Synthesis and antiproliferative activity of 3- and 7-styrylcoumarins

MEDICINAL

CHEMISTRY

RESEARCH

Medicinal Chemistry Research

https://doi.org/10.1007/s00044-018-2202-0

ORIGINAL RESEARCH

Synthesis and antiproliferative activity of 3- and 7-styrylcoumarins

Angie Herrera-R^1,2Wilson Castrillón¹Elver Otero¹Esneyder Ruiz¹Miguel Carda³Raúl Agut³

●

Tonny Naranjo^4,5Gustavo Moreno^1,2Maria Elena Maldonado²Wilson Cardona-G¹

●

Received: 19 February 2018 / Accepted: 1 June 2018

Abstract

A series of styrylcoumarins were obtained via Mizoroki-Heck reactions between 3-bromo-4-methyl-7-(octyloxy)-2H-

chromen-2-one or 2-oxo-2H-chromen-7-yl triﬂuoromethanesulfonate and functionalized styrenes. The structures of the

products were elucidated by spectroscopic analysis. All compounds were evaluated against SW480 and CHO-K1 cell lines.

A number of hybrids showed good antiproliferative activity. Among the tested compounds, hybrids 6e, 10c, and 10d,

exhibited the highest activity (IC_{50- SW480/48h}= 6,92; 1,01 and 5,33 µM, respectively) and selectivity (IS_48h= >400; 67,8 and

7,2, respectively). In addition, these compounds were able to preserve their activities over time. The results achieved by

these hybrids were even better than the lead compounds (coumarin and resveratrol) and the standard drug (5-FU). As regards

structure-activity relationship it seems that the location of the styryl group on the coumarin structure and the presence of the

hydroxyl group on the phenyl ring were determinant for the activity.

●

Keywords Coumarin Styrylcoumarin Antiproliferative activity Colorectal cancer Hybrids

Abbreviations

Introduction

5-FU

5-Fluorouracil

CRC

DMSO

NF-κB

Colorectal Cancer

Dimethylsulfoxide

Nuclear factor-κB

Colorectal cancer (CRC) is the third most commonly

diagnosed malignancy and the fourth leading cause of

cancer death worldwide (Arnold et al. 2016). This pathol-

ogy presents a great geographical distribution and the pat-

terns are very similar among men and women, being the

third most common cancer in men and the second one in

women (Globocan 2012).

Current treatments for CRC include combinations of

chemotherapeuthic agents such as FOLFOX (5-FU/leu-

covorin/oxaliplatin) and FOLFIRI (folic acid/5-FU/irinote-

can) which are composed of 5-ﬂuorouracil as the backbone

of treatment, that, although effective, cause undesirable

gastrointestinal and neurological side-effects which many

times result in dose limitations or cessation of the anticancer

therapy (Pointet and Taieb 2017; McQuade et al. 2014).

These authors contributed equally: Angie Herrera-R, Wilson

Castrillón.

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s00044-018-2202-0) contains supplementary

material, which is available to authorized users.

* Maria Elena Maldonado

School of Dietetics and Human Nutrition, University of Antioquia,

A.A. 1226 Medellín, Colombia

maria.maldonado@udea.edu.co

3

* Wilson Cardona-G

Department of Inorganic and Organic Chemistry, University

Jaume I, E-12071 Castellón, Spain

wilson.cardona1@udea.edu.co

4

Grupo de Micología Médica y Experimental, Corporación para

1

Química de Plantas Colombianas, Institute of Chemistry, Faculty

Investigaciones Biológicas, Medellín, Colombia

of Exact and Natural Sciences, University of Antioquia UdeA,

Calle 70 No. 52–21, A.A 1226 Medellín, Colombia

5

School of Health Sciences, Universidad Pontiﬁcia Bolivariana,

Medellín, Colombia

2

Grupo Impacto de los Componentes Alimentarios en la Salud,

Medicinal Chemistry Research

Thus, it becomes necessary to discover new, more potent,

and selective agents for treating this disease.

These hybrid molecules may display dual activity, but do

not necessarily act on the same biological target (Shaveta

et al. 2016; Tsogoeva 2010). Combining chemotherapy

with agents having different mechanisms of action is one of

the methods adopted for treating cancer (Kerru et al. 2017;

Nepali et al. 2014; Mayur et al. 2009; Solomon et al. 2009).

Several stilbene-coumarin hybrids were identiﬁed as very

promising candidates by their excellent antiproliferative

potency or by their remarkable apoptosis-inducing activity

(see Fig. 1). Compound 1a and 1b showed high activity

against lung carcinoma H460 with IC₅₀values of 0.45 and

0.29 µM, respectively (Belluti et al. 2010). Styrylcoumarin

1c was evaluated for its antitumor activity against MCF-7

and HCT-28 tumor cell lines. This compound showed

varying degrees of growth inhibition on the above men-

tioned cell lines with IC₅₀values of 7.32 and 3.78 µM,

respectively (Shen et al. 2013). 3-Phenylcoumarin 1d

exhibited potent antiproliferative activity with IC₅₀values

of 5.2 and 7.5 µM against HL-60 and A549 cell lines,

respectively (Yang et al. 2011). Benzimidazole-coumarin

hybrid 1e was screened for in vitro antitumor activity on

different cell lines. This compound, tested at a concentration

of 10 µM, induced more than 50% inhibition of most of the

cell lines, with higher selectivity against leukemic cancer

cell lines (Paul et al. 2013). Coumarin-chalcone hybrid 1f

showed around 30 fold more selectivity towards C33A

(cervical carcinoma) cells over normal ﬁbroblast NIH3T3

cells with an IC₅₀value of 3.59 μM (Sashidhara et al. 2010).

The resveratrol-imidazole hybrid 1g showed cytotoxic

activity against leukemia and renal cancer cell lines (4.1 and

8.3 µM, respectively) (Bellina et al. 2015). Resveratrol-

chalcone conjugate 1h showed high selectivity towards

certain ovarian cancer, non-small cell lung cancer and breast

cancer cell lines, with GI₅₀values in the range of 1.28–34.1

µM (Kumar et al. 2014).

In this regard, many pharmacological studies dealing

with coumarins as a key structural motif have been reported.

The pharmacological actions with which coumarins are

endowed include anti-HIV (Kashman et al. 1992), anti-

inﬂammatory (Resch et al. 1998), anticoagulant (Madari

et al. 2003), antimicrobial (Ankleka et al. 2003), antiplatelet

(Vilar et al. 2006), antiprotozoal (Arango et al. 2010;

Pierson et al. 2010) and antioxidant (Gacche and Jadhav

2012). Besides, some coumarins have exhibited anti-

mycobacterial activity which is potentiated when they bear

a fatty acid residue in their structure (Yee et al. 2005).

Furthermore, coumarins have also shown inhibition of cell

proliferation in different types of cancer cell lines (Riveiro

et al. 2008; Yang et al. 2006; Budzisz et al. 2003; Kawaii

et al. 2001; Egan et al. 1997). According to this, some

coumarin antibiotics can bind to the carboxyl terminus of

Hsp90 molecular chaperone, which modulate multiple

oncogenic pathways and is overexpressed in human

malignancies, causing its inhibition (Zhao et al. 2011;

Neckers 2002). On the other hand, natural product resver-

atrol, which belongs to the family of compounds known as

stilbenes, has great signiﬁcance because of its wide variety

of biological properties (Gülçin 2010; Udenigwe et al.

2008; Kedzierski et al. 2007; Baur and Sinclair 2006),

including among them anticancer activity (Borriello et al.

2014). Resveratrol inhibits CRC in all phases acting on

different targets such as NF-κB, AP-1, cyclooxygenase, and

kinases (Athar et al. 2009). This compound enhances the

activation of tumor suppressor p53 which ultimately indu-

ces apoptosis of human colon cancer cells. It also activates

caspases 3 and 8 and increases the BAX/Bcl-2 ratio

(Temraz et al. 2013).

The combination of two pharmacological agents into a

single molecule, which leads to what is known as a hybrid

molecule, has resulted in an emerging strategy in medicinal

chemistry and drug discovery research (Meunier 2008).

Hybridizing the coumarin nucleus with other moieties

has afforded new molecules with improved anticancer

activity proﬁle (Agarwal 2000). In the search for new

OH

H₃CO

OCH₃

H₃C

CH₃

H₃CO

HO

O

1d

O

1c

H₃CO

O

H₃CO

O

1b

1a

O

N

O

N

O

H₃CO

HO

OCH₃

OH

N

H

O

HO

1f

1g

1h

N

H

O

OCH₃

OH

1e

Fig. 1 Hybrid compounds endowed with antitumor activity

Medicinal Chemistry Research

Synthesis of 4-methyl-7-(octyloxy)-2H-chromen-2-one (3)

Coumarin (1 g, 5.7 mmol), potassium hydroxide (955 mg,

17 mmol) and acetonitrile (20 mL) were placed in a 50 mL

ﬂat-bottomed ﬂask equipped with a magnetic stirring bar.

The mixture was stirred and heated to reﬂux for a period of

5 min. Then, 1-bromooctane (1.1 mL, 1.22 g, 6.3 mmol)

was added to the reaction mixture which was then reﬂuxed

for 6 h. The crude reaction mixture was concentrated on a

rotatory evaporator and the residue was puriﬁed by column

chromatography over silica gel. Elution with hexane and a

mixture of hexane-ethyl acetate (9:1 ratio) gave rise to

compound 3 (1.4 g, 85% yield).

Fig. 2 Design of stilbene-coumarin hybrids as anticancer agents

therapeutic alternatives to treat CRC, we have designed and

synthesized a series of stilbene-coumarin hybrids (styr-

ylcoumarins) (Fig. 2). In addition, the anticancer activity of

our hybrid molecules have been evaluated in vitro.

M.p. 48–50 °C; IR (KBr, cm⁻¹): ν_max2924 (C–H), 1718

(C=O), 1620 (C=C), 1512 (C=C_Ar), 1294 (C–O–C), 1199

1

((C=O)–O), 839 (C–H_Ar). H NMR (CDCl₃, 300 MHz): δ

0.93 (CH₃, t, J = 6.5 Hz), 1.35 (3CH₂, m), 1.51 (CH₂, m),

1.64 (CH₂, m), 1.86 (CH₂, m), 2.44 (CH₃, s), 4.05

(–CH₂O–, t, J = 6.5 Hz), 6.17 (H₃, s), 6.85 (H₈, d, J =

2.3 Hz), 6.90 (H₆, dd, J = 8.7, 2.3 Hz) 7.53 (H₅, d, J =

8.7 Hz). ¹³C NMR (CDCl₃, 75 MHz): δ 14.11 (CH₃), 18.70

(CH₃) 22.68 (CH₂), 26.01 (CH₂), 28.99 (CH₂), 29.24 (CH₂),

29.34 (CH₂), 31.80 (CH₂), 68.65 (-OCH₂-), 101.34 (C₈),

111.82 (C₆), 112.74 (C₃), 113.40 (C_4a), 125.46 (C₅), 152.63

(C_8a), 155.32 (C₄), 161.47 (C=O), 162.29 (C₇). EIMS: m/z

289.1800 [M + H]⁺, (calculated for [C₁₈H₂₅O₃]⁺:

289.1804).

Materials and methods

Chemistry

Microwave reactions were carried out in a CEM Discover

microwave reactor in sealed vessels (monowave, maximum

power 300 W, temperature control by IR sensor, ﬁxed

1

temperature). H and ¹³C NMR spectra were recorded on a

Varian instrument operating at 500 (300) and 125 (75)

MHz, respectively. The signals of the deuterated solvent

(CDCl₃) were used as reference (CDCl₃: δ = 7.27 ppm for

¹H NMR and δ = 77.00 ppm for ¹³C NMR). Carbon atom

types (C, CH, CH₂, CH₃) were determined by using the

DEPT or APT pulse sequence. Signals were assigned using

two-dimensional heteronuclear correlations (COSY and

HSQC). High resolution mass spectra were recorded using

electrospray ionization mass spectrometry (ESI-MS). A

QTOF Premier instrument with an orthogonal Z-spray-

electrospray interface (Waters, Manchester, UK) was used

operating in the W-mode. The drying and cone gas was

nitrogen set to ﬂow rates of 300 and 30 L/h, respectively.

Methanol sample solutions (ca. 1 × 10⁻⁵M) were directly

introduced into the ESI spectrometer at a ﬂow rate of 10 µL/

min. A capillary voltage of 3.5 kV was used in the positive

scan mode, and the cone voltage set to Uc = 10 V. For

accurate mass measurements, a 2 mg/L standard solution of

leucine enkephalin was introduced via the lock spray needle

at a cone voltage set to 85 V and a ﬂow rate of 30 μL/min.

IR spectra were recorded on a Spectrum RX I FT-IR system

(Perkin-Elmer, Waltham, MA, USA) in KBr disks. Silica

gel 60 (0.063–0.200 mesh, Merck, Whitehouse Station, NJ,

USA) was used for column chromatography, and

precoated silica gel plates (Merck 60 F254 0.2 mm) were

used for thin layer chromatography (TLC). Monitoring of

the reaction progress and product puriﬁcation was carried

out by TLC.

Synthesis of 3-bromo-4-methyl-7-(octyloxy)-2H-chromen-2-

one (4)

A solution of alkyloxycoumarin 3 (1 g, 3.5 mmol) and NBS

(623 mg, 3.5 mmol) in methanol (20 mL) was stirred in an

ice bath. Then, p-TsOH (170 mg, 1.0 mmol) in methanol

(10 mL) was added to the reaction mixture for 10 min.

Then, the reaction mixture was stirred at room temperature

for 1 h. The crude reaction mixture was concentrated on a

rotatory evaporator and the residue was puriﬁed by column

chromatography over silica gel. Elution with hexane and a

mixture of hexane-ethyl acetate (95:5 ratio) afforded com-

pound 4 (0.96 g, 75% yield).

Yellow oil; IR (KBr, cm⁻¹): ν

2924 (C–H), 1737

max

(C = O), 1614 (C = C), 1468 (C = C_Ar), 1285 (C–O-C),

1201 ((C = O)-O), 811 (C–H_Ar); ¹H NMR (CDCl₃,

300 MHz): δ 0.93 (CH₃, t, J = 6.5 Hz), 1.35 (3CH₂, m),

1.51 (CH₂, m), 1.74 (CH₂, m), 1.86 (CH₂, m), 2.63 (CH₃, s),

4.05 (–CH₂O–, t, J = 6.5 Hz), 6.84 (H₈, d, J = 2.5 Hz), 6.92

(H₆, dd, J = 8.9, 2.5 Hz), 7.53 (H₅, d, J = 8.9 Hz). ¹³C

NMR (CDCl₃, 75 MHz): δ 14.13 (CH₃), 19.50 (CH₃) 22.66

(CH₂), 25.98 (CH₂), 28.97 (CH₂), 29.23 (CH₂), 29.33

(CH₂), 31.81 (CH₂), 68.79 (-OCH₂-), 101.17 (C₈), 109.50

(C₃), 113.20 (C₆), 113.42 (C_4a), 126.01 (C₅), 151.23 (C₄),

153.63 (C_8a), 157.48 (C₇), 162.37 (C=O). EIMS: m/z

Medicinal Chemistry Research

367.0908 [M + H]⁺, (calculated for [C₁₈H₂₄BrO₃]⁺:

367.0909).

pyridine (21 mL). The mixture was heated at reﬂux for a

period of 30 min under microwave irradiation. Then,

toluene (40 mL) was added to the cooled reaction mixture

and the solvent volume reduced in vacuum at 30–40 °C.

Additional toluene (20 mL) was then added and the solvent

again removed in vacuum in order to eliminate all traces of

pyridine. This procedure afforded a residue which was

puriﬁed by column chromatography over silica gel. Elution

with 9:1 hexane/ethyl acetate afforded the corresponding

vinylphenol.

Synthesis of 2-oxo-2H-chromen-7-yl

triﬂuoromethanesulfonate (9)

A solution of 7-hydroxycoumarin (8) (1.0 g, 6.17 mmol) in

pyridine (5 mL) was cooled to 0 °C and treated with triﬂic

anhydride (2.1 mL, 12.34 mmol, 20 min addition). The

reaction mixture was allowed to warm to room temperature

and stirred for an additional 2.5 h. The crude mixture was

partitioned between saturated aqueous CuSO₄and ethyl

acetate. After separation of the fractions, the organic frac-

tion was dried (Na₂SO₄) and concentrated under reduced

pressure. The crude product was puriﬁed by column chro-

matography over silica gel. Elution with a mixture of

hexane-ethyl acetate (8:2 ratio) afforded compound 9 in

85% yield (1.54 mg, 85% yield).

General procedure for the Mizoroki-Heck reactions

Styrene (3 mmol), 3-bromo-7-alkyloxycoumarin (4) or 2-

oxo-2H-chromen-7-yl

triﬂuoromethanesulfonate

(9)

(3 mmol), triethanolamine (3 mL) and Pd(OAc)₂(0.03 g)

were placed in a 25 mL ﬂat-bottomed ﬂask equipped with a

magnetic stirring bar. The mixture was stirred and heated to

reﬂux for a period of 30 min, under microwave irradiation.

The mixture was allowed to cool and then passed through a

pad of silica using ethyl acetate as solvent. Then the ﬁltrate

was evaporated and the crude residue was puriﬁed by pre-

parative chromatography (silica gel, hexane/methylene

chloride (1:1) mixture) affording the coupling products in

35–58% yield.

M.p. 78–80 °C; IR (KBr, cm⁻¹): ν_max1720 (C=O),

1421 y 1220 S(=O)₂, 1130 (CF₃), 1294 (C–O–C), 1110

((C=O)-O), 748–979 (C–O–S). ¹H NMR (CDCl₃,

300 MHz): δ 6.53 (H₃, d, J = 9.7 Hz), 7.25 (H₆, dd, J = 2.3,

8.6 Hz), 7.31 (H₈, d, J = 2.3 Hz), 7.62 (H₅, d, J = 8.6 Hz),

7.76 (H₄, d, J = 9.7 Hz). ¹³C NMR (CDCl₃, 75 MHz): δ

110.60 (C₈), 117.81 (C₃, C₆), 118.66 (–SO₃CF₃, q, J_C–F

=

321 Hz), 118.80 (C_4a), 129.48 (C₅), 142.18 (C₄), 150.84

(C₇), 154.57 (C_8a), 159.35 (C=O). EIMS: m/z 294.9880 [M

+ H]⁺, (calculated for [C₁₀H₆F₃O₅S]⁺: 294.9884).

3-[(E)-2-(4-methoxyphenyl)ethenyl]-4-methyl-7-(octyloxy)-

2H-chromen-2-one (6a) Yield: 0.37 mmol, 155 mg, 37%;

M.p. 114–116 °C; IR (KBr, cm⁻¹): ν_max2926 (C–H), 1710

(C=O), 1610 (C=C), 1512 (C=C_Ar), 1242 (C–O–C), 1178

Styrene synthesis Method 1: Wittig Reaction under

microwave conditions.

1

((C=O)-O), 834 (C–H_Ar); H NMR (CDCl₃, 300 MHz): δ

Potassium carbonate (0.10 mol) and (ethyl)triphenylpho-

sphonium iodide (0.016 mol) were mixed and crushed in a

mortar to a ﬁne powder and transferred into a 250 mL

Erlenmeyer ﬂask. Aldehyde (0.016 mol) was added to the

powdered mixture and thoroughly mixed. The Erlenmeyer

ﬂask was covered with a watch glass and placed into a

microwave oven along with a 1 L beaker of ice (to ensure

that the reaction mixture did not overheat). The microwave

was run at full power for 12 min, making sure to stop the

heating and replace the ice when it had melted. A tiny

sample of the crude reaction product was removed, diluted

in a small amount of hexanes and analyzed by TLC to check

the reaction progress. Upon completion, the crude solids

were extracted with hexanes (3 × 20 mL). The combined

hexane extracts were placed in a round-bottomed ﬂask and

concentrated by rotary evaporation to obtain a yellow oil

which was puriﬁed by ﬂash chromatography on silica gel

using hexanes or a mixture of hexanes/ethyl acetate as

eluent.

0.94 (CH₃, t, J = 6.7 Hz), 1.35 (3CH₂, m), 1.52 (CH₂, m),

1.63 (CH₂, m), 1.86 (CH₂, m), 2.60 (CH₃, s), 3.88 (OCH₃,

s), 4.06 (-CH₂O-, t, J = 6.5 Hz), 6.85 (H₈, d, J = 2.5 Hz),

6.91 (H₆, dd, J = 9.0, 2.5 Hz), 6.95 (H_3´´, H_5´´, d, J =

9.0 Hz), 7.07 (H_1´, d, J = 16.2 Hz), 7.53 (H_2´´, H_6´´, d, J =

9.0 Hz), 7.61 (H_2´, d, J = 16.2 Hz), 7.62 (H₅, d, J = 9.0 Hz).

¹³C NMR (CDCl₃, 75 MHz): δ 14.13 (CH₃), 15.47 (CH₃)

22.68 (CH₂), 25.98 (CH₂), 29.03 (CH₂), 29.23 (CH₂), 29.34

(CH₂), 31.81 (CH₂), 66.73 (OCH₃), 68.59 (-OCH₂-), 100.93

(C₈), 112.90 (C₆), 114.11 (C_3´´, C_5´´), 114.34 (C_4a), 118.89

(C_1´), 119.34 (C₃), 125.86 (C₅), 128.02 (C_2´, C_6´), 130.53

(C_1´´), 135.08 (C_2´), 147.75 (C_8a), 153.57 (C₄), 159.66 (C₄

_´´), 160.57 (C₇), 161.62 (C=O). EIMS: m/z 421.2377 [M +

H]⁺, (calculated for [C₂₇H₃₃O₄]⁺: 421.2379).

3-[(E)-2-(3,4-dimethoxyphenyl)ethenyl]-4-methyl-7-(octy-

loxy)-2H-chromen-2-one (6b) Yield: 0.38 mmol, 171 mg,

38%; M.p. 94–96 °C; IR (KBr, cm⁻¹): ν_max2920 (C–H),

1712 (C=O), 1618 (C=C), 1514 (C=C_Ar), 1251 (C–O–C),

1161 ((C=O)-O), 813 (C–H_Ar); ¹H NMR (CDCl₃,

300 MHz): δ 0.94 (CH₃, t, J = 6.8 Hz), 1.35 (3CH₂, m),

1.52 (CH₂, m), 1.62 (CH₂, m), 1.87 (CH₂, m), 2.61 (CH₃, s),

Method 2: Knoevenagel condensation reactions:

Piperidine (0.75 mL, 7.6 mmol) was added to a solution

of aldehyde (5 mmol) and malonic acid (20 mmol) in

Medicinal Chemistry Research

3.96 (OCH₃, s), 3.99 (OCH₃, s), 4.07 (–CH₂O–, t, J =

6.5 Hz), 6.86 (H₈, d, J = 2.5 Hz), 6.92 (H₆, H_5´´, d, J = 8.8,

2.5 Hz), 7.07 (H_1´, d, J = 16.6 Hz), 7.13 (H_2´´, d, J =

1.8 Hz), 7.15 (H_6´´, dd, J = 8.8, 1.8 Hz), 7.57 (H_2´, d, J =

16.6 Hz), 7.62 (H₅, d, J = 8.8 Hz). ¹³C NMR (CDCl₃,

75 MHz): δ 14.12 (CH₃), 15.54 (CH₃), 22.68 (CH₂), 26.02

(CH₂), 29.03 (CH₂), 29.24 (CH₂), 29.35 (CH₂), 31.81

(CH₂), 53.98 (2OCH₃), 68.71 (-OCH₂-), 100.90 (C₈),

109.37 (C_2´´), 111.21 (C₆), 112.98 (C_5´´), 114.31 (C_4a),

119.17 (C_6´´), 119.30 (C₃), 119.90 (C_1´), 125.87 (C₅),

130.86 (C_1´´), 135.37 (C_2´), 145.90 (C_3´´), 149.11 (C_4´´),

149.27 (C_8a) 153.59 (C₄), 160.61 (C₇), 161.65 (C=O).

EIMS: m/z 451.2483 [M + H]⁺, (calculated for [C₂₈H₃₅O₅]

⁺: 451.2484).

_´´),118.85 (C_6´´), 119.33 (C₃), 120.29 (C_1´), 125.89 (C₅),

130.38 (C_1´´), 135.56 (C_2´), 145.83 (C_4´´), 145.95 (C_3´´),

146.69 (C₄), 153.56 (C_8a) 160.64 (C₇), 161.62 (C=O).

EIMS: m/z 437.2331 [M + H]⁺, (calculated for [C₂₇H₃₃O₅]

⁺: 437.2328).

3-[(E)-2-(2,5-dimethoxyphenyl)ethenyl]-4-methyl-7-(octy-

loxy)-2H-chromen-2-one (6e) Yield: 0.35 mmol, 158 mg,

35%; M.p. 74–77 °C; IR (KBr, cm⁻¹): ν_max2922 (C–H),

1705 (C=O), 1614 (C=C), 1489 (C=C_Ar), 1253 (C–O–C),

1184 ((C=O)-O), 790 (C–H_Ar); ¹H NMR (CDCl₃,

300 MHz): δ 0.94 (CH₃, t, J = 6.9 Hz), 1.35 (3CH₂, m),

1.52 (CH₂, m), 1.63 (CH₂, m), 1.86 (CH₂, m), 2.61 (CH₃, s),

3.87 (OCH₃, s), 3.89 (OCH₃, s) 4.06 (–CH₂O–, t, J =

6.5 Hz), 6.86 (H₈, H_3´´, H_4´´, m), 6.91 (H₆, dd, J = 8.9,

2.5 Hz), 7.19 (H_6´´, d, J = 2.7 Hz), 7.22 (H_1´, d, J =

16.1 Hz), 7.61 (H₅, d, J = 8.9 Hz), 7.81 (H_2´, d, J =

16.1 Hz). ¹³C NMR (CDCl₃, 75 MHz): δ 14.13 (CH₃),

15.64 (CH₃), 22.68 (CH₂), 26.0 (CH₂), 29.05 (CH₂), 29.25

(CH₂), 29.35 (CH₂), 31.81 (CH₂), 55.84 (2OCH₃), 68.61

(–OCH₂–), 100.91 (C₈),112.24 (C₆, C_3´´),112.84 (C_4a),

114.12 (C_4´´), 114.32 (C_6´´),119.80 (C_1´),122.17 (C_1´´),

126.0 (C₃), 127.65 (C₅), 130.80 (C_2´), 146.36 (C_2´´), 151.76

(C_5´´), 153.71 (C_8a), 153.73 (C₄), 160.60 (C₇), 161.66

(C=O). EIMS: m/z 451.2484 [M + H]⁺, (calculated for

[C₂₈H₃₅O₅]⁺: 451.2484).

3-[(E)-2-(2,4-dimethoxyphenyl)ethenyl]-4-methyl-7-(octy-

loxy)-2H-chromen-2-one (6c) Yield: 0.39 mmol, 175 mg,

39%; M.p. 106–108 °C; IR (KBr, cm⁻¹): ν _max2922 (C–H),

1714 (C=O), 1610 (C=C), 1504 (C=C_Ar), 1251 (C–O–C),

1153 ((C=O)-O), 834 (C–H_Ar); ¹H NMR (CDCl₃,

300 MHz): δ 0.94 (CH₃, t, J = 6.8 Hz), 1.35 (3CH₂, m),

1.52 (CH₂, m), 1.62 (CH₂, m), 1.86 (CH₂, m), 2.60 (CH₃, s),

3.89 (OCH₃, s), 3.91 (OCH₃, s) 4.06 (-CH₂O-, t, J =

6.5 Hz), 6.51 (H_3´´, d, J = 2.5 Hz), 6.57 (H_5´´, dd, J = 8.8,

2.5 Hz), 6.85 (H₈, d, J = 2.5 Hz), 6.90 (H₆, dd, J = 8.8,

2.5 Hz), 7.12 (H_1´, d, J = 16.6 Hz), 7.56 (H_6´´, d, J =

8.8 Hz), 7.60 (H₅, d, J = 8.8 Hz), 7.74 (H_2´, d, J = 16.6 Hz).

¹³C NMR (CDCl₃, 75 MHz): δ 14.14 (CH₃), 15.63 (CH₃)

22.68 (CH₂), 26.0 (CH₂), 29.05 (CH₂), 29.25 (CH₂), 29.35

(CH₂), 31.82 (CH₂) 53.43 (OCH₃) 55.59 (OCH₃) 68.61

(–OCH₂–), 98.46 (C_3´´) 100.90 (C₈), 104.93 (C_5´´), 112.81

(C₆), 114.45 (C_4a), 119.76 (C_1´´) 119.92 (C_1´), 120.24 (C₃)

125.82 (C₅) 127.90 (C_6´´) 130.80 (C_2´) 145.38 (C_8a) 153.55

(C₄), 158.39 (C_2´´), 160.76 (C₇), 160.87 (C_4´´) 161.44

(C=O). EIMS: m/z 451.2482 [M + H]⁺, (calculated for

[C₂₈H₃₅O₅]⁺: 451.2484).

3-[(E)-2-(4-hydroxy-3,5-dimethoxyphenyl)ethenyl]-4-

methyl-7-(octyloxy)-2H-chromen-2-one

(6f) Yield:

0.41 mmol, 191 mg, 41%; M.p. 55–57 °C; IR (KBr, cm⁻¹):

ν_max2927 (C–H), 1708 (C=O), 1616 (C=C), 1514

(C=C_Ar), 1253 (C–O–C), 1153 ((C=O)-O), 858 (C–H_Ar);

¹H NMR (CDCl₃, 300 MHz): δ 0.94 (CH₃, t, J = 6.8 Hz),

1.34 (3CH₂, m), 1.52 (CH₂, m), 1.66 (CH₂, m), 1.86 (CH₂,

m), 2.61 (CH₃, s), 3.99 (2OCH₃, s), 4.02 (–CH₂O–, t, J =

6.5 Hz), 6.82 (H_2´´, H_6´´, s), 6.85 (H₈, d, J = 2.0 Hz), 6.91

(H₆, dd, J = 8.9, 2.0 Hz), 7.04 (H_1´, d, J = 16.1 Hz), 7.54

(H_2´, d, J = 16.1 Hz), 7.62 (H₅, d, J = 8.9 Hz). ¹³C NMR

(CDCl₃, 75 MHz): δ 14.12 (CH₃), 15.58 (CH₃), 22.67

(CH₂), 26.0 (CH₂), 29.03 (CH₂), 29.24 (CH₂), 29.34 (CH₂),

31.83 (CH₂), 56.38 (2OCH₃), 68.67 (–OCH₂–), 100.90

(C₈), 103.66 (C_2´´,C_6´´), 112.99 (C₆), 114.26 (C_4a), 119.10

(C_1´),119.18 (C₃), 125.91 (C₅), 129.31(C_2´),135.21 (C₁

_´´),135.78 (C_4´´),145.96 (C₄), 147.22 (C_3´´, C_5´´), 160.61

(C_4a), 161.70 (C₇), 162.47 (C=O). EIMS: m/z 467.2426 [M

+ H]⁺, (calculated for [C₂₈H₃₅O₆]⁺: 467.2434).

3-[(E)-2-(4-hydroxy-3-methoxyphenyl)ethenyl]-4-methyl-7-

(octyloxy)-2H-chromen-2-one (6d) Yield: 0.36 mmol,

157 mg, 36%; M.p. 114–116 °C; IR (KBr, cm⁻¹): ν

max

2924 (C–H), 1716 (C=O), 1610 (C=C), 1512 (C=C_Ar),

1

1257 (C–O–C), 1161 ((C=O)-O), 813 (C–H_Ar); H NMR

(CDCl₃, 300 MHz): δ 0.94 (CH₃, t, J = 6.8 Hz), 1.34

(3CH₂, m), 1.52 (CH₂, m), 1.69 (CH₂, m), 1.86 (CH₂, m),

2.60 (CH₃, s), 3.99 (OCH₃, s), 4.06 (–CH₂O–, t, J =

6.5 Hz), 6.85 (H₈, d, J = 2.5 Hz), 6.91 (H₆, dd, J = 8.8,

2.5 Hz), 6.96 (H_5´´, d, J = 8.8 Hz), 7.04 (H_1´, d, J =

16.3 Hz), 7.10 (H_2´´, d, J = 2.5 Hz), 7.12 (H_6´´, dd, J = 8.8,

2.5 Hz), 7.55 (H_2´, d, J = 16.3 Hz), 7.62 (H₅, d, J = 8.8 Hz).

¹³C NMR (CDCl₃, 75 MHz): δ 14.12 (CH₃), 15.52 (CH₃),

22.66 (CH₂), 25.99 (CH₂), 29.03 (CH₂), 29.24 (CH₂), 29.36

(CH₂), 31.83 (CH₂), 56.0 (OCH₃), 68.67 (-OCH₂-), 100.90

(C₈), 109.03 (C_2´´), 112.96 (C₆), 114.31 (C_4a), 114.58 (C₅

3-[(E)-2-(1,3-benzodioxol-5-yl)ethenyl]-4-methyl-7-(octy-

loxy)-2H-chromen-2-one (6g) Yield: 0.49 mmol, 214 mg,

49%; M.p. 122–125 °C; IR (KBr, cm⁻¹): ν_max2926 (C–H),

1716 (C=O), 1614 (C=C), 1500 (C=C_Ar), 1234 (C–O–C),

1178 ((C=O)-O), 808 (C–H_Ar); ¹H NMR (CDCl₃,

300 MHz): δ 0.94 (CH₃, t, J = 6.8 Hz), 1.35 (3CH₂, m),

Medicinal Chemistry Research

1.51 (CH₂, m), 1.67 (CH₂, m), 1.86 (CH₂, m), 2.58 (CH₃, s),

4.05 (-CH₂O-, t, J = 6.5 Hz), 6.02 (O–CH₂–O) 6.83 (H₈, d,

J = 2.5 Hz), 6.84 (H_5´´, d, J = 8.8, 2.5 Hz), 6.90 (H₆, dd, J

= 8.8, 2.5 Hz), 7.01 (H_1´, H_6´´, m), 7.12 (H_2´´, d, J = 2.5 Hz),

7.59 (H_2´, d, J = 16.1 Hz), 7.60 (H₅, d, J = 9.1 Hz). ¹³C

NMR (CDCl₃, 75 MHz): δ 14.13 (CH₃), 15.37 (CH₃), 22.66

(CH₂), 25.99 (CH₂), 29.03 (CH₂), 29.24 (CH₂), 29.34

(CH₂), 31.81 (CH₂), 68.60 (–OCH₂–), 100.83 (C₈), 101.18

(O–CH₂–O), 105.53 (C_2´´), 108.45 (C_5´´), 112.94 (C₆),

114.20 (C_4a), 118.99 (C_6´´), 119.24 (C_1´), 122.07 (C₃),

125.87 (C₅), 132.33 (C_1´´), 135.07 (C_2´), 146.02 (C_4´´),

147.64 (C_3´´), 148.17 (C_8a) 153.57 (C₄), 160.45 (C₇), 161.66

(C=O). EIMS: m/z 435.2179 [M + H]⁺, (calculated for

[C₂₇H₃₁O₅]⁺: 435.2171).

6.80 (H_2´´, H_6´´, s), 6.97 (H_1´, d, J = 16.1 Hz), 7.15 (H_2´, d, J

= 16.1 Hz), 7.40–7.46 (H₅, H₆, H₈, m), 7.68 (H₄, d, J =

9.5 Hz). ¹³C NMR (CDCl₃, 125 MHz): δ 56.4 (OCH₃),

103.8 (C_2´´, C_6´´), 103.9 (C₈), 115.2 (C₃), 117.8 (C_4a), 122.3

(C₆) 125.0 (C_1´) 127.9 (C₅) 128.0 (C_1´´) 132.1 (C_2´), 135.7

(C_4´´), 141.5 (C₇), 143.0 (C₄), 147.3 (C_3´´, C_5´´), 154.6 (C₉),

160.9 (C=O). EIMS: m/z 325.1076 [M + H]⁺, (calculated

for [C₁₉H₁₇O₅]⁺: 325.1069).

7-[(E)-2-(4-hydroxy-3-methoxyphenyl)ethenyl]-2H-chromen-

2-one (10d) Yield: 0.50 mmol, 147 mg, 52%; yellow solid,

M.p. 204–206 °C; IR (KBr, cm⁻¹): ν_max3402 (OH), 1705

(C=O), 1606 (C=C), 1512 (C=C_Ar), 1267 (C–O–C), 1124

1

((C=O)-O), 835 (C–H_Ar); H NMR (CDCl₃, 500 MHz): δ

3.83 (-OCH₃, s), 6.41 (H₃, d, J = 9.5 Hz), 6.80 (H_5´´, d, J =

8.1 Hz), 7.05 (H_6´´, d, J = 8.3 Hz), 7.16 (H_1´, d, J =

16.3 Hz), 7.25 (H_2´´, s_apparent), 7.38 (H_2´, d, J = 16.3 Hz),

7.54 (H₈, s_apparent), 7.55 (H₆, d, J = 8.9 Hz), 7.67 (H₅, d, J =

8.0 Hz), 8.02 (H₄, d, J = 9.5 Hz). ¹³C NMR (CDCl₃,

125 MHz): δ 56.1 (OCH₃), 110.7 (C_2´´), 113.4 (C₈), 115.4

(C_4a), 116.5 (C₃) 117.9 (C_5´´) 121.5 (C_6´´) 122.9 (C₆) 124.2

(C_1´), 128.4 (C_1´´), 129.0 (C₅), 132.6 (C_2´), 142.3 (C₇), 144.4

(C₄), 148.2 (C_4´´), 148.4 (C_3´´), 154.6 (C₉), 160.6 (C=O).

EIMS: m/z 295.0970 [M + H]⁺, (calculated for [C₁₈H₁₅O₄]

⁺: 295.0964).

7-[(E)-2-(2,5-dimethoxyphenyl)ethenyl]-2H-chromen-2-one

(10a) Yield: 0.51 mmol, 158 mg, 51%; yellow solid, M.p.

125–128 °C; IR (KBr, cm⁻¹): ν_max1724 (C=O), 1606

(C=C), 1494 (C=C_Ar), 1220 (C–O–C), 1041 ((C=O)-O),

1

833 (C–H_Ar); H NMR (CDCl₃, 500 MHz): δ 3.84 (OCH₃,

s), 3.89 (OCH₃, s) 6.39 (H₃, d, J = 9.6 Hz), 6.84–6.88 (H_3´´,

H_4´´, m), 7.12 (H_1´, d, J = 16.5 Hz), 7.16 (H_6´´, s_apparent),

7.43–7.48 (H₅, H₆, H₈, m), 7.59 (H_2´, d, J = 16.5 Hz), 7.68

(H₄, d, J = 9.6 Hz). ¹³C NMR (CDCl₃, 125 MHz): δ 55.8

(OCH₃) 56.2 (OCH₃) 111.9 (C_3´´), 112.4 (C_6´´), 114.3 (C₈),

114.8 (C_4´´), 115.8 (C₃) 117.9 (C_4a), 122.6 (C₆) 126.2 (C_1´´)

126.8 (C_1´) 127.4 (C_2´) 127.9 (C₅) 142.0 (C₇), 143 (C₄),

151.8 (C_2´´), 153.8 (C_5´´), 154.6 (C₉), 160.9 (C=O). EIMS:

m/z 309.1127 [M + H]⁺, (calculated for [C₁₉H₁₇O₄]⁺:

309.1134).

7-[(E)-2-(1,3-benzodioxol-5-yl)ethenyl]-2H-chromen-2-one

(10e) Yield: 0.55 mmol, 161 mg, 55%; yellow solid, M.p.

193–196 °C; IR (KBr, cm⁻¹): ν_max1720 (C=O), 1606

(C=C), 1494 (C=C_Ar), 1249 (C–O–C), 1041 ((C=O)-O),

846 (C–H_Ar); ¹H NMR (CDCl₃, 500 MHz): δ 6.01 (H_6´´, s),

6.38 (H₃, d, J = 9.5 Hz), 6.83 (H_5´´, d, J = 8.0 Hz), 6.95 (H₁

_´, d, J = 16.3 Hz), 6.99 (H_6´´, dd, J = 8.0, 1.5 Hz), 7.10 (H_2´´,

d, J = 1.5 Hz), 7.15 (H_2´, d, J = 16.3 Hz), 7.39 (H₈, d, J =

1.5 Hz), 7.40 (H₆, d, J = 6.0 Hz), 7.44 (H₅, d, J = 8.5 Hz),

7.68 (H₄, d, J = 9.6 Hz). ¹³C NMR (CDCl₃, 125 MHz): δ

101.3 (OCH₂O), 105.7 (C_2´´), 108.5 (C_5´´), 113.9 (C₈), 115.8

(C₄), 117.9 (C_4a) 122.3 (C_6´´) 122.4 (C₆) 125.1 (C₅), 127.9

(C_1´), 131.0 (C_1´´), 131.7 (C_2´), 141.6 (C₇), 143.0 (C₄), 148.1

(C_4´´), 148.3 (C_3´´), 154.6 (C₉), 160.8 (C=O). EIMS: m/z

293.0814 [M + H]⁺, (calculated for [C₁₈H₁₃O₄]⁺:

293.0808).

7-[(E)-2-(2,3-dimethoxyphenyl)ethenyl]-2H-chromen-2-one

(10b) Yield: 0.58 mmol, 179 mg, 58%; orange solid, M.p.

154–157 °C; IR (KBr, cm⁻¹): ν_max1720 (C=O), 1604

(C=C), 1475 (C=C_Ar), 1276 (C–O–C), 1143 ((C=O)-O),

1

837 (C–H_Ar); H NMR (CDCl₃, 500 MHz): δ 3.91 (OCH₃,

s), 3.92 (OCH₃, s) 6.41 (H₃, d, J = 9.4 Hz), 6.91 (H_4´´, dd, J

= 8.1, 1.3 Hz), 7.11 (H_5´´, dd, J = 8.0, 8.0 Hz), 7.17 (H_1´, d,

J = 16.5 Hz), 7.28–7.30 (H_6´´, m), 7.46–7.51 (H₅, H₆, H₈,

m), 7.61 (H_2´, d, J = 16.4 Hz), 7.71 (H₄, d, J = 9.5 Hz). ¹³

C

NMR (CDCl₃, 125 MHz): δ 55.9 (OCH₃), 61.1 (OCH₃)

112.3 (C_4´´), 114.4 (C₈), 115.9 (C₃), 118.0 (C_4a), 122.6 (C₆)

124.2 (C_5´´) 126.4 (C_1´) 127.9 (C_2´) 128.0 (C₅) 130.6 (C_1´´),

142.0 (C₇), 143.0 (C₄), 147.4 (C_2´´), 153.1 (C_3´´), 154.6

(C_8a), 160.8 (C=O). EIMS: m/z 309.1127 [M + H]⁺, (cal-

culated for [C₁₉H₁₇O₄]⁺: 309.1131).

Biological activity assays

Cell lines and culture medium

7-[(E)-2-(4-hydroxy-3,5-dimethoxyphenyl)ethenyl]-2H-chro-

men-2-one (10c) Yield: 0.51 mmol, 165 mg, 51%; yellow

solid, M.p. 159–161 °C; IR (KBr, cm⁻¹): ν_max3433 (OH),

1724 (C = O), 1612 (C=C), 1512 (C=C_Ar), 1211 (C–O–C),

1112 ((C=O)-O), 833 (C–H_Ar); ¹H NMR (CDCl₃,

500 MHz): δ 3.97 (2-OCH₃, s), 6.38 (H₃, d, J = 9.5 Hz),

An adenocarcinoma colon cancer cell line (SW480) and

non-malignant cells (CHO-K1) were used for biological

assays. These were obtained from The European Collection

of Authenticated Cell Cultures (ECACC, England) and

maintained in Dulbecco’s Modiﬁed Eagle Medium, sup-

plemented with 10% heat-inactivated (56 °C) horse serum,

Medicinal Chemistry Research

1% penicillin/streptomycin and 1% non-essential amino

acids (Gibco Invitrogen, Carlsbad, USA). For all experi-

ments, horse serum was reduced to 3%, and the medium

was supplemented with 5 mg/ml transferrin, 5 ng/ml sele-

nium, and 10 mg/ml insulin (ITS-deﬁned medium; Gibco,

Invitrogen, Carlsbad, USA) (Maldonado et al. 2009).

96A) (Pérez et al. 2014). All experiments were performed

in triplicate.

Statistical analysis

All experiments were performed at least three times. Data

are reported as mean SE (standard error). Statistical dif-

ferences between control group (non-treated) and treated

cells were evaluated by one-way ANOVA followed by the

Dunnett’s test. Values with p ≤ 0.05 were considered sig-

niﬁcant. Data were analyzed with GraphPad Prism version

7.03 for Windows (Graph Pad Software, San Diego, Cali-

fornia, USA).

Cell viability by trypan blue dye exclusion assay

To assess the effect of styrylcoumarins on the viability of

both cell lines, a trypan blue dye exclusion test was per-

formed, which allows to differentiate live cells since these

can exclude the dye (Lamy et al. 2007). Cells were seeded

to a ﬁnal density of 1 × 10⁵cells/well in 12-well tissue

culture plates and incubated at 37 °C in a 5% CO₂atmo-

sphere. The cultures were allowed to grow for 24 h and then

were treated for 24 and 48 h with either 0,5% DMSO

(vehicle control) or various concentrations (10–100 µM) of

different styrylcoumarins, as well as coumarin, resveratrol

(lead compounds), and 5-ﬂuorouracil (5-FU; the standard

drug). After treatment, cells were washed with buffer ver-

sene and they were collected by trypsinization (0.5% tryp-

sin/2.6 mM ethylenediaminetetraacetic acid) followed by

centrifugation. Then, trypan blue (1/1, vol/vol) was added to

the pellet. The number of unstained cells (live cells) was

determined by optical microscope, using hemocytometer.

The inhibition percentage was calculated with regard to

untreated cells (vehicle control) and IC₅₀values were

obtained from dose response curves for each compound.

Selectivity index (SI) was calculated by the ratio of IC₅₀

values in non-malignant CHO-K1 cells to IC₅₀of SW480

cells.

Results and discussion

Chemistry

Synthesis of 3-styrylcoumarins

Synthesis of 3-styrylcoumarins was performed following

the Scheme 1, which began with formation of the coumarin

(2) by microwave assisted Pechmann reaction between

resorcinol (1) and ethyl acetoacetate (Manhas et al. 2006).

Compound (3), obtained by microwave assisted Williamson

etheriﬁcation reaction of coumarin (2) (Otero et al. 2014) in

85% yield, was brominated with NBS (Adhikari and

Samant 2002) to give the alkyloxybromocoumarin (4) in

75% yield. This compound was subjected to cross-coupling

with various styrenes (5) under palladium catalysis (Heck

reaction) (Sabrina et al. 2011), leading to the formation of 3-

styrylcoumarins (6a–6g) in 35–49% yields. In order to

improve these results, other methodologies were tested such

as (Pd(Ph₃)₄, AgOAc, DMF) (Martins et al. 2012) or Pd

(AcO)₂, PPh₃, CH₃CO₂Na (Gordo et al. 2011) but the

results were not as expected. Styrenes (5) were synthesized

via microwave assisted Wittig reaction between substituted

aldehydes and methyltriphenylphosphonium bromide

(Martin and Kellen-Yuen 2007) and Knoevenagel con-

densation reactions between 4-hydroxybenzaldehydes with

malonic acid (Simpson et al. 2005).

Antiproliferative activity of styrylcoumarins

The antiproliferative effect of styrylcoumarins was eval-

uated by using Sulforhodamine B (SRB) dye, a colorimetric

assay based on staining of total cellular protein of adherent

cells. Cells were seeded to a ﬁnal density of 2500 cells/well

in 96-well tissue culture plates and incubated at 37 °C in a

5% CO₂atmosphere. The cultures were allowed to grow

for 24 h and then were treated either with increasing con-

centration of styrylcoumarins (0.02–50 µM, ranges depen-

ded on the IC₅₀values) or 0.5% DMSO (control), for 0, 2,

4, 6, and 8 days. Culture media was replaced every 48 h.

After each incubation time, cells were ﬁxed with tri-

chloroacetic acid (50% v/v) (MERCK) for an hour at 4 °C.

Cell proteins were determined by staining with 0.4% (w/v)

SRB (Sigma-Aldrich, United States). Cells were then

washed with 1% acetic acid for the removal of unbound

SRB, and left for air-drying. Protein bound SRB was

solubilized in 10 mM Tris-base and the absorbance was

measured at 492 nm in a microplate reader (Mindray MR-

Synthesis of 7-styrylcoumarins

Scheme 2 shows the synthesis of 7-styrylcoumarins, which

began with the formation of the 7-hydroxycoumarin (8) by

microwave assisted condensation between resorcinol (1)

and malic acid (7) (Symeonidis et al. 2009). Compound (8)

was treated with triﬂuoromethanesulfonic anhydride to

obtain triﬂate 9 (Cano et al. 2013) in 85% yield, that in turn

was subjected to cross-coupling with various styrenes (5)

under palladium catalysis (Sabrina et al. 2011), to afford the

Medicinal Chemistry Research

Scheme 1 Synthetic pathway to 3-styrylcoumarins

styrylcoumarins under study (10a–10e) in 50%–58% yields.

The product of the reaction of coumarin 8 with styrenes 5f

and 5g showed in the NMR duplication of several signals,

which could indicate a mixture of isomers. For this reason,

these compounds were not taken into account in this study.

The structures of all compounds have been established

and C=O, respectively. 7-Styrylcoumarins showed signals

around 116, 145, 124, 132, and 160 ppm, corresponding to

H–C=C–H_cis, H–C=C–H_trans, and C=O, respectively.

Effect of styrylcoumarins on SW480 and CHO K1 cell

viability

1

by a combined study of IR, ESI-MS, H-NMR, ¹³C-NMR,

and COSY spectra. IR spectra exhibited characteristic

absorption peaks corresponding to C=O, C=C, C=C_Ar,

C–O–C, and C–H_Ar. ESI-MS spectra showed characteristic

[M + H]⁺peaks corresponding to their molecular weights.

The assignments of all the signals to individual H or C-

atoms have been performed on the basis of typical δ-values

and J-constants. The ¹H-NMR spectra of 3-styrylcoumarins

dissolved in CDCl₃showed signals of –CH₃(∼2.60 ppm),

–OCH₂– (∼4.00 ppm), H–C=C–H_trans(∼7.00 and

7.60 ppm) and 7-styrylcoumarins showed signals of

H–C=C–H_cis(∼6.50 and 8.00 ppm) and H–C=C–H_trans

(∼7.15 and 7.40 ppm). ¹³C-NMR spectra 3-of styr-

ylcoumarins showed at around 14, 68, 118, 135, and

A series of 3- and 7-styrylcoumarin hybrids were previously

synthesized, and evaluated against SW480 and CHO-K1

cell lines, in order to asses their effect on the viability,

through the trypan blue dye exclusion method. As shown in

Fig. 3, the activity was time-dependent and concentration-

dependent, with a higher cytotoxic effect on SW480 cells in

comparison with CHO-K1 cells. Cytotoxicity was reported

as 50% inhibitory concentration (IC₅₀) values.

All results regarding to the cytotoxic effect are sum-

marized in Table 1. Among the compounds tested, hybrids

6a, 6c, 6g, and 10e exhibited the lowest activity at both 24

and 48 h after treatment. Besides, excluding compound 10e,

the increase of IC₅₀concentration at 48 h suggests a loss in

the cytotoxic activity of these hybrids. On the other hand,

161 ppm, corresponding to –CH₃, –CH₂O–, H–C=C–H_trans

,

Medicinal Chemistry Research

5

8

4

4a

8a

3

2

6

OCH₃

HO

OH

2´

2´´

1

1´´

7

3´´

4´´

O

1´

+

1

6´´

O

OH

5´´

H₂SO₄/MW

OH

OCH₃

10a

HO

O

8

7

O

OCH₃

Tf₂O/Pyridine, 85%

CH₃O

O

Pd(OAc)₂

Triethanolamine

10b

R₁

R₄

R₂

R₃

+

TfO

O

MW (

50 - 58%)

CH₃O

HO

9

10c

5

OCH₃

a. R₁= R₄= OCH_3,R₂= R₃= H

b. R₁= R₂= OCH₃R₃= R₄=H

CH₃O

HO

c. R₂= R₄= OCH_3,R₃= OH, R₁= H

d. R₂=OCH_3,R₃= OH, R₁= R₄= H

e. R₂- R₃= -OCH₂O-, R₁= R₄= H

f. R₁= R₃= OCH_3,R₂= R₄= H

g. R₃= OCH_3,R₁= R₂= R₃= H

O

10d

O

10e

Scheme 2 Synthetic pathway to 7-styrylcoumarins

Fig. 3 Effect of 3- and 7-styrylcoumarins on cell viability of SW480 a,

b and CHO K1 cells c, d, respectively, 48 h post-treatment with

diferent concentrations of styrylcoumarins (10–100 µM). Viable cells

were counted and growth inhibition was calculated using 100%

viability of control. Zero values indicate that there was not inhibition at

that concentration. Data are presented as the mean SE of three

independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001)

Medicinal Chemistry Research

Table 1 Cytotoxic effect (µM)

of styrylcoumarins on SW480

and CHO K1 cell lines

24 h

48 h

Compounds

IC₅₀(µM)

CHO cells

IC₅₀(µM)

SI

IC₅₀(µM)

CHO cells

IC₅₀(µM)

SI

SW480 cells

6a

>3000

66.26

419.69

262.41

>3000

35.88

>3000

254.18

4.55

61.81

40.53

109.49

49.75

11.15

23.77

295.09

0.10

>40

>3000

110.07

213.10

69.81

>3000

45.50

576.91

26.07

6.92***

50.64

>3000

0.68

>1

6b

1.63

2.4

6c

3.83

0.4

6d

5.27

2,7

6e

>200

1.51

>3000****

43.61

>400

0.9

6f

6g

>10

>3000

43.90

>1

10a

2534.80

147.26

0.50

64.8

5.2

10b

0.031

9.40

40.86

7.92

10c

4.66

68.63**

38.14*

>3000

1251.66

2.87

1.01***

5.33***

92.88

3.91

67.8

7.2

10d

9.48

5.47

1.73

10e

1546.22

2541.85

9.00

2020.28

32.12

8.90

0.16

>30

319.7

3.2

Coumarin

Resveratrol

5-Fluorouracil

79.13

1.01

0.89

90.99

61.68

1.48

47.63

35.43

1.34

The IC₅₀values were obtained from dose response curves for each compound. Selectivity index (SI) was

calculated by the ratio of IC₅₀values in non-malignant CHO-K1 cells to IC₅₀of SW480 cells. P-values lower

than 0.05 were considered statistically signiﬁcant. (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

although compound 6f exhibited good activity against

SW480 cancer cell line after 24 h of treatment (IC₅₀

better activity against SW480 cells (6e: IC_50-24h= 11.15

µM, IC_50-48h= 6.92 µM; 10d: IC_50-24h= 5.47 µM, IC_50-48h

= 5.33 µM) in comparison with the non-malignant cell line

(6e: IC_50-24h/48h= >3000 µM; 10d: IC_50-24h= 9.48 µM, IC_50-

_48h= 38.14 µM) and the activity was kept over time. Once

again, these two compounds exhibited higher activity than

the lead and standard compounds, which showed that the

improvement in the activity and selectivity could be due to a

synergistic action of the lead compounds when they are

linked to form a single structure in the hybrid. This study is

consistent with the ﬁndings of Belluti et al. (2010) which

found similar results in the activity of some stilbene-

coumarin hybrids when they were tested in other cancer cell

lines (A431 epidermoid carcinoma, JR8 melanoma cell, and

H460 non-small cell lung cancer).

All compounds that exhibited good activity and selec-

tivity with the trypan blue method (6b, 6d, 6e, 10a, 10b,

10c, and 10d) were tested in order to determine their anti-

proliferative effect with sulforhodamine B assay, which is

based on the measurement of cellular protein content of

adherent cells. After comparing each treatment with the

control, results indicated that the activity was time-

dependent and concentration-dependent. Among the

results obtained, hybrids 6e, 10c, and 10d displayed sig-

niﬁcant antiproliferative activity even at low concentrations

(IC₅₀values lower than 12 µM) from day 4 of the treatment,

while hybrid 6d required a higher concentration (>20 µM)

to exert a signiﬁcant antiproliferative effect in the same

time. Furthermore, molecule 6b required less time (2 days)

=

23.77 µM), this effect decreased after 48 h (IC₅₀= 50.64

µM) as evidenced by the rise in the IC₅₀. In addition,

compound 6f showed a low selectivity index (24 h: 1.51;

48 h: 0.9). Moreover, compounds 6b and 6d exhibited

moderate activity against SW480 cell line after 24 h of

treatment (6b: IC₅₀= 40.53 µM; 6d: IC₅₀= 49.75 µM), and

even though the activity of 6d improved after 48 h (6d: IC₅₀

= 26.07 µM), the selectivity index decreased due to

increased toxicity in the non-malignant cell line (IS = 24 h:

5.27; 48 h: 2,7). The opposite occurred with compounds

10a and 10b which displayed the highest activity after 24 h

of treatment (IC₅₀= 0.10 and 0.031 µM, respectively), but

decreased after 48 h (IC₅₀= 0.68 and 7.92 µM, respec-

tively). Despite the loss in the activity and selectivity of

these two compounds over time, it was worthwhile to

include them in further studies since they were more potent

than the lead and standard compounds at lowest con-

centrations and time. When compound 10c was tested at

24 h, it exhibited higher toxicity against the non-malignant

cell (IC_50-CHOK1= 4.66 µM; IC_50-SW480= 9.40 µM) than the

malignant one. However, after 48 h of treatment the cyto-

toxicity varied in a signiﬁcant way for both cell lines (IC_50-

_CHOK1= 68.63 µM; IC_50-SW480= 1.01 µM), being these

values even better than coumarin and 5-FU (IC_{50- SW480}

=

3.91 µM; IC_50-SW480= 35.43 µM, respectively). This fact

was also reﬂected in the increase of the selectivity index for

compound 10c. Finally, compounds 6e and 10d showed

Medicinal Chemistry Research

Fig. 4 Antiproliferative effect of styrylcoumarins on SW480 colon cancer cell growth. Data are presented as the mean SE of at least three

independent experiments (*P < 0.05; **P < 0.01; ***P < 0.001). Optical density (O.D.) is directly proportional to cell mass of adherent cells

but a higher concentration (50 µM) to display a signiﬁcant

activity (p ≤ 0.05). On the other hand, none of the con-

centrations evaluated for hybrids 10a and 10b caused cell

growth inhibition over time (Fig. 4).

oxygenated positions in 3,4 as well as 2,5 positions were

determinant for the activity. These results are similar to the

ones obtained by Belluti et al. (2010) on H460 cells when

these were treated with 7-methoxycoumarin having 3,5-

disubstitution pattern in trans-vinylbenzene moiety. How-

ever, we observed that the presence of a hydroxyl group in

4-position was necessary to preserve the activity through

time (6d, 10c, and 10d vs 10a) (See Fig. 4). The effect of the

hydroxyl groups may be due to a better molecular recogni-

tion ability towards the target bioreceptors upon hydrogen

bond formation (Patrick 2013). Thus, the presence of spe-

ciﬁc substituents in certain positions proved to be crucial in

endowing the compounds with inhibitory effect and anti-

proliferative activity. The 4-methoxy and methylendioxy

On a structure-activity relationship, it is interesting to

note that in general, the position of styryl group is important

for the activity. Thus, when series 6 and 10 are compared, it

is observed a higher prevalence in the toxicity against

SW480 cells, with better selectivity indexes for 10 series

(see compounds 10a-10d). For instance, when comparing

compounds 6f and 10c (bearing the 4-hydroxy-3,5-dime-

thoxystyryl motif), it is noticed a divergence of activities,

being the hybrid 10c the one which is able to preserve the

best activity over time. On the other hand, we found that

Medicinal Chemistry Research

group were detrimental, since the presence of these groups in

compounds 6a and 6g resulted in a decrease of activity. The

results indicate that the presence of these groups decreases

the selectivity and speciﬁcity with the target.

Budzisz E, Brzezinska E, Krajewska U, Rozalski M (2003) Cytotoxic

effects, alkylating properties and molecular modelling of cou-

marin derivatives and their phosphonic analogues. Eur J Med

Chem 38:597–603

Cano M, Rojas C, Hidalgo W, Sáez J, Gil J, Schneider B, Otálvaro F

(2013) Improved synthesis of 4-phenylphenalenones: the case of

isoanigorufone and structural analogs. Tetrahedron Lett

54:351–354

Conclusions

Egan D, James P, Cooke D, O’Kennedy R (1997) Studies on the

cytostatic and cytotoxic effects and mode of action of 8-nitro-7-

hydroxycoumarin. Cancer Lett 118:201–211

Twelve hybrid molecules combining styryl and coumarin

motifs have been designed and synthesized and their cyto-

toxicity and antiproliferative activity was measured against

SW480 and CHO-K1 cell lines. Among the tested com-

pounds, hybrids 6e, 10c, and 10d exhibited the highest

activity which make them promising candidates for further

studies. The SAR analysis showed that styryl group in the

position 7 and the presence of hydroxyl groups in position 4

or methoxy groups in 2,5 and 3,4-positions were determi-

nant for their biological activity.

Gacche R, Jadhav S (2012) Antioxidant activities and cytotoxicity of

selected coumarin derivatives: preliminary results of

a

structure–activity relationship study using computational tools. J

Exp Clin Med 4:165–169

Globocan (2012) Estimated Cancer Incidente, Mortality and Pre-

valence Worldwide in 2012. International Agency for Research

on Cancer, World Health Organisation. http://globocan.iarc.fr/Pa

ges/fact_sheets_cancer.aspx

Gordo J, Avo J, A. Parola AJ, Lima JC, Pereira A, S. Branco PS

(2011) Convenient synthesis of 3-vinyl and 3-styryl coumarins.

Org Lett 13:5112–5115

Gülçin

I

(2010) Antioxidant properties of resveratrol:

a

structure–activity insight. Innov Food Sci Emerg Technol

11:210–218

Acknowledgements The authors thank Universidad de Antioquia

(grant CODI 2014-808) for ﬁnancial support.

Kashman Y, Gustafson KR, Fuller WR, Cardellina JH, McMahon JB,

Currens MJ, Buckheit Jr RW, Huqhes SH, Cragg GM, Boyd MR

(1992) The calanolides, a novel HIV-inhibitory class of coumarin

derivatives from the tropical rainforest tree, Calophyllum lani-

gerum. J Med Chem 35:2735–2743

Compliance with ethical standards

Conﬂict of interest The authors declare that they have no conﬂict of

interest.

Kawaii S, Tomono Y, Ogawa K, Sugiura M, Yano M, Yoshizawa Y

(2001) The antiproliferative effect of coumarins on several cancer

cell lines. Anticancer Res 21:917–923

Kedzierski L, Curtis J, Kaminska M, Jodynis Liebert J, Murias M

(2007) In vitro antileishmanial activity of resveratrol and its

hydroxylated analogues against Leishmania major promastigotes

and amastigotes. Parasitol Res 102:91–97

Kerru N, Singh P, Koorbanally N, Raj R, Kumar V (2017) Recent

advances (2015-2016) in anticancer hybrids. Eur J Med Chem

142:179–212

Kumar D, Raj KK, Malhotra SV, Diwan S, Rawat DS (2014)

Synthesis and anticancer activity evaluation of resveratrol-

chalcone conjugates. Med Chem Commun 5:528–535

Lamy V, Roussi S, Chaabi M, Gossé F, Schall N, Lobstein A, Raul F

(2007) Chemopreventive effects of lupulone, a hop b-acid, on

human colon cancer-derived metastatic SW620 cells and in a rat

model of colon carcinogenesis. Carcinogenesis 28:1575–1581

Madari H, Panda D, Wilson L, Jacobs RS (2003) Dicoumarol: a

unique microtubule stabilizing natural product that is synergistic

with taxol. Cancer Res 63:1214–1220

Maldonado ME, Bousserouel S, Gossé F, Minker C, Lobstein A, Raul

F (2009) Differential induction of apoptosis by apple procyani-

dins in TRAIL-sensitive human colon tumor cells and derived

TRAIL-resistant metastatic cells. J Cancer Mol 5:21–30

Manhas M, Ganguly S, Mukherjee S, Jain A (2006) Microwave

initiated reactions: Pechmann coumarin synthesis, Biginelli

reaction, and acylation. Tetrahedron Lett 47:2423–2425

Martin E, Kellen-Yuen C (2007) Microwave-assisted organic synthesis

in the organic lab: a simple, greener wittig reaction. J Chem Ed

84:2004–2006

References

Adhikari MV, Samant SD (2002) Sonochemical bromination of

acetophenones

bromosuccinimide. Ultrason Sonochem 9:107–111

using

p-toluenesulfonic

acid

N-

Agarwal R (2000) Synthesis & biological screening of some novel

coumarin derivatives. Biochem Pharmacol 6:1042–1051

Ankleka KY, Lakkannavar CD, Kulkarni GM, Kulkarni MV (2003)

Synthesis, spectral studies and biological evaluation of some new

4-substituted coumarins. Ind J Chem Sect B 42:1548–1550

Arango V, Robledo SM, Seón-Méniel B, Figadere B, Cardona W,

Sáez J, Otálvaro F (2010) Coumarins from Galipea panamensis

and their activity against Leishmania panamensis. J Nat Prod

73:1012–1014

Arnold M, Sierra MS, Laversanne M, Soerjomataram I, Jemal A, Bray

F (2016) Global patterns and trends in colorectal cancer incidence

and mortality. Gut 0:1–9

Athar M, Back JH, Kopelovich L, Bickers DR, Kim AL (2009)

Multiple molecular targets of resveratrol: anticarcinogenic

mechanisms. Arch Biochem Biophys 486:95–102

Baur JA, Sinclair DA (2006) Therapeutic potential of resveratrol: the

in vivo evidence. Nat Rev Drug Discov 5:493–506

Bellina F, Guazzelli N, Lessi M, Manzini C (2015) Imidazole analo-

gues of resveratrol: synthesis and cancer cell growth evaluation.

Tetrahedron 71:2298–2305

Belluti F, Fontana G, Bo LD, Carenini N, Giommarelli C, Zunino F

(2010) Design, synthesis and anticancer activities of stilbene-

coumarin hybrid compounds: Identiﬁcation of novel proapoptotic

agents. Bioorg Med Chem 18:3543–3550

Borriello A, Bencivenga D, Caldarelli I, Tramontano A, Borgia A,

Zappia V, Della Ragione F (2014) Resveratrol:from basic studies

to bedside. Cancer Treat Res 59:167–184

Martins S, Branco P, Pereira A (2012) An efﬁcient methodology for

the synthesis of 3-styryl coumarins. J Braz Chem Soc 23:688–693

Mayur YC, Peters GJ, Prasad VV, Lemo C, Sathish NK (2009) Design

of new drug molecules to be used in reversing multidrug resis-

tence in cancer cells. Curr Cancer Drug Targets 9:298–306

Medicinal Chemistry Research

McQuade RM, Bornstein JC, Nurgali K (2014) Anti-colorectal cancer

chemotherapy-induced diarrhoea: current treatments and side-

effects. Int J Clin Med 5:393–406

Shaveta, Mishra S, Singh P (2016) Hybrid molecules: the privileged

scaffolds for various pharmaceuticals. Eur

J Med Chem

124:500–536

Meunier B (2008) Hybrid molecules with a dual mode of action:

dream or reality? Acc Chem Res 41:69–77

Neckers L (2002) Hsp90 inhibitors as novel cancer chemotherapeutic

agents. Trends Mol Med 8:S55–S61

Shen W, Mao J, Sun J, Sun M, Zhang C (2013) Synthesis and bio-

logical evaluation of resveratrol-coumarin hybrid compounds as

potential antitumor agents. Med Chem Res 22:1630–1640

Simpson CJ, Fitzhenry MJ, Stamford NP (2005) Preparation of

vinylphenols from 2- and 4-hydroxybenzaldehydes. Tetrahedron

Lett 46:6893–6896

Solomon VR, Hu C, Lee H (2009) Hybrid pharmacophore design and

synthesis of isatin-benzothiazole analogs for their anti-breast

cancer activity. Bioorg Med Chem 17:7585–7592

Symeonidis T, Chamilos M, Hadjipavlou-Litina DJ, Kallitsakis M,

Litinas KE (2009) Synthesis of hydroxycoumarins and hydro-

xybenzo[f]- or [h]coumarins as lipid peroxidation inhibitors.

Bioorg Med Chem Lett 19:1139–1142

Temraz S, Mukherji D, Shamseddine A (2013) Potential targets for

colorectal cancer prevention. Int J Mol Sci 14:17279–17303

Tsogoeva SB (2010) Recent progress in the development of synthetic

hybrids of natural or unnatural bioactive compounds for medic-

inal chemistry. Mini Rev Med Chem 10:773–793

Nepali K, Sharma S, Sharma M, Bedi PM, Dhar KL (2014) Rational

approaches, design strategies, structure activity relationship and

mechanistic insights for anticancer hybrids. Eur J Med Chem

22:422–487

Otero E, Vergara S, Robledo SM, Cardona W, Carda M, Vélez ID,

Rojas C, Otálvaro F (2014) Synthesis, leishmanicidal and cyto-

toxic activity of triclosan-chalcone, triclosan-chromone and

triclosan-coumarin hybrids. Molecules 19:13251–13266

Patrick GL (2013) An Introduction to Medicinal Chemistry, ﬁfth edn.,

Oxford University Press, pp. 1–14, England

Paul K, Bindal S, Luxami V (2013) Synthesis of new conjugated

coumarin-benzimidazole hybrids and their anticancer activity.

Bioorg Med Chem Lett 23:3667–3672

Pérez JM, Maldonado ME, Rojano BA, Alzate F, Sáez J, Cardona W

(2014) Comparative antioxidant, antiproliferative and apoptotic

effects of Ilex laurina and Ilex paraguariensis on colon cancer

cells. Trop J Pharm Res 13:1279–1286

Udenigwe CC, Ramprasath VR, Aluko RE, Jones PJ (2008) Potential

of resveratrol in anticancer and anti-inﬂammatory therapy. Nutr

Rev 66:445–454

Pierson J-T, Dumetre A, Hutter S, Delmas F, Laget M, Finet J-P, Azas

N, Combes S (2010) Synthesis and antiprotozoal activity of 4-

arylcoumarins. Eur J Med Chem 45:864–869

Pointet AL, Taieb J (2017) Cáncer de colon. EMC - Tratado De Med

21:1–7

Resch M, Steigel A, Chen ZL, Bauer R (1998) 5-Lipoxygenase and

cyclooxygenase-1 inhibitory active compounds from Atracty-

lodes lancea. J Nat Prod 61:347–350

Riveiro ME, Vazquez R, Moglioni A, Gomez N, Baldi A, Davio C,

Shayo C (2008) Biochemical mechanisms underlying the proa-

poptotic activity of 7,8-dihydroxy-4-methylcoumarin in human

leukemic cells. Biochem Pharmacol 75:725–736

Sabrina A, Horbach R, Deising HB, Siewert B, Csuk R (2011)

Synthesis and antimicrobial activity of (E) stilbene derivatives.

Bioorg Med Chem 19:5155–5166

Sashidhara KV, Kumar A, Kumar M, Sarkar J, Sinha S (2010)

Synthesis and in vitro evaluation of novel coumarin-chalcone

hybrids as potential anticancer agents. Bioorg Med Chem Lett

20:7205–7211

Vilar S, Quezada E, Santana L, Uriarte E, Yanez M, Fraiz N, Alcaide

C, Cano E, Orallo F (2006) Design, sysnthesis and vasorelaxant

and platelet antiaggregatory activities of coumarin-resveratrol

hybrids. Bioorg Med Chem Lett 16:257–261

Yang JY, Della Fera MA, Baile CA (2006) Esculetin induces

mitochondria-mediated apoptosis in 3T3-L1 adipocytes. Apop-

tosis 11:1371–1378

Yang J, Liu GY, Dai F, Cao XY, Kang YF, Hu LM, Tang JJ, Li XZ,

Li Y, Jin XL, Zhou B (2011) Synthesis and biological evaluation

of hydroxylated 3-phenylcoumarins as antioxidants and anti-

proliferative agents. Bioorg Med Chem Lett 21:6420–6425

Yee SW, Shah B, Simons C (2005) Synthesis and antimycobacterial

activity of 7-O-substituted-4-methyl-2H-2-chromenone derivatives

vs Mycobacterium tuberculosis. J Enz Inh Med Chem 20:109–113

Zhao H, Donnelly AC, Kusuma BR, Brandt GE, Brown D, Rajewski

RA, Vielhauer G, Holzbeierlein J, Cohen MS, Blagg BS (2011)

Engineering an antibiotic to ﬁght cancer: optimization of the

novobiocin scaffold to produce antiproliferative agents. J Med

Chem 9:3839–3853

Article Doi

Synthesis and antiproliferative activity of 3- and 7-styrylcoumarins

DOI: 10.1007/s00044-018-2202-0

Source and publish data:

Authors:

Article abstract of DOI:10.1007/s00044-018-2202-0

Full text of DOI:10.1007/s00044-018-2202-0

Products guided by the article

R&D Labs maybe for 108530-10-5

Relevant to this article

Hot Product