Synthesis of biscoumarin and dihydropyran derivatives and evaluation of their antibacterial activity

Article abstract of DOI:10.3390/molecules200917469

In an attempt to find a new class antibacterial agents, a series of biscoumarins (1-4) and dihydropyrans (5-13) were successfully prepared. The molecular structures of four representative compounds, that is, 4, 5, 8 and 12 were confirmed by single crystal X-ray diffraction study. These synthesized compounds were screened for their antibacterial activity in vitro against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), USA 300 (Los Angeles County clone, LAC), Staphylococcus epidermidis (S. epidermidis ATCC 14990), methicillin-resistant S. epidermidis (MRSE XJ 75284) and Escherichia coli (E. coli ATCC 25922). Additionally, there are two classical intramolecular O-H O hydrogen bonds (HBs) in biscoumarins 1-4 and the corresponding HB energies were further performed with the density functional theory (DFT) [B3LYP/6-31G] method.

Full text of DOI:10.3390/molecules200917469

Molecules 2015, 20, 17469-17482; doi:10.3390/molecules200917469
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of Biscoumarin and Dihydropyran Derivatives and
Evaluation of Their Antibacterial Activity
Jing Li 1,†, Chang-Wei Lv 2,†, Xiao-Jun Li 1, Di Qu 3, Zheng Hou 3, Min Jia 3, Xiao-Xing Luo 3,
Xia Li 4,* and Ming-Kai Li 3,*
1
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.)
2
Department of Orthopaedics, Xijing Hospital, the Fourth Military Medical University,
Xi’an 710032, China; E-Mail: lvcwei@fmmu.edu.cn
3
Department of Pharmacology, School of Pharmacy, the Fourth Military Medical University,
Xi’an 710032, China; E-Mails: qu_di1990@126.com (D.Q); hzh_0001@163.com (Z.H.);
yangziyi@fmmu.edu.cn (M.J.); xxluo3@fmmu.edu.cn (X.-X.L.)
4
Department of Neurosurgery, Xijing Hospital, 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: sjwklixia@sina.com (X.L.);
mingkai@fmmu.edu.cn (M.-K.L.); Tel.: +86-29-8477-5539 (X.L.); +86-29-8477-4555 (M.-K.L.).
Academic Editor: Derek J. McPhee
Received: 27 April 2015 / Accepted: 22 July 2015 / Published: 18 September 2015
Abstract: In an attempt to find a new class antibacterial agents, a series of biscoumarins
(1–4) and dihydropyrans (5–13) were successfully prepared. The molecular structures of
four representative compounds, that is, 4, 5, 8 and 12 were confirmed by single crystal X-ray
diffraction study. These synthesized compounds were screened for their antibacterial
activity in vitro against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant
S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699),
USA 300 (Los Angeles County clone, LAC), Staphylococcus epidermidis (S. epidermidis
ATCC 14990), methicillin-resistant S. epidermidis (MRSE XJ 75284) and Escherichia coli
(E. coli ATCC 25922). Additionally, there are two classical intramolecular O–H···O
hydrogen bonds (HBs) in biscoumarins 1–4 and the corresponding HB energies were
further performed with the density functional theory (DFT) [B3LYP/6-31G*] method.

Molecules 2015, 20
Keywords: biscoumarins; dihydropyran; S. aureus; S. epidermidis
17470
1. Introduction
Staphylococcus aureus (S. aureus) is a common pathogen causing serious hospital infections,
community acquired infections and is also the pathogen with the highest drug resistant incidence.
There are still no specific and selective antibacterial agents to kill MRSA (Methicillin-resistant
S. aureus), which leads to a high death rate of patients [1]. Resistance to antibacterial drugs has been
relentlessly increasing over the past two decades, along with a noticeable decrease in the development
of new drugs for infections [2]. Few candidate drugs that offer benefits over existing drugs are
available, and few drugs that will treat infections due to the so-called “ESKAPE” pathogens
(Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,
Pseudomonas aeruginosa, and Enterobacter spp.) are being developed [3]. The need for newer, more
effective treatment options for multidrug-resistant (MDR) pathogens induced the Infectious Diseases
Society of America (IDSA) to launch the 10 × 20 initiative against a range of resistant “ESKAPE”
pathogens including MRSA and to develop 10 new effective antimicrobial drugs to treat the infections
caused by these pathogens by 2020 [4]. Therefore, it is evident and urgent to seek a new defending
strategy against MRSA now.
Bisoumarin and dihydropyran derivatives are widely available in nature. These derivatives exhibit
various biological activities, such as anticoagulant, insecticidal, antihelminthic, hypnotic, and
antifungal activities, phytoalexin production, and HIV protease inhibition [5–8]. Moreover, the two
kinds of compounds are gaining increasing interest because of their versatile activities through
chemical modifications (different substituents on the aromatic ring) [9]. For biscoumarins, the
minimum active harmacophore reportedly consists of a coumarin dimer containing an aryl substituent
on the central linker methylene. The addition of 4-hydroxy substituents to coumarin rings improved the
potency of the compounds [10]. Recognizing the considerable importance of the compounds, the
researchers focused on the synthesis of bisoumarin and dihydropyran derivatives [11].
To identify more active compounds, we prepared a series of biscoumarin and dihydropyran
derivatives (Figure 1) and then evaluated their antibacterial activities. Finally, we calculated the total
HB energies of compounds 1–4 by density functional theory (DFT) method to elucidate a possible
relationship between such hydrogen-bonded structures and their antibacterial activities.
OH HO
O
O
R=
CH
3
OH
Br
F
O
O
R
CH
3
OCH
F
Cl
3
1
3
2
4
Figure 1. Cont.

Molecules 2015, 20
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NH
2
CN
R
O
O
R=
OH
Cl
NO
2
O
5
6
7
NH
2
CN
R
O
O
R=
SO CH
NO
2
2
3
9
10
8
NH
2
NO
2
CN
R
O
R=
OCH
NO
3
2
O
12
11
13
Figure 1. Chemical structures of compounds 1–13.
2. Results and Discussion
2.1. Molecular Structure
The crystal structures of compounds 4, 5, 8 and 12 are given in Figure 2. In the crystal structure of
compound 4, two crystallographically independent molecules are present in the asymmetric unit. The
whole molecule is disordered over two orientations with refined site occupancies of 1:1, and two
4-hydroxycoumarin moieties are linked through a methylene bridge on which one hydrogen atom has
been replaced with a 3-fluoro-4-chlorophenyl group. However, the two components differ with respect
to the reversed twist directions of the 3-fluoro-4-chlorophenyl residue. In the compound, two classical
intramolecular hydrogen bonds were found; each links a coumarin hydroxyl and carbonyl group
[d(O3–O4) = 2.598 Å, d(O1–O6) = 2.676 Å].
Compound 4
Compound 5
Figure 2. Cont.

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Compound 8
Compound 12
Figure 2. Crystal structure of compounds 4, 5, 8 and 12.
In compounds 5, 8 and 12, the new formed pyran ring is essentially planar because the maximum
deviation of the atoms in the skeleton from the C7-O3-C8 plane is only 0.2533 Å in compound 5, the
corresponding value is 0.2006 Å and 0.6686 Å in compounds 8 and 12 respectively. The adjacent
coumarin ring, naphthalene ring and ketone ring is also basically parallel to the pyran ring in the three
compounds. The torsion angle between aromatic ring and pyran ring is about 89.452°, 84.160° and
89.801° in compounds 5, 8 and 12 respectively.
2.2. Quantum Chemical Calculations
2.2.1. Geometric Parameters of Compounds 1–4
The fully optimized molecular structures of biscoumarins 1–4 with atomic numbering calculated at
B3LYP level of theory are shown in Figure 3. For compound 4, experimental geometric parameters
and selected calculated geometric parameters under three different basis sets (6-31G*, 6-31+G**, and
6-311G*) are presented in Table 1.
CH3
OH
OCH3
H3
O3
O4
CH3
R=
R=
R=
R
H
C3
C1
C12
O
1
3
2
F
R=
Cl
O
C19
Br
F
O1
O6
H6
4
Figure 3. Schematic presentation of compounds 1–4.
Under three different basis sets, the calculated results are very close with the average discrepancy of
the selected bond lengths and bond angles between theoretical and experimental data is less than ± 0.02 Å
and ± 2° respectively. B3LYP/6-31G* exhibited sufficient agreement with experimental data and
lower computational cost, so further theoretical study was performed at this level.

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Table 1. Experimental and calculated parameters of the selected bond lengths and bond
angles of 3,3′-(3-fluoro-4-chlorobenzylidene)-bis-(4-hydroxycoumarin) (4).
X-ray 6-31G* 6-31+G** 6-311G*
R(C1—O1)
1.224
1.35
1.234
1.372
1.23
1.236
1.37
1.226
1.371
R(C1—O2)
R(C19—O4)
1.221
1.362
1.508
1.516
1.536
1.233
1.372
1.526
1.524
1.539
121.7
115.9
121.89
115.85
115.94
112.77
114.61
133.05
92.01
130.59
93.85
1.223
R(C19—O5)
1.375
1.526
1.525
1.539
1.374
R(C2—C10)
1.526
R(C10—C11)
1.524
R(C10—C20)
1.537
A(C1—O2—C9)
A(O1—C1—O2)
A(C18—O5—C19)
A(O4—C19—O5)
A(C2—C10—C20)
A(C2—C10—C11)
A(C11—C10—C20)
D(C1—C2—C10—C20)
D(C1—C2—C10—C11)
121.11 121.67
116.05 115.7
121.34 121.9
114.64 115.67
114.84 115.8
113.67 112.59
113.79 114.51
137.5 133.73
121.7
115.95
121.92
115.96
115.88
112.68
114.57
133.63
91.65
89.01
91.83
D(C12—C11—C10—C20) 129.87 131.31
96.14 93.64
130.23
94.44
D(C12—C11—C10—C2)
2.2.2. Estimation of the Single and Total HB Energies in Compounds 1–4
We estimate single and total HB energies of compound 4 as an example. The global minimum
structure is stabilized by two HBs (4ab); two higher energy structures is stabilized by one HB (4a and
4b) respectively.
From Table 2 we can see that, the O6—H6···O1 HB energy was estimated to be −51.402039 kJ/mol
by the equation E(O6 ⎯H6 …Ο1) = Ecoor − Ec4oaor , is from the energy difference between 4ab and 4a (4a
4ab
is a global minimum structure with O3—H3···O4 HB). Similarly, the O3—H3···O4 HB energy was
coor
coor
calculated to be −65.4353365 kJ/mol by the equation E(O3 ⎯H3 …Ο4 ) = E − E , is from the
4ab
4b
energy difference between 4ab and 4b (4b was obtained from the global minimum structure 4ab, but
H3 was rotated around the C3—O3 bond until O3—H3···O4 HB rupture occurred [12,13]).
Table 2. Total electronic energies (in hartree) and hydrogen bond (HB) energies (in
kJ/mol) of hydrogen bonded conformers of compounds 1–4 calculated at B3LYP/6-31G*
level of theory.
System Total Electronic Energies a E(O6—H6···O1) E(O3—H3···O4) E(Total HB)
1ab
1a
−1452.593097
−1491.908094
−1491.888121
−1603.031177
−1603.011081
−1603.004738
−119.118935
−52.4391115
−52.762048
1b
−67.1051545
−69.4155945
2ab
2a
−122.1776425
2b

Molecules 2015, 20
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Table 2. Cont.
System Total Electronic Energies a E(O6—H6···O1) E(O3—H3···O4) E(Total HB)
3ab
3a
−4083.681309
−4083.66182
−4083.656549
−1972.171758
−1972.15218
−1972.146835
−116.1757495
−51.1683695
3b
−65.00738
4ab
4a
−116.8373755
−51.402039
4b
−65.4353365
a ZP corrected.
The total HB energy in compound 4, calculated by the equation E(O3 ⎯H3 …Ο4 ) + E(O6 ⎯H6 …Ο1) ,
was estimated to be −116.8373755 kJ/mol. For compounds 1–3, their total HB energies are
−119.118935, −122.1776425 and −116.1757495 kJ·mol–1, respectively.
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, USA 300 LAC), one drug-sensitive S. epidermidis strain (S. epidermidis
ATCC 14990) and one methicillin-resistant S. epidermidis strain (MRSE XJ 75284) were used in the
systematic analysis of their antibacterial activities in vitro. From Table 3 we can see that in all the
synthesized compounds, biscoumarin derivatives 3 and 4 exerted more potent anti-bacterial activity
against all tested strains expect for E. Coli with the MIC values of 8–16 μg/mL. Compared with
compounds 1–13, the MIC values of levofloxacin, ceftazidime, ceftriaxone, gentamicin, piperacillin
and vancomycin against S. aureus (ATCC 29213) strain were lower (less than 8 μg/mL) but were
higher against other strains at varying degrees.
Table 3. Minimum inhibitory concentrations (MIC) of compounds 1–13 and antibiotics in
Mueller-Hinton broth culture.
MIC (μg/mL)
Drugs
S. aureas MRSA Mu50 LAC S. epidermidis MRSE E. Coli
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
>256
>256
16
>256
>256
16
>256 >256
>256 >256
>256
>256
8
>256
>256
8
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
>256
8
8
8
8
8
8
8
8
>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
>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

Molecules 2015, 20
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Table 3. Cont.
MIC (μg/mL)
LAC S. epidermidis MRSE
Drugs
S. aureas
MRSA
Mu50
E. Coli
Levofloxacin <0.125 (S)
4 (R)
4 (R)
8 (R)
64 (R)
32 (R)
0.25 (S)
8 (R)
0.125 (S)
1 (S)
0.125 (S) 0.25 (S)
Ceftazidime
Ceftriaxone
Gentamicin
Piperacillin
Vancomycin
8 (S)
2 (S)
>256 (R) 256 (R)
>256 (R) 256 (R)
256 (R)
256 (S)
32 (R)
0.25 (S)
0.25 (S)
2 (S)
1 (S)
0.12 (S)
2 (S)
64 (R)
>128 (R) >128 (R)
8 (I) 8 (I)
32 (R)
0.25 (S)
2 (S)
>128 (R)
2 (S)
0.25 (S)
0.5 (S)
0.25 (S)
0.5 (S) >256 (R)
S means drug susceptibility, R means drug resistance, I means intermediate resistance.
2.4. In Vitro Toxicity Measurement
To further explore the safety of the possible development, we investigated the cytotoxicity of
compounds 3 and 4 to human umbilical vein endothelial cells (HUVECs) and human embryonic
cardiomyocyte cell line CCC-HHM-2 (HHHM-2) in vitro. As shown in Figure 4, there was no obvious
inhibitory effect of compounds 3 and 4 on cell viability at a concentration between 6.25 to 200 μg·mL−1,
and the IC50 of compound 3 to the HUVECs and HHHM-2 was 1268.25 μg·mL−1 and 1036.56 μg·mL−1,
the corresponding value of compound 4 was 1145.36 μg·mL−1 and 916.14 μg·mL−1. These results
implied that compounds 3 and 4 had much less toxicity to mammalian cells, and had a relatively wide
safety range for potential antimicrobial applications.
Figure 4. Cytotoxicity measurement of compounds 3 and 4 on the human umbilical vein
endothelial cells (HUVECs) and human embryonic cardiomyocyte cell line CCC-HHM-2
(HHHM-2) in vitro.
2.5. Discussion
Methicillin-resistant S. aureus (MRSA) and is the cause of major outbreaks and epidemics in
hospitalized patients because of the emergence, spread, and rapid evolution of resistance genes
developing among pathogens. As such, the mortality and morbidity rates of patients affected by MRSA

Molecules 2015, 20
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are high. However, the emergence of vancomycin-resistant S. aureus and treatment failure of MRSA
infections urgently requires developing new antimicrobials.
In this work, among two kinds of compounds, novel biscoumarin derivatives 3 and 4 were
demonstrated to be capable of remarkably inhibiting the growth of all tested strains expect for E. coli
with the MIC value of 8–16 μg/mL. However, compared with compounds 3 and 4, other biscoumarins
1 and 2 and dihydropyran derivatives 5–13 almost have no antibacterial activities. In addition, the
results of in vitro toxicity study of compounds 3 and 4 on HUVECs and HHHM-2 revealed that the
two compounds had a relatively wide safety range for potential antimicrobial applications with much
less toxicity to mammalian cells.
X-ray structural analysis also showed that biscoumarins 1–4 were stabilized by two asymmetrical
intramolecular O—H···O HBs, which were considered as important factors in assisting the molecule to
attain the correct configuration for biological activity [14]. The total HB stabilization energies in
compounds 1–4 were estimated to be −119.118935, −122.1776425, −116.1757495 and
−116.8373755 kJ·mol−1, respectively. These values suggest that the most potent antibacterial activity
in compounds 3 and 4 was consistent with their weakest HB strengths.
3. Experimental Section
3.1. Apparatus and Materials
IR spectra (400–4000 cm−1) were obtained using a Brucker Equinox-55 spectrophotometer
1
(Fremont, CA, USA). H-NMR spectra were obtained using a Varian Inova-400 spectrometer (at
400 MHz) (Palo Alto, CA, USA). Mass spectra were obtained using a micrOTOF-Q II mass spectrometer
(Bruker). The melting points were taken on a XT-4 micro melting apparatus (Taike Company, Beijing,
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). All other chemicals and solvents were of analytical grade.
MRSA (XJ 75302) and MRSE (XJ 75284) were isolated from cultures of sputum samples from
patients in Xijing Hospital (Xi’an, China). S. aureus (ATCC 29213), S. epidermidis (ATCC 14990)
and E. coli (ATCC 25922) strains were purchased from the Chinese National Center for Surveillance
of Antimicrobial Resistance. Mu50 (ATCC 700699) and USA 300 (LAC) were purchased from
MicroBiologics (St. Cloud, MN, USA).
3.2. Synthesis and Characterization of Compounds 1–13
Biscoumarins 1–4 were synthesized according to the methods of a previous report [15]. A mixture
of 3,4-dimethylbenzaldehyde (3-hydroxy-4-methoxybenzaldehyde, 3-bromo-4-fluorobenzaldehyde,
3-fluoro-4-chlorobenzaldehyde) (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. The solid was filtered off and then recrystallized from ethanol to give compounds 1–4.

Molecules 2015, 20
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3,3′-(3,4-Dimethylbenzylidene)-bis-(4-hydroxycoumarin) (1): m.p. 227–228 °C. IR (KBr pellet cm−1):
1
2922, 1671, 1601, 1561, 1342, 1303, 1094, 766 cm−1. H-NMR (CDCl3, δ, ppm): 11.507 (s, 1H),
11.268 (s, 1H), 8.015–8.065 (q, 2H), 7.606–7.647 (t, 2H), 7.404–7.425 (d, 4H), 7.067–7.088 (d, 1H),
6.950 (s, 2H), 6.049 (s, 1H), 2.204 (s, 3H), 2.241 (s, 3H). HRMS (ESI+): m/z: calcd for C27H20O6:
463.1152 [M + Na]+; found: 463.1135.
3,3′-(3-Hydroxy-4-methoxybenzylidene)-bis-(4-hydroxycoumarin) (2): m.p. 246–247 °C. IR (KBr
pellet cm−1): 3530, 2608, 1667, 1615, 1567, 1512, 1453, 1350, 1269, 1215, 1095, 1028, 763 cm−1.
1H-NMR (CDCl3, δ, ppm): 11.612 (s, 1H), 11.277 (s, 1H), 8.034–8.070 (d, 2H), 7.623–7.666 (m, 2H),
7.414–7.435 (d, 4H), 6.799–6.819 (t, 2H), 6.708–6.729 (t, 1H), 6.047 (s, 1H), 5.612 (s, 1H), 3.895 (s,
3H). HRMS (ESI+): m/z: calcd for C26H18O8: 481.0894 [M + Na]+; found: 481.7855.
3,3′-(3-Bromo-4-fluorobenzylidene)-bis-(4-hydroxycoumarin) (3): m.p. 215–216 °C. IR (KBr pellet cm−1):
1
1666, 1617, 1567, 1495, 1348, 1097, 1047, 763 cm−1. H-NMR (DMSO-d6, δ, ppm): 7.885–7.901 (d,
3H), 7.763 (s, 2H), 7.562–7.601 (t, 2H), 7.279–7.357 (m, 4H), 6.403 (s, 1H). HRMS (ESI+): m/z: calcd
for C25H14BrFO6: 530.9850 [M + Na]+; found: 530.9865.
3,3′-(3-Fluoro-4-chlorobenzylidene)-bis-(4-hydroxycoumarin) (4): m.p. 259–260 °C. IR (KBr pellet cm−1):
3448, 3083, 1790, 1670, 1611, 1561, 1489, 1413, 1259, 1172, 1064, 863, 767 cm−1. 1H-NMR (CDCl3,
δ, ppm): 11.626 (s, 1H), 11.344 (s, 1H), 8.020–8.107 (m, 2H), 7.656–7.699 (m, 2H), 7.340–7.458 (m,
5H), 6.972–7.055 (m, 2H), 6.054 (s, 1H). HRMS (ESI+): m/z: calcd for C25H14ClFO6: 487.0355 [M + Na]+;
found: 487.0433.
Dihydropyran derivatives (5–13) were also synthesized according to a reported procedure [16].
A mixture of 4-hydroxycoumarin (1-naphthalenol, 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. The solid
was filtered off and then recrystallized from ethanol to give compounds 5–13.
2-Amino-4-(4-hydroxyphenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (5): m.p. 269–270 °C. IR
1
(KBr pellet cm−1): 3511, 3279, 2191, 1694, 1670, 1606, 1382, 1262, 1165, 1069, 757 cm−1. H-NMR
(DMSO-d6, δ, ppm): 9.387 (s, 1H), 7.879–7.902 (q, 1H), 7.684–7.727 (m, 1H), 7.447–7.507 (q, 2H),
7.356 (s, 2H), 7.034–7.055 (d, 2H), 6.679–6.700 (d, 2H), 4.331 (s, 1H). HRMS (ESI+): m/z: calcd for
C19H12N2O4: 355.0689 [M + Na]+; found: 355.0680.
2-Amino-4-(4-chlorophenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (6): m.p. 254–255 °C. IR
1
(KBr pellet cm−1): 3386, 3312, 3190, 2193, 1714, 1675, 1610, 1490, 1377, 1060, 759 cm−1. H-NMR
(DMSO-d6, δ, ppm): 7.889–7.913 (q, 1H), 7.703–7.746 (m, 1H), 7.461–7.521 (m, 4H), 7.359–7.386
(m, 2H), 7.301–7.328 (m, 2H), 4.493 (s, 1H). HRMS (ESI+): m/z: calcd for C19H11ClN2O3: 373.0350
[M + Na]+; found: 373.2438.
2-Amino-4-(4-nitrophenyl)-3-cyano-5-oxo-4H,5H-pyrano[3,2c]chromene (7): m.p. 259–260 °C. IR
1
(KBr pellet cm−1): 3688, 2191, 1718, 1662, 1509, 1341, 1253, 1053, 957, 901, 765 cm−1. H-NMR

Molecules 2015, 20
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(DMSO-d6, δ, ppm): 8.175–8.196 (d, 2H), 7.910–7.933 (q, 1H), 7.723–7.766 (m, 1H), 7.475–7.611 (m,
6H), 4.682 (s, 1H). HRMS (ESI+): m/z: calcd for C19H11N3O5: 384.0591 [M + Na]+; found: 384.0533.
2-Amino-4-(3-phenoxyphenyl)-4H-benzo[h]chromene-3-carbonitrile (8): m.p. 173–174 °C. IR (KBr
1
pellet cm−1): 3337, 1627, 1573, 1488, 1445, 1378, 1240, 1189, 1105, 1025, 892, 758 cm−1. H-NMR
(DMSO-d6, δ, ppm): 8.221–8.241 (d, 1H), 7.899–7.919 (d, 1H), 7.567–7.661 (m, 3H), 7.301–7.389 (m,
3H), 7.112–7.206 (m, 4H), 6.979–7.008 (t, 4H), 6.796–6.822 (q, 1H), 4.938 (s, 1H). HRMS (ESI+):
m/z: calcd for C26H18N2O2: 413.1260 [M + Na]+; found: 413.1255.
2-Amino-4-(4-methanesulfonylphenyl)-4H-benzo[h]chromene-3-carbonitrile (9): m.p. 244–245 °C. IR
(KBr pellet cm−1): 3001, 1629, 1573, 1487, 1412, 1304, 1234, 1190, 1150, 1103, 1016, 764 cm−1.
1H-NMR (DMSO-d6, δ, ppm): 8.246–8.267 (d, 1H), 7.887–7.925 (t, 3H), 7.602–7.683 (m, 3H),
7.523–7.544 (d, 2H), 7.039 (s, 2H), 7.116–7.138 (d, 1H), 5.106 (s, 1H), 3.203 (s, 3H). HRMS (ESI+):
m/z: calcd for C21H16N2O3S: 399.0774 [M + Na]+; found: 399.0723.
2-Amino-4-(4-nitrophenyl)-4H-benzo[h]chromene-3-carbonitrile (10): m.p. 241–242 °C. IR (KBr
pellet cm−1): 3477, 1651, 1512, 1404, 1350, 1185, 1101, 1022, 801, 770 cm−1. 1H-NMR (DMSO-d6, δ,
ppm): 8.198–8.272 (q, 3H), 7.905–7.926 (d, 1H), 7.538–7.685 (m, 5H), 7.343 (s, 2H), 7.106–7.128 (d,
1H), 5.164 (s, 1H). HRMS (ESI+): m/z: calcd for C20H13N3O3: 366.0849 [M + Na]+; found: 366.0831.
2-Amino-4-(4-methoxyphenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran (11):
m.p. 209–210 °C. IR (KBr pellet cm−1): 3320, 3187, 2198, 1685, 1649, 1606, 1582, 1509, 1462, 1411,
1
1370, 1247, 1033, 842 cm−1. H-NMR (DMSO-d6, δ, ppm): 7.045–7.067 (d, 2H), 6.954 (s, 2H),
6.834–6.855 (d, 2H), 4.124 (s, 1H), 3.718 (s, 3H), 2.452–2.508 (q, 2H), 2.229–2.269 (d, 1H), 2.075–2.115
(d, 1H), 1.039 (s, 3H), 0.952 (s, 3H). HRMS (ESI+): m/z: calcd for C19H20N2O3: 347.1366 [M + Na]+;
found: 347.1324.
2-Amino-3-cyano-4-phenyl-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran
(12):
m.p.
219–220 °C. IR (KBr pellet cm−1): 3213, 2961, 2199, 1689, 1603, 1492, 1454, 1413, 1372, 1217,
1
1035, 838 cm−1. H-NMR (DMSO-d6, δ, ppm): 7.273–7.311 (t, 2H), 7.136–7.205 (m, 3H), 6.999 (s,
2H), 4.176 (s, 1H), 2.503–2.526 (m, 1H), 2.240–2.281 (d, 1H), 2.130 (s, 1H), 2.092 (s, 1H), 1.046 (s,
3H), 0.963 (s, 3H). HRMS (ESI+): m/z: calcd for C18H18N2O2: 317.1260 [M + Na]+; found: 317.1344.
2-Amino-4-(2,4-dinitrophenyl)-3-cyano-7,7-dimethyl-5-oxo-4H-5,6,7,8-tetrahydrobenzo[b]pyran (13):
m.p. 217–218 °C. IR (KBr pellet cm−1): 3422, 2202, 1682, 1664, 1602, 1537, 1361, 1216, 1044 cm−1.
1H-NMR (DMSO-d6, δ, ppm): 8.619–8.625 (d, 1H), 8.434–8.462 (m, 1H), 7.707–7.729 (d, 1H), 7.374
(s, 2H), 4.989 (s, 1H), 2.202–2.243 (d, 1H), 2.091 (s, 2H), 2.009–2.050 (d, 1H), 1.027 (s, 3H), 0.898 (s,
3H). HRMS (ESI+): m/z: calcd for C18H16N4O6: 407.0962 [M + Na+]; found: 407.0945.
3.3. X-ray Crystallography
For X-ray diffraction experiments, single crystals of compounds 4, 5, 8 and 12 were both grown
from methanol. The X-ray diffraction data were collected on a Bruker SMART APEX II CCD
diffractometer equipped with a graphite monochromated Mo Kα radiation (λ = 0.71073 Å) by using

Molecules 2015, 20
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the ω-2θ scan technique at room temperature. The structure was solved by direct methods using
SHELXL-97 [17] and refined using the full-matrix least squares method on F2 with anisotropic
thermal parameters for all non-hydrogen atoms by using SHELXL-97. 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.
Table 4. Crystal data, data collection and structure refinement of compounds 4, 5, 8 and 12.
Compound 4 Compound 5 Compound 8 Compound 12
Formula
C25H14ClFO6
464.0463
Monoclinic
P21/c
C19H12N2O4
332.0797
Monoclinic
P21/c
C26H18N2O2
390.1368
Monoclinic
P21/c
C18H18N2O2
294.1368
Monoclinic
P21/c
Mr
Crystal system
Space group
a/Å
b/Å
c/Å
α/°
10.1874(7)
10.1667(6)
20.0790(12)
90
8.8632(6)
13.1743(8)
13.0565(6)
90
11.908(3)
15.4731(17)
11.9343(18)
90
11.2938(5)
9.4591(5)
14.9132(7)
90
β/°
96.571(7)
90
94.484(6)
90
116.15(2)
90
99.304(4)
90
γ/°
V/Å3
Z
calc/g·cm−3
2066.0(2)
4
1519.89(15)
4
1973.9(6)
4
1572.21(13)
4
D
1.491
1.452
1.314
1.244
μ(Mo Kα)/mm−1
θ range/°
0.236
0.104
0.084
0.082
2.70 to 25.00 2.78 to 25.00 2.63 to 25.00
7346 5980 4969
2.56 to 25.00
6337
Reflections collected
No. unique data [R(int)] 3621 [0.0256] 2670 [0.0263] 3099 [0.0186] 2775 [0.0280]
No. data with I ≥ 2σ(I)
2293
0.0501
0.1704
1032644
1991
0.0453
0.1559
1032645
2029
0.0542
0.1561
1032646
2109
0.0465
0.1627
1032647
R1
ωR2(all data)
CCDC
3.4. Quantum Chemical Calculations
All calculations were carried out using the Gaussian 09 package [18]. Density functional theory
(DFT) [19], Becke’s three-parameter hybrid function (B3LYP) [20], and LYP correlation function [21,22]
were used to fully optimize all the geometries on the energy surface without constraints. To obtain
precise results that are in 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.

Molecules 2015, 20
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3.5. Minimal Inhibitory Concentration (MIC) Assay
Based on the CLSI broth microdilution method [23], the determination of minimum inhibitory
concentrations (MICs) via microdilution assay was performed in sterilized 96-well polypropylene
microtiter plates (Sigma-Aldrich, St. Louis, MO, USA) 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 to 256 μg/mL in serial twofold 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. The data were from three repeated experiments.
3.6. Cytotoxicity Assay
The cytotoxicity of compounds 3 and 4 to the human umbilical vein endothelial cells (HUVECs)
and the human embryonic cardiomyocyte cell line CCC-HHM-2 (HHHM-2) was determined by
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) staining. Briefly, cells
(5 × 103 cells per well) were seeded in a 96-well plate with 100 μL DMEM medium with 20% fetal
bovine serum (FBS) in each well for 12 h and then treated with or without compounds at various
concentrations (12.5–800 μg·mL−1) for 24 h. After treatment, MTT solution (final concentration,
0.5%) was added and cells were incubated for another 4 h at 37 °C. 150 μL DMSO was added to each
well after removal of the supernatant and the absorbance at 490 nm was measured with a microplate reader.
4. Conclusions
With the emergence of methicillin-resistant, vancomycin intermediate-resistant, or multi-drug-resistant
S. aureus, more appropriate antibiotics need to be developed. Our results showed that biscoumarin
derivatives 3 and 4 have better anti-bacterial efficiency with minimum MIC value of 8–16 μg/mL in all
synthesized compounds, which is may be that there are two intramolecular O—H···O HBs in their
structures and two strong electron-withdrawing atoms (Br and F; F and Cl) further weakens the total
HB energy.
Acknowledgments
This work was supported by grants from National Natural Science Foundation of China
(No. 81573468, No. 81001460, and No. 81273555), the Development Plan of Science and Technology
Research of Shanxi Province (2012K14-01-04), and the Innovation plan of science and technology of
Shanxi Province (2014KTCL03-03).

Molecules 2015, 20
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Author Contributions
J.L. and X.L. synthesized and characterized the compounds; C.-W.L., D.Q., Z.H. and M.J.
performed the activity assay experiments; X.-J.L. performed the calculation; X.-X.L. and M.-K.L.
designed the study and prepared the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 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/).