Antitumor activities of biscoumarin and dihydropyran derivatives

Article abstract of DOI:10.1016/j.bmcl.2016.07.023

Rising worldwide cancer incidence and resistance to current anti-cancer drugs necessitate the need for new pharmaceutical compounds and drug delivery system. Two novel series of biscoumarin (1–4) and dihydropyran (5–16) derivatives were synthesized via a one-pot multicomponent condensation reaction and evaluated for their antitumor activity in vitro. The X-ray crystal structure analysis of four representative compounds 2, 7, 10 and 13 confirmed the structures of these compounds. Compounds 1–4 showed the most potent antitumor activity among the total 16 derivatives. More interestingly, preliminary mechanism studies revealed that the most potent compound 4 induced apoptosis and arrested the cell cycle at the S phase in HUTU80 cells. Additionally, the increased accumulation of HUTU80 cells in the sub G1 peak further pointed to the occurence of the cell apoptosis. The selectivity index analysis demonstrated that all the biscoumarin compounds (SI?=?3.1–7.5) possess higher selectivity towards intestinal epithelial adenocarcinoma cell line (HuTu80) than positive control drug carboplatin (SI?=?1.6–1.8). The biscoumarin compounds also showed no obvious acute toxicity on mice.

Full text of DOI:10.1016/j.bmcl.2016.07.023

Accepted Manuscript
Antitumor Activities of Biscoumarin and Dihydropyran Derivatives
Hai-Yu Zhou, Feng-Quan Dong, Xin-Liang Du, Zhi-Kun Zhou, Hai-Ru Huo,
Wei-Hao Wang, Hong-Dan Zhan, Yi-Fei Dai, Jing-Meng, Yun-Peng Sui, Jing
Li, Feng Sui, Yun-Hui Zhai
PII:
S0960-894X(16)30736-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.023
BMCL 24067
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 January 2016
9 June 2016
7 July 2016
Please cite this article as: Zhou, H-Y., Dong, F-Q., Du, X-L., Zhou, Z-K., Huo, H-R., Wang, W-H., Zhan, H-D.,
Dai, Y-F., Jing-Meng, Sui, Y-P., Li, J., Sui, F., Zhai, Y-H., Antitumor Activities of Biscoumarin and Dihydropyran
Derivatives, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.023
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Antitumor Activities of Biscoumarin and Dihydropyran
Derivatives
Hai-Yu Zhoua§, Feng-Quan Dong b§, Xin-Liang Duc§, Zhi-Kun Zhoud§, Hai-Ru Huoa§,
Wei-Hao Wanga, Hong-Dan Zhana, Yi-Fei Daia, Jing-Menga, Yun-Peng Suie, Jing Lif, Feng Suia*,
Yun-Hui Zhaif*
a Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
b
Department of Cardiology, The Frist Affliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang
325035, China
c Graduate School of China Academy of Chinese Medical Sciences, Beijing 100700, China;
d Department of Pharmacy, Guangdong Medical College, Dong guan 523808, China;
e Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050, China
f School of Chemical Engineering, The Key Laboratory for Surface Engineering and Remanufacturing in Shaanxi
Province, Xi'an University, Xi'an 710065, China;
§ These authors contributed equally to this work.
Correspondence e-mail: fsui@icmm.ac.cn (Feng Sui); Yunhui_zhai66@yeah.net (Yun-Hui Zhai)
Abstract
Rising worldwide cancer incidence and resistance to current anti-cancer drugs necessitate the need for new
pharmaceutical compounds and drug delivery system. Two novel series of biscoumarin (1-4) and dihydropyran
(5-16) derivatives were synthesized via a one-pot multicomponent condensation reaction and evaluated for their
antitumor activity in vitro. The X-ray crystal structure analysis of four representative compounds 2, 7, 10 and 13
confirmed the structures of these compounds. Compounds 1-4 showed the most potent antitumor activity among
the total 16 derivatives. More interestingly, preliminary mechanism studies revealed that the most potent
compound 4 induced apoptosis and arrested the cell cycle at the S phase in HUTU80 cells. Additionally, the
increased accumulation of HUTU80 cells in the sub G1 peak further pointed to the occurence of the cell apoptosis.
The selectivity index analysis demonstrated that all the biscoumarin compounds (SI=3.1-7.5) possess higher
selectivity towards intestinal epithelial adenocarcinoma cell line (HuTu80) than positive control drug carboplatin
(SI=1.6-1.8). The biscoumarin compounds also showed no obvious acute toxicity on mice.
Key words Biscoumarin, Dihydropyran, X-ray, Antitumor, Apoptosis, Acute toxicity
Cancer is one of the major public health problems worldwide representing the leading cause of morbidity and
mortality in industrialized countries, with approximately 14 million new cases and 8.2 million cancer-related
deaths in 2012, which are expected to rise in the future [1]. In China, according to the Annual Cancer Registry in
2013, more than 3 million new cases were diagnosed with cancer, which is equivalent to 6 patients diagnosed per
minute, and the prevalence has been increasing [2, 3]. This casts great socioeconomic burdens. Despite the fact that
chemotherapy is central to clinical management of cancer, failure in chemotherapy is not uncommon, mainly due
to the dose-limiting toxicities, which is associated with the occurrence of drug resistance [4]
.
Natural products have been used for the treatment of various diseases and are becoming an important
research area for drug discovery. These products, especially phytochemicals have been extensively studies and

have exhibited anti-carcinogenic activities by interfering with the initiation, development and progression of
cancer through the modulation of various mechanisms including cellular proliferation, differentiation, apoptosis,
angiogenesis, and metastasis [5-7]. This concept is gaining attention because it is a cost-effective alternative to
cancer treatment.
However, the naturally occuring compounds generally tend to be less potent when used for prevention and
treatment of cancer [8]. In order to get more effective antitumor agents, it is possible to make modifications on
active chemical structures of title compounds. In the present study, two novel series of biscoumarin (1-4) and
dihydropyran (5-16) derivatives were firstly synthesized (Fig. 1), their antitumor activities on intestinal epithelial
adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line
(PANC1) in vitro were then evaluated. In addition, selective toxicity of the biscoumarin compounds (1-4) was
evaluated in normal human umbilical vein endothelial cell (HUVEC) and human embryonic kidney 293 cell
(HEK293) using the MTT assay. The compound 4 that demonstrated the best antitumour action was chosen to be
assayed by flow cytometry to determine its apoptotic behavior and mechanism. The acute toxicity of compounds
1-4 which have higher antitumour effects than others was also carried out in mice.
O
OH HO
R=
O
O
F
O
CN
F
O
O
R
2
4
1
3
NH
2
CN
R
O
O
R=
NO
Br
CN
2
O
5
8
7
6
NH
2
CN
R
O
CF
F
F
O
3
R=
SO
2
O
CF
F
3
9
10
11
12
NH
2
CN
Cl
O
O
O
CN
R
R=
Cl
O
O
15
13
14
16
Fig. 1 Chemical structures of compounds 1-16.

The compounds were synthesized according to general method as described in Scheme 1 and 2 (see
Supplementary data). Biscoumarin 1-4 were synthesized via a one-pot two-component reaction by condensing
aromatic aldehydes and 4-hydroxycoumarin in the presence of catalytic amount of piperidine in ethanol under
reflux conditions. Pyran derivatives 5-16 were synthesized via a one-pot three-component reaction by condensing
aromatic aldehydes, 4-hydroxycoumarin (1,1-dimethyl-3,5-cyclohexanedione or 1,3-cyclopentadione) and
malononitrile in the presence of 4-(dimethylamino)pyridine (DMAP) as a highly efficient homogenous catalyst.
1
Additionally, chemical structures of all compounds were further characterized by H NMR and ESI-MS (see
Supplementary data).
In order to further confirm the configuration of the products, single crystals of four representative compounds
2, 7, 10 and 13 were cultured for X-ray diffraction analysis. From Fig. 2 we can see that, in crystal structure of
compound 2, two 4-hydroxycoumarin moieties are linked through a methylene bridge, wherein one hydrogen
atom has been replaced with a 2-methoxyphenyl group; and two classical intramolecular hydrogen bonds
(O3–H3···O4 and O6–H6···O1) between a hydroxyl group of one coumarin fragment and a lacton carbonyl group of
another coumarin fragment further stabilize the whole structure.
In crystal structures of compounds 7, 10 and 13, the 4H-pyran ring is nearly planar and the adjacent ketone
ring also adopts a planar conformation. The 4H-pyran ring is almost perpendicular to the benzene ring and is
almost coplanar with the mean plane of the ketone ring.
Compound 2
Compound 7
Compound 10
Compound 13
Fig. 2 Crystal structures of compounds 2, 7, 10 and 13.

Intestinal epithelial adenocarcinoma cell line (HuTu80, human origin), mammary adenocarcinoma cell line
(4T1, mouse origin) and pancreatic cancer cell line (PANC1, human origin) were selected to evaluate the
antitumor activities of the synthesized compounds 1-16 against different tumor types in vitro. Carboplatin, a
standard antitumor drug, was applied to compare the potency of cytotoxicity of the tested compounds under the
same experimental condition. The experimental results demonstrated that all the tested compounds had a certain
degree of cell-killing activities against the three tumor cell lines and their inhibitory actions showed a evident
concentration-activity relationship. Their half maximal inhibitory concentration (IC50) and IC90 values (dose of the
compound which cause a 50% and 90% reduction of survival values, respectively) are shown in Table 1. As can
be seen in Table 1, according to the antitumor activity strength, the tested compounds can be divided into different
groups. Among these compounds, biscoumarins 1-4 showed more potent anticancer activity against the three
tested tumor cells (HuTu80, 4T1 and PANC1) with IC50 and IC90 values of 15-93.5 μg/mL and 28-201.5 μg/mL
respectively; the IC50 and IC90 values of the biscoumarins 1-4 against HuTu80 are much lower than that of the
positive control drug carboplatin with IC50 and IC90 values of 52.5-70.1 μg/mL and 90.7-156.2 μg/mL
respectively.
Table 1 IC50 and IC90 values of compounds 1-16 and carboplatin against three tumor cell lines (μg/mL, n=3)
HUTU80
4T1
PANC1
Drugs
IC50
IC90
IC50
IC90
IC50
IC90
53.8±4.1
17.9±1.8
47.6±1.5
15±1.2
103.2±9.8
85.7±6.4
166.6±12.7
133.2±8.5
102±6.2
98.4±5.5
>500
42.5±3.7
62.1±4.2
93.5±5.6
82.4±7.4
>300
82.5±4.9
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Compound 14
Compound 15
Compound 16
Carboplatin
109.7±8.4
98.3±1.9
28±2.3
52.2±±5.5
51.5±4.8
47.9±1.9
>300
110.3±9.6
201.5±13.8
155±12.1
>500
>300
446.6±21
>500
238.1±16.4
>300
>300
>500
256.1±10.6
>300
>500
>500
>300
>500
>500
97±3.5
187.2±5.6
>500
142.2±10.6
>300
289.4±15.8
>500
218.4±13.4
>300
347.8±21
>500
>300
283.3±21
297.3±18
>300
>500
247.8±10.6
240.9±16.9
>300
>500
>300
>500
>500
>500
282.8±11.5
>300
>500
>500
>500
>500
149.6±10.5
191.3±14
103±11.2
302.3±9.6
381.2±8.9
233.3±13.7
127.2±11.8
125.2±7.9
116.6±8.2
153.4±6.9
52.5±3.8
306.8±21.1
298.6±22
288.5±14.4
364.9±9.1
90.7±2.6
156.2±8.3
142.7±8.8
219.3±12.2
199.7±10.1
70.1±3.5
325.4±14.2
317.4±20.8
435.2±21
391.1±12
156.2±11.4
185.2±16.2 359.8±11.6
54.3±6.9 114.7±10.2
The IC50 (dose of the compound which caused a 50% reduction of survival.) and IC90 (dose of the compound
which caused a 90% reduction of survival) values were calculated from dose-response curves for each compound.
IC50 and IC90 data are an average of at least 3 independent experiments. The variability of the data was less than
10%. Carboplatin was used as positive control.

One of the major hindrances for druggability of compounds with effective antitumor activity is their toxicity to
normal cells. Thus, it is necessary to evaluate cytotoxicity on normal cells in the anticancer drug study. Due to the
higher efficacy of anti-proliferative action against tumour cell lines, compounds 1-4 were chosen for selectivity
test on normal human umbilical vein endothelial cell (HUVEC) and human embryonic kidney 293 cell (HEK293)
using the MTT assay. The selectivity indexes (SI) were calculated by IC50 values in cancer cells divided by IC50
values in normal cells. The results revealed that all the tested compounds were less toxic on HUVEC or HEK293
cells in comparison with the tumor cells (Table 2). The selectivity index (SI) measures the selective cytotoxicity
of a test sample against cancerous cells and the safety of sample towards normal cells. Compounds with a SI value
of more than 3 are considered to have high selectivity towards the particular cancer cell line [9]. Table 3 presents
the SI values of the compounds for various cancer cell lines tested. The analysis showed that all the tested
compounds possess higher selectivity towards intestinal epithelial adenocarcinoma cell line (HuTu80) with SI >3.
Among these compounds, compound 4 demonstrated the highest selectivity towards HuTu80 with SI>7, which
was 3.5 folds better than the positive control compound carboplatin <2, followed by compound 2 with SI>6,
which was 2 folds better than the positive control carboplatin.
Table 2 IC50 and IC90 values of compounds 1-4 against two normal cell lines (μg/mL, n=3)
HUVEC
HEK293
Drugs
IC50
IC90
IC50
IC90
199.1±11
363.2±20.2 165.1±13.2 321.5±16.9
Compound 1
Compound 2
Compound 3
Compound 4
Carboplatin
119.4±6.5
268.7±21.3
116.6±7.7
179.6±5.9
107.3±7.1
89.2±4.1
282.3±8.3
342.6±17.7
220.8±17.6
222.8±15.5
162.2±12.4 321.5±21.4
112.5±5.5
97.4±3.2
276.2±14
241±11.7
The IC50 (dose of the compound which caused a 50% reduction of survival.) and IC90 (dose of the compound
which caused a 90% reduction of survival) values were calculated from dose-response curves for each compound.
IC50 and IC90 data are an average of at least 3 independent experiments. The variability of the data was less than
10%. Carboplatin was used as positive control.
Table 3 Selectivity index for compounds 1-4 representing IC50 for normal cell lines/IC50 for cancerous cell lines
HUVEC
HEK293
Drugs
HUTU80
3.7
4T1
2.3
PANC1
4.7
HUTU80
3.1
4T1
1.9
PANC1
3.9
Compound 1
Compound 2
Compound 3
Compound 4
Carboplatin
6.7
3.4
7.5
1.8
2.3
3.1
2.3
1.9
1.9
1.73
1.36
1.4
6.5
3.8
7.1
1.6
2.2
3.5
2.2
1.7
1.9
1.9
1.3
1.3
Compound 4 shows the highest antiproliferative activity among all the synthesized compounds, so it was
selected to assess whether cell death detected would be due to apoptosis induction. The HUTU80 cells were
stained with annexin V and propidium iodide (PI) and analysized by flow cytometry. The results showed that the

proportion of positively stained cells (regarded as apoptotic) was increased by treatment with compound 4 (Fig. 3).
At 3 μg/mL of compound 4, 1.2% of HUTU80 cells transited to early apoptosis phase; 3.2% of HUTU80 cells
were in late apoptosis phase and 7.5% of HUTU80 cells was in necrosis phase (Fig. 3). These results indicate that
compound 4 could induce apoptosis at a lower concentration. Furthermore, the influence of the compound 4 on
cell-cycle arrest of HUTU80 was also conducted by flow cytometry assay using propidium iodide (PI) staining
(Fig. 4). As is seen from Fig. 4, in comparison with the control group, treatment of compound 4 (3 μg/mL) led to
the obvious decrease in G1 phase (from 43.32% to 26.08%) and dramatic increase in S phase (from 15.79% to
18.48%), indicating that compound 4 is a S cell-cycle arrester. In addition, PI staining of compound 4-treated
HUTU80 cells revealed increased accumulation of cells in the sub G1 peak from 2.27% to 25.06%, indicating cell
apoptosis occured (Fig. 4). All these results indicate that the promising antitumor activity of compound 4 may be
attributed to the S phase arrest and apoptosis.
A
B
Fig. 3 Apoptosis induction in HUTU80 cell line after 48 h treatment with compound 4 at the concentration of 3
μg/mL (B) and no treatment (vehicle control) (A).
M2=sub G0
M1=G0/G1
M3=S
M2=sub G0
M1=G0/G1
M3=S
M4=G2/M
M4=G2/M
A
B
Fig. 4 Effect of compound 4 on apoptosis and cell cycle progression (subG1:G1:S:G2/M) in HUTU80 cells.
Vehicle control (A) and drug treatment (B).

Compounds 1-4 were shown to possess higher antitumour effects among the synthesized chemical
compounds. Thus these four compounds were chosen to be further studied on their acute toxicity in mice. As a
result, all the animals survived the 14 day-treatment period. No physical or abnormal changes was found in the
skin, fur, eyes, mucus membranes, tremors, salivation, behavior patterns, or sleep patterns. No differences were
observed in kidney and liver tissue histopathology analysis between the compound-treated mice and the normal
controls (Fig. 5).
Fig. 5 Histological sections of liver (first row) and kidney (second row) in the acute toxicity test (H&E staining,
40x). Untreated mice (control group) received vehicle only (A and F). Animals treated with 500 mg/kg (B and G),
(C and H), (D and I) and (E and J) are for compounds 1, 2, 3 and 4, respectively. There were no significant
differences in the structures of the liver or kidneys between the treated and untreated groups.
In conclusion, two new series of biscoumarin and dihydropyran derivatives were synthesized in this work;
their antitumour effects against intestinal epithelial adenocarcinoma cell line (HuTu80), mammary
adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) were evaluated by the MTT assay in vitro.
On this basis, the apoptosis-inducing and cell cycle-regulating effects of the synthesized compound 4 on HuTu80
cell line were further performed. The analysis showed that all the biscoumarin compounds possess higher
selectivity towards intestinal epithelial adenocarcinoma cell line (HuTu80), with compound 4 showing highest SI
followed by compound 2. The biscoumarin compounds also showed no obvious acute toxicity on mice. Taken
together, our results suggest that the biscoumarin compounds, such as compound 2 and 4 are potential drug
candidates for cancer chemotherapy.
Of the synthesized compounds, compounds 1-4 had more potent antitumor activity against HuTu80 with the
IC50 and IC90 values (15-53.8 μg/mL and 28.00-109.7 μg/mL) much lower than those of the positive control drug
carboplatin (52.5-70.1 μg/mL and 90.7-156.2 μg/mL) respectively. The reason may be that two classical
intramolecular O—H…O hydrogen bonds in their structures. Furthermore, the apoptosis tests using flow
cytometry assay demonstrated that the best anticancer compound 4 could evoke a significant increase in the
number of HUTU80 cells both in the early phase and in the late phase of cell apoptosis. Further analysis on cell
cycle indicated that compound 4 is a S cell-cycle arrester of HUTU80 cells that could lead to a dramatic decrease
in G1 phase and increase in S phase of the cells. Moreover, the increased accumulation of HUTU80 cells in the
sub G1 peak further pointed to the occurence of the cell apoptosis.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (81274112;
81373986; 81473372; 81403125; 81403322 ), Beijing Municipal Natural Science Foundation (7152106), Science

and Technology Project Foundation Xi’an (No. CXY1352WL06).
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O
OH HO
R=
O
O
F
O
CN
F
O
O
R
2
4
1
3
NH
2
CN
R
O
O
R=
NO
Br
CN
2
O
5
8
7
6
NH
2
CN
R
O
CF
F
F
O
3
R=
SO
2
O
CF
F
3
9
10
11
12
NH
2
CN
Cl
O
O
O
CN
R
R=
Cl
O
O
15
13
14
16