The design, synthesis, and antiviral activity of monofluoro and difluoro analogues of 4′-azidocytidine against hepatitis C virus replication: The discovery of 4′-azido-2′-deoxy-2′-fluorocytidine and 4′-azido-2′-dideoxy-2′,2′-difluorocytidine

Article abstract of DOI:10.1021/jm801595c

The discovery of 4′-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett. 2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC50 = 1.28 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the

Full text of DOI:10.1021/jm801595c

J. Med. Chem. 2009, 52, 2971–2978
2971
The Design, Synthesis, and Antiviral Activity of Monofluoro and Difluoro Analogues of
4′-Azidocytidine against Hepatitis C Virus Replication: The Discovery of
4′-Azido-2′-deoxy-2′-fluorocytidine and 4′-Azido-2′-dideoxy-2′,2′-difluorocytidine
David B. Smith,† Genadiy Kalayanov,‡ Christian Sund,‡ Anna Winqvist,‡ Tatiana Maltseva,‡ Vincent J.-P. Leveque,†
Sonal Rajyaguru,† Sophie Le Pogam,† Isabel Najera,† Kurt Benkestock,‡ Xiao-Xiong Zhou,‡ Ann C. Kaiser,† Hans Maag,†
Nick Cammack,† Joseph A. Martin,† Steven Swallow,† Nils Gunnar Johansson,‡ Klaus Klumpp,† and Mark Smith*,†
Roche Palo Alto LLC, 3431 HillView AVenue, Palo Alto, California 94304, MediVir AB, Department of Medicinal Chemistry, Lunastigen 7,
SE-141 44 Huddinge, Sweden
ReceiVed December 17, 2008
The discovery of 4′-azidocytidine (3) (R1479) (J. Biol. Chem. 2006, 281, 3793; Bioorg. Med. Chem. Lett.
2007, 17, 2570) as a potent inhibitor of RNA synthesis by NS5B (EC50 ) 1.28 µM), the RNA polymerase
encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of several monofluoro
and difluoro derivatives of 4′-azidocytidine. The most potent compounds in this series were 4′-azido-2′-
deoxy-2′,2′-difluorocytidine and 4′-azido-2′-deoxy-2′-fluoroarabinocytidine with antiviral EC50 of 66 nM
and 24 nM in the HCV replicon system, respectively. The structure-activity relationships within this series
were discussed, which led to the discovery of these novel nucleoside analogues with the most potent
compound, showing more than a 50-fold increase in antiviral potency as compared to 4′-azidocytidine (3).
Introduction
Hepatitis C virus (HCV) is a causative agent of chronic liver
disease, estimated to affect over 170 million people worldwide.3
Although often asymptomatic, HCV infection can progress to
fibrosis, reduced liver function, and hepatocellular carcinoma.
Currently, the standard treatment for HCV infection involves
treatment with pegylated R interferon in combination with the
nucleoside analogue ribavirin. This treatment regimen effects
a response in approximately 40-60% of the genotype-1 (GT-
1) population.4 Considering the limitations in efficacy and the
adverse event profile associated with the current standard of
care treatment, there remains a significant unmet clinical need
for the development of new antiviral medication, specific for
HCV activity of monofluoro and difluoro analogues of 4′-
azidocytidine.
HCV. Recently, a number of nucleoside analogues have been
reported to inhibit HCV replication. Among these, 2′-deoxy-
2′-C-methylcytidine (1),5 2′-deoxy-2′-fluoro-2′-C-methylcytidine
(2),6 and 4′-azido-cytidine (3)1,2 have shown most promise and
progressed into clinical development for the treatment of HCV
infection. The identification of 4′-substituted ribonucleosides as
potential inhibitors of HCV replication culminating in the
selection of 3 as a clinical candidate suggested that further
exploration of the SAR may allow further optimization of
phosphorylation efficiency and antiviral potency.1,2 Indeed, 4′-
azido-arabinocytidine (4) (RO-9187) was later identified as an
inhibitor of HCV replication with improved potency as com-
pared to 3, related to improved phosphorylation efficiency
(NS5B EC50 ) 171 nM).7,8
Chemistry
The synthesis of 4′-azido-2′-deoxy-2′-fluorocytidine (10) is
outlined in Scheme 1. Thus, treatment of 4′-azidouridine (5)8
with diphenylcarbonate, according to Reese’s protocol, yielded
6 in 85%.12 Protection with 4-methoxy-3,6-dihydro-2H-pyran,
followed by hydrolysis with NaOH in EtOH, provided the
arabino-derivative 8 in 56% yield over two steps. Fluorination
with (diethylamino)sulfur trifluoride (DAST) furnished 9 as a
single isomer in 56% yield. Standard conversion from uridine
to cytidine was conducted with 1H-tetrazole and 4-chlorophe-
nyldichlorophosphate followed by ammonia. Deprotection with
acetic acid provided 10 in 26% yield over two steps.
4′-Azido-2′-deoxy-2′-fluoroarabinocytidine (17) was prepared
as illustrated in Scheme 2. Thus, treatment of 11 with iodine
and triphenylphosphine and imidazole in THF gave the iodide
12 in 76% yield. Elimination of the hydrogen iodide was
performed by sodium methoxide to furnish 13 in 82%. Treat-
ment of 13 with iodine and benzyltriethylammonium azide,
prepared from the corresponding chloride and sodium azide in
4-methylmorpholine (MMP) and THF, provided 14 in 93% yield
as a single isomer. Oxidative nucleophilic substitution of the
Further work focused on the assessment of fluorinated
derivatives, on the carbohydrate moiety, of 4′-azidocytidine.
Fluoronucleosides have a history of being well phosphorylated
by cellular kinases and can be good substrates for RNA and
DNA polymerases.6,9-11 Here we discuss the synthesis and anti-
* To whom correspondence should be addressed. Phone: +1 650 855
5076. Fax: +1 650 852 1132. E-mail: Mark.Smith@Roche.com.
†
Roche Palo Alto LLC.
Medivir AB.
‡
10.1021/jm801595c CCC: $40.75
2009 American Chemical Society
Published on Web 04/02/2009

2972 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Smith et al.
Scheme 1a
23 cell line as described previously.2,8 It was also determined
if any of the compounds were cytotoxic or inhibited cell
proliferation, an indication of potentially reduced selectivity
against human polymerases. Cell viability was measured using
the WST-1 assay (Roche), and cell proliferation was measured
by quantification of the incorporation of tritiated thymidine into
cellular DNA using the [3H]-thymidine incorporation scintilla-
tion proximity assay (Amersham Biosciences).5-8 Results are
summarized in Table 1.
None of the compounds tested showed significant cytotoxic
or cytostatic properties with the exception of the control
compound, 2′deoxy-2′-fluoroarabinocytidine (dFAraC), which
was a potent inhibitor of cell proliferation inhibiting tritiated
thymidine incorporation with an IC50 value of 174 nM.
Introduction of the 4′-azido moiety into dFAraC resulted in 4′-
azido-2′-deoxy-2′-fluoroarabinocytidine (17), which did not
show any measurable inhibition of cell proliferation. The
introduction of the 4′-azido moiety therefore increased the
selectivity of 17 by over 4000-fold as compared to dFAraC. In
addition, 17 showed exceptionally high antiviral potency in the
HCV replicon system. With an antiviral IC50 value of 24 nM,
the potency of 17 was improved more than 50-fold as compared
to 4′-azido-cytidine and 7-fold as compared to 4′-azido-
arabinocytidine 4.7 Unexpectedly, the lack of a 2′-hydroxy
group, in the ribo configuration, was not only acceptable for
binding and incorporation by the HCV RNA polymerase, but
such 2′-deoxy-nucleoside analogues could become highly potent
without apparent effects on cell viability and proliferation in
the HCV replicon system. Interestingly, inversion of the 2′-
fluoro group to form 4′-azido-2′-deoxy-2′-fluorocytidine (10)
reduced potency by >150-fold as compared to 17. It has not
yet been determined if this reduction in potency is due to
reduced phosphorylation efficiency of 10 or whether incorpora-
tion efficiencies by HCV polymerase are different between 10
and 17 triphosphates. The related compound 2′-deoxy-2-
fluorocytidine (FdC) has previously been characterized in the
HCV replicon system. FdC was found to be a potent inhibitor
of cell proliferation with an IC50 of 0.8 µM.6 Therefore the
introduction of the 4′-azido moiety into FdC to form 10 also
abolished cell proliferation inhibition and increased selectivity
by more than 125-fold.
a (a) (PhO)2CO, NaHCO3, DMF, ∆; (b) CSA, MTHP; (c) NaOH, EtOH;
(d) DAST, pyridine, CH2Cl2; (e) tetrazole, Cl2P(O)C6H4Cl, Et3N, MeCN;
(f) NH3, dioxane; (g) AcOH.
5′-iodine with m-chlorobenzoic acid/m-chloroperoxybenzoic
acid, followed by deprotection and acetylation, gave 15 in 53%
yield. Protection of the 3′,5′-hydroxy groups followed by
standard conversion from uridine to cytidine, via the method
developed by Divakar and Reese, furnished 17 in 88% yield
over two steps.12
Synthesis of 3′-deoxy-3′-fluoroxylocytidine (20) is outlined
in Scheme 3. Herein, protection of 5 by treatment with
triphenylmethyl chloride in pyridine at 100 °C for 3 days gave
18 as the single product in 55% after chromatographic separa-
tion. Fluorination of 18 was achieved by heating with DAST
and pyridine in dichloromethane for 18 h to furnish 19 in 31%
yield. Conversion of 19 to cytidine via the triazole method,12
followed by deprotection with Amberlyst 15 (H+) ion-exchange
resin in MeOH, provided 20 in 47% yield.
4′-Azido-3′-deoxy-3′-fluorocytidine (28) was prepared in an
analogous manner to 17 and is outlined in Scheme 4. Thus,
3′-deoxy-3′-fluorouridine (21), treated with iodine and triph-
enylphosphine and imidazole in THF, gave the iodide 22 in
93% yield. Subsequent elimination by sodium methoxide
provided 23 in 92% yield. Treatment of 23 with iodine and
benzyltriethylammonium azide furnished a 1:1 mixture of
diastereomers that were separated by column chromatography
to give the desired isomer 24 in 31% yield. This lack of
selectivity has previously been observed with a 3′-deoxyuridine
substrate.8 Oxidative nucleophilic substitution of 5′-iodine with
m-chlorobenzoic acid/m-chloroperoxybenzoic acid, followed by
benzoylation, gave 26 in 98% yield. Standard conversion from
uridine to cytidine furnished 28 in a yield of 53% over two
steps.12
Treatment of N-benzoylgemcitabine, prepared according to
the literature13 with iodine and triphenylphosphine, yielded 30
in 90% yield. Elimination followed by protection with 4-meth-
oxybenzoyl chloride and treatment with iodine and benzyltri-
ethylammonium azide gave 32 60% yield over three steps.
Nucleophilic substitution of 5′-iodine 32 with benzoate, followed
by deprotection, led to 34 in 35% yield (Scheme 5).
Gemcitabine is a highly cytotoxic cytidine analogue and
approved for the treatment of several cancer indications,
including nonsmall cell lung cancer and pancreatic cancer. CC50
values for gemcitabine in human hepatoma cells have been
reported to be in the nM range.14,15 As the 2′-difluoro motif
was accepted by kinases and polymerases, it was of interest to
evaluate the effect of 4′-substitutions on the antiviral and
cytotoxicity profile of such compounds. As shown in Table 1,
the introduction of the 4′-azido moiety (34) resulted in a highly
interesting compound with significant antiviral potency in the
HCV replicon system (EC50 ) 66 nM) and no apparent
cytotoxicity, suggesting a substantial increase in selectivity as
compared to gemcitabine. The effect of the 4′-azido moiety to
confer high selectivity has therefore been consistent through 3,
4, 10, 17, and 34.
3′-Fluoro analogues of 4′-azidocytidine, 20 and 28, were
completely inactive as well as nontoxic in the replicon system.
The 3′-hydroxyl group was suggested from structural studies
with deoxycytidine kinase to be important for productive binding
to the kinase, as hydrogen bonds are formed with E197 and
Y86 in the human deoxycytidine kinase active site.16 Removal
of the 3′-hydroxy group in 20 and 28 may prevent these
Results and Discussion
Compounds were characterized as inhibitors of HCV replica-
tion in the subgenomic replicon assay system using the 2209-

Analogues of 4′-Azidocytidine against HCV Replication
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2973
Scheme 2a
a (a) I2, PPh3, imidazole, THF; (b) NaOMe, MeOH; (c) [Bn(Et)3N]N3, I2, MMP, THF; (d) BzCl, DMAP, MMP, THF; (e) (Bu4N)HSO4, K2HPO4, mCPBA,
mCBA, CH2Cl2; (f) NH3, MeOH; (g) 1H-tetrazole, Cl2P(O)OC6H4Cl, pyridine; (h) NH3, dioxane.
Scheme 3a
and N,N-dimethylformamide (1 mL) was heated at 100 °C under
N2. After 14 h, the reaction mixture was cooled to room temperature
and diluted with CH3CN (5 mL). The resulting precipitate was
collected by filtration to provide 6 (0.80 g, 85%) as an off-white
solid. 1H NMR (300 MHz, DMSO-d6): δ 7.94 (d, J ) 7.5 Hz, 1H),
6.62 (d, J ) 5.6 Hz, 1H), 6.46 (d, J ) 6 Hz, 1H), 5.92 (d, J ) 7.5
Hz, 1H), 5.54 (t, J ) 5.7 Hz, 1H), 5.35 (dd, J ) 2.4 and 6 Hz,
1H), 4.50 (br s, 1H), 3.53-3.41 (m, 2H).
2,2′-Anhydro-1-(4′-azido-3′,5′-O-bis-(4-methoxy-tetrahydro-
pyran-4-yl)-ꢀ-D-arabinofuranosyl)uracil (7). To a solution of 6
(0.77 g, 2.88 mmol) in dry N,N-dimethylformamide (15 mL) was
added 4-methoxy-3,4-dihydro-2H-pyran (2.6 mL, 23 mmol), fol-
lowed by camphorsulfonic acid (0.040 g, 0.17 mmol). The reaction
mixture was stirred for 2 days at room temperature, neutralized
with solid K2CO3, and evaporated to dryness under reduced
pressure. Purification by silica gel column chromatography (10-15%
1
MeOH in CHCl3) gave 7 as an off-white solid (0.87 g, 61%). H
a (a) TrCl, pyridine; (b) DAST, pyridine, CH2Cl2; (c) triazole, POCl3,
Et3N, MeCN; (d) NH4OH, dioxane; (e) Amberlyst 15, MeOH.
NMR (400 MHz, CD3Cl): δ 7.36 (d, J ) 7.5 Hz, 1H), 6.29 (d, J
) 6.1 Hz, 1H), 6.09 (d, J ) 7.5 Hz, 1H), 5.34 (dd, J ) 2.0 and 6.1
Hz, 1H), 4.88 (d, J ) 2.0 Hz, 1H), 3.89-3.79 (m, 2H), 3.69-3.56
(m, 6H), 3.45 (q, J ) 10.5 Hz, 2H), 3.32 (s, 3H), 3.14 (s, 3H),
2.10-1.70 (m, 8H).
compounds from binding productively to human dCK and thus
from being phosphorylated to their triphosphate derivatives.
1-(4′-Azido-3′,5′-O-bis-(4-methoxy-tetrahydropyran-4-yl)-ꢀ-
D-arabinofuranosyl)uracil (8). A solution of 7 (0.87 g, 1.76 mmol)
and KOH (0.070 g, 1.25 mmol) in 9:1 mixture of EtOH/H2O (10
mL) was refluxed for 3 h. The reaction mixture was cooled,
neutralized with Amberlyst 15 ion-exchange resin, filtered, and
evaporated to dryness under reduced pressure to give 8 (0.83 g,
Conclusions
We have presented data that shows both 4′-azido-2′-deoxy-
2′,2′-difluorocytidine (34) and 4′-azido-2′-deoxyfluoroarabinocy-
tidine (17) to be highly potent and selective inhibitors of HCV
replication. Potency in this series was increased more than 50-
fold as compared to 4′-azidocytidine (3), which has shown
efficacy in HCV infected patients. Considerable potency is also
observed with 4′-azido-2′-fluorocytidine 10, although ∼3-fold
lower as compared to 4′-azidocytidine (3). Further characteriza-
tion of these compounds is therefore warranted as potential
antiviral agents with low cytotoxicity in human cells. Derivatives
at the 3′ position of 4′-azidocytidine did not show antiviral
potencyincellculture,presumablyduetoinefficientphosphorylation.
1
92%) as a foam. H NMR (400 MHz, CD3Cl): δ 7.52 (d, J ) 7.9
Hz, 1H), 6.36 (d, J ) 5.1 Hz, 1H), 5.58 (d, J ) 7.9 Hz, 1H), 4.53
(t, J ) 5.1 Hz, 1H), 4.47 (d, J ) 5.1 Hz, 1H), 3.80-3.55 (m, 8H),
3.48 (s, 2H), 3.30 (s, 3H), 3.24 (s, 3H), 1.95-1.70 (m, 8H).
1-(4′-Azido-3′,5′-O-bis-(4-methoxy-tetrahydropyran-4-yl)-2′-
deoxy-2′-fluoro-ꢀ-D-ribofuranosyl)uracil (9). To a solution of 8
(0.41 g, 0.8 mmol) in CH2Cl2 (5 mL) was added pyridine (1.2 mL)
followed by DAST (0.64 mL, 4.8 mmol). The reaction mixture
was stirred at room temperature for 24 h, diluted with CH2Cl2,
washed with brine, dried (MgSO4), and evaporated to dryness under
reduced pressure. The residue was purified by silica gel column
chromatography (10-15% MeOH in CHCl3) to give 9 (0.23 g,
Experimental Section
1
56%) as a foam. H NMR (400 MHz, CDCl3): δ 7.54 (d, J ) 7.4
All starting materials, solvents and reagents were reagent grade
and used as purchased. Chromatography solvents were HPLC grade
and used without further purification. Flash column chromatography
was performed on Merck KGaA Silica Gel 60 F254 (particle size
0.040-0.063 mm) unless otherwise stated. Reactions were moni-
tored by thin-layer chromatography (TLC) analysis using precoated
glass plates (Silica Gel 60 F254). The purity of compounds 10, 17,
20, 28, and 34 was determined by high pressure liquid chroma-
tography and were found to be greater than 95%, apart from
compound 20 (94%).
Hz, 1H), 5.83 (d, J ) 21.0 Hz, 1H), 5.80 (d, J ) 7.4 Hz, 1H), 5.25
(dd, J ) 5.5 and 53.9 Hz, 1H), 4.82 (dd, J ) 5.5 and 22.0 Hz,
1H), 3.80-3.55 (m, 10H), 3.33 (s, 3H), 3.21 (s, 3H), 2.00-1.75
(m, 8H). MS (ES+) m/z 516 (MH+).
1-(4′-Azido-2′-deoxy-2′-fluoro-ꢀ-D-ribofuranosyl)cytosine (10).
To a ice-water bath cooled solution of 9 (0.21 g, 0.407 mmol)
and 1H-tetrazole (0.086 g, 1.22 mmol) in dry pyridine (10 mL)
was added chlorophenyldichlorophosphate (0.1 mL, 0.61 mmol)
over 5 min under stirring. The reaction mixture was stirred at room
temperature for 6 h and evaporated to dryness under reduced
pressure (<30 °C). The residue was partitioned between cold
CH2Cl2 and saturated aqueous NaHCO3. The organic phase was
2,2′-Anhydro-1-(4′-azido-ꢀ-D-arabinofuranosyl)uracil (6). A
mixture containing 5 (1.00 g, 3.50 mmol), diphenyl carbonate (0.83
g, 3.85 mmol), sodium hydrogen carbonate (0.030 g, 0.35 mmol),

2974 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Smith et al.
Scheme 4a
a (a) I2, PPh3, Pyr, MeCN; (b) NaOMe, MeOH, 65 °C; (c) I2, [Bn(Et)3N]N3, MeCN, THF, 5 °C; (d) (Bu4N)HSO4, K2HPO4, mCPBA, mCBA, CH2Cl2; (e)
Bz2O, Pyr; (f) 4-ClPhOP(O)Cl2, 1H-tetrazole, Pyr, 5 °C; (g) 0.5 M, NH3/dioxane; (h) 2 M NH3/MeOH.
Scheme 5a
a (a) I2, PPh3, MeCN, Pyr; (b) tBuOK, DMF; (c) I2, [Bn(Et)3N]N3, THF, MeCN; (d) p-MeOBzCl, MMP, DMAP, MeCN, THF; (e) NaBz, 15-crown-5,
DMF, 60 °C; (f) 2 M NH3/MeOH.
washed with brine and then dried over MgSO4, filtered, and
evaporated. The residue was treated with 0.5 M NH3 in 1,4-dioxane
(20 mL), and the resulting solution was stirred for 4 h at room
temperature and evaporated to dryness under reduced pressure. The
residue was dissolved in AcOH/MeOH/H2O [4:2:1 (20 mL)], heated
at 50 °C for 5 h, cooled, and evaporated to dryness under reduced
pressure. Purification by preparative HPLC (Hypercarb column,
Hz, 1H), 3.80 (dd, J ) 12.1 and 39.3 Hz, 2H). HRMS (ES+) calcd
for C9H12FN6O4 [M + H]+ 287.0904; found 287.0908.
1-(2′-Deoxy-2′-fluoro-5′-iodo-ꢀ-D-arabinofuranosyl)uracil (12).
A suspension of 11 (1.61 g, 6.54 mmol) and pyridine (7 mL) in
anhydrous CH3CN (142 mL) was cooled on an ice-water bath.
Iodine (2.00 g, 7.88 mmol) and triphenylphosphine (2.23 g, 8.50
mmol) were added, and the reaction mixture was stirred at 0 °C
for 30 min. After stirring at room temperature for 24 h, the reaction
mixture was again cooled on an ice-water bath and more iodine
(0.40 g, 1.58 mmol) and triphenylphosphine (0.45 g, 1.71 mmol)
were added. The reaction mixture was stirred at room temperature
1
H2O in CH3CN) provided 10 (0.03 g, 26%) as a white solid. H
NMR (400 MHz, methanol-d4): δ 7.87 (d, J ) 7.5 Hz, 1H), 6.12
(dd, J ) 1.3 and 19.4 Hz, 1H), 5.89 (d, J ) 7.5 Hz, 1H), 5.13
(ddd, J ) 1.4, 5.3, and 53.9 Hz, 1H), 4.57 (dd, J ) 5.3 and 22.1

Analogues of 4′-Azidocytidine against HCV Replication
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2975
Table 1. EC50 and CC50 Determination in the HCV Replicon System
1-(4′-Azido-2′-deoxy-2′-fluoro-ꢀ-D-arabinofuranosyl)uracil (15).
3-Chloroperoxybenzoic acid [55% in balance with 3-chlorobenzoic
acid (10%) and water (35%)] (4 × 3.33 g) was added portion-wise
over 20 h to a two-phase suspension containing 14 (0.97 g, 1.94
mmol), tetra-n-butylammonium hydrogensulfate (0.70 g, 2.05
mmol), and 3-chlorobenzoic acid (0.33 g, 2.13 mmol) in CH2Cl2
(125 mL) and 1.7 M aqueous K2HPO4 (45 mL) under stirring at rt.
A solution of Na2SO3 (7.5 g, 59.5 mmol) in saturated aqueous
NaHCO3 (150 mL) was added. The mixture was shaken for 5 min.
The organic layer was separated and the aqueous layer washed with
CH2Cl2 (2 × 50 mL). The combined organic layers were dried
(Na2SO4), filtered, and evaporated to dryness under reduced
pressure. The residue was treated with saturated ammonia in MeOH
(150 mL) for 19 h at room temperature, after which time the solution
was evaporated to dryness under reduced pressure. The residue was
purified by silica gel column chromatography (10% MeOH in
CH2Cl2) followed by reversed phase column chromatography
(5-10% CH3CN in H2O) to give 15 (0.31 g, 56%) as a white solid.
1H NMR (DMSO-d6): δ 7.64 (dd, J ) 1.7 and 8.1 Hz, 1H), 6.40
(d, J ) 6.0 Hz, 1H), 6.34 (dd, J ) 5.7 and 10.6 Hz, 1H), 5.71 (t,
J ) 6.0 Hz, 1H), 5.67 (d, J ) 8.1 Hz, 1H), 5.25 (dt, J ) 5.5 and
53.9 Hz, 1H), 4.45 (dt, J ) 5.6 and 23.1 Hz, 1H), 3.78-3.69 (m,
2H). 13C NMR (DMSO-d6): δ 163.5 (C-4), 150.8 (C-2), 141.8 (C-
6), 102.5 (C-5), 97.34 and 97.26 (C-4′), 95.9 and 94.4 (C-2′), 81.7
(C-1′), 75.3 and 75.1 (C-3′), 62.8 (C-5′). MS (ES+) [MH]+ m/z
288.1.
GT-1b stable
replicon (2209-23)
EC50 (µM)
cytotoxicity
(2209-23)
CC50 (µM)
proliferation
(3H-Thy incorporation)
IC50 (µM)
compd
3
4
10
17
20
28
34
dFAraC
1.28
0.171
4.02
0.024
>100
>100
0.066
0.114
>2000
>1000
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
nd
nd
nd
0.174
for a further 41 h and then concentrated to 1/4 of the volume under
reduced pressure, diluted with CH2Cl2 (200 mL), and washed with
0.5 M Na2S2O3 in saturated aqueous NaHCO3 solution. The aqueous
layer was washed with CH2Cl2 (3 × 50 mL). The combined organic
layers were dried (Na2SO4), treated with activated carbon, and
filtered through celite. The filter cake was then washed with CH2Cl2
(200 mL), followed by azeotropic distillation of the filtrate with
toluene. Chromatography (1-5% MeOH in CH2Cl2) gave 12 as
1
an off-white solid (1.78 g, 76%). H NMR (400 MHz, methanol-
d4): δ 7.71 (dd, J ) 1.8 and 8.2 Hz, 1H), 6.21 (dd, J ) 3.3 and
19.9 Hz, 1H), 5.71 (d, J ) 8.2 Hz, 1H), 5.02 (ddd, J ) 1.8, 3.4,
51.9, 1H), 4.29 (ddd, J ) 1.7, 3.3, 17.5, 1H), 3.99 (td, J ) 3.4,
6.3, 1H), 3.53-3.45 (m, 2H). MS (ES+) [M + H]+ m/z 357.
1-(4′-Azido-2′-deoxy-3′,5′-O-dibenzoyl-2′-fluoro-ꢀ-D-arabino-
furanosyl)uracil (16). A solution of compound 15 (0.244 g, 0.850
mmol), 4-methylmorpholine (0.65 mL, 5.95 mmol), and DMAP
(0.05 g, 0.425 mmol) in anhydrous THF (25 mL) was cooled to 0
°C and benzoyl chloride (0.247 mL, 2.12 mmol) was added
dropwise over 5 min. The reaction mixture was stirred at 0 °C for
a further 1 h and was then diluted with CH2Cl2 and washed with
saturated aqueous NaHCO3. The aqueous layer was washed with
CH2Cl2. The combined organic layers were dried (Na2SO4), filtered,
and concentrated. The residue was purified by silica gel column
chromatography (2-5% MeOH in CH2Cl2) to give compound 16
(0.406 g, 96%) as a white solid. 1H NMR (methanol-d4): δ
8.10-7.39 (m, 11H), 6.63 (dd, J ) 5.1 and 14.4 Hz, 1H), 6.08
(dd, J ) 3.1 and 20.3 Hz, 1H), 5.74-5.72 and 5.61-5.59 (m, 1H),
5.67 (d, J ) 8.2 Hz, 1H), 4.96-4.84 (m, 2H). MS (ES+) [M +
H]+ m/z 496.0.
1-(2′,5′-Dideoxy-2′-fluoro-erytho-ꢀ-D-pent-4-enofuranosyl)ura-
cil (13). Sodium methoxide (0.76 g, 14.1 mmol) was added to a
suspension of compound 12 (1.00 g, 2.81 mmol) in anyhdrous
methanol (21 mL), and the reaction mixture was stirred at 65 °C
for 5 h. The reaction mixture was cooled to room temperature and
diluted with methanol (10 mL). Pyridinium form DOWEX-H+ (4
g) was added, and the mixture stirred until a clear solution was
obtained (ca. 20 min). The mixture was filtered and the resin washed
with methanol and then evaporated to dryness under reduced
pressure. Purification by silica gel column chromatography (10%
MeOH in CH2Cl2 in) to give 13 (0.52 g, 82%) as a white solid. 1H
NMR (methanol-d4): δ 7.46 (dd, J ) 2.1 and 8.2 Hz, 1H), 6.50
(dd, J ) 3.3 and 19.4 Hz, 1H), 5.71 (d, J ) 8.2 Hz, 1H), 5.03
(ddd, J ) 1.9, 3.1, and 51.5 Hz, 1H), 4.67 (d, J ) 2.3 Hz, 1H),
4.66 (dd, J ) 1.7 and 11.7 Hz, 1H), 4.46 (d, J ) 2.3 Hz, 1H). MS
(ES+) [M + H]+ m/z 229.
1-(4′-Azido-3′-O-benzoyl-2′-deoxy-2′-fluoro-5′-iodo-ꢀ-D-ara-
binofuranosyl)uracil (14). Benzyltriethylammonium chloride (0.98
g, 4.29 mmol) and sodium azide (0.28 g, 4.29 mmol) were
suspended in anhydrous CH3CN (22 mL). The suspension was
stirred vigorously for 15 h and was then filtered. The filtrate
containing quarternary ammonium azide was added to a solution
of compound 13 (0.48 g, 2.10 mmol) and 4-methylmorpholine (0.07
mL, 0.64 mmol) in anhydrous THF (15 mL). A solution of iodine
(0.90 g, 3.58 mmol) in anyhdrous THF (19 mL) was added dropwise
over 1 h under stirring at 0 °C. The reaction mixture was stirred at
0 °C for a further 4 h. N-Acetyl-L-cysteine (0.034 g, 0.21 mmol),
4-methylmorpholine (1.16 mL, 10.5 mmol), and DMAP (0.26 g,
2.10 mmol) were added, followed by a dropwise addition of benzoyl
chloride (0.54 mL, 4.63 mmol). Stirring at 0 °C continued for 1 h.
A solution of 0.1 M Na2SO3 in saturated aqueous NaHCO3 (50
mL) was added, and the mixture was shaken. The mixture was
diluted with CH2Cl2 (150 mL) and washed with saturated aqueous
NaHCO3 and water. The organic layer was separated and the
aqueous layer washed with CH2Cl2 (2 × 50 mL). The combined
organic layers were dried (Na2SO4), filtered, and evaporated to
dryness under reduced pressure. The residue was purified by silica
gel column chromatography (3% MeOH in CH2Cl2) to give 14 (0.98
g, 93%) as a white foam. 1H NMR (400 MHz, CDCl3): δ 8.20 (br
s, 1H), 8.10-7.50 (m, 6 H), 6.59 (dd, J ) 4.3 and 17.9 Hz, 1H),
5.87 (dd, J ) 2.1, 11.3 Hz, 1H), 5.84 (t, J ) 1.9 Hz, 1H), 5.43
(ddd, J ) 2.1, 3.9, and 51.7 Hz, 1H), 3.88-3.79 (m, 2H). MS (ES+)
[M + H]+ m/z 501.9.
1-(4′-Azido-2′-deoxy-2′-fluoro-ꢀ-D-arabinofuranosyl)cy-
tosine (17). 4-Chlorophenyldichlorophosphate (0.20 mL, 1.22
mmol) was added dropwise over 5 min to a solution, cooled on an
ice-water bath of 16 (0.40 g, 0.815 mmol) and 1H-tetrazole (0.17
g, 2.45 mmol) in anhydrous pyridine (15 mL). After stirring for 5
min, the reaction mixture was allowed to warm to room temperature.
After 5 h, the reaction mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in CH2Cl2 and washed
with saturated aqueous NaHCO3 and water, dried (4 Å molecular
sieves), and evaporated to dryness under reduced pressure. Azeo-
tropic distillation with toluene removed the remaining traces of
pyridine from the product. The residue was then treated with 0.5
M ammonia in 1,4-dioxane (40 mL) for 5.5 h at room temperature.
The solution was evaporated to dryness under reduced pressure,
and the residue treated with saturated NH3 in methanol (55 mL)
for 15 h at rt. The mixture was evaporated to dryness under reduced
pressure and the residue purified by silica gel column chromatog-
raphy [saturated NH3 in MeOH (0.5%), MeOH (19.5%), CH2Cl2
(80%)] to give 17 as an off-white solid (0.21 g, 93%); mp
1
99.0-100.0 °C. H NMR (methanol-d4): δ 7.75 (dd, J ) 1.2 and
7.6 Hz, 1H), 6.48 (dd, J ) 5.0 and 12.4 hz, 1H), 5.91 (d, J ) 7.4
Hz, 1H), 5.19 (dt, J ) 4.3 and 56.0 Hz, 1H), 4.47 (dd, J ) 4.3 and
21.6 Hz, 1H), 3.83 (s, 2H). 13C NMR (DMSO-d6): δ 166.3 (C-4),
155.3 (C-2), 142.3 (C-6), 97.5 and 97.4 (C-4′), 94.8 (C-5), 96.2
and 94.3 (C-2′), 82.9 (C-1′), 75.8 and 75.6 (C-3′), 63.0 (C-5′). MS
(ES+) [2 M + Na]+ m/z 595.2. HRMS (ES+) calcd for C9H12FN6O4
[M + H]+ 287.0904; found 287.0911.
1-(4′-Azido-2′,5′-di-O-trityl-ꢀ-D-ribofuranosyl)uracil (18). Triph-
enylmethyl chloride (19.30 g, 69.23 mmol) was added to a solution

2976 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Smith et al.
of 5 (5.00 g, 17.53 mmol) in pyridine (50 mL). After stirring at
100 °C under N2 for 72 h, the reaction mixture was cooled to room
temperature and then evaporated to dryness under reduced pressure.
The residue was dissolved in CH2Cl2, washed with brine, dried
(MgSO4), and evaporated to dryness under reduced pressure.
Chromatography (EtOAc in hexanes) provided 18 (7.43 g, 55%)
was removed by filtration and washed with MeOH, and the filtrate
was evaporated to dryness under reduced pressure. The residue was
slurried in 2% EtOH in CH2Cl2 and purified by silica gel column
chromatography (2-6% EtOH in CH2Cl2) to give 23 as a white
1
solid (0.251 g, 92%). H NMR (DMSO-d6): δ 11.50 (br s, 1H),
7.75 (d, J ) 8.1 Hz, 1H), 6.10 (d, J ) 6.2, 1H), 6.04 (d, J ) 7.6
Hz, 1H), 5.70 (dd, J ) 2.2, 8.1 Hz, 1H), 5.26 (dd, J ) 4.4 and
55.8 Hz, 1H), 4.68-4.55 (m, 1H), 4.55 (ddd, J ) 2.2, 6.4, and
18.9 Hz, 2H). MS (ES+) [M + H]+ m/z 229.1.
1
as an off-white foam. H NMR (300 MHz, CDCl3): δ 9.02-8.97
(br s, 1H), 7.32-7.25 and 7.54-7.45 (m, 30H), 6.23 (d, J ) 6 Hz,
1H), 5.18 (d, J ) 8 Hz, 1H), 4.72 (t, J ) 5.5 Hz, 1H), 3.36 (t, J )
4.5 Hz, 1H), 3.10 (s, 2H), 2.79 (d, J ) 4.1 Hz, 1H). MS (ES-) [M
- H]- m/z 768.2.
1-(4′-Azido-3′-deoxy-3′-fluoro-5′-iodo-ꢀ-D-ribofuranosyl)uracil
(24). Benzyltriethylammonium chloride (0.296 g, 1.3 mmol) and
sodium azide (0.084 g, 1.3 mmol) were suspended in anhydrous
CH3CN (7 mL) and sonicated for several minutes. The resulting
fine suspension was stirred for 3 h at room temperature and filtered
under nitrogen into a dry THF solution (4.4 mL) of 23 (0.148 g,
0.65 mmol). 4-Methylmorpholine (0.021 mL, 0.19 mmol) was
added, the resulting solution was cooled on an ice-water bath, and
a solution of iodine (0.28 g, 1.1 mmol) in anhydrous THF (6 mL)
was added dropwise over 60 min. The reaction mixture was stirred
for 16 h at 0-9 °C. N-Acetyl-L-cysteine (0.011 g, 0.065 mmol)
was added, and the solution was stirred until bubbling subsided.
The solvent was concentrated under reduced pressure to half-
volume, and then a solution of 0.1 M Na2S2O3 in saturated aqueous
NaHCO3/brine was added under stirring. The mixture was extracted
with 10% EtOH in CH2Cl2, and the organic layers were dried
(Na2SO4) and evaporated to dryness under reduced pressure. The
4′-epimeric mixture (∼1:1) was separated by silica gel column
chromatography (2-3% EtOH in CH2Cl2) and then by semi-
preparative Hyper Carb HPLC column purification (10-90%
1-(4′-Azido-3′-deoxy-3′-fluoro-2′,5′-di-O-trityl-ꢀ-D-xylofurano-
syl)uracil (19). (Diethylamino)sulfur trifluoride (1.6 mL, 12.40
mmol) was carefully added to a flask containing a solution of 18
(6.40 g, 8.31 mmol) in pyridine (6 mL) and CH2Cl2 (90 mL). Once
complete, the reaction mixture was heated at 50 °C for 18 h under
N2. The cooled reaction mixture was poured onto ice-water and
extracted with CH2Cl2. The organics were washed with brine, dried
(MgSO4), and evaporated to dryness under reduced pressure.
Chromatography (EtOAc in hexanes) provided 19 (2.0 g, 31%) as
1
a white foam. H NMR (300 MHz, CDCl3): δ 8.16-8.14 (br s,
1H), 7.47-7.16 (m, 30H), 6.85 (dd, J ) 1.6 and 8 Hz, 1H), 6.66
(d, J ) 3.1 Hz, 1H), 5.60 (dd, J ) 2.2 and 8 Hz, 1H), 4.14 (dd, J
) 3.2 and 21.1 Hz, 1H), 3.72 (d, J ) 49 Hz, 1H), 3.41-3.33 (m,
2H). MS (ES-) [M - H]- m/z 770.2.
1-(4′-Azido-3′-deoxy-3′-fluoro-ꢀ-D-xylofuranosyl)cytosine (20).
POCl3 (1 mL, 11.82 mmol) was slowly added to a flask, cooled to
0 °C, containing 19 (2.28 g, 2.95 mmol), triazole (3.06 g, 44.31
mmol), triethylamine (8.2 mL, 59.08 mmol), and CH3CN (90 mL).
The resulting mixture was stirred at 0 °C for 15 min and then
allowed to warm to room temperature. After 2.5 h, the crude
reaction mixture was evaporated to dryness under reduced pressure.
The residue was dissolved in CH2Cl2 and washed with saturated
aqueous NaHCO3 solution, dried (MgSO4), and evaporated to
dryness under reduced pressure. The resulting solid was dissolved
in dioxane (60 mL) and treated with NH4OH (10 mL) and left to
stir at room temperature for 20 h. The crude reaction mixture was
evaporated to dryness under reduced pressure. Chromatography
(15% MeOH in CH2Cl2) provided 1-(4′-azido-3′-deoxy-3′-fluoro-
2′,5′-di-O-trityl-ꢀ-D-xylofuranosyl)cytosine (1.78 g, 2.39 mmol) as
a light-brown solid. This material was dissolved in MeOH (60 mL)
and treated with 2 M NH3 in EtOH (7.2 mL). After stirring for 3 h,
the precipitate was removed by filtration and the filtrate evaporated
to dryness under reduced pressure. Chromatography (0-10%
MeOH in CH2Cl2) provided 20 (0.40 g, 47%) as an off-white solid;
1
CH3CN in H2O) to give 24 as a white solid (0.081 g, 31%). H
NMR (CDCl3): δ 8.50 (br s, 1H), 7.34 (d, J ) 8.1 Hz, 1H), 5.81
(d, J ) 8.1 Hz, 1H), 5.70 (d, J ) 4.1 Hz, 1H), 5.38 (dd, J ) 5.7
and 52.1 Hz, 1H), 4.84-4.76 (m, 1H), 3.55 (dd, J ) 11.3 and 28.8
Hz, 2H). MS (ES-) [M - H]- m/z 395.9.
1-(4′-Azido-5′-O-(4-chloro)benzoyl-3′-deoxy-3′-fluoro-ꢀ-D-ri-
bofuranosyl)uracil (25). A solution of 24 (0.081 g, 0.2 mmol) in
CH2Cl2 (12 mL) was combined with a mixture of tetrabutylam-
monium hydrogensulfate (0.073 g, 0.22 mmol) and m-chlorobenzoic
acid (0.048 g, 0.31 mmol) in 1.75 M aqueous K2HPO4 (4.7 mL).
The two-phase system was stirred vigorously at room temperature,
and one portion of m-chloroperbenzoic acid [55% in balance with
3-chlorobenzoic acid (10%) and water, (35%)] (0.360 g) was added.
After 1.5 h, 3 × 120 mg of this reagent mixture was added at 1.5 h
intervals. After the last addition, the mixture was vigorously stir-
red for another 18 h at rt. A solution of 0.1 M Na2SO3 in saturated
aqueous NaHCO3 (10 mL) was added, and the mixture was stirred
vigorously at rt for 15 min. The organic layer was separated, and
the water layer was extracted with CH2Cl2. The combined organic
layers were washed with saturated aqueous NaHCO3. The organic
layer was dried (Na2SO4) and evaporated to dryness under reduced
pressure. The residue was purified by silica gel column chroma-
tography (2-4% EtOH in CH2Cl2) to give 25 as a white solid (0.032
g, 37%). 1H NMR (CDCl3): δ 8.04 (t, J ) 1.8 Hz, 1H), 7.96-7.91
(m, 1H), 7.57 (ddd, J ) 1.1, 2.2, and 8.0 Hz, 1H), 7.42 (t, J ) 7.9
Hz, 1H), 7.29 (d, J ) 8.1 Hz, 1H), 5.75 (d, J ) 8.1 Hz, 1H), 5.74
(d, J ) 2.9 Hz, 1H), 5.52 (dd, J ) 5.7 and 51.8 Hz, 1H), 4.83-4.75
(m, 1H), 4.61 (s, 2H). MS (ES-) [M - H]- m/z 424.
1-(4′-Azido-2′-O-benzoyl-5′-O-(4-chloro)benzoyl-3′-deoxy-3′-
fluoro-ꢀ-D-ribofuranosyl)uracil (26). Benzoic anhydride (0.021
g, 0.09 mmol) was added to a solution of 25 (0.032 g, 0.076 mmol)
in pyridine (0.75 mL). After stirring for 16 h at rt, MeOH (5 mL)
was added. The solvents were evaporated to dryness under reduced
pressure, and the residue was partitioned between CH2Cl2 and
saturated aqueous NaHCO3. The organic phase was separated, dried
(Na2SO4), and evaporated to dryness under reduced pressure.
Purification by silica gel column chromatography (1% EtOH in
CH2Cl2) gave 26 (0.040 g, 98%) as a white foam. 1H NMR (CDCl3):
δ 8.86 (br s, 1H), 8.12-8.08 (m, 3H), 8.00-7.96 (m, 1H),
7.66-7.40 (m, 5H), 7.22 (d, J ) 8.1 Hz, 1H), 5.89-5.76 (m, 4H),
4.75-4-64 (m, 2H). MS (ES+) [M + H]+ m/z 530.6.
1
mp 171.8-172.9 °C. H NMR (300 MHz, methanol-d4): δ 7.66
(d, J ) 7.5 Hz, 1H), 6.26 (d, J ) 4.2 Hz, 1H), 5.95 (d, J ) 7.5 Hz,
1H), 4.77 (dd, J ) 2.5 and 51 Hz, 1H), 4.44 (ddd, J ) 2.5, 4, and
21 Hz, 1H), 3.99-3.87 (m, 2H). HRMS (ES+) calcd for
C9H12FN6O4 [M + H]+ 287.0904; found 287.0896.
1-(3′-Deoxy-3′-fluoro-5′-iodo-ꢀ-D-ribofuranosyl)uracil (22). 3′-
Deoxy-3′-fluorouridine17 21 (0.31 g, 1.28 mmol) and triphenylphos-
phine (0.47 g, 1.79 mmol) were dissolved in CH3CN/pyridine [95:
5 (13 mL)]. Iodine (0.422 g, 1.66 mmol) was added, and the reaction
mixture was stirred for 18 h at room temperature. Water (5 mL)
was added, and the solvents were evaporated to dryness under
reduced pressure. Azeotropic distillation with CH3CN and then with
CHCl3 was performed to remove the water. The residue was purified
by silica gel column chromatography (2-6% EtOH in CH2Cl2) to
1
give 22 (0.426 g, 93%) as a white solid. H NMR (DMSO-d6): δ
11.43 (br s, 1H), 7.70 (d, J ) 8.1 Hz, 1H), 5.95 (d, J ) 6.1 Hz,
1H), 5.85 (d, J ) 8.0 Hz, 1H), 5.72 (d, J ) 8.1 Hz, 1H), 4.90 (dd,
J ) 4.6 and 53.0 Hz, 1H), 4.55-4.40 (m, 1H), 4.25 (dt, J ) 7.5
and 13.7 Hz, 1H), 3.48 (ddd, J ) 6.9, 10.4, 16.5, 2H). MS (ES+)
[M + H]+ m/z 357.1.
1-(3′,5′-Dideoxy-3′-fluoro-erytho-ꢀ-D-pent-4-enofuranosyl)ura-
cil (23). Compound 22 (0.426 g, 1.2 mmol) was dissolved into a
solution of 0.4 M sodium methoxide in MeOH (27 mL). The
reaction mixture was stirred for 5 h at 65 °C. Pyridinium form
DOWEX H+ was added portionwise until neutral pH (2-3 g), and
the mixture was stirred at room temperature for 10 min. The resin

Analogues of 4′-Azidocytidine against HCV Replication
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2977
1-(4′-Azido-3′-deoxy-3′-fluoro-ꢀ-D-ribofuranosyl)cytosine (28).
Compound 26 (0.040 g, 0.075 mmol) and 1H-tetrazole (0.016 g,
0.23 mmol) were azeotroped with pyridine to remove moisture and
then redissolved in anhydrous pyridine (1.3 mL). The solution was
cooled on an ice-water bath, and 4-chlorophenylphosphorodichlo-
ridate (0.018 mL, 0.11 mmol) was added. The reaction mixture
was stirred at 0-5 °C for 5 min and then allowed to warm to room
temperature. After 5 h, the reaction mixture was evaporated to
dryness under reduced pressure and the residue was partitioned
between CH2Cl2 and saturated aqueous NaHCO3. The organic phase
was separated, dried (Na2SO4), and evaporated to dryness under
reduced pressure. The intermediate 27 was then treated with 0.5
M NH3 in 1,4-dioxane (5 mL) for 4 h at rt and then evaporated to
dryness under reduced pressure. The residue was dissolved in
saturated NH3 in MeOH (15 mL) and the mixture was stirred for
16 h at room temperature and then evaporated to dryness under
reduced pressure. Purification by semipreparative Hyper Carb HPLC
column purification (10-90% CH3CN in H2O) gave 28 (0.011 g,
anhydrous THF (62 mL) was added dropwise over 60 min and then
left to stir for 16 h at 0-9 °C. The mixture was concentrated to
half-volume under reduced pressure and the remaining mixture left
to stir for 24 h at 0-9 °C. N-Acetyl-L-cysteine (0.11 g, 0.68 mmol)
was added, and the solution was stirred until bubbling subsided.
The mixture was kept at 5 °C and 4-methylmorpholine (3.72 mL,
33.8 mmol) and DMAP (826 mg, 6.76 mmol) were added, followed
by anisoylchloride (3.45 g, 20.3 mmol) dissolved in MeCN (14
mL). The resulting mixture was stirred for 1 h. MeOH (15 mL)
was added and the solvents were concentrated to half-volume under
reduced pressure, and then a solution of 0.1 M Na2S2O3 in saturated
aqueous NaHCO3 (500 mL) was added and the mixture was stirred
until colorless. The mixture was extracted with CH2Cl2, and the
organic layer was separated and then washed with 5% citric acid,
dried (Na2SO4), and evaporated to dryness under reduced pressure.
The residue was separated twice by silica gel column chromatog-
raphy (0.5-2% EtOH in CH2Cl2) to give 32 (3.33 g, 76%) as a
1
white foam. H NMR (400 MHz, CDCl3): δ 8.10 (d, J ) 8.6 Hz,
1
53%) as a white solid. H NMR (DMSO-d6): δ 7.71 (d, J ) 7.5
2H), 7.95 (br s, 1H), 7.90 (d, J ) 8.0 Hz, 2H), 7.70 (br s, 1H),
7.65 (t, J ) 7.2 Hz, 1H), 7.55 (t, J ) 8.0 Hz, 2H), 6.98 (d, J ) 8.6
Hz, 2H), 6.72 (br s, 1H), 5.90 (br s, 1H), 3.91 (s, 3H), 3.87 (s,
2H). MS (ES+) [M + H]+ m/z 484.
Hz, 1H), 7.32 (br d, J ) 4.8 Hz, 2H), 6.18 (d, J ) 7.6 Hz, 1H),
5.98 (br s, 1H), 5.78 (d, J ) 7.5 Hz, 1H), 5.72 (br s, 1H), 5.07 (dd,
J ) 4.5 and 53.6 Hz, 1H), 4.45 (ddd, J ) 4.4, 7.3, and 22.2 Hz,
1H), 3.49 (q, J ) 12.1 Hz, 2H). MS (ES+) [M + H]+ m/z 287.
HRMS (ES+) calcd for C9H12FN6O4 [M + H]+ 287.0904; found
287.0909.
1-[4′-Azido-4-N-5′-O-dibenzoyl-3′-O-(4-methoxybenzoyl)-2′-
dideoxy-2′,2′-difluoro-ꢀ-D-ribofuranosyl)cytosine (33). Com-
pound 32 (3.33 g, 5.1 mmol) was dissolved in DMF (43 mL),
together with 15-crown-5 (1.72 mL, 8.67 mmol) and sodium
benzoate (1.25 g, 8.67 mmol), and the mixture was stirred for 65 h
at 60 °C. Further sodium benzoate (2.5 g, 17.34 mmol) and 15-
crown-5 (3.4 mL, 17.34 mmol) were added, and the mixture was
stirred for 5 d. As the reaction was not complete, further quantities
of sodium benzoate (1.25 g, 8.67 mmol), 15-crown-5 (1.72 mL,
8.67 mmol), and DMF (5 mL) were added. Stirring continued for
a further 4 d. The thick suspension was filtered through celite and
the filtrate evaporated to dryness under reduced pressure. The
residue was treated with EtOAc, and the mixture was extracted
with 5% aqueous NaHCO3. The organic layer was separated,
washed with water, dried (Na2SO4), and evaporated to dryness under
reduced pressure. Purification by silica gel column chromatography
(20-40% EtOAc in hexanes) provided 33 as an off-white solid
(1.46 g, 44%). 1H NMR (CDCl3): δ 8.12-7.43 and 6.97-6.95 (m,
16 H), 6.77 (m, 1H), 5.86 (m, 1H), 4.87 (s, 2H), 3.89 (s, 3H). MS
(ES+) [M + H]+ m/z 647.
1-(4-N-Benzoyl-2′-dideoxy-2′,2′-difluoro-5′-iodo-ꢀ-D-ribofura-
nosyl)cytosine (30). A solution of 4-N-benzoylgemcitabine (29)15
(3.50 g, 9.54 mmol) in dry CH3CN/pyridine [95:5 (150 mL)] was
cooled on an ice-water bath. Iodine (3.15 g, 12.4 mmol) and
triphenylphosphine (3.38 g, 12.9 mmol) were added, and the
reaction mixture was stirred at 0 °C for 30 min. The ice-water
bath was then removed, and stirring continued at room temperature
for 64 h. The reaction mixture was diluted with CH2Cl2 (300 mL)
and washed with 0.5 M Na2S2O3 in saturated aqueous NaHCO3
(200 mL). The aqueous layer was washed with CH2Cl2 (4 × 25
mL). The combined organic layers were dried (Na2SO4) and filtered.
The filtrate was diluted with toluene (150 mL) and concentrated
under reduced pressure. Purification by silica gel column chroma-
tography (1-5% MeOH in CH2Cl2) gave 30 (4.10 g, 90%) as a
1
white solid. H NMR (methanol-d4): δ 8.20-7.53 (m, 7H), 6.34
(t, J ) 8.2 Hz, 1H), 4.19-4.06 (m, 1H), 3.88-3.82 (m, 1H),
3.72-3.68 (m, 1H), 3.61-3.57 (m, 1H). MS (ES+) [M + H]+ m/z
478.
4′-Azido-2′-dideoxy-2′,2′-difluoro-ꢀ-D-ribofuranosyl)cytosine (34).
Compound 33 (1.46 g, 2.26 mmol) was dissolved in dioxane (10
mL) and then saturated NH3 in methanol (150 mL) was added and
the mixture was stirred for 16 h at room temperature. The residue
after evaporation of the solvents was subjected to purification by
silica gel column chromatography (5-20% EtOH in CH2Cl2) and
by semipreparative Hyper Carb HPLC column chromatography
(10-90% CH3CN in H2O) to give 34 as a white solid (0.548 g,
1-(4-N-Benzoyl-2′,5′-dideoxy-2′,2′-difluoro-ꢀ-D-pent-4-enori-
bofuranosyl)cytosine (31). Compound 30 (4.10 g, 8.59 mmol) was
dissolved in anhydrous N,N-dimethylformamide (125 mL), and the
solution was cooled to 0 °C. A solution of potassium tert-butoxide
(1.93 g, 17.2 mmol) in anhydrous N,N-dimethylformamide (25 mL)
was added dropwise over 20 min. The reaction mixture was stirred
at 0 °C for another 60 min, after which time HPLC analysis showed
some remaining compound 30. A solution of potassium tert-
butoxide (0.19 g, 1.72 mmol) in anhydrous N,N-dimethylformamide
(2.5 mL) was added dropwise over 5 min, and stirring continued
for another 5 min at 0 °C. Pyridinium form DOWEX H+ (16 g)
was added, and the mixture was stirred at room temperature for 10
min. The resin was removed by filtration and washed with DMF.
The filtrate was concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (10% MeOH in
CH2Cl2) to give 31 (2.37 g, 79%) as a white solid. 1H NMR
(methanol-d4): δ 7.99-7.52 (m, 7H), 6.47 (t, J ) 7.3 Hz, 1H),
4.89 (t, J ) 9.7 Hz, 1H), 4.78 (s, 1H), 4.57 (s, 1H). MS (ES+) [M
+ H]+ m/z 350.
1-(4′-Azido-4-N-benzoyl-2′-dideoxy-2′,2′-difluoro-5′-iodo-ꢀ-D-
ribofuranosyl)cytosine (32). Benzyltriethylammonium chloride
(3.08 g, 13.5 mmol) and NaN3 (0.878 g, 13.5 mmol) were suspended
in anhydrous CH3CN (150 mL) and sonicated for 3 min. The
resulting fine suspension was stirred for 3 h at room temperature
and was then filtered under nitrogen into a dry THF solution (47
mL) of 31 (2.36 g, 6.76 mmol). 4-Methylmorpholine (0.223 mL,
2.03 mmol) was added and the resulting solution cooled on an
ice-water bath. A solution of iodine (2.92 g, 11.5 mmol) in
1
80%); mp 143.0-144.0 °C. H NMR (400 MHz, DMSO-d6): δ
7.58 (d, J ) 7.6 Hz, 1H), 7.46 (s, 2H), 6.73 (br s, 1H), 6.30 (br s,
1H), 5.81 (br s, 1H), 5.80 (d, J ) 7.6 Hz, 1H), 4.50 (br s, 1H),
3.80 (s, 2H). MS (ES+) [M + H]+ m/z 305. HRMS (ES+) calcd
for C9H11F2N6O4 [M + H]+ 305.0810; found 305.0812.
Acknowledgment. We thank Marc Cummings and Yanzhou
Liu for analytical support.
Supporting Information Available: Biology methods, analytical
methods, and HPLC spectral data for compounds 10, 17, 20, 28,
and 34. This material is available free of charge via the Internet at
http://pubs.acs.org.
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