Synthesis and biological evaluation of 4-substituted fluoronucleoside analogs for the treatment of hepatitis B virus infection

Article abstract of DOI:10.1021/jm5012963

A series of 4-substituted fluoronucleosides have been synthesized in order to address the toxicity issue of the parent compound 7, and after in vitro evaluation, the cyclopropylamino analog 1f was selected for in vivo study. In mice, this compound exhibit

Full text of DOI:10.1021/jm5012963

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Article
Synthesis and Biological Evaluation of 4-Substituted Fluoronucleoside
Analogs for the Treatment of Hepatitis B Virus Infection
Qinghua Yang, Liyun Zheng, Xue-Jun Wang, Jinfeng Kang, Na Wan, Jie Wu, Yan Qiao, Pengfei
Niu, Sheng-Qi Wang, Youmei Peng, Qingduan Wang, Wenquan Yu, and Junbiao Chang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm5012963 • Publication Date (Web): 23 Apr 2015
Downloaded from http://pubs.acs.org on April 27, 2015
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Synthesis and Biological Evaluation of 4-Substituted Fluoronucleoside Analogs for the
Treatment of Hepatitis B Virus Infection
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Qinghua Yang,1 Liyun Zheng,2 Xue-Jun Wang,3 Jinfeng Kang,1 Na Wan,2 Jie Wu,1 Yan Qiao,1
Pengfei Niu,1 Sheng-Qi Wang,3 Youmei Peng,1,2 Qingduan Wang,2 Wenquan Yu,*, 1 and Junbiao
Chang*, 1
1 College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, P. R. China
2 Henan Academy of Medical and Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450052, P. R.
China
3 Department of Biotechnology, Beijing Institute of Radiation Medicine, Beijing, 100850, P. R. China
ABSTRACT: A series of 4-substituted fluoronucleosides have been synthesized in order to
address the toxicity issue of the parent compound 7, and after in vitro evaluation, the
cyclopropylamino analog 1f was selected for in vivo study. In mice, this compound exhibited a
significantly improved toxicity profile. Administered orally, compound 1f was well tolerated at a
dose up to 3 g/kg, and showed insignificant toxicity on white blood cells and low mutagenic
effect at the dosages up to 80 mg/kg (single) or 20 mg/kg/d (5 days). In duck HBV (DHBV)
infected duck models, both the serum and liver DHBV DNA levels (74.2% and 82.1%
respectively) were markedly reduced by the treatment of 1f at a dose of 1 mg/kg/d for 10 days. In
addition, both the viral DNA levels had a lower degree of recovery after withdrawal of the test
compound for 3 days.
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■ INTRODUCTION
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Despite the availability of effective vaccines and antiviral therapies, hepatitis B is still a major
global health problem, one third of all people on the planet having been at some time infected
with the hepatitis B virus (HBV). In 2014, there are currently more than 240 million people with
chronic hepatitis B, which leads to more than 780,000 deaths per year1 following development of
liver disease, such as cirrhosis, liver cancer or other complications. The therapeutic options for
the patients with chronic hepatitis B include two forms of interferon-α (IFN-α) as immune
system modulators and five nucleoside or nucleotide analogs as inhibitors of the hepatitis viral
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polymerase. IFN therapy is effective for up to 60% of patients, but results in complete cures in
2, 3
only 7%
and also is associated with a high rate of adverse side effects.4, 5 Compared with
interferon products, the nucleoside and nucleotide analogs have the advantages of oral
bioavailability, fewer side effects and superior clinical benefits for almost all the patients, but the
emergence of drug resistance remains a major problem.6
A variety of non-nucleoside anti-HBV compounds have been reported including imino sugars,7,
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phenylpropenamides,9 heteroaryldihydropyrimidines (HAP),10 triazolopyrimidines,11 and
others.12, 13 However, so far no non-nucleoside antiviral drugs have been approved by Food and
Drug Administration (FDA) for the treatment of chronic hepatitis B and all the five small-
molecule anti-HBV drugs in clinical use are derivatives of nucleosides or nucleotides. Currently,
nucleoside/nucleotide antiviral drug discovery is one of the most promising areas for developing
new potential therapeutics for the treatment of chronic hepatitis B.
As a result of its small size, pronounced electronegativity, isosteric mimicking of the hydroxyl
group and strength of the C−F bond, fluorine, when introduced into the structure of nucleosides,
can impart a variety of physical, chemical, and biological properties to the compounds.14-17 There
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are successful cases of drugs discovered by incorporation of fluorine into the structure of
nucleosides (Figure 1). Examples include clofarabine, used for the treatment of acute
lymphoblastic leukaemia, clevudine approved in S. Korea for treatment of hepatitis B,
gemcitabine, used to treat solid cancerous tumors, and emtricitabine (FTC) to combat HIV
infection. It has been widely accepted that nucleosides that contain fluorine, especially at C-2'
position, possess improved acidic and enzymatic stabilities,17 and consequently their biological
activity and other profiles (e.g. F and t1/2) were improved.
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Figure 1. Representative fluorinated nucleoside drugs
This report concerns the development of new fluorinated nucleoside analogs for the treatment
of HBV infection. Previously, starting with the framework presented by 2'-deoxy-2'-β-
fluororibose pyrimidine nucleosides, we have designed and synthesized a series of 4'-substituted
derivatives (5−8, Figure 2) with useful antiviral activity.18-23 Among them, 2'-deoxy-2'-β-fluoro-
4'-azido-β-D-arabinofuranosyl cytidine (FNC) exhibits significant potency against HIV infection
(EC50 = 0.3 nM)18 and is now being studied clinically. Further investigation demonstrated that
compound 7 (FNC) also possesses good antiviral activity against HBV strains, both wild-type
(EC50 = 0.12 μM) and lamivudine-resistant HBV (EC50 = 0.27 μM).22 In the in vivo study, this
nucleoside exhibited good efficacy in duck HBV (DHBV)-infected ducks,21 but indicated certain
toxicity in Kunming (KM) mice. An oral dose of > 200 mg/kg causes animal death. In
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continuation of this research, we therefore performed further lead optimization of this 4'-azido
substituted fluoronucleoside with a view to addressing the toxicity issue.
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Figure 2. 2'-Deoxy-2'-β-fluoro-4'-substituted nucleosides developed in our laboratory
So far, most of the existing therapeutic nucleoside reverse transcriptase inhibitors (NRTIs)
belong to the 2',3'-dideoxynucleosides (ddNs) family.24 However, in the structures of these 2'-
deoxy-2'-β-fluoronucleosides (5−8), a 3'-hydroxy group is retained to mimic the real nucleosides
that viruses need for their DNA synthesis. The structural difference between compounds 5−8 and
the current NRTIs could which could help reduce the risk of the emergence of drug resistance
and/or cross resistance.18, 22, 25 Although bearing this 3'-OH moiety, these nucleosides can still act
as DNA chain terminators owing to the presence of 4'-substitutent.26
Figure 3. Structure-activity relationship analysis of the parent compound 7 and its derivatives 1
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With the arabinofuranose ring optimized in this way, we focused in this current study on
structural modification of the pyrimidine moiety of the parent compound 7. In order to improve
the toxicity profile, a series of 4-N-alkyl substituted derivatives were designed and synthesized.
After the preliminary in vitro screening for anti-HBV activity and cytotoxicity of the newly
synthesized nucleoside analogs, the cyclopropylanimo substituted analog 1f was selected for
further evaluation. The in vitro anti-HBV activity of 1f was further confirmed in the HepG2.2.15
cell line. The toxicity, efficacy, and pharmacokinetics (PK) of this lead compound in vivo were
also examined in KM mice, DHBV infected ducks, and Sprague-Dawley rats, respectively.
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■ CHEMISTRY
The synthesis of the target nucleoside 1 is described in Scheme 1. The key starting material,
the protected bisbenzoyl uridine (2) was prepared from 1,3,5-O-tribenzoyl-2-deoxy-2-fluoro-D-
arabinofuranoside using a previously reported synthetic route.18 Debenzoylation of compound 2
in a solution of ammonia in methanol gave the uridine analog 1a in 86% yield. The 4-carbonyl
group in 2 was selectively activated by reaction with 1,2,4-triazole in the presence of phosphorus
oxychloride and pyridine to form the triazolo-substituted intermediate 3. Substitution of the
triazolo group in compound 3 with primary or secondary, cyclic or acyclic aliphatic amines,
followed by deprotection with ammonia in methanol, afforded compound 1b−k in 36−87% yield
1
over three steps. All these new synthesized nucleoside analogs were fully characterized by H
NMR,27 13C NMR and HRMS. The structure of the cyclopropylamino substituted analog 1f was
further confirmed by X-ray crystallography. Analytical HPLC indicated that all the tested
compounds possess a purity of at least 95%.
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Scheme 1. Synthesis of Compounds 1 from Bisbenzoyl Protected Uridine 2
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■ RESULTS AND DISCUSSION
3.1. In Vitro Anti-HBV Activity Evaluation. The cytotoxicity of the new synthetic
nucleosides 1a−k was examined with an MTT assay in the HepG2.2.15 cell line. No cytotoxicity
was observed for any of these nucleoside analogs at test concentrations up to 125 μM (Table 1).
The anti-HBV activities of 1a−k were evaluated in the HepG2.CW cell line28 using entecavir
(ETV) as a positive control. After the treatment of HepG2.CW cells with the test compounds at
the concentration of 1 μM for 9 days, their inhibitory activity against HBV DNA replication was
measured in an FQ-PCR assay. As shown in Table 1, compounds (1b−c and 1f) with smaller N-
alkyl substituents (e.g. methyl, ethyl and cyclopropyl) showed better inhibitory activity than the
ones with bulkyl substituents (1d−e and 1g−h). Di-N-alkylation caused analogs 1i−k to lose anti-
HBV activity. Overall, five nucleoside analogs (1a−c & 1e−f) at a concentration of 1 μM
exhibited > 50% inhibition rates of the viral DNA replication, with the cyclopropylamino analog
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1f being the best one. Although compound 1f is less active than the potent anti-HBV drug
entecavir, its selectivity may be comparable with this drug owing to the low cytotoxicity (CC50 =
391 μM). To further compare their in vitro anti-HBV activity, both compounds was further
evaluated in HepG2.2.15 cell line, before 1f was selected for in vivo studies.
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Table 1. Inhibition of HBV DNA Replication and Cytotoxicity of Compounds 1
Inhibitiona
Compounds
Structure
CC50b (μM)
(at 1.0 μM)
75.5%
> 125
1a
81.1%
78.0%
> 125
> 125
1b
1c
HN
N
HO
N3
HO
N
O
O
F
HN
N
43.7%
> 125
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HO
N3
HO
N
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O
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HN
N
1e
1f
61.7%
86.6%
37.3%
> 125
HO
N3
HO
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N
O
O
F
391
> 125
1g
HN
N
23.6%
> 125
1h
HO
N3
HO
N
F
O
O
43.6%
7.1%
> 125
> 125
1i
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33.1%
95.7%
> 125
30
1k
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ETV
---
a
HepG2.CW cells were treated with the test compounds at the concentration of 1.0 μM and the inhibition of HBV DNA
b
replication was measured by FQ-PCR assay. CC50: 50% cytotixic concentration, measured by MTT assay in the HepG2.2.15
cell line.
3.2. In Vitro Inhibitory Effect of Compound 1f on HBV DNA in the HepG2.2.15 Cell Line.
The inhibitory effect of compound 1f on HBV DNA replication was further confirmed in
HepG2.2.15 cells. Treatment with 1f at concentrations of 0.2, 1, and 5 μM for 3, 6, or 9 days
resulted in the reduction of both the intracellular and extracellular HBV DNA levels in a time-
and dose-dependent manner. The average inhibition of viral DNA levels at the dosages of 0.2, 1
and 5 μM was 46.5%, 56.9% and 68.2% intracellularly (Table 2) and 52.1%, 76.2% and 87.6%
extracellularly, respectively, on day 9 (Table 3). The inhibition rates of entecavir (at 0.1 μM) on
intracellular and extracellular HBV DNA levels on day 9 were 57.6% and 78.6%, respectively.
These results show that 1f at 1 μM has an inhibitory effect on HBV DNA in HepG2.2.15 cell line
similar to that of the potent anti-HBV drug entecavir (at 0.1 μM), and it was noted that in the
same cell line, compound 1f is 13-times less toxic than entecavir (Table 1).
Table 2. Inhibitory Effect of Compound 1f on Intracellular HBV DNA Levels
3d
×106 copies/mL
6d
×106 copies/mL
9d
×106 copies/mL
Compounds C/µM
Inhibition
Inhibition
Inhibition
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rates
rates
rates
7
8
9
Control
1f
−
0.2
1
6.79 ± 1.52
5.39 ± 0.32
4.13 ± 0.13*
3.65 ± 0.29*
4.22 ± 0.89
−
5.64 ± 0.20
3.85 ± 0.72*
2.85 ± 0.15**
2.65 ± 0.53**
3.35 ± 0.35**
−
8.53 ± 0.15
4.56 ± 0.23**
3.68 ± 0.56**
2.71 ± 0.05**
3.62 ± 0.06**
−
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20.6%
39.2%
46.2%
37.8%
31.7%
49.5%
53.0%
40.6%
46.5%
56.9%
68.2%
57.6%
1f
5
1f
ETV
0.1
*P < 0.05, **P < 0.01 as compared with the control group at the same time point.
Table 3. Inhibitory Effect of Compound 1f on Extracellular HBV DNA Levels
3d
×106 copies/mL
6d
×106 copies/mL
9d
×106 copies/mL
Compounds C/µM
Inhibition
rates
Inhibition
rates
Inhibition
rates
Control
1f
−
0.2
1
14.52 ± 1.23
9.61 ± 0.54**
6.68 ± 0.25**
6.82 ± 0.37**
8.97 ± 0.77**
−
15.54 ± 1.96
7.46 ± 0.60**
3.62 ± 0.32**
2.49 ± 0.11**
8.11 ± 0.89**
−
14.94 ± 1.29
7.15 ± 0.11**
3.56 ± 0.48**
1.86 ± 0.10 **
3.20 ± 0.13**
−
33.8%
54.0%
53.0%
38.2%
52.0%
76.7%
84.0%
47.8%
52.1%
76.2%
87.6%
78.6%
1f
5
1f
ETV
0.1
*P < 0.05, **P < 0.01 as compared with the control group at the same time point.
3.3. Assessment of Acute Toxicity Study in KM Mice. An acute toxicity study of compound
1f was conducted in vivo in KM mice. The animals were administered a single intravenous dose
of the test compound at 200, 300 and 400 mg/kg (dissolved in normal saline). No side effect was
observed at a dose of 200 mg/kg but a dose of 300 mg/kg caused listlessness and reduced activity,
which lasted for only 1 h and dosage of 400 mg/kg led to animal deaths. In a subsequent
experiment, the animals were administered a single oral dose of 1f at 3 g/kg (suspended in 0.5%
CMC-Na solution). This failed to cause any morbidity, mortality, or weight loss during a
subsequent 14-day observation. This acute toxicity study indicated that nucleoside 1f was well
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tolerated in KM mice at dosages of up to 300 mg/kg via intravenous administration and 3 g/kg
via oral gavage, respectively.
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3.4. Assessment of Toxicity on White Blood Cells and Mutagenic Effect in KM Mice. The
KM mice were orally administered single 20, 40 and 80 mg/kg doses of 1f (dissolved in distilled
water). Cyclophosphamide (CP) at 60 mg/kg (dissolved in normal saline) intravenously was used
as a positive control. After 48 h, white blood cell (WBC) counts showed that the peripheral
WBCs were not affected in a statistically significant manner, compared with the negative control
group which was given just water. Then, the animals were sacrificed, and the micronucleus
frequency (MNF) in bone marrow polychromatic erythrocyte (PCE) was analyzed, and found to
be unaffected in a statistically significant manner (Table 4).
Table 4. Toxicity Effect of 1f on Peripheral WBCs and MNF in KM Mice (Single Dose)
Micronucleus frequency
± s, ‰)
Number of
animals
Number of WBCs
(×109/L)
Group
Dose (mg/kg/d)
Gender
(
x
♂
♂
♂
♂
♂
Control
Low dose
Medium dose
High dose
CP
−
5
5
5
5
5
11.10 ± 2.69
8.84 ± 1.54*▲
9.13 ± 3.75*▲
8.26 ± 2.12*▲
3.61 ± 1.51**
2.20 ± 0.84
1.60 ± 1.14*▲
1.40 ± 1.14*▲
1.80 ± 0.84*▲
19.20 ± 1.64**
20 × 1
40 × 1
80 × 1
60 × 1
As compared with the control group: *P > 0.05, **P < 0.01; as compared with the positive control group: ▲P < 0.01.
Furthermore, the mice were given oral doses of compound 1f at 5, 10 and 20 mg/kg/d for 5
days with cyclophosphamide at 30 mg/kg/d intravenously as a positive control. Neither the
peripheral white blood cells nor the micronucleus frequency were affected in a statistically
significant manner when compared with the negative control groups (Table 5). Overall, the
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nucleoside analog 1f exhibits insignificant toxicity effect on white blood cells and low mutagenic
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effect at the dosages up to 80 mg/kg/d (single dose) or 20 mg/kg/d (5 days) in KM mice.
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Table 5. Toxicity Effect of 1f on Peripheral WBCs and MNF in KM Mice (Multiple Doses)
Micronucleus frequency
± s, ‰)
Number of
animals
Number of WBCs
(×109/L)
Group
Dose (mg/kg/d)
Gender
(
x
♂
♂
♂
♂
♂
Control
Low dose
Medium dose
High dose
CP
−
5
5
5
5
5
7.47 ± 3.77
6.05 ± 1.75*▲
8.09 ± 1.79*▲
6.53 ± 1.36*▲
1.76 ± 0.71**
1.80 ± 0.84
1.20 ± 0.45*▲
1.40 ± 0.55*▲
2.80 ± 0.84*▲
23.0 ± 6.08**
5 × 5
10 × 5
20 × 5
30 × 5
As compared with the control group: *P > 0.05, **P < 0.01; as compared with the positive control group: ▲P < 0.01.
3.5. Assessment of Efficacy in DHBV-infected Ducks. The antiviral efficacy of 1f was
examined in DHBV-infected duck models. Compared with the control group, both the DHBV
DNA levels in serum and liver were reduced by the treatment with 1f in a time- and dose-
dependent manner (Table 6−7). The average inhibition of serum DHBV DNA levels with 1f at
the dosages of 0.04, 0.2 and 1 mg/kg/d was 48.1%, 62.5% and 71.9% for day 5, and 43.4%,
69.5% and 74.2% for day 10, respectively. The mean inhibition by 1f of DNA levels in liver
DHBV at the dosages of 0.04, 0.2 and 1 mg/kg/d was 34.8%, 52.7% and 64.1% for day 5, and
62.8%, 68.7% and 82.1% for day 10, respectively. In addition, the levels of both serum and liver
tissue DHBV DNA had a lower degree of recovery after withdrawal of the drug for 3 days.
These results suggested that only 1 mg/kg/d dosage of compound 1f has an effective inhibitory
effect on the viral DNA replication which is comparable to that of anti-HBV drug lamivudine
(3TC, at 20 mg/kg/d) in vivo.
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Table 6. Inhibitory Activity of 1f on DHBV DNA in Duck Serum
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T5
T10
P3
Dose
Compounds
Inhibition
rates
Inhibition
rates
Inhibition
rates
× 106 copies/mL
× 106 copies/mL
× 106 copies/mL
(mg/kg/d)
Control
1f
−
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0.2
1
19.69 ± 5.46
10.22 ± 5.39
7.38 ± 2.69*
5.54 ± 0.93**
2.25 ± 0.86**
−
21.01 ± 2.77
11.90 ± 4.09*
6.41 ± 2.61**
5.41 ± 1.85**
3.74 ± 1.87**
−
27.76 ± 5.26
14.68 ± 10.13
9.44 ± 4.71*
8.35 ± 1.09*
5.75 ± 3.60**
−
48.1%
62.5%
71.9%
88.6%
43.4%
69.5%
74.2%
82.2%
47.1%
66.0%
69.9%
79.3%
1f
1f
3TC
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As compared with the control group:*P < 0.05, **P < 0.01
Table 7. Inhibitory Activity of 1f on DHBV DNA in Duck Liver
T5
T10
P3
Dose
Inhibition
rates
× 106
Inhibition
rates
×106
Inhibition
rates
Compounds
× 106 copies/mL
(mg/kg/d)
copies/mL
copies/mL
Control
1f
−
0.04
0.2
1
12.85 ± 2.67
8.38 ± 3.38
−
41.05 ± 8.09
15.28 ± 3.78**
12.83 ± 3.22**
7.36 ± 4.15**
6.57 ± 1.93**
−
25.86 ± 6.94
10.72 ± 3.91*
9.55 ± 3.59**
6.17 ± 1.56**
6.15 ± 1.59**
−
34.8%
52.7%
64.1%
52.7%
62.8%
68.7%
82.1%
84.0%
58.5%
63.1%
76.1%
76.2%
6.08 ± 2.44*
4.61 ± 0.64**
6.08 ± 1.64*
1f
1f
3TC
20
As compared with the control group:*P < 0.05, **P < 0.01
3.6. Assessment of Pharmacokinetics (PK) Study Sprague-Dawley Rats. A preliminary
pharmacokinetics study was carried out in fasted Sprague-Dawley rats, which were given a 2.5
mg/kg intravenous or 32 mg/kg oral dose of compound 1f. Following intravenous dosing at 2.5
mg/kg, the test compound had an average plasma half life (t1/2) of 1.23 ± 0.38 h, with the total
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body clearance rate (CL) of 2.79 ± 0.67 L/h/kg and the average volume of distribution (Vd) of
4.77 ± 0.33 L/kg. After oral dosing at 32 mg/kg, the test compound was absorbed quickly and
reached the first peak concentration within 0.50 ± 0.00 h, and then it reached the maximum
plasma concentration (Cmax) of 273.97 ± 92.66 ng/mL at an average time (Tmax) of 5.00 ± 1.15 h.
This bimodal curve in the change of drug concentration (see Supporting Information) indicated
that the test compound may remain in enterohepatic circulation for a prolonged period, which
may contribute to the unfavorable oral bioavailability (F = 18.7 ± 7.5%).
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3.7. Assessment of Metabolism Study. In spite of its low oral bioavailability, compound 1f
still exhibited good in vivo efficacy, which indicated that the test compound may have been
partially metabolized to other active metabolites before the phosphorylation. In order to indentify
the real active specie(s) associated with compound 1f, we firstly conducted a preliminary in vitro
metabolism study. After incubation of 1f with rat liver microsomes at 37 ºC for 2 h, a major
metabolite (M1) peak was detected by LC-MS (See Figure 3 in Supporting Information). M1 is
polar than compound 1f with a 16-dalton higher molecular weight, which could be a
hydroxylated derivative of 1f. In the in vivo metabolism study, this primary metabolite (M1) can
also be observed in the plasma, bile and urine of 1f-dosed rats. Further analyzing the urine
sample by LC-MS, another two metabolites (M2 and M3) were indentified. M2 has a 16-dalton
higher molecular weight than M1, and it could be a dihydroxylated derivative of compound 1f.
M3 should be compound 7, as it possesses the same molecular weight and retention time as the
1
latter does (See Figure 4 in Supporting Information). In addition, the H NMR spectrum of
isolated M3 from the urine sample is also identical to that of compound 7. In view of these
metabolites identified, we assume that in the drug-dosed animals compound 1f was firstly
hydroxylated (on the cyclopropyl ring) into metabolites M1 and M2.29 After losing the whole
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cyclopropyl ring,30 it was eventually converted to compound 7 (See Scheme 1 in Supporting
Information), the major active metabolite, which has been demonstrated with potent both in vitro
and in vivo anti-HBV activity in our previous work.21,22 This could be the reason why 1f still
exhibited good efficacy in vivo in spite of its unfavorable oral bioavailability.
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3.8. Molecular Modeling of the Interactions between 1f Triphosphate (1f-TP) and HBV
Polymerase. Although no crystal structure for HBV polymerase is currently available, several
homology models based on the crystal structure of HIV-RT have been reported, and these serve
as a useful guide for understanding the molecular interactions of HBV DNA polymerase with
antiviral drugs.31-34 We performed homology modeling, molecular docking and MD simulations
to investigate the interactions between 1f-TP (the bioactive form of 1f in vivo) and HBV DNA
polymerase. A representative snapshot derived from MD simulations is shown in Figure 4. As
can be seen, 1f-TP fits well in the active site and the complex is stabilized by extensive hydrogen
bonding and salt bridge interactions. The base moiety of 1f-TP forms three hydrogen bonds with
the complementary base pair, dGMP, in the template chain. Moreover, it formed π-π stacking
interaction with the adjacent DNA base in the primer chain. The positively charged side chains
of Arg41 and Lys32 are involved in extensive salt bridge interactions with the negatively
charged phosphate group of 1f-TP. The carboxyl groups of Asp83, Asp205 and Asp 206 together
with the three phosphate groups of 1f-TP formed a metal chelating interaction network with the
two Mg2+ ions. These enzyme-substrate interactions are very similar to those seen in the crystal
structures of HIV-RT (PDB ID: 1T05 and 1RTD),35, 36 suggesting that the pose of 1f-TP in the
active site is favorable. It is noteworthy that the molecular modeling study also demonstrated that
the azido group is positioned well in the hydrophobic pocket enclosed by the side-chains of
Ala87, Phe88, Met204 and Asp205, while the fluorine atom is involved in a C−H---F bond
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interaction with Arg41. In addition, the cyclopropyl group is involved in hydrophobic
interactions with the side-chains of Val30. Further molecular docking study for other alkyl
substituents like methyl, ethyl, isopropyl, cyclopentyl, and cyclohexyl groups indicated that the
hydrophobic interactions for these groups with Val30 are also present. However, for the larger
hydrophobic group like cyclopentyl, it is energetic unfavorable for it to be positioned in the polar
water environment. While for isopropyl, the steric interaction between it and the neighboring
base-pair is disadvantageous for the base-pair interactions. For cyclohexyl group, both steric
hindrance and its unfavorable interaction with water contribute to its lower potential activity. The
molecular modeling results are in agreement with the structure-activity relationship (SAR)
presented in Table 1, and clearly reveal the detailed interactions between 1f-TP and the viral
DNA polymerase, which could assist in the rational design of potential anti-HBV drugs.
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Figure 4. Representative binding complex structure of HBV DNA polymerase and 1f-TP
derived from MD simulations. The HBV DNA polymerase is represented as a cartoon and 1f-TP
is shown in stick form, colored in magenta. The residues interacting with 1f-TP are shown in
yellow, and two green spheres represent the Mg2+ ions.
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■ CONCLUSION
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We have designed and synthesized a series of 4-substituted derivatives of the parent compound 7
in order to address the issue of the toxicity of the parent compound. In vitro evaluation of the
new synthetic nucleosides showed the cyclopropylamino substituted analog 1f to have good anti-
HBV activity. Tests in the HepG2.2.15 cell line indicated that compound 1f at a concentration of
5 μM reduced both the intracellular and extracellular viral DNA levels by 68.2% and 87.6%,
respectively, on day 9. This cyclopropylamino substituted nucleoside exhibited favorable toxicity
profiles in KM mice. It was well tolerated at a dose up to 3 g/kg via oral administration, and
showed insignificant toxicity on white blood cells and low mutagenic effect at oral dosages of up
to 80 mg/kg (single) and 20 mg/kg/d (5 days). Efficacy studies in DHBV-infected ducks
demonstrated that both the serum (74.2%) and liver (82.1%) viral DNA levels were markedly
reduced by the treatment with compound 1f at the dose of 1 mg/kg/d for 10 days, with a lower
degree of recovery after withdrawal of the test compound for 3 days.
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■ EXPERIMENTAL SECTION
1. Chemistry. 1H NMR spectra were recorded on a 400 MHz (13C NMR spectra were recorded
on a 100 MHz) spectrometer. Chemical shift values are given in ppm and referred to the internal
standard of TMS (tetramethylsilane). The peak patterns are indicated as follows: s, singlet; d,
doublet; t, triplet; q, quadruplet; m, multiplet; dd, doublet of doublets and dt, doublet of triplets.
The coupling constants (J) are reported in Hertz (Hz). Flash column chromatography was
performed over silica gel 200-300 mesh, and the eluent was a mixture of MeOH/CH2Cl2 for 1a,
EtOAc/petroleum ether for 4b−k, MeOH/EtOAc for 1b−k. High-resolution mass spectra
(HRMS-ESI) were obtained on a Q-TOF Mass Spectrometer. All the tested compounds possess a
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purity of at least 95%. Analytical HPLC was run on the Agilent 1260 HPLC instrument,
equipped with Agilent XDB-C18 column and UV detection at 270 nm. Eluent system was: 40%
MeOH in H2O; flow rate = 0.8 mL/min.
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1.1. Synthesis of 1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydro-
furan- 2-yl)pyrimidine-2,4(1H,3H)-dione (1a). Compound 2 (529 mg, 1 mmol) was dissolved in
a solution of NH3 in MeOH (10 mL) and stirred at room temperature until TLC indicated the
complete disappearance of starting material 2. The reaction mixture was concentrated and then
purified through silica gel column chromatography (MeOH/CH2Cl2, 5:95) to afford product 1a
(247 mg, 86%) as a white solid. 1H NMR (400 MHz, CD3OD) δ 7.73 (dd, J = 8.0, 1.6 Hz, 1H),
6.44 (dd, J = 11.2, 5.6 Hz, 1H), 5.70 (d, J = 8.0 Hz, 1H), 5.19 (dt, J = 53.6, 5.2 Hz, 1H), 4.49 (dd,
J = 22.4, 5.2 Hz, 1H), 3.83 (s, 2H); 13C NMR (100 MHz, CD3OD) δ 164.3, 150.4, 141.1, 101.1,
96.9 (d, JC-F = 8.4 Hz), 94.7 (d, JC-F = 192.8 Hz), 82.2 (d, JC-F = 16.9 Hz), 74.7 (d, JC-F = 24.6
Hz), 61.7; HRMS (m/z) (M + H) calcd for C9H11FN5O5 288.0739, found 288.0741.
1.2. General Procedure for the Synthesis of Compounds 1b-k. 1,2,4-triazole (760 mg, 11 mmol)
and pyridine (0.81 mL, 10 mmol) were added sequentially to a suspension of compound 2 (529
mg, 1 mmol) in anhydrous CH2Cl2 (20 mL). The resulting mixture was cooled to 0 ºC and treated
slowly with phosphoryl chloride (POCl3, 0.46 mL, 5 mmol). After stirring at 0 ºC for 10 min, the
reaction was heated to reflux for 5 h until TLC indicated the disappearance of starting material 2.
Then it was cooled to 0 ºC, and quenched slowly with H2O (2 mL), followed by addition of brine
(20 mL). After having been stirred for 10 min, the organic layer was separated and the aqueous
layer was extracted with EtOAc (15 mL x 3). The combined organic layer was washed with
sodium bicarbonate solution (20 mL) and brine (20 mL) in sequence, dried over anhydrous
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sodium sulfate, and concentrated to give the crude compound 3 as a pale yellow solid, which
was used directly in the next step without further purification.
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The above intermediate 3 was dissolved in CH2Cl2 (10 mL), and then treated with the
corresponding amine (2.5 mmol) and N,N-diisopropylethylamine (DIPEA, 0.44 mL, 2.5 mmol;
another 2.5 mmol of DIPEA was added, if the HCl salt of the amine was used). After stirring at
room temperature for 5 h, when TLC indicated that the reaction was complete, the reaction was
quenched with 5% citric acid (20 mL) and stirred for 10 min. The organic layer was separated
and the aqueous layer was extracted with CH2Cl2 (15 mL x 3). The combined organic layer was
washed with brine (20 mL), dried over anhydrous sodium sulfate, and concentrated. The
resulting residue was purified by silica gel column chromatography to give the corresponding
intermediate 4, which was stirred in NH3 solution in MeOH (5 mL) until TLC indicated that the
debenzoylation was complete. The solvent was evaporated, and the resulting residue was purified
through silica gel column chromatography to afford products 1b-k in 36−87% yield over three
steps.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(methylamino)pyrimidin-2(1H)-one (1b). The product was obtained according to the general
procedure as a pale yellow solid in 64% yield; 1H NMR (400 MHz, CD3OD) δ 7.62 (dd, J = 7.6,
1.2 Hz, 1H), 6.47 (dd, J = 12.4, 5.2 Hz, 1H), 5.84 (d, J = 7.6 Hz, 1H), 5.18 (dt, J = 53.6, 4.8 Hz,
1H), 4.46 (dd, J = 21.6, 4.8 Hz, 1H), 3.82 (s, 2H), 2.88 (s, 3H); 13C NMR (100 MHz, CD3OD) δ
164.5, 156.7, 139.7, 97.0 (d, JC-F = 7.7 Hz), 95.5, 94.7 (d, JC-F = 192.3 Hz), 83.2 (d, JC-F = 17.1
Hz), 75.0 (d, JC-F = 25.0 Hz), 61.9, 26.4; HRMS (m/z) (M + H) calcd for C10H13FN6O4 301.1055,
found 301.1057.
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1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
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(ethylamino)pyrimidin-2(1H)-one (1c). The product was obtained according to the general
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1
procedure as a white solid in 70% yield; H NMR (400 MHz, CD3OD) δ 7.62 (dd, J = 7.6, 1.2
Hz, 1H), 6.47 (dd, J = 12.4, 5.2 Hz, 1H), 5.83 (d, J = 7.6 Hz, 1H), 5.17 (dt, J = 53.6, 4.8 Hz, 1H),
4.46 (dd, J = 22.0, 4.4 Hz, 1H), 3.82 (s, 2H), 3.38 (q, J = 7.2 Hz, 2H), 1.17 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, CD3OD) δ 163.8, 156.8, 139.7, 97.0 (d, JC-F = 7.7 Hz), 95.6, 94.7 (d, JC-F
=
192.3 Hz), 83.2 (d, JC-F = 17.1 Hz), 75.1 (d, JC-F = 25.0 Hz), 61.9, 35.1, 12.9; HRMS (m/z) (M +
H) calcd for C11H15FN6O4, 315.1212, found 315.1216.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(isopropylamino)pyrimidin-2(1H)-one (1d). The product was obtained according to the general
procedure as a white solid in 85% yield. 1H NMR (400 MHz, CD3OD) δ 7.62 (d, J = 7.6 Hz, 1H),
6.49 (dd, J = 12.4, 5.2 Hz, 1H), 5.83 (d, J = 7.6 Hz, 1H), 5.19 (dt, J = 53.6, 4.8 Hz, 1H), 4.47 (dd,
13
J = 22.0, 4.4 Hz, 1H), 4.24 (heptet, J = 6.4 Hz, 1H), 3.83 (s, 2H), 1.19 (d, J = 6.4 Hz, 6H); C
NMR (100 MHz, CD3OD) δ 162.6, 156.4, 139.3, 96.6 (d, JC-F = 7.7 Hz), 95.3, 94.3 (d, JC-F
=
192.2 Hz), 82.8 (d, JC-F = 17.1 Hz), 74.6 (d, JC-F = 25.0 Hz), 61.5, 41.5, 20.61, 20.56; HRMS
(m/z) (M + H) calcd for C12H18FN6O4 329.1368, found 329.1373.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(sec-butylamino)pyrimidin-2(1H)-one (1e). The product was obtained according to the general
procedure as a white solid in 87% yield. 1H NMR (400 MHz, CD3OD) δ 7.60 (d, J = 7.6 Hz, 1H),
6.47 (dd, J = 12.4, 5.2 Hz, 1H), 5.83 (d, J = 7.6 Hz, 1H), 5.17 (dt, J = 53.6, 4.8 Hz, 1H), 4.45 (dd,
J = 22.0, 4.4 Hz, 1H), 4.10–4.05 (m, 1H), 3.81 (s, 2H), 1.55–1.48 (m, 2H), 1.14 (d, J = 6.4 Hz,
3H), 0.90 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 164.9, 158.3, 141.1, 98.4 (CH, d,
JC-F = 7.6 Hz), 97.1, 96.1 (d, JC-F = 192.3 Hz), 84.6 (d, JC-F = 17.2 Hz), 76.5 (d, JC-F = 25.0 Hz),
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63.3, 48.74, 48.72, 30.2, 30.1, 20.2, 20.1, 10.8; HRMS (m/z) (M + H) calcd for C13H20FN6O4
343.1525, found 343.1525.
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1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(cyclopropylamino)pyrimidin-2(1H)-one (1f). The product was obtained according to the
general procedure as a white solid in 85% yield; 1H NMR (400 MHz, CD3OD): δ 7.67 (d, J = 7.6
Hz, 1H), 6.51 (dd, J = 12.4, 5.2 Hz, 1H), 5.83 (d, J = 7.6 Hz, 1H), 5.22 (dt, J = 53.6, 4.4 Hz, 1H),
4.54-4.45 (m, 1H), 3.85 (s, 2H), 2.89 (heptet, J = 3.6 Hz, 1H), 0.84–0.79 (m, 2H), 0.58–0.54 (m,
2H); tautomer: δ 7.94 (d, J = 7.6 Hz, 1H), 6.51 (dd, J = 12.4, 5.2 Hz, 1H), 6.37 (d, J = 7.6 Hz,
1H), 5.22 (dt, J = 53.6, 4.8 Hz, 1H), 4.54-4.45 (m, 1H), 3.86 (s, 2H), 2.62 (heptet, J = 3.6 Hz,
1H), 0.90–0.86 (m, 2H), 0.66–0.62 (m, 2H); 13C NMR (100 MHz, CD3OD): δ 164.9, 156.2,
139.4, 96.6 (d, JC-F = 7.6 Hz), 94.9, 94.3 (d, JC-F = 192.3 Hz), 82.9 (d, JC-F = 17.1 Hz), 74.6 (d,
JC-F = 25.0 Hz), 61.5, 22.8, 5.2; tautomer: δ 166.5, 155.5, 142.1, 96.8 (d, JC-F = 7.6 Hz), 94.2 (d,
JC-F = 192.8 Hz), 91.1, 83.0 (d, JC-F = 16.5 Hz), 74.6 (d, JC-F = 24.9 Hz), 61.5, 22.6, 6.0, 5.9;
HRMS (m/z) (M + H) calcd for C12H16FN6O4 327.1212, found 327.1215. For the X-ray structure
and data of compound 1f (CIF) see Supporting Information.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(cyclopentylamino)pyrimidin-2(1H)-one (1g). The product was obtained according to the
general procedure as a white solid in 72% yield. 1H NMR (400 MHz, CD3OD) δ 7.60 (d, J = 7.6
Hz, 1H), 6.46 (dd, J = 12.4, 5.2 Hz, 1H), 5.82 (d, J = 7.6 Hz, 1H), 5.17 (dt, J = 53.6, 4.8 Hz, 1H),
4.45 (dd, J = 22.0, 4.8 Hz, 1H), 4.33 (quint, J = 6.8 Hz, 1H), 3.81 (s, 2H), 2.01–1.96 (m, 2H),
1.73–1.45 (m, 6H); 13C NMR (100 MHz, CD3OD) δ 163.0, 156.4, 139.2, 96.6 (d, JC-F = 7.8 Hz),
95.2, 94.3 (d, JC-F = 193.5 Hz), 82.8 (d, JC-F = 17.2 Hz), 74.6 (d, JC-F = 25.0 Hz), 61.5, 51.5, 31.7,
22.9; HRMS (m/z) (M + H) calcd for C14H20FN6O4 355.1525, found 355.1514.
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(cyclohexylamino)pyrimidin-2(1H)-one (1h). The product was obtained according to the
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general procedure as a white solid in 75% yield; H NMR (400 MHz, CD3OD) δ 7.60 (dd, J =
7.6, 1.2 Hz, 1H), 6.46 (dd, J = 12.4, 4.8 Hz, 1H), 5.81 (d, J = 7.6 Hz, 1H), 5.16 (dt, J = 53.6, 4.8
Hz, 1H), 4.45 (dd, J = 21.6, 4.4 Hz, 1H), 3.95–3.88 (m, 1H), 3.81 (s, 2H), 1.95–1,93 (m, 2H),
1.76–1,62 (m, 3H), 1.42–1,33 (m, 2H), 1.26–1,18 (m, 3H); 13C NMR (100 MHz, CD3OD) δ
163.0, 156.8, 139.7, 96.9 (d, JC-F = 7.7 Hz), 95.7, 94.7 (d, JC-F = 192.3 Hz), 83.2 (d, JC-F = 17.1
Hz), 75.0 (d, JC-F = 25.0 Hz), 61.9, 49.0, 32.13, 32.07, 25.2, 24.6; HRMS (m/z) (M + H) calcd
for C15H22FN6O4 369.1681, found 369.1681.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(dimethylamino)pyrimidin-2(1H)-one (1i). The product was obtained according to the general
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procedure as a white solid in 62% yield; H NMR (400 MHz, CD3OD) δ 7.78 (dd, J = 7.6, 1.2
Hz, 1H), 6.48 (dd, J = 12.4, 5.2 Hz, 1H), 6.16 (d, J = 8.0 Hz, 1H), 5.18 (dt, J = 53.6, 4.8 Hz, 1H),
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4.46 (dd, J = 21.6, 4.4 Hz, 1H), 3.82 (s, 2H), 3.16 (s, 3H), 3.11 (s, 3H); C NMR (100 MHz,
CD3OD) δ 163.6, 156.0, 141.0, 97.1 (d, JC-F = 7.6 Hz), 94.7 (d, JC-F = 192.3 Hz), 92.3, 83.3 (d,
JC-F = 17.1 Hz), 75.0 (d, JC-F = 25.1 Hz), 61.9, 36.9, 36.2; HRMS (m/z) (M + H) calcd for
C11H15FN6O4 315.1212, found 315.1215.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(diethylamino)pyrimidin-2(1H)-one (1j). The product was obtained according to the general
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procedure as a white solid in 81% yield; H NMR (400 MHz, CD3OD) δ 7.76 (dd, J = 8.0, 1.2
Hz, 1H), 6.48 (dd, J = 12.4, 5.2 Hz, 1H), 6.12 (d, J = 8.0 Hz, 1H), 5.19 (dt, J = 53.6 Hz, 4.8 Hz,
1H), 4.47 (dd, J = 21.6, 4.4 Hz, 1H), 3.82 (s, 2H), 3.65 (q, J = 6.8 Hz, 2H), 3.46 (q, J = 6.8 Hz,
2H), 1.21–1.15 (m, 6H); 13C NMR (100 MHz, CD3OD) δ 162.4, 156.2, 141.1, 97.1 (d, JC-F = 7.4
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Hz), 94.7 (d, JC-F = 192.4 Hz), 92.5, 83.3 (d, JC-F = 17.1 Hz), 75.0 (d, JC-F = 25.0 Hz), 61.9, 42.9,
42.1, 12.1, 11.5; HRMS (m/z) (M + H) calcd for C13H19FN6O4 343.1525, found 315.1528.
1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-4-
(pyrrolidin-1-yl)pyrimidin-2(1H)-one (1k). The product was obtained according to the general
procedure as a white solid in 36% yield; 1H NMR (400 MHz, DMSO-d6) δ 7.63 (dd, J = 7.6, 1.2
Hz, 1H), 6.41–6.37 (m, 2H), 5.90 (d, J = 7.6 Hz, 1H), 5.70 (t, J = 6.0 Hz, 1H), 5.15 (dt, J = 54.0
Hz, 5.2 Hz, 1H), 4.42 (dt, J = 22.8, 5.2 Hz, 1H), 3.71 (d, J = 5.6 Hz, 2H), 3.44–3.31 (m, 4H),
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1.93–1.82 (m, 4H); C NMR (100 MHz, DMSO-d6) δ 161.2, 154.3, 141.7, 97.2 (d, JC-F = 8.8
Hz), 95.0 (d, JC-F = 191.1 Hz), 93.4, 82.5 (d, JC-F = 17.0 Hz), 75.3 (d, JC-F = 24.1 Hz), 62.5, 47.03,
47.00, 25.5, 24.6; HRMS (m/z) (M + H) calcd for C13H17FN6O4 341.1368, found 341.1371.
2. Cytotoxicity Assay. To determine the cell viability, a MTT-based assay was performed.37
Briefly, HepG2.2.15 cells were seeded at a density of 2 × 104 cells per well on 96-well plates.
After incubation for 24 h, the cells were treated with medium containing the test compound at
various concentrations. The culture medium was replaced with a fresh one on day 3 or day 6
during the 9-day experiment. On day 9, 20 μL of MTT solution (5 mg/mL) was added to each
well. After incubating routinely for 4 h, the supernatants were discarded and 180 μL of DMSO
was added into each well to solubilize the formazan. The absorbance (A) at 490 nm was
measured by using an automatic plate reader (BIO-RAD, 168-1000XC, USA). The survival rate
of HepG2.2.15 cells (%) was calculated as [1 − (A490 of experimental group/A490 of negative
control) × 100%].38
3. In Vitro Anti-HBV Activity Evaluation in HepG2.CW Cell Line. The HepG2.CW cells28
were plated on 24-well plates at a density of 1 × 105 cells per well and routinely cultured for 24 h.
Then, HepG2.CW cells were treated with freshly prepared medium containing the test compound
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at the concentration of 1 μM or ETV as a positive control at 1 μM. Normal control cells were
treated with minimum essential medium (MEM) only. Media were changed every 3 days, and the
supernatants were collected on day 9. HBV DNA was extracted by a viral DNA extraction kit
according to the manufacturer’s instructions and the viral DNA levels were detected by HBV
fluorescent quantitative PCR detection kit according to the manufacturer’s protocol.
Amplification and detection were performed on ABI7500 fluorescent quantitative PCR
instrument. The program was optimized as follows: 37 ºC for 5 min, at 94 ºC for 2 min
(denaturation), followed by 40 cycles of amplification (at 94 ºC for 20 s, 55 ºC for 30 s, 72 ºC for
10 s). Inhibition rate of HBV DNA (%) = [1 − (HBV DNA concentration of experimental
group/HBV DNA concentration of negative control) × 100%]
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4. Assessment of Inhibitory Effect on the HBV DNA level in the HepG2.2.15 Cell Line.
The HepG2.2.15 cells were plated at a density of 2 × 104 cells per well on 24-well plates and
routinely cultured for 24 h. Then, HepG2.2.15 cells were treated with freshly prepared medium
containing compound 1f (0.2, 1, 5 μM) or entecavir (0.1 μM as positive control). Normal control
cells were treated with RPMI 1640 only. Media were changed every 3 days, and both the
supernatants and cells were collected. The extracellular and intracellular HBV DNA was
extracted by viral DNA extraction kit according to the manufacturer’s instructions. The
concentration of extracted HBV DNA was determined by real-time FQ-PCR, performed in
Light-Cycler 1.5 (Roche, Mannheim, Germany) using the HBV fluorescent quantitative PCR
detection kit (Piji Biotechnology Development, Shenzhen, PR China) according to the
manufacturer’s protocol. Inhibition rate of HBV DNA (%) = [1 − (HBV DNA concentration of
experimental group/HBV DNA concentration of negative control) × 100%]39, 40
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5. Formulation. Generally, the test compound (1f) was dissolved in normal saline (or distilled
water) to form the desired concentrations for oral or intravenous administration. High
concentrations of 1f were achieved by sonicating the test compound in normal saline (or distilled
water) at 37 °C until a clear solution was formed. For the oral administration of 1f at 3 g/kg, the
test compound was suspended in 0.5% CMC-Na solution to reach the required concentration.
6. Assessment of Acute Toxicity Study in KM Mice. For the acute toxicity study, twenty
male specefic pathogen free (SPF) KM mice (20 ± 2 g) were randomized into four treatment
groups, with five animals per group. Three groups of the test animals were dosed once with 1f at
400 mg/kg, 300 mg/kg, and 200 mg/kg intravenously, respectively [Formulation: 2.0%, 1.5%,
and 1.0% solution in normal saline (g/mL), dosage volume: 0.2 mL/10 g]. One group of the test
animals were dosed once with 1f at 3 g/kg by oral gavage [Formulation: 7.5% suspension in
0.5% CMC-Na solution (g/mL), dosage volume: 0.4 mL/10 g]. After administration, the mice
were observed for 14 days (general clinical observation, body weight, food consumption) and
sacrificed at day 15.
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7. Assessment of Toxicity on White Blood Cells and Mutagenic Effect in KM Mice.
Twenty-five SPF male KM mice (20 ± 2 g) were randomized into five groups, with five animals
per group. The control group was given just water orally. For the single-dose study, the treatment
groups were dosed with 80, 40, and 20 mg/kg of compound 1f by oral gavage, respectively
[Formulation: 0.4%, 0.2%, and 0.1% solution in distilled water (g/mL), dosage volume: 0.2
mL/10 g]. The positive control group was dosed with 60 mg/kg of cyclophosphamide
intravenously [Formulation: 0.3% solution in normal saline (g/mL), dosage volume: 0.2 mL/10
g]. 48 h after the administration of the test compound, blood (20 μL) was collected from the tail
vein for white blood cell (WBC) counts, and then the animals were sacrificed for micronucleus
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counts. For the multiple-dose study, the treatment groups were dosed with 20, 10, and 5 mg/kg/d
of compound 1f by oral gavage, respectively, for 5 days [Formulation: 0.1%, 0.05%, and 0.025%
solution in distilled water (g/mL), dosage volume: 0.2 mL/10 g]. The positive control group was
dosed with 30 mg/kg/d of cyclophosphamide intravenously for 5 days [Formulation: 0.15%
solution in normal saline (g/mL), dosage volume: 0.2 mL/10 g]. 24 h after the last administration
of the test compound, blood (20 μL) was collected from the tail vein for white blood cell (WBC)
counts, and then the animals were sacrificed for micronucleus counts. Frequencies of
micronucleus (‰) were given as the number of micronuclei in 1000 polychromatic erythrocytes
(PCEs). Results are means ± SD of the indicated number of independent experiments. Statistical
significance was determined using analysis of variance. P-values < 0.01 were considered
statistically significant.
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8. Assessment of Efficacy in DHBV-infected Ducks. Each duck, aged 3 day, was injected in
its tibial vein with 0.2 mL of serum from ducks with positive DHBV DNA serology.41 On
seventh day, their blood was drawn, DHBV DNA of serum samples was extracted and PCR was
performed to screen out positive ducks. Drug treatment experiment was carried out on the
seventh day after the ducks were infected with DHBV. The positive ducks were randomly
divided into five groups with 16 ducks in each group. The treatment groups were administered
doses of 0.04, 0.2, 1 mg/kg of compound 1f orally once daily for 10 days continuously. The
positive control group was given 20 mg/kg/d of lamivudine (3TC) for 10 days, the negative
control group with normal saline. The antiviral activity of compound 1f was assessed by
comparing the serum and liver DHBV DNA levels after 1f-treated at initiation of treatment (T0),
the fifth day of treatment (T5), the tenth day of treatment (T10) and the third day (P3) of post-
treatment follow-up.42-44 The duck serum DHBV DNA was extracted using Viral DNA
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Extraction Kit. The duck liver tissue DHBV DNA was extracted using Tissue DNA Extraction
Kit. FQ-PCR was performed in Light-cycler (Roche) using SYBR Green I.21
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9. Assessment of Pharmacokinetics (PK) Study In Sprague-Dawley Rats. At 12 hours prior
to dosing, Sprague-Dawley rats were fasted, but supplied with water. Two groups of animals
were given 2.5 mg/kg intravenous dose and 32 mg/kg oral dose of compound 1f, respectively.
Blood was harvested from retinal vein at predosing time point, 5 min, 15 min, 30 min, 1 h, 1.5 h,
2 h, 3 h, 4 h, 6 h, 8 h, 12 h, and 24 h. Plasma was prepared, and quantitatively analyzed for the
test compound 1f by LC-MS/MS. Half life (t1/2), clearance rate (CL), volume of distribution (Vd),
the maximum plasma concentration (Cmax), Tmax and oral bioavailability (F) were calculated by
software DAS 3.0.
10. Assessment of Metabolism Study in Rat Liver Microsomes. A 250 μL mixed pool of
compound 1f (5 μg/mL), rat liver microsomes (1 mg/mL), NADPH (2 mM), magnesium chloride
(6.6 mM) and potassium phosphate buffer solution (100 mM, pH = 7.4) was incubated at 37°C
for 2 h. The blank and negative control samples were incubated without compound 1f or
NADPH, respectively. After the incubation, the reaction was cooled by ice-bath and terminated
by ice-cold methanol (750 μL). The solvent was evaporated to dryness under a steam of nitrogen
at 40 ºC; and the resulting residue was re-dissolved in the mobile phase (100 μL) and briefly
vortexed. After centrifuging at 13,000 rpm for 10 min, the supernatant was collected for LC-MS
analysis.
11. Molecular Modeling of the Interactions between 1f Triphosphate (1f-TP) and HBV
DNA Polymerase. Homology modeling, molecular docking and MD simulations were
performed to construct and optimize the 3D structure of HBV polymerase genotype B based on
the X-ray crystal structures of HIV-RT (PDB ID: 1T05 and 1RTD).35, 36 Briefly, the homology
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modeling was carried out by using the automated modeling program, MODELLER 9.13.45 The
sequence alignment between HBV polymerase genotype B and HIV-RT was described by Li et
al.33 After the initial model was prepared, the coordinates of the original ligand tenofovir
diphosphate (TNV-DP), the DNA duplex and the Mg2+ ions were merged into the HBV structure
at a site similar to that in the crystal structures. The ionizable residues were set to the standard
protonated or deprotonated states at the physiological conditions (pH = ~7.4) and the whole
structure of HBV polymerase binding with TNV-DP was solvated in a rectangular box of TIP3P
water molecules with a minimum solute-wall distance of 10 Å. Additional Cl‾ ions were added
as counter ions to neutralize the whole system. Subsequently, energy minimizations were carried
out using the Sander module of the Amber 8 program46 with a non-bonded cutoff of 10 Å, and a
conjugate gradient energy-minimization method. Afterwards, in order to simulate appropriate
base-pair interactions (A−T, G−C), the original base T paired with TNVDP in the crystal
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structure of 1T05 was modified to G. Based on the energy-minimized HBV polymerase structure,
1f-TP was docked into the possible active site of the enzyme by the AutoDock 4.2 program.47
The energy minimization and ~5 ns MD simulations for the resultant binding complex were then
performed with both the ligand and enzyme relaxed to obtain an acceptable binding
conformation.
■ ASSOCIATED CONTENT
1
13
Supporting Information. H and C NMR spectra of compounds 1a−k, X-ray structure and
data of compound 1f (CIF), concentration-time curves of 1f in rats, and also metabolism study
data of 1f. This material is available free of charge via the Internet at http://pubs.acs.org.
■ AUTHOR INFORMATION
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Corresponding Author
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*For J.C.: phone, +86 (0)371-6778-3017; E-mail, changjunbiao@zzu.edu.cn. For W.Y.: phone,
+86 (0)371-6778-1788; E-mail, wenquan_yu@zzu.edu.cn.
Notes
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The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We acknowledge the National Natural Science Foundation of China (Nos. 21172202,
81302637 and 81330075), the China Postdoctoral Science Foundation (Nos. 2013M530341 and
2014T70690) and the Chinese Science and Technology Key Projects Grant (No.
2012ZX10004503-011) for financial support.
■ ABBREVIATIONS USED
HBV, hepatitis B virus; IFN, interferon; FDA, food and drug administration; FTC,
emtricitabine; HIV, human immunodeficiency virus; F, bioavailability; t1/2, half life; EC50, 50%
effective concentration; CC50, 50% cytotoxic concentration; DNA, deoxyribonucleic acid; MK
mice, Kunming mice; DHBV, duck hepatitis B virus; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PCR, polymerase chain reaction; FQ-PCR, fluorescence
quantitative PCR; ETV, entecavir; CP, cyclophosphamide; WBC, white blood cells; PCE,
polychromatic erythrocyte; MNF, micronucleus frequency; 3TC, lamivudine; RT, reverse
transcriptase; MD, molecular dynamics; TP, triphosphate; TLC, thin layer chromatography;
MEM, minimum essential medium; CMC-Na, sodium carboxymethyl cellulose; SPF, specific
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