Adenosine dioxolane nucleoside phosphoramidates as antiviral agents for human immunodeficiency and hepatitis B viruses

Article abstract of DOI:10.1021/ml4001497

There are currently six nucleoside reverse transcriptase inhibitors (NRTI) that are FDA approved for human clinical use and these remain the backbone of current HIV therapy. In order for these NRTIs to be effective they need to be phosphorylated consecutively by cellular kinases to their triphosphate forms. Herein, we report the synthesis of C-6 modified (-)-β-d-(2R,4R)-1,3- dioxolane adenosine nucleosides and their nucleotides including our novel phosphoramidate prodrug technology. We have introduced a side chain moiety on the phenol portion of the phosphoramidate to reduce the toxicity potential. The synthesized phosphoramidates displayed up to a 3600-fold greater potency versus HIV-1 when compared to their corresponding parent nucleoside and were up to 300-fold more potent versus HBV. No cytotoxicity was observed up to 100 μM in the various cell systems tested, except for compounds 17 and 18, which displayed a CC₅₀ of 7.3 and 12 μM, respectively, in Huh-7 cells. The improved and significant dual antiviral activity of these novel phosphoramidate nucleosides was partially explained by the increased intracellular formation of the adenosine dioxolane triphosphate.

Full text of DOI:10.1021/ml4001497

Letter
pubs.acs.org/acsmedchemlett
Adenosine Dioxolane Nucleoside Phosphoramidates As Antiviral
Agents for Human Immunodeficiency and Hepatitis B Viruses
Lavanya Bondada,† Mervi Detorio,† Leda Bassit,† Sijia Tao,† Catherine M. Montero,†
Tyana M. Singletary,† Hongwang Zhang,† Longhu Zhou,† Jong-Hyun Cho,† Steven J. Coats,*,‡
and Raymond F. Schinazi*,†
†Center for AIDS Research, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of
Medicine, and Veterans Affairs Medical Center, Decatur, Georgia 30033, United States
‡RFS Pharma, LLC, Tucker, Georgia 30084, United States
S
* Supporting Information
ABSTRACT: There are currently six nucleoside reverse
transcriptase inhibitors (NRTI) that are FDA approved for
human clinical use and these remain the backbone of current
HIV therapy. In order for these NRTIs to be effective they
need to be phosphorylated consecutively by cellular kinases to
their triphosphate forms. Herein, we report the synthesis of C-
6 modified (−)-β-D-(2R,4R)-1,3-dioxolane adenosine nucleo-
sides and their nucleotides including our novel phosphor-
amidate prodrug technology. We have introduced a side chain
moiety on the phenol portion of the phosphoramidate to
reduce the toxicity potential. The synthesized phosphorami-
dates displayed up to a 3600-fold greater potency versus HIV-1 when compared to their corresponding parent nucleoside and
were up to 300-fold more potent versus HBV. No cytotoxicity was observed up to 100 μM in the various cell systems tested,
except for compounds 17 and 18, which displayed a CC50 of 7.3 and 12 μM, respectively, in Huh-7 cells. The improved and
significant dual antiviral activity of these novel phosphoramidate nucleosides was partially explained by the increased intracellular
formation of the adenosine dioxolane triphosphate.
KEYWORDS: Dioxolane adenosine nucleoside, nucleoside reverse transcriptase inhibitor, phosphoramidate prodrug, ProTide, HIV,
HBV
synthesized and evaluated by the Chu and Schinazi group.7 It
ucleoside reverse transcriptase inhibitors (NRTI) are the
Nbackbone of current HIV highly active antiretroviral
has been shown that some nucleoside analogues are poor
substrates for phosphorylation kinases that convert the
therapy (HAART).1 However, resistance and toxicity with
nucleoside analogue to the corresponding nucleoside mono-
phosphate.8 In order to bypass this rate-limiting step,
nucleoside phosphoramidate derivatives were developed as
more lypophilic monophosphate prodrugs. The phosphorami-
date strategy bypasses the initial nucleoside kinase step, by
intracellular delivery of the monophosphorylated nucleoside
analogue.9−11 The phosphoramidate strategy has been shown
to improve the pharmacological activity of parent nucleosides
both in vitro and in vivo.12−16
Recently, several reports have demonstrated that phosphor-
amidates of dioxolane sugar nucleosides with both purines17 as
well as pyrimidines18,19 improved the in vitro antiviral potency.
These reports demonstrated that phosphoramidates of these
dioxolane analogues displayed enhancement of anti-HIV
activity (100 fold) and anti-HBV activity (38 fold) when
chronic use of NRTI are still a major concern, as is true for all
classes of HIV drugs.2−4 Therefore, exploration of novel
nucleosides with the goal of improved efficacy, resistance
profile, and safety are warranted. Amdoxovir (DAPD), a
prodrug of (−)-β-D-2-amino-6-hydroxy dioxolane guanosine
(DXG) is currently in phase 2 clinical development under a
U.S. Food and Drug Administration investigational new drug
application (Figure 1).5,6
(−)-β-D-(2R,4R)-6-amino dioxolane adenosine (dioxolane-
A) is an adenosine analogue that has been previously
Received: April 19, 2013
Accepted: June 27, 2013
Figure 1. Structure of antiviral dioxolane nucleosides.
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
compared with the parent nucleoside, without an increase in
cytotoxicity. These findings prompted us to explore the
dioxolane series of 6-substituted-2-H-purine nucleosides and
their corresponding phosphoramides.
Scheme 2a
Surprisingly, adenine substituted dioxolane nucleoside
phosphoramidates have not been previously explored. In our
study, the objective was to identify analogues with better
antiviral potency with no enhancement of cytotoxicity. The 6-
modified purine analogues were prepared as more lipophilic
adenine precursors, which would require a two step intracellular
conversion to the adenosine dioxolane analogue. The (−)-β-D-
(2R,4R)-1,3-dioxolane purine modified nucleosides were
synthesized from key intermediate (−)-β-D-1,3-dioxolanyl-2,6-
dichloropurine, 2, which was obtained by the glycosylation
reaction of silylated 2,6-dichloropurine with acetoxy dioxolane
derivative 1 (Scheme 1).20
a
Reagents and conditions: (a) Et3N, THF, −5 to 20 °C.
Scheme 1a
16. Subsequent reaction with the dioxolane nucleoside
analogues provided the desired 5′-phosphoramidates 17−24
as an approximate 1:1 mixture of phosphorus diastereomers
(Scheme 3).
Scheme 3a
a
Reagents and conditions: (a) Ref 20; (i) HMDS, NH4SO4,
nucleoside base; (ii) TMSI, CHCl3, −10 °C; (b) (i) nucleophile,
THF, reflux; (ii) NH3/CH3OH, CH3OH, rt, 12 h; (c) Pd/C, H2,
NaOH, 20 h.
Compound 3 was synthesized by the reaction of 2 with
methanolic ammonia at room temperature for 10 h to give
nucleoside 3 (2-chloro-6-amino) in 63% yield and nucleoside 4
(2-chloro-6-methoxy) in 15% yield. Compound 2 was reacted
with methanol in the presence of K2CO3 at room temperature
for 1 h and gave 4 in 90% yield and 2,6-dimethoxynucleoside in
5% yield. Compound 5 was synthesized in 79% yield by the
reaction of compound 2 with cyclopropylamine in THF at 80
°C for 4 h, followed by treatment with methanolic ammonia at
room temperature to facilitate removal of the 5′-isobutyl ester
group.
Catalytic hydrogenation of the 2-chloro derivatives 3, 4, and
5 was accomplished by dissolving the nucleoside in methanol in
the presence of 2 N NaOH, in an atmosphere of hydrogen in
the presence of catalytic palladium on carbon.21 2-Unsub-
stituted nucleosides 6, 7, and 8 were obtained in 82%, 80%, and
79% yields, respectively (Scheme 2).
Phosphoramidates were synthesized by the reaction of
dioxolane nucleoside analogues 6, 7, and 8 with suitably
substituted phosphoryl chlorides 14, 15, or 16, which were
prepared following previously described chemistry.9 Accord-
ingly, a suitably substituted phenol was reacted with
phosphorus oxychloride to afford an aryloxy phosphoroyl
dichlorides 9, 10, or 11, which were subsequently reacted with
an amino acid ester (ethyl 12 or isopropyl 13) in the presence
of Et3N in THF to afford aryloxy phosphorochloridates 15 and
a
Reagents and conditions: t-BuMgCl, THF, rt, 18 h.
The typical utilization of the phophoramidate prodrug
approach involves the intracellular delivery of phenol. Phenol
is used in some over-the-counter products as an oral
anesthetic/analgesic and commonly used to temporarily treat
pharyngitis (inflammation of the throat). Phenol may cause
harmful effects on the central nervous system, kidneys, and
heart, resulting in dysrhythmia, seizures, and coma.22−24 The
reported LD50 for phenol is 270 mg/kg in mouse, while chronic
exposure in humans may have harmful effects on the liver and
kidneys. We sought to overcome these potential liabilities by
the introduction of a propyl ester side chain at the ortho
postion of the phenol portion of our prodrugs. Cellular
pharmacology in human peripheral blood mononuclear (PBM)
cells showed that unmasking to the monophosphate from this
substituted phosphoramidate results in the formation of
hydroxyphenyl propanoic acid, which is a known nontoxic
B
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Table 1. Antiviral Activity and Cytotoxicity of C-6 Modified Dioxolane-A Nucleosides and Their Corresponding
Phosphoramidates
metabolite25 of dihydrocoumarin, a common flavoring agent
widely used in food26 and cosmetics.27
compounds based on their antiviral profile also displayed
cytotoxicity in one or more of the five tested cell lines that
ranged from a CC50 of 2 to 84 μM. Compound 22 with a 6-
cyclopropyl amine group was the exception and was overall the
best compound with an EC50 of 0.086 μM toward HIV and an
EC50 of 0.8 μM toward HBV and no observed cytotoxicity in
the five tested cell systems.
The antiviral activities of the synthesized nucleosides and
their phosphoramidates were evaluated versus HIV and HBV.
The HIV-1 susceptibility assays were performed using human
PBM cells infected with HIV-1LAI.
28−31 Antiviral activity against
HBV was determined using the HepAD38 cell system, as
described previously.28,32 Cytotoxicity was assessed in human
PBM cells, Vero cells (kidney epithelial cells from the African
green monkey), and CEM cells (a human-T-cell-derived cell
line) as described previously.28,31 The antiviral activity and
cytotoxicity in five cell systems for the C-6 modified dioxolane-
A nucleosides and their phosphoramidates are summarized in
Table 1.
The dioxolane-A analogue 6 (2-H, 6-amino) was found to be
significantly less active versus HIV when compared to the
related clinical compound DAPD; however, their anti-HBV
(low micromolar) and cytotoxic profile (no cyctotoxicity up to
100 μM) were quite similar. Related analogues 7 (2-H, 6-
methoxy) and 8 (2-H, 6-cyclopropylamino) were essentially
not active versus HIV and HBV but also did not display any
cytotoxicity in any of the five tested cell lines. The
corresponding phosphoramidate prodrugs 17, 18, 19, 20, and
22 were up to 3600-fold more active then their parent
nucleosides 6, 7, and 8. Unfortunately, the most interesting
As a first step toward understanding the antiviral data
obtained with these compounds, we undertook a study of their
cellular pharmacology in PBM cells (Table 1). We determined
the levels of nucleoside triphosphates formed and also defined
the relationship between intracellular triphosphate levels and
antiviral activity. Compounds 6 (dioxolane-A), 17, 18, 22, and
23 were evaluated in PBM cells and all were found to form the
dioxolane-A triphosphate. Prodrugs 17 and 18 were found to
give the highest levels of dioxolane-A triphosphate, which
correlates to their exceptional antiviral potency. Interestingly,
the 6-cyclopropylamino prodrugs 22 and 23 were also
converted to the 6-cyclopropylamino purine dioxolane
triphosphate. The effect of this newly identified triphosphate
on antiviral activity is currently under investigation and will be
reported elsewhere.
In conclusion, the phosphoramidate prodrug approach alone
and in combination with modification at the C-6 position of the
purine ring resulted in a significant enhancement of the anti-
C
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ASSOCIATED CONTENT
* Supporting Information
■
S
Biological assays and complete experimental section with full
characterization of all new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*(S.J.C.) Tel: 404-601-1427. E-mail: scoats@rfspharma.com.
(R.F.S.) Tel: 404-728-7711. E-mail: rschina@emory.edu.
■
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Funding
This work was supported in part by CFAR grants NIH 5P30-
AI-50409 and 5R37-AI-025899 and the Department of
Veterans Affairs (to R.F.S.).
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Notes
The authors declare the following competing financial
interest(s): RFS is the founder and a major shareholder in
RFS Pharma, LLC. Dioxolane nucleosides he invented have
been licensed to RFS Pharma, LLC and he may receive future
royaties from these products.
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