Synthesis, fluorescence properties, and antiproliferative potential of several 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives

Article abstract of DOI:10.3390/molecules201018565

In this study, two series of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives (compounds 5a-i and 6a-g) were synthesized. Their in vitro proliferation inhibitory activities against the A549 and NCI-H460 human non-small cell lung cancer (NSCLC) cell lines were evaluated. Their photophysical properties were measured. Among these target compounds, 5e exhibited the strongest antiproliferative activity by inducing apoptosis, arresting cell cycle, and elevating intracellular reactive oxygen species (ROS) level, suggesting that it may be a potent antitumor agent. In addition, compound 6g with very low cytotoxicity, demonstrated excellent fluorescence properties, which could be used as an effective fluorescence probe for biological imaging.

Full text of DOI:10.3390/molecules201018565

Molecules 2015, 20, 18565-18584; doi:10.3390/molecules201018565
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Fluorescence Properties, and Antiproliferative
Potential of Several 3-Oxo-3H-benzo[f]chromene-2-carboxylic
Acid Derivatives
Xiao-Bo Fu †, Xian-Fu Wang †, Jia-Nian Chen *, De-Wen Wu, Ting Li, Xing-Can Shen
and Jiang-Ke Qin
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry & Pharmacy, Guangxi Normal University, Yucai Road 15,
Guilin 541004, China; E-Mails: txzxconstance@163.com (X.-B.F.);
wxf3982296@sina.com (X.-F.W.); wdw3982296@sina.com (D.-W.W.);
litingfendou@163.com (T.L.); xcshen@mailbox.gxnu.edu.cn (X.-C.S.); jiangkeq@sina.com (J.-K.Q.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: cjn288@sina.com;
Tel./Fax: +86-773-2120-958.
Academic Editor: Roman Dembinski
Received: 1 August 2015 / Accepted: 7 October 2015 / Published: 13 October 2015
Abstract: In this study, two series of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives
(compounds 5a–i and 6a–g) were synthesized. Their in vitro proliferation inhibitory activities
against the A549 and NCI-H460 human non-small cell lung cancer (NSCLC) cell lines were
evaluated. Their photophysical properties were measured. Among these target compounds, 5e
exhibited the strongest antiproliferative activity by inducing apoptosis, arresting cell cycle,
and elevating intracellular reactive oxygen species (ROS) level, suggesting that it may be a
potent antitumor agent. In addition, compound 6g with very low cytotoxicity, demonstrated
excellent fluorescence properties, which could be used as an effective fluorescence probe
for biological imaging.
Keywords: 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives; proliferation inhibitory
activity; photophysical property; antitumor; biological imaging

Molecules 2015, 20
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1. Introduction
Coumarin (1,2-benzopyrone or 2H-1-benzopyran-2-one) and its derivatives, best-known as
oxygen-containing heterocyclic compounds, are widely distributed in Nature and many exhibit diverse
biological and interesting pharmacological activities. The numerous therapeutic applications of coumarin
compounds include anti-coagulant effects [1], antitumor therapy [2–5], anti-HIV treatment [6,7], central
nervous system stimulants and protective agents [8,9], antibacterial and anti-inflammatory drugs [10–12].
It has been found that the coumarin nucleus plays an important role as a valuable molecular template for
the development of different structural analogues with improved pharmacological profiles. The different
substituent groups connected with the coumarin nucleus strongly influence the biological activity of the
resulting derivatives [13]. More importantly, the excellent fluorescent properties of coumarin compounds
offer additional development value and they are used in many areas, such as fluorescent brighteners,
fluorescent probes to monitor complex biological events, electroluminescent devices, photochemotherapy,
etc. [14–16].
Recently we focused on anti-tumor activity screening and fluorescent performance of some
coumarin-based compounds. In this report, two series of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid
derivatives including its amide (compounds 5a–i) and ester (compounds 6a–g) were efficiently synthesized
(Scheme 1). Their biological activity and photophysical properties were evaluated. It was found that
compound 5e exhibited the strongest in vitro proliferation inhibitory activity against the A549 and
NCI-H460 cell lines, suggesting that it may be a potent antitumor agent. In addition, compound 6g with
very low cytotoxicity demonstrated excellent fluorescence properties, which could be exploited for
biological and biomedical imaging.
CHO
COCl
O
COOH
O
OH
(a)
(b)
O
O
1
4
3
)
d
(
(c)
COOR2
O
CONHR1
O
O
O
6a−g
5a−i
Scheme 1. Synthetic route to the target compounds 3-oxo-3H-benzo[f]chromene-2-carboxylic
acid derivatives 5a–i and 6a–g. Reagents and conditions: (a) Meldrum’s acid (2), ethanol,
pyridine, 80 °C, 4 h; (b) SOCl2, 1,2-dichloroethane, 60–70 °C, 4 h; (c) R1-NH2, triethylamine,
CH2Cl2, room temperature (r.t.), 10–30 min; (d) R2-OH, triethylamine, CH2Cl2, r.t., 10–30 min.
2. Results and Discussion
2.1. Chemistry
The synthetic route to the target 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives 5a–i and
6a–g is depicted in Scheme 1. Based on the method reported previously [17], we successfully prepared

Molecules 2015, 20
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the key intermediate 3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3) via Knoevenagel condensation
of 2-hydroxy-1-naphthaldehyde (1) with Meldrum’s acid (2). For the first step, cheap ethanol was selected
as the solvent and pyridine as a catalyst. It was noted that an excess of Meldrum’s acid should be avoided
because it could react with 3 [18]. Then 3-oxo-3H-benzo[f]chromene-2-carbonyl chloride (4) was obtained
by chlorination in the presence of thionyl chloride. Compound 4 is easily hydrolyzed and converted back
into the previous carboxylic acid 3 if it is exposed to the air for a long time, therefore 4 was used directly
in the next step without further purification. Finally, different amines and alcohols were reacted with 4
to afford the target compounds 5a–i and 6a–g (Scheme 1). This method is convenient, efficient and can
be used for constructing coumarin-based compound libraries; moreover, the byproducts are easy to
remove, and the intermediate 3 is obtained in high yield and purity.
O
H
N
N
O
O
O
O
H
O
R
(1)
O
O
O
O
O
O
H
H
OH
O
(I)
(2)
H
O
O
O
O
O
N
O
O
N
H2O
O
O
O
R
R
R
O
O
O
OH
OH
OH
(IV)
(II)
(III)
O
O
O
OH
O
R
R
−acetone
CH2CH2
R =
O
O
OH
(V)
(3)
Scheme 2. Proposed mechanism of the pyridine-catalyzed Knoevenagel condensation for
synthesizing 3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3).
After the target compounds were successfully prepared, the reaction mechanism for synthesizing
the key intermediate 3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3) was examined. As shown in
Scheme 2, Meldrum’s acid (2), namely 2,2-dimethyl-1,3-dioxane-4,6-dione, is easily enolized and usually
used as a good nucleophile in many reactions when malonate ester is ineffective [19]. In the presence of
pyridine, Meldrum’s acid (2) is deprotonated to afford its enolate I which can be converted to the
corresponding ketone form. Then the produced carbanion attacks the 2-hydroxy-1-naphthaldehyde substrate
1, and the intermediate II is obtained. However, II is prone to protonate, then lose a water molecule, and
the olefine ketone III is produced [18,20]; meanwhile, the catalyst pyridine restores its initial state.
Subsequently, an intramolecular nucleophilic addition occurs because the carbonyl carbon of III is
susceptible to attack by the hydroxyl group, which gives the hemiacetal IV. The strain of the rightmost
lactone ring combined with instability of the hemiacetal structure forces the loss of an acetone molecule,
and the intermediate V is thus obtained [19,21]. However, because the hydroxyl group is directly linked
with a carbocation, V is also unstable and easily loses a proton, which is captured by the carboxylate
anion to form the carboxyl group and 3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3) is obtained. Of
course, with regard to the conversion from V to 3, there may be another path where the proton of the

Molecules 2015, 20
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carboxyl group comes from a water molecule which was generated previously, rather than the hydroxyl
group of V. From the above analysis, many proton-binding agents can be used as catalysts in the
Knoevenagel condensation. Besides pyridine, maybe other bases, such as piperidine, piperidinium acetate,
piperazine, and ammonium acetate are also appropriate [20,22].
2.2. In Vitro Biological Activity
2.2.1. Antiproliferative Activities of the Target Compounds against A549 and NCI-H460 Cells in Vitro
The in vitro proliferation inhibitory activity of the prepared compounds 5a–i and 6a–g was evaluated
against two NSCLC cell lines including A549 and NCI-H460; 7-hydroxycoumarin (7-HC) and cisplatin
were selected as dual positive reference drugs. The NSCLC cell lines were treated with the target
compounds (final concentrations: 0, 5, 10, 20, 30, 40, 60 μM), and viable cells were measured by an
MTT (3-(4,5-dimethylthiahiazol-2-y1)-2,5-diphenyltetrazolium bromide) assay. As shown in Table 1,
most of the target compounds exhibited very low antiproliferative activities; especially, the IC50 values
(IC50 represented the concentration at which cell growth was inhibited by 50%) of 3-oxo-3H-
benzo[f]chromene-2-carboxylic acid esters 6a–g were over 60 μM. To our delight, among the synthesized
compounds, 5e displayed the best activities with IC50 values of 20.53 ± 1.84 for A549 and 29.19 ± 2.61 for
NCI-H460 cells, respectively. After the co-incubation of A549 cells with 10, 20, and 30 μM compounds
5e for 48 h, the inhibition rates were 22.55% ± 1.97%, 52.59% ± 2.79% and 63.72% ± 3.09%, respectively.
The decline of IC50 values with the prolonged incubation time is shown in Figure 1. At the same
concentration, the proliferation inhibitory activity of 5e was superior to 7-HC, even though neither of
them are comparable with cisplatin which has been extensively used to treat patients with NSCLC within
the last several decades [23–25]. The above results suggest that compound 5e can induce a dose- and
time-dependent proliferation inhibition of A549 and NCI-H460 cells.
(A) 40
(B)
A549
7-HC
Cisplatin
compound 5e
NCI-H460
7-HC
Cisplatin
compound 5e
40
30
20
10
0
30
20
10
0
48 h
72 h
24 h
24 h
48 h
72 h
Incubation time
Incubation time
Figure 1. Antiproliferative effects of compound 5e on NSCLC cell lines. (A) A549 and (B)
NCI-H460 cells were treated with 7-HC, cisplatin, and 5e for the indicated time (24, 48, and
72 h), respectively. After treatment, cell viability was measured by MTT assay, then inhibition
rate and IC50 values were obtained. In the figure, the antiproliferative activity data of 7-HC
after the incubation with A549 and NCI-H460 cell lines for 24 h were omitted because the
IC50 values were over 60 μM under this condition.

Molecules 2015, 20
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Table 1. In vitro antiproliferative activities of 3-oxo-3H-benzo[f]chromene-2-carboxylic
acid derivatives against A549 and NCI-H460 cell lines.
O
R
O
O
IC50 ± SD (μM) a,b
Compound
5a
R
A549
NCI-H460
∗
∗
HN
>60
46.41 ± 4.22
HN
5b
>60
>60
>60
∗
HN
HN
5c
5d
5e
>60
∗
O
38.71 ± 3.62
20.53 ± 1.84
44.39 ± 2.98
29.19 ± 2.61
∗
HN
O
F
F
∗
HN
F
5f
29.72 ± 3.85
>60
H
N
∗
∗
5g
5h
>60
>60
>60
>60
HN
5i
>60
>60
∗
HN
O
6a
6b
6c
>60
>60
>60
>60
>60
>60
∗
O
∗
∗
O
∗
O
6d
6e
6f
>60
>60
>60
>60
>60
>60
O
∗
O
∗
O
6g
∗
>60
>60
7-HC
Cisplatin
—
—
33.82 ± 2.62
15.42 ± 1.06
38.55 ± 2.91
9.61 ± 0.83
a
A549 and NCI-H460 cells were treated with the target compounds for 48 h. Results are expressed as
means ± SD (standard deviation) of four independent experiments; b Compounds with IC50 values >60 μM are
considered to be inactive.
2.2.2. Apoptosis Induced by Compound 5e
To investigate whether the proliferation inhibitory activity of compound 5e was associated with induced
apoptosis, Annexin V-FITC/propidium iodide (Annexin V/PI) dual staining method was used. As shown
in Table 2 and Figure 2, after A549 cells were treated with compound 5e for 48 h, the population of apoptotic
cells (early plus late apoptotic cells, Q2 + Q3 quadrants) increased significantly. Compared with 7-HC, 5e
had more potent capability to induce apoptosis at the same concentration (20 µM). If the incubation time
was extended to 72 h, the population of apoptotic cells was increased from 7.22% ± 0.52% for the control
group to 10.9% ± 2.1% at 10 µM, then to 19.1% ± 1.4% at 20 μM for 5e (Table 2). Therefore, compound
5e can induce apoptosis of A549 cells in a concentration- and time-dependent manner; induced apoptosis
is responsible for the antiproliferative activity of compound 5e against A549 cells.

Molecules 2015, 20
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105
104
103
105
104
103
Q4 Q3
Q1 Q2
4.78
0.91
1.81
5.62
9.05
3.62
102
0
102
0
2
2
10 103 104 105
10 103 104 105
0
0
7-HC, 20 μM
Control
105
104
103
105
104
3.81
6.62
103
6.52
4.68
14.5
3.69
102
0
102
0
0 102 103 104 105
Compd. 5e, 20 μM
0 102 103 104 105
Compd. 5e, 10 μM
Annex V-FITC
Figure 2. Effects of compound 5e on the induction of apoptosis in A549 cells after the
co-incubation for 72 h. The population of apoptotic cells was determined by flow cytometry.
The experiment was repeated thrice. Here only representative flow cytometric graphs are shown.
Table 2. Quantitative apoptosis assay of A549 using Annexin-V/PI dual staining method a.
48 h Incubation
Apoptotic Cells
(Q2 + Q3, %)
4.72 ± 0.41
72 h Incubation
Q2 + Q3 (%)
Conc. b
(µM)
Group
Viable Cells
(Q1, %)
Necrotic Cells
(Q4, %)
Q1 (%)
Q4 (%)
Control
7-HC
0
94.7 ± 2.6
85.7 ± 2.8
91.4 ± 3.1
80.7 ± 2.5
0.62 ± 0.19
3.04 ± 0.29
2.04 ± 0.31
5.41 ± 0.62
91.8 ± 3.3
81.1 ± 2.4
85.3 ± 2.9
73.9 ± 2.7
7.22 ± 0.52
14.2 ± 1.9 *
10.9 ± 2.1
0.97 ± 0.13
4.71 ± 0.49
3.77 ± 0.31
6.98 ± 0.42
20
10
20
11.3 ± 1.4 **
6.51 ± 1.31
Compd. 5e
Compd. 5e
13.9 ± 2.2 **
19.1 ± 1.4 **
a
A549 cells were treated with the indicated concentrations of compound 5e for 48 and 72 h, respectively;
subsequently the cells were stained with Annexin V-FITC and PI. The percentage of viable cells, apoptotic
b
cells and necrotic cells is expressed as the means ± SD from three independent experiments; Conc. is the
abbreviation of concentration; * p < 0.05; ** p < 0.01 vs. the percentage of apoptotic cells of the control.
2.2.3. Cell Cycle Analysis
Cell cycle distribution was examined to determine whether compound 5e inhibited the proliferation
of A549 cells through cell cycle arrest. The cells were co-incubated with 5e and 7-HC for 48 and 72 h,
respectively. Compound 5e treatment increased the population of cells in the G0/G1 phase in a concentration-
and time-dependent manner. This was accompanied by a decrease in the population of cells in S and G2/M
phases, compared with the control. As shown in Table 3 and Figure 3, the population of A549 cells in the
G0/G1 phase was increased from 70.71% ± 2.79% (control) to 80.52% ± 2.29% at 20 μM, then to 85.51% ±
2.03% at 30 μM for 5e; meanwhile, apoptotic cell rate was elevated with increased dose, which was
consistent with the above results of induced apoptosis. These data indicate that, besides induced apoptosis,

Molecules 2015, 20
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cell cycle arrest in the G0/G1 stage is also an important factor by which compound 5e exerts its inhibitory
effects on A549 cells.
Control
7-HC, 20 μM
Compd. 5e,10 μM
Compd. 5e, 20 μM
Compd. 5e, 30 μM
PI
Figure 3. Effects of compound 5e on A549 cell cycle progression. A549 cells were treated
with 5e for 72 h and analyzed by flow cytometry. The experiment was repeated thrice. Here
only representative flow cytometric graphs are shown.
Table 3. Effects of compound 5e on cell cycle progression in A549 cells a.
Group
Control
Conc. (µM) G0/G1 Phase (%) S Phase (%) G2/M Phase (%)
0
70.71 ± 2.79
79.83 ± 2.39 *
72.62 ± 2.67
16.79 ± 1.37
8.38 ± 0.44
15.77 ± 0.84
13.14 ± 1.01
8.82 ± 0.91
12.50 ± 1.41
11.79 ± 1.69
11.60 ± 1.31
6.31 ± 0.62
5.67 ± 0.43
7-HC
20
10
20
30
Compd. 5e
Compd. 5e
Compd. 5e
80.52 ± 2.29 *
85.51 ± 2.03 *
a
A549 cells were treated with compound 5e or 7-HC for 72 h, then DNA content was analyzed through PI
staining. Flow cytometry data were analyzed using the ModFit software. The results are presented as the means
± SD of three independent experiments; * p < 0.05 vs. the percentage of cells in G0/G1 phase of the control.
2.2.4. Induced ROS Generation by Compound 5e
The intracellular ROS levels were measured before and after treatment with compound 5e. As shown
in Figure 4A, exposure to over 20 μM of 5e caused a marked increase in fluorescence intensity of
2′,7′-dichlorofluorescein (DCF), which indicated the production of ROS in the mitochondria of A549
cells. Intracellular fluorescence intensity was further quantitatively analyzed using a luminescence
spectrometer. The mean fluorescence intensities increased by 30.9%, 49.1%, and 66.1% after treatment
with 20, 30, and 40 μM compound 5e, respectively. Similar results were obtained for 7-HC. The relative
fluorescence intensity (% of control) was shown in Figure 4B after the cells were co-incubated with 5e

Molecules 2015, 20
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for 48 h. ROS overproduction is implicated in mediation of apoptosis and has been described as an early
event [26]. Elevated intracellular ROS levels indicate that the balance between ROS generation and
elimination is disrupted, which eventually results in cell apoptosis. Therefore, mitochondrial ROS
overproduction and malfunction are responsible for compound 5e-induced apoptosis.
200
(A)
150
100
50
150
100
50
150
100
50
0
0
0
2
3
4
5
2
3
4
5
2
3
4
5
0
10 10 10 10
10 10 10 10
0
0 10 10 10 10
Control
7-HC, 20 μM
Compd. 5e,10 μM
150
100
50
150
100
50
120
90
60
30
0
0
0
2
3
4
5
2
3
4
5
2
3
4
5
0 10 10 10 10
0 10 10 10 10
0
10 10 10 10
Compd. 5e, 20 μM
Compd. 5e, 30 μM
Compd. 5e, 40 μM
Ros
180
(B)
*
160
140
120
100
80
*
60
1#
2#
3#
4#
5#
6#
Figure 4. Effects of compound 5e on the intracellular ROS level in A549 cells. (A) ROS
level analysis using flow cytometry; (B) Quantitative analysis of relative fluorescence intensity
(% of control) using a luminescence spectrometer. A549 cells were treated with 5e for 48 h
and stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) before monitoring
ROS level. Notes of Figure 4B, 1#: Control; 2#: 7-HC (20 µM); 3#: 5e (10 µM); 4#: 5e
(20 µM); 5#: 5e (30 µM); 6#: 5e (40 µM). Each bar represents the mean ± SD of three
independent experiments; * p < 0.05 vs. control.
2.3. Absorption Spectra, Fluorescence Spectra, and Living Cell Imaging
Fluorescence bioimaging provides a facile and less cell-damaging means of visualizing analytes of
biological interest in living cells. From the viewpoint of the potential application of the synthesized
3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives in biological imaging, we investigated these
compounds’ photophysical performance. Firstly, the ultraviolet-visible (UV-Vis) absorption spectra of
several representative compounds were recorded in 1,2-dichloroethane solution (final concentration:

Molecules 2015, 20
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50 μM). As shown in Figure 5, these compounds have a nearly identical absorption band ranging from
360 to 410 nm. The fluorescence spectra in the excitation of 360 nm were recorded at the concentration
of 5 μM (Figure 6). The compounds 5g and 6a–g produce a strong blue emission band in the range of
430–445 nm, in comparison with 7-HC which has very weak fluorescence emission at 396 nm. At the same
concentration (e.g., 5 μM), the fluorescence intensities of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid
esters 6a–g are sequentially elevated with the increase in the chain length of the terminal alkyl groups
(Figure 6B). Their fluorescence quantum yields (Φf) in the solution state were determined using the
standard procedures, with quinine sulphate (QS) dissolved in 0.1 M sulphuric acid as a reference standard
(Φf = 0.54) [27]. The UV-Vis and fluorescence data including the absorption (λabs) and fluorescence (λem)
maximal values, Stokes shift (νabs − νem), and Φf are summarized in Table 4. Compared with 7-HC, a
remarkable red shift phenomenon in absorption and emission spectra of compounds 5g and 6a–g was
observed. The above eight compounds display good Φf in the range of 0.44–0.66, which is superior to
compounds 5a–f, 5h, 5i, and 7-HC with very low Φf (<0.1). Compound 6g has the highest Φf of 0.66. It
is quite interesting that, as far as 3-oxo-3H-benzo[f]chromene-2-carboxylic acid esters are concerned,
the Φf regularly increase with the prolongation of aliphatic chain, which is coincide with the changing
trend of fluorescence intensity. Moreover, the Φf value of n-propyl ester is higher than i-propyl ester,
suggesting that linear chain alkyl group may lead to the increase of Φf, compared with branched chain
alkyl group if their carbon atoms in the alcoholic portion are equal.
(A)
382 nm
5c
1.0
0.8
0.6
5e
5f
5g
7-HC
0.4
0.2
0.0
300
350
400
450
500
Wavelength (nm)
(B)
0.8
375 nm
6a
6b
6c
6d
6f
0.6
0.4
0.2
0.0
6g
300
350
400
450
500
Wavelength (nm)
Figure 5. The absorption spectra of several representative compounds. (A) Compounds 5c, 5e,
5f, 5g, 7-HC; (B) Compounds 6a, 6b, 6c, 6d, 6f, 6g. The above compounds (final concentration:
50 μM) were dissolved in 1,2-dichloroethane.

Molecules 2015, 20
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441 nm
(A)
4
5c
5e
5f
2
0
450
500
550
600
Wavelength (nm)
(B)
437 nm
6e
5g
6a
6b
6c
6d
6e
6f
800
600
400
200
0
6g
6f
QS
6g
QS
6a
5g
400
450
500
550
600
Wavelength (nm)
Figure 6. The emission spectra of several representative compounds. (A) Compounds 5c, 5e,
5f; (B) Compounds 5g, 6a, 6b, 6c, 6d, 6e, 6f, 6g. The above compounds (final concentration:
5 μM) were dissolved in 1,2-dichloroethane (λex = 360 nm).
Table 4. Optical data of several representative compounds.
Entry Compound λabs, nm a λem, nm a
Stokes Shift b
Φf
1
2
5c
5e
383
383
381
370
375
375
375
375
375
375
380
319
348
442
441
440
434
437
437
437
437
437
437
441
396
445
3485 cm−1 59 nm <0.1
3433 cm−1 58 nm <0.1
3519 cm−1 59 nm <0.1
3986 cm−1 64 nm 0.49
3783 cm−1 62 nm 0.44
3783 cm−1 62 nm 0.51
3783 cm−1 62 nm 0.58
3783 cm−1 62 nm 0.52
3783 cm−1 62 nm 0.60
3783 cm−1 62 nm 0.62
3640 cm−1 61 nm 0.66
6095 cm−1 71 nm <0.1
6264 cm−1 97 nm 0.54
3
5f
4
5g
5
6a
6
6b
7
6c
8
6d
9
6e
10
11
12
13
6f
6g
7-HC
QS c
a Here refers to the λabs and λem values where the UV-Vis absorption and fluorescence intensity are maximum,
respectively; b Stokes shifts are listed in units of both inverse centimeters and nanometers; c QS dissolved in
0.1 M sulphuric acid is selected as a reference standard to calculate the Φf of the synthesized target compounds.
After this, the application of the synthesized target compounds for living cell imaging was investigated.
7-HC, 5e, 5g, and 6g were selected as representative examples. Compounds 5g and 6g, dissolved in

Molecules 2015, 20
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1,2-dichloroethane and exposed to visible light, exhibited conspicuous blue fluorescence. However, almost
no fluorescence was observed for 7-HC and 5e under the same conditions (Figure 7A). The former two
emitted very strong fluorescence once they were exposed to ultraviolet light for a few seconds (Figure 7B).
Living cells images were monitored with a confocal laser scanning microscope (CLSM) after treatment
with the above compounds (Figure 7C–E). Emissions of 430–450 nm were collected when a laser at
360 nm was used as excitation. The cytoplasmic was selectively dyed (blue) by 5g and 6g; the nucleus
was undyed (clear). Moreover, at the same concentration of 20 μM, intracellular fluorescence intensities:
6g > 5g > 7-HC. The cells stained with 7-HC released very weak fluorescence maybe due to its very low
Φf, which was consistent with the previous report [28]. Photophysical studies reveal that at least
compounds 5g and 6g with very low cytotoxicity are potential fluorescent probes for biological imaging
in living cells.
Figure 7. Fluorescence and living cell imaging of representative compounds. (A) Color
responses to visible light; (B) Fluorescence responses after excitation at 365 nm using an UV
lamp; (C–E) CLSM images after A549 cells were treated with 20 μM of 7-HC, 5g, and 6g
for 30 min, respectively.
3. Experimental Section
3.1. Chemistry
3.1.1. Material and Reagents
Proton nuclear magnetic resonance (1H-NMR) and 13C-NMR spectra were recorded in CDCl3
(or DMSO-d6) on a Bruker Avance 500 MHz NMR spectrometer at 500 and 125 MHz, respectively;
chemical shifts were reported in parts per million using tetramethylsilane as an internal standard and were
given in δ units. Uncorrected melting points were determined on an electrothermal melting point apparatus.
High-resolution mass spectra (HRMS) were recorded on a New ultrafleXtreme MALDI TOF/TOF mass
spectrometer (Bruker Daltonics Inc., Billerica, MA, USA). Column chromatography was performed on
silica gel (300–400 mesh). Analytical TLC was carried out on E. Merck 0.25 mm silica gel 60 F-254

Molecules 2015, 20
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precoated plates with detection by UV light. Unless otherwise noted, all solvents and reagents were
commercially available and used without further purification.
3.1.2. Synthesis of 3-Oxo-3H-benzo[f]chromene-2-carboxylic Acid (3)
The synthesis of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3) was described elsewhere [17], but
is summarized here for convenience. Briefly, ethanol (85 mL), Meldrum’s acid (6.5 g, 45 mmol), 2-hydroxy-
1-naphthaldehyde (7.75 g, 45 mmol), and a catalytic amount of pyridine (0.5 mL) were in sequence
added into a 100 mL flask. The mixture was stirred at 80 °C for 4 h. After cooling to room temperature,
the mixture was poured into an ice-water bath and recrystallized. The crude was washed with ethanol
repeatedly and dried in vacuo to afford the desired product 3-oxo-3H-benzo[f]chromene-2-carboxylic acid
(3) as a yellow solid (Yield: 93.1%). M.p.: 235.1–236.5 °C (Lit. 234–236 °C [29]). 1H-NMR (DMSO-d6):
δ 9.34 (s, 1H), 8.57 (d, J = 8.4 Hz, 1H), 8.29 (d, J = 9.0 Hz, 1H), 8.06 (d, J = 8.0 Hz, 1H), 7.78–7.73
(m, 1H), 7.64 (t, J = 7.2 Hz, 1H), 7.57 (d, J = 9.0 Hz, 1H). 13C-NMR (DMSO-d6): δ 164.78, 157.24, 155.49,
144.18, 136.30, 130.27, 129.50, 129.45, 126.88, 122.77, 117.68, 116.94, 112.55. ESI-HRMS (m/z): calcd.
for C14H8O4 [M − H]−: 239.03443; found: 239.03429.
3.1.3. General Procedures for the Preparation of 3-Oxo-3H-benzo[f]chromene-2-amides 5a–i and
3-Oxo-3H-benzo[f]chromene-2-carboxylic Acid Esters 6a–g
First of all, 3-oxo-3H-benzo[f]chromene-2-carbonyl chloride (4) was synthesized by the reaction of
3-oxo-3H-benzo[f]chromene-2-carboxylic acid (3, 2.40 g, 10 mmol) with thionyl chloride (2.2 mL,
30 mmol) in dry 1,2-dichloroethane (20 mL) at 60–70 °C for 4 h. After the removal of excess unreacted
thionyl chloride under reduced pressure, the intermediate 4 was obtained and used directly in the next
step without further purification. This intermediate 4 (1.29 g, 5 mmol) dissolved in dry dichloromethane
(15 mL) was dropwise added to a mixture of a primary amine (6 mmol) and a catalytic amount of
triethylamine (0.1 mL). The reaction mixture was stirred at room temperature for 10–30 min before the
solvent was removed. The crude was purified by flash silica gel column chromatography to afford the final
product 5a–i. Compounds 6a–g were also synthesized using the similar procedures, the only difference
being that the primary amines described above were replaced by different aliphatic alcohols.
N-(3,5-Dimethylphenyl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5a). Yield: 74.4%; m.p.:
277.1–279.2 °C. 1H-NMR (CDCl3): δ 9.82 (s, 1H), 8.50 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 9.1 Hz, 1H),
7.99 (d, J = 7.9 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 8.9 Hz, 1H), 7.45
(s, 2H), 6.85 (s, 1H), 2.38 (s, 6H). 13C-NMR (CDCl3): δ 161.89, 159.64, 155.04, 144.35, 138.79, 137.59,
136.17, 130.46, 129.54, 129.36, 129.21, 126.91, 126.59, 121.98, 118.32, 117.19, 116.34, 113.54, 21.45.
ESI-HRMS (m/z): calcd. for C22H17NO3 [M + Na]+: 366.11061; found: 366.10929.
N-(2,6-Diethylphenyl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5b). Yield: 35.4%; m.p.:
190.3–193.5 °C. 1H-NMR (CDCl3): δ 9.82 (s, 1H), 8.50 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 9.0 Hz, 1H), 7.99
(d, J = 8.0 Hz, 1H), 7.82–7.78 (m, 1H), 7.68 (m, 1H), 7.59 (d, J = 9.1 Hz, 1H), 7.30 (dd, J = 12.4, 5.3 Hz,
1H), 7.22 (d, J = 7.6 Hz, 2H), 2.71 (q, J = 7.6 Hz, 4H), 1.26 (t, J = 7.6 Hz, 6H). 13C-NMR (CDCl3): δ 162.10,
160.85, 155.15, 144.79, 141.22, 136.17, 132.66, 130.45, 129.56, 129.28, 129.17, 128.01, 126.90, 126.42,

Molecules 2015, 20
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122.16, 116.89, 116.36, 113.47, 25.15, 14.52. ESI-HRMS (m/z): calcd. for C24H21NO3 [M + Na]+:
394.14191; found: 394.14231.
N-(4-(Tert-butyl)phenyl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5c). Yield: 22.7%; m.p.:
180.2–181.9 °C. 1H-NMR (CDCl3): δ 9.79 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H),
7.97 (d, J = 8.1 Hz, 1H), 7.84–7.77 (m, 1H), 7.69–7.63 (m, 1H), 7.56 (d, J = 9.0 Hz, 1H), 7.48–7.43
13
(m, 2H), 7.25–7.19 (m, 1H), 6.71–6.64 (m, 1H), 1.38 (s, 9H). C-NMR (CDCl3): δ 161.87, 159.58,
154.98, 147.80, 144.26, 143.70, 141.48, 136.09, 135.18, 130.43, 129.24, 126.89, 125.99, 122.02, 120.30,
117.14, 116.32, 114.98, 113.51, 34.49, 33.93, 31.40. ESI-HRMS (m/z): calcd. for C24H21NO3 [M + Na]+:
394.14191; found: 394.14090.
N-(4-Methoxyphenyl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5d). Yield: 61.2%; m.p.:
259.3–261.8 °C (Lit. 259–261 °C [30]). 1H-NMR (CDCl3): δ 9.82 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.18
(d, J = 9.0 Hz, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.82 (t, J = 7.7 Hz, 1H), 7.73 (d, J = 8.9 Hz, 2H), 7.68
(t, J = 7.5 Hz, 1H), 7.58 (d, J = 9.0 Hz, 1H), 6.97 (d, J = 8.9 Hz, 2H), 3.86 (s, 3H). 13C-NMR (CDCl3):
δ 161.93, 156.70, 154.98, 144.19, 136.08, 131.01, 130.47, 129.53, 129.30, 129.18, 126.89, 122.09,
122.04, 117.18, 116.34, 114.23, 113.56, 55.51. ESI-HRMS (m/z): calcd. for C21H15NO4 [M + Na]+:
368.08988; found: 368.08763.
N-(4-Ethoxyphenyl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5e). Yield: 80.3%; m.p.:
205.1–206.6 °C. 1H-NMR (CDCl3): δ 9.82 (s, 1H), 8.51 (d, J = 8.4 Hz, 1H), 8.17 (d, J = 9.0 Hz, 1H),
7.99 (d, J = 8.1 Hz, 1H), 7.82 (t, J = 7.2 Hz, 1H), 7.77–7.62 (m, 3H), 7.58 (d, J = 9.0 Hz, 1H),
7.04–6.82 (m, 2H), 4.08 (d, J = 7.0 Hz, 2H), 1.46 (s, 3H). 13C-NMR (CDCl3): δ 161.94, 159.45, 156.10,
154.99, 144.17, 136.06, 130.91, 130.48, 129.55, 129.31, 129.19, 126.89, 122.08, 117.22, 116.36, 114.85,
113.58, 63.72, 14.88. ESI-HRMS (m/z): calcd. for C22H17NO4 [M + H]+: 360.12358; found: 360.12223.
3-Oxo-N-(4-(trifluoromethyl)phenyl)-3H-benzo[f]chromene-2-carboxamide (5f). Yield: 73.9%; m.p.:
231.3–233.9 °C. 1H-NMR (CDCl3): δ 9.81 (s, 1H), 8.49 (d, J = 8.3 Hz, 1H), 8.16 (d, J = 9.0 Hz, 1H),
7.98 (d, J = 8.0 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 7.67 (t, J = 7.6 Hz, 1H), 7.57 (d, J = 8.9 Hz, 1H), 7.44
(s, 2H), 7.27 (s, 1H), 6.84 (s, 1H). 13C-NMR (CDCl3): δ 161.97, 160.16, 155.29, 145.02, 140.81, 136.69,
130.50, 129.54, 129.53, 129.50, 129.30, 127.08, 126.36, 126.34, 121.98, 120.23, 116.55, 116.34, 113.50.
ESI-HRMS (m/z): calcd. for C21H12F3NO3 [M − H]−: 382.06910; found: 382.06886.
3-Oxo-N-propyl-3H-benzo[f]chromene-2-carboxamide (5g). Yield: 88.1%; m.p.: 182.3–184.9 °C.
1H-NMR (CDCl3): δ 9.70 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.13 (d, J = 9.0 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H),
7.79 (t, J = 7.5 Hz, 1H), 7.68–7.61 (m, 1H), 7.53 (d, J = 9.0 Hz, 1H), 3.50 (dd, J = 13.0, 7.0 Hz, 2H),
1.77–1.68 (m, 2H), 1.05 (t, J = 13.5 Hz, 3H). 13C-NMR (CDCl3): δ 161.86, 161.60, 154.83, 143.80, 135.70,
130.39, 129.56, 129.13, 126.75, 122.04, 117.08, 116.33, 113.38, 41.67, 22.74, 11.56. ESI-HRMS (m/z):
calcd. for C17H15NO3 [M + Na]+: 304.09496; found: 304.09352.
N-Cyclohexyl-3-oxo-3H-benzo[f]chromene-2-carboxamide (5h). The process for the synthesis of
compound 5h was described previously [31]. Yield: 89.1%; m.p.: 178.6–180.2 °C. 1H-NMR (CDCl3): δ
9.72 (s, 1H), 8.49 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.83–7.77 (m,

Molecules 2015, 20
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1H), 7.69–7.63 (m, 1H), 7.54 (d, J = 9.0 Hz, 1H), 4.12–4.01 (m, 1H), 2.04 (m, 2H), 1.84–1.77 (m, 2H),
1.70–1.55 (m, 2H), 1.52–1.35 (m, 2H), 1.32–1.19 (m, 2H). 13C-NMR (CDCl3): δ 161.60, 160.83, 154.81,
143.76, 135.64, 130.40, 129.58, 129.14, 129.11, 126.73, 122.08, 117.29, 116.36, 113.43, 48.61, 32.80,
29.72, 25.62, 24.70. ESI-HRMS (m/z): calcd. for C20H19NO3 [M + H]+: 322.14432; found: 322.14298.
(Z)-N-(Octadec-9-en-1-yl)-3-oxo-3H-benzo[f]chromene-2-carboxamide (5i). Yield: 60.9%; m.p.:
79.1–81.8 °C. 1H-NMR (CDCl3): δ 9.62 (s, 1H), 8.40 (d, J = 8.5 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H), 7.91
(d, J = 8.1 Hz, 1H), 7.74 (dd, J = 11.3, 4.1 Hz, 1H), 7.61 (t, J = 7.5 Hz, 1H), 7.47 (d, J = 9.0 Hz, 1H),
5.44–5.30 (m, 2H), 3.51 (dd, J = 13.1, 7.0 Hz, 2H), 2.02 (d, J = 4.7 Hz, 4H), 1.72–1.62 (m, 2H),
13
1.38–1.19 (m, 22H), 0.88 (t, J = 6.9 Hz, 3H). C-NMR (CDCl3): δ 161.73, 161.49, 154.73, 143.59,
135.60, 130.31, 129.88, 129.82, 129.08, 126.70, 121.96, 117.01, 116.26, 113.28, 40.00, 31.91, 29.78,
29.76, 29.70, 29.53, 29.45, 29.33, 29.26, 27.22, 27.07, 22.69, 14.13. ESI-HRMS (m/z): calcd. for
C32H43NO3 [M − H]−: 488.31647; found: 488.31728.
Methyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6a). Yield: 92.1%; m.p.: 161.6–163.2 °C (Lit.
162–163 °C [32]). 1H-NMR (CDCl3): δ 9.39 (s, 1H), 8.35 (d, J = 8.3 Hz, 1H), 8.12 (d, J = 9.0 Hz, 1H), 7.94
(d, J = 8.1 Hz, 1H), 7.76 (m, 1H), 7.63 (m, 1H), 7.48 (d, J = 9.1 Hz, 1H), 4.02 (s, 3H). 13C-NMR (CDCl3):
δ 164.24, 156.96, 156.12, 145.16, 136.36, 130.23, 129.45, 129.29, 129.21, 126.63, 121.53, 116.68, 116.03,
112.36, 53.05. ESI-HRMS (m/z): calcd. for C15H10O4 [M + Na]+: 277.04768; found: 277.04710.
Ethyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6b). Yield: 93.1%; m.p.: 116.6–118.1 °C (Lit.
118–119 °C [32]). 1H-NMR (CDCl3): δ 9.30 (s, 1H), 8.31 (d, J = 8.2 Hz, 1H), 8.10 (d, J = 9.0 Hz, 1H),
7.94 (d, J = 8.1 Hz, 1H), 7.76 (m, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.46 (m, 1H), 4.50 (d, J = 7.1 Hz, 2H),
1.48 (s, 3H). 13C-NMR (CDCl3): δ 163.56, 156.89, 155.94, 144.49, 136.14, 130.17, 129.38, 129.26, 129.13,
126.58, 121.47, 116.64, 116.40, 112.26, 62.09, 14.35. ESI-HRMS (m/z): calcd. for C16H12O4 [M + Na]+:
291.06333; found: 291.06174.
n-Propyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6c). Yield: 88.5%; m.p.: 85.2–86.9 °C (Lit.
1
86–88 °C [33]). H-NMR (CDCl3): δ 9.26 (s, 1H), 8.27 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H),
7.92 (d, J = 8.1 Hz, 1H), 7.74 (t, J = 7.7 Hz, 1H), 7.60 (t, J = 7.5 Hz, 1H), 7.43 (d, J = 9.0 Hz, 1H), 4.37 (t,
13
J = 6.8 Hz, 2H), 1.86 (m, 2H), 1.08 (t, J = 7.4 Hz, 3H). C-NMR (CDCl3): δ 163.66, 156.83, 155.93,
144.37, 136.09, 130.15, 129.37, 129.26, 129.13, 126.57, 121.43, 116.64, 116.47, 112.24, 67.56, 22.07,
10.51. ESI-HRMS (m/z): calcd. for C17H14O4 [M + Na]+: 305.07898; found: 305.07756.
Isopropyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6d). Yield: 85.2%; m.p.: 95.2–97.9 °C. 1H-NMR
(CDCl3): δ 9.23 (s, 1H), 8.29 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 9.0 Hz, 1H), 7.92 (d, J = 8.1 Hz, 1H),
7.76–7.72 (m, 1H), 7.62–7.58 (m, 1H), 7.44 (d, J = 9.0 Hz, 1H), 5.32 (m, 1H), 1.44 (d, J = 6.3 Hz, 6H).
13C-NMR (CDCl3): δ 162.97, 156.88, 155.85, 143.96, 135.97, 130.17, 129.33, 129.26, 129.07, 126.54,
121.47, 116.89, 116.69, 112.25, 69.81, 29.72, 21.91. ESI-HRMS (m/z): calcd. for C17H14O4 [M + Na]+:
305.07898; found: 305.07824.
n-Butyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6e). Yield: 83.1%; m.p.: 60.1–62.6 °C (Lit.
1
60–62 °C [33]). H-NMR (CDCl3): δ 9.27 (s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 8.08 (d, J = 9.0 Hz, 1H),

Molecules 2015, 20
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7.92 (d, J = 8.1 Hz, 1H), 7.74 (m, 1H), 7.60 (m, 1H), 7.44 (d, J = 9.0 Hz, 1H), 4.41 (t, J = 6.8 Hz, 2H),
1.94–1.77 (m, 2H), 1.54–1.47 (m, 2H), 1.03 (t, J = 7.4 Hz, 3H). 13C-NMR (CDCl3): δ 163.71, 156.86,
155.96, 144.42, 136.11, 130.18, 129.40, 129.27, 129.13, 126.57, 121.46, 116.68, 116.52, 112.28, 65.93,
30.70, 19.23, 13.82. ESI-HRMS (m/z): calcd. for C18H16O4 [M + Na]+: 319.09463; found: 319.09340.
Tert-pentyl-3-oxo-3H-benzo[f]chromene-2-carboxylate (6f). Yield: 83.6%; m.p.: 49.4–51.8 °C.
1H-NMR (CDCl3): δ 9.32 (s, 1H), 8.32 (d, J = 8.4 Hz, 1H), 8.11 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 8.1 Hz,
1H), 7.77 (m, 1H), 7.66–7.61 (m, 1H), 7.48 (d, J = 9.0 Hz, 1H), 1.56–1.42 (m, 6H), 1.40 (m, 2H), 0.92
13
(t, J = 7.1 Hz, 3H). C-NMR (CDCl3): δ 164.35, 156.87, 151.96, 141.45, 136.11, 132.38, 129.21,
127.12, 121.34, 119.67, 117.18, 114.52, 107.49, 72.22, 36.81, 25.28, 8.73. ESI-HRMS (m/z): calcd. for
C19H18O4 [M + Na]+: 333.11028; found: 333.11101.
1
n-Hexyl 3-oxo-3H-benzo[f]chromene-2-carboxylate (6g). Yield: 88.4%; m.p.: 44.3–46.7 °C. H-NMR
(CDCl3): δ 9.09 (s, 1H), 8.15 (d, J = 8.5 Hz, 1H), 7.99 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.68
(t, J = 8.2 Hz, 1H), 7.58–7.53 (m, 1H), 7.35–7.30 (m, 1H), 4.37 (t, J = 6.9 Hz, 2H), 1.82–1.74
(m, 2H), 1.47–1.29 (m, 6H), 0.92–0.87 (m, 3H). 13C-NMR (CDCl3): δ 163.49, 156.67, 155.73, 144.07,
135.97, 130.00, 129.19, 129.09, 126.52, 121.27, 116.48, 116.26, 112.04, 66.13, 31.44, 28.59, 25.60,
22.55, 14.04. ESI-HRMS (m/z): calcd. for C20H20O4 [M + Na]+: 347.12593; found: 347.12554.
3.2. Biological Activity
3.2.1. Cell Antiproliferative Activity Assay
The proliferation inhibitory activities of the synthesized target compounds 5a–i and 6a–g were
evaluated using NSCLC cell lines (A549 and NCI-H460) by the MTT method in vitro with 7-HC and
cisplatin as positive reference drugs. A549 and NCI-H460 cell lines were purchased from American
Type Culture Collection (Rockville, MD, USA). Both cell lines were cultured in Dulbecco’s modified
Eagle medium, 10% fetal bovine serum, 100 μg/mL penicillin, and 100 μg/mL streptomycin in humidified
air at 37 °C. After the cells were seeded in 96-well culture plates and co-incubated with the compounds
for different time, 20 μL of MTT solution (5 mg/mL) was added to each well and incubated for another
4 h at 37 °C. The formazan precipitate was dissolved in 100 μL DMSO and the absorbance at 495 nm
of each well was measured using a microplate reader. All assays were conducted with four parallel
samples. IC50 values were obtained by nonlinear regression using Origin software (version 8.0).
3.2.2. Apoptosis Detection by Flow Cytometry
The extent of apoptosis was measured quantitatively by Annexin V binding assay [34]. Briefly, A549
cells in 60 mm dishes were treated with the indicated concentrations of compound 5e or positive
reference 7-HC at 37 °C. The cells floating in the supernatant were combined with the adherent fraction,
washed with phosphate buffered saline (PBS) thrice, and incubated with Annexin V-FITC and PI for
15 min at 37 °C in the dark according to the manufacturer’s instructions (BD Biosciences, San Jose, CA,
USA). The samples were immediately analyzed using a FACSCalibur flow cytometry system. The
percentages of viable (Annexin V−/PI−), early apoptotic (Annexin V+/PI−), late apoptotic (Annexin
V+/PI+), and necrotic (Annexin V−/PI+) cells were determined using FlowJo software (Tree Star, Inc.,

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Ashland, OR, USA). For each sample, 1 × 104 cells were collected. All experiments were performed
in triplicate.
3.2.3. Cell Cycle Distribution Analysis
Cell-cycle analysis was carried out by flow cytometry, as previously described [35]. In brief, after
treatment with compound 5e for different time, A549 cells were washed with PBS, fixed with 70% cold
ethanol, and stored at −20 °C overnight. Fixed cells were washed with PBS again and sequentially
incubated with RNase A (50 μg/mL) for 30 min and PI (50 μg/mL) for 15 min in the dark. The
distribution of the cell cycle was determined by flow cytometry. The data were analyzed using ModFit
software (Becton Dickinson, San Jose, CA, USA). All assays were conducted with three parallel samples,
and 1 × 104 cells were collected for each sample.
3.2.4. Determination of Intracellular ROS Production
The DCFH-DA assay was used to determine the intracellular ROS level [36]. After treatment with
compound 5e, A549 cells were trypsinized, washed with PBS, and incubated with DCFH-DA at a final
concentration of 10 μM for additional 20 min at 37 °C. The fluorescence intensity of the cell suspensions
was measured using flow cytometry. For each sample, 1 × 104 cells were collected. All experiments
were performed in triplicate.
A fluorescence spectrometer was used for quantitative evaluation of intracellular ROS level
changes [37]. After A549 cells were treated with compound 5e, harvested and washed with PBS, the
fluorescence intensity was detected using a Perkin Elmer LS55 spectrofluorimeter (PerkinElmer, Inc.,
Waltham, MA, USA). The results are expressed as the relative ROS level with the untreated cells. All
experiments were performed in triplicate.
3.3. UV-Vis Absorption and Fluorescence Experiments
The UV-Vis spectra were recorded on an Agilent Cary 60 (Agilent Technologies, Santa Clara, CA,
USA) spectrometer. The fluorescence emission was measured using the above spectrofluorimeter
(see Section 3.2.4). The fluorescence quantum yield of QS in 0.1 M sulphuric acid solution was used as a
reference standard. The following equation was used to calculate the fluorescence quantum yield of the
target compounds:
ꢀꢁꢂꢃꢄꢁꢅ
ꢀꢃꢂꢁꢄꢃꢅ
Φs = Φr
(1)
where Φ is the fluorescence quantum yield. I stands for the integrated area under the emission curves.
The subscripts s and r stand for sample and reference, respectively. A is the absorbance at a particular
excitation wavelength; n is the refractive index of the medium. For living cell imaging, A549 cells were
seeded on 35 mm glass-bottomed dishes at a density of 1 × 105 cells/dish in culture medium and
incubated overnight. The cells were treated with 10 and 20 μM of 7-HC, 5g, and 6g, respectively. After
washing twice with PBS, the cells were immediately observed under a CLSM (Carl Zeiss LSM710,
Oberkochen, Germany) using the excitation channel (λex = 360 nm).

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3.4. Data Analysis
Except where mentioned, data were presented as means ± SD. One-way analysis of variance test was
performed on the data to assess the impact of the variables on the results (n = 3 or more). A p value of
≤0.05 was considered to be statistically significant.
4. Conclusions
In summary, two series of 3-oxo-3H-benzo[f]chromene-2-carboxylic acid derivatives were successfully
prepared by a simple and convenient method. Among all the target compounds, 5e showed the strongest
proliferation inhibitory activity. In vitro pharmacological analysis demonstrated that compound 5e exerted
its activity against A549 cell line by inducing apoptosis and intracellular ROS generation, arresting cell
cycle at the G0/G1 phase, suggesting it may be a promising lead for antitumor drug discovery in the
future. Photophysical studies revealed that compound 6g exhibited very low cytotoxicity and superior
fluorescence properties, implying it may be a potential candidate for biological imaging.
Supplementary Materials
Supplementary materials include the following items: (1) 1H-NMR spectra of the compounds 5e, 5g,
and 6g; (2) 13C-NMR spectrum of the compound 5g; (3) HRMS spectra of the compounds 5e and 6g.
These data can be accessed at: http://www.mdpi.com/1420-3049/20/10/18565/s1.
Acknowledgments
This work was supported by the National Natural Science Foundation of China (No. 21364002,
21262006, 21462007, 81260472, 201502028, and 81260472), National Basic Research Program of China
(2012CB723501), IRT1225, Guangxi Natural Science Foundation of China (No. 2013GXNSFBA019200,
2013GXNSFGA019001, and 2012GXNSFDA053007), State Key Laboratory Cultivation Base for
the Chemistry and Molecular Engineering of Medicinal Resources (No. CMEMR2012-A01 and
CMEMR2013-C03), Guangxi Education Department Common Project (No. 2013YB025), Guangxi
Pharmaceutical Industry Talent Small Highland Project (No. 1313 and 1105), and Guangxi Normal
University Key Project (No. 2011ZD006).
Author Contributions
The synthesis was done by Xiao-Bo Fu, Xian-Fu Wang, and Jia-Nian Chen. The biological activity
and fluorescence analyses were finished by De-Wen Wu and Ting Li, Xing-Can Shen and Jiang-Ke Qin
contributed reagents/materials/analysis tools. All of the authors contributed equally to the preparation of
the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

Molecules 2015, 20
18582
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Sample Availability: Samples of the compounds 5e, 5g, and 6g are available from the authors.
© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
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(http://creativecommons.org/licenses/by/4.0/).