2′-Deoxy-2′-α-fluoro-2′-β-C-methyl 3′,5′-cyclic phosphate nucleotide prodrug analogs as inhibitors of HCV NS5B polymerase: Discovery of PSI-352938

Article abstract of DOI:10.1016/j.bmcl.2010.10.035

A series of novel 2′-deoxy-2′-α-fluoro-2′-β-C- methyl 3′,5′-cyclic phosphate nucleotide prodrug analogs were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demonstrated a 10-100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to higher cellular triphosphate levels. Our structure-activity relationship (SAR) studies provided compounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).

Full text of DOI:10.1016/j.bmcl.2010.10.035

Bioorganic & Medicinal Chemistry Letters 20 (2010) 7376–7380
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
20-Deoxy-20-
a
-fluoro-20-b-C-methyl 30,50-cyclic phosphate nucleotide prodrug
analogs as inhibitors of HCV NS5B polymerase: Discovery of PSI-352938
⇑
P. Ganapati Reddy , Donghui Bao, Wonsuk Chang, Byoung-Kwon Chun, Jinfa Du, Dhanapalan Nagarathnam,
Suguna Rachakonda, Bruce S. Ross, Hai-Ren Zhang, Shalini Bansal, Christine L. Espiritu, Meg Keilman,
Angela M. Lam, Congrong Niu, Holly Micolochick Steuer, Phillip A. Furman, Michael J. Otto, Michael J. Sofia
Pharmasset, Inc., 303A College Road East, Princeton, NJ 08540, USA
a r t i c l e i n f o
a b s t r a c t
A series of novel 20-deoxy-20- -fluoro-20-b-C-methyl 30,50-cyclic phosphate nucleotide prodrug analogs
a
Article history:
Received 9 September 2010
Revised 6 October 2010
Accepted 8 October 2010
Available online 15 October 2010
were synthesized and evaluated for their in vitro anti-HCV activity and safety. These prodrugs demon-
strated a 10–100-fold greater potency than the parent nucleoside in a cell-based replicon assay due to
higher cellular triphosphate levels. Our structure–activity relationship (SAR) studies provided com-
pounds that gave high levels of active triphosphate in rat liver when administered orally to rats. These
studies ultimately led to the selection of the clinical development candidate 24a (PSI-352938).
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Antiviral
HCV
Nucleoside
Prodrug
Cyclic Phosphate
An estimated 180 million people worldwide are infected with
the hepatitis C virus (HCV).1 The current standard of care (SOC)
characteristics, we investigated purine derivatives of the 20-
a-F-
20-b-C-methyl class of nucleosides.4d The guanosine analog 5
for HCV infection, the combination of pegylated interferon-
a
and
(Fig. 2) was shown to be weakly active in the HCV replicon assay
ribavirin, provides less than 50% response rates among patients
infected with the most prevalent genotype 1 virus.2 There is an
urgent medical need for more effective and well tolerated anti-
HCV agents to treat HCV infections across all genotypes.3 We have
(EC90 = 69
l
M), but its triphosphate 6 was found to be a potent
M). These re-
inhibitor of the HCV NS5B polymerase (IC50 = 5.94
l
sults indicated that weak potency of the parent nucleoside 5 could
be a result of poor phosphorylation at some stage of the nucleoside
phosphorylation cascade. Nucleoside analogs need to be phosphor-
ylated to their corresponding active triphosphate by host cellular
kinases before they can bind to the HCV NS5B polymerase
shown that nucleoside/tide analogs with 20- -F-20-b-C-methyl sub-
a
stitution represent an important class of HCV NS5B polymerase
inhibitors with broad genotype coverage.4 Although several nucle-
oside classes that include NM-2835 (1), RG7128 (3), and R16266 (4)
have been in development as inhibitors of HCV, only RG7128 is still
in development and is currently in Phase 2b clinical trials (Fig. 1).
RG7128 is a 30,50-diisobutyrate ester prodrug of the nucleoside
PSI-6130 (2).4a–c,7 In a 4-week combination study with SOC,
RG7128 demonstrated efficacy in genotype 1, 2, and 3 patients,8
the first direct-acting antiviral to show pan-genotype coverage in
the clinic.9
Since RG7128 has demonstrated positive clinical attributes, we
were interested in identifying second generation agents with im-
proved potency, better resistance profile, enhanced pharmacoki-
netic properties to support QD dosing and with the potential for
generating high concentrations of the active triphosphate in the li-
ver. In our search for second generation nucleosides with unique
O
O
N
N
NH2
NH2
O
O
N
N
HO
O
HO
O
O
O
HO
OH
(NM283)
F
1
2
(PSI-6130)
H2N
O
O
N
N
NH2
O
NH2
O
O
N
O
O
N
N3
O
O
O
O
F
O
3
(RG7128)
4
(RG1626)
⇑
Corresponding author. Tel.: +1 609 613 4151; fax: +1 609 613 4150.
E-mail address: pgreddy@pharmasset.com (P.G. Reddy).
Figure 1. Nucleoside inhibitors of HCV.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.10.035

P. G. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7376–7380
7377
7
N
N
8
N
N
O
N
O
OH
N
9
OBn
a
5'
5
O
O
O
4'
3'
O
O
6
N
N
O
O
P
O
O
P
O
1'
P
O
N
4
HO
O
N
N
P
MeO
N
3
NH
O
NH
NH2
O
MeO
N
OH
N
F
O
2'
P
1
F
O
HO
HO
OH
F
5
F
2
NH2
HO
NH2
OH
15
14
NH2
6
EC90 (replicon): 69µM
CC50: >100µM
IC50 (NS5B polymerase): 5.94µM
Scheme 2. Preparation of guanosine methyl cyclic phosphate 15. Reagents and
conditions: (a) 10% Pd/C, MeOH, H2 (balloon), rt, 30 min, 98%.
Figure 2. 20-Deoxy-20-
a
-F-20-b-C-methyl guanosine triphosphate 6 as an inhibitor
of HCV NS5B polymerase.
and inhibit HCV RNA synthesis. Therefore, to produce the active tri-
phosphate a nucleoside analog must be a substrate for each of the
kinases in the phosphorylation cascade where the first phosphory-
lation step is the most discriminating.10 Neither the active triphos-
phate nor the mono- or diphosphate species can be considered as
possible drug candidates because of their instability and poor cel-
lular permeability. Nucleoside cyclic phosphate prodrugs have
been shown in vitro to be precursors of the nucleoside monophos-
phate with improved cellular permeability.11 We therefore decided
N
b
O
a
O
Cl
OH
N
Cl
N
BzO
BzO
N
N
N
BzO
BzO
F
N
F
9
7
NH2
N
H
NH2
8
N
N
R1
R1
O
O
N
OR2
P
N
to investigate cyclic phosphate prodrugs of the 20-deoxy-20-
b-C-methyl nucleoside derivatives. When 30,50-cyclic phosphate
derivatives of 20-deoxy-20- -F-20-b-C-methyl uridine and cytidine
a
-F-20-
c
O
HO
N
N
+ (iPr)2N
N(iPr)2
N
N
P
O
HO
d
F
F
R2O
a
10
11
12
NH2
NH2
nucleosides were prepared, the resulting prodrugs did provide
compounds that were 10–100-fold more potent than the parent
nucleoside, but they did not offer any potency advantage over
the corresponding phosphoramidate prodrugs already in clinical
development.12 However, unlike the uridine and cytidine analogs,
cis/ trans mixture
N
N
R1
R1
O
O
N
N
O
P
O
O
+
N
N
R2O
O P
O
N
N
O
F
F
OR2
NH2
NH2
20-deoxy-20- -F-20-b-C-methyl purine cyclic phosphate analogs
a
13b
(trans)
13a
(cis)
provided both the desired potency and resistance profiles in our
initial cell-based replicon assay. Here, we report the synthesis,
SAR, and preclinical in vitro and in vivo rat PK data supporting
Scheme 1. Synthesis of cyclic phosphate prodrugs. Reagents and conditions:
(a) DEAD, PPh3, THF, rt, 16 h; (b) NaH, ROH (6-alkoxy derivatives) or NHR0R00
(6-alkylamino derivatives); (c) 0.5 M tetrazole in CH3CN or 4,5-dicyanoimidazole,
CH3CN, rt, 3 h; (d) 77% mCPBA, DCM, rt, 30 min or 1 M tBuOOH in DCM, CH3CN, rt,
20 h.
the nomination of a 20-deoxy-20- -F-20-b-C-methyl purine nucleo-
a
side cyclic phosphate prodrug to preclinical development status.
Figure 3. ORTEP drawing of 24a showing Rp (cis) configuration at phosphorous.

7378
P. G. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7376–7380
Our initial SAR was focused on modifications around the cyclic
phosphate ester and 6-position of 20-deoxy-20- -F-20-b-C-methyl
n-propylamino substituents of the isopropyl cyclic phosphate (28
and 31) offered no advantage over the 6-ethoxy derivates 24a
and 24b.
a
guanosine analog. The desired cyclic phosphate prodrugs of this
class were readily prepared by the method shown in Scheme 1.12
Although several methods for preparing cyclic phosphates have
been reported in the literature,11,13,14 in our case a two step proto-
col via cyclic phosphite intermediate followed by oxidation gave
improved results. The nucleoside starting material 10 was pre-
pared by Mitsunobu coupling of lactol 77 with 6-chloropurine 8
followed by nucleophilic displacement of chlorine with required
alkoxide or amine. Cyclization of the nucleoside with tetraisopro-
pyl phoshorodiamidite 11 using either tetrazole or 4,5-dicyanoim-
idazole (DCI)15 as activators gave cyclic phosphite intermediate 12
as a mixture of cis/trans isomers. The trans isomer of the cyclic
phosphite 12 can be converted to the thermodynamically more
stable cis isomer by heating the reaction mixture at 50 °C for sev-
eral hours.13 However, since there was an interest in obtaining
both isomers the reaction was performed at room temperature.
Subsequent oxidation of the cyclic phosphite mixture with mCPBA
or tBuOOH gave corresponding cyclic phosphates (13a and 13b) in
moderate yield as a mixture of diastereomers. Wherever possible,
the two individual diastereomers were isolated by column chro-
matography and evaluated for their anti-HCV activity. Improved
yields were observed when DCI was used as an activator in the first
step. The tetraisopropyl phosphorodiamidite reagent 11 was pre-
In addition to the activity against HCV, the key compounds were
further evaluated for stability and subjected to extensive cytotox-
icity evaluation. In order to assess the ability of the cyclic phos-
phate prodrugs to survive exposure in the gastrointestinal tract
and preferentially release the nucleotide monophosphate in the
liver, compound stability was determined in simulated gastroin-
testinal fluid (SGF), simulated intestinal fluid (SIF), human plasma
and human liver S9 fraction, a surrogate in vitro model for liver sta-
bility. Tables 2 and 3 list stability and expanded cytotoxicity data,
respectively, for the key compounds (16, 19, 22, 24a, and 24b) that
had passed the initial activity and cytotoxicity filter. Of these, com-
pound 22 had poor stability in the liver S9 fraction. The stability
half life (t1/2) for this compound in human S9 was 2.53 h while
the t1/2 values for the other compounds were >24 h. In addition,
compound 22 was shown to exhibit a cytostatic effect. Compound
19 had poor stability in the SIF (t1/2 = 4 h) and showed cytotoxicity
in the Huh 7 cell line. In addition, this compound was associated
with mitochondrial toxicity (CC50 = 14 lM). Hence neither com-
pound was considered for further development. On the other hand,
compound 16 demonstrated acceptable stability profile and no sig-
nificant cytotoxicity in all four cell lines. Compounds 24a and 24b
were shown to be devoid of any cytotoxicity in the four cell lines
tested and possessed prolonged stability in SGF, SIF, human plasma
pared following
a
literature procedure.16 Treatment of the
bis(diisopropylamino)chlorophosphine with the corresponding
alcohol in the presence of Et3N gave the required tetraisopropyl
phoshorodiamidite 11. The guanosine methyl cyclic phosphate 15
was prepared by the debenzylation of the corresponding 6-benzyl-
oxy methyl cyclic phosphate 14 as shown in Scheme 2.
Table 1
SAR data of cyclic phosphate prodrugs of 20-deoxy-20-
analogs
a
-fluoro-20-b-C-methyl purine
The structure and stereochemistry of one of the cyclic phos-
phates (24a) was determined by single crystal X-ray analysis. An
ORTEP drawing of compound 24a is shown in Figure 3 indicating
the Rp (cis) configuration at phosphorous center. In the 31P NMR
spectra, the 31P resonance for the cis isomer 24a appears upfield
from the trans isomer 24b. This observation was applied by anal-
ogy to the other diastereomeric compounds to assign the
stereochemistry.
N
R1
O
N
O
P
OR2
N
N
O
O
F
NH2
Compd Isomer/ratioa (cis/
trans)
R1
R2
HCV
EC90
M)
Cytotoxicityb
CC50 (lM)
(
l
The anti-HCV activities of these prodrugs were assessed using
the clone A replicon and a quantitative real time PCR assay.17 Cyto-
toxicity (CC50) was simultaneously evaluated by assessing cellular
RNA replication. Representative examples assessed to identify a
subset of lead compounds are listed in Table 1. Our initial SAR
had focused on the 6-position of the base keeping the methyl cyclic
phosphate constant (Table 1, 15–22). Unsubstituted guanosine
methyl cyclic phosphate 15 did not provide the desired submi-
cromolar potency presumably due to its poor cellular penetration.
In order to address this, we chose to increase the lipophilicity by
using simple 6-alkoxy or alkyamine substituents (double prodrug).
As desired, simple alkoxy groups (16 and 17) provided submicrom-
olar potency with no substantial cytotoxicity. Similarly, small
alkylamine and azetidine substituents (19 and 22) were well toler-
ated. However, benzyloxy and cycloalkyl amines (18, 20, and 21)
did not give the desired potency and were not used in subsequent
SAR studies.
The initial SAR investigation around the 6-position of the base
while maintaining the methyl cyclic phosphate moiety constant
demonstrated that small alkoxy, alkyamine and azeditidine were
preferred substituents. Subsequently, SAR at the phosphate ester
was performed using these three substituents at the 6-position of
the base. Linear alkyl, cycloalkyl and phenyl groups with all three
preferred substituents at the 6-postion gave derivatives that were
weak inhibitors of HCV. Both cis and trans isomers of 6-ethoxy
isopropyl ester derivatives (24a and 24b) afforded the desired
potency with no significant cytotoxicity. The 6-azetidinyl and
3
—
cis
—
—
3.9
>100
>65
>30
>30
—
15
16
17
18
19
20
–OH
–OEt
Me
Me
Me
Me
Me
Me
11.75
0.41
0.83
5.05
0.92
1.56
1:1.5
1:1.3
1:1.6
1:1.3
1:1.4
–OPrn
–OBn
–NHPrn
–NH(c-
butyl)
–NH(c-
pentyl)
>30
—
21
22
1:1.3
1:1.1
Me
7.20
—
Me
0.98
>30
N
23
cis
cis
–OEt
–OEt
–OEt
–OEt
nPr
iPr
iPr
Ph
nPr
nPr
Et
13.27
1.37
0.69
1.72
23.83
5.27
48.14
9.28
4.76
—
24a
24b
25
26a
26b
27
>100
>100
—
—
>11
—
trans
2:1
cis
trans
cis
–OPrn
–OPrn
–NHPrn
–NHPrn
–NHPrn
28
29
cis
cis
iPr
Ph
—
—
30
31
32
33
cis
cis
cis
cis
Et
69.19
18.6
7.30
7.70
—
—
—
—
N
N
N
N
iPr
c-
Butyl
Ph
Based on 31P NMR chemical shifts.
CC50 was determined for the compounds with desired potency.
a
b

P. G. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7376–7380
7379
Table 2
Table 4
Stability data for selected purine cyclic phosphate analogs
In vitro/in vivo triphosphate (6) levels of the key compounds in a rat PK study
Compd
Stability t1/2 (h)
Plasma
Compd
Cellular triphosphate (6)
Rat liver triphosphate
(6) levels
levels (mM)
SIF
SGF
S9
Clone A
Human hepatocytes
Cmax
(ng/g)
AUC (0–24 h,
ng h/g)
16
19
22
24a
24b
>20
4
>20
>20
20
>20
>20
>20
>20
18
8.88
15.70
8.33
>24
>24
>24
2.53
>24
>24
16
24a
0.08 (24)
0.41 (72)
0.12 (24)
0.59 (72)
613
4860
4092
41893
>24
analyzing by HPLC. Surprisingly, for compound 24a triphosphate
levels in clone A cells and primary human hepatocytes were shown
to be much higher than that generated by 16, the more potent
compound in the replicon assay. Similar results were seen in vivo
in the rat PK study when each compound was administered as a
single 50 mg/kg oral dose and liver levels of the nucleoside triphos-
phate 6 were determined. The relative levels of triphosphate 6
found in the liver samples were considered an indication of antic-
ipated in vivo potency and were the basis for selecting which com-
pound to progress further. Table 4 shows the PK parameters for
these two compounds. Compound 24a produced the highest tri-
phosphate Cmax and AUC0–24 levels post dose indicating high drug
exposure in the liver.19 In addition, significant exposure to the
parent prodrug 24a was observed in rat liver (Cmax = 2829 ng/g,
and liver S9 fraction. Intriguingly, all of these purine analogs were
equipotent against the NS5B S282T resistant replicon. It was
shown previously that the S282T replicon cells were less suscepti-
ble to certain modified 20-C-methyl nucleoside/tides.18
Based on the replicon potency, stability and cytotoxicity profile,
compounds 16, 24a, and 24b were selected for further evaluation.
Of the two diastereomers, 24a and 24b, the cis isomer 24a was
shown to be the thermodynamically more stable isomer and could
be made efficiently by equilibration of the initial phosphite inter-
mediate mixture (36a and 36b) to 36a followed by oxidation and
crystallization (Scheme 3). Consequently, compound 24a was
selectively prepared by first cyclization of the nucleoside 34 with
isopropyl N,N,N0,N0-tetraisopropylphosphorodiamidite 35 using
DCI as an activator to give a mixture of 36a and 36b in about
85:15 ratio respectively. After heating the reaction mixture at
50 °C for 6 h, 31P NMR indicated >95% of 36a. Further oxidation
of the mixture with tBuOOH followed by crystallization from ethyl
acetate or acetone afforded 24a in high purity (>99%).
Cyclic phosphates 16 and 24a were further evaluated for their
in vitro and in vivo liver levels of the active triphosphate 6
(Table 4). Since triphosphate levels are correlated to in vivo po-
tency of nucleoside/tide analogs, in vitro triphosphate production
in clone A replicon cells and primary human hepatocytes was as-
sessed. In vitro analysis of triphosphate (6) production was accom-
plished by incubating clone A replicon cells and primary human
AUC0–24 = 13234 ng h/g) compared to the compound 16 (Cmax
=
3052 ng/g, AUC0–24 = 8310 ng h/g). This suggests that compound
24a survived through GI exposure, remained intact during the
absorption phase and delivered the active triphosphate into the
liver. Based on the relative liver exposure levels of both the pro-
drug and active triphosphate of the two compounds 16 and 24a,
it appears that the prodrug stability in plasma may correlate to
liver triphosphate levels.
Based on its superior potency against both wild type and resis-
tant mutants (S282T), ability to produce higher intracellular tri-
phosphate levels both in vitro and in vivo and due to the
synthetic accessibility of a single diastereomer, 24a was selected
for further development. Compound 24a (PSI-352938) is currently
in Phase I clinical trials for the treatment of HCV infection.
hepatocytes with 100 lM of each compound at 37 °C for 72 h
and then extracting triphosphate metabolites from the cells and
Table 3
Cytotoxicity data for selected purine cyclic phosphate analogs
Compd
HCV inhibition (clone A): EC90
(
lM)
Cytotoxicity: CC50
(lM)
Mitochondrial toxicity: CC50 (lM)
WT
S282T
Huh 7
HepG2
BxBC3
CEM
CEM
HepG2
16
19
22
24a
24b
0.41
0.92
0.98
1.37
0.69
0.4
96
72
>100
>100
>100
95
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14
>100
>100
>100
3.08
1.53
1.61
0.86
>100
>100
>100
>100
P
N
N
O
P
O
O
O
N
O
O
OEt
O
OEt
+
N
34
O
N
O
P
O
a
O
N
HO
OEt
N
N
F
N
N
N
+
(iPr)2N
N(iPr)2
HO
b
F
N
F
N
36b (trans)
NH2
NH2
35
36a (cis)
NH2
N
N
N
O
O
OEt
OEt
O
N
N
OEt
c
O
P
O
O
P
O
+
N
N
N
N
N
N
P
O
O
O
O
O
O
F
F
F
O
NH2
NH2
24a (>95%)
24a (>99%)
24b (<5%)
NH2
Scheme 3. Synthesis of compound 24a. Reagents and conditions: (a) DCI, CH3CN, rt to 50 °C, 6 h; (b) tBuOOH, CH3CN, rt, 20 h; (c) Crystallization from EtOAc or acetone, 35%
(three steps).

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P. G. Reddy et al. / Bioorg. Med. Chem. Lett. 20 (2010) 7376–7380
E.; Nelson, D.; Everson, G.; Jacobson, I.; Reddy, R.; Hill, G. Z.; Beard, A.;
Acknowledgments
Symonds, W. T.; Berrey, M. M.; McHutchison, J. G. 43rd Annual Meeting of the
European Association for the Study of the Liver (EASL), Milan, April 2008.
9. Rodriguez-Torres, M.; Lalezari, J.; Gane, E.; DeJesus, E.; Nelson, D.; Everson, G.;
Jacobsen, I.; Reddy, R.; McHutchinson, J.; Beard, A.; Walker, S.; Symonds, B.;
Berrey, M. 59th Annual Meeting of the American Association for the Study of
Liver Disease, San Francisco, November 2008, Abstr. No. 1899.
We thank Wuxi AppTec and Dr. Patrick Carroll, University of
Pennsylvania (X-ray analysis) for their contribution to the work de-
scribed in this manuscript.
10. (a) Oeberg, B. Antiviral Res. 2006, 71, 90; (b) Sabini, E.; Ort, S.; Monnerjahn, C.;
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.10.035.
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