The chemical synthesis of ring-expanded nucleosides (RENs) with a β-configuration, including but not limited to 1
, have been previously reported (21
). The procedure for the synthesis of RENs with an α-configuration is described below.
A general procedure for the synthesis of α-deoxy ring-expanded nucleoside (REN) analogs
An appropriate alkylamine (6 mmol) and 3, 5-dimethylpyrazole-1-carboxamidine nitrate (1.244 g, 6 mmol, 97%) was refluxed in methanol (20 ml) for 2 hours. The solvent was evaporated under vacuum, dried in a desiccator over P2
under vacuum for two nights. Without purification, it was dissolved in anhydrous methanol (6.0 mL), cooled to 0°C, and a solution of methanolic sodium methoxide (25% wt in methanol, 3.15 ml) was added drop-wise. The reaction mixture was kept in ice-bath for 0.5 hour. Solution from the reaction mixture was directly filtered into butyl 1-(2’-deoxy-3’,5’-di-O
) (1.38–1.81 mmol) in anhydrous methanol (30 ml). The ring closure reaction went complete after stirring the reaction mixture overnight. The product was purified by silica gel chromatography, eluting successively with a mixture of chloroform:methanol (30:1), chloroform:methanol (10:1), and methanol. During solvent evaporation of the collected fractions, solid started to precipitate from solution. The precipitate was filtered and dried over P2
4,5-Dihydro-8H-6-(N-tetradecyl)amino-1-(2’-deoxy-α-D-erythropentofuranosyl)imidazo[4,5-e]diazepine-4,8-dione (compound 11)
Yield: 92.6 %, mp > 250 °C; 1H NMR (DMSO-d6): δ 10.37 (brs, 1H, NH, exchangeable with D2O), 8.18 (s, 1H, imidazole), 7.10 (brs, 1H, NH, exchangeable with D2O), 6.62 (d, J= 5.9 Hz, 1H, 1’-H), 5.10 (m, 1H, 3’-OH, exchangeable with D2O), 4.87 (m, 1H, 5’-OH, exchangeable with D2O), 4.24 (m, 2H, 3’,4’-H), 3.42 (m, 2H, 5’-H1,2), 3.20 (m, 2H, CH2), 2.62 (m, 1H, 2’-H1), 2.01 (d, J= 14.3 Hz, 1H, 2’-H2), 1.46 (m, 2H, CH2), 1.22 (m, 22H, (CH2)11), 0.83 (t, J= 5.9 Hz, 3H, CH3); Anal. Calcd. for C25H41N5O5·H2O (509.64): C, 58.92; H, 8.50; N, 13.74. Found: C, 58.67; H, 8.29; N, 13.58.
4,5-Dihydro-8H-6-(N-hexadecyl)amino-1-(2’-deoxy-α-D-erythropentofuranosyl)imidazo[4,5-e]diazepine-4,8-dione (compound 12)
Yield: 99.0 %, mp > 250 °C; 1H NMR (DMSO-d6): δ 10.56 (brs, 1H, NH, exchangeable with D2O), 8.19 (s, 1H, imidazole), 7.11 (brs, 1H, NH, exchangeable with D2O), 6.63 (d, J= 6.6 Hz, 1H, 1’-H), 5.11 (m, 1H, 3’-OH, exchangeable with D2O), 4.87 (m, 1H, 5’-OH, exchangeable with D2O), 4.24 (m, 2H, 3’,4’-H), 3.42 (m, 2H, 5’-H1,2), 3.20 (m, 2H, CH2), 2.64 (m, 1H, 2’-H1), 2.01 (d, J= 13.9 Hz, 1H, 2’-H2), 1.47 (m, 2H, CH2), 1.22 (m, 26H, -(CH2)13), 0.83 (t, J= 6.0 Hz, 3H, CH3); Anal. Calcd. for C27H45N5O5·1.5H2O (546.70): C, 59.32; H, 8.85; N, 12.81. Found: C, 59.71; H, 8.60; N, 13.05.
4,5-Dihydro-8H-6-(N-octadecyl)amino-1-(2’-deoxy-α-D-erythropentofuranosyl)imidazo[4,5-e]diazepine-4,8-dione (compound 2)
Yield: 86.7 %, mp > 250 °C; 1H NMR (DMSO-d6): δ 10.30 (brs, 1H, NH, exchangeable with D2O), 8.19 (s, 1H, imidazole), 7.06 (brs, 1H, NH, exchangeable with D2O), 6.62 (d, J= 6.6 Hz, 1H, 1’-H), 5.10 (d, J= 2.2 H, 1H, 3’-OH, exchangeable with D2O), 4.87 (t, J= 5.1 Hz, 1H, 5’-OH, exchangeable with D2O), 4.24 (m, 2H, 3’,4’-H), 3.43 (m, 2H, 5’-H1,2), 3.21 (m, 2H, CH2), 2.64 (m, 1H, 2’-H1), 2.01 (d, J= 14.3 Hz, 1H, 2’-H2), 1.46 (m, 2H, CH2), 1.22 (m, 30H, (CH2)15), 0.84 (t, J= 6.4 Hz, 3H, CH3); Anal. Calcd. for C29H49N5O5·H2O (565.75): C, 61.57; H, 9.09; N, 12.38. Found: C, 61.88; H, 9.19; N, 12.18.
HIV-1 T-cell-tropic molecular clone (NL4-3) and macrophage-tropic molecular clone (AD8) were used. HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum. Infectious virus stocks were generated by transfecting HeLa cells with either pNL4-3 or pAD8 plasmids. Culture supernatant was collected 36 hrs post transfection, and the virus stock was quantified by RT assay (35
). MT4 cells were maintained in RPMI supplemented with 10% fetal bovine serum. Usually 4 × 106
cells were infected with 20,000 normalized RT counts of NL4-3, and compounds were added 4 hrs post infection. Afterwards, 50% of the media was replaced every 2 days, with fresh media containing the compounds at the initial dosing level. Human monocyte derived macrophages (MDMs) were generated by culturing elutriated blood monocytes in RPMI-1640 containing 10% pooled human AB serum and M-CSF. Cells were cultured for 5–7 days before infecting with 105
RT counts of HIV-1 AD8.
RNA unwinding assay
RNA unwinding assay was performed as described previously (9
). Briefly pBluescript plasmid was used to generate partially double stranded RNA for the RNA unwinding assay. The pBluescript vector was digested to completion with Kpn
I and transcribed with T3 polymerase to generate a 120 base long transcript. The vector was also digested with Eco
RI and transcribed with T7 polymerase in the presence of (α32p) UTP. The two complementary transcripts were purified and hybridized at 95° C for 20 minutes. Double stranded RNA was then incubated with purified DDX3 (with or without 4 mM 1
) in ATPase/helicase (20 mm
Tris-HCl, pH 8.0, 70 mm
KCl, 2 mm
, 2 mm
dithiothreitol, 15 units RNasin, and 2 mm
ATP). After incubation for 30 mins at 37 °C, the reactions were stopped by the addition of a solution containing 10 mm
EDTA, 40% glycerol, bromphenol blue, xylene cyanol. The reaction was resolved in a 10% polyacrylamide gel for 2 hours. The gel was dried and exposed to a phosphorimaging plate.
4 × 106 suspension T cells (MT4) were incubated with or without 1 or 2. 50% of the media was removed every 2 days and replaced with an equivalent amount of complete media containing the inhibitors. At time of each harvest, 100 µl of the cell suspension were measured for viablilty and compared to a similar amount of control (untreated) cells using the Cell Counting kit-8 (Dojindo) according to manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader. For in vivo studies 8–10 weeks old BALB/c mice were injected with various concentrations of 1, 2, DMSO, or normal saline twice a week intraperitoneally for 4 weeks and sacrificed for necropsy after week 5. Blood from the mice was collected once per week via the tail vein.
HPLC analysis of mouse serum
Serum samples for HPLC were extracted with 3 volumes of ethyl acetate and lyophilized. Each mouse serum sample was dissolved in a mixture of 100 µl water and 20 µl acetonitrile. The samples were analyzed by reverse-phase HPLC using a C4 Vydac 214TP1010 column (10 × 250 mm). The injection for each run was 20 µl with monitoring at λmax 214 and 254 nm. The column was equilibrated with 75% developer A (water, 0.1%TFA) and 25% developer B (CH3CN, 0.1% TFA), and a gradient was employed to give a final composition of 25% developer A and 75% B over a period of 80 minutes at 3 ml/min. For the HPLC analysis of 1 alone without the mouse serum, the compound was dissolved in a mixture of MeOH:AcCN (1:1).