Design, synthesis, and biological evaluation of AV6 derivatives as novel dual reactivators of latent HIV-1

Article abstract of DOI:10.1039/c8ra01216d

The "shock and kill" strategy might be a promising therapeutic approach for HIV/AIDS due to the existence of latent viral reservoirs. A major challenge of the "shock and kill" strategy arises from the general lack of clinically effective latency-reversing

Full text of DOI:10.1039/c8ra01216d

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Design, synthesis, and biological evaluation of AV6
derivatives as novel dual reactivators of latent HIV-1†
Cite this: RSC Adv., 2018, 8, 17279
a
Mingtao Ao,‡a Zhenrui Pan,‡a Yuqing Qian,‡a Bowen Tang,a Zeming Feng,
c
a
b
a
a
*
*
*
Hua Fang, Zhen Wu, Jingwei Chen, Yuhua Xue and Meijuan Fang
The “shock and kill” strategy might be a promising therapeutic approach for HIV/AIDS due to the existence
of latent viral reservoirs. A major challenge of the “shock and kill” strategy arises from the general lack of
clinically effective latency-reversing agents (LRAs). The 2-methylquinoline derivative, antiviral 6 (AV6) has
been reported to induce latent HIV-1 expression and act synergistically with a HDAC inhibitor VA to
reverse HIV latency. We report herein the design and identification of AV6 analogues which possess the
zinc-binding group of HDAC inhibitors and have dual acting mechanism for the reactivation of HIV-1
from latency. Evaluation of compounds for the reactivation of HIV-1 latency identified two excellent
active compounds 12c and 12d. Further bioassays revealed that these two compounds reactivated latent
HIV-1 through dual mechanism, the inhibition of HDACs and NFAT-required for early HIV-1 gene
expression. Additionally, it was found that 12c and 12d could reactivate HIV-1 transcription by releasing
P-TEFb from the inactive complex 7SK snRNP. At last, molecular docking identified their orientation and
binding interactions at the active site of HDAC2. This experimental data suggests that 12c and 12d can
be served as effective HIV-1 LRAs which can be taken up for further studies.
Received 8th February 2018
Accepted 24th April 2018
DOI: 10.1039/c8ra01216d
rsc.li/rsc-advances
transcription elongation factor b (P-TEFb) activators and other
unclassied HIV-1 latency-reversing agents.14–18 HDAC inhibi-
1. Introduction
tors, such as valproic acid (VA), vorinostat (also known as
SAHA), and panobinostat have been used in clinical trials in
HIV.19–23 However, preclinical and clinical studies suggest that
these LRAs which target a single mechanism involved in latency
may not be efficient enough to reactivate latent HIV-1 in vivo.24–27
Hence, there is an urgent requirement of developing more
effective latency-reversing interventions with a unique structure
and effective mode of action.
Antiviral 6 (AV6, Fig. 1), a novel HIV-1 latency-reversing agent
identied through high-throughput screening, has been re-
ported to reproducibly activate latent HIV-1 in different cell-
based latency model systems without causing general T cell
proliferation or activation.28 Its activation is independent of the
site of provirus integration, which complements the latency
antagonist activity of HDAC inhibitor. For example, AV6 was
demonstrated to act cooperatively with VA (a HDAC inhibitor)
for enhanced induction of HIV-1 gene expression.28 This is
a proof of concept showing that the combination of two or more
inducers of HIV gene expression acting in accord via different
mechanisms may produce a more potent reactivation effect.29,30
In this study, we design the AV6 analogues as LRAs with the goal
of achieving HDACs inhibition. Key to the design is to identify
the attachment appoint and the linker for the introduction of
the essential active group of HDACs. Herein, we performed the
chemical synthesis and biological evaluation of the rst chem-
ical series of AV6 analogues (series I) to determine the suitable
The latent HIV reservoirs in infected patients are the major
barrier to eliminate HIV as reported.1,2 Recently, scientists have
made efforts to use the “shock and kill” strategy to eradicate
latently infected cells.3–10 In the “shock and kill” approach,
latency-reversing agents (LRAs) should be used to force the
replication and expression of latent HIV proviruses in the initial
“shock” phase, and then the reactivated cells would be elimi-
nated (“kill” phase) by a combination of viral cytopathic effects
and the host immune response, as well as concomitant highly
active anti-retroviral therapy (HAART) to prevent new infec-
tions.11–13 Currently, a wide range of small molecule have been
explored as LRAs to reactivate latent provirus, including histone
deacetylase (HDAC) inhibitors (Fig. 1), methyltransferase
inhibitors, protein kinase
C (PKC) activators, positive
aSchool of Pharmaceutical Sciences, Fujian Provincial Key Laboratory of Innovative
Drug Target Research, Xiamen University, South Xiang-An Road, Xiamen, 361102,
China. E-mail: xueyuhua@xmu.edu.cn; fangmj@xmu.edu.cn; Fax: +86-592-
2189868; Tel: +86-592-2189868
bKey Laboratory for Chemical Biology of Fujian Province, College of Chemistry and
Chemical Engineering, Xiamen University, Xiamen 361005, China. E-mail: jwchen@
xmu.edu.cn
cThe Third Institute of Oceanography of the State Oceanic Administration, Xiamen,
361005, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra01216d
‡ These authors contribute equally to this paper.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17279

View Article Online
RSC Advances
Paper
We hypothesized that the substituents on the benzene or
quinoline rings contained in AV6 structure might modulate the
activation activity of the compound. Therefore, we initially
designed series I derivatives (Fig. 2, compounds 7b-7B) of AV6 to
evaluate the inuence of different substituents and various
attachment points on the activation activity and gure out the
best site for the introduction of the functional group ZBG of the
HDACis. There are various ZBGs for HDACi, such as hydroxamic
acids, aminobenzamides, carboxylates (short-chain fatty acids),
electrophilic ketones, thiols, mercaptoacetamides, and 3-
hydroxypyridin-2-thiones. Among all types of ZBGs, hydroxamic
acid and carboxylate are common zinc ion chelating groups. So
Fig. 1 Several HIV reactivators. HDAC inhibitors: short-chain aliphatic
acids (butyric acid and valproic acid), hydroxamic acids (vorinostat and hydroxamic acid and carboxylates with different linker were
givinostat). AV6: its target protein is not available now.
subsequently incorporated into the suitable site of N-phenyl-
quinoline nucleus in order to identify more potent novel LRAs
(Fig. 2, series II). In this paper, we report the design, synthesis,
attachment appoint for the introduction of the functional group
of HDAC inhibitor. Then, a linker optimization strategy was
pursued to obtain effective LRAs bearing AV6 scaffold structure
and the functional group of HDAC inhibitors. These new AV6
derivatives (series II) were evaluated for their reactivation of
latent HIV. The most potent reactivators were then taken up to
do further bioassays.
and biological evaluation of AV6 analogues as novel LRAs with
dual activated mechanism.
2.2. Syntheses of the designed compounds
The synthetic approaches for the two different series of target
compounds are outlined in Scheme 1 and 2. Compounds 7a–7w
were prepared according to a four-step procedure described
previously and shown in Scheme 1.37 p-Anisidine was
condensed with ethyl acetoacetate to form compound 2, which
was cyclized in diphenyl ether affording 6-methoxy-4-hydroxy-2-
methylquinoline (3). The treatment of 3 with phosphorus oxy-
chloride to provide the key intermediate 4-chloro-6-methoxy-2-
methylquinoline (4).38–40 Compounds 7a–7e could be easily ob-
tained through the reaction of 4 with different nucleophilic
amine derivatives in the presence of catalytic amount of HCl.
On the other hand, demethylation of 4 using BBr3 produced 4-
chloro-2-methylquinolin-6-ol (5). Alkylation of 5 with bromo-
ethane or 2-bromopropane provided 6a or 6b, and the reaction
of halides (6a or 6b) with corresponding amine derivatives
produced compounds 7f–7w (Scheme 1).41,42
2. Results and discussion
2.1. Design of the target compounds
Multi-target drugs have emerged as an innovative therapeutic
approach for many diseases due to their complex etiology and
their multifactorial progression.31–33 AV6 and HDAC inhibitors
have been validated as two inducers of HIV gene expression
acting in accord via two different mechanisms. The N-phenyl-
quinoline nucleus of AV6 is considered as ‘Master Key Core’ in
many compounds aimed at different targets to bring out varied
pharmacological properties. Besides, many HDACIs have been
designed and synthesized as dual- or multi-targeted inhibitors
for the cancer therapy,34 and the synergistic effect on the acti-
vation of latent provirus between VA (a carboxylate HDACi) and
AV6 has been conrmed.28 Thus, we developed an idea that the
introduction of the function groups of HDACis in the N-phe-
nylquinoline structural scaffold of AV6 might also result in new
compounds both targeting HDACs and with AV6 related active
mechanism to generate more potent LRAs.
The designed products 9a–9e, 10a–10d, 11a–11d and 12a–
12d (series II) were synthesized according to the pathway shown
There are only three class I HDACs (HDAC 1, HDAC 2, HDAC
3) which are localized at the integrated HIV-1 LTR and are zinc-
dependent deacetylasesas.14,35 The classical pharmacophore for
their HDACi consists of a zinc-binding group (ZBG), an appro-
priate linker and a surface recognition cap group.36 Generally,
ZBG plays a signicant role in the binding efficiency between
HDACi and enzyme. Therefore, we intend to incorporate the
essential active group ZBG of HDACis into the N-phenylquino-
line scaffold to generate new small molecule entities (AV6
analogues) which may display better potency to activate latent
HIV-1. However, this is not a straight-forward procedure, and we
must identify the appropriate common features, substitution
sites, and then growing vectors.
Fig. 2 Design of multi-targeted HIV activator based AV6 and HDAC
inhibitors.
17280 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Scheme 1 Synthesis of 2-methyl-N-arylquinolin-4-amine 7a–7w. Reagents and conditions: (a) ethyl 3-oxobutanoate, EtOH, reflux, 4 h; (b)
diphenyl ether, 1 h; (c) POCl3, 120 ꢁC, 2 h; (d) BBr3, DCM, r.t., 2 h; (e) CH3CH2Br or CH3CHBrCH3, NaH, DMF, r.t., overnight; (f) substituted aromatic
amines, butyl alcohol, conc. HCl, 120 ꢁC, 5 h.
in Scheme 2. Demethylation on C-6 position of compound 7a compound 7a (AV6) with 6-methoxy and 30,40-dichloro substi-
gave corresponding C6–OH quinolone (8). Compound 8 con- tutions induced 57.0 ꢀ 1.2 percent GFP production in J-Lat A2
verted to compounds 9a–9e and 10a–10d by alkylation of the cells at 10 mM. Upon comparison of the activities of 6-ethoxy
C6–OH with appropriate alkyl halide and various lengths of derivatives 7f–7j and 6-isopropoxy derivatives 7o–7s with 6-
ethyl or methyl bromoalkanoate, respectively. Carboxylic acid methoxy derivatives 7a–7e, we found that the reactivation
derivatives 11a–11d were nally obtained by the hydrolysis of potency of the derivatives for latent HIV-1 is very sensitive to the
esters 10a–10d. Conversion of esters 10a–10d to hydroxamic type of R1 substituent. Compounds 7o–7s have no effect on HIV-
acids using freshly prepared hydroxylamine yielded the target 1 gene expression in J-Lat A2 cells, which suggest that the
compounds 12a–12d.43
replacement of the methoxy group with the isoproyloxy group at
The structures of the target compounds were conrmed with C-6 position of quinoline ring is very detrimental for the reac-
1H-NMR, 13C-NMR, and high-resolution mass spectrometry tivation potency of latent HIV-1. While in 6-ethoxy derivatives
(HRMS).
7f–7j, 7f exhibits good activity of latent HIV-1 reversing (47.3 ꢀ
3.3% at 10 mM), and compounds 7g–7i produce enhanced
potency (14.5–39.9% of the GFP expression at 10 mM) compared
to corresponding 6-methoxy derivatives 7b–7d (with GFP%
values ranged from 2.1–8.3% at 10 mM). These results imply that
the C-6 position (R1) of the quinoline ring may be a promising
attachment point for the introducing of the ZBG groups by
using the linear alkyl linker.
On the other hand, the replacement of Cl with linker groups
(CH3 (7d and 7r) and OCH3 (7e, 7j, 7s, and 7z)), ZBG groups
(COOH (7l and 7u), COOR (7m and 7v), and CONHOH (7n and
7y)), or other groups (NO2 (7k, and 7t)) at the C-30 (R3) and C-40
(R4) positions of phenyl ring leads to the disappearance of
activity as shown in Table 1. It indicates that 30,40-dichloro
substitutions at the phenyl ring are essential for potent reac-
tivation of latent HIV-1 gene expression. Moreover, compounds
7A (5.4 ꢀ 0.4%) and 7B (1.3 ꢀ 0.3%) in which the phenyl ring is
2.3. Biological evaluation
2.3.1 Reactivation of latent HIV-1 gene expression in J-Lat
A2 cells and structure–activity relationship. All target
compounds were rst evaluated for their activation of latent
HIV-1 in J-Lat A2 cells, which contained an integrated 50-LTR-
Tat-Flag-iRES-EGFP-30-LTR expression cassette that was nor-
mally silent, the enhanced green uorescent protein (GFP)
would be expressed under the control of 50LTR promoter upon
activation.44 In this screening experiment, AV6 and SAHA were
employed as positive controls. The results are presented in
Tables 1–3. The structure–activity relationship (SAR) was further
explored as described below.
At the rst initial exploration, a variety of substituents and
different attachment points at the benzene or quinoline rings
contained in AV6 structure were examined. As shown in Table 1,
Scheme 2 Synthesis of 3-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin-6-yl)oxy)-alkanoic acid/hydroxyl acid. Reagents and conditions:(a)
BBr3, DCM, r.t., 2 h; (b) appropriate alkyl halide, NaH, DMF, r.t., overnight; (c) ethyl or methyl bromoalkanoate, NaH, DMF, r.t., overnight; (d) EtOH,
40% NaOH aqueous solution, 50 ꢁC, 1 h; (e) NH2OH, NaOH, CH3OH, 0 ꢁC to r.t.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17281

View Article Online
RSC Advances
Paper
changed to the pyridine ring display weak or no induction of J-Lat A2 cells at 10 mM (19.7 ꢀ 0.7%–32.4 ꢀ 2.9%) and 5 mM (11.1
GFP expression in J-Lat A2 cells at 10 mM in comparison with ꢀ 1.8%–29.1 ꢀ 2.2%). It was further conrmed that together
AV6/7a (57.0 ꢀ 1.2%) and 7f (47.3 ꢀ 3.3%), respectively. It with the oxygen connection, the straight alkyl chain could be
implies that the phenyl ring is also essential for the activity. used as linear linker for the introduction of ZBG groups at 6-
Additionally, 7b, 7f–7j, 7m, and 7A also showed certain degree position of quinolone ring.
of activation effect (with GFP% values > 2.0%) at 5 mM. Among
Finally, we investigated the effect of various ZBG groups with
them, 6-ethoxy derivatives 7f–7j could reactivated latent HIV-1 linear alky linker connected to the C-6 position of the quinoline
in a dose-dependent manner better than 6-methoxy deriva- ring on latent HIV-1 reactivation. Different ZBG groups
tives 7a–7e. Above all, the position C-6 (R1) of quinoline ring (COOC2H5, COOH, and CONHOH) were introduced at the
might be better suited for the introduction of ZBG group than terminal of the straight alkyl chain on 6-position of quinolone
the positions R3 and R4 of benzene ring, and the ZBG group ring, and another three series of AV6 analogues (10a–10d, 11a–
would like to be introduced in R1 position by using linear linker. 11d, and 12a–12d) were generated and analyzed. Among these
To explore the effect of chain length of linear linker on new derivatives with various ZBG groups, compounds 10a–10d
activity, a series of ether analogues of AV6 (9a–9e) with 3–7 (carboxylates) and 11a–11d (carboxylic acids) were either weakly
carbons of n-alkyl chain at the C-6 position of the quinolone active or completely inactive at tested concentrations (Table 3).
ring was generated and their reactivation of latent HIV-1 gene Compounds with hydroxamic acid side chain showed varying
expression was evaluated. As shown in Table 2, all ether degrees of potency to reverse HIV latency (12b–12d), except for
analogues of AV6 (9a–9e) with 3–7 carbons of linear alkyl chain compound 12a. Compounds 12c and 12d exerted a remarkable
retained potent activities in the induction of GFP expression in improvement in the latent HIV-1 reactivation activity, inducing
Table 1 Reactivation of latent HIV-1 gene expression by the indicated quinolines in J-Lat A2 cellsa
% GFP(+)
% GFP(+)
Compound
X
R1
R2
R3
R4
cells (10 mM)
cells (5 mM)
DMSO
SAHA
AV6/7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
0.7 ꢀ 0.1
71.2 ꢀ 4.2
57.0 ꢀ 1.2
8.3 ꢀ 2.5
1.8 ꢀ 0.1
2.1 ꢀ 0.3
6.4 ꢀ 0.5
47.3 ꢀ 3.3
39.9 ꢀ 4.5
28.5 ꢀ 1.6
14.5 ꢀ 0.5
5.1 ꢀ 0.9
1.3 ꢀ 0.9
1.0 ꢀ 0.1
5.2 ꢀ 1.1
0.6 ꢀ 0.1
0.9 ꢀ 0.1
0.9 ꢀ 0.1
1.2 ꢀ 0.2
0.9 ꢀ 0.2
1.3 ꢀ 0.4
1.5 ꢀ 0.3
2.8 ꢀ 0.1
1.4 ꢀ 0.3
0.7 ꢀ 0.1
1.0 ꢀ 0.2
1.0 ꢀ 0.2
0.9 ꢀ 0.3
5.4 ꢀ 0.4
1.3 ꢀ 0.3
0.7 ꢀ 0.1
56.8 ꢀ 1.2
3.8 ꢀ 1.1
10.8 ꢀ 0.3
1.2 ꢀ 0.1
0.9 ꢀ 0.1
1.9 ꢀ 0.1
4.1 ꢀ 0.1
11.6 ꢀ 2.3
15.3 ꢀ 2.6
8.5 ꢀ 1.2
2.3 ꢀ 0.6
0.9 ꢀ 1.9
0.9 ꢀ 0.2
3.8 ꢀ 0.9
0.6 ꢀ 0.1
0.8 ꢀ 0.1
0.8 ꢀ 0.1
0.8 ꢀ 0.2
0.8 ꢀ 0.1
0.8 ꢀ 0.3
0.9 ꢀ 0.3
1.5 ꢀ 0.1
0.8 ꢀ 0.2
0.6 ꢀ 0.1
1.2 ꢀ 0.2
0.9 ꢀ 0.2
0.7 ꢀ 0.1
5.1 ꢀ 1.3
0.8 ꢀ 0.1
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
CH3
H
H
H
H
Cl
Br
F
CH3
OCH3
Cl
Br
F
CH3
OCH3
NO2
COOH
COOC4H9
CONHOH
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH3
7l
7m
7n
7o
7p
7q
7r
7s
7t
7u
7v
7w
7x
Br
F
CH3
OCH3
NO2
COOH
COOC4H9
H
H
H
OCH3
Cl
Cl
CH2CH3
CH2CH3
CH(CH3)2
CH3
7y
7z
7A
7B
CONHOH3
OCH3
Cl
N
CH2CH3
Cl
a
Data are presented as mean ꢀ SD of at least three independent experiments performed in triplicate. Cells were treated with the indicated
compounds at 5, 10 mM for 24 h. The expression of GFP were evaluated by FACS.
17282 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Table 2 Reactivation of latent HIV-1 gene expression by the indicated quinolines in J-Lat A2 cellsa
% GFP(+) cells (mM)
10
% Cell viability (mM)
Compound
n
R5
5
1
10
5
1
DMSO
SAHA
AV6/7a
7f
7o
9a
9b
9c
9d
9e
0.7 ꢀ 0.1
0.7 ꢀ 0.2
0.7 ꢀ 0.2
98.6 ꢀ 0.5
43.2 ꢀ 3.2
47.6 ꢀ 5.6
43.2 ꢀ 3.2
67.3 ꢀ 3.3
17.7 ꢀ 3.1
13.1 ꢀ 2.3
19.6 ꢀ 3.9
17.2 ꢀ 3.1
20.4 ꢀ 2.5
98.1 ꢀ 1.9
50.9 ꢀ 5.1
73.4 ꢀ 3.5
50.9 ꢀ 5.1
76.7 ꢀ 2.6
41.5 ꢀ 1.6
34.1 ꢀ 0.4
39.6 ꢀ 0.8
37.6 ꢀ 3.1
52.3 ꢀ 1.0
98.8 ꢀ 0.6
80.4 ꢀ 2.4
80.7 ꢀ 5.4
69.9 ꢀ 2.4
85.2 ꢀ 2.1
56.1 ꢀ 3.8
60.3 ꢀ 0.5
57.6 ꢀ 4.3
58.2 ꢀ 3.8
72.9 ꢀ 3.3
71.2 ꢀ 4.2
57.0 ꢀ 1.2
43.3 ꢀ 0.3
0.9 ꢀ 0.1
56.8 ꢀ 1.2
3.8 ꢀ 1.1
20.1 ꢀ 0.3
0.5 ꢀ 0.5
2.4 ꢀ 0.3
0.8 ꢀ 0.1
2.3 ꢀ 0.3
5.7 ꢀ 1.3
3.1 ꢀ 0.5
5.6 ꢀ 1.2
1.4 ꢀ 0.1
1
1
0
2
3
4
5
6
H
CH3
CH(CH3)2
CH3
CH3
CH3
CH3
CH3
21.1 ꢀ 1.2
0.8 ꢀ 0.1
25.4 ꢀ 4.1
19.9 ꢀ 1.9
19.7 ꢀ 0.7
24.5 ꢀ 2.8
32.4 ꢀ 2.9
18.6 ꢀ 1.8
22.8 ꢀ 1.0
21.6 ꢀ 0.9
29.1 ꢀ 2.2
11.1 ꢀ 1.8
a
Data are presented as mean ꢀ SD of at least three independent experiments performed in triplicate. Cells were treated with the indicated
compounds at 1, 5, 10 mm for 24 h. The expression of GFP were evaluated by FACS.
39.7 ꢀ 4.1% and 49.9 ꢀ 3.5% GFP production in J-Lat A2 cells at its terminal basic group (R5) have important impacts on the
1 mM, respectively. However, they displayed slightly higher compounds regarding their binding activities.
toxicity than SAHA and AV6 according to the cell viability (Table
Among all these AV6 analogues, 12c and 12d showed the
2). In this experiment, we also observed that the length of the most optimal properties in terms of its efficient latency-
alkyl linker with oxygen bonded connection at the C-6 position reversing activity and displayed relatively mild cytotoxicity,
had an important effect on the activity. For example, for thus they were selected for further bioassays as outlined below.
compounds that had the same terminal basic group –CONHOH,
2.3.2 Compounds 12c and 12d promote HIV-1 LTR-driven
the latency-reversing activity of 12c (n ¼ 5) and 12d (n ¼ 6) were GFP expression in different latency cell models. When J-Lat
much better than that of 12a (n ¼ 3) and 12b (n ¼ 4). These A2 cells were treated with different amounts of AV6, SAHA,
results indicate that the length of the linear alkyl linker (n) and 12c, and 12d, the percentage of GFP-positive cells induced by
Table 3 Reactivation of latent HIV-1 gene expression by the indicated quinolines in J-Lat A2 cellsa
% GFP(+) cells (mM)
10
0.7 ꢀ 0.1
% Cell viability (mM)
Compound
n
R5
5
1
10
5
1
DMSO
SAHA
AV6/7a
10a
10b
10c
10d
11a
11b
11c
0.7 ꢀ 0.2
0.7 ꢀ 0.2
98.6 ꢀ 0.5
43.2 ꢀ 3.2
47.6 ꢀ 5.6
94.3 ꢀ 2.5
92.6 ꢀ 3.4
95.4 ꢀ 3.3
96.6 ꢀ 3.2
97.6 ꢀ 2.8
95.6 ꢀ 2.4
94.6 ꢀ 2.8
97.6 ꢀ 3.5
85.7 ꢀ 4.2
54.1 ꢀ 3.7
44.1 ꢀ 3.4
38.6 ꢀ 3.1
98.1 ꢀ 1.9
50.9 ꢀ 5.1
73.4 ꢀ 3.5
97.1 ꢀ 2.9
95.6 ꢀ 3.7
97.3 ꢀ 2.9
96.7 ꢀ 2.5
97.1 ꢀ 1.9
97.2 ꢀ 3.0
95.4 ꢀ 2.9
96.3 ꢀ 1.9
94.2 ꢀ 1.1
89.5 ꢀ 4.2
49.8 ꢀ 3.9
32.7 ꢀ 3.4
98.8 ꢀ 0.6
80.4 ꢀ 2.4
80.7 ꢀ 5.4
86.3 ꢀ 3.4
85.8 ꢀ 4.7
87.0 ꢀ 3.1
86.1 ꢀ 2.3
76.8 ꢀ 2.6
83.7 ꢀ 3.5
94.9 ꢀ 0.6
97.8 ꢀ 1.6
95.3 ꢀ 3.1
89.3 ꢀ 4.8
44.2 ꢀ 3.6
39.1 ꢀ 2.9
71.2 ꢀ 4.2
57.0 ꢀ 1.2
3.3 ꢀ 0.2
1.6 ꢀ 0.7
0.7 ꢀ 0.2
0.9 ꢀ 0.2
0.6 ꢀ 0.1
1.1 ꢀ 0.1
0.8 ꢀ 0.1
1.0 ꢀ 0.1
2.7 ꢀ 0.1
19.7 ꢀ 2.1
87.2 ꢀ 2.0
84.9 ꢀ 3.5
56.8 ꢀ 1.2
3.8 ꢀ 1.1
3.2 ꢀ 0.5
1.4 ꢀ 0.4
1.5 ꢀ 0.5
1.7 ꢀ 0.3
0.7 ꢀ 0.2
1.2 ꢀ 0.1
1.1 ꢀ 0.1
1.1 ꢀ 0.1
1.3 ꢀ 0.9
2.8 ꢀ 1.7
81.5 ꢀ 1.5
77.1 ꢀ 2.4
20.1 ꢀ 0.3
0.5 ꢀ 0.5
0.8 ꢀ 0.1
0.9 ꢀ 0.2
0.9 ꢀ 0.2
0.8 ꢀ 0.1
0.9 ꢀ 0.1
0.7 ꢀ 0.1
0.7 ꢀ 0.2
0.9 ꢀ 0.2
1.1 ꢀ 0.3
0.7 ꢀ 0.6
39.7 ꢀ 4.1
49.9 ꢀ 3.5
1
3
4
5
6
3
4
5
6
3
4
5
6
H
COOC2H5
COOCH3
COOC2H5
COOCH3
COOH
COOH
COOH
COOH
CONHOH
CONHOH
CONHOH
CONHOH
11d
12a
12b
12c
12d
a
Data are presented as mean ꢀ SD of at least three independent experiments performed in triplicate. Cells were treated with the indicated
compounds at 1, 5, 10 mm for 24 h. The expression of GFP were evaluated by FACS.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17283

View Article Online
RSC Advances
Paper
Fig. 3 HIV latency can be reversed by 12c-12d in different latency cell models. (A) J-Lat A2 cells, (B) 2D10 cells, (C) the HeLa-based NH1 cells. (D)
The NH2 cells expressing Tat. All tested cells were treated with 0.1% DMSO or the indicated concentrations of the drugs (12c, 12d, SAHA and AV6)
for 24 h. The expression of GFP for tested J-Lat A2 and 2D10 cells was measured by FACS. Whole cell extracts for tested NH1 and NH2 cells were
prepared and examined for the luciferase activities. The error bars in all panels represent mean ꢀ SD based on at least three independent
experiments. The P value was defined as **p < 0.01, and ***p < 0.001 vs. control.
12c and 12d increased in a dose-dependent manner (Fig. 3(A)). designed by incorporating histone deacetylase (HDAC) inhibi-
The stimulatory effect to reactivate latent HIV-1 was also tory functionality into the pharmacophore of AV6, as dual-
conrmed in 2D10 cell line (Fig. 3(B)), which is another Jurkat- acting HIV LRAs. Previous studies have reported that AV6 acts
based post-integrative latency model that harbors almost the in part at a posttranscriptional level and synergizes with the
complete HIV genome except for encoding a partially attenu- HDAC inhibitor valproic acid to reverse HIV latency.28 So we rst
ated Tat variant H13L and the nef gene that is replaced by the veried that the synergism can also be found between AV6 and
GFP-coding sequence.45 In order to know whether compounds SAHA in J-Lat A2 cells. As shown in Fig. 4(A), the combination of
12c and 12d activated latent HIV-1 depend on viral 50-LTR, we 5 mM AV6 and 1 mM SAHA produced 49.0 ꢀ 3.2 of % GFP
additionally tested the effect of AV6, SAHA, 12c, and 12d at 2.5 expression, which is greater than the simple sum (23.9 ꢀ 1.4) of
mM in NH1 and NH2 cells, which is a pair of HeLa-based the effects produced by 5 mM AV6 (3.8 ꢀ 1.1) and 1 mM SAHA
isogenic cells line and both of which contain an integrated (20.1 ꢀ 0.3). The efficacy is believed to be a result of the synergy
HIV-1 50-LTR-luciferase reporter gene but only the latter stably between AV6 and SAHA. However, there is no synergy between
expresses HIV-1 Tat.46,47 As shown in Fig. 3(C) and (D), AV6 did 12c and AV6/SAHA (Fig. 4(A)). For example, the combination of
not shown any activation effect, while compounds 12c and 12d 1 mM SAHA and 1 mM 12c, which produced 58.4 ꢀ 3.8 of % GFP
induced a signicant increase in luciferase activity in both NH1 expression, almost have the same effect on the activation of
and NH2, which is much better than SAHA at the same latency HIV-1 with the simple sum (59.8 ꢀ 4.4) of 1 mM SAHA
concentration (2.5 mM). Taken together, these results suggest (20.1 ꢀ 0.3) and 1 mM 12c (39.7 ꢀ 4.1). Similarly, the combi-
that compounds 12c and 12d promote HIV-1 LTR-driven GFP nation of 5 mM AV6 and 1 mM 12c produced 44.5 ꢀ 2.9 of % GFP
expression in different latency cell models and give us a hint expression which is almost the same as the simple sum (43.5 ꢀ
that previous design of AV6 analogues is reasonable.
2.3.3 Dual mechanism of 12c for the reactivation of latent these ndings, we can assume that 12c may have similar
HIV-1. Compounds 12c and 12d were derived from AV6 and
5.2) of 5 mM AV6 (3.8 ꢀ 1.1) and 1 mM 12c (39.7 ꢀ 4.1). From
17284 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
Fig. 4 12c retains the reactivation mechanism of AV6. (A) J-Lat A2 cells were treated with AV6, SAHA, 12c alone, or in combination for 24 h, and
then analyzed by flow cytometry to determine the percentages of GFP(+) cells. (B) J-Lat A2 cells were treated with SAHA, AV6 or 12c for 24 h, in
the presence or absence of the calcineurin inhibitor FK506 (20 mM). The percentages of GFP(+) cells were evaluated by FACS and plotted. Results
shown are the means ꢀ SD from three independent experiments. The P value was defined as **p < 0.01, and ***p < 0.001 vs. control.
mechanisms with AV6 and SAHA to induce HIV-1 latency HIV latency induced by 12c was decreased aer pre-treated with
reversal.
FK506, so FK506 also did block 12c-dependent viral gene
2.3.3.1 Activate latent HIV-1 via the NFAT-required mecha- expression to some extent. These results suggest that 12c retains
nism. It was reported that AV6 requires nuclear factor of acti- the reactivation mechanism of AV6, which requires NFAT for
vated T-cells (NFAT) for inducing latent HIV-1 expression, but early HIV-1 gene expression. While compound 12c is not as
not SAHA. The function of NFAT is regulated by calcineurin sensitive to FK506 as AV6 is, it indicates that 12c may activate
(CaN), a calcium and calmodulin dependent serine/threonine latent HIV-1 from latent reservoirs as a HDACs inhibitor.
protein phosphatase, which activates the T cells of the
2.3.3.2 Activate latent HIV-1 as histone deacetylase (HDAC)
immune system and can be blocked by its inhibitors.48 To inhibitors. It is well known that inhibition of HDACs is sufficient
determine whether NFAT is involved in the reversal of HIV to reactivate a fraction of latent HIV in a variety of experimental
latency by 12c, J-Lat A2 cells were pre-treated with the calci- systems. Histone acetylation consists of the addition of an
neurin inhibitor FK506 before the incubation with 12c. As acetyl group to the 3-amino group of lysine residues. Histone
illustrated in Fig. 4(B), the calcineurin inhibitor FK506 could lysine acetylation, on lysine 9 or 14 of histone H3 (H3K9,
block AV6-dependent viral expression but did not affect the H3K14) or on lysine 16 of histone H4 (H4K16), is generally
reactivation of HIV latency induced by SAHA. The reactivation of associated with active gene transcription.15,21 Thus, we
Fig. 5 Western blotting analysis of the acetylation levels of histones H3 and H4 in A549 cells (A), HeLa cells (B) and J-Lat A2 cells (C) after 12c and
12d treatment respectively for 24 h.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17285

View Article Online
RSC Advances
Paper
scaffold generated two excellent LRAs 12c and 12d, which
possessed strong activity on HDACs inhibition to induce HIV-1
latency reversal.
2.3.4 Compounds 12c and 12d release P-TEFb from the
inactive complex 7SK snRNP. Positive transcription elongation
factor b (P-TEFb), which is composed of CDK9 and CycT1, is
a critical component of the cellular transcriptional machinery
that is required for the elongation of HIV transcription.49,50 In
latently HIV infected cells, most of the cellular P-TEFb are
sequestered in inactive complex 7SK snRNP. Besides P-TEFb,
this complex also contains the 7SK snRNA that acts as
a molecular scaffold to hold the complex together and the
protein HEXIM1 and LARP7, which inhibits the CDK9 kinase
activity and binds to the 30 end of 7SK snRNA, respec-
tively.16,46,51–55 Releasing active P-TEFb from 7SK snRNP should
be an excellent strategy to activate viral replication in these
cells.49 To determine whether 12c and 12d are capable of
inducing P-TEFb's dissociation from 7SK snRNP, an anti-Flag
immunoprecipitation was performed in the HeLa-based F1C2
cells which stably express the Flag-tagged CDK9 (CDK9-
Flag),51,56 to examine the association of CDK9 with two signature
7SK snRNP components HEXIM1 and LARP7.57 Using DMSO as
a negative control, it was found that 12c and 12d markedly
reduced the amounts of HEXIM1 and LARP7 bound to the
immunoprecipitated CDK9-Flag (Fig. 6). It indicated that 12c
Fig. 6 The HeLa-based F1C2 cells stably expressing the Flag-tagged
CDK9 were treated with 12c, 12d, or DMSO for 24 h. Nuclear extracts
(NE) and anti-Flag immunoprecipitates (IP) derived from NE were
analyzed by western blotting for the indicated proteins.
examined whether compounds 12c and 12d with the functional
group ZBG of HDACs inhibitors also inhibited intracellular even
nuclear HDACs by monitoring the acetylation levels of histones
H3 and H4 in HeLa and A549, and J-Lat A2 cell lines. In this
assay, SAHA (a clinically approved pan-HDAC inhibitor) was
used as positive control, DMSO and AV6 were used as negative
controls. It was found that aer the treatment of compounds
12c and 12d, the acetylation of histone H3 and H4 was signi-
cantly up regulated in a dose-dependent manner, whereas AV6
which lacked HDAC inhibition activity was not effect on the
acetylation levels of histones H3 and H4 (Fig. 5). It indicated
that the right introduction of a ZBG (–CONHOH) in the AV6
Fig. 7 SAHA (A) and 12c (B) docked into the active site of HDAC2 (PDB entry 4LXZ). SAHA and 12c are shown in stick with yellow and brown
carbon, respectively. The catalytic zinc in magenta CPK representation. The HDAC2 binding site is presented with a coloured ribbon. Neigh-
bouring amino acids were displayed in lines within a distance of 5 A˚ approximately to ligand. 2D binding models of SAHA (C) and 12c (D) with
HDAC2. Hydrogen bonds are indicated with solid arrows, colour lines around ligands (SAHA and 12c) stand for the binding pocket and the
residues in colours nearby established the pocket. The green colour denotes the hydrophobic nature of amino acids, the red colour denotes the
acid amino acids, the purple denotes the alkalinity of amino acids, the cyan denotes the polar amino acids, and the grey points of ligand atoms
denotes the solvent accessibility.
17286 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
and 12d were able to reactivate HIV-1 transcription by releasing and histone H4 increased in a dose-dependent manner. This
P-TEFb from the inactive complex 7SK snRNP.
study implies that 12c and 12d also act as HDAC inhibitors to
reactivate HIV-1 transcription. Positive transcription elongation
factorb (P-TEFb) is a critical component of the cellular tran-
scriptional machinery that is required for the elongation of HIV
transcription. In this work, the co-immunoprecipitation assay
conrmed that 12c and 12d were able to release P-TEFb from
the inactive complex 7SK snRNP to activate HIV transcription.
Molecular docking yielded binding modes for compounds 12c
and 12d with the similar metal coordination with ZN2+ as well as
SAHA, and its larger cap group quinoline core increased the
interactions with HDAC2 and turned in the opposite direction
from the cap group of SAHA. All these results proved the
potential of 12c and 12d as novel dual reactivator of latent HIV-
1. However, further studies are required to determine their roles
in reactivating latent HIV-1.
2.4. Molecular docking study of compound 12c and 12d to
HDAC2
SAHA achieves broad HDAC inhibitor activity through recogni-
tion of conserved structural features within this family of
enzymes. We performed molecular docking study in the HDAC2
to determine the binding mode for AV6 analogues and study the
key interactions between AV6 analogues and HDAC2. The X-ray
protein structure of the human HDAC2 co-crystallized with
SAHA in the ligand binding pocket (PDB code: 4LXZ) was
selected to run the docking calculations. Re-docking was rst
carried out with the co-crystal ligand (SAHA) into the binding
site of HDAC2 and compared with the co-crystal pose (4LXZ) to
assess the docking protocol in this study. The RMSD value of re-
˚
docked SAHA refereeing with the co-crystal pose is 0.773 A (see
Fig. S1 of the ESI†), which indicated our docking results were
4. Experimental section
4.1. Chemistry
reliable as the success rate of docking is usually judged using
˚
the RMSD value lesser than 2.0 A. Then, we performed the
All reagents were commercial available and used without
further purication, unless otherwise indicated. Reactions,
which required the use of anhydrous, inert atmosphere tech-
niques, were carried out under an atmosphere of nitrogen.
Reactions were magnetically stirred and monitored by thin-
layer chromatography (TLC) on Merck silica gel 60F-254 by
uorescence. 1H-NMR and 13C-NMR spectra were obtained
using a Bruker (Bruker Biospin, Zug, Switzerland) AV2600
Ultrashield spectrometer at 600 and 150 MHz, respectively.
Chemical shis were reported in parts per million (ppm) rela-
tive to tetramethylsilane (TMS) as an internal standard. Multi-
plicities were abbreviated as follows: single (s), doublet (d),
doublet of doublets (dd), doublet–triplet (dt), triplet (t), triplet–
triplet (tt), triplet–doublet (td), quartet (q), quartet–doublet (qd),
multiplet (m), and broad signal (brs). High-resolution mass
spectral (HRMS) data were acquired on a Q Exactive. Melting
points were measured on a SGW X-4 micro-melting point
spectrometer and were uncorrected. The purities of synthesized
compounds were assessed by high performance liquid chro-
matography (HPLC) with a COSMOSIL C18-MS-II column
(250 mm ꢃ 4.6 mm i.d., 5 mm). The HPLC analysis was per-
formed on an Agilent Technologies 1100 Series HPLC system.
docking analysis of 12c binding to HDAC2. Molecular docking
study indicated that the docking score of 12c
(ꢂ5.949 kcal molꢂ1) beyond 2-fold to SAHA (ꢂ2.759 kcal molꢂ1).
Comparison of the binding poses of SAHA and 12c in the active
site of HDAC2 (Fig. 7) shows that the ZBGs of 12c and SAHA
both form interactions with the zinc ion and TYR308, but only
the OH group of 12c's ZBG establishes an additional hydrogen
bond which interacted with the side chain of residue HID145
˚
(distance: 1.96 A). In addition, the interaction between the
surface of binding site and their cap groups was apparently
different. The cap group of SAHA phenyl group has p–p stack-
ing interaction with residue HID33, while for 12c binding mode
the quinolone core formed a key p–p stacking interaction with
residue TYR209. These ndings might lead to the better dock-
ing score for 12c than SAHA. In this work, molecular model of
HDAC2 in complex with 12d was also constructed. It was found
that compound 12d showed a similar binding mode with 12c
(see Fig. S2 of the ESI†).
3. Conclusion
In summary, series of 2-methylquinolin-4-amine compound The mobile phase was methanol (A) and water (B) in a linear
were synthesized and their ability to activate latent HIV-1 were gradient mode as follows: (A) from 5% to 100% and (B) from
evaluated in J-Lat A2 cells. Among all synthesized compounds, 95% to 0% during 0–30 min. The ow rate was 1 mL minꢂ1, and
compound 12c and 12d exhibited excellent potency to reverse the detection wavelengths were 254 nm and 280 nm.
HIV latency. Compared with the obviously a lot of synergy
4.1.1 Ethyl 3-((4-methoxyphenyl)imino)butanoate (2). A
between SAHA and AV6, no cooperative effect was observed mixture of p-anisidine (1) (406.3 mmol, 50.0 g), ethyl acetoace-
using the combination of 12c and AV6 for the reactivation of tate (487.2 mmol, 62.0 mL), anhydrous magnesium sulfate
latent HIV-1 gene expression, and to some extent the 12c- (291.0 mmol, 35.1 g) and acetic acid (2.0 mL) in ethanol (100.0
dependent viral gene expression was blocked by the calcineurin mL) was reuxed at 90 ꢁC for 4 h. Aer completion of the
inhibitor FK506 which did block AV6-dependent mRNA reaction, the mixture was ltered and concentrated under
expression.28 These data indicate that 12c retains the reac- reduced pressure to give a brown liquid. The residue was puri-
tivation mechanism of AV6, which requires NFAT for early HIV- ed by ash chromatography on a silica gel (300–400 mesh)
1 gene expression. On the other hand, the western blotting using ethyl acetate/petroleum ether as eluant to afford 2 (86.1 g,
clearly showed that both in the HepG2 cells and A549 cells 90.1%) as a brown liquid. 1H NMR (600 MHz, DMSO-d6): d 10.14
treated with 12c and 12d, the lysine acetylation of histone H3 (s, 1H, NH), 7.11–7.14 (m, 2H, Ph-H), 6.91–6.93 (m, 2H, Ph-H),
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17287

View Article Online
RSC Advances
Paper
4.62 (s, 1H, C]CH), 4.04 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 3.75 (s, petroleum ether/ethyl acetate (4 : 1) to afford 6a as a white solid
3H, OCH3), 1.89 (s, 3H, CH3), 1.19 (t, J ¼ 7.1 Hz, 3H, OCH2CH3). (1.2 g, 93.6%). 1H NMR (600 MHz, CDCl3): d 8.02 (d, J ¼ 9.5 Hz,
13C NMR (150 MHz, DMSO-d6): d 169.9 (C]O), 160.3 (NHCH3– 1H, H-8), 7.39–7.42 (m, 2H, H-5 and H-7), 7.38 (s, 1H, H-3), 4.19
C]CH), 157.4 (C–OCH3), 132.0 (Ph-C), 126.7 (s, 2C, Ph-C), 114.7 (q, J ¼ 7.0 Hz, 2H, OCH2CH3), 2.72 (s, 3H, CH3), 1.51 (t, J ¼
(s, 2C, Ph-C), 84.8 (C]CH), 58.5 (OCH2CH3), 55.7 (OCH3), 20.2 7.0 Hz, 3H, OCH2CH3). 13C NMR (150 MHz, CDCl3): d 157.6 (C-
(OCH2CH3), 15.0 (CH3). ESI-HRMS (+): m/z [M + H]+ calculated 6), 155.7 (C-2), 144.0 (C-9), 141.4 (C-4), 130.1 (C-8), 125.7 (C-7),
+
for C13H18NO3 , 236.1128, found, 236.1281.
123.5 (C-10), 122.1 (C-3), 102.4 (C-5), 64.0 (OCH2CH3), 24.5
4.1.2 6-Methoxy-2-methylquinolin-4(1H)-one (3). A solu- (CH3), 14.7 (OCH2CH3). ESI-HRMS (+): m/z [M + H]+ calculated
tion of ethyl 3-[(4-methoxyphenyl) imino] butanoate (2) for C12H13ClNO+, 222.0680, found, 222.0681.
ꢁ
(85.1 mmol, 20.0 g) in diphenyl ether was heated to 250 C for
4.1.6 4-Chloro-6-isopropoxy-2-methylquinoline (6b).
A
30 min. At the end of the period, the reaction mixture was mixture of 4-chloro-2-methylquinolin-6-ol 5 (20.7 mmol, 4.0 g),
cooled to 25 ꢁC and diluted with ethyl acetate. Then crystalline 2-bromopropane (41.4 mmol, 5.1 g) and potassium carbonate
solid separated out from the mixture, washed with ethyl acetate (62.1 mmol, 8.6 g) in DMF (120.0 mL) was stirred at 40 ꢁC for 4 h.
(30.0 mL) and dried in vacuum to give 3 (8.3 g, 51.5%) as a white The reaction mixture was ltered and the ltrate was concen-
solid. 1H NMR (600 MHz, DMSO-d6): d 7.80 (d, J ¼ 9.0 Hz, 1H, H- trated in vacuo. The residue was puried by column chroma-
8), 7.50 (d, J ¼ 2.8 Hz, 1H, H-5), 7.45 (dd, J ¼ 2.8, 9.1 Hz, 1H, H- tography using petroleum ether/ethyl acetate (4 : 1) to afford 6b
7), 6.49 (s, 1H, H-3), 3.88 (s, 3H, OCH3), 2.54 (s, 3H, CH3). 13C as a white solid (2.9 g, 59.6%). 1H NMR (600 MHz, CDCl3): d 7.91
NMR (150 MHz, DMSO-d6): d 174.6 (C]O), 156.2 (C-6), 150.5 (C- (d, J ¼ 9.2 Hz, 1H, H-8), 7.43 (d, J ¼ 2.8 Hz, 1H, H-5), 7.32–7.36
2), 135.2 (C-9), 124.7 (C-10), 123.1 (C-7), 120.4 (C-8), 107.4 (C-3), (m, 2H, H-3 and H-7), 4.71–4.78 (m, 1H, OCH(CH3)2), 2.67 (s,
104.1 (C-5), 55.9 (OCH3), 19.9 (CH3). ESI-HRMS (+): m/z [M + H]+ 3H, CH3), 1.43 (s, 3H, OCH(CH3)2), 1.42 (s, 3H, OCH(CH3)2). 13
C
+
calculated for C11H12NO2 , 190.0863, found, 190.0862.
NMR (150 MHz, CDCl3): d 156.4 (C-6), 155.7 (C-2), 143.9 (C-9),
4.1.3 4-Chloro-6-methoxy-2-methylquinoline (4). The mixture 141.3 (C-4), 130.2 (C-8), 125.7 (C-7), 124.1 (C-10), 122.1 (C-3),
of 6-methoxy-2-methylquinolin-4-ol (3) (52.9 mmol, 10.0 g) and 104.0 (C-5), 70.4 (OCH(CH3)2), 24.5 (CH3), 21.9 (s, 2C,
POCl3 (60.0 mL) was heated to 110 ꢁC for 2 h. Aer completion OCH(CH3)2). ESI-HRMS (+): m/z [M + H]+ calculated for
of the reaction, the excess of POCl3 was removed under reduced
pressure. The residue was quenched into ice-water and stirred
C
13H15ClNO+, 236.0837, found, 236.0837.
4.1.7 General procedure for the synthesis of 4-substituted-
at 0 ꢁC for 15 min, then the pH was adjusted to 7 with saturated aminoquinoline compounds 7a–7w. A mixture of 4-chlor-
sodium hydroxide solution. The solid obtained was collected by oquinoline derivatives (6a-6b, 0.5 mmol), appropriate
ltration and dried in vacuum to give 4 as a white solid (8.1 g, substituted anilines (0.6 mmol, 1.2 eq.) and 3 drops conc. HC in
73.9%). 1H NMR (600 MHz, CDCl3): d 7.83 (d, J ¼ 9.2 Hz, 1H, H- n-butanol (15.0 mL) was stirred at reux for 8 h. The reaction
8), 7.31 (d, J ¼ 2.6 Hz, 1H, H-5), 7.28–7.30 (m, 1H, H-7), 7.27 (s, mixture was cooled to room temperature and the yellow solid
1H, H-3), 3.87 (s, 3H, OCH3), 2.59 (s, 3H, CH3). 13C NMR (150 formed was ltered, washed with diethyl ether and recrystal-
MHz, CDCl3): d 157.1 (C-6), 155.0 (C-2), 143.7 (C-9), 140.0 (C-4), lized from ethanol to afford the desired product.
129.5 (C-8), 124.6 (C-7), 122.0 (C-10), 121.1 (C-3), 100.7 (C-5),
4.1.7.1 N-(3,4-Dichlorophenyl)-6-methoxy-2-methylquinolin-4-
54.6 (OCH3), 23.7 (CH3). ESI-HRMS (+): m/z [M + H]+ calcu- amine (7a). White solid; yield: 35.8%; HPLC purity: 98.9% (tR ¼
lated for C11H11ClNO+, 208.0524, found, 208.0524.
21.92 min); mp: 266–267 ꢁC. 1H NMR (600 MHz, DMSO-d6):
4.1.4 4-Chloro-2-methylquinolin-6-ol (5). To a cooled (0 ꢁC) d 8.08 (s, 1H, H-5), 7.96 (d, J ¼ 9.2 Hz, 1H, H-8), 7.77 (d, J ¼
solution of 4-chloro-6-methoxy-2-methylquinoline (4) (24.3 mmol, 4.2 Hz, 1H, Ph-H), 7.76 (d, J ¼ 1.7 Hz, 1H, Ph-H), 7.58 (dd, J ¼
5.0 g) in CH2Cl2 (50.0 mL) was added dropwise BBr3 (26.6 mL, 2.6, 9.2 Hz, 1H, H-7), 7.50 (dd, J ¼ 2.4, 8.6 Hz, 1H, Ph-H), 6.89 (s,
26.6 mmol, 1 M in CH2Cl2) under argon atmosphere. The reaction 1H, H-3), 3.97 (s, 3H, OCH3), 2.59 (s, 3H, CH3); 13C NMR (150
mixture was then allowed to warm to room temperature and stir- MHz, DMSO-d6): d 157.9 (C-2), 153.9 (C-6), 151.9 (C-4), 151.8 (C-
red for 4 h. The solvent was then condensed and the residue was 9), 139.2 (C-10), 132.4 (Ph-C), 132.0 (C-8), 128.3 (Ph-C), 126.6 (Ph-
slowly quenched with saturated aqueous NaHCO3 solution. The C), 125.0 (C-7), 124.7 (Ph-C), 123.7 (Ph-C), 118.5 (C-10), 103.0 (C-
precipitate was ltered and washed with water to give 5 as an off- 3), 101.9 (C-5), 56.8 (OCH3), 21.1 (CH3). ESI-HRMS (+): m/z [M +
white solid (3.4 g, 72.5%). 1H NMR (600 MHz, DMSO-d6): d 10.28 (s, H]+ calculated for C17H15Cl2N2O+, 333.0556, found, 333.0552.
1H, OH), 7.85 (d, J ¼ 9.7 Hz, 1H, H-8), 7.56 (s, 1H, H-5), 7.34–7.37
Other target compounds (7b-7B) were synthesized following
(m, 2H, H-3 and H-7), 2.59 (s, 3H, CH3). 13C NMR (150 MHz, the general procedure as described above with the yield of 25.0–
DMSO-d6): d 156.6 (C-6), 155.7 (C-2), 143.6 (C-9), 139.4 (C-4), 130.9 86.0%. Their structures were assigned by the spectroscopic data
(C-8), 125.6 (C-7), 123.3 (C-10), 122.4 (C-3), 104.7 (C-5), 24.6 (CH3). including H NMR, 13C NMR, and ESI-HRMS (see ‘ESI†’).
1
ESI-HRMS (+): m/z [M + H]+ calculated for C10H19ClNO+, 194.0367,
4.1.8 4-((3,4-Dichlorophenyl)amino)-2-methylquinolin-6-ol
(8). To a cooled (0 ꢁC) solution of 7a (10.0 mmol, 3.3 g) in CH2Cl2
found, 194.0368.
4.1.5 4-Chloro-6-ethoxy-2-methylquinoline (6a). A mixture (50.0 mL) was added dropwise BBr3 (11.1 mL, 11.1 mmol, 1 M in
of 4-chloro-2-methylquinolin-6-ol 5 (5.7 mmol, 1.1 g), bromo- CH2Cl2) under argon atmosphere. The reaction mixture was
ethane (10.3 mmol, 1.6 g) and potassium carbonate (17.1 mmol, then allowed to warm to room temperature and stirred for 6 h.
2.4 g) in DMF (25.0 mL) was stirred at 40 ꢁC for 4 h. The reaction The solvent was then condensed and the residue was slowly
mixture was ltered and the ltrate was concentrated in vacuo. quenched with saturated aqueous NaHCO3 solution. The
The residue was puried by column chromatography with precipitate was ltered and washed with water to give 8 as an
17288 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
off-white solid (2.2 g, 68.0%). 1H NMR (600 MHz, DMSO-d6): 10), 118.5 (Ph-C), 103.5 (C-3), 99.4 (C-5), 67.0 (C-12), 50.6
d 8.76 (br. s., 1H, OH), 7.69 (d, J ¼ 9.0 Hz, 1H, H-8), 7.55 (d, J ¼ (COO–CH3), 32.9, 27.7, 24.5, 23.5 (C-11). ESI-HRMS (+): m/z [M +
+
8.6 Hz, 1H, Ph-H), 7.47 (s, 1H, Ph-H), 7.40 (s, 1H, H-5), 7.28 (d, J H]+ calculated for C23H25Cl2N2O3 , 447.1237, found, 447.1236.
¼ 8.4 Hz, 1H, Ph-H), 7.25 (d, J ¼ 9.0 Hz, 1H, H-7), 6.96 (s, 1H, H-
4.1.10.2 Ethyl 7-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin-
3), 2.45 (s, 3H, CH3). 13C NMR (150 MHz, DMSO-d6): d 156.4 (C- 6-yl)oxy)heptanoate (10d). White solid; yield: 59.1%; HPLC purity:
1
2), 153.3 (C-6), 151.9 (C-4), 145.6 (C-9), 139.2 (C-10), 132.4 (Ph-C), 96.4% (tR ¼ 25.76 min); mp: 129–132 C. H NMR (600 MHz,
132.0 (C-8), 128.3 (Ph-C), 126.5 (Ph-C), 124.9 (C-7), 124.7 (Ph-C), CDCl3): d 7.90 (d, J ¼ 9.2 Hz, 1H, H-8), 7.43 (d, J ¼ 8.6 Hz, 1H, Ph-
123.3 (Ph-C), 118.9 (C-10), 105.6 (C-3), 101.7 (C-5), 21.0 (CH3). H), 7.37 (d, J ¼ 2.4 Hz, 1H, H-5), 7.31 (dd, J ¼ 2.6, 9.2 Hz, 1H, H-
ESI-HRMS (+): m/z [M + H]+ calculated for C16H13Cl2N2O+, 7), 7.17–7.20 (m, 1H, Ph-H), 7.14 (dd, J ¼ 2.5, 8.5 Hz, 1H, Ph-H),
ꢁ
319.0399, found, 319.0398.
6.91 (s, 1H, H-3), 4.12 (q, J ¼ 7.0 Hz, 2H, COO–CH2CH3), 3.98 (t, J
4.1.9 General procedure for the synthesis of 9a–9e (general ¼ 6.4 Hz, 2H, 12-CH2), 2.58 (s, 3H, 11-CH3), 2.28–2.35 (m, 2H),
procedure for the alkylation of phenols). A mixture of 4-(3,4- 1.79–1.82 (m, 2H), 1.66 (quin, J ¼ 7.5 Hz, 2H), 1.43–1.51 (m, 2H),
dichlorobenzyl)-2-methylquinolin-6-ol (8) (1.0 mmol), appro- 1.35–1.42 (m, 2H), 1.25 (t, J ¼ 7.2 Hz, 1H, COO–CH2CH3); 13C
priate alkyl halides (1.1 mmol), and potassium carbonate NMR (150 MHz, CDCl3): d 174.0 (C]O), 156.7 (C-2), 156.5 (C-6),
(331 mg, 2.4 mmol) in DMF (6.0 mL) was stirred at 40 ꢁC for 4 h. 146.0 (C-4), 144.4 (C-9), 140.7 (C-10), 133.3 (Ph-C), 131.1 (C-8),
The reaction mixture was ltered and the ltrate was concen- 130.3 (Ph-C), 126.6 (C-7), 123.0 (Ph-C), 121.8 (Ph-C), 120.8 (Ph-
trated in vacuo. The residue was puried by column chroma- C), 119.5 (C-10), 104.5 (C-3), 100.2 (C-5), 68.2 (C-12), 60.4
tography using appropriate mixtures of ethyl acetate and (COO–CH2CH3), 34.2, 28.8, 28.7, 25.6, 25.1, 24.8 (C-11), 14.2
petroleum to yield the titled compound.
(COO–CH2CH3). ESI-HRMS (+): m/z [M + H]+ calculated for
C
4.1.9.1 N-(3,4-Dichlorophenyl)-2-methyl-6-propoxyquinolin-4-
amine (9a). White solid; yield: 30.0%; HPLC purity: 96.1% (tR ¼
25H29Cl2N2O3+, 475.1550, found, 475.1549.
Other target compounds (10a-10b) were synthesized
23.96 min); mp: 266–267 ꢁC. 1H NMR (600 MHz, DMSO-d6): following the general procedure as described above with the
d 7.77 (d, J ¼ 9.2 Hz, 1H, H-8), 7.69 (d, J ¼ 2.0 Hz, 1H, H-5), 7.63 yield of 34.0–64.0%. Their structures were assigned by the
(d, J ¼ 8.6 Hz, 1H, Ph-H), 7.58 (d, J ¼ 2.4 Hz, 1H, Ph-H), 7.36– spectroscopic data including 1H NMR, 13C NMR, and ESI-HRMS
7.40 (m, 2H), 6.94 (s, 1H, H-3), 4.08 (t, J ¼ 6.5 Hz, 2H, OCH2), (see ‘ESI†’).
2.49 (s, 3H, CH3), 1.81 (sxt, J ¼ 7.0 Hz, 2H, OCH2CH2CH3), 1.03
4.1.11 General procedure for the synthesis of 11a–11d. To
(t, J ¼ 7.3 Hz, 3H, OCH2CH2CH3); 13C NMR (150 MHz, DMSO- a solution of 10a–10d (0.5 mmol) in EtOH (5.0 mL) was added
d6): d 156.2 (C-2), 155.9 (C-6), 147.1 (C-4), 143.0 (C-9), 141.7 (C- a 20% aqueous NaOH solution and the reaction mixture was
10), 132.1 (Ph-C), 131.6 (C-8), 129.1 (Ph-C), 125.0 (C-7), 123.5 (Ph- stirred at room temperature for 1 h. The mixture was neutral-
C), 122.3 (Ph-C), 121.9 (Ph-C), 119.6 (C-10), 103.7 (C-3), 102.5 (C- ized by the addition of a 10.0% aqueous HCl solution. The
5), 70.0 (OCH2), 24.4 (OCH2CH2CH3), 22.5 (C-11), 11.0 (OCH2- resulting precipitate was ltered, washed with diethyl ether and
CH2CH3). ESI-HRMS (+): m/z [M
19H19Cl2N2O+, 361.0869, found, 361.0865.
Other target compounds (9b–9e) were synthesized following
+
H]+ calculated for recrystallized from ethanol to afford the corresponding
C
carboxylic acid compound.
4.1.11.1 4-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin-
the general procedure as described above with the yield of 30.0– 6-yl)oxy) butanoic acid (11a). White solid; yield: 70.0%; HPLC
1
ꢁ
55.0%. Their structures were assigned by the spectroscopic data purity: 97.0% (tR ¼ 21.99 min); mp: 266–267 C. H NMR (600
1
including H NMR, 13C NMR, and ESI-HRMS (see ‘ESI†’).
MHz, DMSO-d6): d 7.79 (d, J ¼ 9.2 Hz, 1H, H-8), 7.72 (d, J ¼
4.1.10 General procedure for the synthesis of 10a–10d 2.4 Hz, 1H, H-5), 7.65 (d, J ¼ 8.8 Hz, 1H, Ph-H), 7.59 (d, J ¼
(hydrolysis). A mixture of 4-(3,4-dichlorobenzyl)-2-methylquinolin- 2.6 Hz, 1H, Ph -H), 7.39 (td, J ¼ 2.2, 8.8 Hz, 2H, H-7 and H-60),
6-ol (8) (1.0 mmol), various lengths of ethyl or methyl bro- 6.93 (s, 1H, H-3), 4.14 (t, J ¼ 6.4 Hz, 2H, 12-CH2), 2.49 (s, 3H,
moalkanoate (1.1 mmol), and potassium carbonate (331 mg, 2.4 CH3), 2.45 (t, J ¼ 7.2 Hz, 2H, 14-CH2), 2.03 (quin, J ¼ 6.8 Hz, 2H,
mmol) in acetone (10.0 mL) was stirred at 40 ꢁC for 4 h. The 13-CH2); 13C NMR (150 MHz, DMSO-d6): d 174.6 (C]O), 156.2
reaction mixture was ltered and the ltrate was concentrated in (C-2), 155.8 (C-6), 147.7 (C-4), 142.1 (C-9), 141.3 (C-10), 132.1 (Ph-
vacuo. The residue was puried by column chromatography using C), 131.7 (C-8), 128.4 (Ph-C), 125.5 (C-7), 124.0 (Ph-C), 122.7 (Ph-
appropriate mixtures of ethyl acetate and petroleum to yield the C), 122.4 (Ph-C), 119.3 (C-10), 103.3 (C-3), 102.5 (C-5), 67.7
titled compound.
(OCH2), 30.6 (CH2–COOH), 24.7 (OCH2CH2), 24.0 (C-11). ESI-
ꢂ
4.1.10.1 Methyl 6-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin- HRMS (ꢂ): m/z [M ꢂ H]ꢂ calculated for C20H17Cl2N2O3
6-yl)oxy)hexanoate (10c). White solid; yield: 64.2%; HPLC purity: 403.0622, found, 403.0618.
,
1
ꢁ
93.3% (tR ¼ 24.35 min); mp: 129–131 C. H NMR (600 MHz,
Other target compounds (11b–11d) were synthesized following
CDCl3): d 7.90 (d, J ¼ 9.2 Hz, 1H, H-8), 7.44 (d, J ¼ 8.6 Hz, 1H, Ar- the general procedure as described above with the yield of 70.0–
H), 7.38 (d, J ¼ 2.6 Hz, 1H, H-5), 7.30–7.33 (m, 1H, H-7), 7.13– 91.0%. Their structures were assigned by the spectroscopic data
7.17 (m, 2H, Ar-H), 6.92 (s, 1H, H-3), 3.96–4.04 (m, 2H, 12-CH2), including 1H NMR, 13C NMR, and ESI-HRMS (see ‘ESI†’).
3.68 (s, 3H, COO–CH3), 2.58 (s, 3H, 11-CH3), 2.34–2.41 (m, 2H),
4.1.12 General procedure for the synthesis of 12a–12d from
1.81–1.85 (m, 2H), 1.69–1.73 (m, 2H), 1.47–1.54 (m, 2H); 13C their corresponding esters. The appropriate esters 10a–10d (0.2
NMR (150 MHz, CDCl3): d 173.3 (C]O), 155.7 (C-2), 155.3 (C-6), mmol), NaOH (2.3 mmol) and hydroxylamine solution (6.9
145.0 (C-4), 143.4 (C-9), 139.7 (C-10), 132.2 (Ph-C), 130.0 (C-8), mmol) was added to DCM/CH3OH ¼ 1 : 2 (2.0 mL/4.0 mL) at
129.2 (Ph-C), 125.5 (C-7), 121.9 (Ph-C), 120.9 (Ph-C), 119.7 (C-
0
ꢁC. The reaction mixture was warmed to room temperature
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17289

View Article Online
RSC Advances
Paper
and stirred at 25 C for 24 h. Aer completion of the reaction have been described previously.58,59 All the cell lines used in this
(monitored by TLC), the mixture was neutralized with acetic study were either purchased from American Type Culture
acid. The formed precipitate was collected by ltration, washed Collection (ATCC, Manassas,VA) or described as previously.47
ꢁ
with water and recrystallized from ethanol to give the crude
4.2.2 Cell culture. J-Lat A2 and 2D10 cells were cultured in
product. The crude was puried by column chromatography 1640 medium (Life Technological) with 10% fetal bovine serum
using appropriate mixtures of dichloromethane and methanol and kept in a CO2 incubator which consisted of a humidied
to yield the titled compound.
4.1.12.1 6-((4-((3,4-dichlorophenyl)amino)-2-methylquinolin-6-
atmosphere of 5% CO2 at 37 ꢁC. All the cancer cells (HeLa, A549,
NH1, NH2 and HepG2) were grown in DMEM medium (Life
yl)oxy)-N-hydroxy-hexanamide (12c). White solid; yield: 50.1%; Technological) with 10% fetal bovine serum in a humidied
1
ꢁ
HPLC purity: 97.9% (tR ¼ 22.09 min); mp: 266–267 ꢁC. H NMR atmosphere containing 5% CO2 at 37 C.
(600 MHz, CD3OD): d 7.70 (d, J ¼ 9.2 Hz, 1H, H-8), 7.65 (d, J ¼
4.2.3 Flow cytometry screening. The Flow cytometry
2.4 Hz, 1H, H-5), 7.54 (d, J ¼ 8.6 Hz, 1H, Ph-H), 7.52 (d, J ¼ screening assay was performed in Jurkat-based cells line (J-Lat A2)
2.6 Hz, 1H, Ph-H), 7.41 (dd, J ¼ 2.5, 9.3 Hz, 1H, H-7), 7.30 (dd, J containing LTR-Tat-IRES-GFP-LTR44 and 2D10 cells harboring the
¼ 2.4, 8.6 Hz, 1H, Ph-H), 6.75 (s, 1H, H-3), 4.07 (t, J ¼ 6.3 Hz, 2H, nearly complete HIV-1 genome except for the nef gene that is
12-CH2), 2.48 (s, 3H, CH3), 2.05 (t, J ¼ 7.3 Hz, 2H, 16-CH2), 1.74– replaced by the GFP-coding sequence.45 Aer incubation with the
1.85 (m, 2H, 13-CH2), 1.62 (quin, J ¼ 7.6 Hz, 2H, 15-CH2), 1.43– indicated compounds or 0.1% dimeththyl sulfoxide (DMSO) as
1.51 (m, 2H, 14-CH2); 13C NMR (150 MHz, CD3OD): d 171.4 (C] negative control, cells were harvested and washed twice with 1ꢃ
O), 157.7 (C-2), 153.9 (C-6), 151.8 (C-4), 138.7 (C-9), 136.9 (C-10), phosphate buffered saline (PBS) and analyzed the expression of
133.0 (Ph-C), 131.3 (C-8), 129.1 (Ph-C), 125.8 (C-7), 124.4 (Ph-C), GFP by ow cytometer (Epics Altra, BECKMAN COULTER).
123.8 (Ph-C), 123.4 (Ph-C), 118.3 (C-10), 101.7 (C-3), 101.2 (C-5),
4.2.4 Luciferase assay. The luciferase assay was conducted
68.3 (C-12), 32.3 (C-16), 28.4 (C-13), 25.2 (C-15), 25.0 (C-14), 20.1 in NH1 and NH2, a pair of HeLa-based isogenic cell lines, both of
(C-11). ESI-HRMS (ꢂ): m/z [M ꢂ H]ꢂ calculated for C22H22Cl2- which contain an integrated HIV-1 LTR-luciferase reporter gene
N3O3ꢂ, 446.1044, found, 446.1040.
but only the latter stably expresses HIV-1 Tat.47,51 Cells in twelve-
4.1.12.2 7-((4-((3,4-Dichlorophenyl)amino)-2-methylquinolin-6-
well plate were treated with different concentration of 12c, 12d or
yl)oxy)-N-hydroxy-heptanamide (12d). White solid; yield: 64.9%; DMSO for 24 h, then the cell lysates were normalized and
HPLC purity: 98.4% (tR ¼ 22.76 min); mp: 266–267 ꢁC. 1H NMR measured luciferase activity using luciferase kit from Promega.
(600 MHz, DMSO-d6): d 7.72 (d, J ¼ 9.0 Hz, 1H, H-8), 7.68 (d, J ¼
4.2.5 Western blotting. Equal amounts of the lysates were
2.6 Hz, 1H, H-5), 7.59 (d, J ¼ 0.9 Hz, 1H, Ph-H), 7.58 (d, J ¼ electrophoresed on 8% SDS-PAGE gel and transferred to PVDF
4.8 Hz, 1H, Ph-H), 7.38 (dd, J ¼ 2.5, 8.7 Hz, 1H, Ph-H), 7.30 (dd, J membranes (0.22 mM). The membranes were blocked with 5%
¼ 2.6, 9.2 Hz, 1H, H-7), 6.96 (s, 1H, H-3), 4.11 (t, J ¼ 6.4 Hz, 2H, nonfat milk in TBST (50 mM Tris–HCl, pH7.4, 150.0 mM NaCl
H-12), 2.46 (s, 3H, CH3), 2.17 (t, J ¼ 7.2 Hz, 2H, H-17), 1.74–1.82 and 0.1% Tween 20) buffer for 1 h at room temperature and
(m, 2H, H-13), 1.52 (quin, J ¼ 7.4 Hz, 2H, H-16), 1.45 (quin, J ¼ incubated with various primary antibodies overnight at 4 ꢁC.
7.5 Hz, 2H, H-14), 1.31–1.39 (m, 2H, H-13); 13C NMR (150 MHz, Aer three TBST washes, the membranes were incubated with
DMSO-d6): d 169.6 (C]O), 157.1 (C-2), 154.1 (C-6), 153.0 (C-4), HRP-conjugated anti-mouse or anti-rabbit antibody for 2 h at
139.7 (C-9), 138.2 (C-10), 134.3 (Ph-C), 131.8 (C-8), 130.3 (Ph-C), room temperature. The nal immunoreactive products were
125.6 (C-7), 125.1 (Ph-C), 124.4 (Ph-C), 122.7 (Ph-C), 118.6 (C- detected with an enhanced chemiluminescence (ECL) system.
10), 103.4 (C-3), 100.3 (C-5), 69.0 (C-12), 49.1 (C-17), 32.7 (C-
4.2.6 Co-immunoprecipitation.
The
co-
13), 28.8 (C-15), 25.8 (C-16), 25.5 (C-14), 20.6 (C-11). ESI-HRMS immunoprecipitation assay was performed as described with
(ꢂ): m/z [M ꢂ H]ꢂ calculated for C23H24Cl2N3O3ꢂ, 426.1200, minor modications.60 Briey, to detect the dissociation of 7SK
found, 426.1197.
snRNP aer compounds treatment, nuclear extracts (NEs) were
Other target compounds (12a-12b) were synthesized made from F1C2 cells, a HeLa-based cell line stably expressing
following the general procedure as described above with the CDK9-Flag.59 One day later, the cells were treated with 5 mM 12c,
yield of 35.0–65.0%. Their structures were assigned by the 12d or DMSO as a control for 24 h and then subjected to the
spectroscopic data including 1H NMR, 13C NMR, and ESI-HRMS preparation of NEs. For anti-Flag IP, NEs were incubated with anti-
(see ‘ESI†’).
Flag agarose beads (sigma) for 4 h at a ratio of 10 mL beads per 100
mL NEs. Aer extensive washing, the eluted proteins were analyzed
by western blotting with the indicated antibodies.
4.2. Biological assays
4.2.7 Statistical analysis. The data were expressed as mean
4.2.1 Reagents, antibody and cell lines. SAHA, 3-(4,5- ꢀ SD. Each assay was repeated in triplicate in three indepen-
dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) dent experiments. Statistical signicance of differences between
and DMSO were purchased from MedChemExpress. The anti- groups was analysed by using Student's t-test or analysis of
acetylated histone H3 and H4 antibodies were from Santa variance. P < 0.05 was considered signicant.
Cruz Biotechnology. The horseradish peroxidase (HRP) conju-
gated secondary antibodies were obtained from Thermo Fisher
Scientic and the antibodies against a-tubulin (T6074), poly-
4.3. Molecular modeling
merase (Parp) (9542) and Flag (F1804) were from sigma. The X-ray protein structure of human HDAC2 (PDB code: 4LXZ)
rabbit polyclonal anti-HEXIM1, -CDK9, and -LARP7 antibodies was download from Protein Data Bank (PDB) and prepared
17290 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018

View Article Online
Paper
RSC Advances
with Protein Preparation Wizard panel as implemented in
A. Telenti, C. Van Lint, E. Verdin, A. Woolfrey, J. Zaia and
F. Barre-Sinoussi, Nat. Rev. Immunol., 2012, 12, 607–614.
5 J. D. Siliciano and R. F. Siliciano, J. Clin. Invest., 2016, 126,
409–414.
¨
Maestro 10.5 (Schrodinger Release 2016–1: Maestro,
¨
Schrodinger, LLC, New York, NY, 2016.). Missing hydrogens
and residues were added, bond orders were assigned, and
˚
water molecules beyond 5 A to inhibitor were removed. Both
6 K. M. Barton, B. D. Burch, N. Soriano-Sarabia and
D. M. Margolis, Clin. Pharmacol. Ther., 2013, 93, 46–56.
7 C. Schwartz, S. Bouchat, C. Marban, V. Gautier, C. Van Lint,
O. Rohr and V. Le Douce, Biochem. Pharmacol., 2017, 146, 10–
22.
the native ligand SAHA and compound 12c were prepared
with LigPrep panel with OPLS2005 force eld, generating the
protonation and tautomeric states at 7.0 ꢀ 2.0 pH units. The
glide grid center was setting according the geometrical center
of original inhibitor (SAHA) and the grid size was 20 ꢃ 20 ꢃ
8 S. G. Deeks, Nature, 2012, 487, 439–440.
9 G. Darcis, B. Van Driessche and C. Van Lint, Trends
Immunol., 2017, 38, 217–228.
3
20 A , in which the metal coordination was set for ZN2+ in the
˚
binding site. Prior to docking compound 12c, a re-docking
run was performed to evaluate the docking performance of 10 P. K. Datta, R. Kaminski, W. Hu, V. Pirrone, N. T. Sullivan,
Glide extra precision (Glide XP) mode. Glide XP mode
M. R. Nonnemacher, W. Dampier, B. Wigdahl and
¨
¨
(Schrodinger Release 2016-1: Glide, Schrodinger, LLC, New
K. Khalili, Curr. HIV Res., 2016, 14, 431–441.
York, NY, 2017.) was successful in reproducing the binding 11 D. M. Margolis, J. V. Garcia, D. J. Hazuda and B. F. Haynes,
˚
mode of native ligand SAHA with a RMSD value of 0.773 A.
Science, 2016, 353, aaf6517.
Then based on this docking grid, all inhibitors including the 12 N. M. Archin and D. M. Margolis, Curr. Opin. Infect. Dis.,
co-crystal ligand SAHA were docked exibly by Glide 7.0 in
2014, 27, 29–35.
extra precision with default options.
13 J. T. Kimata, A. P. Rice and J. Wang, Curr. Opin. Immunol.,
2016, 42, 65–70.
14 S. Xing and R. F. Siliciano, Drug discovery today, 2013, 18,
541–551.
Conflicts of interest
There are no conicts to declare.
15 D. M. Margolis, Curr. Opin. HIV AIDS, 2011, 6, 25–29.
16 X. Contreras, M. Barboric, T. Lenasi and B. M. Peterlin, PLoS
Pathog., 2007, 3, 1459–1469.
17 J. Kulkosky, D. M. Culnan, J. Roman, G. Dornadula,
M. Schnell, M. R. Boyd and R. J. Pomerantz, Blood, 2001,
98, 3006–3015.
Acknowledgements
This work was supported by the National Natural Science Foun-
dation of China (No. 81773600, 81672955 and 81302652), the
Natural Science Foundation of Fujian Province of China (No. 18 M. Stoszko, E. De Crignis, C. Rokx, M. M. Khalid, C. Lungu,
2018J01132 and 2016J011172), and the Fundamental Research
Funds for the Central Universities (No. 20720180051 and
20720160062) to Y. X. This research was also sponsored by the
R. J. Palstra, T. W. Kan, C. Boucher, A. Verbon,
E. C. Dykhuizen and T. Mahmoudi, EBioMedicine, 2016, 3,
108–121.
Scientic Research Foundation of Third Institute of Oceanog- 19 N. M. Archin, A. Espeseth, D. Parker, M. Cheema, D. Hazuda
raphy, SOA (Grant No. 2016042) and the Educational Department
of Fujian Province (JA15017).
and D. M. Margolis, AIDS Res. Hum. Retroviruses, 2009, 25,
207–212.
20 (a) M. Dokmanovic and P. A. Marks, J. Cell. Biochem., 2005,
96, 293; (b) D. Demonte, V. Quivy, Y. Colette and C. Van
Lint, Biochem. Pharmacol., 2004, 68, 1231–1238; (c)
K. Shirakawa, L. Chavez, S. Hakre, V. Calvanese and
E. Verdin, Trends Microbiol., 2013, 21, 277–285.
References
1 D. Finzi, J. Blankson, J. D. Siliciano, J. B. Margolick,
K. Chadwick, T. Pierson, K. Smith, J. Lisziewicz, F. Lori,
C. Flexner, T. C. Quinn, R. E. Chaisson, E. Rosenberg, 21 X. Contreras, M. Schweneker, C. S. Chen, J. M. McCune,
B. Walker, S. Gange, J. Gallant and R. F. Siliciano, Nat Med,
1999, 5, 512–517.
S. G. Deeks, J. Martin and B. M. Peterlin, J. Biol. Chem.,
2009, 284, 6782–6789.
2 J. D. Siliciano, J. Kajdas, D. Finzi, T. C. Quinn, K. Chadwick, 22 N. M. Archin, J. L. Kirchherr, J. A. M. Sung, G. Clutton,
J. B. Margolick, C. Kovacs, S. J. Gange and R. F. Siliciano, Nat
Med, 2003, 9, 727–728.
3 A. R. Cillo and J. W. Mellors, Curr. Opin. Virol., 2016, 18, 14–
19.
K. Sholtis, Y. Y. Xu, B. Allard, E. Stuelke, A. D. Kashuba,
J. D. Kuruc, J. Eron, C. L. Gay, N. Goonetilleke and
D. M. Margolis, J. Clin. Invest., 2017, 127, 3126–3135.
23 T. D. Zaikos, M. M. Painter, N. T. Sebastian, V. H. Terry and
K. L. Collins, J. Virol., 2018, 02110–02117.
4 A. S. S. W. G. o. H. I. V. C. International, S. G. Deeks,
B. Autran, B. Berkhout, M. Benkirane, S. Cairns, 24 N. Sagotlerolle, A. Lamine, M. L. Chaix, F. Boufassa,
N. Chomont, T. W. Chun, M. Churchill, M. Di Mascio,
C. Katlama, A. Lafeuillade, A. Landay, M. Lederman,
J. P. Aboulker, D. Costagliola, C. Goujard, C. Pallier,
C. Paller and J. F. Delfraissy, AIDS, 2008, 22, 1125–1129.
S. R. Lewin, F. Maldarelli, D. Margolis, M. Markowitz, 25 J. P. Routy, C. L. Tremblay, J. B. Angel, B. Trottier,
J. Martinez-Picado, J. I. Mullins, J. Mellors, S. Moreno,
U. O'Doherty, S. Palmer, M. C. Penicaud, M. Peterlin,
G. Poli, J. P. Routy, C. Rouzioux, G. Silvestri, M. Stevenson,
D. Rouleau, J. G. Baril, M. Harris, S. Trottier, J. Singer,
N. Chomont, R. P. Sekaly and M. R. Boulassel, HIV Med.,
2012, 13, 291–296.
This journal is © The Royal Society of Chemistry 2018
RSC Adv., 2018, 8, 17279–17292 | 17291

View Article Online
RSC Advances
Paper
26 N. M. Archin, R. Bateson, M. K. Tripathy, A. M. Crooks,
K. H. Yang, N. P. Dahl, M. F. Kearney, E. M. Anderson,
S. K. Puri, D. Launay and D. Martin, Eur. J. Med. Chem.,
2013, 69, 527–536.
J. M. Coffin, M. C. Strain, D. D. Richman, K. R. Robertson, 42 H. Cope, R. Mutter, W. Heal, C. Pascoe, P. Brown, S. Pratt
A. D. Kashuba, R. J. Bosch, D. J. Hazuda, J. D. Kuruc, and B. Chen, Eur. J. Med. Chem., 2006, 41, 1124–1143.
J. J. Eron and D. M. Margolis, J. Infect. Dis., 2014, 210, 728– 43 Y. Wu, W. Dai, X. Chen, A. Geng, Y. Chen, T. Lu and Y. Zhu,
735. RSC Adv., 2017, 7, 52180–52186.
27 N. M. Archin, M. Cheema, D. Parker, A. Wiegand, R. J. Bosch, 44 A. Jordan, D. Bisgrove and E. Verdin, EMBO J., 2003, 22,
J. M. Coffin, J. Eron, M. Cohen and D. M. Margolis, PLoS One,
2010, 5, e9390.
28 S. Micheva-Viteva, Y. Kobayashi, L. C. Edelstein,
1868–1877.
45 Y. K. K., R. Pearson, J. Hokello, K. Lassen, J. Friedman,
M. Tyagi and J. Karn, J. Virol., 2008, 82, 12291–12303.
A. L. Pacchia, H. L. Lee, J. D. Graci, J. Breslin, B. D. Phelan, 46 V. T. Nguyen, T. Kiss, A. A. Michels and O. Bensaude, Nature,
L. K. Miller, J. M. Colacino, Z. Gu, Y. Ron, S. W. Peltz and
J. P. Dougherty, J. Biol. Chem., 2011, 286, 21083–21091.
29 G. M. Laird, C. K. Bullen, D. I. Rosenbloom, A. R. Martin,
2001, 414, 322–325.
47 Z. Li, J. Guo, Y. Wu and Q. Zhou, Nucleic Acids Res., 2013, 41,
277–287.
A. L. Hill, C. M. Durand, J. D. Siliciano and R. F. Siliciano, 48 P. G. Mccaffrey, C. Luo, T. K. Kerppola, J. Jain,
J. Clin. Invest., 2015, 125, 1901–1912.
T. M. Badalian, A. M. Ho, E. Burgeon, W. S. Lane,
30 E. D. Crignis and T. Mahmoudi, Viruses, 2014, 6, 4581–4608.
J. N. Lambert and T. Curran, Science, 1993, 262, 750–754.
´
31 M. Purwin, J. Hernandez-Toribio, C. Coderch, R. Panchuk, 49 D. D. Richman, D. M. Margolis, M. Delaney, W. C. Greene,
N. Skorokhyd, K. Filipiak, B. de Pascual-Teresa and
D. Hazuda and R. J. Pomerantz, Science, 2009, 323, 1304–
A. Ramos, RSC Adv., 2016, 6, 66595–66608.
1307.
32 X. Chen, S. Zhao, Y. Wu, Y. Chen, T. Lu and Y. Zhu, RSC Adv., 50 J. H. N. Yik, R. C. Chen, R. Nishimura, J. L. Jennings,
2016, 6, 103178–103184. A. J. Link and Q. Zhou, Mol. Cell, 2003, 12, 971–982.
33 (a) A. K. Halder, S. Mallick, D. Shikha, A. Saha, K. D. Saha 51 Z. Yang, Q. Zhu, K. Luo and Q. Zhou, Nature, 2001, 414, 317–
and T. Jha, RSC Adv., 2015, 5, 72373–72386; (b) X. Zhang, 322.
J. Zhang, M. Su, Y. Zhou, Y. Chen, J. Li and W. Lu, RSC 52 C. Jeronimo, D. Forget, A. Bouchard, Q. Li, G. Chua,
´
Adv., 2014, 4, 40444–40448.
C. Poitras, C. Therien, D. Bergeron, S. Bourassa and
34 A. Petrelli and G. Valabrega, Expert Opin. Pharmacother.,
2009, 10, 589–600.
35 K. S. Keedy, N. M. Archin, A. T. Gates, A. Espeseth,
D. J. Hazuda and D. M. Margolis, J. Virol., 2009, 83, 4749–
4756.
J. Greenblatt, Mol. Cell, 2007, 27, 262–274.
53 B. J. Krueger, C. Jeronimo, B. B. Roy, A. Bouchard,
C. Barrandon, S. A. Byers, C. E. Searcey, J. J. Cooper,
´
O. Bensaude and E. A. Cohen, Nucleic Acids Res., 2008, 36,
2219–2229.
36 E. Bourguet, K. Ozdarska, G. Moroy, J. Jeanblanc and 54 B. Sobhian, N. Laguette, A. Yatim, M. Nakamura, Y. Levy,
M. Naassila, J. Med. Chem., 2018, 61, 1745–1766. R. Kiernan and M. Benkirane, Mol. Cell, 2010, 38, 439–451.
37 K. Abouzid and S. Shouman, Bioorg. Med. Chem., 2008, 16, 55 J. E. Brogie and D. H. Price, Nucleic Acids Res., 2017, 45, 6864–
7543–7551. 6880.
38 J. A. Joule, K. Mills and G. F. Smith, Quinolines and 56 J. Wu, M. T. Ao, R. Shao, H. R. Wang, D. Yu, M. J. Fang,
isoquinolines: reactions and synthesis, Heterocyclic
Chemistry, 1995, pp. 120–145.
39 X. Q. Wang, C. L. Xia, S. B. Chen, J. H. Tan, T. M. Ou,
S. L. Huang, D. Li, L. Q. Gu and Z. S. Huang, Eur. J. Med.
Chem., 2015, 89, 349–361.
X. Gao, Z. Wu, Q. Zhou and Y. H. Xue, Sci. Rep., 2017, 7.
57 N. He, N. S. Jahchan, E. Hong, Q. Li, M. A. Bayeld,
R. J. Maraia, K. Luo and Q. Zhou, Mol. Cell, 2008, 29, 588–
599.
58 Y. H. Xue, Z. Y. Yang, R. C. Chen and Q. Zhou, Nucleic Acids
Res., 2010, 38, 360–369.
40 E. Pitta, M. K. Rogacki, O. Balabon, S. Huss, F. Cunningham,
E. M. Lopez-Roman, J. Joossens, K. Augustyns, L. Ballell, 59 Z. Y. Yang, J. H. N. Yik, R. C. Chen, N. H. He, M. K. Jang,
R. H. Bates and P. Van der Veken, J. Med. Chem., 2016, 59,
6709–6728.
41 V. S. Gopinath, J. Pinjari, R. T. Dere, A. Verma,
P. Vishwakarma, R. Shivahare, M. Moger, P. S. Kumar
Goud, V. Ramanathan, P. Bose, M. V. Rao, S. Gupta,
K. Ozato and Q. Zhou, Mol. Cell, 2005, 19, 535–545.
60 N. H. He, M. Liu, J. Hsu, Y. H. Xue, S. Chou, A. Burlingame,
N. J. Krogan, T. Alber and Q. Zhou, Mol. Cell, 2010, 38, 428–
438.
17292 | RSC Adv., 2018, 8, 17279–17292
This journal is © The Royal Society of Chemistry 2018