J. Wang et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx
5
Table 5
S-DABOs played an important role in improving anti-HIV-1 activ-
ity. It provided a steric hindrance role in the hydrophobic region
and formed a bond with Tyr181 of HIV-1 RT.27 We speculated that
the C-5 iodine atom might have a similar effect on HP. These phar-
macophore results suggested that the possible binding modes
between target compounds with HBV polymerase and HIV-1 RT
were similar.
Inhibitory activity of compounds 5c1 and 5b4 against DHBV polymerase and HBV
DNA.
3. Conclusion
Compd.
DHBV Pa
HepG2.2.15b
In summary, we have evaluated a series of 2-arylthio-5-iodo
pyrimidine derivatives against HBV DNA replication. In order to
find out the mechanism of these compounds, DHBV polymerase
was isolated from duck livers. Using a chemiluminescent substrate,
we firstly established a non-radioisotopic assay for measuring the
inhibitory activity of our compounds on DHBV polymerase. The
biological results demonstrated that 2-arylthio-5-iodo pyrimidine
derivatives as non-nucleoside inhibitors could be against HBV
DNA replication and targeted HBV polymerase. Further investiga-
tion of structural optimum is ongoing in our laboratories.
5c1
5b4
16.15%
3.66%
32.92%
13.35%
a
Inhibitory rate at the concentration of 100 mM.
Inhibitory rate at the concentration of 20 mM.
b
significantly inhibited HBV DNA replication, displayed no inhibi-
tion on DHBV polymerase.
To investigate the effect of the iodine atom at C-5 position, com-
pounds 5c1 and 5b4 were also evaluated for their inhibitory activ-
ity both on DHBV polymerase and HepG2.2.15 cells. Compared to
the corresponding compounds 6c1 and 6b4, their activity disap-
peared as the iodine atom was removed (Table 5). These biological
results were consistent with their inhibitory activity on HIV-1 RT,
in which the iodine atom at the C-5 position might play a crucial
role in the binding process with HBV polymerase.
4. Experimental
4.1. In vitro cytotoxicity study of target compounds
The HepG2.2.15 cells were incubated in 96-well plates at
2 ꢀ 105 cells per mL (200 mL per well). 24 h later, the cells were
treated with various concentrations of target compounds. 72 h
later, the medium was replcaced by fresh one containing the
compounds. Another 72 h later, 20 mL of MTT solution was added
to each well and was incubated for 4 h. The culture medium was
discarded then and 200 mL DMSO was added to each well to
solubilize formazan. The plate was measured at an absorbance of
490 nm by an automatic plate reader.
2.3. Pharmacophore models
As we know, the high-resolution structure of HBV polymerase
has not been analyzed. Therefore, construction of a pharmacophore
model would be necessary to optimize the structure and identify
lead compounds. Compounds 6a1, 6b1, 6b2, 6b6, 6b7 and 6c1,
which showed the best activity against HBV polymerase, were
selected to generate pharmacophore models. As shown in Fig. 5,
several pharmacophore points were identified for the 2-arylthio-
5-iodo pyrimidine derivatives, including two hydrophobic, one
hydrogen bond acceptor and one hydrogen bond donor points.
Two phenyl groups (R8, R9) were recognized as aromatic rings.
4.2. Assays for measuring the inhibition activity of compounds against
HBV DNA
The HepG2.2.15 cells were incubated in 96-well plates at
2 ꢀ 105 cells per mL (200 mL per well). 24 h later, the cells were
treated with various concentrations of target compounds. 72 h
later, the medium was replcaced by fresh one containing the com-
pounds. Another 72 h later, the medium was removed and the cells
was washed by phosphate-buffered saline (PBS). The cells was har-
vested and the intracellular HBV DNA was extracted from cell lysis.
The levels of HBV DNA was determined by quantitative real-time
PCR (qRT-PCR).
The
p-p conjugated system of phenyl ring on position C-2 (R9)
was consistent with the docking results of these compounds bind-
ing to HIV-1 RT.18 The iodine atom on position C-5 was recognized
as a hydrophobic point. As reported, the C-5 iodine atom of
4.3. Isolation DHBV polymerase from duck livers
Ducklings aged 1 day were infected with DHBV at a dose of
0.5 ml DHBV (+) serum per duckling (containing DHBV DNA). Seven
days later, the duck livers were collected. The liver homogenate was
prepared with 1:1 NEB buffer (Tris-HCl pH 7.5, 20 mM; NaCl
50 mM; MgSO4 7 mM; DTT 0.1%; NP-40 0.5%; Sucrose 8.57%). Most
of the tissue fragments in the homogenate were removed by cen-
trifugation at 11,000 rpm at 4 °C for 1 h. The supernatant portion
was centrifuged at 45,000 rpm at 4 °C for 3 h and the sediment
was collected to separate the DHBV polymerase. The protein was
purified through sucrose density gradient centrifugation. Sucrose
of 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% was added to the tube
from bottom to the top. The diluted sediment was added on the top
of the sucrose. The mixture was centrifuged at 27,000 rpm at 4 °C
for 4 h. After the gradient centrifugation, per fraction was collected
according to different concentration of sucrose. The fractions of 35%
Fig. 5. Pharmacophore models of target compounds. H-bond acceptor: light red,
arrows pointing in the direction of the lone pairs; H-bond donor: light blue, arrow
pointing in the direction of the potential H-bond; hydrophobic is presented by
green sphere, positive by blue sphere and aromatic ring by orange. (For interpre-
tation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Wang, Jie
