Design and synthesis of a novel candidate compound NTI-007 targeting sodium taurocholate cotransporting polypeptide [NTCP]-APOA1-HBx-Beclin1-mediated autophagic pathway in HBV therapy

Article abstract of DOI:10.1016/j.bmc.2015.01.020

Sodium taurocholate cotransporting polypeptide (NTCP) is a multiple transmembrane transporter predominantly expressed in the liver, functioning as a functional receptor for HBV. Through our continuous efforts to identify NTCP as a novel HBV target, we designed and synthesized a series of new compounds based on the structure of our previous compound NT-5. Molecular docking and MD simulation validated that a new compound named NTI-007 can tightly bind to NTCP, whose efficacy was also measured in vitro virological examination and cytotoxicity studies. Furthermore, autophagy was observed in NTI-007 incubated HepG2.2.15 cells, and results of q-PCR and Western blotting revealed that NTI-007 induced autophagy through NTCP-APOA1-HBx-Beclin1-mediated pathway. Taken together, considering crucial role of NTCP in HBV infection, NTCP-mediated autophagic pathway may provide a promising strategy of HBV therapy and given efficacy of NTI-007 triggering autophagy. Our study suggests pre-clinical potential of this compound as a novel anti-HBV drug candidate.

Full text of DOI:10.1016/j.bmc.2015.01.020

Bioorganic & Medicinal Chemistry 23 (2015) 976–984
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of a novel candidate compound NTI-007
targeting sodium taurocholate cotransporting polypeptide [NTCP]–
APOA1–HBx–Beclin1-mediated autophagic pathway in HBV therapy
Jin Zhang a, , Lei-lei Fu b, , Mao Tian b, , Hao-qiu Liu b, Jing-jing Li b, Yan Li a, Jun He b, Jian Huang a,
,
⇑
Liang Ouyang b, , Hui-yuan Gao a, Jin-hui Wang a,c,
⇑
⇑
a Key Laboratory of Structure-Based Drug Design & Discovery of Ministry of Education, School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, People’s Republic of China
b State Key Laboratory of Biotherapy/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, People’s Republic of China
c College of Pharmacy, Xinjiang Medical University, Urumqi 830011, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Sodium taurocholate cotransporting polypeptide (NTCP) is a multiple transmembrane transporter
predominantly expressed in the liver, functioning as a functional receptor for HBV. Through our contin-
uous efforts to identify NTCP as a novel HBV target, we designed and synthesized a series of new com-
pounds based on the structure of our previous compound NT-5. Molecular docking and MD simulation
validated that a new compound named NTI-007 can tightly bind to NTCP, whose efficacy was also
measured in vitro virological examination and cytotoxicity studies. Furthermore, autophagy was
observed in NTI-007 incubated HepG2.2.15 cells, and results of q-PCR and Western blotting revealed that
NTI-007 induced autophagy through NTCP–APOA1–HBx–Beclin1-mediated pathway. Taken together,
considering crucial role of NTCP in HBV infection, NTCP-mediated autophagic pathway may provide a
promising strategy of HBV therapy and given efficacy of NTI-007 triggering autophagy. Our study sug-
gests pre-clinical potential of this compound as a novel anti-HBV drug candidate.
Received 8 December 2014
Revised 10 January 2015
Accepted 12 January 2015
Available online 19 January 2015
Keywords:
Hepatitis B virus (HBV)
Sodium taurocholate cotransporting
polypeptide (NTCP)
Anti-HBV drug candidate
Autophagy
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
The HBV envelope consists of the small (S), middle (M), and
large (L) envelope proteins, which are multiple transmembrane
Hepatitis B virus (HBV), a member of the hepadnaviridae family,
can cause both acute and chronic hepatitis B, which is a major
global health problem and results in more than 780,000 deaths
every year.1 HBV infectious particle consists of an outer envelope
with host-derived lipids and a nucleocapsid which contains a
double-stranded DNA genome.2 The viral genome consists of four
overlapping open reading frames (ORFs), which code for the viral
proteins including the surface antigen (HBsAg), the core antigen
(HBcAg), the polymerase, and the X protein (HBx). Polypeptides
of HBcAg and Hepatitis Be antigen (HBeAg) are encoded from
HBV core gene, which processing two start codons of translation.
Although HBeAg and HBcAg are serologically distinct: the HBcAg
possesses unique immunologic features, while the presence of
HBeAg is a serological marker of viral replication.3
spanners sharing the same C-terminal domain corresponding to
the S protein but differing at their N-terminal domains.4 The L pro-
tein and integrity of S protein are critical for HBV infections, and
the pre-S1 domain of the L protein is a key determinant for entry
of HBV, which is believed to mediate viral interaction with the cel-
lular receptor(s) on hepatocytes.5The inspiring finding of Yan et al.
in 2012 revealed that the receptor-binding region of pre-S1 specif-
ically interacts with sodium taurocholate cotransporting polypep-
tide (NTCP), a multiple transmembrane transporter predominantly
expressed in the liver.6,7 Using a synthetic modified peptide as a
probe and employing biochemical approaches, virological assays
has confirmed NTCP to interact specifically with L proteins of
HBV, functioning as a functional receptor for the virus.7 Numerous
studies have focused on identifying inhibitors of NTCP, hoping to
find a potential drug for HBV therapy, but up to now, the mecha-
nism of inhibiting NTCP expression to cure the HBV-related dis-
eases has not been fully comprehended.
⇑
Corresponding authors. Tel./fax: +86 028 85503817.
E-mail addresses: profhj@163.com (J. Huang), ouyangliang@scu.edu.cn
(L. Ouyang), profwjh@126.com (J.-h. Wang).
Among the HBV gene products, HBx is a multifunctional viral
regulator which is considered as one of the most important
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bmc.2015.01.020
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
977
determinants involved in viral pathogenesis and carcinogenesis.8
HBx processes nearly 127 interacting proteins, and previous study
has confirmed that human apolipoprotein A-I (APOA-I), which
plays a specific role in lipid and cholesterol metabolism, may
potently influence the HBV secretion via interacting with HBx.9
Mature APOA-I, a 243-residue polypeptide, can originate in both
the liver and small intestine.10,11 As a major protein constituent
of high density lipoprotein (HDL), APOA-I plays an important role
in HDL stability, lipid and cholesterol transportation and has a sig-
nificant inhibitory effect on HBV secretion, indicating the crucial
role of the HBx–APOA-1 axis in the HBV life cycle.12,13
Recently, multiple reports have covered that HBx can induce
autophagy through several signaling pathways, such as activating
death-associated protein kinase (DAPK), PI3K/Akt-mTOR pathway
and up-regulation of Beclin-1.14–16 Researches in hepatic or hepa-
toma cell lines revealed that enforced expression of HBx by trans-
fection technology results in starvation-induced autophagy, while
the up-regulation of the endogenous messenger RNA (mRNA)
and protein levels ofBeclin-1 are also detected.15 Therefore, the
interaction between HBx and Beclin-1 is suspected to regulate star-
vation-induced autophagy in HBV-infected hepatic cells. Beclin-1
processes the crucial up-regulation capacity in autophagy,17,18
and the cross-talk role of it plays in autophagy and apoptosis as
a direct substrate of caspase-3, caspase-7 and caspase-8 in apopto-
sis.19,20 Beclin-1 acts as a platform in autophagy to recruit and acti-
vate PI3KCIII/Vps34, and is mediated by its positive regulators
including ATG14L, UVRAG, Bif-1, Ambra1, HMGB1, Survivin,
PINK-1, TRAF6, DAPK1, TRIF, nPIST and SLAM as well as negative
regulators.21,22 In addition, Beclin-1 is commonly overexpressed
in hepatitis B virus (HBV)-infected cancerous liver tissues, suggest-
ing the potential pivotal role Beclin-1 may play in HBV-infected
cells, though it may expressed at reduced levels in other tumors,
such as breast tumors.15
In our previous studies, we used in silico analysis and experi-
mental validation of candidate compounds targeting NTCP in
HBV therapy. We identified six small-molecule compounds that
can directly inhibit NTCP expression, and first reported that
NTCP–APOA1–HBx axis could be an appealing therapeutic target.23
Considering interaction between HBx and Beclin-1 regulates
autophagy in HBV cells, in this study, we optimized the most effec-
tive structure of NT-5 (shown in Fig. 1). Then, we used molecular
modeling and molecular dynamics (MD) simulation of NTI-007 to
verify the function of NTCP inhibitor in autophagy. Moreover,
through several experiments conducting in HepG2.2.15 cells, such
as q-PCR, transmission electron microscopy, fluorescence of GFP–
LC3 and Western blotting, we verified the therapeutic mechanism
of compound NTI-007 as a new lead compound targeting NTCP–
APOA1–HBx–Beclin1-mediated autophagic pathway.
thionyl chloride and then the Friedel–Crafts acylation reaction was
triggered by AlCl3 with substituted benzene or other heterocyclic
compounds to obtain the intermediate 3 in a yield of 32–68%. Then,
the two nitro groups were reduced into amino after hydrogenation
reaction catalyzed by H2–Pd–C, yield was almost 100% without addi-
tional purification. The intermediate 4 was reacted with methoxy
(1-iminoethyl)carbamate to afford the final products 5a–g (yield
67–79%). The synthesis of compound 10 could refer to this method
only changing the starting material into 5,6-dinitropicolinic
acid.35–37 The high degree of symmetry in these molecules enabled
facile confirmation by NMR techniques. The purity of all compounds
determined by HPLC normalization method was above 97.0%. Fur-
thermore, the structures of these compounds were verified by ESI-
MS.
2.2. In vitro virological examination
The potential anti-HBV activity and cytotoxicity of all com-
pounds were tested in HepG2.2.15 cells, and adefovir was used
as positive control. (Table 1) These compounds generally exhibited
moderate and good anti-HBV potency, and the most effective com-
pound 10c (NTI-007) exhibited excellent inhibitory activity to
HBsAg and HBeAg, which is more potent than adefovir. However,
it was little toxic with CC50 value of 197 lM. It is worth noting that
compound NTI-007 was the most potent compound with high SI
value (SI = 11), suggesting that it would be a good lead compound
for further optimization.
2.3. Anti-HBV effects and NTCP of NTI-007
To further evaluate anti-HBV activity of NTI-007, we detected
expression level of HBV DNA in cells by using qPCR techniques.
HBV DNA was extracted and analyzed by qPCR using an HBV
DNA PCR kit. Data are expressed as mean SD. As shown in Table 2,
the results demonstrated that HBV DNA within cells was reduced
markedly in those that had received NTI-007 comparing NT-5
and adefovir. In addition, an HBV ELISA kit was used to test concen-
tration of HBsAg and HBeAg to demonstrate whether NTI-007
inhibits HBV DNA replication, whose results showed concentration
of HBsAg and HBeAg was 258 25 pg/ml and 8.14 1.77 ng/ml
when treating with NTI-007. Compared to positive control and
the lead compound, data pertaining to adefovir were 571 47 pg/
ml and 12.14 1.33 ng/ml, NT-5 were 361 36 and
10.12 2.41 ng/ml.
2.4. Molecular modeling, docking and molecular dynamics (MD)
simulations of NTI-007
Although the cellular target had not been defined in the exper-
imental antiviral investigations of these molecules, it was desirable
to investigate interactions of the active compounds with NTCP tar-
geted by leading compound NT-5. We developed the 3D-model
structure of NTCP and used the molecular docking to measure
affinity between NTCP, NT-5 and NTI-007. Through the result of
molecular docking, NTI-007 revealed a better binding capacity to
NTCP than NT-5. NTI-007 bound to the pocket of NTCP, and the
2. Results and discussion
2.1. Chemistry
The synthesis of these two series of target compounds benzoim-
idazole and imidazo(4,5-b) pyridine (compounds 5 and 10) was
shown in Schemes 1 and 2. 3,4-Dinitrobenzoic was treated with
O
O
NH
O
O
NH
O
N
X
Structure modification
O
N
N
H
R
F
N
H
NT-5
SI of HBsAg=2.5
SI of HBeAg=4.75
New Derivatives
Figure 1. Chemical structure of NT-5 and its structure modification.

978
J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
O2N
O2N
O2N
O2N
Al3Cl3, CH2Cl2
SOCl2
O2N
O2N
R1
O
0oC, DMF
COOH
COCl Friedel-Crafts reaction
O
2
3
1
O
H2N
H2N
O
HCl
H2, Pd-C
CH3OH
N
R1
R1
NH
N
H
O
O
O
5a-g
4
HN
N
H
O
Scheme 1. The synthesis of compounds 5a–g.
O2N
O2N
O2N
O2N
O2N
Al3Cl3, CH2Cl2
SOCl2
R2
O2N
N
8
0oC, DMF
COOH
Friedel-Crafts reaction
COCl
N
N
7
O
6
O
HCl
O
H2N
H2N
O
H2, Pd-C
CH3OH
N
O
N
R2
R2
NH
N
O
N
H
O
HN
N
O
9
H
10a-c
Scheme 2. The synthesis of compounds 10a–c and NTI-007.
binding site of NTI-007 was located in Asn71 and Thr171 of NTCP.
MD simulations of NTI-007 in NTCP were carried out to further
confirm precise binding mechanisms and interaction stability. As
shown in Figure 2b, during 10 ns dynamics, there was a rapid
increase during the first 4 ns of NTCP–NTI-007 complex, while
the backbone RMSD and standard deviation of NTCP over the last
ns was 4 Å. The above data revealed that the NTCP–NTI-007 com-
plex has achieved their stabilities after a period of dynamic varia-
tion. Based on the results of MD simulations, it can be proved that
compound NTI-007 can target the ATP-binding site of NTCP
directly and interact with NTCP steadily.
2.6. Induction of autophagy through NTCP–APOA1–HBx–Beclin
1 pathway
In a previous study of our group, we built the whole PPI net-
work involved in many existing and potential HBx-interacted pro-
teins such as NTCP and APOA1, and APOA1 may have a significant
inhibitory effect on HBV secretion.23 Although the newly discov-
ered functional receptor NTCP has a rather small number of inter-
action proteins and the network is not yet mature, we believed that
NTCP–APOA1–HBx axis may play a crucial role in modulating cell
death in HBV-infection cells.23 Therefore, we examined NTCP,
APOA1 and HBx levels by Western blotting analysis, and as shown
in Figure 4B, decreased expression levels of NTCP, APOA1 and HBx
were verified, indicatingthatNTI-007 exerted its function through
NTCP–APOA1–HBx axis. Considering the protein interaction
between HBx and Beclin-1, we measured Beclin-1 level by Western
blot analysis to affirm the pathway of NTI-007-induced autophagy
(Fig. 4B). Increased level of Beclin-1 suggested that NTI-007 trig-
gered autophagic cell death through activation of Beclin-1 via the
NTCP–APOA1–HBx axis. Accordingly, these results indicated that
treatment of NTCP inhibitor, NTI-007, activated autophagic cell
death in HepG2.2.15 cells through NTCP–APOA1–HBx–Beclin1
pathway, which may provide a new molecular basis on further
exploring a more precise signaling autophagic pathway by which
NTI-007 induces autophagy in HBV-infected cells.
2.5. Emerging of autophagy in NTI-007 treated HepG2.2.15 cells
NTI-007 caused
HepG2.2.15 cell growth in a time-dependent manner with treat-
ment of 20 M NTI-007 for 24 h. Therefore, we examined the
a remarkable anti-proliferative effect on
l
ultra-structure of NTI-007-treated HepG2.2.15 cells by the trans-
mission electron microscopy. As shown in Figure 3A, control cells
displayed normal cell phenotype, while in contrast, NTI-007-trea-
ted HepG2.2.15 cells showed typical autophagic features, including
appearance of autophagosome and extensive cytoplasm vacuoliza-
tion, in which some autophagic vacuoles contained degraded
organelles. Meanwhile, the formation of autophagic vacuoles was
further assessed by GFP–LC3 distribution (Fig. 3B). As shown in Fig-
ure 3B, control cells presented diffused staining, and NTI-007 treat-
ment resulted in extensive GFP–LC3 localization, suggesting that
NTI-007 induces HepG2.2.15 cell autophagy. Next, we measured
autophagic ratio through the fluorescent dye MDC to explore
autophagy-inducing ability of NTI-007 in a time-dependent man-
ner. As shown in Figure 3C, compared with the control group, the
autophagic ratio was significantly increased by the treatment of
NTI-007. Moreover, the autophagic ratio of 48 h NTI-007-treating
cells was higher than 24 h cells, indicating that NTI-007 induced
autophagy in HepG2.2.15 cells in a time-dependent manner. In
addition, we examined levels of some hub proteins in autophagy
by Western blotting analysis to confirm the roles of NTI-007 in
autophagy. As shown in Figure 4A, the apparent increase of LC3-
II and decrease of p62 suggested that NTI-007 induced autophagic
cell death.
3. Discussion
HBx is well characterized by important mediating pathological
effects of HBV through interacting with various cell proteins, which
processes a large protein-protein interaction (PPI) network. And
recently, plentiful researches have reported that HBx can both
induce autophagy through different pathways and inhibit autopha-
gic degradation through impairing lysosome maturation, suggest-
ing its pivotal role of autophagy in HBV infected cells. And
considering that numerous HBV infections happen each year and
its huge social relevance of related diseases, targeting HBx-related
pathway via triggering autophagic cell death in HBV-infected cells
may be a potential strategy both in prevention and therapy of hep-
atitis B and D. Through exploring the mechanism of NTCP inhibitor

J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
979
Table 1
Anti-HBV activity and cytotoxicity of the targeted small molecule candidate compounds
O
O
O
O
NH
N
O
N
O
N
R2
R1
NH
N
H
N
H
5a-g
10a-c
a
Compound
R1
R2
CC50
HBsAg
HBeAg
b
b
IC50
SIc
IC50
SI
—
d
d
5a
5b
—
—
>1000
>1000
—
>1000
O
116.7 4.12
97.5 3.12
189.6 1.2
245.1 3.2
29.7 2.77
1.19
0.25
0.09
3.55
478.7 7.99
312.4 10.12
397.4 9.24
215.8 4.15
0.24
0.15
0.05
0.49
O
O
5c
5d
5e
—
—
—
48.5 6.12
21.2 1.45
105.4 6.17
F
O
HO
N
d
d
5f
—
—
>1000
>1000
—
>1000
—
d
d
5g
>1000
>1000
—
>1000
—
O
O
O
10a
—
—
78.1 3.14
41.2 1.67
1.89
2.74
166.7 6.73
0.47
1.30
10b
102.4 3.25
37.3 2.79
78.4 2.4
O
O
10c (NTI-007)
—
—
197.3 4.2
490
17.9 1.4
257 1.4
11
25.1 1.2
215 1.3
7.86
2.27
HO
—
Adefovir
1.90
a
b
c
CC50 is 50% cytotoxicity concentration in HepG22.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity index (SI = CC50/IC50).
d
No SI can be obtained.
autophagy in NTI-007 treated cells. LC3, as an important biomarker
of autophagy, whose carboxy-terminal lipid modification is
required for autophagosome formation, has proved to increase after
treatment of NTI-007 in Western blotting analysis, suggesting
autophagy being triggered by NTCP inhibitor. What was also
showed in Western blotting analysis were the decrease of HBx,
APOA1 and NTCP, and the increase of Beclin-1. We assumed NTCP
can interact with HBx through APOA1,23 and in this study, we ver-
ified that the inhibition of NTCP can cause the decrease of HBx pro-
tein expression, proving that targeting NTCP inhibition can affect
HBV-infected cell survival. Moreover, experimentation results also
confirmed our earlier research that APOA-1 is an important interac-
tion protein of HBx.3 Furthermore; NTCP was first reported to par-
ticipate in regulation of autophagy, which provided a promising
future to develop targeting drugs in hepatitis B through triggering
autophagic cell death.
Currently, few FDA-approved compounds have been reported to
process inhibitory abilities of NTCP, such as Cyclosporine A, Cyclo-
sporine B,24 and Irbesartan,5 however, the mechanism study of
NTCP inhibitors are remained to be discovered, and the mechanism
of NTCP participating in regulation of autophagy in HBV-infected
cells are unknown. Through molecular docking and MD simulation,
the common feature pharmacophore of NTCP inhibitors that previ-
Table 2
Anti-HBV activity evaluation by qPCR and ELISA
Compound
HBV DNA levela (AU)
HBsAgb (pg/ml)
HBeAgb (ng/ml)
Control
NT-5
NTI-007
Adefovir
6.12 1.46
3.76 0.35
2.46 0.16
4.12 1.27
721 32
361 36
258 25
571 47
14.16 1.92
10.12 2.41
8.14 1.77
12.14 1.33
a
Measured by qPCR.
Measured by ELISA.
b
inducing autophagy in HepG2.2.15 cells, we could further elucidate
the function of HBx in autophagy, as well as the interaction
between HBx and Beclin-1.
NTCP, as a newly identified entry receptor of HBV, has not been
related to autophagy in previous studies; however, based on the PPI
network, we speculated that it may regulate autophagy in HBV-
infected cells.23 We identified NTCP inhibitor may induce autoph-
agy through NTCP–APOA1–HBx–Beclin1 pathway in this study,
and we further research the interaction between HBx–APOA1 and
HBx–Beclin-1. Neither from transmission electron microscopy
results or the increase of GFP–LC3, we observed the emergence of

980
J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
Figure 2. Docking and molecular dynamics (MD) simulation of NT-5 and NTI-007. (A) Docking results of NTI-007 and NT-5. Downloaded amino acid sequence of NTCP and
the lately disclosed active state structure of ASBT were selected as the template to construct the agonistic conformation of NTCP. Then, Accelrys Discovery Studio 3.5 with
CHARMm force-field parameters was used to dock pre-generated conformations of NTI-007 and NT-5 into NTCP for identifying its inhibitory effect. (B) MD simulations were
performed to monitor the binding states between NTCP and NTI-007.
ous studies estimated are two hydrophobes, one hydrogen bond
acceptor and 10 excluded volumes,5 based on which we designed
the new compounds NTI-007. We identified that the structure-
optimized NTI-007 processed a better affinity to NTCP and a better
pharmaco-dynamic effects, comparing to our previous studied
compound, NT-5.23 NTI-007, as a potent inhibitor of NTCP, may
antagonize ATP binding to NTCP by blocking ATP binding site of
NTCP. Moreover, in vitro virological examinations have validated
the HBsAg SI of NTI-007 is 11, while Adefovir is 1.9 and NT-5 is
2.7. Further biological evaluation through qPCR and ELISA vali-
dated the new compound NTI-007 has more potential in HBV ther-
apy. In addition, we found that NTI-007 inhibited expression of
NTCP in the HepG2.2.15 cells, suggesting that NTI-007 could,
indeed, be a potential anti-HBV drug targeting NTCP.
5. Experimental
General: All reactions requiring anhydrous conditions were per-
formed under an Ar or N2 atmosphere. Chemicals and solvents
were either A.R. grade or purified by standard techniques. Thin
layer chromatography (TLC): silica gel plates GF254; compounds
were visualized by irradiation with UV light and/or by treatment
with a solution of phosphomolybdic acid (20% wt in ethanol) fol-
lowed by heating. Column chromatography was performed by
using silica gel with eluent given in parentheses. 1H NMR analysis
was performed using CDCl3 or DMSO-d6 as a solvent at room tem-
perature. The chemical shifts are expressed in relative to TMS
(=0 ppm) and the coupling constants J in Hz. The purity of com-
pound screened in biological assays was determined to be P97%
by HPLC analysis with a photodiode array detector, an atlantis
C18 (150 mm ꢀ 4.6 mm, i.d. 5
lm) (Waters, Milford, Mass, USA)
was used with a gradient elution of methanol and HPLC-grade
4. Conclusion
water as mobile phase at a flow rate of 1 ml/min.
Starvation-induced autophagy caused by up-regulation of
Beclin-1 plays a pro-survival role in HBV-infected cells, and is
dependent on over-expression of HBx. However, NTI-007-medi-
ated autophagy promotes Beclin-1 but inhibits HBx through inhibi-
tion of NTCP, and plays a death-promoting role in HBV infection. In
this study, we used in silico analysis and chemical synthesis to
identify that NTI-007 exhibits better binding capacity than other
drugs. In addition, NTI-007 processed other features to developing
5.1. General procedure for the synthesis of compound 3
The substituted benzene or heterocyclic (1 equiv) in anhydrous
methylene chloride was added aluminum chloride (1 equiv) and
3,4-dinitrobenzoyl chloride (1 equiv added by drop wised). The mix-
ture was heated under reflux for 3 h. After cooling, the precipitate
was filtered and the solvent was rotary-evaporated to give crude
product. Then, the crude product was purified by silica gel column
chromatography using PE–EA as an eluent (yield 54.1–71.2%).
a potential targeting drug candidate, such as low values of IC50
,
high SI and time-dependent stability. The mechanism studies first
reported NTCP could be involved in autophagic cell death, which
revealed the precise pathway of NTI-007 triggering autophagy.
Moreover, unraveling mechanism of NTCP and identifying PPI net-
work of NTCP involved targeting NTCP–APOA1–HBx–Beclin1-med-
iated autophagic pathway. All the results may shed light on
developing the NTI-007 to be a potential anti-HBV drug candidate
and how such a small molecule compound targeting NTCP in the
host cell will lead to novel prevention and therapeutic strategies
against HBV.
5.2. General procedure for the synthesis of compound 4
Compound 3 (1 equiv) was dissolved in methanol (10 ml), and
10% Pd–C (10 equiv) were added into the solution. The reaction
mixture was stirred at room temperature under a H2 atmosphere.
After 24 h, the mixture was passed through a membrane filter to
remove the catalyst and then evaporated under vacuum to afford
compound 4 (yield 87.2–91.7%).

J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
981
Figure 3. NTI-007 induced autophagy in HepG2.2.15 cells. (A) Transmission electron microscope of control cells and NTI-007 treated cells. Control cells were treated with
DMSO and displayed normal cell phenotype. NTI-007-treated HepG2.2.15 cells showed typical autophagic features, including appearance of autophagosome and extensive
cytoplasm vacuolization. (B) GFP–LC3 formation. Control cells presented diffused staining. NTI-007 treatment resulted in extensive GFP–LC3 localization. (C) Flow cytometry
results. Compared with the control group, the autophagic ratio was significantly increased by the treatment of NTI-007. Moreover, the autophagic ratio of 48 h NTI-007-
treating cells was higher than 24 h cells.
5.3. General procedure for the synthesis of compound 5
The methoxy(1-iminoethyl)carbamate (1 equiv)
5.3.1. Compound 5a: methyl(5-benzoyl-1H-benzo(d)imidazol-2-
yl)carbamate
and
1H NMR (400 MHz, CDCl3) d 7.91 (d, J = 15.0 Hz, 1H), 7.86 (d,
J = 3.1 Hz, 1H), 7.82 (s, 1H), 7.80 (d, J = 3.3 Hz, 1H), 7.61 (ddd,
J = 12.8, 7.9, 3.3 Hz, 1H), 7.54 (d, J = 1.0 Hz, 1H), 7.51 (s, 1H), 7.48
(d, J = 3.0 Hz, 1H), 3.73 (s, 3H). 13C NMR (100 MHz, CDCl3) d
197.53, 158.22, 148.36, 138.05, 136.76, 133.39, 133.01, 132.42,
129.91, 128.63, 122.62, 117.16, 107.49, 53.52. MS: m/
z = 318.0861 (M+Na)+.
compound 4 (1 equiv) in chloroform were cooled in an ice bath
and stirred mechanically. To the solution, concentrated
hydrochloric acid (1 equiv) was added dropwise at such rate to
keep the temperature below 10 °C throughout the addition.
Stirring was continued for additional 30 min, and then the
mixture was heated under reflux for overnight. After cooling,
the precipitate was filtered and the solvent was rotary-
evaporated to give crude product. Then, the crude product was
purified by silica gel column chromatography using PE–EA as
an eluent (yield 41.2–66.7%).
5.3.2. Compound 5b: methyl(5-(3-methoxybenzoyl)-1H-benzo(d)
imidazol-2-yl)carbamate
1H NMR (400 MHz, CDCl3) d 7.91 (d, J = 15.0 Hz, 1H), 7.86 (d,
J = 3.1 Hz, 1H), 7.62 (d, J = 14.9 Hz, 1H), 7.48 (d, J = 2.9 Hz, 1H),

982
J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
Figure 4. NTI-007 modulates the NTCP–APOA1–HBx–Beclin1 pathway in HepG2.2.15 cells. (A) After treatment with NTI-007, there are apparent increase of LC3-II and
decrease of p62, suggesting the emerge of autophagy. (B) NTI-007 incubating led to reduce of NTCP, APOA1 and HBx. Expression of key autophagic protein, Beclin-1, is
increased with NTI-007 treatment.
7.43 (s, 1H), 7.24 (t, J = 3.0 Hz, 1H), 7.10 (s, 1H), 3.77 (s, 3H), 3.73
(s, 3H). 13C NMR (100 MHz, CDCl3) d 196.54, 159.41, 158.22,
148.36, 141.33, 136.76, 133.39, 133.01, 129.15, 122.62, 121.35,
117.21, 113.00, 107.49, 56.08, 53.52. MS: m/z = 348.0949
(M+Na)+.
5.4. General procedure for the synthesis of compound 10
The same procedure as described above for preparation of com-
pound 5.
5.4.1. Compound 10a: methyl(5-(3-methoxybenzoyl)-1H-imidazo
(4,5-b)pyridin-2-yl)carbamate
5.3.3. Compound 5c: methyl(5-(3,4-dimethoxybenzoyl)-1H-benzo
(d)imidazol-2-yl)carbamate
1H NMR (400 MHz, CDCl3) d 8.58 (d, J = 14.9 Hz, 1H), 7.99 (d,
J = 14.9 Hz, 1H), 7.84 (d, J = 15.0 Hz, 1H), 7.65 (dt, J = 20.4, 9.3 Hz,
2H), 7.10 (d, J = 14.9 Hz, 1H), 3.77 (s, 3H), 3.73 (s, 3H). 13C NMR
1H NMR (400 MHz, CDCl3) d 7.91 (d, J = 14.9 Hz, 1H), 7.86 (d,
J = 3.1 Hz, 1H), 7.45 (d, J = 3.0 Hz, 2H), 7.24 (d, J = 2.9 Hz, 1H),
7.08 (d, J = 15.0 Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.73 (s, 3H).
13C NMR (100 MHz, CDCl3) d 196.54, 158.22, 153.42, 148.78,
148.36, 136.76, 133.38, 133.02, 131.92, 125.55, 122.62, 117.16,
112.87, 110.52, 107.49, 56.83, 53.52. MS: m/z = 378.1078
(M+Na)+.
(100 MHz, CDCl3)
d 192.90, 160.45, 158.22, 151.09, 148.82,
140.68, 140.21, 130.98, 129.33, 123.81, 118.56, 117.96, 114.62,
109.65, 56.08, 53.52. MS: m/z = 349.0923 (M+Na)+.
5.4.2. Compound 10b: methyl(5-(3,4-dimethoxybenzoyl)-1H-
imidazo(4,5-b)pyridin-2-yl)carbamate
5.3.4. Compound 5d: methyl(5-(3-fluorobenzoyl)-1H-benzo
(d)imidazol-2-yl)carbamate
1H NMR (400 MHz, CDCl3) d 8.58 (d, J = 14.9 Hz, 1H), 7.99 (d,
J = 14.9 Hz, 1H), 7.89–7.81 (m, 1H), 7.66 (d, J = 2.9 Hz, 1H), 7.09
(d, J = 15.1 Hz, 1H), 3.85 (s, 3H), 3.83 (s, 3H), 3.73 (s, 3H). 13C
NMR (100 MHz, CDCl3) d 192.90, 158.22, 152.16, 151.09, 149.65,
148.82, 140.68, 131.06, 126.45, 117.96, 111.41, 110.92, 109.65,
56.83, 53.52. MS: m/z = 379.1023 (M+Na)+.
1H NMR (400 MHz, CDCl3) d 7.90 (s, 1H), 7.86 (d, J = 1.5 Hz, 1H),
7.54–7.45 (m, 2H), 7.44–7.34 (m, 2H), 7.33–7.27 (m, 1H), 3.73 (s,
3H). 13C NMR (100 MHz, CDCl3) d 196.54, 155.96, 148.36, 148.36,
140.06, 136.76, 133.39, 133.01, 131.13, 125.85, 122.62, 119.12,
117.16, 115.18, 107.49, 53.52. MS: m/z = 378.8 (M+Na)+. MS: m/
z = 336.0781 (M+Na)+.
5.4.3. Compound 10c: methyl(5-(4-hydroxy-3-methoxybenzoyl)
-1H-imidazo(4,5-b)pyridin-2-yl)carbamate
5.3.5. Compound 5e: methyl(5-(4-hydroxy-3-methoxybenzoyl)-
1H-benzo(d)imidazol-2-yl) carbamate
1H NMR (400 MHz, CDCl3) d 8.58 (d, J = 14.9 Hz, 1H), 7.99 (d,
J = 15.1 Hz, 1H), 7.68 (dd, J = 15.0, 3.0 Hz, 1H), 7.49 (d, J = 3.0 Hz,
1H), 7.15 (d, J = 15.0 Hz, 1H), 5.50 (s, 1H), 3.83 (s, 3H), 3.73 (s,
3H). 13C NMR (100 MHz, CDCl3) d 192.90, 158.22, 151.09, 149.83,
148.82, 148.15, 140.68, 131.77, 130.98, 125.65, 117.96, 113.12,
111.99, 109.65, 56.83, 53.52. MS: m/z = 365.0871 (M+Na)+.
1H NMR (400 MHz, CDCl3) d 7.90 (s, 1H), 7.86 (d, J = 1.5 Hz, 1H),
7.47 (dd, J = 7.5, 1.5 Hz, 1H), 7.26 (dd, J = 7.5, 1.5 Hz, 1H), 7.15 (s,
1H), 7.07 (d, J = 1.4 Hz, 1H), 4.78 (s, 1H), 3.83 (s, 3H), 3.73 (s, 3H).
13C NMR (100 MHz, CDCl3) d 196.54, 158.22, 150.53, 148.36,
147.44, 136.76, 133.39, 132.98, 124.78, 122.62, 117.16, 115.92,
110.42, 107.49, 56.83, 53.52. MS: m/z = 364.0916 (M+Na)+.
5.5. Modeling and molecular docking
5.3.6. Compound 5f: methyl(5-picolinoyl-1H-benzo[d]imidazol-
2-yl)carbamate
The amino acid sequence of NTCP was downloaded from the
UniProtKB database (Entry code: Q14973), and sequence similarity
search was performed using the NCBI BLAST server.25 The lately
disclosed active state structure (PDB code: 3ZUY) of ASBT26 was
selected as the template to construct the agonistic conformation
of NTCP. Sequence alignment of NTCP and ASBT was carried out
using the ClustalX 2.0 program.27 Homology modeling was per-
formed with Discovery Studio 3.5 (hereafter abbreviated to DS).28
In our previous studies, FDA-approved small molecule compounds
from the latest version of DrugBank (http://www.drugbank.ca/)
were downloaded to construct the screening library for NTCP.
Then, we used Accelrys Discovery Studio version 3.5 with
CHARMm force-field parameters to dock pre-generated conforma-
tions of NTI-007 into NTCP for identifying its inhibitory effect. We
1H NMR (400 MHz, CDCl3) d 8.75 (s, 1H), 8.28 (s, 1H), 8.10 (s,
1H), 7.99 (s, 1H), 7.90 (d, J = 12.0 Hz, 2H), 7.82 (s, 1H), 3.73 (s,
3H). 13C NMR (100 MHz, CDCl3) d 192.88, 158.22, 154.42, 148.36,
147.00, 138.59, 137.12, 134.42, 129.55, 128.04, 123.84, 118.76,
109.44, 53.52. MS: m/z = 296.0911 (M+Na)+.
5.3.7. Compound 5g: methyl(5-(furan-2-carbonyl)-1H-benzo
(d)imidazol-2-yl)carbamate
1H NMR (400 MHz, CDCl3) d 8.18 (s, 1H), 7.98 (d, J = 12.0 Hz,
2H), 7.58 (s, 1H), 7.47 (s, 1H), 6.84 (s, 1H), 3.73 (s, 3H). 13C NMR
(100 MHz, CDCl3)
d 184.35, 158.22, 153.04, 148.36, 144.18,
140.21, 135.69, 132.23, 123.34, 122.71, 114.36, 110.96, 108.64,
53.52. MS: m/z = 308.0660 (M+Na)+.
performed flexible-ligand docking to a rigid receptor with

J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
983
grid-based scoring, in which compound NTI-007 was allowed to be
5.11. Determination of HBsAg and HBeAg levels by ELISA
flexible and structurally rearranged in response to NTCP. In our
docking processes, maximum number of orientations was set to
500.
HBV antigen and HBV DNA inhibitory efficacies of NTI-007 were
evaluated in cultured HepG2.2.15 cells. 9 days after treatment with
different dosages of the compounds, cell culture supernatants were
collected and HBV antigen activities were tested by enzyme linked
immune sorbent assay (ELISA).34
5.6. Molecular dynamics [MD] simulations
MD simulations were performed with GROMACS (version 4.5.5)
software package29 to monitor the binding states between NTCP
and NTI-007. The newly synthetic compound NTI-007 was sub-
jected to PRODRG2 server30 to generate ligand topologies. The
topology of NTCP was edited by Amber force field 99SB and the
small molecule was edited by Amber general force field. The com-
plex was immersed in a cubic box of simple point charge (SPC)
water molecules. Eight and eleven sodium counter-ions were
added by replacing water molecules to ensure the overall charge
neutrality of the receptors simulated system, respectively. In this
MD process, 200 ps simulations with a time step of 10 ns were per-
formed, and the resulting trajectory files were viewed and ana-
lyzed using VMD software.31
5.12. qPCR and Western blotting
The supernatants of HepG2.2.15 cells were collected from
9 days culture after the compounds were added. The HBV DNA in
the supernatants was quantified by using fluorescent PCR. HBV
DNA PCR kit (Shanghai Kehua) was used. Briefly, 50 ll of the super-
natants were added into the extraction buffer, boiled for 10 min
and centrifuged for 5 min, and then proper aliquots were used
for the fluorescent PCR. PCR primers were: P1: 50-ATCCTGCTGC-
TATGCCTCATCTT-30, P2: 50-ACAGTGGGGAAAGCCCTACGAA-30. The
probe was 50-TGGCTAGTTTACTAGTGCCATTTTG-30. PCR reaction
was run at MJ Research PTC-200, and results were analyzed by
software OpticonMonitor v2.01. The HepG2.2.15 cells were treated
with NTI-007. Both adherent and floating cells were collected, and
then Western blotting analysis was carried out by the method as
described previously.32
5.7. Cell culture
The HepG2.2.15 cell lines were provided by Medical Sciences
Center of West China in Sichuan University. The cells were cultured
in DMEM medium with 10% fetal bovine serum (containing 100 U/
5.13. Statistical analysis
ml penicillin, 100 U/ml streptomycin, 3% glutamine and 380
l
g/ml
Results are summarized from three independent experiments
and presented as mean SD.
G418) maintained at 37 °C with 5% CO2 in
a humidified
atmosphere.
Acknowledgments
5.8. Transmission electron microscopy
We are grateful to Prof. Canhua Huang and Dr. Bo Liu (Sichuan
University) for their critical reviews on this manuscript. This work
was supported by grants from the Key Projects of the National Sci-
ence and Technology Pillar Program (No. 2012BAI30B02), National
Natural Science Foundation of China (Nos. 81473091, 81102325),
and Shenyang Science and Technology Project (F12-157-9-00).
The HepG2.2.15 cells were treated with NTI-007 for the indi-
cated time periods. The collected cells were fixed with PBS contain-
ing 3% glutaraldehyde, post fixed with PBS containing 1% OsO4. The
samples were dehydrated in graded alcohol, embedded and sec-
tioned. Ultrathin sections were stained with uranyl acetate and
lead citrate, and were examined by a JEM-1200 transmission elec-
tron microscope (JEOL, Japan).32
References and notes
1. Lozano, R.; Naghavi, M.; Foreman, K.; Lim, S.; Shibuya, K.; Aboyans, V.;
Abraham, J.; Adair, T.; Aggarwal, R.; Ahn, S. Y.; Alvarado, M.; Anderson, H. R.;
Anderson, L. M.; Andrews, K. G.; Atkinson, C.; Baddour, L. M.; Barker-Collo, S.;
Bartels, D. H.; Bell, M. L.; Benjamin, E. J.; Bennett, D.; Bhalla, K.; Bikbov, B.; Bin,
Abdulhak A.; Birbeck, G.; Blyth, F.; Bolliger, I.; Boufous, S.; Bucello, C.; Burch, M.,
et al. Lancet 2012, 380, 2095.
2. Mhamdi, M.; Funk, A.; Hohenberg, H.; Will, H.; Sirma, H. Hepatology 2007, 46,
95.
3. Lee, B. O.; Tucker, A.; Frelin, L.; Sallberg, M.; Jones, J.; Peters, C.; Hughes, J.;
Whitacre, D.; Darsow, B.; Peterson, D. L.; Milich, D. R. J. Immunol. 2009, 182,
6670.
5.9. Measurement of autophagy
After incubation with NTI-007 for fixed times, the cells were
cultured at 37 °C for 60 min. The fluorescence intensity of cells
was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes,
NJ).32 Cells were transfected with GFP–LC3 plasmid by using the
Lipofectamine 2000 reagent (Invitrogen) according to the manu-
facturer’s instructions. The fluorescence of GFP–LC3 was observed
under a fluorescence microscope.
4. Gripon, P.; Rumin, S.; Urban, S.; Le, S. J.; Glaise, D.; Cannie, I.; Guyomard, C.;
Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99,
15655.
5.10. Analysis of cytotoxicity
5. Dong, Z.; Ekins, S.; Polli, J. E. Mol. Pharm. 2013, 10, 1008.
6. Gozalpour, E.; Greupink, R.; Wortelboer, H. M.; Bilos, A.; Schreurs, M.; Russel, F.
G.; Koenderink, J. B. Mol. Pharm. 2014, 11, 1844.
Cytotoxicity of NTI-007 was assessed by MTT assay. Dissolved
in DMSO, it was serially diluted in cell culture media and then
added onto cell monolayers. HepG2.2.15 cells were maintained
in 96-well tissue culture plates for 48 h before being treated
with compounds, and subsequently cells were incubated at
37 °C for 9 days. Cells with media alone were used as the blank
control. MTT reagents were added at a concentration of 5 g/L at
4 h before the cells were harvested and lysed with 10% sodium
dodecyl sulfate (SDS) and 50% N,N-dimethylformamide (DMF)
at pH 7.2. The OD values of cell lysates were read at a wave-
length of 570 nm, and the percentages of cell death were
determined.33
7. Yan, H.; Zhong, G.; Xu, G.; He, W.; Jing, Z.; Gao, Z.; Huang, Y.; Qi, Y.; Peng, B.;
Wang, H.; Fu, L.; Song, M.; Chen, P.; Gao, W.; Ren, B.; Sun, Y.; Cai, T.; Feng, X.;
Sui, J.; Li, W. Elife 2012, 1, e00049.
8. Hu, L.; Chen, L.; Li, L.; Sun, H.; Yang, G.; Chang, Y. J. Virol. 2011, 85, 3214.
9. Zhang, T.; Xie, N.; He, W.; Liu, R.; Lei, Y.; Chen, Y.; Tang, H.; Liu, B.; Huang, C.;
Wei, Y. J. Proteomics 2013, 84, 92.
10. Stoffel, W. J. Lipid Res. 1984, 25, 1586.
11. Norton, P. A.; Gong, Q.; Mehta, A. S.; Lu, X.; Block, T. M. J. Virol. 2003, 77, 3.
12. Laccotripe, M.; Laccotripe, S. C.; Jonas, A.; Zannis, V. I. J. Biol. Chem. 1997, 272,
11.
13. Sviridov, D.; Hoang, A.; Sawyer, W. H.; Fidge, N. H. J. Biol. Chem. 2000, 275,
19707.
14. Wang, P.; Wang, Q. S.; Wang, Z. W.; Qian, H. X. Mol. Cell Biochem. 2013, 372, 1.
15. Tang, H.; Da, L.; Mao, Y.; Li, Y.; Li, D.; Xu, Z.; Li, F.; Wang, Y.; Tiollais, P.; Li, T.;
Zhao, M. Hepatology 2009, 49, 60.

984
J. Zhang et al. / Bioorg. Med. Chem. 23 (2015) 976–984
16. Zhang, H. T.; Chen, G. G.; Hu, B. G.; Zhang, Z. Y.; Yun, J. P.; He, M. L.; Lai, P. B. J.
Viral Hepat. 2014, 21, 2.
17. Lapierre, L. R.; Silvestrini, M. J.; Nuñez, L.; Ames, K.; Wong, S.; Le, T. T.; Hansen,
M.; Meléndez, A. Autophagy 2013, 9, 278.
26. Hu, N. J.; Iwata, S.; Cameron, A. D.; Drew, D. Nature 2011, 478, 408.
27. Larkin, M. A.; Blackshields, G.; Brown, N. P.; Chenna, R.; McGettigan, P. A.;
McWilliam, H.; Valentin, F.; Wallace, I. M.; Wilm, A.; Lopez, R.; Thompson, J. D.;
Gibson, T. J.; Higgins, D. G. Bioinformatics 2007, 23, 2947.
18. Wen, X.; Wu, J. M.; Wang, F. T.; Liu, B.; Huang, C. H.; Wei, Y. Q. Free Radical Biol.
Med. 2013, 65, 402.
28. Accelrys Software Inc., Discovery Studio Modeling Environment, Release 4.0,
San Diego: Accelrys Software Inc., 2013.
19. Wang, R. C.; Wei, Y.; An, Z.; Zou, Z.; Xiao, G.; Bhagat, G.; White, M.; Reichelt, J.;
Levine, B. Science 2012, 338, 956.
29. Pronk, S.; Páll, S.; Schulz, R.; Larsson, P.; Bjelkmar, P.; Apostolov, R.
Bioinformatics 2013, 29, 845.
20. Li, H.; Wang, P.; Sun, Q.; Ding, W. X.; Yim, X. M.; Sobol, R. W. Cancer Res. 2011,
71, 3625.
30. Schüttelkopf, A. W.; van Aalten, D. M. Acta Crystallogr., Sect. D: Biol. Crystallogr.
2004, 60, 1355.
21. Kang, R.; Zeh, H. J.; Lotze, M. T.; Tang, D. Cell Death Differ. 2011, 18, 571.
22. Fu, L. L.; Cheng, Y.; Liu, B. Int. J. Biochem. Cell Biol. 2013, 45, 921.
23. Fu, L. L.; Liu, J.; Chen, Y.; Wang, F. T.; Wen, X.; Liu, H. Q.; Wang, M. Y.; Ouyang,
L.; Huang, J.; Bao, J. K.; Wei, Y. Q. Cell Prolif. 2014, 47, 26.
24. Iwamoto, M.; Watashi, K.; Tsukuda, S.; Aly, H. H.; Fukasawa, M.; Fujimoto, A.;
Suzuki, R.; Aizaki, H.; Ito, T.; Koiwai, O.; Kusuhara, H.; Wakita, T. Biochem.
Biophys. Res. Commun. 2014, 17, 808.
31. Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graphics 1996, 14, 33.
32. Liu, B.; Wu, J.; Li, J.; Liu, J. J.; Li, W. W.; Li, C. Y.; Xu, H. L.; Bao, J. K. Biochimie
2010, 92, 1934.
33. Wu, J. M.; Lin, J. S.; Xie, N.; Liang, K. H. World J. Gastroenterol. 2003, 9, 1840.
34. Zhang, T.; Xie, N.; He, W.; Liu, R.; Lei, Y.; Chen, Y. J. Proteomics 2013, 84, 92.
35. Yang, X. Y.; Xu, X. Q.; Guan, H. Bioorg. Med. Chem. Lett. 2014, 24, 4247.
36. Marimani, M. D.; Ely, A.; Buff, M. C. R. Bioorg. Med. Chem. 2013, 21, 6145.
37. Ouyang, L.; Huang, Y. H.; Zhao, Y. W. Bioorg. Med. Chem. Lett. 2012, 22, 3044.
25. Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.;
Lipman, D. Nucleic Acids Res. 1997, 25, 3389.