5-Iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones as non-nucleoside anti-HBV agents

Article abstract of DOI:10.1039/c5md00181a

A series of 5-iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones, which can be considered as S-DABO derivatives, have been synthesized and their antiviral effect on extracellular HBV DNA was evaluated using the HepAD38 cell system. Compounds 6d1 and 6e3 exhibited more potent anti-HBV activity than lamivudine with EC₅₀ values of 0.376 μM and 0.469 μM, respectively. In addition, inhibition of intracellular HBV DNA, pgRNA, HBeAg and HBsAg of compounds 6d1 and 6e3 was examined to initially infer the action mechanism. RT-PCR analysis of pgRNA demonstrated that these new S-DABO analogues could not interfere with HBV transcription. TRFIA analysis revealed that compounds 6d1 and 6e3 effectively reduced the secretion of HBeAg. These results demonstrated that 5-iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones possess anti-HBV abilities and could be used as potential agents against HBV infection with an additional merit of low cytotoxicity.

Full text of DOI:10.1039/c5md00181a

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5-Iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones as non-
nucleoside anti-HBV agents
Yu Zhang a, Xuefeng Sun a, Ningning Fan a, Jianxiong Zhao a, Jing Tu c, Xiangmei Chen c, Junyi Liu a,b *
and Xiaowei Wang a *
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A series of 5-iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones, which can be considered as S-DABO derivatives, have been
synthesized and their antiviral effect on extracellular HBV DNA was evaluated using the HepAD38 cell system. Compounds
6d1 and 6e3 exhibited more potent anti-HBV activity than lamivudine with EC50 values of 0.376μM and 0.469 μM,
respectively. In addition, inhibition of intracellular HBV DNA, pgRNA, HBeAg and HBsAg of compounds 6d1 and 6e3 were
detected to initially infer the action mechanism. RT-PCR analysis of pgRNA demonstrated that these new S-DABOs
analogues could not interfere with HBV transcription. TRFIA analysis revealed that compounds 6d1 and 6e3 effectively
reduced the secretion of HBeAg. These results demonstrated that 5-iodo-2-arylalkylthio-6-aryl pyrimidin-4(3H)-ones
possess anti-HBV ability and could be used as potential agents against HBV infection with an additional merit of low
cytotoxicity.
Currently, interferon α and nucleoside analogues are widely
used in controlling the progression of chronic hepatitis B.
However, interferon α is less effective in curing HBV infection
and has some adverse effects.4 Nucleoside analogues (Figure
1) could selectively inhibit the viral polymerase, but the
required long-term therapy might lead almost invariably to
resistance problems.5 Under these circumstances, it is urgent
to find effective anti-HBV agents as nonnucleosides with
different action mechanism.
1.Introduction
The hepatitis B virus (HBV) belongs to Hepadnaviridae,
and its infection is a major cause of viral hepatitis worldwide.1
Acute HBV infection occasionally results in fulminant hepatitis
and usually progresses to a chronic state, which could lead to
liver cirrhosis and hepatocellular carcinoma (HCC).2,3
Approximately 350 million people worldwide are chronically
infected with this virus, which results in 0.5 -1.2 million deaths
annually.
So far, the clinical used anti-HBV drugs are nucleoside
analogues (Figure 1), which need to be transformed to their
active triphosphate forms by cellular kinases. As chain-
terminators, the triphosphate nucleoside inhibitors compete
with natural nucleoside substrates for incorporation into the
elongating DNA chain.6
Interestingly, most of these nucleoside analogues are also
efficient against human immunodeficiency virus(HIV).
Lamivudine7, Tenofovir7 and Entricitabine8 have been clinically
approved for the treatment of HIV infection. In addition,
Adefovir dipivoxil9,10, Entecavir11 and Telbivudine12 are also
reported with anti-HIV activity.
As reported in literature, the polymerase of HBV is
structurally related with the reverse transcriptase of HIV-113.
Therefor, some compounds with anti-HIV activity were
screened for anti-HBV. Lee and his co-workers successfully
developed 2,5-pyridinedicarboxylic acid derivatives, which
originated from non-nucleoside HIV-1 RT inhibitors14, as anti
agent of HBV.
Figure 1. Clinical used anti-HBV drugs
a.Department of Chemical Biology, School of Pharmaceutical Sciences, Peking
University, Beijing 100191, China
b.State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing
100191, China
As anti-HIV-1 non-nucleoside reverse transcriptase
inhibitors(NNRTIs), dihydroalkylthio benzyloxopyrimidines(S-
DABO) derivatives have attracted attention due to their high
specificity, excellent potency and low cytotoxicity.15
c.Department of Microbiology & Infectious Disease Center, School of Basic Medical
Science, Peking University, Beijing 100191, China
* Author to whom correspondence should be addressed; E-Mail:
xiaoweiwang@bjmu.edu.cn ; Tel.: +86-10-8280-5203; Fax: +86-10-8280-1706.
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Reagents and conditions: (a) ethyl bromoacetate, Zn/ THF, reflux. (b) MgCl2, Et3N,
CDI, CH3CN, CH2(COOEt)(COOK), rt. 12h and then reflux 2h (c) thiourea,
EtONa/EtOH, reflux. (d) substituted alkyl halide, K2CO3, rt. (e) NIS, DMF, 0°C.
Scheme 1. Synthesis of target compounds 6
However, little attention has been paid to their anti-HBV
activity. According to the SAR of S-DABO derivatives as HIV-
1 RT inhibitors, both substituents on the side chain of C-2 and
C-6 were important for antiviral activity. Especially, the iodine
atom at the 5-position would provide a halogen bond which
could be beneficial for the inhibitory activity.16 To widen the
antiviral spectrum of S-DABOs, we decided to explore the
different structural modifications of the S-DABOs, including
structural diversity at both the side chain on C-2 and C-6
position with bearing a iodine atom at the 5-position. Firstly,
we screened the activity of target compounds against HBV on
extracellular HBV DNA. For the active compounds, their
inhibition of intracellular HBV DNA, pgRNA, HBeAg and HBsAg
were detected to initially infer the action mechanism.
Figure 2. Preliminary viral screening on extracellular HBV DNA
in HepAD38 cells
2.2 Results and discussion
To preliminary screen the antiviral activity of compounds on
extracellular HBV DNA, all the target compounds were treated with
HepAD38 cells at one concentration(10µM) and lamivudine was
used as a reference antiviral drug (Figure 2). The cell supernatants
were collected and analyzed by qRT-PCR (Quantitative Real-time
PCR). The results are summarized in Table 1.
As shown in Table 1, most of the target compounds displayed
moderate activity against HBV replication. Especially, 6d1 (81%)
displayed potent HBV inhibitory activity close to lamivudine (88%).
Results and discussion
2.1 Chemistry
A general synthesis of target compounds is outlined in Scheme
1. The β-ketoesters 3a-d were obtained by reaction of substituted
benzyl cyanide with zinc and ethyl 2-bromoacetate using the
method of Hannick and Kishi.17 However, compounds 3e was
prepared with an alternative method reported by Clay et al.18
through the reaction of benzo[d]triazol-1-yl acetic acid 2 with N,N'-
carbonyldiimidazole (CDI) followed by treatment with ethyl
potassium malonates in the presence of anhydrous MgCl2 and Et3N.
Condensation of 3a-e with thiourea in the presence of sodium
ethoxide in boiling ethanol provided the 6-aryl thiouracil 4a-e. The
S-DABO analogues 5a-e were prepared from S-alkylation with
appropriate substituted alkyl halide using potassium carbonate in
anhydrous DMF or sodium ethoxide in ethanol15,19,20. Subsequent
treatment of 5a-e with NIS in DMF at 0°C afforded the desirable
target compounds 6a-e21. The structure assignments of these
compounds were identified by NMR and mass spectral data.
Figure 3. Dose-dependent effect on extracellular HBV DNA of
six candidate compounds in HepAD38 cells
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Table 1. Inhibition of extracellular HBV DNA by target compounds
com
pd
inhibit
ionab
NAc
com
pd
inhibit
R1
R2
R1
R2
ion
81%
67%
6%
6a1
6a2
6a3
phenyl
phenyl
phenyl
(p-NO2)C6H5CH2
(p-CN)C6H5CH2
6d1
6d2
6d3
6d4
6d5
6d6
6d7
6d8
6d9
6d10
6d11
6d12
6d13
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
3,4-dimethoxyphenyl
(p-NO2)C6H5CH2
(p-CN)C6H5CH2
63%
(p-NO2)C6H5OCH2CH2
13%
(p-NO2)C6H5OCH2CH2
(p-I)C6H5CH2
17%
54%
41%
49%
NA
31%
52%
62%
65%
NA
6b1
6b2
6b3
3,5-dimethylphenyl
3,5-dimethylphenyl
3,5-dimethylphenyl
(p-NO2)C6H5CH2
(p-CN)C6H5CH2
25%
33%
20%
(p-CF3)C6H5CH2
3,5-Dimethoxybenzyl
(p-CH3)C6H5OCH2CH2
(p-Br)C6H5OCH2CH2
cycloheptyl
(p-NO2)C6H5OCH2CH2
6c1
6c2
6c3
3,5-difluorophenyl
3,5-difluorophenyl
3,5-difluorophenyl
(p-NO2)C6H5CH2
(p-CN)C6H5CH2
(p-NO2)C6H5OCH2CH2
47%
14%
9%
cyclohex-2-en-1-yl
(o-NO2)C6H5CH2
(m-Cl)C6H5CH2
6e1
6e2
6e3
6e4
6e5
3TC
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
(p-NO2)C6H5CH2
(p-CN)C6H5CH2
49%
27%
74%
34%
NA
(m-CH3O)C6H5CH2
(p-NO2)C6H5OCH2CH2
(p-I)C6H5CH2
6e6
6e7
6e8
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
benzo[d]triazol-1-yl
3,5-Dimethoxybenzyl
(p-CH3)C6H5OCH2CH2
(p-Br)C6H5OCH2CH2
59%
35%
31%
(p-CF3)C6H5CH2
88%
aThe data are expressed as percent inhibition of viral DNA in the presence of 10µM of the test compounds as compared to untreated infected controls. bData represent
mean values of at least two experiments. cNA=not active at 10µM
Considering the different substitutions at C-2 position for the C-6 position, compound 6e3 (74%) represented the most
compounds 6d, electron withdrawing groups, particularly nitro active derivative.
(
6d1,81%) and cyano (6d2,67%) would be beneficial for the
After the preliminary screen at one concentration, six
compounds with high inhibition rates were selected to
perform in dose response assays (Figure 3) using the same
HepAD38 cells and the concentrations required to inhibit 50%
of HBV DNA (EC50) are summarized in Table 2. We also
determined the cytotoxic effects through the MTT assay and
the cytotoxicity of each compound was expressed as the
concentration of compound required to cause 50% cell death
(CC50) of the HepAD38 cells (Table 2).
inhibitory activity. For 6e subseries, bearing benzotriazole on
Table 2. In vitro anti-HBV activity and cytotoxicity of the target
compounds
Compd
6d1
EC50(μM)a
0.376
4.84
CC50(μM)b
124
SIc
330
19
6d2
92
6d9
40.8
98
2.4
As shown in Table 2, most of the selected compounds
displayed activity against HBV with low cytotoxicity. Among
them, compound 6d1 and compound 6e3 showed excellent
anti-HBV activity with EC50 values of 0.376μM and 0.469
μM,respectively, which were more potent than reference drug
lamivudine(0.731 μM).
6d12
6e3
4.28
119
27.8
111
12
0.469
12.16
0.731
52
6e6
146
3TC
>100
>137
aConcentration inhibiting viral replication by 50%. b50% cytotoxicity
concentration cSelecivity index , defined by CC50/EC50
.
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Figure 4. Pharmacophore model of the 5-iodo-2-arylalkylthio-6-aryl
pyrimidin-4(3H)-ones. A4 is a H-bond acceptor (shown as a pink
ball). D5 is a H-bond donor (shown as a blue ball). H6 and H9 are
hydrophobic points (shown as green balls). R10, R11, R12 are
aromatic rings (shown as orange circles).
The designed and synthesized 5-iodo-2-arylalkylthio-6-aryl
pyrimidin-4(3H)-ones represent a new series of anti-HBV
compounds. The target of our compounds is not clarified,
therefore the construction of a pharmacophoremodel is a
necessary method for further structural optimization.
According to the anti-HBV activities, compounds 6d1 and
6e3 were selected to generate pharmacophore models using
“Develop Common Pharmacophore Hypotheses” module of
Phase (Figure 4). Several pharmacophore points were
identified including three aromatic rings, two hydrophobic
points, one H-bond acceptor and one H-bond donor point.
Three phenyl groups (R10, R11, R12) were recognized as
aromatic ring. R10 is identical among the compounds. R11
highlighted the importance of the substituted phenyl group
attached to the C-2 side chain, which was consistent with the
SAR results. R12 was also recognized as aromatic ring,
indicating that aromatic group at C-6 was beneficial for the
anti-HBV activity. The iodine atom at C-5 was identified as a
hydrophobic point (H6). Moreover, the hydrogen attached to
the N atom was recognized as a H-bond donor point (D5).
However, A4 and H9 points are common structural elements
among the synthesized compounds.
The biological results further demonstrated that some
compounds with electron withdrawing groups at C-2 position,
such as 6d1 and 6e3 (p-NO2), 6d2 (p-CN), 6d12 (m-Cl), showed
significant anti-HBV activity (Table 2). In addition, compound
6d9 was found to be less active when compared to lamivudine,
so the introduction of aliphatic ring at the C-2 side chain would
decrease the antiviral activity. Because the present study is a
preliminary structural modification, the Structure-Activity
Relationships (SARs) of 5-iodo-2-arylalkylthio-6-aryl pyrimidin-
4(3H)-ones remain to be further studied.
The selectivity index(SI), determined as the ratio of CC50
value to EC50 value, was an important pharmaceutical
parameter that estimates possible future clinical development
(Table 2). Therefore, we chose two compounds (6a1,6e3) with
high SI values for further study. Huh7 cell system was used to
detect intracellular HBV DNA, pgRNA, HBsAg and HBeAg
(Figure 5) .
As shown in Figure 5, after the treatment of two compounds
on Huh7 cells, Intracellular HBV DNA analysis was performed
by qRT-PCR (Figure 5A). The result showed that 6d1 and 6e3
could efficiently impede the replication of intracellular HBV
DNA. RT-PCR analysis of pgRNA (Figure 5B) indicated that the
addition of the two compounds did not suppress the
transcription of HBV genome. Figure 5C and Figure 5D showed
the secretion of HBsAg and HBeAg in treated Huh 7 cell culture
media. The tested two compounds affected HBsAg slightly,
whereas the effect on HBeAg secretion could be observed at
25µM. These results suggested that 6d1 and 6e3 would reduce
the secretion of HBeAg in a dose-dependent manner.
Figure 5. (A) HBV core-associated DNA in intracellular core particles;
(B) The levels of pgRNA; (C) Effects on HBV viral HBsAg secretion,
Data represents the mean value ± SD; (D)Effects on HBV viral HBeAg
secretion, Data represents the mean value ± SD.
3.Conclusions
In summary, a series of 5-iodo-2-arylalkylthio-6-aryl
pyrimidin-4(3H)-ones were synthesized and tested for their in
vitro anti-HBV activity, using lamivudine as a reference control.
Most of the compounds revealed anti-HBV activity. The most
promising results were observed for compounds 6d1 and 6e3
with EC50 values against HBV DNA replication of 0.376μM and
0.469μM, respectively. In addition, they have potent
,
a
inhibitory effect on HBeAg secretion. The work of pgRNA
detection demonstrated that 5-iodo-2-arylalkylthio-6-aryl
pyrimidin-4(3H)-ones could not interfere with HBV
transcription. Further investigation of the mechanism of action
are ongoing in our laboratories.
4 | J. Name., 2012, 00, 1-3
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4.2.2Detection of extracellular HBV DNA
4.Experimental
Inhibition of the synthesized target compounds of extracellular
virus was determined in the HepAD38 cell line. Briefly,
HepAD38 cells were plated onto 48-well plates. After 24h, the
medium was removed and was replaced with medium
containing test compound. Two days after the addition of
compound, the medium was removed, the cells were washed
with warmed (37°C) phosphate-buffered saline (PBS),and fresh
medium containing compound was added. 72h later,the
medium was collected,boiled for 10min and clarified at 12000
rpm for 10min. The quantity of viral DNA in the medium was
determined by Quantitative Real-time PCR(qRT-PCR). The
forward primer was 5´-CAACCTCCAATCACTCACCAAC-3´ and
the reverse primer was 5´-ACGGGCAACATACCTTGGTAG-3´.
PCR reactions were performed on Lightcycler 480II Realtime
PCR Detection System. Melt curve analysis was conducted
according to the manufacturer’s instructions to verify that
single PCR product. The Ct values generated from the DMSO-
treated cells were used as control.
4.1 Chemistry
NMR spectra were recorded on a Bruker AVANCE
Ⅲ-400
spectrometer with TMS as an internal standard and CDCl3,
DMSO-d6 as the solvents. The chemical shifts were expressed
in parts per million (δ ppm). Coupling constants (J) were
measured in Hz. MS were taken by Bruker Apex IV FTMS with
methanol as the solvent. Melting points were measured on an
X-4 apparatus without correction. Silica gel H was used for the
column chromatography and Silica gel GF254 was used for TLC
plates, which were used to monitor the reactions. CH3CN and
acetone were dried with CaCl2, other chemicals were
purchased as reagent grade and used without further
purification.
General procedure for the syntheses of compounds 5a-e:
6-substituted benzyl-2-thioxouracil(compound
4, 0.47mmol)
and K2CO3(0.94mmol) were dissolved in DMSO(2 mL) and
stirred at room temperature, then the corresponding
substituted alkyl bromide(0.47mmol) was added to the
solution followed by stirring for 12 h. 7 mL H2O was added and
then the pH of solution was adjusted to 3 with 2N hydrochloric
acid. After the addition of 10 mL ethyl acetate, the solution
was stirred for another 15 min. Ethyl acetate 20 mL×3 was
used to extract the aqueous layer and the organic phases were
combined, dried over MgSO4 and concentrated at reduced
pressure with a rotary evaporator. The crude product was
purified with silica gel H, and EtOAc–petroleum ether was used
4.2.3Determination of viral HBsAg and HBeAg antigens
HBV antigen of target compounds were evaluated in cultured
Huh7 cell line. Two days after the treatment of test
compounds, the medium was removed, the cells were washed
with warmed (37°C) phosphate-buffered saline (PBS), and
fresh medium containing compound was added. After 7 days,
cell culture media were collected. For the HBV antigens
detection, a total of 100μL cell culture media were used for
each test by using time-resolved fluoroimmunoassay (TRFIA)
according to manufacturer's instructions (SY60108A and
SY60105A; PerkinElmer).
as the eluting solvent, to yield the target products 5a-e
.
General procedure for the syntheses of compounds 6a-e:
Compound 5a-e (0.30mmol) was dissolved in DMF (3 mL) and
stirred at 0°C, then NIS (0.36mmol) was added to this solution
followed by stirring for 5h. The product was purified with silica
gel H, and EtOAc-petroleum ether was used as the eluting
4.2.4General Method for intracellular HBV DNA and total
RNA inhibition assays.
The Huh-7 cells were seeded in 24-well plates. Twenty-four
hours later, cells were transiently transfected with 1.2 xHBV
plasmid and vector control using Lipofectamine Plus
(Invitrogen, Carlsbad, CA, USA) following the Manufacturer’s
recommendations. Compounds with different doses were
added in cell culture medium 6h later. After treatment with
compound for 48h, cells were harvested and the intracellular
HBV DNA and total RNA were measured. Briefly, DNA was
extracted from Huh-7 cells by using a commercially available
DNA extraction kit (QIAamp DNA Mini Kit, Qiagen, Germany).
Intracellular HBV DNA analysis was performed by qRT-PCR as
previously described for extracellular HBV DNA detection.
Meanwhile, Total RNA was extracted from the TRIzol reagents
(Invitrogen, Carlsbad, Calif) from the Huh-7cells according to
the manufacturer’sinstructions. The isolated RNA was first
subjected to reverse transcription and cDNA was prepared by
SuperScript® III RT-PCR kits(Invitrogen, Grand Island, NY, USA).
The cDNA was then quantified using the Real-time PCR assay
which was carried out to measure the expression level of 3.5kb
transcripts of HBV. The house keeping gene ACTB was used as
an internal control. The Primer sequences for ACTB
amplification were as follows: the forward primer was 5´-
CTACAGCTTCACCACCACGG-3´ and the reverse primer was 5´-
TCAGGCAGCTCGTAGCTCTTC-3´. The relative expression level
solvent, to yield products 6a-e
.
4.2 Biology
4.2.1In vitro cytotoxicity study of target compounds.
Cytotoxicity of potent compounds was assessed by MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]
assay via measuring the amount of HepAD38 (stable
expression of HBV) cells after exposure to compounds for 7
days. Briefly, logarithmically growing cells were seeded in 96-
well culture plates at 1× 105 cells/mL (200μL/well). Twenty-
four hours later, cells were treated with various concentrations
of each compound (50μM, 100μM, 200μM). Cells treated with
culture medium containing 0.4% DMSO were used as negetive
control. Media were changed every 2 days. After 7 days, 20μL
of MTT solution was added to per well and incubation was
allowed to continue for another 4h. Then, the culture medium
was discarded and 150μL of DMSO was added to each well to
solubilize the formazan. The absorbance (A) of the formazan at
490 nm was measured by using an automatic plate reader. The
survival ratio of HepAD38 cells (%) was calculated using the
following formula: 1- (A490 of experiment group/A490 of
negative control) × 100%.
of HBV RNA was calculated by the 2−ΔCT
.
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5.Acknowledgments
The research work was supported by the National Natural
Science Foundation of China (21172014, 20972011 and 21042009)
and Grants from the Ministry of Science and Technology of China
(2009ZX09301-010) for financial support.
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MedChemComm
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DOI: 10.1039/C5MD00181A
Graphical abstract
Pyrimidone derivatives have been synthesized and evaluated as anti-HBV
agents, two compounds exhibited more potent than lamivudine.