Synthesis and antiviral activity of new acrylamide derivatives containing 1,2,3-thiadiazole as inhibitors of hepatitis B virus replication

Article abstract of DOI:10.1016/j.ejmech.2010.01.032

A series of new acrylamide derivatives containing 1,2,3-thiadiazole were synthesized, characterized, and evaluated for their anti-hepatitis B virus (HBV) activities in vitro. The IC₅₀ of compounds 9b (10.4?μg/mL), 9c (3.59?μg/mL) and 17a (9.00?μg/mL) of the inhibition on the replication of HBV DNA were higher than that of the positive control lamivudine (14.8?μg/mL). Compound 9d exhibited significant activity against secretion of HBeAg (IC₅₀?=?12.26?μg/mL).

Full text of DOI:10.1016/j.ejmech.2010.01.032

European Journal of Medicinal Chemistry 45 (2010) 1919–1926
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antiviral activity of new acrylamide derivatives containing
1,2,3-thiadiazole as inhibitors of hepatitis B virus replication
,
,
Wei-Li Dong a b, Zheng-Xiao Liu a, Xing-Hai Liu a, Zheng-Ming Li a, Wei-Guang Zhao a
*
a State Key Laboratory of Elemento-Organic Chemistry, National Pesticide Engineering Research Center (Tianjin), Nankai University, Tianjin 300071, China
b School of Pharmacy, Tianjin Medical University, Tianjin 300070, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of new acrylamide derivatives containing 1,2,3-thiadiazole were synthesized, characterized, and
Received 25 July 2009
Received in revised form
5 January 2010
Accepted 15 January 2010
Available online 25 January 2010
evaluated for their anti-hepatitis B virus (HBV) activities in vitro. The IC50 of compounds 9b (10.4
9c (3.59 g/mL) and 17a (9.00 g/mL) of the inhibition on the replication of HBV DNA were higher than
that of the positive control lamivudine (14.8
secretion of HBeAg (IC50 ¼ 12.26 g/mL).
mg/mL),
m
m
m
g/mL). Compound 9d exhibited significant activity against
Ó 2010 Elsevier Masson SAS. All rights reserved.
m
Keywords:
1,2,3-Thiadiazole
Acrylamide
Synthesis
Anti-HBV activity
1. Introduction
antitumor activities [20]. Recently, we synthesized a series of
2-substituted-N-methyl-2-(1,2,3-thiadiazol-4-yl)-acetamides and
disclosed their favorable anti-HBV activities first, such as
compound 3 [21], which framework structure is also similar to that
of acrylamide 1. Prompted by the biological properties of 1,2,3-
thiadiazole derivatives and the similar structure feature of antiviral
compounds 1, 2 and 3, we reasoned that the bioisoteric replace-
ment of the phenyl in phenylacrylamide with 1,2,3-thiadiazolyl
would produce a new class of acrylamide analogues with improved
anti-HBV activities. Herein, we designed and synthesized three
series of novel acrylamide derivatives containing 1,2,3-thiadiazole
in which the A or B ring of 1 was replaced by 4-methyl-1,2,3-thi-
diazole (Fig. 2). The synthesized compounds were evaluated for
their anti-HBV activities in vitro for the first time.
Hepatitis B virus (HBV) infection is one of the leading causes of
death due to infectious diseases worldwide. Approximately 400
million people worldwide are chronically infected by HBV, with an
annual global death toll of 1.2 million a year [1,2]. Although inter-
feron-a (IFN-a) and several nucleoside analogues such as lamivudine
(3-TC) and adefovir dipivoxil have been used to treat HBV infections,
still the side effects of interferon and the viral resistance of nucleo-
side analogues make the current treatment regimens far from being
satisfactory [3–5]. To address the toxicity and resistance problems of
nucleoside inhibitors, a major goal in the field of medicinal chem-
istry is to discover and develop non-nucleoside inhibitors [6].
Currently some non-nucleoside inhibitors have also been reported to
inhibit HBV replication [7–10].
A phenylacrylamide derivative, AT-130 (Fig. 1) has been discov-
ered to possess potent anti-HBV activity through random screening
[11–13]. It appears to act by inhibiting the packaging step in the life
cycle of HBV [14]. A natural compound 2 extracted from Dichondra
repens forens had a similar structure with compound 1, exhibiting
high anti-HBV activity [15]. To our knowledge, many compounds
possessing 1,2,3-thiadiazole fragment exhibit useful biological
properties, such as antiviral activities [16–18], antimicrobial [19] and
2. Chemistry
The Series I derivatives were prepared by replacing the B ring of
1 with 4-methyl-1,2,3-thidiazole, as shown in Scheme 1. The acid
intermediate 5-carboxyl-4-methyl-1,2,3-thiadiazole 4, which was
previously synthesized [22], was treated with thionyl chloride at
reflux to give 4-methyl-1,2,3-thiadiazole-5-carbonyl chloride 5,
which was then reacted with glycine to give 2-(4-methyl-1,2,3-
thiadiazole-5-carboxamido) acetic acid 6. Condensation of 6 with
the appropriate substituted benzaldehydes in acetic anhydride
under ultrasound irradiation afforded oxazolones 7a–c, which was
followed by ring-opening reaction with piperidine in CHCl3 to give
* Corresponding author. Tel.: þ86 22 23498368; fax: þ86 22 23505948.
E-mail addresses: zwg@nankai.edu.cn, zhaoweiguang@foxmail.com (W.-G. Zhao).
0223-5234/$ – see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.01.032

1920
W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
irradiation (70 ꢀC, 2 h). The bis-1,2,3-thiadiazole substituted
compound 20 was synthesized in a similar method as described for
the synthesis compounds 9a–d.
3. Results and discussion
The synthesized acrylamide derivatives containing 1,2,3-thia-
diazole were evaluated for their cytotoxicities and anti-HBV activ-
ities using the antiviral drug lamivudine as the reference drug in
2.2.15 cells. The results are summarized in Table 1.
Initial studies were conducted on the Series I derivatives 9a–d,
which has different patterns of substitution on the phenyl ring of
acrylamide derivatives. Among derivatives 9a–c, the chlorine
substituted analogues (9b and 9c) demonstrated good antiviral
activity, and the para-substituent analogue (9c) displayed more
Fig. 1. Chemical structures of some of the reported compounds showing potent anti-
HBV activity.
intermediates 8a–c in high yields. Finally, halogenations of 8a–c
with the appropriate halogens afforded phenylacrylamide deriva-
tives containing 1,2,3-thiadiazole 9a–d.
potent antiviral activity (IC50 ¼ 3.59
mg/mL) than the ortho-substit-
uent analogue 9b (IC50 ¼ 10.4 g/mL). However, compounds (9a and
For the aromatic aldehyde to react with compound 6, the
conventional heating method (Erlenmeyer’s method [23]) was
unsuitable, as two compounds (compounds 7 and 10) would appear
at a higher temperature (100 ꢀC). In the reaction procedure, only
compound 10 would be obtained at 140 ꢀC, similar to the reference
[23]. At 140 ꢀC in the presence of sodium acetate and acetic anhy-
dride, compound 7 was completely converted to the rearranged
product 10. To optimize the synthetic reaction conditions for the key
intermediate 7 and to avoid the formation of the side product 10 at
high temperatures, we used the ultrasound irradiation method for
this transformation. Without much difficulty, the desired 7 from
compound 6 with aromatic aldehyde was successfully obtained by
the ultrasound irradiation (70 ꢀC, 2 h). The new compound 11 was
obtained after aminated ring-opening and halogenation of 10, and
the structure of the rearranged product 10 was confirmed by an
X-ray diffraction analysis of compound 11 (Fig. 3). The mechanism of
this transformation is postulated as follows (Fig. 4). The acetate ion
attacks the carbonyl of substrate compound 7 to generate interme-
diate A. It is followed by the subsequent nucleophilic attack of the
nitrogen negative ion of A on a carbonyl moiety of acetic anhydride
to provide intermediate B. A stable compound 10 is then formed
through intramolecular cyclic reaction.
m
9d), which bear no substituent on the phenyl, proved to be virtually
inactive. This result suggests that the substituent on the phenyl ring
is an important feature in conferring relatively potent inhibitory
activity. Surprisingly, compound 9d, replacing the bromine atom at
the double bond of 9a with a chlorine atom, exhibited excellent
inhibitory activity against secretion of HBeAg (IC50 ¼ 12.26
The most potent compound in this subseries was compound 9c
(IC50 ¼ 3.59 g/mL), which was nearly four times more potent than
the reference drug lamivudine (IC50 ¼ 14.80 g/mL).
mg/mL).
m
m
Replacement of the ring A of 1 with 1,2,3-thiadiazole produced
the Series II derivatives (Table 1, 16a and 17a–d). Among derivatives
17a–c, the compound containing piperidine group (17a) was more
effective than morpholine derivative (17b), but the anti-HBV
activity was eliminated when the amine position was substituted
with the none-ring structural diethylamine group (17c). Besides,
when the bromine atom at the double bond of 17a was replaced
with the hydrogen atom (17a) or chlorine atom (17d), anti-HBV
activities were also eliminated. This result suggests that both the
substituent on the amine and the substituent on the double bond
have significant influence on anti-HBV DNA activity. Of these
compounds, the most active compound 17a exhibited high activity
The Series II derivatives were prepared by replacing the A ring of
1 with 4-methyl-1,2,3-thidiazole, as shown in Scheme 2. 5-Ethox-
ycarbonyl-4-methyl-1,2,3-thiadiazole 12, which was previously
synthesized [22], was reduced with KBH4 in ethanol to give
5-hydroxymethyl-4-methyl-1,2,3-thiadiazole 13. Treatment of 13
with pyridinium chlorochromate (PCC) in methylene chloride
afforded 4-methyl-1,2,3-thiadiazole-5-carboxaldehyde 14, which
was subsequently condensed with hippuric acid in acetic anhydride
at 100 ꢀC to provide oxazolone 15. Finally, the 1,2,3-thiadiazolyla-
crylamide derivatives 17a–d were synthesized with a similar
procedure to that described for the synthesis compounds 9a–d.
The Series III derivative 20 was prepared by simultaneously
replacing the A and B rings of 1 with 4-methyl-1,2,3-thidiazole, as
shown in Scheme 3. The intermediate oxazolone 18 was obtained
by condensation of 6 and 14 in acetic anhydride under ultrasound
against HBV (IC50 ¼ 9.00
m
g/mL), which was more potent than the
reference drug lamivudine (IC50 ¼ 14.80
mg/mL).
The selective indexes of 9b, 9c, and 17a were 8.29, 4.96, and 21.38,
respectively. Although their SIs were lower than that of lamivudine,
their anti-HBV potencies were higher than that of lamivudine.
When rings A and B were simultaneously replaced by 1,2,3-
thiadiazole (Series III, 20), the anti-HBV activity was eliminated, and
the cytotoxicity of the disubstituted 1,2,3-thiadiazole derivative was
higher than that of the corresponding monosubstituted analogues.
4. Conclusions
In summary, a series of acrylamide derivatives containing
1,2,3-thiadiazole based on compound 1 were synthesized and
Fig. 2. The design of novel acrylamide derivatives containing 1,2,3-thiadiazole.

W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
1921
Scheme 1. Synthetic pathway to phenylacrylamide derivatives containing 1,2,3-thiadiazole derivatives 9a–d. Reagents and conditions: (i) SOCl2, reflux; (ii) glycine, NaOH, pH ¼ 9–10;
(iii) ArCHO, Ac2O/NaOAc, ultrasonic/70 ꢀC; (iv) piperidine, CHCl3; (v) X2, CHCl3; (vi) ArCHO, Ac2O/NaOAc, 140 ꢀC; (vii) morpholine, CHCl3; (viii) Br2, CHCl3.
assessed for their anti-HBV activity and cytotoxicity in vitro, using
lamivudine as the reference drug. Compound 9c showed the most
potence anti-HBV activity within all of the tested compounds. Its
chromatography was carried out on silica gel (200–300 mesh).
Melting points were measured on an X-4 binocular microscope
melting point apparatus (Beijing Tech Instruments Co., Beijing,
China) and were uncorrected. 1H NMR spectra were obtained at
300 MHz on a Bruker Avance 300 spectrometer with tetrame-
thylsilane (TMS) as an internal standard. 13C NMR spectra were
obtained at 100 MHz on a Bruker Avance 400 spectrometer with
tetramethylsilane (TMS) as an internal standard. All chemical shifts
were reported in ppm. Elemental analysis was performed on a Vario
EL III automatic elemental analyzer. Mass spectra were recorded on
a Thermo Finnigan LCQ Advantage LC/mass detector instrument. IR
spectra were recorded on an Bruker550 spectrometer. X-ray
diffraction data were collected on a Bruker SMART 1000 charge-
coupled device (CCD) diffractometer. Reagent-grade solvents were
used without further purification unless otherwise specified.
IC50 was 3.59
the control lamivudine. Compound 9b and 17a also exhibited
significant efficacy on the replication of HBV DNA (IC50: 10.4, 9.0 g/
mL). In addition, compound 9d exhibited excellent inhibitory
mg/mL, which was about 3 times higher than that of
m
activity against secretion of HBeAg (IC50 ¼ 12.26
mg/mL).
5. Experimental
5.1. Chemistry
Thin-layer chromatography (TLC) was carried out on silica GF254
plates (Qingdao Haiyang Chemical Co., Ltd, China). Column
5.1.1. 4-methyl-1,2,3-thiadiazole-5-carboxylic acid (4)
Compound 4 was prepared as a white solid using commercially
available acetyl ethyl acetate as the starting material according to
the literature [24]. Yield ¼ 98.2%; m.p. 174–178 ꢀC (m.p. 174–175 ꢀC
[25]).
5.1.2. 4-Methyl-1,2,3-thiadiazole-5-carbonyl chloride (5)
In a 50 mL round-bottomed flask, 4 (7.07 g, 49 mmol) and 28 mL
of thionyl chloride were placed. The resulting mixture was heated
at 70–80 ꢀC for 4 h and then concentrated in vacuo to obtain the
crude residue 5, which was used in the next step without further
characterization.
5.1.3. 2-(4-Methyl-1,2,3-thiadiazole-5-carboxamido)acetic acid (6)
In a 100 mL three-neck round-bottomed flask, glycine (3.0 g,
40 mmol) and 8 mL water were placed. The solution was stirred
until the mixture became clear, and then it was neutralized to pH 10
with 4 N NaOH solution. The crude acid chloride 5 was added
slowly to the solution under a salt-ice bath. The reaction mixture
was stirred for 3 h and left at room temperature overnight. It was
filtered and washed with ethyl acetate. The filtered solution was
acidified to pH 2 and extracted with ethyl acetate. The organic layer
was dried over anhydrous MgSO4 and concentrated to afford 6
Fig. 3. Crystal structure of compound 11.

1922
W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
Fig. 4. The possible reaction mechanism of rearranged product 10.
(6.97 g, 69.0% yield) as a yellow solid, m.p. 141–143 ꢀC; 1H NMR
(100 MHz, CDCl3): d 167.95,161.05,158.48,141.83,133.30,129.13 (2C),
(300 MHz, D6-Acetone):
CH2), 7.34 (s, 1H, NH).
d
3.47 (s, 3H, CH3), 4.10 (d, J ¼ 3.9 Hz, 2H,
128.73, 128.63 (2C), 128.42, 124.14, 49.24, 43.43, 25.68, 25.26, 24.51,
13.92. ESI-MS (m/z): 355.12 (M ꢁ H)ꢁ. Anal. calcd for C18H20N4O2S: C,
60.65; H, 5.66; N, 15.72; found: C, 60.47; H, 5.52; N, 16.01.
5.1.4. General procedure for the preparation of 4-substituted
benzylidene-2-(4-methyl-1,2,3-thiadiazol-5-yl)oxazol-5(4H)-one
(7a–c)
5.1.5.2. N-(1-(2-Chlorophenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-
2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (8b). White solid,
yield 98.0%, m.p. 175–177 ꢀC; IR (KBr, cmꢁ1): 3145 (NH), 2944, 2932,
2859 (CH aliph.), 1674, 1651 (C]O), 1597, 1522, 1473 (C]C, phenyl).
Compound 6 (8.0 g, 38 mmol) and NaAc (3.28 g, 40 mmol) were
porphyrized and added to a 100 mL conical flask, then substituted
benzaldehydes (40 mmol) and acetic anhydride (12.72 g, 120 mmol)
were added. The mixture was rocked rapidly at room temperature,
which was subsequently treated under ultrasound irradiation at
70 ꢀC for 2 h, 15 mL ethanol was added and rocked, and then the
product was precipitated out of solution. The resulting precipitate
was filtered and dried to obtain the intermediates 7, which were
used in the next step without further characterization.
1H NMR (300 MHz, CDCl3):
d 1.66–1.72 (m, 6H, piperidine-3CH2), 2.78
(s, 3H, thiadiazole-CH3), 3.58–3.79 (m, 4H, piperidine-2CH2), 6.40 (s,
1H, C]C–H), 7.30–7.55 (m, 4H, Ph-H), 8.97 (br, 1H, NH). 13C NMR
(100 MHz, CDCl3):
d 167.10, 160.47, 158.06, 141.95, 133.75, 131.98,
130.18–129.70 (4C), 126.78, 121.83, 49.33, 43.51, 25.81, 25.38, 24.47,
13.79. ESI-MS (m/z): 389.18 (M ꢁ H)ꢁ. Anal. calcd for C18H19ClN4O2S:
C, 55.31; H, 4.90; N, 14.33; found: C, 55.44; H, 4.74; N, 14.34.
5.1.5. General procedure for the preparation of intermediates 8a–c
In a 25 mL round-bottomed flask, compound 7 (2.2 mmol) and
30 mL of CHCl3 were placed. After cooling in a ice bath, a solution of
piperidine (0.196 g, 2.3 mmol) in CHCl3 (6 mL) was added. The
resulting solution was stirred at room temperature overnight and
then concentrated in vacuo. The residue was subjected to silica gel
column chromatography (petroleum ether/ethyl acetate: 5/1) to
afford compounds 8a–c.
5.1.5.3. N-(1-(4-Chlorophenyl)-3-oxo-3-(piperidin-1-yl)prop-1-en-
2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (8c). White solid,
yield: 97.9%, m.p. 176–178 ꢀC; IR (KBr, cmꢁ1): 3142 (NH), 2937, 2856
(CH aliph.), 1671, 1646 (C]O), 1600, 1525, 1489 (C]C, phenyl). 1H
NMR (300 MHz, CDCl3):
d 1.49–1.54 (m, 6H, piperidine-3CH2), 2.90
(s, 3H, thiadiazole-CH3), 3.05–3.59 (m, 4H, piperidine-2CH2), 6.85
(s, 1H, C]C–H), 7.09–7.24 (m, 4H, Ph-H), 8.89 (br, 1H, NH). 13C NMR
(100 MHz, CDCl3):
d 167.74, 161.27, 158.45, 141.45, 134.56, 131.69,
130.33 (2C), 128.89 (2C), 128.54, 122.36, 49.32, 43.57, 25.64, 25.31,
24.48, 13.94. ESI-MS (m/z): 389.07 (M ꢁ H)ꢁ. Anal. calcd for
C18H19ClN4O2S: C, 55.31; H, 4.90; N, 14.33; found: C, 55.37; H, 4.86;
N, 14.47.
5.1.5.1. 4-Methyl-N-(3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-
yl)-1,2,3-thiadiazole-5-carboxamide (8a). White solid, yield 88.0%,
m.p. 163–165 ꢀC; IR (KBr, cmꢁ1): 3139 (NH), 2956, 2936, 2859
(CH aliph.), 1665, 1649 (C]O), 1609, 1521, 1474 (C]C, phenyl). 1H
NMR (300 MHz, CDCl3):
d
1.65–1.68 (m, 6H, piperidine-3CH2), 2.86
5.1.6. General procedure for the preparation of derivatives 9a–d
Bromine liquid (0.225 g, 1.4 mmol) was added to a solution of
compounds 8a–c (1.4 mmol) in CHCl3 (15 mL) under a salt-ice bath.
(s, 3H, thiadiazole-CH3), 3.53–3.71 (m, 4H, piperidine-2CH2), 6.07 (s,
1H, C]C–H), 7.36–7.44 (m, 5H, Ph-H), 9.41 (br, 1H, NH). 13C NMR
Scheme 2. Synthetic pathway to 1,2,3-thiadiazolylacrylamide derivatives 17a–d. Reagents and conditions: (i) KBH4, EtOH; (ii) PCC, CH2Cl2; (iii) Hippuric acid, Ac2O/NaOAc, 100 ꢀC;
(iv) Secondary amines, CHCl3; (v) X2, CHCl3.

W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
1923
Scheme 3. Synthetic pathway to 1,2,3-thiadiazolylacrylamide derivative 20. Reagents and conditions: (i) 2-(4-methyl-1,2,3-thiadiazole-5-carboxamido)acetic acid 6, Ac2O/NaOAc,
100 ꢀC; (ii) morpholine, CHCl3; (iii) Br2, CHCl3.
The mixture was stirred at room temperature for 3 h and then
CaCO3 (0.07 g, 0.7 mmol) was added, the resulting solution was
stirred at room temperature overnight. The residue was subjected
to silica gel column chromatography (petroleum ether/ethyl
acetate: 5/1) to afford compounds 9a–c.
The solution of 8a (0.5 g, 1.4 mmol) in CCl4 (15 mL) was stirred
under a salt-ice bath. A solution of Cl2 (0.1 g, 1.4 mmol) in CCl4 was
added and stirred at room temperature for 5 h, after which CaCO3
(0.07 g, 0.7 mmol) was added, the resulting solution was stirred at
room temperature overnight. The residue was subjected to silica gel
column chromatography (petroleum ether/ethyl acetate: 5/1) to
afford compound 9d.
25.12, 24.81, 24.07, 14.34. ESI-MS (m/z): 470.93 (M þ H)þ. Anal.
calcd for C18H18BrClN4O2S: C, 46.02; H, 3.86; N, 11.93; found: C,
45.99; H, 3.73; N, 12.05.
5.1.6.3. N-(1-Bromo-1-(4-chlorophenyl)-3-oxo-3-(piperidin-1-yl)prop-
1-en-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (9c). White
solid, yield 73.9%, m.p. 178–180 ꢀC; IR (KBr, cmꢁ1): 3146 (NH),
2939, 2864 (CH aliph.), 1674, 1655 (C]O), 1614, 1520, 1484 (C]C,
phenyl). 1H NMR (300 MHz, CDCl3):
d
0.98–1.50 (m, 6H, piperi-
dine-3CH2), 3.08 (s, 3H, thiadiazole-CH3), 3.17–3.48 (m, 4H,
piperidine-2CH2), 7.32–7.49 (m, 4H, Ph-H), 8.04 (br, 1H, NH). 13
NMR (100 MHz, CDCl3): 161.96, 159.79, 156.77, 142.43, 135.83,
C
d
134.42, 130.53 (2C), 130.18, 128.59 (2C), 113.09, 47.80, 42.70,
24.75, 24.55, 23.97, 14.22. ESI-MS (m/z): 468.86 (M ꢁ H)ꢁ. Anal.
calcd for C18H18BrClN4O2S: C, 46.02; H, 3.86; N, 11.93; found: C,
45.81; H, 3.74; N, 12.05.
5.1.6.1. N-(1-Bromo-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-yl)
-4-methyl-1,2,3-thiadiazole-5-carboxamide (9a). White solid, yield
83.4%, m.p. 180–182 ꢀC; IR (KBr, cmꢁ1): 3155 (NH), 2942, 2857 (CH
aliph.), 1675, 1659 (C]O), 1608, 1517, 1484 (C]C, phenyl). 1H NMR
(300 MHz, CDCl3):
d
0.53–1.51 (m, 6H, piperidine-3CH2), 3.04 (s, 3H,
5.1.6.4. N-(1-Chloro-3-oxo-1-phenyl-3-(piperidin-1-yl)prop-1-en-2-
yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (9d). White solid,
yield 80.9%, m.p. 176–179 ꢀC; IR (KBr, cmꢁ1): 3150 (NH), 2943, 2856
(CH aliph.), 1671, 1662 (C]O), 1611, 1519, 1486 (C]C, phenyl). 1H
thiadiazole-CH3), 3.02–3.48 (m, 4H, piperidine-2CH2), 7.34–7.53
(m, 5H, Ph-H), 7.95 (br, 1H, NH). 13C NMR (100 MHz, CDCl3):
d
162.53, 159.96, 157.13, 142.48, 135.97, 129.77, 129.52, 129.16 (2C),
128.27 (2C), 116.24, 47.79, 42.65, 24.52, 24.46, 23.93, 14.14. ESI-MS
(m/z): 434.93 (M ꢁ H)ꢁ. Anal. calcd for C18H19BrN4O2S: C, 49.66; H,
4.40; N, 12.87; found: C, 49.72; H, 4.44; N, 12.91.
NMR (300 MHz, CDCl3): d 0.40–1.39 (m, 6H, piperidine-3CH2), 2.91
(s, 3H, thiadiazole-CH3), 3.08–3.38 (m, 4H, piperidine-2CH2), 7.25–
7.42 (m, 5H, Ph-H), 7.75 (br, 1H, NH). 13C NMR (100 MHz, CDCl3):
d
162.59, 159.98, 156.98, 142.45, 134.51, 129.86, 128.62 (2C), 128.31
5.1.6.2. N-(1-Bromo-1-(2-chlorophenyl)-3-oxo-3-(piperidin-1-yl)prop-
1-en-2-yl)-4-methyl-1,2,3-thiadiazole-5-carboxamide (9b). White
solid, yield 99.0%, m.p. 173–175 ꢀC; IR (KBr, cmꢁ1): 3154 (NH),
2936, 2858 (CH aliph.), 1675, 1665 (C]O), 1621, 1517, 1466 (C]C,
(2C), 127.25, 123.99, 47.84, 42.63, 24.48, 24.42, 23.98, 14.08. ESI-MS
(m/z): 389.04 (M ꢁ H)ꢁ. Anal. calcd for C18H19ClN4O2S: C, 55.31; H,
4.90; N, 14.33; found: C, 55.41; H, 4.82; N, 14.27.
phenyl). 1H NMR (300 MHz, CDCl3):
d
0.98–1.50 (m, 6H, piperi-
dine-3CH2), 3.08 (s, 3H, thiadiazole-CH3), 3.17–3.48 (m, 4H,
piperidine-2CH2), 7.32–7.49 (m, 4H, Ph-H), 8.04 (br, 1H, NH). 13
NMR (100 MHz, CDCl3): 162.96, 161.20, 159.13, 156.34, 142.98,
5.1.7. 4-Benzylidene-2-methyloxazol-5(4H)-one (10)
Compound
6 (0.50 g, 2.5 mmol), benzaldehyde (0.25 g,
C
2.5 mmol), NaAc (0.21 g, 2.5 mmol) and acetic anhydride (10 mL)
were added to a 25 mL round-bottomed flask. The mixture was
heated at 140 ꢀC for 2 h. After being cooled to room temperature,
d
134.42, 131.70, 131.19, 130.25, 127.41, 126.97, 108.64, 47.81, 42.70,
Table 1
Anti-HBV activities of title compounds (mg/mL).
a
No.
X
R
MW
TC50
HBsAg
HBeAg
IC50
DNA replication
IC50
SIc
SI
IC50
SI
b
d
9a
9b
9c
9d
16a
17a
17b
17c
Br
Br
Br
Cl
H
Br
Br
Br
Cl
Br
H
435.34
469.78
469.78
390.89
356.44
435.34
437.31
423.33
392.86
459.34
229.26
111.11
86.23
17.81
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
5.23
–
–
–
–
–
–
–
o-Cl
p-Cl
H
piperidine
piperidine
Morpholine
Diethylamine
Morpholine
Morpholine
–
–
–
–
–
–
–
–
–
–
10.4
3.59
–
8.29
4.96
–
64.15
12.26
231.12
192.45
333.33
86.23
577.35
47.72
–
–
–
–
–
–
–
–
–
9.00
30.51
–
–
–
21.38
10.92
–
–
–
17d
20
Lamivudine
–
–
961.50
14.80
64.97
a
TC50 is 50% cytotoxic concentration in 2.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity Index (SI:TC50/IC50).
b
c
d
No antiviral activity at a concentration lower than its TC50
.

1924
W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
30 mL CH2Cl2 was added and rocked. The resulting precipitate was
filtered and washed with water, the filtered solution was concen-
trated in vacuo. The residue was subjected to silica gel column
chromatography (petroleum ether/ethyl acetate: 3/1) to afford the
crude 10, which was used in the next step without further
characterization.
C18H20N4O2S: C, 60.65; H, 5.66; N, 15.72; found: C, 60.76; H, 5.45; N,
15.76.
5.1.13.2. N-(1-(4-Methyl-1,2,3-thiadiazol-5-yl)-3-morpholino-3-oxo-
prop-1-en-2-yl)benzamide (16b). Yellow solid, yield 99.6%, m.p.
187–189 ꢀC; IR (KBr, cmꢁ1): 3198 (NH), 2923, 2855 (CH aliph.), 1732,
1681 (C]O), 1604, 1509, 1474 (C]C, phenyl). 1H NMR (300 MHz,
5.1.8. N-(1-Bromo-3-morpholino-3-oxo-1-phenylprop-1-en-2-
yl)acetamide (11)
Compound 11 was prepared in a similar manner as described for
the synthesis of 9a–c except morpholine was used in place of
piperidine. The compound 11 was confirmed by X-ray diffraction
analysis.
CDCl3):
pholine-4CH2), 6.15 (s, 1H, C]C–H), 7.39–7.85 (m, 5H, Ph-5H), 9.28
(br, 1H, NH). 13C NMR (100 MHz, CDCl3):
166.83, 166.49, 157.54,
141.38, 134.79, 132.68, 131.61, 128.63 (2C), 127.81 (2C), 109.84, 66.37
(2C), 48.74, 42.90, 12.97. ESI-MS (m/z): 357.08 (M ꢁ H)ꢁ. Anal. calcd
for C17H18N4O3S: C, 56.97; H, 5.06; N, 15.63; found: C, 56.92; H,
5.00; N, 15.73.
d 2.67 (s, 3H, thiadiazole-CH3), 3.40–3.79 (m, 8H, mor-
d
5.1.9. Ethyl 4-methyl-1,2,3-thiadiazole-5-carboxylate (12)
Compound 12 was prepared as a yellow oil using commercially
available acetyl ethyl acetate as the starting material according to
the literature [24]. Yield ¼ 67.1%; 1H NMR (300 MHz, CDCl3):
5.1.13.3. N-(3-(Diethylamino)-1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-
oxoprop-1-en-2-yl)benzamide (16c). Red solid, yield: 91.7%, m.p.
164–166 ꢀC; IR (KBr, cmꢁ1): 3169 (NH), 2973, 2874 (CH aliph.), 1731,
1669 (C]O), 1618, 1514, 1477 (C]C, phenyl). 1H NMR (300 MHz,
d
1.39 (t, 3H, CH2CH3), 2.95 (s, 3H, thiadiazole-CH3), 4.49 (q, 2H,
CH2CH3) [26].
CDCl3):
3.40–3.71 (2br, 4H, 2 CH2CH3), 6.14 (s, 1H, C]C–H), 7.32–7.83 (m,
5H, Ph-5H), 9.96 (br, 1H, NH). 13C NMR (100 MHz, CDCl3):
168.08,
d 1.19 (br, 6H, 2 CH2CH3), 2.62 (s, 3H, thiadiazole-CH3),
5.1.10. 5-Hydroxymethyl-4-methyl-1,2,3-thiadiazole (13)
d
Compound 13 was prepared according to the literature [27] as
166.72, 157.05, 141.88, 135.92, 132.20, 131.65, 128.29 (2C), 127.95
(2C), 109.12, 43.77, 39.88, 13.62, 12.82, 11.99. ESI-MS (m/z): 343.07
(M ꢁ H)ꢁ. Anal. calcd for C17H20N4O2S: C, 59.28; H, 5.85; N, 16.27;
found: C, 59.06; H, 5.76; N, 16.26.
a yellow oil. Yield ¼ 41.9%; 1H NMR (300 MHz, CDCl3):
d 2.62 (s, 3H,
thiadiazole-CH3), 3.35 (s, 1H, OH), 5.00 (s, 2H, CH2) [26].
5.1.11. 4-Methyl-1,2,3-thiadiazole-5-carboxaldehyde (14)
Compound 14 was prepared according to the literature [28] as
5.1.14. General procedure for the preparation of derivatives 17a–d
Bromine liquid (0.32 g, 2.0 mmol) was added to a solution of 16
(1.96 mmol) in CHCl3 (20 mL) under a salt-ice bath. The mixture
was stirred at room temperature for 0.5 h, after which CaCO3 (0.1 g,
1 mmol) was added. The resulting solution was stirred at room
temperature overnight. The residue was subjected to silica gel
column chromatography (petroleum ether/ethyl acetate: 5/1) to
afford compound 17a–c.
A solution of 16b (0.5 g, 1.39 mmol) in CHCl3 (10 mL) was stirred
under a salt-ice bath, a solution of Cl2 (0.1 g, 1.4 mmol) in CCl4 was
added. The mixture was stirred at room temperature for 2 h, and
then CaCO3 (0.07 g, 0.7 mmol) was added. The resulting solution
was stirred at room temperature overnight. The residue was sub-
jected to silica gel column chromatography (petroleum ether/ethyl
acetate: 5/1) to afford compound 17d.
a yellow oil. Yield ¼ 43.7%; 1H NMR (300 MHz, CDCl3):
d 3.03 (s, 3H,
thiadiazole-CH3), 10.25 (s, 1H, CHO) [26].
5.1.12. 4-((4-Methyl-1,2,3-thiadiazol-5-yl)methylene)-2-
phenyloxazol-5(4H)-one (15)
Benzoylglycine (3.36 g, 17.9 mmol), 14 (2.32 g, 17.9 mmol),
anhydrous NaAc (1.54 g, 17.9 mmol), and acetic anhydride (5.47 g,
53.6 mmol) were placed in a 50 mL three-necked round-bottomed
flask equipped with a reflux condenser bearing a calcium chloride
tube. The mixture was heated at 138 ꢀC for 10 min, then cooled to
95–100 ꢀC for 2 h. After being cooled to 80 ꢀC, the reaction mixture
was treated with ethanol (3 mL) and cooled to room temperature.
The product was precipitated out of the solution, and after standing
overnight, the solid was collected, washed with ethanol (2 ꢂ 10 mL)
and hot water (3 ꢂ 5 mL), and then dried to obtain 15 (2.81 g, 57.0%
yield) as a red solid, m.p. 233–235 ꢀC; 1H NMR (300 MHz, CDCl3):
5.1.14.1. N-(1-Bromo-1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-oxo-3-
(piperidin-1-yl)prop-1-en-2-yl)benzamide (17a). White solid, Yield
43.0%, m.p. 192–193 ꢀC; IR (KBr, cmꢁ1): 3317 (NH), 2934, 2856 (CH
aliph.), 1725, 1683 (C]O), 1633, 1512, 1466 (C]C, phenyl). 1H NMR
d
2.90 (s, 3H, thiadiazole-CH3), 7.40 (s, 1H, C]C–H), 7.57–8.30 (m,
5H, Ph-H).
5.1.13. General procedure for the preparation of intermediates 16a–c
A solution of 15 (1.4 g, 5.1 mmol) in CHCl3 (20 mL) was stirred
under an ice bath, a solution of appropriate secondary amines
(5.6 mmol) in CHCl3 (10 mL) was added dropwise, and then the
mixture was stirred at room temperature for 1.5 h. After standing
overnight, the resulting solution was filtered and evaporated to
afford the product.
(300 MHz, CDCl3):
thiadiazole-CH3), 3.49–3.65 (m, 4H, piperidine-2CH2), 7.22–7.53
(m, 5H, Ph-H), 9.11 (s, 1H, NH). 13C NMR (100 MHz, CDCl3):
163.79,
160.99, 159.17, 143.96, 136.96, 133.11, 131.98, 129.05 (2C), 127.52
(2C), 90.00, 47.72, 42.69, 25.09, 24.61, 24.05, 13.57. ESI-MS (m/z):
434.79 (M ꢁ H)ꢁ. Anal. calcd for C18H19BrN4O2S: C, 49.66; H, 4.40;
N, 12.87; found: C, 49.73; H, 4.42; N, 13.00.
d 0.90–1.88 (m, 6H, piperidine-3CH2), 2.58 (s, 3H,
d
5.1.13.1. N-(1-(4-Ethyl-1,2,3-thiadiazol-5-yl)-3-oxo-3-(piperidin-1-yl)-
prop-1-en-2-yl)benzamide (16a). Yellow solid, yield: 69.4%, m.p.
175–177 ꢀC; IR (KBr, cmꢁ1): 3236 (NH), 3060 (CH, olefinic), 2925,
2855 (CH aliph.), 1733, 1671 (C]O), 1618, 1533, 1474 (C]C, phenyl).
5.1.14.2. N-(1-Bromo-1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-morpho-
lino-3-oxoprop-1-en-2-yl)benzamide (17b). White solid, yield 58.6%,
m.p. 187–188 ꢀC; IR (KBr, cmꢁ1): 3167 (NH), 2986, 2924, 2849
(CH aliph.),1678,1625 (C]O),1581,1515,1474 (C]C, phenyl).1H NMR
1H NMR (300 MHz, CDCl3):
d
0.92–1.75 (m, 6H, piperidine-3CH2),
(300 MHz, CDCl3):
morpholine-4CH2), 7.24–7.51 (m, 5H, Ph-H), 9.26 (br, 1H, NH). 13C
NMR (100 MHz, CDCl3): 165.19,163.80,156.84,146.96,134.37,132.60,
d 2.54 (s, 3H, thiadiazole-CH3), 3.40–4.23 (m, 8H,
2.59 (s, 3H, thiadiazole-CH3), 3.29–3.72 (m, 4H, piperidine-2CH2),
6.25 (s, 1H, C]C–H), 7.24–7.81 (m, 5H, Ph-5H), 9.15(s, 1H, NH). 13C
d
NMR (100 MHz, CDCl3):
d
166.06, 164.11, 157.01, 142.90, 136.39,
130.89, 128.55 (2C), 127.57 (2C), 98.86, 66.06 (2C), 47.36, 42.26, 13.94.
ESI-MS (m/z): 458.91 (M þ Na)þ. Anal. calcd for C17H17BrN4O3S: C,
46.69; H, 3.92; N, 12.81; found: C, 46.61; H, 4.04; N, 12.98.
132.70, 132.37, 128.43 (2C), 127.38 (2C), 102.84, 47.69, 42.86, 25.77,
24.64, 24.20, 12.63. ESI-MS (m/z): 355.15 (M ꢁ H)ꢁ. Anal. calcd for

W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
1925
5.1.14.3. N-(1-Bromo-3-(diethylamino)-1-(4-methyl-1,2,3-thiadia-
zol-5-yl)-3-oxoprop-1-en-2-yl)benzamide (17c). White solid, yield
56.8%, m.p. 181–182 ꢀC; IR (KBr, cmꢁ1): 3242 (NH), 2988, 2941, 2873
(CH aliph.), 1729, 1670 (C]O), 1633, 1519, 1480 (C]C, phenyl). 1H
3.69–3.97 (m, 8H, morpholine-4CH2), 9.63 (s, 1H, NH). 13C NMR
(100 MHz, CDCl3): 166.42, 162.20, 159.09, 158.25, 140.27, 140.07,
132.96, 112.63, 66.34, 66.37, 29.70 (2C), 13.99, 13.00. ESI-MS (m/z):
379.10 (M ꢁ Br)ꢁ. Anal. calcd for C14H15BrN6O3S2: C, 36.61; H, 3.29;
N, 18.30; found: C, 36.47; H, 3.38; N, 18.41.
d
NMR (300 MHz, CDCl3):
2.75 (s, 3H, thiadiazole-CH3), 3.14–3.42 (m, 4H, CH2CH3), 7.50–7.90
(m, 5H, Ph-H), 8.38 (s,1H, NH). 13C NMR (100 MHz, CDCl3):
163.52,
d 0.91 (t, 3H, CH2CH3), 1.01 (t, 3H, CH2CH3),
d
5.2. Crystal data for 11
161.52, 159.36, 143.82, 137.27, 133.07, 129.06 (2C), 128.39, 127.50
(2C), 89.45, 42.86, 38.48, 13.67, 12.65, 11.44. ESI-MS (m/z): 422.89
(M ꢁ H)ꢁ. Anal. calcd for C17H19BrN4O2S: C, 48.23; H, 4.52; N, 13.23;
found: C, 48.20; H, 4.52; N, 13.33.
The crystal data of the rearranged product are as follows.
C15H17BrN2O3, M ¼ 353.22, Monoclinic, a ¼ 12.201(3), b ¼ 14.079(4),
c ¼ 18.852(5) Å,
a
¼
b
¼
g
¼ 90ꢀ, V ¼ 3238.5(15) Å3, T ¼ 293(2) K,
space group P2(1)2(1)2(1), Z ¼ 8, Dc ¼ 1.449 g/cm3,
m (Mo-
Ka) ¼ 0.71073 mmꢁ1, F(000) ¼ 1440 reflections measured, 6650
unique (Rint ¼ 0.0519), which were used in all calculation. Fine
R1 ¼ 0.0567, wR (F2) ¼ 0.1092 (all data). The full crystallographic
details of the rearranged product have been deposited at the Cam-
bridge Crystallographic Data Center and were allocated the deposi-
tion number CCDC 662310.
5.1.14.4. N-(1-Chloro-1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-mor-
pholino-3-oxoprop-1-en-2-yl)benzamide (17d). White solid, yield
60.3%, m.p. 196–198 ꢀC; IR (KBr, cmꢁ1): 3202 (NH), 2924, 2855 (CH
aliph.), 1723, 1680 (C]O), 1621, 1507, 1478 (C]C, phenyl). 1H NMR
(300 MHz, CDCl3):
morpholine-4CH2), 7.44–7.89 (m, 5H, Ph-H), 9.16 (br, 1H, NH). 13
NMR (100 MHz, CDCl3): 165.63, 163.14, 159.45, 145.44, 134.63,
d 2.61 (s, 3H, thiadiazole-CH3), 3.61–4.05 (m, 8H,
C
d
131.36, 128.99, 128.49, 127.85, 127.67, 127.55, 109.99, 66.36, 66.11,
47.27, 42.33, 14.18. ESI-MS (m/z): 391.19 (M ꢁ H)ꢁ. Anal. calcd for
C17H17ClN4O3S: C, 51.97; H, 4.36; N, 14.26; found: C, 51.78; H, 4.36;
N, 14.46.
5.3. Biological assay
5.3.1. In vitro anti-HBV activity assay
The anti-HBV activities of the target compounds were evaluated
in 2.2.15 cells by previously reported methods [29,30]. This assay
included the ability to inhibit the production of HBsAg and HBeAg,
and the replication of HBV DNA in HBV-infected 2.2.15 cells. Briefly,
confluent cell cultures in 96-well flat-bottomed tissue culture plates
were treated with various doses of the test compounds or lam-
ivudine (purchased by Glaxo & Welcome Co.) in RPMI 1640 medium
supplemented with 2% fetal bovine serum. The cell control was set
up. Medium was changed daily with fresh test compounds and
positive control for 8 days. The excretion of HBsAg and HBeAg in the
cell supernatant was detected by radioimmunoassay (RIA) using
commercial kits (HBsAg, HBeAg, Beijing North Institute of Biological
Technology). The DNA replication of HBV in the cell was detected by
dot blot hybridization. The IC50 and selected index of the evaluated
compounds and lamivudine were calculated, respectively.
5.1.15. 2-(4-Methyl-1,2,3-thiadiazol-5-yl)-4-((4-methyl-1,2,3-
thiadiazol-5-yl)methylene)oxazol-5(4H)-one (18)
Compound 6 (4.21 g, 21.0 mmol), 14 (2.68 g, 21.0 mmol), anhy-
drous NaAc (1.72 g, 21.0 mmol), and acetic anhydride (6.41 g,
62.3 mmol) were placed in a 50 mL three-necked round-bottomed
flask equipped with a reflux condenser bearing a calcium chloride
tube. The mixture was rocked rapidly at room temperature. After
this, the reaction mixture was treated under ultrasound irradiation
at 70 ꢀC for 2 h. The product was precipitated out of the solution.
The solid was collected, washed with ethanol (3 ꢂ 10 mL), hot water
(3 ꢂ 5 mL), and petroleum ether (2 ꢂ 10 mL), and dried to obtain
compound 18 (3.90 g, 63.4% yield) as a yellow solid, m.p. 166–
168 ꢀC; 1H NMR (300 MHz, CDCl3):
d 2.94 (s, 3H, thiadiazole-CH3),
3.24 (s, 3H, thiadiazole-CH3), 7.58 (s, 1H, C]C–H).
5.3.2. Cytotoxicity assay
5.1.16. 4-Methyl-N-(1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-
morpholino-3-oxoprop-1-en-2-yl)-1,2,3-thiadiazole-5-carboxamide
(19)
The cytotoxicities of the test compounds to 2.2.15 cells were
assessed by MTT assay [31]. Briefly, 2.2.15 cells were treated as
described above. Untreated control cultures were also maintained
on each 96-well plate. Toxicity was determined by measuring
neutral red dye uptake, as determined from the absorbance at
510 nm relative to untreated cells, 9 days of treatment.
A solution of 18 (1.33 g, 4.44 mmol) in CHCl3 (20 mL) was stirred
under an ice bath, a solution of morpholine (0.39 g, 4.44 mmol) in
CHCl3 (10 mL) was added dropwise. The mixture was stirred at
room temperature for 4 h. After standing overnight, the residue was
subjected to silica gel column chromatography (petroleum ether/
ethyl acetate: 4/1) to afford compound 19 (0.73 g, 43.3% yield) as
Acknowledgements
a white solid, m.p. 195–198 ꢀC; 1H NMR (300 MHz, CDCl3):
d 2.71 (s,
3H, thiadiazole-CH3), 2.78 (s, 3H, thiadiazole-CH3), 3.75–3.81 (m,
8H, morpholine-4CH2), 6.20 (s, 1H, C]C–H), 9.92 (br, 1H, NH).
The authors are indebted to the National Natural Science
Foundation of China (20202003, 20772069) and the National Basic
Research Program of China (2003CB114400) for the financial
support, and the National Center for Drug Screening of China for the
technical assistance in performing the biological assays.
5.1.17. N-(1-Bromo-1-(4-methyl-1,2,3-thiadiazol-5-yl)-3-
morpholino-3-oxoprop-1-en-2-yl)-4-methyl-1,2,3-thiadiazole-5-
carboxamide (20)
References
Bromine liquid (0.31 g, 1.92 mmol) was added to a solution of 19
(0.73 g, 1.92 mmol) in CHCl3 (20 mL) under a salt-ice bath. The
mixture was stirred at room temperature for 2.5 h, and then CaCO3
(0.1 g, 1 mmol) was added. The resulting solution was stirred at
room temperature overnight. The residue was subjected to silica gel
column chromatography (petroleum ether/ethyl acetate: 4/1) to
afford compound 20 (0.6 g, 67.9% yield) as a white solid, m.p.
181–183 ꢀC; IR (KBr, cmꢁ1): 3076 (NH), 2921, 2856 (CH aliph.), 1671,
1665 (C]O), 1611, 1508, 1477 (C]C, phenyl). 1H NMR (300 MHz,
[1] A.H. Malik, W.M. Lee, Ann. Intern. Med. 132 (2000) 723–731.
[2] A.S. Lok, J. Hepatol. 32 (2000) 89–97.
[3] G. Fattovich, L. Brollo, A. Alberti, P. Pontisso, G. Giustina, G. Realdi, Hepatology
8 (1988) 1651–1654.
[4] C.L. Lai, R.N. Chien, N.W.Y. Leung, T.T. Chang, R. Guan, D.I. Tai, K.Y. Ng, P.C. Wu,
J.C. Dent, J. Barber, S.L. Stephenson, D.F. Gray, N. Engl. J. Med. 339 (1998) 61–68.
[5] J.L. Dienstag, E.R. Schiff, T.L. Wright, R.P. Perrillo, H.L. Hann, Z. Goodman,
L.D. Crowther, M. Woessner, M. Rubin, N.A. Brown, N. Engl. J. Med. 341 (1999)
1256–1263.
[6] T.M. Williams, T.M. Ciccarone, S.C. MacTough, C.S. Rooney, S.K. Balani,
J.H. Condra, E.A. Emini, M.E. Goldman, W.J. Greenlee, L.R. Kauffman,
CDCl3): d 2.55 (s, 3H, thiadiazole-CH3), 2.72 (s, 3H, thiadiazole-CH3),

1926
W.-L. Dong et al. / European Journal of Medicinal Chemistry 45 (2010) 1919–1926
J.A. O’Brien, V.V. Sardana, W.A. Schleif, A.D. Theoharides, P.S. Anderson, J. Med.
Chem. 36 (1993) 1291–1294.
[17] P. Zhan, X.Y. Liu, Y. Cao, Y. Wang, C. Pannecouque, E. De Clercq, Bioorg. Med.
Chem. Lett. 18 (2008) 5368–5371.
[7] D.E. Zembower, Y.M. Lin, M.T. Flavin, F.C. Chen, B.E. Korba, Antiviral Res. 39
(1998) 81–88.
[18] P. Zhan, X.Y. Liu, Z.Y. Li, Z.J. Fang, Z. Li, D.F. Wang, C. Pannecouque, E. De Clercq,
Bioorg. Med. Chem. 17 (2009) 5920–5927.
[8] J.S. Lee, H.S. Shim, Y.K. Park, S.J. Park, J.S. Shin, W.Y. Yang, H.D. Lee, W.J. Park,
Y.H. Chung, S.W. Lee, Bioorg. Med. Chem. Lett. 12 (2002) 2715–2717.
[9] K. Deres, C.H. Schroder, A. Paessens, S. Goldmann, H.J. Hacker, O. Weber, T. Kramer,
U. Niewohner, U. Pleiss, J. Stoltefuss, E. Graef, D. Koletzki, R.N.A. Masantschek,
A. Reimann, R. Jaeger, R. Gro, B. Beckermann, K.-H. Schlemmer, D. Haebich,
H. Ruebsamen-Waigmann, Science 299 (2003) 893–896.
[19] H. Bhojya Naik, M. Ramesha, B. Swetha, T. Roopa, Phosphorus Sulfur Silicon
Relat. Elem. 181 (2006) 533–541.
[20] M.J. Wu, Q.M. Sun, C.H. Yang, D.D. Chen, J. Ding, Y. Chen, L.P. Lin, Y.Y. Xie,
Bioorg. Med. Chem. Lett. 17 (2007) 869–873.
[21] W.G. Zhao, J.G. Wang, Z.M. Li, Z. Yang, Bioorg. Med. Chem. Lett. 16 (2006)
6107–6111.
[10] C.S. Zhao, Y.F. Zhao, H.F. Chai, P. Gong, Bioorg. Med. Chem. 14 (2006)
2552–2558.
[22] W.L. Dong, J.Y. Xu, X.H. Liu, Z.M. Li, B.J. Li, Y.X. Shi, Chem. J. Chin. Univ. 29
(2008) 1990–1994.
[11] R.W. King, S.K. Ladner, T.J. Miller, K. Zaifert, R.B. Perni, S.C. Conway, M.J. Otto,
Antimicrob. Agents Chemother. 42 (1998) 3179–3186.
[12] R.B. Perni, S.C. Conway, S.K. Ladner, K. Zaifert, M.J. Otto, R.W. King, Bioorg. Med.
Chem. Lett. 10 (2000) 2687–2690.
[13] W.E. Delaney, R. Edwards, D. Colledge, T. Shaw, P. Furman, G. Painter,
S. Locarnini, Antimicrob. Agents Chemother. 46 (2002) 3057–3060.
[14] J.J. Feld, D. Colledge, V. Sozzi, R. Edwards, M. Littlejohn, S.A. Locarnini, Antiviral
Res. 76 (2007) 168–177.
[15] G.Y. Liang, Y.M. Liu, B.X. Xu, Faming Zhuanli Shenqing Gongkai Shuomingshu
CN 14 37 (2003) 937.
[23] J.S. Buck, W.S. Ide, Org. Synth. 13 (1933) 8–9.
[24] Z.J. Fan, Z.G. Shi, H.K. Zhang, X.F. Liu, L.L. Bao, L. Ma, X. Zuo, Q.X. Zheng, N. MIi,
J. Agric. Food Chem. 57 (2009) 4279–4286.
[25] H.R. Krueger, F. Arndt, R. Rusch, Ger. Offen. DE 27 28 (1979) 523.
[26] W. Porcal, P. Hernandez, M. Gonzalez, A. Ferreira, C. Olea-Azar, H. Cerecetto,
A. Castro, J. Med. Chem. 51 (2008) 6150–6159.
[27] T. Yamakawa, M. Masaki, H. Nohira, Bull. Chem. Soc. Jpn. 64 (1991) 2730–2734.
[28] F.E. Ziegler, G.R. Reid, W.L. Studt, P.A. Wender, J. Org. Chem. 42 (1997)
1991–2001.
[29] B.E. Korba, G. Milman, Antiviral Res. 15 (1991) 217–228.
[30] M.A. Sells, A.Z. Zelent, M. Shvartsan, G. Acs, Biochem. Biophys. Res. Commun.
156 (1988) 1144–1151.
[16] K. Ijichi, M. Fujiwara, H. Nagano, Y. Matsumoto, Y. Hanasaki, T. Ide, K. Katsuura,
H. Takayama, S. Shirakawa, N. Aimi, S. Shigeta, K. Konno, M. Matsushima,
T. Yokota, M. Baba, Antiviral Res. 31 (1996) 87–94.
[31] T. Mosmann, J. Immunol. Methods 65 (1983) 55–63.