Arylazolyl(azinyl)thioacetanilide. Part 9#: Synthesis and biological investigation of thiazolylthioacetamides derivatives as a novel class of potential antiviral agents

Article abstract of DOI:10.1007/s12272-012-0604-y

In continuation of our endeavor to develop new, potent, selective and less toxic antiviral agents, a novel series of 2-(2-amino/chloro-4-(2,4- dibromophenyl) thiazol-5-ylthio)acetamide derivatives was synthesized via an expeditious route and evaluated for their anti-HIV activities against wild-type virus and clinically relevant mutant strains, and for their anti-influenza virus activities against influenza A (H1N1 and H3N2) and influenza B in cellular assays. The selected active compounds were also assayed for their enzymic inhibitory activities. The results showed that some 2-chloro substituted thiazolylthioacetamide derivatives possessed potent activity against wild type HIV-1 and several key mutant strains (E138K, K103N, L100I) of HIV-1 in MT-4 cells with EC₅₀ values in micromolar range. Two 2-amino substituted thiazole derivatives 8a7 and 8a8 displayed significant potency against influenza A/H1N1 in MDCK cells with EC₅₀ values much lower than that of oseltamivir carboxylate, ribavirin, amantadine and rimantadine. Though the mechanism of actions is still unclear, these novel thiazolylthioacetamides might serve as original leads for further pharmacological investigations as potential therapeutic agents against HIV-1 or influenza virus.

Full text of DOI:10.1007/s12272-012-0604-y

Arch Pharm Res Vol 35, No 6, 975-986, 2012
DOI 10.1007/s12272-012-0604-y
Arylazolyl(azinyl)thioacetanilide. Part 9#: Synthesis and Biological
Investigation of Thiazolylthioacetamides Derivatives as a Novel
Class of Potential Antiviral Agents
Peng Zhan1, Liu Wang1, Hong Liu1, Xuwang Chen1, Xiao Li1, Xin Jiang1, Qiangqiang Zhang1, Xinyong Liu1,
Christophe Pannecouque2, Lieve Naesens2, Erik De Clercq2, Ailin Liu3, and Guanhua Du3
1Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (Ministry of Education),
2
School of Pharmaceutical Sciences, Shandong University, Ji’nan, ShanDong, China, Rega Institute for Medical Research,
3
K.U. Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium, and Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, China
(Received October 30, 2011/Revised December 6, 2011/Accepted December 22, 2011)
In continuation of our endeavor to develop new, potent, selective and less toxic antiviral
agents, a novel series of 2-(2-amino/chloro-4-(2,4-dibromophenyl) thiazol-5-ylthio)acetamide
derivatives was synthesized via an expeditious route and evaluated for their anti-HIV activi-
ties against wild-type virus and clinically relevant mutant strains, and for their anti-influenza
virus activities against influenza A (H1N1 and H3N2) and influenza B in cellular assays. The
selected active compounds were also assayed for their enzymic inhibitory activities. The
results showed that some 2-chloro substituted thiazolylthioacetamide derivatives possessed
potent activity against wild type HIV-1 and several key mutant strains (E138K, K103N,
L100I) of HIV-1 in MT-4 cells with EC50 values in micromolar range. Two 2-amino substituted
thiazole derivatives 8a7 and 8a8 displayed significant potency against influenza A/H1N1 in
MDCK cells with EC50 values much lower than that of oseltamivir carboxylate, ribavirin,
amantadine and rimantadine. Though the mechanism of actions is still unclear, these novel
thiazolylthioacetamides might serve as original leads for further pharmacological investiga-
tions as potential therapeutic agents against HIV-1 or influenza virus.
Key words: HIV, Influenza virus, NNRTIs, Thiazolylthioacetamides, Synthesis, Bioactivity, SAR
membered aromatic rings containing oxygen and sulfur,
with the exclusion of their benzannulated derivatives,
INTRODUCTION
Currently, there has been considerable interest in are key building blocks used in novel antiviral drug
the discovery of original molecules with broad-spec- development (Spector et al., 1998; Liuzzi et al., 2004;
trum antiviral activity and favorable pharmacokinetic Ghaemmaghami et al., 2010; Xia et al., 2010; Gallardo-
profiles, to be used as an alternative to the approved Godoy et al., 2011; Zhan et al., 2011).
antiviral drugs, should they fail as therapeutics.
Heterocyclic compounds continue to play an increas-
Recently, triazole/tetrazole thioacetanilides derivatives
L1 and L2, Fig. 1) were reported as potent and bioav-
(
ingly important role in lead discovery and biological ailable HIV-1 reverse transcriptase (RT) inhibitors,
activities in the antiviral drug research field (Zhan et
al., 2008). Among all the aromatic heterocycles, five-
#Parts 1-8 reported in our previous articles.
Correspondence to: Xinyong Liu, Department of Medicinal
Chemistry, School of Pharmaceutical Sciences, Shandong Uni-
versity, Ji’nan, ShanDong, China
Tel: 86-531-88380270, Fax: 86-531-88382731
E-mail: xinyongl@sdu.edu.cn
Fig. 1. Triazole/tetrazole thioacetanilide based NNRTIs.
975

976
P. Zhan et al.
Silica Gel 60 (230-400 mesh). 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.
General procedure for the synthesis of 2-(2-
Fig. 2. 1,2,3-thiadiazole thioacetanilides (TTAs) based
NNRTIs.
amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
N-
arylacetamide (8a)
To a vigorously stirred mixture of the commercially
with low nanomolar intrinsic activity in cell assay and available 1,3-dibromobenzene ( , 23.6 g, 0.1 mol) and
1
significant activity against wild-type and mutant strains anhydrous aluminum chloride (14.6 g, 0.11 mol) at
of HIV-1 RT (De La Rosa et al., 2006; Muraglia et al., room temperature was added chloroacetyl chloride (13
2006; Wang et al., 2006; Gagnon et al., 2007; Zhang g, 0.1 mol) dropwise. The reaction mixture was stirred
et al., 2007). Consequently, our efforts have been fo- at 60-65oC for 10 h (monitored by TLC). After cooling,
cused on the development of arylazolylthioacetanilide the mixture was then diluted with CH2Cl2 (40 mL).
derivatives as potent anti-HIV-1 agents (Zhan et al., The separated organic phase was washed successively
2008, 2009, 2010, 2011). Especially, 1,2,3-thiadiazole with water, aqueous saturated NaHCO3 and brine.
thioacetanilides (TTAs) (ZP7, Fig. 2) exhibited signi- The organic layer was dried (Na2SO4), filtered and
ficant anti-HIV-1 activities. Inspired by these promis- concentrated under reduced pressure to afford crude
ing results and in continuation of our endeavor to product 2-chloro-1-(2,4-dibromophenyl)ethanone (IX-
obtain more potent compounds and to establish further
2) as light yellow oil (26.7 g, yield: 85.4%), which was
SAR on arylazolylthioacetanilide-based anti-HIV agents used in the next step without any further purification
(Zhan et al., 2008, 2009, 2011), we synthesized of (Zhang et al., 2007).
structurally related novel thiazolylthioacetamides and
2-chloro-1-(2,4-dibromophenyl)ethanone (2, 15.6 g,
evaluated for their in vitro anti-HIV activities. In 0.05 mol) was added to a mixture of methyl ethyl 2-
addition, according to the previous literature report, mercaptoacetate (6.5 mL, 0.06 mol) and Na2CO3 (6.5 g,
substituted thiazole (especially for 2-aminothiazole) 0.06 mol) in 150 mL EtOH. The reaction mixture was
derivatives exhibited broad-spectrum antiviral activities stirred at room temperature for 12 h. The solvent was
(Spector et al., 1998; Liuzzi et al., 2004; Ghaemmaghami evaporated under reduced pressure. The residue was
et al., 2010; Gallardo-Godoy et al., 2011). Therefore, then diluted with CH2Cl2 (100 mL), washed successively
these newly prepared thiazolylthioacetamide derivatives with aqueous 0.1 M HCl solution, aqueous saturated
were also evaluated for anti-influenza virus activities NaHCO3, water and brine. The organic layer was dried
in Madin-Darby canine kidney (MDCK) cells infected (Na2SO4), filtered and concentrated under reduced pres-
with different strains of human influenza virus (influ- sure to afford crude product
enza A/H1N1, A/H3N2 and B viruses). Herein, we report et al., 2007).
3 as light yellow oil (Zhang
the details of our investigation results.
MATERIALS AND METHODS
Chemistry
To a soln. of 3 (19.8 g, 0.05 mol) in glacial AcOH (100
mL) was added a soln. of Br2 (8.0 g, 0.05 mol) in glacial
AcOH (30 mL) at r.t. and over a period of 30 min. The
obtained mixture was further stirred at r.t. for 8 h
(monitored by TLC), and poured into ice water (100 g),
All melting points were determined on a micromelting then extracted with CH2Cl2 (3 × 50 mL). The obtained
point apparatus and are uncorrected. Infrared spectra organic phase was washed successively with water
(IR) were recorded with a Nexus 470FT-IR Spectro- and 5% NaHCO3 and dried to give compound
meter. H-NMR spectra were obtained on a Brucker yellow oil (Zhang et al., 2007).
4 as a
1
Avance-600 NMR-spectrometer in the indicated solvents.
Thiourea (11.42 g, 0.15 mmol) was dissolved in EtOH
-bromoketone
Chemical shifts are expressed in units and TMS as (150 mL) followed by addition of
δ
α
4
internal reference. Mass spectra were taken on a LC (0.05 mmol). The reaction mixture was stirred at room
Autosampler Device: Standard G1313A instrument. temperature and evaluated with TLC (petroleum/
TLC was performed on Silica Gel GF254 for TLC EtOAc = 2:1). The reaction was stopped after about 10
(Merck) and spots were visualized by iodine vapors or h when no starting material spot was detected. The
by irradiation with UV light (254 nm). Flash column reaction mixture was evaporated off, followed by
chromatography was performed on column packed with addition of water (150 mL) and extraction with ethyl

Thiazolylthioacetamides as Potential Antiviral Agents
977
acetate (3 × 50 mL). The organic layer was washed 1H-NMR (CDCl3, ppm):
δ
8.25 (t, 1H, Ph’H), 8.19 (brs,
with saturated aqueous NaHCO3 solution, water, and 1H, NH), 7.66 (d, 1H, J1 = 1.8 Hz, PhH), 7.28 (dd, 1H,
brine, dried over Na2SO4, evaporated to give crude pro- J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.15-7.08 (m, 4H, 1 ×
duct as yellow solid, which was recrystallized from PhH & 3 × Ph’H), 5.27 (s, 2H, NH2), 3.45 (s, 2H, S-
MeOH to provide the pure product ethyl 2-(2-amino-4- CH2). MS (ESI): m/z 516.1 (M+1), 518.1 (M+3), 520.1
(2,4-dibromophenyl)thiazol-5-ylthio)acetate (
lamellar crystals (10 g, yield: 44.3%) (South, 1991).
The solution of the ester (2.98 g, 6.6 mmol), 1 N 2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
NaOH (20 mL), and DMSO was stirred in room tem- -(2-bromophenyl)acetamide (8a3)
perature for 4 h. Ethyl acetate (30 mL) and water (150 White cubic crystals, yield: 55.7%. mp: 176-178oC. 1H-
mL) was added. The water layer was separated and NMR (CDCl3, ppm): 8.59 (s, 1H, NH), 8.30 (d, 1H, J =
5) as yellow (M+5). C17H12Br2FN3OS2 (514.88).
5
N
δ
neutralized with 10% HCl to pH 2. The solid was formed 7.8 Hz, Ph’H), 7.66 (d, 1H, J1 = 1.8 Hz, PhH), 7.55 (d,
and filtered, washed with water and then dried in air 1H, J2 = 8.4 Hz, PhH), 7.34-7.32 (m, 1H, Ph’H), 7.26-
to afford crude product 2-(2-amino-4-(2,4-dibromophenyl) 7.24 (m, 1H, Ph’H), 7.11-7.10 (m, 1H, PhH), 7.03-7.00
thiazol-5-ylthio)acetic acid (
cation.
Phosphorous pentachloride (0.15 g, 0.6 mmol) was 1526, 1436 (
6
) without further purifi- (t, 1H, Ph’H), 5.27 (s, 2H, NH2), 3.48 (s, 2H, S-CH2). IR
(KBr, cm−1): 3308, 3123 (
NH), 1680 ( C=O), 1653, 1592,
N=N), 1307, 1225, 742 ( C-S). MS (ESI): m/z
added dropwise under vigorous stirring to a solution 576.0 (M+1), 578.0 (M+3), 580.0 (M+5). C17H12Br3N3OS2
of acetic acid ( ) (0.2 g, 0.5 mmol) in ether (20 mL) at (574.80).
υ
υ
υ
υ
6
room temperature. The mixture was stirred for 5 h at
room temperature. After evaporation of the solvent, 2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
yellow oil
the preparation of compound 8a
A solution of acid chloride
(0.5 mmol) in dry dichlor- 1H-NMR (CDCl3, ppm):
methane (10 mL) was added dropwise under vigorous = 7.2 Hz, Ph’H), 7.65 (d, 1H,
stirring to a solution of the appropriate amine (0.5 7.15 (m, 4H, 2 × PhH & 2 × Ph’H), 5.24 (s, 2H, NH2),
mmol) and triethylamine (2 mmol) in dichlormethane 3.52 (s, 2H, S-CH2). IR (KBr, cm−1): 3332, 3113 (
NH),
(10 mL) in ice bath. Then, the mixture was stirred at 1677 ( C=O), 1653, 1583, 1519, 1450 ( N=N), 1383, 1372,
room temperature for 10 h. The organic layer was 1308, 1101, 736 ( C-S). MS (ESI): m/z 566.1 (M+1), 568.1
7
was obtained, which was used directly for
N
-(2,4-dichlorophenyl)acetamide (8a4)
White needle crystals, yield: 54.8%. mp: 183-185oC.
8.39 (s, 1H, NH), 8.23 (d, 1H,
= 1.8 Hz, PhH), 7.44-
.
7
δ
J
J
υ
υ
υ
υ
washed with 10% HCl to remove the excess of amine, (M+3), 570.1 (M+5). C17H11Br2Cl2N3OS2 (564.81).
then with saturated aqueous NaHCO3 solution, water,
and brine, dried over Na2SO4, and evaporated to give 2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
the target compounds 2-(2-amino-4-(2,4-dibromophenyl)
thiazol-5-ylthio)-
-arylacetamide (8a) as oil or solid. White needle crystals, yield: 70.4%. mp: 103-106oC.
Purification was performed with column chromatog- 1H-NMR (CDCl3, ppm):
8.68 (d, 1H, J1 = 2.4 Hz, J2
raphy (ethyl acetate/petroleum ether 60-90; 1:2). 7.8 Hz, pyridine-H), 8.60 (brs, 1H, NH), 8.16 (dd, 1H,
= 1.8, 4.8 Hz, pyridine-H), 7.68 (d, 1H, J1 = 1.8 Hz,
2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)- PhH), 7.35 (dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH),
-(2-chlorophenyl)acetamide (8a1) 7.29-7.27 (m, 1H, PhH), 7.14 (d, 1H, = 7.8 Hz, pyridine-
White needle crystals, yield: 42.6%. mp: 192-194oC. H), 5.24 (s, 2H, NH2), 3.48 (s, 2H, S-CH2). IR (KBr,
1H-NMR (CDCl3, ppm): 9.47 (s, 1H, NH), 7.88 (d, 1H, cm−1): 3313, 3173 (
NH), 1685 ( C=O), 1615, 1583, 1510,
= 1.8 Hz, PhH), 7.80 (d, 1H, = 7.8 Hz, Ph’H), 7.52 1454 ( N=N), 1391, 1300, 1078, 737 ( C-S). MS (ESI): m/z
(dd, 1H, J1 = 8.4 Hz, J2 = 1.8 Hz, PhH), 7.37 (d, 1H, 533.2 (M+1), 535.11 (M+3). C16H11Br2ClN4OS2 (531.84).
= 8.4 Hz, PhH), 7.34-7.32 (m, 2H, Ph’H), 7.17 (t, 1H,
Ph’H), 5.26 (s, 2H, NH2), 3.52 (s, 2H, S-CH2). IR (KBr, 2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
cm−1): 3308, 3121 (
NH), 1683 ( C=O), 1654, 1593, 1526, -(4-methyl-2-nitrophenyl)acetamide (8a6)
1440 ( N=N), 1310, 1248, 741 (
C-S). MS (ESI): m/z 532.2 Yellow solid, yield: 61.9%. mp: 218-219oC. H-NMR
(M+1), 534.1 (M+3), 536.2 (M+5). C17H12Br2ClN3OS2 (CDCl3, ppm): 10.83 (s, 1H, NH), 8.54 (d, 1H, = 8.4
(530.85). Hz, Ph’H), 8.04 (d, 1H, J1 = 1.8 Hz, PhH), 7.59 (d, 1H,
= 1.8 Hz, Ph’H), 7.47 (dd, 1H, J1 = 1.8 Hz, J2 = 8.4
2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)- Hz, PhH), 7.25 (d, 1H, = 8.4 Hz, PhH), 7.14 (d, 1H,
-(2-fluorophenyl)acetamide (8a2) = 8.4 Hz, Ph’H), 5.78 (s, 2H, NH2), 3.49 (s, 2H, S-CH2),
White needle crystals, yield: 39.4%. mp: 173-175oC. 2.43 (s, 3H, CH3). IR (KBr, cm−1): 3300, 3088 (
NH), 1681
N-(2-chloropyridin-3-yl)acetamide (8a5)
N
δ
=
J
N
J
δ
υ
υ
J
J
υ
υ
J
υ
υ
N
1
υ
υ
δ
J
J
J
N
J
υ

978
P. Zhan et al.
(
υ
C=O), 1653, 1583, 1516 (
υ
as NO2), 1450 (
υ
N=N), 1340 (υs 1H, J1 = 8.4 Hz, J2 = 1.8 Hz, PhH), 7.50 (d, 1H,
= 7.8 Hz, Ph’H), 7.35 (t, 1H,
Ph’H), 7.22 (t, 1H, Ph’H), 3.78 (s, 2H, S-CH2). IR (KBr,
cm−1): 3312 (
NH2), 1668 ( C=O), 1592, 1532, 1456, 1442
N=N), 1304, 1055, 745 ( C-S). MS (ESI): m/z 551.1
(M+1), 553.1 (M+3), 555.0 (M+5). C17H10Br2Cl2N2OS2
Yellow solid, yield: 58.7%. mp: 186-189oC. H-NMR (549.80).
(CDCl3, ppm): 10.75 (s, 1H, NH), 8.74 (d, 1H, = 8.4
Hz, Ph’H), 8.24 (dd, 1H, J1 = 1.2 Hz, J2 = 8.4 Hz, 2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
Ph’H), 8.11 (d, 1H, J1 = 1.8 Hz, PhH), 7.68 (dt, 1H, J1 -(2-fluorophenyl)acetamide (8b2)
= 1.2 Hz, J2 = 8.4 Hz, Ph’H), 7.40 (dd, 1H, J1 = 1.8 Hz, White needle crystals, yield: 48.6%. mp: 106-109oC. 1H-
J2 = 8.4 Hz, PhH), 7.26 (d, 1H, = 8.4 Hz, PhH), 7.24 NMR (DMSO-d6, ppm): 9.95 (s, 1H, NH), 8.01 (d, 1H,
(m, 1H, Ph’H), 5.75 (s, 2H, NH2), 3.51 (s, 2H, S-CH2). J1 = 1.2 Hz, Ph’H), 7.90-7.87 (m, 1H, PhH), 7.58 (dd, 1H,
MS (ESI): m/z 543.1 (M+1), 545.1 (M+3), 547.1 (M+5). J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.39 (d, 1H, = 8.4 Hz,
C17H12Br2N4O3S2 (541.87). Ph’H), 7.29-7.25 (m, 1H, PhH), 7.20-7.18 (m, 2H, Ph’H),
3.76 (s, 2H, S-CH2). MS (ESI): m/z 535.0 (M+1), 537.0
2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)- (M+3), 539.1 (M+5). C17H10Br2ClFN2OS2 (533.83).
-tolylacetamide (8a8)
White powder, yield: 62.8%. mp: 198-200oC. H-NMR
(CDCl3, ppm): 7.95 (brs, 1H, NH), 7.88 (d, 1H, J1
7.8 Hz, PhH), 7.68 (d, 1H,
= 1.8 Hz, Ph’H), 7.26-7.17 White solid, yield: 62.3%. mp: 160-162oC. H-NMR
(m, 3H, 2 × PhH & 1 × Ph’H), 7.12-7.07 (m, 2H, Ph’H), (DMSO-d6, ppm): 9.66 (s, 1H, NH), 8.02 (d, 1H, = 1.8
5.72 (s, 2H, NH2), 3.86 (s, 2H, S-CH2), 2.17 (s, 3H, Hz, Ph’H), 7.66 (d, 1H, = 7.8 Hz, PhH), 7.62 (dd, 1H,
= 1.8, 7.8 Hz, PhH), 7.60 (d, 1H, = 7.2 Hz, Ph’H),
J = 8.4
NO2), 1276, 1149, 831 ( C-S). MS (ESI): m/z 557.0 (M+1), Hz, PhH), 7.39 (d, 1H,
υ
J
559.1 (M+3), 561.1 (M+5). C18H14Br2N4O3S2 (555.89).
υ
υ
υ
2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
N
(
υ
-(2-nitrophenyl)acetamide (8a7)
1
δ
J
N
J
δ
J
N-o
1
N
-(2-bromophenyl)-2-(2-chloro-4-(2,4-dibromophen-
δ
=
yl)thiazol-5-ylthio)acetamide (8b3)
1
J
δ
J
J
CH3). MS (ESI): m/z 512.2 (M+1), 514.2 (M+3), 516.1
(M+5). C18H15Br2N3OS2 (510.90).
J
J
7.42-7.38 (m, 2H, 1 × Ph’H & 1 × PhH), 7.17-7.15 (t, 1H,
Ph’H), 3.77 (s, 2H, S-CH2). MS (ESI): m/z 595.4 (M+1),
597.1 (M+3), 599.2 (M+5). C17H10Br3ClN2OS2 (593.75).
General procedure for the synthesis of 2-(2-
chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
N-arylacetamide(8b)
To a mixture of 2-(2-amino-4-(2,4-dibromophenyl)thia-
N-(2,4-dichlorophenyl)acetamide (8b4)
zol-5-ylthio)-
cupric chloride (1.5 mmol), and acetonitrile (15 mL) 1H-NMR (DMSO-d6, ppm):
was added dropwise -butyl nitrite (1.5 mmol) over 15 1H, = 8.4 Hz, Ph’H), 7.71 (d, 1H,
min, under nitrogen atomsphere at room temperature. 7.41 (d, 1H, = 1.8 Hz, Ph’H), 7.33 (dd, 1H,
During the addition process, the solution became slight- 8.4 Hz, PhH), 7.27 (dd, 1H, = 1.8, 8.4 Hz, Ph’H), 7.13
ly exothermic and nitrogen gas was evolved. After the (d, 1H, = 8.4 Hz, PhH), 3.55 (s, 2H, S-CH2). MS
N
-arylacetamide (8a) (1 mmol), anhydrous White needle crystals, yield: 69.4%. mp: 173-174oC.
δ
8.40 (s, 1H, NH), 8.23 (d,
= 2.4 Hz, PhH),
= 2.4,
t
J
J
J
J
J
J
addition was complete, the mixture was stirred for 2 (ESI): m/z 585.3 (M+1), 587.1 (M+3), 589.1 (M+5).
h, then added 15 mL of water and 15 mL of chloroform, C17H9Br2Cl3N2OS2 (583.76).
and then acidified with 5 N hydrochloric acid. The aque-
ous layer was separated and extracted with additional 2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
chloroform (2 × 15 mL). The collected organic solvent
was dried over Na2SO4, and evaporated under reduced White solid, yield: 75.8%. mp: 168-170oC. H-NMR
pressure. The residue was chromatographed on silica (DMSO-d6, ppm): 8.64 (d, 1H, = 7.8 Hz, pyridine-
gel using ethyl acetate:petroleum ether (1:4). Pure frac- H), 8.46 (brs, 1H, NH), 8.17 (dd, 1H, = 1.8, 4.8 Hz,
tions were collected and concentrated, giving the desired pyridine-H), 7.74 (d, 1H, = 1.8 Hz, PhH), 7.40 (dd,
compound 2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5- 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.31-7.26 (m, 1H,
ylthio)- -phenylacetamide (8b) (South, 1991). PhH), 7.17 (d, 1H, = 7.8 Hz, pyridine-H), 3.57 (s,
2H, S-CH2). MS (ESI): m/z 552.1 (M+1), 554.1 (M+3).
2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)- C16H9Br2Cl2N3OS2 (550.79).
-(2-chlorophenyl)acetamide (8b1)
White powder, yield: 55.8%. mp: 129-130oC. H-NMR 2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
(DMSO-d6, ppm): 9.71 (s, 1H, NH), 8.02 (d, 1H, -(4-methyl-2-nitrophenyl)acetamide (8b6)
1.8 Hz, PhH), 7.70 (d, 1H,
= 7.8 Hz, Ph’H), 7.60 (dd, Yellow solid, yield: 70.8%. mp: 187-189oC. H-NMR
N-(2-chloropyridin-3-yl)acetamide (8b5)
1
δ
J
J
J
N
J
N
1
δ
J
=
N
1
J

Thiazolylthioacetamides as Potential Antiviral Agents
979
(DMSO-d6, ppm):
1.8 Hz, Ph’H), 7.83 (d, 1H,
1H, J1 = 1.8 Hz, J2 = 8.4 Hz, Ph’H), 7.58-7.54 (m, 2H, of the microtiter tray. Mock-infected cells were used to
PhH), 7.39 (d, 1H, = 8.4 Hz, Ph’H), 3.74 (s, 2H, S- evaluate the effect of test compound on uninfected cells
CH2), 2.38 (s, 3H, CH3). IR (KBr, cm−1): 3281 (
NH2), in order to assess the cytotoxicity of the test compound.
3087 ( NH), 2921, 1691 ( C=O), 1581, 1516 ( as NO2), Exponentially growing MT-4 cells (Miyoshi et al., 1982)
1457 ( N=N), 1341 ( s NO2), 1276, 1056, 837 ( C-S). MS were centrifuged for 5 min at 1000 rpm and the super-
(ESI): m/z 576.2 (M+1), 578.3 (M+3), 580.2 (M+5). natant was discarded. The MT-4 cells were resuspend-
C18H12Br2ClN3O3S2 (574.84). L volumes were trans-
ed at 6 × 105 cells/mL, and 50-
ferred to the microtiter tray wells. Five days after
2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)- infection, the viability of mock- and HIV-infected cells
-(2-nitrophenyl)acetamide (8b7) was examined spectrophotometrically by the MTT
Yellow solid, yield: 56.2%. mp: 137-138oC. H-NMR assay.
(DMSO-d6, ppm): 10.51 (s, 1H, NH), 8.01 (d, 1H, The MTT assay is based on the reduction of yellow
1.8 Hz, Ph’H), 7.99 (d, 1H, = 1.8 Hz, PhH), 7.74 (t, colored 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
1H, Ph’H), 7.70-7.60 (d, 1H, = 8.4 Hz, PhH), 7.60 zolium bromide (MTT) (Acros Organics) by mitochon-
δ
10.42 (s, 1H, NH), 7.99 (d, 1H,
J
=
CCID50 (cell culture infectious dose) or culture medium
J
= 1.8 Hz, PhH), 7.59 (dd, was added to either the infected or mock-infected wells
J
υ
υ
υ
υ
υ
υ
υ
µ
N
1
δ
J =
J
J
(dd, 1H, J1 = 1.8 Hz, J2 = 8.4 Hz, PhH), 7.42-7.40 (m, drial dehydrogenase of metabolically active cells to a
2H, Ph’H), 3.77 (s, 2H, S-CH2). IR (KBr, cm−1): 3341 blue-purple formazan that can be measured spectro-
(
υ
NH2), 3120 (
1489 ( as NO2), 1452 (
749 ( C-S). MS (ESI): m/z 562.2 (M+1), 564.2 (M+3), Ascent Reader, Labsystems), at two wavelengths (540
566.3 (M+5). C17H10Br2ClN3O3S2 (560.82).
υ
NH), 2924, 1703, 1696 (
υC=O), 1605, 1580, photometrically. The absorbances were read in an eight-
υ
υ
υ
N=N), 1334 ( s NO2), 1261, 1062, channel computer-controlled photometer (Multiscan
υ
and 690 nm). All data were calculated using the median
OD (optical density) value of tree wells. The 50% cyto-
2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)- toxic concentration (CC50) was defined as the concen-
-o-tolylacetamide (8b8) tration of the test compound that reduced the absor-
White solid, yield: 47.6%. mp: 174-176oC. H-NMR bance (OD540) of the mock-infected control sample by
(DMSO-d6, ppm): 9.48 (brs, 1H, NH), 8.04 (d, 1H, 50%. The concentration achieving 50% protection from
= 2.4 Hz, PhH), 7.62 (dd, 1H, = 1.8, 8.4Hz, Ph’H), the cytopathic effect of the virus in infected cells was
7.42 (d, 1H, = 8.4 Hz, PhH), 7.37 (d, 1H, = 8.4 Hz, defined as the 50% effective concentration (EC50).
N
1
δ
J
J
J
J
PhH), 7.22-7.17 (m, 2H, Ph’H), 7.10 (t, 1H, Ph’H), 3.74
(s, 2H, S-CH2), 2.12 (s, 3H, CH3). IR (KBr, cm−1): 3255
HIV-1 RT inhibition assay
(
υ
NH2), 1654 (
υ
C=O), 1589, 1542, 1457, 1392 (
C-S). MS (ESI): m/z 431.1(M+1), 533.1 tides linked to microtiter plate with colorimetric detec-
υ
N=N), 1326,
Inhibition of HIV-1 RT was developed using nucleo-
1006, 757 (
(M+3), 535.1 (M+5). C18H13Br2ClN2OS2 (529.85).
υ
tion of incorporated biotin-dUTP into homopolymer
template primers (Suzuki et al., 1993; Wu et al., 2007).
The incorporated quantities of the biotin-dUTP into
the enzyme represented the activity of HIV-1 RT. IC50
Biological activity
Anti-HIV activity assays
Evaluation of the antiviral activity of the compounds values corresponded to the concentration of the substi-
against HIV-1 strain IIIB and HIV-2 strain (ROD) in tuted s-triazole derivatives required to inhibit biotin-
MT-4 cells was performed using the MTT assay as pre- dUTP incorporation into the HIV-1 RT by 50%.
viously described (Pauwels et al., 1988; Pannecouque
et al., 2008). Stock solutions (10 × final concentration)
of test compounds were added in 25- L volumes to and B virus-infected MDCK cells
two series of triplicate wells so as to allow simultane- Madin-Darby canine kidney (MDCK) cells (Matrosovich
Antiviral activities in influenza A/H1N1, A/H3N2,
µ
ous evaluation of their effects on mock- and HIV-in- et al., 2003) were grown in Dulbecco’s modified Eagle
fected cells at the beginning of each experiment. Serial medium (DMEM) supplemented with 10% fetal calf
fivefold dilutions of test compounds were made directly serum (FCS) (Integro), 1 mM sodium pyruvate, and
in flat-bottomed 96-well microtiter trays using a 0.075% sodium bicarbonate. During virus experiments,
Biomek 3000 robot (Beckman instruments). Untreated the MDCK cells were maintained in Ultra MDCK
control HIV- and mock-infected cell samples were in- medium (Lonza), supplemented with 0.0225% sodium
cluded for each sample.
HIV-1(IIIB) (Popovic et al., 1984) or HIV-2 (ROD) phenylalanylchloromethylketon (TPCK)-treated trypsin
(Barré-Sinoussi et al., 1983) stock (50
L) at 100-300 (Sigma). The human influenza virus strains A/PR/8/34
bicarbonate, 2 mM L-glutamine, and 2 µg/mL tosyl-
µ

980
P. Zhan et al.
(A/H1N1) and B/HK/5/72 were purchased from the Fluostar Galaxy (excitation, 360 nm; emission, 450
American Type Culture Collection (ATCC). The strain nm), and substrate blanks were subtracted from the
A/HK/7/87 (A/H3N2) were obtained from J. Neyts sample readings. The IC50 was calculated by plotting
(Katholieke Universiteit Leuven). Virus stocks of these percent inhibition versus the inhibitor concentration,
laboratory-adapted strains were prepared by passag- and determination of each point was performed in
ing them in 10-day-old embryonated chicken eggs.
The following control compounds were included: riba-
virin (Virazole), obtained from ICN Pharmaceuticals;
amantadine (Sigma); rimantadine (Sigma); and oselta-
mivir carboxylate (GS-4071) (a kind gift from T. Cihlar,
Gilead Sciences). The test compounds were evaluated
duplicate.
RESULTS
Chemistry
The synthetic route to the target compounds 2-(2-
for anti-influenza virus activity by a multicycle cyto- amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
pathic effect (CPE) reduction assay. MDCK cells were amides 8a and 2-(2-chloro-4-(2,4-dibromophenyl)thia-
seeded into 96-well plates at 7,500 cells per well 16 h zol-5-ylthio)-
-acetamides 8b is depicted in Scheme 1.
prior to infection and incubated at 35oC. Serial dilu- 2-Chloro-1-(2,4-dibromophenyl)ethanone (
), synthesiz-
tions of the compounds were added to the cells, to- ed by Friedel-Crafts reaction of commercially avail-
gether with the influenza virus (multiplicity of infec- able 1,3-dibromobenzene ( ) with 2-chloroacetyl chlor-
N-acet-
N
2
1
tion, 50% cell culture infective doses [CCID50] per well, ide, were reacted with ethyl 2-mercaptoacetate in
which corresponds to 0.0004 PFU per cell). At 3 days EtOH at ambient temperature for several hours to
postinfection (p.i.), microscopy was performed to deter- obtain the thioether
mine the antiviral activity, expressed as the compound ectly used for the next step.
concentration producing 50% inhibition of virus- ed in good yield, which is an important intermediate
3
which was not purified and dir-
α
-Bromination of provid-
3
4
induced CPE (50% effective concentration [EC50]), as for construction of aminothiazole heterocycle ethyl 2-
well as the cytotoxicity of the compounds, expressed as (2-amino-4-(2,4-dibromo phenyl)thiazol-5-ylthio)acet-
the compound concentration causing minimal changes ate (
in cell morphology (MCC). The data were confirmed was easily converted into the acetic acid derivative
by the formazan-based 3-(4,5-dimethylthiazol-2-yl)-5- by saponification reaction in NaOH-DMSO-H2O solution,
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 - tetra- and was subsequently transformed to acyl chloride
zolium (MTS) cell viability assay (CellTiter 96 AQueous The key intermediate was then converted into various
One Solution Cell Proliferation Assay from Promega), 2-(2-amino-4-(2,4-dibromophenyl)thiazol-5-ylthio)- -acet-
and the spectrophotometric data were used to calculate amide derivatives 8a, by treating with the appropri-
5) by reaction with thiourea (South, 1991). Compound
5
6
H
7.
7
N
the EC50 and 50% cytotoxic concentration (CC50).
ate amine in the presence of triethylamine. Sander-
meyer reaction of 8a by using the reagents of CuCl2/t
BuONO/ MeCN at room temperature gave the 2-(2-
-acetam-
minidase (NA) inhibitory actions has been previously ide derivatives 8b (South, 1991). It should be noted that
described (Liu et al., 2005). The NA enzyme is from an alternative approach from the to 8b via chlorinated
-
Neuraminidase inhibition assay
The methodology of evaluation for in vitro neura- chloro-4-(2,4-dibromophenyl)thiazol-5-ylthio)-
N
5
the influenza virus strain A/H1N1 [strain A/PR/8/34] reaction, hydrolysis and acylation under conditions
(Laver et al., 1984). This spectrofluorometric assay described in Scheme 1 is proved feasible, but not used
(excitation wavelength: 355 nm and emission wave- in the preparation of the title compounds listed in this
length: 460 nm) uses 2'-(4-methylumbelliferyl)-α-D- article. The synthesized compounds were characteriz-
acetylneuraminic acid (MUNANA) as the enzyme sub- ed by physicochemical and spectral means and the IR
strate, which is converted to a fluorescent product upon and NMR spectral data are found in agreement with
cleavage by NA. The intensity of fluorescence can the assigned molecular structures.
reflect the activity of NA sensitively.
In the enzyme reaction system, there were 30
the enzyme in 33 mmol/L MES buffer (pH = 3.5), 10
L of 4 mmol/L CaCl2, 20 L of 20
and 30 L water in a 96-well microplate. The terminal were evaluated for their anti-HIV activity and cyto-
volume was 100 L of toxicity in MT-4 cells using the MTT method. The used
L. After 10 min at 37oC, 150
µL of
Anti-HIV activities evaluation
The novel 2-(2-amino/chloro-4-(2,4-dibromophenyl)
µ
µ
µmol/L MUNANA, thiazol-5-ylthio)-N-acetamide derivatives 8a and 8b
µ
µ
µ
14 mmol/L NaOH in 83% ethanol was added to 0.1 virus strains are HIV-1 strain IIIB and HIV-2 strain
mL of the reaction mixture to terminate the reaction. ROD. Nevirapine (NVP), zidovudine (azidothymidine,
The intensity of the fluorescence was quantitated in AZT), dideoxycitidine (DDC) and dideoxyinosine (DDI)

Thiazolylthioacetamides as Potential Antiviral Agents
981
Scheme 1. Reagents and conditions: (i) ClCOCH2Cl/AlCl3; (ii) HSCH2COOEt, Na2CO3, EtOH; (iii) Br2/AcOH; (iv)
NH2CSNH2, AcONa, EtOH; (v) NaOH, DMSO/H2O; (vi) SOCl2, aniline, THF, rt; (vii) CuCl2, -BuONO, MeCN.
t
were used as the reference drugs. The results were type potency, indicating that the introduction of rela-
summarized in Table I. tively hydrophilic heterocycles in this region could be
The screening results indicated that some 2-chloro a valid strategy to get novel molecules with increased
substituted thiazole derivatives exhibited evident in- antiviral potency. The above findings are partly in
hibition activity against HIV-1, none of the compounds agreement with the results of earlier SAR studies
inhibited the replication of HIV-2 (ROD) in MT-4 cells (Zhan et al., 2009).
at subtoxic concentration. Among the evaluated com-
pounds, a 2-(2-chloro-4-(2,4-dibromophenyl)thiazol-5- fact that all the compounds did not show any activity
ylthio)acetamide derivative 8b7 (EC50 = 2.09 ± 1.14 M) against HIV-2 (ROD) in MT-4 cells, it can be conclud-
emerged as the most active HIV-1 inhibitor. Other ed that this new series of 2-chloro substituted thiazole
three compounds 8b1 8b5 and 8b6 showed EC50 of derivatives (8b) were specific inhibition of HIV-1 repli-
6.59 ± 0.29 M, 2.67 ± 1.16 M and 3.33 ± 0.16 M, cation and belonged to typical non-nucleoside reverse
and resulting in selectivity index (SI) of 3, 9 and 38, transcriptase inhibitor (NNRTIs).
Based on the above SAR analysis, together with the
µ
,
µ
µ
µ
respectively. They inhibited HIV-1 replication in MT-
4 cells more effectively than DDI.
Besides, the results of anti-HIV assay shown in
In addition, it was interesting to note that all the newly
synthesized 2-amino substituted thiazole derivatives
(8a) were not active against HIV-1 or HIV-2 replica-
Table I revealed some important SAR information on tion. These results confirmed the importance of the
the role of different substitutions in the aromatic ring substituents at the 2-position of thiazole in the binding
linked with amide. A clear order of the aromatic amine of the inhibitors to the RT and finding potent HIV re-
moiety for anti-HIV activity was found as the follow- plication inhibitors.
ing trend: 2-chloropyridin-3-yl (8b5) 2-nitrophenyl
Notoriously, antiviral therapy with NNRTIs is usu-
8b7) >4-methyl-2-nitrophenyl (8b6) >2-chlorophenyl ally compromised by the appearance of HIV-1 drug re-
8b1) (black body in Table I). It was suggested that sistance. Improved activity against mutant strains of
(
(
the ortho substitution on the aniline moiety has signi- virus may be beneficial in terms of suppressing the
ficant impact on antiviral potency. In addition, switch- emergence of drug-resistance. Therefore, selected some
ing the aromatic amine moiety from a 2-chlorophenyl active compounds (8b5, 8b6 and 8b7) were also tested
group to a 2-chloropyridin-3-yl homologue, as shown in cell culture for their activity against a panel of
with compound 8b5, gave us a 3-fold increase in wild- NNRTI resistant strains, containing E138K, K103N,

982
P. Zhan et al.
Table I. Anti-HIV activities, cytotoxicities and selectivity indices of 2-(2-amino/chloro-4-(2,4-dibromophenyl)thiazol-5-
ylthio)acetamide derivatives (8a and 8b
)
EC50
HIV-1 IIIB
(µ
M)a
HIV-2 ROD
SIc
CC50
M)b
Compd
R
(
µ
HIV-1 IIIB HIV-2 ROD
8a1
8b1
>23.67
>23.67
>17.45
23.67 1.71
±
<1
3
<1
<1
2-chlorophenyl
2-fluorophenyl
6.59 ± 0.29
17.45 ± 4.77
8a2
8b2
>24.94
>17.07
>24.94
>17.07
24.94 ± 2.53
17.07 ± 3.28
<1
<1
<1
<1
8a3
8b3
>33.18
>8.25
>33.18
>12.50
33.18 ± 5.64
12.50 ± 0.99
<1
2
<1
<1
2-bromophenyl
8a4
8b4
>20.73
>159.13
>20.73
>159.13
20.73 ± 1.85
159.13 ± 11.88
<1
<1
<1
<1
2,4-dichlorophenyl
2-chloropyridin-3-yl
4-methyl-2-nitrophenyl
2-nitrophenyl
8a5
8b5
>45.73
2.67 ± 1.16
>45.73
>23.24
45.73 ± 10.29
23.24 ± 4.46
<1
9
<1
<1
8a6
8b6
>30.81
3.33 ± 0.16
>30.81
>127.40
30.81 ± 6.61
127.40 ± 3.72
<1
38
<1
<1
8a7
8b7
>33.04
2.09 ± 1.14
>33.04
>109.46
33.04 ± 4.80
109.46 ± 33.83
<1
52
<1
<1
8a8
8b8
>23.96
>26.94
>23.96
>26.94
23.96 ± 1.52
26.94 ± 5.01
<1
<1
<1
<1
o-tolyl
NVP
AZT
DDC
DDI
0.19 ± 0.04
0.0076 ± 0.0014 0.0033 ± 0.0006
0.75 ± 0.55
8.86 ± 2.89
>15
>15
>88.26
>94
>80
>11587
>127
>24
>28348
>108
>13
0.88 ± 0.53
16 ± 5.16
>211
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell against HIV-1-induced cytotoxicity, as
b
determined by the MTT method; CC50: concentration required to reduce the viability of mock-infected cells by 50%, as
determined by the MTT method; cSI: selectivity index (CC50/EC50).
L100I, Y181C, Y188L and RES056 (K103N/Y181C), the wild-type virus) and 8b7 (2.23 µM, 1.0-fold shift)
F227L/V106A double mutations. The results are illus- against the L100I mutant virus, which was compar-
trated in Table II with those of AZT, NVP, efavirenz able to that observed with DLV, are of great interest
(EFV), delavirdine (DLV) and etravirine (TMC125) as considering that the K101I is a key mutation asso-
reference standards. As the data demonstrated, the ciated with the current NNRTI resistance. However,
antiviral activities of the tested analogues are sensi- none of these analogues effectively inhibit the replica-
tive to these mutations in the NNRTI binding site, tion of Y181C, Y188L, F227L+V106A and RES056
which suggests that RT is the target of these 2-chloro (K103N+Y181C) mutant virus.
substituted thiazole derivatives. Specifically, we found
that they showed moderate activity against several
key mutant strains (E138K, K103N and L100I) of HIV-
HIV-1 RT inhibition assay
Since arylazolylthioacetanilide derivatives previously
1 with EC50 values in micromolar range and favorable identified to target at the HIV-1 RT, two selected title
fold-resistance values (Table II). In particular, the compounds (8b5 and 8b7) were tested in enzyme
EC50’s for 8b6 (2.58 µM, 0.76-fold shift from that of assays against highly purified recombinant HIV-1 RT

Thiazolylthioacetamides as Potential Antiviral Agents
983
Table II. Anti-HIV mutants activities of selected 2-chloro thiazolthioacetamide derivatives
EC50(
µ
M)a (fold resistance)b
K103N
Compd
8b5
E138K
8.28 (3.14)
L100I
7.68 (3.38)
>23.24 (>8.8)
8b6
8b7
7.89 ± 0.80 (2.34)
7.65 ± 4.42 (3.4)
38.95 ± 9.56 (11.48)
≥13.08 (≥5.9)
2.58 ± 0.02 (0.76)
2.23 ± 0.78 (1.0)
AZT
NVP
EFV
DLV (Mesylate)
TMC125
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)
0.0049 ± 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)
0.00184 ± 0.00021 (0.62)
aEC50: concentration of compound required to achieve 50% protection of MT-4 cell against HIV-1-induced cytotoxicity, as
b
determined by the MTT method; Fold resistance: ratio of EC50 value against drug-resistant strain and EC50 of the wt
HIV-1 IIIB (EC50mutant strains/ EC50wt).
using poly(rC)-oligo(dG) as template primer. Quite un- and the MCC (minimal cytotoxic concentration), repre-
expectedly, the results showed that these compounds senting the compound concentration at which minimal
slightly inhibited HIV-1 RT. All of them proved to be changes in cell morphology were observed (Naesens et
more weak in enzymatic assays than in cellular tests. al., 2009; Vanderlinden et al., 2010). In all experiments,
Accordingly, it may be difficult to explain their anti- oseltamivir carboxylate, ribavirin, amantadine and
viral activity only in terms of RT inhibition. This result rimantadine were used as the reference drugs. The re-
may reflect that the test compounds may interfere sults of this assay are reported in Table IV.
with other viral replication steps, besides the RT.
Interestingly, the results showed that only two
derivatives 8a7 and 8a8 within 2-amino substituted
thiazole series displayed submicromolar potency against
influenza A/H1N1 and high antiviral selectivity index,
Antiviral activities in influenza A/H1N1, A/H3N2
and B virus-infected MDCK cells
Meanwhile, given the fact that the substituted thia- which revealed that the nature of the substituent on
zole derivatives have attracted much attention due to position 2 of the triazole ring markedly influences the
their diverse antiviral activities (Spector et al., 1998; anti-influenza virus activity profiles. However, no
Liuzzi et al., 2004; Ghaemmaghami et al., 2010; Gallardo- activity in 2-amino series (8a) was observed for A/
Godoy et al., 2011). The compounds were evaluated for H3N2 and influenza B viruses. Encouragingly, these
antiviral activity against influenza viruses of the A/ two compounds inhibit the influenza A/H1N1 repli-
H1N1 (strain A/PR/8/34), A/H3N2 (strain A/HK/7/87), cation with EC50 much lower than that of oseltamivir
and B (strain B/HK/5/72) (sub)type, using a cell-culture carboxylate, ribavirin, amantadine and rimantadine.
based assay in Madin-Darby canine kidney (MDCK) However, further investigation of the link between
cells, as previously described (Naesens et al., 2009; inhibition of the influenza A/H1N1 replication and
Vanderlinden et al., 2010). Antiviral activity was ex- cytotoxicity suggests that toxicity to the host cell may
pressed as the 50% antiviral effective concentration be the cause of the inhibition.
(EC50), or compound concentration producing 50% in-
hibition of virus-induced CPE, as determined by micros-
Neuraminidase inhibition assay
copy or MTS assay. Cytotoxicity was expressed as the
In order to identify the target of the two active thia-
50% cytotoxic concentration (CC50), or compound con- zole derivatives. Firstly, the enzymatic neuraminidase
centration causing 50% cytotoxicity in the MTS assay, (NA) inhibitory assay of compounds 8a7 and 8a8 was
performed using the method reported by Guanhua Du
Table III. Inhibitory activity of 2-chloro substituted thia-
zole derivatives against HIV-1 RT
(Liu et al., 2005) and, as the source of NA from the in-
fluenza virus strain A/H1N1 [strain A/PR/8/34] de-
scribed by Laver (Laver et al., 1984). The compound
concentration producing 50% inhibition of NA activity
(IC50) was calculated by plotting the percentage inhib-
ition vs the inhibitor concentration, with each datum
point performed in duplicate. Compounds 8a7 and
8a8 showed almost no activity in the neuraminidase
Compd.
IC50
M)a
8b5
8b7
NVP
4.39
(
µ
>360.94
>354.82
aIC50: 50% of the inhibitory concentration of the 2-chloro
substituted thiazole derivatives required to inhibit biotin
deoxyuridine triphosphate (biotin-dUTP) incorporation into
the HIV-1 RT by 50%.

984
P. Zhan et al.
Table IV. Anti-influenza virus activity and cytotoxicity in MDCK cell cultures
a
Cytotoxicity (µM)
Antiviral EC50
(µM)
Compd
Influenza A/H1N1
Influenza A/H3N2
Influenza B
b
CC50
MCCc
CPE
MTS
CPE
MTS
CPE
MTS
0.73
0.37
0.73
0.37
>183.74
>183.74
>183.74
>183.74
>183.74
>183.74
>183.74
>183.74
42.26
22.05
36.75
8a7
8a8
≥
7.35
38.97
7.79
>100
100
0.97
0.39
1.95
1.75
>194.83
>194.83
>194.83
>194.83
>194.83
>194.83
>194.83
>194.83
50.66
21.43
≥
≥
Oseltamivir carboxylate
Ribavirin
Amantadine
28.5
10.5
134.5
32.5
32.5
9.7
149
60
10.5
2.4
>59
9
3.95
0.05
45
9
>500
>500
27.5
8.4
>500
>500
>100
>100
>500
258
≥
>500
500
Rimantadine
33.6
0.15
aAntiviral activity was expressed as the EC50 value, defined as the compound concentration producing 50% inhibition of
virus replication, as estimated by microscopic scoring of the cytopathic effect (CPE), or by measuring cell viability in the
formazan-based MTS assay. Values are the mean of duplicate experiments; b50% Cytotoxic concentration, as determined
c
by measuring the cell viability with the colorimetric formazan-based MTS assay; Minimum compound concentration
that causes a microscopically detectable alteration of normal cell morphology.
Table V. Evaluation of the thiazole derivatives for inhibi-
almost no activity in the neuraminidase (H1N1) inhib-
ition assay, thus further studies including the elucida-
tion of the mechanism of action on this novel family of
potential antiviral inhibitors are ongoing in our labor-
atories and will be reported in due course.
tory effect on influenza virus neuraminidasea
Inhibition
ratio (%)
b
Compd
Concentration
IC50
8a7
8a8
Zanamivir
73.50
77.93
12 nM
µ
µ
M
M
22.09
17.90
99.00
−
−
ACKNOWLEDGEMENTS
0.29 nM
aInfluenza virus strain used: strain A/PR/8/34; b“
that the IC50 could not be calculated.
−” means
The financial support from the National Natural
Science Foundation of China (NSFC No.81102320, No.
30873133, No.30772629, No.30371686), Key Project of
(H1N1) inhibition assay (Table V). Thus, it is concluded NSFC for International Cooperation (No.30910103908),
that these two compounds might target an additional Research Fund for the Doctoral Program of Higher Edu-
step of the influenza virus multiplication cycle or act cation of China (No. 20110131130005, 20110131120037),
on neuraminidase in a different way other than the Independent Innovation Foundation of Shandong
typical inhibitors. Other pharmacological studies are University (IIFSDU, No.2010GN044), Shandong Post-
in progress to confirm their mechanism of action.
doctoral Innovation Science Research Special Program
(No.201002023), China Postdoctoral Science Founda-
tion funded project (No.20100481282) and KU Leuven
(GOA 10/014) is gratefully acknowledged.
DISCUSSION
In conclusion, a series of novel 2-amino/chloro sub-
We thank K. Erven, K. Uyttersprot and C. Heens for
stituted thiazolylthioacetamide derivatives were pre- technical assistance with the HIV assays.
pared via an expeditious route and profiled as inhib-
itors of HIV-1 or influenza A virus replication in cell
culture. The bioactivity results showed that some 2-
chloro substituted thiazolylthioacetamide derivatives
possessed potent activity against wild type HIV-1 and
several key mutant strains (E138K, K103N and L100I)
of HIV-1 with EC50 values in micromolar range. Two
2-amino substituted thiazole derivatives 8a7 and 8a8
displayed significant potency against influenza A/
H1N1 with EC50 values much lower than that of osel-
tamivir carboxylate, ribavirin, amantadine and riman-
tadine. Additionally, compounds 8a7 and 8a8 showed
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