Antitumor agents 292. Design, synthesis and pharmacological study of S- and O-substituted 7-mercapto- or hydroxy-coumarins and chromones as potent cytotoxic agents

Article abstract of DOI:10.1016/j.ejmech.2011.12.025

Thirty-five S- and O-substituted 7-mercaptocoumarin (9-23) and 7-hydroxy- or 7-mercapto-chromone (24-43) analogs were designed, synthesized and evaluated in vitro against four human tumor cell lines [KB (nasopharyngeal), KB-vin (vincristine-resistant subline), A549 (lung) and DU145 (prostate)] with paclitaxel as the positive control. Many of the synthesized compounds exhibited potent cytotoxic activity. Among them, compounds 10 and 18 showed broad spectrum activity with GI₅₀ values ranging from 0.92 to 2.11 μM and 2.06-14.07 μM, respectively. However, 33, a 3-brominated compound, displayed significant and selective inhibition against MDR KB-vin with a GI₅₀ of 5.84 μM. Regardless of the size of the 7-alkoxy group, 2-α-bromoethyl-8-bromomethyl compounds (40-43) exhibited increased cytotoxicity compared with 2-ethyl-8-bromomethyl compounds (36-39). Moreover, in a preliminary pharmacological study, 10 not only remarkably increased cellular apoptosis in a concentration-dependent manner, but also clearly induced A549 cell cycle arrest at the G2/M phase. Thus, these coumarin derivatives merit investigation as novel potential antitumor agents with further structural modification to produce an optimal lead compound and elucidate the detailed pharmacological mechanism(s).

Full text of DOI:10.1016/j.ejmech.2011.12.025

European Journal of Medicinal Chemistry 49 (2012) 74e85
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antitumor agents 292. Design, synthesis and pharmacological study of S- and
O-substituted 7-mercapto- or hydroxy-coumarins and chromones as potent
cytotoxic agents
,
,
,
c
c
Ying Chen a 1, Hong-Rui Liu b 1, Hong-Shan Liu a, Ming Cheng a, Peng Xia a , Keduo Qian , Pei-Chi Wu ,
*
Chin-Yu Lai c, Yi Xia c, Zheng-Yu Yang c, Susan L. Morris-Natschke c, Kuo-Hsiung Lee c
,d,
**
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
b Department of Pharmacology, School of Pharmacy, Fudan University, Shanghai 201203, China
c Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA
d Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Thirty-five S- and O-substituted 7-mercaptocoumarin (9e23) and 7-hydroxy- or 7-mercapto-chromone
(24e43) analogs were designed, synthesized and evaluated in vitro against four human tumor cell lines
[KB (nasopharyngeal), KB-vin (vincristine-resistant subline), A549 (lung) and DU145 (prostate)] with
paclitaxel as the positive control. Many of the synthesized compounds exhibited potent cytotoxic activity.
Among them, compounds 10 and 18 showed broad spectrum activity with GI50 values ranging from 0.92
Received 11 October 2011
Received in revised form
15 December 2011
Accepted 16 December 2011
Available online 29 December 2011
to 2.11
significant and selective inhibition against MDR KB-vin with a GI50 of 5.84
the 7-alkoxy group, 2- -bromoethyl-8-bromomethyl compounds (40e43) exhibited increased cytotox-
m
M and 2.06e14.07
m
M, respectively. However, 33, a 3-brominated compound, displayed
m
M. Regardless of the size of
Keywords:
Coumarin
a
icity compared with 2-ethyl-8-bromomethyl compounds (36e39). Moreover, in a preliminary pharma-
cological study, 10 not only remarkably increased cellular apoptosis in a concentration-dependent
manner, but also clearly induced A549 cell cycle arrest at the G2/M phase. Thus, these coumarin
derivatives merit investigation as novel potential antitumor agents with further structural modification
to produce an optimal lead compound and elucidate the detailed pharmacological mechanism(s).
Ó 2011 Elsevier Masson SAS. All rights reserved.
Synthesis
Cytotoxicity
Cell cycle arrest
1. Introduction
osteoporosis, anti-HIV, and antitumor activities [2e5]. Accordingly,
many reports have described various structures and biological
Coumarins and their derivatives play an important role in the
agricultural and pharmaceutical industries. They are widely present
in higher plants such as Rutaceae, Apiaceae, Asteraceae, Legumi-
nosae, Thymelaeaceae, as well as occur as animal and microbial
metabolites [1]. Most of them show a wide spectrum of pharma-
cological effects, including antimicrobial, anti-arrhythmic, anti-
evaluations of numerous coumarin analogs newly synthesized or
isolated from plants. For example, in 2005, Andre reported that
coumarin (1, Fig. 1) and its analogs interact directly with cyto-
chrome P450 (CYP) to inhibit the mutagenicity of 2-amino-3-
methylimidazo[4,5-f]quinoline in Salmonella typhimurium TA98
[6], and libanoridin (2, Fig. 1), isolated from Corydalis heterocarpa in
2009, was found to have significant anti-inflammatory potency
against HT-29 human colon carcinoma cells [7]. Since the 1950s,
warfarin (3, Fig. 1) has been successfully used in the clinic to
prevent thromboembolic disease, and its analog tecarfarin (4, Fig.1)
was also found to be a novel orally active vitamin K epoxide
reductase inhibitor that can reduce the levels of vitamin K-depen-
dent coagulation factors (factors II, VII, IX, and X) and prolong
prothrombin time in canine and rabbit thrombosis models [8].
Abbreviations: HIV, Human immunodeficiency virus; CYP, Cytochrome P450;
MDR, Multidrug resistance; DMDCK, (ꢀ)-30-O-40-O-bis(3,4-dimethoxycinnamoyl)-
cis-khellactone; DSP, 30R,40R-disubstituted-20,20-dimethyldihydropyrano [2,3-f]
chromone; DCK, Dicamphanoyl-khellactone; DCP, Dicamphanoyl-dihydropyr-
anochromone; GI, Growth inhibition.
* Corresponding author. Tel./fax: þ86 21 51980116.
** Corresponding author. Natural Products Research Laboratories, UNC Eshelman
School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599-7568, USA.
Tel.: þ1 919 962 0066; fax: þ1 919 966 3893.
Multidrug resistance (MDR) always presents
a
major
obstacle in the treatment of human cancers with chemo-
therapeutic agents. Recently, some natural products, including
E-mail addresses: pxia@fudan.edu.cn (P. Xia), khlee@unc.edu (K.-H. Lee).
These authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.12.025

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
75
Fig. 1. Structures of bioactive natural and synthetic coumarin derivatives.
(ꢀ)-30-O-40-O-bis(3,4-dimethoxycinnamoyl)-cis-khellactone [(ꢀ)
DMDCK, 5, Fig. 1] and certain related synthetic compounds such as
30R,40R-disubstituted-20,20-dimethydihydropyrano[2,3-f]chromone
(DSP, 6, Fig. 1), exhibited significant potency in reversing P-gp-
mediated MDR [9,10]. In our previous research, hundreds of
dicamphanoyl-khellactone (DCK, 7, Fig. 1) and dicamphanoyl-
dihydropyranochromone (DCP, 8, Fig. 1) analogs containing
a coumarin or chromone moiety were designed and synthesized as
anti-HIV agents [5,11e13]. In these studies, we also found that some
7-mercapto- and 7-hydroxy-coumarin or -chromone intermediates
exhibited considerable cytotoxicity. These findings prompted us
to design and synthesize additional new S- and O-substituted
7-mercapo or 7-hydroxy-coumarin and chromone analogs and
screen them for cytotoxicity using paclitaxel as a potent antitumor
reference. The most potent cytotoxic analog 10 was selected for
further pharmacological studies in the A549 human lung cancer
cell line to more fully elucidate the compound’s mechanism of
action and corresponding target. Herein the synthesis, cytotoxicity
against four human tumor cell lines, and preliminary pharmaco-
logical mechanism of the new compounds are reported.
Scheme 1. The synthesis of 7-S-heterocyclic and aryl substituted coumarins (9e23). Reaction conditions: (i) 2,6-dichloropyridine N-oxide/pyridine/rt; (ii) PCl3/CHCl3/reflux;
(iii) variable bromides/K2CO3/acetone/rt.

76
Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
Scheme 2. The synthesis of 2-ethyl-7-S-heterocyclic substituted chromen-4-one analogs (24e27). Reaction conditions: (i) chloromethyl methyl ether/K2CO3/acetone/rt; (ii) ethyl
propionate/NaH/THF/reflux; (iii) conc. HCl/EtOH/reflux; (iv) dimethylthiocarbamoyl chloride/DMF/K2CO3; (v) 220e230 ꢁC; (vi) KOH/MeOH/N2/reflux; (vii) 2,6-dichloropyridine
N-oxide/pyridine/rt; (viii) PCl3/CHCl3/reflux.
2. Design
Bioisosterism is a commonly used technique in drug re-
search, although bioactive sulfur-containing coumarins have
rarely been reported. Therefore, fifteen S-substituted 4-methyl-
7-mercaptocoumarins (9e23), four S-substituted 2-ethyl-7-
mercapto-chromones and four O-substituted analogs (28e31)
were designed, synthesized, screened for cytotoxic activity and
evaluated for the effect of diverse substituents on the potency.
Based on the promising degree of cytotoxicity shown by
certain brominated chromone intermediates obtained in our prior
anti-HIV agents research, twelve new 2-ethyl-7-alkyloxy- and
-aryloxy-chromen-4-one bromides (32e43) were also synthesized
in this study. All synthesized compounds were screened in vitro
R
O
O
28. R=H
30. R=CF3
31. R=CN
29. R=CH3
O
i
iii
28-31
O
O
O
Br
ii
iii
RO
O
RO
O
HO
O
52a. R = CH3; 52b. R = CH2CH3;
32. R = CH3
33. R = CH(CH3)2
34. R = benzyl
48b
32-35
52c. R = CH(CH3)2; 52d. R = CH2CH(CH3)2
35. R = 3-trifluoromethyl-benzyl
iv
O
O
36.40. R = CH3
+
37.41. R = CH2CH3
38.42. R = CH(CH3)2
39.43. R = CH2CH(CH3)2
RO
O
RO
O
Br
Br
40-43
Br
36-39
Scheme 3. The synthesis of 2-ethyl-7-O-alkyl and aryl substituted chromen-4-one analogs and bromides (28e43). Reaction conditions: (i) variable halides/K2CO3/DMF/50e60 ꢁC;
(ii) variable halides/K2CO3/acetone/rt; (iii) N-bromosuccinimide/CH3CN/rt; (iv) N-bromosuccinimide/CCl4/reflux.

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
77
Table 1
In vitro anticancer activity of 9e43 against KB, KB-vin, A549 and DU145 cell lines.
Compd
R
X
R1
R2
R3
R4
GI50
KB
(mM)
KB-vin
24.52
A549
DU145
O
N
N
9
>31.27
0.92
>31.27
>31.27
Cl
10
11
12
0.92
2.11
1.15
Cl
O
>36.19
>35.29
>36.19
>35.29
>36.19
>35.29
>36.19
>35.29
O
N
N
13
14
>35.29
>35.29
>35.29
>35.29
>35.29
>35.29
>35.29
>35.29
N
CF3
15
16
17
18
>28.54
>32.53
>32.01
2.06
>28.54
>32.53
>32.01
5.41
>28.54
>32.53
>32.01
7.70
>28.54
>32.53
>32.01
14.07
CN
OCH3
O
COCH3
CH3
19
20
>33.74
>29.20
>33.74
>29.20
>33.74
>29.20
>33.74
>29.20
OCH
3
3
OCH
OCF3
OCH3
21
22
14.77
15.95
15.28
17.36
15.28
15.48
>25.56
>25.56
Br
OCH3
OCH3
23
>25.81
>25.81
>25.81
>25.81
O2N
O
N
N
24
25
S
S
H
H
H
H
H
>29.96
>28.75
>29.96
>28.75
>29.96
>28.75
>29.96
>28.75
Cl
O
Me
Cl
(continued on next page)

78
Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
Table 1 (continued )
Compd
R
X
R1
R2
R3
R4
GI50
KB
(mM)
KB-vin
A549
DU145
N
N
26
27
28
29
S
H
H
H
H
H
H
H
H
H
>31.47
>30.14
>33.97
28.60
>31.47
>31.47
>31.47
Cl
Cl
S
Me
Me
Me
23.93
24.80
24.91
>30.14
>33.97
24.94
>30.14
27.69
O
O
24.09
30
O
Me
H
H
>27.60
>27.60
>27.60
>27.60
31
32
33
34
O
O
O
O
Me
Me
Me
Me
H
H
H
H
H
>31.31
>33.65
22.60
>31.31
23.19
5.84
>31.31
>33.65
>30.75
>26.79
>31.31
>33.65
>30.75
>26.79
Me
Br
Br
Br
>26.79
>26.79
35
O
Me
H
Br
>22.66
>22.66
>22.66
>22.66
36
37
O
O
Me
Ethyl
CH2Br
CH2Br
H
H
H
H
>33.65
>32.14
>33.65
>32.14
>33.65
>32.14
>33.65
>32.14
38
39
O
O
CH2Br
CH2Br
H
H
H
H
>30.75
>29.48
>30.75
>29.48
>30.75
>29.48
>30.75
>29.48
40
41
O
O
Me
Ethyl
CH2Br
CH2Br
Br
Br
H
H
15.08
15.59
16.99
12.77
>26.59
19.84
>26.59
20.92
42
O
O
CH2Br
CH2Br
Br
Br
H
H
12.77
11.41
9.45
>1
12.79
8.71
15.47
43
10.93
10.52
Paclitaxel
4.12 nM
5.46 nM
3.86 nM
against four different human cancer cell lines, KB (nasopharnygeal),
KB-vin (its vincristine-resistant subline), A549 (lung) and DU145
(prostate). Compound 10 with the best activity was tested inde-
pendently in cell proliferation, apoptosis and cell cycle arrest assays
in the A549 human lung cancer cell line.
the presence of PCl3. Target compounds (11e23) were synthesized
via treatment of 44 with various heterocyclic and aryl bromides in
the presence of potassium carbonate at room temperature.
The synthesis of 2-ethyl-7-S-heterocyclic substituted
chromen-4-ones is described in Scheme 2. The commercially
available compounds 45a,b were selectively protected as the
4-methoxymethyl (MOM) ethers (46a,b), followed by condensation
with ethyl propionate to afford 47a and 47b, which were further
treated with concentrated HCl in EtOH to provide the bicyclic
compounds (48a,b). The intermediate 7-mercapto compounds
(51a,b) were prepared from 48a and 48b in three steps, a-acylation
with dimethylthiocarbamoyl chloride to give 49a and 49b,
followed by NewmaneKwart rearrangement to give 50a and 50b,
3. Chemistry
As shown in Scheme 1, commercially available 7-mercapto-
4-methyl-2H-chromen-2-one (44) was reacted with 2,6-
dichloropyridine N-oxide in pyridine at room temperature to
form compound 9, which was subsequently converted to 7-
(6-chloropyridin-2-ylthio)-4-methyl-2H-chromen-2-one (10) in

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
79
aliphatic halides in acetone at room temperature. Bromides 32e43
were prepared by reaction 28, 29, and 52aed with N-bromo-
succinimide in CH3CN at room temperature or in CCl4 under reflux,
respectively.
4. Results and discussion
Thirty-five newly synthesized compounds (9e43) were
screened for in vitro cytotoxic activity against a panel of human
tumor cell lines, including KB (nasopharyngeal), KB-vin (its
vincristine-resistant subline), A549 (lung) and DU145 (prostate),
with paclitaxel as a reference. The screening results are shown in
Table 1.
Overall, compound 10 [7-(6-chloropyridin-2-ylthio)-4-methyl-
2H-chromen-2-one] was the most potent compound with GI50
values ranging from 0.92 to 2.11 mM, while its N-oxide (9) exhibited
Fig. 2. Anti-proliferative effect of 10 in A549 human lung cancer cells. Each value
represents means ꢀ SD in three independent experiments. *p < 0.05 vs. control group,
#p < 0.01 vs. control group.
only weak cytotoxicity. Interestingly, the four chromones 24e27,
which have an isomeric 2-ethyl-4H-chromen-4-one coumarin
core, with or without an 8-methyl group, showed much lower
cytotoxicity than 10. This result implied that the 4-methyl-2H-
chromen-2-one skeleton may fit more favorably into the target
binding site compared with the 2-ethyl-4H-chromen-4-one struc-
ture. Most of the remaining coumarin (11e17) and chromone
(19e31) derivatives, which were modified at the 7-S or -O position
with heterocyclic and aryl rings substituted with electron-donating
or -withdrawing groups, showed, at most, weak cytotoxic activity;
thus a direct relationship was not found between electronic factors
and activity. However, compound 18, the 10-methoxy-formyl-
benzyl substituted coumarin analog, exhibited promising cancer
cell growth inhibitory activity with GI50 values ranging from 2.06 to
and finally hydrolysis in the presence of methanolic potassium
hydroxide. Subsequently, the nucleophilic alkylation of 51a and
51b with 2,6-dichloropyridine N-oxide yielded the desired
compounds (24e27) using the same procedures as for the synthesis
of 10.
The synthesis of four 7-benzyl ethers (28e31) and twelve
bromides (32e43) is depicted in Scheme 3. Compounds 28e31
were obtained by the etherification of 2-ethyl-7-hydroxy-8-
methyl-4H-chromen-4-one (48b) with four different benzyl
bromides in the presence of K2CO3 in DMF at 50e60 ꢁC. 7-Alkyl
ethers (52aed) were synthesized by the alkylation of 48b with
14.07 mM.
Fig. 3. Compound 10 induced apoptosis in A549 human lung cancer cells. Condensed and fragmented nuclei with bright staining were considered to be apoptotic cells. The results
represent three independent experiments.

80
Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
Fig. 4. G2/M cell cycle arrest in A549 cells by 10. Each value represents mean ꢀ SD from three independent experiments. *p < 0.05 vs. control group, #p < 0.01 vs. control group.
Twelve compounds (32e43) contained either one or
two brominated substituents. While the four 7-O-alkyl-8-
bromomethyl-2-ethyl analogs (36e39) were essentially inactive
against the four tested cancer cell lines, adding a second bromine at
7-alkoxy group and presence of a 2-a-bromine were important for
optimal cytotoxicity of the brominated compounds. Interestingly,
these compounds showed similar potency against the KB and
KB-vin cell lines. Moreover, 3-bromo-2-ethyl-7-isopropoxy-8-
methyl-4H-chromen-4-one (33) selectively inhibited the KB-vin
the a-position of the 2-ethyl group increased the inhibitory activity
of the dibromo analogs (40e43). The size of the 7-O-alkyl group
also affected the activity, with following rank order of potency:
methyl < ethyl < isopropyl < isobutyl. Thus, both the size of the
MDR cancer cell line with a GI50 value of 5.84
with the 1 M GI50 value of the control paclitaxel in the same
assay. Analogs with 7-methoxy (32) and 7-benzyloxy (34)
mM, compared
m

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
81
substitutions did not show the same potency or selectivity against
this cell line.
by filtration, and removal of the solvent in vacuo. The residue was
purified by column chromatography (eluent: CH2Cl2/EtOAc 99:1)
to afford the desired compound (9) as white crystals in 48% yield.
Because compound 10 had a significantly broader spectrum of
activity against the four tested human cancer cell lines, it was
selected for further pharmacological screening including cell
proliferation, apoptosis, and cell cycle arrest assays in the A549
human lung cancer cell line with docetaxel as a control. The data
(Figs. 2 and 3) showed that 10 markedly inhibited the proliferation
rate of A549 cells and increased the cellular apoptosis, respectively,
in a concentration-dependent manner.
Mp 236e237 ꢁC. 1H NMR
d 2.50 (3H, s, 4-CH3), 6.41 (1H, s, 3-H), 6.55
(1H, dd, J1 ¼ 8.1 Hz, J2 ¼ 1.8 Hz, 6-H), 7.01 (1H, t, J ¼ 8.4 Hz, H of
pyridine), 7.28, 7.54 (each 1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.8 Hz, 2H of
pyridine), 7.62 (1H, d, J ¼ 1.8 Hz, 8-H), 7.73 (1H, d, J ¼ 8.1 Hz, 5-H).
13C NMR
d 18.83, 58.42, 116.76, 120.27, 122.30, 124.00, 125.77,
126.45, 131.11, 133.28, 141.80, 151.75, 154.05, 154.91, 159.97. ESI MS
m/z 320 (Mþ þ 1).
Furthermore, the results of 10 on A549 cell cycle progression are
shown in Fig. 4. As compared to the control, G2/M phases of A549
6.3. 7-(6-Chloropyridin-2-ylthio)-4-methyl-2H-chromen-2-one (10)
cells treated with 10 (0, 1, 5 and 10 mg/mL) for 48 h were 6.36%,
83.58%, 88.98%, and 83.86%, while G0/G1 phases were 26.90%,
5.02%, 0.67%, and 0.12%. These results showed that compound 10
could induce A549 cell cycle arrest at the G2/M phase (p < 0.01).
A solution of compound 9 (500 mg, 1.56 mmol) in PCl3 (2 mL)
and CHCl3 (20 mL) was heated at reflux temperature for 1.5 h. The
mixture was diluted with ice-water (200 mL) and extracted with
CH2Cl2 (30 mL ꢂ 4). After drying the organic phase over anhy-
drous Na2SO4, the mixture was filtered and the solvent was
removed under reduced pressure. Then, the residue was purified
by column chromatography (eluent: CH2Cl2/MeOH 20:1) to give
10 as a white crystalline solid in 48% yield. Mp 138e140 ꢁC. 1H
5. Conclusion
Twenty-three novel coumarin and chromone analogs with
heterocyclic- and aryl-substituents at the 7-S and O-positions, and
twelve brominated coumarin derivatives were designed and
synthesized. Among them, compound 10 not only showed a broader
spectrum of activity against four tumor cells, but also remarkably
increased cellular apoptosis in a concentration-dependent manner
and induced A549 cell cycle arrest at the G2/M phase. Moreover,
3-bromo-2-ethyl-7-isopropoxy-8-methyl-4H-chromen-4-one (33)
showed high selective suppression of MDR KB-vin cell growth, sug-
gesting that groups at the 7-position might interact selectively with
different binding pockets of the molecular target. 8-Bromomethyl-
2-ethyl compounds (36e39) were inactive, while the cytotoxic
NMR
d 2.46 (3H, s, 4-CH3), 6.33 (1H, s, 3-H), 7.01, 7.13, 7.52
(each 1H, d, J ¼ 7.8 Hz, 3H of pyridine), 7.45 (1H, d, J ¼ 8.4 Hz,
6-H), 7.48 (1H, s, 8-H), 7.62 (1H, d, J ¼ 8.4 Hz, 5-H). 13C NMR
d
18.94, 116.55, 121.00, 122.05, 122.26, 122.31, 126.24, 129.81, 136.78,
140.25, 152.41, 152.80, 154.75, 160.35, 161.25. ESI MS m/z 304
(Mþ þ 1).
6.4. General procedure for synthesis of 4-methyl-7-heterocyclic and
aryl-substituted 7-thio-chromen-2H-2-ones (11e23)
activity of 2-
a
-bromoethyl-8-bromomethyl compounds (40e43)
A mixture of 44 (300 mg, 1.56 mmol), K2CO3 (372 mg,
2.34 mmol), and halogenated compound (1 equiv) in acetone
(10 mL) was stirred for 0.5e3.5 h at rt. After filtering the mixture
and removing the solvent in vacuo, the residue was purified by
column chromatography (eluent: hexane/EtOAc 85:15) to obtain
the target compounds (11e23) as white solids.
increased according to the size of 7-O-alkyl group. These findings
show that potency is affected by structural change and also indicate
that the binding site of the target cancer cell may have a strict
structural requirement. Based on these preliminary results, further
modification and optimization of the most potent compound, as well
as detailed study of the pharmacological target, are warranted.
6.4.1. 4-Methyl-7-((2-oxo-tetrahydrofuran-3-yl)methylthio)-2H-
6. Experimental
chromen-2-one (11)
Yield 81%, mp 137e138 ꢁC. 1H NMR
d 2.34, 2.80 (2H, m,
6.1. General
J ¼ 6.3 Hz, furanone 3-H2), 2.43 (3H, s, 4-CH3), 4.06 (1H, t, J ¼ 6.3 Hz,
furanone 2-H), 4.40 (2H, m, J ¼ 6.3 Hz, furanone 4-H2), 6.29 (1H, s,
3-H), 7.42 (1H, dd, J1 ¼ 8.4 Hz, J2 ¼ 1.5 Hz, 6-H), 7.44 (1H, d,
J ¼ 1.5 Hz, 8-H), 7.55 (1H, d, J ¼ 8.4 Hz, 5-H). ESI MS m/z 299
(Mþ þ Na).
Melting points were measured with a Fisher Johns melting
apparatus without correction. The proton nuclear magnetic reso-
nance (1H NMR) spectra were measured on 300 MHz Varian Gemini
2000 spectrometer using TMS as internal standard. The solvent
used was CDCl3 unless indicated. Mass spectra were measured on
HP5973N analytical mass spectrometers. High resolution mass
spectra (HRMS) were measured on a Shimadzu LCMSIT-TOF with
ESI interface. HPLC purity determinations were conducted using
a Shimadzu LCMS-2010 with a Grace Alltima 2.1 ꢂ 150 mm HP C18
6.4.2. 4-Methyl-7-(pyridin-2-ylmethylthio)-2H-chromen-2-one (12)
Yield 37%, mp 92e93 ꢁC. 1H NMR
d 2.39 (3H, s, 4-CH3), 4.36 (2H,
s, 7-SCH2e), 6.21 (1H, s, 3-H), 7.17e7.27 (3H, m, J1 ¼ 8.4 Hz,
J2 ¼ 7.8 Hz, 6-H & 8-H & 1H of pyridine), 7.41e7.66 (3H, m,
J1 ¼8.4 Hz, J2 ¼ 7.8 Hz, 2H of pyridine & 5-H), 8.57 (1H, d, J ¼ 7.8 Hz,
1H of pyridine). ESI MS m/z 284 (Mþ þ H).
3 mM column and Shimadzu SPD-M20A detector at 254 nm wave-
length in MeCN/H2O and MeOH/H2O two different solvent condi-
tions. All target compounds had purity greater than 95%.
Commercially available silica gel H was used for column chroma-
tography. Thin-layer chromatography (TLC) was performed on PLC
silica gel 60 F254 plates.
6.4.3. 4-Methyl-7-(pyridin-3-ylmethylthio)-2H-chromen-2-one (13)
Yield 57%, mp 128e129 ꢁC. 1H NMR
d 2.40 (3H, s, 4-CH3), 4.20
(2H, s, 7-SCH2e), 6.24 (1H, s, 3-H), 7.15 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.5 Hz, 6-H), 7.20 (1H, d, J ¼ 1.5 Hz, 8-H), 7.26 (1H, t, J ¼ 7.8 Hz,
1H of pyridine), 7.46 (1H, d, J ¼ 8.4 Hz, 5-H), 7.71 (1H, dt, J1 ¼ 7.8 Hz,
J2 ¼ 1.8 Hz, 1H of pyridine), 8.50, 8.57 (2H, m, J1 ¼ 7.8 Hz,
J2 ¼ 1.8 Hz, 2H of pyridine). ESI MS m/z 282 (Mþ ꢃ H).
6.2. N-Oxide-7-(6-chloropyridin-2-ylthio)-4-methyl-2H-chromen-
2-one (9)
A mixture of 44 (300 mg, 1.56 mmol), 2,6-dichloropyridine
N-oxide (256 mg, 1.56 mmol) in pyridine (6 mL) was stirred for
2 h at rt. Then, 2 N HCl (50 mL) was added to the reaction, followed
6.4.4. 4-Methyl-7-(pyridin-4-ylmethylthio)-2H-chromen-2-one (14)
Yield 67.39%, mp 155e156 ꢁC. 1H NMR
d
2.40 (3H, s, 4-CH3), 4.18
(2H, s, 7-SCH2e), 6.23 (1H, s, 3-H), 7.13 (1H, dd, J1 ¼ 8.7 Hz,

82
Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
J2 ¼ 1.8 Hz, 6-H), 7.17 (1H, d, J ¼ 1.8 Hz, 8-H), 7.31, 8.55 (4H, dd,
J1 ¼ 6.0 Hz, J2 ¼ 1.8 Hz, H of pyridine), 7.45 (1H, d, J ¼ 8.7 Hz, 5-H).
ESI MS m/z 282 (Mþ ꢃ H).
6.4.13. 7-(4,5-Dimethoxy-2-nitrobenzylthio)-4-methyl-2H-
chromen-2-one (23)
Yellow solid, yield 21% (starting from 100 mg of 44), mp
173e175 ꢁC. 1H NMR
d 2.40 (3H, s, 4-CH3), 3.88, 3.96 (each 3H, s,
6.4.5. 4-Methyl-7-(3-trifluoromethyl)benzylthio-2H-chromen-
2-one (15)
2ꢂ OCH3 of aromatic), 4.62 (2H, s, 7-SCH2e), 6.24 (1H, s, 3-H), 6.94,
7.69 (each 1H, s, 2H of aromatic), 7.18 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.8 Hz, 6-H), 7.23 (1H, d, J ¼ 1.8 Hz, 8-H), 7.47 (1H, d, J ¼ 8.4 Hz,
5-H). ESI MS m/z 389 (Mþ þ 2).
Yield 85.96%, mp 139e140 ꢁC. 1H NMR
d 2.40 (3H, s, 4-CH3), 4.25
(2H, s, 7-SCH2e), 6.23 (1H, s, 3-H), 7.13e7.18 (2H, m, J ¼ 8.7 Hz, 6-H
& 8-H), 7.45e7.61 (5H, m, J1 ¼ 8.7 Hz, J2 ¼ 6.6 Hz, 4H of aromatic &
5-H). ESI MS m/z 349 (Mþ ꢃ 1).
6.5. 1-(2-Hydroxy-4-(methoxymethoxy)-3-methylphenyl)ethanone
(46b)
6.4.6. 3-((4-Methyl-2-oxo-2H-chromen-7-ylthio)methyl)
benzonitrile (16)
Chloromethyl methyl ether (9.14 mL, 120 mmol) was added
dropwise into a mixture of 45b (10.0 g, 60.2 mmol) and potassium
carbonate (20.8 g, 150 mmol) in anhydrous acetone (60 mL) in an
ice-bath. The reaction mixture was allowed to warm to rt and
stirred for 1.5 h. The mixture was then filtered, and the filtrate was
dissolved in brine and extracted with EtOAc (35 mL). The organic
layer was dried in vacuo to provide 46b (13.9 g) as brown oil, 91%
Yield 10% (starting from 200 mg of 44), mp 172e173 ꢁC. 1H NMR
d
2.41 (3H, s, 4-CH3), 4.22 (2H, s, 7-SCH2e), 6.23 (1H, s, 3-H),
7.13e7.17 (2H, m, J1 ¼ 9.0 Hz, J2 ¼ 2.1 Hz, 6-H & 8-H), 7.41e7.66 (5H,
m, J1 ¼ 9.0 Hz, J2 ¼ 7.5 Hz, 4H of aromatic & 5-H). ESI MS m/z 330
(Mþ þ Na).
yield. 1H NMR
d 2.14 (3H, s, 3-CH3), 2.56 (3H, s, 1-COCH3), 3.50 (3H,
6.4.7. 7-(3-Methoxybenzylthio)-4-methyl-2H-chromen-2-one (17)
Yield 17% (starting from 200 mg of 44), mp 137e138 ꢁC. 1H NMR
s, 4-OCH2OCH3), 5.27 (2H, s, 4-OCH2OCH3), 6.65 (1H, d, J ¼ 8.7 Hz,
6-H), 7.57 (1H, d, J ¼ 8.7 Hz, 5-H), 12.80 (1H, s, 2-OH). ESI MS m/z
209 (Mþ ꢃ 1).
d
2.39 (3H, s, 4-CH3), 3.79 (3H, s, OCH3 of aromatic), 4.19 (2H, s,
7-SCH2e), 6.21 (1H, s, 3-H), 6.81, 6.68 (3H, dd, J1 ¼ 8.7 Hz,
J2 ¼ 2.7 Hz, H of aromatic), 7.14e7.26 (3H, m, J1 ¼8.4 Hz, J2 ¼ 8.7 Hz,
1H of aromatic and 6-H & 8-H), 7.44 (1H, d, J ¼ 8.4 Hz, 5-H). ESI MS
m/z 311 (Mþ ꢃ H).
6.6. Preparation of 48a and 48b
6.6.1. 2-Ethyl-7-hydroxy-4H-chromen-4-one (48a)
The synthesis of the intermediate 48a from 45a was reported
previously in Ref. [9].
6.4.8. Methyl 3-((4-methyl-2-oxo-2H-chromen-7-ylthio)methyl)
benzoate (18)
Yield 12% (starting from 200 mg of 44), mp 121e123 ꢁC. 1H NMR
6.6.2. 2-Ethyl-7-hydroxy-8-methyl-4H-chromen-4-one (48b)
d
2.40 (3H, s, 4-CH3), 3.92 (3H, s, eOCH3), 4.25 (2H, s, 7-SCH2e), 6.22
Sodium hydride (60% in mineral oil, 9.52 g/15.9 g, 397 mmol)
was added slowly to a mixture of 46b (13.9 mg, 66.1 mmol) and
ethyl propionate (19.0 mL, 165 mmol) in absolute THF under
nitrogen. The mixture was warmed to reflux temperature for 2 h,
cooled, and neutralized to pH 8 with 37% HCl (25 mL). Water
(60 mL) was added and the mixture was extracted with EtOAc
(4 ꢂ 30 mL). The organic layer was collected and evaporated in
vacuo to yield 47b as dark oil. This crude product and 37% HCl
(3 mL) were dissolved in EtOH (60 mL) and refluxed for 45 min to
give 48b, which was used in the next reaction without further
(1H, s, 3-H), 7.14e7.18 (2H, m, J1 ¼ 8.1 Hz, J2 ¼ 2.1 Hz, 6-H & 8-H),
7.38, 7.57, 7.94 (3H, d, J ¼ 7.5 Hz, H of aromatic), 7.45 (1H, d, J ¼ 8.1 Hz,
5-H), 8.06 (1H, s, H of aromatic). ESI MS m/z 339 (Mþ ꢃ H).
6.4.9. 7-(3-Methylbenzylthio)-4-methyl-2H-chromen-2-one (19)
Yield 84.32%, mp 100e101 ꢁC. 1H NMR
d 2.35 (3H, s, CH3 of
aromatic), 2.40 (3H, s, 4-CH3), 4.19 (2H, s, 7-SCH2e), 6.21 (1H, s, 3-H),
7.08 (1H, d, J ¼ 8.1 Hz, 6-H), 7.14e7.22 (5H, m, J ¼ 7.2 Hz, 4H of
aromatic&8-H); 7.45(1H, d, J ¼ 8.1 Hz, 5-H). ESIMSm/z 295 (Mþ ꢃ 1).
purification. Mp 223e224 ꢁC. 1H NMR (DMSO,
d) 1.23 (3H, t,
6.4.10. 7-(3,5-Dimethoxybenzylthio)-4-methyl-2H-chromen-2-one
J ¼ 7.2 Hz, 2-CH2CH3), 2.20 (3H, s, 8-CH3), 2.65 (2H, q, J ¼ 7.2 Hz,
2-CH2CH3), 6.06 (1H, s, 3-H), 6.98 (1H, d, J ¼ 8.7 Hz, 6-H), 7.68 (1H,
d, J ¼ 8.7 Hz, 5-H). ESI MS m/z 203 (Mþ ꢃ 1).
(20)
Yield 14%, mp 146e148 ꢁC. 1H NMR
d 2.39 (3H, s, 4-CH3), 3.77
(6H, s, 2ꢂ OCH3 of aromatic), 4.16 (2H, s, 7-SCH2e), 6.21 (1H, s, 3-H),
6.37, 6.55, 6.81 (3H, s, H of aromatic), 7.16 (1H, dd, J1 ¼ 8.1 Hz,
J2 ¼ 1.5 Hz, 6-H), 7.18 (1H, d, J ¼ 1.5 Hz, 8-H), 7.45 (1H, d, J ¼ 8.1 Hz,
5-H). ESI MS m/z 341 (Mþ ꢃ H).
6.7. Preparation of 49a and 49b
6.7.1. O-2-Ethyl-4-oxo-4H-chromen-7-yl dimethylcarbamothioate (49a)
Compound 48a (1. 0 g, 5.26 mmol) and K2CO3 (1.82 g,
13.15 mmol) were dissolved in DMF (5 mL) and reacted with
dimethylthiocarbamoyl chloride (1.3 g, 10.52 mmol) for 1.5 h at rt.
The mixture was poured into ice-water (200 mL) and filtered to
afford crude product, which was purified by column chromatog-
raphy (eluent: petroleum ether/EtOAc 4:1) to give 49a as a white
6.4.11. 7-(3-(Trifluoromethoxy)benzylthio)-4-methyl-2H-chromen-
2-one (21)
Yield 89%, mp 106e107 ꢁC. 1H NMR
d 2.40 (3H, s, 4-CH3), 4.21
(2H, s, 7-SCH2e), 6.22 (1H, s, 3-H), 7.13e7.18 (3H, m, J ¼ 6.5 Hz, H of
aromatic), 7.22 (1H, s, H of aromatic), 7.33 (1H, dd, J1 ¼ 8.4 Hz,
J2 ¼ 1.5 Hz, 6-H), 7.35 (1H, d, J ¼ 1.5 Hz, 8-H), 7.45 (1H, d, J ¼ 8.4 Hz,
5-H). ESI MS m/z 365 (Mþ ꢃ H).
solid (1.37 mg). Yield 94%, mp 137e139 ꢁC. 1H NMR
d 1.31 (3H, d,
J ¼ 7.5 Hz, 2-CH2CH3), 2.65 (2H, q, J ¼ 7.5 Hz, 2-CH2CH3), 3.38, 3.48
(each 3H, s, Ne(CH3)2), 6.17 (1H, s, 3-H), 7.10 (1H, dd, J1 ¼ 8.7 Hz,
J2 ¼ 2.1 Hz, 6-H), 7.18 (1H, d, J ¼ 2.1 Hz, 8-H), 8.20 (1H, d, J ¼ 8.7 Hz,
5-H). ESI MS m/z 278 (Mþ þ H).
6.4.12. 7-(2-Bromo-5-methoxybenzylthio)-4-methyl-2H-chromen-
2-one (22)
Yield 84%, mp 136e137 ꢁC. 1H NMR
d 2.40 (3H, s, 4-CH3), 3.74
(3H, s, OCH3 of aromatic), 4.28 (2H, s, 7-SCH2e), 6.23 (1H, s, 3-H),
6.71 (1H, d, J1 ¼ 9.0 Hz, J2 ¼ 3.0 Hz, H of aromatic), 6.99 (1H, d,
J ¼ 3.0 Hz, H of aromatic), 7.17e7.20 (2H, m, J ¼ 9.0 Hz, 6-H & 8-H),
7.26 (1H, s, H of aromatic), 7.47 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z
391 (Mþ þ 1).
6.7.2. O-2-Ethyl-8-methyl-4-oxo-4H-chromen-7-yl
dimethylcarbamothioate (49b)
The procedure was the same as that used for the preparation of
49a. Yield 76% (starting from 500 mg of 48b), mp 164e166 ꢁC. 1H

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
83
NMR
d
1.33 (3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.30 (3H, s, 8-CH3), 2.70
6.10.2. N-Oxide-7-(6-chloropyridin-2-ylthio)-2-ethyl-8-methyl-
4H-chromen-4-one (25)
(2H, q, J ¼ 7.5 Hz, 2-CH2CH3), 3.41, 3.49 (each 3H, s, Ne(CH3)2), 6.19
(1H, s, 3-H), 7.05 (1H, d, J ¼ 8.7 Hz, 6-H), 8.05 (1H, d, J ¼ 8.7 Hz, 5-H).
ESI MS m/z 292 (Mþ þ H).
The procedure was the same as that used for the preparation of
9. Yield 53% (starting from 100 mg of 51b), mp 190e192 ꢁC. 1H NMR
d
1.37 (3H, d, J ¼ 7.8 Hz, 2-CH2CH3), 2.60 (3H, s, 8-CH3), 2.74 (2H, q,
J ¼ 7.8 Hz, 2-CH2CH3), 6.27 (1H, s, 3-H), 6.34, 7.27 (each 1H, dd,
J1 ¼ 8.1 Hz, J2 ¼ 1.8 Hz, 2H of pyridine), 6.98 (1H, t, J ¼ 8.1 Hz, H of
pyridine), 7.62 (1H, d, J ¼ 8.4 Hz, 6-H), 8.12 (1H, d, J ¼ 8.4 Hz, 5-H).
6.8. Preparation of 50a and 50b
6.8.1. S-2-Ethyl-4-oxo-4H-chromen-7-yl dimethylcarbamothioate
(50a)
13C NMR
d 11.17, 13.71, 18.57, 27.78, 58.60, 109.37, 119.73, 122.07,
125.14, 132.47, 133.65, 134.32, 141.93, 154.00, 155.34, 171.25, 178.30.
Compound 49a (300 mg, 1.08 mmol) was heated to 220e230 ꢁC
with stirring for 1 h. The crude product was purified by column
chromatography (eluent: CH2Cl2/EtOAc 99:1) to give 50a as light
ESI MS m/z 270 (Mþ þ Na).
yellow crystals (170 mg). Yield 57%, mp 121e123 ꢁC. 1H NMR
d 1.31
6.11. Preparation of 26 and 27
(3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.65 (2H, q, J ¼ 7.5 Hz, 2-CH2CH3),
3.06, 3.12 (each 3H, s, Ne(CH3)2), 6.19 (1H, s, 3-H), 7.46 (1H, dd,
J1 ¼8.4 Hz, J2 ¼ 1.5 Hz, 6-H), 7.67 (1H, d, J ¼ 1.5 Hz, 8-H), 8.16 (1H, d,
J ¼ 8.4 Hz, 5-H). ESI MS m/z 278 (Mþ þ H).
6.11.1. 7-(6-Chloropyridin-2-ylthio)-2-ethyl-4H-chromen-4-one
(26)
The procedure was the same as that used for the preparation of
10. Yield 12% (starting from 60 mg of 24), mp 106e107 ꢁC. 1H NMR
d
1.32 (3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.67 (2H, q, J ¼ 7.5 Hz,
6.8.2. S-2-Ethyl-8-methyl-4-oxo-4H-chromen-7-yl
dimethylcarbamothioate (50b)
2-CH2CH3), 6.21 (1H, s, 3-H), 7.02, 7.14 (each 1H, d, J ¼ 7.8 Hz, 2H of
pyridine), 7.48 (1H, dd, J1 ¼ 8.1 Hz, J2 ¼ 1.8 Hz, 6-H), 7.51 (1H, t,
J ¼ 7.8 Hz, H of pyridine), 7.66 (1H, d, J ¼ 1.8 Hz, 8-H), 8.18 (1H, d,
J ¼ 8.1 Hz, 5-H). ESI MS m/z 318 (Mþ þ H).
The procedure was the same as that used for the preparation of
50a. Yield 34% (starting from 450 mg of 49b), mp 202e204 ꢁC. 1H
NMR
d
1.33 (3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.57 (3H, s, 8-CH3), 2.70
(2H, q, J ¼ 7.5 Hz, 2-CH2CH3), 3.04, 3.16 (each 3H, s, Ne(CH3)2), 6.20
(1H, s, 3-H), 7.50 (1H, d, J ¼ 8.1 Hz, 6-H), 8.01 (1H, d, J ¼ 8.1 Hz, 5-H).
ESI MS m/z 292 (Mþ þ H).
6.11.2. 7-(6-Chloropyridin-2-ylthio)-2-ethyl-8-methyl-4H-
chromen-4-one (27)
The procedure was the same as that used for the preparation of
10. Yield 6% (starting from 50 mg of 25), mp 117e118 ꢁC. 1H NMR
d
6.9. Preparation of 51a and 51b
1.36 (3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.57 (3H s, 8-CH3), 2.73 (2H, q,
J ¼ 7.8 Hz, 2-CH2CH3), 6.24 (1H, s, 3-H), 6.73, 7.08 (each 1H, d,
J ¼ 7.8 Hz, 2H of pyridine), 7.44 (1H, t, J ¼ 7.8 Hz, H of pyridine), 7.55
(1H, d, J ¼ 8.1 Hz, 6-H), 8.06 (1H, d, J ¼ 8.1 Hz, 5-H). ESI MS m/z 332
(Mþ þ H).
6.9.1. 2-Ethyl-7-mercapto-4H-chromen-4-one (51a)
Under nitrogen and in the dark, compound 50a (170 mg,
0.61 mmol) was hydrolyzed in the presence of KOH (103 mg,
1.84 mmol) in MeOH (10 mL) for 1.5 h at reflux temperature. After
cooling to rt, conc. HCl was added to pH 1e2 and ice-water (50 mL)
was added. The mixture was extracted with EtOAc (20 mL ꢂ 3), and
the combined organic layer was dried over anhydrous Na2SO4.
Product 51a (120 mg) was obtained after filtration and removal of
solvent under reduced pressure. Yield 95%, mp 73e75 ꢁC. 1H NMR
6.12. Synthesis of 7-aryloxy-substituted-8-methyl-4H-chromen-
4-one (28e31)
The procedure was similar to that used in the preparation of
11e23 with DMF/50e60 ꢁC instead of reaction in acetone at room
temperature.
d
1.30 (3H, d, J ¼ 7.8 Hz, 2-CH2CH3), 2.64 (2H, q, J ¼ 7.8 Hz,
2-CH2CH3), 3.73 (1H, s, 7-SH), 6.15 (1H, s, 3-H), 7.20 (1H, dd,
J1 ¼8.4 Hz, J2 ¼ 1.8 Hz, 6-H), 7.30 (1H, d, J ¼ 1.8 Hz, 8-H), 8.02 (1H, d,
J ¼ 8.4 Hz, 5-H). ESI MS m/z 205 (Mþ ꢃ H).
6.12.1. 7-(Benzyloxy)-2-ethyl-8-methyl-4H-chromen-4-one (28)
Yield 98% (starting from 340 mg of 48b), mp 98e99 ꢁC. 1H NMR
d
1.33 (3H, t, J ¼ 7.2 Hz, 2-CH2CH3), 2.36 (3H, s, 8-CH3), 2.68 (2H, q,
6.9.2. 2-Ethyl-7-mercapto-8-methyl-4H-chromen-4-one (51b)
The procedure was the same as that used for the preparation of
51a. Yield 88% (starting from 150 mg of 50b), mp 107e109 ꢁC. 1H
J ¼ 7.2 Hz, 2-CH2CH3), 5.21 (2H, s, 7-OCH2e), 6.12 (1H, s, 3-H), 7.01
(1H, d, J ¼ 9.0 Hz, 6-H), 7.37e7.45 (5H, m, H of aromatic), 8.03 (1H,
d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 293 (Mþ ꢃ 1).
NMR
d
1.33 (3H, d, J ¼ 7.8 Hz, 2-CH2CH3), 2.43 (3H, s, 8-CH3), 2.69
(2H, q, J ¼ 7.8 Hz, 2-CH2CH3), 3.62 (1H, s, 7-SH), 6.16 (1H, s, 3-H),
7.23 (1H, d, J ¼ 8.4 Hz, 6-H), 7.88 (1H, d, J ¼ 8.4 Hz, 5-H). ESI MS m/z
219 (Mþ ꢃ H).
6.12.2. 2-Ethyl-8-methyl-7-(3-methylbenzyloxy)-4H-chromen-4-
one (29)
Yield 62% (starting from 322 mg of 48b), mp 93e94 ꢁC. 1H NMR
d
1.33 (3H, t, J ¼ 7.8 Hz, 2-CH2CH3), 2.34 (3H, s, 3-CH3 of aromatic),
6.10. Preparation of 24 and 25
2.38 (3H, s, 8-CH3), 2.69 (2H, q, J ¼ 7.8 Hz, 2-CH2CH3), 5.22 (2H, s,
7-OCH2e), 6.13 (1H, s, 3-H), 7.04 (1H, d, J ¼ 9.0 Hz, 6-H), 7.15 (1H, d,
J ¼ 7.8 Hz, H of aromatic), 7.25 (3H, m, J ¼ 7.8 Hz, H of aromatic), 8.13
(1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 307 (Mþ ꢃ 1).
6.10.1. N-Oxide-7-(6-chloropyridin-2-ylthio)-2-ethyl-4H-chromen-
4-one (24)
The procedure was the same as that used for the preparation of
9. Yield 51% (starting from 130 mg of 51a), mp 163e164 ꢁC. 1H NMR
6.12.3. 2-Ethyl-8-methyl-7-(3-(trifluoromethyl)benzyloxy)-4H-
chromen-4-one (30)
d
1.34 (3H, d, J ¼ 7.5 Hz, 2-CH2CH3), 2.70 (2H, q, J ¼ 7.5 Hz,
2-CH2CH3), 6.26 (1H, s, 3-H), 6.55, 7.29 (each 1H, dd, J1 ¼ 8.1 Hz,
J2 ¼ 1.8 Hz, 2H of pyridine), 7.01 (1H, t, J ¼ 8.1 Hz, H of pyridine),
7.58 (1H, dd, J1 ¼ 8.1 Hz, J2 ¼ 1.8 Hz, 6-H), 7.76 (1H, d, J ¼ 1.8 Hz,
Yield 47% (starting from 500 mg of 48b), mp 122e123 ꢁC. 1H
NMR
d
1.34 (3H, t, J ¼ 7.5 Hz, 2-CH2CH3), 2.38 (3H, s, 8-CH3), 2.69
(2H, q, J ¼ 7.5 Hz, 2-CH2CH3), 5.25 (2H, s, 7-OCH2e), 6.13 (1H, s,
3-H), 6.99 (1H, d, J ¼ 9.0 Hz, 6-H), 7.55 (1H, t, J ¼ 7.2 Hz, H of
aromatic), 7.64 (2H, t, J ¼ 7.2 Hz, H of aromatic), 7.73 (1H, s, H of
aromatic), 8.04 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 363 (Mþ þ 1).
8-H), 8.29 (1H, d, J ¼ 8.1 Hz, 5-H). 13C NMR
d 11.07, 18.58, 27.68,
58.59, 109.66, 120.32, 122.31, 125.28, 127.79, 131.58, 135.47, 141.76,
154.72, 156.83, 171.61, 177.67. ESI MS m/z 256 (Mþ þ Na).

84
Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
6.12.4. 3-((2-Ethyl-8-methyl-4-oxo-4H-chromen-7-yloxy)methyl)
benzonitrile (31)
6.14.3. 7-(Benzyloxy)-3-bromo-2-ethyl-8-methyl-4H-chromen-
4-one (34)
Yield 52% (starting from 500 mg of 48b), mp 176e177 ꢁC. 1H NMR
Yield 34% (starting from 2.23 g of 28), mp 155e157 ꢁC. 1H NMR
d
1.34 (3H, t, J ¼ 7.8 Hz, 2-CH2CH3), 2.38 (3H, s, 8-CH3), 2.69 (2H, q,
d
1.39 (3H, t, J ¼ 7.5 Hz, 2-CH2CH3), 2.36 (3H, s, 8-CH3), 3.00 (2H, q,
J¼ 7.8Hz, 2-CH2CH3), 5.23(2H,s,7-OCH2e),6.13(1H, s,3-H), 6.97(1H,
d, J ¼ 9.0 Hz, 6-H), 7.55e7.67 (3H, t, J ¼ 8.1 Hz, H of aromatic), 7.77 (1H,
s, H ofaromatic), 8.03(1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 320 (Mþ þ 1).
J ¼ 7.5 Hz, 2-CH2CH3), 5.19 (2H, s, 7-OCH2e), 7.04 (1H, d, J ¼ 9.0 Hz,
6-H), 7.35e7.46 (5H, m, H of aromatic), 8.06 (1H, d, J ¼ 9.0 Hz, 5-H).
ESI MS m/z 373 (Mþ þ 1).
6.13. Synthesis of 2-ethyl-7-alkoxy-8-methyl-4H-chromen-4-one
(52aed)
6.14.4. 3-Bromo-2-ethyl-8-methyl-7-(3-(trifluoromethyl)
benzyloxy)-4H-chromen-4-one (35)
Yield 41% (starting from 200 mg of 30), mp 159e161 ꢁC. 1H
The procedure was the same as that used for the preparation of
11e23.
NMR
d
1.39 (3H, t, J ¼ 7.2 Hz, 2-CH2CH3), 2.39 (3H, s, 8-CH3), 3.02
(2H, q, J ¼ 7.2 Hz, 2-CH2CH3), 5.26 (2H, s, 7-OCH2e), 7.03 (1H, d,
J ¼ 9.0 Hz, 6-H), 7.55, 7.63 (3H, t, J ¼ 7.2 Hz, H of aromatic), 7.72
(1H, s, H of aromatic), 8.08 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 441
(Mþ þ 1).
6.13.1. 2-Ethyl-7-methoxy-8-methyl-4H-chromen-4-one (52a)
Yield 75% (starting from 2.0 g of 48b), mp 104e106 ꢁC. 1H NMR
d
1.32 (3H, t, J ¼ 7.5 Hz, 2-CH2CH3), 2.30 (3H, s, 8-CH3), 2.68 (2H, q,
J ¼ 7.5 Hz, 2-CH2CH3), 3.95 (3H, s, 7-OCH3), 6.11 (1H, s, 3-H), 6.96
(1H, d, J ¼ 9.0 Hz, 6-H), 8.10 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 219
(Mþ þ 1).
6.15. Synthesis of bromides (36e43)
A mixture of 52aed (1 equiv) and NBS (1.2 equiv) in CCl4 was
refluxed for 16 h. After filtration and removal of the solvent, the
residue was purified by column chromatography with a gradient
eluent of CH2Cl2/MeOH (40:1 to 30:1) to afford pure mono-
(36e39) and di-brominated (40e43) products in yields of 40e78%
and 11e20%, respectively.
6.13.2. 7-Ethoxy-2-ethyl-8-methyl-4H-chromen-4-one (52b)
Yield 73%, mp 85e87 ꢁC. 1H NMR
d
1.33 (3H, t, J ¼ 7.5 Hz,
2-CH2CH3), 1.48 (3H, t, J ¼ 7.2 Hz, 7-OCH2CH3), 2.31 (3H, s, 8-CH3),
2.68 (2H, q, J ¼ 7.5 Hz, 2-CH2CH3), 4.17 (2H, q, J ¼ 7.2 Hz,
7-OCH2CH3), 6.11 (1H, s, 3-H), 6.93 (1H, d, J ¼ 9.0 Hz, 6-H), 8.01 (1H,
d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 233 (Mþ þ 1).
6.15.1. 8-(Bromomethyl)-2-ethyl-7-methoxy-4H-chromen-4-one
(36)
6.13.3. 2-Ethyl-7-isopropoxy-8-methyl-4H-chromen-4-one (52c)
Yield 79% (starting from 2.0 g of 48b), mp 99e101 ꢁC. 1H NMR
Yield 40% (starting with 300 mg of 52a), mp 162e164 ꢁC. 1H
NMR
d
1.36 (3H, t, J ¼ 7.5 Hz, 2-CH2CH3), 2.72 (2H, q, J ¼ 7.5 Hz,
d
1.33 (3H, t, J ¼ 6.9 Hz, 2-CH2CH3), 1.38 (6H, d, J ¼ 6.0 Hz,
2-CH2CH3), 4.03 (3H, s, 7-OCH3), 4.78 (2H, s, 8-CH2Br), 6.15 (1H, s,
3-H), 6.99 (1H, d, J ¼ 9.0 Hz, 6-H), 8.16 (1H, d, J ¼ 9.0 Hz, 5-H). ESI
MS m/z 297 (Mþ þ 1).
7-OCH(CH3)2), 2.29 (3H, s, 8-CH3), 2.68 (2H, q, J ¼ 6.9 Hz, 2-CH2CH3),
4. 70 (2H, m, J ¼ 6.0 Hz, 7-OCH(CH3)2), 6.11 (1H, s, 3-H), 6.95 (1H, d,
J ¼ 9.0 Hz, 6-H), 8.01 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 247 (Mþ þ 1).
6.15.2. 8-(Bromomethyl)-7-ethoxy-2-ethyl-4H-chromen-4-one
(37)
6.13.4. 2-Ethyl-7-isobutoxy-8-methyl-4H-chromen-4-one (52d)
Yield 82%, mp 61e62 ꢁC. 1H NMR
d
1.07 (6H, d, J ¼ 6.6 Hz,
Yield 58% (starting with 880 mg of 52b), mp 114e116 ꢁC. 1H NMR
7-OCH2CH(CH3)2), 1.33 (3H, t, J ¼ 7.2 Hz, 2-CH2CH3), 2.16 (1H, m,
J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 2.32 (3H, s, 8-CH3), 2.68 (2H,
J ¼ 7.2 Hz, 2-CH2CH3), 3.86 (2H, d, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 6.11
(1H, s, 3-H), 6.93 (1H, d, J ¼ 9.0 Hz, 6-H), 8.01 (1H, d, J ¼ 9.0 Hz, 5-H).
ESI MS m/z 261 (Mþ þ 1).
d
1.36 (3H, t, J ¼ 7.8 Hz, 2-CH2CH3),1.52 (3H, t, J ¼ 6.9 Hz, 7-OCH2CH3),
2.72 (2H, q, J ¼ 7.8 Hz, 2-CH2CH3), 4.25 (2H, q, J ¼ 6.9 Hz, 7-OCH2CH3),
4.79 (2H, s, 8-CH2Br), 6.14 (1H, s, 3-H), 6.96 (1H, d, J ¼ 9.0 Hz, 6-H),
8.13 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 311 (Mþ þ 1).
6.15.3. 8-(Bromomethyl)-2-ethyl-7-isopropoxy-4H-chromen-4-one
(38)
6.14. Synthesis of 3-bromo-2-ethyl-7-alkoxy or 7-aryloxy-8-
methyl-4H-chromen-4-one (32e35)
Yield 45% (starting with 1.2 g of 52c), mp 66e68 ꢁC. 1H NMR
d
1.36 (3H, t, J ¼ 7.8 Hz, 2-CH2CH3), 1.44 (6H, d, J ¼ 6.0 Hz,
A mixture of 28, 30, 52a or 52c (1.0 equiv) and N-bromosucci-
nimide (1.2 equiv) in CH3CN (5 mL) was stirred for 1 day at rt. After
filtration and removal of the solvent, the residue was purified by
column chromatography (eluent: CH2Cl2/MeOH 99:1) to obtain
3-brominated compounds (32e35) as white solids.
7-OCH(CH3)2), 2.71 (2H, q, J ¼ 7.8 Hz, 2-CH2CH3), 4.72e4.81 (3H, m,
J ¼ 6.0 Hz, 7-OCH(CH3)2 & 8-CH2Br), 6.14 (1H, s, 3-H), 6.96 (1H, d,
J ¼ 9.0 Hz, 6-H), 8.12 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 325
(Mþ þ 1).
6.15.4. 8-(Bromomethyl)-2-ethyl-7-isobutoxy-4H-chromen-4-one
(39)
6.14.1. 3-Bromo-2-ethyl-7-methoxy-8-methyl-4H-chromen-4-one (32)
Yield 44% (starting from 50 mg of 52a), mp 115e117 ꢁC. 1H NMR
Yield 78% (starting with 200 mg of 52d), mp 91e93 ꢁC. 1H NMR
d
1.38 (3H, t, J ¼ 7.5 Hz, 2-CH2CH3), 2.31 (3H, s, 8-CH3), 3.01 (2H, q,
d
1.25 (6H, d, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 1.50 (3H, t, J ¼ 7.5 Hz,
J ¼ 7.5 Hz, 2-CH2CH3), 3.96 (3H, s, 7-OCH3), 6.99 (1H, d, J ¼ 8.7 Hz,
2-CH2CH3), 2.35 (1H, m, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 2.85 (2H,
J ¼ 7.5 Hz, 2-CH2CH3), 4.07 (2H, d, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 4.93
(2H, s, 8-CH2Br), 6.28 (1H, s, 3-H), 7.09 (1H, d, J ¼ 9.0 Hz, 6-H), 8.27
(1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 339 (Mþ þ 1).
6-H), 8.08 (1H, d, J ¼ 8.7 Hz, 5-H). ESI MS m/z 297 (Mþ þ 1).
6.14.2. 3-Bromo-2-ethyl-7-isopropoxy-8-methyl-4H-chromen-4-
one (33)
Yield 41% (starting from 50 mg of 52c), mp 96e98 ꢁC. 1H NMR
6.15.5. 2-(1-Bromoethyl)-8-(bromomethyl)-7-methoxy-4H-
chromen-4-one (40)
d
1.35e1.42 (9H, m, J1 ¼ 6.0 Hz, J2 ¼ 7.5 Hz, 2-CH2CH3
&
7-OCH(CH3)2), 2.30 (3H, s, 8-CH3), 3.01 (2H, q, J ¼ 7.5 Hz, 2-CH2CH3),
4.70 (2H, m, J ¼ 6.0 Hz, 7-OCH(CH3)2), 6.98 (1H, d, J ¼ 9.0 Hz, 6-H),
8.04 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 325 (Mþ þ 1).
Yield 14% (starting with 300 mg of 52a), mp 153e156 ꢁC. 1H
NMR
d
2.09 (3H, d, J ¼ 7.2 Hz, 2-CHBrCH3), 4.04 (3H, s, 7-OCH3), 4.81
(2H, s, 8-CH2Br), 4.94 (1H, q, J ¼ 7.2 Hz, 2-CHBrCH3), 6.33 (1H, s,

Y. Chen et al. / European Journal of Medicinal Chemistry 49 (2012) 74e85
85
3-H), 7.02 (1H, d, J ¼ 9.0 Hz, 6-H), 8.16 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS
6.18. A549 cellular apoptosis assay
m/z 377 (Mþ þ 1).
A549 cells were seeded in 24-well plates at a density of
6.15.6. 2-(1-Bromoethyl)-8-(bromomethyl)-7-ethoxy-4H-chromen-
4-one (41)
5 ꢂ 103 cells/well, and incubated with 10 (0, 1, 5 and 10
mg/mL) for
48 h. The cellular monolayer was fixed and stained with DNA
fluorochrome Hoechst 33258 for 20 min. After washing with
phosphate buffered saline (PBS), the morphological features of
apoptosis (including cellular nucleus shrinkage, chromatin
condensation, intense fluorescence and nuclear fragmentation)
were monitored by fluorescence microscopy (Zeiss, German).
Yield 20% (starting with 880 mg of 52b), mp 138e140 ꢁC. 1H
NMR
d
1.53 (3H, t, J ¼ 6.9 Hz, 7-OCH2CH3), 2.09 (3H, d, J ¼ 7.2 Hz,
2-CHBrCH3), 4.26 (2H, q, J ¼ 6.9 Hz, 7-OCH2CH3), 4.82 (2H, s,
8-CH2Br), 4.95 (1H, q, J ¼ 7.2 Hz, 2-CHBrCH3), 6.33 (1H, s, 3-H), 6.99
(1H, d, J ¼ 9.0 Hz, 6-H), 8.12 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 391
(Mþ þ 1).
6.19. A549 human lung cancer cell cycle arrested assay
6.15.7. 2-(1-Bromoethyl)-8-(bromomethyl)-7-isopropoxy-4H-
chromen-4-one (42)
A549 cells (3 ꢂ 105/well) were seeded into 6-well plates and
Yield 15% (starting with 1.2 g of 52c), mp 95e97 ꢁC. 1H NMR
exposed to 10 at various concentrations (0, 1, 5 and 10 mg/mL) for
d
1.45 (6H, d, J ¼ 6.0 Hz, 7-OCH(CH3)2), 2.85 (3H, d, J ¼ 6.9 Hz,
48 h, and then harvested and washed with PBS, and fixed in 70%
EtOH at 4 ꢁC. Staining was performed in PBS containing 40
g/mL
RNaseA and 10 g/mL PI in the dark at rt for 30 min. The cell cycle
2-CHBrCH3), 4.80 (3H, m, J ¼ 6.0 Hz, 7-OCH(CH3)2 & 8-CH2Br), 4.94
(1H, q, J ¼ 6.9 Hz, 2-CHBrCH3), 6.32 (1H, s, 3-H), 7.00 (1H, d,
J ¼ 9.0 Hz, 6-H), 8.12 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 405
(Mþ þ 1).
m
m
was measured using FACScan Flow Cytometry (BD FACSCalibur).
Acknowledgments
6.15.8. 2-(1-Bromoethyl)-8-(bromomethyl)-7-isobutoxy-4H-
chromen-4-one (43)
This research was supported by the grants from the National
Natural Science Foundation of China awarded to P.X. (No.
20272010) and Y.C. (No. 30200348 and 30873164, respectively),
Grant 10DZ1972100 from Traditional Chinese Medicine Moderni-
zation to H.R.L., and Grant CA-17625-32 from the National Cancer
Institute awarded to K.H.L. Thanks are also due to the National Drug
Innovative Program (Grant No. 2009ZX09301-011) and Taiwan
Department of Health, China Medical University Hospital Cancer
Research Center of Excellence (DOH100-TD-C-111-005) for partial
support.
Yield 11% (starting with 200 mg of 52d), mp 108e109 ꢁC. 1H
NMR
d
1.11 (6H, d, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 2.09 (3H, d,
J ¼ 6.9 Hz, 2-CHBrCH3), 2.22 (1H, m, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2),
3.95 (2H, d, J ¼ 6.6 Hz, 7-OCH2CH(CH3)2), 4.83 (2H, s, 8-CH2Br), 4.94
(1H, J ¼ 6.9 Hz, 2-CHBrCH3), 6.33 (1H, s, 3-H), 7.0 (1H, d, J ¼ 9.0 Hz,
6-H), 8.12 (1H, d, J ¼ 9.0 Hz, 5-H). ESI MS m/z 419 (Mþ þ 1).
6.16. Sulforhodamine B (SRB) assay
For the anti-proliferation assay in tumor cells, KB (nasopha-
ryngeal), KB-vin (its vincristine-resistant subline), DU145 (pros-
tate) and A549 (lung) cells were fixed in situ with 10%
trichloroacetic acid (TCA) to represent a measurement of the cell
population at the time of drug addition. Briefly, the plates were
incubated for an additional 72 h after attachment and drug addi-
tion, and the assay was terminated by 10% TCA. Then, 0.4% SRB dye
in 1% HOAc was added to stain the cells for 10 min. Unbound dye
was removed by repeated washing with 1% HOAc, and the plates
were air dried. Bound stain was subsequently solved with 10 mM
trizma base, and the absorbance is read under 515 nm. Growth
inhibition of 50% (GI50) was calculated as the drug concentration
that caused a 50% reduction in the net protein increase in control
cells during the drug incubation.
Appendix. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.ejmech.2011.12.025.
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and replaced with fresh medium (180
mL/well). Twenty mL of
MTT solution (5 mg/mL) were added to each well and the plates
were incubated for an additional 4 h at 37 ꢁC. The medium was
aspirated off, and DMSO (150
mL) was added to each well. The
absorbance was read at 570 nm using a microplate reader (TECAN
Infinite 200). Cell viability was expressed as a percentage of the
untreated cells.