Evaluation of 2′-α-fluorine modified nucleoside phosphonates as potential inhibitors of HCV polymerase

Article abstract of DOI:10.1016/j.bmcl.2013.03.095

Ribonucleoside phosphonate analogues containing 2′-α-fluoro modifications were synthesized and their potency evaluated against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent inhibitio

Full text of DOI:10.1016/j.bmcl.2013.03.095

Accepted Manuscript
Evaluation of 2’-α-Fluorine Modified Nucleoside Phosphonates as Potential
Inhibitors of HCV Polymerase
Jay P. Parrish, Sharon K. Lee, Constantine G. Boojamra, Hon Hui, Darius
Babusis, Brandon Brown, I-hung Shih, Joy Feng, Adrian S. Ray, Richard L.
Mackman
PII:
S0960-894X(13)00423-X
http://dx.doi.org/10.1016/j.bmcl.2013.03.095
BMCL 20331
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
8 February 2013
20 March 2013
22 March 2013
Please cite this article as: Parrish, J.P., Lee, S.K., Boojamra, C.G., Hui, H., Babusis, D., Brown, B., Shih, I-h., Feng,
J., Ray, A.S., Mackman, R.L., Evaluation of 2’-α-Fluorine Modified Nucleoside Phosphonates as Potential
Inhibitors of HCV Polymerase, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/
j.bmcl.2013.03.095
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Evaluation of 2'-α-Fluorine Modified Nucleoside Phosphonates
as Potential Inhibitors of HCV Polymerase
Jay P. Parrish*, Sharon K. Lee, Constantine G. Boojamra, Hon Hui, Darius Babusis,
Brandon Brown, I-hung Shih, Joy Feng, Adrian S. Ray, Richard L. Mackman
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404
This is where the receipt/accepted dates will go; Received Month XX, 2000; Accepted Month XX, 2000 [BMCL RECEIPT]
Abstract—Ribonucleoside phosphonate analogues containing 2’-α-fluoro modifications were synthesized and their potency evaluated
against HCV RNA polymerase. The diphosphophosphonate (triphosphate equivalent) adenine and cytidine analogues displayed potent
inhibition of the HCV polymerase in the range of 1.9–2.1 μM, but only modest cell-based activity in the HCV replicon. Prodrugs of the
parent nucleoside phosphonates improved the cell-based activity. ©2000 Elsevier Science Ltd. All rights reserved.
The current treatments for hepatitis C (HCV) are sub-
optimal resulting in an unmet medical need for the 200
million people that are infected world-wide.1,2 It is
widely expected that similar to the HAART paradigm
for HIV, effective and durable anti-HCV therapy that is
interferon sparing will involve the combination of
inhibitors targeting multiple enzymes and viral life
cycle events.3 Toward this end, the discovery of anti-
HCV nucleosides is an important goal, as it is
anticipated that this compound class will become an
important part of any anti-HCV armature in the future.4
has demonstrated exceptional potency in combination
with ribavirin toward genotype 2 and 3 subjects.5e
The nucleoside phosphonate approach to anti-virals, in
which the phosphonate represents a bioisostere of the
monophosphate group, is a powerful alternative to the
use of phosphate prodrugs. The phosphonate bioisostere
effectively bypasses the first metabolism step and is
more stable chemically and enzymatically than the
corresponding phosphate group. This improved stability
results in a prolonged pool of phosphonate inside the
cells that is available for metabolism to the active
Nucleosides with anti-HCV activity have been reported,
including 2’-deoxy-2’-α-fluoro nucleoside analogues
(e.g. 1–3, Figure 1).5 The nucleoside is activated
intracellularly by cellular kinases to the triphosphate
species which inhibits the HCV NS5b polymerase by
competing with the natural ribonucleside triphosphate
substrates. Further elaboration of the 2’-deoxy-2’-α-
fluoro nucleoside analogues led to the inclusion of a 2’-
C-Me group, in addition to the 2’-α-F, resulting in
compounds 4 (PSI–6130, 2’-C-Me, 2’-α-F dC) and 5
(PSI–6206, 2’-C-Me, 2’-α-F dU). Uridine analogue 5,
although a potent inhibitor of NS5b polymerase,5l has an
inefficient first metabolism step en route to the active
triphosphate, and so a phosphate prodrug has been
developed to bypass this metabolic limitation,
compound 6 (sofosbuvir, GS–7977). Compound 6 is the
most advanced nucleoside in clinical trials for HCV and
diphospho-phosphoate
(triphosphate
equivalent)
species, ultimately leading to long half lives inside cells
for the active species. As a result of these properties,
antiviral phosphonates such as adefovir (7) and
tenofovir (8) are now widely used in convenient once-
daily antiviral regimens.6 Not surprisingly, modified
ribose-based nucleoside phosphonates have also been
evaluated for HCV and shown to have modest cell
based activity.7 In order to design nucleoside
phosphonates that are potent and selective inhibitors of
NS5b, we embarked on an investigation of the 2’-α-F
ribose-based phosphonates. Of particular interest was
compound 11, a phosphonate version of 3 the most
potent of the 2’-α-F modified nucleoside analogues.8
The results of these efforts are reported herein.

inhibition of NS5b polymerase by the DP analogues.
The replicon active adenosine and cytidine analogues, 9
and 11 respectively, are both >10-fold more potent
polymerase inhibitors than the uridine analogue 10
consistent with the replicon activity data. This trend
suggests that inhibition of the NS5b polymerase is the
likely mode of action for these nucleoside
phosphonates. The inhibition of NS5b by the
diphosphate pyrimidines 10 and 11 parallels the
inhibition of NS5b by the corresponding nucleoside
triphosphates, but the purine phopshonate analogue 9 is
a considerably more potent inhibitor of NS5b than the
corresponding nucleoside 1 by 50-fold The reason for
this divergence of SAR is not clear but suggests that the
C=C linkage to the phosphorous in this adenine
analogue is superior to the natural C–O linkage of the
phosphate. A straightforward reduction of the C=C
group for the adenosine analogue to the saturated C–C
(2’-α-F C–C dAP, 12) was performed and the data is
also reported in Table 1. The analogue displayed potent
inhibition, once again more potent than the nucleoside 1
but lacked antiviral activity.
A general procedure for the synthesis of 2’-α-F
modified analogues is shown in Scheme 1. The
synthesis of 9 and 12, the unsaturated (C=C) and
saturated (C–C) phosponoethyl analogues of 2’-deoxy,
2’fluoro adenosine, commenced with a selective 5’-OH
protection of the fluorosugar using MMT-Cl in
pyridine. Protection of the 3’-OH and 6-NH2 with BzCl,
followed by treatment with AcOH to remove the MMT
group then provided 13. The low yield (44%) for the
MMT removal was attributed to the formation of an
acetate ester by-product, but treatment of this with
K2CO3 in MeOH provided 13 in >90%. Oxidation of the
5’-OH was performed with DMSO/DCC in the presence
of catalytic pyridine•TFA. Subsequent same pot
addition of the potassium salt of the HWE reagent
provided the dimethyl phosphonate in 15% yield.
Dealkylation of the methyl esters was accomplished
with TMSBr in the presence of 2,6-lutidine. The
corresponding benzoyl-protected diacid was treated
with concentrated NH4OH overnight to afford diacid 9
in 48% after reverse phase HPLC. Subjecting 9 to
standard hydrogenaton conditions in water afforded
saturated diacid 12 in 47%.
The cytidine phosphonate analogue 11 has the same
inhibition value for the polymerase as the corresponding
nucleoside triphosphate but 5-fold weaker anti-viral
activity. This disconnect in antiviral activity whereby
the nucleoside phosphonates are less potent in cell
based assays, could be due to limited cell penetration of
the phosphonates due to their charged nature at
physiological pH, or poor intracellular metabolism to
the active diphosphate species compared to the
nucleosides.
In order to complete a thorough evaluation of the 2’-α-F
phosphonates, we prepared the diphosphophosphonates
(DP) and the bis-amidate pro-drugs (PD) of each
corresponding diacid. A general procedure for their
synthesis is shown in Scheme 2. Activation of diacid 9
with
CDI
followed
by
treatment
with
tetrabutylammonium pyrophosphate (TBApp) yielded
9-DP in 43% after ion-exchange chromatography.
Subsequent hydrogenation afforded 12-DP in 69%. The
pro-drugs of C=C linked analogues were prepared by
treating the diacids to known coupling conditions.9
Hydrogenation afforded the saturated-linked pro-drugs
in moderate yields.
To address the poor cell penetration properties of the
nucleoside phosphonate diacids we prepared bis-
alanine-N-butyl amidate pro-drugs, which typically
increases the log D from –2 to the range of 3–5
depending on the nucleobase of choice. It was
determined from earlier work on anti-HIV phosphonates
that this pro-drug provided excellent cell penetration
properties in other cell types and release of the parent
diacid.6b Anti-HCV activity for the pro-drugs
dramatically improved for the uridine phosphonate 10
and saturated adenosine phosphonate (12), marginally
improved for the adenosine analogue 9, and did not
improve for the cytidine analogue. The lack of
improvement for the cytidine analogue pro-drug
indicates that cell penetration is likely not the major
limitation in the 5-fold drop in anti-viral activity for the
nucleoside phosphonate. Despite the encouraging
results for the initial pro-drugs, these analogues also
displayed increased cytostatic effects in the MT-4 cell
line, especially for the most potent prodrug 12. The MT-
4 cell line is a useful cell line for selectivity testing
because the cells are sensitive to cytostatic effects of
The profile of the 2’-α-F nucleoside phosphonate
analogues 9–12 toward HCV NS5b is shown in Table 1
and can be compared with the corresponding 2’-α-F
modified nucleosides. From the three examples of C=C
phosphonates (9–11) replicon activity was present for
2’-α-F C=C deoxyadenosine phosphonate (2’-α-F C=C
dAP, 9) and 2’-α-F C=C deoxycytidine phosphonate
(2’-α-F C=C dCP, 11), but not 2’-α-F C=C
deoxyuridine phosphonate (2’-α-F C=C dUP, 10).
Comparison with the corresponding nucleosides 1–3
reveals similar trends in replicon activity except that the
cytidine analogue 3 is 5-fold more potent than the
corresponding nucleoside phosphonate 11. None of the
compounds displayed toxicity in the replicon Huh-7 cell
line up to 250 μM.
The mechanism of action for the nucleoside
phosphonates was examined by evaluating the

compounds. The poor selectivity of the pro-drugs
suggests that the compounds may not be highly
selective inhibitors of HCV, since the HCV replicon
Huh-7 cell system readout of antiviral activity is also
dependent on viable growth of the Huh-7 cells. Thus
the antiviral activity needs to be treated with caution
when the difference between the Huh-7 HCV replicon
data and the MT-4 CC50 data is small as is the case for
these pro-drugs.
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Scheme 1. Representative synthesis of 9 and 12: (i) MMTCl, py, rt, 20 h, 63%; (ii) BzCl, py, rt, 18 h, 95%; (iii) 90% AcOH, 50 ˚C, 48 h, 44%; (iv) DCC,
DMSO, py•TFA, rt, 18 h, then [(MeO)2P(O)] 2CH2, KOtBu, 0 ˚C to rt, 1 h, 15%; (v) TMSBr, 2,6-lutidine, MeCN, rt, 18 h; (vi) conc. NH4OH, rt, 18 h, 48%
(2 steps); (vii) H2, 10% Pd/C, H2O, rt, 18 h, 47%. Note: similar sequence used for the synthesis of other phosphonate analogues.

Scheme 2. Representative diphosphophosphonate (DP) and prodrug synthesis (PD).

Figure 1. Nucleosides and Phosphonates

Table 1. Compound data: HCV Pol inhibition, Replicon activity and cytotoxicity data for nucleosides phosphonate diacids, and
prodrugs.

Compound
HCV Pol 1b
DA Replicon
DA Huh-7
PD Replicon
PD Huh-7
PD MT-4
IC50 (μM)
EC50 (μM)a
CC50 (μM)
EC50 (μM)b
CC50 (μM)
CC50 (μM)
2’-α-F dA (1)
2’-α-F dU (2)
102
134
>250
>250
–
–
–
–
–
–
–
–
–
12.8
>250
2’-α-F dC (3)
1.85
15.0
>250
2’-α-F C=C dAP (9)
2.1
31
132
>250
>250
58
113
18
92
2’-α-F C=C dUP (10)
>250
14.5
>250
2’-α-F C=C dCP (11)
2’-α-F C–C dAP (12)
1.9
4.6
76
>250
>500
134.5
11
>250
24
>250
36
>500
aDA = phosphonic diacid (bis-ammonium salt).
bPD = phosphonate bis-alanine butyl ester bis-amidate prodrug.

Evaluation of 2'-α-Fluorine Modified Nucleoside Phosphonates as Potential Inhibitors of
HCV Polymerase
Jay P. Parrish*, Sharon K. Lee, Constantine G. Boojamra, Hon Hui, Darius Babusis, Brandon
Brown, I-hung Shih, Joy Feng, Adrian S. Ray, Richard L. Mackman
Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA, 94404, USA
Ribonucleoside phosphonate analogues containing 2’-α-fluoro modifications were
synthesized and their potency evaluated against HCV RNA polymerase.