Synthesis and biological evaluation of imidazole thioacetanilides as novel non-nucleoside HIV-1 reverse transcriptase inhibitors

Article abstract of DOI:10.1016/j.bmc.2009.07.028

A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among them, the most potent HIV-1 inhibitors were 4a5 (E

Full text of DOI:10.1016/j.bmc.2009.07.028

Bioorganic & Medicinal Chemistry 17 (2009) 5775–5781
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of imidazole thioacetanilides as novel
non-nucleoside HIV-1 reverse transcriptase inhibitors
a
a
a
b
Peng Zhan a, Xinyong Liu a, , Junjie Zhu , Zengjun Fang , Zhenyu Li , Christophe Pannecouque ,
*
Erik De Clercq b
a Institute of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, No. 44 Wenhuaxi Road, Jinan 250012, PR China
b Rega Institute for Medical Research, Katholieke Universiteit Leuven, 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:
A series of 2-(1-aryl-1H-imidazol-2-ylthio)acetamide [imidazole thioacetanilide (ITA)] derivatives were
Received 3 June 2009
Revised 12 July 2009
Accepted 14 July 2009
Available online 18 July 2009
synthesized and evaluated as potent inhibitors of human immunodeficiency virus type-1 (HIV-1). Among
them, the most potent HIV-1 inhibitors were 4a5 (EC50 = 0.18
lM), and 4a2 (EC50 = 0.20 lM), which were
more effective than the lead compound L1 (EC50 = 2.053 M) and the reference drugs nevirapine and del-
l
avirdine. The preliminary structure–activity relationship (SAR) of the newly synthesized congeners is
discussed.
Keywords:
Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
HIV-1
AIDS
NNRTIs
Imidazole thioacetanilides
Anti-HIV-1 activity
1. Introduction
fication of this region will provide ample opportunity for the
discovery of the next generation of HIV-1 NNRTIs.
The reverse transcriptase (RT) of the human immunodeficiency
virus type 1 (HIV-1) is a key target for inhibition of HIV-1 replica-
tion. The RT can be inhibited by two classes of drug belonging
either to the nucleoside (or nucleotide) reverse transcriptase inhib-
itors (N(t)RTIs) or to the non-nucleoside reverse transcriptase
inhibitors (NNRTIs). HIV-1 NNRTIs have become key components
in the combination regimens of anti-HIV therapy.1–4 Since the first
report of sulfanyltriazole/tetrazoles (L1–L4, Fig. 1) as potent HIV-1
NNRTIs, these novel substituted scaffolds have excited great inter-
est in the development of novel NNRTIs, because of their high po-
tency and low toxicity against HIV-1 wild-type and resistant
strains.5–11 We recently reported that 1,2,3-thiadiazole efficiently
behaves as a triazole/tetrazole surrogate (Fig. 2).12 Therefore, it is
likely that the five-membered heterocycle portion in these inhibi-
tors could simply be acting as a scaffold which orients the pharma-
cophores into the proper geometry for binding to RT. This
conclusion of preliminary SAR analysis was consistent with the re-
sults from molecular modeling studies.11 However, recent research
has also revealed that there are differences in the electronic and
conformational contribution of the heterocyclic group to the bind-
ing of the inhibitors with the HIV-1 RT.13 Therefore, further modi-
In continuation of our research work on understanding the role
of the five-membered heterocycles in the binding of the inhibitors
to the RT and finding potent HIV replication inhibitors, a novel ser-
ies of imidazole thioacetanilide (ITA) derivatives was designed and
synthesized based on the general principle of bioisosterism in
medicinal chemistry.14–16 The triazole, tetrazole or 1,2,3-thiadia-
zole moieties in the corresponding lead compounds were replaced
by the imidazole ring in the ITA analogues (Fig. 2), the other frag-
ments which were considered to be necessary for conserving anti-
X
X
N
N
N N
H
N
H
N
N
S
S
N
N
O
O
Y
R
R
L3. R=2,4,6-TriMe, X=Cl
L1. R=CH3, X=Y=H,
EC50= 2.053µM(WT)
L2. R=H, X=Br, Y=CH3
EC50= 0.1 µM(WT)
IC50= 5 nM (WT)
L4. R-Ph=1-naphthalene, X=NO2
IC50 = 9.5 nM(WT)
IC50 = 766 nM(K103N/Y181C)
EC50= 1.3 µM(K103N/Y181C)
* Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
Figure 1. Sulfanyltriazole and sulfanyltetrazole lead compounds.5,6,8–10
0968-0896/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.028

5776
P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
X
X
N
N
N
H
N
Bioisosterism based design
S
S
N
O
O
Y
Y
R
X
X
N
N
S
N
H
N
H
N
S
S
N
N
N
O
H
N
O
Y
Y
R
R
R'
N
R
Original leads
Previously reported NNRTIs by us
Newly designed ITA scaffold
Figure 2. Bioisosterism based design of imidazole thioacetanilide (ITA) scaffold.
HIV-1 activity, such as the ‘S–CH2–CO–NH’ linker and the 2-substi-
tuted anilides,17 were left unchanged. Here, the synthesis and the
anti-HIV evaluation of the designed ITAs (4a1–4d6) in MT-4 cell
culture, are reported.
2.2. Anti-HIV evaluation
All of the newly synthesized imidazole thioacetanilides (4a1–
4d6) were first evaluated for their anti-HIV activity and cytotoxic-
ity in MT-4 cells infected with wild-type HIV-1 strain IIIB and HIV-
2 strain ROD. The results, expressed as CC50, EC50 and SI, are sum-
marized in Table 1 together with those of nevirapine (NVP), dela-
viridine (DLV), efavirenz (EFV) and zidovudine (azidothymidine,
AZT) as reference drugs for comparative purposes.
2. Results and discussion
2.1. Chemistry
As shown in Table 1, most of the test compounds inhibited HIV-
1 replication in a lower micromolar concentration range and none
of the compounds were active against HIV-2. The most potent HIV-
The title compounds (series 4a–4d) were prepared as shown in
Scheme 1 according to the reported method.18,19 Condensation of
commercially available or self-made20 aryl isothiocyanates (1a–
1d) with 2,2-dimethoxyethanamine afforded the corresponding
N-substituted thioureas (2a–2d), which was cyclized in the pres-
ence of 5 M HCl under reflux to give the N-1 substituted 2-mer-
capto-imidazoles (3a–3d). It must be pointed out that the by-
product 5-methoxy-N-aryl-4,5-dihydrothiazol-2-amine (30) was
also formed during cyclization, which was consistent with the re-
sults previously reported.19 The final imidazole thioacetanilides
(4a1–4d6) were synthesized by reaction of intermediates 3a–3d
with suitable 2-chloro-N-aryl-substituted acetamides in good
yield. All synthesized compounds were characterized by NMR,
MS, and IR. Both analytical and spectral data of all the compounds
are in full agreement with the proposed structures. For example,
the 1H NMR spectra of the synthesized compounds revealed the
cyclization of imidazole, supported by two protons resonating as
double singlets at about 7.3 and 7.2 ppm, respectively, with cou-
pling constant (J) about 1.2 Hz. In addition, the 1H chemical shift
of the SCH2 group was at about 4.00 ppm.
1 inhibitors were 4a5 (EC50 = 0.18
and 4a2 (EC50 = 0.20 M, CC50 = 35.24
ues of these two compounds were lower than those of the lead
compound L1 (EC50 = 2.053 M) and of the reference drugs NVP
and DLV. Other compounds, 4a3, 4a4, 4c5, and 4a1, also showed
higher anti-HIV-1 potency (EC50 = 0.64, 0.73, 1.03, and 1.78 M,
lM, CC50 = 28.81
lM, SI = 162),
l
lM, SI = 170). The EC50 val-
l
l
respectively) compared with the respect to that of lead compound
derivative L1, indicating that the imidazole is an acceptable isos-
teric replacement for the triazole, tetrazole, or 1,2,3-thiadiazole
in the lead compounds.
On the basis of the chemical structure and the fact that these
compounds inhibit HIV-1, but not HIV-2 replication, these mole-
cules can be proposed to act as genuine NNRTIs.
SAR analysis showed that analogues 4a with naphthalen-1-yl at
N-1 of the imidazole ring were found to be the most potent series,
and the active sequence of the aryl groups was as follows:
naphthalen-1-yl > 4-methylphenyl, 4-chlorophenyl > 4-methoxy-
phenyl. Therefore, the aryl linked to the imidazole core is crucial
S
X
N
N
H
N
i
O
ii
ii
iii
HN
Ar
N
H
Ar-N=C=S
S
SH
N
Ar
N
Ar
O
O
Y
1a-1d
2a-2d
3a-3d
4a1-4a6
4b1-4b6
4c1-4c6
4d1-4d6
1a:Ar=naphthalen-1-yl;
1b:Ar=4-chlorophenyl;
1c:Ar=p-tolyl;
O
S
Ar
1d:Ar=4-methoxyphenyl
N
H
N
3'
by-product
Scheme 1. Reagents and conditions: (i) 2,2-dimethoxyethanamine, EtOH/petroleum ether; (ii) 5 M HCl; (iii) ClCH2CONHPh, Na2CO3 or NaOH, EtOH.

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
5777
Table 1
Anti-HIV activities, cytotoxicities and selectivity indices of 2-(1-aryl-1H-imidazol-2-ylthio)acetamides derivatives (series 4a–4d)
X
N
H
N
N
S
O
Ar
Y
No.
Ar
X
Y
EC50
(l
M)a
CC50
(
l
M)b
SIc
HIV-1 IIIB
HIV-2 ROD
HIV-1 IIIB
HIV-2 ROD
4a1
4a2
4a3
4a4
4a5
4a6
4b1
4b2
4b3
4b4
4b5
4b6
4c1
4c2
4c3
4c4
4c5
4c6
4d1
4d2
4d3
4d4
4d5
4d6
NVPd
DLVd
EFVd
AZTd
Naphthalen-1-yl
Naphthalen-1-yl
Naphthalen-1-yl
Naphthalen-1-yl
Naphthalen-1-yl
Naphthalen-1-yl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
4-Chlorophenyl
p-Tolyl
F
Cl
Br
Br
NO2
Me
F
Cl
Br
H
H
H
Me
H
H
H
H
H
Me
H
H
H
H
H
Me
H
H
H
1.78 0.26
0.20 0.08
0.64 0.23
0.73 0.09
0.18 0.05
2.46 0.91
43.81 0.58
P11.71
15.80 4.26
P55.18
4.86 1.60
35.35 4.14
>37.87
4.97 0.64
5.15 0.52
>80.78
1.03 0.19
30.91 8.68
>41.97
32.28 11.07
P18.36
>25.77
>34.66
>35.24
>30.52
>29.36
>28.81
>34.62
>345.48
>330.44
>295.72
>204.03
>321.48
>159.50
>37.87
>33.67
>39.27
>80.78
>130.70
>147.29
>41.97
>334.35
>298.82
>25.77
>160.97
>353.67
34.66 3.23
35.24 1.02
30.52 0.98
29.36 2.81
28.81 3.83
34.62 2.12
>345.48
>330.44
>300.2
220.72 10.62
>321.48
159.50 9.86
37.87 4.48
33.67 2.82
39.27 8.58
80.78 88.87
130.70 21.36
147.29 25.72
P41.97
>334.35
>298.82
25.77 24.59
160.97 8.25
>353.67
20
170
48
40
162
14
<1
<1
<1
<1
<1
<1
ꢀ1
ꢀ1
ꢀ1
<1
>8
ꢀ1
>19
64
Br
NO2
Me
F
Cl
Br
>66
5
<1
7
8
66
126
5
<orꢀ1
>10
>orꢀ16
<1
>9
>2
>72
>12
>1434
>6192
ꢀ1
<1
<1
<1
<1
<1
<1
<1
<orꢀ1
ꢀ1
ꢀ1
<1
p-Tolyl
p-Tolyl
p-Tolyl
p-Tolyl
Br
NO2
Me
F
Cl
Br
Br
NO2
Me
p-Tolyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
4-Methoxyphenyl
H
H
Me
H
H
17.85 1.40
154.62 62.61
0.208
0.320
0.00440
<1
ꢀ1
>15.02
>3.827
>6.336
>93.55
0.0151
a
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). The SI values: ꢀ1 stand for not active.
b
c
d
The antiviral properties of these compounds were previously described.12
for the antiviral activity, and agrees with previous SARs findings in
1,2,3-thiadiazole thioacetanilides series.12 Probably, the hydropho-
bicity of the aryl group helps to improve the binding affinity (
interaction) between the active binding site and the inhibitors, and
thus enhance the biological activity.
RT. Three-dimensional coordinates of the HIV-1 RT/GW564511
(benzophenone based NNRTI) complex (Brookhaven Protein Data
Bank entry 3DLG) were used as the input structure for docking cal-
culations because of the high degree of similarity between sulfa-
nyltriazole/tetrazoles and benzophenones.11 Default parameters
were used as described in the Autodock Vina manual unless other-
wise specified. The theoretical binding mode of 4a2 to the NNIBP is
shown in Figure 3.
Results showed that the naphthalene ring of 4a2 fits into the
aromatic-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 (green color) is parallel
p–p
Just as SAR of 1,2,3-thiadiazoles made in our previous studies,12
the nature of the substituents at ortho-position of phenyl ring of
the anilide moiety essentially influences the anti-HIV-1 activity.
As shown in Table 1, the anti-HIV-1 activity of the same series in-
creased mainly as the ortho-substitution of the anilide moiety was
replaced by a methyl, fluorine atom, bromine atom, chlorine atom
or nitro. It should highlighted that the 2-nitro substitution on the
phenyl ring furnished the most potent compounds of the four ser-
ies (as shown in Table 1).
to the Tyr188 side chain, giving rise to a positive p-stacking inter-
action. The inhibitor’s amide carbonyl forms a key hydrogen bond
with the backbone N–H of Lys103. The anilide moiety of 4a2 is
close to Pro236, and the 4-substituent points toward the solvent
exposed region. Therefore, 4-substituent allows hydrophilic groups
as the preferred substituents, which can explain the SAR. As illus-
trated in Figure 3, the compound 4a2 binds in a ‘kinked’ conforma-
tion which involves a rotation about the ‘S–CH2–CO–NH’ dihedral
angle from 180° in the fully extended free-state conformation to
almost 0° in the bound state. The imidazole is orthogonal to the
naphthalene ring, which orients the pharmacophores into the
proper geometry for binding.
In addition, when the methyl group is introduced to the para-
position of the anilide moiety, the bioactivity was decreased or
completely lost (4a3/4a4, 4b3/4b4, 4c3/4c4). It is noteworthy that
the introduction of a naphthalen-1-yl group (series 4a) enhanced
the biological activity, but led to increased cytotoxicity, thus pro-
viding important information for further design of novel
analogues.
2.3. Molecular modeling analysis
In order to investigate the binding mode of our newly synthe-
sized compounds, molecular modeling study was performed by
means of Autodock Vina for docking. Compound 4a2 was chosen
to be docked into the NNRTIs binding pocket (NNIBP) of HIV-1
In summary, the results of the molecular modeling analysis sup-
ported the SARs elucidation of our newly designed and synthesized
compounds. Further optimization of imidazole analogues will take
into account these aspects in further design attempts.

5778
P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
Cl
HN
O
S
N
4a2
N
Figure 3. Model of 4a2 docked into the RT non-nucleoside binding site (PDB code: 3DLG) using Autodock Vina [http://vina.scripps.edu]. The docking result of 4a2 is showed
by PyMOL[http://pymol.sourceforge.net].
tion. The crude product was boiled with 50 ml of 5 M hydrochloric
acid for 3 h under reflux. The reaction mixture was cooled, alkal-
ized to pH 8 with 4 M NaOH solution, and then filtered. The filtrate
was acidified with 4 M hydrochloric acid, the precipitated white
solid was collected, washed with water and dried under vacuum
to give 1-(naphthalen-1-yl)-1H-imidazole-2-thiol (3a) as key inter-
mediate (white powder), which was pure enough to be used in the
following step. Yield: 27.8%.
To a mixture of 3a (0.45 g, 2 mmol) in ethyl alcohol (30 ml) was
added sodium hydroxide (0.08 g, 2 mmol) followed by various 2-
chloro-N-(substituted aromatic group)acetamides (2 mmol). The
solution was stirred at room temperature for 5–10 h (checked by
TLC). The solvent was removed under reduced pressure, and the
residue was diluted with dichloromethane (30 ml) and washed
with water (3 ꢀ 30 ml) and brine, and dried with anhydrous
Na2SO4. The mixture solution was filtered off, and the solvent
was removed in vacuo. The residue was purified by recrystallisa-
tion from ethanol to yield the title compounds (4a1–4a6).
3. Conclusions
The bioassay results show that our bioisosterism approach has
led to the development of imidazole analogues as novel and potent
anti-HIV agents. Among them, the most potent HIV-1 inhibitors
were 4a5 (EC50 = 0.18
more effective than the lead compound L1 (EC50 = 2.053
the reference drugs NVP and DLV. Preliminary SAR results for the
newly synthesized congeners and docking studies are presented,
expecting to provide the foundation for the rational modification
of novel azole thioacetanilides and accelerate the discovery of
more potent and selective NNRTIs.
l
M), and 4a2 (EC50 = 0.20
l
M), which were
l
M) and
4. Experimental
4.1. Chemistry
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with a Nexus 470FT-IR Spectrometer. NMR spectra were obtained
on a Bruker Avance-600 NMR-spectrometer in the indicated sol-
vents. Chemical shifts are expressed in d units and TMS as internal
reference. Mass spectra were taken on a LC Autosampler Device:
Standard G1313A instrument. TLC was performed on Silica Gel
GF254 for TLC (Merck) and spots were visualized by iodine vapors
or by irradiation with UV light (254 nm). Solvents were reagent
grade and, when necessary, were purified and dried by standard
methods. Concentration of the reaction solutions involved the
use of rotary evaporator at reduced pressure.
4.2.1. N-(2-Fluorophenyl)-2-(1-(naphthalen-1-yl)-1H-imidazol-
2-ylthio)acetamide (4a1)
White needle crystals, yield: 48.5%. Mp: 133–135 °C. 1H NMR
(CDCl3, ppm) d: 11.23 (s, 1H, NH), 8.36 (t, 1H), 7.99 (d, 1H,
J = 8.4 Hz), 7.95 (d, 1H, J = 8.4 Hz), 7.58–7.01 (m, 8H), 7.35 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.21 (d, 1H, J = 1.2 Hz, imidazol-H), 3.93
(s, 2H, S–CH2). IR (KBr, cmꢁ1): 3177 (
mNH), 1683 (mC@O), 1615,
1550, 1492, 1440, 1511 (dNH). ESI-MS: m/z 380.0 (M+2), 755.6
(2M+1). C21H16FN3OS (377.1).
4.2.2. N-(2-Chlorophenyl)-2-(1-(naphthalen-1-yl)-1H-imidazol-
2-ylthio)acetamide (4a2)
4.2. General procedure for the synthesis of 2-(1-(naphthalen-1-
yl)-1H-imidazol-2-ylthio)acetamides (4a1–4a6)
White needle crystals, yield: 54.8%. Mp: 125–127 °C. 1H NMR
(CDCl3, ppm) d: 10.59 (s, 1H, NH), 8.31 (d, 1H, J = 7.8 Hz), 8.02 (d,
1H, J = 8.4 Hz), 7.98 (d, 1H, J = 7.8 Hz), 7.60–7.05 (m, 9H), 3.98
(dd, 2H, S–CH2); 13C NMR (CDCl3, ppm) d: 167.88 (C@O), 144.28
(N@C–N), 135.40, 134.25, 132.48, 130.30, 129.60, 129.29, 129.06,
128.42, 127.86, 127.37, 127.11, 125.33, 125.18, 124.78, 124.13,
124.04, 122.63, 122.65, 36.69 (S–CH2); IR (KBr, cmꢁ1): 3197
To a stirred solution of 1-isothiocyanatonaphthalene (1a, 9.26 g,
50 mmol) in ethyl alcohol (100 ml) was added dropwise the 2,2-
dimethoxyethanamine (5.3 g, 50 mmol) at room temperature.
After further stirring for 30 min, the formed white solid was fil-
tered, washed with cold ethyl alcohol (2 ꢀ 15 ml) and dried to give
1-(2,2-dimethoxyethyl)-3-(naphthalen-1-yl)thiourea (2a), which
was used directly in the next reaction without any further purifica-
(mNH), 1683 (mC@O), 1592, 1538, 1469, 1439. ESI-MS: m/z 394.8
(M+1). C21H16ClN3OS (393.07).

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
5779
4.2.3. N-(2-Bromophenyl)-2-(1-(naphthalen-1-yl)-1H-imidazol-
2-ylthio)acetamide (4a3)
2H, J = 8.4 Hz, PhH), 7.35 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, PhH),
7.32 (d, 2H, J = 8.4 Hz, PhH), 7.25 (dt, 1H), 7.20 (s, 1H), 7.11 (d,
1H, J = 1.2 Hz), 7.03 (dt, 1H), 3.98 (s, 2H, S–CH2); 13C NMR (CDCl3,
ppm) d:167.87 (C@O), 142.61 (N@C–N), 135.40, 134.96, 134.81,
129.90, 129.58, 127.28, 126.35, 124.72, 123.74, 122.52, 122.38,
White needle crystals, yield: 46.7%. Mp: 140–142 °C. 1H NMR
(CDCl3, ppm) d: 10.38 (s, 1H, NH), 8.24 (d, 1H, J = 8.4 Hz), 7.99 (d,
1H, J = 8.4 Hz), 7.95 (d, 1H, J = 8.4 Hz), 7.58–6.96 (m, 8H), 7.38 (d,
1H, J = 1.2 Hz, imidazol-H), 7.20 (d, 1H, J = 1.2 Hz, imidazol-H),
36.76 (S–CH2); IR (KBr, cmꢁ1): 3211 (
mNH), 1705 (mC@O), 1597,
4.00 (s, 2H, S–CH2). IR (KBr, cmꢁ1): 3187 (
mNH), 1684 (
mC@O),
1541, 1512, 1489, 1442. ESI-MS: m/z 379.3 (M+1). C17H13Cl2N3OS
1590, 1533, 1467, 1437. ESI-MS: m/z 439.0 (M+1), 442.3 (M+4),
(377.02).
877.4 (2M+1). C21H16BrN3OS (437.02).
4.3.3. N-(2-Bromophenyl)-2-(1-(4-chlorophenyl)-1H-imidazol-
2-ylthio)acetamide (4b3)
4.2.4. N-(2-Bromo-4-methylphenyl)-2-(1-(naphthalen-1-yl)-1H-
imidazol-2-ylthio)acetamide (4a4)
White needle crystals, yield: 54.3%. Mp: 169–171 °C. 1H NMR
(CDCl3, ppm) d: 10.25 (s, 1H, NH), 8.23 (d, 1H, J = 8.4 Hz, Ph0H),
8.23 (d, 1H, J = 8.4 Hz, Ph0H), 7.47 (d, 2H, J = 8.4 Hz, PhH), 7.32 (d,
2H, J = 8.4 Hz, PhH), 7.28(d, 1H, J = 7.8 Hz, PhH), 7.21 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.12 (d, 1H, J = 1.2 Hz, imidazol-H), 6.97
White needle crystals, yield: 51.6%. Mp: 138–140 °C. 1H NMR
(CDCl3, ppm) d: 10.27 (s, 1H, NH), 8.07 (d, 1H, J = 8.4 Hz), 7.99 (d,
1H, J = 7.8 Hz), 7.95 (d, 1H, J = 7.8 Hz), 7.56 (m, 2H), 7.49 (m, 2H),
7.38 (m, 2H), 7.31 (d, 1H, J = 1.2 Hz, imidazol-H), 7.19 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.10 (d, 1H, J = 8.4 Hz), 3.97 (s, 2H, S–
(t, 1H, Ph0H), 4.02 (s, 2H, S–CH2). IR (KBr, cmꢁ1) 3198 (
m
NH), 1681
C@O), 1594, 1538, 1510, 1492, 1438. ESI-MS: m/z 422.6 (M+1),
425.4 (M+3). C17H13BrClN3OS (420.97).
CH2), 2.30 (s, 3H, CH3). IR (KBr, cmꢁ1): 3195 (
m
NH), 1688 (mC@O),
(m
1582, 1532, 1467, 1440. ESI-MS: m/z 452.9 (M+), 456.1 (M+3),
905.4 (2M+1). C22H18BrN3OS (451.04).
4.3.4. N-(2-Bromo-4-methylphenyl)-2-(1-(4-chlorophenyl)-1H-
imidazol-2-ylthio)acetamide (4b4)
4.2.5. 2-(1-(Naphthalen-1-yl)-1H-imidazol-2-ylthio)-N-(2-nitro-
phenyl)acetamide (4a5)
White needle crystals, yield: 37.6%. Mp: 147–149 °C. 1H NMR
(CDCl3, ppm) d: 10.17 (s, 1H, NH), 8.08 (d, 1H, J = 2.4 Hz, Ph0H),
7.46 (d, 2H, J = 8.4 Hz, PhH), 7.35 (s, 1H, Ph0H), 7.32 (d, 2H,
J = 8.4 Hz, PhH), 7.20 (d, 1H, J = 1.2 Hz, imidazol-H), 7.11 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.09 (m, 1H, Ph0H), 3.98 (s, 2H, S–CH2),
Yellow brown cubic crystals, yield: 68.1%. Mp: 167–169 °C. 1H
NMR (CDCl3, ppm) d: 11.43 (s, 1H, NH), 8.59 (d, 1H, J = 2.4 Hz),
8.11 (d, 1H, J = 2.4 Hz), 7.99–7.17 (m, 9H), 7.32 (d, 1H, J = 1.2 Hz,
imidazol-H), 7.19 (d, 1H, J = 1.2 Hz, imidazol-H), 4.05 (s, 2H, S–
CH2). IR (KBr, cmꢁ1): 3156 (
1503 ( NO2), 1488, 1440, 1347 (
m
NH), 1708(
m
C@O), 1660, 1584, 1553,
2.88 (s, 3H, CH3). IR (KBr, cmꢁ1) 3133 (
mNH), 1682 (mC@O), 1590,
1539, 1511, 1492, 1442. ESI-MS: m/z 437.2 (M+2). C18H15BrClN3OS
m
m
NO2). ESI-MS: m/z 404.1 (M+),
407.3 (M+3), 809.4 (2M+1). C21H16N4O3S (404.09).
(434.98).
4.2.6. 2-(1-(Naphthalen-1-yl)-1H-imidazol-2-ylthio)-N-o-toly-
lacetamide (4a6)
4.3.5. 2-(1-(4-Chlorophenyl)-1H-imidazol-2-ylthio)-N-(2-nitro-
phenyl)acetamide(4b5)
White needle crystals, yield: 45.2%. Mp: 143–145 °C. 1H NMR
(CDCl3, ppm) d: 10.37 (s, 1H, NH), 8.01 (d, 1H, J = 8.4 Hz), 7.96 (d,
1H, J = 7.8 Hz), 7.95 (d, 1H, J = 7.8 Hz), 7.56 (m, 2H), 7.53 (t, 1H),
7.49 (d, 1H, J = 7.2 Hz), 7.35 (d, 1H, J = 7.8 Hz), 7.26 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.22 (d, 1H, J = 1.2 Hz, imidazol-H), 7.19
(d, 2H, J = 8.4 Hz), 7.05 (t, 1H), 3.86 (s, 2H, S–CH2), 2.35 (s, 3H,
Light yellow needle crystals, yield: 38.7%. Mp: 193–195 °C. 1H
NMR (CDCl3, ppm) d: 11.41 (s, 1H, NH), 8.59 (d, 1H, J = 2.4 Hz,
Ph0H), 8.12 (dd, 1H, J = 2.4 Hz, J = 1.2 Hz, Ph0H), 7.62 (dt, 1H, Ph0H),
7.47 (d, 2H, J = 8.4 Hz, PhH), 7.36 (d, 2H, J = 8.4 Hz, PhH), 7.21 (d,
1H, J = 1.2 Hz, imidazol-H), 7.19 (m, 1H, Ph0H), 7.12 (d, 1H,
J = 1.2 Hz, imidazol-H), 4.07 (s, 2H, S–CH2). IR (KBr, cmꢁ1) 3156
CH3). IR (KBr, cmꢁ1): 3235 (
m
NH), 1677 (
m
C@O), 1587, 1541, 1512,
(mNH), 1708 (mC@O), 1660, 1584, 1553, 1503 (dNO2), 1488, 1440,
1456, 1437. ESI-MS: m/z 376.6 (M+3), 747.6 (2M+1). C22H19N3OS
1347 (dNO2). ESI-MS: m/z 390.7 (M+1), 392.1 (M+2). C17H13ClN4O3S
(373.12).
(388.04).
4.3. General procedure for the synthesis of 2-(1-(4-chlorophenyl)-
1H-imidazol-2-ylthio) acetamides (4b1–4b6)
4.3.6. 2-(1-(4-Chlorophenyl)-1H-imidazol-2-ylthio)-N-o-tolyl-
acetamide (4b6)
White needle crystals, yield: 41.2%. Mp: 164–166 °C. 1H NMR
(CDCl3, ppm) d: 10.25 (s, 1H, NH), 7.96 (d, 1H, J = 8.4 Hz, Ph0H),
7.47 (d, 2H, J = 8.4 Hz, PhH), 7.32 (d, 2H, J = 8.4 Hz, PhH), 7.20 (d,
1H, J = 1.2 Hz, imidazol-H), 7.17 (m, 2H, Ph0H), 7.13 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.04 (t, 1H, Ph0H), 3.90 (s, 2H, S–CH2),
With a similar procedure for compounds 4a1–4a6, the 2-(1-(4-
chlorophenyl)-1H-imidazol-2-ylthio)acetamides (4b1–4b6) were
synthesized starting from 1-chloro-4-isothiocyanatobenzene 1b
(50 mmol). Compound 1b was carried out according to the re-
ported procedure.20
2.30 (s, 3H, CH3). IR (KBr, cmꢁ1) 3200 (
mNH), 1684 (mC@O), 1584,
1551, 1507, 1488, 1434. ESI-MS: m/z 359.3 (M+2), 361.2 (M+4).
4.3.1. 2-(1-(4-Chlorophenyl)-1H-imidazol-2-ylthio)-N-(2-fluoro-
phenyl)acetamide (4b1)
C18H16ClN3OS (357.07).
White needle crystals, yield: 45.8%. Mp: 131–133 °C. 1H NMR
(CDCl3, ppm) d: 11.13 (s, 1H, NH), 8.35 (m, 1H, Ph0H), 7.47 (d, 2H,
J = 8.4 Hz, PhH), 7.31 (d, 2H, J = 8.4 Hz, PhH), 7.23 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.12 (d, 1H, J = 1.2 Hz, imidazol-H), 7.10
4.4. General procedure for the synthesis of 2-(1-(p-tolyl)-1H-
imidazol-2-ylthio) acetamides (4c1–4c6)
The starting material 1-isothiocyanato-4-methylbenzene was
synthesized according to a known procedure.20 A solution of 1-
isothiocyanato-4-methylbenzene (1c, 7.46 g, 50 mmol) and 2,2-
dimethoxyethanamine (5.3 g, 50 mmol) in petroleum ether (bp:
60–90 °C, 100 ml) was stirred at room temperature for 2.5 h. The
formed white solid was filtered, washed with petroleum ether
(2 ꢀ 20 ml) and dried in air to give 1-(2,2-dimethoxyethyl)-3-(p-
tolyl)thiourea (2c), which was used directly without any further
purification. The crude product was boiled with 50 ml of 5 M
(m, 3H, Ph0H), 3.90 (s, 2H, S–CH2). IR (KBr, cmꢁ1): 3188 (
m
NH),
C@O), 1625, 1559, 1507, 1490, 1457. ESI-MS: m/z 363.6
(M+2), 365.2 (M+2). C17H13ClFN3OS (361.05).
1679 (
m
4.3.2. N-(2-Chlorophenyl)-2-(1-(4-chlorophenyl)-1H-imidazol-
2-ylthio)acetamide (4b2)
White needle crystals, yield: 48.9%. Mp: 143–145 °C. 1H NMR
(CDCl3, ppm) d: 10.54 (s, 1H, NH), 8.33 (d, 1H, J = 7.8 Hz), 7.46 (d,

5780
P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
hydrochloric acid for 3 h under reflux. The mixture was concen-
trated under reduced pressure. It was triturated with hot ethyl
alcohol to give the compound 1-p-tolyl-1H-imidazole-2-thiol (3c)
as white crystals, Yield: 32.7%.
CH2), 2.41 (s, 3H, CH3). IR (KBr, cmꢁ1): 3289 (
1610, 1584, 1502 ( NO2), 1439, 1336 ( NO2), 1273, 1146, 824, 743.
ESI-MS: m/z 369.3 (M+1). C18H16N4O3S (368.09).
mNH), 1693 (mC@O),
m
m
A mixture of 3c (0.38 g, 2 mmol), sodium carbonate (0.23 g,
2.1 mmol), and 2-chloro-N-(substituted aromatic group)aceta-
mides (2 mmol) in N,N-dimethylformamide (10 ml) was stirred
overnight. The resulting mixture was diluted with water and ex-
tracted with dichloromethane (30 ml). The organic layer was
washed with water (3 ꢀ 30 ml), brine, and dried over Na2SO4. Re-
moval of the solvent in vacuo and recrystallisation from ethanol
afforded the title compounds (4c1–4c6).
4.4.6. N-o-Tolyl-2-(1-p-tolyl-1H-imidazol-2-ylthio)acetamide
(4c6)
White crystals, yield: 59.3%. Mp: 144–146 °C. 1H NMR (CDCl3,
ppm) d: 10.39 (s, 1H, NH), 7.97 (d, 1H, J = 7.8 Hz, Ph0H), 7.28 (d,
2H, J = 8.4 Hz, PhH), 7.24 (d, 2H, J = 8.4 Hz, PhH), 7.20 (m, 1H, Ph0H),
7.17 (m, 1H, Ph0H), 7.13 (d, 1H, J = 1.2 Hz, imidazol-H), 7.12 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.03 (m, 1H, Ph0H), 3.88 (s, 2H, S–CH2), 2.42
(s, 3H, CH3), 2.31 (s, 3H, CH3). IR (KBr, cmꢁ1): 3244 (
m
NH), 1674
C@O), 1593, 1554, 1518, 1459, 819, 761, 730. ESI-MS: m/z 338.4
(M+1). C19H19N3OS (337.12).
(m
4.4.1. N-(2-Fluorophenyl)-2-(1-p-tolyl-1H-imidazol-2-ylthio)aceta-
mide (4c1)
White solid, yield: 88.0%. Mp: 115–117 °C. 1H NMR (CDCl3,
ppm) d: 11.31 (s, 1H, NH), 8.37 (d, 1H, J = 8.4 Hz, Ph0H), 7.28 (d,
2H, J = 8.4 Hz, PhH), 7.24 (d, 2H, J = 8.4 Hz, PhH), 7.21 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.12 (d, 1H, J = 1.2 Hz, imidazol-H), 7.10–
6.99 (m, 3H, Ph0H), 3.88 (s, 2H, S–CH2), 2.41 (s, 3H, CH3). IR (KBr,
4.5. General procedure for the synthesis of 2-(1-(4-methoxy-
phenyl)-1H-imidazol-2-ylthio) acetamides (4d1–4d6)
With a similar procedure for compounds 4c1–4c6, the 2-(1-
(4-methoxyphenyl)-1H-imidazol-2-ylthio)acetamides (4d1–4d6)
were synthesized starting from 1-isothiocyanato-4-methoxyben-
zene 1d (50 mmol). Compound 1d was obtained according to the
reported procedure.20
cmꢁ1): 3194 (
mNH), 1669 (mC@O), 1617, 1548, 1508, 1493, 1449.
ESI-MS: m/z 342.3 (M+1). C18H16FN3OS (341.1).
4.4.2. N-(2-Chlorophenyl)-2-(1-p-tolyl-1H-imidazol-2-ylthio)aceta-
mide (4c2)
4.5.1. N-(2-Fluorophenyl)-2-(1-(4-methoxyphenyl)-1H-imidazol-
2-ylthio)acetamide (4d1)
White solid, yield: 70.1%. Mp: 105–107 °C. 1H NMR (CDCl3,
ppm) d: 10.68 (s, 1H, NH), 8.33 (d, 1H, J = 8.4 Hz, Ph0H), 7.34 (d,
1H, J = 8.4 Hz, Ph0H), 7.28 (d, 2H, J = 8.4 Hz, PhH), 7.26 (m, 1H,
Ph0H), 7.23 (d, 2H, J = 8.4 Hz, PhH), 7.17 (d, 1H, J = 1.2 Hz, imida-
zol-H), 7.11 (d, 1H, J = 1.2 Hz, imidazol-H), 7.01 (dt, 1H, Ph0H),
3.96 (s, 2H, S–CH2), 2.41 (s, 3H, CH3). 13C NMR (CDCl3, ppm) d:
168.17, 142.58, 138.97, 135.54, 134.01, 130.20, 129.26, 127.35,
124.87, 124.62, 123.82, 122.69, 122.43, 36.79, 21.15. IR (KBr, cm
White needle crystals, yield: 28.1%. Mp: 125–127 °C. 1H NMR
(CDCl3, ppm) d: 11.27 (s, 1H, NH), 8.37 (t, 1H, Ph0H), 7.28 (d, 2H,
J = 8.4 Hz, PhH), 7.20 (d, 1H, J = 1.2 Hz, imidazol-H), 7.12 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.10–7.02 (m, 3H, Ph0H), 6.98 (d, 2H,
J = 8.4 Hz, PhH), 3.88 (s, 2H, S–CH2), 3.86 (s, 3H, OCH3). IR (KBr,
cmꢁ1): 3248 (
mNH), 1693 (mC@O), 1624, 1554, 1520, 1458, 1327,
1259, 839, 753. ESI-MS: m/z 358.3 (M+1). C18H16FN3O2S (357.09).
ꢁ1): 3221 (
mNH), 1698 (mC@O), 1578, 1539, 1515, 1490, 1439. ESI-
MS: m/z 358.3 (M+1). C18H16ClN3OS (357.07).
4.5.2. N-(2-Chlorophenyl)-2-(1-(4-methoxyphenyl)-1H-imidazol-
2-ylthio)acetamide (4d2)
4.4.3. N-(2-Bromophenyl)-2-(1-p-tolyl-1H-imidazol-2-ylthio)-
acetamide (4c3)
White needle crystals, yield: 46.9%. Mp: 121–123 °C. 1H NMR
(CDCl3, ppm) d: 10.67 (s, 1H, NH), 8.33 (d, 1H, J = 8.4 Hz, Ph0H),
7.35 (d, 1H, J = 7.8 Hz, Ph0H), 7.27 (d, 2H, J = 8.4 Hz, PhH), 7.25
(m, 1H, Ph0H), 7.17 (d, 1H, J = 1.2 Hz, imidazol-H), 7.09 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.02 (m, 1H, Ph0H), 6.98 (d, 2H, J = 8.4 Hz,
PhH), 3.96 (s, 2H, S–CH2), 3.86 (s, 3H, OCH3). 13C NMR (CDCl3,
ppm) d: 168.18, 159.78, 142.85, 135.56, 129.33, 129.27, 129.22,
127.36, 126.55, 124.61, 123.80, 122.92, 122.42, 114.71, 55.59,
Colorless needle crystals, yield: 46.5%. Mp: 128–130 °C. 1H NMR
(CDCl3, ppm) d: 10.45 (s, 1H, NH), 8.25 (d, 1H, J = 8.4 Hz, Ph0H), 7.52
(m, 1H, Ph0H), 7.29 (d, 2H, J = 8.4 Hz, PhH), 7.25 (d, 1H, J = 8.4 Hz,
Ph0H), 7.24 (d, 2H, J = 8.4 Hz, PhH), 7.18 (d, 1H, J = 1.2 Hz, imida-
zol-H), 7.12 (d, 1H, J = 1.2 Hz, imidazol-H), 6.96 (dt, 1H, Ph0H),
3.97 (s, 2H, S–CH2), 2.41 (s, 3H, CH3). IR (KBr, cmꢁ1): 3432 (
1696 (
402.3 (M+1), 404.3 (M+3). C18H16BrN3OS (401.02).
m
NH),
m
C@O), 1595, 1519, 1438, 1303, 825, 746. ESI-MS: m/z
36.74. IR (KBr, cmꢁ1): 3431 (
mNH), 1700 (mC@O), 1597, 1518, 1442,
1254, 837, 749. ESI-MS: m/z 374.3 (M+1). C18H16ClN3O2S (373.07).
4.4.4. N-(2-Bromo-4-methylphenyl)-2-(1-p-tolyl-1H-imidazol-
2-ylthio)acetamide (4c4)
4.5.3. N-(2-Bromophenyl)-2-(1-(4-methoxyphenyl)-1H-imidazol-
2-ylthio)acetamide (4d3)
White needle crystals, yield: 74.1%. Mp: 138–140 °C. 1H NMR
(CDCl3, ppm) d: 10.33 (s, 1H, NH), 8.08 (d, 1H, J = 8.4 Hz, Ph0H),
7.35 (s, 1H, Ph0H), 7.28 (d, 2H, J = 8.4 Hz, PhH), 7.24 (d, 2H,
J = 8.4 Hz, PhH), 7.17 (d, 1H, J = 1.2 Hz, imidazol-H), 7.11 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.09 (d, 1H, J = 8.4 Hz, Ph0H), 3.95 (s, 2H,
S–CH2), 2.41 (s, 3H, CH3), 2.28 (s, 3H, CH3). IR (KBr, cmꢁ1) 3130
White needle crystals, yield: 24.0%. Mp: 117–119 °C. 1H NMR
(CDCl3, ppm) d: 10.41 (s, 1H, NH), 8.25 (d, 1H, J = 7.8 Hz, Ph0H),
7.52 (dd, 1H, J = 1.2 Hz, J = 8.4 Hz, Ph0H), 7.28 (m, 3H, 1 ꢀ Ph0H
and 2 ꢀ PhH), 7.18 (d, 1H, J = 1.2 Hz, imidazol-H), 7.09 (d, 1H,
J = 1.2 Hz, imidazol-H), 6.96 (m, 3H, 1 ꢀ Ph0H and 2 ꢀ PhH), 3.97
(s, 2H, S–CH2), 3.86 (s, 3H, OCH3). IR (KBr, cmꢁ1): 3428 (
1694 (m
m
NH),
C@O), 1594, 1520, 1437, 1256, 1027, 839, 747. ESI-MS: m/
z 418.4 (M+1). C18H16BrN3O2S (417.01).
(mNH), 1690 (
mC@O), 1588, 1541, 1515, 1489, 1448. ESI-MS: m/z
416.3 (M+1), 418.3 (M+3). C19H18BrN3OS (415.04).
4.4.5. N-(2-Nitrophenyl)-2-(1-p-tolyl-1H-imidazol-2-ylthio)aceta-
mide (4c5)
4.5.4. N-(2-Bromo-4-methylphenyl)-2-(1-(4-methoxyphenyl)-1H-
imidazol-2-ylthio)acetamide (4d4)
Light yellow powder, yield: 61.1%. Mp: 146–148 °C. 1H NMR
(CDCl3, ppm) d: 11.50 (s, 1H, NH), 8.60 (d, 1H, J = 8.4 Hz, Ph0H),
8.10 (d, 1H, J = 8.4 Hz, Ph0H), 7.28 (d, 2H, J = 8.4 Hz, PhH), 7.23 (d,
2H, J = 8.4 Hz, PhH), 7.19 (d, 1H, J = 1.2 Hz, imidazol-H), 7.11 (d,
1H, J = 1.2 Hz, imidazol-H), 7.08 (m, 2H, Ph0H), 4.03 (s, 2H, S–
White needle crystals, yield: 63.8%. Mp: 138–140 °C. 1H NMR
(CDCl3, ppm) d: 10.28 (s, 1H, NH), 8.07 (d, 1H, J = 8.4 Hz, Ph0H),
7.35 (s, 1H, Ph0H), 7.27 (d, 2H, J = 8.4 Hz, PhH), 7.17 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.11 (d, 1H, J = 1.2 Hz, imidazol-H), 7.09
(m, 1H, Ph0H), 6.98 (d, 2H, J = 8.4 Hz, PhH), 3.97 (s, 2H, S–CH2),

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5775–5781
5781
3.86 (s, 3H, OCH3), 2.29 (s, 3H, CH3). IR (KBr, cmꢁ1): 3203 (
1690 ( C@O), 1521, 1127, 1030, 841, 815, 739. ESI-MS: m/z 432.3
(M+1). C19H18BrN3O2S (431.03).
m
NH),
in an eight-channel computer-controlled photometer (Multiscan
Ascent Reader, Labsystems, Helsinki, Finland), at two wavelengths
(540 and 690 nm). All data were calculated using the median OD
(optical density) value of tree wells. The 50% cytotoxic concentra-
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).
m
4.5.5. 2-(1-(4-Methoxyphenyl)-1H-imidazol-2-ylthio)-N-(2-nitro-
phenyl)acetamide (4d5)
Light yellow solid, yield: 32.6%. Mp: 139–141 °C. 1H NMR
(CDCl3, ppm) d: 11.49 (s, 1H, NH), 8.61 (d, 1H, J = 7.8 Hz, Ph0H),
8.11 (d, 1H, J = 8.4 Hz, Ph0H), 7.61 (dt, 1H, Ph0H), 7.30 (d, 2H,
J = 8.4 Hz, PhH), 7.18 (m, 2H, imidazol-H and Ph0H), 7.10 (d, 1H,
J = 1.2 Hz, imidazol-H), 6.97 (d, 2H, J = 8.4 Hz, PhH), 4.02 (s, 2H,
Acknowledgements
S–CH2), 3.86 (s, 3H, OCH3). IR (KBr, cmꢁ1): 3290 (
1503 ( as NO2), 1428, 1336 (
(M+1). C18H16N4O4S (384.09).
m
NH), 1690 (
mC@O),
Research work in the authors’ laboratory has been supported by
the National Natural Science Foundation of China (NSFC
Nos.30371686, 30772629, 30873133), Key Project of The Interna-
tional Cooperation, Ministry of Science and Technology of China
(2003DF000033) and Research Fund for the Doctoral Program of
Higher Education of China (070422083). We are grateful to Kristien
Erven and Kris Uyttersprot for technical assistance.
m
ms NO2), 1271, 1249. ESI-MS: m/z 385.4
4.5.6. 2-(1-(4-Methoxyphenyl)-1H-imidazol-2-ylthio)-N-o-tolyl-
acetamide (4d6)
White needle crystals, yield: 70.8%. Mp: 142–144 °C. 1H NMR
(CDCl3, ppm) d: 10.37 (s, 1H, NH), 7.96 (d, 1H, J = 7.8 Hz, Ph0H),
7.26 (d, 2H, J = 8.4 Hz, PhH), 7.20 (dt, 1H, Ph0H), 7.17 (d, 1H,
J = 7.8 Hz, Ph0H), 7.13 (d, 1H, J = 1.2 Hz, imidazol-H), 7.10 (d, 1H,
J = 1.2 Hz, imidazol-H), 7.04 (dt, 1H, Ph0H), 6.98 (d, 2H, J = 8.4 Hz,
PhH), 3.89 (s, 2H, S–CH2), 3.86 (s, 3H, OCH3), 2.31 (s, 3H, CH3). IR
References and notes
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4.6. Anti-HIV activity assays
Evaluation of the antiviral activity of the compounds against
HIV-1 strain IIIB and HIV-2 strain (ROD) in MT-4 cells was per-
formed using the MTT assay as previously described.21,22 Stock
solutions (10 ꢀ final concentration) of test compounds were added
in 25-ll volumes to two series of triplicate wells so as to allow
simultaneous evaluation of their effects on mock- and HIV-infected
cells at the beginning of each experiment. Serial fivefold dilutions
of test compounds were made directly in flat-bottomed 96-well
microtiter trays using a Biomek 3000 robot (Beckman instruments,
Fullerton, CA). Untreated control HIV- and mock-infected cell sam-
ples were included for each sample.
HIV-1(IIIB)23 or HIV-2 (ROD)24 stock (50
lL) at 100–300 CCID50
(cell culture infectious dose) or culture medium was added to
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 cells25 were cen-
trifuged for 5 min at 1000 rpm and the supernatant was discarded.
The MT-4 cells were resuspended at 6 ꢀ 105 cells/mL, and 50-
lL
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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-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Acros Organics, Geel, Belgium) by mitochondrial dehydrogenase
of metabolically active cells to a blue-purple formazan that can
be measured spectrophotometrically. The absorbances were read
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