Phosphoramidate prodrugs of 2′-C-methylcytidine for therapy of hepatitis C virus infection

Article abstract of DOI:10.1021/jm900447q

The application of a phosphoramidate prodrug approach to 2′-C-methylcytidine (NM107), the first nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 2′-C-Methylcytidine, as its valyl ester prodrug (NM283), was efficacious in r

Full text of DOI:10.1021/jm900447q

5394 J. Med. Chem. 2009, 52, 5394–5407
DOI: 10.1021/jm900447q
Phosphoramidate Prodrugs of 20-C-Methylcytidine for Therapy of Hepatitis C Virus Infection
Cristina Gardelli,*,§ Barbara Attenni,§ Monica Donghi,§ Malte Meppen,§ Barbara Pacini,§ Steven Harper,§ Annalise Di Marco,§
Fabrizio Fiore,§ Claudio Giuliano,§ Vincenzo Pucci,§ Ralph Laufer,§ Nadia Gennari,§ Isabella Marcucci,§ Joseph F. Leone,†
David B. Olsen,‡ Malcolm MacCoss,†, Michael Rowley,§ and Frank Narjes§
§Departments of Medicinal Chemistry and Pharmacology, Istituto di Ricerche di Biologia Molecolare, P. Angeletti S.p.A. (IRBM-MRL Rome),
Via Pontina Km 30,600, 00040 Pomezia, Italy, †Department of Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065,
and ‡Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486. Current address: Schering Plough
Corp., Kenilworth, NJ 07033.
Received April 7, 2009
The application of a phosphoramidate prodrug approach to 20-C-methylcytidine (NM107), the first
nucleoside inhibitor of the hepatitis C virus (HCV) NS5B polymerase, is reported. 20-C-Methylcytidine,
as its valyl ester prodrug (NM283), was efficacious in reducing the viral load in patients infected with
HCV. Several of the phosphoramidates prepared demonstrated a 10- to 200-fold superior potency with
respect to the parent nucleoside in the cell-based replicon assay. This is due to higher levels of 20-C-
methylcytidine triphosphate in the cells. These prodrugs are efficiently activated and converted to the
triphosphate in hepatocytes of several species. Our SAR studies ultimately led to compounds that gave
high levels of NTP in hamster and rat liver after subcutaneous dosing and that were devoid of the toxic
phenol moiety usually found in ProTides.
Introduction
The HCVa nonstructural protein 5B (NS5B) is an RNA
dependent RNA-polymerase (RdRp) which is at the core of
the HCV replication complex. Inhibitors of the polymerase
can be assigned to two broad categories based on their
mechanism of action and their chemical structure: nucleoside
analogues (NI) and allosteric non-nucleoside inhibitors
(NNI). Nucleoside inhibitors are especially attractive and
are expected to play a prominent role in future therapies.
They target the active site of NS5B, which is highly conserved
across the six major HCV genotypes, and are expected to
be broadly effective against infections with HCV genotypes
1-6.11 Another advantage of nucleoside inhibitors is that they
show a higher barrier to the emergence of resistance with
respect to allosteric polymerase inhibitors, whose binding sites
display a considerable polymorphism.9,12 This has been con-
firmed in studies using the standard subgenomic replicon
system13-15 and also on HCV isolates from patients, where
resistance mutations to NNIs were observed at a low fre-
quency within the clinical isolates’ viral quasispecies. This was
not the case for the known mutations against nucleoside
inhibitors.16
An estimated 2% of the world’s population is infected
with the hepatitis C virus.1 Presently there is no vaccine to
prevent the infection and the combination of weekly
injections of pegylated interferon-R (PEG-IFN) with dai-
ly oral administration of ribavirin is the recommended
therapy.2 This regimen gives a sustained viral response
(SVR), which is defined as undetectable viral load
6 months after the end of treatment, in only 50% of the
patients. The response rate is lower in patients infected
with genotype 1 of the virus, where treatment can last up
to 12 months, and considerably higher in infections
associated with genotypes 2 and 3 where 6 months of
treatment can be sufficient to achieve SVR. The current
therapy is often associated with severe side effects
such as depression, fatigue, flulike symptons, and hemo-
lytic anemia that force many patients to discontinue
treatment.3,4 On the basis of the successful paradigm
established for HIV, the development of agents that target
specific viral enzymes is an attractive way to arrive at new
and better tolerated treatment regimens.5-10
Three nucleoside inhibitors, all of them cytidine analogues
with a modified ribose unit, have shown anti-HCV activity as
single agents in clinical trials (Figure 1). Valopicitabine 1
(NM283) was the first polymerase inhibitor to achieve proof
of concept for HCV NS5B inhibitors in the clinic. About 1.2
log10 reduction of plasma HCV RNA was observed in 14 days
uponanoral doseof 800mg b.i.d. Insimilar settings2 (R7128)
achieved 2.7 log10 (1500 mg, bid) and 3 (R1626) achieved 1.2
log10 (1500 mg q.d.). Of these three nucleosides only 2 is still
under development.17
*To whom correspondence should be addressed. Phone: þ39-
0691093279. Fax: þ39-0691093654. E-mail: cristina_gardelli@merck.
com.
a Abbreviations: HCV, hepatitis C virus; SAR, structure-activity
relationship; NTP, nucleoside triphosphate; DCM, dichloromethane;
THF, tetrahydrofuran; pTsOH, p-toluenesulfonic acid; RP-HPLC,
reverse phase high performance liquid chromatography; Py, pyridine;
TFA, trifluoroacetic acid; EC50, compound concentration that returns
50% of inhibition; CC50, compound concentration that returns 50% of
cytotoxicity; SI, selectivity index defined as CC50/EC50; AUC, area
under the curve; PK, pharmacokinetic; BLQ, below the limit of quanti-
fication; RT, room temperature; DMSO, dimethyl sulfoxide; DMA,
dimethylacetamide; ACN, acetonitrile; EDTA, ethylenediaminetetraa-
cetic acid; EGTA, ethylenebis(oxyethylenenitrilo)tetraacetic acid;
NADPH, reduced nicotinamide adenine dinucleotide phosphate.
Nucleoside analogues need to be converted via cellular
kinases to the active triphosphate (NTP), which in the cases
of 1-3 act as a nonobligate chain-terminators, causing in-
hibition of viral replication.12,18,19
r
pubs.acs.org/jmc
Published on Web 08/06/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5395
In the current study we also address issues such as the
replacement of the potentially toxic aryl moiety present in 5.
Results and Discussion
Chemistry. The synthesis of aryl phosphoramidate pro-
drugs of 20-C-methylcytidine was carried out as depicted in
Scheme 1. The previously described chemistry for the
synthesis of ProTide prodrugs, using 1-methylimidazole
as the coupling agent, failed under various conditions.
Uchiyama’s method was investigated next. The nucleoside
4a, prepared as described in the literature,30,31 was used as
such or protected as its 20,30-isopropylidene ketal 9 to
increase solubility and was then phosphorylated at the 50
position with the appropriate aryloxyphosphorochloridate
8,24,32 using a strong organometallic base such as tert-
butylmagnesium chloride to form the corresponding alk-
oxide.
Figure 1. Structures of 1 (NM283, valopicitabine), 2 (R7128), and
3 (R1626).
When the ketal 9 was used, the deprotection of 10 was
performed with a solution of 80% formic acid in water for 4 h
at room temperature. Because of the stereochemistry at the
phosphorus center, the final compounds 5 were isolated as
mixtures of two diastereoisomers that, whenever possible,
were separated by reverse phase high performance liquid
chromatography. The absolute stereochemistry of the dia-
stereomers at phosphorus has not been elucidated, but
HPLC retention times were used as parameters to discrimi-
nate between the two isomers.
For the preparation of benzyloxy phosphoramidates 14a
and 14b and the des-phenol derivative 16 a different chemical
approach based on a final oxidative step (Atherton-Todd
reaction)33 was followed (Scheme 2). Diphenylphosphite was
treated with fluorenylmethyl alcohol (FmOH) or benzyl
alcohol to give the intermediates 12 which were then treated
with the nucleoside 4a, in pyridine at 0 °C. Pyridine was
removed from the solution under reduced pressure, and the
resulting bisphosphites 13 were subsequently reacted with
alanine ethyl ester in the presence of CCl4 and Et3N to give
the desired products 14 and 15. Because of solubility pro-
blems, this step had to be carried out in a mixture of
isopropanol and N,N-dimethylacetamide in order to achieve
good yields. The fluorenylmethyl derivative 15 was sub-
mitted to piperidine deprotection to give the phosphorami-
date 16.
Figure 2. Structures of 20-C-methylcytidine 4a (NM107), the cor-
responding NTP 4b, and the targeted aryl phosphoramidate pro-
drugs 5.
The efficacy of the chain termination depends on the
affinity of the modified NTP for the polymerase, the ratio of
the NTP analogue to endogenous NTP’s, and the relative rate
of incorporation of the polymerase for different nucleotides.
Other factors that potentially limit the activity of the NIs are
poor cell permeability, unwanted metabolic reactions, or poor
conversion to the pharmacologically active triphosphate. In
the latter case the first phosphorylation step that produces the
50-monophosphate has often been found to be rate-limiting.20
This might also be the case for 20-C-methylcytidine. It was
noted by us and others that the triphosphate of 20-C-methyl-
cytidine 4b (20-Me-C-TP, Figure 2) is highly active on the
isolated NS5B enzyme (IC50 = 0.025 μM), but the parent
nucleoside 4a(NM107) shows only modestpotencyin the cell-
based assay (2-7 μM).21 Recent results from the literature
indicated that poor conversion of 4a to its NTP is the reason.
Among a variety of modified nucleosides investigated, 4a was
the poorest substrate for conversion to its monophosphate by
human 20-deoxycytidine kinase.19
Following a literature procedure,34 the alanyl phosphor-
amidate 18 was efficiently prepared by treating a 1:1 diaster-
eomeric mixture 17 with Et3N in water (Scheme 3).
Compound 18 is unstable at room temperature, but it could
be stored at -80 °C for several weeks.
To circumvent this problem, several monophosphate pro-
drug strategies can be applied, where the charged, non-cell-
penetrable phosphate group is masked to give a more lipo-
philicmembrane permeablecompound.20,22 Recent successful
examples applied to HCV include liver-targeted prodrugs
(HepDirect) of 20-C-methyladenosine, where the nucleoside
was protected from rapid metabolism to the inactive inosine
derivative,23 or phosphoramidate (ProTide) prodrugs of 40-
azidouridine, which turned the inactive nucleoside, which was
not monophosphorylated in cells, into a potent inhibitor of
HCV in cell culture.24
For the preparation of the phosphoramidate monoesters
50 and 51 we worked out conditions for a multigram scale
synthesis that are reported in Scheme 4. The protected
nucleoside 9 was treated with biphenyl phosphite and then
with benzyl alcohol to give compound 19. The order of
addition of the benzyl alcohol and the nucleoside is critical,
since treating the phosphite first with benzyl alcohol leads
mainly to addition of two molecules of BnOH to the phos-
phite and consequently low yields of 19. Compound 19 was
then coupled to esterified aminoacid 21-I or aminoalcohol
21-II under oxidative conditions, followed by deprotection
of the acetonide and removal of the benzyl group. Hydro-
genation in the presence of Pd/C 5% was the method of
choice to avoid overreduction of the cytidine residue and to
obtain the final compounds 50 and 51 in excellent purity.
The ProTide approach has also been successful with deri-
vatives of ddA,25 d4T,26,27 LCd4A,28 and d4A.25,29
We started a program with the objective to discover a
monophosphate prodrug of 20-C-methylcytidine 4a, hoping
that by bypassing the initial kinase dependence of4a, we could
generate high levels of NTP 4b in the liver. If successful, this
could lead to a potential new agent for the treatment of HCV
infection or improve the therapeutic index of 4a. As our
starting point, we chose the ProTides 5 (Figure 2).

5396 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Gardelli et al.
Scheme 1. General Synthetic Pathway for the Synthesis of 20-C-Methylcytidine Aryloxy Phosphoramidates 5a
a Reagents and conditions: (a) Et3N, DCM; (b) 4a, t-BuMgCl, dry THF; (c) acetone, pTsOH, 71-87% yield; (d) 8, t-BuMgCl, dry THF; (e) HCOOH
80%, water. For details of the structures 5, see Tables 1-3.
Scheme 2. General Synthetic Pathway for the Synthesis of 20-C-Methylcytidine Aryloxy Phosphoramidates 14a, 14b, 15 and
Monoester 16a
a Reagents and conditions: (a) BnOH or FmOH, Py, 0 °C; (b) 4a, Py, 0-40 °C, 1 h; (c) L-AlaCOOEt HCl, Et3N, CCl4, i-PrOH, N,N-
3
dimethylacetamide, 10 min, 0 °C; (d) piperidine, DCM.
Scheme 3. Preparation of the Metabolic Product 18a
SAR. The phosphoramidates were characterized as in vitro
inhibitors of HCV replication in the subgenomic HCV
replicon assay and compared to the parent nucleoside 4a or
its valyl ester 1. Both reference compounds 1 and 4a had an
EC50 of about 8 μM in our hands. In the first round of SAR
we kept the p-chlorophenyl moiety constant and investigated
the amino acid portion using the corresponding methyl or
ethyl esters. Table 1 shows the most representative results.
a Reagents and conditions: (a) Et3N/water, yield 54%.
Compound 17, as a 1:1 mixture of diastereoisomers, contain-
ing L-alanine ethyl ester already showed a 4-fold improve-
ment in potency with respect to 1 and no sign of cytotoxicity
SAR, Antiviral Activity, and in Vivo NTP Formation. On
the basis of the extensive studies reported in the literature by
McGuigan and co-workers,25,27 we performed a comprehen-
sive SAR study. The amino acid, its stereochemical varia-
tion, the ester functionality of the amino acid, and the
aryloxy moiety were explored by preparing about 250 pro-
drugs. We were aware that a separate ProTide motif opti-
mization process is needed for each nucleoside analogue
versus a given target,24 since cell line dependent en-
zyme expression may determine different phosphoramidate
up to 20 μM.
Other amino acids such as glycine methyl ester (22, EC50
=
0.8 μM) or L-norleucine (23, EC50 = 1.5 μM) gave also
efficient prodrugs. Compound 24, containing β-alanine was
inactive, while compounds with D-alanine (25) or other amino
acids (26-29) offered no advantage with respect to 1. All the
potent compounds (EC50 e 1 μM) showed CC50 > 20 μM.
Compound 17 was separated into its diastereoisomers
using reversed phase HPLC, giving 17a, the first eluting,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5397
Scheme 4. Multigram Scale Synthesis of Compounds 50 and 51a
a Reagents and conditions: (a) (PhO)2POH, Py, anhydrous conditions; (b) BnOH; (c) 21-I or 21-II, Et3N, CCl4, CH3CN, i-PrOH, THF, 50% yield
over three steps; (d) TFA-H2O (8:2); (e) H2, Pd/C 5%, i-PrOH.
Table 1. Cell-Based Activity and Cytotoxicity of 1 and Prodrugs 17, 17a, 17b, and 22-29
compd
R
R1
R2
EC50 ( μM)a
CC50 ( μM)b
SI CC50/EC50
1 (NM283)
17 (1:1)
17a (fe)
7.6
1.8
>100
>20
>20
>20
>20
>20
>20
>20
>20
>50
>20
>50
>13
>11
>2.5
>20
>25
>13
Me
Me
Me
H
H
Et
Et
H
7.9
1.0
17b (se)
22 (1:1)
H
Et
H
Me
Et
0.8
1.5
23 (1:0.8)
24 (1.5:1)
25 (2:1)
n-Bu
β-Ala
H
H
β-Ala
Me
H
Me
Me
Me
Et
>20
7.7
>2.5
26 (1:1.5)
27 (1:1)
Bn
>20
7
i-Bu
Ph
H
>7
>2.5
>3
28 (1:0.8)
29 (1:0.9)
H
Et
Et
7.4
3-indolyl-CH2-
H
17
a EC50 values are quoted for n = 2-9 independent determinations. b Concentration of inhibitor reducing the cell viability by 50%.
and 17b, the more lipophilic second eluting diastereomer.
Interestingly, they showed an 8-fold difference in activity,
the first eluting 17a being the less active. This trend in
activity was observed for all the pairs of diastereoiso-
mers whenever we were able to separate them. The higher
activity of the second eluting, more lipophilic diastereoi-
somer is not unprecedented35 and could be due to a better
diffusion through cell membranes or to a more efficient
stereoselective metabolism of this diastereoisomer. Because
of this trend in the activity, we report from now on only
the activity of the second eluting diastereoisomer. A full
set of replicon data for the first eluting diastereoisomers
can be found in the Supporting Information. Compound
17b was used as a lead to develop further SAR on the
phenol residue with particular focus on more hydrophobic
moieties (Table 2).
The SAR around the phenolic moiety showed that beyond
4-Cl, also 2-Cl and even 2-Me were very good substituents
(compounds 32b and 33b). Substituents with higher electron
withdrawing or electron donating effects gave compounds
(30-31) less active than 17. Very interesting results were
obtained by replacing the phenyl ring with much more
lipophilic groups such as naphthyls (compounds 34b-36)
or an extensively substituted phenyl ring (37b) leading to
submicromolar inhibitors. Similar results were seen with
potential anticancer phosphoramidates of BVdU.24,35 In
the case of compounds 34b and 35b, the contribution (2-
fold) from a more lipophilic ester group, a butyl versus an
ethyl, also needs to be appreciated. All these derivatives
showed EC50 below 0.2 μM, while salicylate derivative
(38), nonaromatic (39b), or benzylic (14b) moieties offered
no advantage. Efforts to release a natural amino acid such as

5398 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Table 2. Cell-Based Activity of Prodrugs 30-42
Gardelli et al.
Figure 3. Time course of triphosphate formation in HBI10A cells.
HBI10A cells were incubated with 100 μM compound 1 (empty
square), compound 17b (filled square), and compound 35b
(triangle). Intracellular NTP was quantified at the indicated time
points, considering that 106 hepatoma cells is 2 μL in volume. Each
point is the mean ( half-range of duplicate determinations.
a EC50 values are quoted for n = 2-9 independent determinations.
b Concentration of inhibitor reducing the cell viability by 50%.
tyrosine instead of phenol (40b) failed, probably due to poor
processing by cellular esterases in the presence of a sterically
more hindered substituent.36 The release of an indole
instead is possible as shown by the activity of prodrug 41b
that is equipotent to the unsubstituted phenol derivative
42b, which is itself more active than 17b. All the most active
compounds (EC50 e 0.5 μM) demonstrated a selectivity
index SI (CC50/EC50) of more than 40-fold. This suggests
that inhibition of the replicon is due to conversion of the
phosphoramidate prodrugs to the triphosphate species
which acts as a nonobligate chain terminator of NS5B
polymerase.12 To assess the selectivity toward other unre-
lated targets with different mechanism of action, the pro-
drug 42b and the active species, the triphosphate 4b, were
also tested in the Pan Laboratories screening against 150
different enzymes, channels, and receptors; the two agents
showed no significant activity up to 10 μM.
On the basis of the encouraging results obtained in the
cell based assay, we wanted to confirm that these prodrugs
generate high levels of triphosphate in the replicon cells (HuH7/
HB1 10a cells) and in hepatocytes. As shown in Figure 3, in the
replicon HuH7/HB1 10a cells, NTP formation from prodrug
17b was much higher than from the valyl ester 1 and the 10-fold
more potent prodrug 35b gave even higher NTP levels.
Figure 4. Kinetics of phosphorylation in primary human hepato-
cytes. Cryopreserved primary human hepatocytes were incubated
with 10 μM compound 17b (filled square), compound 35b (triangle),
and compound 1 (empty square). The intracellular concentration of
NTP is estimated considering that 106 hepatocytes is 4 μL in volume.
Results are the average ( half-range, n = 2.
Table 3. NTP AUC of Compounds 1, 17b, and 35b in HuH7 Cells and in
Human and Rat Hepatocytesa
HuH7/HB1
compd [concn NTP AUC0-24h
human hepatocytes rat hepatocytes
NTP AUC0-4h
NTP AUC0-4h
(μM h)c
(μM)]
(μM h)b
(μM h)d
3
70
3
3
1 [10]
1 [100]
3
7
830
17b [10]
17b [100]
35b [10]
35b [30]
35b [100]
62
698
106
343
553
517
4265
12404
b
a Summary of results reported in Figures 3-5. Intracellular 20-C-
methylcytidine triphosphate (NTP) after incubation in replicon HuH7/
HB1 10a cells. c Intracellular NTP after incubation with human hepa-
tocytes. d Intracellular NTP after incubation with rat hepatocytes.
The same behavior was observed in human hepatocytes
between prodrugs 17b and 35b and reference compound
1 (see Figure 4). In human hepatocytes, the amount of
NTP formed is dose dependent (see Table 3). When com-
pound 17b was incubated at 10 μM, the amount of NTP
and 553 μM h were obtained at incubation concentrations of
3
formed expressed as AUC0-4h was 62 μM h, 10-fold lower
10, 30, and 100 μM, respectively.
3
than when the same compound was incubated at 100 μM,
To validate these prodrugs in vivo, we screened them in rat
hepatocytes, and also in this preclinical species, compound
17b gave high NTP level (see Figure 5).
AUC0-4h = 698 μM h. A good dose dependence was also
3
observed with compound 35b. With it AUC0-4h of 106, 343,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5399
Table 4. Antiviral Activity and Conversion to NTP in Human Hepatocytes of Prodrugs 42b-49b
compda
42b
43b
R
EC50 ( μM)b
CC50 ( μM)c
SI CC50/EC50
[NTP], AUC0-2h ( μM h)d
3
Et
0.45
0.97
0.21
1.9
>20
>20
10
>44
>20
47
65
57
nPr
i-Pr
44b
45b
89
MeO(CH2)3
2EtBu
1-Hep
>20
7
>10
31
115
460
551
638
107
16
46b
47b
0.22
0.10
0.10
0.037
7.6
6
60
90
48b
49b
c-Hep
2-PrPen
9
2
>100
54
13
1 (NM283)
a Second eluting diastereoisomer. b EC50 values are quoted for n = 2-9 independent determinations. c Concentration of inhibitor reducing the cell
viability by 50%. d Intracellular 20-C-methylcytidine triphosphate (NTP) after incubation at 10 μM with cryopreserved human hepatocytes (106 cells/
mL), n = 2-8.
Figure 5. Time course of triphosphate formation in rat hepato-
cytes. Rat hepatocytes were incubated with 10 μM compound 1
(empty symbol) and compound 17b (filled symbol), and NTP levels
were quantified at the indicated time points. Data are the average (
half-range from duplicate experiments.
Figure 6. Conversion of compound 17b (square) to the metabolic
product 18 (rhombus) in rat plasma.
tolerated: not only were the usually reported esters active
(42b-44b) but also longer residues such as compounds 45b-
49b, with the 2-Pr-Pen derivative 49b being one of the most
These results were very encouraging and consistent with
the observations in the replicon assay, indicating that the
prodrugs successfully deliver the monophosphate to the
replicon cells and confirming that a kinase bypass approach
can circumvent the poor conversion of 4a to its NTP 4b.
The plasma stability of prodrug 17b was tested in several
species. No significant degradation occurred in human and
dog plasma, while significant degradation was observed after
5 min of incubation time in rat and mouse plasma. To
facilitate the identification of the degradation products of
these pronucleotides, the hypothesized metabolite 18 was
prepared,34 and it proved to be the main hydrolysis product
of compound 17b in rat plasma (Scheme 2 and Figure 6).
Having shown that these prodrugs deliver NTP in human
and rat hepatocytes, we turned our attention to the releasing
moiety: 1 equiv of phenol is released by each molecule of
pronucleotide administered. Since p-chlorophenol toxicity is
less documented, the studies were focused on the unsubsti-
tuted phenol derivatives 42b-49b, which are also more
potent (Table 4). An extensive SAR study was conducted,
and a very wide range of esters were tested. From the results
in Table 4, it was clear that ester variation was widely
potent phosphoramidates in the replicon assay with EC50
=
0.037 μM. The reported compounds were all tested for their
capacity to generate NTP in human hepatocytes (see
Table 4). All the reported prodrugs were more active than
the reference compound 1 in the replicon assay with EC50
e
1 μM, and these results were confirmed by the values of NTP
measured after incubation of the prodrugs at 10 μM with
cryopreserved human hepatocytes. In every case the NTP
concentration was much higher than with compound 1. The
correlation between replicon activity and NTP concentra-
tion in human hepatocytes is good. Very potent compounds
such as 46b-48b with EC50 < 250 nM generally form very
high NTP concentrations in human hepatocytes with
AUC0-2h above 450 μM h.
3
On the basis of the in vitro potency and of the data collected
in the hepatocytes, compound 46b was profiled further. The
stability of prodrug 46b was measured in plasma samples of
several species. No significant degradation occurred in dog
and human plasma, while a significant degradation occurred
after 5 min of incubation in rat plasma samples. The poor
stability in rat plasma of these prodrugs complicates their

5400 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Gardelli et al.
Figure 7. Disappearance of compound 46b in human (rhombus),
rat (square), dog (triangle), and hamster (cross) plasma.
Figure 8. Profile of the liver concentration of NTP 4b in hamster,
rabbit, and rat after subcutaneous administration of 50 at 1 mg/kg.
Table 5. Hamster Liver NTP Level ( μM) of Compound 46b and 1 after
Different Administration Routes
hamster liver NTP level ( μM)c
compd
administration route
3h
6h
elimination of the phosphoramidate phenol moiety became
the focus of our investigation.
46b
poa
imb
scb
poc
scd
1.1
2.2
The lesson learned so far was that a more lipophilic residue
is necessary to obtain higher NTP levels. With this in mind,
supported by the work already present in the literature by
Wagner and co-workers,42-46 two pairs of compounds with
and without the phenol and based on a relevant difference in
lipophilicity were prepared. The results are reported in
Table 6.
In the case of the less polar pair of compounds 42b and 16
bearing an alanine ethyl ester, the results were in favor of the
phenol derivative 42b, active in the replicon and more
efficient in forming NTP in human hepatocytes. In the case
of the more apolar pair of compounds 49b and 50, bearing
the more lipophilic 2-propylpentyl ester, the behavior was
inverted. We were very pleased to see that the monoester
prodrug 50, although not more active in the replicon assay
than the parent compound 1, generated very high NTP levels
3.6
5.6
10.1
0.48
BLQe
1
a 30.2 μmol/kg. b 1.5 μmol/kg. c n = 3. d 7.55 μmol/kg. e Lower limit of
quantification = 0.2 μM.
development. In the literature very interesting findings are
reported to overcome these kinds of problems such as
conducting the studies in commercially available carboxy-
esteresase deficient mice37 or with the support of carboxy-
esterases inhibitors. However, the compound38 was found to
be moderately stable in hamster plasma (Figure 7).
Because of its acceptable stability in hamster plasma, we
could evaluate the pharmacokinetic profile of compound 46b
in this species. Compound 46b was dosed orally, intramus-
cularly, and subcutaneously using compound 1 as a refer-
ence, and liver levels of NTP 4b were determined at 3 and 6 h
(time of maximum NTP concentration).39
As shown in Table 5, after oral administration of 30 μmol/
kg compound 46b to hamsters, the NTP concentration in the
liver at 6 h was 1.1 μM. This low NTP concentration, albeit
2-fold higher than that obtained with 1, is indicative of a very
low bioavailability of this prodrug. The administration by
intramuscular injection at 20-fold lower dose (1.5 μmol/kg)
gave much higher liver NTP concentrations, 3.6 and 2.2 μM
at 3 and 6 h, respectively. The administration by subcuta-
neous injection at the same dose gave even higher liver
exposure with NTP concentrations of 5.6 and 10.1 μM at
3 and 6 h, respectively, while 1 gave NTP levels below the
lower limit of quantification (0.2 μM). The stability of
prodrug 46b in simulated gastric fluid40 was measured as
well, and the compound was completely stable after 3 h; the
reason for the low bioavailability might be poor intestinal
absorption of the compound or metabolic degradation in the
intestine.
in human hepatocytes with AUC0-2h = 511 μM h. The
3
reason for the difference between the replicon cells and the
hepatocyte data might be the completely different physico-
chemical properties of 50 with respect to the ProTide pro-
drugs. Possibly, nucleoside uptake mechanisms present in
hepatocytes but not in the replicon cells could be involved, as
well as differences in the expression of hydrolytic enzymes
between the two types of cells.47
Compound 50 demonstrated a selectivity index SI (CC50/
EC50) comparable to that of the parent compound. It was
tested in the Pan Laboratories screening as well, and it
showed no significant activity up to 10 μM.
Prodrug 50 was stable in human, dog, rabbit, rhesus,
and hamster plasma but turned over rapidly in rat
plasma, as the corresponding phenol derivative 49b.
Compound 50 was dosed in hamster, rabbit, and rat
subcutaneously at 1 mg/kg (Figure 8). Very efficient
NTP formation was observed in the liver of all species.
In hamster and rabbit, NTP levels were higher than in rat;
at 24 h they were 6.0, 4.1, and 2.9 μM for hamsters, rabbit,
and rat, respectively.
Subcutaneous administration gave a high level of NTP in
the liver, and the remaining issue to be faced was the
compatibility of this approach with the release of an equiva-
lent amount of phenol. In the literature many of the effects of
phenol toxicity are extensively documented;41 therefore, the
Compound 50 was also administered at higher doses, 5
and 10 mg/kg in hamster and rat, respectively (Figure 9).
After 1 and 5 mg/kg subcutaneous dosing, the AUC0-48h in

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5401
Table 6. Antiviral Activity, Cytotoxicity, and Conversion to NTP in Human Hepatocytes of Prodrugs 42b, 16, 49b, and 50
compd
R
R1
EC50 ( μM)a
CC50 ( μM)b
SI CC50/EC50
[NTP] AUC0-2 h ( μM h)c
3
42b
16
Ph
H
Et
Et
0.45
8.8
>20
>20
2
>44
>2
54
65
10
49b
50
Ph
H
2-PrPen
2-PrPen
0.037
8.2
107
511
>100
>12
a EC50 values are quoted for n = 2-9 independent determinations. b Concentration of inhibitor reducing the cell viability by 50%. c Intracellular NTP
after incubation with human hepatocytes.
Table 8. Rat and Hamster Liver NTP Level (μM) of Compounds 50 and
51 after Different Administration Routes
Figure 9. Profile of liver concentration of NTP 4b and plasma
concentration of nucleoside 4a in hamster and rat after subcuta-
neous administration of 50 at 5 and 10 mg/kg, respectively.
Table 7. Comparison of Triphosphate Levels (AUC) between Com-
pounds 50 and 51 in Hepatocytes from Several Species
a Dose, 1.5 μmol/kg. b Dose, 30.2 μmol/kg.
NTP AUC0-4h (μM h)
3
species
50
51
50
The metabolic stability of compound 50 was studied in rat
and human liver S9 fractions in the presence of NADPH, and
the prodrug showed a very high metabolic stability in human
fractions (rat Clint = 12 (μL/min)/mg; human Clint < 1 (μL/
min)/mg). On the basis of these data, the good levels of NTP
found in human hepatocytes, the high stability in human
plasma, and the good correlation between in vitro and in vivo
data in preclinical species, we predict a favorable PK of this
prodrug in humans.
Based on the work carried on in parallel in our labora-
tories,48 we wanted to investigate if the same approach
could be used in the acyloxyethylaminophosphoramidate
series, where the amino acid was replaced by an amino
alcohol, and compound 51 was synthesized following the
same synthetic route applied to obtain compound 50.
The potential for NTP formation was assessed in hepato-
cytes of several species, and although NTP levels were
generally lower with respect to compound 50, significant
NTP concentrations were achieved in human and rat hepa-
dog
rabbit
rat
150
330
70
170
190
470a
100
860
human
rhesus
hamster
a AUC0-3h
1720
2000a
2040
.
hamster were near dose proportional, 700 and 4600 μM h,
respectively.
3
Analysis of hamster and rat plasma showed the nucleoside
4a as the major species, while only traces of 18, prodrug 50,
and nucleoside monophosphate were detected (Figure 9).
The formation of NTP from compound 50 was studied in
hepatocytes of several species and while lower levels of NTP
were observed in dog and rabbit hepatocytes with AUC0-4h
of 150 and 330 μM h, respectively, a similar behavior was
3
observed between human and hamster hepatocytes with
AUC0-4h of 1720 and 2040 μM h, respectively (see Table 7,
compound 50).
3
tocytes, with AUC0-4h of 190 and 170 μM h, respectively
3

5402 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Gardelli et al.
(Table 7, compound 51). Compound 51 showed a higher
stability in rat plasma, and the compound was tested both
in rat and in hamster for comparison (Table 8).
20-C-Methyl-20,30-O-(1-methylethylidene)cytidine (9). 20-C-
Methylcytidine was diluted with acetone (0.04 M), and catalytic
p-toluensulfonic acid and 2,2-dimethoxypropane were added.
The resulting slurry was stirred for 24 h at RT. The solvent was
removed under vacuo, the residue was dissolved in MeOH, and
AmberliteA-26 (previously washed with 2 N NaOH and H2O)
was added. The resulting mixture was stirred for 2 h. The
Amberlite was filtered off, and the solution was evaporated.
The crude product was purified by column chromatography,
using as eluent DCM/MeOH (9/1), to obtain the pure product
9 as a white solid. 1H NMR (300 MHz, CD3OD, δ) 7.96 (d, J=
7.56 Hz, 1H), 6.18 (s, 1H), 5.90 (d, J=7.56 Hz, 1H), 4.51-4.48
(m, 1H), 4.28-4.23 (m, 1H), 3.86 (dd, J=3.04, 12.12 Hz, 1H),
3.78 (dd, J=3.52, 12.12 Hz, 1H), 1.59 (s, 3H), 1.43 (s, 3H), 1.25
(s, 3H). MS (ESþ) m/z 298 (M þ H).þ
The values obtained both in human hepatocytes (NTP
AUC0-2h = 70 μM h) and after subcutaneous administra-
3
tion show that also this type of prodrug should be taken into
consideration when a kinase bypass needs to be addressed.
After subcutaneous administration at 1.5 μmol/kg, high liver
exposure of 4b was obtained in both rat and hamster (36 and
13 μM after 6 h). Compound 51 was also dosed orally in
hamster at 30.2 μmol/kg, and at 6 h only 0.5 nmol/g 20-Me-
Cy-TP was observed in the liver, indicating a low oral
bioavailability as for the phosphoramidate monoester.
General Procedure. Preparation of 20-C-Methylcytidine Phos-
phoramidates (17, 22-49). 20-C-Methylcytidine was diluted with
dry THF (0.1 M). The resulting slurry was cooled to -78 °C, and
then tert-butylmagnesium chloride (as 1.0 M solution in THF,
2.2 equiv) was added. The mixture was immediately warmed to
0 °C, stirred for 30 min, and again cooled to -78 °C. Then the
appropriate aryloxyphosphorochloridate (as 1.0 M solution in
THF, 2.2 equiv) was added dropwise. The mixture was allowed
to reach room temperature overnight, and then the reaction
was quenched by the addition of water. The aqueous phase
was extracted three times with EtOAc, and the combined
organic phases were washed with brine and dried over Na2SO4.
The crude was purified by column chromatography using
as eluent DCM/MeOH (from 9/1 to 8/2), and the resulting
off-white solid was dissolved in DMSO and purified by RP-
HPLC. Fractions containing the pure diastereoisomers were
combined and freeze-dried to afford the title compounds as
a TFA salt.
Conclusions
A series of ProTide phosphoramidate prodrugs of 20-Me-
cytidine have been prepared and tested as HCV replication
inhibitors. Not only were they tested in the replicon and
cytotoxicity assays but their activation to nucleotide tripho-
sphate was measured both inHuH7 cellsand inhepatocytes of
several species. The phenol release was an issue that was
solvedbythepreparation of more lipophilicphosphoramidate
monoesters. The most interesting compound in this series was
compound 50, which gave rise to high NTP concentrations in
human hepatocytes. It demonstrated a selectivity index com-
parable to that of the parent drug. Together with its active
anabolite 4b, it was also tested in the Pan Laboratories
screening where both species showed no significant activity
up to 10 μM. This compound was also dosed subcutaneously
inhamsters, reaching a highNTP exposure in liver, namely, an
50-O-[(4-Chlorophenoxy)[[(1S)-2-ethoxy-1-methyl-2-oxoethyl]-
mino]phosphinyl]-20-C-methylcytidine. First eluting diastereoi-
AUC0-48h of 4600 μM h, after 5 mg/kg dosing.
3
This study demonstrates that it is possible to bypass the
rate limiting first phosphorylation of 20-C-methylcytidine by
delivering directly into the cell the corresponding mono-
phosphate that is then efficiently converted to the active
triphosphate species 4b, responsible for the antiviral activity.
1
somer (17a): H NMR (400 MHz, CD3OD, δ) 8.03 (d, J=7.8
Hz, 1H), 7.41 (d, J=8.9 Hz, 2H), 7.28 (d, J=8.9 Hz, 2H), 6.09
(d, J=7.8 Hz, 1H), 6.03 (s, 1H), 4.66-4.59 (m, 1H), 4.52-4.45 (m,
1H), 4.24-4.16 (m, 3H), 4.04-3.95 (m, 1H), 3.63 (d, J=9.2 Hz,
1H), 1.39 (d, J=7.1 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H), 1.21 (s, 3H).
31P NMR (300 MHz, CD3OD, δ) 5.21. MS (ESþ) m/z 547 (M þ
H)þ. Second eluting diastereoisomer (17b): 1H NMR (300 MHz,
CD3OD, δ) 8.01 (d, J = 7.74 Hz, 1H), 7.43 (d, J = 8.85 Hz,
2H), 7.31 (d, J=8.85 Hz, 2H), 6.06 (d, J=7.74 Hz, 1H), 6.02
(s, 1H), 4.58 (ddd, J=1.82, 6.25, 11.89 Hz, 1H), 4.47-4.38 (m,
1H), 4.24-4.12 (m, 3H), 4.06-3.92 (m, 1H), 3.81 (d, J=9.28 Hz,
1H), 1.41 (d, J=6.85 Hz, 3H), 1.27 (t, J=7.18 Hz, 3H), 1.21
(s, 3H). 31P NMR (300 MHz, CD3OD, δ) 5.10. MS (ESþ)
m/z 547 (M þ H)þ.
Experimental Section
Solvents and reagents were obtained from commercial suppli-
ers and were used without further purification. Flash chroma-
tography purifications were conducted using prepacked
cartridges on a Biotage system, eluting with petroleum ether/
ethyl acetate mixtures. Reactions were carried out under an
atmosphereofnitrogeninoven-dried (110 °C) glassware. Organic
extracts were dried over sodium sulfate (Na2SO4) and were
concentrated (after filtration of the drying agent) on rotary
evaporators operating under reduced pressure. 1H and 31P
NMR spectra were recorded on Bruker AM series spectrometers
operating at (reported) frequencies between 300 and 600 MHz.
Chemical shifts (δ) for signals corresponding to nonexchangeable
protons (and exchangeable protons where visible) are recorded in
parts per million (ppm) relative to tetramethylsilane and are
measured using the residual solvent peak as reference. As a
criterion of purity, two different chromatographic systems with
NMR and MS data were employed. Purity of final compounds
was more than 95% by area. Preparative scale HPLC separations
were carried out on a Waters 2525 pump equipped with a 2487
dual absorbance detector. Compounds were eluted with linear
gradients of water and MeCN, with water containing 5 mM
ammonium bicarbonate. As stationary phase, the following
columns were used: Phenomenex Luna C18 (2) 5 μm, 250 mm ꢀ
21.20 mm; Waters XBridge Pre C18 OBD, 5 μm, 30 mm ꢀ 150
mm; Waters XTerra MS C18 OBD, 5 μm, 19 mm ꢀ 150 mm, and
50 mm ꢀ 100 mm. Compound 50 was analyzed by HPLC on the
column: Waters XTerra MS C18, 5 μm, 3 mm ꢀ 150 mm.
50-O-[(4-Chlorophenoxy)[(2-methoxy-2-oxoethyl)amino]phos-
phinyl]-20-C-methylcytidine. Mixture 1.2:1 of diastereoisomers
at phosphorus [(first eluting, /)/(second eluting)] (22): 1H NMR
(400 MHz, CD3OD, δ) 8.09 (d, J = 7.9 Hz, 1H) and 8.04* (d,
J = 7.9 Hz, 1H), 7.44-7.40 (m, 2H, both), 7.31-7.29 (m. 2H,
both), 6.09* (d, J = 7.9 Hz, 1H) and 6.08 (d, J = 7.9 Hz, 1H),
6.03 (s, 1H, both), 4.67-4.60 (m, 1H, both), 4.52-4.45 (m,
1H, both), 4.20-4.18 (m, 2H, both), 3.87-3.79 (m, 3H, both),
3.76* (s, 3H) and 3.75 (s, 3H), 1.23 (s, 3H) and 1.21* (s, 3H). 31
P
NMR (400 MHz, CD3OD, δ) 5.49 and 5.09*. MS (ESþ) m/z 541
(M þ Na)þ.
50-O-[(4-Chlorophenoxy)[[(1S)-1-(ethoxycarbonyl)pentyl]-
amino]phosphinyl]-20-C-methylcytidine. Mixture 1:0.8* of dia-
stereoisomers at phosphorus (23): 1H NMR (400 MHz,
CD3OD, δ) 8.09-8.03 (m, 1H), 7.45-7.39 (m, 2H), 7.33-7.26
(m, 2H), 6.13 and 6.10* (d, J=7.9 Hz, 1H), 6.02 and 6.01* (s,
1H), 4.65-4.55 (m, 1H), 4.52-4.39 (m, 1H), 4.24-4.11 (m, 3H),
3.91-3.80 (m, 2H), 1.83-1.71 (m, 1H), 1.71-1.57 (m, 1H),
1.40-1.19 (m, 10H), 0.95-0.85 (m, 3H). 31P NMR (400 MHz,
CD3OD, δ) 4.51, 4.24*. MS (ESþ) m/z 589 (M þ H)þ.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5403
50-O-[(4-Chlorophenoxy)[(3-methoxy-3-oxopropyl)amino]-
NMR (300 MHz, CD3OD, δ) 65.9, 79.5. MS (ESþ) m/z 589
(M þ Na)þ.
phosphinyl]-20-C-methylcytidine. Mixture 1.5:1 of diastereoi-
1
somers at phosphorus (24): H NMR (400 MHz, CD3OD, δ)
50-O-[[[(1S)-2-Methoxy-1-methyl-2-oxoethyl]amino](4-meth-
oxyphenoxy)phosphinyl]-20-C-methylcytidine. Mixture 1:1 of
diastereoisomers at phosphorus (31): 1H NMR (400 MHz,
CD3OD, δ) 8.08 (d, J = 7.9 Hz, 0.5H), 8.07 (d, J = 7.9 Hz,
0.5H), 7.25-7.17 (m, 2H), 6.98-6.91 (m, 2H), 6.1 (d, J=7.9 Hz,
0.5H), 6.07 (d, J=7.9 Hz, 0.5H), 6.02 (s, 0.5H), 6.01 (s, 0.5H),
4.65-4.53 (m, 1H), 4.50-4.36 (m, 1H), 4.23-4.15 (m, 1H),
4.05-3.95 (m, 1H), 3.87-3.80 (m, 1H), 3.82 (s, 1.5H), 3.81
(s, 1.5H), 3.75 (s, 1.5H), 3.72 (s, 1.5H), 1.42-1.33 (m, 3H), 1.22
(s, 1.5H), 1.21 (s, 1.5H). 31P NMR (400 MHz, CD3OD, δ) 4.47,
4.29. MS (ESþ) m/z 530 (M þ H)þ.
8.05 (d, J=7.9 Hz, 1H) and 8.01* (d, J=7.9 Hz, 1H), 7.44-7.40
(m, 2H, both), 7.29-7.27 (m, 2H, both), 6.06 (d, J = 7.9 Hz, 1H)
and 6.05* (d, J = 7.9 Hz, 1H), 6.01 (s, 1H, both), 4.59-
4.53 (m, 1H, both), 4.43-4.38 (m, 1H, both), 4.21-4.18
(m, 1H, both), 3.84 (d, J=9.2 Hz, 1H) and 3.82* (d, J=9.2
Hz, 1H), 3.69* (s, 3H) and 3.68 (s, 3H), 3.33-3.28 (m, 2H, both),
2.57-2.54 (m, 2H, both), 1.22 (s, 3H) and 1.21* (s, 3H).
31P NMR (400 MHz, CD3OD, δ) 5.77 and 5.55*. MS (ESþ)
m/z 533 (M þ H)þ.
50-O-[(4-Chlorophenoxy)[[(1R)-2-methoxy-1-methyl-2-oxo-
ethyl]amino]phosphinyl]-20-C-methylcytidine. Mixture 1.9:1* of
diastereoisomers at phosphorus (25). 1H NMR (400 MHz,
CD3OD, δ) 8.12 (d, J=7.9 Hz, 1H) and 8.00* (d, J=7.9 Hz,
1H), 7.45-7.41 (m, 2H, both), 7.32-7.27 (m, 2H, both), 6.11 (d,
J=7.9 Hz, 1H) and 6.10* (d, J=7.9 Hz, 1H), 6.04 (s, 1H) and
6.01* (s, 1H), 4.63-4.56 (m, 1H, both), 4.49-4.37 (m, 1H, both),
4.21-4.16 (m, 1H, both), 4.06-3.98 (m, 1H, both), 3.86 (d, J=
9.2 Hz, 1H) and 3.77* (d, J=9.2 Hz, 1H), 3.73 (s, 3H) and 3.72*
(s, 3H), 1.41* (d, J=7.2 Hz, 3H) and 1.37 (d, J=7.2 Hz, 3H), 1.23
(s, 3H) and 1.21* (s, 3H). 31P NMR (400 MHz, CD3OD, δ) 4.39
and 3.60*. MS (ESþ) m/z 533 (M þ H)þ.
50-O-[[[(1S)-2-Butoxy-1-methyl-2-oxoethyl]amino](2-chloro-
phenoxy)phosphinyl]-20-C-methylcytidine. Second eluting dia-
1
stereoisomer (32b): H NMR (300 MHz, CD3OD, δ) 8.0 (d,
J=7.9 Hz, 1H), 7.6-7.49 (m, 2H), 7.4-7.31 (m, 1H), 7.29-7.19
(m, 1H), 6.06 (d, J=7.8 Hz, 1H), 6.01 (s, 1H), 4.67-4.55 (m, 1H),
4.53-4.42 (m, 1H), 4.23-3.98 (m, 4H), 3.82 (d, J=9.2 Hz, 1H),
1.7-1.57 (m, 2H), 1.48-1.33 (m, 5H), 1.20 (s, 3H), 0.96 (t,
J=7.3 Hz, 3H). 31P NMR (400 MHz, CD3OD, δ) 4.0. MS (ESþ)
m/z 576-578 (M þ H)þ.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino](2-methyl-
phenoxy)phosphinyl]-20-C-methylcytidine. Second eluting dia-
1
50-O-[(4-Chlorophenoxy)[[(1S)-2-methoxy-2-oxo-1-(phenyl-
methyl)ethyl]amino]phosphinyl]-20-C-methylcytidine. Mixture 1:1.5*
of diastereoisomers at phosphorus (26). 1H NMR (300 MHz,
CD3OD, 300 K) δ 7.99* and 7.96 (d, J = 7.9 Hz, 1H), 7.40-
7.00 (m, 9H), 6.10* and 6.02 (d, J=7.9 Hz, 1H), 6.01* and 5.97
(s, 1H), 4.38-4.02 (m, 4H), 3.81-3.74 (m, 1H), 3.73* and 3.66
(s, 3H), 3.21-3.10 (m, 1H), 2.97-2.83 (m, 1H), 1.20 (s, 3H).
31P NMR (300 MHz, CD3OD, 300 K) δ 4.99, 4.79. MS (ESþ)
m/z 609 (M þ H)þ.
stereoisomer (33b): H NMR (400 MHz, CD3OD, δ) 7.96 (d,
J=7.6 Hz, 1H), 7.37-7.12 (m, 4H), 6.02-5.97 (m, 2H), 4.59-
4.51 (m, 1H), 4.44-4.37 (m, 1H), 4.19-4.11 (m, 3H), 4.02-3.92
(m, 1H), 3.79 (d, J=8.8 Hz, 1H), 2.35 (s, 3H), 1.39 (d, J=6.8 Hz,
3H), 1.27-1.24 (m, 3H), 1.19 (s, 3H). 31P NMR (400 MHz,
CD3OD, δ) 3.97. MS (ESþ) m/z 528 (M þ H)þ.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino](1-napht-
halenyloxy)phosphinyl]-20-C-methylcytidine. Second eluting dia-
stereoisomer (34b): 1H NMR (400 MHz, CD3OD, δ) 8.27-8.22
(m, 1H), 7.98-7.94 (m, 1H), 7.92 (d, J = 7.83 Hz, 1H), 7.79
(d, J=7.83 Hz, 1H), 7.65-7.55 (m, 3H), 7.50 (t, J=7.95 Hz, 1H),
6.01 (s, 1H), 5.85 (d, J=7.83 Hz, 1H), 4.65 (ddd, J=1.83, 6.12,
11.81 Hz, 1H), 4.49 (ddd, J=11.87 Hz, J=5.81 Hz, J=3.53 Hz,
1H), 4.23-4.17 (m, 1H), 4.11 (q, J=7.07 Hz, 2H), 4.07-3.99 (m,
1H), 3.84 (d, J=9.09 Hz, 1H), 1.36 (dd, J=6.69 Hz, J = 0.37 Hz,
3H), 1.22 (t, J=7.07 Hz, 3H), 1.17 (s, 3H). 31P NMR (400 MHz,
CD3OD, δ) 4.35. MS (ESþ) m/z 563 (M þ H)þ.
50-O-[(4-Chlorophenoxy)[[(1S)-1-(ethoxycarbonyl)-3-methyl-
butyl]amino]phosphinyl]-20-C-methylcytidine. Mixture 1:0.9* of
diastereoisomers at phosphorus (27): 1H NMR (400 MHz,
CD3OD, δ) 8.06 and 8.03* (d, J = 7.9 Hz, 1H), 7.45-7.39
(m, 2H), 7.33-7.26 (m, 2H), 6.13 and 6.08* (d, J = 7.9 Hz,
1H), 6.02 and 6.01* (s, 1H), 4.66-4.54 (m, 1H), 4.53-4.38 (m,
1H), 4.25-4.10 (m, 3H), 3.95-3.86 (m, 1H), 3.86-3.79 (m, 1H),
1.81-1.51 (m, 3H), 1.33-1.19 (m, 6H), 0.97-0.81 (m, 6H). 31P
NMR (400 MHz, CD3OD, δ) 4.45, 4.20*. MS (ESþ) m/z 589
(M þ H)þ.
50-O-[[[(1S)-2-Butoxy-1-methyl-2-oxoethyl]amino](1-napht-
halenyloxy)phosphinyl]-20-C-methylcytidine. Second eluting dia-
stereoisomer (35b): 1H NMR (400 MHz, CD3OD, δ) 8.28-8.22
(m, 1H), 7.99-7.92 (m, 2H), 7.79 (d, J=8.1 Hz, 1H), 7.65-7.69
(m, 2H), 7.59-7.55 (m, 1H), 7.50 (t, J=8.1 Hz, 1H), 6.00 (s, 1H),
5.86 (d, J=7.83 Hz, 1H), 4.65 (ddd, J=1.6, 6.3, 11.7 Hz, 1H),
4.53-4.46 (m, 1H), 4.24-4.17 (m, 1H), 4.10-4.00 (m, 3H), 3.83
(d, J=9.3 Hz, 1H), 1.62-1.53 (m, 2H), 1.41-1.32 (m, 5H), 1.17
(s, 3H), 0.93 (t, J=7.4 Hz, 3H). 31P NMR: (300 MHz CD3OD, δ)
4.33. MS (ESþ) m/z 591 (M þ H)þ.
50-O-[(4-Chlorophenoxy)[[(1S)-2-ethoxy-2-oxo-1-phenyl-
ethyl]amino]phosphinyl]-20-C-methylcytidine. Mixture 1:0.8* of
diastereoisomers at phosphorus (28): 1H NMR (400 MHz,
CD3OD, δ) 7.90 and 7.82* (d, J=7.9 Hz, 1H), 7.42-7.29 (m,
7H), 7.22 (d, J=7.8 Hz, 1H), 7.15 (d, J=7.8 Hz, 1H), 6.02* and
5.98 (s, 1H), 5.97 and 5.91* (d, J=7.9 Hz, 1H), 5.20-4.96 (m,
1H), 4.58-4.30 (m, 2H), 4.22-4.10 (m, 3H), 3.78-3.69 (m, 1H),
1.21-1.18 (m, 3H), 1.16 (s, 3H). 31P NMR (400 MHz, CD3OD,
δ) 3.78, 3.71*. MS (ESþ) m/z 609 (M þ H)þ.
50-O-[[[(1S)-2-Butoxy-1-methyl-2-oxoethyl]amino][(4-chloro-
1-naphthalenyl)oxy]phosphinyl]-20-C-methylcytidine. Mixture 1:2.5
of diastereoisomers at phosphorus (36): 1H NMR (300 MHz,
CD3OD, δ) 8.33-8.24 (m, 2H), 8.05-7.96 (m, 1H), 7.79-7.48
(m, 4H), 6.00-5.91 (m, 2H), 4.71-4.45 (m, 2H), 4.25-4.16 (m,
1H), 4.11-3.96 (m, 3H), 3.82 (d, J = 9.1 Hz, 1H), 1.62-1.46
(m, 2H), 1.44-1.26 (m, 5H), 1.18 (s, 3H), 0.97-0.85 (m, 3H). 31P
NMR (400 MHz, CD3OD, δ) 4.39, 4.26. MS (ESþ) m/z 626-628
(M þ H)þ.
50-O-[(4-Chlorophenoxy)[[(1S)-2-ethoxy-1-(1H-indol-3-yl-
methyl)-2-oxoethyl]amino]phosphinyl]-20-C-methylcytidine. Mixture
1:0.9* of diastereoisomers at phosphorus (29):1H NMR (400 MHz,
CD3OD, δ) 7.91 and 7.82* (d, J=7.9 Hz, 1H), 7.55-7.49 (m, 1H),
7.42-7.29 (m, 2H), 7.26-7.19 (m, 1H), 7.18-7.06 (m, 3H), 7.05-
6.96 (m, 2H), 6.01 and 5.87* (d, J=7.9 Hz, 1H), 5.95 and 5.91* (s,
1H), 4.41-4.00 (m, 6H), 3.75-3.68 (m, 1H), 3.33-3.24 (m, 1H),
3.16-3.03 (m, 1H), 1.26-1.18 (m, 3H), 1.16 (s, 3H). 31P NMR (400
MHz, CD3OD, δ) 3.75, 3.71*. MS (ESþ) m/z 663 (M þ H)þ.
50-O-[[[(1S)-2-Methoxy-1-methyl-2-oxoethyl]amino][4-(tri-
fluoromethyl)phenoxy]phosphinyl]-20-C-methylcytidine. Mixture
1:1 of diastereoisomers at phosphorus (30): 1H NMR (300 MHz,
CD3OD, δ) 8.05 (d, J=7.7 Hz, 1H, both), 7.76-7.74 (m, 2H,
both), 7.52-7.46 (m, 2H, both), 6.12-6.07 (m, 1H, both), 6.01
(s, 1H, both), 4.65-4.45 (m, 2H, both), 4.25-4.17 (m, 1H, both),
4.07-4.01 (m, 1H, both), 3.85-3.81 (m, 1H, both), 3.74 (s,
3H) and 3.70 (s, 3H), 1.40 (d, J=6.9 Hz, 3H, both), 1.22 (s, 3H,
both). 31P NMR (300 MHz, CD3OD, δ) 4.85 and 4.78. 19F
50-O-[[4-Chloro-5-methyl-2-(1-methylethyl)phenoxy][[(1S)-2-
ethoxy-1-methyl-2-oxoethyl]amino]phosphinyl]-20-C-methylcyti-
dine. Second eluting diastereoisomer (37b): 1H NMR (300 MHz,
CD3OD, δ) 7.96 (d, J=7.83 Hz, 1H), 7.37 (s, 1H), 7.33 (s, 1H),
6.02 (s, 1H), 6.00 (d, J=7.83 Hz, 1H), 4.57 (ddd, J=1.77, 6.45,
11.88 Hz, 1H), 4.41 (ddd, J=4.04, 6.45, 11.81 Hz, 1H), 4.20-
4.14 (m, 3H), 4.00 (dt, J=7.33, 17.18 Hz, 1H), 3.80 (d, J = 9.34
Hz, 1H), 2.34 (s, 3H), 1.43 (d, J=7.08 Hz, 3H), 1.29-1.25 (m,
9H), 1.20 (s, 3H), NH2, NH, 2 ꢀ OH not visible. 31P NMR (300
MHz, CD3OD, δ) 5.03. MS (ESþ) m/z 625 and 627 (M þ Na)þ.

5404 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Gardelli et al.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino][2-(meth-
oxycarbonyl)phenoxy]phosphinyl]-20-C-methylcytidine. Mixture
1:1 of diastereoisomers at phosphorus (38): 1H NMR (300
MHz, CD3OD, δ) 8.17 (d, J=7.9 Hz, 0.5H), 8.11 (d, J=7.8
Hz, 0.5H), 7.99-7.89 (m, 1H), 7.69-7.3 (m, 3H), 6.16 (d, J=7.8
Hz, 0.5H), 6.09 (d, J=7.8 Hz, 0.5H), 6.03 (s, 0.5H), 6.01 (s,
0.5H), 4.73-4.44 (m, 2H), 4.27-3.84 (m, 5H), 3.94 (s, 3H), 1.35
(t, J =7.3 Hz, 3H), 1.3-1.18 (m, 6H). 31P NMR (300 MHz,
CD3OD, δ) 4.78, 4.57. MS (ESþ) m/z 572 (M þ H)þ.
J= 7.2 Hz, 1H), 5.23 (d, J=6.6 Hz, 1H), 5.09 (s, 1H), 4.43-4.30
(m, 1H), 4.29-4.16 (m, 1H), 4.16-3.78 (m, 4H), 3.69-3.52 (m,
1H), 3.43-3.36 (m, 2H), 3.22 (s, 3H), 1.84-1.73 (m, 2H), 1.26 (d,
J = 6.5 Hz, 3H), 0.96 (s, 3H). 31P NMR (400 MHz, DMSO-d6,
δ) 3.75. MS (ESþ) m/z 557 (M þ H)þ.
50-O-[[[(1S)-2-(2-Ethylbutoxy)-1-methyl-2-oxoethyl]amino]-
phenoxyphosphinyl]-20-C-methylcytidine. Second eluting diaster-
eoisomer (46b): 1H NMR (400 MHz, CD3OD, δ) 7.70 (d, J=7.5
Hz, 1H), 7.41-7.37 (m, 2H), 7.30-7.28 (m, 2H), 7.22 (t, J=7.3
Hz, 1H), 6.06 (s, 1H), 5.85 (d, J=7.5 Hz, 1H), 4.52 (dd, J=5.7,
10.3 Hz, 1H), 4.39 (dd, J=5.3, 9.5 Hz, 1H), 4.12-3.95 (m, 4H),
3.74 (d, J = 9.2 Hz, 1H), 1.54-1.48 (m, 1H), 1.39-1.33 (m, 7H),
1.12 (s, 3H), 0.90 (t, J = 7.5 Hz, 6H). 31P NMR (400 MHz,
CD3OD, δ) 3.79. MS (ESþ) m/z 569 (M þ H)þ.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino](2,2,2-tri-
chloroethoxy)phosphinyl]-20-C-methylcytidine. Second eluting
diastereoisomer (39b): 1H NMR (400 MHz, CD3OD, δ) 8.0
(d, J=8.1 Hz, 1H), 6.15 (d, J=7.8 Hz, 1H), 6.01 (s, 1H), 4.74-
4.70 (m, 2H), 4.58-4.49 (m, 1H), 4.47-4.36 (m, 1H), 4.28-4.13
(m, 3H), 4.05-3.95 (m, 1H), 3.81 (d, J=9.3 Hz, 1H), 1.47 (d,
J=6.8 Hz, 3H), 1.31 (t, J=7.1 Hz, 3H), 1.21 (s, 3H). 31P NMR
(400 MHz, CD3OD, δ) 7.59. MS (ESþ) m/z 567 (M þ H)þ.
50-O-[[4-[(2S)-2-Amino-3-methoxy-3-oxopropyl]phenoxy]-
[[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino]phosphinyl]-20-C-
methylcytidine. Second eluting diastereoisomer (40b): 1H NMR
(300 MHz, CD3OD, δ) 8.0 (d, J=7.8 Hz, 1H), 7.32 (m, 4H), 6.1
(d, J=9.8 Hz, 1H), 6.02 (s, 1H), 4.59-4.31 (m, 3H), 4.25-4.13
(m, 3H), 4.06-3.94 (m, 1H), 3.85 (s, 3H), 3.81 (d, J=9.4 Hz, 1H),
3.31-3.14 (m, 2H), 1.42 (d, J=7.2 Hz, 3H), 1.31 (t, J=7.2 Hz,
3H), 1.21 (s, 3H). 31P NMR (300 MHz, CD3OD, δ) 5.23. MS
(ESþ) m/z 615 (M þ H)þ.
50-O-[[[(1S)-2-(Heptyloxy)-1-methyl-2-oxoethyl]amino]phe-
noxyphosphinyl]-20-C-methylcytidine. Second eluting diastereoi-
1
somer (47b): H NMR (300 MHz, CD3OD, δ) 7.72 (d, J=7.7
Hz, 1H), 7.44-7.39 (m, 2H), 7.32-7.22 (m, 3H), 6.08 (s, 1H),
5.86 (d, J=7.7 Hz, 1H), 4.58-4.52 (m, 1H), 4.42-4.39 (m, 1H),
4.15-4.07 (m, 3H), 4.03-3.98 (m, 1H), 3.76 (d, J=9.1 Hz, 1H),
1.67-1.62 (m, 1H), 1.41-1.34 (m, 13H), 1.14 (s, 3H), 0.96-0.91
(m, 3H). 31P NMR (300 MHz, CD3OD, δ) 3.67. MS (ESþ) m/z
583 (M þ H)þ.
50-O-[[[(1S)-2-(Cycloheptyloxy)-1-methyl-2-oxoethyl]amino]-
phenoxyphosphinyl]-20-C-methylcytidine. Second eluting diaster-
eoisomer (48b): 1H NMR (400 MHz, DMSO-d6, δ) 7.52 (d, J=
7.4 Hz, 1H), 7.39 (t, J=7.7 Hz, 2H), 7.30-7.14 (m, 4H), 7.09 (br
s, 1H), 6.08-5.96 (m, 1H), 5.94 (s, 1H), 5.69 (d, J=7.4 Hz, 1H),
5.23 (d, J=6.9 Hz, 1H), 5.09 (s, 1H), 4.87-4.74 (m, 1H), 4.43-
4.29 (m, 1H), 4.29-4.14 (m, 1H), 4.03-3.91 (m, 1H), 3.90-3.71
(m, 1H), 3.66-3.52 (m, 1H), 1.88-1.71 (m, 2H), 1.67-1.44 (m,
8H), 1.44-1.32 (m, 2H), 1.24 (d, J=7.0 Hz, 3H), 0.95 (s, 3H).
31P NMR (400 MHz, DMSO-d6, δ) 3.73. MS (ESþ) m/z 581
(M þ H)þ.
50-O-[[[1-[(1,1-Dimethylethoxy)carbonyl]-1H-indol-5-yl]oxy]
[[(1S)-2-ethoxy-1-methyl-2-oxoethyl]amino]phosphinyl]-20-C-
methylcytidine. Second eluting diastereoisomer (41b): 1H NMR
(300 MHz, DMSO-d6, δ) 8.02 (d, J=8.91 Hz, 1H), 7.72 (d, J=
3.63 Hz, 1H), 7.57 (d, J=7.54 Hz, 1H), 7.49 (s, 1H), 7.20 (d, J=
7.60 Hz, 1H), 7.18 (br s, 1H), 7.10 (br s, 1H), 6.71 (d, J=3.63 Hz,
1H), 6.00 (dd, J = 10.42, 12.34 Hz, 1H), 5.94 (s, 1H), 5.70
(d, J=7.44 Hz, 1H), 5.23 (d, J=7.05 Hz, 1H), 5.09 (s, 1H),
4.43-4.32 (m, 1H), 4.30-4.18 (m, 1H), 4.13-3.93 (m, 3H),
3.92-3.80 (m, 1H), 3.62 (t, J=7.02 Hz, 1H), 1.65 (s, 9H), 1.25 (d,
J=7.02 Hz, 3H), 1.13 (t, J=7.11 Hz, 3H), 0.96 (s, 3H). 31P NMR
(300 MHz, DMSO-d6, δ) 4.16. MS (ESþ) m/z 653 (M þ H)þ.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino]phenoxy-
phosphinyl]-20-C-methylcytidine. Second eluting diastereoi-
50-O-[[[(1S)-1-Methyl-2-oxo-2-[(2-propylpentyl)oxy]ethyl]-
amino]phenoxyphosphinyl]-20-C-methylcytidine. Second eluting
diastereoisomer (49b): 1H NMR (400 MHz, DMSO-d6, δ) 7.50
(d, J = 7.4 Hz, 1H), 7.40-7.34 (m, 2H), 7.24-7.15 (m, 4H),
7.11-7.03 (bs, 1H), 6.08-5.99 (m, 1H), 5.94-5.88 (bs, 1H), 5.66
(d, J=7.4 Hz, 1H), 5.20 (d, J=7.1 Hz, 1H), 5.10-5.06 (bs, 1H),
4.37-4.29 (m, 1H), 4.23-4.16 (m, 1H), 4.00-3.90 (m, 2H),
3.90-3.81 (m, 2H), 3.60-3.52 (m, 1H), 1.64-1.55 (m, 1H),
1.31-1.15 (m, 11H), 0.93 (s, 3H), 0.87-0.79 (m, 6H). 31P NMR
(400 MHz, DMSO-d6, δ) 3.90. MS (ESþ) m/z 597 (M þ H)þ.
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino](9 H-fluo-
ren-9-ylmethoxy)phosphinyl]-20-C-methylcytidine (15). Bisphe-
nyl phosphite was dissolved in pyridine (0.3 M), and a
solution of fluorenylmethyl alcohol in pyridine (0.3 M) was
added. The mixture was stirred at 0 °C for 20 min. Then a
solution of 20-C-methylcytidine in pyridine (0.3 M) was added at
0 °C. The resulting solution was warmed to 40 °C and stirred for
1 h at this temperature. The solvent was evaporated, and the
crude 13-II was dissolved in DMA (0.19 M). The resulting
solution was added to a solution of L-alanine ethyl ester hydro-
chloride (1.2 equiv) and Et3N (2.0 equiv) in i-PrOH/CCl4 (0.24
M, 10/1). The mixture was stirred for 10 min at 0 °C, and then
the solvent was evaporated. The residue was dissolved in EtOAc
and water. The aqueous phase was extracted three times with
EtOAc, and the combined organic phases were washed with
brine and dried over Na2SO4. The crude product was purified by
RP-HPLC. Fractions containing the pure compound were
freeze-dried to afford the title compound 15 as white powder
and as a 1:1 mixture of diastereoisomers. 1H NMR (400 MHz,
CD3OD, δ) 8.1 (d, J = 7.9 Hz, 0.5H), 7.95 (d, J = 7.9 Hz, 0.5H),
7.85-7.81 (m, 2H), 7.74-7.64 (m, 2H), 7.45-7.38 (m, 2H),
7.36-7.29 (m, 2H), 6.02 (d, J=7.8 Hz, 1H), 5.97 (d, J=9.1 Hz,
1H), 4.56-4.2 (m, 4H), 4.2-4.04 (m, 3H), 3.89-3.73 (m, 2H),
1.37 (d, J = 7.2 Hz, 1.5H), 1.34 (d, J = 7.2 Hz, 1.5H), 1.25
(t, J = 7.2 Hz, 3H), 1.19 (s, 1.5H), 1.19 (s, 1.5H). 31P NMR
(400 MHz, CD3OD, δ) 8.46, 8.14. MS (ESþ) m/z 615 (M þ H)þ.
1
somer (42b): H NMR (400 MHz, CD3OD, δ) 8.05 (d, J=7.9
Hz, 1H), 7.43 (t, J=7.6 Hz, 2H), 7.32 (d, J = 7.6 Hz, 2H), 7.26 (t,
J=7.6 Hz, 1H), 6.05 (d, J=7.9 Hz, 1H), 6.01 (s, 1H), 4.62-4.54
(m, 1H), 4.46-4.39 (m, 1H), 4.22-4.13 (m, 3H), 4.04-3.94 (m,
1H), 3.83 (d, J=9.2 Hz, 1H), 1.40 (d, J=7.1 Hz, 3H), 1.27 (t, J=
7.2 Hz, 3H), 1.21 (s, 3H). 31P NMR (400 MHz, CD3OD, δ) 3.85.
MS (ESþ) m/z 513 (M þ H)þ.
50-O-[[[(1S)-1-Methyl-2-oxo-2-propoxyethyl]amino]phenoxy-
phosphinyl]-20-C-methylcytidine. Second eluting diastereoi-
1
somer (43b): H NMR (400 MHz, CD3OD, δ) 8.01 (d, J=7.8
Hz, 1H), 7.45-7.41 (m, 2H), 7.32-7.24 (m, 3H), 6.04-6.02 (m,
2H), 4.60-4.55 (ddd, J=1.8, 5.9, 11.9 Hz, 1H), 4.45-4.39 (ddd,
J=3.4, 5.9, 11.9 Hz, 1H), 4.19-4.17 (m, 1H), 4.10-4.06 (m, 2H),
4.03-3.98 (m, 1H), 3.83-3.81 (d, J=9.2 Hz, 1H), 1.72-1.63
(m, 2H), 1.40 (d, J = 7.1 Hz, 3H), 1.21 (s, 3H), 0.97 (t, J=7.4 Hz,
3H). 31P NMR (400 MHz, CD3OD, δ) 3.84. (ESþ) m/z 527
(M þ H)þ.
50-O-[[[(1S)-1-Methyl-2-(1-methylethoxy)-2-oxoethyl]amino]-
phenoxyphosphinyl]-20-C-methylcytidine. Second eluting diaster-
eoisomer (44b): 1H NMR (300 MHz, CD3OD, δ) 8.06 (d, J= 7.8
Hz, 1H), 7.5-7.19 (m, 5H), 6.06 (d, J=7.8 Hz, 1H), 6.01 (s, 1H),
5.08-4.93 (m, 1H), 4.64-4.37 (m, 2H), 4.23-4.14 (m, 1H),
4.02-3.88 (m, 1H), 3.83 (d, J=9.2 Hz, 1H), 1.39 (d, J=7.0 Hz,
3H), 1.26 (d, J = 6.2 Hz, 6H), 1.22 (s, 3H). 31P NMR (300 MHz,
CD3OD, δ) 5.01. MS (ESþ) m/z 528 (M þ H)þ.
50-O-[[[(1S)-2-(3-Methoxypropoxy)-1-methyl-2-oxoethyl]-
amino]phenoxyphosphinyl]-20-C-methylcytidine. Second eluting
diastereoisomer (45b): 1H NMR (400 MHz, DMSO-d6, δ) 7.53
(d, J=7.2 Hz, 1H), 7.46-7.33 (m, 2H), 7.31-7.14 (m, 4H), 7.09
(br s, 1H), 6.05 (t, J=11.4 Hz, 1H), 5.94 (br s, 1H), 5.68 (d,

Article
50-O-[[[(1S)-2-Ethoxy-1-methyl-2-oxoethyl]amino]hydroxyph-
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5405
30 min at 0 °C, and then the salts were filtrated. The resulting
solution was evaporated and then diluted with EtOAc and
water. The aqueous phase was separated and extracted three
times with EtOAc, and the combined organic phases were
washed with brine and dried (Na2SO4). The crude product
was purified by column chromatography using as eluent
DCM/MeOH (95/5) to give 20-II, a white solid as 1:1 mixture
of diastereoisomers. 1H NMR (300 MHz, DMSO-d6, δ) 8.78 (bs,
1H), 8.01 (bs, 1H), 7.95-7.85 (m, 1H), 7.42-7.30 (m, 5H), 6.02
(s, 1H), 5.93 (d, J=7.26 Hz, 1H), 5.46-5.38 (m, 1H), 5.0 (d, J=
7.44 Hz, 2H), 4.51 (s, 1H), 4.38 (s, 1H), 4.22-4.19 (m, 2H),
4.06-4.01 (m, 2H), 3.09-3.03 (m, 2H), 2.37-2.30 (m, 1H), 1.53
(s, 3H), 1.58-1.21 (m, 8H), 1.39 (s, 3H), 1.21 (s, 3H), 0.86 (t, J=
7.26 Hz, 6H). 31P NMR (300 MHz, DMSO-d6, δ) 10.2, 10.4. MS
(ESþ) m/z 637 (M þ H)þ.
osphinyl]-20-C-methylcytidine (16). Compound 15 was dissolved
in DCM (0.012 M), and piperidine (56 equiv) was added. The
resulting solution was evaporated and the residue washed with
water. The precipitate was discarded, and the solution was
concentrated to give a residue that was purified by RP-HPLC.
Fractions containing the pure compound were freeze-dried to
afford the title compound 16 as a white powder as an NH4 salt.
1H NMR (400 MHz, CD3OD, δ) 8.23 (d, J = 7.6 Hz, 1H), 6.08-
6.06 (m, 2H), 4.25-4.15 (m, 3H), 4.10-4.02 (m, 2H), 3.95-3.87
(m, 2H), 1.36 (d, J=7.1 Hz, 3H), 1.28 (t, J= 7.1 Hz, 3H), 1.15
(s, 3H). 31P NMR (400 MHz, CD3OD, δ) 6.87. MS (ESþ) m/z
436 (M þ H)þ.
50-O-[[[(1S)-1-Methyl-2-oxo-2-[propylpentyl)oxy]ethyl]amino]-
phenylmethoxy)phosphinyl]-20-C-methyl-20,30-O-(1-methylethy-
lidene)cytidine (20-I). Compound 9 was diluted with pyridine
(0.67 M) in the presence of molecular sieves. The resulting
solution was cooled to 0 °C, diphenylphosphite (80%, 1.3 equiv)
was added, and the mixture was stirred for 1 h at 0 °C. To this
solution benzyl alcohol (2.0 equiv) was added, and the mixture
was stirred at RT for 1 h. The solvent was evaporated, and the
residue 19 was dissolved in THF/CCl4 (0.08 M, 12/1). The
resulting solution was cooled to 0 °C, and Et3N (7.0 equiv)
and a solution of L-alanine, 2-propylpentyl ester hydrochloride
21-I (1.3 equiv) in i-PrOH were added. The mixture was stirred
for 30 min at 0 °C and then was quenched by the addition of
water. The aqueous phase was extracted three times with
EtOAc, and the combined organic phases were washed with
brine and dried over Na2SO4. The crude product was purified by
column chromatography using as eluent DCM/MeOH (95/5) to
give 20-I as a white solid as a 1:1 mixture of diastereoisomers. 1H
NMR (400 MHz, CD3OD, δ) 7.76 (d, J=7.6 Hz, 0.5H), 7.75 (d,
J=7.6 Hz, 0.5H), 7.46-7.33 (m, 5H), 6.15 (s, 0.5H), 6.13 (s,
0.5H), 5.88-5.82 (m, 1H), 5.11 (d, J=7.6 Hz, 1H), 5.09-5.05
(m, 1H), 4.54-4.43 (m, 1H), 4.41-4.35 (m, 1H), 4.33-4.21 (m,
2H), 4.14-3.88 (m, 3H), 1.75-1.65 (m, 1H), 1.58 (s, 3H), 1.44-
1.27 (m, 8H), 1.41 (s, 6H), 1.20 (d, J=7.3 Hz, 3H), 0.92 (t, J=5.9Hz,
6H). 31P NMR (400 MHz, CD3OD, δ) 8.68, 8.48. MS (ESþ) m/z
652 (M þ H)þ.
50-O-[Hydroxy[[2-[(1-oxo-2-propylpentyl)oxy]ethyl]amino]ph-
osphinyl]-20-C-methylcytidine (51). Compound 20-II was dis-
solved in a solution of TFA/H2O (0.1 M, 8/2). The result-
ing solution was warmed to 30 °C and stirred for 20 min. The
solvent was evaporated and the residue dissolved in water and
EtOAc. The aqueous phase was extracted three times with
EtOAc, and the combined organic phases were washed with
brine and dried (Na2SO4). The crude product was puri-
fied by column chromatography using as eluent DCM/MeOH
(95/5) to give a white solid as mixture of diastereoisomers. MS
(ESþ) m/z 597 (M þ H)þ. This mixture was dissolved in
methanol (0.08 M), and Pd/C (10%) (20% w/w) was added.
The resulting suspension was stirred under H2 atmosphere for
18 h at RT. The mixture was filtered, and the solvent was
evaporated. The residue was dissolved in acetonitrile and pur-
ified by RP-HPLC. Fractions containing the pure compound
were freeze-dried to afford the title compound 51 as white
1
powder. H NMR (400 MHz, DMSO-d6, δ) 8.76 (s, 1H), 8.08
(d, J=7.8 Hz, 1H), 6.05 (d, J=5.88 Hz, 1H), 5.79 (s, 1H), 4.21-
4.13 (m, 1H), 4.02-3.95 (m, 4H), 3.6 (d, J=9.1 Hz, 1H), 2.96-
2.9 (m, 2H), 2.35-2.27 (m, 1H), 1.51-1.3 (m, 4H), 1.25-1.16
(m, 4H), 1.03 (s, 3H), 0.82 (t, J = 6.3 Hz, 6H). 31P NMR
(300 MHz, DMSO-d6, δ) 7.87. MS (ESþ) m/z 507 (M þ H)þ.
Diammonium 50-O-({[(1S)-1-Carboxylatoethyl]amino}phosp-
hinato)-20-C-methylcytidine (18). Compound 17 (39 mg, 0.072
mmol; as a 1:1 mixture of isomers 17a and 17b) was dissolved in
water (3 mL) and triethylamine (3 mL) and stirred at RT for 15
h. All volatiles were removed in vacuo, and the resulting crude
was redissolved in ACN (0.5 mL) and water (1 mL) and purified
by RP-HPLC (eluent, H2O (containing 5 mM ammonium
bicarbonate)/MeCN). The fractions containing the pure com-
pound were combined and freeze-dried to constant weight to
afford the title compound 18 as a bis-ammonium salt (17.2 mg,
54%) as a white powder. 1H NMR (300 MHz, D2O, δ) 8.05 (d,
J=7.7 Hz, 1H), 6.13 (d, J = 7.6 Hz, 1H), 5.95 (s, 1H), 4.13 (m,
1H), 4.04 (m, 1H), 3.95-3.87 (m, 2H), 3.55 (app t, J = 7.2 Hz,
1H), 1.22 (d, J = 6.7 Hz, 3H), 1.08 (s, 3H). 31P NMR (300 MHz,
D2O, δ) 6.79. MS (ESþ) m/z 409 (M þ H)þ.
50-O-[Hydroxy[[(1S)-1-methyl-2-oxo-2-[(2-propylpentyl)oxy]-
ethyl]amino]phosphinyl]-20-C-methylcytidine (50). Compound
20-I was dissolved in a solution of TFA/H2O (0.1 M, 8/2). The
resulting solution was warmed to 30 °C and stirred for 20 min.
The solvent was evaporated and the residue dissolved in water
and EtOAc. The aqueous phase was extracted three times with
EtOAc, and the combined organic phases were washed with
brine and dried over Na2SO4. The crude product was purified by
column chromatography using as eluent DCM/MeOH (95/5) to
give a white solid as mixture of diastereoisomers. MS (ESþ) m/z
611 (M þ H)þ. This mixture was dissolved in isopropanol (0.08
M), and Pd/C (5%) (20% w/w) was added. The resulting
suspension was stirred under H2 atmosphere for 2 h at RT.
The mixture was filtered, and the solvent was evaporated. The
residue was dissolved in acetonitrile and purified by RP-HPLC.
Fractions containing the pure compound were freeze-dried to
afford the title compound 50 as a white powder. 1H NMR (300
MHz, CD3OD, δ) 8.26 (d, J=7.65 Hz, 1H), 6.11 (d, J=7.47 Hz,
1H), 6.05 (s, 1H), 4.27-4.2 (m, 1H), 4.18-3.90 (m, 6H), 1.75-
1.6 (m, 1H), 1.4-1.35 (m, 11H), 1.16 (s, 3H), 0.95-0.9 (m, 6H).
13C NMR (100 MHz, MeOD, δ) 176.99, 165.61, 155.97, 144.15,
96.19, 93.47, 82.73, 82.64, 80.20, 73.09, 69.95, 68.66, 63.07,
51.71, 38.21, 34.74, 34.67, 21.91, 20.93, 20.87, 20.18, 14.71.
31P NMR (300 MHz, CD3OD, δ) 5.22. MS (ESþ) m/z 521
(M þ H)þ.
Prodrug Conversion to NTP in Hepatic Cells. HBI10A cells
and primary hepatocytes were used for nucleoside triphosphate
quantification. Briefly, HBI10A cells were plated at 800 000
cells/well in six-well tissue culture plates and, 24 h after plating,
incubated with compounds at 37 °C in 5% CO2. Primary
hepatocytes (106 cells/mL) were incubated with compounds in
96-well plates in a shaking water bath at 37 °C, under an
atmosphere of 95% O2/5% CO2. At different times, cells were
then extracted in 70% methanol, 20 mM EDTA, and 20 mM
EGTA and centrifuged. Samples were dried, reconstituted, and
purified by solid-phase extraction, and triphosphate levels were
quantified by LC-MS/MS.
50-O-[[[2-[(1-Oxo-2-propylpentyl)oxy]ethyl]amino]phenylme-
thoxy)phosphinyl]-20-C-methy-20,30-O-(1-methylethylidene)cyti-
dine (20-II). Following the same procedure used to obtain
compound 20-I but using a solution of 2-aminoethyl 2-propyl-
pentanoate hydrochloride 21-II, the desired compound (1.3
equiv) in i-PrOH/THF was added. The mixture was stirred for
Acknowledgment. The authors thank Fabio Bonelli and
Renzo Bazzo for purity checks on compounds 50 and
51, Simona Cianetti for stability and solubility testing on

5406 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Gardelli et al.
compound 50, and Antonella Cellucci for technical support
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Supporting Information Available: Analytical data for target
compounds and biological data for the first eluting diastereoi-
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