Preparation and biological evaluation of 1′-cyano-2′-C-methyl pyrimidine nucleosides as HCV NS5B polymerase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.015

The first synthesis of 1′-cyano-2′-C-methyl pyrimidine nucleosides is described. Anti-HCV activity of these nucleosides and their nucleotide phosphoramidate prodrugs was assessed and compared to the 1′-unsubstituted counterparts and to the related 1′-cyan

Full text of DOI:10.1016/j.bmcl.2014.05.015

Bioorganic & Medicinal Chemistry Letters 24 (2014) 3092–3095
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Preparation and biological evaluation of 10-cyano-20-C-methyl
pyrimidine nucleosides as HCV NS5B polymerase inhibitors
⇑
Michael R. Mish , Aesop Cho, Thorsten Kirschberg, Jie Xu, C. Sebastian Zonte, Martijn Fenaux,
Yeojin Park, Darius Babusis, Joy Y. Feng, Adrian S. Ray, Choung U. Kim
Gilead Sciences Inc., 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
The first synthesis of 10-cyano-20-C-methyl pyrimidine nucleosides is described. Anti-HCV activity of
these nucleosides and their nucleotide phosphoramidate prodrugs was assessed and compared to the
10-unsubstituted counterparts and to the related 10-cyano-20-C-methyl C-nucleoside parent of GS-6620.
Ó 2014 Elsevier Ltd. All rights reserved.
Article history:
Received 4 March 2014
Revised 2 May 2014
Accepted 5 May 2014
Available online 22 May 2014
Keywords:
HCV antivirals
NS5B polymerase inhibitor
Pyrimidine nucleoside analogs
Nucleotide phosphoramidate prodrugs
HCV infection is a worldwide health issue, and considerable
effort has been invested in developing a treatment that is well
tolerated, offers the prospect of good adherence, and is accessible
to the over 150 million people estimated to be infected globally.1
Of late, detailed knowledge relating to the HCV replication cycle
has resulted in the discovery of methods to inhibit HCV.2 By anal-
ogy with the standard of care for HIV treatment, current strategies
for HCV therapy now include a nucleotide inhibitor of the HCV
NS5B polymerase and inhibitors of the HCV NS3 protease.3 In
particular, nucleotide inhibitors have emerged as possible
backbones for HCV therapy based on their broad genotype cover-
age, lack of pre-existing mutation with reduced susceptibility,
and high barrier to selection of resistant variants.4–6
Recently, we reported the discovery and evaluation of GS-6620,
a prodrug of 10-cyano-20-C-methyl 4-aza-7,9-dideaza adenosine
monophosphate that once released in cells is further phosphory-
lated to its triphosphate form, a potent and selective HCV polymer-
ase inhibitor.7 GS-6620 showed the potential for potent antiviral
activity (viral load reductions in excess of 4 logs in a few patients)
in a Phase 1 clinical trial but required high doses of up to 900 mg
BID and showed substantial intra and inter subject pharmacoki-
netic and pharmacodynamic variability.8 As the first 10-substituted,
carbon-nucleotide and adenosine analog to establish clinical proof
of concept, GS-6620 established conceptual validation for the
potential of a 10-substituted nucleotide to serve as an effective
polymerase inhibitor.
To further explore the properties of 10-substituted nucleoside
analogs, 10-cyano-20-C-methyl cytidine (C) 1 and 10-cyano-20-C-
methyl-uridine (U) 2 were prepared (Fig. 1). A differentiating
property of the 10-CN nucleosides reported here is the presence
of 20-C-Me and 10-CN disubstitutions on pyrimidine N-nucleosides.
These structural features invite comparison of these new analogs
with related 20-C-methyl-substituted pyrimidine nucleosides with
a 20-OH or 20-F that lack additional 10 substitution, such as the
nucleoside parents of clinically tested prodrugs NM283 and
GS-7977, respectively.
Synthetic methods to access 10-cyano-20-C-methyl 4-aza-
7,9-dideaza adenosine nucleoside compounds such as 5 have been
described in the recent literature,7 and various successful
approaches to 10-unsubstituted N-nucleosides are known for the
structural class exemplified by compounds 3 and 4.9,10 A means
to obtain 10-CN N-nucleosides in the pyrimidine series that made
use of
a
10-halogenated ribosyl donor had been previously
disclosed. This prior report had shown that the 10CN-substituted
adenosine (A) and C analogs were accessible via use of Hg(II)-
promoted Vorbrüggen coupling employing 10CN-10-Br substituted
ribosyl donors.11 However, examples of substrates that are
sterically hindered at the 20 position have not been reported. In
the case which would ultimately deliver the more hindered
20-C-methyl substituted compound 2, only trace product was
observed after prolonged heating under these literature conditions
(Table 1).
⇑
Corresponding author. Tel.: +1 650 522 5316; fax: +1 650 522 5169.
E-mail address: mmish@gilead.com (M.R. Mish).
http://dx.doi.org/10.1016/j.bmcl.2014.05.015
0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

M. R. Mish et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3092–3095
3093
Figure 1. Novel 10-cyano-20-C-methyl cytidine and uridine (1, 2) and structurally related, anti-HCV nucleosides 3, 4 and 5.
Table 1
from amination followed by global deprotection in 20% overall
yield from 6.13
Lewis acid promoter screen in Vorbrüggen coupling of ribosyl donor 6
Lewis Acid
Result/Yielda
Nucleosides 1 and 2 were evaluated for HCV replicon activity
(EC50), as well as being subjected to nucleoside triphosphate prep-
aration in order to measure HCV polymerase enzyme inhibitory
activity (IC50). The results of these assays are tabulated below
and presented in the context of the comparable data for nucleo-
sides/triphosphates related to clinical leads GS-6620 and
GS-7977. Data presented in Table 2 shows that in the case of the
triphosphates of both analogs 1 and 2, the 10CN substituent was
not as well tolerated by the NS5B polymerase as when this substi-
tution was applied to the 20-C-methyl 4-aza-7,9-dideaza adenosine
C-nucleotide (triphosphate of 5).
In addition to the reduced intrinsic potency of the correspond-
ing triphosphates against the HCV NS5B polymerase, the poor cel-
lular activity noted for nucleosides 1 and 2 may be related to
inefficient intracellular phosphorylation. As an additional means
of evaluating the potential utility of 10CN nucleosides as anti-
HCV therapeutics, compound 1 was subjected to reaction with
the previously reported7 4-nitrophenolate compound 8 to provide
mono-phosphoramidate prodrug 9 in 20% yield, as illustrated in
Scheme 2.
The results from the in vitro evaluation of 9 are shown in
Table 3, where the replicon EC50 and nucleoside triphosphate levels
are presented alongside those of the previously reported phos-
phoramidate prodrug 10 (GS-6620). Note that despite the rela-
tively modest HCV polymerase IC50 value for the nucleoside TP of
1, replicon activity for the corresponding phosphoramidate 9 is rel-
atively robust. This result can be rationalized by noting the high
levels of nucleoside TP measured within the replicon assay in the
presence of 9, indicating that conversion from the monophosphate
Hg(CN)2
SnCl4
Zn(CN)2
Ag(OBz)
Ag(OTf)
Trace product
NR
NR
NR
58%b
a
Reactions were performed in 1:1 dichloroethylene/acetonitrile, heating at
reflux with reaction times from between 30 min to 18 h.
b
Reaction carried out at 135 °C for 30 min in a microwave reactor.
In order to improve this first result, a screen of a series of Lewis
acids including silver perchlorate, silver benzoate, SnCl4, Zn(CN)2,
and silver triflate was undertaken to examine their potential to
serve as promoters for the hindered ribosyl donor 6 (Scheme 1).
It was ultimately discovered that the optimal results for this cou-
pling were provided via use of AgOTf as promoter. Specifically,
reaction of 6 gave the coupling product using bis-TMS-U as nucle-
ophile in 1,2-dichloroethane/acetonitrile at 85 °C, while improved
yields were obtained upon heating at 135 °C for 30 min in a micro-
wave reactor. In this case, adduct 7 was obtained in a yield of 58%,
with >20:1 b-selectivity as judged by the absence of any detectable
a
-anomer via 1H NMR spectroscopy.12
With a practical synthesis of the main 10-CN N-nucleoside
pyrimidine precursor 7 secured, a concise chemical sequence was
applied to advance the Vorbrüggen adduct to obtain the corre-
sponding uridine analog, or alternatively convert from the U to C
nucleoside. Scheme 1 illustrates the chemistry utilized to provide
the protected uridine analog 2, and alternatively the sequence of
transformations which delivers the cytidine analog 1 resulting
Scheme 1. Preparation of uridine analog 2 and cytidine analog 1.

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M. R. Mish et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3092–3095
Table 2
a
b
Selected nucleoside EC50 & nucleoside triphosphate (NTP) IC50
EC50
IC50
[
[
l
l
M]
M]
>89/>89
27
76/>89
17.7
2.5/2.8
1.67
>89/>89
0.29
a
IC50 determined by enzymatic assay using an HCV genotype NS5b protein.
EC50 determined by cell based 384 well format assay using 1b-Rluc cells harboring the subgenomic HCV genotype 1b replicon in the presence of 40% HS.
b
Scheme 2. Conversion of nucleoside 1 to phosphoramidate prodrug 9.
Table 3
Comparison of replicon EC50 and replicon TP concentration14 for novel phosphoramidate prodrug 9 and prodrug 10 (GS-6620)
EC50 (1a/1b)
Rep TP C24h
1.2/2.1
332 pmol/million
0.094/0.24
40 pmol/million
Scheme 3. Preparation of 30-isobutyryl, 50-phosphormamidate 10-CN nucleoside prodrug 11.
to nucleoside triphosphate is highly efficient for this newly dis-
closed 10CN-pyrimidine nucleoside prodrug.
As a final step to further improve the anti-HCV properties for
the series of nucleoside analogs based on 1, compound 9 was
elaborated to the corresponding 30-isobutyryl ester 11 was pre-
pared in three steps and 89% overall yield (Scheme 3). This modi-
fication provided a bis-prodrug that allows direct comparison to
the biological activity of 10-CN nucleoside analog 10 (GS-6620).

M. R. Mish et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3092–3095
3095
In the case of the resulting 10-CN-pyrimidine-20-C-methyl-3-isobu-
tyryl cytidine 11, the 1a/1b replicon EC50 improved to 0.25 M and
0.6 M, respectively.
In contrast to the results previously described for 20-Deoxy-
20-fluoro-20-methylcytidine,15
notable property of the
3. Manns, M. P.; von Hahn, T. Nat. Rev. Drug Disc. 2013, 12, 595. and references
cited therein.
4. Brown, N. A. Expert Opin. Investig. Drugs 2009, 18, 709.
5. Sofia, M. J. Antivir. Chem. Chemother. 2011, 22, 23.
6. The FDA approved GS-7977 (Sofosbuvir) on Dec. 6th, 2013 for treatment of
genotypes 1–4 in HCV infected subjects.
l
l
a
7. (a) Cho, A.; Zhang, L.; Xu, J.; Lee, R.; Butler, T.; Metobo, S.; Aktoudianakis, V.;
Lew, W.; Ye, H.; Clarke, M.; Doerffler, E.; Byun, D.; Wang, T.; Babusis, D.; Carey,
A. C.; German, P.; Sauer, D.; Zhong, W.; Rossi, S.; Fenaux, M.; McHutchison, J. G.;
Perry, J.; Feng, J.; Ray, A. S.; Kim, C. U. J. Med. Chem. 2014, 57. article ASAP; (b)
Murakami, E.; Wang, T.; Babusis, D.; Lepist, E. I.; Sauer, D.; Park, Y.; Vela, J. E.;
Shih, R.; Birkus, G.; Stefanidis, D.; Kim, C.-U.; Cho, A.; Ray, A. S. Antimicrob.
Agents Chemother. 2014, 58. article published online ahead of print.
8. Lawitz, E.; Hill, J.; Marbury, T.; Hazan, L.; Gruener, D. [Abstract 1188]. Presented
at: 47th Annual Meeting of the European Association for the Study of the Liver
(EASL); 2012 April 18–22; Barcelona, Spain. J. Hepatol. 2012, 56, S470.
9. (a) Pierra, C.; Amador, A.; Benzaria, S.; 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; (b) Sofia, M. J.; Bao, D.; Chang, W.; Du,
J.; Nagarathnam, D.; Rachakonda, S.; Reddy, P. G.; Ross, B. S.; Wang, P.; Zhang,
H. R.; Bansal, S.; Espiritu, C.; Keilman, M.; Lam, A. M.; Micolochick-Steuer, H.
M.; Niu, C.; Otto, M. J.; Furman, P. A. J. Med. Chem. 2010, 53, 7202. and
references cited therein.
10CN-pyrimidine C nucleoside 1 is the apparently slow conversion
to the corresponding U analog. Under conditions where related
cytidine analogs lacking 10CN substitution showed extensive
metabolism to uridine derivatives via cytidine deaminase, we did
not observe significant levels of the corresponding U derivatives
in the case of nucleotide prodrugs such as 9 and 11. This observa-
tion suggests that the 10CN modification may play a particularly
useful role in deterring pyrimidine deamination.
Conclusion
The synthesis and characterization of a series of 10-cyano-20-
C-methyl C and U nucleosides was accomplished by means of an
Ag(OTf) promoted Vorbrüggen condensation with a 10-cyano,
10-bromo-20C-methyl ribosyl donor in 58% optimized yield. The
resulting adduct was further elaborated to furnish the correspond-
ing 10-cyano, 20-C-methyl U and C nucleosides, which had not
previously been reported. The nucleoside TPs were assessed for
HCV polymerase enzyme inhibitory activity (IC50), and shown to
have moderate inhibitory activity. Further, phosphoramidate
prodrug derivatives of 1 showed relatively robust replicon EC50
activity, a result consistent with highly efficient conversion of the
prodrugs 9 and 11 to the active triphosphate in cells.
10. Wang, P.; Chun, B.-K.; Rachakonda, S.; Du, J.; Khan, N.; Shi, J.; Stec, W.; Cleary,
D.; Ross, B. S.; Sofia, M. J. J. Org. Chem. 2009, 74, 6819.
11. Uteza, V.; Chen, G.-C.; Le Quan Tuoi, J.; Descotes, G.; Fenet, B.; Grouiller, A.
Tetrahedron 1993, 49, 8579.
12.
A
diagnostic 1H NMR peak consistent with the b-anomer product is the
appearance of the 20-Me group at <1.2 ppm. This was observed in intermediate
7 as well as final compounds 1 and 2. Subsequent 2D NMR structural analysis
confirmed this stereochemical assignment.
13. 1H NMR (400 MHz, D2O) spectral data for compound 1: d 7.74 (d, J = 8.0 Hz, 1H),
5.84 (d, J = 8.0 Hz, 1H), 4.20 (m, 1H), 3.81 (m, 1H), 3.68 (m, 1H), 3.62 (m, 1H),
1.08 (s, 3H) and 2: d 7.91 (d, J = 8.4 Hz, 1H), 5.78 (d, J = 8.4 Hz, 1H), 4.15 (m, 1H),
3.94 (m,1H), 3.75 (m,1H), 3.67 (m,1H), 1.18 (s, 3H).
14. Replicon cells were maintained in Dulbecco’s 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 ꢀ 106 cells/well). Cells were treated for 24 h with a
Acknowledgments
test compound (10 lM). Following 24 h, 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.
M.R.M. thanks Drs. Haolun Jin and Samuel E. Metobo for
insightful discussions and their critical review of this manuscript.
References and notes
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2009, 29, 74; (c) U. S. Department of Health and Human Services: Centers for
Disease Control and Prevention. The ABCs of hepatitis [online], 2012.
2. Kronenberger, B.; Zeuzem, S. J. Viral Hepat. 2012, 19, 48.
15. Murakami, E.; Niu, C.; Bao, H.; Micolochick-Steuer, H. M.; Whitaker, T.;
Nachman, T.; Sofia, M. A.; Wang, P.; Otto, M. J.; Furman, P. A. Antimicrob. Agents
Chemother. 2008, 52, 458.