CHEMMEDCHEM
FULL PAPERS
25.80 mmol) in anhydrous CH3CN (80 mL). The reaction mixture
was heated at 658C for 21 h, after which it was allowed to cool to
room temperature, poured into a saturated solution of NaHCO3
(200 mL), and extracted with CH2Cl2 (3ꢁ100 mL). The combined or-
ganic phase was dried over Na2SO4 and concentrated. The residue
was crystallized from MeOH to give the pure b-anomer as a white
solid (17%, 0.74 g); 19F NMR (DMSO, 470 MHz): d=ꢂ187.82 ppm;
1H NMR (DMSO, 500 MHz): d=8.72 (1H, s, H-8), 8.06–7.50 (10H, m,
Ph), 6.87–6.85 (1H, m, H-1’), 6.72 (2H, bs, NH2), 6.07–5.98 (1H, m,
H-2’), 5.79–5.75 (1H, m, H-3’), 5.31–5.29 (1H, m, H-4’), 4.68–
4.66 ppm (2H, m, H-5’); 13C NMR (DMSO, 126 MHz): d=63.70 (C-5’),
76.47 (d, JC-F =30.5 Hz, C-3’), 83.80 (C-4’), 90.15 (d, JC-F =36.1 Hz, C-
1’), 97.42 (d, JC-F =184.9 Hz, C-2’), 114.39 (C-5), 128.62, 128.78,
129.23, 129.29, 129.44, 133.59, 133.83 (Ph, “ipso” Ph), 142.63 (C-4),
146.41 (C-8), 160.14 (C-6), 164.44 (C-2), 164.69, 165.45 ppm (COPh).
Standard procedure B: synthesis of phosphoramidates (9a–w)
tBuMgCl (1.0m in THF, 1.20–2.00 molequiv) was added to a suspen-
sion/solution of the appropriate nucleoside (1.00 molequiv) in an-
hydrous THF (10 mL) under an argon atmosphere and stirred at
room temperature for 30 min. The appropriate phosphorochlori-
date (2.00 molequiv) dissolved in anhydrous THF was added drop-
wise, and the reaction mixture was stirred at room temperature
overnight. The solvent was removed under reduced pressure, and
the crude residue was purified by flash column chromatography
(gradient elution of CH2Cl2/MeOH) to give the desired product.
Standard procedure C: synthesis of phosphoramidates
(16a–c)
Synthesis of 6-O-methyl-2’-b-fluoro-2’-deoxyguanosine-5’-O-
naphthyl(methoxy-l-alaninyl)phosphate (16a): Prepared accord-
ing to standard procedure C, using 10 (0.19 g, 0.62 mmol), 8b
(0.61 g, 1.86 mmol), NMI (0.25 mL, 3.10 mmol) in anhydrous THF
(10 mL), and anhydrous pyridine (3 mL); the reaction mixture was
stirred at room temperature overnight. After this period 8b (0.31 g,
0.93 mmol) and NMI (0.15 mL, 1.86 mmol) were added, and the re-
action mixture was stirred at room temperature overnight. The
crude was purified by column chromatography (gradient elution of
CH2Cl2/MeOH 98:2, then 96:4). The product was further purified by
preparative TLC (gradient elution of CH2Cl2/MeOH 98:2, then 96:4)
to give a white solid (6%, 0.02 g); 31P NMR (MeOD, 202 MHz): d=
4.10, 3.96 ppm; 19F NMR (MeOD, 470 MHz): d=ꢂ192.52,
NMI (5.00–10.00 molequiv) was added dropwise to a stirring sus-
pension/solution of the appropriate nucleoside (1.00 molequiv)
and phosphorochloridate (3.00 molequiv) in anhydrous THF (10–
20 mL), and the reaction was stirred at room temperature over-
night. The solvent was removed under reduced pressure, and the
crude residue was purified by flash column chromatography (gradi-
ent elution of CH2Cl2/MeOH) and in some cases by preparative TLC
to give the desired product.
Synthesis of 6-O-methyl-2’-fluoro-2’-deoxyguanosine-5’-O-naph-
thyl(methoxy-l-alaninyl)phosphate (9b): Prepared according to
standard procedure B, using 2 (0.20 g, 0.67 mmol) in anhydrous
THF (10 mL), tBuMgCl (1.0m solution THF, 0.80 mL, 0.80 mmol), and
8b (0.44 g, 1.34 mmol); the reaction mixture was stirred at room
temperature overnight. After this period tBuMgCl (1.0m in THF,
0.80 mL, 0.80 mmol) was added, and the reaction mixture was
stirred at room temperature for a further 2 h. The crude was puri-
fied by column chromatography (gradient elution of CH2Cl2/MeOH
98:2, then 96:4). The product was further purified by preparative
TLC (gradient elution of CH2Cl2/MeOH 98:2, then 96:4) to give
a white solid (2%, 0.01 g); 31P NMR (MeOD, 202 MHz): d=4.22,
4.13 ppm; 19F NMR (MeOD, 470 MHz): d=ꢂ204.83, ꢂ205.48 ppm;
1H NMR (MeOD, 500 MHz): d=8.12–8.06 (1H, m, H-8 Naph), 7.96,
7.94 (1H, 2 s, H-8), 7.88–7.33 (6H, m, NaphO), 6.21–6.17 (1H, m, H-
1’), 5.54–5.39 (1H, m, H-2’), 4.93–4.82 (1H, m, H-3’), 4.56–4.39 (2H,
m, H-5’), 4.26–4.27 (1H, m, H-4’), 4.02, 4.00 (3H, 2 s, OCH3), 3.99–
3.88 (1H, m, CHCH3), 3.57, 3.54 (3H, 2 s, COOCH3), 1.28, 1.22 ppm
(3H, 2d, J=7.0 Hz, CHCH3); 13C NMR (MeOD, 126 MHz): d=20.24 (d,
1
ꢂ192.72 ppm; H NMR (MeOD, 500 MHz): d=8.21–7.43 (8H, m, H-
8, NaphO), 6.25–6.20 (1H, m, H-1’), 5.44–5.32 (1H, m, H-2’), 4.57–
4.55 (1H, m, H-4’), 4.50–4.43 (1H, m, H-3’), 4.41–4.28 (2H, m, H-5’),
4.13–4.09 (1H, m, CHCH3), 4.08, 4.06 (3H, 2 s, OCH3), 3.65, 3.63 (3H,
2 s, COOCH3), 1.37–1.34 ppm (3H, m, CHCH3); 13C NMR (MeOD,
126 MHz): d=20.36 (d, JC-P =7.3 Hz, CH3-Ala), 20.44 (d, JC-P =6.6 Hz,
CH3-Ala), 51.63, 51.72 (CH-Ala), 52.73, 52.77 (COOCH3), 54.59, 54.82
(OCH3), 67.12 (d, JC-P =5.4 Hz, C-5’), 67.29 (d, JC-P =5.4 Hz, C-5’),
74.90 (d, JC-F =24.5 Hz, C-3’), 75.00 (d, JC-F =24.5 Hz, C-3’), 85.82,
85.88, 85.92, 85.96, 85.98, 86.02 (C-4’), 91.13 (d, JC-F =35.4 Hz, C-1’),
91.22 (d, JC-F =35.3 Hz, C-1’), 101.00 (d, JC-F =187.2 Hz, C-2’), 101.02
(d, JC-F =187.2 Hz, C-2’), 116.30 (d, JC-P =3.4 Hz, C-2 Naph), 116.40 (d,
J
C-P =3.5 Hz, C-2 Naph), 118.79 (C-5), 122.69, 125.81, 126.01, 126.04,
126.51, 127.41, 127.48, 127.76, 127.81, 127.94, 128.85, 128.87 (C-3
Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph, C-8 Naph, C-8a
Naph), 136.31 (C-4a Naph), 145.16, 145.22 (C-8), 148.00, 148.03,
148.06, 148.08 (‘ipso’ Naph), 159.13 (C-4), 162.00 (C-2), 164.82 (C-6),
J
C-P =6.8 Hz, CH3-Ala), 20.42 (d, JC-P =6.4 Hz, CH3-Ala), 51.50, 51.53
(CH-Ala), 52.63, 52.72 (COOCH3), 54.16 (OCH3), 66.73 (d, JC-P =4.9 Hz,
C-5’), 67.12 (d, JC-P =5.2 Hz, C-5’), 70.28 (d, JC-F =16.5 Hz, C-3’), 70.44
(d, JC-F =17.5 Hz, C-3’), 82.66, 82.72 (C-4’), 88.26 (d, JC-F =33.8 Hz, C-
1’), 88.37 (d, JC-F =34.6 Hz, C-1’), 94.43 (d, JC-F =187.0 Hz, C-2’), 94.46
(d, JC-F =186.7 Hz, C-2’), 115.63 (C-5), 116.08, 116.12, 122.65, 122.69,
125.91, 126.44, 127.43, 127.46, 127.72, 127.84, 128.77, 128.81 (C-2
Naph, C-3 Naph, C-4 Naph, C-5 Naph, C-6 Naph, C-7 Naph, C-8
Naph, C-8a Naph), 136.23 (C-4a Naph), 139.65 (C-8), 147.89, 147.84
(“ipso” Naph), 154.40 (C-4), 161.96 (C-2), 162.66 (C-6), 175.28,
174.48 ppm (COOCH3); ESMS: 591.18 [M+H]+, 613.16 [M+Na]+,
629.14 [M+K]+; HRMS (C25H29N6O8PF) calcd: 591.1796, found:
591.1796; RP-HPLC (flow=1 mLminꢂ1, l=254 nm), eluting with
H2O/MeOH from 90:100 to 0:100 over 30 min, tR =21.81, 22.36 min,
eluting with H2O/CH3CN from 90:100 to 0:100 over 30 min, tR =
14.27, 14.63 min.
175.30 (d,
JC-P =5.2 Hz, COOCH3), 175.60 ppm (d, JC-P =5.0 Hz,
COOCH3); ESMS: 589.17 [MꢂH]+, 625.14 [M+Cl]ꢂ; RP-HPLC (flow=
1 mLminꢂ1, l=254 nm), eluting with H2O/MeOH from 90:100 to
0:100 in 30 min, tR =18.76 min.
Biological experiments
Isolation of viral ribonucleoprotein complexes and RNA elongation
assay: The detailed procedure for isolation of the viral vRNP com-
plexes from disrupted virions, which is based on published proce-
dures,[25,26] has been described elsewhere.[20] Briefly, influenza A/X-
31 virus was pelleted from an allantoic stock by ultracentrifugation,
and, after detergent disruption of the viral pellet, vRNP complexes
were purified by gradient ultracentrifugation. Collected fractions
were subjected to western blot analysis with an anti-PB2 antibody,
and the fractions containing the vRNP complexes were pooled, dia-
lyzed against storage buffer, and stored at ꢂ808C.
Synthesis of 2-amino-6-chloro-9-(2-deoxy-2-fluoro-3,5-di-O-ben-
zoyl-b-d-arabinofuranosyl)-9H-purine (15): TMSOTf (6.24 mL,
34.40 mmol) was added dropwise to a precooled (08C) solution of
13 (4.00 g, 8.60 mmol), 14 (1.60 g, 9.46 mmol), and DBU (3.86 mL,
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 12
&
9
&
ÞÞ
These are not the final page numbers!