Discovery of PSI-353661, a novel purine nucleotide prodrug for the treatment of HCV infection

Article abstract of DOI:10.1021/ml100209f

Hepatitis C virus afflicts approximately 180 million people worldwide, and the development of direct acting antivirals may offer substantial benefit compared to the current standard of care. Accordingly, prodrugs of 2′-deoxy-2′-fluoro-2′-C-methylguanosine

Full text of DOI:10.1021/ml100209f

pubs.acs.org/acsmedchemlett
Discovery of PSI-353661, a Novel Purine Nucleotide
Prodrug for the Treatment of HCV Infection
Wonsuk Chang, Donghui Bao, Byoung-Kwon Chun, Devan Naduthambi,
Dhanapalan Nagarathnam, Suguna Rachakonda, P. Ganapati Reddy, 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, and
Michael J. Sofia*
Pharmasset, Inc., 303A College Road East, Princeton, New Jersey 08540-6608, United States
ABSTRACT Hepatitis C virus afflicts approximately 180 million people world-
wide, and the development of direct acting antivirals may offer substantial benefit
compared to the current standard of care. Accordingly, prodrugs of 20-deoxy-20-
fluoro-20-C-methylguanosine monophosphate analogues were prepared and eval-
uated for their anti-HCV efficacy and tolerability. These prodrugs demonstrated
>1000 fold greater potency than the parent nucleoside in a cell-based replicon
assay as a result of higher intracellular triphosphate levels. Further optimization
led to the discovery of the clinical candidate PSI-353661, which has demonstrated
strong in vitro inhibition against HCV without cytotoxicity and equipotent activity
against both the wild type and the known S282T nucleoside/tide resistant replicon.
PSI-353661 is currently in preclinical development for the treatment of HCV.
KEYWORDS PSI-353661, hepatitis C virus, NS5B polymerase, antivirals, nucleoside,
prodrug, phosphoramidate, triphosphate
epatitis C virus is a 9.6 kb positive-sense single-
stranded RNA virus of the Flaviviridae family. Nearly
1.6% of the U.S. population and an estimated
As part of our continuing efforts to discover novel anti-HCV
agents, we were interested in finding second generation
agents with improved potency, better resistance profile,
enhanced pharmacokinetic properties to support QD dosing,
and the potential for generating high concentrations of the
active triphosphate in the liver. In addition to the cytidine
analogue RG7128, we have investigated derivatives of
2-deoxy-2-R-fluoro-2-β-C-methylribofuranose with other nu-
cleic acid bases.10-13 Among them, the guanosine analogue
1 was shown to be only weakly active (EC90=69.2 μM) in a
HCV replicon assay, but its triphosphate 2 was found to be a
potent inhibitor of the HCV NS5B polymerase (IC50 = 5.94 μM)
(Figure 1).
Nucleoside analogues need to be phosphorylated to their
corresponding triphosphate by host cellular kinases before
they can bind to the HCV NS5B polymerase and inhibit RNA
replication. However, many nucleoside analogues are poor
substrates for nucleoside kinases that are responsible for
their phosphorylation to their active triphosphate derivative.14
The tri-/di-/monophosphate species cannot be considered as
possible drug candidates because of their instability and
poor cellular permeability.15 Phosphoramidate prodrugs have
been shown to enhance nucleoside potency in cell culture,
H
180 million people worldwide are infected with HCV.1 Approxi-
mately 80% of infected individuals become chronically
infected. Untreated HCV infections can progress to cirrhosis,
hepatocellular carcinoma, and liver failure, a primary cause
for liver transplantation.2 The current standard of care (SOC)
for chronic HCV infection, PEG-IFNR and ribavirin, provides
limited sustained virologic response (SVR) rates (e.g., e50%
for genotype 1 patients) and can produce various undesir-
able side effects ranging from flu-like symptoms to severe
adverse effects, including anemia, cardiovascular events,
and psychiatric problems such as suicidal ideation.3 Conse-
quently, there is a clear unmet medical need to discover new
direct-acting antivirals (DAA) to effectively and safely treat
chronic HCV infection.4
Three first generation nucleoside prodrugs (NM283,
R1626, and RG7128) have been reported to show anti-HCV
activity as direct-acting antiviral agents in clinical trials.5,6
Among them, only RG7128 is currently under development
(phase IIb). RG7128 is a 30,50-diisobutyrate ester prodrug of
β-D-20-deoxy-20-R-fluoro-20-β-C-methylcytidine (PSI-6130) and
has already established proof-of-concept in the clinic.7,8 In a
28 days combination study with SOC, RG7128 demon-
strated efficacy in genotype 1, 2, and 3 patients and was
the first direct-acting antiviral to show broad genotype
coverage in the clinic.9
Received Date: September 10, 2010
Accepted Date: December 8, 2010
Published on Web Date: December 17, 2010
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presumably by increasing the intracellular concentrations of
the nucleoside monophosphate, thus bypassing a potentially
nonproductive first phosphorylation step.16-18 We therefore
decided to investigate phosphoramidate derivatives of
20-deoxy-20-R-fluoro-20-β-C-methylguanosine monophosphate.
The study of phosphoramidate prodrugs began with the
investigation of the anti-HCV structure activity relationship
at the 6-position of the purine base, keeping the substitution
on the phosphoramidate moiety constant. A series of 6-(un)-
modified guanosine analogues 6a-k was synthesized starting
from the previously reported19 ribonolactol 3 by preparing
the 6-chloro nucleoside 5 as a key intermediate (Scheme 1).20
The phosphoramidate moiety was introduced onto each
nucleoside by treatment with a phosphorochloridate reagent
in the presence of N-methylimidazole.21 Each purine phos-
phoramidate derivative was prepared as a mixture of dia-
stereomers at phosphorus.
Anti-HCVactivity in the clone A replicon whole cell assayof
the ethyl ester series 8-18 is summarized in Table 1.22-24
The 6-unsubstituted guanine phosphoramidate 8 showed low
micromolar activity: approximately a 25-fold increase in
potency over nucleoside 1. On the other hand, a number of
the phosphoramidates having 6-alkoxy, -alkylsulfane, and
-alkylamine substituents (double prodrug) demonstrated
more dramatic improvements in potency against HCV. Espe-
cially, 6-methoxyguanosine analogue 9and 6-amino substituted
analogues 15 and 16 exhibited single digit nanomolar EC90
values, thus offering >5000-fold increased potency compared
to the parent nucleoside 1.25
Further SAR assessment focused on preferred C-6 sub-
stitutions on the base and small alkyl esters at the terminal
carboxylate group of the phosphoramidate moiety (Table 2).
A survey of substitutions at both the C-6 position of the base
and the terminal ester showed that substituents that in-
creased the overall lipophilicity of the molecule translated
into increased cytotoxicity (data not shown). Consequently,
we chose to minimize the overall lipophilicity and focus our
efforts on the introduction of relatively small substituents
both at C-6 of the base and at the terminal ester group. For
the 6-unmodified guanosine series (19, 20, and 21), im-
proved anti-HCV activity was observed when the terminal
carboxylate ester substituent was either iPr, cyclopentyl, or
cyclohexyl, affording EC90 values of e0.75 μM. None of the
6-OH (natural guanosine) analogues showed cytotoxicity up
to 100 μM against an expanded cell panel. Most of the
6-alkoxy analogues demonstrated a >1000-fold increase
in potency compared to the guanosine analogue 1. Among
Table 1. HCV Clone A Replicon Data
compd
R1
R2
EC90 (μM)
8
-OH
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
Et
2.38
9
-OMe
0.0074
0.010
0.013
0.020
0.027
0.047
0.0070
0.0098
0.024
0.026
10
11
12
13
14
15
16
17
18
-OEt
-OPr
-O(CH2)2OMe
-O(CH2)2F
-SEt
-N(CH2)3
-NH(cPr)
-NH(cBu)
-NH(cPentyl)
Figure 1. β-D-20-Deoxy-20-R-fluoro-20-β-C-methylguanosine (1) and
its anabolite triphosphate 2.
Scheme 1. General Synthetic Pathway for the Phosphoramidates of Guanosine Analoguesa
a Reagents and conditions: (a) 4, DEAD, PPh3, THF, rt, 16 h. (b) for 6a, 2-mercaptoethanol, NaOMe, MeOH, Δ; for 6b to 6g, ROH, NaH or K2CO3, DME, Δ;
for 6h-k, NHR0R00, MeOH or EtOH, Δ. (c) 7, NMI, THF, 0 °C to rt.
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Table 2. Anti-HCVActivity and Cytotoxicity
HCV inhibition EC90 (μM)
cytotoxicity CC50 (μM)
compd
R1
-OH
R2
WT
S282T
Huh7
BxBC3
HepG2
CEM
8
Et
2.38
>100
>100
>100
>100
>100
>100
>100
>100
47
>100
>100
>100
>100
65
>100
>100
>100
>100
71
>100
>100
>100
>100
20
19
20
21
22
9
-OH
iPr
0.75
0.61
0.12
-OH
cPentyl
cHexyl
Me
Et
0.18
-OH
0.446
0.0081
0.0074
0.020
0.016
0.010
0.020
0.0146
0.033
0.090
0.0070
0.019
0.0082
0.0098
0.0088
-OMe
-OMe
-OMe
-OEt
0.011
0.024
0.016
67
62
55
23
24
10
25
26
27
28
15
29
30
16
31
iPr
>100
>100
68
>100
>100
61
>100
74
Me
Et
-OEt
37
-OEt
iPr
59
>100
39
>100
45
93
-OPr
Me
iPr
39
17
-OPr
49
>100
>100
92
>100
>100
75
44
-N(CH2)3
-N(CH2)3
-N(CH2)3
-NH(cPr)
-NH(cPr)
-NH(cPr)
Me
Et
0.04
>100
84
75
39
iPr
53
74
100
74
Me
Et
>100
>100
96
>100
>100
98
>100
>100
>100
34
45
iPr
>100
Table 3. Stability of Key Compounds 20, 23, and 28
stability, t1/2 (h)
the 6-alkoxy derivatives, isopropyl ester 23 and methyl ester
24 showed no significant cytotoxicity. Alkylamine substitu-
tion at the 6-position of the base also produced derivatives
with potent anti-HCV activity. Of the alkylamine derivatives,
the 6-N-azetidine 28 gave the best cytotoxicity profile rela-
tive to the other phosphoramidates in the alkylamine series.
Several analogues (9, 19, 20, 23, 24, and 28) were tested
further against the known nucleoside resistant mutant, S282T,
which has demonstrated reduced sensitivity to the 20-methyl
containing nucleoside analogues RG7128 and NM283.26 It is
noteworthy that all of these purine nucleosides were shown
to be equipotent against both the wild type and the S282T
mutant replicon (Table 2). Based on anti-HCV replicon po-
tency, cytotoxicity profile, and structural diversity, a set of
four guanosine phosphoramidate analogues, 20, 23, 24, and
28, were selected for further study.
Since some compounds previously reported to inhibit the
HCV replicon also inhibited cell growth27 and exhibited
mitochondrial toxicity,28 compounds 20, 23, 24, and 28
were evaluated for effects on cell growth and for mitochon-
drial toxicity. When evaluated for their cytostatic potential,
compounds 20, 23, and 28 showed no significant effect on
cell doubling time up to 100 μM; however, the 6-ethoxy ether
analogue 24 was shown to retard cell growth by 5 h at 50 μM.
When mitochondrial toxicity (CC90) was evaluated by incu-
bating CEM and HepG2 cells with compounds 20, 23, and 28
for 14 days and then measuring the levels of mitochondrial
COXII DNA (mtDNA) and rDNA (rDNA) using real time PCR,
all three compounds showed no measurable mitochondrial
toxicity up to 100 μM in both cell lines.
compd
SGF
SIF
human plasma
S9
20
23
28
15
14
>20
>20
>20
>24
>24
11.6
0.2
0.2
0.4
>20
in simulated gastrointestinal fluid (SGF), simulated intestinal
fluid (SIF), human plasma, and human liver S9 fraction, a
surrogate in vitro model for liver stability, was studied. As
shown in Table 3, the derivatives with bulky ester groups,
20 and 23, provided better stability in human plasma as well
as in SGFand SIF when compared to the methyl ester analogue 28.
Each compound exhibited short half-lives in liver S9 fraction,
indicating that each phosphoramidate had the potential for
rapid conversion to the desired nucleotides in the liver.
Since triphosphate levels are correlated to the in vivo
potency of nucleos(t)ide analogues,29 in vitro triphosphate
production of the remaining compounds 20, 23, and 28 in
primary human hepatocytes was assessed and compared to
that produced in the replicon clone A cells (Table 4). In vitro
analysis of triphosphate 2 production was accomplished by
incubating each compound and then extracting triphos-
phate 2 from the cells and quantifying by HPLC analysis.
Of the three compounds studied, compound 23 produced
the highest level of triphosphate in primary human hepato-
cytes. TriphosphatelevelsforeachofthecompoundsincloneA
cells correlated well with anti-HCV replicon potency. There-
fore, one might anticipate that triphosphate levels observed
in primary human hepatocytes in vitro provide an indication
of anti-HCV potency in human hepatocytes in vivo.
To assess the ability of prodrugs 20, 23, and 28 to survive
exposure in the gastrointestinal tract and preferentially
deliver the nucleotide monophosphate to the liver, stability
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Since 23 was developed as a liver targeted nucleotide
prodrug, the relative liver to plasma exposure and the
production of active triphosphate 2 in the liver was evaluated
when 23 was administered in vivo. To accomplish this,
tritiated phosphoramidate nucleotide 33 was prepared by
first bromination of 23 at C-8 of the purine base followed by
reductive tritiation (Scheme 2). The tritiated derivative 33
was administered orally to rats (60 μCi total dose in peanut
oil), and plasma samples were collected at 1 and 6 h after
dosing. In addition, after administration of tritiated deriva-
tive 33, livers were removed at 1 and 6 h postdosing and
levels of triphosphate and key metabolites were determined
from the liver extracts. Parent prodrug and metabolites were
measured in plasma and liver extracts by HPLC using radio-
detection. As has been described for other nucleotide
phosphoramidates,16 metabolism of phosphoramidate 23
proceeds first to the diacid intermediate 34 and then to the
monophosphate 35, to the diphosphate 36, and finally to the
active triphosphate 2 (Scheme 3). With radiolabeled 33, we
were able to measure the levels of each of these productive
metabolites in both the liver and plasma (Table 5). In plasma
after 1 h, each of the diastereomers of 23 is evident, as is the
diacid 34 and the guanosine nucleoside 1. Nucleoside 1 pre-
sumably arises from decomposition of the monophosphate. At
the 6 h time point, the major metabolite in plasma is the
nucleoside 1, and very little of the parent prodrugs or inter-
mediate metabolite 34 is present. In the liver at 1 h, the mono-,
di-, and triphosphates are observed in addition to the inter-
mediate metabolite 34 and the nucleoside 1. At 6 h postdose,
liver extracts still show the presence of the mono-, di-, and
triphosphates and intermediate metabolite 34 in addition to
large amounts of 1. To determine the liver to plasma ratio, the
total metabolites observed in the liver were compared to the total
metabolites observed in plasma (Ta bl e 5 ). The liver to plasma
ratio was determined to be 3.5/1 and 4.8/1 at 1 and 6 h postdose,
respectively, thus supporting the liver targeting properties of 23.
In addition, the presence of diacid 34, mono-, di-, and tripho-
sphate in the liver indicates that 23 is able to reach the liver and
achieve conversion to the active triphosphate as designed.
As a result of its strong in vitro potency against HCV, good
metabolic stability, very clean in vitro toxicity profile, and high
liver to plasma ratio in a rat in vivo study, the 6-methoxy-
guanosine analogue 23 was chosen for further study. Phos-
phoramidate 23 is a mixture of diastereomers (23a:23b,
∼1:1.8 ratio) at the phosphorus center.30 The diastereomers
of 23 were separated by simulated moving bed chromatogra-
phy to provide the two pure isomers 23a and 23b. The anti-HCV
activity of each diastereomer against the wild type clone A
replicon and the S282T mutant replicon was determined, and
diastereomer 23b was shown to be 3.4-fold more potent than
diastereomer 23a and again retained activity in the mutant
replicon (Tabl e 6 ). The more potent diastereomer 23b also
produced about 3-fold higher concentrations of triphosphate in
both clone A and primary human hepatocytes than did 23a,
consistent with its in vitro potency in clone A cells.
Table 4. Cellular Triphosphate Levels of Key Compounds 20, 23,
and 28
cellular TP levelsa (mM)
compd
clone A (at 48 h)
primary human hepatocytes (at 24 h)
20
23
28
0.06
0.12
0.16
0.04
0.12
0.05
a Incubated at 100 μM compound concentration.
Scheme 2. Synthesis of Radiolabeled Prodruga
Pure 23b was crystallized in dichloromethane and hexane
solution, and a single crystal X-ray structure of 23b was
obtained unambiguously, confirming the configuration of
the phosphorus center as Sp (see Supporting Information).
Subsequently, it was demonstrated that diastereomer 23b
could be selectively crystallized directly from the diastereo-
meric mixture without prior chromatographic separation.
Based on its superior overall potency and safety profile,
a Reagents and conditions: (a) NBS, dioxane, rt, 5 h. (b) 3H2, 10% Pd/C,
Et3N, MeOH, rt, 1 h.
Scheme 3. Activation Pathway for Prodrug 23
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Table 5. Radio Counts of Tritiated 23 (33) and Metabolites in Rat Plasma and Liver Samples
peak areab (cpm)
rat samplesa
sample (volume or weight)
1
34
35
36
2
23a
23b
total (cpm/mL or cpm/g)
plasma
1 h
1.33 mL
1.00 mL
0.75 g
1376
3848
4224
11512
920
288
ND
ND
ND
ND
1160
272
ND
1112
368
ND
3426
4776
6 h
1 h
6 h
ND
1828
1920
ND
988
liver
1648
572
384
460
12096
22977
0.70 g
1620
ND
ND
a Dose: 60 μCi total dose in 2.9 mL of peanut oil. b ND = not detected.
Table 6. Comparison of the Diastereomers 23a and 23b
HCV inhibition EC90 (μM)
cellular TP levelsa (mM)
primary human hepatocytes (at 24 h)
compd
WT
S282T
clone A (at 48 h)
23a
23b
0.027
0.008
0.038
0.011
0.07
0.17
0.14
0.44
a Incubated at 25 μM compound concentration for clone Acells and 100 μM compound concentration for primary human hepatocytes, respectively.
compound 23b (PSI-353661) was selected as a clinical devel-
opment candidate.
(2)
Zeuzem, S.; Berg, T.; Moeller, B.; Hinrichsen, H.; Mauss, S.;
Wedemeyer, H.; Sarrazin, C.; Hueppe, D.; Zehnter, E.; Manns,
M. P. Expert opinion on the treatment of patients with chronic
hepatitis C. J. Viral Hepatitis 2009, 16, 75–90.
In conclusion, a series of β-D-20-deoxy-20-R-fluoro-20-β-C-
methylguanosine phosphoramidate analogues were pre-
pared and their SAR as inhibitors of HCV replication was
studied. Most of the prodrugs showed superior anti-HCV
potency relative to the parent guanosine analogue 1, often
by more than 1000-fold. After a comprehensive evaluation
that included cytotoxicity, resistance profile, cytostatic activ-
ity, metabolic stability, and cellular triphosphate production,
the single diastereomer 23b (PSI-353661) was chosen as a
preclinical development candidate for the treatment of HCV
infection. PSI-353661 exhibited strong inhibition of HCV in
the cell-based replicon assay, equipotent activity against
both the wild type and S282T resistant replicons, and ability
to produce high concentrations of the active triphosphate in
primary human hepatocytes.
(3)
Neukam, K.; Macias, J.; Mira, J. A.; Pineda, J. A. A review of
current anti-HCV treatment regimens and possible future
strategies. Expert Opin. Pharmacother. 2009, 10, 417–433.
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aimed at viral eradication. J. Viral Hepatitis 2010, 17, 77–90.
Furman, P. A.; Lam, A. M.; Murakami, E. Nucleoside analogue
inhibitors of hepatitis C viral replication: recent advances,
challenges and trends. Future Med. Chem. 2009, 1, 1429–1452.
Carroll, S. S.; La Femina, R. L. Nucleoside analogue inhibitors
of hepatitis C viral replication. In Antiviral Research;
La Femina, R. L., Ed.; American Society for Microbiology:
Washington, DC, 2009; pp153-166.
Stuyver, L. J.; McBrayer, T. R.; Tharnish, P. M.; Clark, J. L.;
Hollecher, L.; Lostia, S.; Nachman, T.; Grier, J.; Bennett, M. A.;
Xie, M.-Y.; Schinazi, R. F.; Morrey, J. D.; Julander, J. L.; Furman,
P. A.; Otto, M. J. Inhibition of hepatitis C replicon RNA
synthesis by β-D-20-deoxy-20-fluoro-20-C-methylcytidine: a
specific inhibitor of hepatitis C virus replication. Antiviral
Chem. Chemother. 2006, 17, 79–87.
Murakami, E.; Niu, C.; Bao, H.; Steuer, H. M.; Whitaker, T.;
Nachman, T.; Sofia, M. A.; Wang, P.; Otto, M. J.; Furman, P. A.
The mechanism of action of β-D-20-deoxy-20-fluoro-20-C-
methylcytidine involves a second metabolic pathway leading
to β-D-20-deoxy-20-fluoro-20-C-methyluridine 50-triphosphate,
a potent inhibitor of the hepatitis C virus RNA-dependent RNA
polymerase. Antimicrob. Agents Chemother. 2008, 52, 458–464.
Le Pogam, S.; Kang, H.; Seshaadri, A.; Kosaka, A.; Hu, S.;
Ewing, A.; Yan, J.-M.; Beard, A.; Symons, J.; Cammack, N.;
(4)
(5)
(6)
(7)
SUPPORTING INFORMATION AVAILABLE Experimental
procedures and analytical data for all new compounds. X-ray
structure and cif data for 23b. HPLC radiochromatograms and
triphosphate measurement of active prodrugs. This material is
available free of charge via the Internet at http://pubs.acs.org.
(8)
(9)
AUTHOR INFORMATION
Corresponding Author: *Telephone: 609-613-4123. Fax: 609-
613-4150. E-mail: michael.sofia@pharmasset.com.
ACKNOWLEDGMENT We thank Dr. Patrick Carroll, University
of Pennsylvania for X-ray analysis, and WuXi AppTec and Moravek
Biochemicals for their contribution to the work described in this
manuscript.
ꢀ
Najera, I. No evidence of R7128 drug resistance after up to
4 weeks treatment of GT 1, 2 and 3 hepatitis C virus infected
individuals. 44th Annual Meeting of the European Association
for the Study of the Liver; Copenhagen, Denmark, April, 2009;
Abstr. No. 839.
(10) 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
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ever, the phosphoramidates of L-alanine were strongly
preferred to the others in terms of anti-HCV potency
in vitro.
r
2010 American Chemical Society
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DOI: 10.1021/ml100209f ACS Med. Chem. Lett. 2011, 2, 130–135
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