transfer Jeanne Leblond, Nathalie Mignet, Ce´line Largeau, Maria-Vittoria Spanedda, Johanne Seguin, Daniel Scherman, and Jean Herscovici* Inserm, U640, Paris, F-75006, CNRS, UMR8151, Paris, F-75006, Ecole Nationale Supe´rieure de Chimie de Paris, Paris, F-75005, Unite´ de Pharmacologie Chimique et Ge´ne´tique, France, Universite´ Rene´ Descartes, Faculte´ de Pharmacie, Paris, F-75270 France, and Chimie Mole´culaire de Paris Centre, CNRS, FR 2769, Paris, F-75005. Received May 30, 2006; Revised Manuscript Received November 28, 2006

A DNA-transfection protocol has been developed that makes use of thiourea non-cationic synthetic lipid, N-[1,3bis(carbamothioylamino)propan-2-yl]-2-(dialkycarbamoylmethoxy)acetamide. It was found that these new compounds could be formulated without helper lipid and that the N-decanoyl and N-lauryl derivatives transfected B16 cells in the presence of serum with an efficiency at the same level as cationic lipids, under identical conditions. In vivo transfection using intratumoral injection was also investigated. It was found that compounds 18c and 19 showed an efficiency of the same magnitude as naked DNA and cationic lipid.

INTRODUCTION Plasmid-based gene medicine (1) requires a safe and efficient delivery system to transport DNA to the cell nucleus in order to cure innate or acquired disease. Naked plasmid DNA (2) could be directly administrated to the muscle (3), liver (4), or tumor (5), but in most cases, DNA is delivered by systemic injection and, for increased efficiency, should be protected from degradation in the bloodstream. The use of cationic lipid or polymer is one of the main strategies to deliver therapeutic genes or oligonucleotides to the nucleus (6-8). As compared to viral vectors (9), these gene delivery technologies suffer from a lack of efficiency but offer more flexibility and safety. This lack of efficiency is particularly true for in vivo transfection, due to interaction of cationic complexes with blood proteins (10, 11). Several strategies have been proposed to solve this drawback, like liposome postgrafting (12), polymer shielding (13, 14), and anionic liposomal delivery system (15). Nonelectrostatic systems have also been worked out that might bind DNA by base stacking (16, 17), nucleoside interaction (18), or trapping into a saccharidic cluster (19). But in these last cases, DNA transfection was seldom observed in vitro except for the glycocluster (19), unless the system bore a cationic charge (20). We have previously reported alternative lipids that interact with DNA in a nonelectrostatic fashion (21). These lipids are composed of di- or trithiourea functions that bind DNA. Unfortunately, these complexes did not transfect cells efficiently probably because of their high hydrophobic nature and their poor interaction between uncharged lipopolythiourea (LPT) liposomes and the cell membrane. Targeting LPT-DNA particles to membrane receptors seemed to represent the most suitable way to use these new DNA complexing particles for gene transfer. We recently obtained lipopolythiourea liposomes labeled with a fluorescent agent and formulated with a PEGRGD lipid (22). These targeting thiourea lipoplexes were efficiently internalized by the cells displaying Rvβ3 integrin receptor, as compared to the nontargeting complexes. However, no transfection was achieved by such lipoplexes. This previous study showed that modifications of the lipopolythiourea head from three linear thiourea functions to two branched thiourea functions still allowed efficient DNA compaction. Moreover, * E-mail: [email protected].

we observed that such structural element modification improved the formulation and decreased the hydrophobic properties without destabilizing the lipoplexes. We therefore concentrated our efforts on decreasing the hydrophobicity of LPT lipids. Log P calculation clearly showed that hydrophilicity could be greatly improved by decreasing the length of the hydrophobic anchor. Here, we describe a general synthesis of readily available dithioureas. These derivatives interact with DNA without any helper lipid and exhibit cell transfection properties. These novel delivery agents represent a new example of nonelectrostatic nanosystem with transfecting properties.

EXPERIMENTAL PROCEDURES Materials and Methods. All solvents were purchased from Carlo Erba-SDS (Peypin France). Dichloromethane was distilled from P2O5. DMF was dried over 3 Å molecular sieves and pyridine over KOH. All chemicals were purchased from SigmaAldrich-Fluka or Lancaster. Tosyl chloride was recrystallized in petroleum ether, and diglycolic anhydride was dehydrated (P2O5) prior to use. Other solvents and products were used without further purification. Reactions were monitored by thinlayer chromatography using Merck precoated 60 F254 silica gel plates. Column chromatography was performed over SDS (Peypin France) 35-70 nm silicagel according to the method of Still, Khan, and Mitra (23) or using small column flash chromatography (SFC) according to the following procedure: a plastic syringe was filled with silica gel (product/silica gel 1/5) and connected to a vacuum pump. The column was equilibrated with heptane, and then the sample dissolved in a minimum of dichloromethane was added to the top. The column was eluted by ten fractions of a heptane/ethyl acetate mixture. The volume was equal to the silica gel volume. For each fraction, the amount of ethyl acetate was increased (10% to 100% (v/v) heptane in ethyl acetate). Inverse-phase SPE was performed on Supelco Superclean C8 cartridge using an H2O/ CH3CN/CH3OH mixture. 1H and 13C NMR spectra were recorded on a Bruker Advance DRX-300 spectrometer at 300.13 MHz for 1H and 75.47 MHz for 13C. NMR spectra were processed using Xwinnmr (Bruker) or SwaN-MR (24). MS were carried out on a Shimazu 2010A LC-MS on ESI mode. Highresolution MS and elemental analysis were carried out by the “Service Central de Micro-analyse du CNRS” Vernaison France.

10.1021/bc060141b CCC: $37.00 © 2007 American Chemical Society Published on Web 01/20/2007

Lipopolythioureas: New Non-Cationic Vector for Gene Transfer

Dipalmytoylphosphatidylcholine (DPPC) and phosphatidylethanolamine-lissamine rhodamine were purchased from Aventi Polar Lipids (Alabaster Alabama). Log P calculations were performed using MarVinSketch (Chemaxon, Ltd., Budapest, Hungary) (25). Synthetic Procedures. tert-Butyl 1,3-Dihydroxypropan-2ylaminoformate (2). A solution of 2-aminopropane-1,3-diol (1) (5 g, 55 mmol) in ethanol (200 mL) was added to a solution di-tert-butylcarbonate (12 g, 55 mmol) in ethanol (200 mL). The reaction mixture was stirred for 5 h, then evaporated to dryness to yield a white solid, which was recrystallized in heptane to yield 10 g of 2 (93%). Mp 82 °C. 1H NMR (CDCl3) δ (ppm): 1.45 (s, 9H, CH3), 3.24 (m, 2H, OH), 3.67-3.83 (m, 5H, CH, CH2OH), 5.34 (d, 1H, J ) 6.6 Hz, NH). 13C NMR (CDCl3) δ (ppm): 28.44 (s, CH3), 53.31 (s, CH), 63.45 (s, CH2OH), 80.07 (s, C(CH3)3), 156.14 (s, CdO). Anal. Calcd for C8 H17 NO4 C, 49.96; H, 8.94; N, 7.32. Found C, 50.25; H, 8.96; N, 7.32. tert-Butyl 1,3-Bis(methoxysulfonyloxy)propan-2-ylaminoformate (3). Methanesulfonyl chloride (8.3 mL, 100 mmol) and triethylamine (18.6 mL, 133 mmol) were added to a solution of diol 2 (8.5 g, 44 mmol) in dichloromethane (100 mL). The reaction mixture was stirred for 3 h; then, the solution was diluted (dichloromethane, 500 mL), washed (water, 2 × 150 mL), then dried (MgSO4). After filtration and evaporation of the solvent, 15.5 g of 3 was recovered as a solid (100%). Mp 85 °C. 1H NMR (CDCl3) δ (ppm): 1.43 (s, 9H, CH3), 3.06 (s, 6H, CH3), 4.23-4.37 (m, 5H, CH, CH2OH), 5.07 (d, 1H, J ) 7.6 Hz, NH). 13C NMR (CDCl3) δ (ppm): 28.32 (s, CH3), 37.54 (s, CH3), 48.69 (s, CH), 67.02 (s, CH2OH), 80.05 (s, C(CH3)3), 154.97 (s, CdO). MS (ESI, m/z) 370 (M + Na)+, 391 (M + HCOO)-. Anal. Calcd C, 34.57; H, 6.09; N, 4.03; S, 18.46. Found C, 32.86; H, 5.98; N, 4.32; S, 19.09. tert-Butyl 1,3-Diazidopropan-2-ylaminoformate (4). A solution of dimesyl 3 (1.8 g, 5.1 mmol), sodium azide (2.0 g, 30.6 mmol), and 18-crown-6 (0.14 g, 0.5 mmol) in DMF (25 mL) was refluxed for 20 h. The mixture was coevaporated with toluene and water, then diluted in ethyl acetate, washed three times with brine, dried (MgSO4), and evaporated to yield 4 as a yellow oil that was further used without any purification (1.24 g, 100%). 1H NMR (CDCl3) δ (ppm): 1.43 (s, 9H, CH3), 3.40 (dd, J ) 7.8 Hz, J ) 16.6 Hz, CH2), 3.51 (dd, J ) 6.8 Hz, J ) 16.6 Hz, CH2), 3.85 (m, 1H, CH), 4.91 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 28.36 (s, CH3), 49.61 (s, CH2), 51.84 (s, CH), 80.05 (s, C(CH3)3), 155.19 (s, CdO). 1,3-Diazidopropan-2-amine (5). A TFA/water mixture (3:1, 80 mL) was added to a solution of diazide 4 (4.7 g, 20 mmol) in dichloromethane (20 mL). The reaction mixture was stirred for 4 h, then poured on solid NaHCO3 and filtered. The aqueous phase was extracted by dichloromethane, adjusted to pH 10 (NaOH 2 N), and then extracted again with dichloromethane. The mixed organic layers were dried (MgSO4), filtered, and then evaporated to dryness to give 4 (2.7 g, 96%) that was used without any purification. 1H NMR (CDCl3) δ (ppm): 1.45 (s, 2H, NH), 3.02 (p, 1H, J ) 5.8 Hz, CH), 3.32 (dd, 2H, J ) 5.8 Hz, J ) 12.0 Hz, CH2), 3.40 (dd, 2H, J ) 5.8 Hz, J ) 12.0 Hz, CH2). 13 C NMR (CDCl3) δ (ppm): 50.69 (s, CH), 55.08 (s, CH2). MS (EI DCI) 142 (M + H)+. N′,N′-Ditetradecylsuccinamide (7). Succinic anhydride (10.7 g, 107 mmol) and 4-dimethylaminopyridine (0.48 g, 3.9 mmol) were added to a solution of ditetradecylamine (6a) (8g, 21.5 mmol) in a mixture of dry pyridine (32 mL) and dichloromethane (80 mL). The reaction mixture was stirred at RT for 18 h before addition of 1 N hydrochloride (20 mL). The organic layer was extracted, washed with brine, dried (MgSO4), and evaporated to afford 7 as an oil (9.8 g, 98%). 1H NMR (CDCl3) δ (ppm): 0.85 (t, 6H, J ) 6.3 Hz, CH3), 1.23 (m, 44H, -CH2-

Bioconjugate Chem., Vol. 18, No. 2, 2007 485

), 1.48 (m, 2H, -CH2-), 2.64 (s, 4H, CH2CO), 3.22 (m, 4H, CH2N). 13C NMR (CDCl3) δ (ppm): 14.08 (s, CH3), 22.69 (s, -CH2-), 27.74 (s, -CH2-), 28.10 (s, CH2CO), 28.92 (s, -CH2-), 29.67 (m, -CH2-), 30.07 (s, CH2CO), 31.96 (s, -CH2-), 46.21 and 47.98 (2s, -CH2N), 171.46 (s, CdO), 176.34 (s, CdO). MS (ESI, m/z) 511 (M + H)+. N-(2-Azido-1-azidomethylethyl)-N′,N′-ditetradecylsuccinamide (8). Diazide 5 (0.074 g, 0.53 mmol) was added to solution of 7 (0.26 g, 0.5 mmol) in dichloromethane (3 mL). The reaction mixture was stirred for 1 h before the addition of a solution of DCC (0.16 g, 0.75 mmol) in dichloromethane (1 mL). After overnight stirring, diethyl ether (5 mL) was added, and the resulting precipitate was filtered through a celite pad. The solvent was evaporated to yield a crude oil that was dissolved in THF (10 mL/mmol). Amberlyst-21 (0.27 g, 4 equiv) was added to remove the acid excess. The resin was removed by filtration, and then the filtrate was evaporated to yield 8 as a yellow oil (0.31 g, 93%). 1H NMR (CDCl3) δ (ppm): 0.87 (t, 6H, J ) 6.6 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.47 (m, 4H, -CH2-), 2.60 (dd, 4H, J ) 22 Hz, J ) 8 Hz, CH2CO), 3.23 (dt, 4H, J ) 22 Hz, J ) 8 Hz, CH2N), 3.43 (dd, 4H, J ) 6.0 Hz, J ) 14.4 Hz, CH2N3), 4.15 (m, 1H, CH), 7.23 (d, 1H, J ) 8 Hz, NH). 13C NMR (CDCl3) δ (ppm): 13.95 (s, CH3), 22.66 (s, -CH2-), 27.12 (s, -CH2-), 27.84 (s, CH2CO), 28.97 (s, -CH2-), 29.66 (m, -CH2-), 31.90 (m, -CH2-, CH2CO), 46.42 and 48.09 (2s, -CH2N), 48.55 (s, CH), 51.62 (s, CH2N3), 171.39 (s, CdO), 172.87 (s, CdO). MS (ESI, m/z) 634 (M + H)+. N-(2-Amino-1-aminomethylethyl)-N′,N′-ditetradecylsuccinamide (9). A round-bottom flask equipped with a stirring bar was charged with 8 (0.30 g, 0.48 mmol), ethanol (10 mL), Pd/C (0.06 g), and hydrogen. The suspension was stirred for 19 h at RT; then, the mixture was filtered through a celite pad to yield after evaporation 0.27 g of the diamine 9 (98%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.80 (t, 6H, J ) 8.4 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.49 (m, 4H, -CH2-), 2.43 (m, 2H, CH2CO), 2.63 (m, 2H, CH2CO), 2.75 (m, 4H, CH2NH2), 2.86 (m, 4H, NH2), 3.25 (m, 4H, CH2N), 3.82 (m, 1H, CH), 7.12 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.14 (s, CH3), 22.73 (s, -CH2-), 27.12 (s, -CH2-), 27.82 (s, -CH2), 29.40 (s, CH2CO), 29.66 (m, -CH2-), 31.97 (m, -CH2-, CH2CO), 43.56 (s, CH2NH2), 46.36 and 48.10 (2s, -CH2N), 53.27 (s, CH), 171.39 (s, CdO), 172.87 (s, CdO). MS (ESI, m/z) 582 (M + H)+, 626 (M + HCOO)-. N′-[1,3-Bis(methylthiocarbamoylamino)propan-2-yl]-N,Nditetradecylbutanediamide (10). Methyl isothiocyanate (11 mg, 0.15 mmol) was added to solution of diamine 9 (58 mg, 0.1 mmol) and triethylamine (14 µL, 0.1 mmol) in dichloromethane (1 mL). The solution was stirred overnight. The solvent was evaporated, and the crude was purified by SFC to yield 11 as an oil (0.046 g, 63%). 1H NMR (CDCl3) δ (ppm): 0.87 (t, 6H, J ) 8.4 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.46 (m, 4H, -CH2-), 2.47 (m, 2H, CH2CO), 2.66 (d, 2H, CH2CO), 3.01 (s, 6H, CH3NCS), 3.22 (m, 4H, CH2N), 3.75-3.90 (m, 5H, CH, CH2NCS), 7.59 (m, 5H, NH). 13C NMR (CDCl3) δ (ppm): 14.20 (s, CH3), 22.79 (s, -CH2-), 27.17 (s, -CH2-), 28.01 (s, -CH2-), 29.02 (s, CH2CO), 29.66 (m, -CH2-), 31.21 (s, CH3 NCS), 31.78 (s, CH2CO), 32.06 (s, -CH2-), 44.75 (s, CH2NCS), 46.83 and 48.51 (s, -CH2N), 51.99 (s, CH), 171.88 (s, CdO), 173.67 (s, CdO), 183.61 (s, CdS). HR-ESMS calcd for C39H78N6O2NaS2: 749.5525. Found 749.5487. N-(2-Isothiocyanato-1-isothiocyanatomethylethyl)-N′,N′-ditetradecylsuccinamide (11). Diisopropylethylamine (0.25 mL, 1.4 mmol) and carbon disulfide (0.11 mL, 1.9 mmol) were successively added to a solution of diamine 9 (0.28 g, 0.5 mmol) in dichloromethane (5 mL). The solution was stirred for 3 h, and then a solution of tosyl chloride (0.18 g, 0.9 mmol) in

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dichloromethane was added dropwise. The mixture was stirred for 20 h at RT, diluted with dichloromethane, and washed three times with brine. The organic solution was dried (MgSO4) and evaporated under vacuum. The crude was purified by SFC to yield 11 as a colorless oil (0.07 g, 22%). 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.6 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.51 (m, 4H, -CH2-), 2.56 (m, 2H, CH2CO), 2.69 (m, 2H, CH2CO), 3.25 (m, 4H, -CH2N), 3.76 (dd, 4H, J ) 6.0 Hz, J ) 11.1 Hz, CH2NCS), 4.28 (m, 1H, CH), 7.51 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.18 (s, CH3), 22.77 (s, -CH2-), 27.19 (s, -CH2-), 27.87 (s, -CH2-), 29.03 (s, CH2CO), 29.74 (m, -CH2-), 32.01 (m, -CH2-, CH2CO), 45.98 and 46.69 (2s, -CH2N), 48.10 (s, CH), 49.04 (s, CH2NCS), 171.39 (s, CdO), 172.87 (s, CdO). IR (NaCl) σ (cm-1): 1629 (νN-CdO), 1675 (νNH-CdO), 2089 (νNdCdS), 2852 (ν)C-H2), 2929 (νCH2). Anal. Calcd for C37H68N4O2S2: C, 66.82; H, 10.31; N, 8.42. Found C, 66.94; H, 10.70; N, 7.63. N′-[1,3-Bis(2,3-dihydroxypropylthiocarbamoylamino)propan2-yl]-N,N-ditetradecylbutanediamide (12). 3-Amino-1,2-propanediol (40 mg, 0.44 mmol) was added to a solution of 11 (0.073 g, 0.11 mmol) in dry DMF (1.5 mL). The solution was stirred at RT for 2 h, then heated overnight (50 °C). The solvent was evaporated, and then the resulting oil was dissolved in dichloromethane (4 mL) and washed twice with brine (1 mL). The organic layer was dried (MgSO4), filtrated, and evaporated. The crude was first purified by SPE (C8, 100% H2 O to 100% CH3CN then to 100% CH3OH), then by SFC to yield 12 as a colorless oil (23 mg, 25%).1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.6 Hz, CH3), 1.26 (m, 44H, -CH2-), 1.55 (m, 4H, -CH2-), 2.52 (t, 2H, J ) 6.0 Hz, CH2CO), 2.68 (t, 2H, J ) 6.0 Hz, -CH2CO), 3.11 (t, 2H, J ) 7.5 Hz, -CH2N), 3.18 (t, 2H, J ) 7.5 Hz, -CH2N), 3.21 (m, 2H, CH2NCS), 3.39 (m, 4H, CH2NCS), 3.53 (m, 6H, CH2NCS, CH2OH), 3.77 (t, 2H, J ) 4.5 Hz, CHOH), 3.87 (m, 1H, CH), 6.97 (t, 1H, J ) 6.0 Hz, NH). 13 C NMR (CDCl3) δ (ppm) 14.05 (s, CH3), 22.70 (s, -CH2-), 27.02 (s, -CH2-), 27.81 (s, -CH2-), 28.93 (s, -CH2CO), 29.66 (m, -CH2-), 31.68 (s, CH2CO), 31.91 (s, -CH2-), 44.46 (s, CH2NCS), 46.18 (s, CH2NCS), 46.58 and 48.29 (2s, CH2N), 53.12 (s, CH), 63.44 (s, CH2OH), 70.93 (s, CHOH), 171.66 (s, CdO), 174.76 (s, CdO). Ditetradecylcarbamoylmethoxyacetic Acid (13a). Glycolic anhydride (6.3 g, 53.8 mmol) was added to a solution of ditetradecylamine 6a (8.8 g, 21.5 mmol) and DMAP (1.3 g, 10.7 mmol) in dichloromethane (200 mL). The solution was stirred for 18 h at RT; then, the reaction mixture was diluted with dichloromethane and washed successively with 1 N HCl (3 times) and brine (3 times), then dried (MgSO4). Acid 13a was isolated as a white solid after removal of the solvent (11.3 g, 100%). Mp 62 °C. 1H NMR (CDCl3) δ (ppm): 0.87 (t, 6H, J ) 6.9 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.54 (m, 2H, -CH2-), 3.08 (t, 2H, J ) 7.5 Hz, -CH2N), 3.34 (t, 2H, J ) 7.5 Hz, -CH2N), 4.21 (s, 2H, CH2CO), 4.38 (s, 2H, CH2CO). 13C NMR (CDCl ) δ (ppm): 14.13 (s, CH ), 22.72 (s, 3 3 -CH2-), 27.01 (s, -CH2-), 28.71 (s, -CH2-), 29.68 (m, -CH2-), 31.97 (s, -CH2-), 46.95 (s, -CH2N), 71.21 (s, CH2O), 72.87 (s, CH2O), 170.69 (s, CdO), 172.03 (s, CdO). MS (ESI, m/z) 527 (M + H)+, 95% purity by ELSD. Didodecylcarbamoylmethoxyacetic Acid (13b). Glycolic anhydride (1.6 g, 14.1 mmol) was added to a solution of bis-dodecylamine 6b (8.8 g, 21.5 mmol) and DMAP (0.17 g, 1.4 mmol) in dichloromethane (20 mL). The solution was stirred for 23 h and was treated as for 13a. SFC purification gave 13b as a white solid (1.1 g, 84%). Mp 55 °C. 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.6 Hz, CH3), 1.26 (m, 36H, -CH2-), 1.55 (t, 2H, J ) 6.9 Hz, -CH2-), 3.08 (t, 2H, J ) 7.5 Hz, -CH2N), 3.35 (t, 2H, J ) 7.5 Hz, -CH2N), 4.21 (s, 2H, CH2O), 4.38 (s, 2H, CH2O). 13C NMR (CDCl3) δ (ppm): 14.18 (s, CH3),

Leblond et al.

22.76 (s, -CH2-), 27.02 (s, -CH2-), 27.51 (s, -CH2-), 29.70 (m, -CH2-), 31.98 (s, -CH2-), 46.98 (s, -CH2N), 71.32 (s, CH2O), 73.15 (s, CH2O), 170.68 (s, CdO), 174.13 (s, CdO). MS (ESI, m/z) 470 (M + H)+, 100% purity by ELSD. Anal. Calcd for C28H56NO4: C, 71.59; H, 11.80; N, 2.98. Found C, 70.26; H, 11.65; N, 2.78. Didecylcarbamoylmethoxyacetic Acid (13c). Glycolic anhydride (4.7 g, 40 mmol) was added to a solution of didecylamine 6c (3.0 g, 10 mmol) and DMAP (0.6 g, 5 mmol) in dichloromethane (100 mL). The solution was stirred for 20 h at RT and was treated as for 13a to yield 13c as a white solid (4.14 g, 100%). Mp 55 °C. 1H NMR (CDCl3) δ (ppm): 0.85 (t, 6H, J ) 6.0 Hz, CH3), 1.25 (m, 28H, -CH2-), 1.53 (m, 4H, -CH2-), 3.08 (t, 2H, J ) 7.3 Hz, -CH2N), 3.33 (t, 2H, J ) 7.3 Hz, -CH2N), 4.19 (s, 2H, CH2O), 4.39 (s, 2H, CH2O). 13C NMR (CDCl3) δ (ppm): 14.08 (s, CH3), 22.69 (s, -CH2-), 26.85 (s, -CH2-), 27.45 (s, -CH2-), 28.67 (s, -CH2-), 29.30 (m, -CH2-), 29.51 (s, -CH2-), 31.89 (s, -CH2-), 46.93 (s, -CH2N), 71.20 (s, -CH2O), 72.78 (s, -CH2O), 170.7 (s, Cd O), 171.1 (s, CdO). MS (ESI, m/z) 414 (M + H)+. HPLC purity 100% by ELSD. Anal. Calcd for C24H47NO4: C, 69.69; H, 11.22; N, 3.30. Found C, 69.00; H, 11.35; N, 3.39. N,N-Bis(azidomethyl)-2-(ditetradecylcarbamoylmethoxy)acetamide (14a). A solution of DCC (0.16 g, 0.75 mmol) in dichloromethane (1 mL) was added to a solution of 13a (4.0 g, 7.6 mmol) and 5 (1.1 g, 8 mmol) in dichloromethane (50 mL). The reaction mixture was stirred overnight, and then diethyl ether (70 mL) was added. The resulting suspension was filtered through a celite pad; then, the solvent was evaporated under reduced pressure to yield a crude oil that was first purified by SFC and then dissolved in THF (20 mL/mmol). Amberlyst-21 (4 equiv) was added to remove the acid excess. The resin was removed by filtration, and then the solvent was evaporated under reduced pressure. The amide 14a was isolated as an oil (4.9 g, 98%). 1H NMR (CDCl3) δ (ppm): 0.87 (t, 6H, J ) 6.6 Hz, CH3), 1.25 (m, 44H, -CH2-), 1.52 (m, 4H, -CH2-), 3.06 (t, 2H, J ) 7.5 Hz, -CH2N), 3.31 (t, 2H, J ) 7.5 Hz, -CH2N), 3.47 (dd, 4H, J ) 6.0 Hz, J ) 12.3 Hz, CH2N3), 3.55 (dd, 4H, J ) 5.7 Hz, J ) 12.3 Hz, CH2N3), 4.10 (s, 2H, CH2O), 4.17 (m, 1H, CH), 4.27 (s, 2H, CH2O), 8.75 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.13 (s, CH3), 22.73 (s, -CH2-), 27.06 (s, -CH2-), 27.64 (s, -CH2-), 29.70 (m, -CH2-), 31.98 (s, -CH2-), 46.45 and 46.91 (2s, CH2N), 48.44 (s, CH), 51.54 (s, CH2N3), 70.33 (s, CH2O), 72.68 (s, CH2O), 168.70 (s, CdO), 170.30 (s, CdO). MS (ESI, m/z) 650 (M + H)+, 695 (M + HCOO)-. HPLC purity 94% by ELSD. Anal. Calcd for C35H68N8O3: C, 64.78; H, 10.56; N, 17.27. Found C, 64.84; H, 10.71; N, 16.91. N,N-Bis(azidomethyl)-2-(didodecylcarbamoylmethoxy)acetamide (14b). N-Hydroxysuccinimide (0.12 mg, 1.06 mmol) and DCC (0.55 g, 2.65 mmol) were added to a solution of acid 13b (0.50 g, 1.06 mmol) in dry THF (10 mL). The reaction mixture was stirred for 2 h at RT; then, a solution of 5 (0.13 mg, 0.89 mmol) in THF (2 mL) was added, and the stirring was followed during 19 h. The suspension was evaporated under reduced pressure, diluted with ethyl acetate, and washed successively with water and brine. The organic layer was dried (MgSO4) and evaporated. The crude was purified by SFC to afford 14b as a yellow oil (0.51 g, 81%). 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.6 Hz, CH3), 1.26 (m, 36H, -CH2-), 1.50 (t, 4H, J ) 6.9 Hz, -CH2-), 3.04 (t, 2H, J ) 7.5 Hz, -CH2N), 3.30 (t, 2H, J ) 7.5 Hz, -CH2N), 3.45 (dd, 4H, J ) 6.0 Hz, J ) 12.3 Hz, CH2N3), 3.53 (dd, 4H, J ) 6.0 Hz, J ) 12.3 Hz, CH2N3), 4.09 (s, 2H, CH2O), 4.20 (m, 1H, CH), 4.25 (s, 2H, CH2O), 8.70 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.12 (s, CH3), 22.72 (s, -CH2-), 27.04 (s, -CH2-), 27.62 (s, -CH2-), 29.70 (m, -CH2-), 31.95 (s, -CH2-),

Lipopolythioureas: New Non-Cationic Vector for Gene Transfer

46.42 (s, -CH2N), 46.89 (s, CH2N), 48.43 (s, CH), 51.51 (s, CH2N3), 70.30 (s, CH2O), 72.63 (s, CH2O), 168.68 (s, CdO), 170.28 (s, CdO). Anal. Calcd for C31H60N8O3: C, 62.80; H, 10.20. Found C, 63.37; H, 10.31. N,N-Bis(azidomethyl)-2-(didecylcarbamoylmethoxy)acetamide (14c). A solution of DCC (2.3 g, 11.1 mmol) in dichloromethane (25 mL) was added to solution of 13c (3.0 g, 7.4 mmol) and 5 (1.1 g, 7.7 mmol) in dichloromethane (20 mL). The reaction mixture was stirred overnight, and then diethyl ether (50 mL) was added. The resulting suspension was filtered through a celite pad, and then the solvent was evaporated under reduced pressure. The crude oil that was purified by SFC to yield 14c as an oil (2.3 g, 58%). 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.0 Hz, CH3), 1.25 (m, 28H, -CH2-), 1.51 (m, 4H, -CH2-), 3.05 (t, 2H, J ) 7.3 Hz, -CH2N), 3.31 (t, 2H, J ) 7.3 Hz, CH2N), 3.47 (dd, 2H, J ) 6.0 Hz, CH2N3), 3.55 (dd, 2H, J ) 6.0 Hz, CH2N3), 4.10 (s, 2H, CH2O), 4.22 (m, 1H, CH), 4.27 (s, 2H, CH2O), 8.77 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.12 (s, CH3), 22.71 (s, -CH2-), 26.94 (s, -CH2-), 27.63 (s, -CH2-), 28.94 (s, CH2NH2), 29.36 (m, -CH2-), 29.56 (s, -CH2-), 31.92 (s, -CH2-), 46.43 and 46.90 (2s, CH2N), 48.42 (s, CH), 51.52 (s, CH2N3), 70.31 (s, CH2O), 72.65 (s, CH2O), 168.65 (s, CdO), 170.25 (s, CdO). MS (ESI, m/z) 537 (M + H)+, 559 (M + Na)+. Anal. Calcd for C27H52N8O3: C, 60.42; H, 9.76; N, 20.73. Found C, 60.64; H, 9.88; N, 20.73. N,N-Ditetradecyl-2-(1,3-diaminopropan-2-ylcarbamoylmethoxy)acetamide (15a). A round-bottom flask was charged with 14a (0.5 g, 0.77 mmol), ethanol (10 mL), Pd/C (0.075 g), and hydrogen. The suspension was stirred overnight at RT, and then the mixture was filtered through celite to yield after evaporation the diamine 15a (0.45 g, 97%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.0 Hz, CH3), 1.26 (m, 44H, -CH2-), 1.53 (m, 4H, -CH2-), 2.55 (m, 4H, NH2), 2.712.87 (m, 4H, CH2NH2), 3.08 (t, 2H, J ) 7.5 Hz, -CH2N), 3.30 (t, 2H, J ) 7.5 Hz, -CH2N), 3.93 (m, 1H, CH), 4.09 (d, J ) 7.5 Hz, CH2O), 4.25 (d, J ) 7.5 Hz, CH2O), 8.21 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.09 (s, CH3), 22.69 (s, -CH2-), 27.02 (s, -CH2-), 27.62 (s, -CH2-), 29.66 (m, -CH2-), 31.94 (s, -CH2-), 43.69 (s, CH2NH2), 46.28 and 46.89 (2s, -CH2N), 53.25 (s, CH), 69.63 (s, CH2O), 72.10 (s, CH2O), 168.70 (s, CdO), 170.30 (s, CdO). Anal. Calcd for C35H72N4O3: C, 66.62; H, 11.82; N, 8.88. Found C, 67.02; H, 12.03; N, 7.85. N,N-Didodecyl-2-(1,3-diaminopropan-2-ylcarbamoylmethoxy)acetamide (15b). A round-bottom flask was charged with 14b (0.50 g, 0.77 mmol), ethanol (10 mL), Pd/C (0.075 g), and hydrogen. The suspension was stirred overnight RT, and then the mixture was filtered through celite to yield after evaporation the diamine 15b (0.45 g, 97%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.85 (t, 6H, J ) 6.6 Hz, CH3), 1.24 (m, 36H, -CH2-), 1.49 (m, 4H, -CH2-), 3.06 (m, 4H, CH2NH2), 3.26 (m, 4H, -CH2N), 3.94 (m, 4H, CH), 4.09 (s, 2H, CH2O), 4.26 (s, 2H, CH2O), 8.41 (d, 1H, J ) 8.4 Hz, NH). 13C NMR (CDCl3) δ (ppm): 14.11 (s, CH3), 22.71 (s, -CH2-), 26.99 (s, -CH2-), 27.70 (s, -CH2-) 29.40 (m, -CH2-), 31.96 (s, -CH2-), 42.42 (s, CH2NH2), 46.32 (s, -CH2N), 46.99 (s, -CH2N), 51.12 (s, CH), 69.64 (s, CH2O), 71.82 (s, CH2O), 168.53 (s, CdO), 170.01 (s, CdO). N,N-Didecyl-2-(1,3-diaminopropan-2-ylcarbamoylmethoxy)acetamide (15c). A round-bottom flask was charged with 14c (2.3 g, 4.3 mmol), ethanol (70 mL), Pd/C (0.3 g), and hydrogen. The suspension was stirred overnight RT, and then the mixture was filtered through celite to yield after evaporation the diamine 15c (1.5 g, 72%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.84 (t, 6H, J ) 6.0 Hz, CH3), 1.25 (m, 28H, -CH2-), 1.48 (m, 4H, -CH2-), 1.77 (m, 4H, NH2), 2.54 (dd, 1H, J ) 6.0

Bioconjugate Chem., Vol. 18, No. 2, 2007 487

Hz, J ) 12.0 Hz, CH2NH2), 2.71 (dd, 1H, J ) 5.8 Hz, J ) 12.6 Hz, CH2NH2), 3.05 (t, 2H, J ) 7.3 Hz, -CH2N), 3.26 (m, 3H, -CH2N, CH), 3.39 (s, 2H, CH2O), 4.05 (s, 2H, CH2O), 8.20 (m, 1H, NH). 13C NMR (CDCl3) δ (ppm): 14.11 (s, CH3), 22.69 (s, -CH2-), 26.94 (s, -CH2-), 27.66 (s, -CH2-), 28.98 (s, -CH2-), 29.35 (m, -CH2-), 29.55 (s, -CH2-), 31.90 (s, -CH2-), 43.46 (s, CH2NH2), 46.28 and 46.90 (2s, CH2N), 53.35 (s, CH), 69.78 (s, CH2O), 72.15 (s, CH2O), 168.65 (s, CdO), 170.25 (s, CdO). Anal. Calcd for C27H56N4O3: C, 69.37; H, 12.06; N, 8.67. Found C, 69.39; H, 11.63; N, 9.89. 4-Isothiocyanatomethyl-2,2-dimethyl-(1,3)-dioxolane (16). A solution of DCC (3.2 g, 15.5 mmol) and carbon disulfide (6.3 mL, 104 mmol) in THF (10 mL) was cooled to -5 °C by an ice/NH4Cl (4:1, v/v) bath. Then, 2,2-dimethyl-1,2-dioxolan-4methanamide (2.0 g, 15.5 mmol) diluted in dichloromethane (7 mL) was added dropwise. The reaction mixture was stirred for 4 h at RT. Then, the suspension was filtered, and the filtrate was concentrated and treated with diethylether. The suspension was again filtered, and the filtrate was concentrated. After purification by flash chromatography (heptane/ethyl acetate 8:2), 16 was obtained as a clear liquid (2.2 g, 85%). 1H NMR (CDCl3) δ (ppm): 1.31 (s, 3H, CH3), 1.42 (s, 3H, CH3), 3.54 (dd, 1H, J ) 5.1 Hz, J ) 15.0 Hz, CH2NCS), 3.64 (dd, 1H, J ) 5.1 Hz, J ) 15.0 Hz, CH2NCS), 3.80 (dd, J ) 5.1 Hz, J ) 8.7 Hz, CH2O), 4.07 (dd, J ) 6.3 Hz, J ) 8.7 Hz, CH2O), 4.26 (q, 1H, J ) 5.3 Hz, CHO). 13C NMR (CDCl3) δ (ppm): 25.24 (s, CH3), 26.85 (s, CH3), 47.47 (s, CH2NCS), 66.69 (s, CH2O), 73.76 (s, CHO), 110.49 (s, CO2(CH3)3), 133.12 (s, CdS). MS (ESI, m/z) 174 (M + H)+. Anal. Calcd for C7H11NO2S: C, 48.53; H, 6.40; N, 8.09; S, 18.51. Found C, 48.54; H, 6.52; N, 8.09; S, 18.51. 2-[1,3-Bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methylthiocarbamoylamino]propan-2-ylcarbamoyl-methoxy]-N,N-ditetradecylacetamide (17a). Isothiocyanate 16 (0.22 g, 1.25 mmol) was added to a solution of diamine 15a (0.24 g, 0.5 mmol) and N,Ndiisopropylethylamine (0.87 mL, 5 mmol) in dichloromethane (4 mL). The solution was stirred overnight at RT. The solvent was evaporated under reduced pressure, and then the crude was purified by SFC (standard condition then acetone) to yield 17a (0.37 g, 66%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.86 (t, 6H, J ) 6.6 Hz, CH3), 1.26 (m, 44H, -CH2-), 1.32 (s, 6H, CH3), 1.42 (s, 6H, CH3), 1.50 (m, 4H, -CH2-), 3.08 (t, 2H, J ) 7.2 Hz, CH2NCO), 3.20 (t, 2H, J ) 7.2 Hz, CH2N), 3.67-3.83 (m, 8H, CH2NCS, H-8), 4.02-4.10 (m, 6H, H-3, CH2O), 4.29 (m, 7H, CH2O, CH, CHO), 7.7 (m, 4H, NH). 13C NMR (CDCl3) δ (ppm): 13.98 (s, CH3), 22.59 (s, -CH2-), 25.27 and 26.85 (2s, CH3), 27.62 (s, -CH2-), 28.91 (s, -CH2-), 29.66 (m, -CH2-), 31.84 (s, -CH2-), 44.73 (m, CH2NCS), 46.57 and 46.59 (2s, CH2NCO), 54.29 (s, CH), 68.86 (s, CH2O), 69.81 (s, CH2O), 71.79 (s, CH2O), 74.30 (s, CHO), 109.47 (s, CO2(CH3)3), 168.33 (s, CdO), 171.78 (s, CdO), 183.18 (s, CdS). MS (ESI, m/z) 944 (M + H)+, 966 (M + Na)+. HPLC purity 96% by ELSD. Anal. Calcd for C49H94N6O7S2: C, 62.38; H, 10.04; N, 8.91; S, 6.80. Found C, 61.83; H, 10.21; N, 8.64; S, 5.94. 2-[1,3-Bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methylthiocarbamoylamino]propan-2-ylcarbamoylmethoxy]-N,N-didodecylacetamide (17b). Isothiocyanate 16 (0.087 g, 0.5 mmol) was added to solution of diamine 15b (0.11 g, 0.2 mmol) and N,Ndiisopropylethylamine (0.35 mL, 2 mmol) in dichloromethane (2 mL). The solution was stirred overnight at RT. The solvent was evaporated under reduced pressure, and then the crude was purified by SFC (standard condition then acetone) to yield 17b (0.11 g, 62%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.87 (t, 6H, J ) 6.0 Hz, CH3), 1.26 (m, 36H, -CH2-), 1.33 (s, 6H, CH3), 1.41 (s, 6H, CH3), 1.52 (m, 4H, -CH2-), 3.09 (t, 2H, J ) 7.0 Hz, -CH2NCO), 3.30 (t, 2H, J ) 7.0 Hz, -CH2NCO), 3.67-3.80 (m, 8H, CH2NCS), 4.08 (m, 6H, CH2O), 4.29

488 Bioconjugate Chem., Vol. 18, No. 2, 2007

(m, 7H, CH2O, CH, CHO), 7.68 (m, 4H, NH). 13C NMR (CDCl3) δ (ppm): 14.15 (s, CH3), 22.74 (s, -CH2-), 25.41 and 26.98 (2s, CH3), 27.77 (s, -CH2-), 29.06 (s, -CH2-), 29.68 (m, -CH2-), 31.98 (s, -CH2-), 40.05 (s, CH2NCS), 46.72 and 47.17 (2s, CH2NCO), 49.56 (s, CH), 66.97 (s, CH2O), 70.08 (s, CH2O), 72.06 (s, CH2O), 74.43 (s, CHO), 109.64 (s, CO2(CH3)3), 168.68 (s, CdO), 171.15 (s, CdO), 183.05 (s, Cd S). MS (EI, m/z) 887 (M + H)+, 886 (M - H)-, 934 (M + Cl)-. Anal. Calcd for C45H86N6O7S2: C, 60.91; H, 9.77; N, 9.47; S, 7.23. Found C, 61.05; H, 10.21; N, 9.81; S, 6.93. 2-[1,3-Bis[(2,2-dimethyl-1,3-dioxolan-4-yl)methylthiocarbamoylamino]propan-2-ylcarbamoylmethoxy]-N,N-didecylacetamide (17c). Isothiocyanate 16 (0.22 g, 0.45 mmol) was added to solution of diamine 15c (0.24 g, 0.5 mmol) and N,Ndiisopropylethylamine (0.87 mL, 5 mmol) in dichloromethane (2 mL). The solution was stirred overnight. The solvent was evaporated under reduced pressure, and then the crude was purified by SFC (standard condition then acetone) to yield 17c (0.11 g, 62%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.85 (t, 6H, J ) 6.6 Hz, CH3), 1.23 (m, 28H, -CH2-), 1.30 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.49 (m, 4H, -CH2-), 3.06 (t, 2H, J ) 7.2 Hz, -CH2NCO), 3.27 (t, 2H, J ) 7.2 Hz, -CH2NCO), 3.68 (m, 8H, CH2NCS), 4.04 (m, 6H, CH2O), 4.25 (m, 7H, CH2O, CH, CHO). 13C NMR (CDCl3) δ (ppm): 14.12 (s, CH3), 22.70 (s, -CH2-), 25.39 and 26.95 (2s, CH3), 26.95 (s, -CH2-), 27.72 (s, -CH2-), 29.57 (m, -CH2-), 31.91 (s, -CH2-), 40.02 (s, CH2NCS), 46.64 and 47.11 (2s, CH2NCO), 51.30 (s, CH), 66.92 (s, CH2O), 69.95 (s, CH2O), 71.89 (s, CH2O), 74.43 (s, CHO), 109.55 (s, CO2(CH3)3), 168.56 (s, Cd O), 171.05 (s, CdO), 182.78 (s, CdS). MS (ESI, m/z) 829 (M - H)-, 831 (M + H)+, 853 (M + Na)+. Anal. Calcd for C41H78N6O7S2: C, 59.24; H, 9.46; N, 10.11. Found C, 59.29; H, 9.60; N, 9.42. N-[1,3-Bis(2,3-dihydroxypropylthiocarbamoylamino)propan2-yl]-2-(ditetradecylcarbamoylmethoxy)acetamide (18a). 1 N Hydrochloric acid (1.3 mL) was added to a solution of 17a (0.13 g, 0.13 mmol) in THF (1.3 mL). The reaction mixture was stirred for 3 h, and then most of the THF was evaporated under reduced pressure in a cold water bath. The resulting liquid was lyophilized to yield 18a (0.1 g, 92%) as a white solid. Mp 50 °C. 1H NMR (CDCl3) δ (ppm): 0.86 (t, 6H, J ) 6.6 Hz, CH3), 1.24 (m, 44H, -CH2-), 1.51 (m, 4H, -CH2-), 3.06 (t, 2H, J ) 7.2 Hz, CH2NCO), 3.28 (m, 4H, H-1 CH2NCO, CH2NCS), 3.46 (m, 4H, CH2NCS), 3.55-3.67 (m, 6H, CH2NCS, CH2OH), 3.80 (m, 2H, CHOH), 4.08 (s, 2H, CH2O), 4.17 (m, 1H, CH), 4.25 (s, 1H, CH2O), 7.32-7.53 (m, 4H, NH), 8.29 (t, J ) 8.4 Hz, 1H, NH). 13C NMR (CDCl3) δ (ppm): 14.00 (s, CH3), 22.61 (s, -CH2-), 27.00 (s, -CH2-), 28.89 (s, -CH2-), 29.66 (m, -CH2-), 31.86 (s, -CH2-), 40.31 (s, CH2NCS), 40.81 (s, CH2NCS), 46.73 and 46.96 (2s, CH2NCO), 49.43 (s, CH), 64.03 (s, CH2OH), 69.64 (s, CH2O), 71.17 (s, CHOH), 71.85 (s, CH2O), 168.39 (s, CdO), 170.61 (s, CdO), 183.53 (s, Cd S). Anal. Calcd for C43H86N6O7S2: C, 59.82; H, 10.04; N, 9.73; S, 7.43. Found C, 58.11; H, 9.64; N, 9.17; S, 6.16. HR-ESMS calcd for C43H86N6O7NaS2: 885.5897. Found 885.5887. N-[1,3-Bis(2,3-dihydroxypropylthiocarbamoylamino)propan2-yl]-2-(didodecylcarbamoylmethoxy)acetamide (18b). 1 N Hydrochloric acid (0.5 mL) was added to a solution of 17b (0.08 g, 0.09 mmol) in THF (0.5 mL). The reaction mixture was stirred for 2 h, and then most of the THF was evaporated under reduced pressure in a cold water bath. The resulting liquid was lyophilized to yield 18b (0.08 g, 73%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.0 Hz, CH3), 1.26 (m, 36H, -CH2-), 1.53 (m, 4H, -CH2-), 3.10 (m, 2H, -CH2NCO), 3.29 (m, 2H, -CH2NCO), 3.62 (m, 8H, CH2NCS), 3.92 (m, 1H, CH), 4.17 (m, 6H, CH2O, CH2OH), 4.34 (m, 6H, CH2O, CHOH), 4.72 (m, 4H, OH), 7.60 (m, 4H, NH). 13C NMR

Leblond et al.

(CDCl3) δ (ppm): 14.17 (s, CH3), 22.76 (s, -CH2-), 27.70 (s, -CH2-), 28.91 (s, -CH2-), 29.66 (m, -CH2-), 32.00 (s, -CH2-), 40.61 (s, CH2NCS), 46.79 and 47.36 (2s, CH2NCO), 49.94 (s, CH), 63.92 (s, CH2OH), 69.32 (s, CH2O), 71.13 (m, CH2O, CHOH), 169.05 (s, CdO), 171.70 (s, CdO), 182.85 (s, CdS). Anal. Calcd for C39H78N6O7S2: C, 58.03; H, 9.74. Found C, 56.13; H, 9.95. HR-ESMS calcd for C39H78N6O7NaS2: 829.5271. Found 829.5245. N-[1,3-Bis(2,3-dihydroxypropylthiocarbamoylamino)propan2-yl]-2-(didecylcarbamoylmethoxy)acetamide (18c). 1 N Hydrochloric acid (0.5 mL) was added to a solution of 17c (0.19 g, 0.23 mmol) in THF (2.1 mL). The reaction mixture was stirred for 3 h, and then most of the THF was evaporated under reduced pressure in a cold water bath. The resulting liquid was lyophilized to yield 18c (0.17 g, 96%) as a colorless oil. 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.0 Hz, CH3), 1.23 (m, 28H, -CH2-), 1.54 (m, 4H, -CH2-), 2.14 (s, 4H, OH), 3.09 (m, 2H, CH2NCO), 3.30 (m, 2H, CH2NCO), 3.63 (m, 8H, CH2NCS), 3.92 (m, 1H, CH), 4.13 (m, 6H, CH2O, CH2OH), 4.31 (m, 7H, CH2O, CHOH), 7.50 (m, 5H, NH). 13C NMR (CDCl3) δ (ppm): 14.18 (s, CH3), 22.76 (s, -CH2-), 27.02 (s, -CH2-), 27.70 (s, -CH2-), 29.45 (m, -CH2-), 31.98 (s, -CH2-), 40.36 (m, CH2NCS), 46.83 and 47.32 (2s, -CH2NCO), 51.00 (s, CH), 63.98 (s, CH2OH), 69.52 (s, CH2O), 71.16 (m, CH2O, CHOH), 169.28 (s, CdO), 171.92 (s, CdO), 183.38 (s, CdS). Anal. Calcd for C35H70N6O7S2: C, 55.97; H, 9.39; N, 11.19; S, 8.54. Found C, 56.63; H, 9.59; N, 9.66; S, 5.94. HR-ESMS calcd for C35H70N6O7NaS2: 773.4645. Found 773.4652. N′-[1,3-Bis(methylthiocarbamoylamino)propan-2-yl]-2-(didecylcarbamoylmethoxy)acetamide (19). Methyl isothiocyanate (0.71 mL, 10.3 mmol) was added to solution of diamine 15c (2.0 mg, 4.1 mmol) and triethylamine (0.58 mL, 4.13 mmol) in dichloromethane (41 mL). The solution was stirred overnight at RT. The solvent was evaporated and the crude was purified by SFC to yield 19 as an oil (2.35 g, 90%). 1H NMR (CDCl3) δ (ppm): 0.88 (t, 6H, J ) 6.0 Hz, CH3), 1.25 (m, 28H, -CH2-), 1.52 (m, 4H, -CH2-), 3.01 (s, 6H, CH3NCS), 3.08 (t, 2H, J ) 7.3 Hz, -CH2-), 3.29 (m, 3H, -CH2-), 3.40 (m, 1H, CH), 3.51-3.98 (m, 4H, CH2NCS), 4.13 (s, 2H, CH2O), 4.30 (s, 2H, CH2O), 7.60 (m, 1H, NH), 8.54 (m, 4H, NH). 13C NMR (CDCl3) δ (ppm): 14.88 (s, CH3), 22.79 (s, -CH2-), 27.22 (s, -CH2-), 27.80 (s, -CH2-), 29.07 (s, -CH2-), 29.44 (m, -CH2-), 29.66 (s, -CH2-), 32.01 (m, -CH2-, CH3NCS), 41.39 (s, CH2NCS), 46.63 and 47.15 (2s, CH2NCO), 50.96 (s, CH), 69.89 (s, CH2O), 71.97 (s, CH2O), 168.73 (s, CdO), 171.54 (s, CdO), 184.00 (s, CdS). MS (ESI, m/z) 631 (M + H)+, 666 (M + Cl)-, 676 (M + HCOO)-. HPLC purity 98% by ELSD. Anal. Calcd for C31H62N6O3S2: C, 59.01; H, 9.90; N, 13.32; S, 10.13. Found C, 59.85; H, 9.79; N, 13.13; S, 8.58. Physicochemistry and Biology. Liposome Preparation. Lipids (and colipids if necessary) were dissolved in ethanol and were added dropwise to ten volumes of water under vigorous agitation. The mixture was stirred overnight and then evaporated under reduced pressure at RT to obtain a fairly concentrated solution of liposomes. As an example, 18c (5 mg, 6,4 µmol) was dissolved in 500 µL ethanol. This solution was added dropwise into 5 mL of stirred filtered water. The mixture was stirred overnight, then evaporated under reduced pressure at RT to obtain a clear suspension of 18c (440 µL, 14.5 mM). Size Measurement. Particle diameter was determined by dynamic light scattering on a Zeta Sizer NanoSeries Malvern (Malvern Instruments, France). Liposome concentration, 0.1 mM in H2O, from the mean value of three runs is given (1 min equilibration, 3 min run).

Lipopolythioureas: New Non-Cationic Vector for Gene Transfer

Preparation of LPT-DNA Complexes. Plasmid pVax2 was used for transfection experiments. pVax2 is a derivative of the commercial plasmid pVax1 (InVitrogen), which was digested with the restriction enzymes HincII and BamHI to excise the promoter. The plasmid was then blunted with the Klenow fragment, dephosphorylated with alkaline phosphatase, pCMVbeta (Clontech), and was digested with EcoR1 and BamHI to excise the CMV promoter. The CMV promoter was blunted with Klenow enzyme and ligated into the blunted pVax1 to give pVax2. The plasmid pXL3031 (26) was digested with EcoRI and BamHI and then treated with the Klenow fragment to produce a blunted fragment containing the luciferase cDNA. This fragment was ligated into pVax2 after EcoRV digestion and phosphatase alkaline dephosphorylation to give pVax21Luc (27). Plasmid (100 µL, 0.02 g/L in H2 O) was added dropwise with constant vortexing to various amounts of LPT liposomes (in 100 µL H2O) at RT. TU/P indicates the ratio in nanomoles of thiourea function (2 per lipid) versus nanomoles of DNA phosphates. Gel Retardation Experiments. Samples were described as above (20 µL), and 5 µL of bromophenol blue was added. The mixture was loaded on a 0.8% agarose gel in TBE buffer (1 M Tris, 0.9 M boric acid, 0.01 EDTA) at 80 V/cm. DNA was revealed with ethidium bromide and visualized under UV light. DNA Accessibility. LPT-DNA complexes (corresponding to 40 ng DNA) were loaded on a 96 well plate, and 100 µL of picogreen (diluted 200× in TBE) was added in each well. Fluorescence was measured by a Wallac Victor2 1420 Multilabel Counter Perkin-Elmer. Values are subtracted from the background and are expressed in percentage of the value of free DNA. DNA Protection. 20 µL of culture medium (MEM with 10% or 50% murine fresh serum) were added on 20 µL of LPTDNA complexes (0.1 g/L DNA). Samples were incubated at 37 °C. After 6 h, 10 µL of samples were frozen at -20 °C. After 24 h, all the samples were replaced at RT. SDS 2% (5 µL), EDTA (2 µL, 0.5 M), and bromophenol blue (3 µL) were added on each sample (10 µL). The mixture was loaded on agarose gel 1% containing 0.05% SDS at 80 V/cm. After 24 h washing in water, DNA was revealed with ethidium bromide and visualized under UV light. Transfection Method. B16 murine melanoma cells were grown into DMEM supplemented with 10% fetal bovine serum, 1% antibiotics (penicillin and streptomycin), and 1% glutamine. One day before transfection, cells were treated by trypsin and deposited into 24 well plates (45 000 cells/well) and incubated 24 h at 37 °C in DMEM supplemented with 10% fetal bovine serum. 100 µL of LPT-DNA (corresponding to 0.5 µg DNA) complex was loaded on each well, and the plates were incubated at 37 °C for 48 h. Then, the cells were washed twice with PBS and treated with 200 µL of a passive lysis buffer (Promega). After 15 min, the cells were centrifuged for 5 min at 12 000 rpm. 10 µL of supernatant and 10 µL of iodoacetamide were deposited on a 96 well plate, which was incubated at 37 °C for 1 h. Protein quantification was performed with the BCA Protein Assay Kit (Pierce) and reported to the BSA taken as a reference curve. Luciferase activity was quantified using a commercial kit Luciferase Assay System (Promega). On 10 µL of the lysed cells, 50 µL of the luciferin substrate was injected via an injector, and the absorbance was read immediately at 563 nm on a Wallac Victor2 1420 Multilabel Counter from Perkin-Elmer. Intratumoral Injection. LPT-DNA (40 µL, 10 µg DNA, 40 TU/P) was injected in the tumor of three mice C57BL6 bearing 3LL tumors. Mice were sacrificed 24 h after injection, then tumors were removed, weighed, crushed in 5 mL lysis buffer per tumor, and centrifuged for 10 min (12 000 tr/min, 4 °C). 50 µL of the supernatant was loaded on a 96 well plate, and

Bioconjugate Chem., Vol. 18, No. 2, 2007 489 Scheme 1. Retrosynthetic Analysis of Lipopolythiourea Lipids

luciferase activity was quantified using a commercial kit (Protein Assay). The luminescence was read on a Wallac Victor2 1420 Multilabel Counter from Perkin-Elmer.

RESULTS AND DISCUSSION Chemistry. As we had previously established that two thiourea groups were enough for the lipid to properly interact with DNA, we kept an identical interacting lipid head, but we modified the linker and the lipid length in order to obtain lipids with a suitable hydrophilic-lipophilic balance. We chose to use a propane triamine scaffold that could be readily prepared from 2-amino-1,3-diazidopropane (28). The preparation of these compounds could be envisioned according to the retrosynthetic scheme shown in Scheme 1. Thiourea formation could occur either by the reaction of an isothiocyanate on diamine B or by the condensation of an amine on the bis-isothiocyanate C. Precursor B may be prepared by reduction of diazide D. The synthesis of D could be easily achieved by coupling acid E

490 Bioconjugate Chem., Vol. 18, No. 2, 2007 Scheme 2

a

Leblond et al.

a

(i) Boc2O, EtOH, RT, 93%; (ii) MsCl, Et3N, CH2Cl2, RT, quant.; (iii) NaN3, DMF, 100 °C, quant.; (iv) TFA, CH2Cl2, RT, 96%.

Scheme 3

a

a (i) DMAP, pyridine, CH2Cl2, RT, 98%; (ii) DCC, 5, CH2Cl2, RT, 98%; (iii) H2, Pd/C, EtOH, RT, quant.; (iv) MeNCS, Et3N, CH2Cl2, RT, 63%; (v) CS2, DIPEA, TsCl, CH2Cl2, RT, 22%; (vi) 1-amino-2,3-propanediol, DMF/CH2Cl2, RT, 25%.

Table 1. Formulation Properties and log P Calculation of Lipopolythiourea

a

LPT

anchor

log P

solubility

DPPC

diameter (nm)

PDIa

methyl

10

14 C

8.54

CHCl3

tetraol tetraol tetraol tetraol methyl

12 18a 18b 18c 19

14 C 14 C 12 C 10 C 10 C

6.91 6.38 4.79 3.21 4.84

CHCl3 EtOH EtOH EtOH EtOH

1:1 1:2 1:1

72 ( 0.29 205 ( 15 124 ( 35 89 ( 1 81 ( 16 70 ( 12 108 ( 1

0.449 0.274 0.261 0.194 0.237 0.225 0.158

linker

terminus

succinic succinic glycolic glycolic glycolic glycolic

Polydispersity index.

with the amine F. F could be obtained in two steps from BOCprotected serinol via a sulfonylation/substitution protocol. Accordingly, disconnection of E gives the known dialkylamine and acid anhydride. Therefore, we began the synthesis with the preparation of diazide 5 (Scheme 2), which was obtained in four steps from serinol. First, the amine was BOC protected; then, diol 2 was mesylated to yield 3 quantitatively. Substitution of the mesylate using sodium azide in DMF gave the diazide 4 whose BOCprotecting group was cleaved to afford 5 with an excellent global yield (89%). With the diazide 5 in hand, we turned our attention to the preparation of lipopolythioureas (Scheme 3). The synthesis started with the reaction of ditetradecylamine 6a with succinic anhydride, which provided the acid 7 in 98% yield. Coupling 7 with the diazidoamine 5 was performed using various conditions. Both mixed-anhydride protocol using isobutyl chloroformate (29) and in situ preparation of the acid chloride using thionyl chloride and A-21 resin (30) afforded the amide 8 in 60% yield. However, DCC coupling offered higher yields (93%), and DCC/ NHS was then selected for the syntheses. Catalytic hydrogenation of 8 gave the corresponding amine 9. The first lipopolythio urea 10 was achieved by reaction of amine 9 with methyl isothiocyanate. Besides, treatment of 9 with carbon disulfide in the presence of tosyl chloride (31) afforded the bisisothiocyanate 11. The modest yield of this reaction could be explained by an intramolecular process leading to a cyclic

thiourea as described by Marmillon et al. (32). However, no side product was isolated. Finally reaction of 11 with 1-amino 2,3-propanediol gave the lipopolythiourea 12 in 25% yield. Concerning the preparation of the glycolic derivatives, the diamides 15a, 15b, and 15c were prepared in three steps from the corresponding diamines similarly to 9 (Scheme 4). Regarding the low yields obtained in the last steps of the synthesis of 12, a different strategy was used for the LPT formation in order to improve the reaction efficacy. 15a, 15b, and 15c were treated with isothiocyanate 16 to afford the corresponding thioureas in 62% to 89% yields. Deprotection of 17a, 17b, and 17c with 1 N hydrochloric acid in THF led to the tetraols 18a, 18b, and 18c in good yields. In addition, the diamine 15a was treated with methyl isothiocyanate to give the methyl thiourea 19 in 90% yield. Formulation Characterization. The ability of lipopolythioureas (LPT) to form micellar or liposomal particles was then evaluated. Previous studies have indicated that LPT hydrophobicity prevents its solubilization in H2O and particle formation (22). For this reason, phosphatidylcholine (DPPC) has been added as an amphiphilic colipid because of its high hydration potency and its zwitterionic nature. Then, solubility of the LPT lipids in chloroform and ethanol was first evaluated. The aim of this test was to define the way for the lipid to be formulated and the necessity of adding a colipid. Data are reported in Table 1, in parallel to log P calculation. Both calculation and solubility were in agreement.

Lipopolythioureas: New Non-Cationic Vector for Gene Transfer Scheme 4

Bioconjugate Chem., Vol. 18, No. 2, 2007 491

a

Figure 1. Accessibility of DNA in LPT/DNA complexes measured by picogreen fluorescence. Free DNA corresponds to 100% fluorescence. Measures for the free lipids were taken as the background. TU/P indicates the ratio of thiourea function per DNA phosphate.

a (i) DMAP, CH2Cl2, RT; (ii) DCC or DCC/NHS, CH2Cl2, RT; (iii) H2, Pd/C, EtOH, RT; (iv) 6, DIPEA, CH2Cl2, RT; (v) HCl, THF, RT; (vi) MeNCS, DIPEA, CH2Cl2, RT.

Indeed, the most lipophilic compounds 10 and 12, bearing a succinic linker, exhibited a good solubility only in CHCl3. Interestingly, comparison of compounds 12 and 18a that differ only by an oxygen atom within the linker showed that this atom added interesting physicochemical properties despite the very close values of their log P. A more dramatic influence on log P, as well as solubility, was observed when the lipid length was reduced from myristyl to decanoyl, on the series of glycolic LPTs 18a, 18b, and 18c. The glycolic LPT 19, bearing a methyl group within the thiourea condensing head, clearly showed a higher log P as compared to its tetraol counterpart 18c. Next, we investigated the formulation of LPT with or without the use of the colipid DPPC. The formulations were performed using ethanolic injection, a gentle method leading spontaneously to small particles (see Material and Methods section). Data in Table 1 indicate that the formulation was also closely related to the log P. All the compounds under log P ) 6.4 were soluble in ethanol and could be formulated without helper lipid. Above this value, LPT, namely, LPT with succinic linker 10 and 12, required DPPC. A 1:1 ratio was found to be optimal for 12, but the more lipophilic compound 10 required a higher amount of DPPC in order to obtain a homogeneous suspension as shown by the polydispersity index value (PDI). Formulation of the LPT led to liposomes with diameters ranging from 70 to 205 nm as measured by dynamic light scattering experiments. It is noteworthy that smaller particles were obtained for compounds 18a, 18b, and 18c. These results clearly showed that structural

changes of polar head, linker, and lipidic anchor had a real influence on the formulation properties of the resulting lipid. LPT-DNA Interaction. The degree of DNA accessibility, once complexed with LPT, was assessed by the double-strandedDNA-binding reagent picogreen. Fluorescence assays were performed using a fixed DNA amount and increasing the LPT equivalents. As observed in Figure 1, fluorescence decreased when the amount of lipids increased. However, the intensity was only half-reduced in comparison to the intensity observed with free DNA, even with a large excess of lipids (120 TU/P). This experiment means that DNA was still accessible to picogreen despite its interaction with the LPT. To evaluate if DNA was only partly associated to LPT, we loaded the complexes on an agarose gel (Figure 2). We could observe that all prepared LPT possessed the capacity to fully associate with DNA leading to LPT-DNA complexes, the required lipid amount depending on the LPT structure. Hence, lipopolythiourea 19 required more lipids to complex all DNA (40 TU/P) than compounds 18a, 18b, and 18c, which required a low ratio TU/P ) 15 (Figure 1). From agarose gel retardation experiments, we observed that all prepared LPT totally retained DNA migration at a ratio TU/P ) 15. On the other hand, DNA accessibility experiments suggested that LPT interaction with DNA led to a partially compacted structure that left free access for picogreen as previously described for sphingosine (33). Lipoplex Sensitivity to Serum. DNA protection toward serum degradation was investigated in the absence and presence of LPT. After 30 min incubation at 37 °C in 10% serum containing medium, samples were treated by SDS and loaded on agarose gel containing 0.05% SDS (34) (Figure 3). In these conditions, free DNA was fully degraded in 24 h (lane 3), while DNA integrity was maintained in 10% serum when associated to LPT (TU/P ) 40). Glycolic LPT presented the same capacity as cationic lipopolyamine RPR 209120 (7, 35), taken as reference, to protect DNA (36). Same results have been previously found for the lipopolythiourea DTTU (21). The same experiment performed with compound 18c in 50% serum showed the ability for this lipid, as compared to free DNA, to partially protect DNA in these drastic conditions (data not shown). These data indicate that DNA is partly protected from degradation in serum-containing medium when associated to LPT. This favors the hypothesis of a strong interaction between LPT and DNA that might be mediated not only by the thiourea/phosphate interaction but also by the overall supramolecular lipid structure. These data may also suggest that the LPT-DNA complexes allow accessibility to small molecules like picogreen, but not to large ones, like DNases. In Vitro Transfection Efficiency. We evaluated the lipids in vitro as a preliminary study for in vivo experiments. In this context, all lipids were tested in serum-containing media, which

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Figure 2. Migration of LPT/DNA complexes (18a, 18b, 18c, left; 19, right) on a 0.8% agarose gel in TBE (1×). Complexes were revealed with ethidium bromide staining. From left to right, the lanes correspond to 2.5, 5, 10, and 15 TU/P.

Figure 3. Complexes LPT/DNA (TU/P ) 40) were incubated for 0, 6, or 24 h in 10% serum at 37 °C, then treated with SDS 2% and EDTA 0.5 M and loaded on an 1% agarose gel containing in TBE (1×) 0.05% SDS for electrophoresis (80 V/cm).

appears more appropriate (37). The lipopolythiourea transfection efficiency was tested on B16 cells at various TU/P ratios. The transfection efficiency of the LPT was compared to those of the cationic lipid and PEI (38). Results are presented in Figure 4 as well as the complex size, which might influence the result. Indeed, cationic complexes exhibit higher transfection capacity in serum when precipitated (39). Thus, PEI was formulated in NaCl and taken as a positive control. Although lipopolythiourea 18a showed a poor transfection activity, compounds 18b and 18c led to a more significant transfection level. They proved to be comparable to the efficiency of the cationic systems of nanometric scales in these conditions, in particular, the lipoplex RPR209120/DNA, which exhibited a similar size as compared to LPT-DNA complexes. In addition, the transfection level was found to be dose-dependent. Furthermore, LPT 19 displayed the same activity as LPT 18c with the same hydrophobic anchor. Thus, the length of the chain appeared to be decisive for transfection activity. In Vivo Transfection Efficiency. Interestingly, intratumoral injection of the more efficient compounds as selected by in vitro experiments showed good transfection ability in vivo as compared to the lipopolyamine RPR209120 (Figure 5). No significant difference could be observed between the four samples, meaning that LPT are able to induce gene expression in vivo as well as in this cationic system.

Figure 4. Hydrodynamic diameter (lozenge symbols, right scale) and gene transfection (bars, left scale) measured by luciferase activity of LPT/DNA complexes at 20, 40, and 80 TU/P ratios after 48 h incubation on B16 cells in the presence of serum. LPT/DNA complexes were prepared at 20, 40, and 80 TU/P in H2O. RPR209120/DOPE/DNA lipoplexes was prepared N/P ratio ) 8 in NaCl 150 mM. PEI/DNA polyplexes were prepared at N/P ) 15 in glucose 5% or NaCl 150 mM. The complexes were then added to the cells in DMEM + 10% FBS at 37 °C. Results are expressed as the mean of an experiment performed in triplicate errors bars indicating standard error of the mean. Note: 18c at 20TU/P was found to be precipitated (>1000 nm).

Figure 5. Luciferase activity 24 h after intratumoral injection of 18c/ DNA, 19/DNA (40 TU/P, 10 µg DNA) and RPR209120/DOPE/DNA (ratio N/P ) 5) on three mice previously injected with 3LL carcinoma cells.

CONCLUSION The presented lipopolythioureas provided new tools for nucleic acid delivery using nonelectrostatic vectors with a transfection level comparable to cationic systems in serumcontaining medium. Lipopolythioureas 18a, 18b, 18c, and 19 can be formulated without the use of colipid, leading to small

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stable particles. In addition, these compounds showed efficient interaction with plasmid DNA and can protect it from serum degradation during 24 h. The mechanism of LPT-DNA interaction and transfection is now under investigation.

ACKNOWLEDGMENT This work was financially supported by INSERM, CNRS, MNERT, the Re´gion Ile de France (SESAME, CPER), and the 6th PCRD (MOLEDA). M.-V. S. thanks the EC (MOLEDA) for a postdoctoral fellowship.

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