Paper
Organic & Biomolecular Chemistry
JCF = 8.8, 248.3 Hz, Carom), 171.24, 171.32 (C6), 172.41, 172.56 (ether/petroleum ether, 2 : 1, v/v) to give 6.1 g of 9 as oil. Yield
(COO); 15P NMR (CDCl3) δ: −1.08, −0.98, −0.89, −0.78; HRMS 48% as a mixture of two α-anomers; one of the α-anomers was
1
(MALDI): m/z calcd for C28H35F2N4NaO5P (MNa+) 599.2205, purified by repeated chromatography; H NMR (CDCl3) δ: 1.63
found 599.2180.
(d, 3H, J = 7.2 Hz, CH3CH), 1.87(s, 3H, CH3–C5), 1.95, 2.02,
(2S)-Prop-2-ynyl 2-{[6-(1-(2,6-difluorophenyl)ethyl)-2-(dimethyl- 2.04, 2.05, (4 s, 12 H, CH3CO), 3.07 [s, 6H, (CH3)2N], 3.86–3.91
amino)-5-methylpyrimidin-4-yloxy](phenoxy)phosphorylamino}- (m, 1H, H5′), 4.14 (dd, 1H, J = 12.3, 2.4 Hz, H6′), 4.25 (dd, 1H,
propanoate (8d). Yield 54%; obtained as an oil of diastereo- J = 12.3, 5.1 Hz, H6′), 4.58 (q, 1H, J = 7.1 Hz, CH–CH3), 5.16 (t,
1
mers; H NMR (CDCl3) δ: 1.37–1.48 (m, 3H, CH3CH), 1.64 (d, 1H, J = 9.6 Hz, H4′), 5.27–5.37 (m, 2H, H2′, H3′), 6.04 (d, 1H,
3H, J = 7.2 Hz, CH3CH), 1.78, 1.83 (2s, CH3–C5), 2.17 (s, 1H, J = 8.1 Hz, H1′), 6.74–6.84 (m, 2H, Harom), 7.07–7.17 (m, 1H,
CHuC), 3.14 [s, 6H, (CH3)2N], 4.28–4.40 (m, 1H, CH3CH), 4.54
H
arom); 13C NMR (CDCl3) δ: 8.67 (CH3–C5), 17.75 (CH3CH),
(q, 1H, J = 7.2 Hz, CH3CH), 4.61–4.78 (m, 3H, NH and CH2), 20.55, 20.57, 20.60, 20.67 (Ac), 34.04 (CH3CH), 36.54 [(CH3)2N],
6.78–6.86 (m, 2H, Harom), 7.06–7.32 (m, 6H, Harom); 13C NMR 61.98 (C6′) 68.48, 70.39, 72.42, 72.95 (C2′, C3′, C4′, C5′), 93.43
(CDCl3) δ: 9.05, 9.09 (CH3C5), 17.60 (CH3CH), 21.24, 21.38 (C1′), 100.89 (C5), 111.08–111.43 (m, Carom), 127.72 (t, JCF
=
(CH3CH–NH), 34.31 (CH3CH), 36.93 [(CH3)2N], 50.26, 50.30 10.7 Hz, Carom), 159.45 (C2), 161.45 (dd, JCF = 9.0, 247.8 Hz,
(CH3CH–NH), 52.68, 52.74 (OCH2), 75.32 (CHuC), 76.90
Carom), 165.58 (C4), 169.83 (C6), 169.24, 169.41, 170.24, 170.63
(CHuC), 102.13, 102.19, 102.25, 102.31 (C5), 111.20–111.55 (COO); HRMS (MALDI): m/z calcd for C29H35F2N3NaO10 (MNa+)
(m, Carom), 120.32–120.51 (m, Carom), 124.93, 125.02 (Carom), 646.2183, found 646.2191.
127.97 (t, JCF = 10.7 Hz, Carom), 129.48, 129.54, 150.48, 150.53
Anti-HIV
(Carom), 156.12 (C2), 159.16 (C4), 161.44 (dd, JCF = 8.7, 248.4
Hz, Carom), 171.36, 171.48 (C6), 172.49, 172.81 (COO); 15P NMR
(CDCl3) δ: −1.42, −1.34, −1.28, −1.16; HRMS (MALDI): m/z
calcd for C27H30F2N4O5P (MH+) 559.1916, found 559.1913.
Cell-based assays
Compounds. Compounds were dissolved in DMSO at 100 µM
and then diluted in culture medium.
(2S)-Benzyl
2-{[6-(1-(2,6-difluorophenyl)ethyl)-2-(dimethyl-
Cells and viruses. Cell lines were purchased from American
amino)-5-methylpyrimidin-4-yloxy](phenoxy)phosphorylamino}- Type Culture Collection (ATCC). The absence of mycoplasma
propanoate (8e). Yield 55%; obtained as oil of diastereomers; contamination was checked periodically by the Hoechst stain-
1H NMR (CDCl3) δ: 1.34–1.47 (m, 3H, CH3CH), 1.64 (d, 3H, J = ing method. The cells supporting the multiplication of HIV-1
6.9 Hz, CH3CH), 1.76, 1.77, 1.81, 1.82 (4s, 3H, CH3–C5), 3.09, were the CD4+ human T-cells (MT-4) containing an integrated
3.10 [2s, 6H, (CH3)2N], 4.27–4.43 (m, 1H, CH3CH), 4.55 (q, 1H, HTLV-1 genome.
J = 6.9 Hz, CH3CH), 4.69–4.84 (m, 1H, NH), 5.04, 5.08 (2s, 2H,
Cytotoxicity assays. For cytotoxicity evaluation, exponentially
OCH2), 6.77–6.85 (m, 2H, Harom), 7.05–7.36 (m, 11H, Harom); growing MT-4 cells were seeded at an initial density of 1 × 105
13C NMR (CDCl3) δ: 9.05, 9.09 (CH3–C5), 17.61, 17.61 (CH3CH), cells per ml in 96 well plates, with RPMI-1640 medium sup-
21.45, 21.54 (CH3CH), 34.31 (CH3CH), 36.87 [(CH3)2N], 50.45 plemented with 10% fetal calf serum (FCS), 100 units per mL
(CH3CH–NH), 67.02, 67.10 (OCH2), 102.15, 102.23, 102.27, penicillin G and 100 µg per mL streptomycin. Cell cultures
102.34 (C5), 111.19–111.54 (m, Carom), 120.32–120.54 (m, were then incubated at 37 °C under a humidified 5% CO2
Carom), 124.88, 124.98, 127.84, 128.03, 128.35, 128.52, 128.54, atmosphere in the absence or presence of serial dilutions of
128.55, 135.20, 135.25, 150.40, 150.54 (Carom), 154.24 (C2), test compounds. Cell viability was determined after 96 h at
159.14 (C4), 161.45 (dd, JCF = 8.6, 248.4 Hz, Carom), 171.35, 37 °C by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetr-
171.43 (C6), 173.11, 173.28 (COO); 15P NMR (CDCl3) δ: −1.26, azolium bromide (MTT) method.28
−1.15, −1.10, −0.96; HRMS (MALDI): m/z calcd for
Antiviral assays. The activity of compounds against Human
Immunodeficiency Virus type-1 (HIV-1) was based on inhi-
C31H33F2N4NaO5P (MNa+) 633.2049, found 633.2020.
(2R,3R,4S,5R,6R)-2-(Acetoxymethyl)-6-{6-[1-(2,6-difluoro- bition of virus-induced cytopathogenicity in MT-4 cells acutely
phenyl)ethyl]-2-(dimethylamino)-5-methylpyrimidin-4-yl-oxy}- infected with a multiplicity of infection (m.o.i.) of 0.01. Briefly,
tetrahydro-2H-pyran-3,4,5-triyl triacetate (9). Sodium hydride 50 µL of RPMI containing 1 × 104 MT-4 were added to each
(1.1 g of 55% suspension in paraffin oil, 24.6 mmol) was well of flat-bottom microtiter trays containing 50 µL of RPMI,
added portionwise to a solution of MC-1220 (6 g, 20.5 mmol) without or with serial dilutions of test compounds. Then,
in dry N,N-dimethylformamide (50 mL), and the mixture was 20 µL of an HIV-1 suspension containing 100 CCID50 were
stirred for 1 hour. The sodium salt of MC-1220 was added added. After 4-day incubation, the cell viability was determined
dropwise at 0 °C to a solution of α-acetobromoglucose (9.3 g, by the MTT method.
22.6 mmol) in dry N,N-dimethylformamide (50 mL). After com-
plete addition, the reaction mixture was left to reach room
Stability tests in cell culture medium
temperature with stirring for 2 hours. The reaction mixture 100 µM solutions of 8a–e or 9, prepared in RPMI sup-
was poured in ice-cold water (500 mL), ether (100 mL) was plemented with 10% FCS, 100 units per mL penicillin G and
added and the two layers were separated. The water layer was 100 µg per mL streptomycin, were incubated at 37 °C with
extracted two times with ether (2 × 50) and the combined ether shaking. To follow the rate of hydrolysis into MC-1220, 20 µL
extracts were dried and evaporated under reduced pressure. samples of the various prodrugs, collected at indicated times
The residue was chromatographed using a column of silica gel (Fig. 1), were injected on an SSODS2 reverse phase HPLC
Org. Biomol. Chem.
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