Novel coumarin-containing aminophosphonatesas antitumor agent: synthesis, cytotoxicity, DNA-Binding and apoptosis evaluation

Article abstract of DOI:10.3390/molecules200814791

A series of novel coumarin-containing α-aminophosphonates were synthesized and evaluated for their antitumor activities against Human colorectal (HCT-116), human nasopharyngeal carcinoma (human KB) and human lung adenocarcinoma (MGC-803) cell lines in vit

Full text of DOI:10.3390/molecules200814791

Molecules 2015, 20, 14791-14809; doi:10.3390/molecules200814791
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Novel Coumarin-Containing Aminophosphonatesas
Antitumor Agent: Synthesis, Cytotoxicity, DNA-Binding and
Apoptosis Evaluation
Ya-Jun Li 1,2,†, Cai-Yi Wang 3,†, Man-Yi Ye 1, Gui-Yang Yao 1,* and Heng-Shan Wang 1,*
1
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal
Resources, School of Chemistry & Chemical Engineering of Guangxi Normal University,
Guilin 541004, China; E-Mails: liyajungxnu@163.com (Y.-J.L.); baobei_mayu@163.com (M.-Y.Y.)
2
College of Medicine and Pharmacy, Hunan Polytechnic of Environment and Biology,
Hengyang 421000, China
3
College of Chemical and Material Science, Hebei Normal University, Shijiazhuang 050024, China;
E-Mail: wangcaiyigxnu@163.com
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: yaoguiyangmayu@126.com (G.-Y.Y.);
whengshan@163.com (H.-S.W.); Tel./Fax: +86-773-2120958 (H.-S.W.).
Academic Editor: Jean Jacques Vanden Eynde
Received: 29 June 2015 / Accepted: 10 August 2015 /Published: 13 August 2015
Abstract: A series of novel coumarin-containing α-aminophosphonates were synthesized
and evaluated for their antitumor activities against Human colorectal (HCT-116), human
nasopharyngeal carcinoma (human KB) and human lung adenocarcinoma (MGC-803)
cell lines in vitro. Compared with 7-hydroxy-4-methylcoumarin (4-MU), most of the
derivatives showed an improved antitumor activity. Compound 8j (diethyl 1-(3-(4-methyl-
2-oxo-2H-chromen-7-yloxy) propanamido)-1-phenylethyl-Phosphonate), with IC50 value of
8.68 μM against HCT-116 cell lines, was about 12 fold than that of unsubstituted parent
compound. The mechanism investigation proved that 8c, 8d, 8f and 8j were achieved through
the induction of cell apoptosis by G1 cell-cycle arrest. In addition, the further mechanisms of
compound 8j-induced apoptosis in HCT-116 cells demonstrated that compound 8j induced
the activations of caspase-9 and caspase-3 for causing cell apoptosis, and altered anti- and
pro-apoptotic proteins. DNA-binding experiments suggested that some derivatives bind to
DNA through intercalation. The results seem to imply the presence of an important synergistic

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effect between coumarin and aminophosphonate, which could contribute to the strong
chelating properties of aminophosphonate moiety.
Keywords: 7-hydroxy-4-methylcoumarin; α-aminophosphonates; synthesis; cytotoxicity;
cell cycle; DNA binding
1. Introduction
Chemical modification of bioactive components of medicinal herbs is one of the most common
approaches in drug discovery for new drugs and improved therapeutic properties. Coumarins are an
important class of compounds obtained from nature and synthetic origin that possess diverse
pharmacological and biological activities such as antitumor [1], anti-inflammatory [2], anticoagulant [3],
anti-HIV [4], herbicidal [5] and fungicidal [6] activities. The pharmacological and biochemical properties
of coumarins depend upon the pattern of substitution of the naturally occurring scaffold. The naturally
occurring 7-hydroxy coumarin derivatives have been investigated as potential lead structures for cancer
drug development [7]. Some 4-hydroxycoumarins, bearing an aryl group in the third position, inhibit
cancer cell proliferation [8].
Novobiocin, a member of amino-4-hydroxycoumarin family, has been shown to possess anticancer
activity, owing to its amide side chain, the coumarin ring and the sugar moiety [9]. However, little
attention has been paid to the cytotoxicity and DNA binding affinity studies of 4-MU, which belongs to
the same class of coplanar coumarin as novobiocin [10]. In general, most DNA binding compounds
containing a linear or angular planar chromophore with apolyaromatic ring can influence the structures
and physiological functions of DNA [11]. To enhance antitumor activity, generally the DNA binding
compounds should carry one or two flexible basic side chains on chromophore [12]. The flexible basic
side chains are highly influential in directing the thermodynamic-binding mechanism, geometry of the
ligand-DNA complex and sequence selectivities [13].
Recently, Łukasz Berlicki and his co-workers [14] suggested that the N-C-P molecular fragment and
its chemistry could offer many possibilities for structural modifications, which have resulted in broad
biological relevance. As an important class of compounds containing N-C-P molecular fragment,
α-aminophosphonates (APAs) and their derivatives are an important class of compounds that exhibit
intriguing biological activities [15], especially antitumor [16] and inhibitors of enzyme related to tumor
genesis and invasions [17]. Because alkaline phosphatase is known to be over expressed in the extracellular
space of specific tumor cells such as ovarian and hepatic carcinoma cells, introducing a phosphate group
for targeted delivery appears to be a reasonable strategy to increase solubility and enhance transport
through cellular membrane [18]. Some phosphate groups also exhibit high affinity to calcium ions and
have been used to design targeted drugs for bone cancer [19]. Since phosphonate esters can be hydrolyzed
under biological conditions, aminophosphonate esters are a good choice in designing targeted anticancer
drug [20].
Recently, it was found that APAs group could enhance the antitumor activities of coumarin in our
laboratory [21]. As part of our series of research work, coumarin-bearing α-aminophosphonates were
designed in our recent study to establish more efficient antitumor lead compounds. This work aims to

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synthesize a group of new coumarin derivatives with improved DNA binding affinity and antitumor
activity by incorporating various α-aminophosphonates into the chromophore. The DNA binding
capacity of the new coumarin derivatives was evaluated and their cytotoxicity was assessed on three
types of tumor cell lines. Flow-cytometric analysis was also employed in the study to reveal the
mechanism of how some compounds showing higher activity killed HCT-116 cells.
2. Results and Discussion
2.1. Synthesis of Coumarin Derivatives
The synthetic steps of the target compounds are shown in Scheme 1. The synthesis of 7-hydroxy-4-
methylcoumarin 2 was carried out according to our previous work [21], which included the condensation
of phenols with ethyl acetoacetate in the presence of the catalysis sodium bisulfate. Compound 3 was
then obtained in good yields by the coupling reaction of 2 with 3-bromopropionic acid in the presence
of potassium carbonate.
O,O′-dialky ((N-(phenylmethylene)-α-amino)-α-(substituted phenyl)methyl) phosphonates 5, obtained
by reacting substituted benzaldehyde 4 with ammonium acetate and O,O′-dialkylphosphite, were
converted to O,O′-dialkylα-amino-(α-(substituted phenyl)methyl) phosphonate7 via hydrolysis [22]. The
α-aminophosphonates 7 were then coupled 3 to provide compounds 8a–8j in satisfactory yields, which
was purified by column chromatography over silica gel.
Scheme 1. General synthetic route for compound 8a–8j.
2.2. Cytotoxicity against Tumor Cells
Table 1 shows the IC50 values (cytotoxicity potency indexes) and the inhibitory rates of cell viability
of compounds 8a–8j against HCT-116, human KB and MGC-803 cells in culture. All the compounds were
found to be more toxic against human KB cells than HCT-116 and MGC-803 cells. The lead compound
4-MU exhibited insignificant cytotoxicity with high IC50 values, more than 100 μM on HCT-116 cells
and more than 50 μM on human KB cells. In comparison, most of the derivatives had much more potent

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cytotoxicity with significantly lower IC50 values on the tumor cells. Compound 8j was the most effective
one against HCT-116 and human KB cells with IC50 values being eleven to twelve fold lower than
those for the lead compound under the same experimental conditions. Therefore, it is suggested that the
introduction of α-aminophosphonates to 4-MU can obviously improve the antitumor activity of the lead
compound. In comparison, activities of compounds against normal cells (Human Umbilical Vein Endothelial
Cells, HUVEC) were also examined (Table 1). The IC50 values proved that the anti-proliferative activity
of some compounds were much higher against cancer cells than normal cells. Compound 8j exhibited
more sensitivity to cancer cells compared to normal cells. Cytotoxicity of the some compounds were
much better than 5-fluoro-2,4(1H,3H)pyrimidinedione (5-Fu) against cancer cells. The IC50 values of 8j
against the MGC-803, HCT-116 and human KB cell lines were 14.55 μM, 8.68 μM and 0.08 μM,
respectively, whereas those of 5-Fu were 15.32 μM, 10.05 μM, and 1.23 μM, respectively.
Table 1. Cytotoxic evaluation under no irradiation of compounds 8a–8j.
IC50 a, μM
Compounds
R1
R2
MGC-803
>100
HUVEC
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
HCT-116
>100
KB
2
8a
8b
8c
-
H
-
>50
Ph
>100
>100
>50
H
o-F-Ph
o-Cl-Ph
p-Cl-Ph
o-Br-Ph
m-Br-Ph
p-Br-Ph
o-OCH3-Ph
2-Na
>100
>100
>50
H
79.38 ± 3.82
76.85 ± 3.89
67.49 ± 3.26
74.16 ± 3.36
85.63 ± 2.83
36.85 ± 1.96
41.47 ± 2.86
14.55 ± 1.11
88.68 ± 1.69
86.18 ± 1.86
90.55 ± 1.09
25.75 ± 1.21
88.52 ± 4.62
23.68 ± 5.89
38.90 ± 0.04
8.68 ± 3.20
10.05 ± 2.20
33.4 ± 1.20
7.8 ± 5.36
9.55 ± 0.09
0.50 ± 2.68
7.27 ± 6.18
0.45 ± 1.03
0.79 ± 1.70
0.08 ± 1.12
1.23 ± 1.09
8d
8e
H
H
8f
H
8g
8h
8i
H
H
H
8j
CH3
Ph
5-Fu
15.32 ± 1.96 56 ± 3.21
a IC50 values are presented as the mean ± SD (standard error of the mean) from three separated experiments.
According to the antitumor activity data (Table 1), the IC50 values of the compounds, which have
α-aminophosphonates containing bromo-substituted phenyl (R2) and hydrogen (R1) as side chains, were in
the order of 8e > 8g > 8f. It was the same to derivatives with chloro-substituted phenyl (R2) and hydrogen
(R1) (8c > 8d). The IC50 values of 8h, bearing α-aminophosphonate with ortho-methoxyl-substituted
phenyl (R2) and hydrogen (R1) as side chain, against the HCT-116 and human KB cell lines were 23.68 μM
and 0.45 μM, respectively, whereas those of 8d and 8i were 86.18 μM and 7.8 μM, 38.90 μM and 0.79 μM,
respectively. Compound 8j, which has methyl and phenyl as R1 and R2, respectively, was the most
effective one. From the above results, some interesting structure-activity relationships can be disclosed
that: (i) the substituted position of the phenyl group of α-aminophosphonate have evident effect
on the antitumor activity: meta-substitution > para-substitution > othor-substitution; (ii) the different
substituents of the phenyl group of α-aminophosphonate have different effects on the antitumor activity:
methoxylgroup > bromine atom > chlorine atom > fluorine atom, an explanation for this phenomenon
may be because the differences of their electrical charge cause different lipotropy which can influence

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diffuse through the plasma membrane; (iii) Compared with hydrogen as R1, the methyl as R2 shows
higher cytotoxicity.
2.3. DNA Binding Properties
2.3.1. Fluorescence Emission Titration
The DNA binding properties of compounds 8a–8j were evaluated based on their affinity or interaction
with CT-DNA, measured with fluorescence spectrometric titration [23]. Figure 1a shows the fluorescence
spectrometric titration spectra of 8j, and Figure 1b shows the corresponding peak fluorescence intensity at
increasing CT-DNA concentration. The results show that the fluorescence of 8j could be quenched by
DNA, and increasing the concentration of CT-DNA resulted in a decrease in fluorescence intensity of
8j. The fluorescence quenching of 8j by CT-DNA indicated that 8j had a strong interaction with CT-DNA
as most of the intercalators did [24]. The titration spectra and peaks for other compounds exhibited the
similar trend of changes with the CT-DNA concentration (see Figures S1–S10).
Figure 1. (a) Spectrofluorimetric titration of 8j (2.0 × 10−3 M) with CT-DNA of increasing
concentration (0–360 μM) in 2.5 mL Tris-HCl buffer (pH = 7.2) at room temperature, ex
325 nm; (b) the peak fluorescence intensity in (a) at 383 nm vs. CT-DNA concentration.
The calibration curve for determination of DNA was constructed by adding different proportion of
DNA to a 2.0 × 10−3 M compound 8j solution (pH = 7.2 Tris-HCl buffer). The results exhibited good
consistency with the Stern-Volmer equation [25]:
F0/F = 1 + KSV[Q] = 1 + KQτ0[Q]
(1)
where F0 and F are the steady-state fluorescence intensities in the absence and presence of the quencher,
respectively. [Q] is the concentration of quencher. KSV is the Stern-Volmer constant and KQ is the
bimolecular quenching rate constant. τ0 is the lifetime of the fluorophore in the absence of quencher and
for most biomolecules τ0 is about 10−8 s [26]. The Stern-Volmer constant, KSV, for compound 8j has been
determined from the plot of F0/F-1 vs. [DNA] (see Figure 2) and found to be 5.11 × 102 M−1. The
Stern-Volmer constants KSV for other compounds were listed in Table 2. The plots of F0/F-1 vs. [DNA]
for other compounds were showed in Figures S11–S20.

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Figure 2. Plot of [DNA] × 105 vs. (F0/F-1) of compound 8j.
The Stern-Volmer plot is linear for 8j, indicating that only one type of quenching process occurs. The
Stern-Volmer quenching is largely higher than the maximum collision diffuse quenching constant of the
biomolecules (2.0 × 1010 L·mol−1·s−1), indicating that the fluorescence quenching was mainly arisen from
static quenching by complex formation instead of dynamic quenching [27]. The Stern-Volmer plots are
linear for other compounds, too.
When small molecules binding independently to a set of equivalent sites on a macromolecule, the
equilibrium between free and bound molecules is given by the equation [28]:
lg (F0/F-1) = lgKb + nlg[Q]
(2)
where Kb and n are the binding constant and the number of binding sites, respectively, which could be
got from the curve of lg(F0/F-1) vs. lg[Q]. The plot of lg(F0/F-1) vs. lg[DNA] has been shown in Figure 3
for compound 8j and indicates a simple binding process for the compound. The binding constants Kb
and the number of binding sites n of all the derivatives were listed in Table 2. The plots of lg(F0/F-1) vs.
lg[DNA] for other compounds were showed in Figures S21–S30.
Figure 3. Plot of lg[DNA] vs. lg(F0/F-1), Kb = 7.81 × 104 M−1 of compound 8j.
Typical-binding constant between organic compound and DNA usually ranges from 104–106 M−1,
Table 2 shows that some compounds are actually moderate DNA-intercalators. The relative binding
affinities as indicated by the binding constants Kb are in the order of 8h > 8j > 8i > 8f > 8d > 8g > 4-MU
> 8a > 8e > 8c > 8b. Therefore, more than half of the derivatives had a higher DNA binding constant
than their lead compound 2. This result indicated that introduction of some aminophosphonates side
chains could dramatically increase the DNA binding capacity of 4-MU.

Molecules 2015, 20
Table 2. Stern-Volmer constants and binding data for compounds 8a–8j.
14797
Compound Kb (M−1)
n
KSV (M−1)
4.42 × 102
3.4 × 102
2.99 × 102
4.81 × 102
4.29 × 102
4.75 × 102
4.12 × 102
4.1 × 102
2
4.56 × 102
2.44 × 102
77.5
1.00
0.96
0.83
0.83
1.05
0.84
1.42
1.04
1.95
0.96
1.43
8a
8b
8c
8d
8e
8f
1.30 × 102
5.91 × 102
1.37 × 102
1.16 × 104
5.76 × 102
7.38 × 105
1.24 × 104
1.6 × 104
8g
8h
8i
3.57 × 102
1.81 × 104
5.11 × 102
8j
In most cases, compounds strongly binding to DNA are high cytotoxic agent. Among the derivatives,
those with stronger DNA binding affinities, 8f, 8h, 8i and 8j exhibited lower IC50 values against the tumor
cells. In contrast, the other compounds exhibited no or low activity, showing low-affinity with DNA.
Compound 8h, having ortho-methoxyl-substituted phenyl as R2, had the largest DNA binding constant, it
may be attributed to the strong binding of DNA molecules through electrostatic interaction and hydrogen
bonding by its methoxyl group.
Steady-state competitive binding experiments using 8f, 8h and 8j as quenchers were undertaken to
get further proof for the binding of the compounds to DNA. The binding abilities of these compounds to
CT-DNA were primarily investigated by competitive binding in which they served as an intercalative
binding probe in competition with GelRed. GelRed, which is environmentally safe and ultra-sensitive
for DNA staining, is a newly developed DNA intercalator to replace the classic DNA intercalator EB.
Furthermore, both GelRed and EB bound with CT-DNA emit characteristic fluorescence at around 590 nm
under 350 nm excitation [15].
In competitive binding experiments, GelRed and CT-DNA solutions were pre-incubated for 30min to
ensure sufficient interactions. The concentration ratio of GelRed to DNA was set at [GelRed]/[DNA] = 1:10
for sufficient binding sites of DNA for GelRed. The emission spectra of the GelRed-CT-DNA system in
the absence and presence of 8j are shown in Figure 4 (for those of 8f and 8h, see Figures S31 and S32,
respectively). GelRed-DNA binary solution system gave a characteristic fluorescence emission at around
590 nm when excited at 350 nm, indicating that GelRed molecules intercalated between the adjacent
base pairs of DNA and were sufficiently prevented from fluorescence quenching by polar solvent
molecules. The addition of compounds 8f, 8h or 8j into the GelRed/CT-DNA binary binding system
induced a significant fluorescence quenching of GelRed/CT-DNA. These results strongly suggested the
existence of competitive intercalative binding between GelRed and 8f, 8h or 8j, in which compounds 8f,
8h or 8j could displace the GelRed molecules from the DNA neighboring base pairs and there after push
the GelRed molecules back into the aqueous solution.
The quenching abilities of compounds 8f, 8h and 8j to GelRed fluorescence can be quantitively
estimated by their respective quenching constant, Kq, which was derived from the Stern-Volmer quenching
equation. The quenching of GelRed bound to DNA by the test compounds are in good agreement with
the linear Sterne-Volmer equation. The values of Kq corresponding to the three compounds 8f, 8h and

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8j, obtained by plotting I0/I vs. [Q] , were 1.73 × 103 M−1, 2.41 × 103 M−1 and 2.35 × 103 M−1, respectively.
Meanwhile, it can also be confirmed that the intercalative binding mode of the test compounds to DNA
was similar to that of GelRed [29]. These values suggested that the compound 8h exhibited more intensive
intercalation to DNA than the compounds 8f and 8j which is consistent with the fluorescence spectrometric
titration results.
Figure 4. Emission spectra of DNA-GelRed (165 μM), in the presence of 0, 40, 80, 120,
160, 200, 240, 280, 320, 360, 400 and 440 μM of compound 8j. Arrow indicates the changes
in the emission intensity as a function of compound concentration. Inset: Sterne-Volmer plot
of the fluorescence titration data corresponding to the compound 8j.
2.3.2. Circular Dichroism Spectra
The circular dichroism (CD) is a useful technique to assess whether the nucleic acids undergo
conformational changes as a result of complex formation or changes in environmental conditions [30].
As indicated in Figure 5, the CD spectra of CT-DNA (1.5 × 10−4 M) showed a positive absorption
peak at 281 nm and a negative absorption peak at 247 nm due to π-π base stacking and right-hand helicity,
respectively, which is consistent with the characteristic B conformation of DNA [31]. The CD absorption
of CT-DNA in the presence of compound 8j showed a significant decrease in the intensity at the
[8j]/[DNA] ratio of 1:10. The decrease percentages in the DNA maximal positive and negative
absorption by 8j were of 26.82% and 30.4%, respectively. It suggests that compound 8j may intercalate
between the neighboring base pairs of CT-DNA mainly due to the aromatic planarity of the coumarin,
because the decreases in the intensities of both the positive and negative bands can usually be observed
in the intercalative binding of small molecules to DNA [32]. The CD absorptional curves of CT-DNA
in the presence of compounds 8h and 8f were similar to that of compound 8j (for those of 8f and 8h, see
Figures S33 and S34, respectively), which suggests the comparable DNA binding properties of all the
three compounds due to their same binding mode. The calculated results for the changes on intensities
of both the positive and negative CD absorption peak of CT-DNA bound with compounds 8j, 8h and 8f
were listed in Table 3.The changes in CD signals of DNA after adding 8j, 8h and 8f were consistent
with Scatchard-binding constants, 8h > 8f > 8j, which indicated that DNA-binding ability of 8h is
stronger than those of 8f and 8j. These results were consistent with the competitive binding fluorescence
measurements of 8j, 8h and 8f. Furthermore, no appreciable red-or blue-shifts in wavelengths were
observed and the shape of the CT-DNA absorption curve was unchanged, indicating that there was no
significant transformation in CT-DNA secondary structure [33].

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Figure 5. CD spectra of CT-DNA (3 mL solution, 1.5 × 10−4 M) in the absence and presence
of compound 8j (1.5 × 10−5 M).
Table 3. Calculated changes on intensities of both the positive and negative CD absorption
peak of CT-DNA bound with Compounds 8j, 8h and 8f.
Compounds
8f
8h
8j
Positive AI decrease(%) a
23.1 34.5
26.9 28.5
26.82
30.4
Negative AI decrease(%) a
a AI: Absorption Intensity.
2.4. Apoptosis and Cell Cycle Analysis
Flow-cytometric analysis further confirmed tumor cell apoptosis as shown in Figure 6. Cytometric
profiles of the PI-stained DNA showed cell cycle arrest of HCT-116 cells treated with compounds 8c,
8d, 8f and 8j for 48 h at IC50 concentrations. Compared to the control (untreated cells), changes in the
cell cycle distribution of treated HCT-116 cells were evident. G1-phase populations of 87.24% for 8d,
74.25% for 8f and 82.72% for 8c were observed compared with a G1-phase population of 35.32% for
untreated cells. For the compound 8j, the sub-G1 peak and the accumulation of cells in the G2 phase
accompanying reduction in the G1 phase were observed after the cells were exposed to 8j.
2.5. Release of Cytochrome c and Activation of Caspases were Involved in the Apoptosis Induced by 8j
To confirm the molecular mechanisms involved in the observed apoptosis alterations, we investigated
the effects of compound 8j on the expression of proteins important for mitochondria mediated apoptosis.
Apoptosis occurs through two different pathways, that is the intrinsic and extrinsic pathways. Intrinsic
pathway involves mitochondria that play a pivotal role in apoptosis, whereas extrinsic pathway is
mediated by cell death receptors such as Fas, TNF α after receiving the death signal. It is well-known
that the intrinsic pathway is regulated by the Bcl-2 family of pro- and anti- apoptotic proteins, which
stimulate the permeabilization of the mitochondrial outer membrane and cytochrome c released into the
cytosol, promoting in the activation of the caspase cascade and the induction of apoptotic cell death. The
effects of compound 8j on the constitutive levels of Bax, Bcl-2 and cytochrome c in HCT-116 cells are
given in Figure 7. In comparison with the control cells, 8j induced a significant increase in the levels of
Bax and a decrease in the expression of Bcl-2, in a dose-dependent manner. Compound 8j treatment
caused an accumulation of cytochrome c in the cytosol, most probably due to the release of mitochondrial
cytochrome c. These results indicated an involvement of caspases in the apoptotic process downstream

Molecules 2015, 20
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of mitochondria. Then, the roles of important caspases (caspase-9 and caspase-3) were investigated. As
Figure 7 shown, the treatment of HCT-116 cells with 8j caused a marked increase in the levels of
caspase-3 and caspase-9 proteins compared to the control. These results revealed an involvement of
caspases in the intrinsic apoptotic process downstream of mitochondria. Thus, compound 8j induced
HCT-116 cells apoptosis might decrease the activation of Bcl-2 and stimulate its downstream proteins
which are associated with the mitochondria-dependent apoptotic pathway.
Figure 6. Inhibition of cell cycle progress in HCT-116 cells treated with compounds 8d, 8f,
8c and 8j for 48 h. Cells were fixed with ethanol and stained with PI. Cell cycle distribution
was analyzed by flow cytometry. First peak represents sub-G1 peak, which was taken as the
fraction of the apoptotic cell population.
Figure 7. Effect of compound 8j on cytochrome c release and levels of Bcl-2, Bax, Caspase-9
and Caspase-3. Equal amount of protein was loaded on SDS-PAGE gel for western blot
analysis as described in experimental section. β-actin was used as an internal control.

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3. Experimental Section
3.1. Chemistry
All chemicals (reagent grade) used were commercially available. NMR spectra were measured on a
BRUKER AVANCE AV500 spectrometer (Bruker Corporation, Karlsruhe, Germany) using TMS as the
internal standard. The mass spectra were obtained on a BRUKER ESQUIRE HCT spectrometer (Bruker
Corporation). Melting points were determined using an X-4 apparatus (Huier Corporation, Zhejiang,
China) and were uncorrected. The elemental analyses (C, H and O) were carried out on a Perkin Elmer
Series II CHNS/O 2400 elemental analyzer (Perkin Elmer Corporation, Waltham, MA, USA).
All the chemical reagents and the solvents were analytical grade. Calf thymus DNA (CT-DNA) was
purchased from Sigma-Aldrich (Saint Louis, MO, USA). They were all used as received without further
purification unless noted specifically.
3.1.1. 7-Hydroxy-4-methyl-2H-chromen-2-one (2)
A mixture of phenols (1 mmol) and ethyl acetoacetate (1 mmol) was ground with sodium bisulfate
(2 mmol) in a mortar by pestle for 10 min when a color change of the reaction mixture took place. The
reaction mixture was kept at room temperature for about 20–80 min. The completion of the reaction was
checked by TLC (silica gel using solvent petroleum ether:acetone, 2:1). The reaction mixture was diluted
with ice-cold water. The solid that separated out was filtered at vacuum, washed with water, and
recrystallized from ethanol to give pure 4-MU in 96% yield. White acicular crystal. m.p. 185–187 °C.
1H-NMR (500 MHz, CDCl3) δ 8.45 (dd, J = 9.6, 3.4 Hz, 1H), 6.75 (dd, J = 8.8, 2.5 Hz, 1H), 6.70 (d,
J = 2.5 Hz, 1H), 6.09 (d, J = 1.1 Hz, 1H), 2.34 (d, J = 1.1 Hz, 3H).
3.1.2. 3-(4-Methyl-2-oxo-2H-chromen-7-yloxy)propanoic Acid (3)
In 500 mL of RBF, provide with reflux condenser 4-MU (0.01 mol), 3-bromopropionic acid (0.01 mol)
and K2CO3 (0.03 mol) in 25 mL of distilled acetone. Then resultant solution was refluxed in water bath.
After 1 h, pH of the solution had dropped to 7, and further 1 g of K2CO3 was added. Refluxing was
continued for a 12 h. The hot solution was acidified with dilute hydrochloric acid. The product was
extracted with diethyl ether. Ether layer was washed with 50 mL of saturated solution of sodium
bicarbonate. The aqueous layer was acidified with HCl acid. White colored solid was filtered off, washed
with water, dried and recrystallized by hot water to get the desired white compounds (3), yield 65%.
White solid. m.p. 140–142 °C.1H-NMR (500 MHz, CDCl3) δ 7.42 (d, J = 8.8 Hz, 1H), 6.75 (dd, J = 8.8,
2.5 Hz, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.09 (d, J = 1.1 Hz, 1H), 4.27 (t, J = 6.3 Hz, 2H), 2.76 (t, J = 6.3 Hz,
13
2H), 2.34 (d, J = 1.1 Hz, 3H). C-NMR (125 MHz, CDCl3) δ (ppm) 172.02, 160.05, 159.26, 155.20,
150.18, 127.30, 112.31, 112.26, 110.96, 102.17, 65.67, 34.76, 20.28. Anal. for C13H12O5: calcd C,
62.90%; H, 4.87%; O, 32.23%; found C, 62.88%; H, 4.86%; O, 32.26%.
3.1.3. General Procedure for the Preparation of the O-Substituted Coumarin Derivatives 8a–8j
A suspension of aromatic aldehyde (aromatic ketone) (10 mmol) and ammonium acetate (11 mmol)
was stirred for 6 h at reflux. The reaction mixture was filtered to give the white precipitate 5, to which

Molecules 2015, 20
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the diethyl phosphite (5 mmol) was added and the resulting solution was stirred for 3 h at 70 °C. After
that, hydrochloric acid (4 mmol) in 30 mL ether was added to the reaction mixture, which was stirred
for 2 h at 0 °C to give the precipitate 6. The precipitate was filtered and washed with ether (15 mL), and
then it was added to 10 mL sodium hydroxide solution (15%) and stirred for 30 min at room temperature.
Extraction with dichloromethane (3 × 30 mL) and evaporation of the solvent gave the oils of 7, which
was purified by column chromatography on silica gel with ethyl acetate. Compound 3 (0.6 mmol),
α-aminophosphonates (0.6 mmol), HOBT (0.6 mmol), and EDAC (1.0 mmol) in dry DMSO (10 mL)
were stirred in ice bath. After 10 min, the reaction mixture was stirred for 6–8 h at room temperature
(TLC detection). The mixture was washed by water and ethyl acetate (water/ethyl acetate = 1:2, v/v) for
three times. The solvent was removed under reduced pressure. The residue was purified by chromatography
on silica gel with a solution of petroleum ether and ethyl acetate (water/ethyl acetate = 1:2, v/v) as eluent
to give desired product.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(phenyl)methylphosphonate (8a). Yield
81%. White solid. m.p. 126–128 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 7.53–7.49 (m, 2H), 7.42 (d,
J = 8.8 Hz, 1H), 7.29 (qd, J = 7.9, 1.3 Hz, 3H), 6.75 (dd, J = 8.8, 2.5 Hz, 1H), 6.70 (d, J = 2.5 Hz, 1H),
6.09 (d, J = 1.1 Hz, 1H), 5.61 (dd, J = 21.0, 9.7 Hz, 1H), 4.30–4.23 (m, 2H), 4.18–3.59 (m, 4H), 2.76 (t,
J = 6.3 Hz, 2H), 2.34 (d, J = 1.1 Hz, 3H), 1.31 (t, J = 7.1 Hz, 3H), 1.05 (t, J = 7.1 Hz, 3H). 13C-NMR
(125 MHz, CDCl3) δ (ppm) 170.01, 164.05, 161.33, 161.01, 155.21, 150.58, 128.65, 128.16, 127.24,
126.51, 114.32, 112.53, 112.41, 102.26, 64.98, 64.29, 64.18, 49.23, 36.26, 19.26, 16.84, 16.80.
31P-NMR (202MHz, CDCl3) δ (ppm) 21.49 (s). HRMS for C24H29NO7P ([M + H]+): calcd 474.16816:
found 474.16656.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(2-fluorophenyl)methylphospho-nate (8b).
1
Yield 85%. White solid. m.p. 130–132 °C. H-NMR (500 MHz, CDCl3) δ (ppm) 8.30 (d, J = 7.4 Hz,
1H), 7.46 (ddd, J = 8.6, 5.1, 2.0 Hz, 2H), 7.41 (d, J = 8.8 Hz, 1H), 6.97 (t, J = 8.6 Hz, 2H), 6.73 (dd,
J = 8.8, 2.5 Hz, 1H), 6.70 (d, J = 2.4 Hz, 1H), 6.08 (d, J = 1.1 Hz, 1H), 5.56 (dd, J = 21.0, 9.6 Hz, 1H),
4.25 (ddd, J = 15.5, 9.3, 3.1 Hz, 2H), 4.18–3.65 (m, 4H), 2.74 (t, J = 6.2 Hz, 2H), 2.34 (d, J = 1.1 Hz,
3H), 1.29 (t, J = 7.1 Hz, 3H), 1.07 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3 ) δ (ppm) 170.16,
161.90, 161.27, 155.58, 152.89, 142.28, 134.16, 131.10, 129.79, 127.66, 127.27, 126.16, 112.61, 112.46,
110.98, 102.24, 65.06, 64.16, 64.08, 45.99, 35.68, 20.20, 16.96, 16.73. 31P-NMR (202 MHz, CDCl3) δ
(ppm) 21.20 (d, J = 4.3 Hz). HRMS for C24H28NO7 FP ([M + H]+): calcd 492.15874: found 492.15732.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(2-chlorophenyl)methylphospho-nate (8c).
Yield 82%. White solid. m.p. 115–117 °C. 1H-NMR (500-MHz, CDCl3) δ (ppm) 8.01 (s, 1H), 7.47 (d,
J = 8.8 Hz, 1H), 7.42–7.21 (m, 4H), 6.81 (dd, J = 8.8, 2.5 Hz, 1H), 6.75 (d, J = 2.4 Hz, 1H), 6.22–6.16
(m, 1H), 6.14 (s, 1H), 4.36–4.30 (m, 2H), 4.30–3.64 (m,4H), 2.79 (td, J = 6.1, 2.6 Hz, 2H), 2.39 (d,
J = 0.8 Hz, 3H), 1.36 (t, J = 7.1 Hz, 3H), 1.06 (t, J = 7.0 Hz, 3H). 13C-NMR (125 MHz, CDCl3 ) δ (ppm)
170.04, 161.95, 161.76, 155.65, 153.02, 142.63, 134.08, 130.27, 129.89, 127.57, 127.10, 126.12, 112.69,
31
112.59, 111.10, 102.34, 64.21, 64.04, 63.98, 47.89, 36.35, 19.20, 17.00, 16.62. P-NMR (202 MHz,
CDCl3) δ (ppm) 20.72 (s). HRMS for C24H28NO7PCl ([M + H]+):calcd 508.12919; found 508.12781.

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Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(4-chlorophenyl)methylphospho-nate (8d).
Yield 85%. White solid. m.p. 110–112 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.21 (s, 1H), 7.46 (dd,
J = 8.5, 2.2 Hz, 1H), 7.42 (d, J = 8.3 Hz, 2H), 7.29 (dd, J = 8.3, 1.6 Hz, 2H), 6.78 (d, J = 2.5 Hz, 1H),
6.76 (s, 1H), 6.13 (d, J = 1.0 Hz, 1H), 5.58 (dd, J = 21.3, 9.5 Hz, 1H), 4.40–4.23 (m, 2H), 4.21–3.72 (m,
4H), 2.78 (t, J = 6.0 Hz, 2H), 2.38 (d, J = 1.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H), 1.12 (dd, J = 7.7, 6.4 Hz,
13
3H). C-NMR (125 MHz, CDCl3) δ (ppm) 170.21, 164.45, 161.83, 161.67, 155.51, 150.98, 133.86,
129.95, 129.16, 126.01, 114.23, 112.64, 112.51, 102.05, 64.95, 64.19, 64.13, 49.22, 36.22, 19.07, 16.87,
31
16.83. P-NMR (202 MHz, CDCl3) δ (ppm) 21.21 (s). HRMS for C24H28NO7 PCl ([M + H]+): calcd
508.12919; found 508.12775.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(2-bromophenyl)methylphospho-nate (8e).
Yield 87%. White solid. m.p. 160–162 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.67 (s, 1H), 7.75 (d,
J = 7.8 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.40 (dd, J = 8.8, 1.2 Hz, 1H), 7.27 (dd, J = 12.7, 5.2 Hz, 1H),
7.12 (dd, J = 11.0, 4.3 Hz, 1H), 6.73 (dd, J = 8.8, 2.4 Hz, 1H), 6.67 (d, J = 2.3 Hz, 1H), 6.20 (dd,
J = 21.0, 9.3 Hz, 1H), 6.07 (s, 1H), 4.34–4.23 (m, 2H), 4.24–3.58 (m, 4H), 2.77 (q, J = 6.1 Hz, 2H), 2.33
(s, 3H), 1.34 (t, J = 7.1 Hz, 3H), 1.02 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3) δ (ppm) 170.15,
162.00, 161.86, 155.56, 153.58, 147.69, 132.68, 130.34, 129.80, 128.00, 127.91, 126.35, 112.49, 112.21,
31
111.12, 102.43, 64.20, 64.06, 63.88, 47.86, 36.34, 19.25, 16.50, 16.39. P-NMR (202MHz, CDCl3) δ
(ppm) 20.85 (s). HRMS for C24H28NO7PBr ([M + H]+): calcd 552.07868; found 552.07666.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(3-bromophenyl)methylphospho-nate (8f).
Yield 83%. White solid. m.p. 171–172 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.36 (d, J = 5.7 Hz,
1H), 7.67 (d, J = 1.5 Hz, 1H), 7.44 (dd, J = 15.1, 8.4 Hz, 2H), 7.20 (t, J = 7.8 Hz, 1H), 6.79 (dd, J = 8.8,
2.4 Hz, 1H), 6.74 (d, J = 2.4 Hz, 1H), 6.13 (d, J = 0.7 Hz, 1H), 5.58 (dd, J = 21.3, 9.5 Hz, 1H), 4.30 (dq,
J = 9.3, 6.4 Hz, 2H), 4.26–3.73 (m, 4H), 2.79 (t, J = 6.1 Hz, 2H), 2.38 (d, J = 0.5 Hz, 3H), 1.33 (t,
J = 7.1 Hz, 3H), 1.12 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3 ) δ (ppm) 170.24, 161.84, 161.62,
155.52, 152.93, 137.99, 131.50, 131.45, 127.43, 127.39, 126.01, 123.01, 114.22, 112.60, 112.52, 102.10,
64.99, 64.05, 63.93, 49.42, 36.17, 19.08, 16.88, 16.58. 31P-NMR (202 MHz, CDCl3) δ (ppm) 21.71 (s).
HRMS for C24H28NO7PBr ([M + H]+): calcd 552.07868; found 552.07642.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(4-bromophenyl)methylphosph-onate (8g).
Yield 87%. White solid. m.p. 110.8–112.8 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.23 (dd, J = 9.5,
4.1 Hz, 1H), 7.43 (d, J = 8.1 Hz, 2H), 7.41 (s, 1H), 7.33 (dd, J = 8.5, 2.0 Hz, 2H), 6.75 dd, J = 8.8, 2.4
Hz, 1H), 6.73 (d, J = 2.3 Hz, 1H), 6.10 (d, J = 1.2 Hz, 1H), 5.54 (dd, J = 21.3, 9.5 Hz, 1H), 4.32–4.21
(m, 2H), 4.19–3.70 (m, 4H), 2.75 (t, J = 6.1 Hz, 2H), 2.35 (d, J = 1.2 Hz, 3H), 1.29 (t, J = 7.1 Hz, 3H),
1.09 (t, J = 7.1 Hz, 3H). 13C-NMR (125MHz, CDCl3) δ (ppm) 170.26, 164.49, 161.86, 161.76, 155.67,
150.89, 132.96, 129.98, 129.27, 126.12, 114.36, 112.50, 112.26, 102.06, 64.39, 64.16, 64.12, 47.96,
365.20, 20.12, 16.89, 16.85. 31P-NMR (202 MHz, CDCl3) δ (ppm) 20.87 (s). HRMS for C24H28NO7PBr
([M + H]+): calcd 552.07868; found 552.07629.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(2-methoxyphenyl)methylphos-phonate (8h).
Yield 81%. White solid. m.p. 128.6–130.6 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.08 (dd, J = 9.7,
2.9 Hz, 1H), 7.57–7.45 (m, 1H), 7.39 (d, J = 8.8 Hz, 1H), 7.24–7.18 (m, 1H), 6.91–6.81 (m, 2H), 6.74

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(dd, J = 8.8, 2.5 Hz, 1H), 6.68 (d, J = 2.5 Hz, 1H), 6.14–6.08 (m, 1H), 6.06 (d, J = 1.2 Hz, 1H),
4.30–4.20 (m, 2H), 4.19–3.70 (m, 4H),3.82 (s, 3H), 2.73 (dd, J = 10.4, 6.1 Hz, 2H), 2.32 (d, J = 1.0 Hz,
3H), 1.28 (t, J = 7.1 Hz, 3H), 0.98 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3 ) δ (ppm) 170.11,
162.16, 161.86, 155.67, 153.21, 142.47, 134.15, 130.57, 129.98, 128.01, 127.78, 126.42, 112.88, 112.69,
31
111.86, 102.59, 65.19, 64.25, 64.19, 53.56, 46.90, 35.36, 19.27, 16.70, 16.62. P-NMR (202 MHz,
CDCl3) δ (ppm) 22.05 (s). HRMS for C25H31NO8 P ([M + H]+): calcd 504.17873; found 504.17688.
Diethyl(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)(naphthalen-2-yl)methylphosph-onate (8i).
Yield 82%. White solid. m.p. 177.6–179.6 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.46 (s, 1H), 7.98
(s, 1H), 7.79–7.71 (m, 3H), 7.63 (dd, J = 8.5, 1.4 Hz, 1H), 7.47–7.39 (m, 2H), 7.32–7.27 (m, 1H), 6.67
(d, J = 1.3 Hz, 1H), 6.65 (t, J = 1.9 Hz, 1H), 6.07 (s, 1H), 5.80 (dd, J = 21.0, 9.6 Hz, 1H), 4.27–3.59 (m,
6H) 2.76 (t, J = 6.3 Hz, 2H), 2.29 (s, 3H), 1.34 (t, J = 7.1 Hz, 3H), 1.02 (t, J = 7.1 Hz, 3H). 13C-NMR
(125 MHz, CDCl3 ) δ (ppm) 170.32, 160.89, 159.76, 154.65, 152.23, 136.67, 133.66, 131.82, 129.97,
128.46, 127.76, 127.45, 127.16, 126.69, 126.33, 125.29, 114.62, 113.60, 112.04, 106.58, 64.67, 63.81,
31
63.65, 47.67, 35.69, 19.98, 16.36, 16.29. P-NMR (202 MHz, CDCl3) δ (ppm) 21.44 (s). HRMS for
C28H31NO7P ([M + H]+): calcd 524.18381; found 524.18213.
Diethyl 1-(3-(4-methyl-2-oxo-2H-chromen-7-yloxy)propanamido)-1-phenylethylphosphonate (8j). Yield
88%. White solid. m.p. 83.6–85.6 °C. 1H-NMR (500 MHz, CDCl3) δ (ppm) 8.45 (dd, J = 9.6, 3.4 Hz,
1H), 7.62-7.45 (m, 2H), 7.42 (d, J = 8.8 Hz, 1H), 7.29 (dd, J = 7.9, 4.1 Hz, 3H), 6.75 (dd, J = 8.8, 2.5
Hz, 1H), 6.70 (d, J = 2.5 Hz, 1H), 6.09 (d, J = 1.1 Hz, 1H), 4.34–4.22 (m, 2H), 4.22–3.67 (m, 4H), 2.78
(td, J = 6.2, 2.7 Hz, 2H), 2.34 (d, J = 1.0 Hz, 3H), 2.10 (d, J = 16.1 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H),
1.09 (t, J = 7.1 Hz, 3H). 13C-NMR (125 MHz, CDCl3 ) δ (ppm) 170.28, 160.75, 159.80, 155.26, 152.12,
137.36, 128.87, 128.67, 127.86, 126.96, 113.86, 112.92, 112.58, 104.01, 65.53, 63.72, 63.56, 58.51,
36.39, 21.32, 18.36, 16.20, 16.11. 31P-NMR (202 MHz, CDCl3) δ (ppm) 21.33 (s). HRMS for
C25H31NO7P ([M + H]+): calcd 488.18381; found 488.18231.
3.2. Biological Assays
3.2.1. Cytotoxicityassay in Vitro
Cell Lines. The following in vitro human cancer cell lines were used: HCT-116 (Human colorectal
cells), human KB (human nasopharyngeal carcinoma cells), MGC-803 (human lung adenocarcinoma
cell line), HUVEC (Human Umbilical Vein Endothelial Cells). The cell lines (HCT-116, KB, MGC-803
and HUVEC) were purchased from the Cell Resource Center of Shanghai Institutes for Biological
Sciences, the Academy of Sciences of China.
Cell Culture. HCT-116, human KB, MGC-803 and HUVEC cells were cultured in Dulbecco Modified
Eagle Medium (DMEM; HyClone, Los Angeles, CA, USA), containing 4.0 mM L-Glutamine and
4500 mg/L Glucose, supplemented with 10% (v/v) foetalbovine serum (FBS; HyClone). The cell
culturemedia was supplemented with penicillin/streptomycin at 100 Units/mL as adherent monolayers.
Cell cultures were kept in a humidified incubator with 5% CO2 at 37 °C. Stock solutions were prepared
in dimethyl sulfoxide (DMSO) and further dilutions were made with fresh culture medium. The
concentration of DMSO in the final culture medium was 1%, which had no effect on the cell viability.

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MTT Assay. Chemosensitivity was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay. Briefly, exponentially growing HCT-116 (3000–4000 cells/well),
human KB (2000–3000 cells/well) and MGC 803 (2000–3000 cells/well) were seeded into 96-well
plates and treated with indicated concentrations of samples for 48 h, and then 10 mL of MTT (10 mg/mL)
was added. After incubation for 4 h at 37 °C, the purple formazan crystals (i.e., a reduced form of MTT)
generated from viable cells were dissolved by adding 100 μL DMSO in each well. The plates were
swirled gently for 10 min to dissolve the precipitate, and quantified by measuring the optical density
(OD) of the plates at a wavelength of 490 nm on plate reader (TECAN infinite M1000). Each
concentration was repeated in three wells and the same experimental conditions were provided for all
compounds and MTT analysis was repeated three times for each cell line.
3.2.2. Apoptosis and Cell Cycle Analysis
Apoptosis was quantified by assessing the fraction of cells with sub-G1 DNA content by flow
cytometry. The cells lines were treated with indicated concentrations of compounds 4c, 4d, 4f and 4j.
After incubated for 48 h, cells were washed twice with ice-cold PBS, fixed and permeabilized with
ice-cold 70% ethanol at −20 °C overnight. The cells were treated with 100 μg/mL RNase A at 37 °C for
30 min after washed with ice-cold PBS, and finally stained with 1 mg/mL propidium iodide (PI) in the
dark at 4 °C for 30 min. Analysis was performed with the system software (Cell Quest; BD Biosciences,
Franklin Lakes, NJ, USA).
3.3. Spectrofluorimetric Titration and DNA Binding Assay
All fluorescence experiments were carried out on a Perkin-Elmer LS50B luminescence spectrometer.
Fluorescence spectra were measured at rt using quartz cells of 1cm path. To the solutions of the drugs
(2.0 × 10−3 M) in 2.5 mL Tris-HCl buffer (pH = 7.2) were added aliquots of CT-DNA (2.0 × 10−3 M)
solution containing drugs (2.0 × 10−3 M) in 2.5 mL Tris-HCl buffer (pH = 7.2). This operation ensured
that the concentration of CT-DNA increased gradually from 0 to 360 M, while the concentrations of
drugs were kept constant. The mixing was achieved by stirring for 10min. Then, the corresponding
fluorescence spectra were measured (ex 325 nm, ex/em 1.5 nm/1.5 nm). Binding constants were derived
from the modified.
A solution containing 2 × 10−4 M DNA and 2 × 10−5 M GelRed ([DNA]/[GelRed] = 10:1) was
prepared for GelRed-DNA competitive binding studies. Fluorescence emission spectra were recorded
under slit width as 10 nm/10 nm for ex/em, respectively. The quenching constant for comparing the
efficiency of fluorescence quenching, i.e., Kq, of each compound was obtained by the linear fit of plotting
I0/I vs. [Q], according to the classic Stern-Volmer equation: I0/I = 1 + Kq × [Q] [34], where I0 and I are
the peak emission intensity of the GelRed-DNA system in the absence and presence of each compound
as quencher, and [Q] is the concentration of quencher. In the fluorescence polarization experiment, each
sample was pre-incubated for 40 min before the fluorescence polarization was recorded under the
condition of 595 nm emitting wavelength with 350 nm exciting wavelength, and slit width was set as
5 nm/5 nm for ex/em, respectively.
CD absorption spectra of DNA were measured in TBS at a 100 nm/min scan rate in the wavelength
range from 200 to 500 nm, with 1.5 × 10−4 M DNA in the absence and presence of each compound of

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1.5 × 10−5 M, respectively. The CD signal of TBS was taken as the background and subtracted from the
spectra. All the spectroscopic experiments were performed at 25 °C.
3.4. Statistics
The data were processed by the Student’s t-test with the significance level p ≥ 0.05 using SPSS (IBM,
Armonk, New York, NY, USA).
4. Conclusions
In summary, a series of novel coumarin-containing aminophosphonates derivatives were synthesized
and the cytotoxicity of these derivatives against different cancer cell lines was determined. The
introduction of aminophosphonates side chains is an effective way to improve the antitumor activity of
4-MU, which may be associated with their DNA binding capacity. Compound 8j exhibited best potency
against HCT-116 and KB cells, but low cytotoxicity towards normal human cell. Importantly, it had
excellent cell selectivity in HCT-116 and human KB cells. In addition, it was found that 8c, 8d, 8f and
8j could disturb the cell cycle in HCT-116cells. The results from western-blot analysis provided evidence
for an apoptotic cell death, which may be through up-regulating the caspase-3, caspase-9 and cytochrome
c signaling pathway. DNA-binding experiments suggested that some novel DNA-intercalators were
developed based on coumarin-APA system. This research may provide some new suggestions for the
design of novel antitumor agent based on coumarin.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/08/14791/s1.
Acknowledgments
This project is supported by the National Natural Science Foundation of China (No. 81260472,
21101035, 21362002), the Natural Science Foundation of Guangxi Province (No. CMEMR2012-A,
1355004-3), and Bagui Scholar project and the Foundation of Ministry of Education Innovation Team
(NO. IRT1225).
Author Contributions
G.Y.Y. conceived and designed the study. Y.J.L., C.Y.W. and M.Y.Y. performed the experiments.
G.Y.Y. wrote the paper. Y.J.L., C.Y.W. and H.S.W. reviewed and edited the manuscript. All authors
read and approved the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.

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References
1. Bhattacharyya, S.S.; Mandal, S.K.; Biswas, R.; Paul, S.; Pathak, S.; Boujedaini, N.; Belon, P.;
Khuda-Bukhsh, A.R. A syntheticcoumarin (4-methyl-7-hydroxy coumarin) has anti-cancerpotentials
against DMBA-induced skin cancer in mice. Eur. J. Pharmacol. 2009, 614, 128–136.
2. Arshad, A.; Osman, H.; Bagley, M.C.; Lam, C.K.; Mohamad, S.; Zahariluddin, A.S.M. Synthesis
and antimicrobial properties of somenew thiazolyl coumarin derivatives. Eur. J. Med. Chem. 2011,
46, 3788–3794.
3. Abdelhafez, O.M.; Amin, K.M.; Batran, R.Z.; Maher, T.J.; Nada, S.A.; Sethumadhavan, S. Synthesis,
anticoagulant and PIVKA-IIinduced by new 4-hydroxycoumarin derivatives. Bioorg. Med. Chem.
2010, 18, 3371–3378.
4. Xia,Y.; Yang, Z.Y.; Xia, P.; Hackl, T.; Hamel, E.; Mauger, A.; Wu, J.H.; Lee, K.H. Antitumor agents.
211. Fluorinated 2-phenyl-4-quinolone derivatives as antimitotic antitumor agents 1. J. Med. Chem.
2001, 44, 3932–3936.
5. Suzuki, A.Z.; Watanabe, T.; Kawamoto, M.; Nishiyama, K.; Yamashita, H.; Ishii, M.; Iwamura, M.;
Furuta, T. Coumarin-4-ylmethoxycarbonylsas phototriggers for alcohols and phenols. Org. Lett.
2003, 5, 4867–4870.
6. Sunthitikawinsakul, A.; Kongkathip, N.; Kongkathip, B.; Phonnakhu, S.; Daly, J.W.; Spande, T.F.;
Nimit, Y.; Rochanaruangrai, S. Coumarins andcarbazoles fromClausena excavateexhibited
antimycobacterial andantifungal activities. Planta Med. 2003, 69, 155–157.
7. Park, S.S.; Park, S.K.; Lim, J.H.; Choi, Y.H.; Kim, W.J.; Moon, S.K. Esculetin inhibits cell
proliferation through the Ras/ERK1/2 pathway in human colon cancer cells. Oncol. Rep. 2011, 25,
223–229.
8. Stanchev, S.; Momekov, G.; Jensen, F.; Manolov, I. Synthesis, computational study and cytotoxic
activity of new 4-hydroxycoumarin derivatives. Eur. J. Med. Chem. 2008, 43, 694–706.
9. Zhao, H.; Donnelly, A.C.; Kusuma, B.R.; Brandt, G.E.L.; Brown, D.; Rajewski, R.A.; Vielhauer, G.;
Holzbeierlein, J.; Cohen, M.S.; Blagg, B.S.J. Engineering an antibiotic to fight cancer: Optimization
ofthe novobiocin scaffold to produce anti-proliferative agents. J. Med. Chem. 2011, 54, 3839–3853.
10. Bhattacharyya, S.S.; Paul, S.; De, A.; Das, D.; Samadder, A.; Boujedaini, N.; Khuda-Bukhsh, A.R.
Poly (lactide-co-glycolide) acid nanoencapsulation of a synthetic coumarin: Cytotoxicity and
bio-distribution in mice, in cancer cell line and interaction with calf thymus DNA as target.
Toxicol. Appl. Pharm. 2011, 253, 270–281.
11. Kemp, S.; Wheate, N.J.; Wang, S.Y.; Grant-Collins, J.; Ralph, S.F.; Day, A.I.; Higgins,V.J.;
Aldrich-Wright, J.R. Encapsulation of platinum(II)-based DNA intercalators within cucurbit[6,7,8]urils.
J. Biol. Inorg. Chem. 2007, 12, 969–979.
12. Ye, M.Y.; Yao, G.Y.; Wei, J.C.; Pan, Y.M.; Liao, Z.X.; Wang, H.S. Synthesis, Cytotoxicity, DNA
Binding and Apoptosis of Rhein-Phosphonate Derivatives as Antitumor Agents. Int. J. Mol. Sci.
2013, 14, 9424–9439.
13. Yin, H.; Xu, Y.; Qian, X. Novel antitumor agent family of 1H-benzo [c,d] indol-2-one with flexible
basic side chains: Synthesis and biological evaluation. Bioorg. Med. Chem. 2007, 15, 1356–1362.
14. Mucha, A.; Kafarski, P.; Berlicki, Ł. Remarkable potential of thea-aminophosphonatephosphinate
structural motif in medicinalchemistry. J. Med. Chem. 2011, 54, 5955–5980.

Molecules 2015, 20
14808
15. Yao, G.Y.; Ye, M.Y.; Huang, R.Z.; Li, Y.J.; Pan, Y.M.; Xu, Q.; Liao, Z.X.; Wang, H.S. Synthesis
and antitumor activities of novel rheina-aminophosphonates conjugates. Bioorg. Med. Chem. Lett.
2014, 24, 501–507.
16. Ye, M.Y.; Yao, G.Y.; Pan, Y.M.; Liao, Z.X.; Zhang, Y.; Wang, H.S. Synthesis and antitumor activities
of novel a -aminophosp honatederivatives containing an alizar in moiety. Eur. J. Med. Chem. 2014,
83, 116–128.
17. Liu, W.; Rogers, C.J.; Fisher, A.J.; Toney, M.D. Aminophosphonateinhibitors of dialkylglycine
decarboxylase: Structural basis forslow binding inhibition. Biochemistry 2002, 41, 12320–12328.
18. Kotsikorou, E.; Oldfield, E. A quantitative structure-activity relationship and pharmacophore modeling
investigation of aryl-X and heterocyclic bisphosphonates as bone resorption agents. J. Med. Chem.
2003, 46, 2932–2944.
19. Piccinonna, S.; Margiotta, N.; Pacifico, C.; Lopalco, A.; Denora, N.; Fedi, S.; Corsinic, M.;
Natile, G. DinuclearPt (II)-bisphosphonate complexes: A scaffold for multinuclear or different
oxidation state platinum drugs. Dalton Trans. 2012, 41, 9689–9699.
20. Xie, L.J.; Qian, X.H.; Cui, J.G.; Xiao, Y.; Wang, K.W.; Wu, P.C.; Cong, L.Y. Novel angular
furoquinolinones bearing flexible chain asantitumor agent: Design, synthesis, cytotoxic evaluation,
and DNA-binding studies. Bioorg. Med. Chem. Lett. 2008, 16, 8713–8718.
21. Li, Y.J.; Ye, M.Y.; Huang, R.Z.; Yao, G.Y.; Pan, Y.M.; Liao, Z.X.; Wang, H.S. Coumarin-containing
aminophosphonates bridged with chiralside chain: Synthesis and influence of chirality on cytotoxicity
and DNA binding. Med. Chem. Res. 2014, 23, 3144–3156.
22. Sobhani, S.; Tashrifi, Z. Al (OTf) 3 as an efficient catalyst forone-pot synthesis of primary diethyl
1-aminophosphonates undersolvent-free conditions. Synth. Commun. 2008, 39, 120–131.
23. Sissi, C.; Leo, E.; Moro, S.; Capranico, G.; Mancia, A.; Menta, E.; Krapcho, A.P.; Palumbo, M.
Antitumor AZA–anthrapyrazoles: Bio-physical and biochemical studies on 8-and 9-aza regioisomers.
Biochem. Pharmacol. 2004, 67, 631–642.
24. Kumar, C.V.; Asuncion, E.H. DNA-binding studies and siteselective fluorescence sensitization of
an anthryl probe. J. Am. Chem. Soc. 1993, 115, 8547–8553.
25. Qiu, B.; Guo, L.; Wang, W.; Chen, G. Synthesis of N-4-butylamine acridone and its use as fluorescent
probe for ct-DNA. Biosens. Bioelectron. 2009, 24, 1281–1285.
26. Lakowicz, J.R.; Weber, G. Quenching of fluorescence by oxygen. Probe for structural fluctuations
in macromolecules. Biochemistry 1973, 12, 4161–4170.
27. Khorasani-Motlagh, M.; Noroozifar, M.; Mirkazehi-Rigi, S. Fluorescence and DNA-binding
properties of neodymium(III) and praseodymium(III) complexes containing 1,10-phenanthroline.
Spectrochim. Acta A 2011, 79, 978–984.
28. Kandagal, P.B.; Ashoka, S.; Seetharamappa, J.; Shaikh, S.M.T.; Jadegoud, Y.; Ijare, O.B. Study of
the interaction of an anticancer drug with human and bovine serum albumin: Spectroscopic approach.
J. Pharm. Biomed. Anal. 2006, 41, 393–399.
29. Zhang, G.; Guo, J.; Zhao, N.; Wang, J. Study of interaction between kaempferol-Eu3+complex and
DNA with the use of the neutra l red dye as a fluorescence probe. Sens. Actuators B Chem. 2010,
144, 239–246.

Molecules 2015, 20
14809
30. Rajendiran, V.; Murali, M.; Suresh, E.; Palaniandavar, M.; Periasamy, V.S.; Akbarsha, M.A.
Non-covalent DNA binding and cytotoxicity of certain mixed-ligand ruthenium(II) complexes of
2,2-dipyridylamine and diimines. Dalton Trans. 2008, 16, 2157–2170.
31. Schäfe, S.; Gust, I.; Ott, R.; Sheldrick, W.S. Influence of the polypyridyl (pp) ligand size on the
DNA binding properties, cytotoxicity and cellular uptake of organoruthenium(II) complexes of the
type (η6-C6Me6) Ru(L)(pp)n+ L = Cl, n = 1; L = (NH2)2CS, n = 2. Eur. J. Inorg. Chem. 2007, 19,
3034–3046.
32. Xu, H.; Zheng, K.C.; Chen, Y.; Li, Y.Z.; Lin, L.J.; Li, H.; Zhang, P.X.; Ji, L.N. Effects of ligand
planarity on the interaction of polypyridyl Ru(II) complexes with DNA. Dalton Trans. 2003, 11,
2260–2268.
33. Zhong, W.; Yu, J.S.; Liang, Y.; Fan, K.; Lai, L. Chlorobenzylidine-calf thymus DNA interaction
II: Circulardichroism and nuclearmagneticresonancestudies. Spectrochim. Acta A 2004, 60, 2985–2992.
34. Ghosh, S.; Barve, A.C.; Kumbhar, A.A.; Kumbhar, A.S.; Puranik, V.G.; Datar, P.A.; Sonawane, U.B.;
Joshi, R.R. Synthesis, characterization, X-ray structure and DNA photocleavage by cis-dichloro
bis(diimine) Co(III) complexes. J. Inorg. Biochem. 2006, 100, 331–343.
Sample Availability: Samples of compounds 8 are available from the authors upon request.
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