Development of benzimidazole derivatives to inhibit HIV-1 replication through protecting APOBEC3G protein

Article abstract of DOI:10.1016/j.ejmech.2015.03.050

Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hypermutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor) counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which provided the best potency, with IC₅₀ values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1 replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we suggest that these two benzimidazole derivatives can be further developed as a new category of anti-HIV-1 leads.

Full text of DOI:10.1016/j.ejmech.2015.03.050

European Journal of Medicinal Chemistry 95 (2015) 500e513
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Development of benzimidazole derivatives to inhibit HIV-1 replication
through protecting APOBEC3G protein
,
b
,
,
b
,
,
, b
Ting Pan a 1, Xin He a 1, Bing Chen a, Hui Chen a, Guannan Geng a b, Haihua Luo a
,
Hui Zhang a *, Chuan Bai a
,
b,
, b, *
a Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong, 510080, China
b Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, Guangdong,
510080, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Human APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G, A3G) is a
potent restriction factor against human immunodeficiency virus type 1 (HIV-1) by inducing hyper-
mutation of G to A in viral genome after its incorporation into virions. HIV-1 Vif (Virion Infectivity Factor)
counteracts A3G by inducing ubiquitination and proteasomal degradation of A3G protein. Vif-A3G axis
therefore is a promising therapeutic target of HIV-1. Here we report the screening, synthesis and SAR
studies of benzimidazole derivatives as potent inhibitors against HIV-1 replication via protecting A3G
protein. Based on the steep SAR of the benzimidazole scaffold, we identified compound 14 and 26 which
provided the best potency, with IC50 values of 3.45 nM and 58.03 nM respectively in the anti-HIV-1
replication assay in H9 cells. Compound 14 and 26 also afforded protective effects on A3G protein
level. Both compounds have been proved to be safe in acute toxicological studies. Taken together, we
suggest that these two benzimidazole derivatives can be further developed as a new category of anti-
HIV-1 leads.
Received 4 August 2014
Received in revised form
17 February 2015
Accepted 20 March 2015
Available online 21 March 2015
Keywords:
HIV-1
APOBEC3G
Vif
Benzimidazole
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
undergoing the most frequent mutations, which leads to the highly
frequent drug resistance for presently available drugs targeting the
Human immunodeficiency virus-1 (HIV-1) has been a world-
wide epidemic for more than 30 years. Significant progress has
been made in high active antiretroviral therapy (HAART) which has
been the major clinical method to reduce AIDS mortality and
mobility. However, the life-long cost and serious side effects of
current therapeutics have been one of the obstacles to eradicate
HIV-1 viruses [1e3]. Moreover, HIV-1 virus is one of the viruses
major viral proteins such as reverse transcriptase, protease, or
integrase. Therefore, novel therapeutics targeting other viral pro-
teins become quite attractive in pursuing new clinical therapeutics
against HIV-1 [4].
Human APOBEC3 family is a host restriction factor against vi-
ruses including HIV-1 and simian immunodeficiency virus (SIV)
[5e9]. In non-permissive cell such as H9 cell line where A3G is
highly expressed, but not in permissive cells such as SupT1 and
293T cell line where A3G is not expressed, A3G is encapsidated into
budding virions and removes the NH2- residues from cytidines in
the newly-synthesized negative strand of viral DNA. This process
leaves the uridine in the negative strand, and subsequently, results
in a G to A hypermutation in the plus strand of viral DNA [5,6,10].
The hypermutation causes the pre-terminations of viral protein(s)
during translation or the productions of mutated viral proteins
without any function, and eventually leads to inactivation of viral
infectivity [5,11,12]. To neutralize the function of A3G, HIV-1 virus
encodes virion infectivity factor (Vif) protein which mediates the
ubiquitination of A3G in the complex with elongins B/C, cullin5,
Abbreviations: HIV-1, Human immunodeficiency virus type-1; Vif, Viral infec-
tivity factor; A3G/APOBEC3G, (apolipoprotein B mRNA-editing, enzyme-catalytic,
polypeptide-like 3G); ELISA, enzyme-linked immunosorbent assay; BUN, Blood
urea nitrogen; CRE, Creatine; AST, Aspartate transaminase; ALT, Alanine trans-
aminase; SPR, Surface plasmon resonance; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium.
* Corresponding authors. Key Laboratory of Tropical Disease Control of Ministry
of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou,
Guangdong, 510080, China.
E-mail addresses: zhangh92@mail.sysu.edu.cn (H. Zhang), baichuan@mail.sysu.
edu.cn (C. Bai).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.ejmech.2015.03.050
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
501
Nedd8, Rbx1, and CBF-
b
, and subsequently degrades A3G protein
replication assay was applied in the SAR studies of our synthe-
sized benzimidazole derivatives of compound 13. We first per-
formed the Vif-A3G assay and the cytotoxicity test (MTS Assay) and
secondly the active compounds with low toxicity identified in this
step were further investigated for their inhibition activity on HIV-1
replication in the anti-HIV-1 replication assay.
[10,13e19]. Thus, the strategy to protect A3G protein level via
disruption of Vif-A3G axis opens an avenue to develop new anti-
HIV-1 therapeutics.
Since the high-throughput screening (HTS) protocol was
developed to efficiently screen chemical libraries for compounds
specifically targeting Vif-A3G axis [20], there has been impressive
progress which showed various chemical structures inhibiting HIV-
1 replication by disrupting Vif-A3G axis [21e23]. We screened a
drug-like chemical library by an optimized HTS work flow targeting
Vif-A3G axis, and identified a hit compound 13 (Fig. 1) with benz-
imidazole scaffold. We further studied the SAR of this hit com-
pound and synthesized two new compounds (14, 26) with potent
anti-HIV-1 activity. Our results have shown that the benzimidazole
derivatives are good starting points to further develop anti-HIV-1
lead compounds that specifically protect A3G protein.
2.2. Chemical synthesis
We found that the benzimidazole scaffold was shared by some
hit compounds which had the IC50 value lower than 5.0
mM.
Therefore we took compound 13 as the starting point for the hit-to-
lead optimization. Syntheses of the benzimidazole substructure
including ring B and C was achieved through a facile method [26],
in which the substituted or unsubstituted o-phenylenediamine (1)
was fused with ethylcyanoacetate (2) to afford compound 3. The
alkyl substitution groups were installed on the nitrogen atom of
benzimidazole ring by using alkyl iodide to afford compound 4
(Scheme 1).
2. Results
The syntheses of 1H-Benzimidazole-2-acetonitrile derivatives
were achieved by following the general procedures illustrated in
Scheme 1, in which the solution of compound 4 and compound 5 in
ethanol with catalytic amount of piperidine was heated at 90 ꢁC to
afford the compounds for SAR studies.
2.1. Hit identification by high-throughput screening (HTS) and anti-
HIV-1 replication assay
A cell-based high-throughput screening (Vif-A3G assay) was
performed using an A3G-GFP fusion protein reporter system [20].
In brief, the Vif-expressing plasmid and A3G-GFP-expressing
plasmid were co-transfected into 293T cells. These 293T cells
2.3. SAR study
were treated with
composed of 20,155 compounds (Enamine) at 50
a
commercial drug-like chemical library
M concentration.
Among the active compounds identified by HTS, compound 13
m
(IC50 ¼ 0.51
mM, Table 1) was selected for optimization because its
After 48 h, the wells containing GFP-positive cells were detected by
an Envision system (Perkin Elmer). After the positive hits were
confirmed by the same system, 372 hit compounds were identified
to counteract the Vif-mediated degradation of A3G-GFP fusion
protein.
These 372 hit compounds were further tested for their potential
anti-HIV-1 replication activity by an anti-HIV-1 replication assay. In
brief, H9 and SupT1 cells (1 ꢀ 106) were infected with the equiv-
alent of 5 ng HIV-1 p24 for 3 h and HIV-1 replication was monitored
by the detection of p24 antigen in supernatant after 4 days. The
amount of p24 was quantified by ELISA. Among 372 initial hits, 155
benzimidazole scaffold was widely distributed in drugs and natural
products with potent biological activities [27,28]. Moreover, the
facile methods to synthesize benzimidazole scaffold made it
possible to synthesize the versatile derivatives in large quantities
[30,31]. The structure of compound 13 was divided into ring A, ring
B, ring C and the linker (Fig. 1). Firstly, compounds with different
alkyl groups and electro withdrawing groups in group A were
synthesized (Table 1, 6e12). However, none of these compounds
showed better activities in the Vif-A3G assay than compound 13.
This result indicated that the N,N-alkyl groups should be a key
substitution group to retain the activity. Therefore derivatives
bearing different N,N-alkyl, N,N-cycloalkyl groups and nitrogen
heterocyclic groups were synthesized (Table 1, 14e24). The HIV-1
replication assay showed that the compound 14 with N,N-diethyl
substitution had much better activity (IC50 ¼ 3.45 nM) than the hit
compound 13 (Fig. 2, A). Interestingly, compounds with cyclic alkyl
groups (Table 1, 17, 18) lost their activities in the Vif-A3G assay,
probably due to their constrained conformations in the ring
structure. Compound 20 with hydroxyl groups on the N,N-alkyl
groups was also synthesized to increase its water solubility. How-
ever, the hydroxyl groups significantly decreased its activity. To
further study whether the N,N-alkyl substitutions could be
replaced by hydroxyl or methoxy groups, compounds 25~32 with
modifications on ring A were synthesized (Table 1). Most of the
compounds were active in Vif-A3G assay, but only the compound
26 bearing both p-hydroxyl and m-methoxy groups possessed
potent anti-HIV-1 activity (IC50 ¼ 58.03 nM) (Fig. 2, B). Eventually,
modifications on ring A afforded compound 14 with the highest
anti-HIV-1 replication activity (IC50 ¼ 3.45 nM).
were found to inhibit HIV-1 replication at 50 mM in H9 cells, while
22 compounds in SupT1. It is notable that H9 cells harbor APO-
BEC3G, while SupT1 cells does not. Most of the hits inhibit HIV-1
replication in H9 but not in SupT1, which support that the target
is Vif-A3G system [24,25]. Therefore, 133 compounds were most
likely to specifically inhibit HIV-1 by Vif-A3G pathway. Further-
more, we have also performed a dose response among these
compounds and selected 5 compounds which showed an inhibition
by more than 50% even at 5 mM (Table S1), narrowing down the hits
which were more efficient. Given that the benzimidazole scaffold is
widely distributed in drugs and natural products with potent bio-
logical activities and the facile methods to synthesize benzimid-
azole scaffold made it possible to synthesize the versatile
derivatives in large quantities, compound 13 (Fig. 1) was chosen for
further hit-to-lead optimization.
The work flow including both Vif-A3G assay and anti-HIV-1
There have been reports regarding on the modifications of
benzimidazole substructure to improve biological activities [27].
Ring B was further modified by adding cyclic or acyclic alkyl sub-
stitution groups on nitrogen of benzimidazole (Table 2, 33e35).
However, these modifications led to complete loss of the activities
in Vif-A3G assay and HIV-1 replication assay. The nitrogen atom
was then replaced with oxygen or sulfur atom to produce
Fig. 1. The structure of the hit compound (13). The SAR was studied in ring A, B, C and
the linker respectively.

502
T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
CN
CN
CN
N
N
N
N
N
O
NH2
NH2
c
a
b
R3
R3
R1
R1
+
O
R1
R1
NC
+
NH2
N
H
H
R2
R2
4
2
5
1
3
aReagents and conditions: (a) 200 ºC reflux;(b) R2-Br, DBU, DCM, 0 oC; (c) Piperidine, EtOH , reflux.
Scheme 1. Synthesis of bezimidazole derivatives.
Table 1
Influence of modifications of ring A on A3G level, cell toxicity and anti-HIV-1 replication activity.
Compd. No.
R
Vif-A3G assay
MTS assay (CC50
,
m
M)a
HIV-1 replication assay IC50 (m
M)b
c
6
7
8
9
10
11
12
13
14
15
16
17
4-Me
4-Et
4-i-Pr
4-Ph
4-SO2Me
4-NHCOCH3
4-COOH
4-N(Me)2
4-N(Et)2
4-N(Pr)2
4-N(i-Pr)2
ꢂ
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.51
0.00345
1.09
3.30
>50
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
d
þþ
þþþ
þþ
þþ
ꢂ
N
4
18
ꢂ
>50
>50
N
N
4
4
4
19
20
21
þþþ
þ
>50
>50
>50
>50
6.11
>50
N
N
N
O
OH
ꢂ
N
4
22
23
3-N(Et)2
ꢂ
>50
>50
>50
0.36
þþ
NH
N
3,4
NH
3,4
24
þþþ
>50
0.72
25
26
27
28
29
30
31
32
4-OMe
3-OMe, 4-OH
3,4-OMe
3,4,5-OMe
2,4,5-OMe
2-OH, 4-N(Et)2
2-OH, 4-N(Me)2
3-OMe, 4-N(Me)2
þþ
þþþ
þ
>50
>50
>50
>50
>50
>50
>50
>50
2.85
0.05803
>50
>50
>50
1.50
>50
0.29
þ
þ
þþþ
þþþ
þ
a
Median (50%) cytotoxic concentration.
Half maximal (50%) inhibitory concentration.
Test result did not show activity.
The fold change of A3G-GFP mean fluorescent intensity (MFI). Calculation method was described in method. ꢂ, no or less than 1 fold change; þ, 1~2 fold change; þþ, 2~3
b
c
d
fold change; þþþ, >3 fold change.

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
503
Fig. 2. Compound 14 (A) and compound 26 (B) inhibited HIV-1 replication in H9 cells with IC50 value of 3.45 nM and 58.03 nM respectively.
Table 2
Influence of the modifications of ring B on A3G level, cell toxicity and anti-HIV-1 replication activity.
Compd. No.
X
R
Vif-A3G assay
MTS assay (CC50
,
m
M)
HIV-1 replication assay IC50 (mM)
33
34
35
36
37
N
N
N
O
S
Me
Et
Cyclo-Pr
ꢂ
>50
>50
>50
>50
>50
>50
>50
>50
2.38
5.92
ꢂ
ꢂ
þþ
þþþ
benzoxazole and benzothiazole substructures (Table 2, 36e37).
These modifications maintained the activities in both the Vif-A3G
assay and the HIV-1 replication assay, although their activities
were lower than that of compound 14.
Ring C was modified by substitutions of electro-withdrawing
groups (Table 3, 38e45), alkyl groups and weak electron-
donating groups (Table 3, 46e50) but those compounds did not
show higher activity in HIV-1 replication assay than compound 14.
Table 3
Influence of the modifications of ring C on A3G level, cell toxicity and anti-HIV-1 activity.
Compd. ID
R1
R2
Vif-A3G assay
MTS assay (CC50
,
m
M)
HIV-1 replication assay (IC50,mM)
38
39
40
41
42
43
44
45
46
47
48
49
50
F
Cl
Br
Cl
H
H
H
Cl
H
H
H
H
Me
H
H
H
H
þþþ
þþ
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.25
1.13
1.35
1.68
>50
1.64
1.04
4.42
2.68
0.75
þþ
þþþ
þþ
CF3
OCF3
COOMe
COOH
Me
t-Butyl
OMe
N(Me)2
SMe
þþþ
þþ
þþ
þþþ
þþþ
þþ
þ
þþ
Compd. ID
Structure
Vif-A3G assay
MTS assay (CC50
,
m
M)
HIV-1 replication assay (IC
7.30
50,mM)
51
þþ
>50
CN
N
N
N
H
N
N
H
52
þþ
>50
3.58
CN
N
N
N
H
H

504
T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
Moreover, compounds with pyridine instead of benzene ring in the
benzimidazole structure (Table 3, 51, 52) were synthesized but
afforded no anti-HIV-1 activity. These results indicated that the
unsubstituted benzene structure of ring C was important to
maintain the activity.
In the studies of the effect of the linker on the activity, compound
53 with a rigid structure in the linker (Table 4) showed lower activity
than the hit compound 14. Compound 54 bearing a sigma bond
rather than an alkene bond in the linker was synthesized (Table 4)
but it was inactive in the Vif-A3G assay. These results indicated that
the alkene linker was important for the anti-HIV-1 activity.
From the SAR studies described above, it has been elucidated
that the N,N-alkyl substitution or p-hydroxyl together with m-
methoxy substitution of ring A, benzimidazole substructure
including ring B and ring C, and the alkene linker connecting ring A
and ring B are required for their anti-HIV-1 activity.
with the untreated A3G protein. These results indicate that com-
pound 14 and 26 could disrupt the interaction of A3G and Vif. To
exclude the possibility that compound 14 and 26 could disrupt
other proteineprotein interactions in Vif-A3G axis, we carried out
the co-IP experiments with Vif and ElonginB, ElonginC and Cullin5.
We found that these two compounds did not affect the binding of
Vif/ElonginB, Vif/ElonginC and Vif/Cullin5 (Fig. 4BeD). In a word,
both the SPR and co-IP showed that compound 14 and 26 could
disrupt the binding of A3G and Vif and then protect the A3G from
Vif-mediated degradation.
2.5. Toxicity test
The structure of benzimidazole has been widely distributed in
drugs and natural products and showed potent activities including
some undesired biological effects [27,28]. To address this issue, the
acute toxicities of compound 14 and 26 on mice were tested. Two
weeks after injected intraperitoneally with compound 14 or 26 in
the dose of 0 mg/kg, 500 mg/kg,1000 mg/kg, 5 male Balb/c mice did
not show significant change of body weight. In the 14th day, the
blood samples and the organs of heart, liver, lung, spleen and kidney
were examined and no significant abnormity was found. The func-
tions of the key enzymes including AST, ALT, BUN, and CRE in the
histological sections of these organs were also normal (Fig. 5).
2.4. Compounds 14 and 26 protected A3G protein from degradation
by Vif
To determine whether compound 14 and 26 could rescue A3G
protein from Vif-mediated degradation and thus increase the A3G
protein level in 293T cells, compound 14 and 26 were subjected to a
flow cytometry assay. 293T cells were co-transfected with Vif and
A3G-GFP-expressing plasmids for 48 h, followed by the treatment
of compound 14 and 26 (0.0005e5
mM). The MFI (mean fluorescent
3. Conclusion and discussion
intensity) of Envision showed that the cellular A3G protein level
was protected by compound 14 and 26 in a dose-dependent
manner (Fig. 3A). The similar results were also observed in other
hit compounds (Fig. S4). The western blot and the fluorescence
microscope images (Fig. 3B) also confirmed that compounds 14 and
26 inhibited Vif-mediated degradation of A3G-GFP fusion protein in
293Tcells. In primary cells (PBMCs) compound 14 and 26 could also
markedly inhibit HIV-1 production (Fig. 3C). Meanwhile, com-
pound 14 and 26 showed no effect to inhibit HIV-1 production in
permissive SupT1 cells (Fig. 3D).
We performed the SAR study on benzimidazole derivatives for
their anti-HIV-1 replication activity through protecting A3G protein
level. The benzene ring (Fig. 1, ring A) and its N,N-dialkyl amine
substitution group were required for the anti-HIV-1 replication
activity. The N,N-dialkyl group, m-methoxy and p-hydroxyl groups
were the most favored substitutions. The modifications on benz-
imidazole substructure and the linker (Fig. 1, ring C) also afforded
compounds with higher activity than the hit compound (lowest
IC50 value as 0.24 mM, compound 53), which indicated the possi-
Surface plasmon resonance (SPR) was performed to study
whether the compounds 14 and 26 could decrease the binding
affinity of Vif-A3G complex and thus protected A3G from Vif-
mediated degradation. The recombinant Vif-His protein (purity
was confirmed by SDS-PAGE, Fig. S3) was immobilized on a CM5
Sensor Chip (carboxymethylated dextran covalently attached to a
gold surface) with an amine coupling kit from GE Healthcare. The
recombinant A3G-His protein (purity was confirmed by SDS-PAGE,
bility to increase activity by modifications on this substructure.
However, modifications on alkyl substitutions on the nitrogen atom
of benzimidazole were not favored. These results indicate that the
binding site of Vif-A3G complex for compounds 14 and 26 is highly
strict and there could be strong interactions between N,N-dialkyl
group and the key amino acids. This information could be very
useful when the structure information of was applied to identify
new therapeutics targeting Vif-A3G complex [29,30]. Compound 14
and 26 were two most potent compounds with the IC50 value of
3.45 nM and 58.03 nM respectively. Their anti-HIV-1 activity
mechanism was studied and our data suggested that compound 14
and 26 protected the A3G protein from degradation by Vif in 293T
cells. SPR studies showed that compounds 14 and 26 decreased,
although not completely disrupted, the Vif-A3G interaction. The
compound 14 and 26 showed satisfactory acute toxicity properties.
Fig. S3, 50
m
g/mL) was pre-incubated (25 ꢁC) with (green) or
M) in a PBS buffer (10 mM) with
without (red) compound 14 (4.0
m
5% DMSO (Fig. 4A, up). Then the binding of these A3G proteins with
Vif proteins were tested. Similar experiment was done with com-
pound 26 (9.0 mM) (Fig. 4A, down). The SPR sensorgrams showed
that the Vif-A3G binding was reduced (about 17% of max resonance
unit) after the treatment of compounds 14 or 26 when compared
Table 4
Influence of modification of linker on A3G level, cell toxicity and anti-HIV-1 replication activity.
Compd. ID
Vif-A3G assay
MTS assay (CC50
,
m
M)
HIV-1 replication assay (IC
0.24
50,mM)
53
þþþ
>50
N
N
N
N
H
O
O
54
e
>50
>50
CN
N
N
H

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
505
Fig. 3. Compound 14 and 26 can counteract the Vif-mediated degradation of A3G in 293T cells. (A) The MFI analysis for the effect of compound 14 or 26 on the intensity of A3G-GFP
fluorescence. Vif and A3G-GFP were co-transfected into HEK293T cells and then the cells were treated with DMSO, multiple concentrations of compound 14 or compound 26
respectively. After 48 h, the fluorescence of cells was detected by Microplate Reader. (B) Fluorescence microscope images and western blot results for the effect of A3G-GFP inhibited
by Vif in HEK293T cells under the treatment of compound 14 and compound 26. (C and D) PBMCs (C) and SupT1 cells (D) were infected by HIV-1 and then treated by Compound 14
and compound 26 under varied concentrations. P24 was detected as production of HIV-1.
Taken together, our data demonstrated that the compounds 14 and
26 bearing benzimidazole sub-structure could be a good basis to
develop lead compounds with higher potency to inhibit HIV-1
replication through protecting A3G protein.
from CaH2 under nitrogen respectively. Analytical thin-layer plates
were obtained from Qingdao Haiyang Chemical. Flash chromatog-
raphy was performed with silica gel 60. NMR spectra were recorded
on a Bruker Avance 400 with the solvents indicated. Purity was
analyzed by reverse-phase HPLC performed on Agilent 6130
Quadrupole and Agilent 1200 equipment, or on Agilent ProStar 218
system. The purity of compounds for the SAR study was more than
95% tested by ESI-LCMS. HRMS were recorded at the Instrumental
Analysis & Research Center in Sun Yat-sen University using a
Thermo Scientific LTQ-Orbitrap Elite mass spectrometer.
In light of the essential role of Vif in the infectivity of HIV-1
viruses, the Vif-A3G axis has become an important target for
developing anti-HIV-1 leads [20e23,31,32]. Therefore the working
mechanisms of compound 14 and 26 should be further studied
including the exact target proteins in the Vif-A3G complex and the
corresponding binding sites. In our study, compound 14 and 26
protected the protein level of A3G and inhibited HIV-1 replication.
Meanwhile, the protein level of Vif in 293T cells did not change
significantly (Fig. S1). The stable Vif level was also observed in 293T
cells treated with compound IMB25/36 [22]. However, RN-18, a
potent compound targeting Vif-A3G axis, significantly decreases Vif
protein level in 293FTcells [20,23]. On the contrary, Vif protein level
was elevated by compound VEC-5 which may disrupt the A3G-Vif-
E3 complex and inhibits hydrolysis of Vif by E3, ElonginB/C-Cullin5
complex [31]. These different responses of quantitative changes of
Vif protein after different treatments suggest that these compounds
may have different target sites on Vif-A3G axis or they may have
profound effect on other proteins involved in the Vif-mediated A3G
degradation.
4.2. General procedure for the synthesis 2-(1H-benzimidazol-2-yl)
acetonitrile derivatives (3, 4)
The mixture of o-phenylenediamine (1) and cyanoacetamide (2)
were stirred in 200 ꢁC for 30 min without any solvent. After cooling,
the mixture was dissolved in ethanol. The crude product was pu-
rified by silica gel column chromatography using dichloromethane-
methanol (60:1) as eluant to give the pure compound 3.
To a solution of compound 3 in DMF was added K2CO3 in 0 ꢁC
and the mixture was stirred at 0 ꢁC with adding alkyl iodide drop
wise. After the mixture was stirred at 0 ꢁC for 1 h, the solvent was
removed in vacuo. The residue was diluted with H2O and extracted
with EtOAc. The combined organic phase was dried over anhydrous
Na2SO4 and concentrated. The crude product was purified chro-
matography on silica gel with solvent cyclohexane-acetone (20:1)
to give the compound 4.
4. Experimental section
4.1. General chemistry methods
All reagents and solvents were purchased from SigmaeAldrich,
J&K Chemical, Aladdin. Tetrahydrofuran and dichloromethane were
freshly distilled from sodium/benzophenone under nitrogen and
4.3. 2-(1H-benzimidazol-2-yl)-3-(4-methylphenyl)acrylonitrile (6)
To the solution of 2-(1H-benzimidazol-2-yl)acetonitrile (80 mg,

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T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
Input
IP: HA
+
B
A
-
-
+
-
-
-
-
14
26
1000
900
800
700
600
500
400
300
200
100
0
-
+
-
+
Vif-HA
EB-Flag
actin
A3G
14+A3G
C
Input
+
IP: HA
+
0
50
100
150
Time (s)
-
-
-
-
-
-
14
1000
900
800
700
600
500
400
300
200
100
0
26
Vif-HA
EC-Flag
actin
-
+
-
+
A3G
26+A3G
Input
+
IP: HA
+
D
-
-
-
-
-
-
14
0
50
100
150
Time (s)
26
Vif-HA
-
+
-
+
Cul5-Flag
actin
Fig. 4. (A) Surface plasmon resonance experiment showed that compound 14 (up) and compound 26 (down) reduced the binding of A3G and Vif. (BeD) HEK293T cells were
transfected with pcDNA3.1-Vif-HA and pcDNA3.1-EC/EB/CUL5-Flag and then were treated with DMSO, compound 14 and compound 26 respectively. After 48 h, cells were harvested
for co-IP assay.
0.51 mmol), 4-methylbenzaldehyde (60 mg, 0.50 mmol) in ethanol
(1.5 mL) was added 1,4-diazacyclohexane (45 mg, 0.52 mmol). The
mixture was refluxed for 4 h in room temperature. The crude
product was filtered off and purified by silica gel chromatography
using cyclohexane-acetone-ethyl acetate (10:1:1) as eluant to
afford compound 6 (120 mg, 93%). 1H NMR (400 MHz, DMSO-d6)
4.5. 2-(1H-benzimidazol-2-yl)-3-(4-isopropylphenyl)acrylonitrile
(8)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-isopropylbenzaldehyde
instead of 4-methylbenzaldehyde. Yield: 49%. 1H NMR (500 MHz,
d
8.30 (s, 1H), 7.91 (d, J ¼ 8.2 Hz, 2H), 7.62 (dd, J ¼ 5.4, 3.3 Hz, 2H),
DMSO-d6)
d
13.06 (s, 1H), 8.32 (s, 1H), 7.95 (d, J ¼ 8.3 Hz, 2H),
7.42 (d, J ¼ 8.1 Hz, 2H), 7.29e7.21 (m, 2H), 2.41 (s, 3H). ESI-MS (m/z):
7.79e7.53 (m, 2H), 7.49 (d, J ¼ 8.2 Hz, 2H), 7.27 (s, 2H), 3.14e2.89
260.1 [MþH]þ.
(m, 1H), 1.26 (d, J ¼ 6.9 Hz, 9H). ESI-MS (m/z): 288.1 [MþH]þ.
4.4. 2-(1H-benzimidazol-2-yl)-3-(4-ethylphenyl)acrylonitrile (7)
4.6. 2-(1H-benzimidazol-2-yl)-3-(biphenyl-4-yl)acrylonitrile (9)
The title compound was synthesized according to the procedure
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-phenylbenzaldehyde
instead of 4-methylbenzaldehyde. Yield: 10%. 1H NMR (500 MHz,
of preparing compound
instead of 4-methylbenzaldehyde. Yield: 30%. 1H NMR (400 MHz,
DMSO-d6)
6 except using 4-ethylbenzaldehyde
d
13.03 (s, 1H), 8.31 (s, 1H), 7.93 (d, J ¼ 8.2 Hz, 2H), 7.69
DMSO-d6)
d
13.12 (s, 1H), 8.40 (s, 1H), 8.11 (d, J ¼ 8.4 Hz, 2H), 8.01
(d, J ¼ 6.9 Hz, 1H), 7.56 (d, J ¼ 6.8 Hz, 1H), 7.44 (d, J ¼ 8.3 Hz, 2H),
7.27 (d, J ¼ 8.8 Hz, 2H), 2.70 (q, J ¼ 7.6 Hz, 2H),1.23 (t, J ¼ 7.6 Hz, 3H).
ESI-MS (m/z): 274.1 [MþH]þ.
(d, J ¼ 8.4 Hz, 1H), 7.94 (d, J ¼ 8.5 Hz, 2H), 7.93e7.89 (m, 1H),
7.84e7.80 (m, 2H), 7.79e7.76 (m,1H), 7.72 (d, J ¼ 8.3 Hz, 2H), 7.67 (d,
J ¼ 7.8 Hz,1H), 7.59 (d, J ¼ 7.8 Hz,1H), 7.46 (d, J ¼ 7.4 Hz, 2H). ESI-MS

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
507
Fig. 5. The safety of compound 14 (up) and 26 (down) was examined with acute toxicological study. (A). The measurement of weight of male Balb/c mice after intraperitoneally
injected with compounds. (B). The effect of compounds on the hepatic and renal function of mice (BUN: Blood urea nitrogen, CRE: Creatine, AST: Aspartate Transaminase, ALT:
Alanine transaminase). C. The several vital organs from the mice treated with various doses of compounds were histologically sectioned and HE stained.
(m/z): 322.1 [MþH]þ.
2.5 mmol) and 4-methylsulfonylbenzaldehyde (428 mg, 2.5 mmol)
in ethanol (6 mL) was added with NaOH (200 mg, 5 mmol). The
mixture was stirred at room temperature for 4 h. Then the reaction
mixture was poured into ice-cold water and neutralized with acetic
acid. The mixture was filtered and the filtrate was concentrated and
purified by silica gel chromatography using cyclohexane-acetone
4.7. 2-(1H-benzimidazol-2-yl)-3-(4-methylsulfonylphenyl)
acrylonitrile (10)
To a solution of 2-(1H-benzimidazol-2-yl)acetonitrile (361 mg,

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T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
(10:1) as eluant to afford compound 8 (208 mg, 26%). 1H NMR
(400 MHz, CD3OD)
of preparing compound
benzaldehyde instead of 4-methylbenzaldehyde. Yield: 48%. 1H
NMR (500 MHz, DMSO-d6): 8.18 (br s, 1H), 7.89 (br s, 2H),
6 except using 4-(diisopropylamino)
d
8.33 (s, 1H), 8.24 (d, J ¼ 8.5 Hz, 2H), 8.15 (d,
J ¼ 8.5 Hz, 2H), 7.65 (d, J ¼ 10.6 Hz, 2H), 7.38e7.33 (m, 2H), 3.22 (s,
d
3H). ESI-MS (m/z): 324.1 [MþH]þ.
7.65e7.62 (m, 2H), 7.34e7.32 (m, 2H), 7.01 (br s, 2H), 4.13e4.08 (m,
2H), 1.32e1.25 (m, 12H). ESI-LCMS (m/z): 345.0 [MþH]þ.
4.8. 2-(1H-benzimidazolyl)-3-(4-N-acetamidophenyl)acrylonitrile
(11)
4.14. 2-(1H-benzimidazol-2-yl)-3-[4-(1-pyrrolidinyl)phenyl]
acrylonitrile (17)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-acetamidobenz-aldehyde
instead of 4-methylbenzaldehyde. Yield: 51%. 1H NMR (500 MHz,
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(1-pyrrolidinyl)benzal-
dehyde instead of 4-methylbenzaldehyde. Yield: 21%. 1H NMR
DMSO-d6):
d 13.01 (s, 1H), 10.36 (s, 1H), 8.26 (s, 1H), 7.99e7.97 (m,
2H), 7.82e7.80 (m, 2H), 7.62e7.58 (m, 2H), 7.26e7.25 (m, 2H), 2.11
(400 MHz, DMSO-d6)
d
7.96 (s, 1H), 7.85 (d, J ¼ 9.0 Hz, 2H),
(s, 3H). ESI-MS (m/z): 303.2 [MþH]þ.
7.59e7.34 (m, 2H), 6.82 (d, J ¼ 9.1 Hz, 1H), 6.55 (d, J ¼ 8.9 Hz, 1H),
3.37 (t, J ¼ 6.6 Hz, 4H), 2.13e1.90 (m, 4H). ESI-LCMS (m/z): 315.1
[MþH]þ.
4.9. 4-[2-(1H-benzimidazol-2-yl)-2-cyanoethenyl]benzoic acid (12)
2-(1H-benzimidazol-2-yl)-3-(4-carboxyl-phenyl)-acrylonitrile
(80 mg, 0.27 mmol) and methanol (30 mg, 1 mmol) were dissolved
in DMF (1.5 mL), followed by adding DCC (56 mg, 0.27 mmol) and
DMAP (8 mg, 0.07 mmol). The mixture was stirred at room tem-
perature for 4 h. The crude product was purified by chromatog-
raphy on silica gel with solvent cyclohexane-acetone (1:1). To
afford product as brown solid (68 mg, 83%).1H NMR (400 MHz,
4.15. 2-(1H-benzimidazol-2-yl)-3-[4-(4-methyl-1-piperazinyl)
phenyl]acrylonitrile (18)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(4-methyl-1-piperazinyl)
benzaldehyde instead of 4-methylbenzaldehyde. Yield: 68%. 1H
NMR (500 MHz, DMSO-d6):
d 12.83 (br s, 1H), 8.16 (s, 1H), 7.91 (d,
DMSO-d6):
d
8.46 (s, 1H), 8.19 (d, J ¼ 8.5 Hz, 2H), 8.16e8.12 (m, 2H),
J ¼ 9.0 Hz, 2H), 7.70e7.50 (m, 2H), 7.22 (d, J ¼ 3.0 Hz, 2H), 7.11 (d,
J ¼ 9.0 Hz, 2H), 3.40 (t, J ¼ 5.0 Hz, 4H), 2.44 (t, J ¼ 5.0 Hz, 4H), 2.24 (s,
3H). ESI-MS (m/z): 344.3 [MþH]þ.
7.67 (s, 2H), 7.30 (dd, J ¼ 6.1, 3.2 Hz, 2H). ESI-MS (m/z): 290.1
[MþH]þ.
4.10. 2-(1H-benzimidazol-2-yl)-3-(4-dimethylaminophenyl)
acrylonitrile (13)
4.16. 2-(1H-benzimidazol-2-yl)-3-(4-morpholinophenyl)
acrylonitrile (19)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(dimethylamino)benzal-
dehyde instead of 4-methylbenzaldehyde. Yield: 70%. 1H NMR
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(4-morpholinyl)benzal-
dehyde instead of 4-methylbenzaldehyde. Yield: 43%. 1H NMR
(400 MHz, CDCl3)
d
8.14 (s, 1H), 7.90 (d, J ¼ 8.2 Hz, 2H), 7.81e7.75
(500 MHz, DMSO-d6):
d
12.86 (s, 1H), 8.18 (s, 1H), 7.93 (d, J ¼ 8.5 Hz,
(m, 1H), 7.5 (m, 1H), 7.40e7.32 (m, 1H), 7.30 (dd, J ¼ 7.8, 3.2 Hz, 1H),
2H), 7.58 (br s, 2H), 7.23e7.22 (m, 2H), 7.11 (d, J ¼ 8.5 Hz, 2H),
6.88 (d, J ¼ 8.2 Hz, 2H), 3.08 (s, 6H). ESI-MS (m/z): 289.2 [MþH]þ.
3.76e3.74 (m, 4H), 3.36e3.35 (m, 4H). ESI-MS (m/z): 331.2 [MþH]þ.
4.11. 2-(1H-benzimidazol-2-yl)-3-(4-diethylaminophenyl)
acrylonitrile (14)
4.17. 2-(1H-benzimidazol-2-yl)-3-{4-[ethyl(2-hydroxyethyl)amino]
phenyl}acrylonitrile (20)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(diethylamino)benzalde-
hyde instead of 4-methylbenzaldehyde. Yield: 40%. 1H NMR
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-[N-ethyl-N-(hydrox-
yethyl)amino]benzaldehyde instead of 4-methylbenzaldehyde.
(500 MHz, DMSO-d6):
d
12.73 (br s, 1H), 8.11 (s, 1H), 7.89 (d,
Yield: 68%. 1H NMR (500 MHz, DMSO-d6):
d 8.17 (br s, 1H),7.90 (d,
J ¼ 9.5 Hz, 2H), 7.56 (br s, 2H), 7.22e7.19 (m, 2H), 6.84 (d, J ¼ 9.0 Hz,
J ¼ 9.0 Hz, 2H), 7.64e7.61 (m, 2H), 7.32 (br s, 2H), 6.90 (d, J ¼ 9.0 Hz,
2H), 3.61 (t, J ¼ 6.0 Hz, 2H), 3.57e3.50 (m, 4H), 1.15 (t, J ¼ 7.0 Hz,
3H). ESI-LCMS (m/z): 333.3 [MþH]þ.
2H), 3.46 (q, J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). HRMS (ESI) m/z
calcd
for
C20H21N4 Hþ:
317.1761.
Found:
317.1760
(D
¼ ꢂ0.32 ppm).
4.18. 2-(1H-benzimidazol-2-yl)-3-[4-(1H-imidazol-1-yl)phenyl]
acrylonitrile (21)
4.12. 2-(1H-benzimidazol-2-yl)-3-(4-dipropylaminophenyl)
acrylonitrile (15)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(imidazol-1-yl)benzal-
dehyde instead of 4-methylbenzaldehyde. Yield: 15%. 1H NMR
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(dipropylamino)benzal-
dehyde instead of 4-methylbenzaldehyde. Yield: 80%. 1H NMR
(500 MHz, DMSO-d6):
d 13.11e13.09 (m, 1H), 8.45e8.36 (m, 2H),
(500 MHz, DMSO–d6):
d
8.19 (s,1H),7.91 (d, J ¼ 9.5 Hz, 2H), 7.64 (dd,
8.15e8.13 (m, 2H), 7.95e7.91 (m, 3H), 7.73e7.59 (m, 2H), 7.27e7.17
J ¼ 3.0 and 6.0 Hz, 2H), 7.34 (dd, J ¼ 3.0 and 6.0 Hz, 2H), 6.86 (d,
J ¼ 9.5 Hz, 2H), 3.38 (t, J ¼ 7.5 Hz, 4H), 1.60e1.57 (m, 4H), 0.92 (t,
J ¼ 7.5 Hz 6H). ESI-LCMS (m/z): 345.3 [MþH]þ.
(m, 3H). ESI-MS (m/z): 312 [MþH]þ.
4.19. 2-(1H-benzimidazol-2-yl)-3-(3-diethylaminophenyl)
acrylonitrile (22)
4.13. 2-(1H-benzimidazol-2-yl)-3-(4-diisopropylaminophenyl)
acrylonitrile (16)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 3-(diethylamino)benzalde-
hyde instead of 4-methylbenzaldehyde. Yield: 35%. 1H NMR
The title compound was synthesized according to the procedure

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
509
(400 MHz, CD3OD):
d
8.17 (s, 1H), 7.63 (brs, 2H), 7.49 (s, 1H),
(m, 1H), 8.30 (s, 2H), 7.75e7.50 (m, 2H), 7.39 (s, 2H), 7.26 (br s, 2H),
3.87 (s, 2H), 3.78 (s, 1H). ESI-LCMS (m/z): 336.1 [MþH]þ.
7.40e7.29 (m, 3H), 7.15 (d, J ¼ 8.0 Hz, 1H), 6.93 (dd, J ¼ 2.4 and
8.4 Hz, 1H), 3.48 (q, J ¼ 6.8 Hz, 4H), 1.23 (t, J ¼ 7.2 Hz, 6H) ppm; ESI-
MS (m/z): 317.1 [MþH]þ.
4.26. 2-(1H-benzimidazol-2-yl)-3-(2,4,5-trimethoxyphenyl)
acrylonitrile (29)
4.20. 2-(1H-benzimidazol-2-yl)-3-(1H-indazol-5-yl)acrylonitrile
(23)
The title compound was synthesized according to the procedure
of preparing compound
zaldehyde instead of 4-methylbenzaldehyde. Yield: 69%. 1H NMR
(500 MHz, DMSO-d6): 13.01 (s, 1H), 8.51 (s, 1H), 7.89 (s, 1H),
7.70e7.50 (m, 2H), 7.24 (br s, 2H), 6.85 (s, 1H), 3.97 (s, 3H), 3.94 (s,
3H), 3.80 (s, 3H). ESI-MS (m/z): 336.2 [MþH]þ.
6 except using 2,4,5-trimethoxyben
The title compound was synthesized according to the procedure
of preparing compound 6 except using 1H-indazole-6-acetalde
hyde instead of 4-methylbenzaldehyde. Yield: 72%. 1H NMR
d
(500 MHz, DMSO-d6):
d 13.48 (s, 1H), 13.03e13.00 (m, 1H), 8.47 (s,
2H), 8.33 (s, 1H), 8.09e8.07 (m, 1H), 7.78e7.76 (m, 1H), 7.63 (m, 2H),
7.27e7.26 (m, 2H). ESI-MS (m/z): 286.2 [MþH]þ.
4.27. 2-(1H-benzimidazol-2-yl)-3-(4-diethylamino-2-
hydroxyphenyl)acrylonitrile (30)
4.21. 2-(1H-benzimidazol-2-yl)-3-(1H-indol-5-yl)acrylonitrile (24)
The title compound was synthesized according to the procedure
The title compound was synthesized according to the procedure
of preparing compound
hydroxybenzaldehyde instead of 4-methylbenzaldehyde. Yield:
11%. 1H NMR (500 MHz, DMSO-d6): 12.71 (s, 1H), 8.54 (s, 1H),
7.83e7.58 (m, 2H), 7.60e7.58 (m, 1H), 7.19 (s, 2H), 6.62 (d, J ¼ 8.0 Hz,
1H), 6.41 (s, 1H), 3.45 (q, J ¼ 6.5 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-
MS (m/z): 333.3 [MþH]þ.
6 except using 4-(diethylamino)-2-
of preparing compound
ldehyde instead of 4-methylbenzaldehyde. Yield: 49%. 1H NMR
(500 MHz, DMSO-d6): 12.97 (s, 1H), 11.59 (s, 1H), 8.42 (s, 1H), 8.29
6 except using 1H-indole-6-carboxa
d
d
(s, 1H), 7.88e7.86 (m, 1H), 7.69e7.67 (m, 1H), 7.61e7.60 (m, 1H),
7.56e7.51 (m, 2H), 7.27e7.22 (m, 2H), 6.65 (s, 1H). ESI-MS (m/z):
285.2 [MþH]þ.
4.28. 2-(1H-benzimidazol-2-yl)-3-[4-(dimethylamino)-2-
hydroxyphenyl]acrylonitrile (31)
4.22. 2-(1H-benzimidazol-2-yl)-3-(4-methoxyphenyl)acrylonitrile
(25)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(dimethylamino)-2-
hydroxybenzaldehyde instead of 4-methylbenzaldehyde. Yield:
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(methoxy)benzaldehyde
instead of 4-methylbenzaldehyde. Yield: 50%. 1H NMR (500 MHz,
20%. 1H NMR (500 MHz, DMSO-d6):
d 12.76 (br s, 1H), 8.59e8.56
DMSO-d6)
d
12.98 (s, 1H), 8.28 (s, 1H), 8.01 (d, J ¼ 8.9 Hz, 2H),
(m, 1H), 7.69e7.50 (m, 3H), 7.20 (br s, 2H), 6.64e6.60 (m, 1H),
7.73e7.64 (m, 1H), 7.55 (d, J ¼ 6.7 Hz,1H), 7.34e7.20 (m, 2H), 7.18 (d,
6.46e6.40 (m, 1H), 3.06 (s, 6H). ESI-MS (m/z): 305.1 [MþH]þ.
J ¼ 8.9 Hz, 2H), 3.88 (s, 3H). ESI-MS (m/z): 276.1 [MþH]þ.
4.29. 2-(1H-benzimidazole-2-yl)-3-(4-dimethylamino-3-
methoxyphenyl)acrylonitrile (32)
4.23. 2-(1H-benzimidazol-2-yl)-3-(4-hydroxy-3-methoxyphenyl)
acrylonitrile (26)
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-(dimethylamino)-3-
methoxybenzaldehyde instead of 4-methylbenzaldehyde. Yield:
The title compound was synthesized according to the procedure
of preparing compound 6 except using 4-hydroxy-3-methoxy
benzaldehyde instead of 4-methylbenzaldehyde. Yield: 33%. 1H
24%. 1H NMR (500 MHz, DMSO-d6): d 12.90 (s, 1H), 8.22 (s, 1H), 7.71
NMR (500 MHz, DMSO-d6):
d
12.93 (br s, 1H), 10.21 (br s, 1H), 8.23
(s, 1H), 7.67e7.64 (m, 1H), 7.55e7.49 (m, 2H), 7.30e7.20 (m, 2H),
6.95 (d, J ¼ 9.0 Hz, 1H), 3.88 (s, 3H), 2.91 (s, 6H). ESI-MS (m/z): 319.2
[MþH]þ.
(s, 1H),7.74 (d, J ¼ 2.0 Hz, 1H), 7.66 (br s, 1H), 7.54 (br s, 1H), 7.47 (dd,
J ¼ 2.0 and 8.5 Hz,1H), 7.24 (br s, 2H), 6.98 (d, J ¼ 8.0 Hz,1H), 3.87 (s,
3H). HRMS (ESI) m/z calcd for C17H14O2N3 Hþ: 292.1080. Found:
292.1079 (
D
¼ ꢂ0.34 ppm).
4.30. 2-(1-methyl-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (33)
4.24. 2-(1H-benzimidazol-2-yl)-3-(3,4-dimethoxyphenyl)
acrylonitrile (27)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 1-methyl-2-benzimida
zolylacetonitrile instead of 2-(1H-benzimidazol-2-yl)acetonitrile.
The title compound was synthesized according to the procedure
of preparing compound
benzaldehyde instead of 4-methylbenzaldehyde. Yield: 15%. 1H
NMR (500 MHz, DMSO-d6): 12.98 (s, 1H), 8.28 (s, 1H), 7.74 (d,
6
except using 3,4-dimethoxy
Yield: 51%. 1H NMR (400 MHz, CDCl3)
d 8.14 (s, 1H), 7.89 (t,
J ¼ 6.7 Hz, 2H), 7.89e7.76 (m, 2H), 7.37 (ddd, J ¼ 11.2, 6.7,1.6 Hz, 2H),
7.30 (dd, J ¼ 7.1, 2.5 Hz, 2H), 4.02 (s, 3H), 2.43 (s, 3H). ESI-LCMS (m/
z): 331.2 [MþH]þ.
d
J ¼ 2.5 Hz, 1H), 7.68 (d, J ¼ 8.0 Hz, 1H), 7.59 (dd, J ¼ 2.0 and 8.5 Hz,
1H), 7.54 (d, J ¼ 7.5 Hz, 1H), 7.30e7.18 (m, 3H), 3.88 (s, 3H), 3.86 (s,
3H). ESI-MS (m/z): 306.2 [MþH]þ.
4.31. 2-(1-ethyl-1H-benzimidazol-2-yl)-3-(4-diethylaminophenyl)
acrylonitrile (34)
4.25. 2-(1H-benzimidazol-2-yl)-3-(3,4,5-trimethoxyphenyl)
acrylonitrile (28)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 1-ethyl-2-benzimida
zolylacetonitrile instead of 2-(1H-benzimidazol-2-yl)acetonitrile.
The title compound was synthesized according to the procedure
of
trimethoxybenzaldehyde instead of 4-methylbenzaldehyde. Yield:
11%. 1H NMR (500 MHz, DMSO-d6): 13.02 (br s, 1H), 13.03e13.00
preparing
compound
6
except
using
3,4,5-
Yield: 11% yield. 1H NMR (400 MHz, DMSO-d6)
d 8.14 (s,1H), 7.96 (d,
J ¼ 8.2 Hz, 2H), 7.69 (dd, J ¼ 11.2, 7.7 Hz, 2H), 7.41 (d, J ¼ 8.1 Hz, 2H),
d
7.38e7.25 (m, 2H), 4.49 (q, J ¼ 7.2 Hz, 2H), 2.41 (s, 3H), 1.44 (t,

510
T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
J ¼ 7.2 Hz, 3H). ESI-MS (m/z): 345.0 [MþH]þ.
acetonitrile. Yield: 35%. 1H NMR (500 MHz, DMSO-d6):
d 8.12 (s,1H),
7.90 (d, J ¼ 9.0 Hz, 2H), 7.75 (d, J ¼ 1.5 Hz, 1H), 7.52 (d, J ¼ 8.5 Hz,
1H), 7.36e7.34 (m, 1H), 6.84 (d, J ¼ 9.5 Hz, 2H), 3.48 (q, J ¼ 7.5 Hz,
4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 395.2 [MþH]þ.
4.32. 2-(1-clopropyl-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (35)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 1-cyclopropyl-2-
benzimidazolylacetonitrile instead of 2-(1H-benzimidazol-2-yl)
4.38. 2-(5, 6-dichloro-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (41)
acetonitrile. Yield: 34%. 1H NMR (500 MHz, DMSO-d6):
d 8.07 (s,
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5,6-dichloro-1H-ben-
zimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)
1H), 7.98 (d, J ¼ 9.0 Hz, 2H), 7.71e7.67 (m, 2H), 7.41e7.33 (m, 2H),
7.10 (d, J ¼ 9.0 Hz, 2H), 3.73e3.70 (m, 1H), 3.50e3.47 (m, 4H),
1.66e1.60 (m, 6H), 1.30e1.28 (m, 2H), 1.00e0.98 (m, 2H) ppm. ESI-
MS (m/z): 357.2 [MþH]þ.
acetonitrile. Yield: 35%. 1H NMR (500 MHz, DMSO-d6):
d 8.12 (s,1H),
7.89 (d, J ¼ 8.5 Hz 1H), 7.78 (s,1H), 6.84 (d, J ¼ 9.5 Hz,1H), 3.49e3.37
(m, 4H), 1.14 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 385.1 [MþH]þ.
4.33. 2-(benzoxazole-2-yl)-3-(4-diethylaminophenyl)acrylonitrile
(36)
4.39. 2-[5-(trifluoromethyl)-1H-benzimidazol-2-yl]-3-(4-
diethylaminophenyl)acrylonitrile (42)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(benzoxazolyl)acetoni-
trile instead of 2-(1H-benzimidazol-2-yl)acetonitrile. Yield: 52%. 1H
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-trifluoromethyl-1H-
benzimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-
NMR (500 MHz, DMSO-d6):
d
8.21 (s, 1H), 8.00 (d, J ¼ 9.5 Hz, 2H),
7.75e7.70 (m, 2H), 7.41e7.38 (m, 2H), 6.84 (d, J ¼ 9.0 Hz, 2H), 3.48 (t,
yl)acetonitrile. Yield: 67%. 1H NMR (500 MHz, DMSO- d6):
d 8.17
J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 318.2 [ M þ H
(s, 1H), 7.92 (d, J ¼ 9.0 Hz, 2H) 7.89 (s, 1H), 7.73 (d, J ¼ 8.5 Hz, 1H),
7.53 (d, J ¼ 8.5 Hz, 1H), 6.85 (d, J ¼ 9.5 Hz, 2H), 3.48 (q, J ¼ 7.0 Hz,
4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 384.9 [MþH]þ.
]þ.
4.34. 2-(benzothiazol-2-yl)-3-(4-diethylaminophenyl)acrylonitrile
(37)
4.40. 2-(5-trifluoromethoxy-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (43)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(benzothiazolyl)aceto-
nitrile instead of 2-(1H-benzimidazol-2-yl)acetonitrile. Yield: 61%.
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-trifluoromethoxy-1H-
benzimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-
1H NMR (500 MHz, DMSO-d6):
d 8.11e8.07 (m, 2H), 8.01e7.97 (m,
3H), 7.54e7.51 (m, 1H), 7.46e7.43 (m, 1H), 6.83 (d, J ¼ 9.0 Hz, 2H),
3.47 (q, J ¼ 7.0 Hz, 4H), 1.14 (t, J ¼ 6.5 Hz, 6H). ESI-LCMS (m/z): 334.1
[MþH]þ.
yl)acetonitrile. Yield: 79%. 1H NMR (500 MHz, DMSO-d6):
d 8.13
(s, 1H), 7.90 (d, J ¼ 9.0 Hz, 2H), 7.62 (d, J ¼ 9.0 Hz, 1H), 7.43 (d,
J ¼ 9.0 Hz, 1H), 7.39 (d, J ¼ 9.0 Hz, 1H), 6.85 (d, J ¼ 9.0 Hz, 2H), 3.47
(q, J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 401.1
[MþH]þ.
4.35. 2-(5-fluoro-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (38)
4.41. Methyl 2-[1-cyano-2-(4-diethylaminophenyl)vinyl]-1H-
benzimidazole-5-carboxylate (44)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-fluoro-1H-benzimi-
dazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)aceto-
nitrile. Yield: 64%. 1H NMR (500 MHz, DMSO-d6):
d 8.11 (s, 1H), 7.89
The title compound was synthesized according to the procedure
of preparing compound 14 except using methyl 2-cyanomethyl-5-
benzimidazolecarboxylate instead of 2-(1H-benzimidazol-2-yl)
(d, J ¼ 9.5 Hz, 2H), 7.59e7.56 (m, 1H), 7.41e7.37 (m, 1H),7.12e7.08
(m, 1H), 6.85 (d, J ¼ 9.5 Hz, 2H), 3.47 (q, J ¼ 7.5 Hz, 4H), 1.15 (t,
J ¼ 6.5 Hz, 6H). ESI-LCMS (m/z): 335.2 [MþH]þ.
acetonitrile. Yield: 59%. 1H NMR (500 MHz, DMSO-d6):
d 8.16 (s,
1H), 8.13 (d, J ¼ 1.0 Hz, 1H), 7.92 (d, J ¼ 9.0 Hz, 2H), 7.85 (dd, J ¼ 1.0
and 8.0 Hz, 1H), 7.64 (d, J ¼ 9.0 Hz, 1H), 6.85 (d, J ¼ 9.0 Hz, 2H), 3.87
(s, 3H), 3.47 (q, J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 6.5 Hz, 6H). ESI-LCMS (m/
z): 375.2 [MþH]þ.
4.36. 2-(5-chloro-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (39)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-chloro-1H-benzimi-
dazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)aceto-
4.42. 2-[1-cyano-2-(4-diethylaminophenyl)vinyl]-3H-
benzimidazol-5-carboxylic acid (45)
nitrile. Yield: 54%. 1H NMR (500 MHz, DMSO-d6):
d 8.12 (s, 1H), 7.90
(d, J ¼ 9.0 Hz, 2H), 7.61 (d, J ¼ 2.5 Hz, 1H), 7.57 (d, J ¼ 8.5 Hz, 1H),
7.24 (dd, J ¼ 2.0 and 8.5 Hz, 1H), 6.847 (d, J ¼ 9.0 Hz 2H), 3.478 (q,
J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 351.2
[MþH]þ.
To a solution of Compound 44 (100 mg, 0.28 mmol) in MeOH/
H2O (1:1, 5 mL) was added NaOH (27 mg, 0.68 mmol). After the
solution was stirred at room temperature overnight, the pH was
adjusted to 1 with HCl solution (1N). After the solvent was removed
in vacuo, the residue was purified by reverse phase preparative
HPLC in a gradient increase of methanol in water solution to afford
4.37. 2-(5-bromo-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (40)
compound 45 (67 mg, 72%). 1H NMR (500 MHz, DMSO-d6):
d 8.17 (s,
1H), 8.13 (s, 1H) 7.92 (d, J ¼ 9.5 Hz, 2H), 7.85 (dd, J ¼ 2.0 and 9.0 Hz
1H), 7.63 (d, J ¼ 8.0 Hz, 1H), 6.86 (d, J ¼ 9.5 Hz, 2H), 3.48 (q,
J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 361.0
[MþH]þ.
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-bromo-1H-benzimi-
dazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)

T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
511
4.43. 2-(5,6-dimethyl-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (46)
6.84 (d, J ¼ 9.5 Hz, 2H), 3.50e3.40 (m, 4H), 1.17e1.11 (m, 6H). ESI-
LCMS (m/z): 318.2 [MþH]þ.
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5,6-dimethyl-1H-ben-
zimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)
4.49. 2-(3H-imidazo[4,5-b]pyridin-2-yl)-3-(4-diethylaminophenyl)
acrylonitrile (52)
acetonitrile. Yield: 56%. 1H NMR (500 MHz, DMSO-d6):
d
8.13 (s,
The title compound was synthesized according to the procedure
of preparing compound 14 except using 3H-imidazo[4,5-b]pyri-
dine-2-acetonitrile instead of 2-(1H-benzimidazol-2-yl)acetoni-
1H), 7.89 (d, J ¼ 9.0 Hz, 2H), 7.40 (s, 2H) 6.87 (d, J ¼ 9.5 Hz, 2H), 3.49
(q, J ¼ 7.0 Hz, 4H), 2.34 (s, 6H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/
z): 345.3 [MþH]þ.
trile. Yield: 43%. 1H NMR (500 MHz, DMSO-d6):
d
8.29 (dd, J ¼ 1.5
and 5.0 Hz, 1H), 8.17 (s, 1H), 7.94 (dd, J ¼ 1.5 and 8.0 Hz, 1H), 7.89 (d,
J ¼ 9.0 Hz, 2H), 7.20 (dd, J ¼ 5.0 and 8.0 Hz, 1H), 6.85 (d, J ¼ 9.0 Hz,
2H), 3.47 (q, J ¼ 7.0 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z):
318.2 [MþH]þ.
4.44. 2-[5-(tert-butyl)-1H-benzimidazol-2-yl]-3-(4-
diethylaminophenyl)acrylonitrile (47)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-tert-butyl-1H-benzi-
midazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)
4.50. 7-diethylamino-3-(benzimidazol-2-yl)coumarin (53)
acetonitrile. Yield: 72%. 1H NMR (500 MHz, DMSO-d6):
d
8.12 (s,
The title compound is a byproduct of the synthesis of compound
30. Compound 53 was eluted out when the reaction mixture was
purified by silica gel chromatography using cyclohexane-acetone-
ethyl acetate (10:1:1). Yield: 15%. 1H NMR (500 MHz, DMSO- d6):
1H), 7.90 (d, J ¼ 9.0 Hz, 2H), 7.54e7.51 (m, 2H), 7.39 (d, J ¼ 8.5 Hz,
1H), 6.87 (d, J ¼ 8.5 Hz, 2H), 3.48 (q, J ¼ 7.0 Hz, 4H), 1.33 (s, 9H), 1.15
(t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 373.3 [MþH]þ.
d
12.28 (s, 1H), 8.93 (s, 1H), 7.72 (d, J ¼ 9.0 Hz, 1H), 7.65e7.58 (m,
4.45. 2-(5-methoxy-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (48)
2H), 7.17 (t, J ¼ 4.5 Hz, 2H), 6.83 (dd, J ¼ 2.0 and 9.0 Hz, 1H), 6.67 (d,
J ¼ 2.0 Hz, 1H), 3.48 (q, J ¼ 6.5 Hz, 4H), 1.17 (t, J ¼ 7.0 Hz, 6H). ESI-MS
(m/z): 334.1 [MþH]þ.
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-methoxy-1H-benzi-
midazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-yl)
4.51. 2-(1H-benzimidazol-2-yl)-3-(4-diethylaminophenyl)
propanenitrile (54)
acetonitrile. Yield: 42%. 1H NMR (500 MHz, DMSO-d6):
d 8.07 (s,
1H), 7.88 (d, J ¼ 9.0 Hz, 2H), 7.49 (d, J ¼ 8.5 Hz, 1H), 7.06 (d,
J ¼ 2.0 Hz, 1H), 6.91 (dd, J ¼ 2.0 and 9.0 Hz, 1H), 6.86 (d, J ¼ 9.0 Hz,
2H), 3.81 (s, 3H), 3.48 (t, J ¼ 6.5 Hz, 4H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-
LCMS (m/z): 347.0 [MþH]þ.
To a solution of Compound 14 (80 mg, 0.25 mmol) in MeOH
(5 mL) was added 5% Pd/C (10 mg, 10% wt) under hydrogen atmo-
sphere at room temperature for 12 h. The reaction mixture was
filtered through celite and the solvent was concentrated in vacuo.
The crude product was purified by reverse phase preparative HPLC
in a gradient increase of methanol in water solution to afford
4.46. 2-[5-(dimethylamino)-1H-benzimidazol-2-yl]-3-(4-
diethylaminophenyl)acrylonitrile (49)
compound 54. Yield: 42%. 1H NMR (500 MHz, DMSO-d6):
d 12.68
(br s, 1H), 7.62e7.51 (m, 2H),7.20 (br s, 2H),7.05 (d, J ¼ 8.5 Hz, 2H),
6.57 (d, J ¼ 8.5 Hz, 2H), 4.74 (dd, J ¼ 6.5 and 9.0 Hz, 1H), 3.28 (q,
J ¼ 6.0 Hz, 4H), 3.20 (dd, J ¼ 5.0 and 9.5 Hz, 2H), 1.04 (t, J ¼ 6.0 Hz,
6H). ESI-LCMS (m/z): 319.2 [MþH]þ
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-dimethylamino-1H-
benzimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-
yl)acetonitrile. Yield: 75%. 1H NMR (500 MHz, DMSO-d6):
d 8.14
(s, 1H), 7.90 (d, J ¼ 9.0 Hz, 2H), 7.57 (d, J ¼ 9.0 Hz, 1H), 7.16e7.14 (m,
2H), 6.88 (d, J ¼ 9.5 Hz, 2H), 3.49 (q, J ¼ 7.0 Hz, 4H), 3.07 (s, 6H), 1.15
(t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/z): 360.0 [MþH]þ.
4.52. Plasmids and cells
The sequence for full-length vif was amplified with PCR from an
infectious HIV-1 clone pNL4-3 and inserted into pcDNA3.1-intron
with a fused hemagglutinin (HA) tag at the N terminus. The cDNA
fragment coding for hA3G was cloned into pEGFP-C1, as described
previously [31].The 293T cells were maintained in the conditioned
Dulbecco's modified Eagle's medium (DMEM) supplemented with
10% (v/v) fetal bovine serum (FBS), plus 100
streptomycin. The H9 and SupT1 cells were cultured in conditioned
RPMI 1640 supplemented with 10% FBS and 100
and streptomycin.
4.47. 2-(5-methanethionyl-1H-benzimidazol-2-yl)-3-(4-
diethylaminophenyl)acrylonitrile (50)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 2-(5-methanethionyl-1H-
benzimidazol-2-yl)acetonitrile instead of 2-(1H-benzimidazol-2-
mg/mL penicillin and
yl)acetonitrile. Yield: 33%. 1H NMR (500 MHz, DMSO-d6):
d
8.10
mg/mL penicillin
(s, 1H), 7.89 (d, J ¼ 9.0 Hz, 2H), 7.52 (d, J ¼ 8.0 Hz, 1H), 7.44 (d,
J ¼ 1.0 Hz, 1H), 7.19 (d, J ¼ 8.0 Hz, 1H), 6.86 (d, J ¼ 9.0 Hz, 2H), 3.48
(q, J ¼ 7.5 Hz, 4H), 2.53 (s, 3H), 1.15 (t, J ¼ 7.0 Hz, 6H). ESI-LCMS (m/
z): 363.0 [MþH]þ.
4.53. High-throughput screen
Vif-HA and A3G-GFP plasmids were co-transfected into 293T
cells in 96-well plate (Corning, Costar) and Lipofectamine 2000
(Invitrogen) by following the instructions of manufacturer. After
transfection, 20,155 drug-like set compounds (Enamine, Mon-
mouth Jct, NJ) were added using a Tecan Freedom EVO150 (Tecan,
€
Mannedorf, Schweiz) with a final concentration of 50 mM. Column
12 received only DMSO instead of any compounds. In addition,
column 1 was only transfected with the plasmid expressing A3G-
GFP as a control. The GFP expression was detected with a PE
4.48. 2-(3H-imidazo[4,5-c]pyridin-2-yl)-3-(4-diethylaminophenyl)
acrylonitrile (51)
The title compound was synthesized according to the procedure
of preparing compound 14 except using 3H-imidazo[4,5-c]pyri-
dine-2-acetonitrile instead of 2-(1H-benzimidazol-2-yl)acetoni-
trile. Yield: 46%. 1H NMR (500 MHz, DMSO-d6):
1H), 8.23e8.21 (m, 1H), 7.91 (d, J ¼ 8.5 Hz, 2H), 7.55e7.53 (m, 1H),
d
8.84 (d, J ¼ 0.5 Hz,

512
T. Pan et al. / European Journal of Medicinal Chemistry 95 (2015) 500e513
Envision (PerkineElmer) at 48 h post-transfection [20]. The fold
change of mean fluorescent intensity (MFI) was calculated as: MFI
of cells with compound treatment together with transfection of
A3G-GFP and Vif/MFI of cells only with transfection of A3G-GFP and
Vif.
coupling kit were purchased directly from GE Healthcare. The
suitable pH value of 4.0 for Vif-His immobilization (50 g/mL in
10 mM acetate buffer) was determined first. The CM5 censor chip
was activated and then injected with Vif-His (100 g/mL, in 10 mM
acetate buffer, pH 4.0) for 7 min. The residual activated groups on
the surfaces were blocked with an injection of ethanolamine HCl
(1M) for 7 min A3G-His protein was pre-incubated with DMSO,
compound 14, or compound 26 for 30 min and then was injected
m
m
4.54. Co-immunoprecipitation (Co-IP)
HEK293T cells were transfected with pcDNA3.1-Vif-HA and
pcDNA3.1-EC/EB/CUL5-Flag and were lysed 48 h later with IP lysis
buffer. Then the lysate were incubated with anti-HA beads (Sigma)
overnight at 4 ꢁC. Then IP products were centrifuged and washed
three times with lysis buffer. Western blot was conducted to
analyze the immunoprecipitated samples with the following pri-
mary antibodies: anti-FLAG (rabbit polyclonal, MBL) or anti-HA
(mouse monoclonal, MBL) [33].
(30 ml/min). Binding to the Vif-His protein was monitored for about
120 s. The dissociate time were 200 s for compound 14 and 150s for
26 with running buffer in per cycle.
4.60. Acute toxicological assay
Male Balb/c mice, 4e6weeks, were purchased from Laboratory
Animal Center in Sun Yat-Sen University, Guangzhou, China. These
mice were randomly divided into three groups and were then
intraperitoneally injected with compounds at different doses. After
two weeks, mice were sacrificed. The organs including heart, liver,
spleen, lung, and kidney were fixed in 4% formaldehyde at room
temperature for hematoxylin and eosin staining and the blood
samples were subjected to the analysis of hepatic or renal
functions.
4.55. PBMCs isolation
The peripheral blood mononuclear cells (PBMCs) were isolated
from healthy human donors through Ficoll gradient centrifugation,
followed by culturing in the RPMI 1640 medium (Invitrogen) sup-
plemented with 10% fetal bovine serum (Invitrogen), 100 units/ml
of penicillin and 100 mg/mL of streptomycin (Invitrogen).
Acknowledgment
4.56. HIV-1 production, infection, and IC50 assay
This work was funded by National Special Research Program for
Important Infectious Diseases (No.2013ZX10001004), Guangdong
Recruitment Program of Creative Research Groups (No.2009010058),
National Basic Research Program of China (973 Program)
(No.2010CB912202), National Natural Science Foundation of China
(No.30972620), Natural Science Foundation of Guangdong
(No.9251008901000022), Specialized Research Fund for the Doctoral
Program of Higher Education of China (No.20090171110083),
Guangdong Natural Science Foundation New Ph.D. Start-up
(S20212040006494). The HIV-1 infectious clone pNL4-3 was ob-
tained from the NIH AIDS Research & Reference Reagent Program.
HIV-1 infectious clone, pNL4-3 (X4) was amplified with HB101
competent cells (Promega). To generate viruses, 293T cells were
transfected with 10 mg of infectious clones and Lipofectamine 2000
(Invitrogen) by following the instructions of manufacturer. Culture
supernatants were harvested at 48 h post-transfection and stored
at ꢂ80 ꢁC. To normalize viral inputs, the amount of p24 was
measured by HIV-1 p24 enzyme-linked immunosorbent assay
(ELISA). The target cells (1 ꢀ 106) were infected with the equivalent
of 5 ng HIV-1 p24 in 1 mL for 3 h at 37 ꢁC. The virus-containing
supernatants were then removed by washing 3 times with PBS.
The cells were maintained in conditioned RPMI 1640 medium and
HIV-1 replication was monitored by p24 detection [5].
Appendix A. Supplementary data
4.57. Cell toxicity assay
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2015.03.050.
Cell toxicity assay was performed with the CellTiter-Glo Lumi-
nescent Cell Viability Assay Kit which was purchased from Promega
Company. The instructions of manufacturer were followed. Lumi-
nescence was recorded with a Promega plate reader [34,35].
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