Antiviral activity of novel 2'-fluoro-6'-methylene-carbocyclic adenosine against wild-type and drug-resistant hepatitis B virus mutants

Article abstract of DOI:10.1016/j.bmcl.2011.08.113

Novel 2'-fluoro-6'-methylene-carbocyclic adenosine (9) was synthesized and evaluated its anti-HBV activity. The titled compound demonstrated significant antiviral activity against wild-type as well as lamivudine, adefovir and double lamivudine/entecavir r

Full text of DOI:10.1016/j.bmcl.2011.08.113

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6328–6331
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antiviral activity of novel 20-fluoro-60-methylene-carbocyclic adenosine
against wild-type and drug-resistant hepatitis B virus mutants
Jianing Wang a, Uma S. Singh a, Ravindra K. Rawal a, Massaya Sugiyama b, Jakyung Yoo a, Ashok K. Jha a,
Melissa Scroggin a, Zhuhui Huang c, Michael G. Murray c, Rajgopal Govindarajan a, Yasuhito Tanaka b,
Brent Korba d, Chung K. Chu a,
⇑
a The University of Georgia, College of Pharmacy, Athens, GA 30602, USA
b Nagoya City University, Graduate School of Medical Sciences, Nagoya 467-8601, Japan
c Southern Research Institute, Frederick, MD 21701, USA
d Georgetown University Medical Center, Washington, DC 20057, USA
a r t i c l e i n f o
a b s t r a c t
Novel 20-fluoro-60-methylene-carbocyclic adenosine (9) was synthesized and evaluated its anti-HBV
activity. The titled compound demonstrated significant antiviral activity against wild-type as well as
lamivudine, adefovir and double lamivudine/entecavir resistant mutants. Molecular modeling study indi-
cate that the 20-fluoro moiety by a hydrogen bond, as well as the van der Waals interaction of the carbo-
cyclic ring with the phenylalanine moiety of the polymerase promote the positive binding, even in the
drug resistant mutants.
Article history:
Received 29 June 2011
Revised 25 August 2011
Accepted 26 August 2011
Available online 1 September 2011
Keywords:
Carbocyclic nucleoside
anti-HBV
Published by Elsevier Ltd.
Drug-resistant
Chronic hepatitis B virus (HBV) infection is one of the leading
causes of morbidity and mortality worldwide. Chronic infection
with HBV occurs in approximately 350 million of the world popula-
tion, including 1.7 million in the USA.1 HBV infection can persist for
the life of the host, often leading to severe consequences such as liver
failure, cirrhosis and eventually hepatocellular carcinoma, resulting
in annually 0.5–1.2 million deaths worldwide.2 HBV is an incom-
plete double-stranded DNA virus. Its DNA replication is unique be-
cause it includes a reverse transcription step. The HBV DNA
polymerase/reverse transcriptase is an essential and multifunc-
tional enzyme, which operates as a DNA polymerase/reverse trans-
criptase, an RNAse H, through coordinating the assembly of viral
nucleocapsids, as well as catalyzing the generation of DNA primers.3
Nucleoside analogues can suppress HBV replication by inhibiting
the viral polymerase/reverse transcriptase. The pivotal role of nucle-
oside/nucleotide analogues such as lamivudine, adefovir, telbivu-
dine, entecavir, clevudine, and tenofovir has been demonstrated
by their therapeutic efficacy in clinical practice. However, long-term
therapy with these drugs is often associated with viral resistance,
which significantly compromises the clinical application of these
agents. For example, the extensive use of lamivudine resulted in
the emergence of mutants that are resistant to the anti-HBV activity;
24% after a 1-year therapy, increasing to over 70% after 4 years of
therapy. Adefovir has been used for the patients, who develop
lamivudine-resistant mutants, however, a significant number of
patients (29% after 5 years of use) also develop the adefovir resistant
mutant (N236T).
Entecavir is a carbocyclic 20-deoxyguanosine analog that dem-
onstrates potent anti-HBV activity4 and is recommended for pa-
tients with the wild-type strain as well as for those patients
harboring lamivudine-resistant strains.5 However, a recent study
by Tanaka and his co-workers suggest that the viral breakthrough
was observed in the lamivudine-refractory group in 4.9% of
patients at baseline and increase to 14.6%, 24% and 44.8% at weeks
48, 96 and 144, respectively.6
In view of the fact that currently adefovir and entecavir are the
most prescribed drugs for the treatment of chronic HBV infection,
it is critical to discover the agents that do not confer cross-
resistance with the adefovir and lamivudine/entecavir-mutants
for the future treatment of drug resistant patients. In this report
we try to demonstrate that our newly discovered compound 9
may potentially play a significant role for that purpose.
Carbocyclic nucleosides are an interesting class of compounds
in which the methylene group replaces the oxygen atom of a fura-
nose ring. As a consequence, the glycosidic bond is resistant to
nucleoside phosphorylase as well as nucleoside hydrolase, which
makes the carbocyclic nucleosides more stable towards metabolic
⇑
Corresponding author. Address: Department of Pharmaceutical and Biomedical
Sciences, College of Pharmacy, The University of Georgia, Athens, GA 30602, USA.
Tel.: +1 706 542 5379; fax: +1 706 542 5381.
E-mail addresses: DCHU@rx.uga.edu, DCHU@mail.rx.uga.edu (C.K. Chu).
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2011.08.113

J. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6328–6331
6329
Scheme 1. Synthesis of target compound 9. Reagents and conditions: (a) NaH, BnBr, DMF, 0 °C; (b) TFA/H2O (2:1), 50 °C; (c) TIDPSCl2/imidazole, DMF, 0 °C; (d) DAST, CH2Cl2,
rt; (e) TBAF/AcOH, THF, rt; (f) BzCl, pyridine, rt; (g) BCl3, CH2Cl2, ꢀ78 °C; (h) N,N-dibocprotected adenine, DIAD, Ph3P, THF, 0 °C; (i) TFA, CH2Cl2, rt; (j) DIBAL-H, CH2Cl2, ꢀ78 °C.
degradation.7 Due to these features, carbocyclic nucleosides have
received much attention as potential chemotherapeutic agents.8
Carbovir and entecavir are examples of results of these efforts.
It is also well known that incorporation of a fluorine atom at the
20-position of nucleosides can increase the stability of the glycosyl
bond towards chemical and metabolic degradation.9,10 A fluorine
substitution on the carbocyclic sugar moiety has been proven to
be useful in producing effective antiviral agents as demonstrated
and lamivudine/entecavir-drug resistant mutants in vitro,16 and
the results are summarized in Table 1. As the compound 9 is a
derivative of an adenine analog, we directly compared its antiviral
activity to that of adefovir instead of entecavir (a guanine analog)
although the carbocyclic moiety is similar to that of entecavir. Fur-
thermore, compound 9, an adenine analogue, can interact with the
thymidine moiety in the DNA template–primer site while entecavir
interacts with the cytosine moiety at the same site in the active
site. Thus, the base moiety is the major deciding factor, not the
sugar moiety in determining the mode of action.
by our group in 20-fluoro-5-methyl-b-
L-arabinofuranosyluracil
(L-FMAU or clevudine)11 as well as in clofarabine.12
In view of the 20-F substitution9,10 as well the introduction of an
exocyclic double bond to carbocyclic nucleosides,4 which have
been beneficial for anti-HBV activity such as in entecavir, 20-flu-
oro-60-methylene-carbocyclic adenosine or (+)-9-[(1R,2R,3R,4R)-
2-fluoro-3-hydroxy-4-(hydroxymethyl)-5-methylenecyclopentan-
1-yl] adenine 9 was synthesized and evaluated for its antiviral
activity against wild-type HBV as well as adefovir, lamivudine
and lamivudine/entecavir (double)-resistant mutants in vitro.
The synthesis of the target nucleoside 9 commenced with com-
pound 7 as the key intermediate (Scheme 1). Compound 1 was syn-
thesized according to the reported procedure from our group.13
The allylic hydroxyl group of 1 was protected with a benzyl group
and subsequent deprotection of the acetonide and the t-butyl
group of compound 2 gave 3 in 86% yield. The 3, 5-hydroxy groups
of 3 were selectively protected with 1,3-dichloro-1,1,2,2-tetraiso-
propyl disilazine to give 4 in 95% yield. Transformation of the 2-
The target compound 9 demonstrated a significant antiviral
in vitro activity against wild-type (WT) HBV with an EC50 value
of 1.5 lM. The antiviral potency was similar to that of adefovir,
while being 7-fold less potent than lamivudine. However, the con-
centration of the compound 9 required to inhibit 90% (EC90) of
wild-type HBV is 4.5
lM, which is 1.5-fold more potent than
adefovir (EC50 7.1 M; Table 1).
l
The compound 9 also showed excellent activity against both
lamivudine and adefovir resistant HBV mutants.17 Particularly,
the compound 9 showed a 4.5-fold enhanced potency of EC50
(1.7 lM) and a 7.8-fold more favorable EC90 (4.6 lM) against ade-
fovir mutant rtN236T. For lamivudine mutants, rtM204V and
rtM204I, the compound 9 showed an EC50 value of 1.8 versus
1.6 lM for adefovir, and 1.0 versus 1.9 lM for compound 9 and
adefovir, respectively, while in the EC90 value, compound 9 demon-
strated more favorable anti-HBV activity for both mutants,
b-hydroxyl group to 2-
a
-fluoro was accomplished by treating the
rtM204 V (4.7 vs 7.0
rtL180M, the antiviral activity of compound 9 was similar to that of
lamivudine in the EC50 2.1 versus 1.5 M, while the compound 9
exhibited a 4.3-fold increased antiviral activity in the EC90 value
(5.1 vs 22.0 M).
Compound 9 was also evaluated against the lamivudine double
mutant, rtL180M/rtM204V, and it exhibited the EC50 2.2 M that
was equal to the adefovir, while the EC90 value of 5.5 M of com-
pound 9 was more effective than that of adefovir (8.5 M). In addi-
tion, deamination studies with adenosine deaminase from calf
thymus indicated that the compound 9 was completely stable.18
In preliminary studies, compound 9 was also evaluated against
lamivudine/entecavir double resistant clone (L180M + S202I +
M202V), in which compound 9 demonstrated significant anti-
lM) and rtM204I (5.0 vs 8.0 lM). For mutant
alcohol 4 with DAST to give 47% yield of compound 5. However,
debenzylation of 5 was unsuccessful under the Birch reduction or
the Lewis acid (BCl3) conditions. Therefore, the silyl group of 5
was removed by using tetrabutyl ammonium fluoride (TBAF/
HOAc) to yield 82% of a diol, which was re-protected by benzoyl
chloride in pyridine to give the fully protected intermediate 6 in
86% yield. The compound 6 was then treated with BCl3 at ꢀ78 °C
to obtain the key intermediate 7 in 76% yield. N,N-diboc protected
adenine was synthesized according to the reported protocol in lit-
erature14 and condensed with 7 to obtain 8 in 51% yield. The
deprotection of the Boc group was carried out by TFA to afford
82% yield. Eventually, the treatment of DIBAL-H gave the target
compound 915 in 76% yield.
l
l
l
l
l
The synthesized nucleoside 9 was evaluated for its antiviral
activity against wild-type HBV as well as adefovir, lamivudine
HBV activity (EC50 0.67
lamivudine and entecavir, there are significant decrease in their
lM) against the mutant. In the case of

6330
J. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6328–6331
Table 1
In vitro anti-HBV activity against adefovir, lamivudine and entecavir drug-resistant mutants in the intracellular HBV DNA replication assay16,17
Strains
Compound 9 (
l
M)
Adefovir (
l
M)
Lamivudine (
l
M)
Entecavir (
l
M)
CC50
b
c
d,e
EC50
EC90
CC50
Fold resistancef
EC50 EC90 Fold resistance
EC50
EC90
Fold resitance
EC50 EC90
(EC90
)
(EC90
)
(EC90
)
Wild Type
rtM204V
rtM204I
rtL180M
rtLM/rtMVa
rtN236T
rtLM/rtMV/
rtSGg
1.5
1.8
1.0
2.1
2.2
1.7
0.67
4.5
4.7
5.0
5.1
5.5
4.6
NT
>100
>100
>100
>100
>100
>100
NT
—
1.3
1.6
1.9
5.5
2.1
7.8
NT
7.1
7.0
8.0
7.7
8.5
—
0.2
0.6
—
0.008 0.033 28
1.0
1.1
1.1
1.2
1.0
—
1.0
1.1
1.1
1.2
>100
>100
1.5
>100 >166
>100 >166
22.0
>100 >166
0.9
NTh
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
36.7
NT
NT
>100
0.2
36.0 5.1
NT
1.5
—
NT
—
>500i NT
1.20j
a
b
c
d
e
f
rtLM/rtMV = rtL180M/rtM204V double mutant.
Effective concentration required to inhibit 50% of HBV-DNA.
Concentration required to reduce infectious virus titer by 90%.
The > sign indicates that the 50% inhibition was not reached at the highest concentration tested.
The drug concentration required to reduce the cellular viability by 50% as assayed by an MTT assay.
Fold resistance = (mutant EC90)/(wt EC90).
rtLM/rtMV/rtSG = rtL180M/rtM204V/rtS202G.
NT = not tested.
Ref. 19.
Ref. 20.
g
h
i
j
antiviral potency (EC50 > 500 and 1.2
Table 1.19,20
lM, respectively) as shown in
sides.23 In the homology model of HBV polymerase, the relative po-
sition of -, b- and -phosphates of compound 9 with respect to
a
c
It was of interest to know how the compound 9 demonstrated
the favorable anti-HBV activity in comparison to that of adefovir.
Therefore, molecular modeling studies were conducted to obtain
the insight of the molecular mechanism of compound 9 by using
the Schrodinger suite.21 The homology model of HBV RT was con-
structed based on the published X-ray crystal structure of HIV re-
verse transcriptase (PDB code: 1RTD),22 which was previously used
for molecular mechanism studies of several anti-HBV nucleo-
the catalytic triad were assumed to occupy the similar position
to the dNTP in the crystal structure of the HIV-1 RT-DNA–dNTP
complex. The molecular docking24 of compound 9 shows that the
triphosphate forms all the network of hydrogen bonds with the ac-
tive site residues, S85, A86, A87, R41, K32 (Fig. 1a). The
c-phosphate of compound 9 retains a critical H-bonding with the
OH of S85 with connection of hydrogen bonds between S85 and
N236. Generally, the N236T mutant loses the hydrogen bond to
S85, which results in destabilization of the S85 to
c-phosphate
interaction, thus causes resistance. However, compound 9 (as its
triphosphate) maintains a critical H-bonding with S85 (Fig. 1b)
similar to that as observed in wild type HBV (Fig. 1a).
The carbocyclic ring with an exocyclic alkene of compound 9
occupies the hydrophobic pocket (residues F88, L180 and M204)
and makes the favorable van der Waals interaction with F88
(Fig. 1a and b). The 20-fluorine substituent in the carbocyclic ring
of compound 9 appears to promote an additional binding with
R41 as shown in Figure 1a and b, which corroborates with the anti-
viral activity of compound 9 shown in Table 1. Overall, the model-
ing studies can qualitatively explain the favorable anti-HBV
activity of the newly discovered compound 9 in WT (Fig. 1a) as
well as against adefovir resistant mutant, N236T (Fig. 1b). These
modeling studies are qualitative, and therefore, more quantitative
calculation is warranted in the future.
In summary, a novel carbocyclic adenosine derivative 9 was
synthesized, and evaluated for its anti-HBV activity. From these
studies, the target nucleoside demonstrated significant anti-HBV
activity against both the wild-type as well as the major nucleo-
side-resistant HBV mutants (adefovir and lamivudine), including
the lamivudine/entecavir double mutant. In view of these promis-
ing anti-HBV activities, further biological and biochemical studies
of the nucleoside 9 is warranted to assess the full potential as an
anti-HBV agent.
Acknowledgments
This research was supported by the U.S. Public Health Service
Grant AI-25899 (C.K.C.), NO1-AI-30047 (Z.H. & M.G.M.), NOI-AI-
30046 (B.K.) from the National Institute of Allergy and Infectious
Diseases, NIH and grant from the National Research Foundation
of Korea Grant (JKY) (NRF-2009-352-E00065).
Figure 1. Binding mode and van der Waals interaction of compound 9 (a) in wild-
type HBV and (b) in N236T adefovir mutant HBV. Yellow dotted lines are hydrogen
bonding interactions (<2.5 Å).

J. Wang et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6328–6331
6331
12. Montgomery, J. A.; Shortnacy-Fowler, A. T.; Clayton, S. D.; Riordan, J. M.;
Secrist, J. A. J. Med. Chem. 1992, 35, 397.
Supplementary data
13. Gadthula, S.; Rawal, R. K.; Sharon, A.; Wu, D.; Korba, B.; Chu, C. K. Bioorg. Med.
Chem. Lett. 2011, 21, 3982.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.08.113.
14. Dey, S.; Garner, P. J. Org. Chem. 2000, 65, 7697.
15. Compound 9: ½a D25
+ 151.80° (c 0.23, CH3OH); mp 215–217 °C; UV (H2O) kmax
ꢁ
259.0 nm (e 14,000, pH 2), 260.0 nm (e 15,600, pH 7), 260.0 nm (e 15,600, pH
11); 1H NMR (500 MHz, CD3OD) d 8.26 (s, 1H), 8.10 (d, J = 2.5 Hz, 1H), 5.90 (d,
J = 2.5 Hz, 1H), 5.46 (s, 1H), 5.01–4.89 (m, 2H), 4.46–4.42 (m, 1H), 391–3.81 (m,
2H), 2.81 (br, 1H); 19F NMR (500 MHz, CD3OD) d ꢀ192.93 (m); 13C NMR
(125 MHz, CD3OD) d 155.9, 152.5, 149.9, 146.0, 141.1, (d, J = 5.3 Hz), 117.8,
111.7, 109.8, 95.9 (d, J = 184 Hz) 72.9 (d, J = 23.6 Hz), 61.7, 57.5 (d, J = 17.4 Hz),
51.0; Anal. Calcd For C12H14FN5O2: C, 51.61; H, 5.05; N, 25.08; Found C, 51.74;
H, 5.09; N, 24.92.
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