Synthesis and antiviral evaluation of 2′,2′,3′,3′-tetrafluoro nucleoside analogs

Article abstract of DOI:10.1016/j.tetlet.2017.01.006

Herein, we report the synthesis of novel 2′,2′,3′,3′-tetrafluorinated nucleoside analogs along with their phosphoramidate prodrugs. A tetrafluoro ribose moiety was coupled with different Boc/benzoyl-protected nucleobases under Mitsunobu conditions. After deprotection, tetrafluorinated nucleosides 13b, 14b, 20b-22b were reacted with phenyl-(isopropoxy-L-alaninyl)-phosphorochloridate to afford corresponding monophosphate prodrugs 24b–28b. All synthesized compounds were evaluated against several DNA and RNA viruses including HIV, HBV, HCV, Ebola and Zika viruses.

Full text of DOI:10.1016/j.tetlet.2017.01.006

Tetrahedron Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis and antiviral evaluation of 20,20,30,30-tetrafluoro nucleoside
analogs
Ozkan Sari a, Leda Bassit a, Christina Gavegnano a, Tamara R. McBrayer b, Louise McCormick a, Bryan Cox a,
Steven J. Coats b, Franck Amblard a, Raymond F. Schinazi a,
⇑
a Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
b Cocrystal Pharma, Inc., 1860 Montreal Road, Tucker, GA 30084, USA
a r t i c l e i n f o
a b s t r a c t
Herein, we report the synthesis of novel 20,20,30,30-tetrafluorinated nucleoside analogs along with their
phosphoramidate prodrugs. A tetrafluoro ribose moiety was coupled with different Boc/benzoyl-pro-
tected nucleobases under Mitsunobu conditions. After deprotection, tetrafluorinated nucleosides 13b,
Article history:
Received 29 November 2016
Revised 23 December 2016
Accepted 2 January 2017
Available online xxxx
14b, 20b-22b were reacted with phenyl-(isopropoxy-L-alaninyl)-phosphorochloridate to afford corre-
sponding monophosphate prodrugs 24b–28b. All synthesized compounds were evaluated against several
DNA and RNA viruses including HIV, HBV, HCV, Ebola and Zika viruses.
Ó 2017 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside
Prodrug
Antiviral
Fluorine
Mitsunobu
Incorporation of fluorine(s) into potential drug candidates con-
tinues to be investigated by medicinal chemists and has shown the
potential to confer favorable biological properties.1 Due to the sim-
ilarity in size of fluorine (147 pm) and hydrogen (120 pm), the sub-
stitution of a single hydrogen atom by a fluorine atom induces only
a minor change in steric factors. However, being the most elec-
tronegative atom in the periodic table, its incorporation results in
considerable electronic changes in the molecule through inductive
electron withdrawing effects. Furthermore, introduction of one or
more atoms of fluorine (e.g., CF2) into a molecule is a viable bioisos-
teric replacement for a diverse set of functional groups such as
carbonyl, ether and hydroxyl groups. Finally, a C-F bond is much
stronger than a C-H bond, which can increase the chemical and
biological stability of the fluorine-containing compounds, com-
pared to their hydrogen counterparts.
FEAU, FMAU exhibit potent activities against herpes simplex virus
(HSV), hepatitis B virus (HBV), varicella zoster virus (VZV), cytome-
galovirus (CMV) and Epstein-Barr virus (EBV).3
Based on these precedents, we turned our attention toward a
unique series of 20,20,30,30-tetrafluorinated nucleoside analogs. Even
though this type of highly fluorinated compounds is claimed in a
patent published in 2000,4 their preparation is only described in
a generic scheme and with no mention of any biological evaluation.
Therefore, we report herein the detailed synthesis and antiviral
evaluation of, not only these compounds, but also their phospho-
ramidate prodrugs.
2,2,3,3-Tetrafluorodideoxy ribose derivative 7 was prepared in
four steps from commercially available 4-bromo-3,3,4,4-tetrafluo-
robut-1-ene 4 following a procedure reported by Linclau et al.5
(Scheme 1). It is noteworthy that the Sharpless dihydroxylation
of the perfluorinated substrate 4 is not entirely enantioselective
(reported ee 78%, observed 60%). Consequently, the 40-position of
the lactol 7, obtained after cyclization of the formylated intermedi-
In the field of nucleoside analogs,2 introduction of fluorine has
proven to be successful and has led to several very widely pre-
scribed drugs such as (-)-FTC (HIV), gemcitabine (cancer), or more
recently sofosbuvir (hepatitis C virus, HCV) (Fig. 1). In addition to
the approved fluorinated nucleosides discussed above, several flu-
orine-containing analogs have been reported in the literature to
have attractive antiviral profiles, although some proved too toxic
for human use. Thus, 20-b-fluoro nucleosides analogs such as FIAC,
ate 6, is isolated as a 8:2 mixture at the 40-position while the
a/b
anomers ratio is 1:1.5. The separation of the diastereomers was
not attempted at this stage. Hence, with 7 in hand, the glycosyla-
tion reaction was studied and different approaches were evaluated
in order to identify the most efficient coupling conditions.
Vorbrüggen type coupling of 10-benzoyl, 10-mesyl or 10-triflate
tetrafluorinated ribose derivative 7 with either silylated uracil or
cytosine in presence of SnCl4 (or TMSOTf) only led to the complete
⇑
Corresponding author.
E-mail address: rschina@emory.edu (R.F. Schinazi).
http://dx.doi.org/10.1016/j.tetlet.2017.01.006
0040-4039/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Sari O., et al. Tetrahedron Lett. (2017), http://dx.doi.org/10.1016/j.tetlet.2017.01.006

2
O. Sari et al. / Tetrahedron Letters xxx (2017) xxx–xxx
Scheme 3. Reagents and conditions: (a) 6-chloropurine, DIAD, PPh3, THF, 2 h, rt,
34%; (b) NH3/MeOH, 80 °C, 16 h, 86%.
Figure 1. Structures of FDA approved fluorine containing nucleosides 1–3 and
targeted 20,20,30,30-tetrafluoro derivatives.
Scheme 4. Reagents and conditions: (a) Protected base (Bp), DIAD, PPh3, THF,
ꢀ20 °C to rt, 2 h (overall yields for both anomers); (b) BCl3, DCM, ꢀ20 °C to rt, 3.5 h.
temperatures led to the formation of several unidentified side
products as determined by LC-MS. The isolated b anomers 9b
and 10b were debenzoylated using methanolic ammonia to afford
intermediates 11b (91%) and 12b (96%), which were subsequently
treated with BCl3 to give uracil and thymine analogs 13b (94%) and
14b (85%), respectively. Preparation of the adenine analog was
Scheme 1. Reagents and conditions: (a) K2OSO4ꢁ2H2O, (DHQ)2Pyr, K3Fe(CN)6,
t-BuOH/H2O, 4 °C, 9 days, 81%; (b) (i) Bu2SnO, TBAI, BnBr, toluene, reflux, 82%; (ii)
Formic acid, DCC, DMAP, CH2Cl2, rt, 16 h, 95%; (c) MeLi, THF, ꢀ78 °C, 3 h, 62%; (d)
BzCl, Et3N, CH2Cl2, rt, 1.5 h, 92% or MsCl, Et3N, CH2Cl2, 0 °C to rt, 16 h, 74% or Tf2O,
pyr, 0 °C to rt, 16 h, quant.
initially attempted by coupling of protected lactol
7 with
6-chloropurine using the Mitsunobu conditions described above
(Scheme 3). However, even though the desired coupling product
15 was obtained in 34% yield, separation of the a/b isomers, at this
stage, or after 6-amination of the purine ring (compound 16), was
found to be challenging.
Therefore, we then examined the coupling of lactol 7 with bis-
Boc adenine.6 Under classical Mitsunobu conditions, the reaction
afforded products 17a–b in 58% yield (Scheme 4) as a silica gel
recovery of starting material 8. Attempts to directly react the 10-tri-
flate derivative 8c with the sodium salt of N3-benzoyluracil was
unsuccessful as well.
In contrast, treatment of compound 7 with N3-benzoyluracil or
N3–benzoylthymine under classical Mitsunobu conditions (DIAD
and PPh3 in THF) afforded the desired coupling products 9a–b
(60%) and 10a–b (68%) respectively as a silica gel separable mix-
separable mixture of
a/b anomers (1.5:1). Further treatment of
the b-anomer 17b with BCl3 afforded adenine nucleoside analog
20b in 96% yield. Likewise, the coupling with bis-Boc-2-amino-6-
benzolyloxypurine7 afforded a mixture of anomers 18 (48%) that
were separable by silica gel chromatography. The b-isomer 18b
was then fully deprotected in one step to give 21b in 40% yield
using BCl3. Finally, the preparation of cytosine analogs 19a–b
was performed in a similar manner using bisBoc-cytosine.8 The
separated b-anomer 19b was subsequently deprotected to afford
nucleoside analog 22b in 82% yield.
ture of
a/b anomers (ratio: 1.5:1) (Scheme 2). Remarkably, the
desired coupling products 9 and 10 were only observed when DIAD
was added slowly at ꢀ20 °C. Performing this reaction at higher
Identification of both
a
and b anomers was determined by 1H
NMR and 2D-NOE experiments after Mitsunobu coupling. In all
cases, clear correlations were observed between 10-protons and
40-protons of b anomers while
a anomers exhibited correlations
between 10-protons and 50-protons. Additionally, in the case of
purine analogs 17 and 18, correlations between 8-protons and
50-protons were also noted (Fig. 2).
In order to express their therapeutic effect, nucleoside analogs
usually rely on cellular kinases to be phosphorylated onto their
active 50-triphosphate forms. Among the three consecutive phos-
phorylations, the first phosphate addition has often been identified
as the limiting step which led to the development of ‘‘protected”
monophosphate nucleosides, or nucleoside prodrugs, that can by-
pass this first activation step.9 Unlike, 50-mono, di or triphosphate
nucleosides, theses prodrugs are capable of crossing the cell mem-
brane to deliver a monophosphate nucleoside after enzymatic
Scheme 2. Reagents and conditions: (a) N3-BzU or N3-BzT, DIAD, PPh3, THF, ꢀ20 °C
to rt, 2 h (overall yields for both anomers); (b) NH3, MeOH, 4 °C, 2 h; (c) BCl3, DCM,
ꢀ20 °C to rt, 3.5 h.
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O. Sari et al. / Tetrahedron Letters xxx (2017) xxx–xxx
3
oro-20,30-dideoxyribose U and A analogs preferred the 30-endo
conformation by 0.4–1.2 kcal/mol. This result suggests that the
tetrafluorinated ribose moiety might be a closer mimic of a ribose
rather than a 20,30-dideoxyribose.
Consequently, nucleosides 13b, 14b, 20b–22b and their corre-
sponding phosphoramidate prodrugs 24b–28b were evaluated
against a panel of DNA and RNA viruses. Hence, the screening
was performed against HIV-112 up to 100 and HBV12
lM
(HepAD38), HCV13 (clone B replicon), Ebola14 (Zaire ebolavirus
replicon) and Zika15 (cytopathic effect reduction assay) viruses
Figure 2. Anomer assignment for compound 18a and 18b via NOE experiments.
up to 20 lM. In addition, cytotoxicity was determined in primary
human peripheral blood mononuclear (PBM) cells, human lym-
phoblastoid CEM, African Green monkey Vero cells and HepG2
cells.16 Except for compound 26b that exhibited slight toxicities
degradation of their biolabile group. The concept of monophos-
phate or phosphonate prodrugs has been clinically validated with
the FDA-approval of drugs such as sofosbuvir 3 (HCV) or tenofovir
disoproxyl fumarate (TDF) (HIV and HBV).
in human PBM (CC50 = 27
of these derivatives exhibited significant activity against these
viruses nor toxicities in PBM, CEM and Vero cells up to 100 M.
lM) and CEM (CC50 = 14 lM) cells, none
Thus, 50-monophosphate prodrugs of nucleosides 13b, 14b and
20b-22b were prepared by reaction with the chloro phosphorami-
date 23 via two different strategies, depending on the nature of the
nucleobase (Scheme 5).10 For free-NH2 containing analogs, such as
adenine, guanine and cytosine derivatives 20b, 21b and 22b,
t-BuMgCl was used to favor alkoxide formation and minimize side
reactions with the amino group while the pyrimidine prodrugs 24b
and 25b were prepared by treatment of 13b and 14b with
N-methylimidazole (NMI). Full conversions were usually observed
after 3 h at room temperature affording prodrugs 24b–28b, as a RP
and SP mixture, in yields ranging from 49% to 64%. Due to the fact
that the synthesis of the tetrafluorinated sugar 7 is not completely
diastereoselective at the 40-position, 31P NMR spectra for these
compounds indicated the presence of four non separable
diastereoisomers in a 4:1:1:4 ratio.
l
In conclusion, five new 20,20,30,30-tetrafluorodideoxy nucleoside
analogs along with their corresponding phosphoramidate prodrugs
were synthesized from tetrafluorinated lactol 7 using a key
Mitsunobu reaction. We demonstrated that the use of Boc or
Bz-protected bases as glycosylation partners in this reaction
facilitated the separation of both
a and b anomers. None of the syn-
thesized compounds showed marked activity when tested against
HIV-1, HBV, HCV, Ebola or Zika viruses.
Acknowledgments
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.
To determine the effects of the tetrafluoro substitution, the
energetics of ring conformations were investigated using quantum
mechanical calculations (Fig. 3).
A. Supplementary material
Consistent with previous data, the ribose and 20-deoxyribose
rings exhibited lower energies in the 30-endo and 20-endo conform-
ers, respectively.11 The 30-endo and 20-endo conformers were equal
in energy for the 20,30-dideoxyribose U and A analogs. The tetraflu-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2017.01.006.
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Please cite this article in press as: Sari O., et al. Tetrahedron Lett. (2017), http://dx.doi.org/10.1016/j.tetlet.2017.01.006