Design, synthesis, and biological evaluation of novel 2′-deoxy-2′-fluoro-2′-C-methyl 8-azanebularine derivatives as potent anti-HBV agents

Article abstract of DOI:10.1016/j.bmcl.2019.04.005

Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent. In this paper, a series of novel 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azanebularine analogues (1 and 2a) and N⁴-su

Full text of DOI:10.1016/j.bmcl.2019.04.005

Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and biological evaluation of novel 2′-deoxy-2′-fluoro-2′-C-
methyl 8-azanebularine derivatives as potent anti-HBV agents
a,1
b,1
a
a
a
d
Wu Yang , Youmei Peng , Jingwen Wang , Chuanjun Song , Wenquan Yu , Yubing Zhou ,
b
b
a,⁎
a,c,⁎
Jinhua Jiang , Qingduan Wang , Jie Wu , Junbiao Chang
b
a
College of Chemistry and Molecular Engineering, Zhengzhou University, Henan 450001, PR China
Institute of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, PR China
Collaborative Innovation Center of New Drug Research and Safety Evaluation, Henan Province, Zhengzhou 450001, PR China
Department of Pharmacy, The First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, PR China
c
d
A R T I C L E I N F O
Keywords:
A B S T R A C T
Hepatitis B virus (HBV) is a global health problem requiring more efficient and better tolerated anti-HBV agent.
2
′-Deoxy-2′-fluoro-2′-C-methyl 8-azanebularine
In this paper, a series of novel 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azanebularine analogues (1
analogues
4
and 2a) and N -substituted 8-azaadenosine derivatives (2b-g) were designed, synthesized and screened for in
Anti-HBV activity
vitro anti-HBV activity. Two concise and practical synthetic routes were developed toward the structural motif
construction of 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azainosine from the ribonolactone 3
under mild conditions. The in vitro anti-HBV screening results showed that these 8-azanebularine analogues had
a significant inhibitory effect on the expression of HBV antigens and HBV DNA at a concentration of 20 μM.
Among them, halogen-substituted 8-azaadenosine derivative 2g displayed activities comparable to that of 3TC.
In particular, 2g retained excellent activity against lamivudine-resistant HBV mutants.
Nucleoside reverse transcriptase inhibitor
(
NRTI)
Lamivudine-resistant HBV mutants
Viral DNA replication
Hepatitis B virus (HBV) infection presents a public health problem
worldwide, with about 260 million people infected and nearly 887,000
received considerable attention as purine antagonists because of their
excellent antiviral and antitumour activities, as well as their interesting
1
–3
18–20
deaths per year worldwide . For many years, scientists have been
committed to design new drugs against HBV. Among them, the mod-
ified nucleosides are effective in almost all the patients and have re-
presented one of the most promising classes of polymerase inhibitors of
HBV viruses. Six nucleoside/nucleotide reverse transcriptase inhibitors
biochemical and chemotherapeutic properties
. Our laboratory has
been devoted to continuing interest in the investigation of novel new
nucleoside analogues against the human immunodeficiency virus
2
1–29
(HIV), HBV, and the hepatitis C virus (HCV)
. As part of our re-
search projects, we demonstrated that a 2′-deoxy-2′-β-fluoro-4′-azido-β-
D-arabinofuranosyl 1,2,3-triazole nucleoside analogue (TAA-1a)
showed potential antiviral effects and retained excellent activity against
(
NRTIs) have been approved by Food and Drug Administration (FDA)
4–7
for the treatment of HBV . However, on account of the poor tolera-
tion, frequent side effects, as well as the development of drug-resistant
viruses, the current therapy for HBV treatment still cannot meet the
3
0
lamivudine-resistant HBV mutants . Based on 1,2,3-triazole nucleo-
sides, an efficient linear strategy for the stereospecific formation of
8
–10
31
clinical need
. New and effective drugs are still urgently needed to
some 8-azanebularine analogues had been developed by our group
.
combat HBV infection, especially for nucleoside analogues with un-
reported scaffolds that do not select resistance-associated mutations.
Among derivatives developed in the nucleoside chemistry, some
unnatural heterocycles can be introduced as nucleobases in the design
of novel nucleoside analogues (Fig. 1). They can not only enhance in
vivo stability but also endow interesting biological activities, e.g. riba-
On the other hand, ribonucleoside analogues with a 2′-deoxy-2′-
fluoro-2′-C-methyl substituent have inspired a wide range of research
over the past few years due to the high efficacy of sofosbuvir against the
3
2
HCV NS5B polymerase . In view of the potential biological activity of
8-azaguanosines and 2′-deoxy-2′-fluoro-2′-C-methylribofuranosyl moi-
eties, we provided a full account of the synthesis and biological eva-
luation of 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azai-
1
1–17
virin, a designed guanosine analogue bearing a 1,2,4-triazole
. In
4
addition, the 8-azanebularine analogues, mimicking the natural purine
nucleosides with 8-azapurine derivatives as nucleobases, have also
nosine (1), 8-azaadenosine (2a) and N -substituted 8-azaadenosine
derivatives (2b-g) in this article. Two efficient linear protocols for the
⁎
Corresponding authors at: College of Chemistry and Molecular Engineering, Zhengzhou University, Henan 450001, PR China (J. Chang).
E-mail addresses: wujie@zzu.edu.cn (J. Wu), changjunbiao@zzu.edu.cn (J. Chang).
These authors contributed equally.
1
https://doi.org/10.1016/j.bmcl.2019.04.005
Received 21 January 2019; Received in revised form 29 March 2019; Accepted 3 April 2019
0960-894X/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Wu Yang, et al., Bioorganic & Medicinal Chemistry Letters, https://doi.org/10.1016/j.bmcl.2019.04.005

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Fig. 1. Nucleoside analogues with unnatural heterocycles.
Scheme 1. Reagents and conditions: (a) (i) LTTBA, Ac
2
O, DMAP, THF, −20 °C, 5 h, 90%; (ii) SnCl
4
, TMSN
3
4
, PhCl, 65℃, 6 h, 30%; (b) Cyanoacetamide, EtONa, EtOH,
5
0 °C, 20 h, 80%; (c) TBDMSCl, Imidazole, DMF, rt, 12 h, 95%; (d) HC(OEt)
3
, 145 °C, 48 h, 63%; (f) NH
F, MeOH, 60 °C, 13 h, 89%.
Scheme 2. Reagents and conditions: (a) Ethyl 3-bromopropiolate, Cu(OAc)
2
, CuBr, THF, 80 °C, 48 h, 97%; (b) t-Butylamine, MeOH, rt, 48 h, 70%; (c) TBDMSCl,
Imidazole, DMF, rt, 12 h, 84%; (d) NaN , DMF, 40 °C, 15 h, 85%; (e) (i) NH
3
3
/MeOH, rt, 24 h; (ii) H , Pd/C, MeOH, rt, 6 h, 83% over the two steps.
2
2

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
stereospecific formation of 8-azainosine structural motif were devel-
oped. By screening, 2g has potential antiviral effects against wild-type
HBV and lamivudine-resistant HBV mutants.
3 through reduction, esterification, and followed by substitution with
TMSN . The 1,3-dipolar cycloaddition reaction of 4 with 2-cyanoaceta-
3
mide furnished triazole 5 in a regiospecific manner, which further reacted
with tert-Butyldimethylsilyl chloride (TBDMSCl) to provide intermediate 6
for the following hypoxanthine formation reaction 33. Notably, only
The synthesis of 8-azainosine derivative 1 was illustrated in Scheme 1.
The key starting material 1-β-azido sugar 4 was prepared from the lactone
Scheme 3. Reagents and conditions: (a) BzCl, Et
DIPEA, CH Cl , 60 °C, 3–5 h; (ii) NH /CH
3
N, acetone, rt, 12 h, 98%; (b) SOCl
2
, DMF, DCM, reflux, 24 h, 71%.(c) NH
3
, CH
3
OH, 60 °C, 19 h, 97% for 2a (i) RNH ,
2
2
2
3
3
OH, rt, 24 h, 91% for 2b, 90% for 2c, 87% for 2d, 94% for 2e, 80% for 2f, 36% for 2g, all over the two steps.
3

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 1
The inhibitory effect of 1 and 2a-g on HBsAg and HBeAg secretion and cytotoxicity.
Compounds
Structure
CC50(μM)b
Inhibition percentages (%)a
HBeAg
HBsAg
3
TC
–
39.6 ± 4.2
7.4 ± 1.0
42.4 ± 3.9
8.5 ± 1.7
1
> 600
2
2
a
b
> 600
> 600
10.0 ± 3.3
24.4 ± 6.1
9.9 ± 2.6
20.1 ± 4.2
2
c
> 600
11.6 ± 3.1
17.0 ± 2.5
2
2
2
d
e
f
> 600
16.7 ± 4.3
17.7 ± 2.7
11.9 ± 2.9
11.4 ± 3.8
14.8 ± 3.6
8.5 ± 1.6
501 ± 19
> 600
2
g
245 ± 11
31.6 ± 6.6
39.7 ± 7.3
a
HepG2.2.15 cells were treated with 1, 2a-g and lamivudine at the concentration of 20 μM for 9 days. The levels of HBsAg and HBeAg in the supernatant were
detected by ELISA assays.
b
CC50: 50% cytotoxic concentration, measured by MTT assay in the HepG2 cell line treated with 1 and 2a-g for 9 days.
4

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 2
The inhibitory Effect of 2g on Intracellular HBV DNA in HepG2.2.15 Cell.
Compounds
day 3
day 6
day 9
4
4
4
Concentration (μΜ) HBV DNA2 × 10
Inhibition rates
(%)
HBV DNA × 10
copies/mL
Inhibition rates
(%)
HBV DNA × 10
copies/mL
Inhibition rates
(%)
copies/mL
control
5.7 ± 1.4
6.6 ± 1.7
8.6 ± 2.7
*
*
*
*
*
*
**
**
3
2
2
2
TC
20
10
20
40
3.0 ± 0.7
4.9 ± 1.2
4.1 ± 0.7
3.6 ± 0.8
47.4
14.0
28.1
36.8
2.0 ± 0.5
69.7
31.8
47.0
65.2
1.9 ± 0.5
77.9
44.2
67.4
80.2
*
**
g
4.5 ± 1.4
4.8 ± 1.0
**
**
g
3.5 ± 1.3
2.8 ± 0.5
**
**
g
2.3 ± 0.9
1.7 ± 0.6
Debenzoylation and amination of 13 with NH
3
/CH OH at 60 °C af-
3
forded 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azaade-
nosine 2a in 98% isolated yield (Scheme 3). Likewise, reactions of 13
with different amines in the presence of disopropylethylamine (DIPEA)
followed by deprotection provided 2b-2g in a medium or high yield.
The structure of 2g was further confirmed by X-ray crystallography.
To investigate the inhibitory effect of 1 and 2a-g on the production
of the hepatitis B surface antigen (HBsAg) and hepatitis Be antigen
(
HBeAg), the HepG2.2.15 cells were treated with the test compounds at
a concentration of 20 μM for 9 days and lamivudine (3TC, 20 μM) used
as a positive control. The supernatant was collected and the titers of
HBsAg and HBeAg were determined by ELISA kits. As shown in Tables 1
and 2a-g have a significant inhibitory effect on the expression of HBV
antigens at a concentration of 20 μM and exhibit low cytotoxicity. Re-
placing the carbonyl in 1 with primary amine leads to the formation of
2
a-2g with better inhibitory activity. Incorporation of substituents
(
alkyl, cyclopropane and phenyl) into the primary amine brings better
inhibitory activity, especially 2b has a more obvious inhibitory activity.
If the hydrogen on the benzene ring of 2e is replaced by halogen, the
resulting compound (2g) presents significantly increased inhibitory
activity and better inhibitory activity than other compounds. Specifi-
cally, 2g significantly reduces the production of HBsAg and HBeAg with
inhibitory rates of 39.7% and 31.6% on day 9, respectively, while 3TC-
treated (20 μM) groups show 42.4% and 39.6% inhibition on day 9. The
cytotoxicity (CC50s) of 2g were 409 ± 16 μM and 245 ± 11 μM in the
HepG2 cell treated for 3 and 9 days, respectively, with inhibitory rate of
7
.7% at the concentration of 40 μM for 9 days. These results indicated
that 2g at 20 μM is equally effective compared to 3TC at 20 μM in in-
hibition of both HBsAg and HBeAg secretion with a favorable cyto-
toxicity profile.
Fig. 2. The inhibitory effect of 2g against HBsAg (A) and HBeAg (B) in the
HepG2.2.15 cell. The HepG2.2.15 cell was treated with 2g (10, 20 and 40 μM)
and 3TC (20 μM) for 3, 6 or 9 days. The HBsAg (A) and HBeAg (B) in the su-
pernatants were quantified using ELISA method. Data were presented as
To further confirm the antiviral activity of 2g in HepG2.2.15 cells,
the secretion of HBsAg, HBeAg and HBV DNA levels were evaluated
after treatment with different concentrations of 2g (10, 20 and 40 μM)
for 3, 6 and 9 days. The significant reductions of HBsAg and HBeAg
secretion were observed in a time- and dose-dependent manner (Fig. 2).
Consistent with the inhibitory effects on HBeAg and HBsAg secretion,
treatment with 2g at concentrations of 10, 20, and 40 μM for 3, 6 and
*
**
mean ± SD of three experiments. P < 0.05 and P < 0.01 compared with
the no-drug control group.
stoichiometric TBDMSCl could be added in the protection reaction and the
reaction time should be limited within 12 h. Or else, the amino group of 5
could further react with surplus TBDMSCl. Condensation of amino amide 6
with triethyl orthoformate resulted in the formation of 8-azainosine deri-
vative 7 as a single key product in 63% isolated yield. Further desilylation
with ammonium fluoride furnished 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-
arabinofuranosyl 8-azainosine 1 in a high yield.
9
days results in the reduction of both the intracellular and extracellular
HBV DNA levels in a time- and dose-dependent manner. The mean in-
hibition percentages of HBV DNA level with 2g at the dosages of 10, 20
and 40 μM are 44.2%, 67.4% and 80.2%, respectively, intracellularly
(
(
Table 2) and 36.9%, 65.0% and 82.5%, respectively, extracellularly
Table 3), on day 9. The inhibition rate of 3TC (20 μM) on intracellular
To achieve the key intermediate 6, we also could make use of an-
and extracellular HBV DNA level is 77.9% and 78.6%, on day 9. The
results show that 2g has a significant inhibitory effect on HBsAg, HBeAg
and HBV DNA in the HepG2.2.15 cell line. The inhibitory effect of 2g at
the concentration of 40 μM on HBV DNA is equivalent to 3TC at the
concentration of 20 μM. Although the inhibitory effect of 2g (20 μM) on
HBV DNA is lower than that of 3TC (20 μM), 2g retains activity against
the lamivudine-resistant HBV mutant.
3
1
other synthetic protocol (Scheme 2) . Copper-catalyzed [3+2] cy-
cloaddition of 4 with ethyl 3-bromopropiolate resulted in the formation
of triazole 8. Transesterification and protection with TBDMS group
yielded 10. The following nucleophilic aromatic substitution of 10 with
sodium azide provided 11 in an excellent isolated yield. Ammonolysis
of the ester followed by hydrogenation of the azide group afforded the
key intermediate 6 in 83% isolated yield in two steps.
To determine the inhibitory effect of 2g against lamivudine-re-
sistant HBV, the HBeAg in the supernatants and HBV DNA were de-
tected in L180M/M204V mutant cell lines after 2g treatment at 10, 20
Starting from 1, 6-chloro-8-azapurine 13 was prepared by benzoy-
lation and then chlorination the carbonyl group with SOCl .
2
5

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
Table 3
The inhibitory Effect of 2g on Extracellular HBV DNA in HepG2.2.15 Cell.
Compounds
day 3
day 6
day 9
4
4
4
Concentration (μΜ) HBV DNA × 10
Inhibition rates
(%)
HBV DNA × 10 copies/ Inhibition rates
HBV DNA × 10 copies/ Inhibition rates
copies/mL
mL
(%)
mL
(%)
control
4.9 ± 1.2
6.1 ± 1.5
2.8 ± 0.7
4.2 ± 0.9
3.5 ± 1.2
1.9 ± 0.5
10.3 ± 3.0
*
**
*
**
**
**
3
2
2
2
TC
20
10
20
40
3.3 ± 0.9
4.0 ± 1.1
3.7 ± 0.9
3.2 ± 1.3
32.7
18.4
24.5
34.7
54.1
31.1
42.6
68.9
2.2 ± 0.6
6.5 ± 2.0
3.6 ± 1.3
1.8 ± 0.4
78.6
36.9
65.0
82.5
*
*
*
*
**
**
g
g
g
dependent manner. The inhibitory effects on HBeAg secretion of wild-
type and lamivudine-resistant HBV have no obvious difference.
Consistent with its inhibitory effects on HBeAg secretion, the HBV
DNA level decreased in a time-and dose-dependent manner after 3, 6 or
9
days treatment with 2g at the concentrations of 10, 20 and 40 μM. The
mean inhibition percentage of HBV DNA level with 2g at the dosages of
1
0, 20, and 40 μM is 36.6%, 65.2% and 76.4%, respectively, in-
tracellularly (Table 4) and 32.1%, 61.4% and 83.6%, respectively, ex-
tracellularly (Table 5), on day 9. In contrast, the mutant rtL180M/
M204V is less susceptible to 3TC than wild-type HBV, with an inhibi-
tion rate of only 11.2% and 19.3%, intracellularly and extracellularly,
on day 9. The results show that 2g is effective against both wild-type
and lamivudine-resistant HBV.
In summary, we have developed an efficient route for the synthesis
of 2′-deoxy-2′-fluoro-2′-C-methyl-β-D-arabinofuranosyl 8-azanebularine
analogues, including 8-azainosine (1) and 8-azaadenosine derivatives
(2a–g), in a highly stereo- and regioselective manner. The synthetic
compounds were tested for anti wild-type and lamivudine-resistant
HBV activity. The results showed that these compounds had a sig-
nificant inhibitory effect on the expression of HBV antigens and HBV
DNA at a concentration of 20 μM. Among them, halogen-substituted 8-
azaadenosine derivative (2g) displays activities comparable to that of
3TC in inhibition of wild-type HBV antigens and HBV DNA. In parti-
cular, 2g is also effective against lamivudine-resistant HBV. These re-
sults provide strong support for the development of 2g and its 8-azaa-
denosine derivatives as a potential alternative or complementary
therapy for the treatment of HBV infection.
Fig. 3. The inhibitory effect of 2g against HBeAg in the L180M/M204V mutant
cell line. The cell line was treated with 2g (10, 20 and 40 μM) and 3TC (20 μM)
for 3, 6 and 9 days. The HBeAg in the supernatants were quantified using ELISA
*
method. Data were presented as mean ± SD of three experiments. P < 0.05
*
*
and P < 0.01 compared with the no drug control group.
and 40 μM for 3, 6 and 9 days. The level of HBsAg from lamivudine-
resistant cell line transfected with the L180M/M204V mutant is lower
limit of detection, and then it was excluded from the antigen cri-
3
4
terion . The antigen inhibitory efficiency of 2g was assessed only
based on HBeAg. As shown in Fig. 3, lamivudine showed lower in-
hibitory effect on HBeAg production from lamivudine-resistant HBV
than that from wild-type HBV. However, 2g significantly reduces the
secretion of HBeAg of lamivudine-resistant HBV in a time- and dose-
Table 4
Inhibitory Effect of 2g on Intracellular HBV DNA Levels in lamivudine-resistant cell.
compounds
day 3
day 6
day 9
Concentration (μΜ) HBV
Inhibition rates
(%)
HBV
Inhibition rates
(%)
HBV
Inhibition rates
(%)
4
4
4
DNA × 10 copies/mL
DNA × 10 copies/mL
DNA × 10 copies/mL
16.1 ± 5.8
control
8.0 ± 2.1
7.5 ± 1.8
7.6 ± 2.2
10.1 ± 3.5
9.3 ± 3.8
*
3
2
2
2
TC
20
10
20
40
6.3
7.9
14.3 ± 5.0
11.2
36.6
65.2
76.4
*
**
**
*
g
5.0
7.8 ± 2.0
22.8
41.6
66.3
10.2 ± 2.1
*
*
**
**
g
6.6 ± 1.6
17.5
31.3
5.9 ± 1.3
5.6 ± 1.5
g
5.5 ± 1.8
3.4 ± 1.0
3.8 ± 0.9
Table 5
Inhibitory Effect of 2g on Extracellular HBV DNA Levels in lamivudine-resistant cell.
compounds
day 3
day 6
day 9
4
4
4
Concentration (μΜ) HBV DNA × 10 copies/ Inhibition rates
HBV DNA × 10 copies/ Inhibition rates
HBV DNA × 10 copies/ Inhibition rates
mL
(%)
mL
(%)
mL
(%)
control
7.3 ± 2.1
7.0 ± 1.6
6.5 ± 1.9
4.7 ± 0.9
4.0 ± 2.2
9.5 ± 3.3
8.7 ± 2.6
6.9 ± 2.0
4.8 ± 1.3
3.7 ± 0.9
14.0 ± 4.5
11.3 ± 3.2
*
3
2
2
2
TC
20
10
20
40
4.1
8.4
19.3
32.1
61.4
83.6
*
**
**
*
g
11.0
35.6
45.2
27.4
49.5
61.1
9.5 ± 2.7
*
*
**
**
g
5.4 ± 1.4
2.3 ± 0.5
*
g
6

W. Yang, et al.
Bioorganic & Medicinal Chemistry Letters xxx (xxxx) xxx–xxx
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Appendix A. Supplementary data
1
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Experimental details, H and C NMR spectra of compounds 1–13,
X-ray structure and data of 2g (PDF). Supplementary data to this article
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