2′-deoxy-2′-spirocyclopropylcytidine revisited: A new and selective inhibitor of the hepatitis C virus NS5B polymerase

Article abstract of DOI:10.1021/jm101050a

The current therapy for hepatitis C virus (HCV) infection has limited efficacy, in particular against the genotype 1 virus, and a range of side effects. In this context of high unmet medical need, more efficacious drugs targeting HCV nonstructural protein

Full text of DOI:10.1021/jm101050a

8150 J. Med. Chem. 2010, 53, 8150–8160
DOI: 10.1021/jm101050a
20-Deoxy-20-spirocyclopropylcytidine Revisited: A New and Selective Inhibitor of the Hepatitis C Virus
NS5B Polymerase
Tim H. M. Jonckers,*,† Tse-I Lin,† Christophe Buyck,† Sophie Lachau-Durand,† Koen Vandyck,† Steven Van Hoof,†
Leen A. M. Vandekerckhove,† Lili Hu,† Jan Martin Berke,† Leen Vijgen,† Lieve L. A. Dillen,† Maxwell D. Cummings,†
‡
‡
‡
Herman de Kock,† Magnus Nilsson,‡ Christian Sund,‡ Christina Rydega
˚
rd, Bertil Samuelsson, Asa Rosenquist,
Gregory Fanning,† Kristof Van Emelen,† Kenneth Simmen,† and Pierre Raboisson*,†
˚
†Tibotec BVBA, A Division of Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Belgium, and
‡Medivir AB, P.O. Box 1086, SE-141 22 Huddinge, Sweden
Received August 13, 2010
The current therapy for hepatitis C virus (HCV) infection has limited efficacy, in particular against the
genotype 1 virus, and a range of side effects. In this context of high unmet medical need, more efficacious
drugs targeting HCV nonstructural proteins are of interest. Here we describe 20-deoxy-20-spirocyclo-
propylcytidine (5) as a new inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying
an EC50 of 7.3 μM measured in the Huh7-Rep cell line and no associated cytotoxicity (CC50 > 98.4 μM).
Computational results indicated high similarity between 5 and related HCV inhibiting nucleosides.
A convenient synthesis was devised, facilitating synthesis of multigram quantities of 5. As the exposure
measured after oral administration of 5 was found to be limited, the 30-mono- and 30,50-diisobutyryl ester
prodrugs 20 and 23, respectively, were evaluated. The oral dosing of 23 led to substantially increased
exposure to 5 in both rats and dogs.
Introduction
treatment discontinuations observed during HCV treatment.4
The anticipated introduction of HCV NS3/4A protease
inhibitors (PIs) (with telepravir (VX-950)5 and boceprevir
(SCH 503034)6 being the most advanced) is expected to result
in a more successful SOC in the near future. However, several
patient populations, e.g., partial or null responders, will still
remain untreatable and the emergence of resistance mutations
as a result of treatment with PIs remains a risk. Consequently,
there is still a need for new and more effective treatment
options for HCV.
The HCV genome consists of a 9.6 kb positive-sense, single-
stranded RNA. It encodes at least 10 proteins, including both
structural and nonstructural (NS) proteins, the latter being
the replication enzymes.7 The HCV NS5B polymerase is an
RNA-dependent RNA polymerase that catalyzes the synthe-
sis of positive (genomic) and negative (template) strand HCV
RNA.8,9 Like other polymerases, NS5B has the typical “right-
hand” three-dimensional motif with a thumb and fingers
domain surrounding the catalytic site in the base of the palm
domain.10 Six classes of NS5B inhibitors have been identified.
Non-nucleoside inhibitors (NNI) are chemically diverse and
bind at one of five allosteric binding sites (NNI-1, NNI-2,
NNI-3, NNI-4, and NNI-5).11 Following metabolism to their
50-triphosphate derivatives by cellular kinases, nucleoside
inhibitors (NI) compete with the binding of natural nucleotide
50-triphosphates at the NS5B active site, which, after incor-
poration in the nascent nucleic acid, leads to a chain termina-
tion. Because of relatively strict conservation of the nucleoside
binding site across HCV genotypes, NIs are generally ex-
pected to show high genotypic coverage.12 In in vitro experi-
ments NIs were found to have a higher genetic barrier to
Over the past decade, the number of hepatitis C patients has
steadily increased, and current estimates are that approxi-
mately 175 million people are infected with hepatitis C virus
(HCVa) worldwide. The infection is thought to be the major
cause of liver diseases including fibrosis, cirrhosis, and hepa-
tocellular carcinoma.1 There are at least six major HCV
genotypes and multiple subtypes.2
The current standard of care (SOC) for the treatment of
HCV infection consists of a combination of pegylated inter-
feron-Randthe broad-spectrum antiviral nucleoside analogue
ribavirin. In patients infected with genotype 2 or 3, a sustained
virological response (SVR) is observed in 80% of treated
patients, while for genotype 1 patients only around 50% SVR
isachieved.3 Moreover, the side effects associated withcurrent
therapy are responsible for the relatively high percentage of
*To whom correspondence should be addressed. For T.H.M.J.:
phone, þþ32(0)14641168; e-mail, tjoncker@its.jnj.com. For P.R.:
phone, þþ32(0)14641823; e-mail, praboisson@its.jnj.com.
a Abbreviations: HCV, hepatitis C virus; SVR, sustained virological
response; SOC, standard of care; NNI, non-nucleoside inhibitor; NI,
nucleoside inhibitor; PI, protease inhibitor; rmsd, root-mean-square
deviation; RDPR, ribonucleotide diphosphate reductase; NTP, 20-R-
hydroxy moiety of ribonucleoside 50-triphosphate; dNTP, 20-deoxyri-
bonucleoside 50-triphosphate; TBAF, tetra-n-butylammonium fluoride;
MMTr, monomethoxy tritylchloride; EC50, 50% effective concentra-
tion in Huh-7-Luc replicon cells; CC50, 50% cytotoxic concentration;
Cmax, mean maximum concentration; tmax, time to Cmax; LC-MS,
liquid chromatography-mass spectrometry; HPLC, high-performance
liquid chromatography; TLC, thin-layer chromatography; ESI, electro-
spray ionization; LRMS, low-resolution mass spectrometry; TIPDSCl,
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane; THF, tetrahydrofuran; DIPEA,
diisopropylethylamine; DIAZALD, N-methyl-N-nitroso-p-toluene-
sulfonamide; DMAP, 4-dimethylaminopyridine; CMV, cytomegalovirus.
r
pubs.acs.org/jmc
Published on Web 10/29/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8151
Figure 1. 20- and 40-modified nucleosides.
Scheme 1. Synthesis of 20-Deoxy-20-spirocyclopropylcytidine (5)a
a (a) TIPDSCl, pyridine, room temp, 12 h, 70%; (b) Dess-Martin periodinane, DCM, room temp, 12 h, 87%; (c) methyltriphenylphosphonium
€
bromide, s-BuLi, THF, room temp, 12 h, 60%; (d) BzCl, Hunig’s base, DCM, room temp, 12 h, 86%; (e) CH2N2, diethyl ether, room temp, 48 h, 84%;
(f) hν, toluene/CH3CN, benzophenone, room temp, 3 h, 84%; (g) NH3, MeOH, room temp, 2 h, 40%; (h) (1) 2,4,6-triisopropylbenzenesulfonyl chloride,
DMAP, Et3N, CH3CN, room temp, 12 h; (2) NH3/H2O, CH3CN, room temp, 3 h, 93%; (i) TBAF, THF, then Dowex WX40 and recrystallization
(MeOH), 62%.
the development of resistance than NNIs and protease
inhibitors.13
derivatives, with 20-β-C-methyl analogues being particularly
important. Among these derivatives, the combination of a
20-β-methyl substituent with an R-fluorine or R-hydroxyl led
to the most promising HCV NIs. Although larger groups have
been reported (e.g., 20-β-ethyl, 20-R-OCH3), these modifica-
tions resulted in less effective inhibitors.28,20 The 40-modified
ribonucleoside derivatives are the second distinct structural
class of HCV NIs. Various substituents at the 40-position
of the sugar are accommodated by NS5B polymerase. In the
cytidine series, however, it has been shown that only the
40-azido group yields an acceptable activity vs toxicity index.29
From a viral resistance perspective, the 20-modified com-
pounds (NM-107 (1)30 and PSI-6130 (2),31 Figure 1) are
susceptible to the S282T mutation of the enzyme while the
40-modified analogue R-1479 (3)32 is impacted by the S96T
and M142T mutations. It is noteworthy that the combination
of the 20-methyl and 40-azido substituents leads to the inactive
hybrid 4.33
The structure of NS5B polymerase has been studied
extensively,14 although detailed understanding of the structural
basis for nucleotide recognition has not yet been attained.
Although RNA polymerases are assumed to interact with the
20-R-hydroxy moiety of ribonucleoside 50-triphosphates
(NTPs), thereby discriminating among 20-deoxyribonucleo-
side 50-triphosphates (dNTPs),15,16 the HCV NS5B polymer-
ase promiscuously accommodates diversity in both the base
and pentose moieties of the nucleotide, and chain-terminating
20-deoxynucleotides that are incorporated into nascent RNA
have been described.17 As a result, numerous 20-substituted
nucleoside derivatives have been studied as potential HCV
drugs.
To date, despite numerous approaches and the exploration
of many novel scaffolds18-21 including carbocyclic deriva-
tives,22 only two different classes of HCV-active nucleosides
have progressed through preclinical evaluation to establish
proof of concept in HCV-infected patients.23-27 The first such
class of HCV NIs encompasses 20-modified ribonucleoside
As stated, several nucleoside derivatives that are inhibitors
of the HCV NS5B RNA-dependent RNA polymerase have
progressed into the clinical phase. However, the occurrence of

8152 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Scheme 2. Synthesis of the Monoester (20)a
Jonckers et al.
a (a) TBAF, THF, room temp, 1 h, then Dowex WX40, 84%; (b) MMTrCl, pyridine, 0 °C to room temp, 12 h; (c) isobutyric anhydride, pyridine, 10 °C
to room temp, 12 h; (d) POCl3, 1H-1,2,4-triazole, triethylamine, DCM, room temp, 4 h, 74%; (e) NH4OH, THF, room temp, 12 h; (f) AcOH (80%),
room temp, 9 h, 62%.
severe unwanted side effects has resulted in the discontinuation
of their development. NM-283, the 30-valine ester prodrug of
1, was halted because of severe GI tract toxicity and hemato-
logical disorders,34,35 while R-1626, the 2,3,5-triisobutyryl
ester prodrug of 3, was stopped mainly because of excessive
neutropenia observed in some patients.36 Consequently, there
is still a need for safe and well tolerated derivatives of this class
of compounds.
Our laboratories are actively involved in several research
programs aimed at identifying new HCV inhibitors. These
efforts have led to the discovery of TMC435, a macrocyclic
NS3/4Aserine protease inhibitor currently in phase 2b clinical
evaluation,37,38 as well as two new classes of benzodiazepines
found to be allosteric NS5B NNI-3 site inhibitors.39,40 We now
report our findings on 20-deoxy-20-spirocyclopropylcytidine (5),
a new inhibitor of the HCV NS5B RNA-dependent RNA poly-
merase with a previously unreported 20-substitution pattern.
group of 7 yielded ketone 8. Introduction of the exocyclic
double bond was accomplished through Wittig olefination
using methyltriphenylphosphonium bromide to give 9 in 60%
yield. To prevent methylation of the N3-atom of the pyrimi-
dine, 9 was benzoylated to yield intermediate 10. The creation
of the spirocyclopropyl fragment was subsequently achieved
through a 1,3-dipolar cycloaddition reaction of diazomethane
on the vinylic precursor 10. The mixture of spiropyrazolines
11a and 11b thus obtained was subjected to a photochemically
induced nitrogen extrusion reaction to provide 12 in 84%
isolated yield. Notably, attempts to synthesize the cyclopropyl
moiety via Simmons-Smith carbene-mediated approaches
were either unsuccessful or inferior to the diazomethane-photo-
chemistry sequence. Similar failure for structurally related
2-deoxy-2-methylenearabinopyranose has been reported, attri-
buting the ineffectiveness of this approach to the highly electron
deficient nature of the double bond.43 Additionally, the exo-
cyclic double bond in 10 might be sterically hindered, thus
hampering the reaction with a bulky carbene reagent. With the
fully protected spirocyclopropyl intermediate 12 in hand, the
synthesis of 5 was completed by debenzoylation, yielding 13,
followed by conversion of the uracil moiety to the correspond-
ing cytidine derivative. For the latter reaction, a two-step
procedure (conversion of 13 into its 1-(4-dimethylamino)-
pyridinium derivative, followed by ammonolysis)44,45 was used,
yielding intermediate 14, which was desilylated by tetra-
n-butylammonium fluoride (TBAF) to provide the target nucleo-
side (5), obtained in an analytically pure form after treatment
with a Dowex cation-exchange resin46 followed by recrystalli-
zation from methanol.
Chemistry
While the 20-deoxy-20-spirocyclopropylcytidine 5 is novel
to the HCV field, it was previously proposed as a potential
inhibitor of ribonucleotide diphosphate reductase (RDPR), a
key enzyme in the de novo synthesis of deoxynucleoside
precursors for DNA synthesis.41 No biological data on its
RDPR inhibitory activity have been reported previously. We
postulated that 5 could provide a useful analogue in the class
of HCV-inhibiting nucleosides and considered it to be parti-
cularly attractive because the 20-carbon of the 20-spirocyclo-
propyl derivative is not stereogenic, unlike that of 1 and 2. On
the basis of earlier work reported by Samano and Robins,42
a new and convenient procedure, enabling multigram-scale
synthesis of 5, was elaborated. As depicted in Scheme 1, the
3-benzoyl-1-{(6a0R,80R,9a0S)-20,20,40,40-tetraisopropyltetrahydro-
spiro[cyclopropane-1,90-furo[3,2-f][1,3,5,2,4]trioxadisilocine]-
80-yl}pyrimidine-2,4(1H,3H)-dione (12) is a key intermediate
for the synthesis of 5.
It is well-documented that the oral administration of nucleo-
sides in preclinical studies often results in low overall expo-
sure.47 Nucleosides are highly polar, resulting in low intestinal
permeability, and unless their uptake is mediated through
nucleoside-specific activetransport systems, loworalbioavail-
ability is often encountered.48 In anticipation of such
difficulties, the 30-mono and 30-50-diisobutyryl derivatives of
5 were synthesized and their pharmacokinetic properties were
compared tothoseof the parent nucleoside. Approaches using
The reaction sequence starts with the simultaneous protec-
tion of the 30- and 50-OH groups of uridine 6, capturing them
as a cyclic disilylether. Dess-Martin oxidation of the 20-OH

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8153
mono- and diisobutyryl derivativesto achievehigher exposure
following oral administration have been reported for related
nucleosides.23,24,49,50 Schemes 2 and 3 illustrate the synthesis
of these ester prodrugs.
The synthesis of the monoisobutyryl ester of 5 is shown in
Scheme 2. Intermediate13was desilylated using TBAF, giving
20-deoxy-20-spirocyclopropyluridine 15. Selective protection
of the primary 50-OH group was achieved by reaction with
monomethoxy tritylchloride (MMTr), giving 16 in 84% yield.
Subsequent esterification with isobutyric anhydride led to 17,
which was converted into the cytidine analogue 19 by succes-
sive treatment with phosphorus oxychloride and 1H-1,2,4-
triazole, followed by ammonolysis of intermediate18. Finally,
acidic removal of the MMTr protecting group afforded
monoester 20.
Scheme 3. Synthesis of the Diester (23)a
As shown in Scheme 3, the synthesis of the diisobutyryl
ester of 5 started from compound 15, which was first esterified
with isobutyric anhydride followed by uracil-to-cytidine con-
version, similar to the sequence described for intermediate 19.
Biological and Pharmacokinetic Results
Antiviral Activity. The parent nucleoside 5 was tested on a
Huh7 replicon cell line containing the subgenomic bicistro-
nic replicon clone ET with a luciferase readout51 to evaluate
its HCV-inhibitory properties. Gratifyingly, 5 showed un-
ambiguous inhibition of HCV replication in a range compar-
able to that of 1, 2, and 3, with no apparent cellular toxicity
(Table 1). In addition, ester prodrugs 20 and 23 showed clear,
albeit lesser, inhibitory properties. These results indicate that
both prodrugs were hydrolyzed in the cellular medium
during the course of the assay, ultimately releasing the parent
nucleoside 5.
Pharmacokinetic Profile. Plasma kinetics and liver-plasma
distribution after a single oral administration of 5, 20, and 23
using 20% hydroxypropyl-β-cyclodextrin as a vehicle were
determined in male Sprague-Dawley rats (Figure 2, Table 2).
Although only low plasma concentrations of 5 were achieved
after administration of the parent compound 5 or its mono-
ester 20, they were quantifiable up to 7 h postadministration.
For the diester 23, high mean maximum concentrations
(Cmax) of 5 in the plasma were achieved at 0.7-0.8 h postdose
(tmax), indicating rapid absorption and hydrolysis of 23 to 5.
At lower dose, the oral exposure of 5 increased approxi-
mately 10-fold after administration of 20 and more than
24-fold after administration of 23. A dose-proportional
increase in AUC of 5 was observed upon dose escalation
a (a) Isobutyric anhydride, pyridine, room temp, 12 h, 96%; (b) POCl3,
1H-1,2,4-triazole, triethylamine DCM, room temp, 2.5 h, 86%; (c) NH4-
OH, THF, 3.5 h, 84%.
Table 1. Inhibition of HCV Replication in Huh-7-Rep Cells (EC50
,
Luciferase Assay) for Reference Nucleosides 1-3 and Cyclopropyl
Analogues 5, 20, and 23, and Cytotoxicity (CC50) Measured in Huh7-
CMV-Luc and MT4-LTR-Luc Cells
CC50 (μM)a
compd
EC50 (μM)a
Huh7-CMV-Luc
MT4-LTR-Luc
1
4.6
1.9
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
>98.4
2
3
4.3
7.3
5
20
23
42.8
20.3
from 15 to 90 mg/kg of 23 (AUC of 7.22 and 40 μg h/mL
3
after administration of 15 and 90 mg/kg, respectively). Com-
pound 20 was undetectable in plasma after administration.
a Average values of at least four repeats.
Figure 2. Mean plasma concentrations of 5 after single oral administration of parent 5, its monoisopropyl prodrug 20, or its diisopropyl
prodrug 23 to Sprague-Dawley rats.

8154 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Jonckers et al.
Following administration of 23, low concentrations of 20
were detected in plasma (around 0.2% of the plasma con-
centrations of 5) and 23 was not detected. The liver-to-
plasma AUC0-7h ratio of 5 was found to be approximately 1
for all of the tested compounds.
The diisopropyl ester 23 was evaluated in dogs (Figure 3,
Table 3). Plasma concentrations were detectable up to 48 h
postadministration. Exposure increased proportionally for
the two doses tested (15 and 75 mg/kg). The diester was
undetectable in plasma at the low dose, while at high dose
diester plasma concentrations below 13 ng/mL were mea-
sured at the earlier time points. The corresponding mono-
ester 20 exposure represented less than 0.4% of the parent 5
exposure after administration of either dose.
and 5, underscoring the overall similarity of the three analogues
and also highlighting the difference at the 20-position.
Superposition of the non-hydrogen atoms common to the
three nucleosides (i.e., omitting the 20-substituents) yields differ-
˚
ences less than 0.1 A rmsd for both the 1-5 and 2-5 pairs. To
further quantify the conformational match discussed above and
shown in Figure 4, a three-dimensional shape comparison be-
tween the low energy conformations of 5 and those of 1 and 2
was performed with the Tanimoto shape algorithm ROCS.54,55
ROCS compares molecules on the basis of shape Tanimoto score,
a quantitative measure of three-dimensional overlap where a
value of 1 means complete overlap (i.e., identical shape) and a
fraction indicates partial overlap.54-56 The shape similarity be-
tween the cyclopropyl analogue 5 and the reference compounds 1
Table 3. Mean Noncompartmental PK Parameters after Single Oral
Administration of the Diisopropyl Prodrug 23 to Beagle Dogs
Computational Modeling
parameter
After demonstrating that 5 selectively inhibited HCV NS5B
RNA-dependent RNA polymerase, we decided to study its
properties computationally. The 20-modified ribose derivatives 1
and 2 are sterically similar, but the 20-hydroxyl group of 1 is
hydrophilic and can act as a hydrogen bond donor and/or
acceptor while the 20-fluorine of 2 is hydrophobic and can only
act as a weak hydrogen bond acceptor. Compound 5 differs from
1 and 2 at the 20-position, with its bulkier and completely
hydrophobic cyclopropyl substituent. The lowest energy confor-
mation for each of the nucleosides 1, 2, and 5 was calculated with
the modeling package MOE (version 2009.10, Chemical Computing
Group, Montreal, Canada) using MMFF94x (unpublished but
see Halgren52 and references therein) and a Born solvation
model.53 Compounds 1 and 5 share a slight energetic preference
for the 30-endo (Northern) conformation (less than 1 kcal mol-1),
consistent with both conformational states being significantly
populated in solution. Under these conditions 2 shows a 4 kcal
mol-1 preference for the 30-endo conformation. Figure 4 shows
the superposition of the calculated low energy conformers of 1, 2,
dose (μmol/kg)
dose (mg/kg)
Cmax (μg/mL)
tmax (h)
36
15
180
75
6.3
1.5
37
42.8
1.3
AUC0-24h (μg h/mL)
204
3
Table 2. Mean Noncompartmental PK Parameters of 5 after Single
Oral Administration of the Parent 5, its Monoisobutyryl Prodrug 20,
and its Diisobutyryl Prodrug 23 to Sprague-Dawley Rats
compound administered
5
20
23
36
5
23
dose (μmol/kg)
doses (mg/kg)
Cmax (μg/mL)
tmax (h)
36
10
28.8
10
216
60
216
90
15
0.085
1
0.248
0.184
0.7
2.62
2.39
0.8
0.527
1.3
1.64
11.4
0.7
40.0a
Figure 4. Superimposed low energy conformations of nucleosides
1, 2, and 5. For clarity, only polar hydrogens are shown. The
30-hydroxyl of 5 was manually adjusted to optimize the overlay of
this group over the three nucleosides.
AUC0-7h (μg h/mL)
7.22
3
a AUC0-24h
.
Figure 3. Mean plasma concentrations of parent 5 after single oral administration of its diisopropyl prodrug 23 to Beagle dogs.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8155
Figure 5. Electron density of 20-substituted ribonucleosides 1 (left), 5 (middle), and 2 (right). Electrostatic potentials are mapped onto
Connolly surfaces. Red represents negative values, while blue represents positive values. Values close to zero are green.
and 2 was extremely high; for both comparisons a Tanimoto
similarity score of 0.98, indicating 98% overlap, was calculated.
Having established the structural similarity of 1, 2, and 5,
electronic propertiesof the three nucleosideswerethencompared.
Electrostatic potentials calculated for the aligned lowest energy
conformations were mapped onto Connolly surfaces57 (calcula-
tions with MOE; Figure 5). The overall electrostatic potential
surfaces of the three scaffolds were found to be strikingly similar
(Figure 5). To quantify electrostatic similarity, the aligned con-
formationof5 was comparedtothoseof1 and 2 withthe Openeye
package EON. Generally, Coulombic electrostatic Tanimoto
indexes range from 1 (identical) to negative values resulting from
the overlap of positive and negative charges.56 In this case, the
Coulombic electrostatic Tanimoto indices were 0.91 between 5
and 1 and 0.92 between 5 and 2, indicating high overall electro-
static similarity. The only region of the surfaces showing visible
difference is the 20-position, consistent with the observation that
the 20-position can accommodate some variation.28
Our computational analyses confirmed the high overall simi-
larity of 1, 2, and 5, consistent with the 20-spirocyclopropyl
substituent being a useful surrogate in this series of anti-HCV
nucleosides. The significance of the slight-to-moderate energetic
preference for the 30-endo (Northern) conformation in these
molecules is unclear. Since both the Northern and Southern
conformers likely exist in solution, both may be available in the
therapeutically relevant biological setting. Furthermore, addi-
tional factors including nucleoside uptake, polymerase affinity,
efficiency of phosphorylation (to the therapeutically relevant
nucleotide; manuscript in preparation), and susceptibility to
deamination may contribute to the observed antiviral activities of
nucleosides. A cogent discussion of the interplay between various
factors that contribute to nucleoside antiviral activity has been
presented by Eldrup.58
supported the potential of the 20-spirocyclopropyl moiety as a
surrogate for the 20-methyl group. The further biological,
pharmacological, and pharmacokinetic evaluation of 20-deoxy-
20-spirocyclopropylcytidine 5 and its related diester prodrug
23 is ongoing, results of which will be reported elsewhere.
Experimental Section
General Experimental Procedures. NMR spectra were recorded
on a Bruker Avance 400 spectrometer operating at 400 MHz
1
for H. Chemical shifts are given in ppm and J values in Hz.
Multiplicity is indicated using the following abbreviations: d for
doublet, t for a triplet, m for a multiplet, etc. Low-resolution mass
spectra were measured on a Waters Alliance 2695 high-performance
liquid chromatography (HPLC) coupled to a single quadrupole
(Waters ZQ) or a time-of-flight (Waters LCT) mass spectrometer
using electrospray ionization (ESI) in positive or negative mode.
The purity of all tested compounds was confirmed to be
g95% using two of four independent chromatographic systems.
In method 1, an Atlantis T3 column (3.0 μm, 4.6 mm ꢀ 50 mm)
and mobile phases A (10 mM NH4OOCH3 þ 0.1% HCOOH in
H2O) and B (CH3OH) were used. Analyses were run at 50 °C
using a flow rate of 2 mL/min applying the following gradient:
0 min, 0% B; 4.2 min, 95% B; 5.4 min, 0% B. Method 2 was
performed on a Vision HT C18P column (1.5 μm, 2.1 mm ꢀ
20 mm) using mobile phases A (0.1% HCOOH in H2O/CH3CN
95:5) and B (CH3CN). Analyses were run at 50 °C using a flow
rate of 0.6 mL/min applying the following gradient: 0 min, 0%
B; 1.5 min, 100% B; 2.1 min, 100%; 2.7 min, 0% B. For Method
3, a SunFire C18 column (3.5 μm, 4.6 mm ꢀ 100 mm) and mobile
phases A (10 mM NH4OOCH3 þ 0.1% HCOOH in H2O) and B
(CH3OH) were employed. Analyses were run at 50 °C using a
flow rate of 1.5 mL/min applying the following gradient: 0 min,
35% B; 7.0 min, 95% B; 9.6 min, 95% B; 9.8 min, 35% B; 12 min,
35% B. The fourth system (method 4) used an XS Strategy
column (1.7 μm, 2.1 mm ꢀ 20 mm) and mobile phases A (0.1%
HCOOH in H2O/CH3CN 95/5) and B (CH3CN). Analyses were
run at 50 °C using a flow rate of 0.6 mL/min applying the
following gradient: 0 min, 0% B; 1.5 min, 100% B; 2.1 min,
100%; 2.7 min, 0% B. Eluted peaks were detected at a single
wavelength (273 nm).
Conclusions
20-Deoxy-20-spirocyclopropylcytidine 5 was discovered as a
new member of the class of 20-modified nucleoside derivatives
that have HCV inhibiting properties. Compound 5 is a novel,
potent (EC50 = 7.3 μM), and selective (CC50 > 98.4 μM)
inhibitor of the HCV NS5B RNA-dependent RNA polymerase,
a clinically validated target for HCV treatment. A convenient
synthesis of 5 was developed in which the 20-spirocyclopropyl
fragment was constructed via a dipolar cycloaddition/photo-
chemistry sequence. Pharmacokinetic evaluation of 5 as well
as its prodrugs 20 and 23 revealed an increased exposure to 5
after administration of the prodrug derivatives, especially so
for the diester prodrug 23. A dose-proportional increase in
exposure to 5 was seen in rats and dogs after administration of
the diisobutyryl ester derivative 23. Molecular modeling studies
indicated close structural similarity between 1, 2, and 5 and
Thin-layer chromatography (TLC) was performed on 5 cm ꢀ
10 cm aluminum sheets coated with silica gel 60 F254 (Merck
KGaA). Preparative HPLC was done on a Synergi 250 mm ꢀ
77 mm ꢀ 10 μm column, using 95% MeCN in water (0.2% FA)
as the mobile phase. The flow rate was 150 mL/min. Detection
was done using a Gillson 215 detector operating at 220 nm. All
reagents were purchased from commercial sources and were
used as received.
Preparation of 1-((6aR,8R,9R,9aS)-9-Hydroxy-2,2,4,4-tetra-
isopropyl-6a,8,9,9a-tetrahydro-6H-furo[3,2-f ][1,3,5,2,4]trioxadisilocin-
8-yl)pyrimidine-2,4(1H,3H)-dione (7). To a solution of uridine 6

8156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Jonckers et al.
(1.0 equiv, 100 g) in pyridine (1000 mL) was added dropwise
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (TIPDSCl; 1.02 equiv,
131.6 g) over 20 min. The reaction mixture was stirred at room
temperature overnight. The solvent was evaporated by distillation,
and the residue was dissolved in CH2Cl2 (500 mL). The CH2Cl2
solution was washed with water (3 ꢀ 400 mL), dried over MgSO4,and
concentrated by rotavapor. The crude product was purified by
column chromatography on silica gel using petroleum ether/ethyl
acetate 4:1 to give intermediate 7 as a white foam (139 g, 70% yield).
Liquid chromatography-mass spectrometry (LC-MS): tR = 3.23
min, m/z = 487 (M þ H)þ. 1H NMR (400 MHz, CDCl3) δ ppm
0.78-1.23 (m, 28 H), 3.30 (br s, 1 H), 4.01 (dd, J = 13.20, 2.05 Hz,
1 H), 4.12 (d, J = 8.61 Hz, 1 H), 4.15-4.25 (m, 2 H), 4.36 (dd, J =
8.61, 4.89 Hz, 1 H), 5.70 (d, J = 8.22 Hz, 1 H), 5.74 (s, 1 H), 7.71 (d,
J = 7.82 Hz, 1 H), 9.09 (br s, 1 H).
Preparation of 1-((6aR,8R,9aR)-2,2,4,4-Tetraisopropyl-9-oxo-
tetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)pyrimidine-
2,4(1H,3H)-dione (8). To a solution of 7 (1.0 equiv, 159 g) in dry
CH2Cl2 (1600 mL) at 0 °C was added Dess-Martin periodinane
(1.2 equiv, 162 g). The reaction mixture was stirred at 25-30 °C
overnight. Then 2000 mL of diethyl ether was added to the
reaction mixture and the precipitates were filtered off over
diatomite. The resulting filtrate was washed with 3000 mL of
saturated NaHCO3 solution containing 185 g of Na2S2O3. The
organic layer was collected and washed with brine, dried over
anhydrous Na2SO4, and evaporated to give intermediate 8 as a
white foam (138 g, 87% yield) which was used as such in the next
step. LC-MS: tR = 3.24-3.45 min, m/z = 485 (M þ H)þ
(hydrate of the ketone is observed under the LC-MS con-
ditions). 1H NMR (400 MHz, DMSO-d6) δ ppm 0.70-1.30
(m, 28 H), 3.90-4.02 (m, 1 H), 4.05 (br s, 2 H), 4.99 (d, J = 9.04
Hz, 1H), 5.49 (s, 1H), 5.70 (d, J = 7.90 Hz, 1 H), 7.77 (d, J =
7.96 Hz, 1 H), 11.60 (br s, 1 H).
Preparation of 1-((6aR,8R,9aS)-2,2,4,4-Tetraisopropyl-9-methyl-
enetetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-8-yl)-
pyrimidine-2,4(1H,3H)-dione (9). Under N2 atmosphere, sec-
butyllithium in hexane (293 mL, 1.4 N, 2.0 equiv, 0.41 mol) was
added dropwise to a solution of triphenylmethylphosphonium
bromide (2.5 equiv, 183 g) in dry THF (1000 mL) cooled at -78 °C.
Then a solution of 8 (1.0 equiv, 100 g, 0.2 mol) in dry THF (300 mL)
was added to the reaction mixture under N2. The cooling bath
was removed, and the reaction mixture was stirred for 16 h at
room temperature. THF was evaporated by rotavapor, and
CH2Cl2 (500 mL) was added to dissolve the residue. The CH2Cl2
solution was washed with water (3 ꢀ 300 mL), dried over
MgSO4, and concentrated by rotavapor. The crude product
was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate 8:1 to 5:1 to give intermediate 9 as
a white foam (60 g, 60% yield). LC-MS: tR = 3.55 min, m/z =
483 (M þ H)þ. 1H NMR (400 MHz, CDCl3) δ ppm 1.02-1.13
(m, 28 H), 3.69 (ddd, J = 8.8, 2.6, 2.0 Hz, 1 H), 4.05 (dd, J =
13.3, 2.6 Hz, 1 H), 4.14 (dd, J = 13.3, 2.0 Hz, 1 H), 4.82 (dd, J =
8.8, 1.2 Hz, 1 H), 5.46 (t, J = 1.7 Hz, 1 H), 5.53-5.55 (m, 1 H),
5.70 (dd, J = 8.1, 2.1 Hz, 1 H), 6.51 (d, J = 0.8 Hz, 1 H), 7.44
(d, J = 8.1 Hz, 1 H), 8.69 (br s, 1 H).
Preparation of 3-Benzoyl-1-((6aR,8R,9aS)-2,2,4,4-tetraisopropyl-
9-methylenetetrahydro-6H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-
8-yl)pyrimidine-2,4(1H,3H)-dione (10). To a solution of 9 (1.0
equiv, 190 g) in CH2Cl2 (2000 mL) was added N,N-diisopropyl-
ethylamine (DIPEA; 1.02 equiv, 61 g). Then benzoyl chloride
(1.1 equiv, 60 g) was added dropwise and the reaction mixture
was stirred at room temperature overnight. The reaction mixture
was washed with a saturated NaHCO3 solution (3 ꢀ 500 mL),
dried with MgSO4, and concentrated by rotavapor. The crude
product was purified by column chromatography on silica gel
using petroleum ether/ethyl acetate 8:1 to give intermediate 10 as a
white foam (200 g, 86% yield). LC-MS: tR = 3.83 min, m/z= 587
(M þ H)þ. 1H NMR (400 MHz, CDCl3) δ ppm 1.04-1.13 (m, 28
H), 3.71 (ddd, J = 8.9, 2.7, 1.8 Hz, 1 H), 4.07 (dd, J = 13.3, 2.7 Hz,
1 H), 4.18 (dd, J = 13.3, 1.8 Hz, 1 H), 4.85 (dd, J = 8.9, 1.3 Hz,
1 H), 5.47 (t, J = 2.0 Hz, 1 H), 5.57-5.58 (m, 1 H), 5.82 (d, J =
8.2 Hz, 1 H), 6.51 (d, J = 1.0 Hz, 1 H), 7.51 (t, J = 7.6 Hz, 2 H),
7.60 (d, J = 8.2 Hz, 1 H), 7.66 (t, J = 7.6 Hz, 1 H), 7.94 (d, J =
7.6 Hz, 2 H).
Preparation of 3-Benzoyl-1-((30R,6aR,8R,9aS)-2,2,4,4-tetra-
isopropyl-40,50,6,6a,8,9a-hexahydrospiro[furo[3,2-f][1,3,5,2,4]-
trioxadisilocine-9,30-pyrazole]-8-yl)pyrimidine-2,4(1H,3H)-dione (11a)
and Its Epimer 3-Benzoyl-1-((30S,6aR,8R,9aS)-2,2,4,4-tetraisopropyl-
40,50,6,6a,8,9a-hexahydrospiro[furo[3,2-f][1,3,5,2,4]trioxadisilocine-
9,30-pyrazole]-8-yl)pyrimidine-2,4(1H,3H)-dione (11b). 2-(2-Ethoxy-
ethoxy)ethanol (200 mL) and diethyl ether (80 mL) were added to
a flask containing a solution of KOH (90 g, 30.6 equiv) in water
(100 mL). The flask was heated to 60 °C, and a solution of N-methyl-
N-nitroso-p-toluenesulfonamide (DIAZALD; 152 g 13.7 equiv) in
diethyl ether (500 mL) was added dropwise in about 2 h. During this
period, the generated diazomethane was condensed with diethyl
ether and dropped into another flask which was cooled by dry ice.
The diazomethane/diethyl ether solution was then added to a
solution of 10 (30 g) in diethyl ether (50 mL) cooled on an ice bath.
The reaction mixture was stirred at room temperature for 48 h, and
the reaction was checked by LC-MS and TLC until completion.
Acetic acid was added dropwise until the yellow color disappeared.
The reaction mixture was evaporated on a rotavapor. The residue
was dissolved in CH2Cl2 and evaporated to dryness. The crude
product was purified by column chromatography on silica gel using
petroleum ether/ethyl acetate 5:1 to 2:1 to give a mixture of inter-
mediates 11a and 11b as a white foam (27 g, 85% yield). LC-MS:
tR = 3.67 min, m/z = 629 (M - H)þ. 1H NMR (400 MHz, CDCl3)
δ ppm 0.82-1.19 (m, 28 H), 1.57-1.64 (m, 2 H), 3.82-5.06 (m,
6 H), 5.84-5.94 (m, 2 H), 7.47-7.53 (m, 2 H), 7.60-7.69 (m, 1 H),
7.88-7.94 (m, 2 H), 8.05-8.15 (m, 1 H).
Preparation of 3-Benzoyl-1-((6a0R,80R,9a0S)-20,20,40,40-tetraiso-
propylhexahydrospiro[cyclopropane-1,90-furo[3,2-f][1,3,5,2,4]-
trioxadisilocine]-80-yl)pyrimidine-2,4(1H,3H)-dione (12). The
mixture of intermediates 11a and 11b (8 g, 1.0 equiv) was
dissolved in dry toluene (400 mL) and acetonitrile (400 mL),
and benzophenone (0.23 g, 0.1 equiv) was added. The mixture
was stirred and illuminated by a medium pressure mercury
lamp for 3 h until 11a and 11b were consumed as monitored by
TLC and LC-MS. The solvent was removed by rotavapor,
and the residue (intermediate 12) was used in the next step
without further purification. LC-MS: tR = 3.90 min, m/z =
601 (M þ H)þ. 1H NMR (400 MHz, CDCl3) δ ppm 0.57-0.64
(m, 1 H), 0.66-0.73 (m, 1 H), 0.86-0.94 (m, 1 H), 0.98-1.14
(m, 28 H), 1.16-1.22 (m, 1 H), 3.86 (dd, J = 8.7, 2.3 Hz, 1 H),
4.07 (dd, J = 13.5, 2.3 Hz, 1 H), 4.21 (d, J = 13.5 Hz, 1 H),
4.51 (d, J = 8.7 Hz, 1 H), 5.83 (d, J = 8.2 Hz, 1 H), 5.86 (s,
1 H), 7.50 (t, J = 7.5 Hz, 2 H), 7.65 (t, J = 7.5 Hz, 1 H),
7.89-7.95 (m, 3 H).
Preparation of 1-((6a0R,80R,9a0S)-20,20,40,40-Tetraisopropyl-
hexahydrospiro[cyclopropane-1,90-furo[3,2-f][1,3,5,2,4]tri-
oxadisilocine]-80-yl)pyrimidine-2,4(1H,3H)-dione (13). Crude
intermediate 12 (110 g) was dissolved in CH2Cl2 (200 mL),
and NH3/methanol (6 N, 1100 mL) was added to the mixture.
The reaction mixture was stirred at room temperature for 2 h.
The solvent was evaporated to dryness by rotavapor, and the
residue was purified by column chromatography on silica gel
using petroleum ether/ethyl acetate 5:1 to 2:1 to give inter-
mediate 13 as a white foam (48 g, 91% purity on LC-MS and
NMR). Intermediate 13 was then repurified by preparative
HPLC (water/CH3CN, with formic acid as modifier). Frac-
tions containing 13 were combined and neutralized to pH 8
with NH3. CH3CN was evaporated by rotavapor, and the
water phase was extracted with ethyl acetate. The organic
phase was dried over MgSO4 and evaporated to give pure
intermediate 13 as a white foam (35 g, 40% yield). LC-MS:
1
tR = 3.66 min, m/z = 497 (M þ H)þ. H NMR (400 MHz,
CDCl3) δ ppm 0.54-0.62 (m, 1 H), 0.70-0.76 (m, 1 H),
0.82-0.90 (m, 1 H), 0.95-1.12 (m, 28 H), 1.16-1.24 (m, 1 H),
3.83 (d, J = 8.7 Hz, 1 H), 4.04 (d, J = 13.5 Hz, 1 H), 4.17

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8157
(d, J = 13.5 Hz, 1 H), 4.47 (d, J = 8.7 Hz, 1 H), 5.71 (d, J =
8.2 Hz, 1 H), 5.87 (s, 1 H), 7.76 (d, J = 8.2 Hz, 1 H), 8.19 (br s, 1 H).
Preparation of 4-Amino-1-((6a0R,80R,9a0S)-20,20,40,40-Tetra-
isopropylhexahydrospiro[cyclopropane-1,90-furo[3,2-f ][1,3,5,2,4]-
trioxadisilocine]-80-yl)pyrimidin-2(1H)-one (14). Triethylamine
(0.41 g, 2.0 equiv) was added to a stirring solution of intermediate
13 (10 g, 1.0 equiv) in CH3CN (300 mL). 2,4,6-Triisopropylbenzene-
sulfonyl chloride (10.2 g, 2.0 equiv) and 4-dimethylamino-
pyridine (DMAP; 5.1 g, 2.0 equiv) were added. The reaction
mixture was stirred at room temperature for 16 h. The mixture was
evaporated to dryness on a rotavapor. The residue was taken into
CH3CN (100 mL), and NH3 (28% aqueous solution, 200 mL) was
added. This reaction mixture was stirred at room temperature for
3 h. Then the solvent was removed by rotavapor and the residue
was dissolved in CH2Cl2 and washed with brine. The organic phase
was dried over Na2SO4 and evaporated to dryness. The crude
product was purified by column chromatography on silica gel
using ethyl acetate/methanol 9:1 to give intermediate 14 as a white
foam (9.28 g, 93% yield) that was used directly in the next step.
LC-MS: tR = 3.44 min, m/z = 496 (M þ H)þ.
dry pyridine (15 mL) was cooled on an ice-water bath, and
monomethoxytrityl chloride (MMTrCl; 900 mg, 2.91 mmol)
was added. The reaction mixture was left on a melting ice-water
bath and was then stirred at room temperature overnight.
Excess methanol was added, and after 30 min, the reaction
mixture was concentrated, dried, and used as such in the next
1
reaction. LC-MS: tR = 2.48 min, m/z = 525 (M - H)-. H
NMR (400 MHz, DMSO-d6) δ ppm 0.54-0.61 (m, 3 H), 1.04-
1.09 (m, 1 H), 3.25-3.32 (m, 2 H), 3.74 (s, 3 H), 3.85-3.91 (m,
1 H), 4.21 (dd, J = 7.5, 5.9 Hz, 1 H), 5.25 (d, J = 5.9Hz, 1 H), 5.38
(d, J = 8.0 Hz, 1 H), 5.90 (s, 1 H), 6.90 (d, J = 8.4 Hz, 2 H),
7.23-7.28 (m, 4 H), 7.29-7.35 (m, 4 H), 7.37-7.42 (m, 4 H),
7.74 (d, J = 8.0 Hz, 1 H), 11.32 (br s, 1 H).
Preparation of (4R,6R,7S)-(4-(2,4-Dioxopyrimidin-1(2H)-yl)-
6-[(4-methoxyphenyldiphenyl)methoxymethyl]-5-oxaspiro[2.4]-
hept-7-yl) Isobutyrate (17). Intermediate 16 (250 mg, 0.475 mmol)
was dissolved in dry pyridine (10 mL), and the solution was cooled
in a with cold water bath. To the solution was added via syringe
isobutyric anhydride (236 μL, 1.424 mmol), and the mixture
was stirred at room temperature for 2 h. More isobutyric anhydride
(236 μL, 1.424 mmol) was added, and the mixture was stirred
further for 2 h. Then additional isobutyric anhydride (236 μL,
1.424 mmol) was added and the mixture was stirred overnight.
After this time, the excess isobutyric anhydride was quenched by
addition of methanol. The solution was stirred for 20 min at room
temperature and then concentrated to dryness. The residue was
taken into ethyl acetate (30 mL) and the solution washed with
saturated aqueous NaHCO3 (2 ꢀ 20 mL). The organic phase was
dried over Na2SO4, the solid was filtered off, and the solvent removed
by evaporation, resulting in 18 as a colorless oil which was used
as such in the next reaction. LC-MS: tR = 3.07 min, m/z = 273
(M þ H)þ.
Preparation of (4R,6R,7S)-4-Amino-1-(7-hydroxy-6-hydroxy-
methyl-5-oxaspiro[2.4]hept-4-yl)-1H-pyrimidin-2-one (5). Inter-
mediate 14 (20 g, 1.0 equiv) was dissolved in THF (600 mL), and
TBAF (21.62 g, 2.05 equiv) was added to the mixture. The
reaction mixture was stirred at room temperature for 1 h. Then
Dowex resin (Dowex 50WX4, 202 g) and CaCO3 (67 g) were
added, and the mixture was stirred at room temperature for 1 h.
The reaction mixture was filtered, and the Dowex resin was
washed twice with a mixture of pyridine, methanol, and water
(1600 mL, 3:1:1). The combined filtrates were concentrated to
dryness by rotavapor. The residue was purified twice by column
chromatography (10-25% methanol in CHCl3) to give com-
pound 5 as a white solid (7.5 g, 92% purity).
Preparation of (4R,6R,7S)-(4-(2-Oxo-4-([1,2,4]triazol-1-yl)-
pyrimidin-1(2H)-yl)-6-[(4-methoxyphenyldiphenyl)methoxy-
methyl]-5-oxaspiro[2.4]hept-7-yl) Isobutyrate (18). POCl3 (102 μL,
1.089 mmol) was added to a cooled mixture of intermediate 17
(250 mg, 0.419 mmol), 1H-1,2,4-triazole (327 mg, 4.73 mmol),
triethylamine (661 μL, 4.73 mmol), and CH2Cl2 (6.0 mL) while the
reaction temperature was maintained below 25 °C (resulting in a
white precipitate). The reaction mixture was stirred at room tem-
perature for 4 h. When the reaction was completed, the excess
POCl3 was quenched by careful addition of cold H2O. The organic
layer was separated and concentrated by evaporation under
vacuum. The mixture was purified by silica gel chromatography
using gradient elution CH2Cl2/ethyl acetate 90:10 to 85:15, resulting
in intermediate 18 as an oil (200 mg, 74%). LC-MS: tR = 3.15
min, m/z = 648 (M þ H)þ. 1H NMR (400 MHz, CDCl3) δ ppm
0.67-0.73 (m, 1 H), 0.78-0.85 (m, 1 H), 0.97-1.05 (m, 1 H),
1.08-1.13 (m, 1 H), 1.15 (d, J = 7.0 Hz, 3 H), 1.18 (d, J = 7.0 Hz,
3 H), 2.57 (spt, J = 7.0 Hz, 1 H), 3.48 (dd, J = 10.7, 3.3 Hz, 1 H),
3.55 (dd, J = 10.7, 2.5 Hz, 1 H), 3.81 (s, 3 H), 4.26 (ddd, J = 4.7,
3.3, 2.5 Hz, 1 H), 5.42 (d, J=4.7Hz, 1H),6.48(s, 1H), 6.66(d, J=
7.2 Hz, 1 H), 6.87 (d, J = 8.6 Hz, 2 H), 7.27-7.36 (m, 8 H),
7.40-7.48 (m, 4 H), 8.10 (s, 1 H), 8.59 (d, J = 7.2 Hz, 1 H), 9.23
(s, 1 H).
Preparation of (4R,6R,7S)-(4-(4-Amino-2-oxopyrimidin-1(2H)-yl)-
6-[(4-methoxyphenyldiphenyl)methoxymethyl]-5-oxaspiro[2.4]-
hept-7-yl) Isobutyrate (19). Intermediate 18 (200 mg, 0.309 mmol)
was dissolved in THF (5 mL) and treated with concentrated
aqueous NH4OH (0.6 mL). After 4 h, more concentrated aqueous
NH4OH (0.3 mL) was added and the mixture was stirred overnight.
The solvent was removed in vacuo, and the oil was taken up in ethyl
acetate and washed with water and brine. After drying with
Na2SO4, filtration, and evaporation of the volatiles, the residue
19 was used as such in the next reaction. LC-MS: tR = 2.86 min,
m/z = 594 (M - H)-.
In order to increase the purity, recrystallization was carried
out. Compound 5 (7.5 g, 92% purity) was dissolved in HPLC-
grade methanol (6 mL), and the solvent was allowed to evapo-
rate slowly at room temperature until less than 1 mL remained.
White crystals were formed and collected by filtration and the
crystals were washed with cold methanol to give compound 5
as white crystals (6.13 g, >98% purity, 62% yield). MS: m/z =
254 (M þ H)þ. 1H NMR (400 MHz, DMSO-d6) δ ppm
0.31-0.61 (m, 3 H), 0.86-1.09 (m, 1 H), 3.49-3.63 (m, 1 H),
3.63-3.78 (m, 2 H), 3.85-4.02 (m, 1 H), 4.98 (br s, 1 H), 5.12
(d, J = 5.28 Hz, 1 H), 5.72 (d, J = 7.24 Hz, 1 H), 6.00 (s,1 H),
6.96-7.32 (m, 2 H), 7.77 (d, J = 7.24 Hz, 4 H). HPLC purity
determination: method 1, tR = 0.22, purity 99.4%; method 2,
t
R = 0.68, purity 98.7%.
Preparation of (4R,6R,7S)-1-(7-Hydroxy-6-hydroxymethyl-
5-oxaspiro[2.4]hept-4-yl)-1H-pyrimidin-2,4(1H,3H)-dione (15).
Intermediate 13 (11.00 g, 22.14 mmol) was dissolved in THF
(280 mL), and TBAF (59.8 mL, 59.8 mmol) was added. The
mixture was stirred at room temperature for 1 h. A mixture of
pyridine, methanol, and water (80 mL, 3:1:1) was added, fol-
lowed by a strongly acidic cation exchanger, Dowex 50WX4
(128 g), in a mixture of pyridine, methanol, and water (320 mL,
3:1:1). The reaction mixture was stirred for 45 min and filtered.
The Dowex residue was washed twice with a mixture of pyridine,
methanol, and water (320 mL, 3:1:1), and the combined filtrates
were concentrated under reduced pressure. The mixture was puri-
fied by silica gel chromatography with gradient elution of 0-10%
methanol in ethyl acetate, resulting in intermediate 15 (5.597 g,
84%) as a white foam. LC-MS: tR =2.05min,m/z= 253 (M - H)-.
1H NMR (400 MHz, DMSO-d6) δ ppm 0.50-0.60 (m, 3 H),
1.01-1.09 (m, 1 H), 3.55-3.62 (m, 1 H), 3.68-3.76 (m, 2 H),
4.03 (t, J = 4.5 Hz, 1 H), 5.02 (t, J = 4.5 Hz, 1 H), 5.20 (d, J =
5.1 Hz, 1 H), 5.64 (d, J = 8.0 Hz, 1 H), 5.92 (s, 1 H), 7.82 (d, J =
8.0 Hz, 1 H), 11.28 (br s, 1 H).
Preparation of (4R,6R,7S)-(4-(4-Amino-2-oxopyrimidin-1(2H)-yl)-
6-hydroxymethyl-5-oxaspiro[2.4]hept-7-yl) Isobutyrate (20).
Intermediate 19 (180 mg, 0.302 mmol) was dissolved in 80%
aqueous acetic acid (5 mL), and the reaction mixture was stirred
Preparation of (4R,6R,7S)-1-(7-Hydroxy-6-((4-methoxyphenyl-
diphenyl)methoxymethyl)-5-oxaspiro[2.4]hept-4-yl)-1H-pyrimidin-
2,4(1H,3H)-dione (16). A solution of 15 (350 mg, 1.377 mmol) in

8158 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Jonckers et al.
at room temperature. After 9 h, the volatiles were removed and the
mixture was purified by silica gel chromatography by gradient
elution with 5-15% methanol in CH2Cl2. The obtained residue
format for 3 days. Cellular activity was then detected by the
measurement of luciferase activity, and 50% effective concen-
trations (EC50) of compounds were determined.
i
Cellular Toxicity. Cellular toxicity was determined in Huh7-
CMV-Luc and MT4-LTR-Luc assays. In brief, Huh7 cells,
stably transformed with a luciferase reporter gene under control
of the cytomegalovirus (CMV) constitutive promoter, or MT4
cells expressing a luciferase reporter controlled by the HIV long
terminal repeat (LTR) promoter, were cultured in the presence
or absence of test compound concentrations. After 3 days of
incubation at 37 °C in a humidified 5% CO2 atmosphere, cell
proliferation was quantified by measuring the luciferase activity
and expressed as CC50 values (cytotoxicity, 50% inhibitory
concentration of cell growth). Tests were performed in 384-well
plates.
Pharmacokinetic Studies. Pharmacokinetic studies were per-
formed in Sprague-Dawley rats and Beagle dogs. Three animals
per dose regimen were administered a single oral solution of test
compound using 20% hydroxylpropyl-β-cyclodextrin. Rats were
dosed at around 36 and 216 μmol/kg with compound 5 and
its prodrug 23, while prodrug 20 was only dosed at around
36 μmol/kg. In dogs, only 20 was given at 36 and 180 μmol/kg.
Blood samples were collected on EDTA and put on ice protected
from light, at 0.5, 1, 2, 4, 7, and 24 h in rats and at 0.5, 1, 2, 4, 6, 8,
24, and 48 h in dogs. Blood samples were centrifuged at 4 °C
within 1 h after collection of blood to prepare plasma. Plasma
samples were stored at -20 °C until further analysis.
Analytical Method for Parent 5. After solubilization and
protein precipitation (with acetonitrile), plasma samples were
quantified on an Atlantis HILIC LC column (3 μm, 50 mm ꢀ 4.6 mm;
Waters). Mobile phases consisted of 0.01 M NH4OOCH3
(solvent A) and CH3CN (solvent B). Chromatographic separa-
tion was obtained by gradient elution (from 5% solvent A/95%
solvent B as starting conditions to 50% solvent A/50% solvent B
in 2.5 min) at a flow rate of 1.2 mL/min.
Analytical Method for Prodrugs 20 and 23. After solubiliza-
tion and protein precipitation (with CH3CN), plasma samples
were quantified on X-bridge C18 LC-column (3.5 μm, 50 mm ꢀ
4.6 mm; Waters). Mobile phases consisted of 0.01 M CH3COO-
NH4 (solvent A) and CH3CN (solvent B). Chromatographic separa-
tion was obtained by gradient elution (from 45% solvent A/55%
solvent B as starting conditions to 5% solvent A/95% solvent B
in 3 min) at a flow rate of 1.2 mL/min.
was triturated with Pr2O and dried in vacuo, resulting in com-
pound 20 (60.8 mg, 62%). MS: m/z = 324 (M þ H)þ. 1H NMR
(400 MHz, DMSO-d6) δ ppm 0.33-0.41 (m, 1 H), 0.62-0.71 (m,
1 H), 0.74-0.82 (m, 2 H), 1.08-1.14 (m, 6 H), 2.55-2.64 (1H, m),
3.62-3.68 (m, 2H), 3.99-4.04 (m, 1 H), 4.98-5.03 (m, 1 H), 5.12
(t, J = 5.2 Hz, 1 H), 5.76 (d, J = 7.4 Hz, 1 H), 6.27 (s, 1 H),
7.14-7.33 (m, 2 H), 7.80 (d, J = 7.4 Hz, 1 H). HPLC purity
determination: method 3, tR = 2.40, purity 97.2%; method 4, tR =
0.51, purity 95.5%.
Preparation of (4R,6R,7S)-(4-(2,4-Dioxopyrimidin-1(2H)-yl)-
6-(isobutyryloxymethyl)-5-oxaspiro[2.4]hept-7-yl) Isobutyrate (21).
Intermediate 15 (5.16 g, 20.30 mmol) was dissolved in dry pyridine
(100 mL), and the solution was externally cooled with cold water.
Isobutyric anhydride (16.85 mL, 101 mmol) was added, and the
mixture was allowed to proceed at room temperature overnight.
The mixture was externally cooled with cold water, and the excess
isobutyric anhydride was quenched by addition of methanol. After
the mixture was stirred for 20 min at room temperature and after
evaporation of the volatiles, ethyl acetate was added and the
mixture was washed with saturated aqueous NaHCO3 (2ꢀ). The
organic phase was dried with MgSO4 and concentrated in vacuo to
give intermediate 21 (7.68 g, 96%) as a white solid. LC-MS: tR =
2.26 min, m/z = 393 (M - H)-. 1H NMR (400 MHz, DMSO-d6)
δppm 0.57-0.63 (m, 1 H), 0.66-0.72 (m, 1 H), 0.87-0.96 (m, 2 H),
1.10 (d, J = 6.8 Hz, 12 H), 2.59 (m, 2 H), 4.21-4.32 (m, 3 H), 5.10
(d, J = 4.3 Hz, 1 H), 5.69 (d, J = 8.1 Hz, 1 H), 6.13 (s, 1 H), 7.61
(d, J = 8.1 Hz, 1 H), 11.37 (br s, 1 H).
Preparation of (4R,6R,7S)-(4-(4-([1,2,4]Triazol-1-yl)-2-oxo-
pyrimidin-1(2H)-yl)-6-(isobutyryloxymethyl)-5-oxaspiro[2.4]-
hept-7-yl) Isobutyrate (22). POCl3 (4.72 mL, 50.6 mmol) was
added to a cooled mixture of 21 (7.68 g, 19.47 mmol), 1H-1,2,4-
triazole (15.20 g, 220 mmol), and triethylamine (30.7 mL,
220 mmol) in dry CH2Cl2 (50 mL). The mixture was stirred at
room temperature for 2.5 h. The excess POCl3 was quenched by
addition of cold water, and the organic layer was separated and
concentrated in vacuo. The mixture was purified by silica gel
chromatography by gradient elution with CH2Cl2/ethyl acetate
90:10 to 85:15, resulting in intermediate 22 (7.5 g, 86%). LC-
MS: tR = 2.38 min, m/z = 446 (M þ H)þ. 1H NMR (400 MHz,
CDCl3) δ ppm 0.56-0.64 (m, 1 H), 0.75-0.83 (m, 1 H), 1.00-
1.06 (m, 1 H), 1.13-1.19 (m, 1 H), 1.19-1.26 (m, 12 H), 2.63
(spt, J = 6.8 Hz, 2 H), 4.35-4.41 (m, 2 H), 4.51 (dd, J = 12.7,
5.7 Hz, 1 H), 4.97 (d, J = 3.1 Hz, 1 H), 6.60 (s, 1 H), 7.09 (d, J =
7.2 Hz, 1 H), 8.14 (s, 1 H), 8.27 (d, J = 7.2 Hz, 1 H), 9.26 (s, 1 H).
Preparation of (4R,6R,7S)-(4-(4-Amino-2-oxopyrimidin-1(2H)-yl)-
6-(isobutyryloxymethyl)-5-oxaspiro[2.4]hept-7-yl) Isobutyrate (23).
Intermediate 22 (7.49 g, 16.81 mmol) was dissolved in THF (200
mL) and treated with concentrated aqueous NH4OH (15 mL).
After 3.5 h, the volatiles were removed under reduced pressure. The
mixture was purified by silica gel chromatography with gradient
elution of 0-5% methanol in CH2Cl2. The product was dissolved
in ethyl acetate, and the mixture was washed with water (2ꢀ) and
brine (2ꢀ). The organic phase was dried with MgSO4 and after
filtration concentrated in vacuo, resulting in compound 23 (5.597 g,
84%) as a white foam. MS: m/z = 394 (M þ H)þ. 1H NMR (400
MHz, DMSO-d6) δ ppm 0.39-0.44 (m, 1 H), 0.67-0.73 (m, 1 H),
0.80-0.89 (m, 2 H), 1.09-1.13 (m, 12 H), 2.54-2.65 (m, 2 H),
4.21-4.35 (m, 3 H), 5.01 (d, J = 3.1 Hz, 1 H), 5.77 (d, J = 7.4 Hz,
1 H), 6.25 (s, 1 H), 7.22-7.31 (m, 2 H), 7.55 (d, J = 7.4 Hz, 1 H).
HPLC purity determination: method 3, tR = 4.58, purity 99.1%;
method 4, tR = 0.71, purity 97.5%.
For all the different compounds, LC-MS/MS analysis
was carried out on an API-4000 MS/MS instrument (Applied
Biosystems), which was coupled to a HPLC system (Agilent).
The API-4000 MS/MS instrument, operating in the positive ion
mode using the TurboIonSpray interface (electrospray ioniza-
tion), was optimized for the quantification of the compounds.
The limit of quantification was at least 5.00 ng/mL for plasma
samples. The accuracy (intrabatch accuracy from independent
quality control samples) was between 85% and 115% of the
nominal value over the entire range for plasma samples. The
typical range of the standard curve was 5-2000 ng/mL.
Pharmacokinetic Analysis. Noncompartmental PK analysis
was performed on the individual plasma concentration-versus-
time data (WinNonlin, version 5.2.1; Pharsight Corp.).
Acknowledgment. Theauthors thank Sylvain Demanzefor
his assistance with NMR experiments; Geert Pille, Nathalie
Kindermans, and Liesbeth Vereyken for their analytical support;
and Luc Geeraert for his help in preparing this manuscript.
References
Replicon Activity. The HCV 1b subgenomic luciferase reporter
replicon (replicon clone ET obtained from R. Bartenschlager,
adapted from Lohmann59 with adaptive mutations E1202G,
T1280I, K1846T) was used to measure anti-HCV activity as
described before.37 Briefly, 2500 cells were incubated with com-
pounds plated in a 384-well nine-point dilution (1/4 dilutions)
(1) Ghobrial, R. M.; Steadman, R.; Gornbein, J.; Lassman, C.; Holt,
C. D.; Chen, P.; Farmer, D. G.; Yersiz, H.; Danino, N.; Collisson,
E.; Baquarizo, A.; Han, S. S.; Saab, S.; Goldstein, L. I.; Donovan,
J. A.; Esrason, K.; Busuttil, R. W. A 10-year experience of liver
transplantation for hepatitis C: analysis of factors determining
outcome in over 500 patients. Ann. Surg. 2001, 234, 384–393.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22 8159
(2) Simmonds, P. Genetic diversity and evolution of hepatitis C
virus;15 years on. J. Gen. Virol. 2004, 85, 3173–3188.
Synthesis and anti-hepatitis B virus and anti-hepatitis C virus
activities of 7-deazaneplanocin A analogues in vitro. J. Med. Chem.
2009, 52, 206–213.
(3) Strader, D. B.; Wright, T.; Thomas, D. L.; Seeff, L. B.; American
Association for the Study of Liver Diseases. Diagnosis, management,
and treatment of hepatitis C. Hepatology 2004, 39, 1147–1171.
(4) Manns, M. P.; Foster, G. R.; Rockstroh, J. K.; Zeuzem, S.; Zoulim,
F.; Houghton, M. The way forward in HCV treatment;finding
the right path. Nat. Rev. Drug Discovery 2007, 6, 991–1000.
(23) Gane, E. J.; Rodriguez-Torres, M.; Nelson, D. R.; Jacobson, I. M.;
McHutchison, J. G.; Jeffers, L.; Beard, A.; Walker, S.; Shulman,
N.; Symonds, W.; Albanis, E.; Berrey, M. M. Antiviral activity of
the HCV nucleoside polymerase inhibitor R7128 in HCV genotype
2 and 3 prior non-responders: interim results of R7128 1500mg bid
with peg-IFN and ribavirin for 28 days. Hepatology 2008, 48,
1024A.
ꢀ
(5) Hezode, C.; Forestier, N.; Dusheiko, G.; Ferenci, P.; Pol, S.;
Goeser, T.; Bronowicki, J. P.; Bourliere, M.; Gharakhanian, S.;
ꢁ
(24) Lalezari, J.; Gane, E.; Rodriguez-Torres, M.; De Jesus, E.; Nelson,
D.; Everson, G.; Jacobson, I.; Reddy, R.; Hill, G. Z.; Beard, A.;
Symonds, W. T.; Berrey, M. M.; McHutchison, J. G. Potent
antiviral activity of the HCV nucleotiside polymerase inhibitor
R7128 with PEG-IFN and ribavirin: interim results of R7128
500 mg BID for 28 days. J. Hepatol. 2008, 48, S29.
(25) Pockros, P. J.; Nelson, D.; Godofsky, E.; Rodriguez-Torres, M.;
Everson, G. T.; Fried, M. W.; Ghalib, R.; Harrison, S.; Nyberg,
L.; Shiffman, M. L.; Najera, I.; Chan, A.; Hill, G. R1626 plus
peginterferon alfa-2a provides potent suppression of hepatitis C
virus RNA and significant antiviral synergy in combination with
ribavirin. Hepatology 2008, 48, 385–397.
(26) Roberts, S. K.; Cooksley, G.; Dore, G. J.; Robson, R.; Shaw, D.;
Berns, H.; Hill, G.; Klumpp, K.; Najera, I.; Washington, C. Robust
antiviral activity of R1626, a novel nucleoside analog: a rando-
mized, placebo-controlled study in patients with chronic hepatitis
C. Hepatology 2008, 48, 398–406.
(27) Zhou, X. J.; Afdhal, N.; Godofsky, E.; Dienstag, J.; Rustgi, V.;
Schick, L.; McInery, D.; Fielman, B. A.; Brown, N. A. Pharma-
cokinetics and pharmacodynamics of valopicitabine (NM283), a
new nucleoside HCV polymerase inhibitor: results from a phase I/II
dose-escalation trial in patients with HCV-1 infection. J. Hepatol.
2005, 42, 229A.
(28) Carroll, S. S.; Olsen, D. B. Nucleoside analog inhibitors of hepatitis
C virus replication. Infect. Disord.: Drug Targets 2006, 6, 17–29.
(29) Smith, D. B.; Martin, J. A.; Klumpp, K.; Baker, S. J.; Blomgren,
P. A.; Devos, R.; Granycome, C.; Hang, J.; Hobbs, C. J.; Jiang,
W.-R.; Laxton, C.; Le Pogam, S.; Leveque, V.; Ma, H.; Maile, G.;
Merrett, J. H.; Pichota, A.; Sarma, K.; Smith, M.; Swallow, S.;
Symons, J.; Vesey, D.; Najera, I.; Cammack, N. Design, synthesis,
and antiviral properties of 40-substituted ribonucleosides as inhibi-
tors of hepatitis C virus replication: the discovery of R1479. Bioorg.
Med. Chem. Lett. 2007, 17, 2570–2576.
(30) Pierra, C.; Amador, A.; Benzaria, S.; Cretton-Scott, E.; D’Amours,
M.; Mao, J.; Mathieu, S.; Moussa, A.; Bridges, E. G.; Standring,
D. N.; Sommadossi, J.-P.; Storer, R.; Gosselin, G. Synthesis and
pharmacokinetics of valopicitabine (NM283), an efficient prodrug
of the potent anti-HCV agent 20-C-methylcytidine. J. Med. Chem.
2006, 49, 6614–6620.
(31) Clark, J. L.; Hollecker, L.; Mason, J. C.; Stuyver, L. J.; Tharnish,
P. M.; Lostia, S.; McBrayer, T. R.; Schinazi, R. F.; Watanabe,
K. A.; Otto, M. J.; Furman, P. A.; Stec, W. J.; Patterson, S. E.;
Pankiewicz, K. W. Design, synthesis, and antiviral activity of
20-deoxy-20-fluoro-20-C-methylcytidine, a potent inhibitor of hepa-
titis C virus replication. J. Med. Chem. 2005, 48, 5504–5508.
(32) Klumpp, K.; Leveque, V.; Le Pogam, S.; Ma, H.; Jiang, W.-R.;
Kang, H.; Granycome, C.; Singer, M.; Laxton, C.; Hang, J. Q.;
Sarma, K.; Smith, D. B.; Heindl, D.; Hobbs, C. J.; Merrett, J. H.;
Symons, J.; Cammack, N.; Martin, J. A.; Devos, R.; Najera, I. The
novel nucleoside analog R1479 (40-azidocytidine) is a potent
inhibitor of NS5B-dependent RNA synthesis and hepatitis C virus
replication in cell culture. J. Biol. Chem. 2006, 281, 3793–3799.
(33) Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Pinho, P.;
Maltseva, T.; Morisson, V.; Leveque, V.; Rajyaguru, S.; Le Pogam,
S.; Najera, I.; Benkestock, K.; Zhou, X.-X.; Maag, H.; Cammack,
N.; Martin, J. A.; Swallow, S.; Johansson, N. G.; Klumpp,
K.; Smith, M. The design, synthesis, and antiviral activity of
40-azidocytidine analogues against hepatitis C virus replication:
the discovery of 40-azidoarabinocytidine. J. Med. Chem. 2009, 52,
219–223.
Bengtsson, L.; McNair, L.; George, S.; Kieffer, T.; Kwong, A.;
Kauffman, R. S.; Alam, J.; Pawlotsky, J.-M.; Zeuzem, S. Telaprevir
and peginterferon with or without ribavirin for chronic HCV infec-
tion. N. Engl. J. Med. 2009, 360, 1839–1850.
(6) Kwo, P. Y.; Lawitz, E. J.; McCone, J.; Schiff, E. R.; Vierling, J. M.;
Pound, D.; Davis, M. N.; Galati, J. S.; Gordon, S. C.; Ravendhran,
N.; Rossaro, L.; Anderson, F. H.; Jacobson, I. M.; Rubin, R.;
Koury, K.; Pedicone, L. D.; Brass, C. A.; Chaudhri, E.; Albrecht,
J. K. Efficacy of boceprevir, an NS3 protease inhibitor, in combi-
nation with peginterferon alfa-2b and ribavirin in treatment-naive
patients with genotype 1 hepatitis C infection (SPRINT-1): an
open-label, randomised, multicentre phase 2 trial. Lancet 2010,
376 (9742), 705–716.
(7) Lindenbach, B. D.; Rice, C. M. Unravelling hepatitis C virus
replication from genome to function. Nature 2005, 436, 933–938.
(8) Behrens, S.-E.; Tomei, L.; De Francesco, R. Identification and
properties of the RNA-dependent RNA polymerase of hepatitis C
virus. EMBO J. 1996, 15, 12–22.
(9) Lohmann, V.; Korner, F.; Herian, U.; Bartenschlager, R. Bio-
chemical properties of hepatitis C virus NS5B RNA-dependent
RNA polymerase and identification of amino acid sequence motifs
essential for enzymatic activity. J. Virol. 1997, 71, 8416–8428.
(10) Lesburg, C. A.; Cable, M. B.; Ferrari, E.; Hong, Z.; Mannarino,
A. F.; Weber, P. C. Crystal structure of the RNA-dependent RNA
polymerase from hepatitis C virus reveals a fully encircled active
site. Nat. Struct. Biol. 1999, 6, 937–943.
(11) Legrand-Abravanel, F.; Nicot, F.; Izopet, J. New NS5B polymer-
ase inhibitors for hepatitis C. Expert Opin. Invest. Drugs 2010,
19 (8), 963–975.
(12) Sarrazin, C.; Zeuzem, S. Resistance to direct antiviral agents in
patients with hepatitis C virus infection. Gastroenterology 2010,
138, 447–462.
(13) McCown, M. F.; Rajyaguru, S.; Le Pogam, S.; Ali, S.; Jiang,
W.-R.; Kang, H.; Symons, J.; Cammack, N.; Najera, I. The hepatitis
C virus replicon presents a higher barrier to resistance to nucleoside
analogs than to non nucleoside polymerase or protease inhibitors.
Antimicrob. Agents Chemother. 2008, 52, 1604–1612.
(14) Kim, J.; Chong, Y. Understanding the RNA-specificity of HCV
RdRp: implications for anti-HCV drug discovery. Bull. Korean
Chem. Soc. 2006, 27, 59–64.
(15) Tan, S.-L. Hepatitis C Viruses: Genomes and Molecular Biology;
Horizon Bioscience: Norwich, U.K., 2006.
(16) Bressanelli, S.; Tomei, L.; Rey, F. A.; De Francesco, R. Structural
analysis of the hepatitis C virus RNA polymerase in complex with
ribonucleotides. J. Virol. 2002, 76, 3482–3492.
(17) Klumpp, K.; Kalayanov, G.; Ma, H.; Le Pogam, S.; Leveque, V.;
Jiang, W.-R.; Inocencio, N.; De Witte, A.; Rajyaguru, S.; Tai,
E.; Chanda, S.; Irwin, M. R.; Sund, C.; Winqist, A.; Maltseva,
T.; Eriksson, S.; Usova, E.; Smith, M.; Alker, A.; Najera, I.;
Cammack, N.; Martin, J. A.; Johansson, N. G.; Smith, D. B.
20-Deoxy-40-azido nucleoside analogs are highly potent inhibitors
of hepatitis C virus replication despite the lack of 20-alpha-hydroxyl
groups. J. Biol. Chem. 2008, 283, 2167–2175.
(18) Chiacchio, U.; Borrello, L.; Crispino, L.; Rescifina, A.; Merino, P.;
Macchi, B.; Balestrieri, E.; Mastino, A.; Piperno, A.; Romeo,
G. Stereoselective synthesis and biological evaluations of novel
30-deoxy-40-azaribonucleosides as inhibitors of hepatitis C virus
RNA replication. J. Med. Chem. 2009, 52, 4054–4057.
(19) Gunic, E.; Chow, S.; Rong, F.; Ramasamy, K.; Raney, A.; Li,
D. Y.; Huang, J.; Hamatake, R. K.; Hong, Z.; Girardet, J.-L.
6-Hydrazinopurine 20-methyl ribonucleosides and their 50-mono-
phosphate prodrugs as potent hepatitis C virus inhibitors. Bioorg.
Med. Chem. Lett. 2007, 17, 2456–2458.
(20) Koch, U.; Narjes, F. Recent progress in the development of
inhibitors of the hepatitis C virus RNA-dependent RNA polymer-
ase. Curr. Top. Med. Chem. 2007, 7, 1302–1329.
(34) Pockros, P. J. New drugs for hepatitis C virus. Expert Rev.
Gastroenterol. Hepatol. 2007, 1, 145–154.
(35) Flisiak, R.; Parfieniuk, A. Investigational drugs for hepatitis C.
Expert Opin. Invest. Drugs 2010, 19 (1), 63–75.
(36) Pockros, P.; Nelson, D.; Godofsky, E.; Rodriguez-Torres, M.;
Everson, G. T.; Fried, M. W.; Ghalib, R.; Harrison, S.; Nyberg,
L.; Shiffman, M. L.; Chan, A.; Hill, G. High relapse rate seen at
week 72 for patients treated with R1626 combination therapy.
Hepatology 2008, 48, 1349–1350.
(21) Liao, X.; Butora, G.; Olsen, D. B.; Carroll, S. S.; McMasters, D. R.;
Leone, J. F.; Stahlhut, M.; Doss, G. A.; Yang, L.; MacCoss, M.
Synthesis of 20-β-C-methyl-neplanocin derivatives as anti-HCV
agents. Tetrahedron Lett. 2008, 49, 4149–4152.
(37) Lin, T.-I.; Lenz, O.; Fanning, G.; Verbinnen, T.; Delouvroy,
˚
F.; Scholliers, A.; Vermeiren, K.; Rosenquist, A.; Edlund, M.;
Samuelsson, B.; Vrang, L.; de Kock, H.; Wigerinck, P.; Raboisson,
(22) Kim, H.-J.; Sharon, A.; Bal, C.; Wang, J.; Allu, M.; Huang, Z.;
Murray, M. G.; Bassit, L.; Schinazi, R. F.; Korba, B.; Chu, C. K.

8160 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 22
Jonckers et al.
P.; Simmen, K. In vitro activity and preclinical profile of
TMC435350, a potent hepatitis C virus protease inhibitor. Anti-
microb. Agents Chemother. 2009, 53, 1377–1385.
chondrin synthesis. Org. Lett. 2007, 9, 723–726.Letters 1989, 30,
659-662.
(47) Li, F.; Maag, H.; Alfredson, T. Prodrugs of nucleoside analogues
for improved oral absorption and tissue targeting. J. Pharm. Sci.
2008, 97, 1109–1134.
˚
(38) Raboisson, P.; de Kock, H.; Rosenquist, A.; Nilsson, M.;
€
Salvador-Oden, L.; Lin, T.-I.; Roue, N.; Ivanov, V.; Wahling, H.;
Wickstrom, K.; Hamelink, E.; Edlund, M.; Vrang, L.; Vendeville, S.;
€
(48) Balimane, P. V.; Sinko, P. J. Involvement of multiple transporters
in the oral absorption of nucleoside analogues. Adv. Drug Delivery
Rev. 1999, 39, 183–209.
Van de Vreken, W.; McGowan, D.; Tahri, A.; Hu, L.; Boutton, C.;
Lenz, O.; Delouvroy, F.; Pille, G.; Surleraux, D.; Wigerinck, P.;
Samuelsson, B.; Simmen, K. Structure-activity relationship study on
a novel series of cyclopentane-containing macrocyclic inhibitors of the
hepatitis C virus NS3 protease leading to the discovery of TMC435350.
Bioorg. Med. Chem. Lett. 2008, 18, 4853–4858.
(49) Brandl, M.; Wu, X.; Holper, M.; Hong, L.; Jia, Z.; Birudaraj, R.;
Reddy, M.; Alfredson, T.; Tran, T.; Larrabee, S.; Hadig, X.;
Sarma, K.; Washington, C.; Hill, G.; Smith, D. B. Physicochemical
properties of the nucleoside prodrug R1626 leading to high oral
bioavailability. Drug Dev. Ind. Pharm. 2008, 34, 683–691.
(50) Wang, P.; Chun, B.-K.; Rachakonda, S.; Du, J.; Khan, N.; Shi, J.;
Stec, W.; Cleary, D.; Ross, B. S.; Sofia, M. J. An efficient and
diastereoselective synthesis of PSI-6130: a clinically efficacious
inhibitor of HCV NS5B polymerase. J. Org. Chem. 2009, 74,
6819–6824.
(51) Lohmann, V.; Hoffmann, S.; Herian, U.; Penin, F.; Bartenschlager,
R. Viral and cellular determinants of hepatitis C virus RNA replica-
tion in cell culture. J. Virol. 2003, 77, 3007–3019.
(52) Halgren, T. A. MMFF VI. MMFF94s option for energy mini-
mization studies. J. Comput. Chem. 1999, 20, 720–729.
(53) Wojciechowski, M.; Lesyng, B. Generalized Born model: analysis,
refinement, and applications to proteins. J. Phys. Chem. B 2004,
108, 18368–18376.
(54) Rush, T. S. r.; Grant, J. A.; Mosyak, L.; Nicholls, A. A shape-based
3-D scaffold hopping method and its application to a bacterial
protein-protein interaction. J. Med. Chem. 2005, 48, 1489–1495.
(55) Grant, J. A.; Gallardo, M. A.; Pickup, B. T. A fast method of
molecular shape comparison: a simple application of a Gaussian
description of molecular shape. J. Comput. Chem. 1996, 17, 1653–1666.
(56) Nicholls, A.; MacCuish, N. E.; MacCuish, J. D. Variable selection
and model validation of 2D and 3D molecular descriptors.
J. Comput.-Aided Mol. Des. 2004, 18, 451–474.
(39) Vandyck, K.; Cummings, M. D.; Nyanguile, O.; Boutton, C. W.;
Vendeville, S.; McGowan, D.; Devogelaere, B.; Amssoms, K.;
Last, S.; Rombauts, K.; Tahri, A.; Lory, P.; Hu, L.; Beauchamp,
D. A.; Simmen, K.; Raboisson, P. Structure-based design of a
benzodiazepine scaffold yields a potent allosteric inhibitor of
hepatitis C NS5B RNA polymerase. J. Med. Chem. 2009, 52,
4099–4102.
(40) Nyanguile, O.; Pauwels, F.; Van den Broeck, W.; Boutton, C. W.;
Quirynen, L.; Ivens, T.; van der Helm, L.; Vandercruyssen, G.;
Mostmans, W.; Delouvroy, F.; Dehertogh, P.; Cummings, M. D.;
Bonfanti, J.-F.; Simmen, K. A.; Raboisson, P. 1,5-Benzodiazepines, a
novel class of hepatitis C virus polymerase non-nucleoside inhibitors.
Antimicrob. Agents Chemother. 2008, 52, 4420–4431.
ꢀ
(41) Czernecki, S.; Mulard, L.; Valery, J.-M.; Commerc-on, A. Synthesis
of 20-deoxy-20-spirocyclopropyl cytidine as potential inhibitor of
ribonucleotide diphosphate reductase. Can. J. Chem. 1993, 71,
413–416.
(42) Samano, V.; Robins, M. J. Nucleic acid and related compounds.
75. Synthesis and radical-induced ring-opening reactions of
20-deoxyadenosine-20-spirocyclopropane and its uridine analogs.
Mechanistic probes for ribonucleotide reductases. J. Am. Chem.
Soc. 1992, 114, 4007–4008.
(43) Petter, R. C.; Powers, D. G. Synthesis of L-2-spirocyclopropyl-
2-deoxyarabinose. Tetrahedron Lett. 1989, 30, 659–662.
(44) Ichikawa, S.; Minakawa, N.; Shuto, S.; Tanaka, M.; Sasaki,
T.; Matsuda, A. Synthesis of 30-beta-carbamoylmethylcytidine
(CAMC) and its derivatives as potential antitumor agents. Org.
Biomol. Chem. 2006, 4, 1284–1296.
(45) Kodama, T.; Shuto, S.; Nomura, M.; Matsuda, A. An efficient
method for the preparation of 10alpha-branched-chain sugar pyr-
imidine ribonucleosides from uridine: the first conversion of a
natural nucleoside into 10-substituted ribonucleosides. Chem.;
Eur. J. 2001, 7, 2332–2340.
(57) Connolly, M. L. Analytical molecular surface calculation. J. Appl.
Crystallogr. 1983, 16, 548–558.
(58) Eldrup, A. B.; Allerson, C. R.; Bennett, C. F.; Bera, S.; Bhat, B.;
Bhat, N.; Bosserman, M. R.; Brooks, J.; Burlein, C.; Carroll, S. S.;
Cook, P. D.; Getty, K. L.; MacCoss, M.; McMasters, D. R.; Olsen,
D. B.; Prakash, T. P.; Prhavc, M.; Song, Q.; Tomassini, J. E.; Xia,
J. Structure-activity relationship of purine ribonucleosides for
inhibition of hepatitis C virus RNA-dependent RNA polymerase.
J. Med. Chem. 2004, 47, 2283–2295.
€
(59) Lohmann, V.; Korner, F.; Koch, J.; Herian, U.; Theilmann, L.;
(46) Kaburagi, Y.; Kishi, Y. Operationally simple and efficient workup
Bartenschlager, R. Replication of subgenomic hepatitis C virus
RNAs in a hepatoma cell line. Science 1999, 285, 110–113.
procedure for TBAF-mediated desilylation: application to hali-