Novel 1,2,3-thiadiazole derivatives as HIV-1 NNRTIs with improved potency: Synthesis and preliminary SAR studies

Article abstract of DOI:10.1016/j.bmc.2009.07.004

A novel synthetic route and anti-HIV activity evaluation of a new series of 2-(4-(2,4-dibromophenyl)-1,2,3-thiadiazol-5-ylthio)acetamide (TTA) derivatives are described. Bioactivity assay indicated that most of the title compounds showed good activities a

Full text of DOI:10.1016/j.bmc.2009.07.004

Bioorganic & Medicinal Chemistry 17 (2009) 5920–5927
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Bioorganic & Medicinal Chemistry
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Novel 1,2,3-thiadiazole derivatives as HIV-1 NNRTIs with improved potency:
Synthesis and preliminary SAR studies
a
a
a
a
Peng Zhan a, Xinyong Liu a, , Zhenyu Li , Zengjun Fang , Zhong Li , Defeng Wang ,
*
Christophe Pannecouque b, 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 novel synthetic route and anti-HIV activity evaluation of a new series of 2-(4-(2,4-dibromophenyl)-
1,2,3-thiadiazol-5-ylthio)acetamide (TTA) derivatives are described. Bioactivity assay indicated that most
of the title compounds showed good activities against HIV-1. In particular, compound 7c displayed the
most potent anti-HIV-1 activity (EC50 = 36.4 nM), inhibiting HIV-1 replication in MT-4 cells more effec-
tively than NVP (by sevenfold) and DLV (by eightfold). The preliminary structure–activity relationships
(SAR) of the newly synthesized congeners are discussed, and molecular modeling of compound 7c in
complex with HIV-1 RT is described, allowing rationalization of some SAR conclusions.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 1 June 2009
Revised 30 June 2009
Accepted 2 July 2009
Available online 7 July 2009
Keywords:
HIV-1
AIDS
NNRTIs
Thiadiazole thioacetanilides
Heterocycle
Antiviral activity
1. Introduction
based on the previous SAR analysis, with the aim to improve the
interaction between the inhibitors and RT and to obtain new bio-
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 reverse transcriptase inhibitors (NRTIs)
or to the non-nucleoside reverse transcriptase inhibitors (NNRTIs).
NNRTIs have become key components in the combination regi-
ments of cocktail therapy.1–4
However, a number of problems still remain with these agents,
in particular, the therapeutic potential of this class of drugs has
been compromised by the rapid development of resistance, but
their encouraging usage in combination therapy has rekindled
interest in the search for novel and potent NNRTIs.1–4
Among the representatives of the NNRTIs, triazole/tetrazole
thioacetanilides derivatives (L1 and L2, Fig. 1) were reported as
potent HIV-1 inhibitors, with low nanomolar intrinsic activity
against the RT and submicromolar antiviral activity in HIV-infected
cells.5–12 Recently, we developed a series of 1,2,3-thiadiazole
thioacetanilides (TTAs) (L3–L7, Fig. 2), which exhibited significant
anti-HIV-1 activities.13 Inspired by these promising results and in
continuation of our work on the research of novel NNRTIs,13–15
we thought it is worthwhile to synthesize new compounds of TTAs
logically active molecules.
The previous SAR and molecular modeling of azole thioacetan-
ilides indicated that increasing volume of the substituted phenyl
linked with azole was beneficial for strengthening
p–p stacking
interaction between inhibitor and Tyr188 or Tyr181 of RT.12,13 This
background prompted us to further explore novel TTAs bearing a
larger 2,4-dibromo substituted phenyl attached to the 1,2,3-thiadi-
azole ring. Additionally, in view of the fact that the strength of hal-
ogen bonding,16,17 one of the important interactions in biological
molecules, decreases in the order Br > Cl, it is likely that the 2,4-di-
bromo derivatives could form potentially enhanced halogen bond
with carbonyl oxygen in the main chain of residues nearby, and
lastly increasing the overall RT-NNRTI binding affinity.
On the other hand, it has been demonstrated that the N-substi-
tuted phenyl moiety sits at the protein/solvent interface near a re-
gion of the protein known to be flexible,12,13 it might tolerate
substitution by structurally diverse groups. Therefore, in several
novel molecules, the anilide phenyl ring was also replaced by sev-
eral substituted heterocycles to further investigate the SARs in this
region.
Herein we reported a facile synthetic pathway and biological
evaluation of
a series of 2-(4-(2,4-dibromophenyl)-1,2,3-thia-
diazol-5-ylthio)-N-phenylacetamide derivatives (Fig. 3) as novel
NNRTIs. It is a further object of this paper to develop our under-
* Corresponding author. Tel.: +86 531 88380270; fax: +86 531 88382731.
E-mail address: xinyongl@sdu.edu.cn (X. Liu).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.004

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
5921
X
X
N
N
N
N
H
H
N
N
N
S
S
R'
N
N
O
O
Y
Y
R
R
L1
L2
Figure 1. Triazole/tetrazole thioacetanilide based NNRTIs.
N
X
compound 4 with thionyl chloride according to the method re-
ported by Hurd and Mori,18–20 and purification by flash chromatog-
raphy produced the thiadiazole derivative 5. Treatment of
compound 5 with sodium methoxide afforded the desired sodium
salt of 1,2,3-thiadiazole-5-thiolate 6 as a result of the b-elimination
of the propyl ester group at C5–S group.21 The final 1,2,3-thiadia-
zole thioacetanilides 7 were synthesized by reaction of 6 with var-
ious 2-chloro-N-(substituted aromatic group)acetamides in high
yields. These reactions are summarized in Scheme 1. The structures
of all the synthesized title compounds were confirmed on the basis
of spectral and analytical data.
It must be pointed out that we once reported a synthetic method
for TTAs in our previous literature which requires the 1,2,3-thiadi-
azole-5-thioacetic acid (9), produced by hydrolysis of compounds 8
in KOH–EtOH–H2O solution, as the key intermediates (Scheme 2).13
In the last step, the title compounds 10 were obtained by acylation
reaction of 9 with substituted anilines in the presence of 1.1 equiv
mol of PCl5. However, this route did not give us satisfactory results
due to the low overall yield and complicated procedure in these two
reaction steps. Consequently, a modified method was disclosed
herein, which not only improved overall yield, but also provide pos-
sibility of further developing TTA derivatives with mild reaction
condition and simple operation.
S
H
N
N
S
O
Cl
Y
Cl
L3: X= NO2, Y= H.
L4: X= Br, Y= CO2Et. EC50 = 0.099 μM, SI > 2298
L5: X= Cl, Y= H.
L6: X= Br, Y= H.
L7: X= Br, Y= Me.
EC50 = 0.059 μM, SI > 4883
EC50 = 0.118 μM, SI = 943
EC50 = 0.149 μM, SI ≥981
EC 50 = 0.204 μM, SI > 1265
Figure 2. 1,2,3-Thiadiazole based NNRTIs.
standing of the SAR among TTAs and to afford information to the
ongoing modification of this scaffold.
2. Results and discussion
2.1. Chemistry
2.2. Anti-HIV cell-based assays and discussions
We described a convenient and original synthetic route for title
compounds which requires the salt of 5-thiol-1,2,3-thiadiazole as
the key intermediate. In this approach, 2-chloro-1-(2,4-dibrom-
ophenyl)ethanone (2), synthesized by Friedel–Crafts reaction of
1,3-dibromobenzene (1) with 2-chloroacetyl chloride, were re-
acted with 3-mercaptopropanoate in EtOH at ambient temperature
for several hours to obtain the methyl 3-(1-(2,4-dibromophe-
nyl)ethanone)propanoate (3) which was not purified and used for
the next step directly. The compound 4 was synthesized from
semicarbazide by reaction with the compound 3. Ring-closure of
The newly synthesized TTA derivatives (7a–r) were evaluated for
their anti-HIV activity and cytotoxicity in MT-4 cells infected with
wild-type HIV-1 strain IIIB and HIV-2 strain ROD in comparison with
nevirapine (NVP), delaviridine (DLV), efavirenz (EFV) and zidovu-
dine (azidothymidine, AZT), which were used as reference drugs.
The results, expressed as CC50 (50% cytotoxic concentration), EC50
(50% HIV-1 replication inhibitory concentration) and SI (selectivity
index given by the CC50/EC50 ratio), are summarized in Table 1.
N
X
S
H
N
N
S
O
Br
Y
N
X
S
H
N
N
Br
S
O
Cl
N
S
Y
H
N
N
S
Ar
O
Br
Cl
Heterocycle
Br
Figure 3. The design of novel 1,2,3-thiadiazole derivatives.

5922
P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
O
O
S
O
Cl
i
ii
iii
O
S
Br
Br
Br
Br
Br
Br
1
2
3
N
S
O
N
S
N
NNHCONH2
S
H
N
N
N
N
O
S
R
iv
S
O
SNa
Br
v
vi
O
Br
Br
O
Br
Br
Br
Br
Br
4
5
6
7
Scheme 1. Reagents: (i) ClCOCH2Cl/AlCl3; (ii) HSCH2CH2COOCH3, Na2CO3, EtOH; (iii) NH2NHCONH2ÁHCl, AcONa, EtOH; (iv) SOCl2; (v) CH3ONa/CH3OH; (vi) ClCH2CONHR,
EtOH.
N
X
N
S
N
S
S
H
N
N
N
N
O
OH
S
S
S
i
ii
R1
R1
O
R1
O
O
Y
R2
R2
8
9
10
R2
Scheme 2. Reagents and conditions: (i) KOH, EtOH–H2O, 50–70 °C; (ii) PCl5, aniline, DCM, rt, 1–2 h, Ref. 13.
The experimental results indicated that the majority of the
tested TTA derivatives were found to be active against HIV-1 in
the range of 0.0364–4.53 lM and none of the compounds was ac-
4-acetyl in 7d led to 7f with slightly improved activity, indicating
that the nature of the substituent at the para position influenced
the anti-HIV-1 activity remarkably.
tive against HIV-2. In particular, 10 compounds showed anti-HIV-1
activities at submicromolar concentrations. Interestingly, the cyto-
toxicity of TTAs was generally low. Owing to the above reasons,
their SIs were in many cases similar to that of the reference drug.
Compound 7c was the most active derivative of this series, with
Significantly increased cytotoxicities were observed in 7j, 7k and
7l characterized by the absence of the ortho substitution at the phe-
nyl ring of the anilide moiety. The result indicates that the ortho sub-
stitutions played a determinant role in their cytotoxicity.
In the N-substituted heterocycle acetamide derivatives, pyri-
dine derivative 7c showed an improved anti-HIV-1 profile superior
to that of 7b, with a higher selectivity index. Moreover, pyridine
derivative 7o, thiazole derivative 7p and thiophene derivative 7r
demonstrated reasonable antiviral activity. These results con-
firmed the idea that the introduction of structurally diverse hetero-
cycles in this region could be a valid strategy to get novel
molecules with increased or appreciable antiviral potency.
In addition, the finding that none of the title compounds was
found effective against HIV-2 (Table 1) showed that this new series
of TTAs was specific for HIV-1 and belonged to typical NNRTIs.
EC50 = 36.4nM, CC50 > 240.1 lM, SI > 6460. 7c was about sevenfold
more active than NVP and eightfold more active than DLV.
In the N-substituted phenyl acetamide derivatives, as expected,
we observed that 2,4-dibromo substitution on the phenyl of the
thiadiazole ring increased the antiviral activity compared to the
2,4-dichloro substituted phenyl series.12 Based on previous SAR
studies on the phenyl-thiadiazole, we found that the antiviral
potency of TTAs decreases in the order 2,4-dibromo > 2,4-di-
chloro > 2,4-difluorine.
Table 1 also reveals the potency order of the ortho substitution at
the phenyl ring of the anilide moiety: NO2 (7g, EC50 = 0.040
(7b, EC50 = 0.083 M) > Br (7d, EC50 = 0.12 M) > F (7a, EC50
0.14 M) > Me (7i, EC50 = 0.52 M) > H (7j, EC50 = 0.54 M), indicat-
lM) > Cl
l
l
=
2.3. Molecular modeling analysis
l
l
l
ing that electron withdrawing groups, such as nitro and halogen,
were the preferred substituents compared with electron-donating
group methyl or hydrogen atom. Thus, the activity data of the TTAs
are affected by the electronic nature or the steric demand of the
ortho substitution. In addition, a decrease of activity was observed
in the para mono-substituted isomer of TTAs with the ortho single
substitution at the anilide moiety (7k vs 7i; 7l vs 7b).
In the case of compounds 7d and 7g, introduction of methyl
group on the para position at the phenyl ring of the anilide moiety
led to 7e and 7h with decreased activity, whereas introduction of
Docking calculations were also performed by means of Autodock
Vina to investigate the binding mode of compound 7c into the NNR-
TIs binding pocket (NNIBP) of HIV-1 RT and to rationalize some SARs.
Three-dimensional coordinates of the HIV-1 RT/GW564511 (benzo-
phenone based NNRTI) complex (Brookhaven Protein Data Bank en-
try 3DLG) were used as the input structure because of the high
degree of similarity between sulfanyltriazole/tetrazole leads and
benzophenones.11 Default parameters were used as described in
the AutoDock manual unless otherwise specified. The theoretical
binding mode of 7c to the NNIBP is shown in Figure 4.

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
5923
Table 1
Anti-HIV activities, cytotoxicities and selectivity indices of 2-(4-(2,4-dibromophenyl)-1,2,3-thiadiazol-5-ylthio)acetamide derivatives (7a–r)
N
S
H
N
N
S
R
O
Br
Br
SIc
a
b
No.
R
EC50
(lM)
CC50
(l
M)
HIV-1 IIIB
0.14 0.01
0.083 0.015
0.0364 0.0038
0.12 0.01
0.22 0.05
0.058 0.026
0.040
0.079 0.017
0.52 0.18
0.54 0.02
1.52 0.96
2.19 0.60
1.44 0.27
>198.73
1.17 0.51
4.53 0.33
>23.55
HIV-2 ROD
HIV-1 IIIB
HIV-2 ROD
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
2-Fluorophenyl
2-Chlorophenyl
>27.82
P221.30
>240.08
171.78 52.90
>216.21
>206.22
>235.76
>229.68
>201.87
>24.83
>24.04
>23.34
>240.95
>198.73
>42.43
P27.82
>240.54
>240.08
>221.59
>216.21
>206.22
>235.76
>229.68
201.87 35.85
24.83 1.26
24.04 0.28
23.34 0.69
240.95 2.98
P198.73
42.43 9.26
87.76 43.37
23.55 1.42
>227.57
>15.02
>3.827
>195
>2904
>6460
>1786
>958
>3546
>5841
>2934
381
47
16
11
166
<orÂ1
>orÂ1
Â1
2-Chloropyridin-3-yl
2-Bromophenyl
2-Bromo-4-methylphenyl
4-Acetyl-2-bromophenyl
2-Nitrophenyl
4-Methyl-2-nitrophenyl
o-Tolyl
Phenyl
p-Tolyl
4-Chlorophenyl
2,3-Dimethylphenyl
2,6-Dimethylphenyl
Pyridin-2-yl
>1
Â1
Â1
Â1
Â1
<1
<1
<1
<1
<1
<orÂ1
<orÂ1
36
<1
Thiazol-2-yl
5-Methylbenzo[d]thiazol-2-yl
3-Methyl acetate-thiophen-2-yl
>87.76
>23.55
>227.57
19
<1
>57
>72
>12
>1434
>6192
<1
<1
Â1
7r
4.01 1.55
0.208
0.320
0.00440
0.0151
NVPd
DLVd
EFVd
AZTd
>6.336
>93.55
a
EC50: concentration of compound required to achieve 50% protection of MT-4 cell from 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: Â1 stand for P1or<1.
b
c
d
The antiviral properties of these compounds were previously described, Ref. 13.
Results showed that the 2,4-dibromophenyl ring of 7c fits into
nyl points directly at, and perpendicular to, the highly conserved
residue W229. The highly conserved amino acid residues in the
NNIBP are essential for viral replication, and improved binding at
these amino acids could result in the development of novel NNRTIs
not sensitive to the common RT mutations.22 Therefore, the activ-
ity was strongly affected by the chemical features of halogen atoms
the aromatic-rich binding pocket, surrounded by the aromatic side
chains of Tyr188, Phe227 and Trp229. Detailed analysis of the
binding mode shows that the phenyl ring is parallel to Tyr188 side
chain, giving rise to a positive
p-stacking interaction, and an inter-
esting but indefinite interaction in which the 4-bromo of the phe-
Figure 4. Model of 7c docked into the RT non-nucleoside binding site (PDB code: 3DLG) using Autodock Vina [http://vina.scripps.edu]. The docking result of 7c is showed by
PyMOL. (a) Overview; (b) View is looking inward along the enzyme–solvent interface under Pro236, showing the solvent exposed lower surface of the 2-chloropyridin-3-yl
group in 7c.

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P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
in the 2,4-bisubstituted phenyl ring linked to thiadiazole and we
can presume that replacing the 2,4-dibromo by 2,4-diiodine phenyl
group may lead to even superior anti-HIV-1 activities.
4.1.1. General procedure for the synthesis of sodium 4-(2,4-
dibromophenyl)-1,2,3-thiadiazole-5-thiolate (6)
To a vigorously stirred mixture of the commercially available
1,3-dibromobenzene (1, 23.6 g, 0.1 mol) and anhydrous aluminum
chloride (14.6 g, 0.11 mol) at room temperature was added chloro-
acetyl chloride (13 g, 0.1 mol) dropwise. The reaction mixture was
stirred at 60–65 °C for 10 h (monitored by TLC). After cooling, the
mixture was then diluted with CH2Cl2 (40 mL). The separated or-
ganic phase was washed successively with water, aqueous satu-
rated NaHCO3 and brine. The organic layer was dried (Na2SO4),
filtered and concentrated under reduced pressure to afford crude
product 2-chloro-1-(2,4-dibromophenyl)ethanone (2) as a light
yellow oil (26.7 g, yield: 85.4%), which was used in the next step
without any further purification.
The inhibitor’s amide carbonyl forms a key hydrogen bond with
the backbone N–H of Lys103 (not shown). In compound 7n, it is
likely that the steric factor of 2,6-dimethyl diminishes this hydro-
gen bond, yielding an overall negative effect on the activity.
The 2-chloropyridin-3-yl group of 7c is close to the flexile Pro236
‘hairpin loop’ of the protein/solvent interface, and the nitrogen atom
in the pyridine ring points toward the solvent exposed region
(Fig. 4b), suggesting that this plasticity region could accommodate
substantial modifications of the inhibitor molecule. The hydrophilic
groups (atoms) has been proved to be the preferred substituents,
which can explain the SAR conclusion of this region.
Overall, our studies suggest that this class of compounds share
the similar bound conformation with sulfanyltriazoles5 and sulfa-
nyltetrazoles-based NNRTIs.9,10 As illustrated in Fig. 4a, the com-
pound 7c binds in a ‘kinked’ conformation 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 thiadiazole is orthogonal to the 2,4-dibromophe-
nyl ring, which orients the pharmacophores into the proper geom-
etry for binding. This brings the two substituted aryl rings into
close proximity and results in the amide N–H pointing toward
the sulfur atom (probably forming intramolecular hydrogen bond).
In summary, the results of the AutoDocking analysis supported
our newly designed and synthesized compounds. Further optimi-
zation of TTA analogues will consider these aspects in further de-
sign attempts.
2-Chloro-1-(2,4-dibromophenyl)ethanone (2, 0.1 mol) was
added to a mixture of methyl 3-mercaptopropanoate (11.4 mL,
0.1 mol) and Na2CO3 (5.3 g, 0.05) in 100 mL EtOH. The reaction
mixture was stirred at room temperature for 12 h. The solvent
was evaporated under reduced pressure. The residue was then di-
luted with CH2Cl2 (100 mL), washed successively with aqueous
0.1 M HCl solution, aqueous saturated NaHCO3, water and brine.
The organic layer was dried (Na2SO4), filtered and concentrated un-
der reduced pressure to afford crude product 3 as a light yellow oil.
A mixture of semicarbazide hydrochloride (12.2 g, 0.12 mol) and
sodium acetate (8.2 g, 0.1 mol) was dissolved in absolute ethanol
(50 mL). The mixture was heated for 60 min under reflux, and then
filtered while hot to remove precipitated sodium chloride. The fil-
trate was then mixed with compound 3 (39.6 g, 0.1 mol), and the
resulting mixtures were heated to reflux, a few drops of concen-
trated hydrochloric acid were added and heating under reflux with
continuous removal of the generated water was continued over-
night. Finally, the solvent was cooled and filtered. The obtained solid
was washed successively with water and diethyl ether and dried to
give compound 4 as white solid, yield: 74.7%, mp 186–188 °C.
A 4.5 g (0.01 mol) portion of semicarbazone (4) was diluted with
10 mL of dry methylene chloride and this was added, fairly rapidly,
using a dropping funnel, to 10 mL of thionyl chloride with rapid stir-
ring which was continued for 6 h after addition was completed. The
reaction mixture was then evaporated under reduced pressure and
two portions of methylene chloride were added and removed under
reduced pressure. The residue was dissolved in ethyl acetate, filtered
and the filtrate was evaporated to a residue which was chromato-
graphed on silica gel using ethyl acetate:petroleum ether (1:4). Frac-
tions were collected and evaporated, giving the desired compound
(5) as brilliant yellow oil (80.7%).
3. Conclusions
In this article, we have described a improved synthetic route
with easy operation and higher yield and anti-HIV-1 activity eval-
uation of a new series of 2-(4-(2,4-dibromophenyl)-1,2,3-thia-
diazol-5-ylthio) acetamides. The bioassay results revealed that
the majority of the title compounds exhibited potent HIV-1 inhib-
itory activity. In particular, the N-(2-chloropyridin-3-yl) analogue
7c turned out to be the most potent with an EC50 value of
36.4 nM, being about sevenfold more active than the reference
compounds NVP and DLV. Based upon the preliminary SAR studies
of these new 2,4-dibromophenyl substituted TTAs, some structural
requirements for high potency against HIV-1 were identified and
rationalized by the molecular modeling studies. Further synthesis
of new TTA derivatives and quantitative SAR (QSAR) studies are
currently performed in our laboratories.
A solution of sodium methoxide (0.54 g, 0.01 mol) in 15 mL of
methanol was added to a solution of methyl 3-(4-(2,4-dibromophe-
nyl)-1,2,3-thiadiazol-5-ylthio)propanoate (5) (4.4 g, 0.01 mol) in
100 mL of methanol. After about 1 h, the reaction solution was
evaporated in vacuo to about 10 mL. Dry methylene chloride
(50 mL) was added, causing precipitation of a white solid. This solid
was collected, wash with methylene chloride, giving the desired
product 6 as white powder (90.4%), mp: 98–100 °C.
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. 1H NMR spectra were ob-
tained on a Brucker Avance-600 NMR-spectrometer in the indi-
cated solvents. 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 va-
pors or by irradiation with UV light (254 nm). Flash column chro-
matography was performed on column packed with Silica Gel 60
(230–400 mesh). Solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods. Concentration
of the reaction solutions involved the use of rotary evaporator at
reduced pressure.
4.1.2. General procedure for the synthesis of 2-(4-(2,4-dibrom-
ophenyl)-1,2,3-thiadiazol-5-ylthio) acetamide (7a–r)
To the solution of sodium 4-(2,4-dibromophenyl)-1,2,3-thiadia-
zole-5-thiolate (6) (1.05 mmol, 0.4 g) in ethanol (30 mL) was added
2-chloro-N-(substituted aromatic group)acetamides (1.0 mmol).
The reaction mixture was stirred at room temperature overnight.
Upon completion of the reaction, the solvent was evaporated, leav-
ing a residue which was treated with methylene chloride (30 mL)
and washed with water (3 Â 30 mL). The organic layer was dried
over anhydrous sodium sulfate, the drying agent was filtered off,
and the solvent was removed in vacuo. The residue was purified by
recrystallization using ethanol to yield the title compounds (7a–r).

P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
5925
4.1.2.1. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-N-(2-
fluorophenyl)acetamide (7a). White needle crystals, yield: 49.8%.
Mp: 121–123 °C. 1H NMR (CDCl3, ppm) d: 8.21 (t, 1H, Ph’H), 8.10 (br
s, 1H, NH), 7.92 (d, 1H, J1 = 1.8 Hz, PhH), 7.57 (dd, 1H, J1 = 1.8 Hz,
J2 = 8.4 Hz, PhH), 7.32 (d, 1H, J2 = 8.4 Hz, PhH), 7.15 (m, 3H, Ph’H),
7.54 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, PhH), 7.34 (d, 1H, J2 = 8.4 Hz,
PhH), 7.27 (m, 2H, Ph’H), 3.85 (s, 2H, S–CH2). IR (KBr, cmÀ1):
3298 (
m
NH), 1691 (
m
C@O), 1500 (mas
), 1431 (
m
N@N), 1338 (ms
),
NO2
NO2
1277, 1221, 742 (mC–S). MS (ESI): m/z 529.2 (M+1), 531.1 (M+3),
533.1 (M+5). C16H10Br2N4O3S2 (527.86).
3.81 (s, 2H, S–CH2). IR (KBr, cmÀ1): 3271 (
1548, 1489, 1456 ( N@N), 744 (
mNH), 1684 (mC@O), 1624,
m
mC–S). MS (ESI): m/z 502.1 (M+1), 504.1
4.1.2.8. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-N-
(4-methyl-2-nitrophenyl)acetamide (7h). Light yellow needle
crystals, yield: 84.7%. Mp: 186–188 °C (decompose). 1H NMR
(CDCl3, ppm) d: 10.99 (s, 1H, NH), 8.56 (d, 1H, J = 8.4 Hz, Ph’H),
8.04 (m, 1H, J = 1.8 Hz, Ph’H), 7.89 (d, 1H, J1 = 1.8 Hz, PhH), 7.55
(dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.49 (dd, 1H, J = 1.8 Hz,
J = 8.4 Hz, Ph’H), 7.34 (d, 1H, J2 = 8.4 Hz, PhH), 3.83 (s, 2H, S–CH2),
(M+3), 506.1 (M+5). C16H10Br2FN3OS2 (500.86).
4.1.2.2. N-(2-Chlorophenyl)-2-(4-(2,4-dibromophenyl)-1,2,3-thia-
diazol-5-ylthio)acetamide (7b). Light yellow cubic crystals, yield:
59.4%. Mp: 161–163 °C. 1H NMR (CDCl3, ppm) d: 8.52 (s, 1H, NH),
8.28 (d, 1H, J = 8.4 Hz, PhH), 7.90 (d, 1H, J = 1.2 Hz, Ph’H), 7.54 (dd,
1H, J1 = 8.4 Hz, J2 = 1.2 Hz, PhH), 7.37 (d, 1H, J = 1.2 Hz, PhH), 7.29 (m,
2H, Ph’H), 7.10 (t, 1H, Ph’H), 3.83 (s, 2H, S–CH2). 13C NMR (CDCl3,
ppm) d: 163.7, 158.4, 147.9, 135.8, 133.5, 133.0, 131.0, 129.9, 129.2,
127.9, 125.7, 124.8, 124.7, 123.2, 121.5, 41.6. IR (KBr, cmÀ1): 3365
2.42 (s, 3H, CH3). IR (KBr, cmÀ1): 3304 (
1514 (mas ), 1445 ( N@N), 1336 (ms ), 1277, 1220, 739 (
m
NH), 2914, 1690 (
m
m
C@O),
C–S).
m
NO2
NO2
MS (ESI): m/z 543.2 (M+1), 545.1 (M+3). C17H12Br2N4O3S2 (541.87).
(
m
NH), 1699 (
m
C@O), 1592, 1528, 1440 (
m
N@N), 1306, 1217, 750 (
m
C–S).
4.1.2.9. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-N-
o-tolylacetamide (7i). White lamella crystals, yield: 70.8%. Mp:
121–123 °C. 1H NMR (CDCl3, ppm) d: 7.92 (s, 1H, J1 = 1.2 Hz, PhH),
7.87 (br s, 1H, NH), 7.72 (d, 1H, J = 8.4 Hz, Ph’H), 7.56 (dd, 1H,
J1 = 1.2 Hz, J2 = 8.4 Hz, PhH), 7.31 (d, 1H, J2 = 8.4 Hz, PhH), 7.23 (t,
1H, Ph’H), 7.19 (m, 1H, Ph’H), 7.12 (t, 1H, Ph’H), 3.84 (s, 2H, S–CH2),
MS (ESI): m/z 518.1 (M+1), 520.1 (M+3), 522.1 (M+5).
C16H10Br2ClN3OS2 (516.83).
4.1.2.3. N-(2-Chloropyridin-3-yl)-2-(4-(2,4-dibromophenyl)-1,2,3-
thiadiazol-5-ylthio)acetamide (7c). White needle crystals, yield:
75.8%. Mp: 161–163 °C. 1H NMR (CDCl3, ppm) d: 8.65 (d, 1H,
J = 7.8 Hz, pyridine-H), 8.52 (br s, 1H, NH), 8.17 (dd, 1H, J = 1.2 Hz,
J = 4.8 Hz, pyridine-H), 7.92 (d, 1H, J1 = 1.8 Hz, PhH), 7.58 (dd, 1H,
J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.32 (d, 1H, J2 = 8.4 Hz, PhH), 7.28 (m,
2.13 (s, 3H, CH3). IR (KBr, cmÀ1): 3242 (
2851, 1640 ( C@O), 1532, 1490 ( N@N), 1216, 751 (m
m
NH), 3026, 2980, 2936,
C–S). MS (ESI):
m
m
m/z 498.1(M+1), 500.1 (M+3). C17H13Br2N3OS2 (496.89).
1H, pyridine-H), 3.84 (s, 2H, S–CH2). IR (KBr, cmÀ1): 3305 (
1680 ( C@O), 1523, 1454 ( N@N), 1392, 1212, 741( C–S). MS (ESI): m/z
519.1 (M+1), 521.1 (M+3). C15H9Br2ClN4OS2 (517.83).
m
NH),
4.1.2.10. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-phenylacetamide (7j). White solid, yield: 78.4%. Mp: 119–
121 °C. 1H NMR (CDCl3, ppm) d: 7.92 (d, 1H, J1 = 1.2 Hz, PhH),
10.37 (br s, 1H, NH), 7.57 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, PhH),
7.41 (d, 1H, J2 = 8.4 Hz, PhH), 7.32 (m, 4H, Ph’H), 7.17 (dt, 1H,
m
m
m
4.1.2.4. N-(2-Bromophenyl)-2-(4-(2,4-dibromophenyl)-1,2,3-thia-
diazol-5-ylthio)acetamide (7d). White cubic crystals, yield: 75.8%.
Mp: 147–149 °C. 1H NMR (CDCl3, ppm) d: 8.54 (s, 1H, NH), 8.25 (d,
1H, J = 7.8 Hz, Ph’H), 7.91 (d, 1H, J1 = 1.8 Hz, PhH), 7.55 (m, 1H, PhH),
7.54 (m, 1H, Ph’H), 7.36 (m, 1H, Ph’H), 7.30 (m, 1H, PhH), 7.03 (t, 1H,
Ph’H), 3.78 (s, 2H, S–CH2). IR (KBr, cmÀ1): 3274 (
1605, 1557, 1444 ( N@N), 1220, 746 (
m
NH), 1676 (
mC@O),
m
mC–S). MS (ESI): m/z 484.2
(M+1), 486.1 (M+3). C16H11Br2N3OS2 (482.87).
Ph’H), 3.83 (s, 2H, S–CH2). IR (KBr, cmÀ1): 3351 (
1527, 1436 ( N@N), 1303, 1217, 748 (
564.0 (M+3), 566.0 (M+5). C16H10Br3N3OS2 (560.78).
m
NH), 1690 (
m
C@O),
4.1.2.11. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-p-tolylacetamide (7k). White crystals, yield: 80.7%. Mp: 155–
157 °C. 1H NMR (CDCl3, ppm) d: 7.92 (d, 1H, J1 = 1.8 Hz, PhH), 7.86
(br s, 1H, NH), 7.57 (dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.32 (d,
1H, J2 = 8.4 Hz, PhH), 7.28 (d, 2H, J = 8.4 Hz, Ph’H), 7.13 (d, 2H,
J = 8.4 Hz, Ph’H), 3.77 (s, 2H, S–CH2), 2.33 (s, 3H, CH3). IR (KBr,
m
mC–S). MS (ESI): m/z 562.1(M+1),
4.1.2.5. N-(2-Bromo-4-methylphenyl)-2-(4-(2,4-dibromophenyl)-
1,2,3-thiadiazol-5-ylthio)acetamide (7e). White solid, yield: 84.5%.
Mp: 169–171 °C. 1H NMR (CDCl3, ppm) d: 8.45 (s, 1H, NH), 8.10 (d,
1H, J = 8.4 Hz, Ph’H), 7.91 (d, 1H, J1 = 1.8 Hz, PhH), 7.54 (dd, 1H,
J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.36 (s, 1H, Ph’H), 7.32 (d, 1H, J = 8.4 Hz,
PhH), 7.14 (s,1H, J = 8.4 Hz, Ph’H), 3.51 (s, 2H, S–CH2), 2.32 (s, 3H,
cmÀ1): 3249 (
N@N), 1374, 1232, 815, 738 (m
m
NH), 3033, 2921, 2854, 1646 (
mC@O), 1541, 1421
(m
C–S). MS (ESI): m/z 498.1 (M+1),
500.1 (M+3). C17H13Br2N3OS2 (496.89).
CH3). IR (KBr, cmÀ1): 3275 (
1488 ( N@N), 1226, 1184, 740 (
(M+3). C17H12Br3N3OS2 (574.8).
mNH), 3039, 2951, 2910, 1681 (
mC@O), 1529,
4.1.2.12. N-(4-Chlorophenyl)-2-(4-(2,4-dibromophenyl)-1,2,3-
thiadiazol-5-ylthio)acetamide (7l). Light yellow solid, yield:
58.7%. Mp: 122–124 °C. 1H NMR (CDCl3, ppm) d: 7.92 (d, 1H,
J1 = 1.2 Hz, PhH), 7.91 (br s, 1H, NH), 7.58 (dd, 1H, J1 = 1.2 Hz,
J2 = 8.4 Hz, PhH), 7.36 (d, 2H, J = 9 Hz, Ph’H), 7.33 (d, 1H,
J2 = 8.4 Hz, PhH), 7.30 (d, 2H, J = 9 Hz, Ph’H), 3.77 (s, 2H, S–CH2).
m
mC–S). MS (ESI): m/z 576.1 (M+1), 578.1
4.1.2.6. N-(4-Acetyl-2-bromophenyl)-2-(4-(2,4-dibromophe-
nyl)-1,2,3-thiadiazol-5-ylthio)acetamide (7f). White needle
crystals, yield: 74.1%. Mp: 175–177 °C (decompose). 1H NMR
(CDCl3, ppm) d: 8.74 (s, 1H, NH), 8.44 (d, 1H, J = 8.4 Hz, Ph’H),
8.17 (s, 1H, Ph’H), 7.92 (d,1H, J1 = 1.2 Hz, PhH), 7.90 (d, 1H,
J = 8.4 Hz, Ph’H), 7.54 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, PhH), 7.31
(d, 1H, J2 = 8.4 Hz, PhH), 3.84 (s, 2H, S–CH2), 2.59 (s, 3H, CH3). IR
IR (KBr, cmÀ1): 3307 (
1400, 1217, 823, 740 (
m
NH), 1672 (
mC@O), 1541, 1492 (mN@N),
m
C–S). MS (ESI): m/z 518.1 (M+1), 520.1
(M+3). C16H10Br2ClN3OS2 (516.83).
4.1.2.13. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-(2,3-dimethylphenyl)acetamide (7m). White needle crystals,
yield: 80.7%. Mp: 120–122 °C. 1H NMR (CDCl3, ppm) d: 7.93 (s,
1H, J1 = 1.2 Hz, PhH), 7.86 (s, 1H, NH), 7.56 (dd, 1H, J1 = 1.2 Hz,
J2 = 8.4 Hz, PhH), 7.38 (d, 1H, J = 7.8 Hz, Ph’H), 7.31 (d, 1H,
J2 = 8.4 Hz, PhH), 7.11 (t, 1H, Ph’H), 7.05 (d, 1H, J = 7.8 Hz, Ph’H),
3.84 (s, 2H, S–CH2), 2.29 (s, 3H, CH3), 2.00 (s, 3H, CH3). IR
(KBr, cmÀ1): 3237 (
N@N), 1260, 1229, 736 (m
mNH), 1681(mC@O), 1668 (mC@O), 1527, 1391
(m
C–S). MS (ESI): m/z 604.0 (M+1), 606.0
(M+3), 608.0 (M+5). C18H12Br3N3O2S2 (602.79).
4.1.2.7. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-N-
(2-nitrophenyl)acetamide (7g). Yellow needle crystals, yield:
74.1%. Mp: 156–158 °C. 1H NMR (CDCl3, ppm) d: 11.10 (s, 1H,
NH), 8.71 (d, 1H, J = 8.4 Hz, Ph’H), 8.24 (dd, 1H, J = 1.2 Hz,
J = 8.4 Hz, Ph’H), 7.89 (d, 1H, J1 = 1.2 Hz, PhH), 7.70 (t, 3H, Ph’H),
(KBr, cmÀ1): 3292 (
1542, 1469 ( N@N), 1216, 773, 741 (
(M+1), 514.2 (M+3). C18H15Br2N3OS2 (510.9).
m
NH), 3058, 2961, 2920, 2854, 1680 (
mC@O),
m
m
C–S). MS (ESI): m/z 512.2

5926
P. Zhan et al. / Bioorg. Med. Chem. 17 (2009) 5920–5927
4.1.2.14. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-(2,6-dimethylphenyl)acetamide (7n). White lamella crystals,
yield: 84.5%. Mp: 156–158 °C. 1H NMR (CDCl3, ppm) d: 7.94 (d,
1H, J1 = 1.2 Hz, PhH), 7.60 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, PhH),
7.55 (br s, 1H, NH), 7.33 (d, 1H, J2 = 8.4 Hz, PhH), 7.13 (q, 1H,
Ph’H), 7.06 (d, 1H, J = 7.8 Hz, Ph’H), 3.86 (s, 2H, S–CH2), 2.10 (s,
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 cells27 were cen-
trifuged for 5 min at 1000 rpm and the supernatant was discarded.
The MT-4 cells were resuspended at 6 Â 105 cells/mL, and 50-
lL
6H, CH3). IR (KBr, cmÀ1): 3273 (
1645 ( C@O), 1518, 1473, 1437 ( N@N), 1214, 765, 740 (m
m
NH), 3020, 2962, 2924, 2851,
C–S). MS
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.
m
m
(ESI): m/z 512.2(M+1), 514.1(M+3). C18H15Br2N3OS2 (510.9).
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 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 concentration
(CC50) was defined as the concentration of the test compound that
reduced the absorbance (OD540) of the mock-infected control sam-
ple 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).
4.1.2.15. 2-(4-(2,4-dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-(pyridin-2-yl)acetamide (7o). White needle crystals, yield:
58.7%. Mp: 128–130 °C. 1H NMR (CDCl3, ppm) d: 8.82 (br s, 1H,
NH), 8.27 (d, 1H, J = 4.8 Hz, pyridine-H), 8.15 (d, 1H, J = 8.4 Hz, pyr-
idine-H), 7.91 (d, 1H, J1 = 1.8 Hz, PhH), 7.76 (m,1H, pyridine-H), 7.56
(dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.33 (d, 1H, J2 = 8.4 Hz, PhH),
7.11 (m, 1H, pyridine-H), 3.81 (s, 2H, S–CH2). IR (KBr, cmÀ1): 3195
(mNH), 1686 (mC@O), 1586, 1535, 1438 (mN@N), 1318, 789, 740 (mC–S).
MS (ESI): m/z 585.1(M+1), 587.1(M+3). C15H10Br2N4OS2 (483.87).
4.1.2.16. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-(thiazol-2-yl)acetamide (7p). Yellow cubic crystals, yield:
54.2%. Mp: 217–219 °C. 1H NMR (CDCl3, ppm) d: 12.48 (s, 1H,
NH), 8.15 (d, 1H, J1 = 1.2 Hz, PhH), 7.77 (dd, 1H, J1 = 1.2 Hz,
J2 = 8.4 Hz, PhH), 7.52 (d, 1H, J2 = 8.4 Hz, PhH), 7.51 (d, 1H,
J = 3.6 Hz, thiazole-H), 7.28 (d,1H, J = 3.6 Hz, thiazole-H), 4.19 (s,
Acknowledgements
2H, S–CH2). IR (KBr, cmÀ1): 3436 (
N@N), 1330, 1227, 1176, 1163, 817, 740 (m
m
NH), 1684 (
mC@O), 1585, 1429
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 International
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
C–S). MS (ESI): m/z
491.1(M+1), 493.1(M+3). C13H8Br2N4OS3 (489.82).
4.1.2.17. 2-(4-(2,4-Dibromophenyl)-1,2,3-thiadiazol-5-ylthio)-
N-(5-methylbenzo[d]thiazol-2-yl)acetamide (7q). Yellow solid,
yield: 74.5%. Mp: 125–127 °C. 1H NMR (CDCl3, ppm) d: 12.65
(s,1H, NH), 8.15 (d, 1H, J1 = 1.8 Hz, PhH), 7.78 (s, H, Ph’H), 7.76
(m, 1H, PhH), 7.65 (d, 1H, J = 8.4 Hz, Ph’H), 7.52 (d, 1H,
J2 = 8.4 Hz, PhH), 7.26 (d, 1H, J = 8.4 Hz, Ph’H), 4.24 (s, 2H, S–
CH2), 2.50 (s, 3H, CH3). IR (KBr, cmÀ1): 3435 (
2854, 1655 ( C@O), 1608, 1550, 1435 ( N@N), 815, 738 (m
m
NH), 2991, 2920,
C–S). MS
References and notes
m
m
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HIV-1(IIIB)25 or HIV-2 (ROD)26 stock (50
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lL) at 100–300 CCID50

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