Crystal structure and activities of three biscoumarin derivatives against Staphylococcus aureus

Article abstract of DOI:10.1016/j.molstruc.2015.05.021

Three new biscoumarin derivatives, namely, 3,3′-[(4-nitrophenyl)methylene]bis(4-hydroxy-2H-chromen-2-one) (NBH), 3,3′-[(4-methoxyphenyl)methylene]bis(4-hydroxy-2H-chromen-2-one) (MBH) and 3,3′-[(4-chloromethylphenyl)methylene]bis(4-hydroxy-2H-chromen-2-on

Full text of DOI:10.1016/j.molstruc.2015.05.021

Journal of Molecular Structure 1097 (2015) 117–123
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstruc
Crystal structure and activities of three biscoumarin derivatives against
Staphylococcus aureus
Fen Li a,1, Chang-wei Lv b,1, Zi-dan Zhang c,1, Jing Li d, Zheng Hou a, Xiao-hui Yang d, Jiang-tao Li d,
Xiao-xing Luo a, , Ming-kai Li a,
⇑
⇑
a Department of Pharmacology, School of Pharmacy, The Fourth Military Medical University, Xi’an, China
b Department of Orthopaedics, Xijing Hospital, The Fourth Military Medical University, Xi’an, China
c Department of Physics, School of Science, Tianjin University, Tianjin, China
d College of Chemistry and Chemical Engineering, The Key Laboratory for Surface Engineering and Remanufacturing in Shaanxi Province, Xi’an University, Xi’an, China
h i g h l i g h t s
ꢀ
ꢀ
ꢀ
Three new biscoumarin derivatives were successfully synthesized.
Their structures were verified by single crystal X-ray crystallography.
The antibacterial activities of the three compounds were further investigated.
a r t i c l e i n f o
a b s t r a c t
Three new biscoumarin derivatives, namely, 3,30-[(4-nitrophenyl)methylene]bis(4-hydroxy-2H-chrom-
en-2-one) (NBH), 3,30-[(4-methoxyphenyl)methylene]bis(4-hydroxy-2H-chromen-2-one) (MBH) and 3,
30-[(4-chloromethylphenyl)methylene]bis(4-hydroxy-2H-chromen-2-one) (CBH) were successfully syn-
thesized and their structures were verified by single crystal X-ray crystallography. In their structures,
there are two intramolecular H-bonds and the corresponding H-bond energies were calculated by DFT
method. The antibacterial activities of NBH, MBH and CBH in vitro against drug-sensitive
Staphylococcus aureus (ATCC 29213) and methicillin-resistant S. aureus (isolated MRSA strains) were fur-
ther investigated.
Article history:
Received 27 November 2014
Received in revised form 8 May 2015
Accepted 11 May 2015
Available online 19 May 2015
Keywords:
Biscoumarin
Crystallography
DFT
Ó 2015 Elsevier B.V. All rights reserved.
S. aureus
Introduction
Coumarin (2H-chromen-2-one) derivatives, containing aro-
matic d-lactones system, are an important class of heterocycles
Staphylococcus aureus (S. aureus) is one of the most important
pathogens that leads to various illnesses in humans, including
wound infections, pneumonia, sepsis, and toxic shock [1–3].
Methicillin-resistant S. aureus (MRSA) is the cause of most antibi-
otic resistant healthcare associated infections, because it spreads
rapidly and cause illness more severe, which further leads to mor-
tality and morbidity rates of patients affected by MRSA are high
[4,5], and the related proportion of such cases increased signifi-
cantly [6,7]. In addition, the emergence of MRSA with decreased
susceptibility to vancomycin, which is customarily used as the
most effective antibiotic to treat for MRSA, induced to the urgent
necessity of developing new antimicrobials.
that have attracted significant importance in the field of organic
and natural product chemistry [8–11]. For example, biscoumarin
and its derivatives have received considerable attention in the past
few years for their versatile biological and medical activities
because of the ease of fine-tuning the aromatic ring with different
substituents on leading to multiple chemical modifications and
activities such as antioxidant, anti-inflammatory, antibacterial
and anticancer [12–14]. Recognizing the considerable importance
of the compounds, the researchers focused on the synthesis of bis-
coumarin derivatives [15].
In this study, we successfully synthesized three novel bis-
coumarin derivatives, namely, 3,30-[(4-nitrophenyl)methylene]bi
s(4-hydroxy-2H-chromen-2-one) (NBH), 3,30-[(4-methoxyphenyl)
methylene]bis(4-hydroxy-2H-chromen-2-one) (MBH) and 3,30-[(4
-chloromethylphenyl)methylene]bis(4-hydroxy-2H-chromen-2-
one) (CBH) (Fig. 1), tested their anti-bacterial activities on
drug-sensitive and drug-resistant S. aureus in vitro, and then
⇑
Corresponding authors.
E-mail addresses: xxluo3@fmmu.edu.cn (X.-x. Luo), mingkai@fmmu.edu.cn
(M.-k. Li).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.molstruc.2015.05.021
0022-2860/Ó 2015 Elsevier B.V. All rights reserved.

118
F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
calculated their total HB energies by density functional theory
(DFT) method.
X-ray crystallography
For compounds NBH, MBH and CBH, three white crystals with
approximate dimensions of 0.22 ꢂ 0.20 ꢂ 0.16 mm3, 0.20 ꢂ
0.20 ꢂ 0.12 mm3, and 0.20 ꢂ 0.18 ꢂ 0.10 mm3 were selected for
data collection respectively. The X-ray diffraction data were col-
lected on a Bruker SMART APEX II CCD diffractometer equipped
with a graphite monochromated Mo Ka radiation (k = 0.71073 A)
Experimental
Apparatus and materials
IR spectra were measured using a Brucker Equinox-55 spec-
trophotometer. 1H NMR spectra, 13C NMR spectra and mass spectra
were tested using the Varian Inova-400 spectrometer, Bruker
Avance III spectrometer and micrOTOF-Q II mass spectrometer,
respectively. The melting points were determined on a XT-4 micro
melting apparatus.
S. aureus strain (ATCC 29213) and isolated MRSA strains (1-3)
were obtained from Chinese National Center for Surveillance of
Antimicrobial Resistance and Xijing Hospital (Xi’an, China), respec-
tively. Antibiotics including levofloxacin, ceftazidime, ceftriaxone,
gentamicin and piperacillin 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.
by using
x–2h scan technique at room temperature. The structures
were solved by direct methods (SHELXS-97) and refined using the
full-matrix leastsquares method on F2 with anisotropic thermal
parameters for all non-hydrogen atoms using SHELXL-97 [17].
The hydrogen atoms were placed in calculated positions and
refined using the riding model. The crystal data, details concerning
data collection and structure refinement are given in Table 1.
Molecular illustrations were prepared using the XP package.
Quantum chemical calculations
Theoretical studies based on density functional theory (DFT)
calculations using the Gaussian 09 package [18–20]. In order to
obtain precise results that are in good agreement with experimen-
tal results, three level of theories have been carried out, they are
B3LYP/6-31G⁄, B3LYP/6-31 + G⁄⁄ and B3LYP/6-311G⁄, respectively
[21–23].
General procedure for preparation of biscoumarin derivatives NBH,
MBH and CBH
NBH, MBH and CBH were prepared from a reported procedure
[16]. In
a
100 mL round bottom flask,
a
mixture of 4-
Bacterial susceptibility testing
nitrobenzaldehyde (4-methoxybenzaldehyde or 4-chloromethyl-
benzaldehyde) (10 mmol) and 4-hydroxycoumarin (20 mmol)
were placed over a magnetic stirrer and the contents were stirred.
To this stirred mixture, a few drops of piperidine were added. The
reaction mixture was heated at 90 °C for 3–5 h and the progress
was monitored by TLC using hexane–chloroform–ethyl acetate
mixture (1:1:1) as eluent. After completion of the reaction, the
reaction mixture was allowed to cool in room temperature
(25 °C) until precipitation occurred. The precipitates were filtered
and then washed with ethanol to get pure products.
The minimum inhibitory concentration (MIC) values were
determined according to the previously reported method [24], S.
aureus strains were grown in 100 lL of nutrient Mueller–Hinton
(MH) broth in the concentration of 5 ꢂ 105 CFU/mL, then 100
lL
–
of MH medium containing the NBH, MBH or CBH (0.12
256 lg/mL in serial two-fold dilutions) was added to the wells of
plates. After they were incubated at 37 °C for 20 h, the lowest con-
centration of compound without visible bacterial growth in the
wells was taken as the MIC value. The experiments were done
independently five times, using duplicate samples each time.
3,30-[(4-Nitrophenyl)methylene]bis(4-hydroxy-2H-chrome-
n-2-one) (NBH): Yield: 60%. mp 251–252 °C.
mmax(KBr): 1658, 1616,
1564, 1521, 1348, 1109, 763 cmꢁ1 1H NMR (CDCl3): 11.57(s, 1H),
.
Bacterial growth rate assay
11.38(s, 1H), 8.18–8.20(d, J = 8.8 Hz, 2H), 8.00–8.10(q, J = 7.6 Hz,
J = 7.6 Hz, 2H), 7.67–7.69(t, 2H), 7.40–7.44(t, 6H), 6.12(s, 1H). 13C
NMR (CD3Cl) d: 169.1, 167.0, 166.4, 164.9, 152.6, 152.3, 146.9,
143.4, 133.4, 127.6, 125.2, 125.2, 124.5, 124.5, 123.9, 116.8,
116.8, 116.7, 116.2, 104.8, 103.3, 36.5. HRMS (ESI+): m/z: calcd
for C25H15NO8: 480.0690 [M + Na+]; found: 480.0489.
The effect of NBH, MBH and CBH to the growth rate of S. aureus
and MRSA were determined as reported method [25], S. aureus and
MRSA were cultured in the 150
mated Bioscreen C system (Lab systems Helsinki, Finland), then
150 L coumarin derivatives solution were added to culture med-
ium at 32 or 128 g/mL in 35 °C, then the culture medium were
lL MH broth each well using auto-
l
3,30-[(4-Methoxyphenyl)methylene]bis(4-hydroxy-2H-chrome-
l
n-2-one) (MBH): Yield: 66%. mp 269–270 °C.
m
max(KBr): 1670,
shaken for 1 min. As the optical density of each sample, OD600
was obtained in regular intervals of 10 min for 20 h at a wave-
length of 600 nm to estimate the concentration of bacterial.
1604, 1560, 1510, 1350, 1256, 1093, 769 cmꢁ1
.
1H NMR (CDCl3):
11.51(s, 1H), 11.30(s, 1H), 7.99–8.08(q, J = 6.8 Hz, J = 6.8 Hz, 2H),
7.61–7.65(t, 2H), 7.40–7.42(d, J = 8.0 Hz, 4H), 7.12–7.14(d,
J = 8.4 Hz, 2H), 6.84–6.87(d, J = 8.8 Hz, 2H), 6.05(s, 1H), 3.80(s,
3H). 13C NMR (CD3Cl) d: 158.4, 132.8, 127.6, 126.9, 124.9, 124.4,
116.6, 114.0, 55.3, 35.5. HRMS (ESI+): m/z: calcd for C26H18O7:
465.0945 [M + Na+]; found: 465.0967.
Cytotoxicity assay
For NBH, MBH and CBH, the cytotoxicity with respect to the
human umbilical vein endothelial cells (HUVECs) were investi-
gated by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazoli
um bromide (MTT) assay. Firstly, HUVECs cells (5 ꢂ 103/well) were
grown for 24 h at 37 °C to confluence in 96-well plates in DMEM
media with 20% fetal bovine serum, then treated with NBH, MBH
or CBH at different concentrations (6.25, 12.5, 25, 50 and
3,30-[(4-Chloromethylphenyl)methylene]bis(4-hydroxy-2H-ch-
romen-2-one) (CBH): Yield: 63%. mp 254–255 °C.
mmax(KBr): 1666,
1616, 1567, 1354, 1095, 908, 767 cmꢁ1 1H NMR (CDCl3): 11.54(s,
.
1H), 11.32(s, 1H), 8.00–8.09(q, J = 6.4 Hz, J = 6.4 Hz, 2H), 7.63–
7.66(m, 2H), 7.35–7.44(m, 6H), 7.22–7.23(d, J = 6.0 Hz, 2H),
6.08(s, 1H), 4.59(s, 2H). 13C NMR (CD3Cl) d: 169.2, 166.9, 165.9,
164.6, 152.5, 152.3, 136.1, 135.7, 133.0, 128.9, 128.0, 126.9,
126.5, 125.0, 124.4, 116.9, 116.7, 116.4, 105.4, 103.8, 45.8, 36.3.
HRMS (ESI+): m/z: calcd for C26H17ClO6: 483.0606 [M + Na+];
found: 483.0601.
100
the cultured HUVECs and incubated for 4 h. Finally, the super-
natant was removed and 150 L DMSO was added to each well,
the absorbance was read at 490 nm.
lg/mL) for 24 h. After that, 0.5% MTT solution was added into
l

F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
119
OH
HO
OH
OH
HO
HO
O
O
O
O
O
O
O
O
O
O
O
O
NBH
MBH
CBH
NO2
OCH3
Fig. 1. Chemical structures of NBH, MBH and CBH.
CH2Cl
Table 1
Crystal data and structure refinement for NBH, MBH and CBH.
calculated parameters of the selected bond lengths and bond
angles are presented in Table 3.
Under three different basis sets, the calculated geometric
parameters of NBH, MBH, and CBH are very close and agree with
the experimental findings. The maximum deviation of the selected
bond lengths between theoretical and experimental data are (NBH)
0.01363, 0.01142, 0.01243; (MBH) 0.02248, 0.0203, 0.02151; and
(CBH) 0.03752, 0.03553, 0.03646, respectively. The maximal devi-
ation of the selected bond angles between theoretical and experi-
mental data are (NBH) 4.202, 4.876, 5.3; (MBH) 1.495, 2.194,
1.672; and (CBH) 2.511, 3.318, 3.663, respectively.
NBH
MBH
CBH
Formula
Mr
Crystal system
Space group
a/Å
b/Å
c/Å
C
25H15NO8
C26H18O7
442.40
Monoclinic
P21/c
9.9800(12)
10.4058(14)
20.042(2)
90
94.693(10)
90
C26H17ClO6
460.85
Monoclinic
P21/c
13.788 (4)
10.233 (3)
15.439 (4)
90
95.633(4)
90
457.38
Orthorhombic
Pna21
13.906(3)
14.159(4)
10.294(3)
90
90
90
2026.7(9)
4
1.499
a
/°
b/°
B3LYP/6-31G⁄ exhibited sufficient agreement with experimen-
tal data and lower computational cost, so further theoretical study
was performed at this level.
c
/°
V/Å3
2074.4(4)
4
1.417
2167.9(10)
4
1.412
Z
Dcalc/g cmꢁ3
l(Mo K
a
)/mmꢁ1
0.114
0.104
0.218
Hydrogen bonds energies in NBH, MBH, and CBH
h range/°
Reflections collected
No. unique data [R(int)]
2.05 to 27.90
20617
4769[0.0432]
4127
1.073
0.0380
0.0699
2.04 to 25.02
17150
3658[0.0591]
3151
1.132
0.0690
1.3 to 27.9
17272
3827[0.0317]
3250
1.117
0.0658
We only used NBH as an example to estimate single and total
HB energies. The global minimum structure is stabilized by two
HBs (NBH); two higher energy structures is stabilized by one HB
(NBH1 and NBH2) respectively. The corresponding values are listed
in Table 4. The O6AH6ꢃ ꢃ ꢃO1 HB energy was calculated to be
No. data with I P 2r(I)
Goodness-of-fit on F2
R1
xR2 (all data)
0.1978
0.1982
ꢁ50.85593 kJ/mol
by
the
equation
EðO6 ꢁ H6 ꢃ ꢃ ꢃ O1Þ ¼
coor
NBH
coor
ER
ꢁ E
, from the energy difference between NBH and
NBH1
NBH1, where NBH1 is
a global minimum structure with
Results
Crystal structure description
O3AH3ꢃ ꢃ ꢃO4 HB. Similarly, the O3AH3ꢃ ꢃ ꢃO4 HB energy was calcu-
lated to be ꢁ62.16659 kJ/mol from the energy difference between
NBH and NBH2 by the equation EðO3 ꢁ H3 ꢃ ꢃ ꢃ O4Þ ¼ ERcNoBoHr ꢁ Ecoor
,
NBH2
The molecular geometry and the atom labeling scheme of NBH,
MBH and CBH are presented in Fig. 2. Most of the bond distances
and bond angles are of the expected values, and in good accordance
with the corresponding values obtained in case of the related bis-
coumarin derivatives [26]. According to X-ray diffraction structural
determination under mild conditions, it can be seen that Michael
addition reaction took place between coumarin C (2 and 11) and
aldehyde C (10). As a result, a new compound was obtained.
In the crystal structures of NBH (MBH and CBH), the C10AC20
distance of 1.532 (1.552 and 1.535) Å is longer than an unstrained
C(sp3)AC(Ar) bond, which indicates significant electron delocaliza-
tion in the benzene ring system. In addition, two
4-hydroxycoumarin moieties are linked through a methylene
bridge, wherein one hydrogen atoms has been replaced with a phe-
nyl ring including p-nitro, p-methoxy and p-chloromethyl groups,
respectively. Two 4-hydroxycoumarin residues are arranged in a
position that permits the formation of two classical intramolecular
hydrogen bonds between a hydroxyl group of one coumarin frag-
ment and a lacton carbonyl group of another coumarin fragment
helping stabilize the whole structure. The corresponding values
of intramolecular hydrogen bonds are listed in Table 2.
in which NBH2 was obtained from the global minimum structure
NBH, but H3 was rotated around the C3AO3 bond until
O3AH3ꢃ ꢃ ꢃO4 HB rupture occurred [27,28]. We can see that
O3AH3ꢃ ꢃ ꢃO4 HB is stronger than O6AH6ꢃ ꢃ ꢃO1 HB, which is consis-
tent with the fact that the distance of O3AO4 (2.594 Å) is shorter
than that of O6AO1 (2.610ꢃ ꢃ ꢃÅ). The total HB energy was calculated
À
Á
coor
NBH
coor
NBH1
coor
NBH2
to be ꢁ113.02252 kJ/mol by the equation 2E
ꢁ E
ꢁ E
.
Similar to NBH, the O3AH3ꢃ ꢃ ꢃO4 HB energy for MBH and CBH is also
stronger than O6AH6ꢃ ꢃ ꢃO1 HB energy [d(O3AO4) < d(O1AO6)]. Their
total HB energies are ꢁ119.12156 and ꢁ115.9132 kJ molꢁ1
,
respectively.
Electron density and Laplacian in NBH, MBH and CBH
For NBH, MBH, and CBH, the electron density (qb) and the cor-
responding Laplacian (
2qb) at bond critical points are calculated,
which are presented in Table 5. From Table 5 we can see that both
2qb values, and values in
»
Oꢃ ꢃ ꢃH bondings have low qb and positive
»
the upper exocyclic ring are higher than those in the lower exo-
cyclic ring. The results indicate that the electrostatic character of
the Oꢃ ꢃ ꢃH bonding in the upper exocyclic ring is stronger than that
in the lower exocyclic ring [29], which is consistent with the fact
that the stronger HB strength in the upper exocyclic ring.
Quantum chemical calculations
Minimal inhibitory concentration (MIC) assay
Geometric parameters of NBH, MBH and CBH
For NBH, MBH and CBH, the fully optimized structures with
atomic numbering are shown in Fig. 3; experimental and
The antibacterial activities of NBH, MBH and CBH in vitro were
systematically studied by drug-sensitive S. aureus and

120
F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
Fig. 2. Crystal structure of the compound NBH (a), MBH (b) and CBH (c).
Table 2
values of reference antibiotics against S. aureus (ATCC 29213) were
Intramolecular hydrogen bond length (Å) and angles (°) for NBH, MBH and CBH.
lower less than 8
varying level.
lg/mL, but were higher against MRSA strains at
DAHꢃ ꢃ ꢃA
DAH/Å
Hꢃ ꢃ ꢃA/Å
Dꢃ ꢃ ꢃA/Å
\(DAHꢃ ꢃ ꢃA)/°
NBH
O6AH6ꢃ ꢃ ꢃO1
O3AH3ꢃ ꢃ ꢃO4
MBH
0.839
0.840
1.775
1.769
2.610 (17)
2.594 (16)
172.89
166.94
Bacterial growth inhibition
In order to explore the relationship between the activity and
time of NBH?MBH and CBH, we further investigated their growth
inhibitory and bactericidal effects by determine the growth rate
of S. aureus in liquid medium containing the three compounds.
As shown in Fig. 4, NBH significantly inhibited the growth of the
O6AH6ꢃ ꢃ ꢃO1
O3AH3ꢃ ꢃ ꢃO4
CBH
0.841
0.840
1.847
1.778
2.686 (8)
2.612 (7)
176.18
172.32
O6AH6ꢃ ꢃ ꢃO1
0.840
0.840
1.778
1.766
2.618 (3)
2.604 (3)
179.13
174.73
O3AH3ꢃ ꢃ ꢃO4
drug-sensitive S. aureus ATCC 29213 at 32
this pathogen completely at 128 g/mL. NBH also effectively inhib-
ited the growth of the three drug-resistant S aureus strains at
32 g/mL and inhibited the growth of these pathogens signifi-
cantly at 128 g/mL (Figs. 5–7). Similar to the results of the MIC
values, the other two compounds hardly showed any inhibitory
activities on the bacterials at 32 or 128 g/mL (Figs. 4–7). S. aureus
growth in MH broth without any compounds, which was used as
the control sample, could not inhibit the growth rate significantly.
The analysis of bacterial growth inhibition showed that aside
from exerting antibacterial activities on S. aureus, NBH also
lg/mL and inhibitied
l
methicillin-resistant S. aureus. Because of the liposolubility of the
three compounds, they were dissolved into the solution with 1%
Dimethyl sulfoxide (DMSO) at final concentration. As shown in
l
l
Table 6, the MIC values of NBH ranged from 16
lg/mL to
32 g/mL, exerting the best bactericidal activities against nearly
l
l
all kinds of S. aureus investigated. By contrast, MBH and CBH
exerted weaker bactericidal effects against S. aureus with their
MIC values exceed 64
lg/mL for drug-susceptive S. aureus and
the MRSA strains. Compared with NBH, MBH and CBH, the MIC

F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
121
H
3
O
O
3
4
R=
R=
R=
NO
2
NBH
MBH
CBH
R
H
C
C
O
C
C
3
19
OCH
3
O
1
12
CH Cl
2
O
O
1
6
H
6
Fig. 3. Schematic presentation of NBH, MBH and CBH.
Table 3
Experimental and calculated parameters of the bond lengths and bond angles of NBH, MBH and CBH.
Name definition
NBH
MBH
CBH
X-ray
6-31G⁄
6-31+G⁄⁄
6-311G⁄
X-ray
6-31G⁄
6-31+G⁄⁄
6-311G⁄
X-ray
6-31G⁄
6-31+G⁄⁄
6-311G⁄
R(C1AO1)
R(C1AO2)
R(C19AO4)
R(C19AO5)
1.227
1.23374
1.37083
1.23071
1.37362
1.52648
1.52504
1.53806
121.676
115.801
121.870
115.755
115.881
112.652
114.536
133.247
92.117
1.23636
1.36862
1.23383
1.37096
1.52639
1.52439
1.53812
121.707
115.988
121.861
115.928
116.083
112.832
114.625
132.465
92.345
1.22650
1.36963
1.22324
1.37292
1.52607
1.52411
1.53696
121.716
116.044
121.888
116.026
115.988
112.757
114.611
133.010
92.007
1.2338
1.3509
1.2249
1.3726
1.5179
1.5285
1.5518
120.600
115.487
120.956
114.538
115.056
113.740
113.297
135.723
91.234
131.739
94.384
1.23359
1.37338
1.22906
1.37754
1.52766
1.52590
1.53948
121.647
115.550
121.930
115.468
115.568
112.245
114.759
134.406
91.385
1.23628
1.37120
1.23215
1.37498
1.52743
1.52522
1.53995
121.672
115.750
121.928
115.632
115.702
112.456
114.912
133.529
91.660
1.22633
1.37241
1.22155
1.37715
1.52715
1.52495
1.53851
121.678
115.801
121.954
115.739
115.628
112.339
114.846
134.312
91.183
1.2285
1.3349
1.1975
1.3581
1.5088
1.5308
1.5348
120.433
116.283
122.889
117.461
114.532
112.852
114.678
134.707
91.632
134.178
92.231
1.23376
1.37242
1.22949
1.37585
1.52691
1.52534
1.53935
121.647
115.648
121.896
115.633
115.705
112.468
114.751
133.449
91.971
1.23635
1.37043
1.23250
1.37347
1.52684
1.52466
1.53959
121.680
115.826
121.894
115.787
115.847
112.617
114.912
132.837
92.020
1.22649
1.37136
1.22197
1.37541
1.52650
1.52433
1.53813
121.684
115.892
121.915
115.907
115.763
112.539
114.883
133.396
91.683
1.3572
1.2255
1.3628
1.5186
1.5212
1.5323
121.083
115.859
120.557
115.736
115.364
111.630
115.335
135.129
90.65
R(C2AC10
)
R(C10AC11
R(C10AC20
)
)
A(C1AO2AC9)
A(O1AC1AO2)
A(C18AO5AC19
A(O4AC19AO5)
A(C2AC10AC20
A(C2AC10AC11
A(C11AC10AC20
D(C1AC2AC10AC20
D(C1AC2AC10AC11
)
)
)
)
)
)
D(C12AC11AC10AC20
D(C12AC11AC10AC2)
)
136.073
89.692
131.871
92.858
131.197
92.934
130.773
93.600
131.245
94.157
130.501
94.314
130.067
95.055
131.667
93.300
130.860
93.560
130.515
94.149
Table 4
Single and total HB energies in NBH, MBH and CBH.
inhibited the growth rate of both antibiotic susceptive and resis-
tant bacterial strains.
System Total electronic
energiesa,b
E(O6AH6ꢃ ꢃ ꢃO1) E(O3AH3ꢃ ꢃ ꢃO4) E(total HB)c
In Vitro Toxicity measurement
NBH
ꢁ1617.827809
ꢁ1617.808439
ꢁ1617.804131
ꢁ1527.817795
ꢁ1527.797835
ꢁ1527.792384
ꢁ1912.220395
ꢁ1912.200641
ꢁ1912.196000
ꢁ113.02252
NBH1
NBH2
MBH
MBH1
MBH2
CBH
ꢁ50.85593
ꢁ62.16659
ꢁ119.12156
ꢁ52.40498
ꢁ66.71658
ꢁ115.9132
ꢁ51.86413
ꢁ64.04907
To further explore the safety for the possible development, we
investigated the cytotoxicity of NBH, MBH and CBH to cultured
HUVECs in vitro. Compared with the group, there was no signifi-
cant difference on cell viability in 200
treated group (P > 0.05), and the IC50 to the HUVECs was 796.24,
755.38 and 648.17 g/mL for NBH, MBH and CBH, respectively
lg/mL NBH, MBH or CBH
CBH1
CBH2
l
a
b
c
ZP corrected;
hartree;
kJ/mol.
(Fig. 8). These results implied that these compounds had much less
toxicity to mammalian cells, and had a relatively less toxicity for
potential clinical applications.
Table 5
Electron density (qb) and Laplacian (»2qb) in NBH, MBH and CBH.
Bond
NBH
MBH
CBH
qb
»
2qb
qb
»
2qb
qb
»
2qb
Upper exocyclic ring
O3AH3
C3AO3(AH3)
0.30352
ꢁ1.546053
ꢁ0.337381
ꢁ0.094182
0.154738
0.304622
0.315551
0.400664
0.047717
ꢁ1.562589
ꢁ0.335714
ꢁ0.076412
0.150724
0.303716
0.315882
0.400403
0.048612
ꢁ1.551914
ꢁ0.334815
ꢁ0.0823166
0.1531226
0.315425
0.399455
0.049313
C
19@O4
O4ꢃ ꢃ ꢃH3
Lower exocyclic ring
O6AH6
0.30639
0.31154
0.397246
0.0446953
ꢁ1.57103
ꢁ0.351326
ꢁ0.120685
0.138151
0.306789
0.310623
0.397213
0.044462
ꢁ1.575185
ꢁ0.356691
ꢁ0.116372
0.137486
0.306658
0.310841
0.397169
0.044526
ꢁ1.573283
ꢁ0.353791
ꢁ0.119333
0.137514
C
12AO6(AH6)
C1@O1
O1ꢃ ꢃ ꢃH6

122
F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
Table 6
MIC values of NBH, MBH, CBH and antibiotics.
Compounds/Drugs
MIC (lg/mL)
S. aureas
MRSA
MRSA 2
MRSA 3
NBH
MBH
CBH
Levofloxacin
Ceftazidime
Ceftriaxone
Gentamicin
Piperacillin
16–32
128–256
128–256
<0.125 (S)
8 (S)
4 (S)
0.5 (S)
4 (S)
16–32
64–28
16–32
16–32
>256
>256
64–128
128–256
8 (R)
256 (R)
256 (R)
32 (R)
128–256
8 (R)
>256 (R)
>256 (R)
64 (R)
8 (R)
64 (R)
32 (R)
0.5 (S)
8 (S)
>256 (R)
>256 (R)
S means drug susceptibility, R means drug resistance.
Discussion
To get ahead of the problem on resistance, we must look for
first-in-class antibacterials and new targets. Anti-bacterial activity
and drug resistance of bacteria have a close relationship with the
structure of antibiotics, hence the discovery of antibiotics with
novel structures and the development of drugs against MRSA are
highly active fields. The number of new antibacterials reaching clin-
ical practice has significantly reduced in the last 20 years. Linezolid
and daptomycin are the only two new antibiotics whose structures
are different compared with previous antibiotics, and daptomycin
bacterial resistance has not been solved since the development of
the drug in 2003 [30]. Biscoumarins are widely distributed in nat-
ure and exhibit a broad pharmacological profile. No antibiotic that
has a similar structure as biscoumarin derivatives is clinically being
used for infection treatment. In this work, three novel biscoumarin
derivatives were demonstrated to be capable of inhibit the growth
of MRSA using MICs and bacterial growth inhibitory rate measure-
ment. However, compared with NBH, MBH and CBH exerted almost
no effect on any strain of S. aureus.
Fig. 5. The time-kill curves with two different concentrations (A: 128
32 g/mL) of NBH, MBH and CBH on MRSA strain1.
lg/mL; B:
l
Our results also showed that there are two asymmetrical
intramolecular H-bonds in the three compounds, which is
Fig. 6. The time-kill curves with two different concentrations (A: 128
32 g/mL) of NBH, MBH and CBH on MRSA strain 2.
lg/mL; B:
l
considered as an important factor for the biological activity [31].
The molecular structures of the three compounds were also calcu-
lated by DFT method and the results were in agreement with the
experimental data. In addition, the total HB stabilization energies
in NBH, MBH, and CBH were estimated to be ꢁ113.02252,
ꢁ119.12156, and ꢁ115.9132 kJ/mol, respectively. These values
Fig. 4. The time-kill curves with two different concentrations (A: 128
32 g/mL) of NBH, MBH or CBH on S. aureus ATCC 29213.
lg/mL; B:
l

F. Li et al. / Journal of Molecular Structure 1097 (2015) 117–123
123
paper. These data can be obtained free of charge via http://www.
ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge
Crystallographic Data Center, 12, Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033).
Acknowledgments
This study was supported by National Natural Science
Foundation of China (No. 81473252), the Innovation plan of
science and technology of Shaanxi Province (2014KTCL03-03),
and Xi’an science and technology project (CXY1443WL07).
The authors thank the High Performance Computing Center of
Tianjin University and Prof. Xuehao He for the services provided.
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Supplementary material
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CCDC 872212, 888353 and 843711 of compounds NBH, MBH
and CBH contain the supplementary crystallographic data for this