Synthesis of biscoumarin and dihydropyran derivatives with promising antitumor and antibacterial activities

Article abstract of DOI:10.1016/j.bmcl.2015.10.063

Two series of biscoumarin (1-3) and dihydropyran (4-12) derivatives were successfully synthesized as new antitumor and antibacterial agents. The molecular structures of four representative compounds 2, 4, 7 and 10 were confirmed by single crystal X-ray di

Full text of DOI:10.1016/j.bmcl.2015.10.063

Accepted Manuscript
Synthesis of biscoumarin and dihydropyran derivatives with promising antitu-
mor and antibacterial activities
Jing Li, Yun-Peng Sui, Jia-Jia Xin, Xin-Liang Du, Jiang-Tao Li, Hai-Ru Huo,
Hai Ma, Wei-Hao Wang, Hai-Yu Zhou, Hong-Dan Zhan, Zhu-Ju Wang, Chun
Li, Feng Sui, Xia Li
PII:
S0960-894X(15)30173-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2015.10.063
BMCL 23219
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
7 July 2015
28 September 2015
21 October 2015
Please cite this article as: Li, J., Sui, Y-P., Xin, J-J., Du, X-L., Li, J-T., Huo, H-R., Ma, H., Wang, W-H., Zhou, H-
Y., Zhan, H-D., Wang, Z-J., Li, C., Sui, F., Li, X., Synthesis of biscoumarin and dihydropyran derivatives with
promising antitumor and antibacterial activities, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://
dx.doi.org/10.1016/j.bmcl.2015.10.063
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of biscoumarin and dihydropyran derivatives with
promising antitumor and antibacterial activities
a§
b, c§
d§
e
a
b
b
b
Jing Li , Yun-Peng Sui , Jia-Jia Xin , Xin-Liang Du , Jiang-Tao Li , Hai-Ru Huo , Hai Ma , Wei-Hao Wang ,
b
b
b
b
b*
f*
Hai-Yu Zhou , Hong-Dan Zhan , Zhu-Ju Wang , Chun Li , Feng Sui , Xia Li
a
School of Chemical Engineering, the Key Laboratory for Surface Engineering and Remanufacturing in Shaanxi
Province, Xi'an University, Xi’an 710065, China
b
c
d
e
f
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China
Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China
Department of Blood Transfusion, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032, China
Graduate School of China Academy of Chinese Medical Sciences, Beijing 100700, China
Department of Neurosurgery, Xijing Hospital, the Fourth Military Medical University, Xi’an 710032, China
§
*
These authors contributed equally to this work.
Corresponding authors: fsui@icmm.ac.cn (Feng Sui); sjwklixia@sina.com (Xia Li)
Abstract
Two series of biscoumarin (1-3) and dihydropyran (4-12) derivatives were successfully synthesized as new
antitumor and antibacterial agents. The molecular structures of four representative compounds 2, 4, 7 and 10 were
confirmed by single crystal X-ray diffraction study. The synthesized compounds (1-12) were evaluated for their
antitumor activities against human intestinal epithelial adenocarcinoma cell line (HuTu80), mammary
adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1) and antibacterial activities against one
drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three MRSA strains (MRSA XJ 75302, Mu50, USA
300 LAC). The further mechanism study demonstrated that the most potent compound 1 could obviously inhibit
the proliferation of cancer cells via the mechanism to induce apoptosis.
Key Words: Bisoumarin, Dihydropyran, Antitumor, Antibacterial
[1, 2]
Cancer is one of the most serious threats against human health in the world
. According to World Cancer
Report from the International Agency for Research on Cancer, cases of cancer doubled globally between 1975 and
[3]
2
000, will double again by 2020, and will nearly triple by 2030 . In addition, Staphylococcus aureus is a main
pathogen responsible for a number of diseases from serious hospital infections and community acquired infections,
such as folliculitis, impetigo, and cellulitis, which is the main cause of in-hospital mortality as high as 15% to
[
4-6]
6
0%
toxicity, low selectivity and severe drug resistance
Heterocyclic compounds attract special attention in chemical literature because of their abundance in natural
. However, most of clinical drugs used for tumor and infection treatment show poor curative effect, high
[7]
.
[8-11]
products and the diverse biological properties associated with them
. There are a large variety of heterocycles
[
12-15]
known and among them coumarin and pyran ring systems are of particular importance
. Numerous
interesting arrays of biological activities have been linked to natural and unnatural compounds possessing a
substituted coumarin or pyran nucleus, making it a suitable building block for many therapeutic agents including
[16-18]
[19, 20]
[21, 22]
antimicrobial activity
, growth stimulating effects
, antifungal and plant growth regulation effects
,

[23, 24]
[25]
[26]
antitumor activity
, central nervous system (CNS) activity
and hypotensive effect
.
In the present studies, two novel series of biscoumarin (1-3) and pyran derivatives (4-12) were reported (Fig.
1), all of which were evaluated for their in vitro antitumor activities against intestinal epithelial adenocarcinoma
cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1); and for
their in vitro antibacterial activities against one drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three
MRSA strains (MRSA XJ 75302, Mu50, USA 300 LAC). In addition, compound 1 with the most promising
antitumor activity was further studied to demonstrate whether the growth inhibition induced by compound 1 in the
cells derived from apoptosis or necrosis.
OH HO
R=
O
O
Cl
Br
F
O
O
R
1
3
2
NH2
CN
R
OCH3
O
O
R=
Br
Br
CN
OCH3
O
4
5
6
NH2
CN
R
O
F
OCH3
OH
R=
O
F
9
7
8
NH2
CN
R
CN
O
CF3
O
R=
O
1
1
1
0
12
Fig. 1 Chemical structures of compounds 1-12
The compounds were synthesized according to general method as described in Scheme 1 and 2 (see
Supplementary data). Biscoumarin 1-3 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 4-12 were synthesized via a one-pot three-component reaction by condensing
aromatic aldehydes, 4-hydroxycoumarin (3,5-cyclohexanedione, or 1,1-dimethyl-3,5-cyclohexanedione) and

malononitrile in the presence of 4-(dimethylamino)pyridine (DMAP) as a highly efficient homogenous catalyst.
1
13
Additionally, chemical structures of all compounds were further characterized by H NMR, C NMR and ESI-MS
(
see Supplementary data).
In order to further confirm the configuration of the products, single crystals of four representative compounds
2
, 4, 7 and 10 were cultured for X-ray diffraction analysis. From Fig. 2 we can see that, in crystal structure of
compound 2, in order to stabilize the whole structure, there are two classical intramolecular hydrogen bonds
–H ···O and O –H ···O ) between a hydroxyl group of one coumarin fragment and a lacton carbonyl group of
(
O
3
3
4
6
6
1
another coumarin fragment, which are further linked by a methylene bridge having a 4-tertbutylphenyl group on
the center.
Compound 2
Compound 4
Compound 7
Compound 10
Fig. 2 Crystal structures of compounds 2, 4, 7 and 10.
In the crystal structures of compounds 4, 7 and 10, the new formed pyran ring and the adjacent coumarin (or
ketone) ring are both basically planar, and the two planes are also essentially parallel each other. However, the
aromatic ring makes a torsion angle to the pyran ring in the three compounds.

For compounds 1-12, the antitumor effect in vitro on the growth of three human tumor cell lines, namely,
human intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1) and
pancreatic cancer cell line (PANC1), was evaluated after a continuous 48h exposure. For comparison purpose, the
cytotoxicity of carboplatin, a standard antitumor drug, was evaluated under the same condition.
As shown in Table 1, all the tested compounds exhibited antitumor activity against the cancer cell lines
HuTu80, 4T1 and PANC1 to a certain degree, and the degree of their inhibitory action get stronger accompanied
with the increased concentration of these compounds. The related IC50 and IC90 values of compounds 1-12 (dose
of the compound which cause a 50% and 90% reduction of survival values respectively), were calculated and
shown in Table 1. The results showed that the differences in antitumor activity among the four different groups of
the tested compounds is significant. Biscoumarins 1-3 from the first group showed more potent antitumor activity
against all the tested cancer cell lines with the IC50 value ranged from 19.26 μg/mL to 32.60 μg/mL and the IC90
value ranged from 36.68 μg/mL to 64.45 μg/mL, which is much lower than the IC50 and IC90 values (45.85-65.62
μg/mL; 102.14-126.24 μg/mL) of the positive control drug carboplatin. However, the compounds 4-12 in other
three groups showed lower antitumor activity with relatively higher IC50 and IC90 values.
Table 1 IC50 and IC90 values of compounds 1-12 and carboplatin against three tumor cell lines (μg/mL)
HUTU80
4T1
PANC1
Drugs
IC50
IC90
IC50
IC90
36.68
IC50
IC90
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Carboplatin
29.70
31.15
32.60
52.64
19.26
31.80
61.41
55.53
24.56
47.13
27.97
53.16
64.45
22.93
43.19
30.77
55.77
140.33
121.25
132.50
281.10
445.97
208.05
414.4
273.67
221.25
232.5
96.17
172.00
207.77
194.03
286.62
307.65
218.65
503.32
575.18
927.38
102.136
176.42
149.75
168.45
368.38
318.99
232.24
241.84
248.08
383.11
52.94
327.7
114.70
106.68
157.00
164.23
113.55
248.55
286.78
480.95
45.85
285.99
310.4
543.15
824.40
398.52
814.4
703.67
624.10
461.99
512.29
502.69
759.05
109.94
388.00
899.80
65.62
788.00
1699.8
126.24
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 done in triplicate for
each compound. Carboplatin was used as positive control.
On the basis of the anti-proliferative data, the most potent compound 1 was selected for further mechanistic
studies to determine whether the growth inhibition induced by compound 1 in the cells derived from apoptosis or
necrosis. To assess the effect of compound 1 on apopsis of 4T1 (the most senstive cell line to compound 1), the
flow cytometry technique was used and the cell culture was not only treated with vehicle alone as control but also
dealt with compound 1 at the concentration of 5 μg/mL for 48 h and stained with Annexin V/Alexa Fuor 488 and

propidium iodide (PI). The percentages of apoptotic cells were determined by flow cytometry analysis. As shown
in Fig. 3, compound 1 displayed a significant action on the induction of apoptosis to the tested cells; apoptotic
4T1 cells (early and late apoptosis) increased more than 8 folds after 48 h treatment with compound 1, as
compared to the untreated vehicle control. Apparently, compound 1 mediated apoptosis of the tested cells, at least
in part, contributes to its antiproliferative effects.
(
a)
Fig. 3 Apoptosis induction in 4T1 cell line after 48 h treatment with compound 1 at the concentration of 5 μg/mL
b) and no treatment (Control) (a).
(b)
(
The antibacterial activity of compounds 1-12 in vitro was investigated by one drug-sensitive S. aureus (S.
aureus ATCC 29213) strain and three MRSA strains (MRSA XJ 75302, Mu50, USA 300 LAC) respectively. From
Table 2 we can see that compared with pyran derivatives 4-12, biscoumarins 1-3 exerted the most bactericidal
effects against nearly all types of S. aureus tested with the value of 8-32 μg/mL. In addition, the MIC values of
levofloxacin, ceftazidime, ceftriaxone, gentamicin and piperacillin against S. aureus (ATCC 29213) strains were
lower (less than 8 μg/mL) but were higher against resistant strains at varying degrees.
Table 2 MIC of compounds 1-12 and antibiotics in Mueller-Hinton Broth Culture.
MIC (ug/mL)
Drugs
S. aureas
MRSA
Mu50
LAC
(
ATCC 29213)
(XJ 75302)
(ATCC 700699)
(USA 300)
Compound 1
Compound 2
Compound 3
Compound 4
Compound 5
16
16
16
16
8
16
8
32
16
16
8
8
>256
>256
>256
>256
>256
>256
>256
>256

Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Levofloxacin
Ceftazidime
Ceftriaxone
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
<0.125 (S)
8 (S)
4 (R)
4 (R)
8 (R)
>256 (R)
>256 (R)
64 (R)
>128 (R)
256 (R)
256 (R)
32 (R)
>128 (R)
64 (R)
32 (R)
0.25 (S)
8 (R)
2 (S)
Gentamicin
0.12 (S)
2 (S)
Piperacillin
S means drug susceptibility, R means drug resistance.
In conclusion, we prepared biscoumarin and pyran derivatives using simple reaction conditions in a single
pot and evaluated for their in vitro antitumor activities against intestinal epithelial adenocarcinoma cell line
(
HuTu80), mammary adenocarcinoma cell line (4T1) and pancreatic cancer cell line (PANC1); and for their in
vitro antibacterial activities against one drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three MRSA
strains (MRSA XJ 75302, Mu50, USA 300 LAC). In the series of biscoumarin and pyran derivatives,
biscoumarins 1-3 showed very potent antitumor and antibacterial activities, possibly because of two asymmetrical
intramolecular O—H···O HBs in their structures in assisting the molecule to attain the correct configuration for
[
27]
biological activity
. The flow cell assay further demonstrated that the proportions of apoptosic cells in the
groups treated with compound 1 increased significantly, indicating that the promising antitumor activity of
compound 1 may be attributed to the induction of the cells to apoptosis. The above data is helpful to further
exploit these compounds for antitumor and antibacterial activity mechanistic studies and also helpful to design
and synthesize more such derivatives to be taken-up for further studies.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (81274112;
81373986; 81473372; 81573468), Beijing Municipal Natural Science Foundation (7152106), Inheritance Program
of China Academy of Chinese Medical Sciences (CM2014GD3002), Voluntary Program of China Academy of
Chinese Medical Sciences (ZZ2014056) and the Innovation Plan of Science and Technology of Shaanxi Province
(
2014KTCL03-03).
Reference
1.
Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. Cancer J. Clin. 2011, 61, 69.
2.
Murray, C. J. L.; Lopez, A. D. Lancet 1997, 349, 1498.

3
4
5
.
.
.
Mathers, C. D.; Loncar, D. PLoS Med. 2006, 3, e442.
Frisina, P. G.; Ann, A. M.; Kutlik, M.; Barrett, A. M. Arch. Phys. Med. Rehabil. 2013, 94, 516.
Climo, M. W.; Yokoe, D. S.; Warren, D. K.; Perl, T. M.; Bolon, M.; Herwaldt, L. A.; Weinstein, R. A.;
Sepkowitz, K. A.; Jernigan, J. A.; Sanogo, K.; Wong, E. S. N. Engl. J. Med. 2013, 368, 533.
Kutcher, M. E.; Ferguson, A. R.; Cohen, M. J. J. Trauma Acute Care Surg. 2013, 74, 1223.
Chen, H. X.; Cleck, J. N. Nat. Rev. Clin. Oncol. 2009, 6, 465.
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
.
.
.
.
Lacy, A.; O'Kennedy, R. Curr. Pharm. Des. 2004, 10, 3797.
Peng, X. M.; Damu, G. L.; Zhou, C. Curr. Pharm. Des. 2013, 19, 3884.
0. Musa, M. A.; Cooperwood, J. S.; Khan, M. O. Curr. Med. Chem. 2008, 15, 2664.
1. Abraham, K.; Wöhrlin, F.; Lindtner, O.; Heinemeyer, G.; Lampen, A. Mol. Nutr. Food Res. 2010, 54, 228.
2. Pagliarini, R.; Shao, W.; Sellers, W. R. EMBO Rep. 2015, 16, 280.
3. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem. 2005, 12, 887.
4. Khan, M. T. Curr. Med. Chem. 2012, 19, 2262.
5. Bates, R. H.; Chen, M.; Roush, W. R. Curr. Opin. Drug Discov. Devel. 2008, 11, 778.
6. Georgiadis, M. P.; Cauladouros, E. A.; Delitheos, A. K. J. Pharm. Sci. 1992, 81, 1126.
7. Mohr, S. J.; Chirigos, M. A.; Fuhrman, F. S.; Pryor, J. W. Cancer Res. 1975, 35, 3750.
8. Peng, X. M.; Damu, G. L.; Zhou, C. Curr. Pharm. Des. 2013, 19, 3884.
9. Abraham, K.; Wöhrlin, F.; Lindtner, O.; Heinemeyer, G.; Lampen, A. Mol. Nutr. Food Res. 2010, 54, 228.
0. Inoue, Y.; Kondo, H.; Taguchi, M.; Jinbo, Y.; Sakamoto, F.; Tsukamoto, G. J. Med. Chem. 1994, 37, 586.
1. Jamkhandi, P. S.; Rajagopal, S. Arch. Pharm. Der. Dtsch.Pharm. Ges. 1967, 300, 561.
2. Borges, F.; Roleira, F.; Milhazes, N.; Santana, L.; Uriarte, E. Curr. Med. Chem. 2005, 12, 887.
3. Bates, R. H.; Chen, M.; Roush, W. R. Curr. Opin. Drug Discov. Devel. 2008, 11, 778.
4. Chauthe, S. K.; Mahajan, S.; Rachamalla, M.; Tikoo, K.; Singh, I. P. Med. Chem. Res. 2015, 24, 2476.
5. Colotta, V.; Cecchi, L.; Melani, F.; Catarzi, D.; Filacchioni, G.; Martini, C.; Giannaccini, G.; Tonelli,
M.; Lucacchini, A. Drug Des. Discov. 1992, 8, 325.
2
2
6. Colotta, V.; Cecchi, L.; Melani, F.; Filacchioni, G.; Martini, C.; Gelli, S.; Lucacchini, A. J. Pharm. Sci. 1991,
80, 276.
7. Schiøtt, B.; Iversen, B. B.; Madsen, G. K. H.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120, 12117.

Graphical abstract
OH HO
R=
O
O
Cl
Br
F
O
O
R
1
3
2
NH2
CN
R
OCH3
O
O
R=
Br
Br
CN
OCH3
O
4
5
6
NH2
CN
R
O
F
OCH3
OH
R=
O
F
9
7
8
NH2
CN
R
CN
O
CF3
O
R=
O
1
1
1
0
12