Identification of benzenesulfonamide quinoline derivatives as potent HIV-1 replication inhibitors targeting Rev protein

Article abstract of DOI:10.1039/c4ob02247e

Human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the export of viral RNA from nucleus to cytoplasm, which is a key step in HIV-1 pathogenesis and transmission. In this study, we have screened a commercial library and identified the hit compound 1 bearing a benzenesulfonamide quinoline scaffold that inhibited Rev activity and HIV-1 infectivity. Compounds bearing this scaffold were synthesized and their SAR was studied. We identified compound 20 with low toxicity and potent activity to inhibit HIV-1 replication by affecting Rev function.

Full text of DOI:10.1039/c4ob02247e

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DOI: 10.1039/C4OB02247E
ARTICLE
Identification of Benzenesulfonamide quinoline
derivatives as potent HIV-1 replication inhibitors
targeting Rev protein
Cite this: DOI: 10.1039/x0xx00000x
Fudi Zhong c, Guannan Geng c, Bing Chen,Ting Pan, Qianwen Li, Hui Zhang b
and Chuan Bai a
Received 00th January 2012,
Accepted 00th January 2012
DOI: 10.1039/x0xx00000x
Human immunodeficiency virus type 1 (HIV-1) Rev protein facilitates the export of viral RNA
from nucleus to cytoplasm, which is a key step in HIV-1 pathogenesis and transmission. In this
www.rsc.org/
study, we screened a commercial library and identified a hit compound
1 bearing
benzenesulfonamide quinoline scaffold that inhibited Rev activity and HIV-1 infectivity.
Compounds bearing this scaffold were synthesized and the SAR was studied. We identified
compound 20 with low toxicity and potent activity to inhibit HIV-1 replication through
affecting Rev function.
drug targeting Rev is in the clinical trial, the discovery and
development of new chemical compounds targeting Rev with
Introduction
potent anti-HIV-1 activity and low toxicity are of great
significance.
AIDS has been a worldwide epidemic for more than 30 years
and it has caused 1.6 million deaths every year. The agents in
high active antiretroviral therapy (HAART) protocol target
several HIV-1 enzymes which play key roles in the virus life
cycle and HAART has become the standard therapy for AIDS.
However, the serious side effects, the fast mutation of HIV-1
virus and high cost of life-long HAART have raised the
requirement of new therapeutics targeting other viral proteins
other than integrase, protease or reverse transcriptase. Rev is a
regulatory protein encoded by HIV-1 which plays a key role in
the nuclear export of unspliced or incompletely spliced HIV-1
mRNA from nucleus to cytoplasm.1-3 Rev specifically binds to
an RNA stem-loop structure termed RRE which locates in all
unspliced or incompletely spliced HIV-1 transcripts.4, 5 Rev also
contains a leucine-rich nuclear export signal (NES) that allows
the RNA-protein complex to access a nuclear export pathway.6
Multiple lines of evidence indicate that Rev-RRE transport
system requires cellular co-factors such as crm-1, Ran, DDX1,
Results and discussion
Hit Identification by High-throughput Screening (HTS) and
Anti-HIV-1 Replication Assay
I
n our study, High-throughput screening (HTS) system was
developed in 293T cells to test the anti-Rev activity and it was
applied to screen a commercial available drug-like chemical
library of 20155 compounds (Enamine). In the HTS system,
PDM628 and PCDNA3.1-Rev plasmids were co-transfected
into 293T cells, followed by the treatment of compounds in 50

M concentration for 48 h and then the luciferase expression
18
was recorded. The HTS results was summarized in Table 1
and 2,4-Difluoro- -8-quinolinyl-benzenesulfonamide (1) was
N
identified as a hit compound to inhibit Rev function (Figure 1,
A).
DDX3, Nup214 and Sam68.
7-10 The crucial role of Rev in viral
replication and activation makes it a good target to develop new
chemotherapy methods against HIV-1. There have been reports
This compound was further tested in the anti-HIV-1 infectivity
of Rev inhibitors from natural products, peptide mimics and assay. In brief, In brief, H9 and SupT1 cells (1×106) were
infected with the equivalent of 5 ng HIV-1 p24 for 3 h. HIV-1
synthesized compounds 11-17
These reported Rev inhibitors
.
replication was monitored by the quantification of p24 antigen
in supernatant by ELISA after 4 days. The IC50 value of
showed the IC50 of anti-HIV-1 activity at hundreds of
micromolar to submicromolar level. Given that no anti-HIV-1
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compound
1 was 0.23 M (Figure 2). Although compound 1
showed potent anti-HIV-1 replication activity, it also showed
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DOI: 10.1039/C4OB02247E
high toxicity with the CC50 value of 5.1
293T cells (Table 2).
M in MTT test in
Table 1. Results of the screening of compound libraries
Scheme 1. Reagents and conditions: (a) 25 ºC -60 ºC, pyridine. (b)
Methyl iodide /ethyl iodide, ethanol, reflux
SAR
In SAR studies, synthesized compounds were first tested for their
anti-Rev activity with the protocol similar with HTS. The active
compounds with low toxicity (CC50>50 M) were then tested their
anti-HIV infectivity activities. The structure of compound 1 was
divided into substituted quinoline (A), benzne ring (B), and the
linker (Figure 1, B). These substructures were investigated their
influences on the activities in both assays.
Figure 1. (A) Hit compound (1) identified from HTS. (B) Hit
substructures for SAR studies.
Firstly, the effects of ring A’s modifications on the anti-Rev activity
was studied. The quinoline was replaced with naphthalene, benzene
or pyridine to test the effects of the size and the H-bond acceptor
atom of the quinoline structure (compound 2-4, Table 2). The
nucleobases or their analogues have been widely used in drugs
which target RNA, DNA or their interactions with proteins.
Therefore compounds 5-7 bearing adenine, cytosine or guanine were
synthesized to replace quinoline substructure. However, these
modifications led to the loss of activities. These results showed that
the quinoline substructure was required for their anti-Rev and anti-
HIV-1 replication activities.
Figure 2. Dose–response curve for hit compound
1 and
It was thought that the 2,4-difluoride substitution on ring B
could be replaced with other halogen atoms that reserved the
compound 20 yielded IC50 values of 0.23
M and 0.27M
respectively.
electron withdrawing property. Compound
substitution showed similar activity with compound
compound 11 with other mono- or di-halogen substitutions
resulted in a loss of the activities, as did the compounds (12 14)
8
with 2,4-dichloro
1
. However,
Chemical Synthesis
9
-
The synthesis of series of compounds bearing the
benzenesulfonamide quinoline scaffold or similar structures
was carried out as outlined in Scheme 1. The derivatives of 8-
aminoquinoline or neucleobases were coupled with aromatic
-
with non-halogen electron-withdrawing groups on ring B
(Table 2). Then the compounds with no substitution (15) and
with electron-donating groups (16-17) were synthesized and
tested but these modifications abolished the activities (Table 2).
These results indicated that the 2,4-difluoro substitution was
vital for the anti-Rev activity and the exact mechanism should
be more complicated than their electron withdrawing property.
sulfonamide compounds in pyridine.
The alkylation of
benzenesulfonamide quinoline was achieved with alkyl iodide
with reflux in ethanol. The alkyl substitution on nitrogen of
sulfonamide linker did not obviously affect the followed
reactions.
The influences of the linker were then studied (Table 3). Firstly,
the sulfonamide linker was replaced by amide structure (18).
Compound 18 was active in anti-Rev assay, but it showed
2 | J. Name., 2012, 00, 1‐3
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lower activity in anti-HIV-1 infectivity assay than compound
1
.


M, Figure 2) and more than 10 fold lower toxicity (CC50>50
Interestingly, compounds (19 20) with methyl or ethyl
,
M) than hit compound 1.
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substitutions on the nitrogen atom of sulfonamide were active
in both anti-Rev assay and anti-HIV-1 infectivity assay. The
compound 20 showed potent anti-HIV-1 activity (IC50= 0.27
DOI: 10.1039/C4OB02247E
Table 2. Influence of modifications of ring A on anti-Rev activity.
CC50 (M) a IC50 (M)b
Compd No.
1
R
Anti-Rev
Assay
5.1
0.23
+
2
3
>30
>20
7.1
+
c
>20
>20
>20
−
4
5
>20
>50
−
−
6
7
>50
>50
>20
>20
−
−
CC50 (M)a : median (50%) cytotoxic concentration ;IC50(M)b: half maximal (50%) inhibitory concentration
−c: test result did not show activity
Table 3. Influence of modifications of ring B on anti-Rev activity.
CC50 (M)
IC50 (M)
Compd No.
R
Anti-Rev
Assay
c
8
9
2-Cl,4-Cl
2-F
+
>50
>50
0.47d
>20
−
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10
11
12
13
14
15
16
17
4-F
4-Cl
−
−
−
−
−
−
−
−
>50
>20
>20
>50
>50
>30
>30
>50
>50
>50
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DOI: 10.1039/C4OB02247E
4-CF3
3-NO2
4-NO2
H
>20
>20
>20
>20
>20
>20
4-Me
4-OMe
Table4. Influence of modifications of linker on anti-Rev activity.
CC50 (M)
IC50 (M)
Compd No.
18
Linker
Anti-Rev
Assay
+
+
+
>50
48
19
20
>50
>50
1.18
0.27
Compound 1 and 20 affected the Rev function
A
The substructures of both 8-aminoquinoline and sulfonamide
are distributed in drugs and natural products with potent
biological activities. Therefore it is crucial to further clarify the
anti-HIV-1 mechanism and the corresponding targets of
compound 1 and 20. Firstly, the quantitative changes of relative
RRE between nucleus and cytoplasm after the treatment of
compound 1 and 20 were measured by real-time PCR. In Brief, the
293T cells were transfected with vector PDM628 or co-transfected
with Rev, and then treated with compound 1. Fractionation of
cytoplasmic and nuclear components was isolated from 293T cells
and RNA was then extracted from each of the fractions. Reverse
transcription reactions were performed with RT reagent and the
cytoplasmic/nuclear ratios of RRE were detected with real-time
PCR.As shown in Figure 3, compound 1 (A) and 20 (B) caused
about 3-fold decrease of RRE-containing mRNA in cytoplasm while
2-fold increase in nucleus. This result showed that the potent activity
of compound 1 and 20 was related with the inhibition of Rev
function of nuclear export of HIV-1 mRNA from nucleus to
B
cytoplasm
.
Figure 3. (A) The expression of RRE in the cytoplasm (left) and
nucleus (right) was changed by compound 1. (B) The similar test
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was applied to compound 20. Data represent three independent
expriments. ** P<0.01 using a student’s t test.
chromatography was performed with silica gel 60. NMR
spectra were recorded on a Bruker Avance 400 with the
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solvents indicated. Purity was analyzed bDyOrIe:v10e.r1s0e3-9p/hCa4sOeBH02P2L47CE
To test whether compound 20 inhibited HIV-1 activity in a Rev
dependent way, the RRE sequence in the plasmid pMDLg/RRE
was replaced by a constitutive transport element (CTE) of the
type D Mason-Pfizer monkey virus (MPMV) to construct the
plasmid pMDLg/CTE, which expresses protein in a Rev-
independent manner. 19 The 293T cells were co-transfected with
pMDLg/RRE and Rev or pMDLg/CTE and Rev, and treated
with compound 20. The p-24 level showed that compound 20
totally lost its activity to block the nuclear export of viral RNA
in the CTE/REV system (Figure 4 A) but inhibited the nuclear
export of viral RNA in the RRE/Rev system (Figure 4 B).
These data demonstrated that the compound 20 could inhibit
HIV-1 replication by targeting the export of Rev/RRE from
nucleus.
performed on Agilent 6130 Quadrupole and Agilent 1200
equipment, or on Agilent ProStar 218 system. HRMS were
recorded at the Instrumental Analysis & Research Center in
Sun Yat-sen University using a Thermo Scientific LTQ-
Orbitrap Elite mass spectrometer.
2,4-Difluoro-
the solution of 8-aminoquinoline (119 mg, 0.83 mmol) and 2,4-
Difluorobenzenesulfonyl chloride (93 L, 0.69 mmol) in
N-(quinolin-8-yl)benzenesulfonamide (1). To

pyridine (3 mL). The mixture was stirred at room temperature
for 1 h. The reaction mixture was washed with HCl solution
(2N) (3
×20 mL) and extracted with hexane. The combined
organic phase was dried over anhydrous Na2SO4 and
concentrated under vacuum. The product was purified by silica
gel chromatography using cyclohexane-ethyl acetate (6:1) to
B
A
1
give
CDCl3)
= 8.3, 1.6 Hz, 1H), 8.00 (td,
7.6, 1.1 Hz, 1H), 7.52–7.37 (m, 3H), 6.94–6.86 (m, 1H), 6.78
(ddd,
= 10.0, 8.5, 2.4 Hz, 1H). 113C NMR (101 MHz, CDCl3)
167.29 (dd, = 257.9, 11.3 Hz), 161.23 (dd, = 259.8,
13.0 Hz), 150.27,139.93, 137.55, 134.57, 134.03 (d,
10.6 Hz), 129.58, 128.09, 125.06 (d, = 14.0 Hz), 123.81,
123.48, 116.09, 112.94 (dd, = 22.0, 3.4 Hz), 107.05 (t,
= 25.4 Hz). HRMS (ESI-) m/z: calcd for C15H9O2N2F2S
1
as white powder (204 mg, 92%). H NMR (500 MHz,
δ
9.50 (s, 1H), 8.81 (dd,
J= 4.2, 1.6 Hz, 1H), 8.11 (dd,
J
J
= 8.4, 6.2 Hz, 1H), 7.75 (dd, J=
J
δ
J
J
J
=
J
J
J
[M - H]-: 319.03583. Found: 319.03584 (Δ = 0.04 ppm).
Figure 4. The compound 20 affects the export of Rev/RRE but
not CTE-dependent gene expression. (A) The 293T cells were co-
transfected with pMDLg/CTE and Rev, and treated with compound
20. The expression of P24 was detected by ELISA. (B) The 293T
cells were co-transfected with pMDLg/RRE and Rev and treated
with compound 20. The expression of P24 was detected by ELISA.
Data represent three independent expriments. *P<0.05 using a
student’s t test.
2,4-Difluoro-N-(naphthalen-1-yl)benzenesulfonamide (2). The
title compound was synthesized according to the procedure of
preparing compound
1 except using 8-aminonaphthalene
instead of 4-methylbenzaldehyde.White powder (42% yield). 1H
NMR (500 MHz, CDCl3) δ 8.01 (d, J= 8.3 Hz, 1H), 7.83 (d, J=
7.5 Hz, 1H), 7.79–7.69 (m, 2H), 7.58–7.47 (m, 2H), 7.44–7.31
(m, 2H), 7.10 (s, 1H), 6.89 (dddd, J= 10.1, 9.0, 8.2, 1.9 Hz, 2H).
13C NMR (126 MHz, DMSO-d6) δ 165.53 (dd, J = 254.1, 11.8
Hz), 159.54 (dd, J = 256.9, 13.3 Hz), 134.32, 132.54 (d, J =
10.8 Hz), 132.05, 130.11, 128.51, 127.69, 126.75 (d, J = 6.2
Hz), 126.02, 125.06 (d, J = 14.3 Hz), 124.35, 123.19, 112.67
(d, J = 22.2 Hz), 106.43 (t, J = 26.1 Hz). HRMS (ESI+) m/z:
calcd for C16H11O2NF2NaS [M + Na]+ : 342.03708. Found:
342.03715 (Δ = 0.21 ppm).
Conclusions
From the HTS hit (1) with potent anti-HIV-1 activity but high
toxicity, we identified compound 20 to inhibit HIV-1 infectivity
by SAR studies. For compound 20, the toxicity was decreased
more than 10 fold, while the potent anti-HIV-1 activity was still
similar to compound 1. Moreover, their anti-HIV-1 mechanism
was clarified by measuring the quantitative change of RRE
between nucleus and cytoplasm in a Rev/RRE dependent way.
Together, compound 20 could be a good starting point to develop
new chemical compounds with potent anti-HIV-1 activity
through specifically targeting the Rev function.
2,4-Difluoro-N-phenylbenzenesulfonamide
(3).
The
title
compound was synthesized according to the procedure of
preparing compound 1 except using aniline instead of 4-
methylbenzaldehyde.White powder (75% yield). 1H NMR (500
MHz, CDCl3) δ 7.83 (ddd, J= 8.3, 7.2, 3.9 Hz, 1H), 7.31–7.19
(m, 2H), 7.17–7.07 (m, 3H), 6.99–6.89 (m, 2H), 6.80 (s, 1H).
Experimental
13C NMR (101 MHz, CDCl3)
δ 167.40 (dd, J = 258.3, 11.6
Hz), 160.87 (dd, J = 257.3, 12.9 Hz), 136.97, 134.15 (d, J =
10.5 Hz), 2×130.84, 127.28, 124.60 (d, J = 13.5 Hz), 2×122.86 ,
113.38 (dd, J = 21.9, 3.3 Hz), 106.90 (t, J = 25.6 Hz). HRMS
(ESI+) m/z: calcd for C12H9O2NF2NaS [M + Na]+ : 292.02143.
Found: 292.02169 (Δ = 0.9 ppm).
Chemistry
General Chemistry Methods.
All reagents and solvents were purchased from Sigma-Aldrich,
J&K Chemical and Aladdin. Analytical thin-layer plates were
obtained from Qingdao Haiyang Chemical.
Flash
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2,4-Difluoro-N-(pyridin-2-ylmethyl)benzenesulfonamide (4). The 1.6 Hz, 1H), 8.20–8.05 (m, 2H), 7.70 (dd, J= 7.6, 1.2 Hz, 1H), 7.45
title compound was synthesized according to the procedure of (dd, J= 8.3, 4.2 Hz, 2H), 7.37 (dd, J= 12.8, 4.8 Hz, 2H), 7.32 (dd, J=
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DOI: 10.1039/C4OB02247E
preparing compound
1
except using pyridin-2-yl-methylamine 8.5, 2.0 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 150.17, 141.16,
instead of 4-methylbenzaldehyde.White powder (38% yield). 1H 139.72, 137.52, 136.54, 134.52, 134.22, 133.00, 129.64,
NMR (500 MHz, CDCl3) δ 8.46 (d, J= 4.4 Hz, 1H), 7.88 (td, J= 8.5, 128.44, 128.09, 123.56 (d, J = 8.3 Hz), 115.31. HRMS (ESI+)
6.2 Hz, 1H), 7.60 (td, J= 7.7, 1.8 Hz, 1H), 7.16 (dd, J= 11.5, 6.5 Hz, m/z: calcd for C15H11O2N2Cl2S [M + H]+ : 352.99128. Found:
2H), 6.98–6.90 (m, 1H), 6.89–6.81 (m, 1H), 6.21 (s, 1H), 4.33 (d, J= 352.99136 (Δ = 0.23 ppm).
5.4 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 167.04 (dd, J =
256.8, 11.8 Hz), 161.04 (dd, J = 257.7, 13.0 Hz), 155.84, 2-Fluoro-N-(quinolin-8-yl)benzenesulfonamide (9). The title
150.53, 138.12, 133.32 (d, J = 10.3 Hz), 126.00, 124.08, compound was synthesized according to the procedure of preparing
123.18, 112.94 (d, J = 18.5 Hz), 106.79 (t, J = 25.6 Hz), compound 1 except using 2-fluoro-benzenesulfonyl chloride instead
48.93.HRMS (ESI+) m/z: calcd for C12H11O2N2F2S [M + H]+ : of 2,4-difluoro-benzenesulfonyl chloride. White powder (56%
1
285.05038. Found: 285.05045 (Δ = 0.24 ppm).
yield). H NMR (500 MHz, CDCl3) δ 9.61 (s, 1H), 8.81 (dd, J= 4.1,
1.2 Hz, 1H), 8.13 (dd, J= 8.2, 1.0 Hz, 1H), 8.01 (td, J= 7.8, 1.6 Hz,
1H), 7.8 –7.73 (m, 1H), 7.52–7.38 (m, 4H), 7.20 (t, J= 7.6 Hz, 1H),
7.04 (t, J= 9.2 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ
160.10 (d, J = 254.5 Hz), 151.37, 140.70, 138.58, 137.95 (d, J
= 8.7 Hz), 134.65, 132.27, 129.93, 128.63, 128.50, 126.67,
125.57, 124.30, 119.38, 119.06 (d, J = 20.9 Hz). HRMS (ESI+)
m/z: calcd for C15H12O2N2FS [M + H]+ : 303.05980. Found:
303.06004 (Δ = 0.78 ppm).
2,4-Difluoro-N-(7H-purin-6-yl)benzenesulfonamide (5). The title
compound was synthesized according to the procedure of
preparing compound
1 except using adenine instead of 4-
1
methylbenzaldehyde.White powder (36% yield). H NMR (500
MHz, DMSO-d6) δ 8.67 (s, 1H), 8.33 (td, J= 8.7, 6.0 Hz, 1H), 8.12
(s, 1H), 7.70 (s, 2H), 7.62 (ddd, J= 11.2, 9.1, 2.4 Hz, 1H), 7.46 (td,
J= 8.4, 2.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 168.81 (dd,
J = 258.7, 12.3 Hz), 161.33 (dd, J = 259.7, 14.1 Hz), 158.08,
156.20, 150.18, 140.10, 135.85 (d, J = 11.7 Hz), 122.79 (d, J
= 16.1 Hz), 120.41, 115.11 (d, J = 25.6 Hz), 108.65 (t, J =
25.9 Hz). HRMS (ESI+) m/z: calcd for C11H8O2N5F2S [M + H]+ :
312.03613. Found: 312.03640 (Δ = 0.87 ppm).
4-Fluoro-N-(quinolin-8-yl)benzenesulfonamide (10). The title
compound was synthesized according to the procedure of
preparing compound
1 except using 4-fluoro-benzenesulfonyl
chloride instead of 2,4-difluoro-benzenesulfonyl chloride.
White powder (30% yield). H NMR (500 MHz, CDCl3) δ 9.20 (s,
2,4-Difluoro-N-(2-oxo-1,2-dihydropyrimidin-4-
1
yl)benzenesulfonamide (6). The title compound was synthesized
according to the procedure of preparing compound 1 except using
cytosine instead of 4-methylbenzaldehyde.White powder (36%
1H), 8.76 (dd, J= 4.2, 1.6 Hz, 1H), 8.11 (dd, J= 8.3, 1.6 Hz, 1H),
7.98–7.88 (m, 2H), 7.83 (dd, J= 7.3, 1.6 Hz, 1H), 7.50–7.38 (m, 3H),
7.05–6.93 (m, 2H). 13C NMR (101 MHz, CDCl3) δ 166.53 (d, J
= 255.1 Hz), 150.19, 140.02, 137.65, 136.80, 134.94, 131.35,
131.26, 129.60, 128.17 , 123.86, 123.40, 117.58, 117.35,
116.86. HRMS (ESI+) m/z: calcd for C15H12O2N2FS [M + H]+ :
303.05980. Found: 303.06008 (Δ = 0.91 ppm).
1
yield). H NMR (500 MHz, DMSO-d6) δ 8.16–7.95 (m, 4H), 7.71–
7.56 (m, 1H), 7.40 (td, J= 8.6, 2.1 Hz, 1H), 6.00 (d, J= 7.9 Hz, 1H).
13C NMR (101 MHz, DMSO-d6) δ 167.99 (dd, J = 256.8, 12.3
Hz),167.82, 160.68 (dd, J = 257.6, 13.9 Hz), 152.52, 141.14,
136.34 (d, J = 11.3 Hz), 123.79 (d, J = 13.0 Hz), 114.53 (d, J
= 20.2 Hz), 107.96 (t, J = 26.1 Hz), 99.61. HRMS (ESI+) m/z:
calcd for C10H8O3N3F2S [M + H]+ : 288.02489. Found: 288.02523 (Δ
= 1.16 ppm).
4-Chloro-N-(quinolin-8-yl)benzenesulfonamide (11). The title
compound was synthesized according to the procedure of
preparing compound
1 except using 4-chloro-benzenesulfonyl
chloride instead of 2,4-difluoro-benzenesulfonyl chloride. White
powder (91% yield). H NMR (500 MHz, CDCl3) δ 9.22 (s, 1H),
2,4-Difluoro-N-(6-oxo-6,7-dihydro-1H-purin-2-
yl)benzenesulfonamide (7). The title compound was synthesized
1
8.76 (dd, J= 4.2, 1.6 Hz, 1H), 8.11 (dd, J= 8.3, 1.5 Hz, 1H), 7.89–
7.75 (m, 3H), 7.53–7.38 (m, 3H), 7.39–7.29 (m, 2H). 13C NMR
(101 MHz, DMSO-d6) δ 151.27, 140.96 , 140.32, 139.76,
138.34, 135.22, 2×131.05, 2×130.70, 130.00, 128.48, 125.37,
124.15, 119.56. HRMS (ESI+) m/z: calcd for C15H12O2N2ClS [M +
H]+ : 319.03025. Found: 319.03057 (Δ = 1 ppm).
according to the procedure of preparing compound
1 except
using guanine instead of 4-methylbenzaldehyde.White powder
(6% yield). 1H NMR (500 MHz, DMSO-d6) δ 10.97 (s, 1H), 8.22
(td, J= 8.7, 6.1 Hz, 1H), 8.16 (s, 1H), 7.65 (ddd, J= 11.3, 9.2, 2.4 Hz,
1H), 7.43 (td, J= 8.4, 2.2 Hz, 1H), 6.73 (s, 2H). 13C NMR (126
MHz, DMSO-d6) 167.33 (dd, J = 258.5, 12.1 Hz), 159.86 (dd,
J = 259.9, 14.1 Hz), 156.60 , 155.14, 150.94, 134.87, 134.37
(d, J = 11.5 Hz), 121.52 (d, J = 12.6 Hz), 117.28, 113.56 (d, J
= 20.6 Hz), 107.25 (t, J = 25.8 Hz). HRMS (ESI+) m/z: calcd for
C11H8O3N5F2S [M + H]+ : 328.03104. Found: 328.03148 (Δ = 1.33
ppm).
N-(quinolin-8-yl)-4-(trifluoromethyl)benzenesulfonamide (12).
The title compound was synthesized according to the procedure
of preparing compound
benzenesulfonyl chloride
1
except using 4-trifluoromethyl-
instead of 2,4-difluoro-
1
2,4-Dichloro-N-(quinolin-8-yl)benzenesulfonamide (8). The title
compound was synthesized according to the procedure of preparing
compound 1 except using 2,4-dichloro-benzenesulfonyl chloride
instead of 2,4-difluoro-benzenesulfonyl chloride. White powder (56%
benzenesulfonyl chloride. White powder (72% yield). H NMR
(500 MHz, DMSO-d6) δ 10.55 (s, 1H), 8.79 (dd, J= 4.2, 1.7 Hz, 1H),
8.34 (dd, J= 8.3, 1.6 Hz, 1H), 8.31–8.25 (m, 2H), 8.15–8.09 (m, 2H),
7.71 (ddd, J= 11.8, 8.0, 1.2 Hz,2H), 7.55 (dd, J= 8.2, 4.1 Hz, 2H).
13C NMR (101 MHz, CDCl3) δ 150.30, 144.33, 139.96,
1
yield). H NMR (500 MHz, CDCl3) δ 9.75 (s, 1H), 8.81 (dd, J= 4.2,
6 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 2012

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Journal Name
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ARTICLE
137.72, 136.05, 135.73, 134.57 , 129.64, 129.05, 128.18, (ESI+) m/z: calcd for C16H15O2N2S [M + H]+ : 299.15497. Found:
127.40 (d, J = 3.5 Hz), 125.80 , 124.18, 123.49, 123.09, 299.15503 (Δ = 0.21 ppm).
View Article Online
DOI: 10.1039/C4OB02247E
116.94. HRMS (ESI+) m/z: calcd for C16H12O2N2F3S [M + H]+ :
353.05661. Found: 353.05700 (Δ = 1.11 ppm).
4-Methoxy-N-(quinolin-8-yl)benzenesulfonamide (17). The title
compound was synthesized according to the procedure of preparing
3-Nitro-N-(quinolin-8-yl)benzenesulfonamide (13). The title compound 1 except using 4-methoxy-benzenesulfonyl chloride
compound was synthesized according to the procedure of instead of 2,4-difluoro-benzenesulfonyl chloride. White powder (91%
1
preparing compound
1
except using 3-nitro-benzenesulfonyl yield). H NMR (500 MHz, CDCl3) δ 9.19 (s, 1H), 8.75 (dd, J= 4.2,
chloride instead of 2,4-difluoro-benzenesulfonyl chloride. 1.6 Hz, 1H), 8.08 (dd, J= 8.3, 1.6 Hz, 1H), 7.87–7.83 (m, 2H), 7.81
1
White powder (36% yield). H NMR (500 MHz, DMSO-d6) δ (dd, J= 6.4, 2.5 Hz, 1H), 7.49–7.38 (m, 3H), 6.86–6.76 (m, 2H), 3.75
8.78 (dd, J= 4.2, 1.7 Hz, 1H), 8.68 (t, J= 1.9 Hz, 1H), 8.40–8.31 (m, (s, 3H). 13C NMR (101 MHz, CDCl3) δ 164.39,150.03,
2H), 8.24 (ddd, J= 7.9, 1.7, 1.0 Hz, 1H), 7.79–7.68 (m, 3H), 7.60– 139.96,137.56,135.36,132.46,2×130.75,129.56,128.19,123.30
7.49 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 151.21, ,116.31,2×115.41,56.83. HRMS (ESI+) m/z: calcd for
149.51, 143.29, 141.46, 138.85, 134.64, 132.83 , 130.20, C16H15O3N2S [M + H]+ : 315.07979. Found: 315.08006 (Δ = 0.86
129.26, 128.63, 126.52, 124.10, 123.76, 122.46 . HRMS (ESI+) ppm).
m/z: calcd for C15H12O4N3S [M + H]+ : 330.05430. Found:
330.05464 (Δ = 1.02 ppm).
2,4-Difluoro-N-(quinolin-8-yl)benzamide (18). To the solution of
8-Aminoquinoline (280 mg, 1.94 mmol) and 2,4-
4-Nitro-N-(quinolin-8-yl)benzenesulfonamide (14). The title Difluorobenzoyl chloride (200 L, 1.61 mmol) in pyridine (3
compound was synthesized according to the procedure of mL). The mixture was stirred at room temperature for 1 h. The
preparing
benzenesulfonylchloride
compound
1
except
instead
using
4-nitrro- reaction mixture was washed with HCl solution (2N) (3
2,4-difluoro- mL) and extracted with dichloromethane (20 mL). The organic
×20
of
benzenesulfonyl chloride. Gray powder (36% yield). 1H NMR phase was collected and concentrated under vacuum. The
(500 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.79 (dd, J= 4.2, 1.7 Hz, 1H), product was purified by silica gel chromatography using
8.35 (dd, J= 8.3, 1.6 Hz, 1H), 8.08 (d, J= 8.2 Hz, 2H), 7.86 (d, J= 8.3 cyclohexane-ethyl acetate (10:1) as eluate to give 18 as white
Hz, 2H), 7.76–7.67 (m, 2H), 7.62–7.49 (m, 2H). 13C NMR (101 powder (311 mg, 68%). H NMR (500 MHz, CDCl3) δ 11.11 (d, J=
1
MHz, DMSO-d6) δ151.65, 151.36, 147.22, 141.43, 138.34, 11.8 Hz, 1H), 8.95 (dd, J= 7.2, 1.7 Hz, 1H), 8.87 (dd, J= 4.2, 1.6 Hz,
134.95, 2×130.35, 130.10, 128.48, 2×126.16, 126.03, 124.13, 1H), 8.26 (td, J= 8.8, 6.6 Hz, 1H), 8.19 (dd, J= 8.3, 1.6 Hz, 1H),
120.99. HRMS (ESI+) m/z: calcd for C15H12O4N3S [M + H]+ : 7.63–7.53 (m, 2H), 7.48 (dd, J= 8.2, 4.2 Hz, 1H), 7.12–7.04 (m, 1H),
330.05430. Found: 330.05438 (Δ = 0.23 ppm).
6.99 (ddd, J= 11.2, 8.5, 2.4 Hz, 1H). 13C NMR (101 MHz, CDCl3)
δ 166.42 (dd, J = 255.5, 12.6 Hz), 162.35 (dd, J = 251.8, 12.2
N-(quinolin-8-yl)benzenesulfonamide (15). The title compound Hz), 162.04, 149.88, 140.19, 137.66, 136.11, 135.22 (d, J =
was synthesized according to the procedure of preparing 10.2 Hz), 129.38, 128.76, 123.50, 123.08, 119.97 (d, J = 11.8
compound
1 except using benzenesulfonylchloride instead of Hz), 118.67 , 113.81 (dd, J = 21.3, 2.9 Hz), 105.89 (t, J =
2,4-difluoro-benzenesulfonyl chloride. White powder (60% yield). 28.2 Hz). HRMS (ESI+) m/z: calcd for C16H11ON2F2 [M + H]+ :
1H NMR (500 MHz, CDCl3) δ 9.24 (s, 1H), 8.75 (dd, J= 4.2, 1.6 Hz, 285.08340. Found: 285.08371 (Δ = 1.1 ppm).
1H), 8.09 (dd, J= 8.3, 1.6 Hz, 1H), 8.01–7.89 (m, 2H), 7.83 (dd, J=
6.8, 2.0 Hz, 1H), 7.47–7.39 (m, 4H), 7.36 (dd, J= 10.5, 4.8 Hz, 2H). N-methylquinolin-8-amine (19-0). To the solution of 8-
13C NMR (101 MHz, CDCl3) δ 150.08, 140.83, 139.94, Aminoquinoline (500 mg, 3.5 mmol) was added iodomethane (199
137.58, 135.15, 134.21,2× 130.22, 129.56, 2×128.55, 128.19, L, 3.2 mmol) in ethanol (3 mL). The mixture was refluxed for 20h.
123.53, 123.31, 116.49. HRMS (ESI+) m/z: calcd for The reaction mixture was concentrated under vacuum and purified
C15H13O2N3S [M + H]+ : 285.06922. Found: 285.06932 (Δ = 0.33 by silica gel chromatography using cyclohexane-ethyl acetate (20:1)
ppm).
as eluate to give 19-0 as yellow oil (310 mg, 61%). 1H NMR (400
MHz, DMSO-d6) δ 8.69 (dd, J= 4.1, 1.7 Hz, 1H), 8.17 (dd, J= 8.3,
4-Methyl-N-(quinolin-8-yl)benzenesulfonamide (16). The title 1.6 Hz, 1H), 7.46 (dd, J= 8.3, 4.2 Hz, 1H), 7.35 (t, J= 7.9 Hz, 1H),
compound was synthesized according to the procedure of 7.02 (dd, J= 8.1, 0.9 Hz, 1H), 6.56 (t, J= 7.4 Hz, 2H), 2.89 (d, J= 5.2
preparing compound
1 except using 4-methyl-benzenesulfonyl Hz, 3H).
chloride instead of 2,4-difluoro-benzenesulfonyl chloride.
White powder (69% yield). H NMR (500 MHz, DMSO-d6) δ 9.86
2,4-Difluoro-N-methyl-N-(quinolin-8-yl)benzenesulfonamide (19).
To the solution of N-methylquinolin-8-amine (142 mg, 0.90
mmol) and 2,4-Difluorobenzenesulfonyl chloride (101 mL,
0.75 mmol) in pyridine (3 mL). The mixture was stirred at
room temperature for 1 h. The reaction mixture was washed
1
(s, 1H), 8.87 (dd, J= 4.2, 1.7 Hz, 1H), 8.35 (dd, J= 8.3, 1.6 Hz, 1H),
7.80 (d, J= 8.3 Hz, 2H), 7.66 (ddd, J= 15.0, 7.9, 1.1 Hz, 2H), 7.59
(dd, J= 8.3, 4.2 Hz, 1H), 7.55–7.48 (m, 1H), 7.28 (d, J= 8.0 Hz, 2H),
2.27 (s, 3H). 13C NMR (101 MHz, DMSO)
δ
with HCl solution (2N) (3
×20 mL) and extracted with
151.18,145.42,140.40,138.37,135.45,2×131.43,
129.91,2×128.78,128.51,124.66,124.19,117.98,22.72. HRMS
dichloromethane (20 mL). The organic phase was collected
and concentrated under vacuum. The product was purified by
This journal is © The Royal Society of Chemistry 2012
J. Name., 2012, 00, 1‐3 | 7

Organic & Biomolecular Chemistry
Page 8 of 9
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silica gel chromatography using cyclohexane-ethyl acetate (6:1) instead of any compounds. In addition, column 1 was only
as eluate to give 19 as white powder (60 mg, 24%). 1H NMR (400 transfected with the plasmid PDM628 as a control. The
MHz, DMSO-d6) δ 8.53 (dd, J= 4.2, 1.7 Hz, 1H), 8.35 (dd, J= 8.3, luciferase expression was detected with aDPOrIo: m10e.1g0a3V9Rie/wCe4aAOdrtieBclr0e2aO2tn4l4i7n8Ee
1.7 Hz, 1H), 7.98 (dd, J= 8.2, 1.3 Hz, 1H), 7.72 (dd, J= 7.4, 1.4 Hz, hour post-transfection. 20
1H), 7.65–7.57 (m, 1H), 7.57–7.49 (m, 1H), 7.48–7.38 (m, 2H), 7.02
(d, J= 2.4 Hz, 1H), 3.50 (d, J= 1.8 Hz, 3H). 13C NMR (101 MHz, HIV-1 Production, Infection, and IC50 Assay.
CDCl3) δ 166.81 (dd, J = 255.7, 11.4 Hz), 161.89 (dd, J = H9 cells were infected with the HIV-1 virus (pNL4-3) in
258.6, 12.5 Hz), 150.94, 146.37, 138.94, 137.49, 135.71, accordance with the amount of 10ng p24/1*10^6 cells. After 3
133.86 (d, J = 10.5 Hz), 133.64, 130.72, 130.21, 127.82, hours, the infected cells were washed by PBS 3 times. Then the
122.63, 112.10 (dd, J = 21.6, 3.3 Hz), 106.38 (t, J = 25.8 Hz), cells were plated in 96-well plates (10^4 cells/well) and
41.18 (s). HRMS (ESI+) m/z: calcd for C16H13O2N2F2S [M + H]+ : incubated with the compounds by 10-fold gradient
335.06603. Found: 335.06622 (Δ = 0.56 ppm).
concentrations. 96h later all the supernatant were conducted to
p24 ELISA assay. The readout of p24 antigen level in the
control group (infected cells and DMSO) should higher than 1.
The IC50 values were determined by plotting the logarithm of
compound concentration versus p24 amount to obtain
concentration that produced 50% inhibition.21
N-ethylquinolin-8-amine (20-0). To the solution of 8-
Aminoquinoline (500 mg, 3.5 mmol) and Iodoethane (256 L,
3.2 mmol) in ethanol (3 mL). The mixture was refluxed for 19h.
The reaction mixture was concentrated under vacuum and
purified by silica gel chromatography using cyclohexane-ethyl
acetate (30:1) as eluate to give 20-0 as yellow oil (232 mg, 42%).
1H NMR (400 MHz, CDCl3) δ 8.71 (dd, J= 4.1, 1.6 Hz, 1H), 8.05
(dd, J= 8.3, 1.6 Hz, 1H), 7.37 (dt, J= 8.3, 6.0 Hz, 2H), 7.26 (s, 1H),
7.03 (d, J= 8.2 Hz, 1H), 6.67 (d, J= 7.6 Hz, 1H), 3.36 (d, J= 6.6 Hz,
2H), 0.88 (dd, J= 12.0, 5.0 Hz, 3H).
Cell Toxicity Assay.
Cell toxicity assay was performed with the CellTiter-Glo
Luminescent Cell Viability Assay Kit which was purchased
from Promega Company. The instructions of manufacturer
were followed. Luminescence was recorded with a Promega
plate reader 22, 23
.
N
-ethyl-2,4-difluoro-N-(quinolin-8-yl)benzenesulfonamide
(20). To the solution of N-ethylquinolin-8-amine (232 mg, 1.35 Cell Fractionation and RNA Isolation
mmol) and 2,4-Difluorobenzenesulfonyl chloride (165 L, Cytoplasm and nuclear fractions were isolated by following the
1.23 mmol) in pyridine (3 mL). The mixture was stirred at manufacturer’s instructions of PARIS kit (Ambion). 293T cells
room temperature for 1 h. The reaction mixture was washed were washed with PBS and collected to 1.5mL tube. The
with HCl solution (2N) (3×20 mL) and extracted with fractionation buffer was added to the cells. The cells were
dichloromethane (20 mL). The organic phase was collected and gently agitated on ice for 5min. The lysate was then centrifuged
concentrated under vacuum.The product was purified by silica for 5min at 500g. The cytoplasmic fraction was collected from
gel chromatography using cyclohexane-ethyl acetate (30:1) as the upper 400L. The 400L ice-cold disruption buffer was
1
eluate to give 20 as white powder (135 mg, 32%). H NMR added to the nuclear fraction and vortex vigorously to lysate the
(500 MHz, CDCl3)
8.3, 1.7 Hz, 1H), 7.81 (d,
7.27 (dd, = 8.3, 4.1 Hz, 1H), 6.84 (ddd,
1H), 6.69 – 6.61 (m, 1H), 4.16 (s, 2H), 1.10 (t,
δ
8.46 (dd,
= 7.4 Hz, 2H), 7.61–7.44 (m, 2H), the supplied buffers and columns. Quantity of total RNA was
= 10.0, 8.8, 2.4 Hz, determined using NanoDrop 2000 spectrophotometer
= 7.2 Hz, 3H). (Thermo Fisher Scientific).24
J
= 4.1, 1.7 Hz, 1H), 8.10 (dd,
J
=
nuclei. RNA was then isolated from each of the fractions using
J
J
J
a
J
13C NMR (126 MHz, CDCl3)
11.3 Hz), 160.51 (dd, = 258.6, 12.7 Hz), 149.59, ELISA assay
145.12 , 136.18, 134.63, 134.37, 132.35 (d, = 10.1 Hz), The 293T cells were co-transfected with pMDLg/RRE and Rev,
129.34, 129.03, 126.30, 125.28 (dd, = 14.9, 3.4 Hz), or pMDLg/CTE and Rev, and treated with compound 20 for 2
121.24, 110.63 (dd, = 21.7, 3.2 Hz), 104.94 (t, J = 25.9 days. The supernatant was collected for ELISA assay. The p24
δ 165.36 (dd, J = 255.5,
J
J
J
J
Hz), 46.71 (d,
J
= 2.4 Hz), 14.98 (s).HRMS (ESI+) m/z: ELISA assays were performed according to manufacturer's
5
calcd for C17H15O2N2F2S [M + H]+ : 349.08168. Found: protocol
.
349.08198 (Δ = 0.86 ppm).
RT-PCR
Biology
We used about 1000ng total RNA for reverse transcriptions by
PrimeScript RT ragent kit (TaKaRa) according to the
High-Throughput Screen.
PDM628 and PCDNA3.1-Rev plasmidswere co-transfected into
293T cells in 96-well plate (Corning, Costar) and
Lipofectamine 2000 (Invitrogen) by following the instructions
of manufacturer. After transfection, 20155 drug-like set
compounds (Enamine, Monmouth Jct, NJ) were added using a
Tecan Freedom EVO150 (Tecan, Männedorf, Schweiz) with a
manufacturer’s
instructions,
followed
by
real-time
amplification using SYBR Premix Ex Taq (TaKaRa). The
cytoplasm fractions were normalized by actin and the nuclear
fractions were normalized by U6.
Acknowledgements
final concentration of 50 M. Column 12 received only DMSO
8 | J. Name., 2012, 00, 1‐3
This journal is © The Royal Society of Chemistry 2012

Page 9 of 9
Journal Name
Organic & Biomolecular Chemistry
ARTICLE
binding of alpha-helical HIV-1 Rev protein to the rev response element
RNA. Angew Chem Int Ed Engl 2007, 46, 9101-4.
This work was supported by National Special Research
Program for Important Infectious Diseases
(No.2013ZX10001004), Guangdong Recruitment Program of
Creative Research Groups (No.2009010058), National Basic
13. Tamura, S.; Kaneko, M.; Shiomi, A.; Yang, G. M.; Yamaura, T.;
View Article Online
Murakami, N. Unprecedented NES non-antagonistic inhibitor for nuclear
DOI: 10.1039/C4OB02247E
export of Rev from Sida cordifolia. Bioorg Med Chem Lett 2010, 20,
1837-9.
Research
Program
of
China
(973
Program)
(No.2010CB912202), National Natural Science Foundation of
China (No.30972620), Natural Science Foundation of
Guangdong(No.9251008901000022), Ministry of Education of
China New Teacher Fund (No.20130171120058), Natural
Science Foundation of Guangdong New Ph.D. Start-up
(S2012040006494). The HIV-1 infectious clone pNL4-3 was
obtained from the NIH AIDS Research & Reference Reagent
Program.
14. Luedtke, N. W.; Hwang, J. S.; Glazer, E. C.; Gut, D.; Kol, M.; Tor, Y.
Eilatin Ru(II) complexes display anti-HIV activity and enantiomeric
diversity in the binding of RNA. Chembiochem 2002, 3, 766-71.
15. Riguet, E.; Desire, J.; Boden, O.; Ludwig, V.; Gobel, M.; Bailly, C.;
Decout, J. L. Neamine dimers targeting the HIV-1 TAR RNA. Bioorg
Med Chem Lett 2005, 15, 4651-5.
16. Murakami, N.; Ye, Y.; Kawanishi, M.; Aoki, S.; Kudo, N.; Yoshida, M.;
Nakayama, E. E.; Shioda, T.; Kobayashi, M. New Rev-transport
inhibitor with anti-HIV activity from Valerianae Radix. Bioorg Med
Chem Lett 2002, 12, 2807-10.
17. Tamura, S.; Shiomi, A.; Kaneko, M.; Ye, Y.; Yoshida, M.; Yoshikawa,
M.; Kimura, T.; Kobayashi, M.; Murakami, N. New Rev-export inhibitor
from Alpinia galanga and structure-activity relationship. Bioorg Med
Chem Lett 2009, 19, 2555-7.
18. J. Fang, S. Kubota, B. Yang, N. Zhou, H. Zhang, R. Godbout, R.J.
Pomerantz, A DEAD box protein facilitates HIV-1 replication as a
cellular co-factor of Rev, Virology, 2004, 330, 471-480.
19. Zhou, X.; Luo, J.; Mills, L.; Wu, S.; Pan, T.; Geng, G.; Zhang, J.; Luo,
H.; Liu, C.; Zhang, H.: DDX5 facilitates HIV-1 replication as a cellular
co-factor of Rev. PloS one 2013, 8, e65040.
20. Fang, J; Kubota ,S; Yang, B; Zhou, N; Zhang, H; Godbout, R;
Pomerantza, R. A DEAD box protein facilitates HIV-1 replication as a
cellularco-factor of Rev. Virology. 2004, 330,471-480.
Notes
a Corresponding author : Institute of Human Virology, Key Laboratory of
Tropical Diseases Control of Ministry of Education of China, Zhongshan
School of Medicine, Sun Yat-sen University, Guangzhou, 510080,
Tel.:+86-20-87332588;fax:+86-20-87332588;e-mail:
baichuan@mil.sysu.edu.cn
b Corresponding author; Institute of Human Virology, Key Laboratory of
Tropical Diseases Control of Ministry of Education of China, Zhongshan
School of Medicine, Sun Yat-sen University, Guangzhou, 510080,
21. Zhang, H.; Yang, B.; Pomerantz, R. J.; Zhang, C.; Arunachalam, S. C.;
Gao, L. The cytidine deaminase CEM15 induces hypermutation in newly
synthesized HIV-1 DNA. Nature. 2003, 424, 94-98.
22. John A. Barltro, T. C. O., Ann H. Cory, Joseph G.Cory. 5-(3-
carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-
sulfophenyl)tetrazolium, inner salt (MTS) and related analogs of 3-(4,5-
dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reducing to
purple water-soluble formazans As cell-viability indicators. Bioorg. Med.
Chem. Lett. 1991, 1, 611-614
Tel.:
+86-20-87332588;
fax:
+86-20-87332588;
e-mail:
zhangh92@mail.sysu.edu.cn.
c These authors contributed to this work equally.
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