Synthesis and in vitro evaluation of 3-(4-nitrophenyl)coumarin derivatives in tumor cell lines

Article abstract of DOI:10.1016/j.bioorg.2014.11.009

Coumarins are naturally-occurring compounds that have attracted considerable interest due to their numerous biological activities depending on their pattern of substitution on the coumarin molecule. In this present investigation, we synthesized 3-(4-nitro

Full text of DOI:10.1016/j.bioorg.2014.11.009

Accepted Manuscript
Synthesis and in vitro evaluation of 3-(4-nitrophenyl)coumarin Derivatives in
Tumor Cell Lines
Musiliyu A. Musa, Lekan M. Latinwo, Clifford Virgile, Veera L.D. Badisa,
Akintunde J. Gbadebo
PII:
S0045-2068(14)00126-6
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.11.009
YBIOO 1777
Reference:
Bioorganic Chemistry
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31 May 2014
Please cite this article as: M.A. Musa, L.M. Latinwo, C. Virgile, V.L.D. Badisa, A.J. Gbadebo, Synthesis and in
vitro evaluation of 3-(4-nitrophenyl)coumarin Derivatives in Tumor Cell Lines, Bioorganic Chemistry (2014), doi:
http://dx.doi.org/10.1016/j.bioorg.2014.11.009
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Synthesis and in vitro evaluation of 3-(4-nitrophenyl)coumarin Derivatives in
Tumor Cell Lines
Musiliyu A. Musa a,*, Lekan M. Latinwo b, Clifford Virgile a, Veera L. D. Badisa b,
Akintunde J. Gbadebo b
a Departments of Chemistry, Florida A and M University, Tallahassee, FL 32307, USA
b Departments of Biology, Florida A and M University, Tallahassee, FL 32307, USA
*Correspondence to: Musiliyu A. Musa, Ph.D., Department of Chemistry, College of Arts and
Sciences, Florida A&M University, 219 Jones Hall, Tallahassee, FL 32307, USA. Tel: +1
8505993509, Fax: +1 8505612388, email: musiliyu.musa@famu.edu
Keywords: 3-Arylcoumarin; Cytotoxicity; Anticancer; Cell cycle, Mitochondrial Membrane
Potential.
1

Abstract: Coumarins are naturally-occurring compounds that have attracted considerable
interest due to their numerous biological activities depending on their pattern of substitution on
the coumarin molecule. In this present investigation, we synthesized 3-(4-nitrophenyl)coumarin
derivatives (9a-e) and evaluated their in-vitro cytotoxic effect on human lung (A549), breast
(MDA-MB-231) and prostate (PC3) cancer cell lines for 48 h using crystal violet dye binding
assay. Cytotoxic effects of the most active compound on normal human lung (MRC-9) and
breast (MCF-10A) cell lines, cell cycle analysis using flow cytometry and mitochondrial
membrane potential (MMP) using Tetramethyl Rhodamine Methyl Ester (TMRM; rhodamine-
123) fluorescent dye were also examined. Among the compounds that were evaluated, 9c
showed cytotoxic effect (active), caused significant cells arrest (p<0.05) in G0/G1 and S phases
of cell cycle and loss of MMP in A459, MDA-MB-231 and PC3 cell lines. Additionally, the
cytotoxic effect of 9c was compared to reference drugs (Coumarin and Docetaxel) for
comparative study. These results further demonstrate that acetoxy group at C-7 and C-8
positions of 9c are responsible for the observed cytotoxic effect in these cancer cell lines.
2

1. Introduction
Coumarins are important class of naturally-occurring heterocyclic compounds known as
benzo-α-pyrones, a pyrone ring fused with a benzene nucleus (1, 2). Most coumarin compounds
occurred as secondary metabolites in green plants, while some are produced by fungi and
bacteria (3). They exhibit diverse array of pharmacological and biochemical properties such as
anti-coagulant (4), anti-tumor (5, 6), anti-thrombotic (7), anti-viral (8, 9), anti-bacterial (10),
anti-microbial (11), anti-inflammatory (12, 13) and anti-oxidant (14, 15) properties. However,
the anti-proliferative and anti-tumor activities of various coumarins have been extensively
reported, e.g. simplest coumarin (1) and its metabolite, 7-hydroxycoumarin (2) have been
reported to inhibit cell growth proliferation in various human cancer cell lines, such as A549
(lung), ACHN (renal), H727 (lung), MCF-7 (breast) and HL-60 (leukemia) (16-190). They have
also been reported in clinical trials to demonstrate activity against breast cancer, prostate cancer,
malignant melanoma, and metastatic renal cell carcinoma (5, 20, 21). In addition to their
biological activities, coumarins are used as additives in food and cosmetics, and as optical
brightening agents (22, 23). These properties of coumarins have led to considerable interest in
their synthesis and possible applications as therapeutic agents for the treatment of various
diseases.
Recent studies have shown that therapeutic applications, including pharmacological and
biochemical properties of coumarins depends on the nature of the group present and its pattern of
substitution on the core coumarin molecule (24, 25). For examples, acetoxycoumarin such as
7,8-diacetoxy-4-methylcoumarin (DAMC; 3) exhibits anti-cancer and pro-oxidant activities in
human tumor cell lines (26, 27); nitrocoumarins exhibit potent anti-microbial, anti-allergic and
anti-cancer properties (528-30); hydroxycoumarins exhibit diverse biological properties such as
3

anti-coagulants, anti-arthritis, anti-inflammatory, anti-pyretic, anti-bacterial, anti-viral, and anti-
cancer properties (5, 31, 32); and 3-arylcoumarins commonly produced by plants have shown
different types of biological properties such as antioxidant, anti-inflammatory, anticancer, anti-
HIV and antimicrobial activities (33−36). As part of our on-going investigation on coumarins as
anticancer agents, we recently reported that 3-arylcoumarin containing acetoxy group (4) and
basic amino side chain (5 and 6) exhibits cytotoxic activity in various cancer cell lines (37, 38).
These studies and others not mentioned here strongly support the potential therapeutic
applications of coumarin and its derivatives, making them attractive for further evaluation as
novel therapeutic agents for cancer treatment.
Recent advances in anti-cancer studies have shown that heterocyclic compound
containing p-nitrophenyl pharmacophore offers great potential for the development of novel anti-
cancer agents (39, 40). This finding aroused our interest in the presence cytotoxicity studies of
coumarins containing p-nitrophenyl groups substituted at the 3-position with the aim of obtaining
more potent biologically active coumarins. Therefore, we herein report the synthesis of 3-(4-
nitrophenyl)coumarin derivatives (9a-e, Table I); and evaluation of their in-vitro cytotoxic effect
on A549, MDA-MB-231 and PC3 cancer cell lines. In addition, cytotoxic effect of the most
active compound on normal (MRC-9 and MCF-10A) cell lines, cell cycle and MMP were also
evaluated. Finally, cytotoxic effect of the most active compound was also compared to Coumarin
and Docetaxel drugs.
Fig. 1.
2.0. Materials and Methods
2.1. Chemistry
4

2.1.1. Materials
Commercial grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis,
MO, USA) or Alfa Aeser (Ward Hill, MA, USA) and used without further purification. The
NMR spectra were recorded on Varian 300 MHz spectrometer (1H: 300 MHz, 13C: 75MHz,).
The appropriate deuterated solvents are indicated in the experimental procedure and line
positions recorded in (δ) ppm from the reference signal. ESI-TOF Mass Spectrometer was
recorded on Finnigan LCQ – Quadrupole Ion Trap (Thermo Finnigan, San Jose, CA), and the
HPLC pump was an Agilent (HP) 1100 series pump or Applied Biosystems model 400 pump.
CHN elemental analysis was performed by combustion using automatic analyzers. To estimate
the degree of purity of the active compound (9c), HPLC analysis was conducted with Waters
Alliance 2996 HPLC system equipped with Wasters 2996 Photodiode array detector. HPLC
grade acetonitrile and HPLC water (Merck) was used for the chromatography. Melting points
were determined on a Gallenkamp (UK) apparatus.
2.1.2. General Experimental Procedures
To a mixture of 2-hydroxybenzaldehyde (7a) [or 2-hydroxyacetophenone (7b), 2,3,4-
trihydroxy benzaldehyde (7c), 2-hydroxy-4-methoxybenzophenone (7d) or 2-hydroxy-
benzophenone (7e)] (7.4 mmol) and 4-nitrophenylacetic acid (8) (8mmol) in acetic anhydride
(3mL) was added triethylamine (99%, 1 mL). The reaction mixture was then refluxed overnight
at 90oC, allowed to cool to r.t. and 10mL of ether was added with occasional stirring for another
2-3hrs to afford the desired product as a precipitate that was collected using vacuum filtration.
The synthesis and structural characterization of 9a has been previously reported in
literature (41-43).
5

2.1.2.1.
3-(4-nitrophenyl)coumarin (9a)
This title compound was prepared according to the above experimental procedure starting
from 2-hydroxybenzaldehyde (7a) to afford 9a as a yellow precipitate (1.02g, 52%) with m.p. =
o
1
267.2-268.4 C (Literature reported m.p. = 267-268 oC). H-NMR (300 MHz, CDCl3) δ: 7.33-
7.43 (2H, ArH), 7.59-7.65 (2H, ArH), 7.91-7.96 (3H, ArH) and 8.30-8.33(2H, ArH); HRMS
(ESI) (m/z): for C15H9NO4 calcd: 268.0604; [M+H]; found: 268.0605.
2.1.2.2.
4-methyl-3-(4-nitrophenyl)coumarin (9b)
This title compound was prepared according to the above experimental procedure starting
from 2-hydroxyacetophenone (7b) to afford 9b as a yellow precipitate (1.52g, 77%) with m.p. =
o
1
200.6-201.8 C. H-NMR (300 MHz, CDCl3) δ: 2.36 (3H, s. CH3), 7.35-7.41 (2H, ArH), 7.52-
7.55 (2H, ArH), 7.58-7.64 (1H, ArH), 7.72-7.45 (1H, ArH) and 8.30-8.33 (2H, ArH). 13C-NMR
(75MHz, CDCl3) 16.73 (CH3), 11.7.03, 120.00, 123.63, 124.70, 125.21, 125.35, 131.43, 132.23,
141.35, 147.55, 148.96, 152.74 (Ar-C) and 160.25 (C=O); HRMS (ESI) (m/z): for C16H11NO4
calcd: 282.0768; [M+H]; found: 282.0767.
2.1.2.3.
7,8-diacetoxy-3-(4-nitrophenyl)coumarin (9c)
This title compound was prepared according to the above experimental procedure starting
from 2,3,4-Trihydroxy benzaldehyde (7c) to afford 9c as a yellow precipitate (2.66g, 97%) with
m.p. = 224.4-226.1 oC. 1H-NMR (300 MHz, CDCl3) δ: 2.37-245 (6H, s, 2 x OCOCH3), 7.17-7.20
13
(1H, ArH), 7.46-7.49 (1H, ArH), 7.85-7.92 (3H, ArH) and 8.28-8.30 (2H, ArH). C-NMR
(75MHz, CDCl3) 20.34 and 20.68 (2 x OCOCH3), 117.88, 119.62, 123.70, 125.49, 125.83,
129.45, 140.56, 146.00, 145.85, 146.86, 147.87, 158.39 (Ar-C), and 167.33 and 167.72 (C=O);
6

HRMS (ESI) (m/z): for C19H13NO8 calcd: 384.0714; [M+H]; found: 384.0716. Anal. Calcd for
C19H13NO8: C, 59.54; H, 3.42; N, 3.65. Found: C, 59.70; H, 3.41; N, 3.69. Purity (HPLC):
99.17%.
2.1.2.4.
7-methoxy-3-(4-nitrophenyl)-4-phenylcoumarin (9d)
This title compound was prepared according to the above experimental procedure starting
from 2-Hydroxy-4-Methoxybenzophenone (7d) to afford 9d as a yellow precipitate (1.45g, 52%)
o
1
with m.p. = 214.9-216.6 C. H-NMR (300 MHz, CDCl3) δ: 3.91 (3H, s. OCH3), ), 6.78-6.82
(1H, ArH), 6.94-6.95 (1H, ArH), 7.01-7.17 (3H, ArH), 7.27-7.36 (5H, ArH) and 8.01-8.06 (2H,
ArH). 13C-NMR (75MHz, CDCl3) 55.91 (OCH3), 100.69, 112.78, 113.50, 121.41, 132.91,
128.66, 128.99, 129.16, 131.90, 133.93, 141.32, 146.72, 153.28, 155.21, 160.86 (Ar-C) and
163.22 (C=O); HRMS (ESI) (m/z): for C22H15NO5 calcd: 374.1023; [M+H]; found: 374.1035.
2.1.2.5.
3-(4-nitrophenyl)-4-phenyl-coumarin (9e)
This title compound was prepared according to the above experimental procedure starting
from 2-hydroxybenzophenone (7e) to afford 9e as a yellow precipitate (1.43g, 56%) with m.p. =
o
1
236.1-239.0 C. H-NMR (300 MHz, CDCl3) δ: 7.11-7.14 (2H, ArH), 7.24-7.28 (2H, ArH),
7.31-7.37 (5H, ArH), 7.45-7.48 (1H, ArH), 7.58-7.63 (1H, ArH) and 8.04-8.07(2H, ArH). 13C-
NMR (75MHz, CDCl3) 116.97, 119.99, 122.97, 124.56, 124.80, 128.10, 128.72, 129.10, 129.18,
131.77, 132.41, 133.60, 140.97, 146.92, 153.08, 153.38 (Ar-C) and 160.49 (C=O); HRMS (ESI)
(m/z): for C21H13NO4 calcd: 344.0917; [M+H]; found: 344.0927).
2.2.
Biology
2.2.1. Chemicals
7

F12 K medium, RPMI medium, penicillin-streptomycin anti-biotic solution (100x), fetal
bovine serum (FBS), Trypsin-EDTA solution (1x), phosphate buffer (PBS), 50% glutaraldehyde,
crystal violet, propidium iodide, Rhodamine-123, IGPAL CA-630, Coumarin, Docetaxel and
RNase were obtained from Sigma-Aldrich Company (St. Louis, MO, USA). The potassium
phosphate, EDTA, D-glucose, ethanol were obtained from Thomas Scientific Company
(Swedesboro, NJ, USA).
2.2.2. Cell line maintenance
The cell lines (A549, MRC-9, MDA-MB-231, MCF-10A and PC3) were obtained from
the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured as per the
guidelines supplied. The cells were maintained in F12K (A549, PC3) or MEM (MRC-9) or
RPMI (MDA-MB-231) or DMEM-F12 (MCF-10A) medium containing 100 units of
penicillin/ml, 100 µg of streptomycin/ml, 2 mM L-glutamine and 10% FBS in T-75 cm2 flasks at
37oC in a 5% CO2 incubator.
2.2.3. Treatment of cells
The cells (A549, MRC-9, MDA-MB-231, MCF-10A or PC3 ) were plated at a density of
5 x 104 cells per well in polystyrene, flat bottom 24-well microtiter plates (Corning Costar,
Rochester, NY, USA) in complete medium containing 10% FBS and allowed to stabilize
overnight in a CO2 incubator at 37oC. Next, the cells were treated with compounds (9a-e) at
different concentrations (0, 10, 25, 50, 75 and 100 µM) in a final volume of 1 ml per well in
triplicate wells for each treatment for 48 h at 37oC in a 5% CO2 incubator. All studies were
repeated at least twice.
8

2.2.4. Evaluation of cell viability
At the end of incubation period, the cell viability was evaluated by dye uptake assay
according to previous reported method by Badisa et al. (44). Glutaraldehyde (400 μl of 0.25%)
was added to each well and incubated for 30 min at room temperature to fix the cells. The
glutaraldehyde (0.07% final concentration in the well) in the crystal violet dye staining assay
procedure fixed the viable cells after the treatment with compounds. The plates were rinsed with
water to wash off the dead cells and dried under airflow inside a laminar hood for 5-10 min.
Crystal violet (400 μl of 0.1%) was added to each well, incubated for 15 min, washed and dried.
To solubilize the dye, 1 ml of 0.05 M sodium phosphate solution (monobasic) in 50% ethyl
alcohol was added to each well and the plates were read at 540 nm in a plate reader (Bio-Tek
EL800 Plate Reader). The mean absorbance value of control was considered as 100% and the
treated sample percentages were calculated by comparing the treated samples absorbance with
the mean absorbance of control.
2.2.5. Cell cycle analysis
The cell cycle analysis was evaluated using Accuri flow cytometer (BD Biosciences, San
Jose, CA). Cells (A549, MDA-MB-231 and PC3) at a density of 0.65 x 106 cells per T-25 cm2
flask were plated and incubated overnight. The cells were then treated with 0, 10 and 20 µM of
the active compound in triplicate flasks and incubated for 48 h in a 5% CO2 incubator at 37oC. At
the end of incubation, the cells were trypsinized and centrifuged at 2,500 rpm for 10 min at room
temperature. The pellet was suspended in 1 ml of PBS and transferred to eppendorff tube. The
cells were pelleted by centrifugation at 2,500 rpm for 5 min. Then the cells were stained with
500 µl of Vindelov’s reagent containing 0.01mg/ml of ribonunuclease A and 0.075mg/ml of
9

propidium iodide and 1 µl/ml of IGPAL CA-630 in phosphate buffered saline for overnight at
4oC in dark. The distribution of cells in each phase was analyzed with the Accuri flow cytometer
(Becton Dickinson, San Jose, CA). In each sample, a total of 50,000 events were analyzed
separately. The Accuri C6 software was used for acquisition and analysis of the data and the
percentage of cells in each phase.
2.2.6. Measurement of MMP
The loss of MMP was evaluated using rhodamine-123 fluorescent dye in A549, MDA-
MB-231 and PC3 cells. At the end of incubation, cells were fixed with 400 µl of 0.25% aqueous
glutaraldehyde containing rhodamine-123 to yield a final concentration of 1 µM for 30 min at
room temperature. The supernatant was discarded, and the plates were washed with tap water
and air dried in the hood. Finally, 500 µl of 0.1% Triton X 100 in dPBS was added per well and
incubated at 370C for 1 h. The plates were read with the excitation filter set at 485 nm and the
emission filter at 538 nm in TECAN microplate Fluorometer.
2.2.7. Statistical analysis
The viability values were presented as mean standard deviation (n=3). All treated cells
data were presented as percentage values in comparison to the untreated control (100%). The
viability and CC50 graphs were plotted in Prizm 3.00 software (GraphPad Software, Inc., San
Diego, CA, USA). The CC50 value was calculated from the CC50 graph where the two lines meet
(the dose at which 50% of the cells dies) using Prizm 5 software.
3.0 RESULT
3.1
Synthesis of 3-(4-nitrophenyl)coumarin derivatives (9a-e)
10

In this present investigation, we reported the synthesis of 3-(4-nitrophenyl)coumarin
derivatives (9a-e) via modified reported based-catalyzed Perkin condensation reaction (45-47)
(Scheme 1). This process involved refluxing a mixture of benzaldehyde (acetophenone or
benzophenone) and 4-nitrophenylacetic acid in the presence of triethylamine in acetic anhydride
overnight at 90oC to afford the desired compounds as solid between 52-97% yields. The
synthesized compounds were characterized using proton (1H) and carbon (13C) NMR, HRMS
and CHN elemental analyses as indicated in the experimental section. The HPLC analysis was
also conducted to estimate the degree of purity of the active compound (9c) and result indicated
99.17% purity.
Scheme 1.
Table 1.
3.2. Cytotoxicity Studies
The synthesized compounds (9a-e) were evaluated for their in-vitro cytotoxic effect using
crystal violet dye-staining assay. The cytotoxic concentration values (CC50, the concentration of
tested drug where 50% dead cells is observed in the treated sample compared to the untreated
control) for 9a-e, Coumarin and Docetaxel were calculated according to the Ipsen’ method (48)
and are given in Table 1. Result indicated that 9c showed cytotoxic effect in A549 (CC50 = 12.2
µM), MDA-MD-231 (CC50 = 27.6 µM) and PC3 (CC50 =18.2µM) cancer cell lines, while 9a, 9b,
9d and 9e didn’t show cytotoxic effect (CC50 > 100 µM; inactive). The compound 9c was most
cytotoxic to A549 cells, causing dose-dependent cell death in comparison to the untreated control
cells (Fig. 2). Comparison of the cytotoxic effect of 9c with Coumarin and Docetaxel revealed
11

that (1) Coumarin was not active in the three cancer cell lines (CC50 >100) and (2) 9c showed
comparable cytotoxicity to Docetaxel on A549 cell line and lesser cytotoxicity compared to
Docetaxel on both MDA-MD-231 and PC3 cell lines (Table 1). Additionally, 9c showed similar
cytotoxic effect on both cancer (A549 and MDA-MB-231) and normal (MRC-9 and MCF-10A)
cell lines (Table 1).
Fig. 2.
3.3.
Cell cycle Analysis
Cells (A549, MDA-MB-231 and PC3) were treated with 9c for 48 h, followed by
staining with propidium iodide. The percentage of cells in G1, G2/M and S phases of the cell
cycle was analyzed using a flow cytometer. Result indicated that treatment of A549, MDA-MB-
231 and PC3 cells with 9c caused cell cycle arrest (p<0.05) in concentration dependent manner
(Fig. 3A-C). Furthermore, it was observed that 9c arrest (i) A549 cells in G0/G1 phase at 10 µM
concentration (2.17%) (Fig. 3A), (ii) MDA-MB-231 cells in G0/G1 phase at 30 µM concentration
(2.90%), and S phase at both 15 µM concentration (4.72 %) and 30 µM concentration (2.23%)
(Fig. 3B) and (iii) PC3 cells in G0/G1 phase at both 10 µM concentration (3.04%) and 20 µM
concentration (2.84%) with respect to the untreated control cells (Fig. 3C).
Fig. 3.
3.4. Measurement of MMP
Cells (A549, MDA-MB-231 and PC3) were treated with 9c for 48 h and then fixed with
rhodamine-123 dye. Results indicated that 9c caused loss of MMP in the following order; A549
(lung) > PC 3 (prostate) >MDA-MB-231 (breast) cells (Fig. 4a). The percentage of MMP in
12

comparison to the untreated control cells (100%) in (i) A549 cells at 10 μM (74.2% 3.86), 25
μM (38.7% 0.99), 50 μM (35.8% 1.21), 75 μM (34.9% 1.45) and 100 μM (32.2% 2.82); (ii)
MDA 231 cells at 10 μM (90.8% 2.56), 25 μM (82.4% 1.87), 50 μM (81.9% 2.52), 75 μM
(80.7% 2.772) and 100 μM (72.4% 2.47) and (iii) PC3 cells at 10 μM (91.1% 4.22), 25 μM
(70.9% 3.07), 50 μM (59.6% 0.66), 75 μM (55.2% 4.29) and 100 μM (55.0% 0.66); indicating
loss of MMP respectively. Additionally, cells exposed to 9c treatment at shorter period of time
(4, 6 and 24 h) also showed dose and time dependent loss of MMP in all three cancer cell lines as
indicated in Figure 4b.
Fig. 4.
4.0. DISCUSSION
Interest in the synthesis and biological evaluation of coumarins has increased over the
years due to their diverse pharmaceutical activities. As part of our ongoing investigation
involving coumarins aims at evaluating the role of substituent effect in modulating cytotoxicity,
we synthesized series of 3-(4-nitrophenyl)coumarin derivatives (9a-e, Table 1) and evaluated
their cytotoxic effect on A549, MDA-MB-231 and PC3 cancer cell lines. The in-vitro
cytotoxicity results indicate that 9c bearing acetoxy group at C-7 and C-8 positions on the
benzenoid ring of coumarin molecule showed significant cytotoxic effect with respect to
untreated cells, while absence of this group as indicated in 9a, 9b, 9d and 9e resulted in loss of
cytotoxic activity (Table 1). This finding is supported by previous reports indicating that the
presence of acetoxy group at C-7 and C-8 positions of the coumarin molecule (3) enhanced drug
activity such as anti-cancer, antioxidant and radicals scavenging properties (26, 27, 49).
Interestingly, Coumarin was relatively non-toxic in all tested cancer cell lines; indicating that it’s
13

cytotoxic effect was metabolism and dose-time dependent (50, 51). Additionally, compound 9c
showed similar cytotoxicity on A549, and two-fold (PC3) and fourth-fold (MDA-MB-231)
decreased in cytotoxicity compared to Docetaxel. Docetaxel (formerly called taxotere) belongs to
a new class of cytotoxic agents known as taxanes and is used in the treatment of breast cancer,
lung cancer, prostate cancer, stomach cancer, and head/neck cancer (52). Comparison of 9a and
9c based on structural relationship (SAR) activity indicates that presence of p-nitrophenyl group
at the C-3 position did not make any contribution to the observed cytotoxic effect of 9c; further
supporting the beneficial effect of acetoxy group at C-7 and C-8 positions on the coumarin
molecule in modulating cytotoxicity. The order of 9c cytotoxic effect based on CC50 values is
A549 > PC3 > MDA-MD 231 cells. However, the non-selectivity of 9c in normal MRC-9 (CC50
= 17.1µM 1.07) and MCF-10A (CC50 = 9.6µM 1.41) cell lines is of utmost concern, since one
of the important criteria in the development of therapeutic drugs for cancer treatment is to have
few or no side-effects on normal cells of patients undergoing chemotherapy. A possible reason
for the lack of 9c selective cytotoxicity could be due that the two selected non-malignant lines
are female-origin cells (53).
One effective strategy for controlling tumor growth is the inhibition of cell cycle
progression in cancer cells (54). Previous studies have demonstrated that toxic effect of
compound on different phases of the cell cycle can activate or inhibit of cell growth and
proliferation of cancer cells (55, 56). In the present investigation, it was observed that 9c caused
cell arrest of (i) A549, MDA-MB-231 and PC3 cells in G0/G1 phase, indicating apoptotic mode
of cell death and (ii) MDA-MB 231 cells in S phase, indicating inhibition of DNA synthesis (Fig.
3). This finding is consistent with previous investigations indicating that coumarins caused G0/G1
and S phases cell arrest in different cancer cell lines (57, 58).
14

Rhodamine-123, a green-fluorescent dye, has been used to measure MMP changes in
cells (59). This dye is selectively taken up by mitochondria and the amount taken up by the cells
is directly proportional to its membrane potential (59). Thus, evaluation of MMP changes in
living cells is of critical important in accessing the effect of chemicals on mitochondria function
(60). The present investigation indicates loss of MMP in the following order: MDA-MB-231 >
PC3 >A549 cell lines after 48 h treatment with 9c (Fig. 4a). Furthermore, 9c caused dose-time
dependent loss of MMP in A549 cells during the early stages of the treatment; suggesting that it
might be using intrinsic apoptotic pathway to inflict cell death (Fig. 4b) (61). Interestingly, the
observed correlation between cytotoxic effect and loss of MMP in these cancer cell lines (Fig. 1
and 4) indicates that the elicited differential cytotoxicity of 9c may be associated with the loss of
MMP.
5.0. CONCLUSION
A series of 3-(4-nitrophenyl)coumarin derivatives (9a-e) were synthesized, characterized
and evaluated for their in vitro cytotoxic effect on A549, MDA-MB-231 and PC3 cancer cell
lines. Compound 9c bearing acetoxy group at C-7 and C-8 positions showed cytotoxic effect
compared to other synthesized analogs (9a, 9b, 9d and 9e). The cytotoxic effect of 9c is
associated with cell arrest in G0/G1 and S phases of the cell cycle, and loss of MMP.
Additionally, the elicited differential cytotoxicity of 9c is associated with the loss of MMP based
on the observed correlation between cytotoxic effect and loss of MMP. This present investigation
further correlates the importance of acetoxy group at C-7 and C-8 positions on the coumarin
molecule with respect to modulating cytotoxic effect and thus indicates that 9c could serve as an
15

attractive leads in the future development of new cytotoxic compounds that are more potent and
selective.
Declaration of interest statement
The authors declare that they have no financial or non-financial competing interests.
Acknowledgements
The authors gratefully acknowledge Florida A & M University TITLE III PROGRAM for their
financial support.
References
[1]
[2]
R. D. H. Murray, J. Mendez, S. A. Brown, John Wiley, New York, pp. 282-289, 1982.
F. Borges, F. Roleira, N. Milhazes, L. Santana, E. Uriarte, Curr. Med. Chem. 12(8) 887-
916.
[3]
[4]
[4]
[6]
[7]
[8]
Z. E. Nazari, M. Iranshahi, Phytother Res. 25 (2011) 315-323.
I. Manolov, C. Maichle-Moessmer, N. Danchev, Eur. J. Med. Chem. 41 (2006) 882-890.
I. Kostova, Curr Med Chem Anticancer Agents 5 (2005) 29-46.
H. Zhao, B. Yan, L. B. Peterson, B. S. Blagg, ACS Med. Chem. Lett. 3 (2012) 327-331.
J. R. Hoult, M. Paya, Gen. Pharmacol. 27 (1996) 713-722.
L. Xie, Y. Takeuchi, L. M. Cosentino, A. T. McPhail, K. H. Lee, J. Med. Chem. 44
(2001) 664-671.
[9]
Z. M. Nofal, M. I. El-Zahar, S. S. AbdEl-Karim, Molecules 5 (2000) 99-113.
[10] P. Aragade, V. Maddi, S. Khode, M. Palkar, P. Ronad, S. Mamledesai, D.
Satyanarayana, Arch Pharm (Weinheim) 342 (2009) 361-366.
[11] D. A. Ostrov, J. A. Hernandez Prada, P. E. Corsino, K. A. Finton, N. Le, T.C. Rowe,
Antimicrob. Agents Chemother. 51 (2007) 3688-3698.
[12] S. Khode, V. Maddi, P. Aragade, M. Palkar, P. K. Ronad, S. Mamledesai, A. H.
Thippeswamy, D. Satyanarayana, Eur. J. Med. Chem. 44 (2009) 1682-1688.
[13] I. E. Bylov, M. V. Vasylyev, Y. V. Bilokin, Eur. J. Med. Chem. 34 (1999) 997-1001.
[14] O. M. Singh, N. S. Devi, D. S. Thokchom, G. J. Sharma, Eur. J. Med. Chem. 45 (2010)
2250-2257.
[15] S. Stanchev, V. Hadjimitova, T. Traykov, T. Boyanov, I. Manolov, Eur. J. Med. Chem.
44 (2009) 3077-3082.
[16] M. Baba, Y. Jin, A. Mizuno, H. Suzuki, Y. Okada, N. Takasuka, H. Tokuda, H.
Nishino, T. Okuyama, Biol. Pharm. Bull 25 (2002) 244-246.
[17] D. Thornes, L. Daly, G. Lynch, H. Browne, A. Tanner, F. Keane, S. O'Loughlin, T.
Corrigan, P. Daly, G. Edwards, J. Henley, T. Healy, F. Keane, F. Lennon, N. McMurray,
16

S. O'Loughlin, M. Shine, A. Tanner, Eur. J. Surg. Oncol. 15 (1989) 431-435.
[18] S. Stanchev, G. Momekov, F. Jensen, I. Manolov, Eur. J. Med. Chem. 43 (2008) 694-
706.
[19] M. E. Marshall, K. Kervin, C. Benefield, A. Umerani, S. Albainy-Jenei, Q. Zhaoand M.
B. Khazaeli, J. Cancer Res. Clin. Oncol. 120 Suppl (1994), S3-10.
[20] J. L. Mohler, L.G. Gomella, E. D. Crawford, L. M. Glode, C. D. Zippe, W.R. Fair, M. E.
Marshall, Prostate 20 (1992) 123-131.
[21] A. Lacy, R. O'Kennedy, Curr. Pharm. Des. 10 (2004) 3797-3811
[22] R. O'Kennedy, R. D. Thornes, Coumarins: Biology, Applications and Mode of
Action, John Wiley & Sons, Chichester, 1997.
[23] D. Bogdal, J. Chem. Res. (S) (1998) 468-469
[24] M. A. Musa, J. S. Cooperwood, M. O. Khan, Curr. Med. Chem. 15 (2008) 2664-2679.
[25] A. Carotti, A. Carrieri, S. Chimichi, M. Boccalini, B. Cosimelli, C. Gnerre, P. A.
Carrupt, B. Testa, Bioorg. Med. Chem. Lett. 12 (2002) 3551-3555.
[26] L. Koshy, B. S. Dwarakanath, H. G. Raj, R. Chandra, T. L. Mathew, Indian J. Exp. Biol.
41 (2003) 1273-1278.
[27] P. Khurana, R. Kumari, P. Vohra, A. Kumar, Seema, G. Gupta, H. G. Raj, B. S.
Dwarakanath, V. S. Parmar, D. Saluja, M. Bose, A. Vij, N. K. Chaudhary, J. S.
Adhikari, Y. K. Tyagi, E. Kohli, Bioorg. Med. Chem. 14 (2006) 575-583.
[28] I. Fujii, Y. Mano, N. Hirayama, Analytical Sciences 22 (2006) 117-118.
[29] R. S. A. de Araújo, F. Q. S. Guerra, E. de O. Lima, C. A. de Simone, J. F. Tavares, L.
Scotti, M. T. Scotti, T. M. de Aquino, R. O. de Moura, F. J. B. Mendonça Jr., J. M.
Barbosa-Filho, Int. J. Mol. Sci. 14 (2013) 1293-1309.
[30] Ž. Debeljak, A. Škrbo, I. Jasprica, A. Mornar, V. Plec ko, M. Banjanac, M. Medić-
Šarić,
J. Chem. Inf. Model. 47 (2007) 918-926.
[31] J-C. Jung, O-S. Park, Molecules 14 (2009) 4790-4803.
[32] S. Thaisrivongs, P. K. Tomich, K. D. Watenpaugh, K. T. Chong, W. J. Howe, C. P.
Yang, J. W. Strohbach, S. R. Turner, J. P. McGrath, J. Med. Chem. 37 (1994) 3200-
3204.
[33] S. Demizu, K. Kajiyama, K. Takahashi, Y. Hiraga, S. Yamamoto, Y. Tamura, K. Okada,
T. Kinoshita, Chem. Pharm. Bull. 36 (1988) 3474−3479.
[34] H. R. J. El-Seedi, Nat. Prod. 70 (2007) 118−120.
[35] L. S. You, R. An, X. H. Wang, Y. M. Li, Bioorg. Med. Chem. Lett. 20 (2010)
7426−7428.
[36] D. Olmedo, R. Sancho, L. M. Bedoya, J. L. Lopez-Perez, E. Del Olmo, E. Munoz,
J. Alcami, M.P. Gupta, A. San Feliciano, Molecules 17(2012) 9245-9257.
[37] M. A. Musa, V. L. Badisa, L. M. Latinwo, J. Cooperwood, A. Sinclair, A. Abdullah,
Anticancer Res. 31 (6) 2017-2022.
[38] M. A. Musa, V.L. Badisa, L. M. Latinwo, C. Waryoba, N. Ugochukwu, Anticancer
Res. 30 (11) 4613-4617.
[39] S. S. Shah, G. Rivera, M. Ashfaq, Mini. Rev. Med. Chem. 13 (2013) 70-86.
[40] S. Ibsen, E. Zahavy, W. Wrasdilo, M. Berns, M. Chan, S. Esener, Pharm. Res. 27 (2010),
1848-1860.
[41] S. H. Mashraqui, D. Vashi, H. D. Mistry, Synth. Commun. 34 (2004) 3129-3134.
17

[42] M. J. Matos, S. Vazquez-Rodriguez, F. Borges, L. Santana, E. Uriarte, Tetrahedron
Letters 52 (2011) 1225-1227.
[43] C. Rondestvedt, S. Christian, O. Vogl, J. Am. Chem. Soc. 77 (1955) 3401-3402.
[44] R. B. Badisa, O. Tzakou, M. Couladis, E. Pilarinou, Phytother Res. 17 (2003) 472-476.
[45] R. M. Kanojia, N. Jain, J. Xu, Z. Sui, Tetrahedron Lett. 45 (2004) 5837
[46] A. Horvath, K. De Smet, D. Ormerod, D. Depre´, C. Pe´rez-Balado, T. Govaerts, D. Van
den Heuvel, I. Schorpion, I. Org. Process Res. Dev. 9 (2005) 356
[47] M.A. Musa, A. Zhou, O.A. Sadik, Med. Chem. 7 (2011) 112-120.
[48] J. Ipsen, P. Feigl, Bancroft’s Introduction to Biostatistics, 2nd edition. Harper and Row,
New York, p. 164, 1970.
[49] H. G. Raj, V. S. Parmar, S. C. Jain, S. Goel, P. Himanshu, S. Malhotra, A. Singh, C. E.
Olsen, J. Wengel, Bioorg. Med. Chem. 6 (1998) 833-839.
[50] M. Kawase, H. Sakagami, K. Hashimoto, S. Tani, H. Hauer, S. S. Chatterjee, Anticancer
Res. 23 (2003) 3243-3246
[51] S. L. Born, D. Caudill, B. J. Smith, L. D. Lehman-McKeeman, Toxicol. Sci., 58 (2000)
23-31.
[52] N. J. S. Fauzee, Z. Dong, Y. Wang, Asian Pacific J Cancer Prev. 12 (2011) 837-851
[53] L. M. Nunes, E. Robles-Escajeda, Y. Santiago-Vazquez, N. M. Ortega, C. Lema, A.
Muro, G. Almodovar, U. Das, S. Das, J. R. Dimmock, R. J. Aguilera, A. Varela-
Ramirez, Aaps J 16 (2014) 872-874.
[54] J. K. Buolamwini, Curr. Pharm. Des. 6 (2000) 379-392.
[55] J. I. Chao, P.C. Kuo, T.S. Hsu, J. Biol. Chem. 279 (2004) 20267-20276.
[56] X. Xu, Y. Zhang, D. Qu, T. Jiang, S. Li, J. Exp. Clin. Cancer Res. 30 (2011) 33.
[57] J. Kahn, P. Preis, F. Waldman, A. Tseng, Jr., J. Cancer Res. Clin. Oncol. 120 Suppl
(1994) S19-22.
[58] A. Rasul, M. Khan, B. Yu, T. Ma, H. Yang, Asian Pac. J. Cancer Prev. 12 (2011) 1219-
1223
[59] J. M. Millot, S. Sharonov, M. Manfait, Cytometry 17 (1994) 50-58.
[60] S. Jayaraman, J. Immunol. Methods 306 (2005) 68-79.
[61] E. Robles-Escajeda, D. Lerma, A. M. Nyakeriga, J. A. Ross, R. A. Kirken, R. J.
Aguilera, A. Varela-Ramirez, PLoS One, 8 (2013), e73508.
18

Figure Captions
Scheme 1. Reagents and Conditions: (i) AC2O, NEt3, refluxed overnight at 90oC
Fig. 1. Structures of (1) Coumarin, (2) 7-hydroxycoumarin. (3) 7,8-diacetoxy-4-methyl coumarin
(DAMC), (4) 4-(7-(diethylamino)-4-methyl-2-oxo-2H-chromen-3-yl)phenyl acetate (5) 3-(4-(2-
(dimethylamino)ethoxy)phenyl)-7-methoxy-4-phenylcoumarin
(6)
3-(4-(2-(pyrrodin-1-yl)-
ethoxy)phenyl)-7-methoxy-4-phenylcoumarin
Fig. 2. Effect of compound 9c on A549, MDA-MB-231 and PC3 cell viability. Data are
represented as the mean and SD, n=6.
Fig. 3: Effect of compound 9c on (A) A549, (B) MDA-MB-231 and (C) PC3 cell cycle. Data are
represented as mean and SD, n=3. *Statistically significant difference from the control (p<0.05)
using Dunnett’s multiple comparison test.
Fig. 4. Effect of compound 9c on mitochondrial membrane potential in (A) A549, MDA-MB-
231 and PC3 cell lines for 48 h and (B) A549 cell lines for 4, 6, 24 h. Data are represented as
mean and SD, n=3.*Statistically significant difference from the control (p<0.05) using Dunnett’s
multiple comparison test.
R
H
R1
NO2
O
NO2
a
H
R1
b
c
H
CH3
H
R1
OH
(i)
R
R
7,8-OAc
COOH
O
O
d
e
7-OAc C6H5-
C6H5-
8
7a-e
9a-e
H
Scheme 1.
19

Fig. 1.
O
4
5
8
O
O
6
7
3
2
O
O
O
O
O
N
O
O
O
O
O
1
(4)
(3)
(1)
O
HO
O
O
N
N
(2)
O
O
O
O
O
O
(6)
(5)
Fig. 2.
120
100
80
60
40
20
0
A549
MDA-MB- 231
PC3
0
20
40
60
80
100
120
Compound 9c (μM)
20

Fig. 3.
A.
100
80
60
40
20
0
Control
5 μM
*
10 μM
A549 Cells
G0/G1
S
G2/M
21

B.
100
75
50
25
0
Control
15 μM
30 μM
*
MDA-MB-231 Cells
*
G0/G1
S
G2/M
22

C.
100
80
60
40
20
0
Control
10 μM
20 μM
*
*
PC3 Cells
G0/G1
S
G2/M
23

Fig. 4.
A.
160
140
120
100
80
A549
MDA 231
PC3
60
40
20
0
0
20
40
60
80
100
120
Compound 9c (μM)
B.
150
100
50
0
10 μM
A549 cells
25 μM
50 μM
75 μM
100 μM
*
*
*
*
*
*
*
*
*
*
*
*
*
0
4 h
6 h
24 h
24

Table 1. The CC50 values (μΜ) for compounds 9a-e and reference drugs (Coumarin and
Docetaxel) tested on lung (A549), breast (MDA-MD-231) and prostate (PC3) cancer cell lines
for 48 h.
______________________________________________________________________________
CC50 (µM) mean ± SD
Compounds
A549
MRC-9
MDA-MB-231
MCF-10A
PC-3
NO2
NO2
>100.0
>100.0
ND
ND
>100.0
>100.0
ND
ND
>100.0
>100.0
O
O
9a
9b
O
O
9c
NO2
12.2 1.85 17.1µM 1.07 27.6 3.39 9.6µM 1.41 18.2 0.15
O
O
O
O
O
O
NO2
>100.0
>100.0
ND
ND
>100.0
>100.0
ND
ND
>100.0
>100.0
O
O
O
9d
NO2
O
O
9e
Docetaxel
Coumarin
9.40 0.07
>100
ND
ND
7.30 0.03
>100
ND
ND
9.44 0.44
>100
1
Data represent the average of two values (n = 6).
2The cytotoxic concentration (CC50) value was determined from the graph where the live and dead cells line graphs
meet in the Graph pad Prism.
3 Drugs effects were determined for 48 h exposure
4 ND: Not Determined
25

Graphical Abstract:
R
H
R1
H
9a
9b
9c
H
CH3
H
NO2
R1
O
7,8-OAc
9d
9e
7-OAc -C6H5
-C6H5
R
O
H
OAc = acetoxy group
26

Highlights
ꢀ 3-(4-nitrophenyl)coumarin derivatives (9a-e) were synthesized
ꢀ
ꢀ
ꢀ
9a-e were evaluated for in-vitro cytotoxic effect in cancer cell
lines
9c bearing acetoxy group at C-7 and C-8 positions exhibited
cytotoxic effect
9c caused G0/G1 and S phases cell arrest and loss of MMP
27