Synthesis, crystal structures, and anti-drug-resistant Staphylococcus aureus activities of novel 4-hydroxycoumarin derivatives

Article abstract of DOI:10.1016/j.ejphar.2013.09.040

Four novel 4-hydroxycoumarin derivatives (4-MBH, 3-MBH, 4-MDT and 3-MDT) were successfully synthesized and their structures were verified by single-crystal X-ray crystallography. All target compounds were evaluated for their in vitro antibacterial activit

Full text of DOI:10.1016/j.ejphar.2013.09.040

European Journal of Pharmacology 721 (2013) 151–157
Contents lists available at ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar
Molecular and cellular pharmacology
Synthesis, crystal structures, and anti-drug-resistant Staphylococcus
aureus activities of novel 4-hydroxycoumarin derivatives
Ming-kai Li a,1, Jing Li b,1, Bao-hui Liu c, Ying Zhou a, Xia Li d, Xiao-yan Xue a,
a,
n
Zheng Hou a, , Xiao-xing Luo
n
a Department of Pharmacology, School of Pharmacy, The Fourth Military Medical University, Xi′an, China
b School of Chemistry and Chemical Engineering, Xi′an University of Arts and Sciences, Xi′an, China
c Department of Cardiac Surgery, Xijing Hospital, The Fourth Military Medical University, Xi′an, China
d Department of Neurosurgery, Xijing Hospital, The Fourth Military Medical University, Xi′an, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel 4-hydroxycoumarin derivatives (4-MBH, 3-MBH, 4-MDT and 3-MDT) were successfully
synthesized and their structures were verified by single-crystal X-ray crystallography. All target compounds
were evaluated for their in vitro antibacterial activity 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). The minimum inhibitory concentration and time–kill curves
were obtained for the test compounds and antibiotics. Among the tested compounds, 3-MBH showed the
most potent antibacterial activities.
Received 13 May 2013
Received in revised form
6 September 2013
Accepted 19 September 2013
Available online 25 September 2013
Keywords:
4-Hydroxycoumarin
Single crystal
& 2013 Elsevier B.V. All rights reserved.
Staphylococcus aureus
Minimum inhibitory concentration
1. Introduction
losing its effectiveness against MRSA and the prevention of MRSA
infections at the surgical site (Haill et al., 2013). Thus, S. aureus
Staphylococcus aureus, an important pathogen widely presents in
the natural environment, can cause a variety of human tissue and
organ infections (Dukic et al., 2013). Methicillin-resistant S. aureus
(MRSA) emerged in the 1960s and has been widely disseminated
since then. MRSA is the major source of nosocomial infections
worldwide, causing Z50% of the hospital-acquired S. aureus infec-
tions in several countries (Sandora and Goldmann, 2012; Kinnevey
et al., 2013; Blomfeldt et al., 2013; Babakir-Mina et al., 2012). The
prevalence rates of MRSA in hospitals in some Asian countries, such
as Taiwan, China, Japan, and South Korea, range from 70% to 80%
(Song et al., 2011). Although the predominant clinical manifestations
of the pathogen are skin and soft tissue infections (SSTIs), severe life-
threatening infections, such as necrotizing fasciitis, necrotizing pneu-
monia, and severe sepsis, have been reported (Miller et al., 2005). In
the 48 contiguous states of the USA, community-associated MRSA
skin and soft tissue infections are predominantly caused by the MRSA
strain USA 300 (Amini and Salzman, 2013; Moran et al., 2006).
Vancomycin, a representative of the glycopeptide class of clini-
cal antibiotics for serious Gram-positive bacterial infections, is
widely used to treat MRSA infection. However, vancomycin is
continues to challenge surgeons as an adaptable pathogen that can
defy all treatment efforts (Pitz et al., 2011; Koyama et al., 2012). To
prevent the spread of both methicillin-sensitive S. aureus and
MRSA, antibiotics that can effectively eradicate this pathogen need
to be developed urgently.
4-Hydroxycoumarin is an important component of numerous
synthetic and natural products with wide-ranging biological activities,
including anticoagulant, insecticidal, anthelminthic, hypnotic, anti-
fungal, phytoalexin, and HIV protease inhibition (Jung and Oh, 2011;
Su et al., 2006). These special properties of 4-hydroxycoumarin have
stimulated considerable interest in this class of compounds, and
various biscoumarins and epoxydicoumarins have been synthesized.
Epoxydicoumarins are a derivative of biscoumarins resulting from the
removal of a water molecule. Epoxydicoumarins and biscoumarins
possess versatile activities through chemical modifications (different
substituents on the aromatic ring). Single-crystal X-ray diffraction,
which can reveal molecular conformation, intramolecular and inter-
molecular interactions in the solid state, is among the most versatile
techniques used to study coumarins (Khan et al., 2004; Hamdi et al.,
2008).
A number of coumarin derivatives (novobiocin and analogs) have
proven to be highly active antibiotics. Among synthetic coumarin
derivatives, several antibacterial 4-hydroxycoumarins have been
described (Lin et al., 2012). However, the effects of biscoumarins
and epoxydicoumarins on bacteria, especially drug-resistant S. aureus,
n
Corresponding authors. Tel./fax: þ86 29 84774591.
E-mail addresses: hzh_0001@163.com (Z. Hou),
xxluo3@fmmu.edu.cn (X.-x. Luo).
1
These authors contributed equally to this work.
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejphar.2013.09.040

152
M.-k. Li et al. / European Journal of Pharmacology 721 (2013) 151–157
7.099–7.145 (q, 4H), 6.076 (s, 1H), 2.342 (s, 3H). HRMS (ESIþ): m/z:
calcd for C26H18O6: 449.0996 [MþNaþ]; found: 449.0941.
3,3′-(3-Methylbenzylidene)-bis-(4-hydroxycoumarin) (3-MBH):
m.p. 237–238 1C. IR (KBr pellet cmꢀ1): 1674, 1604, 1560, 1348,
1101, 763 cmꢀ1 1H NMR (CDCl3, δ, ppm): 11.528 (s, 1H), 11.285
.
(s, 1H), 8.009–8.088 (q, 2H), 7.623–7.654 (t, 2H), 7.415–7.432 (d, 4H),
7.206–7.236 (t, 1H), 7.082–7.097 (d, 1H), 7.012–7.042 (t, 2H), 6.080
(s, 1H), 2.312 (s, 3H). HRMS (ESIþ): m/z: calcd for C26H18O6:
449.0996 [MþNaþ]; found: 449.0985.
The compounds 4-MBH and 3-MBH were dissolved by heating
in anhydride acetic. The reaction mixture was heated to reflux
under magnetic stirring for 4 h. Then, the solution was cooled to
room temperature, and the separated white solid was filtered off.
The solid was subsequently recrystallized from ethanol to obtain
4-MDT and 3-MDT.
9-(4-Methylphenyl)-1,8-dioxo-9H-dibenzo[c,h]-2,7,10-trioxanthene
(4-MDT): m.p. 319–320 1C. IR (KBr pellet cmꢀ1): 1735, 1668, 1606,
1456, 1365, 1182, 1058, 887, 763 cmꢀ1 1H NMR (DMSO-d6, δ, ppm):
.
8.394–8.417 (q, 2H), 7.750–7.793 (m, 2H), 7.506–7.573 (m, 4H), 7.253–
7.273 (d, 2H), 7.066–7.086 (d, 2H), 4.848 (s, 1H), 2.208–2.223 (d, 3H).
HRMS (ESIþ): m/z: calcd for C26H16O5: 431.0890 [MþNaþ]; found:
431.0838.
Fig. 1. Chemical structures of 4-MBH, 3-MBH, 4-MDT and 3-MDT.
remain unclear. In this work, a new series of biscoumarins and
epoxydicoumarins (Fig. 1) were synthesized and their corresponding
crystal structures were successfully obtained. Furthermore, the anti-
bacterial properties of the compounds were also investigated.
A possible relationship between the spatial structure and antibacterial
function of these kinds of compound was then proposed.
9-(3-Methylphenyl)-1,8-dioxo-9H-dibenzo[c,h]-2,7,10-triox-
anthene (3-MDT): m.p. 333–334 1C. IR (KBr pellet cmꢀ1): 1735,
1669, 1608, 1456, 1365, 1178, 1058, 887, 759 cmꢀ1. 1H NMR (CDCl3,
δ, ppm): 8.088–8.106 (d, 2H), 7.621–7.657 (t, 2H), 7.441–7.478
(t, 2H), 7.370–7.390 (d, 2H), 7.218–7.238 (d, 2H), 7.140–7.178 (t, 1H),
7.005–7.023 (d, 1H), 5.113 (s, 1H), 2.287 (s, 3H). HRMS (ESIþ): m/z:
calcd for C26H16O5: 431.0890 [MþNaþ]; found: 431.0899.
2. Materials and methods
2.3. X-ray crystallography
2.1. Chemicals and instruments
Single crystals of 4-MBH, 3-MBH, 4-MDT and 3-MDT for X-ray
diffraction experiments were grown from methanol. The X-ray
diffraction data were collected on a Bruker SMART APEX II CCD diffra-
ctometer equipped with a graphite monochromated Mo Kα radiation
(λ¼0.71073 Å) using ω–2θ scan technique at room temperature. The
structure was solved by direct methods with SHELXS-97 and refined
using the full-matrix leastsquares method on F2 with anisotropic
thermal parameters for all non-hydrogen atoms using SHELXL-97
(Sheldrick, 1997). Hydrogen atoms were generated geometrically. The
crystal data, details concerning data collection and structure refine-
ment are given in Table 1. Molecular illustrations were prepared using
the XP package. Parameters in CIF format are available as Electronic
Supplementary Publication from Cambridge Crystallographic Data
Center.
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 analytical grade.
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. Mu50 (ATCC 700699) and
USA 300 (LAC) were purchased from MicroBiologics (MN, USA).
IR spectra (400–4000 cmꢀ1) were measured on a Brucker
Equinox-55 spectrophotometer. 1H NMR spectra were obtained using
a Varian Inova-400 spectrometer (at 400 MHz). Mass spectra were
recorded on a micrOTOF-Q II mass spectrometer. Melting points
were taken on a XT-4 micro melting apparatus; the thermometer
was uncorrected.
2.4. Bacterial susceptibility assays
2.2. Synthesis and characterization of 4-MBH, 3-MBH, 4-MDT
and 3-MDT
According to the CLSI broth microdilution method, the mini-
mum inhibitory concentrations (MICs) were determined by micro-
dilution assay 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 culture medium containing the test compound
(0.12–256 μg/mL in serial two-fold dilutions). The plates were
incubated at 37 1C for 20 h in an incubator. About 50 mL of 0.2%
triphenyl tetrazolium chloride (TTC), a colorimetric indicator, was
added to each well of microter plates and incubated at 35 1C for
1.5 h. The TTC-based MIC was determined as the lowest concen-
tration of oxacillin that showed no red color change indicating
complete growth inhibition.
4-MBH and 3-MBH were synthesized according to a previous
report (Kontogiorgis and Hadjipavlou-Litina, 2005; Kidwai et al.,
2007). A mixture of 4-methylbenzaldehyde (or 3-methylbenzalde-
hyde) (10 mmol) and 4-hydroxycoumarin (20 mmol) was dissolved in
100 mL of EtOH. A few drops of piperidine were added, and the mix-
ture was stirred for 4 h at room temperature. After reaction comple-
tion 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 under a
vacuum.
3,3′-(4-Methylbenzylidene)-bis-(4-hydroxycoumarin)
(4-MBH):
m.p. 291–292 1C. IR (KBr pellet cmꢀ1): 1670, 1618, 1564, 1352, 1095,
906, 763 cmꢀ1. 1H NMR (CDCl3, δ, ppm): 11.521 (s, 1H), 11.296 (s, 1H),
8.000–8.082 (q, 2H), 7.615–7.649 (m, 2H), 7.408–7.425 (d, 4H),
To obtain time–kill curves for methicillin-susceptible S. aureus
and MRSA, the synthetic compounds and antibiotics were added to

M.-k. Li et al. / European Journal of Pharmacology 721 (2013) 151–157
153
Table 1
Crystal data, data collection and structure refinement.
Parameter
4-MBH
3-MBH
4-MDT
3-MDT
Formula
Mr
C26H18O6 ꢂ CHCl3
545.77
C26H18O6
426.4
C26H16O5
408.39
C26H16O5
408.39
Temperature (K)
113(2)
113(2)
113(2)
294(2)
Crystal size (mm)
0.20 ꢁ 0.20 ꢁ 0.16
Monoclinic
P21/c
13.1846(9)
10.5242(8)
18.6990(12)
90
110.4520(10)
90
2431.1(3)
4
0.20 ꢁ 0.10 ꢁ 0.01
Monoclinic
P21/c
9.881(4)
10.094(4)
20.535(7)
90
97.553(7)
90
2030.4(13)
4
0.18 ꢁ 0.16 ꢁ 0.14
0.22 ꢁ 0.18 ꢁ 0.16
Monoclinic
P21/c
11.716(2)
11.775(2)
14.833(3)
90
107.51(3)
90
1951.5(7)
4
Crystal system
Space group
a (Å)
b (Å)
c (Å)
Triclinic
̄
P1
8.5390(10)
10.4500(15)
11.3367(18)
75.75(4)
73.44(2)
84.73(4)
939.6(2)
2
α (1)
β (1)
γ (1)
V (Å3)
Z
Dcalc (g cmꢀ3
)
1.491
1.395
1.444
1.39
0.42
1.65–27.87
24385
0.1
2.00–27.90
20751
0.823
4.18–72.70
10627
0.097
2.51–26.00
17224
μ(Mo Kα) (mmꢀ1
θ range (1)
)
Reflections collected
No. unique data [R(int)]
No. data with IZ2s(I)
R1
5763 [0.0391]
4037
0.0596
4847 [0.0425]
3851
0.0486
3517 [0.0677]
2699
0.0714
3842 [0.0519]
2633
0.0597
0.1755
0.1291
0.1466
0.155
ωR2 (all data)
CCDC
872211
843712
874730
895385
strain cultures to a final concentration of 128 μg/mL or 32 μg/mL.
The strains were cultivated in the automated Bioscreen C system
(Lab systems Helsinki, Finland) using MH broth culture medium.
The working volume in the wells of the Bioscreen plate was 300 mL,
comprising 150 mL of MH broth and 150 mL of drug solution. The
temperature was controlled at 35 1C, and the optical density of the
cell suspensions was measured automatically at 600 nm in regular
intervals of 10 min for 20 h. Before each measurement, the culture
wells were automatically shaken for 60 s. Statistical data for each
experiment were obtained from at least two independent assays
performed in duplicate.
essentially planar because the maximum deviation of the atoms in
the skeleton from the molecular plane (C1–O1/C19) was only
0.2970 Å in 4-MDT and 0.3464 Å in 3-MDT. This phenomenon
resulted in the formation of a large, essentially planar heterocyclic
ring system with a 2.716(46)1 dihedral angle between rings B and
A (C8/C9/C1/C2/C7/O2) as well as a 3.822(44)1 dihedral angle
between rings B and C (C12/C11/C19/C18/C13/O5) in 4-MDT. The
corresponding values in 3-MDT are 6.211(61)1 and 3.295(59)1. The
dihedral angles between the mean planes of the heterocyclic ring
system and the 4-methylphenyl and 3-methylphenyl rings were
86.379(51)1 and 84.686(65)1. In the two whole stabilized struc-
tures, the 4-methylphenyl and 3-methylphenyl groups “stand” on
the plane of the heterocyclic ring system using a non-classical
intramolecular hydrogen bond C25–H25⋯Cg. The former H-bond
parameters are C25–H25: 0.95 Å, H25⋯Cg: 2.71 Å, and C25⋯Cg:
3.0785(17) Å, and the angle is 1041. The respective latter H-bond
parameters are 0.93 Å, 2.65 Å, 3.033(2) Å, and 1061 (Cg refers to
the center of gravity of ring B).
3. Results
3.1. Crystal structure description
The crystal structures of 4-MBH, 3-MBH, 4-MDT, and 3-MDT
are presented in Fig. 2. Compounds 4-MBH and 3-MBH have
similar structural features to those of dicoumarols. From the
diagram of the asymmetric unit including the atomic numbering
scheme of 4-MBH, we can see a chloroform solvent molecule
incorporated in the asymmetric unit. In the crystal structure of
3-MBH, two crystallographically independent molecules are pre-
sent in the asymmetric unit. Two 4-hydroxycoumarin fragments
are linked by a methylene bridge, wherein one hydrogen atom is
replaced with an m-methylphenyl residue with identical confor-
mations between the two molecules. However, these fragments
differ with respect to the reversed twist directions of the m-
methylphenyl ring. In each compound, two classical asymmetrical
intramolecular O–H⋯O hydrogen bonds [d(O1–O6)¼2.677(3) Å,
d(O3–O4)¼2.631(3) Å for 4-MBH; d(O1–O6)¼2.6729(15) Å, d(O3–
O4)¼2.6119(14) Å for 3-MBH] between a hydroxyl group of a
coumarin fragment and a lacton carbonyl group of another
coumarin fragment were used to stabilize the whole structure.
Compounds 4-MDT and 3-MDT were obtained from two
hydroxyl groups forming an ether bond by the removal of the
water molecule from the corresponding compounds 4-MBH and 3-
MBH. The newly formed ether ring B (C1/C9/C10/C11/C19/O1) was
3.2. MIC
3-MBH exerted potent bactericidal effects against almost all
S. aureus tested including USA 300 (LAC), a highly virulent and
widespread clinical isolate responsible for the recent epidemic of
MRSA infections; most MIC values were around 64 μg/mL and
32 μg/mL (Table 2). By contrast, 4-MBH, 4-MDT and 3-MDT exerted
no effect against S. aureus, with MIC values of more than 256
μg/mL for S. aureus (ATCC 29213) and the other three MRSA
strains. The MIC values for levofloxacin, ceftazidime, ceftriaxone,
gentamicin, vancomycin, and piperacillin against S. aureus (ATCC
29213) strains were o8 μg/mL, but were much higher against the
resistant strains of varying degrees (Table 2).
3.3. Time–kill curves
The MICs showed 3-MBH had the most potent and broad-
spectrum antibacterial activity on S. aureus. Thus, further investi-
gations on the growth inhibitory and bactericidal effects of 3-MBH

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M.-k. Li et al. / European Journal of Pharmacology 721 (2013) 151–157
Fig. 2. Crystal structures of 4-MBH (a), 3-MBH (b), 4-MDT (c) and 3-MDT (d).
were conduct. To evaluate the growth inhibitory effects on S.
aureus ATCC 29213 (Fig. 3), MRSA XJ 75302 (Fig. 4), Mu50 (Fig. 5)
and MRSA USA 300 LAC (Fig. 6), 4-MBH, 3-MBH, 4-MDT and 3-
MDT were added to cultures at 128 μg/mL or 32 μg/mL,
respectively. As shown in Figs. 3–6, 3-MBH inhibited the growth of
these pathogens and exhibited almost completely growth inhibi-
tion on these pathogens at 128 μg/mL or 32 μg/mL. This finding is
consistent with the result of MIC, 4-MBH, 4-MDT and 3-MDT had

M.-k. Li et al. / European Journal of Pharmacology 721 (2013) 151–157
155
Table 2
MIC of 4-MBH、3-MBH、4-MDT、3-MDT and antibiotics in Mueller–Hinton broth
culture.
Drugs
MIC (μg/mL)
S. aureas
MRSA
Mu50
LAC
(ATCC 29213)
(XJ 75302)
(ATCC 700699)
(USA 300)
3-MBH
64
64
32
32
3-MDT
4-MBH
4-MDT
Levofloxacin
Ceftazidime
Ceftriaxone
Gentamicin
Vancomycin
Piperacillin
4256
4256
4256
o0.125 (S)
8 (S)
4256
4256
4256
4 (R)
4256 (R)
4256 (R)
64 (R)
128 (R)
4128 (R)
4256
4256
4256
4 (R)
256 (R)
256 (R)
32 (R)
8(I)
4256
4256
4256
8 (R)
64 (R)
32 (R)
0.25 (S)
0.5 (S)
8 (R)
2 (S)
0.12 (S)
0.5 (S)
2 (S)
4128 (R)
S means drug susceptibility, R means drug resistance, and I means intermediate
resistance. Levofloxacin, ceftazidime, ceftriaxone, gentamicin, vancomycin, and
piperacillin as control antibiotics exert antibacterial effects on the drug-
susceptible S. aureas strain (ATCC 29213). MRSA (XJ 75302) and Mu50 (ATCC
700699) are resistant to all of the control antibiotics, whereas USA 300
is susceptible to vancomycin and gentamicin but resistant to the other control
antibiotics. Compared with 3-MDT, 4-MBH, and 4-MDT, 3-MBH has more potent
effects on the bacterials listed above.
Fig. 4. Concentration-dependent inhibition of compounds on the growth of MRSA
XJ 75302. The cells were cultured in liquid culture medium and treated with
different concentrations of 4-MBH, 3-MBH, 4-MDT and 3-MDT. The growth curves
for MRSA XJ 75302 were measured using a Bioscreen C™ instrument in the absence
(blue line) and presence of 3-MBH (black line), 3-MDT (green line), 4-MBH (red
line), and 4-MDT (purple line). The sample frequency was 10 min. The responses to
128 μg/mL compound (A) and 32 μg/mL compound (B) treatments of MRSA XJ
75302 were investigated. (For interpretation of the references to color in this figure
caption, the reader is referred to the web version of this paper.)
4. Discussion
As a result of the emergence, spread, and rapid evolution of
resistance genes developing among pathogens, MRSA is the cause
of major outbreaks and epidemics among hospitalized patients,
with high mortality and morbidity rates. Recently, some schemes
have led to both a dramatic rise in the total numbers of cases of S.
aureus bacteremia reported annually and an increase in the
proportion of such cases that involve MRSA (from 2% in 1990 to
440% in the early 2000s) (Murray et al., 2008; Brennan et al.,
2013). However, the emergence of vancomycin-resistant S. aureus
and treatment failure of MRSA infections urgently require devel-
oping new antimicrobials.
In this work, 4-hydroxycoumarin derivatives were demon-
strated as able to inhibit the growth of multi-drug resistant S.
aureus significantly. The MIC values showed the efficacy of 3-MBH
at around 32 μg/mL or 64 μg/mL to S. aureus (ATCC 29213), MRSA
(XJ 75302), Mu50, and USA 300. To prove the anti-MRSA activity of
3-MBH, the growth inhibition rate of bacterial using time–kill
curves was evaluated. The concentration-dependent inhibition of
3-MBH on the growth of S. aureus (ATCC 29213), MRSA (XJ 75302),
Mu50, and USA 300 was consistent with the MIC results, in which
128 or 32 μg/mL 3-MBH significantly inhibited the growth of
MRSA. However, 4-MBH, 4-MDT and 3-MDT have no effect on
any S. aureus in the MIC or growth inhibition rate experiments.
X-ray structural analysis showed that the biscoumarins 4-MBH and
3-MBH have two classical intramolecular O–H⋯O hydrogen bonds in
their structures whereas the epoxydicoumarins 4-MDT and 3-MDT
have non-classical intramolecular hydrogen bonds in their structures.
The formation of intramolecular hydrogen bonds is considered as an
Fig. 3. Concentration-dependent inhibition of compounds on the growth of S.
aureus ATCC 29213. The cells were cultured in liquid culture medium and treated
with different concentrations of 4-MBH, 3-MBH, 4-MDT and 3-MDT. The growth
curves for S. aureus ATCC 29213 were measured using a Bioscreen C™ instrument
in the absence (blue line) and presence (black line) of 3-MBH, 3-MDT (green line),
4-MBH (red line), and 4-MDT (purple line). The sample frequency was 10 min. The
responses to 128 μg/mL compound (A) and 32 μg/mL compound (B) treatments of S.
aureus ATCC 29213 were investigated. (For interpretation of the references to color
in this figure caption, the reader is referred to the web version of this paper.)
no inhibitory effects on these pathogens at 128 μg/mL or 32 μg/mL.
S. aureus growth in MH broth without any compound as the
control did not show any significant growth inhibitory effect.

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M.-k. Li et al. / European Journal of Pharmacology 721 (2013) 151–157
Fig. 5. Concentration-dependent inhibition of compounds on the growth of Mu50
ATCC 700699. The cells were cultured in liquid culture medium and treated with
different concentrations of 4-MBH, 3-MBH, 4-MDT and 3-MDT. The growth curves
for Mu50 ATCC 700699 were measured using a Bioscreen C™ instrument in the
absence (blue line) and presence of 3-MBH (black line), 3-MDT (green line), 4-MBH
(red line), and 4-MDT (purple line). The sample frequency was 10 min. The
responses to 128 μg/mL compound (A) and 32 μg/mL compound (B) treatments of
Mu50 ATCC 700699 were investigated. (For interpretation of the references to color
in this figure caption, the reader is referred to the web version of this paper.)
Fig. 6. Concentration-dependent inhibition of compounds on the growth of USA
300 LAC. The cells were cultured in liquid culture medium and treated with
different concentrations of 4-MBH, 3-MBH, 4-MDT and 3-MDT. The growth curves
for USA 300 LAC were measured using a Bioscreen C™ instrument in the absence
(blue line) and presence (black line) of 3-MBH, 3-MDT (green line), 4-MBH (red
line), and 4-MDT (purple line). The sample frequency was 10 min. The responses to
128 μg/mL compound (A) and 32 μg/mL compound (B) treatments of USA 300 LAC
were investigated. (For interpretation of the references to color in this figure
caption, the reader is referred to the web version of this paper.)
important factor in assisting the molecule to attain the correct
configuration for biological activity. In biological systems, strong
intramolecular hydrogen bonds stabilize and hold the compounds in
a suitable configuration for binding to an enzyme. Weak, non-classical
intramolecular hydrogen bonds in the structures of 4-MDT and 3-MDT
caused their weaker structural stability compared with those of 4-
MBH and 3-MBH. Thus, 4-MDT and 3-MDT have weaker antibacterial
activity than 4-MBH and 3-MBH. The antibacterial activity of 4-MBH
has almost no effect compared with 3-MBH, although both have the
same substituent groups. Differences in antibacterial effects may be
due to different positions (para and meta) of these substituent groups.
Since the antimicrobial activity of some antimicrobial agents is
related to the rate of bacterial growth, this may be a major
limitation of in vitro models. As an example, β-lactams are known
to display bactericidal activity that is dependent on the rate of
growth. Thus, this class of antibacterial agents may be expected to
be more effective in vitro than in vivo. The present study shows
that the novel biscoumarin derivative 3-MBH has potent antibacter-
ial effects using the two different but equally important in vitro
methods of MIC determination and time–kill curve analysis. Future
applications of the present work are expected to ultimately provide
insights into the development of treatments for antibiotic-resistant
infectious diseases.
appropriate antibiotics have to be developed to treat infected
patients. Our results confirmed that the novel biscoumarin deri-
vative 3-MBH has excellent antibacterial efficiency. However,
additional studies that aim to define the mechanism underlying
the antibacterial activity of the derivative and evaluate correla-
tions with its drug efficacy in vivo are necessary.
Acknowledgments
This work was supported by grants from the National Natural
Science foundation of China (no. 81001460 and no. 81273555).
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