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M. Quintiliani et al. / Bioorg. Med. Chem. 19 (2011) 4338–4345
C19H22N3O9F2PI required 632.0107, found 632.0123. HPLC (H2O/
MeOH from 90/10 to 0/100 in 30 min): tR = 18.92, 18.93 min.
tion of H2O/MeOH from 90/0 to 0/100 in 30 min) to give a white
solid (0.0214 g, 7%). 31P NMR (MeOD, 202 MHz): d 3.89, 3.69. 19F
NMR (MeOH, 471 MHz): d ꢀ117.61 (d, J = 241.5 Hz), ꢀ117.73 (d,
J = 243.1 Hz), ꢀ119.71 (broad), ꢀ120.21 (broad). 1H NMR (MeOD,
500 MHz): d 7.94, 7.90 (2s, 1H, H-6), 7.40–7.20 (m, 10H, PhO,
OCH2Ph), 6.16–6.12 (m, 1H, H-10), 5.19–5.13 (m, 2H, OCH2Ph),
4.51–4.22 (m, 3H, H-50, H-30), 4.13–4.02 (m, 2H, H-40, CH-Ala),
1.45–1.34 (m, 3H, CH3-Ala). 13C NMR (MeOD, 126 MHz): d 20.31
(d, JC-P = 7.1 Hz, CH3-Ala), 20.44 (d, JC-P = 6.5 Hz, CH3-Ala), 51.70,
51.82 (CH-Ala), 65.54 (d, JC-P = 4.4 Hz, C-50), 65.90 (d, JC-P = 4.3 Hz,
C-50), 68.05 (OCH2Ph), 70.79, 70.96, 71.01, 71.06, 71.17, 71.22,
71.28, 71.44 (C-30), 80.92 (C-40), 85.58, 85.98 (C-10), 98.19 (CBr),
121.47, 121.51, 121.56 (Ph), 123.33 (t, JC-F = 259.4 Hz, CF2),
126.29, 126.31, 129.26, 129.38, 129.39, 129.62, 130.83 (Ph),
137.19, 137.23 (‘ipso’ OCH2Ph), 140.91, 141.12 (C-6), 151.14 (‘ipso’
Ph), 152.04, 152.09 (C-2) 160.97 (C-4), 174.62 (d, JC-P = 4.9 Hz, C@O
ester), 174.79 (d, JC-P = 4.4 Hz, C@O ester). MS (ES+) m/z: 682.04
(M+Na); Accurate Mass: C25H25N3O9F2NaPBr required 682.0378,
found 682.0390. HPLC (H2O/MeOH from 90/10 to 0/100 in
30 min): tR = 23.72, 24.21 min.
4.1.19. 20-Deoxy-20-fluoro- -5-iodouridine 50-O-naphthyl-
D
(benzoxy-alaninyl)-phosphate (18)
Prepared according to standard procedure B, from 8 (0.1527 g,
0.39 mmol), 14 (0.4742 g, 1.17 mmol), anhydrous NMI (0.15 mL,
1.95 mmol) and anhydrous THF (10 mL). The crude was purified
by column chromatography (DCM/MeOH 98:2). The product was
further purified by preparative reverse phase HPLC (gradient elu-
tion of H2O/MeOH from 90/0 to 0/100 in 30 min) to give a white
solid (0.0171 g, 6%). 31P NMR (MeOD, 202 MHz): d 4.15, 4.01. 19F
NMR (MeOH, 471 MHz): d ꢀ117.31 (d, J = 242.7 Hz), ꢀ117.44 (d,
J = 242.1 Hz), ꢀ119.53 (broad). 1H NMR (MeOD, 500 MHz): d
8.21, 8.19 (2s, 1H, H-6), 7.98–7.26 (m, 12H, NaphO, OCH2Ph),
6.14–6.03 (m, 1H, H-10), 5.13–5.08 (m, 2H, OCH2Ph), 4.55–4.36
(m, 2H, H-50), 4.34–4.20 (m, 1H, H-30), 4.18–4.05 (m, 2H, H-40,
CH-Ala), 1.40–1.32 (m, 3H, CH3-Ala). 13C NMR (MeOD, 126 MHz):
d 20.37 (d, JC-P = 7.3 Hz, CH3-Ala), 20.47 (d, JC-P = 6.7 Hz, CH3-Ala),
51.85, 51.87 (CH-Ala), 66.02 (d, JC-P = 4.8 Hz, C-50), 66.19 (d, JC-
P = 4.9 Hz, C-50), 68.06 (OCH2Ph), 69.81, 69.85 (C–I), 71.05, 71.23,
71.43, 71.61 (C-30), 79.47 (C-40), 80.91 (C-10), 116.26, 116.28,
116.42, 116.45, 122.69, 122.87 (Ph, Naph), 123.30 (CF2, t,
JC-F = 259.3 Hz), 126.12, 126.53, 126.55, 127.51, 127.56, 127.81,
127.84, 127.89, 127.94, 128.89, 128.93, 129.28, 129.36, 129.59,
136.33, 136.35 (Ph, Naph), 137.12, 137.15 (‘ipso’ OCH2Ph), 146.07,
146.22 (C-6), 147.90, 147.95 (‘ipso’ Naph), 151.51, 151.53 (C-2),
162.20 (C-4), 174.58 (d, JC-P = 4.8 Hz, C=O ester), 174.78 (d,
JC-P = 4.4 Hz, C=O ester). MS (ES+) m/z: 780.04 (M+Na); Accurate
Mass: C29H27N3O9F2NaPI required 780.0395, found 780.0416. HPLC
(H2O/MeOH from 90/10 to 0/100 in 30 min): tR = 26.68, 26.89 min.
4.1.22. 20-Deoxy-20,20-difluoro- -5-chlorouridine 50-O-phenyl-
D
(benzoxy-alaninyl)-phosphate (21)
Prepared according standard procedure B, from 10 (0.1491 g,
0.50 mmol), 12 (0.5295 g, 1.50 mmol), anhydrous NMI (0.2 mL,
2.49 mmol) and anhydrous THF (10 mL). The crude was purified
by column chromatography (DCM/MeOH 98:2). The product was
further purified by preparative reverse phase HPLC (gradient elu-
tion of H2O/MeOH from 90/0 to 0/100 in 30 min) to give a white
solid (0.0118 g, 4%). 31P NMR (MeOD, 202 MHz): d 3.92, 3.73. 19F
NMR (MeOH, 471 MHz): d ꢀ117.75 (d, J = 234.1 Hz), ꢀ117.92 (d,
J = 239.3 Hz), ꢀ119.83 (broad), ꢀ120.34 (broad). 1H NMR (MeOD,
500 MHz): d 7.86, 7.83 (2s, 1H, H-6), 7.45–7.19 (m, 10H, PhO,
OCH2Ph), 6.20–6.11 (m, 1H, H-10), 5.23–5.11 (m, 2H, OCH2Ph),
4.53–4.21 (m, 3H, H-50, H-30), 4.14–4.00 (m, 2H, H-40, CH-Ala),
1.47–1.34 (m, 3H, CH3-Ala). 13C NMR (MeOD, 126 MHz): d 20.30
(d, JC-P = 7.0 Hz, CH3-Ala), 20.41 (d, JC-P = 6.5 Hz, CH3-Ala), 51.70,
51.83 (CH-Ala), 65.54 (d, JC-P = 4.6 Hz, C-50), 65.88 (d, JC-P = 4.8 Hz,
C-50), 68.04 (OCH2Ph), 70.74, 70.89, 70.98, 71.02, 71.13, 71.40 (C-
30), 80.89 (C-40), 85.51, 85.71 (C-10), 110.36 (C–Cl), 121.45,
121.48, 121.52 (Ph), 123.33 (t, JC-F = 259.7 Hz, CF2), 126.29,
129.25, 129.37, 129.61, 130.82 (Ph), 137.21, 137.23 (0ipso’ OCH2Ph),
138.34, 138.56 (C-6), 151.00 (‘ipso’ Ph), 152.04, 152.09 (C-2),
160.99 (C-4), 174.61 (d, JC-P = 4.9 Hz, C@O ester), 174.80 (d,
JC-P = 4.3 Hz, C@O ester). MS (ES+) m/z: 638.09 (M+Na); Accurate
Mass: C25H25N3O9F2NaPCl required 638.0883, found 638.0898.
HPLC (H2O/MeOH from 90/10 to 0/100 in 30 min): tR = 24.33,
24.72 min.
4.1.20. 20-Deoxy-20-fluoro- -5-iodouridine 50-O-naphthyl-
D
(methoxy-alaninyl)-phosphate (19)
Prepared according to standard procedure B, from 8 (0.2000 g,
0.51 mmol), 15 (0.5040 mg, 1.54 mmol), anhydrous NMI
(0.61 mL, 7.70 mmol,) and anhydrous THF (10 mL). The crude
was purified by column chromatography (DCM/MeOH 98:2). The
product was further purified by preparative reverse phase HPLC
(gradient elution of H2O/MeOH from 90/0 to 0/100 in 30 min) to
give a white solid (0.0044 g, 1%). 31P NMR (MeOD, 202 MHz): d
4.17, 4.08. 19F NMR (MeOH, 471 MHz):
d
ꢀ117.30 (d,
J = 241.1 Hz), ꢀ117.44 (d, J = 237.5 Hz), ꢀ119.38 (broad). 1H NMR
(MeOD, 500 MHz): d 8.22, 8.21 (2s, 1H, H-6), 8.03–7.42 (m, 7H, Na-
phO), 6.17–6.07 (m, 1H, H-10), 4.61–4.41 (m, 2H, H-50), 4.40–4.25
(m, 1H, H-30), 4.16–4.02 (m, 2H, H-40, CH-Ala), 3.64, 3.64 (2s, 3H,
OCH3), 1.38–1.30 (m, 3H, CH3-Ala). 13C NMR (MeOD, 126 MHz): d
20.36 (d, JC-P = 7.2 Hz, CH3-Ala), 20.48 (d, JC-P = 6.5 Hz, CH3-Ala),
51.71, 51.80 (CH-Ala), 52.81 (OCH3), 65.65, 65.95 (C-50), 69.83
(C–I), 70.92, 71.06, 71.06 (C-30), 80.85 (C-40), 85.70 (C-10), 116.24,
116.38, 122.59, 122.67, 123.42 (Naph), 123.33 (t, JC-F = 259.4 Hz,
CF2), 126.18, 126.58, 127.26, 127.56, 127.66, 127.82, 127.90,
127.93, 128.52, 128.96, 128.98, 136.20, 136.34 (Naph), 146.06,
146.18 (C-6), 147.90, 147.95 (‘ipso’ Naph), 152.02, 152.10 (C-2),
162.24 (C-4), 174.62, 175.78 (C=O ester). MS (ES+) m/z: 704.01
(M+Na); Accurate Mass: C23H23N3O9F2NaPI required 704.0082,
found 704.0110. HPLC (H2O/MeOH from 90/10 to 0/100 in
30 min): tR = 23.03, 23.27 min.
4.2. Antiviral assays
The antiviral assays [except anti-human immunodeficiency
virus (HIV) assays] were based on inhibition of virus-induced cyto-
pathicity in HEL [herpes simplex virus type 1 (HSV-1), HSV-2 (G),
vaccinia virus and vesicular stomatitis virus], Vero (parainflu-
enza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus),
HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respira-
tory syncytial virus), MDCK (influenza virus A and B) and CRFK (fe-
line corona virus and feline herpes virus) cell cultures. Confluent
cell cultures in microtiter 96-well plates were inoculated with
100 cell culture infective dose-50 (CCID50) of virus (1 CCID50 being
the virus dose to infect 50% of the cell cultures) in the presence of
4.1.21. 20-Deoxy-20,20-difluoro- -5-bromouridine 50-O-phenyl-
D
(benzoxy-alaninyl)-phosphate (20)
Prepared according to standard procedure B, from 9 (0.1660 g,
0.48 mmol), 12 (0.5135 g, 1.45 mmol), anhydrous NMI (0.19 mL,
2.4 mmol) and anhydrous THF (10 mL). The crude was purified
by column chromatography (DCM/MeOH 98:2). The product was
further purified by preparative reverse phase HPLC (gradient elu-
varying concentrations (200, 40, 8, . . . lM) of the test compounds.
Viral cytopathicity was recorded as soon as it reached completion
in the control virus-infected cell cultures that were not treated
with the test compounds. The methodology of the anti-HIV assays