Arylazolyl(azinyl)thioacetanilides. Part 20: Discovery of novel purinylthioacetanilides derivatives as potent HIV-1 NNRTIs via a structure-based bioisosterism approach

Article abstract of DOI:10.1016/j.bmc.2016.07.041

By means of structure-based bioisosterism approach, a series of novel purinylthioacetanilide derivatives were designed, synthesized and evaluated as potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs). Some of the tested compounds were found to be active against wild-type (WT) HIV-1(IIIB) with EC₅₀in the range of 0.78–4.46?μM. Among them, LAD-8 displayed the most potent anti-HIV activity (EC₅₀?=?0.78?μM, SI?=?24). In addition, LBD-6 showed moderate activity against L100I mutant (EC₅₀?=?5.64?μM) and double mutant strain RES056 (EC₅₀?=?22.24?μM). Preliminary structure–activity relationships (SARs) were discussed in detail. Molecular modeling study was used to predict the optimal conformation in the NNRTI binding site, which may play a guiding role in further rational optimization.

Full text of DOI:10.1016/j.bmc.2016.07.041

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Arylazolyl(azinyl)thioacetanilides. Part 20: Discovery of novel
purinylthioacetanilides derivatives as potent HIV-1 NNRTIs via a
q
structure-based bioisosterism approach
a
a
a
a
a
a
a
Xueyi Lu , Xiao Li , Jiapei Yang , Boshi Huang , Dongwei Kang , Fabao Zhao , Zhongxia Zhou ,
b
b
b,
⇑
a,
⇑
a,
⇑
Erik De Clercq , Dirk Daelemans , Christophe Pannecouque , Peng Zhan , Xinyong Liu
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44 West Culture
Road, 250012 Ji’nan, Shandong, PR China
Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
b
a r t i c l e i n f o
a b s t r a c t
Article history:
By means of structure-based bioisosterism approach, a series of novel purinylthioacetanilide derivatives
were designed, synthesized and evaluated as potent HIV-1 non-nucleoside reverse transcriptase inhibi-
tors (NNRTIs). Some of the tested compounds were found to be active against wild-type (WT) HIV-1
Received 17 May 2016
Revised 19 July 2016
Accepted 20 July 2016
Available online xxxx
(
IIIB) with EC50 in the range of 0.78–4.46
activity (EC50 = 0.78 M, SI = 24). In addition, LBD-6 showed moderate activity against L100I mutant
EC50 = 5.64 M) and double mutant strain RES056 (EC50 = 22.24 M). Preliminary structure–activity
lM. Among them, LAD-8 displayed the most potent anti-HIV
l
(
l
l
Keywords:
HIV-1
AIDS
NNRTIs
relationships (SARs) were discussed in detail. Molecular modeling study was used to predict the optimal
conformation in the NNRTI binding site, which may play a guiding role in further rational optimization.
Ó 2016 Elsevier Ltd. All rights reserved.
Purinylthioacetanilide
Anti-HIV activity
Drug design
Bioisosterism
1
. Introduction
derivatives which exhibited potent antiretroviral activity against
WT and mutant HIV-1 strains, were selected as lead compounds
for structural modifications in our lab.
1
5
The reverse transcriptase (RT), on account of its multifunctional
roles in the replication of human immunodeficiency virus type 1
HIV-1), is an appealing target for exploring anti-HIV drugs with
Our lab has performed extensive optimizations on the aryla-
zolylthioacetanilide motif based on structure-based bioisosterism
principle, which lead to the identification of novel back-up deriva-
(
1,2
high efficiency and low toxicity. As one of the major components
of highly active antiretroviral therapy (HAART), non-nucleoside
reverse transcriptase inhibitors (NNRTIs) have shown notable
activity in clinical use.3 Unfortunately, the therapeutic potency
of NNRTIs is weakened by the rapid emergence of drug-resistant
1
6–25
tives as potent HIV-1 NNRTIs.
The results indicated that when
the five-membered azole moiety was superseded by a bunch of six-
membered heteroaromatic bioisosteres (such as pyrimidine, pyra-
zine and so on), most of the derivatives possessed potent anti-HIV
activities in cell culture assay (Fig. 1). Especially, compound
SPZ8b15 displayed the best anti-HIV (WT) activity with an EC50
,4
5
–8
HIV-1 mutants.
Thus, it is undoubtedly required of new NNRTIs
9
–12
with high efficacy and higher genetic barrier to resistance.
1
6
Owing to remarkable activities against HIV-1 wild-type (WT)
and some mutant strains, arylazolylthioacetanilides show attrac-
value of 18 nM. In continuation of these efforts, further attempts
using a scaffold hopping strategy to modify the central heterocycle
in our lab over the past few years led to the discovery of novel sub-
stituted imidazopyridinylthioacetanilides as potent HIV-1 inhibi-
1
3,14
tive structural characteristics as agents of HIV-1 NNRTIs.
VRX-480733 and RDEA-806 (in phase IIa clinical trials reported
in 2008) (Fig. 1), two representative 1,2,4-triazolythioacetanilide
2
6,27
tors (exemplified by compound XL5b16).
These encouraging
results further proved that modification of central heterocycle por-
tion of arylazolyl(azinyl)thioacetanilides appeared the most
promising strategy to optimize drug-target interactions and to
obtain novel NNRTIs with potent anti-HIV activity.
q
Parts 1–19: Refs. 16–27.
Corresponding authors. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail addresses: Christophe.Pannecouque@rega.kuleuven.be (C. Pannecouque),
⇑
zhanpeng1982@sdu.edu.cn (P. Zhan), xinyongl@sdu.edu.cn (X. Liu).
http://dx.doi.org/10.1016/j.bmc.2016.07.041
0
968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

2
X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
N
N
Cl
N
N
Cl
H
N
H
N
Br
Br
N
S
N
S
O
O
SO NH
2 2
COOK
VRX-480773
RDEA-806
Bioisosterism
N
N
N
NO2
N
NO2
N
Br
H
N
H
H
N
N
N
N
S
Cl
S
S
O
O
O
2 2
SO NH
SPZ6K
EC50 = 1.7 uM, SI = 23
SPZ8K
EC50 = 46 nM, SI = 2149
SPZ8b15
EC50 = 18 nM, SI = 1293
Scaffold hopping
N
N
Br
H
N
N
S
O
2 2
SO NH
R
2
R
2
XL5b16
H
N
R
1
H
N
R1
N
EC50 = 0.21 uM, SI = 46
N
N
N
R3
R
3
S
S
Cl
N
N
O
Cl
N
N
O
Bioisosterism
Target compounds
Figure 1. Design strategies of our previously reported arylazolyl(azinyl)thioacetanilides-based NNRTIs and the newly designed 2-chloro-purinylthioacetanilides series.
Herein, with the aim to discover novel NNRTIs with high
nitropyrimidin-4-amine (LAB). Then the nitro group of intermedi-
ate LAB was converted to amino LAC in the presence of SnCl . The
potency and enhanced interactions within the RT hydrophobic site,
a novel series of purin-8-ylthioacetanilides derivatives were
designed via a structure-based bioisosterism approach based on
the analysis of the ligand-receptor interactions, in which the five/
six-membered heterocycles was replaced by purine as a central
ring to orient other pharmacophore elements into the proper
geometry for binding. Notably, in the newly designed analogues,
the favorable naphthyl or a 4-cyclopropyl naphthyl were adapted
from lead compounds to better occupy the aromatic-rich sub-
pocket in NNRTI binding pocket (NNIBP). Meanwhile, diverse sub-
stituents endowed of various electronic and steric properties were
introduced in the phenyl ring of the anilide moiety to further
investigate potential interactions. Moreover, a methyl substituted
acetamide linker (propanamide) was also employed to probe SARs
in this region (Fig. 1). The synthesis, anti-HIV activities, prelimi-
nary SARs, and molecular modeling studies of novel substituted
2
pivotal intermediate LAD was achieved from LAC by cyclization
0
with 1,1 -thiocarbonyldiimidazole (TCDI). Treatment of LAD with
a set of 2-chloro-N-aryl-substituted acetamides or propanamides
in a rapid and convenient way to afford the target compounds
LAD-1 ꢀ LAD-11. In terms of LBD-2 ꢀ LBD-11, the key intermedi-
ate 4-cyclopropylnaphthalen-1-amine (LBA) was prepared from
commercially available 4-bromonaphthalen-1-amine (LAA) and
2
8
cyclopropylboronic acid via Suzuki reaction. Afterwards, the
same route of LAB was also used to give the target compounds
LBD-2 ꢀ LBD-11. The synthesized compounds (Table 1) were char-
1
acterized by proton nuclear magnetic resonance ( H NMR), carbon
1
3
nuclear magnetic resonance ( C NMR), and electrospray ionization
mass spectrometry (ESI-MS) spectral data.
2.2. Anti-HIV activity evaluation
2
-chloro-purin-8-ylthioacetanilides will be discussed in detail.
The newly synthesized purinylthioacetanilides derivatives were
evaluated for anti-HIV activity in MT-4 cell cultures infected by
HIV-1 IIIB strain (WT), a panel of NNRTIs-resistant HIV-1 single
RT mutant strains (L100I, K103N, E138K, Y181C, Y188L), two dou-
ble RT mutant strains (RES056: K103N/Y181C; F227L/V106A) and
HIV-2 strain ROD, respectively. The FDA-approved drugs nevirap-
ine (NVP), delavirdine (DLV), efavirenz (EFV) and etravirine (ETV)
were chosen as reference drugs. Comparisons of the antiviral inhi-
bitory concentration (EC50), cytotoxic concentration (CC50), and SI
values (selectivity index, given by the CC50/EC50 ratio) for different
compounds are depicted in Tables 1 and 2.
2
2
. Results and discussions
.1. Chemistry
The general synthetic steps adopted to obtain the desired com-
pounds LAD-1 ꢀ LAD-11 and LBD-2 ꢀ LBD-11 were straightfor-
wardly outlined in Scheme 1. The compounds LAD-1 ꢀ LAD-11
were synthesized from the commercially available starting mate-
rial naphthalen-1-amine (LAA). Firstly LAA was reacted with 2,4-
dichloro-5-nitropyrimidine in the presence of trimethylamine
As shown in Table 1, most of the test compounds inhibited HIV-
1 IIIB replication at micromolar concentrations. In particular, the
(
3
Et N) in THF at 0 °C to afford 2-chloro-N-(naphthalen-1-yl)-5-
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X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
R2
NH2
N
NO2
N
H
N
N
N
R1
N
N
NH
2
Cl
Cl
N
SH
R3
iii Cl
N
NH
iv
v
N
S
ii
Cl
N
NH
N
N
O
R
R
R
R
R
LAA: R = H;
LBA: R = cyclopropyl
i
LAB: R = H;
LBB: R = cyclopropyl
LAC: R = H;
LBC: R = cyclopropyl
LAD: R = H;
LBD: R = cyclopropyl
LAD-1~LAD-11: R = H;
LBD-2~LBD-11: R = cyclopropyl
Scheme 1. Reagents and conditions: (i) cyclopropylboronic acid, Pd(PPh
3
)
4
, K
3
PO
4
, toluene/H
2
O (25:1), N
2
atmosphere, 100 °C, 6 h; (ii) 2,4-dichloro-5-nitropyrimidine, Et
3
N,
0
THF, 0 °C, 30 min, rt, 10 h; (iii) SnCl
2
, EtOH, 80 °C, 6 h; (iv) 1,1 -thiocarbonyldiimidazole (TCDI), Et
3
N, THF, 70 °C, overnight; (v) K
2
CO
3
2
, acetone, substituted ClCH CONHAr or
3
substituted BrCH(CH )CONHAr, rt, 8 h (parallel reaction).
Table 1
Anti-HIV activity, cytotoxicity and selectivity index of target compounds
R2
N
R1
H
N
N
Cl
N
S
R3
N
O
R
LAD-1~LAD-11: R = H;
LBD-2~LBD-11: R = cyclopropyl
Compds
R1
R2
R3
HIV-1(IIIB)
RES056
M)
CC50b
(lM)
EC50a
(l
M)
SIc
EC50a
(l
SIc
LAD-1
LAD-2
LAD-3
LAD-4
LAD-5
LAD-6
LAD-7
LAD-8
H
H
H
H
H
H
H
CONH
2
P28.84
4.46 ± 0.16
>14.26
65
23
<1
12
5
>156.31
>103.70
ND
<1
<1
156.30 ± 18.28
103.70 ± 78.59
14.25 ± 6.17
21.56 ± 1.05
22.74 ± 1.93
21.83 ± 2.39
23.56 ± 2.57
18.99 ± 2.93
21.51 ± 3.40
156.47 ± 29.60
109.00 ± 32.09
112.60 ± 28.93
16.17 ± 4.09
11.22 ± 5.32
6.39 ± 1.82
55.85 ± 47.09
25.07 ± 9.72
15.90 ± 1.40
17.61 ± 1.33
153.42 ± 14.07
150.21 ± 27.71
15.02
SO
CF
2
NH
2
d
3
Br
Cl
Br
Cl
Br
Cl
Br
Br
H
CONH
CONH
CONH
CONH
2
2
2
2
1.80 ± 0.54
4.56 ± 1.13
1.86 ± 0.64
2.73 ± 0.80
0.78 ± 0.04
1.07 ± 0.15
1.66 ± 0.56
3.43 ± 0.59
18.70 ± 3.17
>16.17
>21.56
>22.74
>21.83
>23.56
>18.98
>21.52
>156.47
>109.00
>112.61
<1
<1
<1
<1
<1
<1
<1
<1
<1
H
Me
Me
Me
Me
H
H
H
H
H
12
9
SO
SO
2
NH
NH
2
24
20
94
32
6
<1
<1
<1
8
<1
6
4
<1
<1
>58
>1212
733
733
LAD-9
2
2
LAD-10
LAD-11
LBD-2
LBD-3
LBD-4
LBD-5
LBD-6
LBD-7
LBD-8
COOMe
COOEt
SO NH
2 2
d
H
CF
3
ND
d
Br
Cl
Br
Cl
Br
Cl
Br
Br
CONH
CONH
CONH
CONH
2
>11.22
>6.39
ND
d
H
2
2
2
ND
Me
Me
Me
Me
H
7.22 ± 1.98
>25.07
2.75 ± 0.41
4.04 ± 0.63
>153.42
>150.22
0.25836
0.00523
0.00335
22.24 ± 11.06
10
d
ND
SO
SO
2
NH
NH
2
>15.90
P5.74
<1
63
LBD-9
2
2
d
LBD-10
LBD-11
Nevirapine
Efavirenz
Etravirine
Delavirdine
COOMe
COOEt
ND
d
H
ND
d
ND
d
ND
6.34
2.46
36.8
d
ND
d
0.03834
ND
a
b
c
EC50: concentration of compound required to achieve 50% protection of MT-4 cell against HIV-1-induced cytotoxicity, as determined by the MTT method.
CC50: concentration required to reduce the viability of mock-infected cells by 50%, as determined by the MTT method.
SI: selectivity index (CC50/EC50).
ND: not determined.
d
most potent compound LAD-8 had an EC50 value of 0.78
SI value of 28, which was similar to that of reference drug nevirap-
ine (EC50 = 0.258 M, SI > 58). Besides, some other compounds,
l
M and a
newly synthesized purinylthioacetanilides demonstrated compa-
rable potency. Thus, the purine ring is a meaningful attempt for
providing new opportunities for further optimization.
l
LAD-2, LBD-9, LAD-5 ꢀ LAD-11, LBD-8 and LAD-4 also exhibited
It is worth noting that among all the tested compounds, only
moderate to good activities against HIV-1 strain IIIB with EC50 val-
LBD-6 showed moderate activity against L100I mutant (EC50
5.64 M) and double mutant strain RES056 (EC50 = 22.24 M), a
frequently encountered double mutant.
=
ues in the range of 1.07–4.46
l
M. Overall, compared with the pre-
l
l
2
6
vious reported imidazopyridinylthioacetanilides in our lab, the
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

4
X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
1
Table 2
(5) For the SCRs, when the H at R position was replaced by the
methyl group, the cytotoxicity was enhanced by about four
to five-times in LBD series (LBD-4/LBD-6, LBD-5/LBD-7),
while it changed a little in LAD series (LAD-4/LAD-6, LAD-
5/LAD-7). The difference was probably affected by the bind-
ing mode of 4-cyclopropylnaphthalenyl group to RT. What’s
more, the ortho-substitution at the phenyl ring of the anilide
Anti-HIV mutant strain activity, cytotoxicity and selectivity index of LBD-6 and
control drugs
Compds
Strains
EC50a
(lM)
CC50b
(lM)
SIc
LBD-6
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
5.64 ± 0.52
P48.08
>55.84
>55.84
P65.12
>55.84
55.85 ± 47.09
55.85 ± 47.09
55.85 ± 47.09
55.85 ± 47.09
55.85 ± 47.09
55.85 ± 47.09
8
61
<1
<1
61
<1
2
moiety (R group) was found to exert a large impact on the
cytotoxicity, which might result from the electronic nature
or the steric demand of the ortho substitution as well.
Nevirapine
Efavirenz
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
0.83 ± 0.32
6.78 ± 0.08
6.21 ± 0.08
9.61 ± 1.86
0.23 ± 0.06
7.17 ± 1.86
>15.02
>15.02
>15.02
>15.02
>15.02
>15.02
>18
>2
>2
>2
>64
>2
To sum up, these studies provide some constructive information
for further modification of purinylthioacetanilides as potent
NNRTIs.
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
0.10 ± 0.01
0.12 ± 0.04
0.0068 ± 0.0006
0.34 ± 0.02
0.0051 ± 0.0007
0.21 ± 0.008
>6.34
>6.34
>6.34
>6.34
>6.34
>6.34
>62
>52
>928
>19
>1235
>30
2
.3. Inhibition of HIV-1 RT
For the sake of further confirming the drug target, LAD-8 was
selected to be tested in an enzymatic assay against highly purified
recombinant HIV-1 RT using hybrid poly (A)-oligo (dT)15
(9A260 nm/mL), lyophilizate as template primer. The result indi-
cated that LAD-8 exhibited moderate inhibition of enzymatic activ-
Etravirine
Delavirdine
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
0.0066 ± 0.002
0.0032 ± 0.0002
0.015 ± 0.0003
0.022 ± 0.0004
0.014 ± 0.002
0.017 ± 0.0004
2.46
2.46
2.46
2.46
2.46
2.46
370
773
159
111
179
143
ity with an IC50 value of 6.42
reference drug NVP (IC50 = 8.78
l
M, which is slightly lower than the
M) (Table 3). This result indicated
l
that the newly designed purinylthioacetanilides could inhibit the
HIV-1 replication by targeting HIV-1 RT, and they can be classified
as typical NNRTIs.
L100I
K103N
Y181C
Y188L
E138K
F227L + V106A
2.54 ± 0.31
3.00
2.46 ± 0.54
0.84 ± 0.01
0.051 ± 0.006
2.27 ± 0.14
36.80
36.80
36.80
36.80
36.80
36.80
14
12
15
44
715
16
2
.4. Molecular modeling
In purpose of better understanding the structural basis of their
a
EC50: concentration of compound required to achieve 50% protection of MT-4
binding mode and accounting for SARs of the newly synthesized
compounds, molecular modeling analysis of compounds LAD-8,
LAD-6, VRX-480773 were performed by means of software Auto-
Dock 4.2 and MGL tools 1.5.6, and the docking results were shown
by PyMOL. The hypothetical binding mode in the NNIBP is shown
in Figure 2.
cell against HIV-1-induced cytotoxicity, as determined by the MTT method.
b
CC50: concentration required to reduce the viability of mock-infected cells by
5
0%, as determined by the MTT method.
c
SI: selectivity index (CC50/EC50).
The SARs and structure-cytotoxicity relationships (SCRs) of
As indicated in Figure 2a, the naphthalene ring of LAD-8 located
in the hydrophobic binding pocket, forming a p–p stacking interac-
independent sections of purinylthioacetanilides were summarized
as follows:
tion with the aromatic residues of Tyr188, Phe227, and Trp229. The
anilide moiety was close to Pro236, and the sulfonamide moiety
points toward the solvent exposed region. Besides, carbonyl oxy-
gen of LAD-8 forms a hydrogen bond with the backbone N–H of
Lys103. In addition, the oxygen of 4-substituent at the phenyl ring
interacts to Val106 by a hydrogen bond. These two hydrogen bonds
improved the affinity between inhibitor and RT. The purine moiety,
which settled down in the middle of NNIBP, played an important
role in anchoring the naphthalene ring and the anilide moiety.
In Figure 2b, LBD-6 exhibited similar binding mode to the lead
compound VRX-480773. As described above, LBD-6 showed mod-
(
1) Unexpectedly, it is the naphthalenyl group rather than the 4-
cyclopropylnaphthalenyl group that helps to increase the
anti-HIV activities and decrease cytotoxicities (compared
LAD series to LBD series).
(
2) When the methyl group was introduced into R1 group, it
looks like that the anti-HIV activities and cytotoxicities were
slightly increased (LAD-5 < LAD-7).
2
(
3) It is the ortho-substitution at the phenyl ring (R group) of
the anilide moiety that contribute to a potent activity,
according to the sequence: Br (LAD-4, EC50 = 1.80
LAD-5, EC50 = 4.56 M) > H (LAD-1, EC50 = 28.84
Briefly, consistent with the investigations in other aryla-
lM) > Cl
erate activity against L100I mutant (EC50 = 5.64
lM) and double
(
l
lM).
mutant strain RES056 (EC50 = 22.24 M). The residue of isoleucine
l
in L100I mutant strain was more flexible than the residue of
leucine in WT, which was likely to loosen up the rejection of the
aromatic-rich sub-pocket to 4-cyclopropyl. On the other side,
double mutant strain RES056 perhaps took advance of a larger
zolylthioacetanilide series,1
7,18
these results implied that
the electronic nature or the steric demand of the ortho sub-
stitution has an effect on the activities of the purinylthioac-
etamide derivatives.
(
4) It is also interesting to find that the nature of the para posi-
3
tion of the anilide moiety (R group) plays an important role
Table 3
in activity against HIV-1. When the sulfonamide group was
in charge of the para position of the anilide moiety, the
Inhibitory activity of LAD-8 against HIV-1 RT
bioactivity was strikingly increased (LAD-2, EC50 = 4.46
LAD-8, EC50 = 0.78 M; LAD-9, EC50 = 1.07 M; LBD-8,
EC50 = 2.75 M; LBD-9, EC50 = 4.04 M). It suggested that
the nature of the sulfonamide group acclimated to the
chemical environment in this domain of RT.
l
M;
Compd
LAD-8
NVP
8.78
TMC-125
3.10
l
l
IC50 a
(l
M)
6.42
l
l
a
IC50: 50% of the inhibitory concentration of tested compound required to inhibit
biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by
0%.
5
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X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 2. (a) Predicted binding mode and molecular docking of LAD-8 into the allosteric site of HIV-1 RT (PDB code: 3DLG). (b) Predicted binding mode and molecular
docking of LBD-6 (purple) with VRX-480773 (blue) into the allosteric site of HIV-1 RT (PDB code: 3DOL).
aromatic-rich sub-pocket to accommodate 4-cyclopropyl. Accord-
ingly, the introduction of 4-cyclopropyl was probably beneficial
to inhibiting mutant strains.
All in all, the docking simulation results helped to interpret the
binding mode of the purinylthioacetanilides to WT RT, which pro-
vided us valuable information for further design of more potent
NNPTIs with this scaffold.
0.045 mmol), K
26 mL) was stirred at 100 °C for 6 h under N
3
PO
4
(0.33 g, 1.58 mmol) in toluene/H
2
O (25:1,
2
. After cooling, toluene
was evaporated under reduced pressure and then a small amount
of water was added. The mixture was extracted with ethyl acetate
(3 ꢁ 10 mL). The combined extracts were dried with anhydrous
sodium sulfate, filtered off, and then the solvent was removed
under reduced pressure to give the crude product, which was puri-
fied by chromatography on silica gel eluted with acidified (several
drops of acetic acid) ethyl acetate–petroleum ether (1:4) to afford
crude intermediate LBA. Dark purple oil, yield: 63.2%.
3
. Conclusion
In summary, in this study, a series of novel purinylthioac-
4
.1.2. General procedure for the synthesis of 2-chloro-N-
etanilides were discovered as potent NNRTIs via a structure-based
bioisosterism approach. Some compounds displayed higher
potency in inhibiting HIV-1 replication, among which LAD-8 was
classified as the most active compound with favorable inhibitory
(
naphthalen-1-yl)-5-nitropyrimidin-4-amine (LAB) and 2-
chloro-N-(4-cyclopropylnaphthalen-1-yl)-5-nitropyrimidin-4-
amine (LBB)
A mixed solution of naphthalen-1-ylboronic acid (0.074 g,
potency against HIV-1IIIB (EC50 = 0.78
of enzymatic activity (IC50 = 8.78 M). In addition, LBD-6 displayed
moderate activity against a frequently encountered HIV-1 double
mutant strain RES056 (EC50 = 22.24 M). The valuable information
lM) and moderate inhibition
0
.52 mmol) (or crude intermediate LBA (0.094 g, 0.52 mmol)) and
l
Et N (79.04 mmL, 0.57 mmol) in THF (10 mL) was dropwised into
a solution of 2,4-dichloro-5-nitropyrimidine (0.10 g, 0.52 mmol)
in THF (10 mL) at 0 °C. The reaction mixture was stirred from
0
3
l
from preliminary SARs, SCRs and docking studies are beneficial to
further optimization of this versatile scaffold.
°C to room temperature for about 10 h. The resulting solution
was concentrated and chromatographed on silica gel using ethyl
acetate–petroleum ether (5:1) to give an orange solid LAB, yield:
4
4
. Experimental section
8
5.8%; LBB: off-red solid, yield: 81.30%, m/z 341.4 [M+1], 343.5
[
M+3], C17 13ClN [Exact Mass: 340.07].
H
4 2
O
.1. Synthetic procedures and analytical data
4
4
.1.3. General procedure for the synthesis of 2-chloro-N -
Melting points (mp) were achieved by a micromelting point
(
naphthalene-1-yl)pyrimidine-4,5-diamine (LAC) and 2-chloro-
-(4-cyclopropylnaphthalen-1-yl)pyrimidine-4,5-diamine
(LBC)
A solution of LAB (0.072 g, 0.21 mmol) (or LBB (0.063 g,
.21 mmol)) and SnCl (0.20 g, 1.05 mmol) in ethanol (20 mL)
was stirred at 80 °C for 6 h under N . After cooling, ethanol was
apparatus (Tian Jin Analytical Instrument Factory, Nankai, Tianjin,
China). Mass spectra were carried out on a G1313A Standard LC
Autosampler (Agilent). 1H NMR and C NMR spectra were per-
formed on a Bruker AV-400 spectrometer (Bruker BioSpin, Switzer-
N
4
13
0
2
6
land), making use of solvents as indicated (DMSO-d ). Chemical
shifts were recorded in d values (ppm) with tetramethylsilane as
the internal reference, while J values were recorded in hertz (Hz).
2
removed under reduced pressure and then ethyl acetate (10 mL)
was added. Afterwards, aqueous phase was modulated to pH = 8
by adding concentrated solution of NaHCO . The mixture was
3
Thin layer chromatography was performed on pre-coated HUAN-
GHAIÒ HSGF254, 0.15–0.2 mm TLC-plates (Yantai Jiangyou Silica
extracted by ethyl acetate (2 ꢁ 10 mL). The combined organic layer
was dried with anhydrous magnesium sulfate, filtered off, and then
the solvent was evaporated under vacuo. The residue was dis-
solved with dichloromethane and chromatographed on silica gel
using methanol-dichloromethane system. Pure fractions were col-
lected and concentrated, giving the desired solid LAC, yield: 54.0%.
Gel Development Co., Ltd., Yantai, Shandong, China). Flash column
chromatography took advantage of columns packed with silica gel
6
0 (200–300 mesh) (Qingdao waves silica gel desiccant Co., Ltd,
Qingdao, China).
4
.1.1. General procedure for the synthesis of 4-cyclopropylnap-
ESI-MS: m/z 271.5 [M+1], 273.4 [M+3], C14
70.07]; LBC: solid, yield: 55.2%, ESI-MS: m/z 311.5 [M+1], 313.4
M+3], C17 15ClN [Exact Mass: 310.10].
4
H11ClN [Exact Mass:
hthalen-1-amine (LBA)
A solution of 4-bromonaphthalen-1-amine (0.10 g, 0.45 mmol),
3 4
cyclopropylboronic acid (0.05 g, 0.59 mmol), Pd(PPh ) (0.051 g,
2
[
H
4
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

6
X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4
.1.4. General procedure for the synthesis of 2-chloro-9-
6
DMSO-d , ppm) d: 10.82 (s, 1H, NH), 9.05 (s, 1H, purine-H), 8.31
(
naphthalen-1-yl)-9H-purine-8-thiol (LAD) and 2-chloro-9-(4-
(d, J = 8.1 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
thalene-H), 7.85 (dd, J = 7.3, 1.1 Hz, 1H, naphthalene-H), 7.82–
7.76 (m, 3H, 1 ꢁ naphthalene-H, 2 ꢁ Ph-H), 7.72–7.66 (m, 3H,
1 ꢁ naphthalene-H, 2 ꢁ Ph-H), 7.58 (td, J = 7.6, 1.2 Hz, 1H, naph-
thalene-H), 7.30 (d, J = 8.4 Hz, 1H, naphthalene-H), 4.46 (s, 2H,
cyclopropylnaphthalen-1-yl)-9H-purine-8-thiol (LBD)
To a solution of LAC (0.38 g, 1.40 mmol) (or LBC (0.44 g,
0
1
.40 mmol)) and 1,1 -thiocarbonyldiimidazole (1.00 g, 5.61 mmol)
3
in anhydrous THF (100 mL) was added Et N (0.78 mL, 5.61 mmol)
at once. The reaction solution was stirred at room temperature
overnight. The solvent was evaporated in vacuo and ethyl acetate
1
3
2
CH ).
6
C NMR (100 MHz, DMSO-d , ppm) d: 166.07, 158.98,
156.93, 152.51, 147.08, 142.71, 134.47, 133.94, 131.77, 129.63,
129.13, 128.65, 128.49, 128.06, 127.76, 126.69, 126.65, 126.35,
126.12, 124.23, 123.90, 123.43, 122.45, 119.53 (2 ꢁ C), 36.97. ESI-
(
30 mL) was added. The mixture was acidified with diluted
hydrochloric acid and then extracted by ethyl acetate
(
3 ꢁ 15 mL). After washing with saturated brine (2 ꢁ 10 mL), the
3 5
MS: m/z 513.9 [M+1], 515.7 [M+3], C24H15ClF N OS [Exact Mass:
organic phase was dried over anhydrous magnesium sulfate, fil-
tered off, and then the solvent was removed under reduced pres-
sure to give the crude product, which were purified by
chromatography on silica gel eluted with acidified ethyl acetate–
petroleum ether system (1:1) to afford intermediate LAD. Yield:
513.06].
4.1.5.4. 3-Bromo-4-(2-((2-chloro-9-(naphthalen-1-yl)-9H-purin-
8-yl)thio)acetamido)benzamide (LAD-4). White powder,
yield: 67.2%. Mp: 218–221 °C. H NMR (400 MHz, DMSO-d
1
6
,
4
0.0%. ESI-MS: m/z 311.3 [Mꢂ1], 313.3 [M+1], C15
H
9
ClN
4
S [Exact
ppm) d: 10.04 (s, 1H, NH), 9.12 (s, 1H, purine-H), 8.31 (d,
J = 8.2 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
Mass: 312.02]; LBD: solid yield: 38.7%, ESI-MS: m/z 351.4 [Mꢂ1],
3
4
0
53.4 [M+1], C18
4
H13ClN S [Exact Mass: 352.05].
thalene-H), 8.16 (s, 1H, Ph-H), 8.05 (s, 1H, CONH
H, 1 ꢁ naphthalene-H, 2 ꢁ Ph-H), 7.79 (t, J = 8 Hz, 1H, naph-
thalene-H), 7.69 (td, J = 7.6, 0.8 Hz, 1H, naphthalene-H), 7.56 (td,
J = 8, 0.8 Hz, 1H, naphthalene-H), 7.48 (s, 1H, CONH ), 7.30 (d,
J = 8.4 Hz, 1H, naphthalene-H), 4.55–4.35 (m, 2H, CH
(100 MHz, DMSO-d , ppm) d: 166.44, 166.36, 158.95, 156.94,
2
), 7.91–7.82 (m,
3
.1.5. General procedure for the synthesis of target compounds
A solution of LAD (0.163 g, 0.60 mmol) (or LBD (0.187 g,
2
1
3
.60 mmol)), K
2
CO
3
(0.10 g, 0.72 mmol), and different 2-chloro-N-
2
). C NMR
(
2
substituted aromatic group)acetamides (0.72 mmol) or different
-bromo-N-(substituted aromatic group)acetamides (0.72 mmol),
6
152.59, 147.25, 138.78, 134.47, 133.90, 132.63, 132.34, 131.79,
129.61, 129.14, 128.66, 128.45, 128.08, 127.87, 127.77, 126.36,
124.98, 122.44, 116.11, 36.29. ESI-MS: m/z 565.1 [Mꢂ1], 567.3
in acetone (20 mL) was stirred at room temperature for 8 h and
then evaporated in vacuo. The residue was dissolved with a small
amount dichloromethane to chromatograph on silica gel with ethyl
acetate–petroleum ether system or methanol-dichloromethane
system. Pure fractions were collected and concentrated to afford
the desired compounds.
6 2
[M+1], 569.3 [M+3], C24H16BrClN O S [Exact Mass: 565.99].
4.1.5.5. 3-Chloro-4-(2-((2-chloro-9-(naphthalen-1-yl)-9H-purin-
8-yl)thio)acetamido)benzamide (LAD-5). White powder,
yield: 76.4%. Mp: 217–221 °C. H NMR (400 MHz, DMSO-d
1
6
,
4
.1.5.1. 4-(2-((2-Chloro-9-(naphthalen-1-yl)-9H-purin-8-yl)thio)
acetamido)benzamide (LAD-1). White powder, yield:
3.0%. Mp: 258–261 °C. H NMR (400 MHz, DMSO-d , ppm) d:
0.67 (s, 1H, NH), 9.06 (s, 1H, purine-H), 8.31 (d, J = 8.1 Hz, 1H,
ppm) d: 10.15 (s, 1H, NH), 9.08 (s, 1H, purine-H), 8.31 (d,
J = 8.2 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
thalene-H), 8.05 (s, 1H, CONH ), 8.00 (d, J = 1.8 Hz, 1H, Ph-H),
2
7.97 (d, J = 8.5 Hz, 1H, naphthalene-H), 7.90–7.81 (m, 2H, naph-
thalene-H, Ph-H), 7.81–7.76 (m, 1H, naphthalene-H), 7.69 (t,
J = 7.4 Hz, 1H, naphthalene-H), 7.57 (t, J = 8 Hz, 1H, naphthalene-
1
7
1
6
naphthalene-H), 8.31 (d, J = 8.1 Hz, 1H, naphthalene-H), 7.88 (s,
1
1
7
2
H, CONH ), 7.87–7.82 (m, 3H, 1naphthalene-H, 2 Ph-H), 7.79 (t,
H, naphthalene-H), 7.70 (td, J = 8.4, 0.8 Hz, 1H, naphthalene-H),
.64 (s, 1H, Ph-H), 7.62 (s, 1H, Ph-H), 7.58 (td, J = 8.4, 0.8 Hz, 1H,
H), 7.48 (s, 1H, CONH
2
), 7.30 (d, J = 8.4 Hz, 1H, naphthalene-H),
). C NMR (100 MHz, DMSO-d , ppm) d:
6
1
3
4.59–4.37 (m, 2H, CH
2
naphthalene-H), 7.31 (d, J = 8.5 Hz, 1H, naphthalene-H), 7.28 (s,
166.51, 166.45, 159.00, 156.93, 152.58, 147.14, 137.52, 134.47,
133.90, 132.05, 131.79, 129.61, 129.13 (2 ꢁ C), 128.66, 128.46,
128.08, 127.77, 127.35, 126.35, 125.12, 124.20, 122.41, 36.38.
13
1
H, CONH
2 2 6
), 4.45 (s, 2H, CH ). C NMR (100 MHz, DMSO-d ,
ppm) d: 167.76, 165.72, 159.04, 156.93, 152.50, 147.07, 141.72,
1
1
34.47, 133.95, 131.76, 129.63, 129.13, 128.95 (3 ꢁ C), 128.65,
ESI-MS: m/z 523.3 [M+1], 525.3 [M+3], 527.8 [M+5], 540.7 [M
+
28.51, 128.07, 127.77, 126.36, 122.46, 118.71 (2 ꢁ C), 37.01. ESI-
4
+NH ], C24H16Cl
2
N
6
O
2
S [Exact Mass: 522.04].
+
MS: m/z 489.3 [M+1], 491.3 [M+3], 511.4 [M+Na ], C24
6
H17ClN O
2
S
[
Exact Mass: 488.08].
4.1.5.6. 3-Bromo-4-(2-((2-chloro-9-(naphthalen-1-yl)-9H-purin-
-yl)thio)propanamido)benzamide (LAD-6). White powder,
yield: 62.1%. Mp: 258–261 °C. 1H NMR (400 MHz, DMSO-d
ppm) d: 10.18 (d, J = 23.3 Hz, 1H, NH), 9.17 (d, J = 7.5 Hz, 1H, pur-
ine-H), 8.30 (d, J = 8.2 Hz, 1H, naphthalene-H), 8.23–8.12 (m, 2H,
8
4
.1.5.2. 2-((2-Chloro-9-(naphthalen-1-yl)-9H-purin-8-yl)thio)-
White powder,
yield: 68.3%. Mp: 268–271 °C. H NMR (400 MHz, DMSO-d
6
,
N-(4-sulfamoylphenyl)acetamide (LAD-2).
1
6
,
ppm) d: 10.80 (s, 1H,NH), 9.05 (s, 1H, purine-H), 8.31 (d,
J = 8.1 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
thalene-H), 7.85 (dd, J = 7.3, 1.2 Hz, 1H, naphthalene-H), 7.82–
1 ꢁ naphthalene-H, 2 ꢁ Ph-H), 8.06 (s, 1H, CONH
2
), 7.92–7.73 (m,
4H, 2 ꢁ naphthalene-H, 2 ꢁ Ph-H), 7.68 (dd, J = 16.1, 8.2 Hz, 1H,
naphthalene-H), 7.62–7.51 (m, 1H, naphthalene-H), 7.49 (s, 1H,
7
.71 (m, 5H), 7.69 (td, J = 8, 1.2 Hz, 1H, naphthalene-H), 7.59 (td,
CONH
J = 6.9 Hz, 1H, CH), 1.66 (d, J = 8 Hz, 3H, CH
DMSO-d ppm) d: 169.83, 166.34, 158.39, 156.75, 152.68,
2
), 7.28 (dd, J = 25.1, 8.4 Hz, 1H, naphthalene-H), 5.12 (q,
1
3
J = 8.0, 1.2 Hz, 1H, naphthalene-H), 7.30 (d, J = 10.8 Hz, 3H, 1 naph-
thalene-H, 2 Ph-H), 4.45 (s, 2H, CH
3
)
C NMR (100 MHz,
13
2
). C NMR (100 MHz, DMSO-d
6
,
6
,
ppm) d: 165.98, 158.99, 156.92, 152.52, 147.08, 142.02, 139.20,
147.36, 138.63, 134.45, 133.93, 132.82, 132.33, 131.80, 129.56,
129.14, 128.70, 128.41, 128.13, 127.89, 127.78, 126.34, 125.27,
122.39, 116.85, 45.83, 18.86. ESI-MS: m/z 581.2 [M+1], 583.1 [M
1
1
34.47, 133.94, 131.77, 129.63, 129.14, 128.66, 128.49, 128.06,
27.77, 127.28 (2C), 126.36, 122.44, 119.22 (2 ꢁ C), 36.93. ESI-
MS: m/z 525.3 [M+1], 527.2 [M+3], C23
H17ClN
6
O
3
S
2
[Exact Mass:
6 2
+3], 585.2 [M+5], C25H18BrClN O S [Exact Mass: 580.01].
5
24.05].
4
.1.5.7. 3-Chloro-4-(2-((2-chloro-9-(naphthalen-1-yl)-9H-purin-
8-yl)thio)propanamido)benzamide (LAD-7). White powder,
yield: 73.0%. Mp: 257–259 °C. 1H NMR (400 MHz, DMSO-d
ppm) d: NMR (400 MHz, DMSO) d: 10.28 (d, J = 28.5 Hz, 1H, NH),
4
.1.5.3. 2-((2-Chloro-9-(naphthalen-1-yl)-9H-purin-8-yl)thio)-
N-(4-(trifluoromethyl)phenyl)acetamide
powder, yield: 78.2%. Mp: 218–221 °C.
(LAD-3).
White
6
,
1
H
NMR (400 MHz,
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
9
(
(
.13 (s, 1H, purine-H), 8.30 (d, J = 8.2 Hz, 1H, naphthalene-H), 8.18
dd, J = 8.2, 4.0 Hz, 1H, naphthalene-H), 8.05 (s, 1H, CONH ), 8.00
dd, J = 5.8, 1.2 Hz, 1H, Ph-H), 7.95 (d, J = 8.5 Hz, 1H, naphthalene-
4.1.5.11. Ethyl 3-bromo-4-(2-((2-chloro-9-(naphthalen-1-yl)-
9H-purin-8-yl)thio)acetamido)benzoate (LAD-11). White
NMR (400 MHz,
, ppm) d: 10.09 (s, 1H, NH), 9.12 (s, 1H, purine-H), 8.31
(d, J = 8.2 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
thalene-H), 8.15 (d, J = 1.9 Hz, 1H, Ph-H), 8.01 (d, J = 8.6 Hz, 1H,
Ph-H), 7.94 (dd, J = 8.5, 1.9 Hz, 1H, Ph-H), 7.86 (dd, J = 7.3, 1.0 Hz,
1H, naphthalene-H), 7.81–7.75 (m, 1H, naphthalene-H), 7.71–
7.66 (m, 1H, naphthalene-H), 7.60–7.53 (m, 1H, naphthalene-H),
7.30 (d, J = 8.4 Hz, 1H, naphthalene-H), 4.46 (q, J = 15.5 Hz, 2H,
2
1
powder, yield: 73.0%. Mp: 187–190 °C.
DMSO-d
H
H), 7.91–7.81 (m, 2H, Ph-H), 7.77 (dd, J = 15.3, 7.4 Hz, 1H, naph-
thalene-H), 7.68 (dd, J = 16.6, 8.3 Hz, 1H, naphthalene-H), 7.62–
7
J = 29.4, 8.4 Hz, 1H, naphthalene-H), 5.15 (q, J = 7.0 Hz, 1H, CH),
1
6
2
.51 (m, 1H, naphthalene-H), 7.49 (s, 1H, CONH ), 7.28 (dd,
13
.65 (d, J = 8.0 Hz, 3H, CH
3
)
6
C NMR (100 MHz, DMSO-d , ppm)
d: 170.00, 166.43, 158.42, 156.74, 152.69, 147.23, 137.34, 134.45,
1
1
1
33.93, 132.30, 131.81, 129.56, 129.13 (2 ꢁ C), 128.70, 128.37,
28.11, 127.78, 127.37, 126.33, 125.69, 124.54, 122.38, 45.87,
SCH
2
), 4.31 (q, J = 7.1 Hz, 2H, OCH
C NMR (100 MHz, DMSO-d , ppm) d: 166.64, 164.55, 158.92,
156.93, 152.60, 147.28, 140.57, 134.46, 133.89, 133.82, 131.79,
29.60, 129.53, 129.13, 128.66, 128.43, 128.08, 127.89, 127.77,
126.35, 124.82, 122.43, 115.70, 61.58, 36.31, 14.57. ESI-MS: m/z
594.3 [Mꢂ1], 596.2 [M+1], C26 19BrClN S [Exact Mass: 595.01].
2 3
), 1.32 (t, J = 7.1 Hz, 3H, CH ).
1
3
8.91. ESI-MS: m/z 537.3 [M+1], 539.3 [M+3], 541.4 [M+5], C25
S [Exact Mass: 536.06].
H
18
-
6
2 6 2
Cl N O
1
4
.1.5.8. N-(2-Bromo-4-sulfamoylphenyl)-2-((2-chloro-9-(naph-
thalen-1-yl)-9H-purin-8-yl)thio)propanamide (LAD-
). White powder, yield: 56.7%. Mp: 168–170 °C. H NMR
400 MHz, DMSO-d , ppm) d: 10.29 (d, J = 19.5 Hz, 1H, NH), 9.17
d, J = 8.1 Hz, 1H, purine-H), 8.30 (d, J = 8.2 Hz, 1H, naphthalene-
H
5 3
O
1
8
(
(
6
4.1.5.12.
2-((2-Chloro-9-(4-cyclopropylnaphthalen-1-yl)-9H-
purin-8-yl)thio)-N-(4-sulfamoylphenyl)acetamide (LBD-
1
H), 8.18 (dd, J = 8.2, 3.7 Hz, 1H, naphthalene-H), 8.06 (d,
J = 6.7 Hz, 1H Ph-H), 7.96 (d, J = 8.5 Hz, 1H, Ph-H), 7.91–7.79 (m,
2).
White powder, yield: 68.4%. Mp: 240–241 °C. H NMR
6
(400 MHz, DMSO-d , ppm) d: 10.78 (s, 1H, NH), 9.04 (s, 1H, pur-
2
H, 1 naphthalene-H, 1 Ph-H), 7.77 (dd, J = 13.3, 5.3 Hz, 1H, naph-
thalene-H), 7.73–7.64 (m, 1H, naphthalene-H), 7.62–7.51 (m, 1H,
naphthalene-H), 7.49 (d, J = 6.6 Hz, 2H, SO NH ), 7.29 (dd,
J = 24.8, 8.4 Hz, 1H, naphthalene-H), 5.12 (q, J = 7.0 Hz, 1H, CH),
ine-H), 8.62 (d, J = 8.5 Hz, 1H, naphthalene-H), 7.78 (d, J = 8.9 Hz,
2H, Ph-H), 7.72 (d, J = 7.8 Hz, 4H, Ph-2H, naphthalene-2H), 7.58
(t, J = 7.7 Hz, 1H, naphthalene-H), 7.50 (d, J = 7.6 Hz, 1H, naph-
2
2
thalene-H), 7.27 (d, J = 7.3 Hz, 3H, naphthalene-H, SO
(s, 2H, SCH ), 2.67–2.53 (m, 1H, CH), 1.17 (dd, J = 8.0, 4.6 Hz, 2H,
CH CH ), 1.01–0.80 (m, 2H, CH
, ppm) d: 164.91, 158.10, 155.91, 151.41, 145.96, 142.41,
2 2
NH ), 4.44
13
1
1
1
1
.65 (d, J = 8 Hz, 3H, CH
3
). C NMR (100 MHz, DMSO-d
6
, ppm) d:
2
1
3
70.04, 158.32, 156.74, 152.70, 147.39, 142.25, 139.16, 134.46,
33.92, 131.81, 130.49, 129.56, 129.15, 128.70, 128.36, 128.12,
27.79, 126.33, 126.03 (2 ꢁ C), 122.39, 116.91, 45.74, 18.75. ESI-
2
2
2 2
CH ). C NMR (100 MHz, DMSO-
d
6
140.93, 138.13, 133.08, 132.86, 128.46, 127.23, 126.62, 126.59,
MS: m/z 617.1 [M+1], 619.2 [M+3], 621.2 [M+5], C24
6
H18BrClN O
S
3 2
126.21 (3 ꢁ C), 125.63, 124.50, 122.22, 121.89, 118.13 (2 ꢁ C),
[
Exact Mass: 615.98].
35.85, 12.41, 6.80, 6.48. ESI-MS: m/z 565.4 [M+1], 567.4 [M+3], C26-
6 3 2
H21ClN O S [Exact Mass: 564.08].
4
.1.5.9.
N-(2-Chloro-4-sulfamoylphenyl)-2-((2-chloro-9-(naph-
White
powder, yield: 54.3%. Mp: 186–191 °C. H NMR (400 MHz, DMSO-
, ppm) d: 10.41 (s, 1H, NH), 9.13 (s, 1H, purine-H), 8.29 (d,
J = 8.2 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
thalene-H), 8.05 (d, J = 8.6 Hz, 1H, naphthalene-H), 7.91 (d,
J = 2.0 Hz, 1H, Ph-H), 7.82 (dd, J = 7.3, 1.1 Hz, 1H, Ph-H), 7.79–
thalen-1-yl)-9H-purin-8-yl)thio)propanamide (LAD-9).
4.1.5.13.
2-((2-Chloro-9-(4-cyclopropylnaphthalen-1-yl)-9H-
1
purin-8-yl)thio)-N-(4-(trifluoromethyl)phenyl)acetamide (LBD-
1
d
6
3).
White powder, yield: 73.3%. Mp: 221–225 °C. H NMR
, ppm) d: 10.82 (s, 1H, NH), 9.04 (s, 1H, pur-
(400 MHz, DMSO-d
6
ine-H), 8.62 (d, J = 8.5 Hz, 1H, naphthalene-H), 7.78 (d, J = 8.6 Hz,
2H, Ph-H), 7.74 (t, J = 4.1 Hz, 1H, naphthalene-H), 7.72 (s, 1H, naph-
thalene-H), 7.70 (d, J = 8.8 Hz, 2H, Ph-H), 7.58 (dd, J = 11.2, 4.1 Hz,
1H, naphthalene-H), 7.50 (d, J = 7.6 Hz, 1H, naphthalene-H), 7.28
(d, J = 8.4 Hz, 1H), 4.45 (s, 2H, SCH
(dt, J = 8.2, 4.3 Hz, 2H, CH CH ), 1.00–0.80 (m, 2H, CH
NMR (100 MHz, DMSO-d , ppm) d: 166.08, 159.16, 156.99,
7
.73 (m, 2H, Ph-H, and naphthalene-H), 7.69 (t, J = 7.6 Hz, 1H,
naphthalene-H), 7.62–7.54 (m, 1H, naphthalene-H), 7.48 (s, 2H,
SO NH ), 7.31 (d, J = 8.4 Hz, 1H, naphthalene-H), 5.15 (q,
J = 7.0 Hz, 1H), 1.65 (d, J = 6.8 Hz, 3H, CH
DMSO-d , ppm) d: 170.15, 158.34, 156.73, 152.70, 147.26, 141.78,
2
2
2
), 2.65–2.54 (m, 1H, CH), 1.18
13
13
3
). C NMR (100 MHz,
2
2
2 2
CH ).
C
6
6
1
1
4
37.85, 134.46, 133.92, 131.82, 129.56, 129.15, 128.70, 128.36,
28.11, 127.79, 127.34, 126.33, 126.09, 125.55, 125.31, 122.39,
152.49, 147.04, 143.49, 142.69, 134.16, 133.93, 129.54, 128.30,
127.70, 127.67, 126.70, 126.65, 126.12, 125.57, 124.23, 123.92,
5.80, 18.82. ESI-MS: m/z 573.2 [M+1], 575.2 [M+3], C24
[Exact Mass: 572.03].
H18Cl
2
N
6
-
123.42, 123.29, 123.29, 122.96, 119.53 (2 ꢁ C), 36.96, 13.48, 7.87,
+
O
S
3 2
7.55. ESI-MS: m/z 552.3 [MꢂH ], C27
3 5
H19ClF N OS [Exact Mass:
5
53.10].
4
9
.1.5.10. Methyl 3-bromo-4-(2-((2-chloro-9-(naphthalen-1-yl)-
H-purin-8-yl)thio)acetamido)benzoate (LAD-10).
White
4.1.5.14. 3-Bromo-4-(2-((2-chloro-9-(4-cyclopropylnaphthalen-
1-yl)-9H-purin-8-yl)thio)acetamido)benzamide (LBD-4). White
powder, yield: 65.1%. Mp: 238–241 °C. H NMR (400 MHz, DMSO-d
ppm) d: 10.03 (s, 1H, NH), 9.10 (s, 1H, purine-H), 8.61 (d, J = 8.5 Hz,
1H, naphthalene-H), 8.15 (s, 1H, Ph-H), 8.05 (s, 1H, CONH ), 7.86 (s,
2H, Ph-H), 7.74 (t, J = 7.6 Hz, 2H, naphthalene-H), 7.60–7.53 (m, 1H,
naphthalene-H), 7.49 (d, J = 7.8 Hz, 2H, naphthalene-H, CONH ), 7.27
(d, J = 8.4 Hz, 1H, naphthalene-H), 4.51–4.35 (m, 2H, SCH ), 2.66–2.54
(m, 1H, CH), 1.28–1.07 (m, 2H, CH CH ), 1.01–0.78 (m, 2H, CH CH ).
, ppm) d: 166.45, 166.35, 159.14,
1
powder, yield: 73.6%. Mp: 215–217 °C.
DMSO-d
d, J = 8.2 Hz, 1H, naphthalene-H), 8.19 (d, J = 8.2 Hz, 1H, naph-
H
NMR (400 MHz,
1
6
, ppm) d: 10.09 (s, 1H, NH), 9.12 (s, 1H, purine-H), 8.30
6
,
(
thalene-H), 8.16 (d, J = 1.9 Hz, 1H, Ph-H), 8.02 (d, J = 8.6 Hz, 1H,
Ph-H), 7.94 (dd, J = 8.6, 1.9 Hz, 1H, Ph-H), 7.85 (dd, J = 7.3, 1.0 Hz,
2
1
H, naphthalene-H), 7.82–7.75 (m, 1H, naphthalene-H), 7.68 (dd,
2
J = 11.5, 4.3 Hz, 1H, naphthalene-H), 7.60–7.51 (m, 1H, naph-
thalene-H), 7.30 (d, J = 8.4 Hz, 1H, naphthalene-H), 4.45 (q,
J = 15.5 Hz, 2H,CH
2
2
2
2
2
), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-d
13
C NMR (100 MHz, DMSO-d
6
2
6
,
ppm) d: 166.66, 165.06, 158.91, 156.93, 152.61, 147.28, 140.62,
157.00, 152.56, 147.20, 143.52, 138.77, 134.15, 133.90, 132.62,
132.33, 129.51, 128.31, 127.87, 127.72, 127.67, 126.66, 125.58,
124.95, 123.30, 122.96, 116.08, 36.27, 13.48, 7.89, 7.55. ESI-MS: m/z
1
1
5
34.47, 133.89 (2 ꢁ C), 131.79, 129.60, 129.57, 129.13, 128.66,
28.44, 128.08, 127.77, 127.60, 126.35, 124.78, 122.43, 115.68,
2.85, 36.30. ESI-MS: m/z 580.1 [Mꢂ1], 582.1 [M+1], C25
H
17BrClN
5
-
6 2
607.1 [M+1], 609.1 [M+3], 611.2 [M+5], C27H20BrClN O S [Exact Mass:
O
3
S [Exact Mass: 580.99].
606.02].
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

8
X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4
.1.5.15. 3-Chloro-4-(2-((2-chloro-9-(4-cyclopropylnaphthalen-
White
powder, yield: 63.2%. Mp: 256–257 °C. H NMR (400 MHz, DMSO-d
ppm) d: 10.14 (s, 1H, NH), 9.07 (s, 1H, purine-H), 8.61 (d, J = 8.5 Hz,
H, naphthalene-H), 8.05 (s, 1H, CONH ), 8.00 (d, J = 1.9 Hz, 1H, Ph-
H), 7.97 (d, J = 8.5 Hz, 1H, Ph-H), 7.83 (dd, J = 8.6, 1.9 Hz, 1H, Ph-H),
.74 (t, J = 7.6 Hz, 2H, naphthalene-H), 7.57 (t, J = 7.7 Hz, 1H, naph-
thalene-H), 7.49 (d, J = 7.3 Hz, 2H, naphthalene-H, CONH ), 7.27 (d,
J = 8.4 Hz, 1H, naphthalene-H), 4.54–4.40 (m, 2H, SCH ), 2.68–2.53
m, 1H, CH), 1.24–1.09 (m, 2H, CH CH ), 1.01–0.81 (m, 2H, CH CH ).
, ppm) d: 166.53, 166.45, 159.20,
56.99, 152.56, 147.10, 143.52, 137.52, 134.15, 133.89, 132.05,
29.52, 129.13, 128.31, 127.72, 127.67, 127.35, 126.67, 125.58,
25.10, 124.18, 123.30, 122.93, 36.37, 13.48, 7.89, 7.55. ESI-MS: m/z
61.3 [Mꢂ1], 563.3 [M+1], C27 S [Exact Mass: 562.07].
133.92, 130.48, 129.46, 128.35, 127.76, 127.69, 126.57, 126.02
1-yl)-9H-purin-8-yl)thio)acetamido)benzamide (LBD-5).
(2 ꢁ C), 125.59, 123.29, 122.92, 116.91, 45.70, 18.77, 13.47, 7.85,
1
6
,
6 3 2
7.55. ESI-MS: m/z 657.2 [M+1], 659.0 [M+3], C27H22BrClN O S
[Exact Mass: 656.01].
1
2
4.1.5.19.
N-(2-Chloro-4-sulfamoylphenyl)-2-((2-chloro-9-(4-
7
cyclopropylnaphthalen-1-yl)-9H-purin-8-yl)thio)propanamide
1
2
(LBD-9).
White powder, yield: 70.6%. Mp: 222–224 °C.
H
2
NMR (400 MHz, DMSO-d
6
, ppm) d: 10.38 (d, J = 24.5 Hz, 1H, NH),
(
2
2
2
2
9.11 (s, 1H, purine-H), 8.61 (dd, J = 8.4, 4.0 Hz, 1H, naphthalene-
H), 8.02 (dd, J = 26.0, 8.6 Hz, 1H, Ph-H), 7.93–7.88 (m, 1H, Ph-H),
7.80–7.67 (m, 3H, Ph-H, naphthalene-2H), 7.62–7.43 (m, 4H, naph-
1
3
C NMR (100 MHz, DMSO-d
6
1
1
1
5
thalene-2H, SO
2 2
NH ), 7.25 (dd, J = 29.1, 8.4 Hz, 1H, naphthalene-
H), 5.14 (q, J = 6.9 Hz, 1H, SCH), 2.66–2.53 (m, 1H, CH), 1.65 (dd,
H
20Cl
2
N
6
O
2
J = 22.1, 7.0 Hz, 3H, CH
by solvent peak), 0.91 (dd, J = 17.5, 8.4 Hz, 2H, CH
(100 MHz, DMSO-d
3
), 1.28–1.08 (m, 2H, CH
2
CH
2
, overlapped
13
2
2
CH ). C NMR
4
1
6
.1.5.16. 3-Bromo-4-(2-((2-chloro-9-(4-cyclopropylnaphthalen-
-yl)-9H-purin-8-yl)thio)propanamido)benzamide (LBD-
). White powder, yield: 58.4%. Mp: 238–241 °C. H NMR
, ppm) d: 10.17 (d, J = 23.4 Hz, 1H, NH), 9.15
d, J = 7.0 Hz, 1H, purine-H), 8.61 (dd, J = 8.5, 3.9 Hz, 1H, naph-
), 7.89–
.81 (m, 2H, Ph-H), 7.73 (ddd, J = 12.6, 11.6, 5.8 Hz, 2H, naph-
thalene-H), 7.62–7.51 (m, 1H, naphthalene-H), 7.49–7.46 (m, 2H,
naphthalene-1H, CONH -1H), 7.25 (dd, J = 25.2, 8.4 Hz, 1H, naph-
thalene-H), 5.10 (q, J = 6.9 Hz, 1H, SCH), 2.63–2.53 (m, 1H, CH),
.65 (m, 7.0 Hz, 3H, CH ), 1.23–1.10 (m, 2H, CH CH ), 0.97–0.79
). C NMR (100 MHz, DMSO-d , ppm) d: 169.90,
6
, ppm) d: 170.13, 158.54, 156.79, 152.67,
147.21, 143.53, 141.84, 141.76, 137.85, 137.78, 134.13, 133.93,
129.50, 128.36, 127.74, 127.34, 126.57, 125.55, 125.30, 123.29,
122.91, 122.80, 45.92, 18.95, 13.47, 7.86, 7.57. ESI-MS: m/z 613.3
1
(
(
400 MHz, DMSO-d
6
2 6 3 2
[M+1], 614.8 [M+3], C27H22Cl N O S [Exact Mass: 612.06].
thalene-H), 8.19–8.12 (m, 1H, Ph-H), 8.05 (s, 1H, CONH
2
7
4.1.5.20. Methyl 3-bromo-4-(2-((2-chloro-9-(4-cyclopropyl-
naphthalen-1-yl)-9H-purin-8-yl)thio)acetamido)benzoate (LBD-
1
2
10).
White powder, yield: 78.2%. Mp: 193–194 °C. H NMR
(400 MHz, DMSO-d
6
, ppm) d: 10.08 (s, 1H, NH), 9.11 (s, 1H, pur-
1
3
2
2
ine-H), 8.61 (d, J = 8.5 Hz, 1H, naphthalene-H), 8.15 (d, J = 1.9 Hz,
1H, Ph-H), 8.01 (d, J = 8.6 Hz, 1H, Ph-H), 7.94 (dd, J = 8.6, 1.9 Hz,
1H, Ph-H), 7.74 (t, J = 7.6 Hz, 2H, naphthalene-H), 7.56 (t, J = 7.6 Hz,
1H, naphthalene-H), 7.49 (d, J = 7.6 Hz, 1H, naphthalene-H), 7.28 (d,
1
3
(
m, 2H, CH
2
CH
2
6
1
1
1
1
66.33, 158.58, 156.81, 152.64, 147.31, 143.54, 138.62, 134.13,
33.93, 132.82, 132.33, 129.47, 128.35, 127.88, 127.77, 127.68,
26.58, 125.59, 125.28, 123.30, 122.91, 116.65, 45.79, 18.87,
3.47, 7.85, 7.55. ESI-MS: m/z 619.2 [Mꢂ1], 621.5 [M+1], 623.4
J = 8.4 Hz, 1H, naphthalene-H), 4.44 (q, J = 15.5 Hz, 2H, SCH
(s, 3H, OCH ), 2.66–2.52 (m, 1H, CH), 1.17 (td, J = 4.8, 1.9 Hz, 2H, CH
2 2 6
CH ), 0.99–0.82 (m, 2H, CH CH ). C NMR (100 MHz, DMSO-d ,
2
), 3.85
3
2
-
13
[
M+3], C28
6
H22BrClN O
2
S [Exact Mass: 620.04].
2
ppm) d: 166.69, 165.06, 159.11, 156.99, 152.57, 147.24, 143.52,
140.62, 134.14, 133.89 (2 ꢁ C), 129.57, 129.51, 128.31, 127.73,
127.68, 127.57, 126.64, 125.58, 124.76, 123.30, 122.95, 115.66,
52.86, 36.28, 13.48, 7.89, 7.54. ESI-MS: m/z 622.2 [M+1], 624.2 [M
4
1
7
.1.5.17. 3-Chloro-4-(2-((2-chloro-9-(4-cyclopropylnaphthalen-
-yl)-9H-purin-8-yl)thio)propanamido)benzamide (LBD-
). White powder, yield: 61.6%. Mp: 246–249 °C. H NMR
, ppm) d: 10.30 (s, 1H, NH), 9.11 (s, 1H, pur-
ine-H), 8.61 (d, J = 8.5 Hz, 1H, naphthalene-H), 8.05 (s, 1H, CONH ),
.00 (d, J = 1.7 Hz, 1H, Ph-H), 7.95 (d, J = 8.5 Hz, 1H, Ph-H), 7.83 (dd,
J = 8.5, 1.6 Hz, 1H, Ph-H), 7.75 (t, J = 7.7 Hz, 1H, naphthalene-H),
.70 (d, J = 7.6 Hz, 1H, naphthalene-H), 7.59 (t, J = 7.3 Hz, 1H, naph-
thalene-H), 7.49 (s, 1H, CONH ), 7.47 (d, J = 7.6 Hz, 1H), 7.28 (d,
J = 8.4 Hz, 1H, naphthalene-H), 5.14 (q, J = 6.9 Hz, 1H, SCH), 2.64–
.54 (m, 1H, CH), 1.62 (d, J = 7.0 Hz, 3H, CH ), 1.24–1.09 (m, 2H, CH
CH , overlapped by solvent peak), 0.98–0.80 (m, 2H, CH
NMR (100 MHz, DMSO-d , ppm) d: 170.01, 166.42, 158.61, 156.80,
52.65, 147.19, 143.54, 137.34, 134.14, 133.92, 132.30, 129.46,
1
(
400 MHz, DMSO-d
6
5 3
+3], 626.0 [M+5], C28H21BrClN O S [Exact Mass: 621.02].
2
8
4.1.5.21. Ethyl 3-bromo-4-(2-((2-chloro-9-(4-cyclopropyl-naph-
thalen-1-yl)-9H-purin-8-yl)thio)acetamido)benzoate (LBD-
1
7
11).
White powder, yield: 75.7%. Mp: 258–261 °C. H NMR
2
6
(400 MHz, DMSO-d , ppm) d: 10.09 (s, 1H, NH), 9.11 (s, 1H, pur-
ine-H), 8.61 (d, J = 8.5 Hz, 1H, naphthalene-H), 8.15 (d, J = 1.9 Hz,
1H, Ph-H), 8.01 (d, J = 8.6 Hz, 1H, Ph-H), 7.94 (dd, J = 8.5, 1.9 Hz,
1H, Ph-H), 7.74 (t, J = 7.0 Hz, 2H, naphthalene-H), 7.56 (dd,
J = 11.3, 4.0 Hz, 1H, naphthalene-H), 7.49 (d, J = 7.6 Hz, 1H, naph-
thalene-H), 7.28 (d, J = 8.4 Hz, 1H, naphthalene-H), 4.45 (q,
2
3
2
-
1
3
2
2
CH
2
).
C
6
1
1
1
5
29.12, 128.35, 127.76, 127.68, 127.36, 126.58, 125.68, 125.59,
24.53, 123.29, 122.91, 45.82, 18.91, 13.47, 7.85, 7.55. ESI-MS: m/z
75.2 [Mꢂ1], 577.2 [M+1], C28
J = 15.5 Hz, 2H, SCH
(m, 1H, CH), 1.32 (t, J = 7.1 Hz, 3H, CH
0.99–0.81 (m, 2H, CH
1
2
), 4.31 (q, J = 7.1 Hz, 2H, OCH
), 1.23–1.10 (m, 2H, CH
). C NMR (100 MHz, DMSO-d , ppm) d:
66.66, 164.54, 159.12, 156.99, 152.57, 147.23, 143.52, 140.56,
2
), 2.67–2.54
3
2
CH ),
2
1
3
2 6 2
H22Cl N O S [Exact Mass: 576.09].
2
CH
2
6
4
.1.5.18. N-(2-Bromo-4-sulfamoylphenyl)-2-((2-chloro-9-(4-
134.14, 133.88, 133.82, 129.53, 129.51, 128.31, 127.86, 127.72,
127.67, 126.64, 125.58, 124.78, 123.30, 122.95, 115.68, 61.58,
36.29, 14.57, 13.48, 7.89, 7.54. ESI-MS: m/z 634.2 [Mꢂ1], 636.4
cyclopropyl naphthalen-1-yl)-9H-purin-8-yl)thio)propanamide
1
(
LBD-8).
NMR (400 MHz, DMSO-d
m, 1H, purine-H), 8.61 (d, J = 8.5 Hz, 1H, naphthalene-H), 8.06 (d,
White powder, yield: 66.8%. Mp: 176–180 °C.
H
6
, ppm) d: 10.30 (s, 1H, NH), 9.22–9.11
[M+1], 638.3 [M+3], C29
5 3
H23BrClN O S [Exact Mass: 635.04].
(
J = 2.0 Hz, 1H, Ph-H), 7.95 (d, J = 8.5 Hz, 1H, Ph-H), 7.79 (dd,
J = 8.5, 2.1 Hz, 1H, Ph-H), 7.77–7.72 (m, 1H, naphthalene-H), 7.70
4.2. In vitro anti-HIV assay
(
d, J = 7.6 Hz, 1H, naphthalene-H), 7.58 (t, J = 7.7 Hz, 1H, naph-
thalene-H), 7.47 (d, J = 7.1 Hz, 3H, naphthalene-H, and SO NH ),
.28 (d, J = 8.4 Hz, 1H, naphthalene-H), 5.11 (q, J = 7.0 Hz, 1H,
It has been previously described that how to evaluate the
antiviral activity of the compounds against HIV-1 strain IIIB, dou-
ble RT mutant strains of HIV-1 IIIB (RES056 and F227L/V106A), five
single RT mutant strains of HIV-1 IIIB (L100I, K103N, E138K, Y181C,
2
2
7
3
SCH), 2.63–2.53 (m, 1H, CH), 1.62 (d, J = 7.1 Hz, 3H, CH ), 1.24–
2
9
1
2
1
.11 (m, 2H, CH
2
CH
2
, overlapped by solvent peak), 0.97–0.81 (m,
C NMR (100 MHz, DMSO-d , ppm) d: 170.05,
58.51, 156.80, 152.66, 147.35, 143.55, 142.25, 139.15, 134.14,
Y188L), and HIV-2 strain (ROD).
Stock solutions (10 ꢁ final concentration) of test compounds
were added in 25- l volumes to two series of triplicate wells so
1
3
H, CH
2
2
CH ).
6
l
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
as to allow simultaneous evaluation of their effects on mock- and
HIV-infected cells at the beginning of each experiment. Serial five-
fold dilutions of test compounds were made directly in flat-bot-
tomed 96 wells microtiter trays using a Biomek 3000 robot
complexes with GWI999 (PDB code: 3DOL) were retrieved from
the Protein Data Bank and were used for the docking experiment
by means of AutoDock 4.2 and MGL tools 1.5.6. For the docking
study, water and GW564511 (GWI999) were deleted, charges
and protonation state were assigned, energy minimization was
done using OPLS2005 force field. AutoDock grids were generated
using a grid box with 46 ꢁ 30 ꢁ 40 grid points in xyz with grid
spacing of 0.375 Å. The grid center was designated at dimensions
(x, y, z): ꢂ7.204, ꢂ36.360, 25.496. The Lamarckian genetic algo-
rithm was used for docking simulations of LAD-8 (LAD-6 and
VRX-480773). After that, the ligands were AutoDocked into the
grids. Top-scoring pose was shown by the software of pymol ver-
sion 1.5 (www.pymol.org). The secondary structure of RT is shown
in cartoons, and only the key residues for interactions with the
inhibitor were shown in sticks and labeled. The potential hydrogen
bonds were presented by dashed lines.
(
Beckman instruments, Fuller-ton, CA). Untreated control HIV-
and mock-infected cell samples were included for each sample.
HIV-1(IIIB) or HIV-2 (ROD) 24 stock (50 L) at 100–300 CCID50
cell culture infectious dose) or culture medium was added to
l
(
either the infected or mock-infected wells of the microtiter tray.
Mock-infected cells were used to evaluate the effect of test com-
pound on uninfected cells in order to assess the cytotoxicity of
the test compound. Exponentially growing MT-4 cells were cen-
trifuged for 5 min at 1000 rpm and the supernatant was discarded.
5
The MT-4 cells were resuspended at 6 ꢁ 10 cells/mL, and 50-
lL
volumes were transferred to the microtiter tray wells. Five days
after infection, the viability of mock- and HIV-infected cells was
examined spectrophotometrically by the MTT assay.
The MTT assay is based on the reduction of yellow colored 3-
Acknowledgments
(
(
4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase
The financial support from the National Natural Science Foun-
dation of China (NSFC Nos. 81273354, 81573347), Key Project of
NSFC for International Cooperation (No. 81420108027), Science
and Technology Major Project of Shandong Province (Major
key technology, No. 2015ZDJS04001), and the Science and
Technology Development Project of Shandong Province (Nos.
2014GSF118175, 2014GSF118012) and KU Leuven (GOA 10/014)
is gratefully acknowledged. We thank K. Erven, K. Uyttersprot
and C. Heens for technical assistance with the HIV assays.
of metabolically active cells to a blue-purple formazan that can
be measured spectrophotometrically. The absorbances were read
in an eight-channel computer-controlled photometer (Multi-scan
Ascent Reader, Labsystems, Helsinki, Finland), at two wave-lengths
(
(
tion (CC50) was defined as the concentration of the test compound
that reduced the absorbance (OD540) of the mock-infected control
sample by 50%. The concentration achieving 50% protection from
the cytopathic effect of the virus in infected cells was defined as
the 50% effective concentration (EC50).
540 and 690 nm). All data were calculated using the median OD
optical density) value of tree wells. The 50% cytotoxic concentra-
References and notes
1.
2.
3.
Ragno, R.; Frasca, S.; Manetti, F.; Brizzi, A.; Massa, S. J. Med. Chem. 2005, 48, 200.
Zhan, P.; Pannecouque, C.; De Clercq, E.; Liu, X. J. Med. Chem. 2016, 59, 2849.
Zhan, P.; Chen, X.; Li, D.; Fang, Z.; De Clercq, E.; Liu, X. Med. Res. Rev. 2013, 33,
E1.
4
.3. HIV-1 RT inhibition assay
4
5
.
.
Li, D.; Zhan, P.; De Clercq, E.; Liu, X. J. Med. Chem. 2012, 55, 3595.
MacArthur, R. D.; Novak, R. M.; Peng, G.; Chen, L.; Xiang, Y.; Hullsiek, K. H.;
Kozal, M. J.; van den Berg-Wolf, M.; Henely, C.; Schmetter, B.; Dehlinger, M.
Lancet 2006, 368, 2125.
Hauser, A.; Sewangi, J.; Mbezi, P.; Dugange, F.; Lau, I.; Ziske, J.; Theuring, S.;
Kuecherer, C.; Harms, G.; Kunz, A. PLoS ONE 2012, 7, e32055.
Iyidogan, P.; Anderson, K. S. Viruses 2014, 6, 4095.
A RT assay kit produced by Roche was selected for the RT inhi-
bition assay. All the reagents for performing the RT reaction came
with the kit, and the ELISA procedures for RT inhibition assay was
performed following the description in the kit protocol.3
0,31
Briefly,
6
.
.
the reaction mixture containing template/primer complex, viral
nucleotides (dNTPs) and RT in the incubation buffer with or with-
out inhibitors was incubated for 1 h at 37 °C. After that, the reac-
tion mixture was transferred to a streptavidin-coated microtiter
plate and incubated for another 1 h at 37 °C to make sure retran-
scriptional cDNA chain that consisted biotin-labeled dNTPs bound
to streptavidin. Then removed un-bound dNTPs using washing buf-
fer and added anti-DIG-POD working solution. After incubation for
7
8. Zhang, H.; Qin, F.; Ye, W.; Li, Z.; Ma, S.; Xia, Y.; Jiang, Y.; Zhu, J.; Li, Y.; Zhang, J.;
Chen, H. F. Chem. Biol. Drug Des. 2011, 78, 427.
9.
Li, X.; Zhang, L.; Tian, Y.; Song, Y.; Zhan, P.; Liu, X. Expert Opin. Ther. Pat. 2014,
4, 1199.
0. Zhan, P.; Liu, X. Expert Opin. Ther. Pat. 2011, 21, 717.
11. Zhan, P.; Liu, X.; Li, Z. Curr. Med. Chem. 2009, 16, 2876.
2
1
1
2. Zhan, P.; Liu, X.; Li, Z.; Pannecouque, C.; De Clercq, E. Curr. Med. Chem. 2009, 16,
903.
3. Zhan, P.; Li, Z.; Liu, X.; De Clercq, E. Mini-Rev. Med. Chem. 2009, 9, 1014.
3
1
1
h at 37 °C, the DIG-labeled dNTPs incorporated in cDNA were
14. Li, W.; Li, X.; De Clercq, E.; Zhan, P.; Liu, X. Eur. J. Med. Chem. 2015, 102, 167.
1
5. Moyle, G.; Boffito, M.; Stoehr, A.; Rieger, A.; Shen, Z.; Manhard, K.; Sheedy, B.;
Hingorani, V.; Raney, A.; Nguyen, M.; Nguyen, T.; Ong, V.; Yeh, L. T.; Quart, B.
Antimicrob. Agents Chemother. 2010, 54, 3170.
bound to the anti-DIG-POD antibody. The unbound anti-DIG-PODs
were removed and the peroxidesubstrate (ABST) solution was
added to the MTPs. A colored reaction proceed during cleavage of
the substrate catalyzed by POD. The absorbance of the sample
was determined at OD 405 nm using a microtiter plate ELISA
reader. The percentage inhibitory activity of RT inhibitors was cal-
culated by formula as given below:
16. Zhan, P.; Li, X.; Li, Z.; Chen, X.; Tian, Y.; Chen, W.; Liu, X.; Pannecouque, C.; De
Clercq, E. Bioorg. Med. Chem. Lett. 2012, 22, 7155.
1
1
7. Song, Y.; Zhan, P.; Liu, X. Curr. Pharm. Des. 2013, 19, 7141.
8. Song, Y.; Fang, Z.; Zhan, P.; Liu, X. Curr. Med. Chem. 2014, 21, 329.
19. Rai, D.; Chen, W.; Zhan, P.; Liu, H.; Tian, Y.; Liang, X.; De Clercq, E.;
Pannecouque, C.; Balzarini, J.; Liu, X. Chem. Biol. Drug Des. 2014, 84, 420.
2
2
0. Li, Z.; Cao, Y.; Zhan, P.; Pannecouque, C.; Balzarini, J.; Clercq, E. D.; Shen, Y.; Liu,
X. Med. Chem. 2013, 9, 968.
1. Zhan, P.; Chen, W.; Li, Z.; Li, X.; Chen, X.; Tian, Y.; Pannecouque, C.; Clercq, E. D.;
Liu, X. Bioorg. Med. Chem. 2012, 20, 6795.
%
Inhibition ¼ ½OD value with RT but without inhibitors OD
value with RT and inhibitorsꢃ=½OD value with RT
and inhibitors OD value without RT and inhibitorsꢃ:
22. Song, Y. n.; Zhan, P.; Kang, D.; Li, X.; Tian, Y.; Li, Z.; Chen, X.; Chen, W.;
Pannecouque, C.; De Clercq, E.; Liu, X. MedChemComm 2013, 4, 810.
2
3. Li, X.; Lu, X.; Chen, W.; Liu, H.; Zhan, P.; Pannecouque, C.; Balzarini, J.; De
Clercq, E.; Liu, X. Bioorg. Med. Chem. 2014, 22, 5290.
4. Huang, B.; Li, X.; Zhan, P.; De Clercq, E.; Daelemans, D.; Pannecouque, C.; Liu, X.
Chem. Biol. Drug Des. 2016, 87, 283.
The IC50 values corresponded to the concentrations of the inhi-
bitors required to inhibit biotin-dUTP incorporation by 50%.
2
4
.4. Molecular simulation experiment
25. Jiang, X.; Zhan, P.; Liu, X.; Clercq, E. D. Lat. Am. J. Pharm. 2012, 31, 751.
2
6. Li, X.; Zhan, P.; Liu, H.; Li, D.; Wang, L.; Chen, X.; Liu, H.; Pannecouque, C.;
Balzarini, J.; De Clercq, E.; Liu, X. Bioorg. Med. Chem. 2012, 20, 5527.
7. Li, X.; Huang, B.; Zhou, Z.; Gao, P.; Pannecouque, C.; Daelemans, D.; De Clercq,
E.; Zhan, P.; Liu, X. Chem. Biol. Drug Des. 2016, 88, 241.
The crystallographic structures of HIV-1 RT complexes with
2
GW564511 (PDB code: 3DLG) and HIV-1 L100I mutant strain RT
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041

1
0
X. Lu et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
2
8. D’Angelo, N. D.; Bellon, S. F.; Booker, S. K.; Cheng, Y.; Coxon, A.; Dominguez, C.;
Fellows, I.; Hoffman, D.; Hungate, R.; Kaplan-Lefko, P.; Lee, M. R.; Li, C.; Liu, L.;
Rainbeau, E.; Reider, P. J.; Rex, K.; Siegmund, A.; Sun, Y.; Tasker, A. S.; Xi, N.; Xu,
S.; Yang, Y.; Zhang, Y.; Burgess, T. L.; Dussault, I.; Kim, T. S. J. Med. Chem. 2008,
29. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protoc. 2008, 3, 427.
30. Suzuki, K.; Craddock, B. P.; Okamoto, N.; Kano, T.; Steigbigel, R. T. J. Virol.
Methods 1993, 44, 189.
31. Reverse Transcriptase Assay, Colorimetric, Roche Diagnostics GmbH, Roche
Applied Science, Version 13.0 ed., 2010.
51, 5766.
Please cite this article in press as: Lu, X.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.041