Synthesis and anti-HCV activity of a series of β-D-2′-deoxy-2′-dibromo nucleosides and their corresponding phosphoramidate prodrugs

Article abstract of DOI:10.1016/j.bmcl.2017.10.024

Several β-D-2′-deoxy-2′-substituted nucleoside analogs have displayed potent and selective anti-HCV activities and some of them have reached human clinical trials. In that regard, we report herein the synthesis of a series of 2′-deoxy,2′-dibromo substituted U, C, G and A nucleosides 10a–d and their corresponding phosphoramidate prodrugs 13a–d. The synthesized nucleosides 10a–d and prodrugs 13a–d were evaluated for their inhibitory activity against HCV as well as cellular toxicity. The results showed that the most potent compound was prodrug 13a, which exhibited micromolar inhibitory activity (EC₅₀ = 1.5 ± 0.8 μM) with no observed toxicity. In addition, molecular modeling and free energy perturbation calculations for the 5′-triphosphate formed from 13a and related 2′-modified nucleotides are discussed.

Full text of DOI:10.1016/j.bmcl.2017.10.024

Accepted Manuscript
Synthesis and anti-HCV activity of a series of β-D-2’-deoxy-2’-dibromo nu-
cleosides and their corresponding phosphoramidate prodrugs
Zhe Chen, Bryan D. Cox, Ethel C. Garnier-Amblard, Tamara R. McBrayer,
Steven J. Coats, Raymond F. Schinazi, Franck Amblard
PII:
S0960-894X(17)31009-0
https://doi.org/10.1016/j.bmcl.2017.10.024
BMCL 25353
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 September 2017
10 October 2017
11 October 2017
Please cite this article as: Chen, Z., Cox, B.D., Garnier-Amblard, E.C., McBrayer, T.R., Coats, S.J., Schinazi, R.F.,
Amblard, F., Synthesis and anti-HCV activity of a series of β-D-2’-deoxy-2’-dibromo nucleosides and their
corresponding phosphoramidate prodrugs, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/
10.1016/j.bmcl.2017.10.024
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Synthesis and anti-HCV activity of a series of β-
-2’-deoxy-2’-dibromo nucleosides and their
corresponding phosphoramidate prodrugs
Leave this area blank for abstract info.
D
Zhe Chena, Bryan D. Cox a, Ethel C. Garnier-Amblardb, Tamara R. McBrayerb, Steven J. Coatsb, Raymond F. Schinazi,a,*,
and Franck Amblarda,*
HCV EC50 = 1.7 ± 0.8 µM
Huh-7 CC50 > 100 µM
PBMC CC50 > 100 µM
CEM CC50 = 95 µM
Vero CC50 > 100 µM

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Synthesis and anti-HCV activity of a series of β-D-2’-deoxy-2’-dibromo
nucleosides and their corresponding phosphoramidate prodrugs
Zhe Chena, Bryan D. Cox a, Ethel C. Garnier-Amblardb, Tamara R. McBrayerb, Steven J. Coatsb, Raymond
F. Schinazia,∗ and Franck Amblarda,*
aCenter for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics,
Emory University, School of Medicine, Atlanta, Georgia 30322, United States
bCocrystal Pharma, Inc., Tucker, Georgia 30084, United States.
ARTICLE INFO
ABSTRACT
Several ꢀ-D-2’-deoxy-2’-substituted nucleoside analogs have displayed potent and selective anti-
Article history:
Received
Revised
Accepted
Available online
HCV activities and some of them have reached human clinical trials. In that regard, we report
herein the synthesis of a series of 2’-deoxy,2’-dibromo substituted U, C, G and A nucleosides
10a-d and their corresponding phosphoramidate prodrugs 13a-d. The synthesized nucleosides
10a-d and prodrugs 13a-d were evaluated for their inhibitory activity against HCV as well as
cellular toxicity. The results showed that the most potent compound was prodrug 13a, which
exhibited micromolar inhibitory activity (EC50 = 1.5 0.8 µM) with no observed toxicity. In
addition, molecular modeling and free energy perturbation calculations for the 5’-triphosphate
formed from 13a and related 2’-modified nucleotides are discussed.
Keywords:
Synthesis
Nucleoside
Prodrug
Hepatitis C virus
2009 Elsevier Ltd. All rights reserved
.
———
∗ Corresponding authors (RFS and FA) Tel.: +1-404-727-1414; e-mail: rschina@emory.edu and famblar@emory.edu

Hepatitis C virus (HCV) is a blood-borne pathogen infecting
an estimated 180 million subjects worldwide with 55-85% of
infected individuals progressing to chronic HCV infection.1 More
concerning is that approximately 30% of those chronically
infected persons will develop liver cirrhosis and 1-7% of these
will go on to develop hepatocellular carcinoma.2,3 Just a few
years ago, the standard of care for HCV patients was pegylated
interferon-ꢁ (PEG-IFN) and ribavirin (RBV) in combination with
boceprevir, telaprevir, (two first-generation NS3/4A protease
inhibitors) and simeprevir (a second-generation NS3/4A protease
inhibitor), which demonstrated limited e cacy and often
intolerable side e ects.4 Since their initial discovery, nucleoside
analogs, as inhibitors of HCV NS5B polymerase, have been
favored due to their high genetic barrier to drug resistance and
pan-genotypic activity.5 To date, sofosbuvir is the only approved
nucleoside analog for HCV treatment and remains the backbone
of several combination therapies. Compared with previous
treatments, sofosbuvir-based regimens provide a durable cure
rate along with fewer side effects. However, the duration of
approved sofosbuvir treatment regimens remains at 8-12 weeks.6
Therefore, there is still an unmet need to develop novel pan-
genotypic and more e cacious nucleoside analogs, which could
lead to new short to ultra-short combination therapies with
improved safety profiles and higher barrier to resistance.7
complexed to the RNA primer/template reveal dynamic
rearrangement of residues contacting the 2’-OH versus 2’-F
moieties12 and based on the ꢀ-
D-2’-Me, 2’-F-uridine diphosphate
HCV RdRp structure (PDBID 4WTG), we predicted that ꢀ- -2’-
D
di-Br-uridine triphosphate in the active site would incur only
minimal steric clash (Figure 2). However, because reliable
energy calculations require the use of methods that account for
the flexibility in the polymerase active site we next performed
alchemical free energy perturbation (FEP) calculations. FEP
calculates relative binding free energy between two ligands by
mutating one to the other over a series of molecular dynamics
13
simulations in solution and bound to the protein.
FEP
calculations yield accurate ꢂꢂG as the change of binding energy
between the first and second ligands.14 A negative ꢂꢂG value
suggests a gain in binding energy. To evaluate this approach for
2’-modified HCV nucleotide analogs, FEP calculations were
performed on known inhibitors relative to the UTP-bound
complex (PDBID 4WTA) using Desmond MD with the OPLS-
2005 force field on a GPU-enabled EXXACT MD server. It must
be noted that the FEP ꢂꢂG results do not reflect the overall
antiviral activity as many other pharmacologic steps are not
included (cell penetration, prodrug processing, phosphorylation
by host kinases and etc.), nor do these calculations necessarily
recapture the enzymatic IC50 as they do not account for the chain-
terminating chemical steps. However, these calculations predict
how well the 5’-triphosphate analog binds to the active site; a
critical step for antiviral activity. The calculations were
performed in the physiologically relevant tri-anion and tetra-
Inspired by the success of 2’-halogenated nucleoside analogs
such as sofosbuvir 18 or gemcitabine 2,9 several groups, including
ours, studied extensively 2’-dihalogenated nucleosides as
potential anti-HCV agents. Thus, Pinho et al. reported the
anion protonation states.15 The FEP ꢂꢂG for ꢀ-
D-2’-Me,2’-F-
uridine triphosphate (the active form of sofosbuvir) was -2.52
0.18 kcal/mol relative to UTP (Table 1), which agrees with the
potent inhibitory activity of the drug. Two other 2’-halogenated
discovery of
prodrug 3 inhibiting HCV replication 10 while we recently
reported new ꢀ- -2’-Cl, 2’-F-uridine phosphoramidate
a ꢀ-D-2’-deoxy-2’-dichlorouridine nucleotide
a
D
nucleotide 4 as a non-toxic, pan-genotypic, potent and specific
anti-HCV NS5B polymerase agent.11 As part of our continuing
efforts in this area, we report herein the synthesis and antiviral
evaluation of a series of 2’-dibromo nucleosides 10a-d and their
corresponding phosphoramidate prodrugs 13a-d.
analogs recently reported as HCV inhibitors, ꢀ-D-2’-Cl-2’’-F and
ꢀ- -2’-Cl-2’’-Cl -uridine, display negative FEP ꢂꢂG relative to
D
UTP in both protonation states in agreement with their
experimental antiviral activity. Interestingly, the FEP ꢂꢂG for
the proposed 2’-dibromo UTP analog was large and negative for
both tri- and tetra-anions (-2.87 0.26 and -4.21 0.18 kcal/mol,
respectively) suggesting this analog can favorably bind to the
active site relative to UTP thereby meriting synthesis and testing.
O
NH2
NH
N
O
P
O
N
H
O
HO
N
O
N
O
O
O
O
O
Me
F
HO
F
HO
F
sofosbuvir
gemcitabine
2
1
O
O
N
NH
NH
O
P
O
O
O
N
H
O
N
H
P
O
N
O
O
O
O
O
O
O
O
Table 1. Results of FEP calculations on 2’-modified nucleotide
Cl
Cl
triphosphates in the HCV NS5 RdRp active site.
HO
Cl
HO
F
4
3
FEP ∆∆G (kcal/mol)
2’
Group
2”
Group
O
P
O
Tri-anion
Tetra-anion
N
H
O
B
O
O
O
Br
-CH3
-Cl
-F
-F
-2.52
0.18
0.25
0.27
0.26
0.44
-0.67
-3.16
-4.21
0.13
0.23
0.25
0.18
HO Br
13a-d
-0.45
-1.89
-2.87
B = U, C, A and G
Figure 1. Selected ꢀ-D-2’-deoxy-2’-disubstituted nucleoside and nucleotide
analogs and targeted 2’-dibromo nucleotide analogs 13a-d.
-Cl
-Cl
-Br
-Br
To determine if the HCV NS5 RdRp active site can
accommodate 2’-dibromo substitutions, molecular modeling
studies were initiated. Crystal structures of the HCV RdRP

Figure 2. Models of 2’-modified uridine triphosphate analogs in the HCV NS5 active site (PDBID 4WTG) refined using Prime MMGBSA (5 Å cut-off). The
active site surface is rendered in grey, and the Van der Waals radii of the 2’ substitutions are shown as spheres. A) ꢀ- -2’-Me,2’-F-uridine 5’-triphosphate, the
-2’-diCl-uridine 5’-triphosphate (-29.9 kcal/mol), and C) ꢀ- -2’-diBr-uridine 5’-
D
active form of sofosbuvir (Prime MMGBSA ꢂGbind = -25.3 kcal/mol), B) ꢀ-
D
D
triphosphate (-30.7 kcal/mol).
Synthesis of nucleosides 10a-d is described in Scheme 1.
Oxidation of commercially available 2-deoxy-D-ribose 5 with Br2
TBAF, followed by debenzylation in TFA afforded ꢀ isomer 10d
and ꢁ isomer 11d in 30-35% (ratio = 1:1.2).
followed by silylation with tert-butyldimethylchorosilane
(TBDMSCl) yielded the di-silyl protected lactone 6 in 70%
yield.16 Reaction of 6 with 2.2 equivalent of N-bromosuccinimide
(NBS) in presence of LiHMDS gave the lactone 7 in 80% yield.
Reduction of lactone 7 with lithium diisobutylaluminium hydride
(DIBAL-H) provided the desired lactol intermediate, which was
subjected to benzoylation with BzCl in presence of Et3N yielding
benzoylate 8 in 81% yield. Initial glycosylation of 8 with
HO
TBSO
TBSO
O
O
O
OH
O
O
b
a
Br
HO
TBSO
Br
OTBS
5
7
6
TBSO
TBSO
O
O
B
OBz
Br
d
silylated protected nucleobases (Uracil, N4-Bz-cytosine17 , N6-
c
Br
18
diBoc-adenine or N4-diBoc-O6-Bn-Guanine
) to obtain
TBSO
Br
TBSO
9a
Br
B = uracil
9b B = N4-Bz-cytosine
9c
8
nucleoside intermediates 9a-d was attempted using standard
Vorbruggen conditions in the presence of TMSOTf. However,
these coupling reactions, especially for C and G nucleosides (9b
and 9d), needed long reaction times (> 24 h) and elevated
B = adenine
9d B = O6-Bn-guanine
B
HO
HO
H
O
O
e
Br
Br
o
temperatures (> 100 C), which resulted in low yields probably
HO
Br B
HO
Br
due to the instability of these nucleosides to the reaction
conditions. In order to improve the yields for formation of
nucleosides 9a-d, a set of experiments were carried out under
microwave irradiation (MW) using CH3CN as the solvent. After
screening several temperatures and reaction times, it was found
11
10
10a, 11a
B = Uracil
10b, 11b
10c, 11c
B = Cytosine
B = Adenine
10d, 11d B = Guanine
Scheme 1. Reagents and conditions: (a) (i) Br2, H2O, rt, 5 d; (ii) TBDMCl,
imidazole, DMF, rt, 24 h, 70% over two steps; (b) NBS, LiHMDS, THF, -78
oC to -10oC , 4-6 h, 80%; (c) (i) DIBAL-H, toluene, -78 oC, 2 h, rt ; (ii) BzCl,
Et3N, DCM, 0 oC to rt, 12 h, 81%; (d) (i) nucleobase (Uracil, N4-Bz-cytosine,
N6-diBoc-adenine or N4-diBoc-O6-Bn-Guanine), BSA, CH3CN, 60 oC, 30
min; (ii) TMSOTf, CH3CN, MW, 120 oC, 10 min; (e) for 9a and 9c: TBAF,
THF, 0oC, 1 h, 40-50% yield; for 9b: (i) NH3/MeOH, rt, 16-24 h; (ii) TBAF,
THF, 0oC, 1 h, 33% yield; for 9d: (i) TBAF, THF, 0 oC, 1 h; (ii) TFA, rt, 48-
72 h, 33% yield.
o
that the coupling reactions did not proceed at 80 C, while a
messy mixture was obtained when the temperature was increased
to 140 oC. Best results were obtained when reactions were
irradiated at 120 oC for 10 min with a maximum power of 200W.
Under these conditions, compounds 9a-d were obtained as
inseparable mixtures of α/β anomers, in yields ranging from 40%
to 60%. It is noteworthy that the silylated purine nucleobases
need to be Boc protected (N6-diBoc-adenine or N2-diBoc-O6-Bn-
guanine) in order for the glycosyslation step to work however,
these group fall off either during the reaction or during workup.
α
and ꢀ anomers were identified using 1H 2D-NOESY
experiments. For example, clear NOE enhancements were
observed between H5 of the uracil nucleobase and H3’ of the
sugar as well as between H1’ and H4’ for ꢀ -isomer 10a. While
obvious NOE interactions between the H5 and H4’, H1’ and H3’
were observed for its ꢁ-isomer 11a (Figure 2).
Finally, deprotection of compounds 9a-d was performed and
separation of each α and β isomers was achieved with the
unprotected nucleoside analog using preparative reverse phase
HPLC. Thus, treatment of compounds of 9a and 9c with TBAF
in THF gave, after purification, ꢀ isomers 10a and 10c and ꢁ
isomers 11a and 11c as anomer mixtures in 40-50% yield
(10a:11a and 10c:11c ratio: 3:5 and 1:2 respectively).
Debenzoylation of 9b in a saturated solution of NH3 in MeOH,
followed by treatment with TBAF in THF yielded ꢀ isomer 10b
and ꢁ isomer 11b in 30-35% (ratio: 5:7). Desilylation of 9d with

Figure 3. Anomer assignment for nucleosides 10a and 11a via NOE
experiments.
Scheme 2. Synthesis of nucleoside phosphoramidates 13a-d. Reagents and
conditions: (a) tBuMgCl, THF, 0 oC to rt, 24 h, 18-25%.
In order to be a substrate for HCV NS5B, nucleosides analogs
need to be activated to their triphosphate form. This multistep
process is often limited by the first phosphorylation step which
led to the development of nucleoside monophosphate prodrugs.19
These prodrugs allow for the intracellularly delivery of
monophosphate species after enzymatic and/or chemical cleavage
of different masking groups. Among all these prodrugs, the
phosphoramidate/phosphonamidate approach is now, by far, the
most popular and lead to the approval of compounds like
sofosbuvir (HCV) and tenofovir alafenamide (HIV).20 As both of
these compounds are Rp isomers, we have decided to prepare
pure monophosphate prodrugs of nucleosides 10a-d using chiral
pentafluorophenyl reagent 12 synthesized according to Ross et
al’ procedure.21 Thus, reaction of nucleosides 10a-d with 12 in
the presence of tert-butyl magnesium chloride provided the
desired Rp prodrugs 13a-d in 18-25% yield.
Compounds 10a-d and their corresponding phosphoramidate
prodrugs 13a-d were evaluated for inhibition of HCV genotype
1b RNA replication in Huh-7 cells using a subgenomic HCV
replicon system.22 Cytotoxicity in Huh-7 cells was determined
simultaneously by extraction and amplification of both HCV
23
RNA and cellular ribosomal RNA (rRNA).
In addition,
cytotoxicity was determined in primary human peripheral blood
mononuclear (PBM) cells, human lymphoblastoid CEM, and
African Green monkey Vero cells.24,25 None of the nucleosides
synthesized showed antiviral activity at concentration up to 10
µM (10b-d) or 33 µM (10a). However, while prodrugs 13b-d did
not display any anti-HCV activity, uracil prodrug 13a was found
weakly active (EC50 = 1.5 µM) and non-toxic versus all four
tested cell lines. The discrepancy, in term of activity, between
nucleoside 10a and its monophosphate prodrug 13a can most
probably be attributed to a lack of phosphorylation by the cellular
kinases.
In addition, all of the nucleosides 10a-d and prodrugs 13a-d
were evaluated for inhibitory activities against West Nile,
Dengue, Chikungunya, RSV, Influenza, Ebola, Norovirus and
HIV, respectively. However, none of them exhibited obvious
activities when tested up to 10 µM (Data not shown).
Table 2. HCV genotype 1b replicon activity and cytotoxicity of nucleosides 10a-d and their phosphoramidate prodrugs 13a-d
Compound
Anti-HCV activity (µM)
Cytotoxicity, CC50 (µM)
EC50
EC90
Huh-7
PBM
CEM
Vero
> 33
> 10
> 33
> 10
> 33
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 100
> 100
> 100
> 100
> 100
> 100
57
> 100
82
> 100
> 100
> 100
> 100
> 100
> 100
60
10a
10b
10c
10d
13a
13b
13c
13d
> 10
> 10
> 100
> 100
95
> 10
> 10
1.5 0.8
> 10
2.7 0.3
> 10
73
> 10
> 10
33
> 10
> 10
> 100
34
> 100
2’C-Me C
sofosbuvir
1.7 0.8
0.2 0.1
6.5 1.9
0.5 0.3
> 10
> 10
> 100
> 100
> 100
> 100
> 100
> 100
All assays were performed in replicates. Only means + SD of three replicates are shown
In summary, a series of ꢀ-D-2’-deoxy-2’-dibromo substituted
U, C A and G nucleosides 10a-d and their corresponding
phosphoramidate prodrugs 13a-d were synthesized and evaluated
against HCV along with other viruses. Molecular modeling
studies and FEP calculations indicated that the 2’-deoxy-2’-
dibromo sugar ring modification binds favorably to the HCV
RdRp active site. However, all four nucleosides 10a-d and three
prodrugs 13b-d displayed no apparent inhibitory activities
against HCV. Notably, the ꢀ-D-2’-Br2-uridine phosphoramidate
prodrug analog 13a exhibited moderate inhibitory activity against
HCV (EC50= 1.5 0.8 µM). Since FEP calculations suggest that
this agent may be a potent substrate to the HCV RdRp, the
modest activity is likely due to other limiting factors unaccounted
for by the model (such as prodrug processing or low host kinase
phosphorylation). Despite the weak potency of prodrug 13a as
HCV inhibitor, further modifications of these 2’-

dihalogenonucleosides are currently being investigated and will
be subject of future publications.
24 Schinazi, R. F.; Sommadossi, J. P.; Saalmann, V.; Cannon, D. L.; Xie, M.-
W.; Hart, G. C.; Smith, G. A.; Hahn, E. F. Antimicrob. Agents Chemother.
1990, 34, 1061-1067.
Acknowledgments
25 Stuyver, L. J.; Lostia, S.; Adams, M.; Mathew, J.; Pai, B. S.; Grier, J.;
Tharnish, P.; Choi, Y.; Chong, Y.; Choo, H.; Chu, C. K.; Otto, M. J.;
Schinazi, R. F. Antimicrob. Agents Chemother. 2002, 46, 3854-3860.
This work was supported in part by NIH grant 5P30-AI-50409
(CFAR). Dr. Schinazi is the Chairman and a major shareholder of
Cocrystal Pharma, Inc. Emory received no funding from
Cocrystal Pharma, Inc. to perform this work and vice versa.
Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at http://xxxx.
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