Synthesis and characterization of 2′-C-Me branched C-nucleosides as HCV polymerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.04.065

A series of 2′-C-methyl branched purine and pyrimidine C-nucleosides were prepared. Their anti-HCV activity and pharmacological properties were profiled, and compared with known 2′-C-Me N-nucleoside counterparts. In particular, 2′-C-Me 4-aza-7,9-dideazaad

Full text of DOI:10.1016/j.bmcl.2012.04.065

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4127–4132
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of 20-C-Me branched C-nucleosides as HCV
polymerase inhibitors
⇑
Aesop Cho , Lijun Zhang, Jie Xu, Darius Babusis, Thomas Butler, Rick Lee, Oliver L. Saunders, Ting Wang,
Jay Parrish, Jason Perry, Joy Y. Feng, Adrian S. Ray, Choung U. Kim
Gilead Sciences, 333 Lakeside Drive, Foster City, CA 94044, USA
a r t i c l e i n f o
a b s t r a c t
A series of 20-C-methyl branched purine and pyrimidine C-nucleosides were prepared. Their anti-HCV
activity and pharmacological properties were profiled, and compared with known 20-C-Me N-nucleoside
counterparts. In particular, 20-C-Me 4-aza-7,9-dideazaadenosine C-nucleoside (2) was found to have
potent and selective anti-HCV activity in vitro as well as a favorable pharmacokinetic profile and
in vivo potential for enhanced potency over the corresponding N-nucleoside.
Article history:
Received 19 March 2012
Revised 4 April 2012
Accepted 10 April 2012
Available online 20 April 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
C-Nucleoside
Antiviral
HCV
Polymerase inhibitor
Search for effective direct-acting antiviral agents (DAAs) for
treatment of HCV infection has been intense for the last decade.
A number of compounds have been advanced to the development,
and many have demonstrated antiviral activity clinically.1 Among
them, nucleoside polymerase inhibitors are promising in that they
offer pan-genotype activity and a high barrier to the selection of
viral resistance.2
branched purine and pyrimidine C-nucleosides for their anti-HCV
activity. Here, we report (1) preparation of a series of novel
20-C-Me C-nucleoside analogs, (2) their in vitro anti-HCV activity
compared with the N-nucleoside counterparts, and (3) relevant
pharmacological properties.
Adenosine analog 1 was prepared in a highly convergent way
(Scheme 1).8 An O-benzyl protected 20-C-Me ribonolactone 69 and
a fully elaborated bromo heterocycle 710 were prepared first and
then coupled together. Addition of ꢀ3 equiv of BuLi to a cold
suspension of 7 in THF in the presence of ‘stabase’ resulted in both
protection of the free amino group and exchange of lithium–
bromine, which was then reacted with the lactone 6, affording the
adduct 8. Compound 8 appears to exist in equilibrium with the
two anomers in solutions. Remarkably, subsequent reduction of 8
with triethylsilane and boron trifluoride etherate was highly
stereoselective, leading to formation of 9 as an exclusive product.
Final O-debenzylation either via hydrogenolysis or using BCl3
provided the desired nucleoside 1. Similarly, 20-C-Me 4-aza-7,9-
dideaza A (2) was prepared starting the bromo pyrrolotriazine
10.11 The stereochemistry at 10-C was assigned based on NOE
experiments.
Preparation of a guanosine C-nucleoside analog 3 started with
coupling of the 20-C-Me ribonolactone 6 and a bis(thiomethoxy)
bromo imidazolotriazine 13 (Scheme 2).12 The resulting 10-OH
intermediate 14 was reduced to a b-C-nucleoside derivative 15.
Similar to the previous observations for compounds 1 and 2, the
reduction was highly stereoselective. Next step was to properly
replace the two thiomethoxy groups in 15 with the amino group
at 2-C and the hydroxyl group at 6-C as in compound 3. Treatment
Several nucleoside polymerase inhibitors have been in develop-
ment. Compounds shown in Figure 1 are ones that are reported to
be under active clinical developments at the present time.2 Struc-
turally, all of these compounds contain a 20-C-Me branched sugar.
20-C-Me N-nucleosides, the syntheses of which was initially
described in the late 1960s3, were among the first to show
anti-HCV activity in vitro.4 These inhibitors are metabolized to
the active triphosphorylated nucleosides, which upon incorpora-
tion into RNA, inhibit further elongation catalyzed by the viral
RNA-dependent RNA polymerase, nonstructural protein 5B
(NS5B). The presence of the 20-C-Me group prevents an incoming
nucleoside triphosphate from binding to the active site of NS5B.5
Butora et al. reported HCV inhibitory activity of C-nucleosides
containing a 20-C-Me.6 Although only moderately active, the results
demonstrated that the C-nucleoside scaffold could be tolerated for
HCV inhibitory activity. Very recently, we also reported a series of
10-substituted 4-aza-7,9-dideazaadenosine C-nucleosides display-
ing antiviral activity against various RNA viruses including HCV.7
As continuing efforts in this area, we decided to evaluate 20-C-Me
⇑
Corresponding author. Tel.: +1 650 522 5177; fax: +1 650 522 5899.
E-mail address: acho@gilead.com (A. Cho).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.065

4128
A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
NH2
O
N
N
N
O
O
N
N
O
P
N
F
NH2
O
O
O
HN
O
O
O
O
O
OH
HO
O
Mericitabine
INX-189
O
O
N
N
N
O
O
NH
NH2
NH
O
O
P
O
P
O
N
F
HN
O
O
S
O
O
O
O
NH
HO
HO
OH
HO
GS-7977
IDX-184
Figure 1. Nucleoside polymerase inhibitors under active clinical developments.
rane afforded compound 19. The best way in our hands to construct
the pyrimidine ring from 19 was to treat it sequentially with Brede-
reck0s reagent and guanidine. The resulting 21 was found to be a
ꢀ1:1 mixture of the two anomers. After hydrogenolysis, the stereo-
isomeric mixture was separated by HPLC to obtain 4.
Uridine analog 5 was prepared by a modified method for prep-
aration of the 20-C-unsusbtituted peudouridine (Scheme 4).14 The
lactone 6 and the bromo pyrimidine 2215 were coupled, which
was reduced to the diol 23. Treatment of the diol with hydrochloric
acid resulted in dehydrative ring closure and removal of the two
t-butyl groups. The product 24 was a ꢀ1:1 mixture of the two ano-
mers. Final hydrogenolysis followed by HPLC separation of the two
isomers afforded 5.
The newly synthesized C-nucleosides, together with the
corresponding known N-nucleosides, were evaluated for their
anti-HCV activity (EC50) using a subgenomic GT1b replicon and
cytotoxicity (CC50) from Huh-7 cells (Table 1).7 In addition, enzy-
matic activity (IC50) of the nucleoside triphosphates was deter-
mined using GT1b NS5B recombinant protein.7 Consistent with
the literature reports, all the N-nucleosides displayed selective
NH2
N
X
NH2
N
N
N
BnO
O
O
X
BnO
O
(a)
OH
OBn
+
N
N
OBn
BnO
BnO
Br
6
7
, X = N
8
, X = N
10
, X = CH
11
, X = CH
(b)
NH2
N
NH2
N
X
N
X
N
N
N
(c)
BnO
O
HO
O
OBn
BnO
OH
HO
1
, X = N
9
, X = N
anti-HCV activity in a range of EC50 of 0.08–15 lM in our replicon
2
, X = CH
12
, X = CH
assay.2 The activity varied depending on the nucleobase of the
ribonucleosides, where the 7-deazaadenine base (i.e., MK-608)
afforded the most potent activity. In the case of C-nucleoside
analogs, the nucleobase was found to cause much greater impact
on antiviral activity. Only adenosine analogs 1 and 2 showed
inhibition of HCV. The EC50s for 1 and 2 were poor relative to the
corresponding N-nucleosides (up to EC50 >20-fold greater for
C-nucleosides). Importantly, however, the intrinsic enzyme activity
measured for the adenosine and guanosine C-nucleoside triphos-
phates were comparable to the corresponding N-nucleoside
triphosphates. Consistent with their lack of cellular activity, the
pyrimidine C-nucleosides 4 and 5 were found to inhibit NS5B only
very weakly as their triphosphates and further studies showed no
evidence of their functional incorporation into elongating RNA.
Encouraged by the potent enzyme activity, further pharmacologi-
cal properties for the purine C-nucleosides were profiled.
Scheme 1. Reagents and conditions: (a) 7 or 10, 1,1,4,4-tetramethyl-1,4-dichloro-
disilylethylene (‘stabase’, 1.2 equiv) and n-BuLi (3.3 equiv), THF, ꢁ78 °C followed by
addition of 6, 1 h, 56% for 8/60% for 11; (b) Et3SiH (4 equiv), BF3_OEt2 (2 equiv),
CH2Cl2, 0 °C to rt, 16 h, 35–85%; (c) H2, 10% Pd/C, acetic acid, rt, alternatively BCl3
(4–8 equiv), CH2Cl2, ꢁ78 °C, 1 h.
of 15 with 7 N ammonia in methanol at 45 °C gave the monosub-
stituted product 16 where the substitution occurred only at 6-C.
Attempt to achieve further substitution to the diamino product
17 by elevating the reaction temperature (up to 110 °C) was unsuc-
cessful. Ultimately, the resilient 2-thiomethoxy group was oxi-
dized to the more reactive sulfone using oxone. Subsequent
treatment with ammonia gas at 110 °C in a high-pressure steel
reactor afforded 17. After de-benzylation, a DAP analog 18 was
treated with adenosine deaminase to give 3.
Cytidine analog 4 was prepared in a manner similar to the meth-
od reported by Chu, et al (Scheme 3).13 Accordingly, treatment of
the lactone 18 with (ethoxycarbonylmethylene) triphenylphospho-
Strikingly, the guanosine analog 3 did not inhibit HCV
replication despite the potent enzyme activity observed for its
triphosphate metabolite. This lack of replicon activity could be

A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
4129
S
N
N
N
S
N
S
BnO
O
(a)
O
N
BnO
O
N
OH
+
N
S
N
OBn
BnO
OBn
14
BnO
Br
6
13
(b)
N
S
N
NH2
N
N
N
N
N
S
S
N
(c)
BnO
O
BnO
O
OBn
15
OBn
16
BnO
BnO
(d)
O
N
NH2
N
N
N
NH
N
N
NH2
(f)
NH2
N
HO
O
RO
O
OH
HO
OR
RO
(e)
3
17
(R = Bn)
(R = H)
18
Scheme 2. Reagents and conditions: (a) 13, n-BuLi, (1.0 equiv), THF, ꢁ78 °C followed by addition of 6, 1 h, 85%; (b) Et3SiH (4 equiv), BF3–OEt2 (2 equiv), CH2Cl2, 0 °C to rt, 16 h,
86%; (c) 7 N ammonia in methanol, 45 °C, 24 h; (d) oxone, acetone–water, 0 °C to rt; then liquid ammonia (5 mL/g), 110 °C, 40 h, 81% in three steps; (e) BCl3 (4–8 equiv),
CH2Cl2, ꢁ78 °C, 1 h, 69%; (e) adenosine deaminase from bovine spleen, water, 37 °C, 5 h, 100%.
N
O
O
O
OEt
OEt
BnO
O
BnO
O
BnO
O
OH
OBn
(a)
(b)
N
N
O
OH
OBn
O
OBn
BnO
20
BnO
19
BnO
BnO
BnO
O
O
(a)
(b)
N
OH
N
18
+
OBn
O
BnO
BnO
OBn
Br
6
23
22
(c)
NH2
(c)
O
NH2
HN
HN
N
N
O
HN
O
HN
(d)
HO
O
BnO
O
NH
O
O
NH
O
O
(d)
BnO
HO
O
OH
OBn
HO
BnO
4
21
OBn
OH
BnO
HO
5
24
Scheme 3. Reagents and conditions: (a) (ethoxycarbonylmethylene)tripheylphos-
phorane (1.5–2.5 equiv), MeCN, microwave 180 °C, 1 h, 55–65%; (b) t-butoxy
bis(dimethylamino)methane (1.5 equiv), toluene, 120 °C, 6 h; (c) guanidine
(10 equiv freshly prepared by treatment of sodium methoxide in methanol),
methanol, rt, 24 h, 30% in two steps; (d) H2, 10% Pd/C, 54%.
Scheme 4. Reagents and conditions: (a) 22, n-BuLi, (1.0 equiv), THF, ꢁ78 °C
followed by addition of 6, 1 h; (b) NaBH4 (4 equiv), methanol, 0 °C, 1 h; (c) c-HCl
and ethanol (1/10, v/v), rt, 2 h, 43% in three steps; (d) H2, 10% Pd/C, 30%.
due to the inability of 3 to be converted to its biologically active tri-
phosphate derivative by cellular kinases. In fact, it is well estab-
lished that the first phosphorylation of 20-C-Me G N-nucleoside
to its monophosphate derivative is inefficient in the phosphoryla-
tion cascade,5 even though it still shows an appreciable level of
activity (EC50 of 3.25 lM). Thus, monophosphate prodrug strate-

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A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
Table 1
Antiviral activity of the 20-C-Me branched nucleosides
Base
A
Nucleoside
GT1b EC50
(
lM)
Huh-7 CC50
(lM)
GT1b NS5B TP IC50 (lM)
20-C-Me A
0.35
1.28
0.08
1.98
3.25
>89
4.07
>89
15.2
>89
66
6.83
2.10
0.30
0.31
0.25
0.19
0.73
13.4a
1.32
13.4a
20-C-Me 4-aza-9-deaza A (1)
20-C-Me 7-deaza A
20-C-Me 4-aza-7,9-dideaza A (2)
20-C-Me G
>89
>89
85
>89
>89
>89
>89
>89
>89
7-deaza A
G
C
20-C-Me 4-aza-9-deaza G (3)
20-C-Me C
20-C-Me iso-
20-C-Me U
wC (4)
U
20-C-Me
w
U (5)
a
No incorporation observed for triphosphates by NS5B.
gies have been devised to by-pass the first phosphorylation step.
IDX-184 and INX-189 are such examples, where much improved
Table 2
Degradation of adenosine analogs in the presence of bovine adenosine deaminase
antiviral activity was achieved (EC50 of <0.5 lM) as a result of effi-
Nucleoside
(%) Remaininga
cient formation of the triphosphate metabolite.16,17 In order to see
if 3 can be rescued by the monophosphate prodrug strategy, an S-
acetylthioethanol (SATE) monophosphate prodrug (26) was pre-
pared and evaluated (Fig. 2). This C-nucleoside prodrug showed
20-C-Me A
94%b
55%
20-C-Me 4-aza-9-deaza A (1)
20-C-Me 7-deaza A
98%c
100%
20-C-Me 4-aza-7,9-dieaza A (2)
significant increase in activity (EC50 of 9.54
However, the effect was less pronounced compared to the corre-
sponding N-nucleoside prodrug (25, EC50 of 0.05 from
3.25 M). To understand these results, intracellular levels of the
lM from >89 lM).
a
Results are the mean of 2 independent experiments done in duplicate. Percent
nucleoside remaining measured after a 60 min incubation.21
lM
b
Formation of 20-C-Me inosine correlating to the decrease in 20-C-Me A was
l
detected.
c
No formation 20-C-Me 7-deaza inosine detected corresponding to slight loss of
phosphorylated metabolites from 25 and 26 were measured. Incu-
bation of 26 in replicon cells resulted in accumulation of high lev-
els of the monophosphate metabolite over time, with only minor
20-C-Me 7-deaza A.
A
O
O
N
N
NH
NH2
S
O
P
N
O
O
O
O
O
OH
HO
S
25
B
O
O
N
NH
NH2
S
O
P
N
N
O
O
O
O
O
OH
HO
S
26
Figure 2. Intracellular metabolism of bis(SATE) prodrugs of 20-C-Me G N-Nuc (25; A) and C-Nuc (26; B) during continuous 24 h incubations with Huh-7 replicon cells. Results
are the average of duplicate wells from a single side-by-side experiment.

A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
4131
Figure 3. Comparison of intracellular tirphosphate levels formed following in vitro
incubation of 20-C-Me 7-deaza A and C-nucleoside 2 with Huh-7 replicon cells (A)
and primary human hepatocytes (B). Results are the mean standard deviation
from 2 independent experiments done in duplicate. Results in panel B are in
primary human hepatocytes from two separate donors.
Figure 4. Comparison of hepatic triphosphate levels formed 20-C-Me 7-deaza A and
C-nucleoside 2 following oral administration to hamsters (A) and rats (B). Orally
administered doses in rats were 2 mg/kg and 5 mg/kg and in hamsters were 20 mg/
A and 2, respectively. Results are the
mean standard deviation from intracellular triphosphate measurements made
from the livers from 3 animals at each time point.
kg and 10 mg/kg for 20-C-Me 7-deaza
levels of its di-and triphosphate metabolites. This suggests that 26
was efficiently metabolized to the monophosphate, but further
phosphorylation leading to the triphosphate was not efficient. In
contrast to this, high levels of the triphosphate were observed
when cells were incubated with the N-nucleoside prodrug 25.
An adenosine N-nucleoside, 20-C-Me A, was reported to be
susceptible to degradation by adenosine deaminase (ADA) and
purine nucleoside phosphorylase (PNP), resulting in poor pharma-
cokinetic properties (e.g., plasma clearance >200 mL/min/kg, oral
bioavailability 0% in rats).18 Our C-nucleosides 1 and 2 should
not be susceptible to PNP due to the incorporation of the car-
bon–carbon glycosidic bond. Accordingly, their instability to ADA
was examined only relative to the corresponding N-nucleoside
analogs. As summarized in Table 2, compound 1 was found to be
degraded by ADA faster than 20-C-Me A suggesting that it will have
a poor pharmacokinetic profile and, for that reason, 1 was excluded
from further characterization in vivo. In contrast to the analogs
containing nitrogen at the 7 position of the respective heterocyclic
base, both 20-C-Me 7-deaza A and the C-nucleoside analog 2 were
observed to be stable to ADA degradation. 20-C-Me 7-deaza A has
been reported to have a favorable pharmacokinetic profile and
potent anti-HCV activity in chimpanzees.19,20 Therefore, we
decided to further characterize 2 for its potential as an orally
administered therapeutic agent for chronic HCV infections.
liver blood flow (0.4 L/hr/kg), a volume of distribution in large ex-
cess of extracellular water (1.66 L/kg) and a resulting long terminal
elimination half life in plasma in excess of 7 h. Rapid absorption
(time to maximum concentration of approximately 30 min) and
high oral bioavailability (70%) were observed following oral
administration at 1 mg/kg. The plasma pharmacokinetic profile
for 2 was similar to that reported previously for 20-C-Me 7-deaza
A (MK-608).19
To cause antiviral activity, nucleosides must first permeate the
target cells and be phosphorylated to their active triphosphate
forms. The phosphorylation of 2 was compared to that of its N-
nucleoside counterpart in Huh-7 replicon cells and primary human
hepatocytes.23,24 Compound 2 was not phosphorylated to its tri-
phosphate form as efficiently in replicon cells as 20-C-Me 7-deaza
A (Fig. 3A), which is consistent with the relative potency observed
in the replicon assays. In contrast to this, 2 was more efficiently
phosphorylated in primary human hepatocytes (Fig. 3B). This im-
proved phosphorylation in primary human hepatocytes suggest
the potential for enhanced activation of C-nucleoside 2 over 20-C-
Me 7-deaza A in the liver where HCV replication takes place.
To gain a better understanding of the combined effect of oral
absorption and intracellular activation described above on liver
loading with the active triphosphate metabolite, the liver pharma-
cokinetic profiles of the triphosphate formed by 2 was compared to
that of its N-nucleoside counterpart in rodents (hamsters and
As an initial step in characterizing the pharmacokinetic profile
of 2, the pharmacokinetic profile following intravenous and oral
administration to dogs was assessed.22 Intravenous administration
of 2 at 0.5 mg/kg resulted in plasma clearance markedly below
rats).25
triphosphate levels were 2- and 5-fold higher for 2 than for its
Following
oral
administration,
dose-normalized

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A. Cho et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4127–4132
19. Olsen, D. B.; Eldrup, A. B.; Bartholomew, L.; Bhat, B.; Bosserman, M. R.; Ceccacci,
A.; Colwell, L. F.; Fay, J. F.; Flores, O. A.; Getty, K. L.; Grobler, J. A.; LaFemina, R.
L.; Markel, E. J.; Migliaccio, G.; Prhavc, M.; Stahlhut, M. W.; Tomassini, J. E.;
MacCoss, M.; Hazuda, D. J.; Carroll, S. S. Antimicrob. Agents Chemother. 2004, 48,
3944.
20. Carroll, S. S.; Ludmerer, S.; Handt, L.; Koeplinger, K.; Zhnag, N. R.; Graham, D.;
Davies, M.-E.; MacCoss, M.; Hazuda, D.; Olsen, D. B. Antimicrob. Agents
Chemother. 2009, 53, 926.
corresponding N-nucleoside (Fig. 4). Consistent with the high oral
bioavailability observed in dogs for 2, high nucleoside levels were
observed in hamster plasma and, relative to a separate intravenous
study, the oral bioavailability in rats was 50% (data not shown).
In summary, characterization of a series of 20-C-Me C-nucleo-
sides has identified nucleoside analogs with potent anti-HCV activ-
ity. While the pyrimidine C-nucleosides (4 and 5) lacked replicon
activity due to poor enzymatic potency and, despite excellent
activity as its triphosphate, the guanosine C-nucleoside (3) and
its nucleotide prodrug (26) were inefficiently metabolized, adeno-
sine C-nucleosides (1 and 2) were found to have selective replicon
activity and potent inhibition of NS5B as their triphosphates. Fur-
thermore, 2 was found to have a favorable pharmacokinetic profile
and in vivo potential for enhanced potency over the corresponding
N-nucleoside 20-C-Me 7-deaza A. These results have prompted the
subsequent further characterization of the toxicological profile of
2, which will be published in due course. C-Nucleosides may hold
promise as potential future therapies for HCV infection.
21. Nucleosides (10 lM) were incubated at 37 °C with 0.0125 U/mL bovine
adenosine deaminase (Sigma A5168) in 0.01 M potassium phosphate buffer
pH 6.0 and samples collected over a 1 hour time course. The reactions were
stopped by adding 2.5 volumes of 0.2% formic acid in ice cold methanol.
Nucleoside levels were then measured using liquid chromatography (0.2%
formic acid in water and an acetonitrile gradient) coupled to triple quadrapole
mass spectrometry running in positive ion multiple reaction monitoring mode.
22. Groups of 3 non-naïve male beagle dogs were administered a single dose of 2
formulated as
a
solution in 5/5/5/85 ethanol/polypropylene glycol/
polyethylene glycol/aqueous 50 mM citrate buffer (pH 3.3) either by
intravenous infusion at 0.5 mg/kg or orally at 1 mg/kg. Blood samples were
collected over a 24 h period postdose into VacutainerTM tubes containing
EDTA-K3 (BD Biosciences) and plasma was isolated by the manufacturers
suggested protocol. Plasma samples were processed via protein precipitation
by adding acetonitrile to a final concentration of 60%. Following filtration to
remove precipitated proteins, samples were dried and reconstituted in 20%
acetonitrile in water. Samples were then analyzed by reversed phase liquid
chromatography coupled to triple quadrapole mass spectrometry (LC/MS/MS)
in positive ion and multiple reaction monitoring mode.
References and notes
23. Replicon cells were maintained in Dulbecco0s modified eagle medium
containing glutamax supplemented with 10% heat inactivated fetal bovine
serum, penicillin–streptomycin, and G418 disulphate salt solution. Cells were
transferred to 12-well tissue culture treated plates by trypsonization and
grown to confluency (0.88 ꢂ 106 cells/well). Cells were treated for 24 h with a
1. (a) Vachon, M.-L.; Dieterich, D. T. Semin. Liver Dis. 2011, 31, 399; (b)
Kronenberger, B.; Zeuzem, S. J. Viral Hepatitis 2012, 19, 48.
2. Sofia, M. J. Antiviral Chem. Chemother. 2011, 22, 23.
3. (a) Jenkins, S. R.; Arison, B.; Walton, E. J. Org. Chem. 1968, 33, 2490; (b) Walton,
E.; Jenkins, S. R.; Nutt, R. F.; Holly, F. W. J. Med. Chem. 1969, 12, 306.
4. Carroll, S. S.; Tomassini, J. E.; Bosserman, M.; Getty, K.; Stahlhut, M. W.; Eldrup,
A. B.; Bhat, B.; Hall, D.; Simcoe, A. L.; LaFemina, R.; Rutkowski, C. A.; Wolanski,
B.; Yang, Z.; Migliaccio, G.; Francesco, R. D.; Kuo, L. C.; MacCoss, M.; Olsen, D. B.
J. Biol. Chem. 2003, 278, 11979.
5. Migliaccio, G.; Tomassini, J. E.; Carroll, S. S.; Tomei, L.; Altamura, S.; Bhat, B.;
Bartholomew, L.; Bosserman, M. R.; Ceccacci, A.; Colwell, L. F.; Cortese, R.;
Francesco, R. D.; Eldrup, A. B.; Getty, K. L.; Hou, Z. S.; LaFemina, R. L.; Ludmerer,
S. W.; MacCoss, M.; McMasters, D. R.; Stahlhut, M. W.; Olsen, D. B.; Hazuda, D.
J.; Flores, O. A. J. Biol. Chem. 2003, 278, 49164.
test compound (10 lM). Following 24 hours, cells were washed twice with
2.0 mL ice cold 0.9% sodium chloride saline. Cells were then scraped into
0.5 mL 70% methanol and frozen overnight to facilitate the extraction of
nucleotide metabolites. Extracted cell material in 70% methanol was
transferred into tubes and dried. After drying, samples were re-suspended in
1 mM ammonium phosphate pH 8.5. TP levels were quantified using liquid
chromatography coupled to triple quadrapole mass spectrometry by methods
similar to those previously reported in Durand-Gasselin, L.; Van Rompay, K.K.;
Vela, J.E.; Henne, I.N.; Lee, W.A.; Rhodes, G.R. Mol. Pharm. 2009, 6, 1145.
24. Primary human hepatocytes were maintained in Williams
containing Cellz Direct0s proprietary supplement cocktail. Cells were
purchased from Cellz Direct as twelve well plate format grown to
confluency (0.88 ꢂ 106 cells/well). Cells were treated for 1 h with test
E medium
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a
a
compound (10 M) followed by a 23 h treatment with non-drug containing
media. Cells were collected at 0, 0.5, and 1 h during compound incubation and
at 0, 0.5, 1, 3, 6, 8 and 23 h following compound removal. Cells were collected
and intracellular metabolites were analyzed as described for replicon cells.
25. Groups of 12 Golden Syrian hamsters or Sprague–Dawley rats were
administered nucleosides formulated in water at respective doses. At 1, 4, 8
and 12 h, livers were harvested under isoflurane anesthesia. Collected livers
were wrapped in aluminum foil and snap-frozen in liquid nitrogen
immediately following removal to avoid sample dephosphorylation. Livers
were processed by sectioning into smaller pieces with a razor blade and
collecting into pre-weighed 15 mL conical tubes kept on dry ice. Four volumes
of ice-cold extraction buffer (0.1% KOH and 67 mM EDTA in 70% MeOH,
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containing 0.25 lM Cl-ATP) were added and samples were promptly
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homogenized using an Omni-Tip THTM with disposable, hard tissue
homogenizer probes (Omni International). Aliquots of homogenate were then
centrifuged (20,000ꢂg at 4 °C for 10 min). Aliquots of supernatant were
transferred to clean tubes, evaporated to dryness in a heated centrifugal
evaporator, and reconstituted with an equal volume of 1 mM ammonium
phosphate (pH 7.0). Intracellular triphosphate levels were measured by LC/MS/
MS as described for samples generated in vitro. Levels of endogenous
adenosine triphosphate were also determined to assure the sample stability.
Nucleoside levels in rat plasma were determined by the same methods
described for dogs.
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