Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin derivatives as potential antitumor agents

Article abstract of DOI:10.1016/j.bmcl.2013.12.095

In this research, a series of 4-(1,2,3-triazol-1-yl)coumarin conjugates were synthesized and their anticancer activities were evaluated in vitro against three human cancer cell lines, including human breast carcinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell. To increase the biological potency, structural optimization campaign was conducted focusing on the C-4 position of 1,2,3-triazole and the C-6, C-7 positions of coumarin. In addition, to further evaluate the role of 1,2,3-triazole and coumarin for antiproliferative activity, 9 compounds possessing 4-(piperazin-1-yl)coumarin framework and 3 derivatives baring quinoline core were also synthesized. By MTT assay in vitro, most of the compounds display attractive antitumor activities, especially 23. Further flow cytometry assays demonstrate that compound 23 exerts the antiproliferative role through arresting G₂/M cell-cycle and inducing apoptosis.

Full text of DOI:10.1016/j.bmcl.2013.12.095

Bioorganic & Medicinal Chemistry Letters 24 (2014) 799–807
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 4-(1,2,3-triazol-1-yl)coumarin
derivatives as potential antitumor agents
Wenjuan Zhang, Zhi Li, Meng Zhou, Feng Wu, Xueyan Hou, Hao Luo, Hao Liu, Xuan Han, Guoyi Yan,
⇑
Zhenyu Ding, Rui Li
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041 Sichuan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this research, a series of 4-(1,2,3-triazol-1-yl)coumarin conjugates were synthesized and their antican-
cer activities were evaluated in vitro against three human cancer cell lines, including human breast car-
cinoma MCF-7 cell, colon carcinoma SW480 cell and lung carcinoma A549 cell. To increase the biological
potency, structural optimization campaign was conducted focusing on the C-4 position of 1,2,3-triazole
and the C-6, C-7 positions of coumarin. In addition, to further evaluate the role of 1,2,3-triazole and cou-
marin for antiproliferative activity, 9 compounds possessing 4-(piperazin-1-yl)coumarin framework and
3 derivatives baring quinoline core were also synthesized. By MTT assay in vitro, most of the compounds
display attractive antitumor activities, especially 23. Further flow cytometry assays demonstrate that
compound 23 exerts the antiproliferative role through arresting G2/M cell-cycle and inducing apoptosis.
Ó 2014 Elsevier Ltd. All rights reserved.
Received 9 October 2013
Revised 21 November 2013
Accepted 23 December 2013
Available online 29 December 2013
Keywords:
Coumarin
1,2,3-Triazole
Antiproliferative activity
G2/M cell-cycle arrest
Apoptosis
Coumarin derivatives which are widely distributed in the
plants1,2 have been extensively investigated as anticoagulation,
antiviral,3–5 anti-inflammatory,6,7 antibacterial8 and anticancer9–
While the conjugates of 1,2,3-triazole and coumarin fighting
against different diseases have made significant progress, efficient
molecules against cancer are still urgently needed. As for 4-(1,2,3-
triazol-yl)coumarin derivatives, the synthesis methods have been
reported,30 however, so far their anticancer activities have rarely
been investigated. In our preliminary experiments, we found
6-bromo-4-(4-phenyl-1,2,3-triazol-1-yl)coumarin (compound 1)
showed a moderate anticancer activity with the IC50 values of
17
agents. Another promising group, 1,2,3-triazole, has emerged
as one of the most important heterocycles in current medicinal
chemistry18–20 and its applications have also been extended to
widespread diseases.21–24 In recent years, a library of coumarin
derivatives conjugated with 1,2,3-triazole were synthesized and
proved to possess different bioactivity. As far as anticancer activity
was concerned, H.M. Liu and his coworkers discovered 4-((1,2,3-
triazol-1-yl)methyl)coumarin derivatives exhibiting obvious anti-
cancer activity through inducing apoptosis.25 Besides, novobiocin
analogues with 1,2,3-triazole at the C-3 position of coumarin
displayed potent cytotoxic activity against two breast cancer cell
lines (SKBr-3 and MCF-7).26 For anti-inflammatory activity, a
family of 3-(1,2,3-triazol-1-yl)coumarins, effecting on inducible
nitric oxide synthase, were synthesized and proved to reduce neu-
trophils in the LPS-inflamed subcutaneous tissue,27 while, through
inhibiting 5-lipoxygenase, triazole connected to C-7 position of
coumarin by a methene can also cause remarkable anti-inflamma-
tory activity.28 In addition, compounds with 1,2,3-triazol-1-yl at
C-3 position of coumarin revealed the inhibition of amyloid-b
aggregation which plays a pathogenic role in the progression
of Alzheimer’s disease.29 Compounds mentioned above are sum-
marized in Figure 1.
9.45, 6.66, 11.23 lM against MCF-7, SW480 and A549, respec-
tively, which inspired us to conduct the structural optimization
of compound 1 for more potent anticancer agents. Our optimiza-
tion focused on the following two aspects: (1) in view of the
planarity caused by the conjugated system, introducing a series
of chemical bridges (–CH2–NH–, –CH2–O– or –CH2–S–) between
phenyl and 1,2,3-triazole may be helpful to eliminate the planar-
ity, thereby ameliorate the druggability. Meanwhile, the rotatable
bonds may also facilitate the compounds binding to its possible
receptor through induced fit. (2) To improve the potency, different
substitutions introduced to C-6 and C-7 positions of coumarin
were taken into account. Moreover, it should be mentioned that
in order to evaluate the contribution of 1,2,3-triazole and
coumarin to the antiproliferative activity, respectively, the
replacements of 1,2,3-triazole with piperazine and coumarin with
quinoline were conducted and their biological activity were also
measured.
The general procedures for the preparation of the 4-(1,2,3-tria-
zol-1-yl)coumarin derivatives 1–23 and 6e were efficiently synthe-
sized according to the protocol outlined in Schemes 1–3. Different
⇑
Corresponding author.
E-mail address: lirui@scu.edu.cn (R. Li).
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.095

800
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
O
O
N
N
N
O
O
O
O
HO
F3C
Me
N
N
N
N
N
N
F
anti-inflammatory agent
5-lipoxygenase inhibitor
amyloid- aggregation inhibitor
β
HO
O
O
N
O
O
O
N
N
N
N
O
NH
O
O
N
N
S
N
S
novobiocin analogue
anticancer agent
anticancer agent
Figure 1. Structures of coumarin derivatives conjugated with 1,2,3-triazole.
OH
R2
i
R2
R1
OH
O
R2
R1
O
iii
R2
R1
O
O
ii
R1
O
OH
2a R1=Br
1a R1=Br
R2=H
3a R1=Br
R2=H
R2=H
2b R1=OCH3
2c R1=H
1
1b R =
R2=H
R2=OCH3
3b R1=OCH3
3c R1=H
R2=H
R2=H
OCH
3
1c R1=H
R2=OCH3
R2=OCH3
R2
R1
O
O
iv
v
R2
R1
O
O
vi
R2
R1
O
O
R
Cl
N3
N
N
N
4a R1=Br
R2=H
R2=H
5a R1=Br
R2=H
4b R1=OCH3
5b R1=OCH3
R
R2=H
4c R1=H
R2=OCH3
R2=H
5c R1=H
,
-
R2=OCH3
R2=H
6e 1 23
1
4d
4e
4f
R =OH
1
5d
5e
5f
R =OH
R =H
R =H
1
R2=OH
R2=H
R =H
1
R2=OH
R2=H
1
R =H
1
Scheme 1. Reagents and conditions: (i) acetic anhydride, pyridine, 100 °C, 3.5 h, 82–98%; (ii) AlCl3, 150 °C, 3.5 h, 70–75%; (iii) diethyl carbonate, NaH, Toluene, 100 °C, 4 h,
67–86%; (iv) POCl3, Triethylamine, reflux, 1 h, 70–85%; (v) NaN3, NMP, rt, 69–82%; (vi) t-BuOH, Cu, CuSO4Á5H2O, 65 °C, 49–80%.
phenol derivatives were reacted with acetic anhydride at 100 °C to
prepare the esters 1a–1c, which were further subjected to the Fries
rearrangement reaction with o-hydroxyacetophenone 2a–2c in the
presence of aluminum chloride at 150 °C. Compounds 3a–3c,
which are crucial to the synthesis of all 4-(1,2,3-triazol-1-yl)cou-
marin derivatives, were synthesized from o-hydroxyacetophenone
derivatives by reacting with diethyl carbonate and sodium hydride
at 100 °C. Compounds 4a–4c, with a chlorine group at the C-4 po-
sition of the coumarin ring, were synthesized in 60–97% yield by
treatment of 3a–3c compounds with phosphorus oxychloride
(POCl3) and triethylamine. The product (4b and 4c) in dichloro-
methane was then hydrolyzed with BBr3 at cooled temperature
to obtain the 4-chloro-6-hydroxycoumarin (4d and 4e) (Scheme 2).
The treatment of 4-azido-coumarin compounds (5a–5f) which
were obtained through 4-chloro-coumarin derivatives in the pres-
ence of NaN3 at room temperature, and alkynes (Scheme 3), affor-
ded the 4-(1,2,3-triazol-1-yl) coumarin derivatives in tert-butyl
alcohol at 65 °C with copper sulfate pentahydrate and copper as
CuAAC.31 Furthermore, 4-fluorobenzene-1-sulfonyl chloride, meth-
anesulfonyl chloride or morpholine derivative (12a) were reacted
with compound 6e or 16 to prepare target compounds 18, 19
and 17 (Scheme 2).
To further measure the contribution of 1,2,3-triazole and cou-
marin to the antiproliferative activity, we next replaced them with
piperazine and quinoline, respectively (Scheme 4 and 5). As
outlined in Scheme 4, each candidate was obtained starting from
4-chlorocoumarin (4f) and the synthesis began with the substitu-
tion of piperazine or 2-(piperazin-1-yl)ethanol to obtain 6a and
6b. The 4-(4-benzylpiperazin-1-yl)coumarin analogues 24–30
were prepared by the reaction of the corresponding benzaldehyde
with 6a. On the other hand, analogues 31 and 32 were synthesized
from 6b in two steps. In addition, the syntheses of 6-bromoquino-
lin-4-ol, 2g, was accomplished according to the cyclization of 1g in
boiling diphenyl ether, which was achieved with 4-bromoaniline in

W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
801
O
O
O
O
R
22 R=
HO
O
O
O
O
O
O
F
N
N
N
ii
iii
i
O
HO
HO
16
R=
Cl
Cl
N3
R
16, 22
4b
4d
5d
O
N
O
O
N
O
R
O
S
O
HO
O
18 R=
19 R=
O
O
O
F
HO
O
O
10a
HO
O
O
N
N
N
N
ii
i
iv
iii
O
S
O
O
O
Cl
N3
Cl
4c
4e
5e
OH
OTs
H
N
N
16
N
O
17
v
vi
vii
O
O
12b
12a
Scheme 2. Reagents and conditions: (i) BBr3, DCM, À22 °C (2 h), rt (5 h), 95–97%; (ii) NaN3, NMP, rt, 69–80%; (iii) t-BuOH, Cu, CuSO4Á5H2O, 65 °C, 49–53%; (iv) CH3SO2Cl
or 4-fluorobenzene-1-sulfonylchloride, Et3N, THF, 0 °C, 78–80%; (v) K2CO3, MeCN, reflux, 3 h, 78%; (vi) TsCl, Et3N, DMAP, DCM, rt, overnight, 65%; (vii) K2CO3, DMF, 60 °C,
24 h, 57%.
X
X
Br
9a R5=p-F,
8a R5=o-COOCH3,
8b R5=o-Br,
X=O
X=O
R5
R5
X=O
X=O
X=S
9b R5=p-C2H5,
9c R5=p-Br,
9d R5=p-NH2,
DMF/K2CO3
8c R5=o-OH,
X=O
8a-8d,9a-9i,10a-10e
X=O
10a R5=H,
X=OH,SH or NH2
X=O
9e R5=p-COOCH ,
X=O
X=O
X=O
X=O
10b R5=o-COOCH3, X=O
3
9f R5=p-CF3,
10c R5=H,
10d R5=H,
X=S
X=N
9g R5=p-NO2,
9h R5=p-COOEt,
Br
N
Br
N
Br
DMF/K2CO3
N
O
N
Br
O
DMF/K2CO3
OH
OH
11b
11a
Scheme 3. Reagents and conditions: 3-bromopropyne, K2CO3, DMF, rt, overnight, 60–98%.
the presence of 2,2-dimethyl-1,3-dioxane-4,6-dione and triethyl
orthoformate in ethanol at 90 °C. Nitration occurring at C-3 posi-
tion of quinoline resulted in 6-bromo-3 -nitroquinolin-4-ol (3g).
Compound 4g with a chlorine at C-4 position of quinoline was syn-
thesized by 3g, POCl3 and triethylamine and used to prepare com-
pound 5g in the presence of NaN3 in N-methylpyrrolidone at room
temperature. All the 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl)quino-
line derivatives 33–35 were obtained from 5g and alkynes via
CuAAC (Scheme 5). The general reaction conditions and processing
and the Supplementary Data (MS, NMR, yield and purity) are de-
scribed in Supporting Information.
The IC50 values (concentration required to inhibit tumor cell
proliferation by 50%) for the synthesized compounds against three
human cancer cell lines including human breast carcinoma MCF-7
cell, colon carcinoma SW480 cell and lung carcinoma A549 cell
were measured by MTT assay (Tables 1–5). All of our antiprolifer-
ation tests were performed three times using each agent.
bridges, more specifically, –CH2–NH–, –CH2–O– and –CH2–S– were
inserted to phenyl and 1,2,3-triazole. As for the –CH2–NH– and –
CH2–S– bridges, compounds 2 and 3 displayed improved inhibitory
activity against three tested cancer cell lines with IC50 values of
ꢀ3.51
(compound 4) authorized the potential inhibitory against MCF-7
with IC50 value of 1.72 M. Subsequently, the replacement of phe-
lM. To our delight, the introduction of –CH2–O– bridge
l
nyl with heterocycles, including pyridine (5) and quinoline (6),
generally resulted in loss in bioactivity on three cancer cell lines.
Hence, we use compound 4 for further optimization.
Furthermore, we decided to optimize the substituents of the
phenyl ring, and the IC50 values were summarized in Table 2. First
of all, compounds 7–9 with different substitutions (methoxycar-
bonyl, bromine and hydroxy, respectively) at the C-2 position
showed drastically decreased or equivalent activity against lead
compound 4, suggesting that the modification at C-2 position is
not allowed. Then we turned our attention to the C-4 position of
phenyl. The results in Table 2 show that a lipophilic group, ethyl
(10), and polar groups, including amino (11), bromine (12) and
Consideration of the few rotatable bonds of compound 1, which
may not bind to the possible target well, a series of chemical

802
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
O
H
O
O
H
N
O
N
O
O
O
N
H
i
N
N
R4
ii
Cl
R4
N
H
4f
24-30
6a
HN
iii
N
O
O
O
OH
O
O
R3
O
N
N
HO
N
N
N
N
v
iv
O
OH
OTs
R3
6b
7b
31,32
Scheme 4. Reagents and conditions: (i) ethanol, rt, 70%; (ii) Na(OAc)3BH, DCM, rt, 70–78%; (iii) ethanol, rt, 70%; (iv) TsCl, Et3N, DMAP, DCM, rt, 31%; (v) 60–71%.
OH
N
OH
N
Br
Br
O
O
Br
Br
NO2
i
ii
iii
N
O
NH2
O
O
O
1g
2g
3g
O
O
R
N
N3
N
Cl
N
N
N
Br
Br
NO2
NO2
iv
v
vi
Br
NO2
R
N
4g
5g
33-35
Scheme 5. Reagents and conditions: (i) triethyl orthoformate, 90 °C, 10 min, EtOH, 30 min, 66%; (ii) diphenyl oxide, 220 °C, 5 min, 83.5%; (iii) nitric acid, Propionic acid,
125 °C, 2 h, 50.2%; (iv) POCl3, triethylamine, reflux, 1 h, 68%; (v) NaN3, NMP, rt, 59%; (vi) t-BuOH, Cu, CuSO4Á5H2O, 65 °C, 67–75%.
trifluoromethyl (13), led to the equivalent potent or slightly in-
creased activity. However fluorine and methoxycarbonyl at the
C-4 position endowed compounds (14 and 15, respectively) with
prominent activity against all the three cancer cell lines as low as
0.66 lM, indicating that the hydrogen acceptor at the C-4 position
of phenyl is essential to the activity.
have been expected, compound 23, with fluorine retained at the
C-4 position of phenyl, showed higher bioactivity with IC50 values
of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respec-
tively. It is worth noting that compound 23 is more potent than
compound 20, which can be ascribed to the additional hydrogen
bond caused by the methoxy at C-7 position. Lastly, with hydrogen
at C-7 position of coumarin and fluorine at the C-4 position of phe-
nyl, it was discovered that the replacement of bromine with meth-
oxy (21) or hydroxy (22) group at the C-6 position of coumarin
resulted in the approximate activity to compound 14, reconfirming
that there may be a steric hindrance at the C-6 region.
To further measure the contribution of the triazole and couma-
rin motifs to the biological activity, a number of 6-bromo-4-(pip-
erazin-1-yl)coumarin and 6-bromo-3-nitro-4-(1,2,3-triazol-1-yl)
quinoline derivatives were prepared (Tables 4 and 5). First, the
coumarin analogues substituted by piperazine exhibited enormous
reduction of potency, especially compounds 24–30. However, the
extended chemical bridge (compounds 31, and 32) between phenyl
and piperazine could give a slight improvement in inhibitory activ-
ity, probably because the rotatable bonds can facilitate the
compounds binding to the possible target. On the other hand,
Next, we turned our attention to the substituents at the C-6 and
C-7 positions of coumarin. Compared with the activity of com-
pound 4, complete loss of antiproliferative activity were observed
in Table 3 when the bromine was replaced with hydroxy (16) or
2-morpholinoethoxy (17). Nevertheless, when the bromine was re-
placed with hydrogen, compound 20, with fluorine at the C-4 posi-
tion of phenyl, showed more potency than compound 14 in the
MCF-7 and A549, indicating that bulky group may be unfavorable.
Subsequently, with hydrogen at the C-6 position, the introduction
of bulky group, 4-fluorobenzenesulfonate (18), to C-7 position led
to total loss of potency, while the introduction of smaller group,
methylmethanesulfonate (19), only caused the partial loss of the
bioactivity, suggesting that small group at C-7 position may also
be advantageous to the bioactivity. Then, methoxy introducing to
the C-7 position of coumarin was taken into account. As might

W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
803
Table 1
In Vitro Inhibition of Tumor Cell Growth of compounds 1–6
R
N
N
N
Br
O
O
a
Compd
R
IC50
(lM)
MCF-7
SW480
A549
1
2
9.45 0.55
>50
6.66 0.31
11.23 2.0
18.41 3.5
15.43 0.33
4.99 0.42
13.2 4.2
HN
S
3
4
36.83 2.3
1.72 0.64
3.51 0.98
12.31 0.11
24.79 2.3
O
5
O
39.82 1.8
39.77 2.4
N
N
6
41.56 1.4
3.51 0.31
16.95 2.3
2.43 0.25
13.29 4.9
1.65 0.09
O
Br
Doxorubicin
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard
deviation (n = 3).
Table 2
In Vitro Inhibition of Tumor Cell Growth of compounds 7–15
R5
O
N
N
N
Br
O
O
a
Compd
R5
IC50 (lM)
MCF-7
SW480
A549
7
8
9
10
11
12
13
14
2-COOCH3
2-Br
2-OH
4-CH2CH3
4-NH2
4-Br
4-CF3
4-F
4-COOCH3
34.14 2.2
28.91 1.3
24.82 0.89
12.67 1.4
2.04 1.1
1.92 0.71
11.41 2.3
7.00 0.25
3.13 0.24
3.51 0.31
20.23 3.4
20.13 1.7
19.14 3.3
10.82 0.60
2.09 0.13
1.31 3.2
5.25 0.24
8.43 0.51
7.94 3.4
11.58 0.36
11.85 0.24
24.47 2.5
1.62 1.1
0.85 0.09
0.66 0.03
1.65 0.09
3.09 2.0
1.77 0.03
3.62 0.25
2.43 0.25
15
Doxorubicin
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard
deviation (n = 3).
compounds 33–35, with coumarin replaced by quinoline, can
maintain a part of antiproliferative activity, possibly due to the
nitrogen atom of the quinolone which is similar to the oxygen
atom of coumarin that can form hydrogen bond with the corre-
sponding acceptor. The fact that the coumarin derivatives are more
potent than quinoline can be interpreted by the additional
carbonyl group of coumarin, which may form another hydrogen
bond with its possible receptor. Structure activity relationships
(SAR) were summarized in Figure 2.
Because compound 23 shows the highest antiproliferative
activity among all the synthesized compounds, it was selected to
assess the effect on cell-cycle arrest of MCF-7 by flow cytometry

804
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
Table 3
In Vitro Inhibition of Tumor Cell Growth of compounds 16–23
R5
O
N
N
N
R1
R2
O
O
a
Compd
R1
R2
H
R5
IC50 (lM)
MCF-7
>50
SW480
>50
A549
>50
16
OH
H
O
N
O
17
H
H
>50
>50
>50
>50
O
S
O
O
F
18
19
H
H
H
H
>50
>50
O
O
S
45.81 1.4
10.67 0.98
9.14 0.58
O
20
21
22
23
H
H
H
H
4-F
4-F
4-F
4-F
5.84 0.22
8.56 0.23
4.62 0.65
5.89 0.14
3.51 0.31
3.82 0.40
25.87 1.4
2.72 0.30
1.99 0.38
2.43 0.25
0.68 0.08
3.15 0.39
2.01 0.14
0.52 0.21
1.65 0.09
OCH3
OH
H
OCH3
Doxorubicin
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard
deviation (n = 3).
Table 4
In Vitro Inhibition of Tumor Cell Growth of compounds 24–32
R0
N
N
O
O
a
Compd
R0
IC50 (lM)
MCF-7
SW480
>50
A549
>50
24
>50
F
25
26
>50
>50
>50
>50
>50
>50
HO
27
28
>50
>50
>50
>50
>50
>50
O
O
HO

W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
805
Table 4 (continued)
a
Compd
R0
IC50
(l
M)
MCF-7
SW480
>50
A549
>50
O
29
30
>50
O
>50
>50
>50
OH
O
31
32
24.20 2.5
35.01 5.0
21.58 4.8
O
O
>50
24.01
11.92
F
Doxorubicin
3.51 0.31
2.43 0.25
1.65 0.09
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard
deviation (n = 3).
Table 5
In Vitro Inhibition of Tumor Cell Growth of compounds 33–35
R5
X
N
N
N
Br
NO2
N
a
Compd
X
R5
IC50
(lM)
MCF-7
33
34
35
S
O
O
H
H
COOEt
22.72 3.3
7.69 4.3
13.88 1.3
3.51 0.31
Doxorubicin
a
Inhibitory activity was assayed by exposure for 48 h to substances and expressed as concentration required to inhibit tumor cell proliferation by 50% (IC50). SD, standard
deviation (n = 3).
assay using propidium iodide (PI) staining. As is seen from
Figure 3a, in comparison with the control group, treatment of
compound 23 (5 lM) led to the obvious decrease in G1 phase (from
52.82% to 3.06%) and dramatic increase in G2 phase (from 15.78% to
84.21%), indicating that compound 23 is a G2/M cell-cycle arrester.
A much stronger accumulation of cell in G2/M phase caused by
compound 23 inspired us to test whether it can induce the apopto-
sis of A549 by flow cytometry analysis using annexin V-FITC and
propidium iodide (PI) staining. The percentage of apoptotic cells
induced by compound 23 (1.25 lM) is increased by more than
6-fold (Fig. 3b). All these results indicate that the promising antitu-
mor activity of compound 23 may be attributed to the G2/M phase
arrest and apoptosis.
In summary, a family of 4-(1,2,3-triazol-1-yl)coumarin deriva-
tives were synthesized and their antiproliferative activities were
evaluated. Among these compounds, 23, 4-(4-((4-fluorophenoxy)
methyl)-1,2,3-triazol-1-yl)-7-methoxycoumarin, exhibited excel-
lent broad spectrum anticancer activity in vitro with IC50 values
Figure 2. Structure-activity relationship revealed by optimization.

806
W. Zhang et al. / Bioorg. Med. Chem. Lett. 24 (2014) 799–807
Figure 3. (a) Flow cytometry analyses of cell cycle distribution of breast cancer cell MCF-7 after treatment of compound 23 (5
control for 48 h. (b) Apoptosis induction in lung carcinoma cell A549 after 48 h treatment with 23 (1.25 M) and no treatment.
lM) and no treatment (Ctrl) as reference
l
of 5.89, 1.99 and 0.52 lM against MCF-7, SW480 and A549, respec-
References and notes
tively. The further mechanism study demonstrated that compound
23 could obviously inhibit the proliferation of cancer cells through
inducing apoptosis and arresting the cell-cycle at G2/M phase. In
addition, structure activity relationship research revealed that –
CH2–O– bridge at C-4 position of 1,2,3-triazole core is the best
optimal for bioactivity; a hydrogen bond acceptor at C-4 position
of phenyl is indispensable for the improvement of potency; the
hydrogen bond acceptor at C-7 position of coumarin can make a
positive contribution to the activity. All the conclusions have
encouraged us to continue the development, and the further
validation of mechanism at the molecular level is undergoing.
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