Discovery of 4′-azido-2′-deoxy-2′-C-methyl cytidine and prodrugs thereof: A potent inhibitor of Hepatitis C virus replication

Article abstract of DOI:10.1016/j.bmcl.2012.03.021

4′-Azido-2′-deoxy-2′-methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-dependent RNA polymerase, displaying an EC₅₀ value of 1.2 μM and showing moderate in vivo bioavailability in rat (F = 14%). Here we describe the synthesis and biological evaluation of 4′-azido-2′-deoxy-2′-methylcytidine and prodrug derivatives thereof.

Full text of DOI:10.1016/j.bmcl.2012.03.021

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3265–3268
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 40-azido-20-deoxy-20-C-methyl cytidine and prodrugs thereof:
A potent inhibitor of Hepatitis C virus replication
Magnus Nilsson a, , Genadiy Kalayanov a, Anna Winqvist a, Pedro Pinho a, Christian Sund a,
⇑
Xiao-Xiong Zhou a, Horst Wähling a, Anna-Karin Belfrage a, Michael Pelcman a, Tatiana Agback a,
Kurt Benckestock a, Kristina Wikström a, Mirva Boothee a, Anneli Lindqvist a, Christina Rydegård a,
Tim H.M. Jonckers b, Koen Vandyck b, Pierre Raboisson b, Tse-I Lin b, Sophie Lachau-Durand b,
Herman de Kock b, David B. Smith c, Joseph A. Martin c, Klaus Klumpp c, Kenneth Simmen b, Lotta Vrang a,
Ylva Terelius a, Bertil Samuelsson a, Åsa Rosenquist a, Nils Gunnar Johansson a
a Medivir AB, PO Box 1086, SE-141 22 Huddinge, Sweden
b Tibotec-A Division of Janssen Pharmaceutical Companies of Johnson & Johnson, Turnhoutseweg 30, 2340 Beerse, Belgium
c Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, CA 94304, USA
a r t i c l e i n f o
a b s t r a c t
40-Azido-20-deoxy-20-methylcytidine (14) is a potent nucleoside inhibitor of the HCV NS5B RNA-depen-
Article history:
Received 26 January 2012
Revised 5 March 2012
Accepted 6 March 2012
Available online 16 March 2012
dent RNA polymerase, displaying an EC50 value of 1.2 lM and showing moderate in vivo bioavailability
in rat (F = 14%). Here we describe the synthesis and biological evaluation of 40-azido-20-deoxy-20-meth-
ylcytidine and prodrug derivatives thereof.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Hepatitis C virus
NS5B polymerase
Antivirals
Nucleoside
Prodrug
Phosphoramidate
Hepatitis C virus (HCV) was first discovered in 1989,1 and is an
infectious agent affecting about 170 million people worldwide.2
HCV establishes a chronic infection in a majority of affected people,
and of these approximately 20% will develop cirrhosis of the liver
over a 20–25 year period, and with increased risk of developing
end-stage liver disease and hepatocellular carcinoma.3 Standard
of care (SoC) for HCV infection involves treatment with pegylated
viral diseases and have contributed to a significant reduction in
morbidity, mortality and suffering caused by viral infections,
including human immunodeficiency virus (HIV), hepatitis B virus
and herpes virus infections. The lessons learned from the develop-
ment of treatment regimens for human immunodeficiency virus
(HIV) suggest that drug combinations addressing different targets
of viral replication will be required to combat HCV infections,
underpinning the need for new and more efficacious treatment op-
tions in HCV. The use of nucleoside analogs as one component of a
combination of two or three direct acting antiviral (DAA) agents for
the treatment of HCV shows emerging promise, as in particular
their broad genotypic coverage9 and the relatively high barrier to
resistance are attractive features.10
a-interferon in combination with the nucleoside analogue ribavi-
rin and results in, after a 48 week treatment, approximately 40–
50% sustained virological response (SVR) in the HCV genotype 1
population.4 Recently two HCV NS3/4A protease inhibitors (PIs),
Telaprevir5 (VX-950) and Boceprevir6 (SCH 503034) were approved
by the FDA, which are expected to improve the outcome of HCV
treatment when added to SoC in genotype 1 patients. However,
in difficult to treat patients, for example, prior partial and null
responders to SoC, the new triple therapies of Telaprevir or Boce-
previr added to SoC provide only moderate SVR rates.7,8 Nucleoside
analogues have become cornerstone components in treatment of
The antiviral properties of 40-substituted nucleosides in HIV,
and in particular 40-azido nucleosides, has been described by Maag
et al.11 Figure 1 depicts structures of compounds 1–6 which are 20-
modified 40-azidocytidines. The discovery of 40-azidocytidine,
R147912 (1), as a potent and non-cytotoxic inhibitor of HCV repli-
cation (replicon IC50 = 1.28
>2000
M), and its 20,30,50-triisobutyryl ester prodrug, R1626 (2)
have indeed inspired further modifications in the 20-position of
lM, cytotoxicity in Huh7-cells
l
⇑
Corresponding author.
E-mail address: magnus.k.nilsson@telia.com (M. Nilsson).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.021

3266
M. Nilsson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3265–3268
O
O
N
NH2
O
O
N
O
NH2
O
N
NH2
HO
N3
HO
O
HO
N3
HO
O
O
O
N
N
N
N3
OH
O
O
OH
R1479 (1)
R1626 (2)
RO-9187 (3)
O
O
N
N
NH2
O
O
N
NH2
O
O
N
NH2
HO
N3
HO
HO
N3
HO
HO
N3
HO
N
N
F
OH
F
(5)
Figure 1. 20-Modified 40-azidocytidines.
(4)
(6)
the 40-azidocytidines for the exploration of SAR and ultimately
optimization of phosphorylation efficiency and antiviral potency.
The 20-modified 40-azidocytidines 3, 4, 5 and 6 have been re-
ported.13,14 Nucleoside analogs 3, 4 and 5 are all potent inhibitors
of HCV replication in the HCV replicon system,15 however com-
pound 6 was found to be inactive, most likely due to being a poor
substrate for kinases involved in the first phosphorylation step
leading up to its corresponding active 50-triphosphate, as indicated
by Schinazi and co-workers.16 We now report on the discovery of
40-azido-20-deoxy-20-methylcytidine (14) and the identification of
the clinical candidate TMC649128 (16). The synthesis of 40-azido-
20-deoxy-20-methyluridine (13) and 40-azido-20-deoxy-20-meth-
ylcytidine (14) are outlined in Scheme 1 and were prepared from
compound 717,18 similar as described for analogous
compounds.13,14
The di-isobutyryl ester 16 was obtained by treating 40-azido-20-
deoxy-20-methyluridine (13) with isobutyric anhydride in pyri-
dine, which yielded crude compound 15. This was subsequently
treated with 4-chlorophenylchlorophosphate and 1-H-tetrazole in
pyridine, followed by ammonia in dioxane, which afforded final
product 16 in 35% yield. For the synthesis of 3,5-di-O-Boc-valinyl
ester 19, compound 13 was treated with N,N0-diisopropylcarbodi-
imide (DipCDI) and L-Boc-valine which gave diester 17 in 50% yield.
The conversion of 17 into the cytidine derivative 18 was effected in
40% yield over two steps, as described, vide supra. Subsequent N-
deprotection of 18 using trifluoroacetic acid in dichloromethane
afforded compound 19 as its trifluoroacetic acid salt in 98% yield.
The synthesis of 50-mono ester prodrugs 25 and 29 are outlined
in Scheme 3. Monomethoxytritylation of 13 using MMTrCl (4-
methoxytrityl chloride) in pyridine afforded compound 20 in 88%
yield. Treatment of 20 with TBDMSCl (tert-Butyl(chloro)dimethyl-
silane) and imidazole in DMF resulted in intermediate 21 in 56%
yield. Removal of the MMTr protecting group with trichloroacetic
acid yielded the common intermediate 22 in 75% yield. For the syn-
thesis of derivative 24, compound 22 was treated with isobutyric
anhydride furnishing 23 in 99% yield followed by conversion into
its corresponding cytidine derivative 24, similar as described, vide
supra. Finally, deprotection of the TBDMS group with TBAF (tetra-
butylammonium fluoride) gave 25 in 44% yield over three steps.
In order to overcome the observed moderate bioavailability of
14, a selected set of prodrugs were synthesized according to
Schemes 2–5 with the intention to compare their pharmacokinetic
properties to those of the parent nucleoside.
O
O
R
O
O
N
NH
N
NH
ii
iii
Compound 29 was prepared from 22 by esterification with Boc-L-
O
O
HO
i
HO
valine which afforded 26 in 74% yield. Compound 26 was con-
verted into its cytidine derivative 27 similar as above, followed
by deprotection of the TBDMS group using TBAF which furnished
28 in 28% yield over three steps. N-Boc-deprotection of 28 yielded
the target valine ester 29 in 98%.
7: R = OH
8: R = I
9
O
O
The 30-prodrug esters 32 and 36 were prepared as outlined in
Scheme 4. Treatment of the common intermediate 20 with isobu-
tyric anhydride gave 30 in 96% yield, which subsequently was con-
verted into its corresponding cytidine derivative 31 in 75% yield
I
O
N
NH
m-ClBzO
O
v
N
NH
N3
O
N3
BzO
R
O
10: R = OH
11: R = OBz
iv
12
vii, viii, vi
O
NH2
vi
RO
N3
RO
O
RO
N3
RO
O
N
NH
N
N
ii, iii
iv
O
NH2
HO
N3
HO
O
O
HO
N3
HO
O
O
N
N
NH
N
O
O
13 R = H
15 R = i-butyryl
17 Boc-L-valinyl ester
16 R = i-butyryl
i
13
14
18 R = Boc-L-valinyl ester
v
19 R = L-valinyl ester - TFA-salt
Scheme 1. Reagents and conditions: (i) I2, PPh3, imidazole, THF, 76%; (ii) NaOMe,
MeOH, 75%; (iii) [Bn(Et)3]NN3:I2, NMO, THF; (iv) BzCl, DMAP, NMP, THF, 81% over
two steps; (v) m-CPBA, m-CBA, (NH4)HSO4, CH2Cl2, 68%; (vi) NH3, MeOH, 85% for 13,
61% for 14 over three steps; (vii) pyridine, 2-chlorophenyldichlorophosphate, 1-H-
tetrazole; (viii) dioxane, NH3.
Scheme 2. Reagents and conditions: (i) pyridine, isobutyric anhydride; (ii)
pyridine, 4-chlorophenylchlorophosphate, 1-H-tetrazole; (iii) dioxane, NH3, 35%
for 16 over 3 steps, 40% for 18 over 2 steps ; (iv) N-Boc-Val-OH, DipCDI, DMAP, DMF,
50%; (v) TFA, DCM, 98%.

M. Nilsson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3265–3268
3267
NH2
O
R1O
N3
R2O
R1O
N3
R2O
v
O
O
N
N
N
NH
O
O
13 R1 = R2 = H
24 R1 = isobutyryl, R2 = TBDMS
i
ii
iii
20 R1 = MMTr, R2 = H
vi
25 R1 = isobutyryl, R2 = H
21 R1 = MMTr, R2 = TBDMS
22 R1 = OH, R2 = TBDMS
27 R1 = Boc-L-Valinyl ester, R2 = TBDMS
28 R1 = Boc-L-Valinyl ester, R2 = H
29 R1 = L-Valinyl ester - TFA-salt, R2 = H
vi
viii
23 R1 = isobutyryl, R2 = TBDMS
iv
vii
26 R1 = Boc-L-Valinyl ester, R2 = TBDMS
Scheme 3. Reagents and conditions: (i) MMTrCl, pyridine, 88%; (ii) TBDMSCl, imidazole, DMF, 56%; (iii) TCA, DCM, 75%; (iv) pyridine, isobutyric anhydride, 99%; (v) First
pyridine, 4-chlorophenylchlorophosphate, 1-H-tetrazole, followed by dioxane, NH3; (vi) NH4F, MeOH, 44% for 25 over three steps, 28% for 28 over three steps ; (vii) N-Boc-
Val-OH, DipCDI, DMAP, DMF, 74%; (viii) TFA, DCM, 98%.
NH2
O
R1O
N3
R2O
R1O
N3
R2O
ii
O
O
N
N
N
NH
O
O
20 R1 = MMTr, R2 = H
31 R1 = MMTr, R2 = isobutyryl
i
30 R1 = MMTr, R2 = isobutyryl
iii
32 R1 = H, R2 = isobutyryl
iv
33 R1 = MMTr, R2 = Boc-L-valinyl ester
34 R1 = MMTr, R2 = Boc-L-Valinyl ester
35 R1 = H, R2 = Boc-L-Valinyl ester
36 R1 = H, R2 = L-Valinyl ester - TFA-salt
iii
v
Scheme 4. Reagents and conditions: (i) pyridine, isobutyric anhydride, 96%; (ii) first pyridine, 4-chlorophenylchlorophosphate, 1-H-tetrazole, then dioxane, NH3, 75% of 31
over two steps, 72% of 34 over two steps; (iii) aq. 80% acetic acid, 89% for 32, 84% for 35; (iv) N-Boc-Val-OH, DipCDI, DMAP, DMF, 95%; (v) TFA, DCM, quantitative.
In Table 1, we present data from the HCV replicon assay.15 Com-
pound 13 did not display any activity at the concentrations tested,
probably due to being a poor substrate for kinases, which was sup-
ported by the activity of the corresponding nucleotide prodrug 37
O
O
O
N P O
O
i
O
N
NH
H
13
14
O
N3
HO
O
(EC50 = 6.2
(EC50 = 1.2
l
M). Compound 14 displayed good activity
37
lM) with no cytotoxic activity at the concentrations
tested. The activity of 14 was not improved by its nucleotide pro-
drug derivative 39, indicating that the first phosphorylating step in
replicon cells is not rate-limiting. Prodrug derivatives 16, 19, 25,
29, 32 and 36 showed similar or slightly lower activity compared
with compound 14, reflecting the ease of hydrolysis in the replicon
assay, ultimately releasing the parent nucleoside 14.
R
O
O
N P
O
N3
HO
O
ii, i
N
N
H
O
O
O
In Table 2 a selection of in vitro PK data are presented. The parent
nucleoside 14 displayed excellent stability in human plasma (<3
ll/
38 R = -N=CH-N(CH3)2
39 R = -NH2
min/mg) and in human liver microsomes (<6 l/min/mg), however
l
iii
only moderate permeability in Caco-2 cells (Papp = 1.7 cm/s
ꢀ 10ꢁ6). As expected, the diester prodrugs 16, 19, 25, and 29 all
displayed low stability in human plasma, most likely related to the
facile cleavage of the 50-esters. Compound 16 showed a good perme-
ability in Caco-2 cells (Papp = 7.7 cm/s ꢀ 10ꢁ6), possibly related to
its increased lipophilicity compared to the parent nucleoside 14.
The 30-ester prodrugs 32 and 36 both had good stability in human
Scheme 5. Reagents and conditions: (i) N-methyl imidazole, THF, 1-naphtyl(benz-
oxy-
-alaninyl)phosphorochloridate, (ii) N,N0-dimethylformamide dimethylacetal,
methanol; (iii) Acetonitrile–aq HCl, 4% yield over 3 steps.
L
using standard conditions. Finally, removal of the MMTr group of
31 afforded compound 32 in 89% yield. For the synthesis of com-
pound 36, compound 20 was esterified with Boc-
L-valine yielding
plasma (<3 and 4 ll/min/mg respectively).
33 in 95% yield. Compound 33 was converted into the cytidine
derivative 34 in 72% over two steps, then removal of the MMTr
group gave 35 in 84% yield and N-Boc deprotection quantitatively
afforded prodrug ester 36.
Table 1
Inhibition of HCV replication in Huh7-Rep Cells (EC50
, Luciferase Assay) and
The synthesis of a selection of phosphoramidate prodrugs is out-
lined in Scheme 5. Uridinederivative 37 was obtained in 31% yield by
cytotoxicity (CC50) measured in Huh7-CMV-Luc and MT4-LTR-Luc cells
Compound
CC50 (lM)
reacting compound 13 with 1-naphthyl(benzoxy-L-alaninyl)phos-
phorochloridate.19 The cytidine compound 39 was synthesized from
compound 14 by first protection of the amino group using dimethyl-
formamide dimethylacetal, followed by reaction with 1-naph-
EC50
(lM)
Huh7-CMV-Luc
MT4-LTR-Luc
13
14
16
19
25
29
32
36
37
39
>50
1.2
6.6
1.9
3.6
0.79
6.5
0.72
6.2
3
>100
>98
>100
>98
>50
NA
NA
NA
NA
NA
41
>32
>100
>100
>100
>100
>100
>100
>100
>100
thyl(benzoxy-L-alaninyl)phosphorochloridate which resulted in
compound 38. Finally deprotection of the amino group of 38 under
acidic conditions afforded 39 in an unoptimized 4% overall yield.
The phosphoramidate prodrug of compounds 37 and 39 was chosen
because of its beneficial effect on the anti HCV activity of 40-azidouri-
dine reported by McGuigan and co-workers.20

3268
M. Nilsson et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3265–3268
Table 2
details on the advancement of 16 into clinical phase I studies in our
ambition towards improved HCV therapies will be reported
elsewhere.
In vitro data: Stability in human plasma, human liver microsomes (HLM) and
permeability in Caco-2 cells
Compound Human plasma Clint
l/min/mg)
HLM Clint
min/mg)
(l
l/
Caco-2 Papp (cm/
(l
s ꢀ 10ꢁ6
)
Acknowledgment
14
16
19
25
29
32
36
< 3
17
36
> 40
17
< 3
4
< 6
> 300
ND
ND
ND
12
1.7
7.7
ND
ND
ND
1.6
ND
We thank Ann-Louise Hagbjörk and Anders Eneroth for assis-
tance with DMPK analysis.
References and notes
ND
1. Choo, Q.-L.; Kuo, G.; Weiner, A. J.; Overby, L. R.; Bradley, D. W.; Houghton, M.
Science 1989, 244, 359.
2. Szabó, E.; Lotz, G.; Páska, C.; Kiss, A.; Schaff, Z. Pathol. Oncol. Res. 2003, 9, 215.
3. Rustgi, V. K. J. Gastroenterol. 2007, 42, 513.
4. Neyts, J. Antiviral Res. 2006, 71, 363.
Table 3
Mean plasma levels (n = 2) together with pharmacokinetic parameters after a single
intravenous (5 mg base-equiv/kg) administration of the parent nucleoside 14 and oral
administration (20 mg base-equiv/kg) of 14, 16, 19, 25, 29, 32 and 36 in the fed male
Sprague–Dawley rats. PK parameters based on the plasma concentration of 14
5. Jacobson, I. M.; McHutchison, J. G.; Dusheiko, G.; Di Bisceglie, A. M.; Reddy, K.
R.; Bzowej, N. H.; Marcellin, P.; Muir, A. J.; Ferenci, P.; Flisiak, R.; George, J.;
Rizzetto, M.; Shouval, D.; Sola, R.; Terg, R. A.; Yoshida, E. M.; Adda, N.;
Bengtsson, L.; Sankoh, A. J.; Kieffer, T. L.; George, S.; Kauffman, R. S.; Zeuzem, S.
N. Engl. J. Med. 2011, 364, 2405.
6. Poordad, F.; McCone, J., Jr.; Bacon, B. R.; Bruno, S.; Manns, M. P.; Sulkowski, M.
S.; Jacobson, I. M.; Reddy, K. R.; Goodman, Z. D.; Boparai, N.; DiNubile, M. J.;
Sniukiene, V.; Brass, C. A.; Albrecht, J. K.; Bronowicki, J.-P. N. Engl. J. Med. 2011,
364, 1195.
7. Zeuzem, S.; Andreone, P.; Pol, S.; Lawitz, E.; Diago, M.; Roberts, S.; Focaccia, R.;
Younossi, Z.; Foster, G. R.; Horban, H.; Ferenci, P.; Nevens, F.; Müllhaupt, B.;
Pockros, P.; Terg, R.; Shouval, D.; van Hoek, B.; Weiland, O.; Van Heeswijk, R.;
De Meyer, S.; Luo, D.; Boogaerts, G.; Polo, R.; Picchio, G.; Beumont, M. N. Engl. J.
Med. 2011, 364, 2417.
8. Bacon, B. R.; Gordon, S. C.; Lawitz, E.; Marcellin, P.; Vierling, J. M.; Zeuzem, S.;
Poordad, F.; Goodman, Z. D.; Sings, H. L.; Boparai, N.; Burroughs, M.; Brass, C. A.;
Albrecht, J. K.; Esteban, R. N. Engl. J. Med. 2011, 364, 1207.
Compound
Dose ( mol/kg)
Administration
AUC0–t
AUC0–inf
Cmax
tmax
CL/F (L/h/kg)
F (%)
14
14
16
19
25
29
32
36
l
5
IV
20
PO
20
PO
20
PO
20
PO
20
PO
20
PO
20
PO
(
l
M ꢀ h)
M ꢀ h) 4.98 2.87 12.9 1.35 1.72 1.68 15.8 4.06
0.61 4.65 0.29 0.53 0.5 4.56 1.14
2.00
1.03 7.15 1.55 14.8 11.8 12.0 1.3
100 14 65 78
4.97 2.66 12.7 1.22 1.59 1.56 15.3 3.66
(l
(
lM)
—
—
1
1
1
1
1
1
4.93
20
7
8
8
9. Sarrazin, C.; Zeuzem, S. Gastroenterology 2010, 138, 447.
In Table 3 in vivo rat plasma levels and pharmacokinetic param-
eters of the parent nucleoside 14 and its ester prodrugs are pre-
sented. In addition to the in vivo data in Table 3, the stability of
14 in in vitro rat liver microsomes (RLM) was found to be high (Clint
10. McCown, M. F.; Rajyaguru, S.; Le Pogam, S.; Ali, S.; Jiang, W.-R.; Kang, H.;
Symons, J.; Cammack, N.; Najera, I. Antimicrob. Agents Chemother. 2008, 52,
1604.
11. Maag, H.; Rydzewski, R. M.; McRoberts, M. J.; Crawford-Ruth, D.; Verheyden, J.
P. H.; Prisbe, E. J. J. Med. Chem. 1992, 35, 1440.
12. 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.; LePogam, S.;
Leveque, V.; Ma, H.; Maile, G.; Merret, J. H.; Pichota, A.; Sarma, K.; Smith, M.;
Swallow, S.; Symons, J.; Vesey, D.; Najera, I.; Cammack, N. Bioorg. Med. Chem.
Lett. 2007, 17, 2570.
13. Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Pinho, P.; Maltseva, T.;
Morisson, V.; Leveque, V.; Rajyaguru, S.; LePogam, S.; Najera, I.; Benkestock, K.;
Zhou, X.-X.; Maag, H.; Cammack, N.; Martin, J. A.; Swallow, S.; Johansson, N. G.;
Klumpp, K.; Smith, M. J. Med. Chem. 2009, 52, 219.
14. Smith, D. B.; Kalayanov, G.; Sund, C.; Winqvist, A.; Maltseva, T.; Leveque, V. J.-
P.; Rajyaguru, S.; LePogam, S.; Najera, I.; Benkestock, K.; Zhou, X.-X.; Kaiser, A.
C.; Maag, H.; Cammack, N.; Martin, J. A.; Swallow, S.; Johansson, N. G.; Klumpp,
K.; Smith, M. J. Med. Chem. 2009, 52, 2971.
15. Lohmann, V.; Hoffman, S.; Herian, U.; Penin, F.; Bartenschlager, R. J. Virol. 2003,
77, 3007.
16. Rondla, R.; Coats, S. J.; McBrayer, T. R.; Grier, J.; Johns, M.; Tharnish, P. M.;
Whitaker, T.; Zhou, L.; Schinazi, R. F. Antivir. Chem. Chemother. 2009, 20, 99.
17. Li, N.-S.; Piccirilli, J. A. J. Org. Chem. 2003, 68, 6799.
18. Lemaire, S.; Houpis, I.; Wechselberger, R.; Langens, J.; Vermeulen, W. A. A.;
Smets, N.; Nettekoven, U.; Wang, Y.; Xiao, T.; Qu, H.; Liu, R.; Jonckers, T. H. M.;
Raboisson, P.; Vandyck, K.; Nilsson, K. M.; Farina, V. J. Org. Chem. 2011, 76, 297.
19. Derudas, M.; Brancale, A.; Naesens, L.; Neyts, J.; Balzarini, J.; McGuigan, C.
Bioorg. Med. Chem. 2010, 18, 2748.
<6 ll/min/mg). Based on IV administration, a clearance of 14, in
line with the in vitro data, was found (CL/F = 1.03 L/h/kg). Follow-
ing the results of the oral administration, the low to moderate oral
bioavailability of 14 (F = 14%) could be calculated. Prodrugs poten-
tially targeting peptidyl transporters, which previously have been
shown to have beneficial effect on oral bioavailability for 20-C-
Methylcytidine, such as compounds 19, 29 and 36 all gave lower
or similar oral bioavailability (F = 7%, 8% and 20% respectively)
compared with the parent nucleoside 14.21 For the isobutyryl ester
series, compound 25 gave similar oral bioavailability (F = 8%) to the
parent nucleoside. However, the isobutyryl esters 16 and 32 both
demonstrated greatly improved mean maximum plasma concen-
trations (Cmax = 4.65 and 4.56
lM respectively) and hence a larger
area under the curve (AUC0ꢁt = 12.7 and 15.3
lM/h respectively)
and oral bioavailability (F = 65% and 78% respectively).
In conclusion, the data reported here supports that the 40-azido-
20-deoxy-20-methylcytidine (14) is a potent (in the HCV replicon
assay) and non-cytotoxic agent (in Huh-7 and MT4 cells). A nucle-
otide prodrug of 14, such as compound 39 did not improve the
activity of the compound in the HCV replicon assay, indicating that
further exploration of nucleotide prodrugs of 14 may be futile. Fur-
thermore, it has been possible to improve the rat PK properties by
introducing prodrug esters, that is, compounds 16 and 32. Further
20. Perrone, P.; Luini, G. M.; Kelleher, M. R.; Daverio, F.; Angell, A.; Mulready, S.;
Congiatu, C.; Rajyaguru, S.; Martin, J. A.; Levêque, V.; Le Pogam, S.; Najera, I.;
Klumpp, K.; Smith, D. B.; McGuigan, C. J. Med. Chem. 1840, 2007, 50.
21. 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. J. Med. Chem. 2006, 49, 6614.