Novel fluorescent cephalosporins: Synthesis, antimicrobial activity and photodynamic inactivation of antibiotic resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2012.11.019

Two novel fluorescent cephalosporins, TCA and TBCA, were synthesized and characterized by ¹H NMR, ¹³C NMR, UV-vis, and fluorescence spectroscopies. Biological activity assays demonstrated that TCA inactivated a Klebsiella pneumonia strain that expressed extended-spectrum β-lactamases. Incubation of 6 μM TCA with K. pneumonia cultures resulted in cell death for 84% of the cells after 126 J/cm² of light irradiation. In vitro, TCA exhibited a MIC = 0.5 μg/mL with Staphylococcus aureus. Kinetic evaluation revealed that TCA and TBCA were substrates for B1 and B3 subclass metallo-β-lactamases. TBCA exhibited stronger binding affinities to the Gram-positive bacterial strains MRSA1, MRSA2, and S. aureus with value of 2.95-6.59 μM per 10⁸ cells/mL.

Full text of DOI:10.1016/j.ejmech.2012.11.019

European Journal of Medicinal Chemistry 59 (2013) 150e159
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Novel fluorescent cephalosporins: Synthesis, antimicrobial activity and
photodynamic inactivation of antibiotic resistant bacteria
*
Jian-Min Xiao, Lei Feng, Li-Sheng Zhou, Hui-Zhou Gao, Yi-Lin Zhang, Ke-Wu Yang
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710069, PR
China
a r t i c l e i n f o
a b s t r a c t
Two novel fluorescent cephalosporins, TCA and TBCA, were synthesized and characterized by 1H NMR,
13C NMR, UVevis, and fluorescence spectroscopies. Biological activity assays demonstrated that TCA
Article history:
Received 7 August 2012
Received in revised form
24 October 2012
Accepted 12 November 2012
Available online 21 November 2012
inactivated a Klebsiella pneumonia strain that expressed extended-spectrum
M TCA with K. pneumonia cultures resulted in cell death for 84% of the cells after 126 J/cm2 of light
irradiation. In vitro, TCA exhibited a MIC ¼ 0.5 g/mL with Staphylococcus aureus. Kinetic evaluation
revealed that TCA and TBCA were substrates for B1 and B3 subclass metallo- -lactamases. TBCA
exhibited stronger binding affinities to the Gram-positive bacterial strains MRSA1, MRSA2, and S. aureus
with value of 2.95e6.59
M per 108 cells/mL.
b-lactamases. Incubation of
6
m
m
b
Keywords:
m
Fluorescent cephalosporins
Antimicrobial activity
Photoinactivation
Substrate
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
There are no antibiotics available for the successful treatment of
the antibiotic resistant bacterial infections caused by Klebsiella
The
b
-lactam antibiotics, such as penicillins, cephalosporins and
pneumonia that produce extended-spectrum b-lactamases (ESBLs) or
carbapenems, have been developed as a major class of antimicro-
bial agents to treat bacterial infection in clinics. However, the
overuse of these antibiotics has resulted in a large number of
methicillin-resistant Staphylococcus aureus (MRSA). However,
photodynamic therapy (PDT) may be a promising option. PDT is
a photochemistry-based technology that relies on the wavelength-
specific light activation of certain nontoxic photosensitizers (PSs) to
produce active molecular species that are toxic to targeted biomole-
cules/compounds [7,8]. PSs have been conjugated to antibodies or
peptides for the treatment of bacteria infections, but the inter- or
intracellular targets are large, and labeling of antibodies or peptides
with PSs is experimentally challenging [9]. Therefore, developing the
specific compounds with PSs is very necessary. Xing and co-workers
bacteria with
metallo- -lactamase 1 (NDM-1) becoming resistant to the most
commonly used antibiotics [1e4]. The most common way that
bacteria become resistant to antibiotics is by the production of
lactamases, which hydrolyze the -lactam ring of -lactam-con-
taining antibiotics. The -lactamases have been categorized into
four classes: A, B, C, and D [5]. The B class enzymes, called the
metallo- -lactamase (M Ls), are Zn(II)-dependent and hydrolyze
nearly all known -lactam-containing antibiotics. There are no
known clinical inhibitors of the M Ls [6]. The M Ls have been
further categorized into the B1, B2, and B3 subclasses [5]. M Ls
belonging to the B1 and B3 subclasses hydrolyze all common
b-lactamases including recently reported New Delhi
b
b
-
b
b
b
b
b
reported a simple and specific conjugation of vancomycine
b
porphyrins to use for fluorescent imaging and antibacterial studies
on VRE [2]. Recently, we reported a novel norvancomycin-fluorescein
for photoinactivation of Bacillus subtilis [10].
In this paper, we constructed unique fluorescent cephalospo-
rins, which we hypothesized could be used to block bacterial
growth by generating reactive oxygen species (ROS) [11]. Clinically
important strains, including Gram-negative K. pneumonia
(ATCC 700603) and Gram-positive MRSA1, MRSA2, and S. aureus
(ATCC 25923), were used in studies with these fluorescent
cephalosporins.
b
b
b
b
-
lactam-containing antibiotics, including penicillins, cephalospo-
rins, and carbapenems. In contrast, the B2 subclass enzymes pref-
erentially hydrolyze carbapenems.
* Corresponding author. Tel./fax: þ86 29 8830 2429.
E-mail address: kwyang@nwu.edu.cn (K.-W. Yang).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.019

J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
151
2. Result and discussion
Cephalosporins, TCA and TBCA, were characterized using UVe
vis spectroscopy. The UVevis spectra (Fig. 1) showed that TCA
absorbs more strongly than 3a. TCA exhibited two absorption peaks
centered at 280 and 336 nm, and the maximum absorption peak of
3a was red-shifted 13 nm in comparison to that of TCA. TBCA dis-
played weaker multiple absorption features, and the maximum
absorption peak at 382 nm was red-shifted by 11 nm in comparison
to 3b.
The fluorescence quantum yields (FF) of TCA and TBCA were
determined. The data listed in Table 1 indicate that TCA and TBCA
have lower FF values (0.0521 and 0.0544) than 3a and 3b (0.0848
and 0.2137), demonstrating that the fluorescence of fluorescent
groups in TCA and TBCA has been partially quenched. Fluorescence
emission spectra of TCA and TBCA are shown in Fig. 2. The fluo-
rescence intensity of TCA is reduced 2.5-fold when compared to 3a,
while that of TBCA is reduced 9-fold as compared to 3b; the greater
fluorescence of 3b is likely due to the three rigid rings in 3b. The
fluorescence spectra of TCA and TBCA hydrolysis by CcrA and L1 are
shown in Fig. 3. Clearly, after the fluorescent cephalosporins were
2.1. Synthesis and spectral characterization of fluorescent
cephalosporins
The protected cephalosporin, 7-amino-3-chloromethyl-3-
cephem-4-carboxylic acid p-methoxybenzyl ester (ACLE), was
chosen as the scaffold to which we attached the PS (Scheme 1). ACLE
was modified with 2-thienylacetyl chloride and amino thiophenol to
yield compound 5. The luminophores, coumarin-3-carboxylic acid
(3a) and benzocoumarin-3-carboxylic acid (3b), were linked to
the primary amino group of modified ACLE to yield 6. After removing
the protection group (4-methoxybenzyl) using phenol, the target
compounds 8-oxo-3-((4-(2-oxo-2H-chromene-3-carboxamido)phe-
nylthio)methyl)-7-(2-(thiophen-2-yl)acetamido)-5-thia-1-aza-bicy
cle[4.2.0]oct-2-ene-2-carboxylic acid (TCA) and 8-oxo-3-((4-(3-oxo-
3H-benzo[f]chromene-2-carboxamido)phenylthio)methyl)-7-(2-(thi-
ophen-2-yl)acetamido)-5-thia-1-aza-bicyclo[4.2.0]oct-2-ene-2-carbo
xylic acid (TBCA) were obtained (Scheme 1). Hydrolysis of TCA and
TBCA by
b-lactamases L1 and CcrA causes an obvious change in fluo-
hydrolyzed with M
bLs, the fluorescence intensity of TCA and TBCA
rescence emission spectra.
b
-lactamase cleaves the -lactam ring of
b
obviously decreased.
TCA and TBCA to generate an unstable intermediate, which sponta-
neously rearranges to generate product a and thiophenol b (Scheme 2)
[8]. Product a does not fluoresce, and thiophenol b may undergo
partial quenching [12]. Therefore, the greater fluorescence of TCA and
TBCA, as compared to the hydrolysis products, allows for us to use
2.2. Steady-state kinetic evaluations and biological activity assay
To further probe the interactions of TCA and TBCA with the
Mb
Ls, we performed steady-state kinetic studies using TCA and
these fluorescent cephalosporins to assay M
bLs.
TBCA as substrates and M Ls CcrA (subclass B1) and L1 (subclass
b
O
O
a
O
O
b
O
O
O
H
O
H
O
H
COO
O
1
a
3
H
COO
H
C
O
H
O
H
O
O
d
c
O
2
3b
H
N
O
H
O
lH HH N
C
N
S
2
2
S
S
N
l
C
N
O
l
C
N
S
e
f
S
O
O
S
O
O
O
O
NH2
O
O
H l
C
A
LE
C
4
5
O
O
O
H
O
N
S
H
O
N
S
N
O
S
S
O
g
h
N
S
R
N
H
O
S
O
H
COO
R
N
H
O
6
H
COO
O
O
O
=
T A R
C
=
TB A R
C
O
H
COO
Scheme 1. Synthetic route of fluorescent cephalosporins. Reagents and conditions: (a) H2SO4, acetic anhydride, acetone, rt, 4 h; (b) salicylaldehyde, 70 ꢀC, 6 h; (c) CHCl3, 50% NaOH,
ethanol, HCl, 80 ꢀC; (d) pyridine, ethanol, 80 ꢀC, 2e4 h; (e) 2-thienylacetyl chloride, potassium trimethylsilanolate, CH2Cl2, CH3CN, rt, 12 h; (f) 4-amino thiophenol, N-methyl-
morpholine, CHCl3, rt, 12 h; (g) DIPEA, HATU, CH2Cl2, rt, 12 h; (h) phenol, 60 ꢀC, 4 h. DIPEA ¼ N, N0-diisopropylethylamine, NMM ¼ 4-methylmorpholine, HATU ¼ O-(7-
azabenzotriazol-1-yl)-N, N, N0, N0-tetramethyluronium hexafluorophosphate.

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J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
Table 1
H
N
O
O
O
Fluorescence quantum yield of 3a, 3b, TCA and TBCA.a
S
=
=
R
R
H
Compounds
3a
3b
TCA
TBCA
COO
N
O
S
S
O
FF
0.0848
0.2137
0.0521
0.0544
H
COO
R
H
COO
a
N
H
TCA and TBCA were dissolved in DMSO; 3a and 3b were dissolved in MeOH. The
standard of quinine sulfate (FF ¼ 0.54) was used to determine the fluorescence
quantum yield. All samples were sequentially diluted (O.D. < 0.05 at 360 nm in UVe
vis absorption) and excited in 360 nm in fluorescence area to conduct the yield by
comparison to the standard quinine sulfate.
O
O
beta-lactamase
H
N
S
B3). CcrA and L1 were over-expressed and purified as previously
described [13e15]. The Michaelis constant (Km), maximum reaction
velocity (Vmax), and catalytic constant (kcat) were determined and
calculated by fitting data for initial velocity versus substrate
concentration directly to the MichaeliseMenten equation. TCA and
HN
O
S
O
S
R
COO
N
H
TBCA were hydrolyzed by CcrA (5 nM) exhibited a Km ¼ 38.3
mM
and a kcat ¼ 209 sꢁ1 when using TCA as the substrate and
O
H
S
NH
S
a Km ¼ 24.4
m
M and a kcat ¼ 121 sꢁ1 when using TBCA as substrate;
+
L1 (20 nM) exhibited a Km ¼ 6.02
m
M and a kcat ¼ 23.5 sꢁ1 when
R
S
N
OOC
N
H
using TCA as a substrate and a Km ¼ 20.2
m
M and a kcat ¼ 29.5 sꢁ1
COO
when using TBCA as substrate. The steady state kinetic data are
a
b
Scheme 2. Hydrolysis of the fluorescent cephalosporins with b-lactamases.
Fig. 2. Fluorescence spectra of TCA, 3a (a) and TBCA, 3b (b). TCA (50
mM) and TBCA
(5 M) were dissolved in DMSO; 3a (50 M) and 3b (5 M) were dissolved in methanol.
m
m
m
Fig. 1. UVevis spectra of TCA, 3a (a) and TBCA, 3b (b). Concentrations of all the
samples were 50 M in DMSO.
Each sample of TCA, TBCA, 3a and 3b was excited at 363, 360, 324 and 361 nm,
respectively.
m

J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
153
Fig. 3. Fluorescence spectra of TCA (50
mM) and TBCA (5
m
M) in 50 mM of Tris (pH 7.0) before and after hydrolysis with 5
mM CcrA and L1. Ex ¼ 363 and 360 nm.
shown in Table 2 and indicate that TCA and TBCA are effective
photoinactivation of a K. pneumonia (ATCC 700603) strain with
more than 65% lethality at 10 M compound; control experiments
without irradiation showed less than 5% lethality at 10
substrates, which can be used to assay M
b
Ls from the B1 and B3
m
subgroups.
mM
Photodynamic inactivation of an antibiotic resistant strain by
TCA, 3a, TBCA, and 3b was investigated by a traditional surface
plating method [16]. Briefly, all the bacterial suspensions were
incubated with the selected compounds, exposed to white light
irradiation (400e800 nm, 350 mW/cm2), and spread on Luriae
Bertani (LB) agar plates. All tests were performed three times,
and the colonies on the agar plates were counted. The number of
colony forming units (CFUs) on plates of cells that had been treated
with the compounds and irradiated was divided by the number of
CFUs on plates of cells that had not been treated with compounds
nor irradiated. As shown in Figs. 4 and 5, TCA displayed effective
compound. Furthermore, the energy density of illumination was
Table 2
Kinetic constants of TCA and TBCA hydrolysis with metallo-
b
-lactamases.a
kcat (sꢁ1
)
Mb
Ls Vmax M/s) Km M)
(
m
(
m
TCA
L1
CcrA
0.471 ꢂ 0.069
1.05 ꢂ 0.04
6.02 ꢂ 1.90
38.3 ꢂ 35.1
23.5 ꢂ 3.5
209 ꢂ 72
TBCA
L1
CcrA
0.590 ꢂ 0.437
0.604 ꢂ 0.244
20.2 ꢂ 3.6
29.5 ꢂ 21.8
121 ꢂ 49
24.4 ꢂ 23.8
a
Steady-state kinetic studies were conducted in 50 mM Tris, pH 7.0, at 25 ꢀC,
concentrations of L1 and CcrA were 20 and 5 nM, respectively. Kinetic constants
were determined in triplicate with L1 or CcrA.
Fig. 4. Photodynamic inactivation of K. pneumonia with different concentrations of 3a
and TCA. The light dose was 105 J/cm2 (exposure for 5 min at a rate of 350 mW/cm2).
Bacteria treated with compounds but no light illumination as control groups.

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J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
growth of cells. In contrast, 3a exhibited no distinct mortality on
the K. pneumonia strain.
The in vitro antibacterial activities of TCA and TBCA were
investigated by determining their minimum inhibitory concentra-
tions (MIC) on bacteria [17]. Three antibiotic resistant bacterial
strains, K. pneumonia (ATCC 700603), MRSA1, and MRSA2, and
a S. aureus strain were used to the MIC for the two fluorescent
cephalosporins. TCA was shown to be an effective antimicrobial
agent on the Gram-positive S. aureus, exhibiting a MIC ¼ 0.5
however, TCA exhibited MIC values greater than 128 g/mL on the
MRSA1, MRSA2, and K. pneumonia strains. TBCA exhibited MIC
values greater than 128 g/mL on all tested bacterial strains.
mg/mL;
m
m
The binding affinities of TCA, 3a, TBCA, and 3b to several
bacteria were determined as shown in Fig. 6. The data listed in
Table 3 indicate that TBCA exhibits binding affinities to Gram-
positive bacteria MRSA1 and MRSA2 and S. aureus with values
ranging from 2.95 to 6.59
larger than those exhibited by TCA, which exhibited values ranging
from 1.82 to 2.15
M per 108 cells/mL. TBCA was used to label the
m
M per 108 cells/mL; these values are
Fig. 5. Light dose-dependent photoinactivation of
K. pneumonia. Bacteria treated with light illumination without any compounds as
6 mM 3a and TCA against
m
Gram-positive strains, MRSA1, MRSA2, and S. aureus, and confocal
fluorescence images (Fig. 7) showed that TBCA, as a fluorescent
agent, could be used to label these Gram-positive bacterial strains.
TCA could also be used to label all tested strains; however, the
labeling signal was weaker than observed with TBCA (data not
shown). This result is in agreement with the binding affinities of the
two fluorescent cephalosporins.
control groups.
positively correlated to photoinactivation (Fig. 5). Bacterial cell
death reached to 84% after 126 J/cm2 of light irradiation with 6
mM
TCA. When the sample was irradiated with 189 J/cm2, more than
95% mortality of the bacterial strain was observed. However, TBCA
and 3b showed no obvious photodynamic inactivation on the
tested bacterial strain (data not shown). These experimental results
suggest that irradiated TCA generates sufficient quantities of
cytotoxic species, which are able to diffuse into and affect the
3. Conclusion
Two novel fluorescent cephalosporins reported in this paper
were synthesized and characterized by 1H NMR, 13C NMR, UVevis
Fig. 6. Binding affinities of TCA (a), 3a (b), TBCA (c) and 3b (d) with S. aureus (ATCC 25923) (SA), MRSA1 and MRSA2. TCA, 3a, TBCA (50 mM) and 3b (5 mM) were dissolved in 0.01 M
PBS buffer (pH 7.4).

J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
155
Table 3
against a S. aureus strain. Both TCA and TBCA are substrates of two
dinuclear Zn(II)-containing M Ls. Studies showed that TBCA is
The binding affinity (m
M per 108 cells/mL) values of TCA, 3a, TBCA, and 3b to
b
S. aureus (ATCC 25923), MRSA1, and MRSA2.a
a potential fluorescent labeling reagent for Gram-negative MRSA1
and MRSA2 strains and for a S. aureus strain. The information
gleaned from this study can be used to construct a wide variety of
fluorogenic antibiotics for photoinactivation and intracellular
imaging of the antibiotic resistant bacteria.
Compounds
S. aureus
MRSA1
MRSA2
TCA
3a
TBCA
3b
2.13
0.68
6.59
0.044
2.15
0.71
3.14
0.030
1.82
0.81
2.95
0.033
a
Binding affinity was quantized by comparing the fluorescence intensity of the
TCA, 3a, TBCA and 3b labeling bacterial strains with the separated compounds TCA,
4. Experiments
3a, TBCA (50 mM) and 3b (5 mM).
4.1. Materials and instruments
and fluorescence spectroscopies, and their biological activities
were evaluated with several bacterial strains. These studies
demonstrated that TCA exhibited photodynamic inactivation of
General chemicals were purchased from SigmaeAldrich and
used without further purification. 7-Amino-3-chloromethyl-3-
cephem-4-carboxylic acid p-methoxybenzyl ester hydrochloride
(ACLE$HCl) was gift from Otsuka Chemical Co. Ltd. Escherichia coli
BL21 (DE3) cells were purchased from Wolsen Co. Ltd. LuriaeBertani
a strain of K. pneumonia that expresses extended-spectrum
b-lac-
tamases. TCA was also shown to exhibit good antimicrobial activity
Fig. 7. Confocal microscopic (left) and differential interference contrast (right) images of incubated Gram-positive S. aureus (a), MRSA1 (b) and MRSA2 (c) with TBCA.

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J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
(LB) medium and MuellereHinton Broth (MHB) used as the growth
medium for all bacterial strains were obtained from OXOID.
Melting points were determined using a X-4 microscopic
melting point meter from Shanghai Jingke Scientific Instrument Co.
Ltd. NMR spectra were recorded on a Varian INOVA400 instrument
at 400 MHz for 1H and at 100.1 MHz for 13C NMR, using tetrame-
thylsilane as an internal standard. UVevis absorbance spectra were
recorded on an Agilent UV8453 spectrometer. Fluorescence spectra
were recorded on a Hitachi F-4500 spectrometer. Confocal fluo-
rescence images were collected on an Olympus FV1000 confocal
scanning microscope. The tested compounds were dissolved in
DMSO (biochemical grade, Sigma) before fluorescence measure-
ment in order to be soluble in the aqueous solvents.
8.06 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.76 (t, J ¼ 7.4 Hz, 1H, Ar-H), 7.64 (t,
J ¼ 7.3 Hz, 1H, Ar-H), 7.57 (d, J ¼ 9.0 Hz, 1H, Ar-H). 13C NMR
(100.1 MHz, [d6]DMSO, TMS, d ppm): 111.6, 116.1, 116.7, 121.9, 126.0,
128.6, 129.4, 135.4, 143.3, 154.6, 156.4, 163.9.
4.2.5. Synthesis of p-methoxybenzyl (6R,7R)-3-chloromethyl-7b-(2-
thienylacetamido)-3-cephem-4-carboxylate (4)
To a suspension of ACLE hydrochloride (1.0 g, 2.46 mmol) in
dichloromethane (15 mL) on an ice bath was added potassium
trimethylsilanolate (0.63 g, 4.94 mmol) in 10 mL acetonitrile, and 2-
thienylacetyl chloride (0.304 mL, 2.46 mmol) in 8 mL dichloro-
methane. The resulting mixture was stirred at room temperature
(rt) for 1 h, and the solvent was evaporated under reduced pressure.
The resulting residue was dissolved in chloroform and washed with
water and brine. The organic layer was dried over MgSO4, filtered,
and evaporated. The resulting solid was recrystallized from ethyl
acetate and hexane to give 0.36 g of 4 with a yield of 27.55%. 1H
4.2. Chemistry
4.2.1. Synthesis of 2,2-dimethyl-1,3-dioxane-4,6-dione (1)
Concentrated sulfuric acid (0.17 mL, 3.23 mmol) was added to
a suspension of malonic acid (4.20 g, 40.36 mmol) and acetic
anhydride (4.96 mL, 52.47 mmol) with constant stirring in an ice-
water bath. Acetone (4.16 mL, 56.51 mmol) was added dropwise;
the reaction mixture was stirred for 4 h and then allowed to stand
in fridge overnight. The final crystals were filtered and washed with
ice water to yield 3.53 g of product as a flaky white solid with yield
of 60.68%. Product 1: m.p.: 94e95 ꢀC. 1H NMR (400 MHz, CDCl3,
NMR (400 MHz, CDCl3, TMS, d ppm): 7.38e7.27 (m, 3H, Ar-H,
thiophene-H), 7.00 (d, J ¼ 5.1 Hz, 2H, thiophene-H), 6.89 (d,
J ¼ 8.7 Hz, 2H, Ar-H), 6.33 (d, J ¼ 9.2 Hz, 1H, CH), 5.83 (dd, J ¼ 9.2,
5.0 Hz, 1H, CH), 5.21 (s, 1H, CH2Ar), 4.85 (dd, J ¼ 10.2, 5.0 Hz, 2H,
SCH2), 3.86 (s, 2H, thiophene-CH2), 3.81 (s, 3H, OCH3), 3.53 (dd,
J ¼ 72.8, 18.3 Hz, 2H, CH2Cl). 13C NMR (100.1 MHz, CDCl3, TMS,
d
ppm): 27.1, 37.1, 43.2, 55.3, 57.6, 59.1, 68.3, 114.0, 125.5, 126.0,
126.2, 126.5, 127.6, 127.9, 130.7, 134.6, 159.9, 161.0, 164.5, 170.0.
TMS,
d ppm): 3.64 (s, 2H, CH2), 1.79 (s, 6H, CH3).
4.2.6. Synthesis of 5
4.2.2. Synthesis of 2-hydroxy-1-naphthaldehyde (2)
N-methylmorpholine (0.10 mL, 0.93 mmol) was added into
a suspension of compound 4 (0.4 g, 0.76 mmol) in 20 mL chloro-
form. After the flask was purged with nitrogen, 4-amino thiophenol
(0.094 g, 0.75 mmol) was introduced slowly, the reaction mixture
was stirred for 2 h on an ice-salt bath. The resulting product was
purified using column chromatography (PE/EA ¼ 2:1 as eluent) to
afford 0.28 g of 5 with a yield of 59.95%. 1H NMR (400 MHz, CDCl3,
To a suspension liquid of 2-naphthol (5.0 g, 35 mmol) and
ethanol (20 mL) was added 50% NaOH aqueous solution (10 g,
20 mL) slowly with stirring. The reaction mixture was heated to
80 ꢀC, chloroform (7 g) was slowly added, and the reaction mixture
was stirred at 80 ꢀC for 1 h. After removing the solvent under
vacuum, concentrated HCl (17 mL) and water (20 mL) were added
sequentially in an ice-salt bath. The organic layer was washed with
hot water several times and then extracted with hexane. The
organic layer was dried over sodium sulfate, and the crude product
was purified by column chromatography (silica gel, PE/EA ¼ 15:1 as
eluent) to offer compound 2.89 g 2 with yield of 48.05%. m.p.: 82e
TMS,
d
ppm) 7.28 (d, J ¼ 8.7 Hz, 2H, Ar-H, thiophene-H), 7.12 (d,
J ¼ 8.6 Hz, 2H, Ar-H), 6.98 (d, J ¼ 5.1 Hz, 2H, thiophene-H), 6.87 (d,
J ¼ 8.7 Hz, 2H, Ar-H), 6.50 (d, J ¼ 8.6 Hz, 2H, Ar-H), 6.40 (d, J ¼ 9.1 Hz,
1H, Ar-H), 5.70 (dd, J ¼ 9.1, 4.8 Hz,1H, CH), 4.98 (dd, J ¼ 46.0,11.9 Hz,
2H, CH2Ar), 4.83 (d, J ¼ 4.8 Hz, 1H, CH), 4.21, 3.48 (dd, J ¼ 13.3,
13.0 Hz, 2H, SCH2), 3.82 (d, J ¼ 4.3 Hz, 2H, thiophene-CH2), 3.79 (s,
3H, OCH3), 3.63, 3.27 (dd, J ¼ 18.0, 18.1 Hz, 2H, CH2S).
84 ꢀC. 1H NMR (400 MHz, CDCl3, TMS,
d ppm) 13.18 (s, 1H, CHO),
10.83 (s,1H, OH), 8.36 (d, J ¼ 8.5 Hz,1H, Ar-H), 8.00 (d, J ¼ 9.1 Hz,1H,
Ar-H), 7.81 (d, J ¼ 8.0 Hz, 1H, Ar-H), 7.63 (t, J ¼ 7.5 Hz, 1H, Ar-H), 7.45
(t, J ¼ 7.5 Hz, 1H, Ar-H), 7.15 (d, J ¼ 9.1 Hz, 1H, Ar-H).
4.2.7. Synthesis of 6a
A mixture of compounds 5 (0.12 g, 0.19 mmol), 3a (0.046 g,
0.23 mmol), and HATU (0.12 g, 0.29 mmol) in 30 mL anhydrous
dichloromethane was stirred constantly for 2 h on ice-salt bath.
DIPEA (0.05 mL, 0.29 mmol) was added slowly, and the reaction
mixture was stirred at rt overnight. The solvent was evaporated
under reduced pressure, and the resulting residue was dissolved in
chloroform, washed several times with 5% hydrochloric acid, 5%
saturated NaHCO3 and brine, and dried over sodium sulfate. The
crude product was purified using column chromatography (silica
gel, PE/EA ¼ 1:1 as eluent), and after removing the solvent under
vacuum, 76 mg of 6a was obtained with a yield of 63%. 1H NMR
4.2.3. Synthesis of 2-oxo-2H-chromene-3-carboxylic acid (3a)
Compound 1 (2.0 g, 13.88 mmol) and water (25 mL) were
sequentially added to salicylaldehyde (1.45 mL, 13.88 mmol), the
reaction mixture was heated to 70 ꢀC, stirred for 6 h, cooled to room
temperature, and filtered. The resulting solid was washed with ice
water and dried to afford 2.27 g of 3a with a yield of 86.03%. m.p.:
189e190 ꢀC. 1H NMR (400 MHz, [d6]DMSO, TMS,
d ppm): 13.35 (s,
1H, OH), 8.77 (s, 1H, CHCOH), 7.93 (d, J ¼ 7.6 Hz, 1H, Ar-H), 7.75 (m,
J ¼ 7.7 Hz, 1H, Ar-H), 7.45 (m, J ¼ 9.5 Hz, 2H, Ar-H). 13C NMR
(100.1 MHz, [d6]DMSO, TMS, d ppm):115.6, 117.5, 117.7, 124.3, 129.7,
133.8, 148.0, 154.0, 156.3, 163.5.
(400 MHz, CDCl3, TMS,
d
ppm): 10.85 (s,1H, NH), 9.00 (t, J ¼ 13.4 Hz,
1H, Ar-H), 7.85e7.30 (m, 10H, Ar-H, thiophene-H), 6.96 (m, 4H,
thiophene-H, Ar-H), 6.34 (d, J ¼ 9.0 Hz, 1H, Ar-H), 5.74 (dd, J ¼ 9.0,
4.7 Hz, 1H, CH), 5.05 (q, J ¼ 11.9 Hz, 2H, CH2Ar), 4.87 (d, J ¼ 4.8 Hz,
1H, CH), 4.23, 3.80 (dd, J ¼ 13.4, 13.4 Hz, 2H, SCH2), 3.86 (s, 2H,
thiophene-CH2), 3.73 (s, 3H, OCH3), 3.68, 3.34 (dd, J ¼ 18.0, 18.0 Hz,
2H, CH2S).
4.2.4. Synthesis of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid
(3b)
Ethanol (30 mL) was added into a 100-mL flask with compounds
1(1.67 g, 11.6 mmol) and 2 (2.00 g, 11.6 mmol). A catalytic amount of
pyridine (20 drops) was added, and the mixture was stirred at 80 ꢀC
for 2e4 h. A yellow solid separated out during the reaction was
filtered off to give 1.89 g of 3b with a yield of 67.83%. 1H NMR
4.2.8. Synthesis of 6b
(400 MHz, [d6]DMSO, TMS,
CHCOO), 8.55 (d, J ¼ 8.4 Hz,1H, Ar-H), 8.30 (d, J ¼ 12.5 Hz,1H, Ar-H),
d
ppm): 13.36 (s, 1H, COOH), 9.32 (s, 1H,
A mixture of compounds 5 (0.10 g, 0.16 mmol), 3b (0.046 g,
0.19 mmol), and HATU (0.093 g, 0.24 mmol) in 30 mL anhydrous

J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
157
dichloromethane was stirred for 2 h on ice-salt bath. DIPEA
(0.04 mL, 0.24 mmol) was added slowly, and the reaction mixture
was stirred at rt overnight. The solvent was evaporated under
reduced pressure, and the resulting residue was dissolved in
chloroform, washed with 5% HCl, 5% saturated NaHCO3 and brine,
and dried over sodium sulfate. The crude product was purified
using column chromatography (silica gel, PE/EA ¼ 2:1 as eluent) to
afford 64 mg of 6b with a yield of 49.8%. 1H NMR (400 MHz, CDCl3,
density < 0.05 at 360 nm). All samples were excited at 360 nm and
the absorption versus the fluorescence area was plotted, and the
yield was determined by comparison to the standard quinine
sulfate, according to the equation below [19].
N
Z
IFðlE
lFÞdlF
;
lE
Þ
R
FF
FFR
n2
nR2
1 ꢁ 10ꢁA ð
0
¼
ꢃ
ꢃ
N
lE
Þ
1 ꢁ 10ꢁAð
Z
TMS,
d
ppm):
d
¼ 10.93 (s, 1H, Ar-H), 9.79 (s, 1H, Ar-H), 8.54e7.75
(m, 2H, Ar-H), 7.55 (m, 5H, Ar-H), 7.28 (d, J ¼ 9.4 Hz, 4H,
thiophene-H, Ar-H), 6.84 (d, J ¼ 8.6 Hz, 4H, Ar-H, thiophene-H), 6.30
(d, J ¼ 9.0 Hz, 1H, Ar-H), 5.75 (dd, J ¼ 9.0, 4.8 Hz, 1H, CH), 5.07 (q,
J ¼ 11.9 Hz, 2H, CH2Ar), 4.88 (d, J ¼ 4.8 Hz, 1H, CH), 4.25, 3.74 (dd,
J ¼ 13.4, 13.1 Hz, 2H, SCH2), 3.86 (s, 2H, thiophene-CH2), 3.71 (s, 3H,
OCH3), 3.65, 3.35 (dd, J ¼ 18.0, 18.0 Hz, 2H, CH2S).
IFR
ð
lE
;
lFÞdlF
0
4.4. Over-expression and purification of M
bLs
Mb
L CcrA was over-expressed and purified as described previ-
ously [20]. Briefly, over-expression plasmid, pMSZ02-CcrA, was
transformed into E. coli BL21 (DE3) cells. A 10 mL overnight culture
of these cells in LuriaeBertani (LB) medium was used to inoculate
4.2.9. Synthesis of TCA
Compound 6a (70 mg, 0.0929 mmol) and phenol (0.327 g,
3.71 mmol) were heated at 60 ꢀC for 4 h. The resulting mixture was
diluted with chloroform (10 mL) and extracted with saturated
aqueous NaHCO3 (10 mL). The aqueous layer was cooled on an ice
bath, acidified to pH 1.0 with 10% HCl, and extracted with chloro-
form several times. The organic phase was dried over MgSO4 and
concentrated in vacuo to afford 38 mg of TCA as a solid with yield of
4 ꢃ 1 L of LB medium containing 25
mg/mL kanamycin. The cells
were allowed to grow at 37 ꢀC with shaking until the cells reached
an optical density at 600 nm (O.D.600) of 0.6e0.8. The cultures were
subsequently cooled to 25 ꢀC for 15 min. Protein production was
induced with 1 mM isopropyl-b-thiogalactopyranoside (IPTG), and
the cells were shaken at 25 ꢀC for 20 h. Cells were removed by
centrifugation, and the cleared supernatant was concentrated with
an Amicon concentrator equipped with a YM-10 membrane and
loaded onto a Q-Sepharose column. Bound proteins were eluted
64.59%. 1H NMR (400 MHz, [d6]DMSO, TMS,
d ppm): 10.73 (s, 1H,
NH), 9.12 (d, J ¼ 8.3 Hz, 1H, Ar-H), 8.92 (s, 1H, Ar-H), 8.02 (d,
J ¼ 7.6 Hz, 1H, Ar-H), 7.84e7.65 (m, 3H, Ar-H), 7.60e7.32 (m, 4H, Ar-
H, thiophene-H), 6.95 (dd, J ¼ 9.1, 4.2 Hz, 2H, thiophene-H), 5.66e
5.60 (m, 1H, CH), 5.08 (d, J ¼ 4.6 Hz, 1H, CH), 4.19, 3.95 (dd, J ¼ 13.3,
13.1 Hz, 2H, SCH2), 3.76 (s, 2H, thiophene-CH2), 3.68, 3.52 (dd,
J ¼ 17.7, 17.1 Hz, 2H, CH2S). 13C NMR (100.1 MHz, [d6]DMSO, TMS,
with a 0e400 mM NaCl gradient in 30 mM Tris containing 100 mM
ZnSO4 at pH 7.6. Fractions containing CcrA were pooled, concen-
trated, and loaded onto a Sephadex G-75 column for further puri-
fication. Protein purity was ascertained by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE). Protein concen-
trations were determined using Beer’s law and an extinction
coefficient of 39,000 Mꢁ1 cmꢁ1 [20].
d
ppm): 35.4, 48.2, 56.9, 57.9, 78.8, 115.9, 118.1, 124.5, 124.9, 125.8,
126.2, 128.7, 129.9, 131.8, 133.7, 133.9, 135.3, 136.7, 137.3, 139.6,
139.8, 146.9, 159.4, 163.1, 162.2, 163.5, 169.5, 183.5.
Mb
L L1 was over-expressed and purified as described previously
[21]. An overnight starter culture was prepared by inoculating
50 mL of LB medium containing 25 g/mL kanamycin and 25 g/mL
chloramphenicol with a single colony of E. coli BL21(DE3) con-
taining the pET26b-L1 plasmid, and this culture was allowed to
shake overnight at 37 ꢀC. The preculture was used to inoculate
4.2.10. Synthesis of TBCA
Compound 6b (35 mg, 0.044 mmol) and phenol (0.205 g,
2.20 mmol) were heated at 60 ꢀC for 4 h, and the mixture was
diluted with chloroform (10 mL) and extracted with saturated
aqueous NaHCO3 (10 mL). The aqueous layer was cooled on an ice
bath, acidified to pH 1.0 with 10% HCl, and extracted with chloro-
form. The organic phase was dried over MgSO4 and concentrated in
m
m
4 ꢃ 1 L of LB medium containing 25
mg/mL kanamycin and 25 mg/mL
chloramphenicol, and the cells were shaken at 37 ꢀC until the cells
reached O.D.600 ¼ 0.6e0.8. Protein production was induced with
1 mM IPTG, and the cells were shaken for 3 h at 37 ꢀC. The cells
were collected and resuspended in 15 mL of 50 mM Tris, pH 8.5,
containing 500 mM NaCl. The cells were ruptured by ultrasonic and
separated by centrifugation. The resulting supernatant was purified
with Q-Sepharose chromatography. Proteins were identified by
SDS-PAGE, pooled, and concentrated. L1 was quantitated by
monitoring its absorbance at 280 nm and using extinction coeffi-
cient of 54,614 Mꢁ1 cmꢁ1 [21].
1
vacuo to afford 18 mg of TBCA as solid with a yield of 59.83%. H
NMR (400 MHz, [d6]DMSO, TMS, d ppm): 10.73 (s, 1H, Ar-H), 9.55 (s,
1H, Ar-H), 9.14e8.02 (m, 4H, Ar-H), 7.97e7.55 (m, 5H, Ar-H), 7.53e
7.31 (m, 2H, Ar-H, thiophene-H), 6.94 (dd, J ¼ 9.4, 4.4 Hz, 1H,
thiophene-H), 5.42 (dd, J ¼ 8.3, 4.7 Hz, 1H, CH), 4.88 (d, J ¼ 4.7 Hz,
1H, CH), 4.35, 4.11 (dd, J ¼ 12.8, 12.8 Hz, 2H, SCH2), 3.75 (s, 2H,
thiophene-CH2), 3.49, 3.28 (dd, J ¼ 17.1, 17.0 Hz, 2H, CH2S). 13C NMR
(100.1 MHz, [d6]DMSO, TMS,
d
ppm):
d
¼ 26.9, 36.1, 57.7, 58.7, 79.5,
113.2, 115.1, 116.8, 117.8, 118.2, 119.4, 122.8, 124.1, 125.3, 127.0, 129.4,
129.5, 129.6, 130.1, 132.4, 133.3, 134.0, 134.6, 136.2, 137.4, 142.9,
154.7, 160.5, 160.6, 163.0, 164.2, 170.4.
4.5. Steady-state kinetic evaluation of TCA and TBCA
4.3. Spectroscopic characterization and fluorescence quantum yield
determination of TCA and TBCA
Steady-state kinetic experiments were conducted on an Agilent
UV8453 spectrometer, and absorbance changes were monitored at
282 and 403 nm. The buffer used in these studies was 50 mM Tris,
pH 7.0, and assays were conducted at 25 ꢀC. The initial concentra-
TCA and 3a were dissolved in DMSO and diluted to 50
mM; while
the concentrations of TBCA and 3b were diluted to 5 M because of
m
the high fluorescence intensity of 3b. Measurements relative to the
standard of quinine sulfate (FF ¼ 0.54 [18]) were used to determine
the fluorescence quantum yield of each sample. TCA and TBCA were
dissolved in DMSO and 3a and 3b were dissolved in MeOH to give
5.0 mM stock solutions. Four samples and quinine sulfate were
sequentially diluted to the desired final concentrations (optical
tions of L1 and CcrA were 2.0 and 0.5
state kinetic parameters were determined on at least three
different preparations of the enzymes. series of different
concentrations of substrates (5e75 M for CcrA and 4e30 M for
L1) were added into the Tris buffer, and M Ls were added at time
zero. The initial rates of hydrolysis were determined in triplicate.
mM, respectively. All steady
A
m
m
b

158
J.-M. Xiao et al. / European Journal of Medicinal Chemistry 59 (2013) 150e159
4.6. Fluorescence emission spectra of TCA and TBCA hydrolysis by
CcrA and L1
overnight, and sonicated for 30 min. Binding affinity was evaluated
by comparing the fluorescence intensity of the samples.
Fluorescence spectra were obtained at 25 ꢀC in 50 mM Tris, pH
4.10. Confocal images of S. aureus, MRSA1 and MRSA2 strains
labeled with TCA and TBCA
7.0. The concentrations of TCA, TBCA, and M
M, respectively. Fluorescence emission spectra of the substrates
TCA and TBCA and the two M Ls were determined using an exci-
bLs were 50, 5 and
5
m
b
Overnight bacterial cultures were harvested by centrifugation
(4 ꢀC, 4000 rpm for 10 min), and cell pellets were washed with
0.01 M PBS buffer 3e6 times and then diluted with the same buffer
to O.D.600 of 0.5. The suspensions of the bacterial cultures in PBS
tation at 363 nm and 360 nm, respectively.
4.7. Determination of minimum inhibitory concentration (MIC)
were incubated with 50 mM TCA and TBCA, respectively, in dark
Four bacterial strains, S. aureus (ATCC 25923), K. pneumonia
(ATCC 700603), MRSA1, and MRSA2, were used to assay antibac-
terial activities of the fluorescent cephalosporins. A single colony of
the above described bacteria on solid LuriaeBertani (LB) plates was
transferred to 10 mL of MuellereHinton culture medium, and
cultures were grown at 35 ꢀC for 2e6 h to obtain 108 CFU/mL
bacterium cultures according to Maxwell turbidimetry [22]. TCA
and TBCA were dissolved in ddH2O to prepare 1280 mg/mL stock
solutions. The prepared solutions of TCA and TBCA were diluted to
1 mL by MuellereHinton medium to offer solutions with 256, 128,
with shaking. The cultures were washed with 0.01 M PBS buffer
twice and centrifuged (4 ꢀC, 4000 rpm for 10 min) to give the final
samples for confocal images.
Acknowledgment
We are grateful for assistance in the photodynamic studies from
Dr. Dan Sun at the Laboratory of Photonics and Photon Technique at
Northwest University. We thank Dr. Michael Crowder at Miami
University for the plasmids containing metallo-b-lactamase genes.
64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 mg/mL, respectively. The 1 mL
We also acknowledge the financial support from National Natural
Science Foundation of China (20972127, 21272186), Doctoral
Foundation of China (200806970005), Natural Science Foundation
of Shaanxi Province (2009JM2002), Key Fund for International
Cooperation of Shaanxi Province (2010KW-16), Shaanxi Provincial
Department of Education Fund (09JK786) and Shaanxi Provincial
Bureau of Foreign Experts Fund (SLZ2008013).
bacterial cultures (w106 CFU/mL) in MuellereHinton medium were
added sequentially into the prepared solutions. The mixture was
incubated at 37 ꢀC for 24 h.
4.8. Photodynamic inactivation of TCA, 3a, TBCA and 3b against
K. pneumonia
A single colony of K. pneumonia on LB-agar plates was trans-
ferred to 10 mL of LB culture medium, and the culture was grown at
37 ꢀC overnight. Bacterial cells were collected by centrifugation
(4 ꢀC, 4000 rpm for 10 min), washed 3e6 times with 0.01 M
phosphate-buffered saline (PBS) buffer (pH 7.4), and diluted with
the same buffer to O.D.600 of 0.5. A series of TCA, 3a, TBCA and 3b
samples with various concentrations were prepared as stock solu-
tions. 0.5 mL of the samples were added into 0.5 mL diluted
bacterium fluid and co-cultures were incubated in dark at 37 ꢀC for
30 min with shaking. All of the samples were illuminated with light
(400e800 nm, 350 mW/cm2), which was produced by a Xenon
lamp and separated by optical filters, for 5 min. The photo-
illuminated samples were centrifuged (4 ꢀC, 4000 rpm for
10 min), and the supernatants were removed. Following irradiation,
the bacterial cells were suspended and sequentially diluted 5 ꢃ104-
Appendix A. Supplementary material
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2012.11.019.
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