Discovery of a luoro-2′-β- C -methyluridine Nucleotide Prodrug (PSI-7977) for the treatment of hepatitis C virus

Article abstract of DOI:10.1021/jm100863x

Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the development of HCV therapies. β-d-2′-Deoxy-2′-α-fluoro-2′-β-C-methyl nucleosides are selective inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic. Phosphoramidate prodrugs of the 5′-phosphate derivative of the β-d-2′-deoxy-2′-α- fluoro-2′-β-C-methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.

Full text of DOI:10.1021/jm100863x

7202 J. Med. Chem. 2010, 53, 7202–7218
DOI: 10.1021/jm100863x
Discovery of a β-D-20-Deoxy-20-r-fluoro-20-β-C-methyluridine Nucleotide Prodrug (PSI-7977) for the
Treatment of Hepatitis C Virus
Michael J. Sofia,* Donghui Bao, Wonsuk Chang, Jinfa Du, Dhanapalan Nagarathnam, Suguna Rachakonda,
P. Ganapati Reddy, Bruce S. Ross, Peiyuan Wang, Hai-Ren Zhang, Shalini Bansal, Christine Espiritu, Meg Keilman,
Angela M. Lam, Holly M. Micolochick Steuer, Congrong Niu, Michael J. Otto, and Phillip A. Furman
Pharmasset, Inc., 303A College Road East, Princeton, New Jersey 08540
Received July 10, 2010
Hepatitis C virus (HCV) is a global health problem requiring novel approaches for effective treatment
of this disease. The HCV NS5B polymerase has been demonstrated to be a viable target for the
development of HCV therapies. β-D-20-Deoxy-20-R-fluoro-20-β-C-methyl nucleosides are selective
inhibitors of the HCV NS5B polymerase and have demonstrated potent activity in the clinic.
Phosphoramidate prodrugs of the 50-phosphate derivative of the β-D-20-deoxy-20-R-fluoro-20-β-C-
methyluridine nucleoside were prepared and showed significant potency in the HCV subgenomic
replicon assay (<1 μM) and produced high levels of triphosphate 6 in primary hepatocytes and in the
livers of rats, dogs, and monkeys when administered in vivo. The single diastereomer 51 of diastereomeric
mixture 14 was crystallized, and an X-ray structure was determined establishing the phosphoramidate
stereochemistry as Sp, thus correlating for the first time the stereochemistry of a phosphoramidate
prodrug with biological activity. 51 (PSI-7977) was selected as a clinical development candidate.
Introduction
HCV is a plus strand RNA virus of the Flaviviridea family
witha 9.6kbgenomeencodingfor10proteins: threestructural
proteins and seven nonstructural proteins. The nonstructural
proteins, which include the NS5B RNA dependent RNA
polymerase (RdRp), provide several attractive targets for
the development of anti-HCV therapy.7,8 The HCV RdRp is
part of a membrane associated replication complex that is
composed of other viral proteins, viral RNA, and altered
cellular membranes.3 The NS5B polymerase is responsible for
replicating the viral RNA genome and thus is absolutely
required for HCV replication. As in the case of other viral
polymerases, two approaches have been pursued to identify
small molecule HCV NS5B polymerase inhibitors. These
approaches include the identification of nucleoside analogues
that function as alternative substrate inhibitors that induce a
chain termination event and non-nucleoside inhibitors that
bind to allosteric sites on the polymerase leading to a non-
functional enzyme.5,6
Several nucleoside classes have been or continue to be in
development as inhibitors of HCV. These classes include the
β-D-20-deoxy-20-R-F-20-β-C-methylribose, the β-D-20-β-
methylribose, and the 40-azidoribose classes.9-13 These classes
are represented by the clinical candidates 1 (RG7128), 2 (NM-
283), and 3 (R1626), respectively (Figure 1).
Compound 1 is a 30,50-diisobutyrate ester prodrug of the
cytidine nucleoside 4. In clinical studies when administered at
1000 mg b.i.d., 1 demonstrated efficacy in genotype 1 infected
patients (reduction of HCV RNA levels) in a 14-day mono-
therapy study (-2.7 log10 decrease in HCV RNA) and
produced a 88% RVR in a 4-week combination study with
SOC-pegylated interferon plus ribavirin.14,15 In addition, 1
wasshowntobeefficaciousinHCVgenotype 2,3patientswho
had not responded to prior therapy, the first direct-acting
The hepatitis C virus (HCVa) presents a global health
problem with approximately 180 million individuals infected
worldwide with 80% of those progressing to chronic HCV
infection.1 Of those chronically infected individuals, approxi-
mately 30% will develop liver cirrhosis and 10% will go on to
develop hepatocellular carcinoma.2 The current standard of
care (SOC) for HCV infected patients consists of regular
injections of pegylated interferon (IFN) and oral ribavirin
(RBV) administration. However, SOC has proven to be
effective in producing a sustained virological response in only
40-60% of patients treated, dependent on viral genotype and
other predictors of host immune responsiveness. In addition,
drug discontinuations may be high because of adverse side
effects associated with the SOC treatment regimen.3,4 Conse-
quently, the development of alternative treatment options is
greatly needed. The search for novel therapies for the treat-
ment of HCV infection has focused on the development of
direct acting antiviral agents (DAAs).5,6
*To whom correspondence should be addressed. Phone: (609) 613-
4123. Fax: 609-613-4150. E-mail: michael.sofia@pharmasset.com.
a Abbreviations: HCV, hepatitis C virus; SOC, standard of care; IFN,
interferon; RBV, ribavirin; DAA, direct acting antiviral; RdRp, RNA
dependent RNA polymerase; b.i.d., twice daily; q.d., once daily; YMPK,
uridine-cytidine monophosphate kinase; NDPK, nucleoside diphos-
phate kinase; SAR, structure-activity relationship; NMI, N-methyl-
imidazole; DCM, dichloromethane; EC90, compound concentration that
returns 90% of inhibition; SGF, simulated gastric fluid; SIF, simulated
intestinal fluid; NTP, nucleoside triphosphate; PK, pharmacokinetic;
Cmax, maximum concentration; AUC, area under the curve; tmax, time at
maximum concentration; PAMPA, parallel artificial membrane perme-
ability assay; IC50, compound concentration that returns 50% of
inhibition; NOAEL, no observed adverse effect level; TFA, trifluoro-
acetic acid, THF, tetrahydrofuran; IPE, isopropyl ether; DMSO,
dimethylsulfoxide.
r
pubs.acs.org/jmc
Published on Web 09/16/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7203
antiviral to show multiple genotype coverage in the clinic.16
However, even with the positive clinical attributes of 1, we
were interested in investigating second generation agents with
improved potency, enhanced pharmacokinetic properties
(i.e., q.d. dosing), and the potential for generating high concen-
trations of the active triphosphate in the liver to enable low
doses and potentially fixed-dose combinations of DAAs. To
achieve this objective, we focused on β-D-20-deoxy-20-R-F-20-
β-C-methyluridine (5) (Figure 2). Earlier studies had shown
that while 5 was inactive in the HCV replicon assay, its
triphosphate was a potent inhibitor of HCV NS5B with a
Ki of 0.42 μM.17-19 In addition, metabolism studies with 4
showed that the monophosphate of 4 can be deaminated to
the uridine monophosphate derivative and subsequently ana-
bolized to the triphosphate 6 by uridine-cytidine mono-
phosphate kinase (YMPK) and nucleoside diphosphate kinase
(NDPK) (Figure 2). This uridine triphosphate was shown to
have an intracellular half-life of 38 h.17-19 Therefore, in order
to leverage the desired attributes of the uridine derivative, we
needed to deliver the monophosphate of uridine nucleoside 5.
To accomplish this, we required a monophosphate prodrug
that would bypass the nonproductive phosphorylation step
and that would potentially accomplish ourotherobjective: the
delivery of high liver concentrations of the desired triphos-
phate 6.
Phosphoramidate prodrug strategies had been shown
to enhance nucleoside potency in cell culture presumably
by increasing intracellular concentrations of the active
nucleotide.20-22 However, at the time we began our work
there was no example where phosphoramidate prodrug tech-
nology was applied to the inhibition of HCV. We speculated
that application of the phosphoramidate prodrug method
would be an ideal approach for delivering the desired uridine
monophosphate to hepatocytes in an in vivo setting (Figure 3).
We hoped to take advantage of first pass metabolism where
the liver enzymes would hydrolyze the terminal carboxylic acid
Figure 1. Structures of HCV nucleoside inhibitors 1, 2, 3, and 4.
Figure 2. Metabolism of 4 leads to both the active triphosphate and the inactive nucleoside 5. The monophosphate metabolite of 4 is also
metabolized to the uridine monophosphate derivative which is then further phosphorylated to the active uridine triphosphate 6.
Figure 3. First pass metabolism of the phosphoramidate prodrug derivative of the monophosphate of 5 releases the monophosphate in the
liver at the desired site of action.

7204 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
Scheme 1a
a (a) 70% aqueous acetic acid, 100 °C; (b) 25% methanolic ammonia, 0-15 °C.
Scheme 2a
a NMI, DCM, -5 to 5 °C; (b) NMI, DCM, 5-25 °C.
ester of the phosphoramidate moiety triggering a cascade of
chemical and enzymatic events that would produce the
desired uridine monophosphate at the desired site of action,
the liver. Subsequently, several reports demonstrated that
phosphoramidates of several other anti-HCV nucleosides
were able to improve potency, but these reports did not
translate this improved in vitro potency into a clinical
development candidate.23-25 Here we describe the discov-
ery of phosphoramidate prodrugs of 20-deoxy-20-R-F-20-β-
C-methyluridine 50-monophosphate and the selection of 14
and ultimately of its single isomer 51 as clinical develop-
ment candidates.
moiety was appended by reacting 5 with a freshly prepared
chlorophosphoramidate reagent 11 in the presence of NMI.20
Each chlorophosphoramidate reagent was prepared by stir-
ring an amino acid ester 9 with the appropriate phosphoro-
dichloridate reagent 10 in the presence of an amine base (Et3N
or NMI) in either THF or dichloromethane. The reaction
provided the desired 50-phosphoramidates12-49asthemajor
product with lesser amounts of the 30- and 30,50-phosphor-
amidate derivatives. The desired 50-phosphoramidates were
purified by chromatography as a 1:1 mixture of diastereomers
at the phosphorus center.
The anti-HCV activity of these prodrugs was assessed using
the clone A replicon and a quantitative real time PCR assay.12
Each compound was simultaneously evaluated for cytotox-
icity by assessing for the levels of cellular rRNA.12 The
objective was to identify phosphoramidate prodrugs that
exhibited submicromolar activity with the hope that this
increased activity would translate into reduced drug load in
the clinic relative to 1. A survey of the terminal carboxylic acid
ester of the phosphoramidate moiety in which the amino acid
was alanine and the phosphate ester was simply phenyl
showed that small simple alkyl and branched alkyl groups
provided the desired submicromolar activity; however, in the
case of the n-butyl (15), 2-butyl (16), and n-pentyl (17) esters,
cytotoxicity was observed (Table 1). Small cycloalkyl (18) and
benzyl (22, 23) esters were also compatible; however, phenyl
(21) and halogenated alkyl groups (19, 20) did not provide the
desired potency enhancement.
Results and Discussion
The development of phosphoramidate prodrugs of 5 began
with the investigation of the anti-HCV SAR around the
phosphoramidate portion of the molecule. The synthesis of
the 20-deoxy-20-R-F-20-β-C-methyluridine phosphoramidates
began with the preparation of the uridine nucleoside 5. To
obtain the uridine nucleoside 5, we started with the benzoyl
protected 20-deoxy-20-R-F-20-β-C-methylcytidine (7). We
recently reported on the efficient synthesis of 7.26 From 7,
preparation of the uridine nucleoside was efficiently accom-
plished by a two-step process (Scheme 1). The benzoyl
cytidine 7 was heated with 80% acetic acid overnight to afford
the protected uridine 8, which was then treated at room
temperature with methanolic ammonia to provide 5 in 78%
yield.
The phosphoramidate derivatives of the uridine nucleoside
were prepared as shown in Scheme 2. The phosphoramidate
A survey of the phosphoramidate phosphate ester sub-
stituent (Table 2) demonstrated that the 1-naphthyl ester 29

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7205
Table 1. HCV Replicon Activity of Phosphoramidate Prodrugs 12-23: Modification of the Phosphoramidate Ester Moiety
compd
R2
EC90 cloneA (μM)a
inhibition of cellular rRNA replication at 50 μM (%)b
1
3.9
0
12
13
14
15
16
17
18
19
20
21
22
23
Me
Et
0.91
0.98
0.52
0.09
0.06
>50
0.25
1.72
6.80
18.50
0.13
0.24
0
36.9
25.9
79.6
93.8
92
i-Pr
n-Bu
2-Bu
n-Pen
c-Hex
FCH2CH2
F2CHCH2
Ph
61
43.8
38.3
0
Bn
4-F-Bn
74.3
0
a Each value is a result of n = 2 determinations. b Clone A cells.
Table 2. HCV Replicon Activity of Phosphoramidate Prodrugs 12 and 24-30: Modification of the Phosphoramidate Phenolic Ester Substituent
compd
R3
EC90 cloneA (μM)a
inhibition of cellular rRNA replication at 50 μM (%)b
12
24
25
26
27
28
29
30
Ph
0.91
0.69
0.58
2.11
0.45
0.69
0.09
>50
0.0
4-F-Ph
4-Cl-Ph
4-Br-Ph
3,4-Cl-Ph
2,4-Cl-Ph
1-Napth
Et
16.8
62.8
30.8
63.7
10.9
95.4
16.8
a Each value is a result of n = 2 determinations. b Clone A cells.
provided the greatest potency and that mono- and dihalo-
genated phenolic esters also gave inhibitors with submicromolar
potency. The derivative with a simple alkyl phosphate ester
(30) was not active against HCV. Although the 1-naphthol
ester substitution produced the most potent HCV inhibitor,
this substitution also led to substantial cytotoxicity and was
therefore not considered a viable substituent.
Study of the amino acid side chain demonstrated that a
small alkyl group (12, 32) was accommodated, but R-sub-
stitution larger than ethyl showed substantial reduction in
potency (Table 3). R-Disubstituted amino acids that included
an R-cyclopropanylamino acid derivative 39 did not provide
the target submicromolar potency (Table 4). Additionally, it
was shown that the natural L-amino acid was required for
activity, since the D-alanine derivative 40 was inactive
(Table 4).
acid moiety, that methyl, ethyl, isopropyl, or cyclohexyl
carboxylate esters provided the desired potency enhancement,
and that the phosphate ester accommodated simple phenyl or
halogenated phenyl substituents. Subsequently, select combi-
nations of these preferred substitutions were prepared in
which only the phenyl or para-halogenated phenyl phosphate
ester analogues were examined. Polyhalogenated phosphate
esters were excluded from further evaluation in order to
preempt any potential toxicity issues that may arise from the
release of polyhalogenated phenols upon conversion of the
phosphoramidate to the desired nucleoside monophosphate
(Table 5).27-29 The most dramatic difference observed in the
SAR for the phosphoramidate substituent combinations was
associated with the terminal carboxylic acid ester substituent
where the cyclohexyl ester derivatives (18, 47-49) showed as
much as a 10-fold improvement in potency relative to their
methyl, ethyl, or isopropyl analogues. On the basis of replicon
potency, initial cytotoxicity profile, and structural diversity,
Results of the phosphoramidate moiety single substituent
modifications showed that L-alanine was the preferred amino

7206 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
Table 3. HCV Replicon Activity of Phosphoramidate Prodrugs 12 and 31-37: Modification of the Amino Acid Side Chain
compd
R1
EC90 clone A (μM)a
inhibition of cellular rRNA replication at 50 μM (%)b
31
12
32
33
34
35
36
37
H
22.11
0.91
1.61
>50
5.4
0.0
0.0
Me
Et
0.0
0.0
Me2CH
Me2CHCH2
MeSCH2CH2
PhCH2
0.0
60.13
57.65
15.6
24.1
20.6
68.4
indole-3-CH2
a Each value is a result of n = 2 determinations. b Clone A cells.
Table 4. HCV Replicon Activity of Phosphoramidate Prodrugs 24 and 38-40: R-Disubstituted Amino Acid Side Chains
compd
EC90 clone A (μM)a
inhibition of cellular rRNA replication at 50 μM (%)b
24
38
39
40
0.69
2.20
>50
>50
16.8
0.0
0.0
0.09
a Each value is a result of n = 2 determinations. b Clone A cells.
a select set of seven compounds (12, 14, 18, 41, 44, 45, and 47)
was chosen for further evaluation.
improved potency relative to 4 in the HCV replicon and
stability in SGF, SIF, and plasma but showed a short half-
life in liver S9, which could indicate rapid release in hepato-
cytes. Table 6 shows the in vitro stability data for the key
compounds selected from combinations of the preferred
phosphoramidate substituents. Each of these compounds
exhibited prolonged stability (t1/2>15 h) in SGF, SIF, and
human plasma but decomposed quickly to the monophos-
phate when incubated with human liver S9 fraction.
Having the desired target activity and stability profile,
compounds 12, 14, 18, 41, 44, 45, and 47 were evaluated in
vivo to determine liver levels of the active uridine triphosphate
6 after oral administration. Since HCV replicates in liver cells,
measurable levels of nucleoside triphosphate (NTP) should be
a strong indication of in vivo efficacy, the assumption being
To achieve the objective of identifying a phosphoramidate
prodrug of the 20-R-F-20-β-C-methyluridine monophosphate
suitable for clinical studies as a treatment for HCV, the
prodrug moiety would need to survive exposure in the gastro-
intestinal tract and preferentially release the nucleotide mono-
phosphate in the liver. Consequently, compounds 12, 14, 18,
41, 44, 45, and 47 were further evaluated for gastrointestinal
stability using simulated gastric fluid (SGF) and simulated
intestinal fluid (SIF). In addition, stability in human plasma
and stability on exposure to human liver S9 fraction were
also evaluated (Table 6). The human liver S9 fraction was
chosen as a surrogate in vitro model to test for liver stability.
The ultimate objective was to select compounds that showed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7207
Table 5. HCV Replicon Activity of Phosphoramidate Prodrugs: Simultaneous Carboxylate and Phenolic Ester Modification of the
Phosphoramidate Moiety
compd
R2
Me
R3
Ph
EC90 cloneA (μM)a
inhibition of cellular rRNA replication at 50 μM (%)b
12
24
25
26
13
41
42
43
14
44
45
46
18
47
48
49
1.62
0.69
0.58
2.11
0.98
0.76
0.39
0.36
0.52
0.77
0.42
0.57
0.25
0.04
0.054
0.039
0.0
Me
Me
4-F-Ph
4-Cl-Ph
4-Br-Ph
Ph
16.8
62.8
30.8
36.9
55.3
0.0
Me
Et
Et
Et
4-F-Ph
4-Cl-Ph
4-Br-Ph
Ph
Et
80.5
25.9
0.0
i-Pr
i-Pr
i-Pr
i-Pr
c-Hex
c-Hex
c-Hex
c-Hex
4-F-Ph
4-Cl-Ph
4-Br-Ph
Ph
0.0
0.0
61.1
52.1
66.9
91.5
4-F-Ph
4-Cl-Ph
4-Br-Ph
a Each value is a result of n = 2 determinations. b Clone A cells.
Table 6. Stability Assessment in SGF, SIF, Human Plasma, and
Human Liver S9 Fraction for Compounds 12, 14, 18, 41, 44, 45, and 47
Table 7. PK Parameters of Uridine Triphosphate 6 in Rat Liver after an
Oral Dose of 50 mg/kg Phosphoramidate Prodrugs 12, 14, 18, 41, 44, 45,
and 47a
stability t1/2 (h)
Cmax
(ng/g)
tmax
(h)
AUC(0-t)
(ng h/g)
AUC(inf)
(ng h/g)
compd
SGFa
SIFb
human plasmac
human S9d
compd
3
3
12
14
18
41
44
45
47
15.5
22
>20
>24
>20
>20
>20
>20
>20
16.7
>24
>24
>8
0.18
0.57
1.4
12
14
18
41
44
45
47
1985
1934
557
291
519
339
716
6.00
4.00
2.00
4.00
6.00
1.00
4.00
14206
16796
6487
4191
6140
5143
8937
18968
18080
8831
5423
7375
8468
9888
17
17
0.23
0.42
0.35
0.18
>20
>20
20
>24
>24
>24
a SGF = simulated gastric fluid, pH 1.2, 50 μg/mL concentration,
37 °C, 20 h. b SIF = simulated intestinal fluid, pH 7.5, 50 μg/mL concen-
tration, 37 °C, 20 h. c 100 μM, 37 °C, 24 h. d 100 μM, 37 °C, 24 h, pH 7.4.
a Livers were removed at time points 0.5, 1, 2, 4, 6, and 12 h postdose.
of liver exposure to determine whether a dramatic species
difference in PK behavior existed among compounds 12, 14,
and 47. Comparison of the in vivo PK characteristic in dogs
and cynomolgus monkeys was accomplished by evaluating
both plasma and liver exposures upon oral q.d. dosing over
4 days with a 50 mg/kg daily dose. Plasma samples were taken
on day 3, and liver samples were taken on day 4. Liver levels
were determined at a single time point 4 h postdose on day 4
and therefore reflect a single concentration at a single point in
time. Plasmaand liverlevelsof phosphoramidate prodrug and
liver triphosphate levels were analyzed by LC/MS/MS. In the
dog study, compound 14 showed a 16- and 110-fold higher
overall plasma exposure (AUC) of the parent prodrug than
compounds 12 and 47, respectively (Table 8). In the monkey
compound 14 also provided greater (>3-fold) plasma expo-
sure than did compounds 12 and 47 (Table 9). Similarly, liver
exposures in dog and monkey of the parent prodrugs were
higher for compound 14 than for compounds 12 and 47.
Triphosphate levels in the liver demonstrated the same rela-
tive trends. In dog and monkey compound 14 produced
higher triphosphate levels relative to compounds 12 and 47.
the larger the amount of NTP, the greater the potential
efficacy. Therefore, each of the seven key compounds was
evaluated in a screening rat PK study where each compound
was administered as a single 50 mg/kg oral dose, livers were
removed, and liver extracts were assayed for levels of the
20-deoxy-20-R-F-20-β-C-methyluridine triphosphate. The rela-
tive levels of triphosphate found in the liver samples would be
an indication of anticipated in vivo potency and would be
used to select which of the key compounds would progress
further. Table 7 shows the PK parameters for each of the key
compounds. Among the seven compounds, compounds 12,
14, and 47 produced the highest Cmax and AUC values. These
results strongly suggestthateach of thesecompounds was able
to traverse the GI tract, remain intact during the absorption
phase, and arrive intact at the target organ, ultimately result-
ing in high drug exposure in the liver.
Since little is known about which species is predictive
of human exposure for nucleoside phosphoramidates, addi-
tional in vivo PK assessment was undertaken in dog and
cynomolgus monkeys to provide a cross-species comparison

7208 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
Table 8. Dog Plasma and Liver PK Profile after Oral Administration of Compounds 12, 14, and 47
plasma (prodrug)b
liverc
AUC(inf)
(ng h/mL)
AUC(0-t)
(ng h/mL)
prodrug
(ng/g liver)
uridine triphosphate 6
(ng/g liver)
compda
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
3
3
12
14
47
50
50
50
317
6179
36
1.00
0.50
0.25
420
6903
62
418
6894
54
5.24
612
4960
10560
476
8.72
a Dose of 50 mg/kg for 4 consecutive days. b Blood samples collected at 1, 2, 4, 6, 12, and 24 h postdose on day 3. c Livers removed at 4 h time point
postdose on day 4.
Table 9. Cynomolgus Monkey Plasma and Liver PK Profile after Oral Administration of Compounds 12, 14, and 47
plasma (prodrug)b
liverc
AUC(inf)
(ng h/mL)
AUC(0-t)
(ng h/mL)
prodrug
(ng/g liver)
uridine triphosphate 6
(ng/g liver)
compda
dose (mg/kg)
Cmax (ng/mL)
Tmax (h)
3
34
3
27
12
14
47
50
50
50
19
33
1.8
0.25
1.00
6.00
4.66
177
13
26
57
170
NA
86
NA
NA
a Dose of 50 mg/kg for 4 consecutive days. b Blood samples collected at 1, 2, 4, 6, 12, and 24 h postdose on day 3. c Livers removed at 4 h time point
postdose on day 4.
Table 10. AlogP and PAMPA Profile of Compounds 12, 14, and 47
compd
AlogP a
PAMPA permeability (nm/s)b
12
14
47
0.19
0.92
2.26
0.07
0.46
4.88
a Reference 31. b pH 7.4, incubated for 12-24 h.
generated by cells when incubated with cytidine nucleoside 4.
These enhanced triphosphate levels in clone A cells are
consistent with the increased potency observed for com-
pounds 12, 14, and 47 when compared to 4. 20-F,20-C-methyl-
uridine 50-triphosphate levels in primary hepatocytes of
human, rat, dog, and monkey incubated with compounds 12,
14, and 47 were also shown to be high, whereas in each case
the triphosphate levels following incubation with 4 were
shown to be below the limit of detection. Compound 47
consistently showed the lowest triphosphate levels of the three
phosphoramidate analogues. Compound 14 demonstrated 3-
to 4-fold higher triphosphate levels in human primary hepa-
tocytes relative to compounds 12 and 47. Compounds 12 and
14 produced comparable amounts of triphosphate in monkey
and rat hepatocytes, whereas in dog hepatocytes compound
12 showed 2-fold higher levels than 14. The interspecies
differences observed regarding conversion of phosphor-
amidates 12, 14, and 47 to the active triphosphate 6 may be
attributed to either cross-species differences in the level of
enzymes needed to convert the phosphoramidates to the
intermediate monophosphate or to differences in cell penetra-
tion. Similarly, since each of the phosphoramidates12, 14, and
47 is converted to the same triphosphate metabolite, intra-
species differences could be attributed to the selectivity of
processing enzymes or to the ability to penetrate the species
specific hepatocytes by each of the different analogues. To
assess relative membrane permeability characteristics, both
calculated AlogP values and parallel artificial membrane
permeability (PAMPA) were evaluated. Both AlogP and
PAMPA results indicate that the predicted order of cell
permeability would be 47 > 14 >12, in line with lipophilic
character (Table 10).31,32 This relative order tracks well with
the relative replicon EC90 values; however, the order does not
necessarily correlate to triphosphate levels observed in hepato-
cytes. Therefore, it is possible that other factors such as
Figure 4. Uridine triphosphate (6) levels in human, rat, dog, and
monkey primary hepatocytes when treated with 4 and uridine
phosphoramidates 12, 14, and 47.
However, it should be noted that the liver triphosphate data
were only used for an intraspecies comparison of compounds.
One should not compare the relative dog to monkey liver
triphosphate levels because of the liver single time point
sampling protocol.
Since the generation of triphosphate levels is critical to
predicting in vivo potency of nucleos(t)ide analogues, in vitro
triphosphate production in primary human hepatocytes was
examined and compared to in vitro triphosphate levels deter-
mined in primary hepatocytes from rat, dog, and monkey
when incubated with compounds 12, 14, and 47. An analysis
of uridine triphosphate (6) production in these primary
hepatocyte studies would provide a comparison of the inher-
ent capacity of each nucleotide phosphoramidate to both
enter hepatocytes and be converted to the target triphosphate.
In vitro analysis of triphosphate production was accom-
plished by incubating each compound for 48 h with primary
hepatocytes from rat, dog, monkey, and human and then
extracting the cells and analyzing the extracts by HPLC.19,30
The triphosphate levels generated in the primary hepatocytes
were compared to triphosphate levels generated in clone A
replicon cells and to the triphosphate levels generated on
exposure to a known clinically efficacious agent, 4. Figure 4
shows that for compounds 12, 14, and 47 20-F,20-C-methylur-
idine 50-triphosphate levels in clone A cells were 6- to 16-fold
greater than 20-F,20-C-methylcytidine 50-triphosphate levels

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7209
selectivity for processing enzymes or relative levels of proces-
sing enzymes in hepatocytes from different species are con-
tributing to the relative difference in cellular triphosphate
levels observed.
Nucleos(t)ide toxicity can be correlated to several cellular
effects. Mitochondrial toxicity has been reported to be asso-
ciated with the long-term use of certain nucleos(t)ide ana-
logues resulting in myopathy, peripheral neuropathy, and
pancreatitis.33,34 Hematotoxicity, which can lead to neutro-
penia, severe anemia, and thrombocytopenia, has also been
associated with several nucleoside analogues including the
HCV polymerase inhibitor 3.35,36 One causeof hematotoxicity
is bone marrow toxicity.35,36 To examine for these possible
toxicities, compounds 12, 14, and 47 were evaluated for
general cytotoxicity against an expanded cell panel and
evaluated in vitro for both mitochondrial toxicity and toxicity
to bone marrow progenitor cells. In an expanded cell panel
that included two human hepatocyte cell lines, Huh7 and
HepG2, a human pancreatic cell line, BxBC3, and a human T
lymphoblast cell line, CEM, compounds 12, 14, and 47 were
found to show no cytotoxicity up to 100 μM, the highest
concentrationtested. Compounds12, 14, and 47wereassessed
for mitochondrial toxicity in both CEM and HepG2 cells by
incubating each compound for 14 days. Percentage inhibition
of mitochondrial DNA production was determined relative to
a no drug control. No inhibition of mitochondrial DNA
synthesis up to the highest concentration tested (50 μM) was
observed for the three compounds. To evaluate the potential
for bone marrow toxicity, compounds 12, 14, and 47 were
screened from 0.1 to 50 μM for their effect on human
erythroid and myeloid progenitor cell colony proliferation.
Differentiation of hematopoietic progenitorsintoerythroid or
granulocyte-myeloid cell lineages over 14 days was measured.
The results showed that compounds 12 and 14 had IC50 values
of >50 μM for both erythroid and myeloid progenitor cells.
Erythroid and myeloid progenitor cells were more sensitive to
compound 47 which showed IC50 values of 37 ( 5 μM and
30 ( 5 μM for erythroid and myeloid progenitor cells, res-
pectively. Since each phosphoramidate derivative leads to the
same metabolic intermediates, it is unlikely that the metabolic
intermediates leading to the triphosphate active metabolite
contribute to the effects on erythroid or myeloid progenitor
cells. Therefore, since the only difference between compounds
12, 14, and 47 resides in the nature of the carboxylic acid ester
and the phenolic ester substituents, it would appear that the
character of the carboxylate and/or phenolic esters contributes
significantly to the effect on erythroid and myeloid progenitor
cells and therefore the potential for bone marrow toxicity. The
effect on progenitor cells observed for compound 47 could be a
result of the phosphoramidate itself or from the released ester
moieties after prodrug metabolism.
Figure 5. X-ray crystal structure of 51 crystallized from CH2Cl2.
In vitro hepatocyte triphosphate levels and in vivo PK
profiles and effects on bone marrow progenitor cells were
factors that differentiated compounds 12, 14, and 47. Com-
pound 14 consistently produced high levels of triphosphate
across all species and in particular showed the greatest triphos-
phate levels in primary human hepatocytes by as much as
3-fold over compounds 12 and 47. The in vivo PK assessment
showed that compound 14 consistently demonstrated the
highest liver triphosphate levels across species. Bone marrow
toxicity studies showed that no difference in safety profile was
observed between compounds 12 and 14; however, bone
marrow progenitor cells were more sensitive to the presence
of compound 47. On the basis of the overall profile, com-
pound 14 (PSI-7851) was selected for further development.37
14 is a 1:1 mixture of diastereomers at the phosphorus
center of the phosphoramidate moiety and is a low melting
point (mp = 66-75 °C) amorphous solid. The diastereomers
of 14 were separated by HPLC chromatography to give the
two pure diastereomers 50 (fast moving isomer) and 51 (slow
moving isomer). In the clone A replicon assay, compounds 50
and 51 produced anti-HCV activity with EC90 values of 7.5
and 0.42 μM, respectively, thus demonstrating a >10-fold
difference in activity between the two isomers. Diastereomer
51 was subsequently crystallized using methylene chloride as
the solvent, and a single crystal X-ray structure of 51 was
obtained, definitively establishing the configuration of the
phosphorus center as Sp and by corollary the configuration of
50 as Rp (Figures 5 and 6). This is the first demonstrated
crystallization and X-ray structure determination of a phos-
phoramidate nucleotide prodrug and the first example where
stereochemistry at phosphorus could be correlated unequiv-
ocally to nucleotide phosphoramidate activity.
Each diastereomer was also evaluated against replicons
containing known nucleoside resistant mutations. The S282T
NS5B polymerase mutation was shown to afford a low level
of resistance to the 20-deoxy-20-R-F-20-β-C-methylcytidine
nucleoside 4, and the S96T mutation was shown to afford
resistance to the 40-azidocytidine nucleoside 3.38-40 The
individual uridine phosphoramidate isomers 50 and 51 were
evaluated against these known nucleoside resistant mutants,
and as expected, the S282T mutant was resistant to both
isomers 50 and 51 with EC90 fold increases of >13 and 18.6,
respectively. No cross-resistance was observed when each
isomer was tested against the S96T mutation (Table 11).
Diastereoisomers 50 and 51 were evaluated for their ability
to generate intracellular levels of the active uridine triphos-
phate 6 in clone A and primary human hepatocytes.19,30
A comparison of in vivo acute toxicity of compounds 12, 14,
and 47 was undertaken by single dose oral administration in rats
with a 14-day postdose observation period. Rats (three males
and three females) were dosed with each compound at doses of
50, 300, and 1800 mg/kg. Fourteen days after dose administra-
tion, all rats were euthanized and daily clinical observations,
body weights, macroscopic pathology including kidney and
liver weights were assessed. For each of the compounds, no test-
article-related mortality, clinical signs of toxicity, body weight
changes, macroscopic pathology, or organ weight changes for
liver and kidney were observed in any of the treatment groups.
Consequently, for each of the compounds studied the NOAEL
was established at >1800 mg/kg.

7210 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
Figure 6. Structures of diastereomers 50 and 51.
Table 11. Cross-Resistance Studies Using Replicon Mutants (S282T and S96T/N142T) Resistant to Nucleotide Inhibitors 50 and 51
clone A: EC90 (μM)a
ET-lunet: EC90 (μM)a
EC90 fold increaseb
S282T/WT
EC90 fold increaseb
S96T/WT
compdc
WT S282T
WT
S96T
50
51
7.5 ( 3.0
0.42 ( 0.23
>100
>13
18.6
3.3 ( 1.4
0.23 ( 0.15
1.3 ( 0.3
0.11 ( 0.039
0.4
0.5
7.8 ( 5.3
a Increasing concentrations of 50 or 51 were added to wild type (WT), S282T, or S96T/N142T replicon cells (clone A or ET-lunet). The concentration
at which 90% inhibition (EC90) occurred was calculated. b The fold change in EC90 of the inhibitor against the S282T or S96T/N142T mutant replicon
compared to the wild type replicon was determined. c At least three independent experiments were performed for the compounds in each cell line.
When clone A cells were incubated for 24 h in the presence
of 50 or 51 (5 μM), diastereoisomer 51 produced 29.3 μM
of triphosphate while compound 50 produced only 2.7 μM, a
10-fold difference in favor of 51. When incubated with pri-
mary human hepatocytes under the same conditions, the level
of triphosphate produced with diastereomer 51 (45.0 μM)
was 14% greater than that produced with diastereomer 50
(39.5 μM). It is interesting to note that the overall levels of
triphosphate produced in human hepatocytes is significantly
greater than seen in clone A replicon cell line. In addition, it is
clear that there is a diastereomer preference for triphosphate
formation favoring diastereomer 51 over 50.
with potent anti-HCV activity and that these phosphoramidates
were effective at delivering high levels of active triphosphate
both in vitro and in vivo. 14 was selected as a development
candidate and demonstrated early clinical proof of concept for
the treatment of HCV infection by a 20-R-F-20-β-C-methyl-
uridine phosphoramidate. Identification of 51 as the preferred
single diastereomer resulted in the first crystallization and X-ray
structure determination of a nucleotide phosphoramidate and
correlation of phosphoramidate stereochemistry with biological
potency. The Sp diastereomer 51 is currently in phase II clinical
development for the treatment of HCV infection.
Comparison of cytotoxicity profiles for each isomer (50
and 51) demonstrated that both compounds were essentially
devoid of cytotoxicity when assessed in an 8-day cytotoxicity
assay against an expanded cell panel (Huh7, HepG2, BxPC3,
and CEM cells) at concentrations up to 100 μM. When
mitochondrial toxicity (CC90) was evaluated by incubating
CEM and HepG2 cells with compounds 50 and 51 for 14 days
and then measuring the levels of mitochondrial COXII DNA
(mtDNA) and ribosomal DNA (rDNA) using real time PCR,
both compounds demonstrated no measurable mitochondrial
toxicity up to 100 μM in CEM cells but compound 51 showed
a CC90 of 72.1 and 68.6 μM for the inhibition of mtDNA and
rDNA, respectively, in HepG2 cells.
On the basis of its superior potency and ability to produce
higher intracellular triphosphate levels, diastereomer 51 was
selected as the preferred diastereomer for further study.
Subsequently, we have applied the above crystallization con-
ditions to selectively crystallize the more active diastereomer
51 from the diastereomeric mixture 14. These crystallization
conditions have been applied on multikilogram scale to sup-
port further development of 51. The uridine phosphoramidate
51 (PSI-7977) is currently in phase II clinical trials for the
treatment of HCV infection.37
Experimental Section
Nuclear magnetic resonance (NMR) spectra were recorded on
a Bruker advance II 400 MHz and a Varian Unity Plus 400 MHz
spectrometers at room temperature, with tetramethylsilane as an
internal standard. Chemical shifts (δ) are reported in parts per
million (ppm), andsignals are reported as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), or br s (broad singlet). Purities
of the final compounds were determined by HPLC and were
greater 95%. HPLC conditions to assess purity were as follows:
Shimadzu HPLC 20AB Sepax HP-C18 4.6 mm ꢀ 50 mm (5 μm);
flow rate, 3.0 mL/min; acquire time, 6 min; wavelength, UV
220 nm; oven temperature, 40 °C. Chiral HPLC was conducted
onShimadzuHPLC 20A. The preparative HPLCsystemincludes
twosetsofGilson306pumps, a Gilson156 UV/visdetector, anda
Gilson 215 injector and fraction collector, with Unipoint control
software. A YMC25mmꢀ 30 mm ꢀ 2 mmcolumnwas used. The
mobile phase was HPLC grade water (A) and HPLC grade
acetonitrile (B) system. SFC was conducted on Berger Multi-
Gram SFC from Mettler Toledo Co, Ltd. LC/MS was conducted
on Shimadzu LCMS 2010EV using electrospray positive [ESþve
to give MHþ] equipped with a Shim-pack XR-ODS 2.2 μm
column (3.0 mm ꢀ 30 mm, 3.0 mm i.d.), eluting with 0.0375%
TFA in water (solvent A) and 0.01875% TFA in acetonitrile
(solvent B). High-resolution mass spectra were obtained on a
Agilent G1969a spectrometer.
20-Deoxy-20-fluoro-20-C-methyluridine (5). 30,50-O-Dibenozyl-
20-deoxy-20-fluoro-20-C-methyl-N4-benzoylcytidine (7, 500 g,
0.874 mol) was added to 70% aqueous acetic acid (7.5 L). The
solution was heated to reflux (110 °C) for 20 h. The mixture was
cooled to ambient temperature and diluted with water (2 L).
After the mixture was stirred for 2 h, the resulting precipitate
Conclusion
We have demonstrated that application of the phosphor-
amidate prodrug approach to the 50-monophosphate of 20-deoxy-
20-R-F-20-β-C-methyluridine produced nucleotide derivatives

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7211
was collected by filtration and the solid was rinsed with water
(5 L) and dried in the atmosphere at ambient temperature for
12 h to afford 360 g (88%) of the dibenzoyluridine intermediate
8. This in turn was used as is in the next step by adding it all to
freshly prepared 25% methanolic ammonia (5.4 L) at 0 °C. This
temperature was maintained for 3 h and then allowed to warm to
15 °C for 24 h. The reaction mixture was filtered through a Celite
bed and concentrated under reduced pressure to give the crude
product (216 g). The crude product was stirred with ethyl acetate
(325 mL) for 3 h at ambient temperature. The resulting solid was
collected by filtration and washed with ethyl acetate (200 mL).
The solid was dried under vacuum at ambient temperature for
4 h to afford 160 g (78%) of the desired product 5 in 98.7%
HPLC purity. 1H NMR (DMSO-d6) d 11.44 (br s, 1H, NH), 7.95
(d, 1H, C-6H), 5.97 (d, 1H, C-10H), 5.64 (d, 1H, C-5H),
3.84-3.77 (m, 3H, C-50-Ha, C-30H, C-40H), 3.63-3.60 (m, 1H,
C50-Hb), 1.23 (d, 3H, C-20-CH3). ES-MS M - 1, 259.
General Procedure for the Preparation of Compounds 12-49.
Preparation of Intermediates 11. To a solution of compound 101
(5 mmol) and compound 9 (5 mmol) in dichloromethane
(25 mL), triethylamine or N-methylimidazole (1 g, 10 mmol)
in anhydrous dichloromethane (25 mL) was added dropwise
with vigorous stirring at -78 or -5 °C over a period of 2 h. The
mixture was allowed to warm to 5 °C or room temperature
gradually and stirred for an additional 0.5 h. Solvent was
evaporated under reduced pressure, and the residue was treated
with anhydrous ether (20 mL). The precipitated triethylammo-
nium chloride salt was filtered and washed with ether (10 mL).
Solvent was evaporated from the combined filtrate to give the
crude product 11 as colorless oil, and they were used in the next
step without further purification.
5.53-5.58 (m, 1H), 5.84-6.15 (m, 3H), 7.18-7.23 (m, 3H),
7.35-7.39 (m, 2H), 7.54-7.57 (m, 1H), 11.50 (s, 1H); 13C NMR
(DMSO-d6) δ 173.70, 173.66, 173.59, 173.54, 163.70, 151.60, 151.40,
140.25, 130.63, 125.55, 121.04, 120.99, 120.95, 103.28, 103.21,
102.20, 100.37, 89.42, 80.33, 72.33, 68.98, 68.95, 65.60, 65.14,
50.88, 50.72, 27.79, 22.37, 22.32, 20.77, 20.71, 20.64, 20.57, 17.60,
17.36; MS, m/e 530.2 (M þ 1)þ. HRMS, calcd for C22H30FN3O9P
(Mþ þ H), 530.1698; found, 530.1685. The two diastereomers were
separated using column chromatography on silica gel using 2-5%
isopropanol in dichloromethane as the eluent.
(S)-2-{(R)-[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-
pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-
2-ylmethoxy]phenoxyphosphorylamino}propionic Acid Isopropyl
1
Ester (50). H NMR (DMSO-d6) δ 11.53 (s, 1H), 7.56 (d, J =
8.0 Hz, 1H), 7.39-7.35 (m, 2H), 7.20-7.15 (m, 3H), 6.13-6.07
(m, 1H), 5.91 (d, J = 7.2 Hz, 1H), 5.57 (dd, J = 2.0, 8.0 Hz, 1H),
4.85 (sextet, J = 6.4 Hz, 1H), 4.43-4.38 (m, 1H), 4.30-4.23 (m,
1H), 4.05 (dm, J = 6.4 Hz, 1H), 3.80-3.72 (m, 2H), 1.24 (d, J =
22.4 Hz, 3H), 1.20 (d, J = 6.8 Hz, 3H), 1.15 (dd, J = 2.8, 6.0 Hz,
6H); 31P NMR (DMSO-d6) δ 4.76; MS (ESI) (M þ H)þ 530.1.
HRMS, calcd for C22H30FN3O9P (Mþ þ H), 530.1698; found,
530.1686.
(S)-2-{[(1R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-
1-yl)-4-(R)-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid (S)-Isopropyl
1
Ester (51). H NMR (DMSO-d6) δ 11.50 (s, 1H), 7.54 (d, J =
8.0 Hz, 1H), 7.38-7.33 (m, 2H), 7.21-7.15 (m, 3H), 6.06-5.99
(m, 2H), 5.82 (m, 1H), 5.52 (d, J = 8.0 Hz, 1H), 4.84 (quintet, J =
6.4 Hz, 1H), 4.38-4.32 (m, 1H), 4.23-4.18 (m, 1H), 4.01-3.96
(m, 1H), 3.84-3.72 (m, 2H), 1.24 (d, J = 17.2 Hz, 3H), 1.21 (d,
J = 6.4 Hz, 3H), 1.13 (d, J = 6.0 Hz, 6H); 31P NMR (DMSO-d6)
δ 4.89; MS (ESI) (M þ H)þ 530.1. HRMS, calcd for C22H30-
FN3O9P (Mþ þ H), 530.1698; found, 530.1683.
Preparation of Products 12-49. To a well-stirred mixture
of 20-deoxy-20-fluoro-20-C-methyluridine (5, 0.15 g,1 equiv) and
N-methylimidazole (0.3 g, 8 equiv) in THF (3 mL) at room
temperature, the phosphorochloridate intermediate (11, 6.5
equiv) in THF (3 mL) was added in one lot and the mixture was
vigorously stirred at room temperature overnight. Solvent was
evaporated under reduced pressure. The resulting crude product
was dissolved in methanol and purified by preparative HPLC on a
YMC 25 mm ꢀ 30 mm ꢀ 2 mm column using a water/acetonitrile
gradient elution mobile phase. The acetonitrile and water were
removed under reduced pressure to give the desired product. The
final products were characterized by LC/MS and 1H NMR.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid Methyl Ester (12). Yield:
15.6%. 1H NMR (DMSO-d6) δ 1.20-1.27 (m, 6H), 3.58 (d, J =
16.0 Hz, 3H), 3.76-3.92 (m, 2H), 4.02-4.38 (m, 2H), 5.54 (t, J =
10.2 Hz, 1H), 5.84-5.91 (m, 1H), 6.00-6.16 (m, 1H), 7.18 (d, J =
8.0 Hz, 2H), 7.22 (s, 1H), 7.35 (t, J= 4.4 Hz, 2H), 7.55 (s, 1H), 11.52
(s, 1H); 13C NMR (DMSO-d6) δ 174.61, 174.57, 174.51, 174.46,
163.69, 151.60, 151.54, 151.39, 140.27, 130.61, 125.55, 121.04,
120.99, 120.96, 103.26, 103.18, 102.17, 100.37, 89.50, 80.36, 72.34,
65.55, 65.28, 52.82, 50.73, 50.58, 27.77, 20.71, 20.65, 20.58, 20.51,
17.60, 17.35; MS, m/e 502 (Mþ1)þ. HRMS, calcd for C20H26-
FN3O9P (Mþ þ H), 502.1385; found, 502.1372.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid Butyl Ester (15). Yield:
1
12.7%. H NMR (DMSO-d6) δ 0.81-0.85 (m, 3H), 1.20-1.30
(m, 8H), 1.47-1.51 (m, 2H), 3.78-3.88 (m, 2H), 3.95-4.08 (m,
3H), 4.22-4.45 (m, 2H), 5.56-5.58 (t, 1H), 5.85-6.18 (m, 3H),
7.18-7.23 (m, 3H), 7.37-7.39 (m, 2H), 7.51-7.60 (d, 1H), 11.50
(s.1H); MS, m/e 544.2 (M þ 1)þ. HRMS, calcd for C23H32FN3O9P
(Mþ þ H), 544.1855; found, 544.1839.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid sec-Butyl Ester (16). Yield:
6.3%. 1H NMR (DMSO-d6) δ 0.89 (d, J = 6.8 Hz, 6H), 1.20-1.26
(m, 6H), 1.79-1.86 (m, 1H), 3.37-3.90 (m, 4H), 4.01 (t, J = 11.2
Hz, 1H), 4.21-4.28(m, 1H), 4.33-4.42 (m, 1H), 5.54 (t, J=7.6Hz,
1H), 5.85-5.92 (m, 1H), 5.99-6.13 (m, 2H), 7.19 (t, J = 8 Hz, 3H),
7.36(t, J= 7.6 Hz, 2H), 7.53 (d, J= 7.6 Hz, 1H), 11.52 (s, 1H); MS,
m/e 544.00 (M þ 1)þ. HRMS, calcd for C23H32FN3O9P (Mþ þ H),
544.1855; found, 544.1840.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid Pentyl Ester (17). Yield:
32.2%. 1H NMR (DMSO-d6) δ 0.80-0.81 (m, 3H), 1.17-1.23 (m,
10H), 1.47-1.50 (m, 2H), 3.77-3.81 (m, 2H), 3.94-3.96 (m, 3H),
4.21-4.36 (m, 2H), 5.52-5.54 (m, 1H), 5.87-6.01 (m, 3H),
7.14-7.19 (m, 3H), 7.32-7.35 (m, 2H), 7.51 (d, J = 8 Hz, 1H),
11.49 (s, 1H); MS, m/e 557.9 (M þ 1)þ; 1136.88 (2M þ 23)þ.
HRMS, calcd for C24H34FN3O9P (Mþ þ H), 558.2011; found,
558.1998.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid Cyclohexyl
Ester (18). Yield: 19.3%. 1H NMR (DMSO-d6) δ 1.18-1.41
(m, 12H), 1.59-1.67 (m, 4H), 3.74-3.80 (m, 1H), 3.96-4.02 (m,
1H), 4.19-4.26 (m, 1H), 4.31-4.39 (m, 1H), 4.59 (s, 1H), 5.52 (t,
J = 7.8 Hz, 1H), 5.80-6.09 (m, 3H), 7.15-7.20 (m, 3H), 7.32-
7.36 (m, 2H), 7.52 (d, J = 8 Hz, 1H), 11.50 (s, 1H); 13C NMR
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid Ethyl Ester (13). Yield:
19.6%. 1H NMR (DMSO-d6) δ 1.13-1.16 (m, 3H), 1.20-1.28
(m, 6H), 3.70-3.90 (m, 2H), 4.00-4.08 (m, 3H), 4.18-4.39 (m,
2H), 5.53-5.58 (m, 1H), 5.86-5.98 (m, 1H), 6.01-6.13 (m, 2H),
7.16-7.23 (m, 3H), 7.37-7.40 (m, 2H), 7.54-7.57(m, 1H), 11.54
(s,1H); MS, m/e 516.1 (M þ 1)þ. HRMS, calcd for C21H28-
FN3O9P (Mþ þ H), 516.1542; found, 516.1526.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid Isopropyl Ester (14). Yield:
6%. 1H NMR (DMSO-d6) δ 1.13-1.28 (m, 12H), 3.75-3.81 (m,
2H), 3.95-4.08 (m,1H), 4.20-4.45 (m, 2H), 4.83-4.86 (m, 1H),

7212 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
(DMSO-d6) δ 173.61, 173.57, 173.50, 173.45, 163.68, 151.64,
151.58, 151.40, 140.18, 130.63, 125.55, 121.04, 120.99, 120.95,
103.28, 103.22, 102.19, 100.39, 89.37, 80.37, 73.48, 72.35, 65.15,
50.92, 50.75, 31.80, 31.72, 25.76, 23.94, 20.86, 20.80, 20.73, 20.66,
17.60, 17.35; MS, m/e569.98 (M þ 1)þ; 592.14 (M þ 23)þ. HRMS,
calcd for C25H34FN3O9P (Mþ þ H), 570.2011; found, 570.1999.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}propionic Acid 2-Fluoroethyl Ester (19).
Yield: 7%. 1H NMR (DMSO-d6) δ 1.18-1.25 (m, 6H), 3.71-3.89
(m, 2H), 3.92-3.99 (m, 1H), 4.19-4.27 (m, 4H), 4.48-4.61 (m,
2H), 5.52 (m, 1H), 5.90-6.14 (m, 2H), 7.15-7.21 (m, 3H),
7.32-7.36 (m, 2H), 7.46-7.57 (m, 1H), 11.49 (s,1H); MS, m/e
533.86 (M þ 1)þ. HRMS, calcd for C21H27F2N3O9P (Mþ þ H),
534.1447; found, 534.1434.
4.24-4.39 (m, 2H), 5.56-5.61 (t, 1H), 5.86-6.10 (m, 2H),
6.17-6.20 (m, 1H), 7.21-7.26 (m, 2H), 7.43-7.46 (m, 2H),
7.54-7.57 (d, 1H), 11.50 (s.1H); MS, m/e 536.1 (M þ 1)þ.
HRMS, calcd for C20H25ClFN3O9P (Mþ þ H), 536.0995; found,
536.0981.
(S)-2-{(4-Bromophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Methyl
1
Ester (26). Yield: 12%. H NMR (DMSO-d6) δ 1.20-1.26 (m,
6H), 3.57 (d, J = 2.8 Hz, 3H), 3.84 (s, 1H), 3.97-4.03 (m, 1H),
4.21-4.25 (m, 1H), 4.33-4.37 (m, 2H), 5.54-5.60 (m, 1H),
5.83-5.89 (m, 1H), 5.98-6.19 (m, 1H), 7.16 (t, J = 10.2 Hz,
2H), 7.52-7.57 (m, 3H), 11.52 (s, 1 H); MS, m/e 580 (M þ 1)þ.
HRMS, calcd for C20H25BrFN3O9P (Mþ þ H), 580.0490; found,
580.0490.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid 2,2-Difluoro-
(S)-2-{(3,4-Dichlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyl-
tetrahydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid
Methyl Ester (27). Yield: 4%. 1H NMR (DMSO-d6) δ 1.13 (m,
6H), 3.49 (s, 3H), 3.61-3.85 (m, 2H), 3.90-3.93 (m, 1H),
4.16-4.22 (m, 1H), 4.27-4.31 (m, 1H), 5.47-5.52 (m, 1H),
5.82 (d, J = 11.6 Hz, 1H), 5.93 (d, J = 19.2 Hz, 1H), 6.15-6.25
(m, 1H), 7.13 (t, J = 9.6 Hz, 1H), 7.43 (d, J = 12 Hz, 2H), 7.57
(d, J = 6.0 Hz, 1H), 11.43 (s, 1H); MS, m/e 569.85 (M þ 1)þ.
HRMS, calcd for C20H24Cl2FN3O9P (Mþ þ H), 570.0606;
found, 570.0595.
(S)-2-{(2,4-Dichlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-
3,4-dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyl-
tetrahydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid
Methyl Ester (28). Yield: 11.5%. 1H NMR (DMSO-d6) δ
1.22-1.28 (m, 6H), 3.58-3.60 (m, 3H), 3.85-3.92 (m, 2H),
4.00-4.04 (m, 1H), 4.31-4.44 (m, 2 H), 5.55-5.61 (m, 1H),
5.86-6.10 (m, 2H), 6.33-6.39 (m, 1H), 7.44-7.54 (m, 3H),
7.72-7.75 (m, 1H), 11.54 (s.1H); MS, m/e 570.2 (M þ 1)þ.
HRMS, calcd for C20H24Cl2FN3O9P (Mþ þ H), 570.0606;
found, 570.0597.
(S)-2-[[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-
1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
(naphthalen-1-yloxy)phosphorylamino]propionic Acid Methyl
Ester (29). Yield: 18.3%. 1H NMR (DMSO-d6) δ 1.16-1.27
(m, 6H), 3.51-3.55 (d, 3H), 3.85-3.96 (m, 2H), 4.06-4.09
(m, 1H), 4.31-4.46 (m, 2H), 5.31-5.39 (m, 1H), 5.90-6.04 (m,
2H), 6.22-6.34 (m, 1H), 7.45-7.59 (m, 5H), 7.73-7.77 (m, 1H),
7.94-7.96 (m, 1H), 8.12-8.14 (m, 1H), 11.50 (s.1H); MS, m/e
552.1 (M þ 1)þ. HRMS, calcd for C24H28FN3O9P (Mþ þ H),
552.1542; found, 552.1529.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]ethoxyphosphorylamino}propionic Acid Methyl Ester
(30). Yield: 43.8%. 1H NMR (DMSO-d6) δ 1.18-1.27 (m, 9H),
3.62 (s, 3H), 3.64-3.85 (m, 2H), 3.90-4.10 (m, 4H), 4.19-4.23
(m, 1H), 5.60-5.67 (m, 2H), 5.82-6.10 (m, 2H), 7.58-7.63 (m,
1H), 11.52 (s, 1H); MS, m/e 454.05 (M þ 1)þ. HRMS, calcd for
C16H26FN3O9P (Mþ þ H), 454.1385; found, 454.1366.
{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-1-yl)-
4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}acetic Acid Methyl Ester (31). Yield:
22%. 1H NMR (DMSO-d6) δ 1.19-1.28 (m, 3H), 3.59 (s, 3H),
3.63-3.70 (m, 2H), 3.74-3.80 (m, 1H), 4.02 (d, J = 11.2 Hz, 1H),
4.23-4.28 (m, 1H), 4.40-4.43 (m, 1H), 5.57-5.59 (m, 1H), 5.89
(d, J = 6.8 Hz, 1H), 6.01-6.05 (m, 2H), 7.16-7.23 (m, 3H),
7.35-7.39 (m, 2H), 7.52(d, J = 8 Hz, 1H), 11.52 (s, 1H); MS, m/e
487.97 (M þ 1)þ. HRMS, calcd for C19H24FN3O9P (Mþ þ H),
488.1229; found, 488.1215.
1
ethyl Ester (20). Yield: 7%. H NMR (DMSO-d6) δ 1.17-1.24
(m, 6H), 3.67-3.81 (m, 1H), 3.89-3.98 (m, 2H), 4.21-4.36 (m,
4H), 5.48-5.53 (m, 1H), 5.82-6.05 (m, 2H), 6.18-6.22 (m, 2H),
7.15-7.20 (m, 3H), 7.32-7.36 (m, 2H), 7.51(s, 1H), 11.50 (s, 1H);
MS, m/e 551.92 (M þ 1)þ. HRMS, calcd for C21H26F3N3O9P
(Mþ þ H), 552.1353; found, 552.1342.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid Phenyl
1
Ester (21). Yield: 21%. H NMR (DMSO-d6) δ 1.19-1.25
(m, 3H), 1.38-1.41 (m, 3H), 3.72-3.91 (m, 1H), 4.04-4.14
(m, 2H), 4.28-4.31 (m, 1H), 4.41-4.43 (m, 1H), 5.49-5.55
(m, 1H), 5.88-6.07 (m, 2H), 6.33-6.36 (m,1H), 7.03-7.06
(m, 2H), 7.16-7.27 (m, 4H), 7. 35-7. 39 (m, 4H), 7.41-7.54
(m, 1H), 11.51 (s, 1H); MS, m/e 563.91 (M þ 1)þ, 1148.82
(2M þ 23)þ. HRMS, calcd for C25H28FN3O9P (Mþ þ H),
564.1542; found, 564.1529.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid Benzyl Ester
(22). Yield: 12.5%. 1H NMR (DMSO-d6) δ 1.20-1.28 (m, 6H),
3.72-4.01 (m, 3H), 4.23-4.26 (m, 1H), 4.37-4.38 (m, 1H),
5.08-5.09 (t, 2H), 5.52-5.56 (t, 1H), 5.86-6.04 (m, 2H),
6.16-6.19 (m,1H), 7.15-7.21 (m, 3H), 7.30-7.36 (m, 7H),
7.54-7.56 (d, 1H), 11.50 (s.1H); MS, m/e 578.2 (M þ 1)þ.
HRMS, calcd for C26H30FN3O9P (Mþ þ H), 578.1698; found,
578.1689.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionicAcid4-Fluorobenzyl
Ester (23). Yield: 27.6%. 1HNMR (DMSO-d6): δ 1.20-1.27 (m,
6H), 3.80-3.98 (m, 2H), 4.01 (s, 1H), 4.25-4.26 (m, 1H), 4.37-
4.38 (m, 1H), 5.07 (s, 2H), 5.52-5.56 (m, 1H), 5.86-5.87 (m, 1H),
5.99-6.05 (m, 1H), 6.15-6.17 (m, 1H), 7.15-7.21 (m, 5H), 7.36
(dd, J = 20.0, 8.0 Hz, 4H), 7.54 (s, 1H), 11.55 (s, 1H); MS, m/e
595.95 (M þ 1)þ. HRMS, calcd for C26H29F2N3O9P (Mþ þ H),
596.1604; found, 596.1594.
(S)-2-[[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]propionic Acid Methyl
Ester (24). Yield: 5.8%. 1H NMR (DMSO-d6) δ 1.28-1.34 (m,
6H), 3.65 (d, J = 4 Hz, 3H), 3.85-3.96 (m, 2H), 4.06-4.12 (m,
1H), 4.30-4.34 (m, 1H), 4.40-4.47 (m, 1H), 5.62-5.67 (m, 1H),
5.94-6.01 (m, 1H), 6.09 (d, J = 18.8 Hz, 1H), 6.17-6.26 (m,
1H), 7.27-7.33 (m, 4H), 7.62 (d, J = 7.6 Hz, 1H), 11.61 (s, 1H);
MS, m/e 519.94 (M þ 1)þ. HRMS, calcd for C20H25F2N3O9P
(Mþ þ H), 520.1291; found, 520.1276.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}butyric Acid Methyl Ester
(S)-2-{(4-Chlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Methyl
Ester (25). Yield: 4.4%. 1H NMR (DMSO-d6) δ 1.23-1.28 (m,
6H), 3.58 (d, 2H), 3.81-3.89 (m, 2H), 3.99-4.02 (m,1H),
1
(32). Yield: 14%. H NMR (DMSO-d6) δ 0.72-0.79 (m, 3H),
1.18-1.25 (m, 3H), 1.42-1.61 (m, 2H), 3.53-3.57 (m, 3H),
3.58-3.80 (m, 2H), 3.91-4.02 (m, 1H), 4.12-4.38 (m, 2H),

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7213
5.54-5.59 (m,1H), 5.90-6.03 (m, 1H), 7.14-7.18 (m, 3H),
7.33-7.37 (m, 2H), 7.49 (t,1H), 11.45 (s, 1H); MS, m/e 515.95
(M þ 1)þ; 1052.82 (2M þ 23)þ. HRMS, calcd for C21H28-
FN3O9P (Mþ þ H), 516.1542; found, 516.1526.
1-[(S)-[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]cyclopropane-
carboxylic Acid Methyl Ester (39). Yield: 40%. 1H NMR
(DMSO-d6) δ 0.97-0.99 (m, 1H), 1.06-1.12 (m, 1H), 1.21-
1.29 (m, 5H), 3.55 (s, 3H), 3.73-3.90 (m, 1H), 4.01 (s, 1H),
4.25-4.29 (m, 1H), 4.38-4.43 (m, 1H), 5.56-5.60 (m, 1H), 5.88
(s, 1H), 5.92-6.10 (m, 1H), 6.59 (t, J = 16 Hz, 1H), 7.21-7.23
(m, 4H), 7.56 (dd, J = 8.0, 28.0 Hz, 1H), 11.55 (s, 1H); MS, m/e
532.00 (M þ 1)þ. HRMS, calcd for C21H25F2N3O9P (Mþ þ H),
532.1291; found, 532.1280.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimidin-
1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-ylmethoxy]-
phenoxyphosphorylamino}-3-methylbutyric Acid Methyl Ester
1
(33). Yield: 20%. H NMR (DMSO-d6) δ 0.75-0.85 (m, 6H),
1.21-1.29 (m, 3H), 1.89-1.94 (m, 1H), 3.51-3.55 (m, 1H), 3.58
(d, J = 10.4 Hz, 3H), 3.72-3.95 (m, 1H), 4.03-4.05 (m, 1H),
4.23-4.37 (m, 2H), 5.56 (t, J = 16.0 Hz, 1H), 5.85-5.92 (m,
1H), 6.01-6.07 (m, 1H), 7.16-7.21 (m, 3H), 7.37 (t, J = 8 Hz,
2H), 7.55-7.60 (m, 1H), 11.52 (s, 1H); MS, m/e 530 (M þ 1)þ.
HRMS, calcd for C22H30FN3O9P (Mþ þ H), 530.1698; found,
530.1684.
(R)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}propionic Acid Isopropyl
1
Ester (40). Yield: 8.57%. H NMR (DMSO-d6) δ 1.19-1.29
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}-4-methylpentanoic Acid Methyl
(m, 12H), 3.65-3.75 (m, 2H), 3.95-4.05 (m, 1H), 4.24-4.25
(m, 1H), 4.35-4.37 (m, 1H), 4.84-4.86 (m, 1H), 5.53-5.61 (m,
1H), 5.88 (m, 1H), 6.01-6.14 (m, 2H), 7.14-7.39 (m, 5H),
7.48-7.60 (m, 1H), 11.53 (s, 1H); MS, m/e 529.96 (M þ 1)þ.
HRMS, calcd for C22H30FN3O9P (Mþ þ H), 530.1698; found,
530.1684.
1
Ester (34). Yield: 21%. H NMR (DMSO-d6) δ 0.68-0.89 (m,
6H), 1.26 (d, J = 7.2 Hz, 3H), 1.41-1.64 (m, 3H), 3.70 (s, 3H),
3.73-3.79 (m, 2H), 3.99-4.04 (m, 1H), 4.19-4.27 (m, 1H),
4.34-4.38 (m, 1H), 5.55 (t, J = 9.0 Hz, 1H), 5.86-5.92 (m, 1H),
5.99-6.12(m, 2H), 7.15-7.20 (m, 3H), 7.34-7.39 (m, 2H), 7.55 (s,
1H), 11.53 (s, 1H); MS, m/e 543.99 (M þ 1)þ. HRMS, calcd for
C23H32FN3O9P (Mþ þ H), 544.1855; found, 544.1841.
(S)-2-[[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]propionic Acid Ethyl
Ester (41). Yield: 15.4%. 1H NMR (DMSO-d6) δ 1.13-1.28
(m, 9H), 3.72-3.94(m, 2H), 3.98-4.10 (m, 3H), 4.21-4.42 (m,
2H), 5.56-5.61 (m, 1H), 5.85-6.20 (m, 3H), 7.21-7.24 (m, 4H),
7.55-7.58 (d, 1H), 11.50 (s, 1H); 13C NMR (DMSO-d6) δ 174.01,
173.97, 173.87, 163.60, 160.87, 158.48, 151.32, 147.65, 140.34,
130.52, 122.73, 122.68, 122.65, 120.92, 117.14, 116.90, 103.21,
102.10, 100.29, 89.53, 80.32, 72.40, 72.22, 65.41, 61.46, 61.43,
50.72, 50.60, 20.70, 20.63, 20.56, 20.49, 17.53, 17.28, 14.82;
MS, m/e 533.90 (M þ 1)þ; HRMS, calcd for C21H27F2N3O9P
(Mþ þ H), 534.1453; found, 534.1434.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}-4-methylsulfanylbutyric Acid
1
Methyl Ester (35). Yield: 27.4%. HNMR (DMSO-d6): δ 1.25
(dd, J = 22.8 Hz, 5.6 Hz, 3H), 1.77-1.85 (m, 2H), 1.95 (d, J =
10.0 Hz, 3H), 2.31-2.36 (m, 1H), 2.40-2.44 (m, 1H), 3.59-3.64
(m, 3H), 3.88-3.91 (m, 2H), 4.05 (m, 1H), 4.25-4.26 (m, 1H),
4.29-4.40 (m, 1H), 5.56 (t, J = 8.0 Hz, 1H), 5.90-5.92 (m, 1H),
6.13-6.16 (m, 2H), 7.16-7.23 (m, 3H), 7.36-7.40 (m, 2H), 7.55
(d, J = 8.4 Hz, 1H), 11.51 (s, 1H); MS, m/e 562.52(M þ 1)þ.
HRMS, calcd for C22H30FN3O9PS (Mþ þ H), 562.1419; found,
562.1406.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}-3-phenylpropionic Acid
Methyl Ester (36). Yield: 6.6%. 1H NMR (DMSO-d6) δ
1.19-1.34 (m, 3H), 2.79-2.97 (m, 2H), 3.51-3.57 (m, 3H),
3.60-3.85 (m, 1H), 3.91-4.13 (m, 4H), 5.50-5.52 (m, 1H),
5.80-6.10 (m, 2H), 6.20-6.35 (m, 1H), 7.01-7.05 (m, 2H),
7.14-7.32 (m, 8H), 7.47 (s, 1H), 11.50 (s.1H); MS, m/e 578.2
(M þ 1)þ. HRMS, calcd for C26H30FN3O9P (Mþ þ H),
578.1698; found, 578.1687.
(S)-2-{(4-Chlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Ethyl
Ester (42). Yield: 30.17%. 1H NMR (DMSO-d6) δ 1.14 (t, J =
7.0 Hz, 3H), 1.21-1.28 (m, 6H),3.77-3.89 (m, 2H), 4.00-4.08
(m, 3H), 4.24-4.27 (m, 1H), 4.34-4.43 (m, 1H), 5.56-5.61 (m,
1H), 5.86-6.13 (m, 2H), 6.17-6.24 (m, 1H), 7.21-7.26 (m, 2H),
7.44 (d, J = 7.6 Hz, 2H), 7.55 (d, J = 7.6 Hz, 1H), 11.55 (s, 1H);
MS, m/e 549.11 (M þ 1)þ; HRMS, calcd for C21H27ClFN3O9P
(Mþ þ H), 550.1157; found, 550.1141.
(S)-2-{(4-Bromophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Ethyl
Ester (43). Yield: 35.86%. 1H NMR (DMSO-d6) δ 1.13 (t, J =
7.2 Hz, 3H), 1.16-1.28 (m, 6H), 3.78-3.86 (m, 2H), 4.01-4.07
(m, 3H), 4.24-4.28 (m, 1H), 4.39-4.40 (m, 1H), 5.56-5.61 (m,
1H), 5.82-5.90 (m, 1H), 5.93-6.07 (m, 1H), 6.14-6.19 (m, 1H),
7.18 (dd, J = 11.2, 8.8 Hz, 2H), 7.53-7.58 (m, 3H), 11.53 (s, 1H);
MS, m/e 593.90(M þ 1)þ. HRMS, calcd for C21H27BrFN3O9P
(Mþ þ H), 594.0652; found, 594.0640.
(S)-2-{[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]phenoxyphosphorylamino}-3-(1H-indol-3-yl)propionic
1
Acid Methyl Ester (37). Yield: 8.1%. H NMR (DMSO-d6) δ
0.96 (d, J = 22.8 Hz, 3H), 2.90-2.94 (m, 1H), 3.03-3.08 (m,
1H), 3.42-3.48 (m, 3H) 3.60-3.65 (m, 1H), 3.92-3.94 (m, 2H),
4.17-4.19 (m, 2H), 5.48 (d, J = 8.4 Hz, 1H), 5.87 (d, J = 6.4
Hz, 1H), 5.95-6.00 (m, 1H), 6.21 (t, J = 11.4 Hz, 1H), 6.87-
6.91 (m, 1H), 6.99-7.03 (m, 4H), 7.07-7.11 (m, 1H), 7.22-7.30
(m, 3H), 7.37 (d, J = 8 Hz 1H), 7.45 (d, J = 8 Hz, 1H), 10.80 (s,
1H), 11.48 (s, 1H); MS, m/e 616.96 (M þ 1)þ; 1254.84 (2M þ
23)þ. HRMS, calcd for C28H31FN4O9P (Mþ þ H), 617.1807;
found, 617.1799.
2-[(S)-[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]-2-methylpropio-
nic Acid Methyl Ester (38). Yield: 22%. 1H NMR (DMSO-d6)
δ 1.19-1.36 (m, 9H), 3.54 (s, 3H), 3.56-3.89 (m, 1H), 4.00
(s, 1H), 4.22-4.26 (m, 1H), 4.35-4.38 (m, 1H), 5.52-5.55
(m, 1H), 5.87-6.04 (m, 3H), 7.17-7.23 (m, 4H), 7.58-7.60
(m, 1H), 11.55 (s, 1H); MS, m/e 534.01 (M þ 1)þ. HRMS,
calcd for C21H27F2N3O9P (Mþ þ H), 534.1447; found,
534.1435.
(S)-2-[[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]propionic Acid Iso-
propyl Ester (44). Yield: 11.3%. 1H NMR (DMSO-d6) δ
1.14-1.28 (m, 12H), 3.74-3.84 (m,2H), 3.99-4.06 (m, 1H),
4.23-4.39 (m, 2H), 4.83-4.86 (m, 1H), 5.56-5.60 (t, 1H),
5.85-6.12 (m, 3H), 7.19-7.24 (m, 4H), 7.56-7.57 (d, 1H),
11.50 (s, 1H); MS, m/e 547.91 (M þ 1)þ. HRMS, calcd for
C22H29F2N3O9P (Mþ þ H), 548.1604; found, 548.1591.
(S)-2-{(4-Chlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Isopro-
1
pyl Ester (45). Yield: 24%. H NMR (DMSO-d6) δ 1.05-1.19
(m, 12H), 3.76-3.80 (m, 2H), 3.98-4.08 (m, 1H), 4.31 (m, 2H),
4.73-4.77 (m, 1H), 5.49-5.54 (m, 1H), 5.80-6.20 (m, 3H), 7.20-
7.2 5 (m, 2H), 7.43 (d, J = 8.8 Hz, 1H), 7.54 (d, J = 8 Hz, 1H), 11.51

7214 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
(s, 1H); 13C NMR (DMSO-d6) δ 173.63, 173.59, 173.54, 173.48,
163.71, 151.39, 150.47, 150.42, 140.30, 130.51, 129.61, 122.95,
122.90, 122.83, 103.31, 103.23, 102.18, 100.39, 89.43, 80.32,
72.37, 69.04, 68.99, 65.85, 65.43, 50.86, 50.71, 22.35, 22.34,
22.31, 20.78, 20.71, 20.64, 20.57, 17.62, 17.37; MS, m/e 563.88
(M þ 1)þ; 1148.73 (2M þ 23)þ. HRMS, calcd for C22H29-
ClFN3O9P (Mþ þ H), 564.1308; found, 564.1297.
mixture of diisopropyl ether/dichloromethane (40 mL, 1:1 v/v),
and dried to give white solid (16.6 g, 89.35% pure 51 by 31P
NMR). This solid was suspended in dichloromethane (83 mL)
and heated to reflux for 3 h. The suspension was cooled to
ambient temperature and stirred overnight. The solid was
filtered and washed with cold dichloromethane (10 mL). The
solid was dried under vacuum to give 51 (13.1 g, 99.48% pure by
HPLC). Then 11 g of this solid was redissolved in DCM (330
mL), and the mixture was heated to reflux. The solution was
cooled to ambient temperature and left at this temperature
overnight without stirring. The product was filtered and dried
(25 °C, 0.2 mm, 24 h) to give 10.5 g of 51 (99.74% by HPLC) as
large white needles in 53% recovered theoretical yield of the
single isomer.
(S)-2-{(4-Bromophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Iso-
propyl Ester (46). Yield: 21.2%. 1H NMR (DMSO-d6): δ 1.10-
1.14 (m, 6H), 1.20-1.28 (m, 6H), 3.77-3.82 (m, 2H), 3.99-
4.01 (m, 1H), 4.21-4.25 (m, 1H), 4.37-4.38 (m, 1H), 4.81-
4.86 (m, 1H), 5.58 (dd, J = 8.0, 4.0 Hz, 1H), 5.82-5.95 (m, 1H),
5.96-6.09 (m, 1H), 6.10-6.14 (m, 1H), 7.18 (dd, J = 12.0, 8.0
Hz, 2H), 7.54-7.57 (m, 3H), 11.52 (s, 1H); MS, m/e 607.90
Crystallization of 51 from Crude Mixture of 14. To a stirred
solution of L-alanine isopropyl ester hydrochloride (10.5 g,
61.5 mmol, azeotropically dried, two times, with 50 mL of
toluene each time) in dichloromethane (100 mL) was added
phenyl dichlorophosphate (7.5 mL, 50 mmol) at ambient tem-
perature. The mixture was cooled to -10 °C, and then a solution
of NMI (30.5 mL, 384 mmol) in 30 mL of dichloromethane was
added over a period of 30 min. After completion of the addition,
the mixture was stirred between -10 and -15 °C for 1 h. To the
above mixture was added 20-deoxy-20-fluoro-20-C-methyl-
uridine (5, 10 g, 38.4 mmol) in one lot, and the mixture was stirred
below -10 °C for 3 h and then slowly allowed to warm to 20 °C
(6 h). The mixture was stirred at this temperature overnight
(15 h) and then quenched with 10 mL of methanol. The solvent
was evaporated, and the residue was redissolved in EtOAc (200
mL). The EtOAc layer was washed with water (100 mL), 1 N
HCl (3 ꢀ 75 mL), 2% aqueous NaHCO3 solution (50 mL), and
brine (50 mL). The organic layer was dried over Na2SO4,
filtered, and concentrated. The residue was dried under high
vacuum for 2 h to give white foam (22 g). The above foam was
dissolved in 33 mL of DCM, and then 65 mL of IPE (isopropyl
ether) was added to give a saturated solution. The solution was
filtered though a small pad of Celite, and the filtrate was stirred
with 51 seeds for 72 h at ambient temperature. The white solid
was filtered, washed with IPE (20 mL), and dried to give 4.58 g of
a white powder which was determined by 31P NMR to be an
85:15 mixture of 51/50. The above solid was suspended in DCM
(23 mL), and then the mixture was refluxed for 3 h. The mixture
was cooled to room temperature and stirred for 15 h. The white
solid was filtered, washed with cold DCM (4.5 mL), and dried
under high vacuum at 45 °C to give 3.11 g of 51 (HPLC purity
99.74%, mp 93.9-104.7 °C) in 15.2% yield from 6.
(M þ 1)þ; 1238.64 (2M þ 23)þ. HRMS, calcd for C22H29
BrFN3O9P (Mþ þ H), 608.0803; found, 608.0795.
-
(S)-2-[[(2R,3R,4R,5R)-5-(2,4-Dioxo-3,4-dihydro-2H-pyrimi-
din-1-yl)-4-fluoro-3-hydroxy-4-methyltetrahydrofuran-2-yl-
methoxy]-(4-fluorophenoxy)phosphorylamino]propionic Acid
Cyclohexyl Ester (47). Yield: 9.5%. 1H NMR (DMSO-d6) δ
1.20-1.44 (m, 12H), 1.62-1.71 (m, 4H), 3.75-4.02 (m, 2H),
3.94-4.02 (m, 1H), 4.19-4.26 (m, 2H), 4.59-4.61 (m, 1H),
5.57 (t, J = 8.4 Hz, 1H), 5.85-6.06 (m, 3H), 7.17-7.23 (m,
4H), 7.54 (d, J = 8.4 Hz, 1H), 11.51 (s, 1H); 13C NMR
(DMSO-d6) δ 173.58, 173.54, 163.70, 160.94, 158.55, 151.40,
147.76, 140.20, 122.85, 122.81, 122.76, 122.72, 122.68,
117.25, 117.02, 103.30, 102.18, 100.39, 89.40, 80.32, 73.53,
73.48, 72.38, 65.86, 65.36, 50.90, 50.74, 31.79, 31.72, 25.75,
23.94, 20.88, 20.81, 20.74, 20.68, 17.60, 17.35; MS, m/e
587.92 (M þ 1)þ. HRMS, calcd for C25H33F2N3O9P (Mþ þ
H), 588.1917; found, 588.1906.
(S)-2-{(4-Chlorophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Cyclo-
hexyl Ester (48). Yield: 13.7%. 1HNMR (DMSO-d6): δ
1.21-1.46 (m, 12H), 1.62-1.69 (m, 4H), 3.77-3.83 (m, 2H),
3.98-4.10 (m, 1H), 4.26 (s, 1H), 4.32-4.45 (m, 1H), 4.61 (s,
1H), 5.55-5.60 (m, 1H), 5.82-5.95 (m, 1H), 5.98-6.20 (m,
2H), 7.21-7.26 (m, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.54 (d, J =
8.0 Hz, 1H), 11.55 (s, 1H); MS, m/e 603.97 (M þ 1)þ, 626.10
(M þ 23)þ. HRMS, calcd for C25H33ClFN3O9P (Mþ þH),
604.1621; found, 604.1615.
(S)-2-{(4-Bromophenoxy)-[(2R,3R,4R,5R)-5-(2,4-dioxo-3,4-
dihydro-2H-pyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyltetra-
hydrofuran-2-ylmethoxy]phosphorylamino}propionic Acid Cyclo-
hexyl Ester (49). Yield: 13.7%. 1HNMR (DMSO-d6): δ
1.19-1.46 (m, 12H), 1.62-1.69 (m, 4H), 3.76-3.83 (m, 2H),
3.95-4.08 (m, 1H), 4.25-4.28 (m, 1H), 4.38 (s, 1H), 4.60-4.62
(m, 1H), 5.56-5.61 (m, 1H), 5.82-5.95 (m, 1H), 6.02-6.20 (m,
2H), 7.09-7.20 (m, 2H), 7.53-7.57 (m, 3H), 11.53 (s, 1H);
MS, m/e 650.0 (M þ 3)þ. HRMS, calcd for C25H33BrFN3O9P
(Mþ þ H), 648.1116; found, 648.1114.
X-ray Structure Determination of Compound 51. Compound
51, C23H31N3PO9FCl2, crystallizes in the monoclinic space
group P21 (systematic absences 0k0, k = odd) with a =
˚
˚
˚
12.8822(14) A, b = 6.1690(7) A, c = 17.733(2) A, β =
92.045(3)°, V = 1408.4(3) A , Z = 2, and dcalc = 1.449 g/cm3.
3
˚
X-ray intensity data were collected on a Rigaku mercury
CCD area detector employing graphite-monochromated Mo
˚
KR radiation (λ = 0.710 73 A) at 143 K. Preliminary indexing
Crystallization of 50 from Column Purified Amorphous 50. The
chromatographed fraction containing the first eluting isomer
(50, 3.8 g, 97% pure) was dissolved in isopropanol (36 g) and
diluted with heptanes until cloudy (72 g). The solution was
seeded and stirred at ambient temperature for 5 h. The resulting
solid was collected by vacuum filtration, washed with heptanes
(2 ꢀ 20 mL), and dried (50 °C, 0.2 mm, 24 h) to 2.3 g of very small
white needles that were not suitable for single crystal X-ray
analysis: mp 136.2-137.8 °C; HPLC purity, 99.02%.
was performed from a series of 12 0.5° rotation images with
exposures of 30 s. A total of 648 rotation images were collected
with a crystal to detector distance of 35 mm, a 2θ swing angle
of -12°, rotation widths of 0.5°, and exposures of 30 s: scan no. 1
was a φ-scan from 315° to 525° at ω = 10° and χ = 20°; scan no.
2 was an ω-scan from -20° to 5° at χ = -90° and φ = 315°; scan
no. 3 was an ω-scan from -20° to 4° at χ = -90° and φ = 135°;
scan no. 4 was an ω-scan from -20° to 5° at χ = -90° and φ =
225°; scan no. 5 was an ω-scan from -20° to 20° at χ = -90° and
φ = 45°. Rotation images were processed using CrystalClear
(Rigaku Corp., 1999), producing a listing of unaveraged F2 and
σ(F2) values which were then passed to the CrystalStructure
(Crystal Structure Analysis Package, Rigaku Corp., 2002)
program package for further processing and structure solution
on a Dell Pentium III computer. A total of 7707 reflections were
measured over the ranges 5.48° e 2θ e 50.04°, -14 e h e 15,
Crystallization of 51 from Column Purified Mixture 14. An
amount of 40.0 g of an approximately 1:1 mixture of 14 prepared
in a similar manner was dissolved in dichloromethane (90 mL).
Diisopropyl ether (70 mL) was added to the above solution to
give a saturated solution. The solution was seeded with pure 51,
and the mixture was gently stirred with a stir bar at room
temperature for 20 h.. The solid was filtered, washed with a

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7215
-7 e k e 6, -19 e l e 21, yielding 4253 unique reflections
(Rint = 0.0180). The intensity data were corrected for Lorentz
and polarization effects and for absorption using REQAB
(minimum and maximum transmission of 0.824 and 1.000).
The structure was solved by direct methods (SIR97).41 Re-
finement was by full-matrix least squares based on F2 using
SHELXL-97.42 All reflections were used during refinement.
The weighting scheme used was w=1/[σ2(Fo2) þ 0.0472P2 þ
derived from normal bone marrow (Lonza, Walkersville, MD)
was assessed in a semisolid methylcellulose-based medium
(R&D Systems, Minneapolis, MN) containing recombinant
human (rh-) stem cell factor (50 ng/mL), rh-interleukin-3 (10 ng/
mL), rh-granulocyte/monocyte-colony stimulating factor (10 ng/
mL), and rh-erythropoietin (3 U/mL). Increasing concentra-
tions of each compound (up to 50 μM) diluted in DMSO were
added to progenitor cells seeded at 2 ꢀ 104 cells per culture in
the methylcellulose-based medium. Each compound or the
DMSO (0.1%) control were incubated with the progenitor
cells for 14-16 days in culture, after which the proliferated
colonies were assessed and scored based on size and morphol-
ogy to determine 50% inhibition concentration as described
previously.35,36
Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid
(SIF) Stability Assay. Stock solutions of each compound
(10 mg/mL) were prepared in DMSO and stored at -20 °C.
An amount of 5 μL of the stock solution was added to 995 μL
of simulated gastric fluid (SGF without pepsin, RICCA
Chemical Co.) or simulated intestinal fluid (SIF without
pancreatin, RICCA Chemical Co.) to give the working solu-
tion (50 μg/mL). The working solution was immediately
analyzed by HPLC to obtain the initial (T = 0) absorbance
value for the compound (peak area at 260 nm). The sample was
repeatedly injected every 2 h over a 20 h time period with the
autosampler set at 37 °C. Each compound was analyzed by
reverse phase HPLC with an Eclipse XDB-C18 5 μm column
(Agilent) using a Alliance system (Waters). The mobile phase
consisted of solvent A (Water with 50 mM triethylammonium
acetate) and solvent B (acetonitrile). Elution was performed
using a linear gradient of solvent B from 20% to 98% for
5 min. The amount of parent compound was determined on
the basis of the peak area at each time point, and the per-
centage remaining was calculated on the basis of the initial
amount measured at 0 h.
Compound Stability in Human Liver S9 Fraction. Stock solu-
tions of each compound (50 mM) were prepared in DMSO and
stored at -20 °C. The reaction mixture was prepared in a total
volume of 1 mL containing 5 mM of MgCl2, 50 mM of K2HPO4
(pH 7.4), and 100 μM compound. The reaction was initiated by
adding 4 mg/mL human liver S9 fraction (CellzDirect) to the
reaction mixture and incubated at 37 °C. At the desired times
(0, 0.5, 1, 2, 4, 6, 8, and 24 h), 100 μL aliquots were taken and the
reaction was stopped by mixing the reaction mixture sample
with 300 μL of acetonitrile. The samples were centrifuged at
14 000 rpm for 30 min at 4 °C. Then 100 μL of the supernatant
was mixed with 100 μL of LC/MS solvent A (95% water, 5%
acetonitrile, 10 mM ammonium acetate, and 0.01% formic acid)
and frozen at -20 °C. The standard solutions of each compound
was prepared from 10 mg/mL of stock solution and diluted to a
final concentration of 1 μg/mL in methanol. The standard
solutions were injected into the LC/MS to determine the daugh-
ter ion, cone voltage, and collision energy in order to develop the
LC/MS/MS method. Each compound was separated by using a
Phenomenex Luna 5 μm C18 column with a Waters Alliance
system. The mobile phase consisted of solvent A (95% water,
5% acetonitrile, 10 mM ammonium acetate, and 0.01% formic
acid) and solvent B (95% acetonitrile, 5% water, 10 mM
ammonium acetate, and 0.01% formic acid). Elution was per-
formed using a linear gradient of solvent B from 0% to 100% for
2.5 min. The MS/MS detection was performed by using Micro-
mass Quattro Micro with cone voltage 35 and collision energy
20 eV. The samples collected at the desired time points were
thawed, and 10 μL aliquots were used for LC/MS/MS analysis.
The amount of parent compound was determined on the basis of
the peak area for each time point, and the percentage remaining
was calculated on the basis of the initial amount measured at 0 h.
The half-life of each compound, which is a measure of the
stability of the compound, was calculated by using the Graph-
Pad Prism software.
2
2
0.4960P] where P = (Fo þ 2Fc )/3 . Non-hydrogen atoms were
refined anisotropically, and hydrogen atoms were refined using
a “riding” model. Refinement converged to R1 = 0.0328 and
wR2 = 0.0817 for 4046 reflections for which F > 4σ(F) and
R1=0.0348, wR2 = 0.0838, and GOF = 1.056 for all 4253
unique, nonzero reflections and 358 variables. The maximum
Δ/σ in the final cycle of least squares was 0.000, and the two most
prominent peaks in the final difference Fourier were þ0.312
3
˚
and -0.389 e/A . The Flack absolute structure parameter was
refined to -0.06(6), thus corroborating the stereochemistry of
the title compound.
Biological Assays. HCV Replicon Assay. HCV replicon as-
says using clone A cells and ET-lunet cells were performed as
described previously .13 Briefly, clone A cells and ET-lunet cells
were seeded at a density of 1500 and 3000 cells per well in a 96-
well plate, respectively. Test compounds serially diluted in
culture medium without G418 were added to cells. Plates were
incubated at 37 °C in a 5% CO2 atmosphere for 4 days.
Inhibition of HCV RNA replication was determined by quanti-
tative real time PCR.12
To express the antiviral effectiveness of a compound, the
threshold RT-PCR cycle of the test compound was subtracted
from the average threshold RT-PCR cycle of the no-drug
control (ΔCtHCV). A ΔCt of 3.3 equals a 1 - log 10 reduction
(equal to the 90% effective concentration [EC90]) in replicon
RNA levels. The cytotoxicity of the test compound could also be
expressed by calculating the ΔCtrRNA values. The ΔΔCt speci-
ficity parameter could then be introduced (ΔCtHCV - ΔCtrRNA),
in which the levels of HCV RNA are normalized for the rRNA
levels and calibrated against the no-drug control.
Cell Cytotoxicity Assays. Each compound (serially diluted
from 100 μM) was added to Huh7 (2 ꢀ 103 cells/well), HepG2
(2 ꢀ 103 cells/well), BxPC3 (2 ꢀ 103 cells/well), or CEM (5 ꢀ 103
cells/well) cells and allowed to incubate for 8 days at 37 °C. A
medium only control was used to determine the minimum absor-
bance value and an untreated cell. At the end of the growth period,
MTS dye from the CellTiter 96 AQueous One Solution Cell
Proliferation Assay kit (Promega) was added to each well, and
the plate was incubated for an additional 2 h. The absorbance at
490 nm was read with a Victor3 plate reader (Perkin-Elmer) using
the medium only control wells as blanks. The 50% inhibition value
(CC50) was determined by comparing the absorbance in wells
containing cells and test compound to untreated cell control wells.
Mitochrondria Toxicity Assay. Each compound or ddC was
serially diluted from 100 μM in assay medium and was added to
HepG2, BxPC3, or CEM cells seeded at 1 ꢀ 104 cells/well in a 24-
well plate. Cells were incubated at 37 °C in a humidified 5% CO2
atmosphere for 14 days. On days 7 and 11 medium was removed
and replenished with fresh medium containing the appropriate
concentration of compound. On day 14, cells were harvested
and total cellular DNA was extracted as recommended by the
manufacturer (Qiagen). The mitochondrial cytochrome C oxi-
dase subunit II (COXII) gene and rDNA were amplified using a
multiplex quantitative RT-PCR protocol. The ΔCt of mito-
chondrial COXII DNA (mtDNA) and ΔCt of rDNA (rDNA)
for each sample were determined as described previously.43 ΔCt
was converted to log reduction of mtDNA or rDNA, and
percent inhibition was determined relative to the untreated cell
controls. Toxicity levels were expressed as the concentration
inhibiting mtDNA and rDNA by 90%.
Human Bone Marrow Cytotoxicity Assay. The proliferation
of human erythroid and myeloid hematopoietic progenitor cells

7216 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Sofia et al.
Compound Stability in Human Plasma. Stock solutions of
each compound were prepared in DMSO and stored at -20 °C.
Then 1 mL of human plasma containing 5 mM MgCl2 was used
for the reaction. The reaction was started by adding 2 μL of a
50 mM stock solution of each compound to give a final concentra-
tion of 100 μM and incubated at 37 °C. At the desired times
(0, 0.5, 1, 2, 4, 6, 8, and 24 h), 100 μL aliquots were taken and the
reaction was stopped by mixing the reaction mixture sample
with 200 μL of acetonitrile. The samples were centrifuged at
14 000 rpm for 30 min at 4 °C. Then 100 μL of the supernatant
was mixed with 100 μL of LC/MS solvent A (95% water, 5%
acetonitrile, 10 mM ammonium acetate, and 0.01% formic acid)
and frozen at -20 °C. The standard solution of each compound
was prepared from 10 mg/mL stock solution and diluted to a
final concentration of 1 μg/mL in methanol. The standard
solutions were injected into the LC/MS to determine the daugh-
ter ion, cone voltage, and collision energy in order to develop the
LC/MS/MS method. Each compound was separated by using
a Phenomenex Luna 5 μm C18 column (Phenomenex) with a
Waters Alliance system (Waters). The mobile phase consisted of
solvent A (95% water, 5% acetonitrile, 10 mM ammonium
acetate, and 0.01% formic acid) and solvent B (95% acetonitrile,
5% water, 10 mM ammonium acetate, and 0.01% formic acid).
Elution was performed using a linear gradient of solvent B from
0% to 100% for 2.5 min. The MS/MS detection was performed
by using Micromass Quattro Micro (Waters) with cone voltage
35 and collision energy 20 eV. The samples collected at the
desired time points were thawed, and 10 μL aliquots were used
for LC/MS analysis. The amount of parent compound based
on the peak area was determined for each time point and the
percentage remaining was calculated on the basis of the initial
amount measured at 0 h. The half-life of each compound was
calculated by using the GraphPad Prism software.
Parallel Artificial Membrane Permeability Assay (PAMPA).
A 96-well filtration plate (PVDF membrane, pore size 0.45 μm,
Millipore, Billerica, MA) coated with 5 μL of lecithin in
dodecane (20 mg/mL) was used as donor plate. The donor plate
was filled with 200 μL of PBS buffer (pH 7.4, Gibco) containing
200 μg/mL test compound and 2% of DMSO. The acceptor
plate was filled with 300 μL of PBS buffer with 2% of DMSO.
The donor filter plate was carefully put on the acceptor plate to
form a sandwich, which was left undisturbed for 24 h. The
concentration of compounds in the donor and acceptor wells
after incubation was determined by LC/MS/MS, which con-
sisted of a Waters Alliance HT 2795 separations module,
a Waters Quattro Micro API triple quadrupole mass spectro-
meter (Milford, MA), and a Ascentis phenylhexyl column
(4.6 mm ꢀ 30 mm, 2.7 μm partical, Supelco, Bellefonte, PA).
The mass spectrometer was operated in MRM mode for quan-
titation. The permeability is determined using the following
concentrator, then stored at -20 °C until they were to be
analyzed by HPLC. Residues were suspended in 100 μL of
water, and 50 μL aliquots were injected into the HPLC instru-
ment. HPLC analysis was performed as previously described.19
Rat in Vivo Liver Triphosphate Analysis. Each compound was
dosed orally by gavage with a single dose of compound at 50 mg/
kg to rats in a volume of 5 mL/kg in 0.5% carboxymethylcellu-
lose sodium. Liver samples were removed at time points 0.5, 1, 2,
4, 6, and 12 h postdose by first sacrificing the animal by CO2
inhalation, perfusing the liver with ice cold saline and removing
approximately 1 g of liver, which was then snap-frozen in liquid
nitrogen and stored at -80 °C. Live samples were homogenized
in 3 volumes of ice cold 70% methanol containing 20 mM
EDTA/EGTA. Samples were centrifuged at 3000g for 15 min,
and aliquots of the supernatant (100 μL) were evaporated to
dryness. The resulting dried residue was reconstituted with
100 μL of mobile phase and analyzed for 6 by LC/MS/MS. LC
conditions were as follows: Shiseido SI-2 3301 HPLC instru-
ment; column, Agela venusil XBP C8 2.1 mm ꢀ 50 mm; 30 °C
column temperature; mobile phase of (A) 0.1% formic acid in
water, (B) 0.1% formic acid in acetonitrile; flow rate of 0.45 mL/
min; gradient of 0-3 min; time, 0 min at 5% B and 95% A, 1.4
min at 100% A, 2 min at 5% A and 95% B, 3 min at 5% A and
95% B. Mass spectometer conditions were as follows: Thermo
TSQ Quantum Ultra, electospray ionization, MRM negative
mode, spray voltage 3500 V. Liver concentrations versus time
data were analyzed by noncompartmental approaches using the
WinNonlin software, version 5.0.1.
Dog and Cynomolgus Monkey in Vivo Pharmacokinetic Ana-
lysis. Compounds 12, 14, and 47 were each dosed orally by
gavage (dog, gastric; monkey, nasogastric) at a dose of 50 mg/kg
in hydroxypropyl-β-cyclodextrin in water (20% w/v) for 4
consecutive days. Dose volume was 2.5 mL/kg. Serial blood
samples were collected at 1, 2, 4, 6, 12, and 24 postdose after dose
administration on day 3. All blood samples were obtained by
venipuncture into polypropylene tubes containing K2EDTA
(10 mL, 0.5 M) and kept on ice for processing by centrifuga-
tion. Plasma samples were quick-frozen over dry ice and kept at
-70 °C until LC/MS/MS analysis. At the 4 h time point
postdose on day 4, the animals were anesthetized using pento-
barbital. Ice cold saline was perfused into the liver via a portal
cannula. Five samples from various liver lobes (10 g each) were
harvested, weighed, and snap-frozen in liquid nitrogen. Frozen
liver samples were homogenized in three volumes of ice cold
70% methanol containing 20 mM EDTA/EGTA. The amount
of compound 6 was quantified by LC/MS/MS. LC conditions
were as follows: Shiseido SI-2 3301 HPLC instrument; column,
Agela venusil XBP C8 2.1 mm ꢀ 50 mm; 30 °C column temper-
ature; mobile phase of (A) 0.1% formic acid in water, (B) 0.1%
formic acid in acetonitrile; flow rate of 0.45 mL/min; gradient of
0-3 min; time, 0 min at 5% B and 95%A, 1.4 min at 100% A,
2 min at 5% A and 95% B, 3 min at 5% A and 95% B. Mass
spectometer conditions were as follows: Thermo TSQ Quantum
Ultra, electospray ionization, MRM negative mode, spray
voltage 3500 V. Plasma and liver concentrations versus time
data were analyzed by noncompartmental approaches using the
WinNonlin software program, version 5.0.1.
equations: Pe = [-ln(1 - CA/Ceq)]/[A(1/VD þ 1/VA)t], Ceq
=
(CDVD þ CAVA)/(VD þ VA), where CD is the compound
concentration in donor well, CA is the compound concentration
in acceptor well, VD is the donor well volume, VA is the acceptor
well volume, Ceq is the concentration at equilibrium, A is the
filter area, and t is the incubation time.
In Vitro Determination of Triphosphate Levels in Primary
Human, Rat, Dog, and Monkey Hepatocytes. Clone A cells
and primary hepatocytes were seeded (5 000 000 cells) into T75
flasks in DMEM containing 10% FBS and primary cell plating
medium (CellzDirect, Inc.), respectively. After overnight incu-
bation to allow the cells to attach, the cells were incubated for up
to 24 h at 37 °C in a 5% CO2 atmosphere in the fresh medium
containing 100 μM test compound. At selected times, extra-
cellular medium was removed and the cell layer was washed with
cold PBS. After trypsinization, cells were counted and centri-
fuged at 1200 rpm for 5 min. The cell pellets were resuspended in
1 mL of cold 60% methanol and incubated overnight at -20 °C.
The samples were centrifuged at 14 000 rpm for 5 min, and
the supernatants were collected and dried using a SpeedVac
Single Dose in Vivo Rat Toxicology Assessment. For each
compound, 24 (12 per sex) rats (Rattus norvegicus; breed,
CDIGS-Crl:CD(SD); source, Charles River Laboratories,
Wilmington, MA; age, 4-5 weeks; body weight, 76-100 g) were
dosed with compound or vehicle (30% PEG400/30% Tween 20/
20% corn oil/20% water) as a single dose at a dose volume of
10 mg/mL. Doses administered were 50, 300, and 1800 mg/kg of
body weight. All toxicology animals were euthanized for post-
mortem examinations on study day 15, which was 14 days after
dose was administered. A general physical exam was conducted
on all animals prior to assignment to study. Subsequently, all
animals were examined twice daily for morbidity, mortality,
injury, and availability of food and water. Beginning on day 1

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 7217
after administration and continuing through the day of sched-
uled euthanasia, examinations of general signs of toxicity,
including fecal and urine quality, were conducted once daily.
Beginning on day 1 of dosing, body weights were recorded daily
until euthanasia. Post-mortem examinations were performed on
all toxicology animals. At necropsy, the animals were examined
visually for external abnormalities. The abdominal, thoracic,
and cranial cavities and their contents were examined for
abnormalities and the liver and kidneys removed and examined.
Prenecropsy fasted body weights were examined as well as
necropsy weights of the liver and paired kidneys.
the Study of Liver Disease, Boston, November 2007; Abstract LB9;
unpublished results.
(15) Lalezari, J.; Gane, E.; Rodriguez,-Torres, M.; DeJesus, E.; Nelson,
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Symonds, W. T.; Berrey, M. M.; McHutchison, J. G. Potent
Antiviral Activity of the HCV Nucleoside Polymerase Inhibitor
R7128 with PEG-INF and Ribavirin: Interim Results of R7128 500
mg BID for 28-Days. Presented at the 43rd Annual Meeting of the
European Association for the Study of the Liver, Milan, Italy,
April 2008; unpublished results.
(16) Rodriguez-Torres, M.; Lalezari, J; Gane, E.; DeJesus, E.; Nelson,
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to the HCV Nucleoside Polymerase Inhibitor R7128 for 28 Days
with PEG-INF and Ribavirin: Subanalysis by Race/Ethnicity,
Weight and HCV Genotype. Presented at the 59th Annual Meeting
of the American Association for the Study of Liver Disease, San
Francisco, CA, November 2008; Abstract No. 1899; unpublished
results.
Acknowledgment. We acknowledge the following indi-
viduals and organizations for their contributions to the work
described in this manuscript: Wuxi AppTec; ReachBio LLC;
BASi; and Dr. Patrick Carroll, University of Pennsylvania.
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