Anti-hepatitis B virus and anti-cancer activities of novel isoflavone analogs

Article abstract of DOI:10.1016/j.ejmech.2012.09.017

We have synthesized a series of novel isoflavone analogs and evaluated their anti-HBV and anti-cancer activities in vitro. The bioassays showed that the majority of the resultant compounds exerted inhibitory effects on HBsAg and HBeAg levels, HBV DNA replication, as well as the growth of four human cancer cell lines to various extents, which supported the rationale of the design. In particular, compound 8f showed the highest activity against HBV infection and HBV-related liver cancer. Compound 7l (IC₅₀ = 0.47 μM) also exerted remarkable inhibitory effect on the growth lung cancer cell line A-549.

Full text of DOI:10.1016/j.ejmech.2012.09.017

European Journal of Medicinal Chemistry 62 (2013) 158e167
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Anti-hepatitis B virus and anti-cancer activities of novel isoflavone
analogs
Yikai Zhang a, Hanyu Zhong a, Zhiliang Lv a, Mingfeng Zhang a, Tao Zhang a,
,
Qisheng Li b, Ke Li a
*
a Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, 325 Guohe Road, Shanghai 200433,
People’s Republic of China
b Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized a series of novel isoflavone analogs and evaluated their anti-HBV and anti-cancer
activities in vitro. The bioassays showed that the majority of the resultant compounds exerted inhibitory
effects on HBsAg and HBeAg levels, HBV DNA replication, as well as the growth of four human cancer cell
lines to various extents, which supported the rationale of the design. In particular, compound 8f showed
Received 15 July 2012
Received in revised form
9 September 2012
Accepted 11 September 2012
Available online 20 September 2012
the highest activity against HBV infection and HBV-related liver cancer. Compound 7l (IC50 ¼ 0.47
mM)
also exerted remarkable inhibitory effect on the growth lung cancer cell line A-549.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Keywords:
Isoflavone analogs
Heck reaction
Synthesis
Anti-HBV activity
Anti-cancer activity
1. Introduction
class of compounds could mildly mimic estrogen (Fig. 1) because of
their similarities at the molecular level. Isoflavones (Fig. 1), which
are found in high concentration in soy bean and soy bean products,
represent the most active subfamily of phytoestrogen and have
been shown to possess anti-viral, anti-cancer, anti-oxidative, anti-
hyperglycemic, anti-inflammatory, insecticidal, anti-fungal and
anti-microbial activities [2e8]. Isoflavones exert their effects
primarily through binding to estrogen receptors (ER). They also
entangle with gamma-aminobutyric acid (GABA) receptors,
peroxisome proliferator-activated receptors (PPARs), nuclear factor
The hepatitis B virus (HBV), a hepatotropic member of the
hepadnavirus family is a principal causative agent of acute and
chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC)
{Dienstag 2008 NEJM, Liang 2009 Hepatology, National Institutes of
Health Consensus Development Conference Statement 2009}. HCC
accounts for 80e90% of all primary liver cancers and is the third
leading cause of cancer death worldwide {El-Serag 2011 NEJM}. This
type of cancer usually occurs in patients with chronic liver disease.
Infection with HBV or HCV is the leading cause of HCC, with each
virus increasing the risk of cancer for more than 10-fold [1]. Despite
drugs such as adefovir dipivoxil and 5-fluorouracil (5-Fu) have been
used to combat HBV infection and HBV-mediated HCC, respectively,
a versatile drug with inhibitory effects on both viral infection and
HCC has not been discovered yet. In addition, drug resistance,
toxicity and side effects have made current therapeutic regimens
suboptimal. Therefore, it is a pressing need to develop innovative,
multi-target drugs that could be used not only to cure patients with
HBV infection but also to prevent and treat HBV-induced HCC.
Phytoestrogen, also known as "dietary estrogen", is a naturally
occurring nonsteroidal xenoestrogen extracted from plants. This
kB (NF-kB), serotonin (5-HT), dopamine (DA), etc. [9e12]
Our design protocol was shown in Scheme 1. In our previous
studies, certain isoflavone analogs (II) derived from isoflavone (I)
have been synthesized and evaluated for their anti-HBV and cyto-
toxic activities. What encouraged us was that a few synthesized
analogs were found to possess potent efficacies against HBV
infection and HCC [13]. In view of the novel structural template and
to discover more selective and effective anti-HBV or anticancer
agents, we intended to synthesize additional analogs of isoflavone
and determine their structureeactivity relationships (SARs). Acrylic
acid derivatives have been reported to be of high importance in
therapeutics due to their antitumor, antioxidant and antibacterial
activities [14e16]. According to the design proposal, in this study,
we introduced the acrylic amide pharmacophore (III) to keep the
nitrogen atom and conjugation system, and also prolonged the
* Corresponding author. Tel.: þ86 21 81871237.
E-mail address: proflike@sina.com (K. Li).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.09.017

Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
159
3. Pharmacology
OH
O
OH
In vitro cytotoxicities of all target compounds in HepG2 2.2.15
cells were assessed by MTT assays. Effects on HBsAg and HBeAg
levels by these compounds were evaluated in cultured HepG2
2.2.15 cells by enzyme linked immunosorbent assay (ELISA). Effects
on HBV DNA replication were evaluated by fluorescence PCR.
In the cytotoxicity assays, all the synthesized compounds
(7ae7w, 8ae8w) were evaluated for their in vitro cytotoxic activi-
ties against one human normal cell line and four human cancer cell
lines, including L02 (normal human liver cell line), QGY-7703
(human hepatoma cell line), Huh-7 (human hepatoma cell line),
HeLa (human cervical carcinoma cell line) and A-549 (human lung
adenocarcinoma cell line). Human cervical carcinoma cell line was
recruited because of the important estrogen-mimic function of
isoflavones in reproductive system; human lung adenocarcinoma
cell line was studied to provide another cancer type to oversee the
efficacies of the compounds. In all studies, 5-Fluorouracil (5-Fu)
was used as a positive control.
H
OH
HO
H
H
H
H
H
HO
HO
Estriol
Estradiol
O
H
H
H
O
Estron
e
Isoflavone
Fig. 1. The three major naturally occurring estrogens and the isoflavone.
distance between the benzene ring B and nitrogen atom with one
or two carbon atom (IV) and (V). Herein, SAR studies on anti-HBV
and cytotoxic activities were then investigated in this paper.
4. Results and discussion
2. Chemistry
4.1. In vitro virological assays of anti-HBV activities
The synthetic route of compound 5 was depicted in Scheme 2
according to a reported procedure [13]. Compound 5 was treated
with AlCl3 in toluene to obtain compound 6 in 93% yield. Coupling
of compound 5 (6) with substituted acrylamide by Heck reaction to
produce compound 7ae7w (8ae8i) in 36e73% yield, as illustrated
in Scheme 3. In order to establish a robust synthetic procedure of
resultant compounds, the Heck reaction conditions were opti-
mized. Microwave heating method was employed and various
solvents, bases, metal catalysts, temperatures and heating methods
were explored. The DMF was found to be the best choice, with
triethylamine as the base and Pd(OAc)2 as the metal catalyst. The
optimal microwave power, reaction temperature and time were
500 w, 100 ꢀC and 7 min, respectively. The chemical structures of
these compounds were confirmed by HRMS, NMR spectroscopy
and elemental analyses. The purity of final compounds was
assessed by analytical HPLC (ꢁ95%). (See Supplementary data).
Analyses of 1H NMR spectrum of all the final products suggested
The potential anti-HBV activities and cytotoxicities of the iso-
flavone analogs were studied in HepG2 2.2.15 cells, with adefovir
applied as a positive control drug. The results were summarized in
Table 1. The anti-HBV activity of each compound was expressed as
the concentration of compound that achieved 50% inhibition (IC50
)
of HBsAg, HBeAg, and HBV DNA replication. The cytotoxicity of each
compound was manifested as the concentration of the compound
required to kill 50% (CC50) of the HepG2 2.2.15 cells. The selectivity
index (SI), an important pharmaceutical parameter that facilitates
the estimation of possible future clinical development, was deter-
mined as the ratio of CC50 value to IC50 value. The bioactivity of each
compound was evaluated by a combination of its IC50 value and
corresponding SI.
Compounds 7ae7m had different patterns of substitution on
the phenyl ring attached to the nitrogen atom of acrylamide. As
shown in Table 1, these compounds generally exhibited poor to
none anti-HBV activity, except compounds 7a, 7b, 7m (with higher
inhibitory effect against HBsAg and HBeAg than adefovir) and 7h
(with moderate effect against HBV DNA replication). Nevertheless,
compound 7h had high CC50 value against HepG2 2.2.15 cells,
which led to a moderate SI value (SI ¼ 50.38).
a doublet of substituted acrylamide at around
respectively (7ae7w with CDCl3 as solvent) or around
7.4, respectively (8ae8i with DMSO-d6 as solvent). The coupling
d
7.4 and
d 7.7,
d
7.2 and
d
constant (J ¼ 15.0e15.6 Hz) of the two olefinic protons confirmed
the trans-double bond.
Scheme 1. Schematics of the design for the target compounds.

160
Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
O
O
O
O
O
O
a
b
OH
H
O
OH
O
OH
O
O
OH
2
3
1
O
O
O
O
O
e
d
c
I
I
N
HO
O
OH
O
6
5
4
Reagents and conditions: (a) Na, Ethyl Formate, Ethyl Ether, 0˚C-r.t., 18h; (b) HOAc/HCl, reflux,
30min;(c) Piperidine, CH3OH, reflux, 3h; (d) I2, Pyridine, CH2Cl2, r.t., 20h (e) AlCl3, Toluene, 100˚C,
Overnight
Scheme 2. Synthesis of the key intermediates.
Significantly increased anti-HBV activity was observed with the
Based on the biological data above, eight compounds (7a, 7b, 7h,
7n, 7o, 7p, 7s and 7w) were selected for further modifications, of
which 7-OCH3 were replaced by 7-OH (8ae8i). It is worthy noting
that some modified compounds exhibited increased anti-HBV
activity and decreased cytotoxicity. Particularly in the case of
compound 8f, which gained good activities against HBsAg
replacement of the substituted phenyl groups of compounds by the
substituted benzyl groups (compounds 7ne7t). It is noteworthy that
almost all compounds in this subseries showed moderate inhibitory
effects on HBV DNA replication and moderate to strong effect against
HBsAg, except compound 7n, of which HBsAg and HBeAg inhibiting
effect was not observed. Therefore, these demonstrated that substi-
tutions on the phenyl ring might facilitate antiviral activities.
Although compounds 7p and 7t showed better activities against
(IC50 ¼ 10.22
excellent activity against HBeAg (IC50 ¼ 2.34
toxicity (CC50 ¼ 717.95 M). Likewise, compound 8e also showed
m
M) and HBV DNA replication (IC50 ¼ 4.07
mM),
M) and very low
m
m
HBsAg (IC50 value were 13.97 and 14.29
(IC50 value were 14.41 and 14.82 M, respectively) than the control
drug did, their relatively high cytotoxicity (CC50 value were 43.01 and
79.25 M, respectively) led to a low value of SI.
mM, respectively) and HBeAg
high anti-HBV activity which is only lower than compound 8f,
indicating that the methoxyl at position 4 of the benzyl or methyl
group was important for the antiviral activity. Although compound
8i showed good activity against both HBsAg and HBeAg, its rela-
tively high cytotoxicity resulted in a low value of SI.
m
m
The introduction of substituted phenylethyl groups on the N
atom resulted in the loss of anti-HBV DNA replication activity
(compounds 7ue7w). However, compound 7w maintained high
activity against both HBsAg and HBeAg, making it more potent than
adefovir.
According to the data analysis above, preliminary structuree
activity relationship (SAR) could be concluded. Firstly, benzyl
analogs appeared to be more active than phenyl and phenylethyl
analogs. Secondly, different substitution position on the benzene
O
O
O
I
R2
a
O
N
H
+
R2
N
H
O
R1
O
7a - 7w
8a - 8i
R1
Trans-double bond
1H NMR Coupling Constant: J=15-15.6 Hz
5, 6
Reagents and conditions: (a) Pd(OAc)2, Et3N, DMF, Microwave, 100˚C, 500w, 7min
Compd.
R1
R2
Compd.
R1
R2
Compd.
R1
R2
-OMe
-OMe
-OMe
-OMe
-Ph
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-3’-Cl-Ph
-OMe -CH2CH2-4’-F-Ph
7a
7b
7c
7d
7e
7f
7l
7m
7n
7o
7p
7q
7r
7s
7w
8a
8b
8c
8d
8e
8f
-2’-Me-Ph
-3’-Me-Ph
-4’-Me-Ph
-4’-Cl-Ph
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-OH
-Ph
-CH2Ph
-2’-Me-Ph
-CH2-4’-Me-Ph
-CH2-4’-OMe-Ph
-CH2-2’-F-Ph
-CH2-3’-F-Ph
-CH2-4’-F-Ph
-CH2-2’,4’-F2-Ph
-4’-OMe-Ph
-OMe -2’,5’-Me2-Ph
-CH2Ph
-OMe
-OMe
-OMe
-OMe
-OMe
-OMe
-2’-OMe-Ph
-3’-OMe-Ph
-4’-OMe-Ph
-4’-OEt-Ph
-4’-F-Ph
-CH2-4’-Me-Ph
-CH2-4’-OMe-Ph
-CH2-4’-F-Ph
-CH2-2’,4’-F2-Ph
-CH2CH2-4’-F-Ph
7g
7h
7i
8g
8h
8i
7t
-OMe -CH2CH2-2’-F-Ph
-OMe -CH2CH2-3’-F-Ph
7j
7u
7v
-2’-Cl-Ph
7k
Scheme 3. Synthesis of the title compounds.

Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
161
Table 1
In vitro anti-HBV activity of the target compounds.
Compd.
CC50
(m
M)a
HBsAg
IC50 ꢂ SEM (
HBeAg
DNA replication
m
M)b
SIc
IC50 ꢂ SEM (
m
M)b
SIc
IC50 ꢂ SEM (
m
M)b
SIc
d
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
522.55
59.70
553.73
978.97
15.98 ꢂ 0.63
14.93 ꢂ 2.85
>100
>100
>100
>100
>100
>100
>100
32.70
16.92 ꢂ 1.38
15.82 ꢂ 2.33
>100
>100
>100
>100
>100
>100
>100
30.85
>100
>100
34.93 ꢂ 2.55
28.22 ꢂ 0.21
>100
d
4.00
3.77
d
d
15.85
d
d
d
d
d
d
d
d
d
34.69
d
d
d
1098.65
1045.34
1125.22
717.95
459.56
743.36
1007.04
1104.90
271.66
376.12
257.24
43.01
d
d
d
d
d
d
d
d
d
>100
>100
14.25 ꢂ 0.88
50.38
d
>100
d
d
d
>100
>100
>100
>100
>100
>100
>100
>100
>100
91.70 ꢂ 4.00
2.96
20.95 ꢂ 0.59
12.97
67.46 ꢂ 1.68
56.81 ꢂ 1.65
53.45 ꢂ 0.91
24.27 ꢂ 2.34
68.56 ꢂ 3.22
75.44 ꢂ 2.31
11.24 ꢂ 4.27
37.03 ꢂ 1.36
>100
4.03
6.62
4.81
1.77
2.13
6.30
11.84
d
d
>100
>100
d
82.61 ꢂ 0.31
13.97 ꢂ 1.45
52.15 ꢂ 1.98
83.80 ꢂ 0.99
58.81 ꢂ 3.01
14.29 ꢂ 2.13
47.87 ꢂ 0.96
>100
14.66 ꢂ 3.95
45.54 ꢂ 0.21
>100
>100
>100
16.54 ꢂ 0.78
10.22 ꢂ 0.82
>100
14.53 ꢂ 2.11
14.56 ꢂ 0.92
305
3.11
3.08
2.80
5.67
2.26
5.55
>100
14.41 ꢂ 1.02
2.98
d
145.89
475.18
133.06
79.25
>100
d
d
>100
>100
7s
7t
14.82 ꢂ 0.19
5.35
2.14
d
d
7u
7v
7w
8a
8b
8c
8d
8e
8f
457.06
1030.46
786.29
820.85
374.00
975.84
435.69
500.72
717.95
357.45
469.86
102.41
540
9.55
>100
d
d
d
d
>100
>100
>100
53.64
14.71 ꢂ 5.01
13.43 ꢂ 0.45
>100
>100
>100
19.14 ꢂ 3.67
2.34 ꢂ 3.34
>100
12.35 ꢂ 1.33
15.43 ꢂ 1.27
286
53.45
18.02
61.12
29.02 ꢂ 0.34
28.29
d
d
d
>100
d
d
d
d
7.01 ꢂ 2.34
>100
139.21
d
30.27
26.16
14.18 ꢂ 1.69
4.70 ꢂ 1.03
33.46 ꢂ 0.84
23.31 ꢂ 0.91
>100
35.31
152.76
10.68
70.25
306.82
d
d
8g
8h
8i
32.34
7.03
1.77
38.05
6.64
1.89
20.16
d
Adefovir
0.517
1044.5
a
CC50 is 50% cytotoxicity concentration in HepG2 2.2.15 cells.
IC50 is 50% inhibitory concentration.
Selectivity index (SI ¼ CC50/IC50).
b
c
d
No SI can be obtained.
ring of the side chain could produce different bioactivity to some
extent. Either electron-withdrawing or electron-donating groups
at position-4 had better activities than other synthesized
compounds, with the exception of compound 7i, which means
small-volume para-substituted group at benzene ring was the
best choice for anti-HBV activity. However, substitution at
position-2 could increase the compound’s cytotoxicity. Thirdly,
demethylation of position-7 could not only increase activity but
reduce cytotoxicity.
Overall, our preliminary SAR studies demonstrated that the
most potent anti-HBV compounds in this category would contain
a position-4 substituted benzyl ring attached to the nitrogen atom
of acrylamide, accompanied by a hydrogen group on the benzene
ring of the benzopyran-4-one. Further optimizations are being
carried on to discover optimized compounds.
based on the anti-HBV activity. Among them, compound 8f, iden-
tified to be a promising HBV antiviral drug candidate, also showed
strong inhibitory effect against the growth of both QGY-7703 and
Huh-7 cells, with IC50 values of 10.01 mM and 8.43 mM, respectively,
which were comparable to those of 5-Fu. More impressively,
compound 8f had no effect against the growth of either HeLa or A-
549 cells, implicating the hepatoma cell line-specific activity of this
compound. In addition, compounds 7a, 7o, 7p, 7r, 7s, 7v, 8a, 8b and
8f all showed moderate to strong growth inhibitory effect against
the growth of both of the human hepatoma cell lines, particularly in
the case of compound 8a, which exerted a 4-fold higher effect in
inhibiting Huh-7 cells than the positive control and all these
compounds had good anti-HBV activity except compounds 7v and
8b. In contrast, compound 7i showed no activity against HBV and
poor effect in three of the cancer cell lines. However, it exerted
a strongest inhibitory effect against the growth of Huh-7 cells
among all the tested compounds, implying that this compound may
have unique mechanisms of action in Huh-7 cells.
4.2. In vitro cytotoxic effects of the compounds in various cancer
cell lines
Our results also showed that compounds 7b, 7g, 7k, 7r, 7v and
8a had broad anti-tumor activity against all four cancer cell lines
(Table 2). Unexceptionally, these compounds possess substituted
groups at either the position-2 or position-3 of the benzene ring.
Five of them possess a structural feature of with a benzene ring
directly attached to the N atom, suggesting that the benzyl ring is
not necessary for the broad anti-cancer features. We also found that
compound 7b was the most potent compound in inhibiting the
The results of in vitro cytotoxic analyses in various cancer cell
lines are summarized in Table 2. The IC50 value is defined as the
concentration of a compound that corresponds to a 50% growth
inhibition. We showed that the majority of the synthesized
compounds exerted antitumor activities against the four human
cancer cell lines to various extents and low toxicity to human
normal liver cell line.
growth of HeLa cells, with IC50 value of 7.27
mM, that is even lower
For the reason that the main role of these synthesized
compounds is to against the HBV, we first discussed cytotoxic effect
than that of 5-Fu (IC50 ¼ 16.32 M). A possible mechanism for these
m

162
Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
Table 2
In vitro anti-cancer activity of target compounds.
Compd.
Cytotoxicity IC50 ꢂ SEM (
L02a
m
M)b
QGY-7701a
Huh-7a
HeLaa
A-549a
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
7q
7r
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
46.57 ꢂ 0.68
14.02 ꢂ 0.47
22.45 ꢂ 0.099
>100
9.05 ꢂ 1.45
26.88 ꢂ 2.11
15.47 ꢂ 0.92
>100
11.12 ꢂ 9.11
32.27 ꢂ 3.13
11.13 ꢂ 2.18
>100
3.23 ꢂ 1.13
33.85 ꢂ 5.23
58.42 ꢂ 7.50
>100
37.10 ꢂ 4.73
13.58 ꢂ 0.86
14.32 ꢂ 1.65
7.57 ꢂ 2.22
8.45 ꢂ 0.091
10.07 ꢂ 1.60
20.95 ꢂ 0.73
>100
>100
51.00 ꢂ 1.87
23.40 ꢂ 2.22
>100
17.54 ꢂ 0.15
15.47 ꢂ 1.79
>100
12.80 ꢂ 3.95
22.98 ꢂ 4.09
>100
26.77 ꢂ 0.65
29.94 ꢂ 3.32
0.47 ꢂ 0.058
>100
7.27 ꢂ 0.64
>100
39.29 ꢂ 6.08
24.13 ꢂ 2.14
58.42 ꢂ 3.28
>100
19.23 ꢂ 0.031
13.03 ꢂ 2.56
>100
9.68 ꢂ 0.55
22.42 ꢂ 3.45
11.85 ꢂ 3.34
>100
>100
17.62 ꢂ 5.18
39.09 ꢂ 0.44
>100
>100
>100
22.97 ꢂ 0.66
>100
>100
>100
33.39 ꢂ 1.41
11.82 ꢂ 1.45
10.23 ꢂ 0.36
15.67 ꢂ 1.56
33.99 ꢂ 0.49
28.57 ꢂ 5.18
7.96 ꢂ 1.33
>100
81.67 ꢂ 8.22
23.44 ꢂ 0.02
24.84 ꢂ 4.55
17.94 ꢂ 0.49
9.32 ꢂ 1.93
>100
62.96 ꢂ 8.33
24.86 ꢂ 2.47
10.01 ꢂ 1.09
>100
>100
>100
>100
>100
>100
>100
22.49 ꢂ 1.76
>100
17.58 ꢂ 0.58
26.88 ꢂ 5.34
7s
7t
>100
>100
>100
13.33 ꢂ 1.13
10.30 ꢂ 0.33
9.77 ꢂ 0.44
18.86 ꢂ 5.67
24.38 ꢂ 3.10
>100
7u
7v
7w
8a
8b
8c
8d
8e
8f
>100
12.23 ꢂ 2.56
15.29 ꢂ 2.86
>100
9.70 ꢂ 1.01
8.67 ꢂ 0.40
>100
>100
>100
>100
>100
8.96 ꢂ 2.66
29.18 ꢂ 4.09
3.88 ꢂ 0.023
11.12 ꢂ 3.32
>100
12.82 ꢂ 2.34
4.69 ꢂ 0.38
>100
>100
17.43 ꢂ 1.49
>100
>100
6.61 ꢂ 0.18
6.84 ꢂ 0.67
5.64 ꢂ 0.55
8.43 ꢂ 0.21
>100
>100
>100
15.85 ꢂ 0.21
8g
8h
8i
>100
>100
16.32 ꢂ 0.36
5-Fuc
14.30 ꢂ 0.055
a
Abbreviations: L02: normal human liver cell line; QGY-7701: human hepatoma cell line; Huh-7: human hepatoma cell line; HeLa: human cervical carcinoma cell line; A-
549: human lung adenocarcinoma cell line.
b
The IC50 value was defined as the concentration at which 50% survival of cells was observed.
Used as a positive control.
c
findings is that the estrogen-like structure of the isoflavone analogs
may contribute to the potencies of the compounds.
549 cells in vitro, suggesting that it could be a candidate for the
exploration of novel anti-lung cancer drugs, which is worthwhile
for further study and optimization. Overall, the phenotypes exerted
new vistas of research for treatment of both HBV and HBV-induced
HCC. Further studies are needed for us to better understand the
nature and mechanisms of these compounds.
As shown in Table 2, compound 7l exerted a 12-fold stronger
effect against A-549 cells (IC50 ¼ 0.47
mM) than the reference drug
M), making it an ideal candidate for the development
(IC50 ¼ 5.64
m
of anti-lung cancer new drugs, which are worthwhile for further
investigation.
Preliminary SAR studies showed that the inhibitory effect
against HCC was basically in agreement with the results of anti-
HBV effect. Moreover, neither benzyl nor para-substituted
benzene ring in the side chain appeared to be good for the broad
anti-cancer features. And even without any substitution at benzene
ring, compound 8a exhibited best broad anti-tumor spectrum.
Finally, what excited us most is that these features about anti-HBV
and anti-HCC are not contradictory.
6. Experimental procedures
Melting points were measured with an uncorrected X-5 digital
melting point apparatus (Gongyi City Yuhua Instrument Co., Ltd.;
China). 1H NMR and 13C NMR spectra were recorded with a BRUKER
AVANCE 300 spectrometer (Bruker Company, Germany), with TMS
used as an internal standard and CDCl3 (99.8% D, Cambridge Isotope
Laboratories) or DMSO-d6 (99.9% D, Cambridge Isotope Laborato-
ries) as solvents. Chemical shifts (d values) and coupling constants
5. Conclusion
(J values) were given in ppm and Hz, respectively. Elemental
analyses were performed with a MOD-1106 instrument and were
consistent with theoretical values within 0.4% range. The mass
spectra were recorded with an Esquire 3000 LC-MS mass spec-
trometer. Purity analysis was determined using an Agilent 1100
Serial HPLC (Agilent Company, US); A C18 column (ZORBAX(tm)
In this study, we designed and synthesized a series of isoflavone
analogs and evaluated their anti-HBV and anti-cancer activities
in vitro. Among them, compound 8f seems to be the most promising
drug candidate, due to its potent anti-HBV activity and relatively
low toxicity in HepG2 2.2.15 and L02 cell lines. This compound also
exerted strong inhibitory effect on the growth of two human
hepatoma cell lines (QGY-7703 and Huh-7) further supporting the
rationale of the design. As such, compound 8f, a representative
isoflavone analog, seems like a promising drug candidate for the
treatment of both HBV infection and HBV-related HCC. In addition,
compound 7l exhibited high activity in inhibiting the growth of A-
4.6 ꢃ 150 mm, 5
mm, PN: 993967906 SN: USRK042041). TLC anal-
ysis was carried out on silica gel plates GF254 (Qindao Haiyang
Chemical, China). Silica gel thin-layer chromatography was per-
formed with Silica gel 60 G (Qindao Haiyang Chemical, China). All
solvents and reagents were analytically pure, and no further puri-
fication was needed. All starting materials were commercially
available.

Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
163
6.1. 3-Iodo-7-methoxy-4H-chromen-4-one (5)
1H, J ¼ 2.4 Hz, AreH), 6.93 (d, 1H, J ¼ 7.5 Hz, AreH), 7.00e7.03 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, AreH), 7.19e7.24 (t, 1H, AreH), 7.37 (d,
1H, J ¼ 15.0 Hz, O]CeCH]C), 7.48 (d, 2H, J ¼ 12.9 Hz, AreH), 7.71
(d, 1H, J ¼ 15.0 Hz, O]CeC]CH), 8.07 (s, 1H, OeCH]C), 8.18 ppm
Color: Yellow; m.p.: 158.5e159.2 ꢀC; Yield: 92%; 1H NMR:
(300 MHz, CDCl3)
H), 7.01 (dd,1H, J1 ¼9.0 Hz, J2 ¼ 2.4 Hz, AreH), 8.15 (d,1H, J ¼ 9.0 Hz,
AreH), 8.23 ppm (s, 1H, OeCH]C); 13C NMR (75 MHz, DMSO-d6)
d
: 3.91 (s, 3H, CH3eO), 6.84 (d, 1H, J ¼ 2.4 Hz, Are
(d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d: 21.40, 55.83,
d:
100.17, 115.07, 116.84, 118.01, 119.06, 120.36, 125.03, 125.26, 127.57,
128.74,133.08, 137.96,138.83,157.16,157.78,164.26 ppm; HRMS (m/
z): 336.12 (M þ 1)þ; Anal. (C20H17NO4): C, H, N.
56.16, 86.93, 100.54, 114.92, 115.38, 127.03, 157.52, 158.59, 163.93,
171.96 ppm; HRMS (m/z): 302.95 (M þ 1)þ; Anal. (C10H7IO3): C, H.
6.2. 7-Hydroxy-3-iodo-4H-chromen-4-one (6)
6.3.4. (E)-3-(7-methoxy-4-oxo-4H-chromen-3-yl)-N-p-
tolylacrylamide (7d)
To a solution of 3-iodo-7-methoxy-4H-chromen-4-one (33 mmol,
10 g) in dry toluene (250 ml) was added AlCl3 (50 mmol, 6.6 g) with
stirring at 100 ꢀC for 10 h under N2 protection. The reaction mixture
was concentrated under reduced pressure to remove toluene. Then
the reaction residue was diluted with EtOAc and poured into 10% HCl
iceewater with stirring. The organic layer was separated and the
aqueous layer was extracted with EtOAc (200 ml) twice. The
combined organic layers were dried over MgSO4 and concentrated
under reduced pressure to dryness to provide compound 6. Color:
Yellow; m.p.: 192.2e193.5 ꢀC; Yield: 93%; 1H NMR: (300 MHz, DMSO-
Color: Yellow; m.p.: 242.2e243.3 ꢀC; Yield: 54%; 1H NMR:
(300 MHz, CDCl3) d: 2.33 (s, 3H, CH3), 3.93 (s, 3H, CH3eO), 6.87 (d,1H,
J ¼ 2.1 Hz, AreH), 7.01e7.04 (dd, 2H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, AreH),
7.15 (d, 1H, J ¼ 8.1 Hz, AreH), 7.37 (d, 1H, J ¼ 15.0 Hz, O]CeCH]C),
7.50 (d, 2H, J ¼ 8.4 Hz, AreH), 7.69 (d, 1H, J ¼ 15.0 Hz, O]CeC]
CH), 8.07 (s, 1H, OeCH]C), 8.19 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C
NMR (75 MHz, CDCl3) d: 21.36, 55.88, 100.24, 115.07, 118.02, 119.10,
121.64, 122.84, 127.31, 128.96, 133.23, 135.94, 141.52, 150.65, 157.14,
164.01, 164.34, 176.54; HRMS (m/z): 336.12 (M þ 1)þ; Anal.
(C20H17NO4): C, H, N.
d6)
d
: 6.85(d,1H, J ¼ 2.1 Hz, AreH), 6.94(dd,1H, J1 ¼2.4 Hz, J2 ¼ 2.1 Hz,
AreH), 7.89 (d,1H, J ¼ 8.7 Hz), 8.67 (s,1H, OeCH¼ C),10.95(s,1H, OH);
6.3.5. (E)-N-(2,5-Dimethylphenyl)-3-(7-methoxy-4-oxo-4H-
chromen-3-yl)acrylamide (7e)
13C NMR (75 MHz, DMSO-d6)
d: 68.9, 101.5, 104.6, 114.2, 131.1, 150.5,
158.5,186.5 ppm; HRMS (m/z):288.93(M þ 1)þ; Anal. (C9H5IO3): C, H.
Color: Yellow; m.p.: 248.7e249.0 ꢀC; Yield: 60%; 1H NMR:
(300 MHz, CDCl3) d: 2.26 (s, 3H, CH3), 2.34 (s, 3H, CH3), 3.93 (s, 3H,
6.3. General procedure for the synthesis of compounds 7 and 8
CH3eO), 6.87e6.91 (t, 2H, AreH), 7.01e7.09 (m, 2H, AreH), 7.14 (s,
1H, AreH), 7.39 (d, 1H, J ¼ 15.0 Hz, O]CeCH]C), 7.75 (d, 1H,
J ¼ 14.7 Hz, O]CeC]CH), 8.08 (s, 1H, OeCH]C), 8.19 ppm (d, 1H,
To a solution of compound 5 (6) (3 mmol) and substituted
acrylamide (3.3 mmol) in dry DMF was added Pd(OAc)2 (3%) and
triethylamine (6 mmol). The reaction mixture was stirred under
100 ꢀC via microwave (500 w) for 7 min. Then the mixture was
poured into 10% HCl ice water (200 ml) slowly with stirring. The
suspension was filtered through a filter and filter cake was collected
and dried. The crude product was purified by chromatography over
silica gel to give compounds 7 or 8.
J ¼ 8.7 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d: 17.41, 21.13, 55.90,
100.24, 115.18, 118.07, 119.10, 123.31, 125.14, 125.77, 127.56, 130.22,
133.26, 135.62, 136.47, 157.24, 157.90, 164.34, 175.95 ppm; HRMS
(m/z): 350.13 (M þ 1)þ; Anal. (C21H19NO4): C, H, N.
6.3.6. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-(2-
methoxyphenyl)acrylamide (7f)
Color: Yellow; m.p.: 196.6e198.5 ꢀC; Yield: 66%; 1H NMR:
6.3.1. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-
phenylacrylamide (7a)
(300 MHz, CDCl3) d: 3.94 (s, 3H, CH3eO), 3.97 (s, 3H, CH3eO), 6.90
(d, 2H, J ¼ 7.5 Hz, AreH), 6.93 (d, 1H, J ¼ 7.2 Hz, AreH), 7.01e7.14 (m,
3H, AreH), 7.37 (d, 1H, J ¼ 15.3 Hz, O]CeCH]C), 7.77 (d, 1H,
J ¼ 15.3 Hz, O]CeC]CH), 8.01 (s, 1H, OeCH]C), 8.20 ppm (d, 1H,
Color: Yellow; m.p.: 222.5e223.9 ꢀC; Yield: 79%; 1H NMR:
(300 MHz, CDCl3)
d
: 3.93 (s, 3H, CH3eO), 6.87 (d, 1H, J ¼ 3.0 Hz, Are
H), 7.02e7.05 (dd, 2H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, AreH), 7.30e7.43 (m,
5H, AreH, O]CeCH]C), 7.72 (d, 1H, J ¼ 12.0 Hz, O]CeC]CH),
8.09 (s, 1H, OeCH]C), 8.19 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C
J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d: 55.56, 55.85, 100.17,
109.80, 115.13, 118.02, 119.09, 119.75, 120.99, 123.67, 125.55, 127.49,
127.85, 132.86, 147.88, 157.17, 157.83, 164.25, 175.88 ppm; HRMS (m/
z): 352.12 (M þ 1)þ; Anal. (C20H17NO5): C, H, N.
NMR (75 MHz, CDCl3) d: 55.83,100.16,114.95,116.48,119.56,120.36,
121.01, 127.55, 128.32, 128.64, 133.86, 137.20, 149.64, 157.26, 157.77,
164.74; HRMS (m/z): 322.11 (M þ 1)þ; Anal. (C19H15NO4): C, H, N.
6.3.7. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-(3-
methoxyphenyl)acrylamide (7g)
6.3.2. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-o-
tolylacrylamide (7b)
Color: Yellow; m.p.: 188.9e189.1 ꢀC; Yield: 63%; 1H NMR:
(300 MHz, CDCl3) d: 3.85 (s, 3H, CH3eO), d: 3.93 (s, 3H, CH3eO),
Color: Yellow; m.p.: 213.1e215.7 ꢀC; Yield: 69%; 1H NMR:
6.86 (d, 1H, J ¼ 2.4 Hz, AreH), 6.93 (d, 1H, J ¼ 7.5 Hz, AreH), 7.00
(d, 1H, J ¼ 2.4 Hz, AreH), 7.03 (d, 1H, J ¼ 2.4 Hz, AreH), 7.18e7.24 (t,
1H, AreH), 7.37 (d, 1H, J ¼ 15.0 Hz, O]CeCH]C), 7.41 (d, 2H,
J ¼ 3.9 Hz, AreH), 7.48 (d, 2H, J ¼ 15.0 Hz), 7.72 (d, 1H, J ¼ 15.0 Hz,
O]CeC]CH), 8.07 (s, 1H, OeCH]C), 8.18 ppm (d, 1H, J ¼ 9.0 Hz,
(300 MHz, CDCl3) d: 3.32 (s, 3H, CH3), 3.94 (s, 3H, CH3eO), 6.89 (d,
1H, J ¼ 2.4 Hz, AreH), 7.02e7.06 (dd, 1H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, Are
H), 7.11 (d, 1H, J ¼ 6.9 Hz, AreH), 7.19e7.28 (m, 2H, AreH), 7.41 (d,
1H, J ¼ 15.0 Hz, O]CeCH]C), 7.77 (d, 1H, J ¼ 15.3 Hz, O]CeC]
CH), 8.04e8.07 (m, 1H, AreH), 8.09 (s, 1H, OeCH]C),
d8.20 ppm
AreH); 13C NMR (75 MHz, CDCl3)
d: 55.55, 55.85, 100.15, 109.80,
(d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d
: 18.33, 55.85,
114.65, 117.00, 118.61, 118.97, 121.16, 123.67, 126.94, 129.44, 136.55,
139.28, 142.06, 150.48, 157.17, 160.61, 166.03, 167.64, 176.12 ppm;
HRMS (m/z): 352.11 (M þ 1)þ; Anal. (C20H17NO5): C, H, N.
100.20, 114.05, 114.45, 116.84, 118.06, 121.76, 125.09, 127.57, 128.74,
129.26, 130.86, 131.94, 133.62, 140.95, 148.55, 157.78, 164.26, 167.86,
172.17; HRMS (m/z): 336.12 (M þ 1)þ; Anal. (C20H17NO4): C, H, N.
6.3.8. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-(4-
methoxyphenyl)acrylamide (7h)
6.3.3. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-m-
tolylacrylamide (7c)
Color: Yellow; m.p.: 236.3e236.8 ꢀC; Yield: 74.6%; 1H NMR:
Color: Yellow; m.p.: 199.8e200.6 ꢀC; Yield: 65%; 1H NMR:
(300 MHz, CDCl3)
d: 3.81 (s, 3H, CH3eO), 3.93 (s, 3H, CH3eO), 6.87e
(300 MHz, CDCl3) d: 2.35 (s, 3H, CH3), 3.92 (s, 3H, CH3eO), 6.87 (d,
6.90 (m, 2H, AreH), 7.02 (dd, 2H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, AreH), 7.36

164
Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
(s, 1H, AreH), 7.36 (d, 1H, J ¼ 14.7 Hz, O]CeCH]C), 7.54 (d, 1H,
J ¼ 9.0 Hz, AreH), 7.70 (d,1H, J ¼ 15.0 Hz, O]CeC]CH), 8.06 (d, 1H,
J ¼ 3.0 Hz, OeCH]C), 8.19 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR
1H, OeCH]C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR
(75 MHz, CDCl3) d: 55.65, 100.03, 114.95, 117.82, 118.75, 121.34,
122.51, 124.30, 124.54, 127.41, 128.76, 133.81, 134.60, 156.96, 157.81,
(75 MHz, CDCl3)
d
: 56.74, 57.17,101.50,115.45, 116.40,122.75, 126.51,
164.15, 175.55 ppm; HRMS (m/z): 357.06 (M
(C19H14ClNO4): C, H, N.
þ
1)þ; Anal.
128.90, 134.16, 158.50, 159.04, 165.59 ppm; HRMS (m/z): 352.11
(M þ 1)þ; Anal. (C20H17NO5): C, H, N.
6.3.14. (E)-N-benzyl-3-(7-methoxy-4-oxo-4H-chromen-3-yl)
6.3.9. (E)-N-(4-Ethoxyphenyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7i)
acrylamide (7n)
Color: Yellow;
(300 MHz, CDCl3)
M.P
.: 257.1e257.5 ꢀC; Yield: 82%; 1H NMR:
Color: Yellow; m.p.: 213.3e213.9 ꢀC; Yield: 44%; 1H NMR:
d
: 3.93 (s, 3H, CH3eO), 4.53 (d, 2H, J ¼ 6.0 Hz,
(300 MHz, CDCl3)
d: 1.39e1.43 (t, 3H, CH3eCH2), 3.93 (s, 3H, CH3e
CH2eAr), 5.96 (s,1H, NH), 6.85e6.90 (dd,1H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz,
AreH), 6.98e7.03 (m, 1H, AreH), 7.28e7.37 (m, 6H, AreH, O]Ce
CH]C), 7.74 (d, 1H, J ¼ 15.0 Hz, O]CeC]CH), 8.03 (s, 1H, Oe
CH]C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz,
O), 4.04 (dd, 2H, J1 ¼ 6.9 Hz, J2 ¼ 6.9 Hz, CH2eCH3), 6.86e6.89 (m,
2H, AreH), 7.02 (dd, 2H, J1 ¼ 2.1 Hz, J2 ¼ 2.1 Hz, AreH), 7.36 (d, 1H,
J ¼ 15.3 Hz, O]CeCH]C), 7.40 (s, 1H, AreH), 7.52 (d, J ¼ 15.0 Hz,
AreH), 7.70 (d, 1H, J ¼ 15.3 Hz, O]CeC]CH), 8.07 (s, 1H, OeCH]
CDCl3) d: 43.01, 55.87, 100.20, 115.20, 116.45, 119.11, 125.74, 126.01,
C), 8.19 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d:
127.82, 129.33,129.44, 130.05, 135.54, 150.65, 157.85,164.54, 166.25,
15.10, 56.71, 57.15 100.95, 115.45, 116.61, 122.84, 126.88, 129.11,
134.55, 158.94, 159.16, 165.64, 166.56, 177.26 ppm; HRMS (m/z):
366.13 (M þ 1)þ; Anal. (C21H19NO5): C, H, N.
175.79 ppm; HRMS (m/z): 336.12 (M þ 1)þ; Anal. (C20H17NO4): C, H,
N.
6.3.15. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-(4-
methylbenzyl)acrylamide (7o)
6.3.10. (E)-N-(4-Fluorophenyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7j)
Color: Yellow; m.p.: 192.1e193.4 ꢀC; Yield: 55%; 1H NMR:
Color: Yellow; m.p.: 240.1e240.8 ꢀC; Yield: 73%; 1H NMR:
(300 MHz, CDCl3)
d
: 2.33 (d, 3H, J ¼ 6.0 Hz, CH3eAr), 3.92 (s, 3H,
(300 MHz, CDCl3)
d
: 3.93 (s, 3H, CH3eO), 6.88 (d, 2H, J ¼ 2.4 Hz, Are
CH3eO), 4.53 (d, 2H, J ¼ 6.0 Hz, CH2eAr), 5.92 (s, 1H, NH), 6.86 (d,
1H, J ¼ 3.0 Hz, AreH), 7.00 (dd, 2H, J1 ¼ 2.1 Hz, J2 ¼ 2.4 Hz, AreH),
7.18 (dd, 2H, J1 ¼ 8.1 Hz, J2 ¼ 7.5 Hz), 7.31 (d, 1H, J ¼ 15.3 Hz, O]Ce
CH]C), 7.54 (d, 1H, J ¼ 14.7 Hz, O]CeC]CH), 8.03 (s, 1H, OeCH]
H), 7.03 (dd, 2H, J1 ¼1.2 Hz, J2 ¼ 2.1 Hz, AreH), 7.06 (d,1H, J ¼ 7.2 Hz,
AreH), 7.37 (d, 1H, J ¼ 15.0 Hz, O]CeCH]C), 7.58 (dd, 2H,
J1 ¼ 5.1 Hz, J2 ¼ 5.4 Hz, AreH), 7.70 (d, 1H, J ¼ 15.0 Hz, O]CeC]
CH), 8.08 (s, 1H, OeCH]C), 8.19 ppm (d, 1H, J ¼ 9.0 Hz, AreH);
C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d:
13C NMR (75 MHz, CDCl3)
d
: 55.65, 100.03, 115.18, 117.23, 118.12,
29.65, 43.02, 55.87, 100.19, 115.32, 119.05, 124.47, 127.54, 129.33,
129.44, 132.62, 133.97, 157.19, 157.58, 164.26, 166.25, 175.83 ppm;
HRMS (m/z): 350.13 (M þ 1)þ; Anal. (C21H19NO4): C, H, N.
121.30, 122.51, 125.65, 125.95, 126.32, 128.66, 133.81, 134.76, 156.89,
157.81, 166.24, 175.55; HRMS (m/z): 340.08 (M þ 1)þ; Anal.
(C19H14FNO4): C, H, N.
6.3.16. (E)-3-(7-Methoxy-4-oxo-4H-chromen-3-yl)-N-(4-
methoxybenzyl)acrylamide (7p)
6.3.11. (E)-N-(2-Chlorophenyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7k)
Color: Yellow; m.p.: 219.4e220.6 ꢀC; Yield: 36%; 1H NMR:
Color: Yellow; m.p.: 205.5e205.8 ꢀC; Yield: 54%; 1H NMR:
(300 MHz, CDCl3) d: 3.81 (s, 3H, CH3eO), 3.93 (s, 3H, CH3eO), 4.50
(300 MHz, CDCl3)
d
: 3.93 (s, 3H, CH3eO), 6.89 (d, 1H, J ¼ 2.1 Hz, Are
(d, 2H, J ¼ 6.0 Hz, CH2eAr), 5.92 (s, 1H, NH), 6.91 (d, 2H, J ¼ 3.0 Hz,
AreH), 7.02e7.05 (m, 1H, AreH), 7.29e7.41 (m, 2H, AreH, O]Ce
CH]C), 7.55 (d, 2H, J ¼ 8.7 Hz, AreH), 7.70 (d, 1H, J ¼ 15.0 Hz,
O]CeC]CH), 8.08 (s, 1H, AreH), 8.20 ppm (d, 1H, J ¼ 9.0 Hz, Oe
H), 7.03e7.08 (m, 2H, AreH), 7.27e7.31 (m, 1H, AreH), 7.38 (s, 1H,
AreH) 7.41 (d,1H, J ¼ 15.0 Hz, O]CeCH]C), 7.76 (d,1H, J ¼ 15.0 Hz,
O]CeC]CH), 7.80 (s, 1H, AreH), 8.10 (s, 1H, OeCH]C), 8.19 ppm
(d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d
: 55.64, 100.10,
CH]C); 13C NMR (75 MHz, CDCl3)
d: 43.02, 55.87, 57.05, 100.19,
114.65, 117.48, 119.54, 121.24, 122.51, 124.48, 127.44, 129.07, 132.71,
141.61, 149.69, 157.45, 164.15, 167.15, 175.50 ppm; HRMS (m/z):
357.06 (M þ 1)þ; Anal. (C19H14ClNO4): C, H, N.
116.11, 119.05, 125.23, 127.55, 129.24, 131.45, 132.58, 135.82 157.19,
157.58, 165.49, 166.25, 175.83 ppm; HRMS (m/z): 366.13 (M þ 1)þ;
Anal. (C21H19NO5): C, H, N.
6.3.12. (E)-N-(3-Chlorophenyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7l)
6.3.17. (E)-N-(2-Fluorobenzyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7q)
Color: Yellow; m.p.: 222.8e223.1 ꢀC; Yield: 43%; 1H NMR:
Color: Yellow; m.p.: 228.6e229.3 ꢀC; Yield: 77%; 1H NMR:
(300 MHz, CDCl3)
d
: 3.93 (s, 3H, CH3eO), 6.88 (d, 1H, J ¼ 2.4 Hz, Are
(300 MHz, CDCl3)
d
: 3.92 (s, 3H, CH3eO), 4.63 (d, 2H, J ¼ 6.0 Hz,
H), 7.01e7.08 (m, 1H, AreH), 7.11 (dd, 1H, J1 ¼ 6.0 Hz, J2 ¼ 6.0 Hz,
AreH), 7.23e7.28 (m, 1H, AreH), 7.38 (d, 1H, J ¼ 14.7 Hz, O]Ce
CH]C), 7.43e7.47 (m, 2H, AreH) 7.72 (d, 1H, J ¼ 15.3 Hz, O]Ce
C]CH), 8.09 (s, 1H, OeCH]C), 8.18 ppm (d, 1H, J ¼ 9.0 Hz, Are
CH2eAr), 6.03 (d, 1H, J ¼ 5.1 Hz, NH), 6.86 (d, 1H, J ¼ 5.1 Hz, AreH),
6.99e7.13 (m, 2H, AreH), 7.23 (d, 1H, J ¼ 1.5 Hz, AreH), 7.30 (d, 1H,
J ¼ 15.0 Hz, O]CeCH]C), 7.35e7.40 (m, 1H, AreH), 7.54 (d, 1H,
J ¼ 15.3 Hz, O]CeC]CH), 8.03 (s, 1H, OeCH]C), 8.16 ppm (d, 1H,
H); 13C NMR (75 MHz, CDCl3)
d: 55.64, 100.10, 114.66, 117.88,
J ¼ 12.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d: 38.32, 55.88,
119.01, 121.55 122.56, 124.84, 127.54, 129.41, 132.75, 141.66, 149.41,
157.45, 164.18, 167.15, 175.50 ppm; HRMS (m/z): 357.06 (M þ 1)þ;
Anal. (C19H14ClNO4): C, H, N.
100.09, 113.53, 115.99, 117.38, 119.32, 125.32, 126.53, 127.55, 128.84,
129.11, 130.92, 144.53, 149.84, 158.89, 162.43, 163.28, 165.44,
176.03 ppm; HRMS (m/z): 354.11 (M þ 1)þ; Anal. (C20H16FNO4): C,
H, N.
6.3.13. (E)-N-(4-Clorophenyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7m)
6.3.18. (E)-N-(3-Fluorobenzyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7r)
Color: Yellow; m.p.: 213.9e214.1 ꢀC; Yield: 65%; 1H NMR:
(300 MHz, CDCl3)
d
: 3.92 (s, 3H, CH3eO), 6.88 (d, 1H, J ¼ 2.4 Hz, Are
Color: Yellow; m.p.: 98e100 ꢀC; Yield: 76%; 1H NMR: (300 MHz,
H), 7.06 (dd, 2H, J1 ¼ 7.5 Hz, J2 ¼ 8.7 Hz, AreH), 7.27e7.33 (m, 1H,
AreH), 7.40 (d,1H, J ¼ 15.0 Hz, O]CeCH]C), 7.38e7.43 (m,1H, Are
H), 7.77 (d, 1H, J ¼ 15.0 Hz, O]CeC]CH), 7.85 (s, 1H, AreH), 8.11 (s,
CDCl3)
d
: 3.93 (s, 3H, CH3eO), 4.58 (d, 1H, J ¼ 6.0 Hz, CH2eAr), 6.03
(s, 1H, NH), 6.74 (s, 1H, AreH), 6.87 (d, 1H, J ¼ 2.4 Hz, AreH), 6.94e
7.00 (m, 2H, AreH), 7.09 (d, 1H, J ¼ 7.5 Hz, AreH), 7.28e7.36 (m, 2H,

Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
165
AreH, O]CeCH]C), 7.58 (d, 1H, J ¼ 15.0 Hz, O]CeC]CH), 8.05 (s,
1H, OeCH]C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR
AreH), 6.99e7.03 (m, 3H, AreH), 7.15e7.20 (m, 2H, AreH), 7.27 (d,
1H, J ¼ 15.0 Hz, O]CeCH]C), 7.45 (d, 1H, J ¼ 15.0 Hz, O]CeC]
CH), 8.03 (s, 1H, OeCH]C), 8.15 ppm (d, 1H, J ¼ 8.7 Hz, AreH);
(75 MHz, CDCl3)
d: 44.53, 55.32, 100.10, 113.43, 116.33, 118.96,
119.83, 120.32, 122.58, 127.07, 127.76, 140.43, 143.32, 149.99, 160.22,
162.48, 163.02, 165.89, 175.88 ppm; HRMS (m/z): 354.11 (M þ 1)þ;
Anal. (C20H16FNO4): C, H, N.
13C NMR (75 MHz, CDCl3)
d: 35.30, 40.75, 55.40, 100.11, 114.99,
115.13, 116.45, 119.03, 123.35, 127.54, 131.00, 135.53.53, 141, 156.32,
158.44, 161.78, 166.32, 177.83 ppm; HRMS (m/z): 368.13 (M þ 1)þ;
Anal. (C21H18FNO4): C, H, N.
6.3.19. (E)-N-(4-Fluorobenzyl)-3-(7-methoxy-4-oxo-4H-chromen-
3-yl)acrylamide (7s)
6.3.24. (E)-3-(7-Hydroxy-4-oxo-4H-chromen-3-yl)-N-
phenylacrylamide (8a)
Color: Yellow; m.p.: 241.6e243.1 ꢀC; Yield: 53%; 1H NMR:
(300 MHz, CDCl3)
d
: 3.92 (s, 3H, CH3eO), 4.55 (d, 2H, J ¼ 6.0 Hz,
Color: Yellow; m.p.: 282.8e229.1 ꢀC; Yield: 72%; 1H NMR:
CH2eAr), 5.92 (s, 1H, NH), 6.86 (d, 1H, J ¼ 2.4 Hz, AreH), 6.99e7.05
(m, 3H, AreH), 7.32 (d, 1H, J ¼ 15.3 Hz, O]CeCH]C), 7.28e7.34 (m,
2H, AreH), 7.54 (d, 1H, J ¼ 15.3 Hz, O]CeC]CH), 8.04 (s, 1H, Oe
CH]C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz,
(300 MHz, DMSO-d6)
d
: 6.86 (d, 1H, J ¼ 2.1 Hz, AreH), 6.93 (dd, 1H,
J1 ¼2.1 Hz, J2 ¼ 2.1 Hz, AreH), 7.01 (dd,1H, J1 ¼2.1 Hz, J2 ¼ 2.1 Hz, Are
H), 7.04e7.28 (m, 2H, AreH), 7.33 (s,1H, AreH), 7.65 (d,1H, J ¼ 7.5 Hz),
7.30 (d,1H, J ¼ 15.0 Hz, O]CeCH]C), 7.77 (d,1H, J ¼ 15.0 Hz, O]Ce
C]CH), 8.03 (d,1H, J ¼ 8.7 Hz, AreH), 8.81 (s,1H, OeCH]C),10.30 (s,
CDCl3) d: 43.53, 55.32, 100.10, 114.49, 115.88, 116.59, 119.48, 123.08,
126.18, 129.99, 133.54, 143.15, 149.46, 155.87, 160.48, 165.11,
175.88 ppm; HRMS (m/z): 354.11 (M þ 1)þ; Anal. (C20H16FNO4): C,
H, N.
1H, NH), 10.97 ppm (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6)
d:
102.38, 115.59, 116.37, 118.22, 119.27, 123.20, 124.65, 127.25, 128.70,
132.67, 139.47, 156.87, 159.18, 162.90, 164.18, 174.76 ppm; HRMS (m/
z): 308.10 (M þ 1)þ; Anal. (C18H13NO4): C, H, N.
6.3.20. (E)-N-(2,4-Difluorobenzyl)-3-(7-methoxy-4-oxo-4H-
chromen-3-yl)acrylamide (7t)
6.3.25. (E)-3-(7-Hydroxy-4-oxo-4H-chromen-3-yl)-N-o-
tolylacrylamide (8b)
Color: Yellow; m.p.: 130.6e131.3 ꢀC; Yield: 65%; 1H NMR:
(300 MHz, CDCl3)
d
: 3.92 (s, 3H, CH3eO),
d
4.58 (d, 2H, J ¼ 6.3 Hz,
Color: White; m.p.: 316.8e317.3 ꢀC; Yield: 52%; 1H NMR:
CH2eAr), 6.02 (m,1H, NH), 6.82e6.87 (m, 3H, AreH), 6.99e7.03 (dd,
1H, J1 ¼ 2.4 Hz, J2 ¼ 2.4 Hz, AreH), 7.25e7.42 (m, 2H, AreH, O]Ce
CH]C), 7.53 (d, 1H, J ¼ 15.3 Hz, O]CeC]CH), 8.03 (s, 1H, OeCH]
(300 MHz, DMSO-d6)
d
: 2.22 (s, 1H, CH3eAr), 6.90 (d, 1H, J ¼ 8.7 Hz,
AreH), 6.92e6.96 (m, 2H, AreH), 7.03 (d, 1H, J ¼ 7.5 Hz, AreH),
7.23e7.28 (m, 2H, AreH, O]CeCH]C), 7.60 (d, 1H, J ¼ 15.0 Hz,
O]CeC]CH), 7.94 (d, 1H, J ¼ 8.7 Hz, AreH), 8.71 (s, 1H, OeCH]C),
9.52 (s, 1H, NH), 10.92 ppm (s, 1H, OH); 13C NMR (75 MHz, DMSO-
C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d:
36.16, 55.17, 101.25, 104.22, 111.16, 114.98, 116.34, 119.51, 121.65,
125.18, 127.46, 130.87, 131.94, 157.92, 159.32, 161.81, 163.87, 165.48,
175.87 ppm; HRMS (m/z): 372.10 (M þ 1)þ; Anal. (C20H15F2NO4): C,
H, N.
d6)
d: 18.26, 102.40, 114.65, 115.60, 116.21, 118.14, 120.03, 123.35,
125.01, 127.25, 128.88, 129.32, 130.11, 139.15, 156.55, 160.44, 162.39,
164.20, 175.76 ppm; HRMS (m/z): 322.10 (M
(C19H15NO4): C, H, N.
þ
1)þ; Anal.
6.3.21. (E)-N-(2-Fluorophenethyl)-3-(7-methoxy-4-oxo-4H-
chromen-3-yl)acrylamide (7u)
6.3.26. (E)-3-(7-Hydroxy-4-oxo-4H-chromen-3-yl)-N-(4-
Color: White; m.p.: 250.2e250.8 ꢀC; Yield: 80%; 1H NMR:
methoxyphenyl)acrylamide (8c)
(300 MHz, CDCl3)
d
: 2.91e2.96 (m, 2H, CH2eAr), 3.63e3.69 (m, 2H,
Color: Yellow; m.p.: 324.1e324.5 ꢀC; Yield: 48%; 1H NMR:
CH2eN), 3.92 (s, 3H, CH3eO), 5.72 (s,1H, NH), 6.86 (d,1H, J ¼ 2.4 Hz,
AreH), 7.00e7.11 (m, 3H, AreH), 7.19e7.25 (m, 2H, AreH), 7.28 (d,
1H, J ¼ 15.3 Hz, O]CeCH]C), 7.45 (d, 1H, J ¼ 15.0 Hz, O]CeC]
CH), 8.03 (s, 1H, OeCH]C), 8.16 ppm (d, 1H, J ¼ 9.0 Hz, AreH);
(300 MHz, DMSO-d6) d: 3.91 (s, 3H, CH3eO), 6.91e7.05 (m, 2H, Are
H), 7.11e7.31 (m, 3H, AreH), 7.42 (d, 1H, J ¼ 15.0 Hz, O]CeCH]C),
7.67 (d, 1H, J ¼ 8.7 Hz, AreH), 7.76 (d, 1H, J ¼ 15.3 Hz, O]CeC]CH),
8.07 (d, 1H, J ¼ 9.0 Hz, AreH), 8.23 (s, 1H, OeCH]C), 10.02 (s, 1H,
13C NMR (75 MHz, CDCl3)
d: 35.26, 40.75, 55.38, 100.10, 114.75,
NH), 10.93 ppm (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6)
d: 55.05,
115.23, 116.88, 119.04, 121.87, 124.98, 127.61, 127.95, 132.80, 141.17,
149.04, 158.00, 163.76, 166.90, 177.70 ppm; HRMS (m/z): 368.13
(M þ 1)þ; Anal. (C21H18FNO4): C, H, N.
102.33, 114.67, 115.60, 116.40, 119.43, 122.58, 123.22, 127.31, 129.53,
140.01,158.14,158.89,159.20,162.90,164.19,174.77 ppm; HRMS (m/
z): 328.10 (M þ 1)þ; Anal. (C19H15NO5): C, H, N.
6.3.22. (E)-N-(3-Fluorophenethyl)-3-(7-methoxy-4-oxo-4H-
chromen-3-yl)acrylamide (7v)
6.3.27. (E)-N-Benzyl-3-(7-hydroxy-4-oxo-4H-chromen-3-yl)
acrylamide (8d)
Color: White; m.p.: 252.6e253.0 ꢀC; Yield: 72%; 1H NMR:
Color: Yellow; m.p.: 352.6e253.5 ꢀC; Yield: 77%; 1H NMR:
(300 MHz, CDCl3)
d
: 2.86e2.91 (m, 2H, CH2eAr), 3.65e3.67 (m, 2H,
(300 MHz, DMSO-d6)
d
: 4.38 (d, 2H, J ¼ 6.0 Hz, CH2eAr), 6.88 (d, 1H,
CH2eN), 3.92 (s, 3H, CH3eO), 5.62 (s, 1H, NH), 6.86 (d,1H, J ¼ 2.4 Hz,
AreH), 6.86e6.95 (m, 2H, AreH), 6.99e7.03 (m, 2H, AreH), 7.26 (d,
1H, J ¼ 15.3 Hz, O]CeCH]C), 7.27 (s, 1H, AreH), 7.45 (d, 1H,
J ¼ 15.3 Hz, O]CeC]CH), 8.03 (s, 1H, OeCH]C), 8.16 ppm (d, 1H,
J ¼ 2.1 Hz, AreH), 6.95 (dd,1H, J1 ¼ 2.1 Hz, J2 ¼ 2.1 Hz, AreH), 7.21 (s,
1H, AreH), 7.23 (d, 1H, J ¼ 2.4 Hz, AreH), 7.23e7.35 (m, 4H, AreH,
O]CeCH]C), 7.40 (d, 1H, J ¼ 15.3 Hz, O]CeC]CH), 7.96 (d, 1H,
J ¼ 8.7 Hz, AreH), 8.63 (s,1H, OeCH]C), 8.73 (s,1H, NH),10.89 ppm
J ¼ 8.7 Hz, AreH); 13C NMR (75 MHz, CDCl3)
d: 35.31, 40.39, 55.74,
(s, 1H, OH); 13C NMR (75 MHz, DMSO-d6)
d: 42.22, 102.31, 115.50,
100.07, 113.48, 114.93, 115.30, 115.58, 117.94, 118.96, 124.25, 124.51,
127.40, 130.02, 132.12, 141.16, 157.07, 157.40, 164.13, 166.21,
175.72 ppm; HRMS (m/z): 368.13 (M þ 1)þ; Anal. (C21H18FNO4): C,
H, N.
116.32, 118.29, 124.36, 126.71, 127.22, 128.25, 131.31, 139.54, 156.87,
158.41, 162.80, 165.44, 174.68 ppm; HRMS (m/z): 322.09 (M þ 1)þ;
Anal. (C19H15NO4): C, H, N.
6.3.28. (E)-3-(7-Hydroxy-4-oxo-4H-chromen-3-yl)-N-(4-
methylbenzyl)acrylamide (8e)
6.3.23. (E)-N-(4-Fluorophenethyl)-3-(7-methoxy-4-oxo-4H-
chromen-3-yl)acrylamide (7w)
Color: Yellow; m.p.: 279.6e281.2 ꢀC; Yield: 63%; 1H NMR:
Color: Yellow; m.p.: 177.5e178.5 ꢀC; Yield: 83%; 1H NMR:
(300 MHz, DMSO-d6)
CH2eAr), 6.87 (d, 1H, J ¼ 2.1 Hz, AreH), 6.95 (dd, 1H, J1 ¼ 2.4 Hz,
d
: 2.25 (s, 3H, CH3eAr), 4.32 (d, 2H, J ¼ 6.0 Hz,
(300 MHz, CDCl3)
d
: 2.83e2.87 (m, 2H, CH2eAr) 3.60e3.66 (m, 2H,
CH2eN), 3.92 (s, 3H, CH3eO), 5.70 (s, 1H, NH), 6.86 (d, 1H, J ¼ 2.1 Hz,
J2 ¼ 2.1 Hz, AreH), 7.07e7.14 (m, 4H, AreH), 7.22 (d, 1H, J ¼ 15.6 Hz,

166
Y. Zhang et al. / European Journal of Medicinal Chemistry 62 (2013) 158e167
O]CeCH]C), 7.39 (d, 1H, J ¼ 15.6 Hz, O]CeC]CH), 7.96 (d, 1H,
J ¼ 8.7 Hz, AreH), 8.62 (s,1H, OeCH]C), 8.66 (s,1H, NH),10.89 ppm
antigens were determined by ELISA. Levels of HBV-DNA were
quantified by fluorescence PCR on an MJ Research PTC-200
instrument as previously described [17]. PCR primers used were
as follows: forward, 50-TGTCCTGGTTATCGCTGG-30; reverse,
50-CAAACGGGCAACATA-CCTT-30. Probe used was 50-(FAM)-
TGTGTCTGCGGCGTTTTATCAT-(TAMRA)-30. Data were analyzed by
Opticon Monitor software (version 2.01). The antiviral activities of
various compounds are summarized in Table 1.
(s, 1H, OH); 13C NMR (75 MHz, DMSO-d6)
d: 20.75, 42.11, 102.43,
115.64, 116.43, 118.41, 124.48, 127.36, 128.92, 131.40, 135.91, 136.56,
157.01, 158.43, 162.93, 165.57, 174.85 ppm; HRMS (m/z): 336.12
(M þ 1)þ; Anal. (C20H17NO4): C, H, N.
6.3.29. (E)-3-(7-Hydroxy-4-oxo-4H-chromen-3-yl)-N-(4-
methoxybenzyl)acrylamide (8f)
Color: Yellow; m.p.: 270.0e270.3 ꢀC; Yield: 61%; 1H NMR:
6.4.2. In vitro cytotoxicity study of target compounds
(300 MHz, DMSO-d6)
d
: 3.71 (s, 3H, CH3eO), 4.29 (d, 2H, J ¼ 5.7 Hz,
Cytotoxicity of all synthesized compounds was assessed by MTT
assays as previously described [18]. The compounds, dissolved in
DMSO, were serially diluted in cell culture media and then added
onto cell monolayers. HepG2 2.2.15 cells were maintained in 96-well
tissue culture plates for 48 h before being treated with compounds.
The cells were subsequently incubated at 37 ꢀC for 9 days. Cells with
media alone were used as the blank control, while adefovir was used
as positive controls. MTT reagents were added at a concentration of
5 g/L at4 h beforethecellswereharvested andlysedwith10%sodium
dodecyl sulfate (SDS) and 50% N,N-dimethylformamide (DMF), pH
7.2. The ODvalues of cell lysates were readat awavelength of 570 nm,
and the percentages of cell death were determined.
CH2eAr), 6.83e6.88 (m, 2H, AreH), 6.92e7.00 (m, 3H, AreH), 7.13
(d, 1H, J ¼ 15.6 Hz, O]CeCH]C), 7.17e7.24 (m, 1H, AreH), 7.38 (d,
1H, J ¼ 15.6 Hz, O]CeC]CH), 7.92e7.97 (m, 1H, AreH), 8.62 (s, 1H,
OeCH]C), 8.66 (s, 1H, NH), 10.89 ppm (s, 1H, OH); 13C NMR
(75 MHz, DMSO-d6) d: 41.69, 55.03, 102.31, 113.66, 115.49, 116.33,
118.32, 124.48, 127.20, 128.60, 131.19, 131.50, 156.88, 158.17, 158.33,
162.80, 165.31, 174.69 ppm; HRMS (m/z): 352.11 (M þ 1)þ; Anal.
(C20H17NO5): C, H, N.
6.3.30. (E)-N-(4-Fluorobenzyl)-3-(7-hydroxy-4-oxo-4H-chromen-
3-yl)acrylamide (8g)
Color: Brown; m.p.: 297.7e298.2 ꢀC; Yield: 73%; 1H NMR:
(300 MHz, DMSO-d6)
d
: 4.25 (d, 2H, J ¼ 6.0 Hz, CH2eAr), 6.83e7.08
6.4.3. Screening of the in vitro anti-cancer activities
(m, 5H, AreH), 7.10e7.27 (m, 2H, AreH, O]CeCH]C), 7.50 (d, 1H,
J ¼ 15.6 Hz, O]CeC]CH), 8.46 (d, 1H, J ¼ 9.0 Hz, AreH), 8.75 (s, 1H,
OeCH]C), 8.77 (s, 1H, NH), 10.88 ppm (s, 1H, OH); 13C NMR
In vitro anti-cancer activity was determined by MTT
[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]
assay and manifested by IC50 of the compounds [18e20]. Various
cancer cell lines were obtained from Shanghai Institute of Biological
Sciences (SIBS), CAS. IC50 calculation was done according to the
National Committee for Clinical Laboratory Standards (NCCLS). All
compounds were dissolved in DMSO with the stock concentration
of 10 g/L (stored at 4 ꢀC), and diluted with fresh medium promptly
before drug administration. Cells were seeded in 96-well plates at
the density of 8000 cells/well. 24 h later, compounds at various
concentrations were applied in duplicate to cells, which were
incubated at 37 ꢀC in a humidified incubator with 5% CO2. After
(75 MHz, DMSO-d6)
d: 35.81, 102.33, 111.23, 115.55, 116.33, 118.30,
122.21, 127.20, 128.26, 132.11, 156.43, 149.65, 158.50, 159.11, 162.71,
165.58, 177.68 ppm; HRMS (m/z): 340.10 (M
(C19H14FNO4): C, H, N.
þ
1)þ; Anal.
6.3.31. (E)-N-(2,4-Difluorobenzyl)-3-(7-hydroxy-4-oxo-4H-
chromen-3-yl)acrylamide (8h)
Color: Yellow; m.p.: 268.7e269.1 ꢀC; Yield: 47%; 1H NMR:
(300 MHz, DMSO-d6)
d: 4.36 (d, 2H, J ¼ 5.4 Hz, CH2eAr), 6.88 (d, 1H,
J ¼ 2.1 Hz, AreH), 6.95e7.08 (m, 2H, AreH), 7.17e7.24 (m, 2H, AreH,
O]CeCH]C), 7.38 (d, 1H, J ¼ 15.6 Hz, O]CeC]CH), 7.42 (d, 1H,
J ¼ 6.6 Hz, AreH), 7.96 (d, 1H, J ¼ 5.7 Hz, AreH), 8.62 (s, 1H, OeCH]
C), 8.73 (s, 1H, NH), 10.89 ppm (s, 1H, OH); 13C NMR (75 MHz,
48 h, 20 mL of MTT (Sigma) at 5 mg/ml in PBS (filter sterilized, light
protected, and stored at 4 ꢀC) was added to each well. After 4 h of
incubation at 37 ꢀC, MTT was converted to a blue formazan product
by mitochondrial succinate dehydrogenase. The yielding product
was eluted from cells by adding 150 ml of DMSO. The absorbance at
570 nm wavelength was determined with an ELX800 microplate
spectrophotometer. The IC50 value was defined as the concentration
under which 50% of the cells could survive.
DMSO-d6) d: 35.72, 102.31, 103.59, 111.10, 111.40, 115.50, 116.33,
118.24, 122.32, 122.53, 124.05, 127.20, 130.86, 131.56, 156.87, 158.51,
162.82, 165.58, 174.68 ppm; HRMS (m/z): 358.08 (M þ 1)þ; Anal.
(C19H13F2NO4): C, H, N.
6.3.32. (E)-N-(4-Fluorophenethyl)-3-(7-hydroxy-4-oxo-4H-
chromen-3-yl)acrylamide (8i)
Acknowledgments
Color: Brown; m.p.: 217.9e218.3 ꢀC; Yield: 77%; 1H NMR:
This work was supported by the National Natural Science
Foundation of China (Grant No. 21172259) and Shanghai Rising-Star
Program (Grant No. 09QA1407000). We also thank staffs at the Key
Laboratory of Drug Research for Special Environments of PLA.
(300 MHz, DMSO-d6) d: 2.72e2.85 (m, 2H, CH2eAr), 3.35e3.47 (m,
2H, CH2eN), 6.87 (d, 1H, J ¼ 2.1 Hz, AreH), 7.07e7.14 (m, 3H, AreH),
7.19e7.25 (m, 2H, AreH), 7.29 (d,1H, J ¼ 15.6 Hz, O]CeCH]C), 7.45
(d,1H, J ¼ 15.3 Hz, O]CeC]CH), 7.96 (s, 1H, OeCH]C), 8.46 (d,1H,
J ¼ 1.5 Hz, AreH), 8.65 (s, 1H, NH), 10.88 ppm (s, 1H, OH); 13C NMR
Appendix A. Supplementary data
(75 MHz, DMSO-d6) d: 34.23, 40.90, 102.40, 115.17, 115.65, 116.38,
118.35, 124.53, 127.16, 127.29, 128.36, 130.53, 135.72, 156.99, 158.41,
162.97, 165.57, 174.84 ppm; HRMS (m/z): 354.11 (M þ 1)þ; Anal.
(C20H16FNO4): C, H, N.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2012.09.017.
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