Design, synthesis and biological evaluation of novel furoxan-based coumarin derivatives as antitumor agents

Article abstract of DOI:10.1007/s00044-018-2140-x

In order to find new anticancer drugs, a series of novel furoxan-based coumarin derivatives (10a–k) were synthesized and evaluated for their antiproliferative activities in vitro. All compounds displayed more potent inhibition on human cervical cancer HeLa cell proliferation than coumarin-3-carboxylic acid, and compounds 10b, 10c, 10f, 10h, and 10i with IC₅₀ values ranging from 0.88 to 5.95 μM were even stronger than doxorubicin (IC₅₀ = 10.21 μM). The further study showed that compound 10i exerted the highest antiproliferative activity (IC₅₀ = 0.60 μM) against human breast cancer MCF-7 cells, and compound 10f had broader spectrum antiproliferative activity against five cancer cells with IC₅₀ values in the low micromolar range of 1.86–9.85 μM. More interestingly, compound 10f had little effect on normal intestinal epithelial CCD841 cells. Our findings suggest that these novel furoxan-based coumarin derivatives may provide a new framework for the discovery of novel antitumor agents for the intervention of human carcinoma cells.

Full text of DOI:10.1007/s00044-018-2140-x

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-018-2140-x
ORIGINAL RESEARCH
Design, synthesis and biological evaluation of novel furoxan-based
coumarin derivatives as antitumor agents
Zhuo Zhang1 Zhi-Wei Bai1 Yong Ling2 Li-Qin He1 Peng Huang1 Hong-Xia Gu3 Rong-Feng Hu1
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Received: 21 September 2017 / Accepted: 20 January 2018
© Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
In order to find new anticancer drugs, a series of novel furoxan-based coumarin derivatives (10a–k) were synthesized and
evaluated for their antiproliferative activities in vitro. All compounds displayed more potent inhibition on human cervical
cancer HeLa cell proliferation than coumarin-3-carboxylic acid, and compounds 10b, 10c, 10f, 10h, and 10i with IC50 values
ranging from 0.88 to 5.95 μM were even stronger than doxorubicin (IC50 = 10.21 μM). The further study showed that
compound 10i exerted the highest antiproliferative activity (IC50 = 0.60 μM) against human breast cancer MCF-7 cells, and
compound 10f had broader spectrum antiproliferative activity against five cancer cells with IC50 values in the low
micromolar range of 1.86–9.85 μM. More interestingly, compound 10f had little effect on normal intestinal epithelial
CCD841 cells. Our findings suggest that these novel furoxan-based coumarin derivatives may provide a new framework for
the discovery of novel antitumor agents for the intervention of human carcinoma cells.
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Keywords Coumarin Furoxan Antitumor activity Synthesis
Introduction
efficacy. Coumarin (Fig. 1) is an important classes of nat-
ural products, which isolated from Umbelliferae, Aster-
aceae, Rutaceae, Leguminoase sp, etc (Wang et al. 2017;
Cui et al. 2007; Vilar et al. 2006), showed broad spectrum
biological activity such as antidepressant, antimicrobial,
antioxidant, antitumor, anti-HIV, and anticoagulant (Chen
et al. 2007; Adams et al. 2006; Piao et al. 2004; Appendino
et al. 2004; Cardellina et al. 1995; Zhou et al. 2009). Several
of natural, semisynthetic, or synthetic lead molecules
bearing coumarin scaffold have been discovered in various
phase of drug development in the recent past. Recently,
many coumarin derivatives were widely reported to possess
antitumor activity. For instance, psoralen (Fig. 1), a com-
pound extracted from the seeds of psoralea corylifolia,
displayed antiproliferative and cytotoxic activity to breast
cancer cell EMT6 (Wu et al. 1998) and lung cancer cell
A549 (Hsieh et al. 2014). Compound A (Fig. 1) (Kamath
et al. 2015), indole-3-carboxylic acid substituent at the C3-
position of coumarin, had significant antiproliferative
activity on human breast cancer MCF-7 (IC50 = 5.5 μM)
and low inhibition activity on Africa green monkey kidney
cell Vero (IC50 > 100 μM), displayed superior selectivity.
Compound B (Fig. 1), 3,4-2H-pyrazole-substituent at the
C3-position of coumarin, showed strong inhibition activity
on human gastric cancer cell SGC-7901 (IC50 = 2.69 μM)
Malignant tumor is one of the leading causes of mortality
worldwide. Drug resistance and low selectivity is a common
issue to cancer. Hence, it is important to searching for novel
anticancer drugs to address these problems.
It is well-known that natural products play a critical role
in the drug discovery of virtually all therapeutic areas,
especially in the lead identification and drug discovery in
oncology, and nearly half of the new drugs introduced into
the market are natural products or their derivatives (New-
man et al. 2016). Structural modification of a natural
molecule is common strategy to improve pharmacological
These authors contributed equally: Zhuo Zhang, Zhi-Wei Bai.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-018-2140-x) contains supplementary
material, which is available to authorized users.
* Li-Qin He
hlq661125@126.com
1
Anhui University of Chinese Medicine, 230012 Anhui China
2
School of Pharmacy, Nantong University, 226001 Nantong China
3
West Anhui Health Vocational College, 237005 Anhui China

Medicinal Chemistry Research
O O
S
O
O
O
O
N
O
N
O
Compound C
Fig. 2 Chemical structure of hybrid of 7-hydrocoumarin and furoxan
established by infrared (IR), high resolution mass spectro-
metry (HRMS), and 1H and 13C nuclear magnetic resonance
(NMR), and these target compounds were evaluated for
their inhibitory activity against some cancer cells pro-
liferation. Herein, we report the synthesis and biological
evaluation of these derivatives.
Fig. 1 Chemical structure of coumarin and coumarin related
compound
(Wang et al. 2013). Therefore, coumarins were considered
as a privileged structure for designing novel agents.
Materials and methods
Nitric oxide is one of the most important signaling
molecular, which plays important role in physiological
activity of human body. In recent years, it has been found
that high concentration of NO displayed cytotoxic effect
and could induce apoptosis of tumor cells, hold back tumor
cells proliferation, and assist macrophage to devour tumor
cells (Wink et al. 1998; Hofseth et al. 2003; Huang et al.
2017). Furoxans is an important class of NO-donors, which
can produce high concentration of NO and display strong
antitumor activity (Cerecetto et al. 2005; Boiani et al. 2001).
Compound C, a furoxans-based nitric oxide releasing cou-
marin derivative (Liu et al. 2014), displayed strong anti-
proliferation activities on A549 (IC50 = 0.024 μM), HeLa
(IC50 = 0.11 μM), A2780 (IC50 = 0.014 μM), and HUVEC
(IC50 = 0.034 μM) cell lines and three drug resistant tumor
cell lines MDA-MB-231/Gem (IC50 = 0.14 μM), A2780/
CDDP (IC50 = 0.062 μM), and SKOV3 (IC50 = 0.083 μM)
cell lines in vitro, superior than cisplatin. More importantly,
compound C exhibited lower cytotoxicity on normal cell
T29 (IC50 > 2.0 μM), showing good selectivity against
tumor cells (Fig. 2).
Our research group once synthesized a series of NO-
donating N-alkyl matrinol and NO-donating N-alkyl
matrinic acid derivatives (Wu et al. 2015; He et al. 2010,
2015). Their antitumor activity in vitro showed that some of
compounds exhibited high anti-proliferative activity on
human hepatocellular carcinoma cells Bel-7402 (IC50
values ranging from 2.55 to 3.26 μM) and SMMC-7721
(IC50 values ranging from 5.26 to 5.79 μM) (Fig. 3). With
the aim to develop a new lead possessing efficient phar-
macological activities, using the molecular hybridization
approach, we designed new analogs of coumarin, contain-
ing the furoxan NO donor moiety. It was expected that the
novel furoxan-based coumarin derivatives would improve
the potency and spectrum. All chemical structures were
General
Melting points were measured using a WRS-1B apparatus
without any correction. 1H and 13C NMR spectra were
recorded on 400 MHz Bruker Avance DPX spectrometers
and referenced with tetramethylsilane as an internal stan-
dard. All NMR spectra were recorded in CDCl3 or dimethyl
sulfoxide (DMSO)-d6 at room temperature. IR spectra were
collected on Nicolet Avatar 6700 spectrometer using KBr
film. ESI mass spectra were acquired using a Thermo Fisher
LTQ Orbitrap XL Liquid chromatography-mass spectro-
metry instrument.
Ethyl 2-oxo-2H-chromene-3-carboxylate (2)
The mixture of Salicylaldehyde (4.2 mL, 40 mmol), diethyl
malonate (8.5 mL, 56 mmol), anhydrous EtOH (20 mL),
piperidine (0.5 mL, 5 mmol), and AcOH (0.1 mL) was
heated under 80 °C. After 2 h, water (30 mL) was added to
the reaction mixture, and the resulting suspension was
placed in 0 °C. The crystalline was collected by filtration,
washed with 50% of EtOH, and dried. Finally, the crude
product was purified by recrystallized from 25% EtOH to
give white solid 2 (7.5 g, 85.4%), m.p. 92.0–93.1 °C (Gao
et al. 2014).
2-Oxo-2H-chromene-3-carboxylic acid (3)
The mixture of compound 2 (8.0 g, 28 mmol), NaOH (6.0 g,
150 mmol), 95% EtOH (30 mL) and water (20 mL) was
refluxed and stirred for 20 min. After cooling, the mixture
was added to a solution of 20 mL concentrated hydrochloric
acid in 100 mL water under stirring. Then the crude product
was reduced pressure suction filter and washed with ice
water. Finally, the crude product was purified by

Medicinal Chemistry Research
Fig. 3 Chemical structure of
NO-donation N-(n-Butyl)
matrinic acid and N-(n-Butyl)
matrinol derivatives
1
recrystallized from 50 % EtOH to give white crystal 3 (5.0
1751, 1709, 1626, 1556, 1447, 1255, 1165; H NMR (400
g, 95.6%), m.p. 193.2–194.7 °C (Gao et al. 2014).
MHz, CDCl3) δ 8.58 (s, 1H, =CH), 8.07 (s, 2H, Ph-H), 7.76
(s, 1H, Ph-H), 7.65 (s, 3H, Ph-H), 7.37 (d, J = 4.5 Hz, 2H,
Ph-H), 4.65 (s, 2H, O–CH2), 4.57 (s, 2H, O=CCH2), 2.39
(s, 2H, CH2); 13C NMR (100 MHz, CDCl3) δ 163.11,
158.85, 156.57, 155.21, 149.06, 138.00, 135.69, 134.57,
129.73 (3C), 128.48 (2C), 124.95, 117.87, 117.81, 116.74,
110.49, 68.13, 61.80, 27.90.
2-Oxo-2H-chromene-3-carbonyl chloride (4)
Compound 3 (4 g, 19 mmol) and pyridine (0.3 mL) was
dissolved in sulfoxide chloride (15 mL) and refluxed 6 h.
After cooling, the mixture was removed redundant sulf-
oxide chloride by reduced pressure to give crude product.
Finally, the crude product was purified by recrystallized
from petroleum ether to give faint yellow solid 4 (3.3 g,
81.2%), m.p. 143.6–145.8 °C (Mo et al. 2006).
4-(4-((2-oxo-2H-chromene-3-carbonyl)oxy)butoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10c)
White solid; yield: 80.6%; m.p. 134.6–135.9 °C; APCI-
HRMS (m/z): calcd for C22H18N2O9S (neutral M + H)
487.0733; found 48 7.0845; IR (νmax/cm−1): 2959, 2850,
General procedure for the synthesis of compound
10a–k
1
1764, 1623, 1559, 1453, 1360, 1248, 1165; H NMR (400
Compound 9a–k (1.0 mmol) and triethylamine (TEA) (0.83
mL, 6.0 mmol) was dissolved in dichloromethane (DCM)
(10 mL), then added drop wise the solution of 4 (312 mg,
1.5 mmol) in CH2Cl2 at room temperature. The mixture was
stirred at room temperature for 2 h, then the reaction mix-
ture was concentrated under reduced pressure. The residue
was purified by column chromatography to give white solid
10a–k.
MHz, CDCl3) δ 8.58 (s, 1H, =CH), 8.08 (d, J = 0.9 Hz, 2H,
Ph-H), 7.77 (s, 1H, Ph-H), 7.66 (s, 4H, Ph-H), 7.37 (d, J =
3.3 Hz 2H, Ph-H), 4.51 (d, J = 24.6 Hz, 4H, O=CCH2,
O–CH2), 2.18–1.96 (m, 4H, CH2); 13C NMR (100 MHz,
CDCl3) δ 163.23, 158.94, 156.59, 155.21, 148.86, 138.05,
135.65, 134.47, 129.67 (3C), 128.51 (2C), 124.91, 118.14,
117.85, 116.75, 110.47, 70.99, 65.09, 25.30, 25.02.
4-((5-((2-oxo-2H-chromene-3-carbonyl)oxy)pentyl)oxy)-3-
4-(2-((2-oxo-2H-chromene-3-carbonyl)oxy)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10d)
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10a)
White solid, yield: 81.3%, m.p. 119.9–121.1 °C. ESI-
HRMS (m/z): calcd for C23H20N2O9S (neutral M + H)
501.0923; found 523.0775 (M + Na); IR (νmax/cm−1): 3061,
2961, 2926, 1754, 1619, 1562, 1449, 1392, 1248, 1212,
White solid; yield: 81.5%; m.p. 165.4–166.6 °C. APCI-
HRMS (m/z): calcd for C20H14N2O9S (neutral M + H)
459.0454; found 459.0494; IR (νmax/cm−1): 1764, 1628,
1
1
1349, 1167, 1565; H NMR (400 MHz, DMSO-d6) δ 8.58
1158; H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H, =CH),
(s, 1H, =CH), 8.06 (s, 2H, Ph-H), 7.76 (s, 1H, Ph-H), 7.65
(s, 3H, Ph-H), 7.37 (d, J = 4.0 Hz, 2H, Ph-H), 4.63 (s, 2H,
O–CH2), 4.52 (s, 2H, O=CCH2); 13C NMR (400 MHz,
DMSO-d6) δ 162.68, 159.30, 156.19, 155.07, 149.89,
137.63, 136.40, 135.23, 130.71, 130.31 (2C), 128.75 (2C),
125.39, 118.14, 117.43, 116.69, 111.00, 69.56, 63.01.
8.05 (s, 2H, Ph-H), 7.77 (s, 1H, Ph-H), 7.64 (s, 4H, Ph-H),
7.34 (s, 2H, Ph-H), 4.45 (d, J = 16.8 Hz, 4H, O=CCH2,
O–CH2), 1.96 (d, J = 29.5 Hz, 4H, CH2), 1.69 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d6) δ 163.19, 159.35, 156.46,
154.99, 149.16, 137.66, 136.55, 134.98, 130.73, 130.45
(2C), 128.74 (2C), 125.28, 118.24, 118.16, 116.62, 110.92,
71.77, 66.42, 27.98, 27.89, 22.06.
4-(3-((2-oxo-2H-chromene-3-carbonyl)oxy)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10b)
4-((6-((2-oxo-2H-chromene-3-carbonyl)oxy)hexyl)oxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10e)
White solid; yield: 82.3%; m.p. 119.9–120.2 °C. APCI-
HRMS (m/z): calcd for C21H16N2O9S (neutral M + H)
473.0577; found 473.0695; IR (νmax/cm−1) 2917, 2850,
White solid; 79.5%, m.p. 113.7–114.2 °C. ESI-HRMS (m/
z): calcd for C24H22N2O9S (neutral M + H) 515.1080; found

Medicinal Chemistry Research
537.0928 (M + Na); IR (νmax/cm−1): 2959, 2933, 2853,
1745, 1697, 1617, 1559, 1450, 1309, 1245, 1162; 1H NMR
(400 MHz, CDCl3) δ 8.53 (s, 1H, =CH), 8.06 (d, J = 3.7
Hz, 2H, Ph-H), 7.77 (s, 1H, Ph-H), 7.71–7.60 (m, 4H, Ph-
H), 7.36 (d, J = 5.7 Hz, 2H, Ph-H), 4.45 (s, 2H, O–CH2),
4.40 (s, 2H, O=CCH2), 1.93 (d, J = 1.3 Hz, 2H, CH2), 1.86
(d, J = 9.9 Hz 2H, CH2), 1.57 (s, 4H, CH2); 13C NMR (100
MHz, CDCl3) δ 163.25, 159.04, 156.63, 155.16, 148.61,
138.11, 135.62, 134.37, 129.65, 129.57 (2C), 128.47 (2C),
124.86, 118.34, 117.87, 116.74, 110.48, 71.42, 65.69,
28.44, 28.28, 25.43, 25.25.
O=CCH2, O–CH2), 2.10 (d, J = 4.7 Hz, 2H, O–CH2), 2.02
(d, J = 2.6 Hz, 2H, O–CH2); 13C NMR (100 MHz, DMSO-
d6) δ 163.01, 159.36, 156.36, 155.00, 149.29, 137.65,
136.52, 135.04, 130.71 (2C), 130.41, 128.71 (2C), 125.27,
118.16, 117.90, 116.61, 110.92, 71.33, 68.77, 68.29, 64.96.
4-(2-(2-oxo-2H-chromene-3-carboxamido)ethoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10i)
White solid, yield: 64.2%; m.p. 142.3–145.7℃; APCI-
HRMS (m/z): calcd for C20H15N3S (neutral M + H)
458.0580; found 458.0658; IR (νmax/cm−1): 3273, 3022,
2924, 1710, 1650, 1632, 1531, 1450, 1362, 1254, 1154 cm
−1; 1H NMR (400 MHz, CDCl3) δ 9.20 (s, 1H, =CH), 8.93
(s, 1H, O=C–NH), 8.14 (s, 2H, Ph-H), 7.76–7.70 (m, 3H,
Ph-H), 7.65 (d, J = 5.0 Hz, 2H, Ph-H), 7.43 (d, J = 8.7 Hz,
2H, Ph-H), 4.61 (s, 2H, O–CH2), 3.98 (d, J = 2.9 Hz, 2H,
O=C–NH–CH2); 13C NMR (100 MHz, DMSO-d6) δ
162.13, 160.78, 159.26, 154.43, 148.44, 134.76, 132.95,
130.82, 130.43, 129.37, 128.86, 126.95, 126.69, 125.31,
119.01, 118.89, 116.67, 111.08, 70.31, 38.28.
4-((4-((2-oxo-2H-chromene-3-carbonyl)oxy)but-2-yn-1-yl)
oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10f)
White solid; yield: 78.7%; m.p. 135.1–138.1 °C; APCI-
HRMS (m/z): calcd for C22H14N2O9S (neutral M + H)
483.0420; found 483.0532; IR (νmax/cm−1): 2914, 1762,
1
1609, 1556, 1490, 1372, 1208, 1168, 998 cm−1; H NMR
(400 MHz, CDCl3) δ 8.64 (s, 1H, =CH), 8.11 (d, J = 1.8
Hz, 2H, Ph-H), 7.79 (s, 1H, Ph-H), 7.68 (dd, J = 3.4, 0.8
Hz, 4H, Ph-H), 7.40 (d, J = 4.3 Hz, 2H, Ph-H), 5.16 (d, J =
1.0 Hz, 2H, O–CH2), 5.04 (d, J = 1.4 Hz, 2H, O=CCH2);
13C NMR (100 MHz, CDCl3) δ 149.85, 137.81, 137.79,
135.80, 135.76, 134.93, 129.83, 129.76, 129.73, 128.61
(2C), 125.08, 117.73, 116.85, 110.62, 88.28, 83.51, 58.90,
53.05, 50.90, 50.18, 29.68.
4-(3-(2-oxo-2H-chromene-3-carboxamido)propoxy)-3-
(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10j)
White solid, yield: 67.1%; m.p. 148.5–156.3 °C; APCI-
HRMS (m/z): calcd for C21H17N3O8S (neutral M + H)
472.0770; found 472.0805; IR (νmax/cm−1): 3355, 3045,
2913, 1699, 1648, 1533, 1449, 1369, 1248, 1161; 1H NMR
(400 MHz, DMSO-d6) δ 9.00 (s, 1H, =CH), 8.92 (s, 1H,
O=C–NH), 8.10 (d, J = 6.0 Hz, 2H, Ph-H), 7.74–7.63 (m,
5H, Ph-H), 7.42 (d, J = 7.0 Hz, 2H, Ph-H), 4.55 (s, 2H,
O–CH2), 3.70 (s, 2H, O=C–NH–CH2), 2.25 (s, 2H, CH2);
13C NMR (100 MHz, DMSO-d63) δ 161.94, 160.68, 159.38,
154.32, 147.75, 137.62, 136.61, 134.50, 130.69 (2C),
130.50, 128.90 (2C), 125.58, 119.69, 118.92, 116.59,
110.99, 69.89, 36.27, 28.67.
4-((1-((2-oxo-2H-chrom3ene-3-carbonyl)oxy)propan-2-yl)
oxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10g)
White solid, yield: 80.4%; m.p. 152.9–154.7 °C. ESI-MS
(m/z): calcd for C21H16N2O9S (neutral M + H) 473.0; IR
(νmax/cm−1): 2917, 2850, 1761, 1613, 1553, 1460, 1392,
1293, 1248, 1165, 1085, 1005; 1H NMR (400 MHz, CDCl3)
δ 8.57 (s, 1H, =CH), 8.06 (d, J = 2.0 Hz, 2H, Ph-H), 7.76
(s, 1H, Ph-H), 7.65 (dd, J = 2.8, 1.0 Hz, 4H, Ph-H), 7.36 (s,
J = 0.5 Hz, 2H, Ph-H), 4.61 (d, J = 32.7 Hz, 4H, O=CCH2,
O–CH), 2.38 (d, J = 5.2 Hz, 2H, CH3); 13C NMR (100
MHz, CDCl3) δ 163.14, 158.85, 156.51, 155.24, 149.05,
138.02, 135.66, 134.56, 129.72 (2C), 129.67, 128.50 (2C),
124.93, 117.93, 117.82, 116.78, 110.49, 68.11, 61.80,
27.91.
4-((1-(2-oxo-2H-chromene-3-carboxamido)propan-2-yl)oxy)-
3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10k)
White solid, yield: 65.2%; m.p. 152.9–154.7 °C; APCI-
HRMS (m/z): calcd for C21H17N3O8S (neutral M + H)
472.0770; found 472.0807; IR (νmax/cm−1) 3356, 2917,
2850, 1703, 1623, 1655, 1527, 1453, 1354, 1162; 1H NMR
(400 MHz, CDCl3) δ 8.98 (s, 1H, =CH), 8.90 (s, 1H,
O=C–NH), 8.10 (d, J = 4.8 Hz, 2H, Ph-H), 7.74–7.63 (m,
5H, Ph-H), 7.42 (d, J = 6.0 Hz, 2H, Ph-H), 4.50 (s, 1H,
O–CH), 3.52–3.37 (s, 2H, O=C–NH–CH2), 1.52 (s, 3H,
CH3); 13C NMR (100 MHz, DMSO-d6) δ 162.20, 160.81,
158.79, 154.39, 136.49, 134.73, 130.38 (3C), 128.93,
128.07, 125.95, 125.69, 125.55, 119.17, 118.88, 116.66,
111.10, 78.41, 43.17, 17.27.
4-(2-(2-((2-oxo-2H-chromene-3-carbonyl)oxy)ethoxy)
ethoxy)-3-(phenylsulfonyl)-1,2,5-oxadiazole-2-oxide (10h)
White solid, yield: 77.5%; m.p. 98.9–101.0℃; ESI-HRMS
(m/z): calcd for C22H18N2O10S (neutral M + H) 503.0682;
found 503.0797; IR (νmax/cm−1): 2834, 2895, 1723, 1564,
1
1449, 1345, 1169, 1020; H NMR (400 MHz, CDCl3) δ
8.58 (s, 1H, =CH), 8.08 (s, 2H, Ph-H), 7.77 (s, 1H, Ph-H),
7.66 (s, 4H, Ph-H), 7.38 (s, 2H, Ph-H), 4.54–4.43 (m, 4H,

Medicinal Chemistry Research
O
O
O
O
O
COONa
COONa
i
H
ii
iii
O
OH
OH
ONa
O
O
2
3
1
O
O
iv
Cl
O
4
O
O
O
S
O
O
O
O
N
SH
v
O
S
S
vi
OH
vii
S
OH
N
O
O
5
6
7
O
S
O
HRO
viii
O
N
O
S
N
O
O
O
x
O
R
8a~8h
N
N
O
O
O
O
O
S
O
9a~9k
HRO
ix
N
N
O
O
8i~8k
9b,10b
9d,10d
9f,10f
RO=O(CH2)3O
RO=O(CH2)5O
9a,10a
9c,10c
9e,10e
9g,10g
9i,10i
RO=O(CH2)2O
RO=O(CH2)4O
RO=O(CH2)6O
RO=OCH2C CCH2O
RO=O(CH2)2O(CH2)2O
9h,10h
RO=OCH(CH3)CH2O
RO=NH(CH2)2O
9j,10j
RO=NH(CH2)3O
9k,10k
RO=NHCH(CH3)CH2O
Scheme 1 Synthesis of NO-donating coumarin derivatives 10a–k.
reagents and conditions: (i) diethyl malonate, piperidine (cat.), EtOH,
reflux, 2 h; (ii) NaOH, H2O, EtOH, reflux, 20 min; (iii) HCl, rt; (iv)
SOCl2, pyridine, reflux, 6 h; (v) Chloroacetic acid, NaOH, Na2CO3, rt,
4 h and reflux 1 h; (vi) HOAc, H2O2, rt. 4 h; (vii) 98% HNO3, reflux,
30 min; (viii) HO-R-OH, THF, 25%NaOH, rt. 40 min; (ix) HO-R-
NH2, dry THF, NaH, −5 °C, 30 min; (x) Compound 4, DCM, TEA,
2 h
Anticancer activity study (MTT assay)
in 96-well flat-bottom microplates overnight. The cells were
incubated in triplicate with, or without, different con-
centrations of each test compound for 72 h. Then 20 μL of 5
mg/mL MTT solution was added to each well and incubated
for 4 h. Moved 150 μL DMSO in each well and detection
absorbent value in 470 nm wavelength. The inhibition rate
Human hepatocellular carcinoma HepG2, human colorectal
cancer SW620, HCT116, human cervical cancer HeLa and
human breast cancer MCF-7, human intestinal epithelial
CCD841 cells per well at 104 cell were cultured in 10%
FBS DMEM high glucose solution and a 37 ℃ in 5% CO2

Medicinal Chemistry Research
of each compound in different concentrations was expressed
as a percentage.
Especially, compound 10i (IC50 = 0.88 μM) showed extra-
ordinary antitumor activity, with IC50 values below 1 μM,
being much more active than coumarin-3-carboxylic acid
and more effective than doxorubicin (IC50 = 10.21 μM).
These data suggest that these compounds possess significant
antiproliferative activity suggesting that the furoxan phar-
macophores play crucial roles in manifesting anticancer
activity. This finding is coherent with previous conclusions
made by Liu MM group (Liu et al. 2014).
Results and discussion
Chemistry
The synthetic route of these target compounds 10a–k is
outlined in Scheme 1. Salicylaldehyde 1 which stirred with
diethyl malonate in the presence of piperidine in ethanol
afforded ethyl coumarin-3-carboxylate 2. The ethyl ester
was hydrolyzed in the presence of NaOH to give coumarin-
3-carboxylic acid 3, which was then converted to related
acid chloride 4 by using thionyl chloride. Subsequently, the
obtained acid chloride was reacted with various mono
(phenylsulfonyl) furoxans, synthesized in a four-step
sequence as previously described, in the presence of TEA
in DCM to afford the final compounds. All target com-
pounds were purified by column chromatography, and their
In vitro antitumor activity evaluation
To examine how the target compounds affected other cancer
cells, we selected the most promising compounds 10b–c,
10f, and 10h–i and evaluated their respective cytotoxicity
against on the other four different human cancer cells,
including human hepatocellular carcinoma (HepG2), human
colorectal cancer (HCT116 and SW620) and human breast
cancer (MCF-7) by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphe-
nyl-2-H-tetrazolium bromide (MTT) assay. As shown in
Table 2, that compounds 10c and 10h displayed much less
inhibition on the growth of HepG2, HCT116 and SW620
cells, likewise, 10b was weaker antiproliferative activity on
HCT116 and SW620 cells, with IC50 values exceed 25 μM.
On the contrary, compound 10b showed selective cytotoxic
effect against HepG2 cancer cell line with an IC50 of 3.86
μM. Moreover, all five compounds displayed potent cyto-
toxic activity against breast cancer cell lines, with IC50
values ranging from 0.61 to 8.93 μM, and showed selective
cytotoxic effect against MCF-7 cancer cell line. Notably,
compound 10i was found to be the most potent compound
with an IC50 value of 0.61 μM against MCF-7 cells and 0.88
μM against HeLa cells. Especially, among all the com-
pounds, compound 10f had higher anti-proliferative activity
on MCF-7 (IC50 = 7.90 μM), SW620 (IC50 = 1.86 μM),
HCT-116 (IC50 = 3.46 μM), and HepG2 (IC50 = 9.85 μM),
displayed the prominent and broader spectrum of cytotoxic
activities against all the five human tumor cell lines.
1
structures were confirmed by IR, H and 13C NMR, MS,
and HRMS.
The in vitro activity of target compounds 10a−k was
examined against human cervical cancer HeLa cell by MTT
assay, using coumarin-3-carboxylic acid and doxorubicin as
control. The activity was expressed by half maximal inhi-
bitory concentration (IC50) and was provided in Table 1.
The results revealed that all the coumarin–furoxan hybrids
possessed higher activity than coumarin-3-carboxylic acid,
and five out of eleven compounds, 10b–c, 10f, and 10h–i,
displayed significantly inhibitory effects, which were
superior to one classical anticancer drug, Doxorubicin.
Table 1 The structure and cytotoxic activity of the target compounds
against HeLa human cervical cancer cells
Compounds
–R–
IC50 (μM)
10a
–(CH2)2O–
–(CH2)3O–
-(CH2)4O–
32.32
4.85
10b
10c
5.16
Table 2 In vitro cytotoxicity (IC50, μM) for compounds 10b, 10c, 10f,
10h, and 10i against four human cancer cell lines
10d
10e
–(CH2)5O–
–(CH2)6O–
–CH2C≡CCH2O–
–CH2CH(CH3)O–
–(CH2)2O(CH2)2O–
–(CH2)2NH–
–(CH2)3NH–
–CH2CH(CH3)NH–
/
38.61
34.96
5.95
Compounds
In vitro cytotoxicity (IC50, μM)
10f
SW620
HepG2
HCT116
MCF-7
10g
14.82
4.72
10h
10i
10b
>25
>25
1.86
>25
12.61
2.32
3.86
>25
9.85
>25
17.32
3.54
>25
>25
3.46
>25
14.43
0.48
7.72
8.93
7.92
7.68
0.61
3.25
0.88
10c
10j
17.75
44.12
460
10f
10k
3
10h
10i
Doxorubicin
/
10.21
Doxorubicin
IC50: a concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean IC50 from the does-response
curves of at least three independent experiments
IC50: a concentration required to inhibit tumor cell proliferation by
50%. Data are expressed as the mean IC50 from the does-response
curves of at least three independent experiments

Medicinal Chemistry Research
As seen in Table 1, the antiproliferative activity of the
novel coumarin–furoxan hybrids was influenced by the lin-
ker. For example, variation in the length of alkyl chain
affected significantly the in vitro anticancer activity of these
derivatives. Compounds 10b–c with a 2–4 methylenes linker
displayed stronger anticancer activity in comparison to those
possessing with a 5–6 methylenes linker. For example,
compound 10i containing the aminoethanol linkage showed
better selectivity and enhanced activity. Particularly, it was
also observed that compound 10f with the butynelene linker
enhanced broader spectrum antitumor potency.
the most antiproliferative activities for human breast and
cervical cancer cell lines. More importantly, compound 10f
selectively inhibited colorectal tumor cell proliferation but
not normal cells. Together, given their potent antitumor
activities, these furoxan/coumarin hybrids warrant further
investigation as candidates for the potential treatment of
human cancer.
Acknowledgements We gratefully acknowledge the financial support
by the Nature and Science Foundation of Department of Education,
Anhui province in China (No. KJ2016B03; No.KJ2014A134), Jiangsu
Province Postdoctoral Science Foundation (1601136B), and the
National Natural Science Foundation of China (No. 8127400).
Cancer cell selectivity
Compliance with ethical standards
Given the significant antiproliferative activity of 10f
in vitro, the selectivity profile was investigated by exam-
ining the inhibitory effects of 10f on the growth of human
colorectal cancer SW620 cells and normal intestinal epi-
thelial CCD841 cells. It was found that treatment with
increased dose of 10f had no significant effect on the sur-
vival of non-tumor CCD841 cells, whereas the same treat-
ment induced death of the majority of SW620 cells (Fig. 4).
These results suggest that 10f possesses selectivity in anti-
proliferation activity against colorectal tumor cell over
normal cell.
Conflict of interest The authors declare that they have no conflict of
interest.
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