Discovery of novel 2-(3-(2-chlorophenyl)pyrazin-2-ylthio)-N-arylacetamides as potent HIV-1 inhibitors using a structure-based bioisosterism approach

Article abstract of DOI:10.1016/j.bmc.2012.09.058

The present work is an extension of our ongoing efforts towards the development and identification of new molecules with anti-HIV activity which have previously led to the discovery of arylazolylthioacetanilides as highly active NNRTIs. In this article, a series of 2-2-(3-(2-chlorophenyl)pyrazin-2- ylthio)-N-arylacetamide derivatives were synthesized and evaluated for in vitro anti-HIV activity. Most of the tested compounds exhibited moderate activities against wild-type HIV-1. Among them, compound 6k showed significant activity against wild-type HIV-1 with an EC₅₀ value of 1.7 μM, along with moderate activity against wild-type reverse transcriptase (RT). The preliminary structure-activity relationship (SAR) and docking calculations of this new series of compounds were also investigated, which may help designing more potent molecules.

Full text of DOI:10.1016/j.bmc.2012.09.058

Bioorganic & Medicinal Chemistry 20 (2012) 6795–6802
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel 2-(3-(2-chlorophenyl)pyrazin-2-ylthio)-N-arylacetamides
as potent HIV-1 inhibitors using a structure-based bioisosterism approach
Peng Zhan a, Wenmin Chen a, Zhenyu Li a, Xiao Li a, Xuwang Chen a, Ye Tian a, Christophe Pannecouque b,
Erik De Clercq b, Xinyong Liu a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road,
250012 Jinan, Shandong, PR China
b Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
The present work is an extension of our ongoing efforts towards the development and identification of
new molecules with anti-HIV activity which have previously led to the discovery of arylazolylthioacetan-
ilides as highly active NNRTIs. In this article, a series of 2-2-(3-(2-chlorophenyl)pyrazin-2-ylthio)-N-ary-
lacetamide derivatives were synthesized and evaluated for in vitro anti-HIV activity. Most of the tested
compounds exhibited moderate activities against wild-type HIV-1. Among them, compound 6k showed
Received 13 August 2012
Revised 29 September 2012
Accepted 29 September 2012
Available online 6 October 2012
significant activity against wild-type HIV-1 with an EC50 value of 1.7 lM, along with moderate activity
Keywords:
HIV reverse transcriptase
NNRTIs
Heterocycle
Synthesis
against wild-type reverse transcriptase (RT). The preliminary structure–activity relationship (SAR) and
docking calculations of this new series of compounds were also investigated, which may help designing
more potent molecules.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Anti-HIV-1 activity
SAR
Pyrazinylthioacetamide
Structure-based bioisosterism strategy
1. Introduction
acetanilide derivatives.15–23 Most of these compounds displayed
potent anti-HIV properties in cell lines infected with either wild-
Non-nucleoside reverse transcriptase inhibitors (NNRTIs) play
an essential role in the highly active antiretroviral therapy
(HAART) for acquired immunodeficiency syndrome (AIDS) due to
their potent antiviral activity, high specificity and low toxicity.
Unfortunately, NNRTI potency is dramatically reduced by the
emergence of drug-resistant HIV-1 mutants. Thus, the develop-
ment of novel chemical entities with improved activity profiles
has been a very active research field in recent years.1–4
Among the representatives of the NNRTIs, arylazolylthioacetan-
ilides have interesting structural characteristics and highly anti-
HIV-1 specific activities that have induced many kinds of structural
modifications on the skeleton of the five-membered lead com-
pounds.5–11 Several triazolylthioacetanilide NNRTIs, such as VRX-
48073312 and RDEA806 (in phase IIa clinical trials)13, have been
or are currently being evaluated in clinical trials (Fig. 1).
type or mutant HIV-1. In particular, 1,2,3-thiadiazole derivative
ZP7 displayed the most potent anti-HIV-1 activity (EC50 = 36 nm),
inhibiting HIV-1 replication in MT-4 cells more effectively than
NVP (by sevenfold) and DLV (by eightfold).16 Molecular modeling
studies demonstrated that the five-membered heterocycle portion
of these inhibitors could be acting as a scaffold which orients the
pharmacophores into the proper geometry for effective interac-
tions with the target and as key structural element to form hydro-
gen bond with K103 of the RT pocket (as predicted by the
modeling studies).15 Therefore, there are differences in the elec-
tronic and conformational contribution of the heterocyclic groups
to the binding of the inhibitors with the HIV-1 RT.15 Supported by
these promising results and with the aim of obtaining more potent
compounds and establishing further SAR on this class of NNRTIs,
we designed and synthesized a series of novel pyrazin-2-ylthioac-
etamide derivatives (6a–6q) (Fig. 2), based on the structure-based
bioisosterism strategy in medicinal chemistry24, in which the
‘–SCH2CO–’ linker was maintained in view of its paramount
importance. Their anti-HIV activities against HIV-1 (IIIB) and
HIV-2 (ROD) as well as their HIV RT inhibitory activities were also
presented.
In connection with our research program focusing on the prep-
aration of new therapeutic agents, our group previously designed
and synthesized a number of ‘follow-on’-based14 arylazolylthio-
⇑
Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0968-0896/$ - see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.09.058

6796
P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
Cl
Cl
Cl
N
N
N
S
N
N
N
H
N
H
N
H
N
S
S
N
S
Br
Br
N
N
O
O
O
Br
SO2NH2
COOK
VRX-480773
EC50 = 0.14 µM (WT)
EC50 = 0.23 µM (K103N/Y181C)
RDEA-806
ZP7
Br
EC50 =36.4 nM, SI>6460
Figure 1. Azolylthioacetanilide-based NNRTIs.
R2
N
R2
N
S
N
H
N
H
N
N
R1
S
S
X1
N
O
O
X2
R4
R4
Ar
R
Structure-Based
Bioisosterism Design
Triazole thioacetanilide
N
R2
N
N
N
H
H
N
N
N
N
N
S
S
X3
S
Het
O
O
O
R4
Ar
Ar
R
X3 = CH, N
1,2,3-Thiadiazole thioacetanilide
Target compounds
Lead structures
Figure 2. The structure-based bioisosterism replacement of azoles by pyrazine.
glycinamide with glyoxal.26 Treatment of 4 with phosphorus
pentasulfide in pyridine under reflux afforded 3-(2-chloro-
phenyl)pyrazine-2(1H)-thione (5). The final pyrazin-2-ylthioace-
tanilides (6) were synthesized by reaction of intermediate 5 with
suitable 2-chloro-N-aryl-substituted acetamides in good yields,
using the same conditions as for other alkyl halides. 2-Chloro-N-
phenyl acetamides (or other alkyl halides) were synthesized
according to the reported literature.27 The synthesized compounds
were characterized by the MS, IR and 1H NMR spectral data to-
gether with TLC analysis.
2. Results and discussion
2.1. Chemistry
To achieve the synthesis of the target compounds 6a–6q, the
steps outlined in Scheme 1 were adopted. The key intermediate,
(+/À)-2-amino-2-(2-chlorophenyl) acetamide (3) was prepared
from readily available racemic 2-chlorophenyl glycine (1) accord-
ing to a reported procedure.25 The synthesis of the pyrazinone 4
was achieved in one step by condensation of 2-chlorophenyl
NH
H2N
COOH
Cl
H2N
HCl
COOMe
Cl
H2N
CONH2
N
O
Cl
ii
i
iii
Cl
1
2
3
4
NH
R2
N
N
H
N
Het
N
N
N
S
Cl
S
Cl
X1
S
Cl
iv
v
O
O
X2
R4
6
5
Scheme 1. Reagents and conditions: (i) SOCl2, MeOH, rt, overnight; (ii) NH3ÁH2O, rt, overnight; (iii) glyoxal, 12.5 N NaOH; (iv) P2S5, Pyridine, reflux; (v) ClCH2CONHAr (other
alkyl halides), Na2CO3 or NaOH, EtOH.

P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
6797
2.2. Biological activities
derivative 6q retained its anti-HIV activity in the submicromolar
concentration range (EC50 = 4.0 M).
l
Therefore, based on the above SAR conclusions and the fact that
all the derivatives did not show any activity against HIV-2 (ROD) in
MT-4 cells, it can be concluded that this new series of pyrazin-2-
ylthioacetamide derivatives was specific for HIV-1 and could be
classified as typical NNRTIs.
2.2.1. Anti-HIV activities evaluation
The newly synthesized pyrazin-2-ylthioacetamide derivatives
were evaluated for anti-HIV activity by determining their ability
to inhibit the replication of the HIV-1 IIIB strain28 and HIV-2 ROD
strain29 in MT-4 cell cultures30 in comparison with nevirapine
(NVP), zidovudine (azidothymidine, AZT), dideoxycytidine (DDC),
delavirdine (DLV) and efavirenz (EFV) used as reference drugs.
The methodology of the cell-based anti-HIV assay has been previ-
ously described.31,32 The cytotoxicity of these compounds was
determined in parallel. Comparisons of the antiviral inhibitory con-
centration (EC50), cytotoxic concentration (CC50), and SI (selectiv-
ity, given by the CC50/EC50 ratio) values for different compounds
are depicted in Table 1.
2.2.2. HIV-1 RT inhibition assay
With the aim to further confirm the drug target of pyrazin-2-
ylthioacetamide derivatives, the selected title compound 6k was
tested in enzymatic assays against highly purified recombinant
HIV-1 RT using poly(rC)-oligo(dG) as template primer.33 As shown
in Table 2, this compound exhibited moderate inhibition of enzy-
matic activity with an IC50 value of 50
lM, which was 18 fold high-
er than that of NVP (2.7
lM). This result effectively verified the
The experimental results indicated that 13 members of the
tested pyrazin-2-ylthioacetamide derivatives were found to be
active against HIV-1 (IIIB) with EC50 values in the range of
same drug target for 6k.
3. Molecular modeling
1.7–9.0
2 (ROD). In particular, the most potent compound was found to
be 6k having an EC50 value of 1.7 M against HIV-1(IIIB) with a
lM, and none of the compounds was active against HIV-
To better elucidate the HIV inhibitory potencies of the newly
synthesized compounds at a molecular level and to understand
the structural basis of their binding mode, a molecular modeling
analysis of compound 6k was performed by means of Autodock
Vina [http://vina.scripps.edu]. The X-ray crystal structure of HIV-
1 RT with benzophenone taken from PDB (3DLG) was used as the
input structure for docking calculations because of the high degree
of structural similarity between arylazolylthioacetanilides and
benzophenones.5 Default parameters were used as described in
the Autodock Vina manual unless otherwise specified. The theoret-
ical binding mode of 6k to the NNIBP is shown in Figure 3.
Results suggested that this class of compounds shares a similar
binding mode with previous arylazolylthioacetanilides.17 As illus-
trated in Figure 3, the 2-chlorophenyl ring of 6k fitted into the aro-
matic-rich binding pocket, surrounded by the aromatic side chains
of Tyr188, Phe227, and Trp229. Detailed analysis of the binding
mode showed that one phenyl ring interacts favorably with the
l
selectivity index (SI) of 23. These results indicated that the pyra-
zine ring is an appropriate bioisosteric group instead of the five-
membered heterocycles in the lead compounds.
Table 1 also reveals the potency order of the ortho substitution
at the phenyl ring of the anilide moiety: NO2 (6k,
EC50 = 1.7
l
M) > Br (6e, EC50 = 4.2
l
M) > Cl (6c, EC50 = 4.6
lM) > F
M). It is very clear that sig-
(6b, EC50 = 5.3
lM) > H (6a, EC50 >35
l
nificantly decreased potency was observed for 6a characterized
by the absence of the ortho substitution at the phenyl ring of the
anilide moiety. Consistent with the observations in the arylazolyl-
thioacetanilide series15,17, these results indicated that the activity
data of the pyrazin-2-ylthioacetamide derivatives are affected by
the electronic nature or the steric demand of the ortho substitution.
In addition, it is worth noting that the anti-HIV activity was
also strongly dependent on the nature of the para position of
the anilide moiety. For instance, it has been shown that the intro-
duction of 4-acetyl or a methoxy carbonyl group led to com-
pounds with slightly improved or similar activity (6h, 6i),
whereas, when the methyl group or chlorine atom was intro-
duced into the para position of the anilide moiety, the bioactivity
was strikingly decreased (6e/6f, 6k/6l, 6d/6c) suggesting that the
nature (probably hydrophobicity) of the methyl or chlorine atom
did not accommodate the chemical environment in this region of
RT.
Tyr188 side chain, giving rise to a
p–p stacking interaction. One
N atom in the pyrazine ring and the nitro group at the phenyl ring
of the anilide moiety form multiple key hydrogen bonds with their
surrounding amino acid residues, which is important for the affin-
ity between inhibitor and RT.
Taken together, our docking calculations allowed us to rational-
ize the activity profile of the pyrazin-2-ylthioacetamide against
wild-type RT, which provided valuable information for further de-
sign of novel effective anti-HIV drugs.
In the 1,2,3-thiadiazole series, we16 have demonstrated that the
introduction of the pyridine moiety at the aromatic amine domain
led to substantial improvement in potency and selectivity. Inter-
estingly, this beneficial effect was also observed in this closely re-
lated pyrazin-2-ylthioacetamide series (as shown in 6c and 6m).
Thus again, these results confirmed the idea that the introduction
of structurally diverse heterocycles in this region could be a valid
strategy to get novel molecules with increased or appreciable anti-
viral potency. Meanwhile, it was clear that the anti-HIV activities
of the pyrazin-2-ylthioacetamides are very sensitive to these struc-
tural modifications.
From the structure–activity relationship (SAR) point of view, we
found that the SAR features of the pyrazin-2-ylthioacetamides
were highly consistent with the previously observed arylazolyl-
thioacetanilide-typed NNRTIs.15–21
Moreover, in order to obtain comprehensive SAR indications
and to identify more potent NNRTIs, the replacement of the
N-substituted heterocycle acetamide moiety afforded 3,4-dihy-
dro(iso)quinolin-2(1H)-yl ethanone derivatives 6p and 6q. It was
also observed that 3,4-dihydroisoquinolin-2(1H)-yl ethanone
4. Conclusion
In summary, in the present investigation, we have designed a
series of novel 2-2-(3-(2-chlorophenyl)pyrazin-2-ylthio)-N-arylac-
etamide derivatives as inhibitors of HIV-1 RT based on the struc-
ture-based bioisosterism approach and described the convenient
and efficient method of synthesis for the first time. The preliminary
SAR was discussed. Some derivatives proved to be highly effective
in inhibiting HIV-1 replication at low micromolar concentrations.
Among them, compound 6k was identified as the most promising
candidate with favorable inhibitory activity against the wild-type
HIV-1. The RT inhibitory potency was also assayed. Although the
IC50 value of the tested compound were suboptimal comparing to
that of nevirapine, the valuable information from SAR analysis
and molecular simulation encouraged us toward the rational de-
sign of new derivatives. Further structure modifications on the
six-membered heterocycle section of arylazinylthioacetanilide
scaffold are underway.

6798
P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
Table 1
Anti-HIV activity, cytotoxicity and selectivity indices of 2-(3-(2-chlorophenyl)pyrazin-2-ylthio)-N-arylacetamide derivatives (6a–6q)
N
R2
H
N
N
S
X1
Cl
O
X2
R4
Code
X1
X2
R2
R4
EC50
(
l
M)a
CC50
HIV-1 IIIB
(
l
M)b
SIc
HIV-1 IIIB
HIV-2 ROD
HIV-2 ROD
HIV-1 IIIB
HIV-2 ROD
6a
6b
6c
6d
6e
6f
6g
6h
6i
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
N
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
H
F
Cl
Cl
Br
Br
Br
Br
Br
Br
NO2
NO2
Cl
H
H
H
Cl
H
Me
Cl
COMe
COOMe
COOEt
H
Me
H
>35
5.3
4.6
5.2
4.2
4.3
5.1
2.5
3.7
9.0
1.7
2.9
2.4
>33
>91
>46
>34
>41
>35
>29
>31
>108
>62
>55
>148
>166
35
48
34
29
31
31
26
30
95
45
39
54
152
33
91
46
34
41
35
29
31
108
62
55
148
166
<1
9
7
6
7
<1
<1
< 1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
9
5
12
25
5
23
19
64
6j
6k
6l
6m
N
N
COOMe
S
H
N
N
S
Cl
6n
6o
6p
>68
>32
>33
>48
>30
>34
68
32
33
48
30
34
<1
<1
<1
<1
<1
<1
O
O
O
H
N
N
S
S
Cl
N
N
N
N
N
N
S
Cl
N
S
6q
4.10
>50
33
50
8
<1
O
Cl
NVPd
AZTd
DDCd
EFVd
DLVd
0.090
0.021
1.0
0.005
0.032
>15
249
>95
>6.3
>36
>15
249
>95
>6.3
>36
>168
12,221
>93
>1187
>1096
0.004
1.3
56,907
>74
In bold are the values of active compounds.
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cells against HIV-1-induced cytopathicity, 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). The SI values: X1 stands for P1 or <1.
b
c
d
The data were obtained from the same laboratory (Rega Institute for Medical Research, K.U. Leuven, Belgium).
with a Nexus 470FT-IR Spectrometer. 1H NMR spectra were re-
5. Experimental
5.1. Chemistry
corded on a Bruker Avance 600 or 400 spectrometer at 600 or
400 MHz, using DMSO-d6 as solvent and tetramethylsilane (TMS)
as internal standard. Chemical shifts are reported in parts per mil-
lion (d), and signals are expressed as s (singlet), d (doublet), t (trip-
let), q (quartet) or m (multiplet). Mass spectra were taken on a LC
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded

P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
6799
Table 2
eral hours, 1.0 mL of glacialacetic acid was added with stirring. The
melicera precipitate was formed and extracted with ethyl acetate.
The organic layer was dried over anhydrous sodium sulfate and fil-
tered; then, the filtrate was removed under vacuo. The residue was
purified by recrystallization from ethanol-petroleum ether to yield
the 3-(2-chlorophenyl)pyrazin-2(1H)-one (4) as brown-yellow so-
lid. Mp: 176–179 °C. MS (ESI): m/z 207.2 (M+1), 209.2 (M+3).
Inhibitory activity of compound 6k against HIV-1 RT
Compd
IC50
M)a
6k
50
NVP
2.7
(
l
a
50% of the inhibitory concentration of tested compound required to inhibit
biotin deoxyuridine triphosphate (biotin-dUTP) incorporation into the HIV-1 RT by
50%.
C10H7ClN2O (Exact Mass: 206.02).
To a solution of 3-(2-chlorophenyl)pyrazin-2(1H)-one (4) (0.4 g,
1.93 mmol) in anhydrous pyridine (20 mL), phosphorous pentasul-
fide (1.0 g, 4.5 mmol) was added. The mixture was refluxed for
10 h. The pyridine was removed by evaporation under reduced
pressure and the residue hydrolyzed by the addition of 100 mL
water followed by heating the mixture in a steam bath until the
evolution of hydrogen sulfide had ceased.
The mixture was allowed to cool to room temperature and ex-
tracted by ethyl acetate (3 Â 15 mL). The combined orange solution
was washed with 10% aqueous potassium hydroxide (3 Â 15 mL).
The extract was acidified with diluted hydrochloric acid, the saf-
fron yellow precipitated solid was collected and dried to afford
the 3-(2-chlorophenyl)pyrazine-2(1H)-thione (5) as reddish brown
powder mp: 138–141 °C. MS (ESI): m/z 223.2 (M+1), 225.3 (M+3).
N
NO2
H
N
N
S
O
Cl
6k
EC50 = 1.7 µM
C
10H7ClN2S (Exact Mass: 222).
Figure 3. Predicted binding mode and molecular docking of compound 6k into the
allosteric site of HIV-1 RT (PDB code: 3DLG). The docking results are shown by
PyMOL. Hydrogen bonds are indicated by dashed lines.
5.2.2. General procedure for the synthesis of 2-(3-(2-
chlorophenyl)pyrazin-2-ylthio) acetamides (6a–o)
To the mixture solution of 3-(2-chlorophenyl)pyrazine-2(1H)-
thione (5) (1.0 mmol, 0.22 g) and Na2CO3 (1.5 mmol) in ethanol
(30 mL) were added ClCH2CONHPh (other alkyl halides)
(1.0 mmol). The reaction mixture was stirred at room temperature
overnight. Upon completion of the reaction, the solvent was evap-
orated, leaving a residue which was treated with methylene chlo-
ride (30 ml) and washed with water (3 Â 30 ml). The organic layer
was dried over anhydrous sodium sulfate, filtered off, and then the
solvent was removed under vacuo. The residue was chromato-
graphed on silica gel using ethyl acetate:petroleum ether. Pure
fractions were collected and concentrated, giving the desired com-
pounds (6a–6q) in good yield.
Autosampler Device: Standard G1313A instrument. TLC was per-
formed on Silica Gel GF254 for TLC and spots were visualized by io-
dine vapors or by irradiation with UV light (254 nm). Flash column
chromatography was performed on a column packed with Silica
Gel 60 (200–300 mesh). Solvents were reagent grade and, when
necessary, were purified and dried by standard methods. Concen-
tration of the reaction solutions involved the use of rotary evapo-
rator at reduced pressure.
5.2. General procedure for the synthesis of 2-(3-(2-
chlorophenyl)pyrazin-2-ylthio) acetamides (6a–o)
5.2.1. General procedure for the synthesis of 3-(2-
5.2.3. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-
chlorophenyl)pyrazine-2(1H)-thione (5)
phenylacetamide (6a)
To a vigorously stirred mixture of the commercially available 2-
amino-2-(2-chlorophenyl)acetic acid (1, 9.28 g, 0.05 mmol) and
anhydrous MeOH (150 mL) at 0 °C was added thionyl chloride
(4.4 mL) dropwise. The resulting solution was stirred at room tem-
perature for 24 h (monitored by TLC), and then concentrated to
40 mL. The residue was triturated with diethyl ether to produce
the white solid, which was collected and dried to afford the
(+/À)-2-chlorophenylglycine methyl ester HCl salt (2) as gray
powder. Compound 2 was used in the next step without any
further purification.
White powder, yield: 76.4%. Mp: 152–154 °C. 1H NMR (CDCl3,
ppm) d: 9.66 (s, 1H, NH), 8.51 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.47
(d, 1H, J = 2.4 Hz, pyrazine-H), 7.74 (dd, 1H, J1 = 0.6 Hz, J2 = 8.4 Hz,
PhH), 7.67–7.64 (m, 2H, PhH), 7.63 (dd, 1H, J1 = 0.6 Hz, J2 = 7.0 Hz,
PhH), 7.55 (d, 2H, J = 7.8 Hz, Ph’H), 7.34 (t, 2H, J = 8.4 Hz, Ph’H),
7.10 (d, 1H, J = 7.0 Hz, Ph’H), 3.93 (s, 2H, CH2-S). IR (KBr, cmÀ1):
3257 (tNH), 3085, 2957, 2924, 1647 (
t
C@O), 1600, 1558, 1498,
1445 (tN@N), 1331, 1239, 1117, 754 (
tC–S). MS (ESI): m/z 356.3
(M+1), 358.3 (M+3). C18H14ClN3OS (Exact Mass: 355.05).
The prepared methyl ester HCl salt was taken up in NH3ÁH2O
(45 mL), and the reaction mixture was stirred at room temperature
for 24 h. The solution was concentrated and the obtained solid was
collected and recrystallized from ethyl acetate to afford the desired
product (+/À)-2-amino-2-(2-chlorophenyl)acetamide (3) as white
powder, which was pure enough for the condensation with
glyoxal.25
5.2.4. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(2-
fluorophenyl)acetamide (6b)
White powder, yield: 72.8%. Mp: 107–109 °C. 1H NMR (CDCl3,
ppm) d: 9.64 (s, 1H, NH), 8.52 (d, 1H, J = 3.0 Hz, pyrazine-H), 8.48
(d, 1H, J = 3.0 Hz, pyrazine-H), 8.35 (t, 1H, J = 7.8 Hz), 7.53 (d, 1H,
J = 7.8 Hz), 7.47–7.45 (m, 1H), 7.42–7.37 (m, 2H), 7.13–7.11 (m,
1H), 7.07–7.02 (m, 2H), 3.90 (s, 2H, CH2-S). IR (KBr, cmÀ1): 3303
To 25 g (16.6 mmol) of 40% aqueous glyoxal solution diluted
with 25 mL of water was added 1.84 g. (0.010 mol) of finely pow-
dered 2-amino-2-(2-chlorophenyl)acetamide (3). The mixture
was cooled in an ice bath while kept stirring, was added dropwise
1.0 mL (0.0125 mol) of 12.5 N sodium hydroxide solution The
resulting solution was allowed to stand at room temperature for
a few minutes, which had set to a semisolid mass of finely-divided
crystals (sodium salt of 3-(2-chlorophenyl)pyrazin-2-ol). After sev-
(
t
NH), 3118, 3064, 2925, 1693 (
t
C@O), 1621, 1547, 1454 (
tN@N),
1361, 1330 ( C–F), 1132, 1070, 755, 751 (
374.2 (M+1), 376.3 (M+3). C18H13ClFN3OS (Exact Mass: 373.05).
t
tC–S). MS (ESI): m/z
5.2.5. N-(2-Chlorophenyl)-2-(3-(2-chlorophenyl)pyrazin-2-
ylthio)acetamide (6c)
White powder. 1H NMR (CDCl3, ppm) d: 9.29 (s, 1H, NH), 8.50 (d,
1H, J = 2.4 Hz, pyrazine-H), 8.45 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.36

6800
P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
(dd, 1H, J1 = 8.4 Hz, J2 = 0.6 Hz), 7.55–7.54 (m, 1H), 7.48–7.45 (m,
1H), 7.43–7.38 (m, 2H), 7.38–7.33 (m, 1H), 7.32–7.25 (m, 1H),
7.04–7.02 (m, 1H), 4.38 (s, 2H, CH2-S). MS (ESI): m/z 390.3 (M+1),
392.3 (M+2). C18H13Cl2N3OS (Exact Mass: 389.02).
PhH), 7.44–7.38 (m, 2H, PhH), 3.98 (s, 2H, CH2-S), 2.57 (s, 3H,
CH3). IR (KBr, cmÀ1): 3282 (
NH), 3260, 2955, 2923, 2853, 1698
C@O), 1673 ( C@O), 1595, 1572, 1524, 1467, 1357 ( N@N), 1309,
1266, 1128, 1070, 830, 750 ( C–S). MS (ESI): m/z 476.3 (M+1),
t
(t
t
t
t
478.2 (M+3), 480.2 (M+5). C20H15BrClN3O2S (Exact Mass: 474.98).
5.2.6. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(2,4-
dichlorophenyl)acetamide (6d)
5.2.11. Methyl 3-bromo-4-(2-(3-(2-chlorophenyl)pyrazin-2-
ylthio)acetamido)benzoate (6i)
Yellow powder, yield: 64.8%. Mp: 148–150 °C. 1H NMR (CDCl3,
ppm) d: 9.31 (s, 1H, NH), 8.48 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.47
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.33 (d, 1H, J = 9.0 Hz, Ph’H), 7.54
(dd, 1H, J1 = 9.0 Hz, J2 = 0.6 Hz), 7.46 (dt, 1H, J1 = 7.8 Hz,
J2 = 1.8 Hz), 7.42 (dt, 1H, J1 = 7.2 Hz, J2 = 1.2 Hz), 7.37 (dd, 1H,
J1 = 7.8 Hz, J2 = 1.8 Hz), 7.48 (d, 1H, J = 2.4 Hz), 7.25–7.23 (m, 1H),
White powder, yield: 78.5%. Mp: 101–103 °C. 1H NMR (CDCl3,
ppm) d: 9.31 (s, 1H, NH), 8.51 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.47
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.46 (d, 1H, J = 3.6 Hz), 8.19 (d, 1H,
J = 3.6 Hz), 7.96 (dd, 1H, J1 = 8.4 Hz, J2 = 3.6 Hz), 7.55–7.54 (m,
1H), 7.46 (dt, 1H, J1 = 7.8 Hz, J2 = 1.8 Hz), 7.43–7.38 (m, 2H), 3.98
3.94 (s, 2H, CH2-S). IR (KBr, cmÀ1): 3256 (
1581, 1522, 1386 ( N@N), 1359, 1301, 1132, 1071, 865, 826, 750
C–S). 13C NMR (100 MHz, DMSO-d6, ppm): 167.3 (C@O), 154.6,
tNH), 3073, 1693 (
tC@O),
(s, 2H, CH2-S), 3.90 (s, 3H, CH3). IR (KBr, cmÀ1): 3254 (
2997, 2945, 1719 ( O–C@O), 1700 (
1475, 1436, 1391 ( N@N), 1360, 1288, 1272, 1227, 1119, 1072,
762 C–S), 749. MS (ESI): m/z 492.1 (M+1), 494.2 (M+3).
20H15BrClN3O3S (Exact Mass: 490.97).
t
NH), 3058,
t
t
tNH–C@O), 1599, 1577, 1523,
(t
t
150.9, 143.8, 139.9, 135.8, 134.3, 132.6, 131.8, 131.4, 130.2,
129.6, 129.4, 128.1, 128.1, 126.9, 126.5, 34.53 (CH2-S). MS (ESI):
m/z 424.3 (M+1), 426.3 (M+3), 428.2 (M+5). C18H12Cl3N3OS (Exact
Mass: 422.98).
(t
C
5.2.12. Ethyl 3-bromo-4-(2-(3-(2-chlorophenyl)pyrazin-2-
ylthio)acetamido)benzoate (6j)
5.2.7. N-(2-Bromophenyl)-2-(3-(2-chlorophenyl)pyrazin-2-
ylthio)acetamide (6e)
Yellow powder, yield: 77.6%. Mp: 128–130 °C. 1H NMR (CDCl3,
ppm) d: 9.31 (s, 1H, NH), 8.51 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.47
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.46 (s, 1H, Ph’H), 8.19 (d, 1H,
J = 1.8 Hz), 7.97 (dd, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz), 7.71 (d, 1H,
J = 7.2 Hz), 7.47 (dt, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz), 7.44–7.38 (m, 2H),
4.38 (s, 2H, CH2-S), 4.37 (q, 2H, J = 1.2 Hz, CH2), 1.38 (t, 3H,
White powder, yield: 58.4%. Mp: 120–122 °C. 1H NMR (CDCl3,
ppm) d: 9.11 (s, 1H, NH), 8.51 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.45
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.30 (d, 1H, J = 7.8 Hz), 7.54 (d, 1H,
J = 7.8 Hz), 7.51–7.45 (m, 2H), 7.42–7.39 (m, 2H), 7.32–7.30 (m,
1H), 6.99–6.96 (m, 1H), 3.97 (s, 2H, CH2-S). IR (KBr, cmÀ1): 3346
J = 1.2 Hz, CH3). IR (KBr, cmÀ1): 3257 (
2928, 2898, 1712 ( O–C@O), 1705 ( NH–C@O), 1599, 1579, 1521,
1477, 1439, 1391 ( N@N), 1359, 1283, 1273, 1229, 1128, 1117,
1072, 761 ( C–S), 748. MS (ESI): m/z 506.2 (M+1), 508.2 (M+3),
tNH), 3073, 3074, 2984,
(tNH), 3055, 1688 (
t
C@O), 1593, 1579, 1518, 1434 (
t
N@N), 1359,
t
t
1296, 1117, 1072, 761 (
t
C–S), 750. MS (ESI): m/z 434.3 (M+1),
t
436.3 (M+3). C18H13BrClN3OS (Exact Mass: 432.97).
t
510.2 (M+5). C21H17BrClN3O3S (Exact Mass: 504.99).
5.2.8. N-(2-Bromo-4-methylphenyl)-2-(3-(2-
chlorophenyl)pyrazin-2-ylthio)acetamide (6f)
5.2.13. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(2-
nitrophenyl)acetamide (6k)
Light yellow brown powder, yield: 66.2%. Mp: 124–126 °C. 1H
NMR (CDCl3, ppm) d: 9.56 (s, 1H, NH), 8.15 (d, 1H, J = 2.4 Hz, pyra-
zine-H), 7.92 (d, 1H, J = 2.4 Hz, pyrazine-H), 7.65 (dd, 1H,
J1 = 7.8 Hz, J2 = 0.6 Hz, Ph’H), 7.58–7.49 (m, 4H, PhH), 7.47 (d, 1H,
J = 0.6 Hz, Ph’H), 7.16 (d, 1H, J = 7.8 Hz, Ph’H), 4.08 (s, 2H, CH2-S),
2.27 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6, ppm): 166.9
(C@O), 154.7, 150.9, 143.9, 139.9, 137.0, 135.8, 133.8, 133.1,
132.6, 131.8, 131.4, 130.2, 129.1, 128.1, 126.1, 117.1, 34.53
Yellow powder, yield: 48.6%. Mp: 107–109 °C. 1H NMR (CDCl3,
ppm) d: 11.12 (s, 1H, NH), 8.70 (dd, 1H, J1 = 8.4 Hz, J2 = 1.2 Hz,
Ph’H), 8.53 (d, 1H, J = 3.0 Hz, pyrazine-H), 8.44 (d, 1H, J = 3.0 Hz,
pyrazine-H), 8.17 (d, 1H, J1 = 8.4 Hz, J2 = 1.2 Hz, Ph’H), 7.65–7.63
(m, 1H), 7.55–7.54 (m, 1H), 7.47–7.41 (m, 3H), 7.21–7.18 (m,
1H), 3.79 (s, 2H, CH2-S). IR (KBr, cmÀ1): 3350 (
C@O), 1608, 1586, 1497 (tas NO2), 1457 (
NO2), 1284, 1118, 1070, 752 ( C–S), 743. MS (ESI): m/z 401.3
tNH), 3093, 1697
(t
tN@N), 1436, 1344 (ts
(CH2-S), 20.5 (CH3). IR (KBr, cmÀ1): 3348 (
1697 ( C@O), 1684, 1574, 1519, 1431 ( N@N), 1361, 1294, 1119,
1071, 765 ( C–S). MS (ESI): m/z 448.2 (M+1), 450.2 (M+3), 452.2
t
NH), 3030, 2987, 2922,
t
t
t
(M+1), 402.3 (M+3). C18H13ClN4O3S (Exact Mass: 400.04).
t
(M+5). C19H15BrClN3OS (Exact Mass: 446.98).
5.2.14. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(4-methyl-2-
nitrophenyl)acetamide (6l)
5.2.9. N-(2-Bromo-4-chlorophenyl)-2-(3-(2-
chlorophenyl)pyrazin-2-ylthio)acetamide (6g)
White powder, yield: 74.1%. Mp: 145–147 °C. 1H NMR (CDCl3,
ppm) d: 11.01 (s, 1H, NH), 8.57 (d, 1H, J = 8.4 Hz, Ph’H), 8.56 (d,
1H, J = 2.4 Hz, pyrazine-H), 8.52 (d, 1H, J = 2.4 Hz, pyrazine-H),
7.96 (s, 1H, Ph’H), 7.54 (d, 1H, J = 8.4 Hz, Ph’H), 7.47–7.41 (m, 4H,
PhH), 3.99 (s, 2H, CH2-S), 2.40 (s, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6, ppm): 167.1 (C@O), 154.3, 150.9, 143.8, 141.2, 139.9,
135.8, 135.5, 132.6, 131.8, 131.4, 130.2, 129.6, 128.0, 125.4,
Brown powder, yield: 59.4%. Mp: 140–142 °C. 1H NMR (CDCl3,
ppm) d: 9.12 (s, 1H, NH), 8.50 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.46
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.28 (d, 1H, J = 8.4 Hz, Ph’H), 7.54
(d, 1H, J = 8.4 Hz, PhH), 7.51 (d, 1H, J = 2.4 Hz), 7.47 (dt, 1H,
J1 = 7.8 Hz, J2 = 1.2 Hz), 7.43–7.38 (m, 2H, PhH), 7.27 (dd, 1H,
J1 = 9.0 Hz, J2 = 2,4 Hz, Ph’H), 4.95 (s, 2H, CH2-S). IR (KBr, cmÀ1):
125.0, 34.6 (CH2-S), 20.4 (CH3). IR (KBr, cmÀ1): 3327 (
2987, 2924, 2854, 1703, 1690 ( C@O), 1574, 1497 (tas NO2), 1456
N@N), 1361, 1337 ( s NO2), 1276, 1121, 766 ( C–S). MS (ESI): m/z
tNH), 3075,
3277 (
t
NH), 3068, 1692 (
t
C@O), 1573, 1517, 1383 (
t
N@N), 1358,
t
1295, 1131, 1071, 866, 824, 750 (
t
C–S). C18H12BrCl2N3OS (Exact
(t
t
t
Mass: 466.93).
415.4 (M+1), 417.3 (M+3). C19H15ClN4O3S (Exact Mass: 414.06).
5.2.10. N-(4-Acetyl-2-bromophenyl)-2-(3-(2-
5.2.15. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(2-
chlorophenyl)pyrazin-2-ylthio)acetamide (6h)
chloropyridin-3-yl)acetamide (6m)
White powder, yield: 66.7%. Mp: 159–160 °C. 1H NMR (CDCl3,
ppm) d: 9.34 (s, 1H, NH), 8.51 (d, 1H, J = 3.0 Hz, pyrazine-H), 8.50
(d, 1H, J = 8.4 Hz, Ph’H), 8.47 (d, 1H, J = 3.0 Hz, pyrazine-H), 8.14
(d, 1H, J = 2.4 Hz, Ph’H), 7.88 (dd, 1H, J1 = 8.4 Hz, J2 = 2.4 Hz, Ph’H),
7.55 (d, 1H, J = 7.8 Hz, PhH), 7.48 (dt, 1H, J1 = 7.8 Hz, J2 = 1.8 Hz,
White powder, yield: 58.7%. Mp: 148–150 °C. 1H NMR (CDCl3,
ppm) d: 9.47 (s, 1H, NH), 8.73 (d, 1H, J = 7.8 Hz), 8.52 (d, 1H,
J = 2.4 Hz, pyrazine-H), 8.50 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.10
(d, 1H, J = 4.2 Hz), 7.55 (d, 1H, J = 8.4 Hz), 7.47 (t, 1H, J = 7.8 Hz),
7.42–7.38 (m, 2H), 7.25 (d, 1H, J = 9.6 Hz), 3.95 (s, 2H, CH2-S). IR

P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
6801
(KBr, cmÀ1): 3251 (
N@N), 1391, 1359, 1300, 1128, 1078, 1071, 801, 746 (t
(ESI): m/z 391.3 (M+1), 393.2 (M+3). C17H12Cl2N4OS (Exact Mass:
t
NH), 3075, 2996, 1691, 1583, 1526, 1455
C–S). MS
Mock-infected cells were used to evaluate the effect of the test
compounds on uninfected cells in order to assess the cytotoxicity
of the test compounds. Exponentially growing MT-4 cells30 were
centrifuged for 5 min at 1000 rpm and the supernatant was dis-
carded. The MT-4 cells were resuspended at 6 Â 105 cells/mL, and
(t
390.01).
5.2.16. Methyl 3-(2-(3-(2-chlorophenyl)pyrazin-2-
50-lL volumes were transferred to the microtiter tray wells. Five
ylthio)acetamido)thiophene-2-carboxylate (6n)
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
White powder, yield: 77.3%. Mp: 148–150 °C. 1H NMR
(DMSO-d6, ppm) d: 10.99 (s, 1H, NH), 8.52 (d, 1H, J = 2.4 Hz,
pyrazine-H), 8.40 (d, 1H, J = 2.4 Hz, pyrazine-H), 8.13 (d, 1H,
J = 5.4 Hz, thiophene-H), 7.55–7.52 (m, 2H), 7.47–7.40 (m, 3H),
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) (Acros Organics, Geel, Belgium) by mitochondrial dehydro-
genase 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 (540 and 690 nm). All data were calculated using the
median OD (optical density) value of three wells. The 50% cytotoxic
concentration (CC50) was defined as the concentration of the test
compound that reduced the absorbance (OD540) of the mock-in-
fected control sample by 50%. The 50% effective concentration
(EC50) was defined as the compound concentration required to in-
hibit virus-induced syncytium formation by 50%.
4.02 (s, 2H, CH2-S), 3.86 (s, 3H, Me). IR (KBr, cmÀ1): 3211 (
t
NH),
NH–C@O), 1569,
N@N), 1279, 1259, 1122, 1094, 1074, 783,
C–S). MS (ESI): m/z 420.2 (M+1), 422.3 (M+3).
18H14ClN3O3S2 (Exact Mass: 419.02).
3092, 2947, 2919, 2850, 1689 (
1442, 1420, 1376 (
7445
tO–C@O), 1662 (t
t
(t
C
5.2.17. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-N-(5-
methylbenzo[d]thiazol-2-yl)acetamide (6o)
White powder, yield: 74.3%. Mp: 102–104 °C. 1H NMR (CDCl3,
ppm) d: 10.67 (s, 1H, NH), 8.58 (d, 1H, J = 2.4 Hz, pyrazine-H),
8.52 (d, 1H, J = 2.4 Hz, pyrazine-H), 7.70 (d, 1H, J = 7.8 Hz), 7.60
(s, 1H, Ph’H), 7.55 (d, 1H, J = 7.8 Hz), 7.48–7.38 (m, 3H), 7.12 (d,
1H, J = 8.4 Hz), 3.49 (s, 2H, CH2-S), 2.43 (s, 3H, CH3). IR (KBr,
5.3.2. HIV-1 RT inhibition assay
cmÀ1): 3425 (
1464, 1432, 1358 (
C–S). MS (ESI): m/z 427.2 (M+1), 429.3 (M+3). C20H15ClN4OS2
tNH), 3138, 3053, 2920, 1695 (
t
C@O), 1607, 1547,
Inhibition of HIV-1 RT was developed using nucleotides linked
to microtiter plate wells with colorimetric detection of incorpo-
rated biotin-dUTP into homopolymer template primers.33 The
incorporated quantities of the biotin-dUTP into the enzyme repre-
sented the activity of HIV-1 RT. IC50 values corresponded to the
concentration of the pyrazin-2-ylthioacetamide derivatives re-
quired to inhibit biotin-dUTP incorporation into the HIV-1 RT by
50%.
t
N@N), 1273, 1154, 1119, 1071, 1015, 813, 757
(t
(Exact Mass: 426.04).
5.2.18. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-1-(3,4-
dihydroquinolin-1(2H)-yl)ethanone (6p)
White powder, yield: 67.3%. Mp: 102–104 °C. 1H NMR (CDCl3,
ppm) d: 8.31 (d, 1H, J = 3.0 Hz, pyrazine-H), 8.26 (d, 1H,
J = 3.0 Hz, pyrazine-H), 7.50–7.16 (m, 8H), 4.16 (s, 2H, CH2-S),
3.83 (s, 2H, CH2), 2.75 (s, 2H, CH2), 1.62 (s, 2H, CH2). IR (KBr, cm
Acknowledgments
À1): 3041, 2947, 2888, 1653 (
1198, 1169, 1071, 1015, 850, 759 (
t
C@O), 1491, 1375 (
tN@N), 1358,
The financial support from the National Natural Science Foun-
dation of China (NSFC No. 81102320, No. 30873133, No.
30772629, No. 30371686), Key Project of NSFC for International
Cooperation (No. 30910103908), Research Fund for the Doctoral
Program of Higher Education of China (No. 20110131130005,
20110131120037), Independent Innovation Foundation of Shan-
dong University (IIFSDU, No. 2010GN044), Shandong Postdoctoral
Innovation Science Research Special Program (No. 201002023),
China Postdoctoral Science Foundation funded project (No.
20100481282, 2012T50584) and KU Leuven (GOA 10/014) is grate-
fully acknowledged.
tC–S), 757. MS (ESI): m/z 396.2
(M+1), 398.2 (M+3). C21H18ClN3OS (Exact Mass: 395.09).
5.2.19. 2-(3-(2-Chlorophenyl)pyrazin-2-ylthio)-1-(3,4-
dihydroisoquinolin-2(1H)-yl)ethanone (6q)
White powder. 1H NMR (CDCl3, ppm) d: 8.37–8.32 (m, 2H), 8.26
(d, 1H, J = 2.4 Hz, pyrazine-H), 8.22 (d, 1H, J = 2.4 Hz, pyrazine-H),
7.51–7.43 (m, 2H), 7.41–7.35 (m, 4H), 4.79 (s, 1H, CH), 4.73 (s,
1H, CH), 4.31 (s, 2H, CH2-S), 3.83 (t, 1H, J = 6.0 Hz, CH), 3.74 (t,
1H, J = 6.0 Hz, CH), 2.94 (t, 1H, J = 6.0 Hz, CH), 2.85 (t, 1H,
J = 6.0 Hz, CH). IR (KBr, cmÀ1): 3046, 2925, 1644 (
1358 ( N@N), 1119, 1071, 1014, 758 (
tC@O), 1432,
We thank K. Erven, K. Uyttersprot and C. Heens for technical
assistance with the anti-HIV assays.
t
tC–S), 730. MS (ESI): m/z
396.2 (M+1), 398.3 (M+3). C21H18ClN3OS (Exact Mass: 395.09).
The authors have declared no conflict of interest.
5.3. Biological activity
References and notes
1. Zhan, P.; Liu, X.; Li, Z. Curr. Med. Chem. 2009, 16, 2876.
2. Zhan, P.; Liu, X.; Li, Z.; Pannecouque, C.; De Clercq, E. Curr. Med. Chem. 2009, 16,
3903.
3. Zhan, P.; Chen, X.; Li, D.; Fang, Z.; De Clercq, E.; Liu, X. Med. Res. Rev. 2011.
http://dx.doi.org/10.1002/med.20241.
4. Zhan, P.; Liu, X. Expert. Opin. Ther. Pattern 2011, 5, 717.
5.3.1. In vitro anti-HIV assay
The methodology of the anti-HIV assay has been previously de-
scribed.31,32 Stock solutions (10 Â final concentration) of test com-
pounds were added in 25-lL volumes to two series of triplicate
wells in 96-well microtiter plates so as to allow simultaneous eval-
uation of their effects on mock- and HIV-infected cells at the begin-
ning of each experiment. Serial fivefold dilutions of test
compounds were made directly in flat-bottomed 96-well microti-
ter trays using a Biomek 3000 robot (Beckman instruments, Fuller-
ton, CA). Untreated control HIV- and mock-infected cell samples
were included for each sample.
5. Zhan, P.; Li, Z.; Liu, X.; De Clercq, E. Mini-Rev. Med. Chem. 2009, 9, 1014.
6. Wang, Z.; Wu, B.; Kuhen, K. L.; Bursulaya, B.; Nguyen, T. N.; Nguyen, D. G.; He,
Y. Bioorg. Med. Chem. Lett. 2006, 16, 4174.
7. De La Rosa, M.; Kim, H. W.; Gunic, E.; Jenket, C.; Boyle, U.; Koh, Y. H.; Korboukh,
I.; Allan, M.; Zhang, W.; Chen, H.; Xu, W.; Nilar, S.; Yao, N.; Hamatake, R.; Lang,
S. A.; Hong, Z.; Zhang, Z.; Girardet, J. L. Bioorg. Med. Chem. Lett. 2006, 16, 4444.
8. Muraglia, E.; Kinzel, O. D.; Laufer, R.; Miller, M. D.; Moyer, G.; Munshi, V.;
Orvieto, F.; Palumbi, M. C.; Pescatore, G.; Rowley, M.; Williams, P. D.; Summa,
V. Bioorg. Med. Chem. Lett. 2006, 16, 2748.
HIV-1(IIIB)28 or HIV-2 (ROD)29 stock (50
lL) at 100–300 CCID50
9. O’Meara, J. A.; Jakalian, A.; LaPlante, S.; Bonneau, P. R.; Coulombe, R.; Faucher,
A. M.; Guse, I.; Landry, S.; Racine, J.; Simoneau, B.; Thavonekham, B.; Yoakim, C.
Bioorg. Med. Chem. Lett. 2007, 17, 3362.
(cell culture infectious dose) or culture medium was added to
either the infected or mock-infected wells of the microtiter tray.

6802
P. Zhan et al. / Bioorg. Med. Chem. 20 (2012) 6795–6802
10. Gagnon, A.; Amad, M. H.; Bonneau, P. R.; Coulombe, R.; DeRoy, P. L.; Doyon, L.;
Duan, J.; Garneau, M.; Guse, I.; Jakalian, A.; Jolicoeur, E.; Landry, S.; Malenfant,
E.; Simoneau, B.; Yoakim, C. Bioorg. Med. Chem. Lett. 2007, 17, 4437.
11. Gagnon, A.; Landry, S.; Coulombe, R.; Jakalian, A.; Guse, I.; Thavonekham, B.;
Bonneau, P. R.; Yoakim, C.; Simoneau, B. Bioorg. Med. Chem. Lett. 2009, 19, 1199.
12. 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.
13. Li, D.; Zhan, P.; De Clercq, E.; Liu, X. J. Med. Chem. 2012, 55, 3595.
14. Zhao, H.; Guo, Z. Drug Discovery Today 2009, 14, 516.
15. Zhan, P.; Liu, X.; Cao, Y.; Wang, Y.; Pannecouque, C.; De Clercq, E. Bioorg. Med.
Chem. Lett. 2008, 18, 5368.
16. Zhan, P.; Liu, X.; Li, Z.; Fang, Z.; Li, Z.; Wang, D.; Pannecouque, C.; De Clercq, E.
Bioorg. Med. Chem. 2009, 17, 5920.
17. Zhan, P.; Liu, X.; Fang, Z.; Li, Z.; Pannecouque, C.; De Clercq, E. Eur. J. Med. Chem.
2009, 44, 4648.
18. Zhan, P.; Liu, X.; Li, Z.; Fang, Z.; Pannecouque, C.; De Clercq, E. Chem. Biodivers.
2010, 7, 1717.
19. Zhan, P.; Liu, X.; Zhu, J.; Fang, Z.; Li, Z.; Pannecouque, C.; De Clercq, E. Bioorg.
Med. Chem. 2009, 17, 5775.
24. (a) Patani, G. A.; LaVoie, E. J. Chem. Rev. 1996, 96, 3147; (b) Olesen, P. H. Curr.
Opin. Drug Discovery Dev. 2001, 4, 471; (c) Lima, L. M.; Barreiro, E. J. Curr. Med.
Chem. 2005, 12, 23; (d) Sheng, C.; Che, X.; Wang, W.; Wang, S.; Cao, Y.; Miao, Z.;
Yao, J.; Zhang, W. Eur. J. Med. Chem. 2011, 46, 5276.
25. Maarten, W.; van der, M.; Michel, L.; Edith, G.; Wim, L. N.; Hugo, M.; van
Enckevort, W. J. P.; Bernard, K.; Elias, V.; Richard, M. K. Org. Process Res. Dev
2009, 13, 1195.
26. (a) Jones, R. G. J. Am. Chem. Soc. 1949, 71, 78; (b) Semple, G.; Andersson, B. M.;
Chhajlani, V.; Georgsson, J.; Johansson, M. J.; Rosenquist, A.; Swanson, L. Bioorg.
Med. Chem. Lett. 2003, 13, 1141.
27. Baraldi, P. G.; Preti, D.; Tabrizi, M. A.; Fruttarolo, F.; Saponaro, G.; Baraldi, S.;
Romagnoli, R.; Moorman, A. R.; Gessi, S.; Varani, K.; Andrea Borea, P. Bioorg.
Med. Chem. 2007, 15, 2514.
28. Popovic, M.; Sarngadharan, M. G.; Read, E.; Gallo, R. C. Science 1984, 224, 497.
29. Barré-Sinoussi, F.; Chermann, J. C.; Rey, F.; Nugeyre, M. T.; Chamaret, S.; Gruest,
J.; Dauguet, C.; Axler-Blin, C.; Vézinet-Brun, F.; Rouzioux, C.; Rozenbaum, W.;
Montagnier, L. Science 1983, 220, 868.
30. Miyoshi, I.; Taguchi, H.; Kobonishi, I.; Yoshimoto, S.; Ohtsuki, Y.; Shiraishi, Y.;
Agaki, T. Gann. Monogr. 1982, 28, 219.
31. Pauwels, R.; Balzarini, J.; Baba, M.; Snoeck, R.; Schols, D.; Herdewijn, P.;
Desmyter, J.; De Clercq, E. J. Virol. Methods 1988, 20, 309.
32. Pannecouque, C.; Daelemans, D.; De Clercq, E. Nat. Protoc. 2008, 3, 427.
33. (a) Suzuki, K.; Craddock, B. P.; Okamoto, N.; Kano, T.; Steigbigel, R. T. J. Virol.
Methods 1993, 44, 189; (b) Wu, J.; Liu, X.; Cheng, X.; Cao, Y.; Wang, D.; Li, Z.; Xu,
W.; Pannecouque, C.; Witvrouw, M.; De Clercq, E. Molecules 2007, 12, 2003.
20. Zhan, P.; Liu, X.; Fang, Z.; Pannecouque, C.; De Clercq, E. Bioorg. Med. Chem.
2009, 17, 6374.
21. Zhan, P.; Liu, H.; Liu, X.; Wang, Y.; Pannecouque, C.; Witvrouw, M.; De Clercq, E.
Med. Chem. Res. 2009, 19, 652.
22. Zhan, P.; Chen, X.; Li, X.; Li, D.; Tian, Y.; Chen, W.; Pannecouque, C.; De Clercq,
E.; Liu, X. Eur. J. Med. Chem. 2011, 46, 5039.
23. Zhan, P.; Wang, L.; Liu, H.; Chen, X.; Li, X.; Jiang, X.; Zhang, Q.; Liu, X.;
Pannecouque, C.; Naesens, L.; De Clercq, E.; Liu, A.; Du, G. Arch. Pharm. Res.
2012, 35, 1037.