Arylazolylthioacetanilide. Part 8*: Design, synthesis and biological evaluation of Novel 2-(2-(2,4-Dichlorophenyl)-2H-1,2,4-triazol-3- ylthio)-N-arylacetamides As Potent HIV-1 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2011.08.011

The development of novel HIV-1 NNRTIs offers the possibility of generating novel structures with increased potency. Based on the bioisosteric principle, a novel series of 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N- arylacetamide derivatives were designed, synthesized using a simple and efficient synthetic route, structurally confirmed by spectral analysis, evaluated for their anti-HIV activity in MT-4 cells and their inhibitory effect on HIV-1 RT. The results showed that some of the new compounds displayed low micromolar potency for inhibiting HIV-1 replication and promising activities against several selected resistant strains that confer resistance to current NNRTIs. However, all newly synthesized derivatives were not active against HIV-2 replication.

Full text of DOI:10.1016/j.ejmech.2011.08.011

European Journal of Medicinal Chemistry 46 (2011) 5039e5045
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Arylazolylthioacetanilide. Part 8q: Design, synthesis and biological evaluation
of Novel 2-(2-(2,4-Dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-arylacetamides
As Potent HIV-1 inhibitors
Peng Zhan a, Xuwang Chen a, Xiao Li a, Dongyue Li a, Ye Tian a, Wenwen Chen a, Christophe Pannecouque b,
,
Erik De Clercq b, Xinyong Liu a
*
a Department of Medicinal Chemistry, School of Pharmaceutical Sciences, Shandong University, 44, West Culture Road, 250012 Jinan, Shandong, P.R.China
b Rega Institute for Medical Research, K.U.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 development of novel HIV-1 NNRTIs offers the possibility of generating novel structures with
increased potency. Based on the bioisosteric principle, a novel series of 2-(2-(2,4-dichlorophenyl)-2H-
1,2,4-triazol-3-ylthio)-N-arylacetamide derivatives were designed, synthesized using a simple and effi-
cient synthetic route, structurally confirmed by spectral analysis, evaluated for their anti-HIV activity in
MT-4 cells and their inhibitory effect on HIV-1 RT. The results showed that some of the new compounds
displayed low micromolar potency for inhibiting HIV-1 replication and promising activities against
several selected resistant strains that confer resistance to current NNRTIs. However, all newly synthe-
sized derivatives were not active against HIV-2 replication.
Received 13 April 2011
Received in revised form
19 June 2011
Accepted 5 August 2011
Available online 12 August 2011
Keywords:
HIV
NNRTIs
Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
1,2,4-Triazole
Synthesis
Bioactivity
SAR
1. Introduction
improved pharmacokinetic profiles, is a constant goal for drug
development [4].
Human immunodeficiency virus type-1 (HIV-1) infection affects
close to 40 million individuals worldwide. Since 1981, when the
first case reports of individuals dying from a rare opportunistic
infection were published, 20 million people have died from this
epidemic [1]. The introduction of highly active anti-retroviral
therapy (HAART) has dramatically decreased the morbidity and
mortality resulting from the infection with HIV. However, the AIDS
prevalence remains one of the world’s most serious health prob-
lems [2]. Non-nucleoside reverse transcriptase inhibitors (NNRTIs)
nowadays represent very potent anti-AIDS drugs that specifically
target to HIV-1 reverse transcriptase (RT) [3]. However, to a great
extent, the effectiveness of NNRTI drugs can be hampered by rapid
emergence of drug-resistant viruses and severe side effect in the
long-term usage. Therefore, the identification of novel NNRTIs,
characterized by high potency against both the wild-type HIV-1
and the clinically relevant mutant strains of HIV-1, as well as
Five-membered azoles heterocycles represent an important
class of lead structures for novel antiviral drug development [5].
Arylazolylthioacetanilide derivatives have been of great interest for
the development of novel NNRTIs, because of their high potency
against HIV-1 wild-type and resistant strains, favorable bioavail-
ability and low toxicity [6e12]. Several triazolylthioacetanilide
NNRTIs, such as VRX-480733 [13] and RDEA806 (in phase IIa clin-
ical trials) [14], have been or are currently being entered into
clinical trials (Fig. 1).
Meanwhile, during the course of our investigations on potential
NNRTIs, we embarked on in the chemical and biological studies of
a number of novel arylazolylthioacetanilide derivatives [15e21].
Most of these compounds displayed potent anti-HIV properties in
cell lines infected with either wild type or mutant HIV-1. In
particular, 1,2,3-thiadiazole derivative ZP7 displayed the most
potent anti-HIV-1 activity (EC50 ¼ 36.4 nM), inhibiting HIV-1
replication in MT-4 cells more effectively than nevirapine (NVP)
(by sevenfold) and delavirdine (DLV) (by eightfold) [16]. Molecular
modeling studies demonstrated that the five-membered hetero-
cycle portion of these inhibitors could be acting as a scaffold which
q
Parts 1-7 were reported in references 15-21.
* Corresponding author. Tel.: þ86 531 88382005; fax: þ86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0223-5234/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.08.011

5040
P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
Fig. 1. Triazole/1,2,3-thiadiazole thioacetanilide-based NNRTIs.
orients the pharmacophores into the proper geometry for binding
and key structural element to form hydrogen bond with K103
[6,12]. Therefore, there are remarkable differences in the electronic
and conformational contribution of the heterocyclic groups to the
binding of the inhibitors with the HIV-1 RT [12]. In the light of the
above promising results and our continued interest in the study of
antiviral activity of arylazolylthioacetanilide derivatives, a series of
novel 1,2,4-triazole thioacetanilide derivatives were designed and
synthesized based on the general principle of bioisosterism in
medicinal chemistry [22]. In the newly synthesized analogues,
novel 1,2,4-triazole ring (2-aryl) was substituted for the 1,2,4-
triazole (4-aryl) or 1,2,3-thiadiazole moiety in the corresponding
parent leads (see Fig. 2), the other fragments which were consid-
ered to be necessary for conserving anti-HIV-1 activity, such as the
“eSCH2CONH-” linker and the substituted arylamines, were
maintained. The synthesis, the anti-HIV-1 activities evaluation of
these novel derivatives against HIV-1 (IIIB), HIV-2 (ROD), and
several key mutant HIV-1 strains as well as HIV RT inhibitory assay
are presented in the following sections.
was treated with phosphoryl trichloride under reflux condition to
afford 5-chloro-1-(2,4-dichlorophenyl)-1H-1,2,4-triazole (4) The
ethyl 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)acetate (5)
was synthesized by reaction of 4 with ethyl 2-mercaptoacetate in
high yield. The thioacetic acid derivative 6 was obtained by saponi-
fication of compound 5 in NaOH-DMSO-H2O solution. The final tri-
azole thioacetanilides 7 were synthesized by reaction of 6 with
substituted arylamines in the presence of 1.1 equiv mole of PCl5. Both
analytical and spectral data of all the newly synthesized compounds
are in full agreement with the proposed structures.
2.2. Biological activity
2.2.1. Anti-HIV activities evaluation
The newly synthesized 1,2,4-triazolacetamide derivatives were
evaluated for anti-HIV activity by determining their ability to
inhibit the replication of HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells
in comparison with nevirapine (NVP), zidovudine (azidothymidine,
AZT), dideoxycitidine (DDC) and dideoxyinosine (DDI) used as
reference drugs. The cytotoxicity of the compound was determined
in parallel. The results of inhibitory concentration (EC50), cytotoxic
concentration (CC50), and SI (selectivity, given by the CC50/EC50
ratio) values for different compounds are depicted in Table 1. Some
active compounds were also evaluated against a number of clini-
cally significant NNRTIs resistant strains, containing E138K, K103N,
L100I, Y181C, Y188L and RES056 (K103N þ Y181C), F227L þ V106A
double mutations, using AZT, NVP, efavirenz (EFV), delavirdine
(DLV), and etravirine (TMC125) as standard drugs and the positive
results are presented in Table 2.
2. Results and discussion
2.1. Chemistry
The general synthetic route utilized to obtain the desired
compounds 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-
arylacetamide was straightforward and is outlined in Scheme 1. The
commercially available 1-(2,4-dichlorophenyl)hydrazine (1) was
converted to the hydrazone (2) by reactionwith 2-oxoacetic acid. The
hydrazone was treated with triethylamine and diphenylphosphoryl
azide in toluene to give 2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-
3(4H)-one (3). During the reaction of Curtius Rearrangement,
nitrogen evolution began at 60e65 ꢀC and occasionally became
vigorous if temperature was not gradually risen [23]. Compound 3
The experimental results indicated that more than half of the
tested 1,2,4-triazolacetamide derivatives were found to be active
against HIV-1 and none of the compounds was active against HIV-2.
In particular, six of these compounds exhibited moderate to good
activities against HIV-1 strain IIIB with EC50 values in the range of
Fig. 2. The design of novel 1,2,4-triazole thioacetanilide based on bioisosterism principle.

P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
5041
Scheme 1. Reagents and conditions: (i) HCOCO2H, H2O; (ii) DPPA, TEA, Tol; (iii) POCl3; (iv) NaH, HSCH2COOEt, THF; (v) NaOH, H2O/DMSO, HCl; (vi) PCl5, aniline, THF, rt.
Table 1
Anti-HIV activities, cytotoxicities and selectivity indices of N-aryl-2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)acetamide derivatives (7aeo).
N
H
N
N
S
R
N
O
Cl
Cl
No.
R
EC50
(m
M)a
CC50
(
m
M)b
SIc
HIV-1
IIIB
HIV-2
ROD
HIV-1
IIIB
HIV-2
ROD
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
2-chlorophenyl
2-fluorophenyl
2-bromophenyl
2,4-dichlorophenyl
2-chloropyridin-3-yl
4-methyl-2-nitrophenyl
2-nitrophenyl
6.21 ꢁ 0.87
>38.39
4.85 ꢁ 0.83
34.36 ꢁ 13.57
4.99 ꢁ 0.92
>285.20
2.78 ꢁ 0.97
32.42 ꢁ 1.25
>114.21
>184.25
>188.90
4.13 ꢁ 0.41
4.68 ꢁ 0.72
>26.27
>29.13
>38.39
>95.12
>171.88
>98.46
>285.2
>186.91
>106.41
>114.21
>184.25
>188.9
>59.34
>33.06
>26.27
>22.82
>240.08
>15
29.13 ꢁ 3.36
38.36 ꢁ 6.72
95.12 ꢁ 54.87
171.88 ꢁ 9.93
98.46 ꢁ 13.96
>285.20
186.91 ꢁ 9.62
106.41 ꢁ 8.90
114.21 ꢁ 4.86
>184.25
188.90 ꢁ 2.78
59.34 ꢁ 28.60
33.06 ꢁ 2.58
26.27 ꢁ 8.74
22.82 ꢁ 4.36
>240.08
5
<1
20
5
20
X1
67
3
<1
X1
<1
14
7
<1
<1
>6460
>80
>11587
>127
>24
<1
<1
<1
<1
<1
X1
<1
<1
<1
X1
<1
<1
<1
<1
<1
X1
o-tolyl
4-acetyl-2-bromophenyl
2-bromo-4-methylphenyl
2-bromo-4-chlorophenyl
2-bromo-4-methoxy carbonylphenyl
2-bromo-4-ethoxy carbonylphenyl
4-methoxy carbonylphenyl
4-ethoxy carbonylphenyl
>22.82
ZP7d
NVP
AZT
DDC
DDI
0.0364 ꢁ 0.0038
0.19 ꢁ 0.04
0.0076 ꢁ 0.0014
0.75 ꢁ 0.55
8.86 ꢁ 2.89
>15
>88.26
>94
>211
0.0033 ꢁ 0.0006
0.88 ꢁ 0.53
16 ꢁ 5.16
>28348
>108
>13
Bold represents the values of active compounds.
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: X 1 stand for ꢂ1or<1.
b
c
d
The antiviral properties of these compounds were previously described, Ref. [16].

5042
P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
Table 2
Anti-HIV mutants activities of selected 1,2,4-triazolacetamide derivatives.
Compd.
EC50
(m
M)a (fold-resistance)b
E138K
K103N
L100I
7a
7c
7e
7g
7l
7m
AZT
NVP
20.86 ꢁ 0.87 (3.6)
28.22 ꢁ 15.65 (6.6)
6.79 ꢁ 0.31 (4.3)
9.45 ꢁ 4.57 (2.3)
ꢂ15.25 (ꢂ4.3)
16.05 ꢁ 0.56 (2.8)
21.42 ꢁ 1.31 (3.7)
8.36 ꢁ 0.33 (1.9)
10.49 ꢁ 0.41 (2.8)
7.42 ꢁ 2.76 (1.8)
25.69 ꢁ 20.17 (6.0)
15.38 ꢁ 3.11 (4.4)
8.51 ꢁ 2.85 (2.1)
7.73 ꢁ 1.67 (2.2)
6.95 ꢁ 1.86 (2.0)
>38.66 (>6.7)
9.54 ꢁ 0.91 (1.6)
10.75 ꢁ 1.72 (1.9)
0.0049 ꢁ 0.0015 (0.32)
1.92 ꢁ 1.20 (12.75)
0.054 ꢁ 0.025 (14.2)
2.23 ꢁ 0.33 (68.3)
0.0046 ꢁ 0.0016 (1.54)
0.02 ꢁ 0.009 (1.4)
0.18 ꢁ 0.057 (1.2)
0.0055 ꢁ 0.00078 (1.4)
0.06 ꢁ 0.023 (1.9)
0.008 ꢁ 0.002 (2.6)
0.011 ꢁ 0.0041 (0.71)
7.25 ꢁ 0.64 (48.25)
0.108 ꢁ 0.022 (28.3)
1.72 ꢁ 0.29 (52.8)
0.00184 ꢁ 0.00021 (0.62)
EFV
DLV (Mesylate)
TMC125
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.
ratio of EC50 value against drug-resistant strain and EC50 of the wt HIV-1 IIIB (EC
b
mutant strains
wt
/EC ).
50
50
2.78e6.21
m
M, which are much better than that of DDI. The most
From the SAR studies, we found that the SAR features of the
1,2,4-triazolacetamides were highly consistent with the previously
observed arylazolylthioacetanilide-typed NNRTIs [15e21].
Most NNRTIs are specific for HIV-1 RT and demonstrate minimal
inhibition of HIV-2 RT. Therefore, based on the above SAR conclu-
sions 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 1,2,4-triazolacetamide derivatives was specific for HIV-1
and belonged to typical NNRTIs.
potent and selective compound was found to be 7g having an EC50
value of 2.78 ꢁ 0.97
mM against HIV-1(IIIB) with a selectivity index
(SI) of 67.
Table 1 also reveals the potency order of the ortho substitution at
the phenyl ring of the anilide moiety as following: NO2 (7g,
EC50 ¼ 2.78 ꢁ 0.97
m
M) > Br (7c, EC50 ¼ 4.85 ꢁ 0.83
m
M) > Cl (7a,
EC50 ¼ 6.21 ꢁ 0.87
mM) > Me (7h, EC50 ¼ 32.42 ꢁ 1.25
mM) >F (7b,
EC50 > 38.39 mM), indicating that the activities data of the 1,2,4-
triazolacetamide derivatives are affected by the electronic nature
or the steric demand of the ortho substitution. Significantly
decreased potency was observed in 7n and 7o characterized by the
absence of the ortho substitution at the phenyl ring of the anilide
moiety. The result demonstrated that the ortho substitutions played
a determinant role in keeping anti-HIV activity. This conclusion is
grossly in agreement with the previous structure-activity rela-
tionship (SAR) studies in 1,2,3-thiadiazole series [15,17]
It is worth noting that, in the case of compound 7c, introduction
of methoxy carbonyl group and ethoxy carbonyl group on the para
position at the phenyl ring of the anilide moiety led to 7l and 7m
with the retained activity. Whereas introduction of 4-acetyl in 7c
led to 7i with deprived activity, indicating that the nature of the
substituent at the para position influenced the anti-HIV-1 activity
remarkably. Furthermore, additional substitution with methyl and
chlorine atom at the para position of anilide phenyl ring in
compounds 7g and 7a led to derivatives 7f and 7d respectively,
with strikingly reduced or deprived antiviral potency, suggesting
that the nature (probably hydrophobicity) of methyl and chlorine
atom did not accommodate the chemical environment in this
region of RT.
The six active compounds 7a, 7c, 7e, 7g, 7l and 7m were further
tested for their inhibitory effects against the HIV-1 mutant strains
(Table 2). To define the resistance profile of these compounds, both
the absolute activity against the HIV-1 mutants (EC50 values) and the
relative activity (fold-resistance) need to be considered. In this assay,
the antiviral activities of the tested analogues are sensitive to these
mutations in the NNRTI active site, which once again suggests that RT
is the target of these 1,2,4-triazolacetamide derivatives. As the data
demonstrated, the selected compounds retained in part their activi-
ties against the E138K, K103N and L100I mutant strains, with EC50
values at the low micromolar level and lower fold-resistance values,
whereas they did not show activity against the HIV-1 Y181C, Y188L,
F227L þ V106A, and RES056 (K103N þ Y181C) mutant strains.
Especially, the 7g showed the highest inhibitory activity against the
HIV-1 K103N strain, which is the most frequent drug resistance
mutation observed in patents failing therapy with NNRTIs [24].
2.2.2. HIV-1 RT inhibition assay
Since arylazolylthioacetanilide derivatives was previously
confirmed to target at the HIV-1 RT, three selected title compounds
(7g, 7l and 7m) were tested in enzyme assays against highly
purified recombinant HIV-1 RT using poly(rC)-oligo(dG) as
template primer. The results showed that the enzymatic inhibitory
activity of these compounds is very weak, regardless of the struc-
tural pattern (Table 3). Thus, the possibility cannot be excluded that
these new compounds might interfere with another target or act on
reverse transcriptase in a different way other than the typical
NNRTIs. Studies are in progress to determine the exact mechanism
for the anti-HIV activity of these compounds.
In the 1,2,3-thiadiazole series, we have demonstrated that the
introduction of pyridine moiety at the aromatic amine domain led to
substantial improvement in potency and selectivity [16]. Interest-
ingly, this beneficial effect was also observed in this closely related
1,2,4-triazolyl series (as shown in 7e and 7a). 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 antiviral potency.
3. Conclusion
Table 3
Inhibitory activity of selected 1,2,4-triazolacetamide derivatives against HIV-1 RT.
In summary, our ‘bioisosterism’ approach has led to the
discovery of novel 1,2,4-triazolacetamide-based anti-HIV agents.
The preliminary SAR was discussed. Some derivatives proved to be
effective in inhibiting HIV-1 replication at low micromolar
concentrations. Among them, compound 7g was identified as the
most promising lead with favorable inhibitory activity against the
Compd.
IV-7g
IV-7l
IV-7m
NVP
4.39
IC50
(m
M)a
>471.41
385.32
297.42
a
50% of the inhibitory concentration of selected 1,2,4-triazolacetamide deriva-
tives required to inhibit biotin deoxyuridine triphosphate (biotin-dUTP) incorpo-
ration into the HIV-1 RT by 50%.

P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
5043
wild-type HIV-1 and the viral strain that carry the K103N mutation.
The RT inhibitory potency was also assayed. All of them proved to
be more weak in enzymatic assays than in cellular tests. Accord-
ingly, it may be difficult to explain their antiviral activity only in
terms of RT inhibition. This result may reflect the interference of
the test compounds with viral replication steps other than reverse
transcription. These studies provide more insight into the SARs of
1,2,4-triazolacetamide derivatives as HIV inhibitors, and should
help in designing new arylazolylthioacetanilide derivatives, which
could be useful for the development of antiviral agents.
filtered and concentrated to afford crude product 5-chloro-1-(2,4-
dichlorophenyl)-1H-1,2,4- triazole (4) as a light yellow solid
(0.21 g, yield: 84.6%), which was used in the next step without any
further purification. 1H-NMR (DMSO-d6, ppm)
d: 8.38 (s, 1H,
triazole-H), 8.04 (d, 1H, J1 ¼ 2.4 Hz, PhH), 7.84 (dd, 1H, J1 ¼ 2.4 Hz,
J2 ¼ 8.4 Hz, PhH), 7.73 (d, 1H, J2 ¼ 8.4 Hz, PhH).
5-chloro-1-(2,4-dichlorophenyl)-1H-1,2,4-triazole (4) (0.5 g,
2 mmol) was added to a mixture of ethyl 2-mercaptoacetate
(0.23 mL, 2.1 mmol) and NaH (0.084 g, 2.1 mmol) in 10 mL THF.
The reaction mixture was stirred at 0 ꢀC for 10 min and then
refluxed for 8 h. The solvent was evaporated under reduced pres-
sure and the residue was then diluted with CH2Cl2 (30 mL), washed
successively with water (2 ꢃ 30 mL), aqueous saturated NaHCO3
and brine. The organic layer was dried (Na2SO4), filtered and
concentrated under reduced pressure to afford crude product 5 as
a light yellow oil, which was used in the next step without any
further purification. ESI-MS: m/z 232.3 (M þ 1), 234.4 (M þ 3).
C12H11Cl2N3O2S (Exact Mass: 330.99).
4. Experimental
4.1. Chemistry
All melting points were determined on a micromelting point
apparatus and are uncorrected. Infrared spectra (IR) were recorded
with
obtained on a Brucker Avance-600 NMR-spectrometer in the indi-
cated solvents. Chemical shifts are expressed in units and TMS as
a
Nexus 470FT-IR Spectrometer. 1H-NMR spectra were
Ester 5 (0.4 g, 1.2 mmol) was dissolved in DMSO (1 mL) and
aqueous 1 N NaOH (2.0 mL, 2.0 mmol) solution was added to the
solution. The reaction mixture was stirred at room temperature for
1 h and acidified with aqueous 1 N HCl. The mixture was then
diluted with EtOAc (100 mL) and successively washed with water
and brine, dried with Na2SO4, filtered and concentrated under
vacuum to give compound 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetic acid (6) as light yellow oil (0.35 g, 97%
yield).
d
internal reference. Mass spectra were taken on an 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). Flash column
chromatography was performed on column packed with Silica Gel
60 (200e300 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.
4.2.2. General Procedure for the synthesis of 2-(2-(2,4-
dichlorophenyl)-2H-1,2,4- triazol-3-ylthio)acetamides (7ae7o)
PCl5 (0.23 g, 1.1 mmol) was added to an ice-cold solution of
compound 6 (0.30 g, 1 mmol) in ether (15 mL). The reaction
mixture was stirred at room temperature for 2 h and then was
concentrated under reduced pressure. The crude product was
dissolved in THF (15 mL). The arylamine (1 mmol) and triethyl-
amine (1.2 mmol) was added to the solution successively. The
reaction mixture was stirred at room temperature for 2 h and then
was then evaporated under reduced pressure. The residue was
chromatographed on silica gel using ethyl acetate:petroleum ether
(1:4). Pure fractions were collected and concentrated, giving the
desired compound (7ae7o).
4.2. General procedure for the synthesis of triazole thioacetanilides
(7aeo)
4.2.1. General Procedure for the synthesis of 2-(2-(2,4-
dichlorophenyl)-2H-1,2,4-triazol-3- ylthio)acetic acid (6)
To
a stirred solution of 4.9 g (27.7 mmol) of 1-(2,4-
dichlorophenyl)hydrazine (1) in 30 mL of water was added 3 mL
of concentrated hydrochloric acid followed by a solution of 4.5 g
(30.5 mmol, 50%) of 2-oxoacetic acid in 5 mL of water. The
precipitate that formed was collected by suction filtration and dried
at reduced pressure to afford 2-(2-(2,4-dichlorophenyl)hydrazono)
acetic acid (2) 4.8 g (75%) of a yellow solid, mp: 158 ꢀC 1H NMR
(DMSO-d6, ppm)
(s, 1H, CO2H), 11.9 (bs, 1H, NH). ESI-MS: m/z 233.1 (M þ 1).
d
: 6.91e7.05 (m, 3H, Ph), 7.55 (s, 1H, N]CH), 10.5
4.2.3. N-(2-chlorophenyl)-2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetamide (7a)
To a stirred suspension of 3.0 g (12.9 mmol) of 2 in 80 mL of dry
toluene was added 1.8 mL (1.3 g, 12.9 mmol) of triethylamine and
2.8 mL (3.6 g, 12.9 mmol) of diphenylphosphoryl azide. This
mixture was slowly heated to reflux temperature with stirring.
Rapid gas evolution began at 60 ꢀC and continued for 1 h. The clear
orange solution was cooled and then poured into 100 mL of 10%
aqueous potassium hydroxide. The basic extract was acidified with
concentrated hydrochloric acid and the precipitated solid was
recrystallized from ethyl acetate/heptane to afford 2-(2,4-
dichlorophenyl)-2H-1,2,4-triazol-3(4H)-one (3) 2.55 g (86%) of
White needle crystals, yield: 45.7%. Mp: 118e121 ꢀC. 1H NMR
(CDCl3, ppm)
d
: 9.76 (s, 1H, NH), 8.33 (d, 1H, J ¼ 8.4 Hz, PhH), 8.08 (s,
1H, triazole-H), 7.60 (d,1H, J ¼ 2.4 Hz, PhH), 7.43e7.26 (m, 4H, PhH),
7.05 (dt, 1H, PhH), 4.03 (s, 2H, SeCH2). ESI-MS: m/z 413.5 (M þ 1),
415.4 (M þ 3), 417.5 (M þ 5), 435.4 (M þ Na). C16H11Cl3N4OS
(411.97).
4.2.4. 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-(2-
fluorophenyl)acetamide (7b)
White powder, yield: 50.4%. mp: 108e110 ꢀC. 1H NMR (CDCl3,
a tan solid, mp: 189e190 ꢀC. 1H-NMR (DMSO-d6, ppm)
d: 12.1 (s,
ppm) d: 10.17 (s, 1H, NH), 8.34e8.31 (m, 1H, PhH), 8.11 (s, 1H,
1H, NH), 8.11 (s, 1H, triazole-H), 7.65 (d, 1H, J ¼ 2.4 Hz, PhH), 7.47
triazole-H), 7.61 (d, 1H, J ¼ 2.4 Hz, PhH), 7.41 (dd, 1H, J1 ¼ 2.4 Hz,
J2 ¼ 8.4 Hz, PhH), 7.37 (d, 1H, J ¼ 8.4 Hz, PhH),7.14e7.05 (m, 3H,
PhH), 3.96 (s, 2H, SeCH2). IR (KBr): 3262 (NH), 1691 (C]O), 1624,
1557, 1496, 1456 (N]N), 758 (CeS). ESI-MS: m/z 397.1 (M þ 1),
399.0 (M þ 3). C16H11Cl2FN4OS (396).
(dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.39 (d, 1H, J ¼ 8.4 Hz, PhH).
13C-NMR (DMSO-d6, ppm)
d: 153.35, 137.76, 134.68, 133.96, 132.81,
131.62, 130.46, 128.89. ESI-MS: m/z 230.4 (M þ 1), 232.3 (M þ 3).
The reaction mixture of 3 (0.23 g, 1 mmol) and 5 mL phos-
phoryl trichloride was stirred at reflux for 10 h (monitored by
TLC). After cooling, the mixture was concentrated under reduced
pressure to give crude residue, then diluted with EtOAc (40 mL).
The separated organic phase was washed successively with water,
aqueous saturated NaHCO3 and brine, and then dried over Na2SO4,
4.2.5. N-(2-bromophenyl)-2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetamide (7c)
White cubic crystals, yield: 35.2%. mp: 115e116 ꢀC. 1H NMR
(CDCl3, ppm)
d
: 9.55 (s, 1H, NH), 8.26 (d,1H, J ¼ 8.4 Hz, PhH), 8.08 (s,

5044
P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
1H, triazole-H), 7.60 (d,1H, J ¼ 2.4 Hz, PhH), 7.60 (dd,1H, J1 ¼ 2.4 Hz,
J2 ¼ 8.4 Hz, PhH), 7.54 (dd,1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.42 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.37 (d, 1H, J ¼ 8.4 Hz, PhH), 7.05
(dt, 1H, PhH), 4.04 (s, 2H, SeCH2). IR (KBr): 3338 (NH), 1684 (C]O),
1591, 1527, 1496, 1436 (N]N), 1060, 744 (CeS). ESI-MS: m/z 457.0
(M þ 1), 459.0 (M þ 3). C16H11BrCl2N4OS (455.92).
7.91 (d, 1H, J ¼ 8.4 Hz, Ph0H), 7.64 (dd, 1H, J1 ¼ 1.2 Hz, J2 ¼ 8.4 Hz,
PhH), 7.45 (d, 1H, J2 ¼ 8.4 Hz, PhH), 3.85 (s, 2H, SeCH2), 2.58 (s, 3H,
CH3). MS (ESI): m/z 199.1 (M þ 1), 202.1 (M þ 3), 204.1 (Mvþ 5).
C18H13BrCl2N4O2S (497.93).
4.2.12. N-(2-bromo-4-methylphenyl)-2-(2-(2,4-dichlorophenyl)-
2H-1,2,4-triazol-3-ylthio)acetamide (7j)
4.2.6. N-(2,4-dichlorophenyl)-2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetamide (7d)
White solid, yield: 48.6%. mp: 125e126 ꢀC. 1H-NMR (CDCl3,
ppm)
d
: 9.55 (s, 1H, NH), 8.17 (d, 1H, J ¼ 8.4 Hz, Ph0H), 8.04 (s, 1H,
White needle crystals, yield: 44.7%. mp: 115e118 ꢀC. 1H NMR
triazoleeH), 7.88 (d, 1H, J1 ¼ 1.8 Hz, PhH), 7.59 (dd, 1H, J1 ¼ 1.8 Hz,
J2 ¼ 8.4 Hz, PhH), 7.43 (s, 1H, Ph0H), 7.38 (d, 1H, J ¼ 8.4 Hz, PhH),
7.20 (s, 1H, J ¼ 8.4 Hz, Ph0H), 3.57 (s, 2H, SeCH2), 2.39 (s, 3H, CH3).
MS (ESI): m/z 471.2 (M þ 1), 473.2 (M þ 3). C17H13BrCl2N4OS
(469.94).
(CDCl3, ppm)
d
: 9.90 (s, 1H, NH), 8.26 (d, 1H, J ¼ 9.6 Hz, PhH), 8.07
(s, 1H, triazole-H), 7.43e7.37 (m, 4H, PhH), 7.25 (dd, 1H, J1 ¼ 2.4 Hz,
J2 ¼ 8.4 Hz, PhH), 4.04 (s, 2H, SeCH2). IR (KBr): 3345 (NH), 1692
(C]O), 1580, 1518, 1496, 1413 (N]N), 1385, 1364, 1061, 821, 776
(CeS). ESI-MS: m/z 447.1 (M þ 1), 449.1 (M þ 3). C16H10Cl4N4OS
(445.93).
4.2.13. N-(2-bromo-4-chlorophenyl)-2-(2-(2,4-dichlorophenyl)-
2H-1,2,4-triazol-3-ylthio)acetamide (7k)
4.2.7. N-(2-chloropyridin-3-yl)-2-(2-(2,4-dichlorophenyl)-2H-
1,2,4-triazol-3-ylthio)acetamide (7e)
White solid, yield: 49.8%. mp: 163e165 ꢀC. 1H-NMR (CDCl3,
ppm)
d
: 9.67 (s, 1H, NH), 8.24 (d, 1H, J ¼ 8.4 Hz, Ph0H), 8.08 (s, 1H,
White needle crystals, yield: 65.4%. mp: 143e145 ꢀC. 1H NMR
triazole-H), 7.61 (d, 1H, J ¼ 1.8 Hz, PhH), 7.54 (d, 1H, J ¼ 2.4 Hz,
Ph0H), 7.42 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, Ph0H), 7.38 (d, 1H,
J ¼ 8.4 Hz, PhH), 7.28 (dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 8.4 Hz, PhH), 4.02 (s,
2H, SeCH2). MS (ESI): m/z 491.1 (M þ 1), 493.1 (M þ 3), 495.0
(M þ 5). C16H10BrCl3N4OS (489.88).
(DMSO, ppm)
d: 10.15 (s, 1H, NH), 8.68 (d, 1H, J ¼ 7.8 Hz, pyridine-
H), 8.28 (d, 1H, J1 ¼1.8 Hz, PhH), 8.20 (dd, 1H, J ¼ 1.2 Hz, J ¼ 4.8 Hz,
pyridine-H), 8.01 (s, 1H, triazole-H), 7.61 (dd, 1H, J1 ¼ 1.8 Hz,
J2 ¼ 8.4 Hz, PhH), 7.40 (d, 1H, J2 ¼ 8.4 Hz, PhH), 7.33e7.29 (m, 1H,
pyridine-H), 4.28 (s, 2H, SeCH2). IR (KBr): 3287 (NH), 1652 (C]O),
1582, 1544, 1520, 1493, 1453 (N]N), 1395, 1072, 809, 799, 785
(CeS). ESI-MS: m/z 414.1 (M þ 1), 416.1 (M þ 3), 418.3 (M þ 3), 436.3
(M þ Naþ), 438.2 (M þ 2 þ Naþ). C15H10Cl3N5OS (412.97).
4.2.14. Methyl 3-bromo-4-(2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetamido) benzoate (7l)
White needle crystals, yield: 59.3%. mp: 164e166 ꢀC. 1H NMR
(CDCl3, ppm)
d
: 9.84 (s, 1H, NH), 8.24 (d, 1H, J ¼ 8.4 Hz, Ph0H), 8.23
4.2.8. 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-(4-
methyl-2-nitrophenyl)acetamide (7f)
(d, 1H, J ¼ 1.8 Hz, Ph0H), 8.09 (s, 1H, triazole-H), 7.97 (dd, 1H,
J1 ¼ 1.8 Hz, J2 ¼ 8.4 Hz, Ph0H), 7.61 (d, 1H, J ¼ 2.4 Hz, PhH), 7.41 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.38 (d,1H, J ¼ 8.4 Hz, PhH), 4.05 (s,
2H, SeCH2), 3.91 (s, 3H, OCH3). MS (ESI): m/z 515.3 (M þ 1), 517.4
(M þ 3), 537.0 (M þ Naþ). C18H13BrCl2N4O3S (513.93).
Light yellow powder, yield: 37.4%. mp: 185e186 ꢀC. 1H-NMR
(CDCl3, ppm)
d
: 11.07 (s, 1H, NH), 8.55 (d, 1H, J ¼ 8.4 Hz, PhH), 8.01
(s, 1H, triazole-H), 7.97 (s, 1H, PhH), 7.61 (d, 1H, J ¼ 1.8 Hz, PhH),
7.46e7.42 (m, 3H, PhH), 4.11 (s, 2H, SeCH2), 2.40 (s, 3H, CH3). MS
(ESI): m/z 438.2 (M þ 1), 540.1 (M þ 3). C17H13Cl2N5O3S (437.01).
4.2.15. Ethyl 3-bromo-4-(2-(2-(2,4-dichlorophenyl)-2H-1,2,4-
triazol-3-ylthio)acetamido) benzoate (7m)
4.2.9. 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-(2-
nitrophenyl)acetamide (7g)
White needle crystals, yield: 67.1%. mp: 103e105 ꢀC. 1H-NMR
(CDCl3, ppm)
d
: 9.82 (s, 1H, NH), 8.45 (d, 1H, J ¼ 8.4 Hz, Ph0H), 8.23
Yellow needle crystals, yield: 53.8%. mp: 153e154 ꢀC. 1H-NMR
(d, 1H, J ¼ 1.8 Hz, Ph0H), 8.09 (s, 1H, triazole-H), 7.99 (dd, 1H,
J1 ¼ 1.8 Hz, J2 ¼ 8.4 Hz, Ph0H), 7.61 (d, 1H, J ¼ 2.4 Hz, PhH), 7.42 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.38 (d,1H, J ¼ 8.4 Hz, PhH), 4.36 (s,
2H, OCH2), 4.05 (s, 2H, SeCH2), 1.39 (s, 3H, CH3). IR (KBr): 3228
(NH), 1720 (C]O), 1684, 1600, 1584, 1529, 1494, 1458 (N]N), 1365,
1285, 1271, 1131, 1106, 763 (CeS). MS (ESI): m/z 529.2 (Mþ1), 531.1
(M þ 3), 533.2 (M þ 5), 551.2 (M þ Naþ), 551.2 (Mþ2 þ Naþ).
C19H15BrCl2N4O3S (527.94).
(CDCl3, ppm)
d
: 11.19 (s, 1H, NH), 8.68 (d, 1H, J ¼ 8.4 Hz, PhH), 8.18
(dd, 1H, J ¼ 1.8 Hz, J ¼ 8.4 Hz, PhH), 8.06 (s, 1H, triazole-H), 7.65 (t,
1H, PhH), 7.61 (d, 1H, J1 ¼1.8 Hz, PhH), 7.46e7.42 (m, 2H, PhH), 7.22
(m, 1H, PhH), 4.13 (s, 2H, SeCH2). IR (KBr): 3295 (NH), 1685 (C]O),
1606, 1585, 1497 (NO2), 1436 (N]N), 1362 (NO2), 1337, 1279, 742
(CeS). MS (ESI): m/z 424.1 (M þ 1), 426.3 (M þ 3). C16H11Cl2N5O3S
(423).
4.2.10. 2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-ylthio)-N-o-
tolylacetamide (7h)
4.2.16. Methyl 4-(2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-
ylthio)acetamido)benzoate (7n)
White lamella crystals, yield: 50.7%. mp: 121e122 ꢀC. 1H-NMR
White needle crystals, yield: 68.2%. mp: 153e154 ꢀC. 1H-NMR
(CDCl3, ppm)
d
: 9.42 (brs, 1H, NH), 8.06 (s, 1H, triazole-H), 7.91 (d,
(CDCl3, ppm) d: 10.43 (s,1H, NH), 8.16 (s,1H, triazole-H), 8.01 (d, 2H,
1H, J1 ¼ 7.8 Hz, PhH), 7.61 (d, 1H, J ¼ 2.4 Hz, Ph0H), 7.43 (dd, 1H,
J1 ¼ 7.8 Hz, J2 ¼ 2.4 Hz, PhH), 7.37 (d, 1H, J2 ¼ 2.4 Hz, PhH), 7.22 (t,
1H, Ph0H), 7.18 (d, 1H, J ¼ 7.2 Hz, Ph0H), 7.08 (t, 1H, Ph0H), 3.96 (s,
2H, SeCH2), 2.27 (s, 3H, CH3). IR (KBr): 3258 (NH), 1687 (C]O),
1591, 1549, 1494, 1458 (N]N), 1365, 1314, 999, 828, 771 (CeS).
MS (ESI): m/z 393.1(M þ 1), 395.1 (M þ 3). C17H14Cl2N4OS
(392.03).
J ¼ 8.4 Hz, Ph0H), 7.64 (d, 2H, J ¼ 8.4 Hz, Ph0H), 7.61 (d,1H, J ¼ 2.4 Hz,
PhH), 7.43 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.37 (d, 1H,
J ¼ 8.4 Hz, PhH), 3.90 (s, 2H, SeCH2), 3.88 (s, 3H, OCH3). MS (ESI): m/
z 437.3 (M þ 1), 439.3 (M þ 3), 441.4 (M þ 5), 559.3 (M þ Naþ), 461.3
(M þ 2 þ Naþ). C18H14Cl2N4O3S (436.02).
4.2.17. Ethyl 4-(2-(2-(2,4-dichlorophenyl)-2H-1,2,4-triazol-3-
ylthio)acetamido)benzoate (7o)
4.2.11. N-(4-acetyl-2-bromophenyl)-2-(2-(2,4-dichlorophenyl)-2H-
1,2,4-triazol-3-ylthio)acetamide (7i)
White needle crystals, yield: 71.4%. mp: 147e149 ꢀC. 1H-NMR
(CDCl3, ppm) d: 10.43 (s,1H, NH), 8.16 (s,1H, triazole-H), 8.01 (d, 2H,
White needle crystals, yield: 55.4%. mp: 131e133 ꢀC. 1H-NMR
J ¼ 8.4 Hz, Ph0H), 7.63 (d, 2H, J ¼ 8.4 Hz, Ph0H), 7.61 (d, 1H, J ¼ 2.4 Hz,
PhH), 7.42 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 8.4 Hz, PhH), 7.37 (d, 1H,
J ¼ 8.4 Hz, PhH), 4.35 (s, 2H, OCH2), 4.04 (s, 2H, S-CH2), 1.41 (s, 3H,
(CDCl3, ppm)
d
: 9.27 (s, 1H, NH), 8.36 (d, 1H, J ¼ 8.4 Hz, Ph0H), 8.24
(s, 1H, Ph0H), 8.09 (s, 1H, triazoleeH), 7.98 (d, 1H, J1 ¼ 1.2 Hz, PhH),

P. Zhan et al. / European Journal of Medicinal Chemistry 46 (2011) 5039e5045
5045
CH3). IR (KBr): 3319 (NH), 1700 (C]O), 1684, 1604, 1529, 1495, 1435,
Acknowledgment
1411 (N]N), 1369, 1317, 1290, 1169, 1156, 1117, 1001, 768 (CeS). MS
(ESI): m/z 451.2 (M þ 1), 453.2 (M þ 3), 455.1 (M þ 5), 473.3
(M þ Naþ), 475.3 (M þ 2 þ Naþ). C19H16Cl2N4O3S (450.03).
The financial support from the National Natural Science Founda-
tion 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.070422083), Independent Innovation Foun-
dation of Shandong University (IIFSDU, No.2010GN044), Shandong
Postdoctoral Innovation Science Research Special Program
(No.201002023) and China Postdoctoral Science Foundation funded
project (No.20100481282) is gratefully acknowledged.
4.3. Biological activity
4.3.1. In vitro anti-HIV assay
The methodology of the anti-HIV assay has been previously
described [25,26]. Stock solutions (10 ꢃ final concentration) of
test compounds were added in 25
mL volumes to two series of
The authors have declared no conflict of interest.
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 samples were
included for each sample.
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