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4.3.2. Ethyl (3SR,5RS)-3-(diethoxyphosphoryl)-5-(5-fluoro-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-
5-carboxylate 11b
J = 7.0 Hz, 3H). 13C NMR (CDCl3): d = 165.19, 163.46 (C4), 150.13
(C2), 134.61 (C6), 109.92 (C5), 92.69 (d, J = 14.2 Hz, C10), 63.33
(CH2–O), 63.24 (d, J = 6.9 Hz), 62.15 (d, J = 163.2 Hz, C40), 62.70
(d, J = 6.9 Hz), 45.43 (CH3N), 43.95 (C50), 16.43 (d, J = 4.5 Hz),
13.95 (d, J = 7.5 Hz), 13.80 (CH3–CH@), 12.86. 31P NMR
(121.5 MHz, CDCl3): d 20.79. Anal. Calcd for C16H26N3O8P: C,
45.82; H, 6.25; N, 10.02. Found: C, 45.87; H, 6.27; N, 9.98.
Yield: 68%; white solid, mp 200–201 °C. 1H NMR (CDCl3):
d = 8.91 (s, 1H), 7.77 (d, J = 6.5 Hz, 1H), 4.30–4.10 (m, 6H), 3.83
(ddd, J = 14.0, 8.5 and 4.0 Hz, 1H), 3.25 (ddd, J = 10.0, 8.5 and
3.0 Hz, 1H), 2.95 (s, 3H), 2.93 (ddd, J = 16.5, 14.0 and 10.0 Hz,
1H), 1.35 (t, J = 7.5 Hz, 3H), 1.28 (m, 6H). 13C NMR (125 MHz,
CDCl3): d = 164.12, 156.93 (d, J = 26.7 Hz), 148.74, 139.90 (d,
J = 235.1 Hz), 123.73 (d, J = 35.2 Hz), 92.63 (d, J = 10.6 Hz), 65.13
(d, J = 163.7 Hz), 63.43 (d, J = 7.3 Hz), 63.35, 62.61 (d, J = 6.8 Hz),
46.20, 45.17, 16.41, 16.21, 13.84. Anal. Calcd for C15H23FN3O8P: C,
42.56; H, 5.48; N, 9.93. Found: C, 42.59; H, 5.50; N, 9.91.
4.3.7. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-5-(5-fluoro-2,4-
dioxo-3,4-dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-
5-carboxylate 12b
Yield: 12%; white solid, mp 193–195 °C. 1H NMR (CDCl3):
d = 8.65 (bs s, 1H), 7.86 (d, J = 6.6 Hz), 4.29–4.14 (m, 6H), 3.81
(ddd, J = 15.0, 14.6 and 1.3 Hz, H-C50, 1H), 3.18–3.02 (bm, H-C40
and H-C50, 2H), 3.13 (s, N–CH3, 3H), 1.38 (t, J = 8.5 Hz, 3H), 1.35
(t, J = 8.2 Hz, 3H), 1.25 (t, J = 8.2 Hz, 3H). 13C NMR (CDCl3):
d = 164.66, 159.82 (d, J = 30.4 Hz), 156.20, 139.95 (d, J = 237.0 Hz),
123.60 (d, J = 35.8 Hz), 92.55 (d, J = 12.5 Hz), 64.07 (d, J = 6.5 Hz),
63.88 (d, J = 175.0 Hz), 63.80, 62.90 (d, J = 6.5 Hz), 53.43, 42.62,
16.48, 16.37, 13.87. Anal. Calcd for C15H23FN3O8P: C, 42.56; H,
5.48; N, 9.93. Found: C, 42.53; H, 5.51; N, 9.94.
4.3.3. Ethyl (3SR,5RS)-3-(diethoxyphosphoryl)-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-5-
carboxylate 11c
Yield: 47.61%; white solid, mp 171–174 °C. 1H NMR (CDCl3):
d = 8.61 (bs s, 1H), 7.72 (d, J = 8.2 Hz, 1H), 5.77 (d, J = 8.2 Hz, 1H),
4.37–4.02 (m, 6H), 3.92 (ddd, J = 14.2, 8.5 and 4.1 Hz, Ha
–C50,
1H), 3.31 (ddd, J = 10.1, 8.5 and 2.7 Hz, H–C40, 1H), 3.00 (ddd,
J = 16.3, 14.2 and 10.1 Hz, Hb–C50, 1H),. 2.98 (s, 3H),), 1.34 (t,
J = 7.1 Hz, 3H), 1.32 (m, 6H). 13C NMR (125 MHz, CDCl3):
d = 164.42, 163.31, 150.28, 139.01, 100.96, 92.84 (d, J = 12.0 Hz),
65.04 (d, J = 164.9 Hz), 63.69 (d, J = 6.0 Hz), 63.44, 62.49 (d,
J = 7.2 Hz), 46.11, 45.28, 16.50, 16.41, 16.31, 13.84. Anal. Calcd for
4.3.8. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-5-(2,4-dioxo-3,4-
dihydropyrimidin-1(2H)-yl)-2-methylisoxazolidine-5-
carboxylate 12c
Yield: 23.8%; white solid, mp 178–180 °C. 1H NMR (CDCl3):
d = 8.49 (bs s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 5.79 (d, J = 8.3 Hz, 1H),
C15H24N3O8P: C, 44.45; H, 5,97; N, 10.37. Found: C, 44.41; H,
5.96 N, 10.40.
4.37–4.10 (m, 6H), 4.02 (ddd, J = 14.3, 8.2 and 1.3 Hz, Ha
–C50,
1H), 3.16–3.05 (m, H–C40, and H–C50, 2H), 3.13 (s, 3H), 1.35 (t,
J = 7.2 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t, J = 7.0 Hz, 3H). 13C
NMR (125 MHz, CDCl3): d = 169.33, 166.44, 150.16, 138.88,
101.53, 90.77 (d, J = 12.3 Hz), 64.07 (d, J = 6.0 Hz), 63.14 (d,
J = 176.8 Hz), 63.31, 62.81 (d, J = 7.1 Hz), 47.27, 42.67, 16.41 (d,
J = 4.5 Hz), 14.13, 13.86. Anal. Calcd for C15H24N3O8P: C, 44.45; H,
5,97; N, 10.37. Found: C, 44.47; H, 5.94 N, 10.41.
4.3.4. Ethyl (3SR,5RS)-5-[4-(acetylamino)-2-oxopyrimidin-
1(2H)-yl]-3-(diethoxyphosphoryl)-2-methylisoxazolidine-5-
carboxylate 11d
Yield: 41.27%; yellow sticky oil. 1H NMR (CDCl3): d = 8.91 (bs s,
1H), 8.10 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 7.6 Hz, 1H), 4.22–4.08 (m,
6H), 3.95 (ddd, J = 13.5, 8.4 and 3.4 Hz, Ha
–C50, 1H), 3.28 (ddd,
J = 11.2, 8.4 and 2.9 Hz, H–C40, 1H), 3.07 (s, 3H), 2.92 (ddd,
J = 16.2, 13.5 and 10.3 Hz, Hb–C50, 1H), 2.26 (s, 3H), 1.35 (t,
J = 7.1 Hz, 3H), 1.33 (m, 6H). 13C NMR (125 MHz, CDCl3):
d = 170.96, 164.32, 163.51, 155.18, 143.69, 95.92, 92.84 (d,
J = 10.6 Hz), 65.23 (d, J = 162.6 Hz), 63.61 (d, J = 69 Hz), 63.15,
62.41 (d, J = 6.6 Hz), 46.12, 44.86, 24.76, 16.41 (d, J = 4.4 Hz),
16.30 (d, J = 5.4 Hz), 13.77. Anal. Calcd for C17H27N4O8P: C, 45.74;
H, 6.10; N, 12.55. Found: C, 45.78; H, 6,16 N, 12.53.
4.3.9. Ethyl (3SR,5SR)-5-[4-(acetylamino)-2-oxopyrimidin-
1(2H)-yl]-3-(diethoxyphosphoryl)-2-methylisoxazolidine-5-
carboxylate 12d
Yield: 19.84%; yellow sticky oil. 1H NMR (CDCl3): d = 8.89 (bs s,
1H), 7.75 (d, J = 7.3 Hz, 1H), 7.52 (d, J = 7.3 Hz, 1H), 4.27–4.03 (m,
6H), 4.02 (ddd, J = 13.9, 7.8 and 1.7 Hz, Ha
–C50, 1H), 3.33–3.22
(m, H–C40, and H–C50, 2H), 3.08 (s, 3H), 2.24 (s, 3H), 2.24 (s, 3H),
1.29 (t, J = 7.2 Hz, 3H), 1.27 (t, J = 7.2 Hz, 3H), 1.25 (t, J = 7.2 Hz,
3H). 13C NMR (125 MHz, CDCl3): d = 170.16, 164.30, 162. 22,
155.10, 143.87, 113.88, 93..82 (d, J = 11.9 Hz), 65.85 (d,
J = 105.0 Hz), 63.53 (d, J = 6.0 Hz), 63.21, 62.42 (d, J = 6.5 Hz),
46.17, 44.87, 24.95, 16.39 (d, J = 6.3 Hz), 16.41, 13.96 (d,
J = 16.0 Hz). Anal. Calcd for C17H27N4O8P: C, 45.74; H, 6.10; N,
12.55. Found: C, 45.70; H, 6.14; N, 12.58.
4.3.5. Ethyl (3SR,5RS)-5-[4-amino-2-oxopyrimidin-1(2H)-yl]-3-
(diethoxyphosphoryl)-2-methylisoxazolidine-5-carboxylate 11e
Yield: 41.42%; yellow sticky oil. 1H NMR (CDCl3): d = 8.15 (d,
J = 7.5 Hz, 1H), 7.50 (d, J = 7.5 Hz, 1H), 6.70 (bs s, 1H), 4.20–4.08
(m, 6H), 4.01 (ddd, J = 13.4, 8.2 and 3.4 Hz, Ha
–C50, 1H), 3.30
(ddd, J = 11.2, 8.2 and 3.0 Hz, H–C40, 1H), 3.02(s, 3H), 2.80 (ddd,
J = 16.2, 13.4 and 10.3 Hz, Hb–C50, 1H), 1.35 (t, J = 7.1 Hz, 3H),
1.29 (m, 6H). 13C NMR (125 MHz, CDCl3): d =165.06, 163.50,
154.89, 142.12, 96.28, 94.544 (d, J = 10.4 Hz), 66.02 (d,
J = 162.4 Hz), 64.05 (d, J = 67 Hz), 63.14, 62.38 (d, J = 6.4 Hz),
48.89, 46.85, 15.89 (d, J = 4.4 Hz), 16.22 (d, J = 5.5 Hz), 12.70. Ana-
l.Calcd for C15H25N4O7P: C, 44.56; H, 6.20, N, 13.88. Found: C,
44.59; H, 6.29 N, 13.84.
4.4. Biological assay
The compounds were tested for their inhibitory activity on
HTLV-1 and HIV infection. HTLV-1 infection was carried out as pre-
viously shown.26 Peripheral blood mononuclear cells were
co-cultivated with a HTLV-1 chronically infected cell line and
infection was evaluated as production of the viral core protein
p19. HIV infection was carried on by using a stable T cell line
(CEM) containing a plasmid encoding a green fluorescence protein
(GFP) driven by the HIV-1 long terminal repeat.27 Infection was
carried on as previouslyb shown with some modification.28 Briefly,
5 ꢂ 105 CEM-GFP were infected with a volume of supernatant from
HIV chronically infected H9 cells equivalent to 20 ng/mL of HIV
4.3.6. Ethyl (3SR,5SR)-3-(diethoxyphosphoryl)-2-methyl-5-(5-
methyl-2,4-dioxo-3,4-dihydro-pyrimidin-1(2H)-yl)isoxazolidi-
ne-5-carboxylate 12a
Yield: 28.8%; white solid, mp 198–200 °C. 1H NMR (CDCl3):
d = 8.23 (br s, NH, 1H), 7.61 (br q, J = 0.9 Hz, CH@, 1H), 4.30–4.11
(m, 6H), 3.84 (ddd, J = 15.0, 10.2 and 2.8 Hz, Ha
–C50, 1H), 3.15–
3.08 (m, H–C40 and Hb–C50, 2H), 3.13 (s, 3H), 1.97 (d, J = 0.9 Hz,
3H), 1.34 (t, J = 7.0 Hz, 3H), 1.26 (t, J = 7.0 Hz, 3H), 1.24 (t,
p24, for 2 h in 100
ll CM in presence of 1000, 100, 10 and 1 lM
concentration of compounds. Then medium was added and the