Antibacterial and antitumor activities of biscoumarin and dihydropyran derivatives

Article abstract of DOI:10.3390/molecules200917614

A novel series of biscoumarin (1-4) and dihydropyran (5-13) derivatives were synthesized via a one-pot multicomponent condensation reaction and evaluated for antibacterial and antitumor activity in vitro. The X-ray crystal structure analysis of four repre

Full text of DOI:10.3390/molecules200917614

Molecules 2015, 20, 17614-17626; doi:10.3390/molecules200917614
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antibacterial and Antitumor Activities of Biscoumarin and
Dihydropyran Derivatives
Yun-Peng Sui 1,2,†, Hai-Ru Huo 1,†, Jia-Jia Xin 3,†, Jing Li 4, Xiao-Jun Li 4, Xin-Liang Du 5,
Hai Ma 1, Hai-Yu Zhou 1, Hong-Dan Zhan 1, Zhu-Ju Wang 1, Chun Li 1, Feng Sui 1,*
and Ming-Kai Li 6,*
1
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700,
China; E-Mails: suiyp8888@sina.com (Y.-P.S.); hairuhuo@gmail.com (H.-R.H.);
hma@icmm.ac.cn (H.M.); zhouhaiyu1992@foxmail.com (H.-Y.Z.);
baiyihongdan@163.com (H.-D.Z.); zjwang@icmm.ac.cn (Z.-J.W.); cli@icmm.ac.cn (C.L.)
2
Beijing Chao-Yang Hospital, Capital Medical University, Beijing 100020, China
3
Department of Blood Transfusion, Xijing Hospital, the Fourth Military Medical University,
Xi’an 710032, China; E-Mail: xinjiajia@hotmail.com
4
The Key Laboratory for Surface Engineering and Remanufacturing in Shaanxi Province,
School of Chemical Engineering, Xi’an University, Xi’an 710065, China;
E-Mails: lijing3157@aliyun.com (J.L.); xjli@xawl.edu.cn (X.-J.L.)
5
Graduate School of China Academy of Chinese Medical Sciences, Beijing 100700, China;
E-Mail: dxl906@163.com
6
Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University,
Xi’an 710032, China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: fsui@icmm.ac.cn (F.S.);
mingkai@fmmu.edu.cn (M.-K.L.); Tel./Fax: +86-10-6404-1008 (F.S.); +86-29-8477-4591 (M.-K.L.).
Academic Editor: Derek J. McPhee
Received: 7 August 2015 / Accepted: 14 September 2015 / Published: 23 September 2015
Abstract: A novel series of biscoumarin (1–4) and dihydropyran (5–13) derivatives
were synthesized via a one-pot multicomponent condensation reaction and evaluated for
antibacterial and antitumor activity in vitro. The X-ray crystal structure analysis of four
representative compounds, 3, 7, 9 and 11, confirmed the structures of these compounds.
Compounds 1–4 showed the most potent antitumor activity among the total 13 derivatives;
especially for compounds 1 and 2, they also emerged as promising antibacterial members

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with better antibacterial activity. In addition, the results of density functional theory
(DFT) showed that compared with compounds 3 and 4, biscoumarins 1 and 2 had lower
intramolecular hydrogen bonds (HB) energy in their structures.
Keywords: biscoumarin; dihydropyran; antibacterial; antitumor
1. Introduction
Natural products have a profound impact on both chemical biology and drug discovery, and the great
structural diversity of natural products with various interesting biological characteristics has always
provided medicinal chemists an important source of inspiration in their search for new molecular entities
with pharmacological activity [1,2]. Among them, biscoumarin and dihydropyran derivatives are two
important groups of compounds covering a wide range of biological properties, including anti-oxidant,
anti-inflammatory [3] and anti-microbial [4] as well as anticancer activities [5,6]. However, many
of them are not suitable for therapeutic application due to their relatively lower activity or evident
side-effect properties; and for the already marketable antibacterial and antitumor drugs [7,8], resistance
has become one of main reasons for their failure in chemotherapy. Hence the search for high-quality
novel antibacterial and anticancer agents has always been advisable and emergent [9].
OH
OH
HO
R=
F
F
Cl
Cl
O
O
F
SCH
OH
3
O
O
R
1
3
4
2
NH
2
CN
R
O
O
O
OCH
CF
3
3
R=
OH
O
5
6
7
NH
2
CN
R
O
CF
3
R=
OH
O
CF
3
10
8
9
NH
2
CN
O
Cl
Cl
OCH
OCH
Br
3
R=
R
3
O
OCH
3
11
12
13
Figure 1. Chemical structures of compounds 1–13.
In order to get more effective antibacterial and antitumor agents, it is possible to make modifications
on active chemical structures of title compounds. In the present study, a novel series of biscoumarin (1–4)

Molecules 2015, 20
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and dihydropyran (5–13) derivatives were firstly synthesized (Figure 1), their antibacterial activities were
then measured in vitro against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant
S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), and USA
300 (Los Angeles County clone, LAC), and finally 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.
2. Results and Discussion
2.1. Molecular Structure
The crystal structures of compounds 3, 7, 9 and 11 are given in Figure 2. In the crystal structure of
compound 3, two 4-hydroxycoumarin moieties are linked through a methylene bridge, wherein one
hydrogen atom has been replaced with a 4-methylthiophenyl 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.
Compound 3
Compound 7
Compound 9
Compound 11
Figure 2. Crystal structures of compounds 3, 7, 9 and 11.

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In the crystal structures of compounds 7, 9 and 11, the new formed pyran ring and the adjacent ketone
(coumarin) ring are both basically planar, and the two planes are also essentially parallel to each other.
However, the aromatic ring makes a torsion angle to the pyran ring in the three compounds.
2.2. Hydrogen Bonds Energies in Biscoumarins 1–4
We only used compound 1 as an example to estimate single and total intramolecular hydrogen
bonds (HB) energies. The global minimum structure is stabilized by two HBs (1ab); two higher energy
structures are stabilized by one HB (1a and 1b, respectively). The corresponding values are listed in
Table 1.
Based on our previous calculation results [10], B3LYP/6-31G* exhibited sufficient agreement with
experimental data and lower computational cost, so further theoretical study was performed at this level.
The O6–H6 O1 HB energy was calculated to be −51.3364015 kJ/mol by the equation E(O6–H6···O1) =
coor
1ab
coor
1a
E
– E , from the energy difference between 1ab and 1a, where 1a is a global minimum structure
with O3–H3···O4 HB. Similarly, the O3–H3···O4 HB energy was calculated to be −64.8682285 kJ/mol
coor
from the energy difference between 1ab and 1b by the equation E(O3–H3···O4) = Ec1oaobr − E , in which
1b
1b was obtained from the global minimum structure 1ab, but H3 was rotated around the C3–O3 bond until
O3–H3···O4 HB rupture occurred [11,12]. The total HB energy was calculated to be −116.20463 kJ/mol
by the equation E(O3–H3···O4)+ E(O6–H6···O1). For compounds 2–4, the total HB energies are
−115.7031595, −118.0346035 and −121.970228 kJ/mol, respectively.
Table 1. Single and total intramolecular hydrogen bonds (HB) energies in biscoumarins 1–4.
System Total Electronic Energies a,b E(O6–H6···O1) E(O3–H3···O4) E(Total HB) c
1ab
1a
−1711.038294
−1711.018741
−1711.013587
−2332.538245
−2332.518779
−2332.513642
−2025.68685
−2025.666896
−2025.661847
−1625.791083
−1625.770804
−1625.764906
−116.20463
−115.7031595
−118.0346035
−121.970228
−51.3364015
−51.107983
−52.389227
−53.2425145
1b
−64.8682285
−64.5951765
−65.6453765
−68.7277135
2ab
2a
2b
3ab
3a
3b
4ab
4a
4b
a ZP corrected; b hartree; c kJ/mol.
2.3. Minimal Inhibitory Concentration (MIC) Assay
For compounds 1–13, one drug-sensitive S. aureus (S. aureus ATCC 29213) strain and three MRSA
strains (MRSA XJ 75302, Mu50, and USA 300 LAC) were used in the systematic analysis of their
antibacterial activities in vitro. Because of the liposolubility of these compounds, they were dissolved
into the solution with 1% dimethyl sulfoxide (DMSO) at final concentration. From Table 2, we can see
that, among these compounds, compounds 1 and 2 exerted more potent anti-bacterial activity against the

Molecules 2015, 20
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tested S. aureus with minimum inhibitory concentration (MIC) values in the range of 2–16 μg/mL.
Compared with compounds 1–13, 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 other three strains at varying degrees.
Table 2. Minimum inhibitory concentration (MIC) of compounds 1–13 and antibiotics in
Mueller–Hinton Broth Culture.
MIC (µg/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
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
Compound 11
Compound 12
Compound 13
Levofloxacin
Ceftazidime
Ceftriaxone
16
4
16
4
8
2
8
2
64
64
64
64
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
<0.125 (S)
8 (S)
2 (S)
0.12 (S)
2 (S)
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
4 (R)
>256 (R)
>256 (R)
64 (R)
>128 (R)
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
4 (R)
256 (R)
256 (R)
32 (R)
>128 (R)
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
8 (R)
64 (R)
32 (R)
0.25 (S)
8 (R)
Gentamicin
Piperacillin
S means drug susceptibility, R means drug resistance.
2.4. In Vitro Antitumor Activity
Intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line (4T1)
and pancreatic cancer cell line (PANC1) representing three different tumor types were used in the
systematic analysis of the antitumor activities of the newly synthesized compounds 1–13 in vitro. For
comparison purpose, the cytotoxicity of carboplatin, a standard antitumor drug, was evaluated under the
same condition.
The results showed that all the tested compounds possessed a certain degree of antitumor activities
against the three tumor cell lines and their inhibitory action get stronger with the corresponding higher
concentration. The related 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 3.
As can be seen in Table 3, there is great difference in the antitumor activity between the four different
groups of the tested compounds. Biscoumarins 1–4 from the first group showed more potent antitumor
activity against the three tested tumor cells (HuTu80, 4T1 and PANC1) with IC50 and IC90 values of
18.78–32.63 μg/mL and 36.05–64.55 μg/mL, respectively, which is much lower than the IC50 and IC90

Molecules 2015, 20
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values (45.85–65.62 μg/mL and 102.14–126.24 μg/mL) of the positive control drug carboplatin.
However, the compounds in other three groups demonstrated lower antitumor activity with relatively
higher IC50 and IC90 values.
Table 3. IC50 and IC90 values of compounds 1–13 and carboplatin against three tumor cell
lines (μg/mL).
HUTU 80
4T1
PANC1
Drugs
IC50
IC90
IC50
IC90
IC50
IC90
Compound 1 32.63
Compound 2 28.94
Compound 3 28.42
Compound 4 28.55
64.55
55.87
55.31
55.61
22.09
18.78
20.08
19.33
39.58
36.05
38.21
36.92
27.52
25.05
28.01
26.11
51.33
46.67
51.75
48.56
Compound 5 116.00 216.00 138.00 248.07 79.23 154.62
Compound 6 130.67 264.00 163.67 304.96 149.42 302.09
Compound 7 209.00 409.00 91.44 186.16 174.71 339.00
Compound 8 266.79 483.83 214.87 330.92 172.99 317.39
Compound 9 582.88 1079.16 333.93 638.81 744.59 1340.72
Compound 10 493.58 942.01 303.98 601.82 566.14 1055.13
Compound 11 268.72 490.94 316.54 569.07 305.25 592.40
Compound 12 509.78 985.97 621.34 1117.62 604.45 1143.73
Compound 13 374.50 707.83 481.83 900.82 205.17 410.09
Carboplatin
65.62 126.24 45.85 102.13 52.94 109.94
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.
3. Experimental Section
3.1. Apparatus and Materials
IR spectra (400–4000 cm−1) were obtained using a Brucker Equinox-55 spectrophotometer (Bruker
1
13
Optics, Ettlingen, Germany). H-NMR (Nuclear Magnetic Resonance) spectra, C-NMR spectra and
mass spectra were tested using the Varian Inova-400 spectrometer (Varian Inc., Palo Alto, CA, USA),
Bruker Avance III (Bruker Optics) spectrometer and micrOTOF-Q II (Bruker Optics) mass spectrometer,
respectively. The melting points were taken on a XT-4 micro melting apparatus (Ledon, Suzhou, China),
and the thermometer was uncorrected.
All antibiotics used were purchased from the National Institute for the Control of Pharmaceutical and
Biological Products (Beijing, China). MRSA (XJ 75302) was isolated from cultures of sputum samples
from patients in Xijing Hospital (Xi’an, China). S. aureus strain (ATCC 29213) was purchased from the
Chinese National Center for Surveillance of Antimicrobial Resistance (Beijing, China). Mu50 (ATCC
700699) and USA 300 (LAC) were purchased from MicroBiologics (Saint Cloud, MN, USA).
RPMI 1640 medium, trypsin, and heat inactivated fetal bovine serum (HIFBS) were obtained
from Gibco (New York, NY, USA). 5-Fluorouracil 99% HPLC grade, phosphate-buffered saline (PBS),
penicillin/streptomycin (PS) solution, and 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide

Molecules 2015, 20
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(MTT) reagent were purchased from Sigma-Aldrich (St. Louis, MO, USA). The MTT assay was performed
by using FlexStation 3 benchtop multi-mode microplate reader (Molecular Devices, San Jose, CA, USA).
Human intestinal epithelial adenocarcinoma cell line (HuTu80) was purchased from Institute of Basic
Medical Sciences (IBMS) of Chinese Academy of Medical Sciences (CAMS) (Beijing, China). The cells
were cultured in RPMI supplemented with 10% HIFBS and 1% PS. Cells were cultured in a 5% CO2 in a
humidified atmosphere at 37 °C.
3.2. Synthesis and Characterization of Compounds 1–13
Biscoumarins 1–4 were synthesized according to the methods of a previous report [13].
A mixture of 3,4,5-trifluorobenzaldehyde (3,5-dichlorobenzaldehyde, 4-methylthiobenzaldehyde or
2,4-dihydroxybenzaldehyde) (10 mmol) and 4-hydroxycoumarin (20 mmol) was dissolved in 100 mL of
EtOH. A few drops of piperidine were added, and the mixture was stirred for 3 h at room temperature.
After reaction completion as determined by TLC, water was added until precipitation occurred. After
filtering the precipitates, they were sequentially washed with ice-cooled water and ethanol and then
dried in a vacuum.
3,3′-(3,4,5-Trifluorobenzylidene)-bis-(4-hydroxycoumarin) (1): Yield: 57%. 230–231 °C. IR (KBr
pellet cm−1): 3446, 2360, 1675, 1512, 1265, 1139, 767 cm−1. 1H-NMR (CDCl3, δ, ppm): 6.020 (s, 1H,
CH), 6.854–6.890 (q, 2H), 7.434–7.464 (q, 4H), 7.669–7.708 (t, 2H), 8.031–8.108 (q, 2H), 11.337 (s,
13
1H, OH), 11.665 (s, 1H, OH). C-NMR (DMSO-d6) δ: 36.462, 103.773, 111.689, 111.729, 111.895,
116.431, 118.642, 124.072, 124.439, 132.355, 152.868, 164.907, 166.325. HRMS (ESI+): m/z: calcd
for C25H13F3O6: 489.0556 [M + Na]+; found: 489.0533.
3,3′-(3,5-Dichlorobenzylidene)-bis-(4-hydroxycoumarin) (2): Yield: 55%. 239–240 °C. IR (KBr pellet cm−1):
1
3446, 2360, 1666, 1546, 1353, 1091, 761 cm−1. H-NMR (CDCl3, δ, ppm): 6.036 (s, 1H, CH),
7.106–7.113 (t, 2H), 7.307–7.309 (d, 1H), 7.432–7.468 (t, 4H), 7.666–7.705 (q, 2H), 8.042–8.114 (m,
13
2H), 11.314 (s, 1H, OH), 11.630 (s, 1H, OH). C-NMR (DMSO-d6) δ: 36.672, 103.422, 116.339,
119.160, 123.933, 124.530, 125.667, 126.059, 132.169, 134.153, 146.825, 152.896, 164.774, 167.058.
HRMS (ESI+): m/z: calcd for C25H14Cl2O6: 503.0060 [M + Na]+; found: 503.0069.
3,3′-(4-Methylthiobenzylidene)-bis-(4-hydroxycoumarin) (3): Yield: 50%. 232–233 °C. IR (KBr pellet cm−1):
1
2605, 1668, 1614, 1349, 1211, 1100, 1029, 910, 767 cm−1. H-NMR (CDCl3, δ, ppm): 2.493 (s, 3H,
SCH3), 6.073 (s, 1H, CH), 7.149–7.170 (d, 2H), 7.223–7.244 (d, 2H), 7.427–7.447 (d, 4H), 7.634–7.677
13
(m, 2H), 8.015–8.104 (q, 2H), 11.323 (s, 1H, OH), 11.545 (s, 1H, OH). C-NMR (DMSO-d6) δ:
15.408, 36.064, 104.671, 116.528, 118.068, 124.354, 124.373, 126.513, 127.932, 132.557, 135.395,
136.867, 152.635, 165.316, 165.392. HRMS (ESI+): m/z: calcd for C26H18O6S: 481.0716 [M + Na]+;
found: 481.0779.
3,3′-(2,4-Dihydroxybenzylidene)-bis-(4-hydroxycoumarin) (4): Yield: 55%. m.p. 262–263 °C. IR (KBr):
3260, 2230, 1681, 1611, 1539, 1392, 1184, 910, 757 cm−1. 1H-NMR (DMSO-d6, δ, ppm): 5.635 (s, 1H),
6.568–6.595 (q, 1H), 6.713–6.718 (d, 1H), 6.978–6.999 (d, 1H), 7.318–7.373 (m, 1H), 7.445–7.505 (q,
2H), 7.582–7.624 (m, 1H), 7.683–7.726 (m, 1H), 8.086–8.108 (q, 2H), 9.792 (s, 1H). 13C-NMR

Molecules 2015, 20
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(DMSO-d6) δ: 103.214, 114.327, 116.652, 116.936, 123.182, 124.434, 125.018, 129.643, 132.579,
132.912, 152.422, 157.792, 160.985. HRMS (ESI+): m/z: calcd for C25H16O8: 467.0737 [M + Na]+;
found: 467.0779.
Dihydropyran derivatives (5–13) were also synthesized according to a reported procedure [14]. A
mixture of 4-hydroxycoumarin (3,5-cyclohexanedione, or 1,1-dimethyl-3,5-cyclohexanedione) (10 mmol),
aromatic aldehydes (10 mmol), malononitrile (10 mmol) and 4-(dimethylamino)pyridine (DMAP)
(1 mmol) in ethanol (100 mL) was refluxed for 2–3 h and then cooled to room temperature. After filtering
the precipitates, they were sequentially washed with ice-cooled water and ethanol and then dried under
a vacuum.
2-Amino-4-(3-phenoxyphenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (5): Yield: 60%. 233–234 °C.
1
IR (KBr pellet cm−1): 3688, 2198, 1710, 1670, 1574, 1486, 1374, 1238, 1062, 821 cm−1. H-NMR
(DMSO-d6, δ, ppm): 4.477 (s, 1H, CH), 6.821–6.841 (d, 1H), 6.978–7.041 (m, 4H), 7.112–7.149 (t,
1H), 7.300–7.392 (m, 3H), 7.451–7.519 (m, 4H), 7.707–7.747 (t, 1H), 7.879–7.899 (d, 1H). 13C-NMR
(DMSO-d6) δ: 37.241, 58.105, 104.086, 113.430, 117.067, 117.293, 118.560, 119.068, 119.655, 122.981,
123.034, 123.984, 125.159, 130.488, 130.648, 133.463, 146.071, 152.645, 154.038, 156.800, 157.033,
158.476, 160.047. HRMS (ESI+ ): m/z: calcd for C25H16N2O4: 431.1002 [M + Na]+; found: 431.1033.
2-Amino-4-(3-methoxy-4-hydroxyphenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (6): Yield: 60%.
236–237 °C. IR (KBr pellet cm−1): 3415, 3303, 2191, 1686, 1598, 1518, 1374, 1262, 1085, 749 cm−1.
1H-NMR (DMSO-d6, δ, ppm): 3.728 (s, 3H, OCH3), 4.359 (s, 1H, CH), 6.594–6.619 (q, 1H),
6.690–6.710 (d, 1H), 6.811–6.816 (d, 1H), 7.354 (s, 2H), 7.457–7.511 (q, 2H), 7.690–7.733 (m, 1H),
7.880–7.903 (q, 1H), 8.953 (s, 1H). 13C-NMR (DMSO-d6) δ: 31.181, 36.951, 56.139, 58.781, 104.800,
112.502, 113.532, 115.970, 117.015, 119.889, 120.350, 122.910, 125.097, 133.264, 134.801, 146.201,
147.778, 152.540, 153.516, 158.385, 160.068. HRMS (ESI+): m/z: calcd for C20H14N2O5: 385.0795
[M + Na]+; found: 385.0770.
2-Amino-4-(3-trifluoromethylphenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (7): Yield: 65%.
240–241 °C. IR (KBr pellet cm−1): 3408, 3321, 2205, 1701, 1672, 1594, 1381, 1313, 1167, 1109, 1041,
1
770 cm−1. H-NMR (DMSO-d6, δ, ppm): 4.654 (s, 1H, CH), 7.467–7.659 (m, 8H), 7.713–7.756 (m,
1H), 7.902–7.926 (q, 1H). 13C-NMR (DMSO-d6) δ: 31.169, 37.242, 57.707, 103.550, 113.473, 117.079,
119.517, 123.065, 124.465, 124.502, 124.841, 124.879, 125.159, 126.020, 129.319, 129.632, 130.165,
132.477, 133.525, 145.164, 152.718, 154.304, 158.506, 160.087. HRMS (ESI+): m/z: calcd for
C20H11F3N2O3: 407.0614 [M + Na]+; found: 407.0480.
2-Amino-4-(4-hydroxyphenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (8): Yield: 60%.
1
238–239 °C. IR (KBr pellet cm−1): 3198, 2200, 1647, 1512, 1367, 1215, 1009, 836 cm−1. H-NMR
(DMSO-d6, δ, ppm): 1.854–1.973 (m, 2H), 2.214–2.302 (m, 2H), 2.573–2.606 (q, 2H), 4.074 (s, 1H, CH),
13
6.644–6.665 (q, 2H), 6.926–6.947 (q, 4H), 9.263 (s, 1H). C-NMR (DMSO-d6) δ: 20.307, 26.931,
31.184, 35.032, 36.860, 59.101, 114.735, 115.464, 120.442, 128.612, 135.710, 156.465, 158.846,
164.475, 196.359. HRMS (ESI+): m/z: calcd for C16H14N2O3: 305.0897 [M + Na]+; found: 305.0980.

Molecules 2015, 20
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2-Amino-4-(3,5-ditrifluoromethylphenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (9): Yield:
69%. 209–210 °C. IR (KBr pellet cm−1): 3397, 2194, 1683, 1651, 1419, 1362, 1330, 1211, 700 cm−1.
1H-NMR (DMSO-d6, δ, ppm): 1.889−1.960 (m, 2H), 2.258–2.318 (m, 2H), 2.607–2.709 (m, 2H), 4.543
(s, 1H, CH), 7.239 (s, 2H), 7.857 (s, 2H), 7.968 (s, 1H). 13C-NMR (DMSO-d6) δ: 20.183, 27.055, 31.096,
35.898, 36.679, 57.089, 112.623, 119.735, 121.012, 121.049, 121.082, 122.434, 125.145, 128.644,
130.201, 130.527, 130.851, 131.176, 148.674, 159.034, 166.000, 196.552. HRMS (ESI+): m/z: calcd
for C18H12F6N2O2: 425.0695 [M + Na]+; found: 425.0663.
2-Amino-5-oxo-4-phenyl-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (10): Yield: 65%. 221–222 °C.
IR (KBr pellet cm−1): 3309, 3170, 2194, 1685, 1261, 1064, 999, 702 cm−1. IR (KBr pellet cm–1): 3309,
1
3170, 2194, 1685, 1261, 1064, 999, 702 cm–1. H-NMR (DMSO-d6, δ, ppm): 1.876−1.988 (m, 2H),
2.2262.318 (m, 2H), 2.599–2.640 (q, 2H), 4.183 (s, 1H, CH), 7.019 (s, 2H), 7.146–7.205 (m, 3H),
7.269–7.306 (t, 2H). 13C-NMR (DMSO-d6) δ: 20.295, 26.955, 31.186, 35.922, 36.808, 58.652, 114.255,
120.289, 127.015, 127.609, 128.822, 145.278, 158.948, 164.971, 196.348. HRMS (ESI+): m/z: calcd
for C16H14N2O2: 289.0947 [M + Na]+; found: 289.0933.
2-Amino-4-(3,4-dichlorophenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran (11):
Yield: 66%. 232–233 °C. IR (KBr pellet cm−1): 3325, 2958, 2194, 1639, 1608, 1465, 1357, 1249, 1215,
1033, 898 cm−1. 1H-NMR (DMSO-d6, δ, ppm): 0.965 (s, 3H, CH3), 1.039 (s, 3H, CH3), 2.114–2.155 (d,
1H), 2.234–2.274 (d, 1H), 2.529 (s, 2H), 4.255 (s, 1H, CH), 7.137–7.172 (m, 3H), 7.390–7.395 (d, 1H),
7.756–7.582 (d, 1H). 13C-NMR (DMSO-d6) δ: 27.412, 28.689, 31.183, 32.323, 35.439, 50.387, 57.649,
112.193, 119.902, 128.170, 129.652, 129.773, 131.118, 131.286, 146.380, 158.994, 163.461, 196.238.
HRMS (ESI+): m/z: calcd for C18H16Cl2N2O2: 385.0481 [M + Na]+; found: 385.0422.
2-Amino-4-(3-bromophenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran (12): Yield:
68%. 239–240 °C. IR (KBr pellet cm−1): 3344, 3168, 2963, 2191, 1686, 1651, 1605, 1469, 1427, 1367,
1
1250, 1216, 1036, 695 cm−1. H-NMR (DMSO-d6, δ, ppm): 0.967 (s, 3H, CH3), 1.043 (s, 3H, CH3),
2.111–2.151 (d, 1H), 2.242–2.82 (d, 1H), 2.534 (s, 2H), 4.210 (s, 1H, CH), 7.096 (s, 2H), 7.154–7.174
13
(d, 1H), 7.2567.312 (m, 2H), 7.385–7.409 (m. 1H). C-NMR (DMSO-d6) δ: 27.272, 28.820, 31.195,
32.337, 35.816, 50.398, 58.091, 112.565, 120.011, 122.047, 126.858, 130.012, 130.417, 131.122,
147.967, 159.002, 163.338, 196.187. HRMS (ESI+): m/z: calcd for C18H17BrN2O2: 395.0366 [M + Na]+;
found: 395.0450.
2-Amino-4-(3,4,5-trimethoxyphenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran (13):
Yield: 60%. 184−185 °C. IR (KBr pellet cm−1): 3313, 3186, 2961, 2189, 1656, 1588, 1505, 1459, 1420,
1367, 1322, 1212, 1124, 536 cm−1. 1H-NMR (DMSO-d6, δ, ppm): 1.029 (s, 3H, CH3), 1.060 (s, 3H, CH3),
2.091 (s, 2H), 2.131–2.172 (d, 1H), 2.271–2.311 (d, 1H), 3.633 (s, 3H), 3.727 (s, 6H), 4.145 (s, 1H, CH),
13
6.389 (s, 2H), 6.975 (s, 2H). C-NMR (DMSO-d6) δ: 27.039, 29.098, 31.179, 32.234, 36.098, 50.456,
56.247, 58.802, 60.398, 104.590, 112.816, 120.232, 136.569, 140.966, 153.232, 158.828, 163.272,
196.219. HRMS (ESI+): m/z: calcd for C21H24N2O5: 407.1577 [M + Na]+; found: 407.1565.

Molecules 2015, 20
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3.3. X-ray Crystallography
For X-ray diffraction experiments, single crystals of compounds 3, 7, 9 and 11 were grown from
methanol. The X-ray diffraction data were collected on a Bruker SMART APEX II CCD diffractometer
(Bruker Optics, Ettlingen, Germany) equipped with a graphite monochromated Mo Kα radiation (λ =
0.71073 Å) by using the ω-2θ scan technique at room temperature. The structure was solved by direct
methods using SHELXS-97 (Sheldrick 1997, University of Gottingen, Germany) and refined using the
full-matrix least squares method on F2 with anisotropic thermal parameters for all non-hydrogen atoms by
using SHELXL-97 [15]. Hydrogen atoms were generated geometrically. The crystal data and details
concerning data collection and structure refinement are given in Table 4. Molecular illustrations were
prepared using the XP package. Parameters in CIF format are available as Electronic Supplementary
Publication from Cambridge Crystallographic Data Centre.
CCDC 1048441-1048444 for compounds 3, 7, 9 and 11 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/
retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44-1223-336033;
E-mail: deposit@ccdc.cam.ac.uk)
Table 4. Crystal data, data collection and structure refinement of compounds 3, 7, 9 and 11.
Compound 3 Compound 7 Compound 9 Compound 11
Formula
C26H18O6S
458.08
C20H11F3N2O3 C18H12F6N2O2 C18H16Cl2N2O2
Mr
384.31
Triclinic
Pī
402.3
Triclinic
Pī
362.06
Triclinic
Pī
Crystal system
Triclinic
Pī
Space group
a/Å
b/Å
c/Å
α/°
10.947 (3)
20.402 (7)
20.694 (7)
109.44 (3)
90.09 (3)
93.29 (3)
4350 (3)
2
7.9416 (6)
10.9917 (9)
11.4755 (6)
112.429 (7)
105.094 (6)
97.364 (6)
864.45 (11)
2
8.5762 (6)
8.8891 (8)
12.1026 (8)
109.583 (7)
92.163 (6)
97.257 (7)
859.13 (11)
2
8.2045 (5)
14.1658 (8)
23.3891 (15)
94.540 (5)
97.474 (5)
98.928 (5)
2648.9 (3)
2
β/°
γ/°
V/Å3
Z
D
calc/g·cm−3
1.401
1.447
1.555
1.357
μ(Mo Kα)/mm−1
θ range/°
0.191
0.122
0.146
0.38
2.43 to 25.00
18355
2.74 to 27.68
5790
2.43 to 27.35
5819
2.76 to 27.51
18090
Reflections collected
No. unique data [R(int)] 13442 [0.0377] 3045 [0.0233] 3030 [0.0213] 9317 [0.0442]
No. data with I ≥ 2σ(I)
5431
2170
2318
6552
0.0824
0.261
R1
0.0783
0.2469
0.0595
0.1774
0.0745
0.2276
ωR2(all data)
3.4. Quantum Chemical Calculations
All calculations were carried out using the Gaussian 09 package [16]. Density functional theory (DFT),
Becke’s three-parameter hybrid function (B3LYP), and LYP correlation function were used to fully
optimize all the geometries on the energy surface without constraints. To obtain precise results that are in

Molecules 2015, 20
17624
conjunction with experimental results, three basis sets, namely 6-31G*, 6-31 + G**, and 6-311G*, were
tested. Frequency calculations at the B3LYP (with basis sets 6-31G*) level of theory were carried out to
confirm stationary points as minima and to obtain the zero-point energies and the thermal correlation data
at 1 atm and 298 K.
3.5. Minimal Inhibitory Concentration (MIC) Assay
Based on the CLSI broth microdilution method [17], the determination of minimum inhibitory
concentrations (MICs) via microdilution assay was performed in sterilized 96-well polypropylene microtiter
plates (Sigma-Aldrich) in a final volume of 200 μL. Bacteria were grown overnight in nutrient broth.
Mueller–Hinton (MH) broth (100 μL) containing bacteria (5 × 105 CFU/mL) was added to 100 μL of the
culture medium containing the test compound (0.12 μg/mL–256 μg/mL in serial 2-fold dilutions). The
plates were incubated at 37 °C for 20 h in an incubator. About 50 µL of 0.2% triphenyl tetrazolium
chloride (TTC), a colorimetric indicator, was added to each well of microtiter plates and incubated at
35 °C for 1.5 h. The TTC-based MIC was determined as the lowest concentration of oxacillin that
showed no red color change indicating complete growth inhibition.
3.6. Cell Viability Assay
Viability of intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell
line (4T1) and pancreatic cancer cell line (PANC1) was determined by using the MTT assay as described
by Mosmann [18]. Cells reaching 70%–80% confluency were treated with various concentrations of the
synthesized compounds with 1% dimethyl sulfoxide (DMSO) as a negative control. After 48 h incubation,
20 μL of MTT solution (5 mg/mL in PBS) was added and incubated for an additional 4 h. Subsequently,
the medium was aspirated carefully, and 150 μL of DMSO was added. After incubation for 15 min, the
optical density was measured at 490 nm using FlexStation 3 benchtop multi-mode microplate reader
(Molecular Devices). Data were recorded and analyzed for the assessment of the effects of the test
substances on cell viability and growth inhibition. The IC50 and IC90 values were calculated using
regression equation as explained before. The results are presented as the average percentage viability to
the negative control (1% DMSO). The percentage of cell viability was calculated using the following
formula: % cell viability = (absorbance of treated/absorbance of untreated) × 100. The percentage of
inhibition was plotted against the concentration in Microsoft excel and the IC50 was calculated using the
regression equation.
4. Conclusions
In this work, we synthesized two new series of biscoumarin and dihydropyran derivatives; evaluated
their antibacterial activities in vitro against one-drug-sensitive S. aureus (S. aureus ATCC 29213) strain
and three MRSA strains (MRSA XJ 75302, Mu50, and USA 300 LAC); and then measured their antitumor
activities on intestinal epithelial adenocarcinoma cell line (HuTu80), mammary adenocarcinoma cell line
(4T1) and pancreatic cancer cell line (PANC1) in vitro.

Molecules 2015, 20
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In addition, X-ray structural analysis showed that biscoumarins 1–4 had two classical intramolecular
O–H···O hydrogen bonds in their structures. Their corresponding intramolecular hydrogen bonds energy
was calculated to be −116.20463, −115.7031595, −118.0346035 and −121.970228 kJ/mol, respectively.
Among the synthesized compounds, compounds 1–4 had more potent antitumor activity against the
tested three cancer cell lines with the IC50 and IC90 values of 18.78–32.63 μg/mL and 36.05–64.55 μg/mL,
respectively, which is much lower than that of the positive control drug carboplatin; compounds 1 and 2,
with lower intramolecular hydrogen bonds energy, also showed the most potent antibacterial effect on four
S. aureus bacterial strains with the MIC values of 2–16 μg/mL. The reason may be that intramolecular HB
strength is related to the stability of chemical structure, which further affects the binding affinity between
molecules and target protein.
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).
Author Contributions
Feng Sui, Ming-Kai Li, Yun-Peng Sui, Hai-Ru Huo and Jia-Jia Xin conceived and designed the
experiments; Jing Li, Yun-Peng Sui, Xiao-Jun Li, Xin-Liang Du, Hai Ma, Hai-Yu Zhou, Hong-Dan Zhan,
Zhu-Ju Wang and Chun Li performed the experiments; Feng Sui, Jing Li, Yun-Peng Sui, Hai-Yu Zhou
and Hong-Dan Zhan analyzed the data; Yun-Peng Sui and Jing Li wrote the paper; All authors read and
approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–13 are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).