Novel 3-aryl-5-substituted-coumarin analogues: Synthesis and bioactivity profile

Article abstract of DOI:10.1002/ddr.21639

Eighteen 3-aryl-5-substituted-coumarins—six 5-acetyloxy-derivatives, six 5-hydroxy-derivatives, and six 5-geranyloxy-derivatives—were synthesized, structurally characterized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their

Full text of DOI:10.1002/ddr.21639

Received: 3 September 2019
DOI: 10.1002/ddr.21639
Revised: 2 December 2019
Accepted: 28 December 2019
R E S E A R C H A R T I C L E
Novel 3-aryl-5-substituted-coumarin analogues: Synthesis and
bioactivity profile
Eleni Kavetsou1 | Annita Katopodi1,2 | Letta Argyri2 | Eirini Chainoglou3
|
Eleni Pontiki3 | Dimitra Hadjipavlou-Litina3 | Angeliki Chroni2 | Anastasia Detsi1
1Laboratory of Organic Chemistry, School of
Abstract
Chemical Engineering, National Technical
University of Athens, Athens, Greece
Eighteen 3-aryl-5-substituted-coumarins—six 5-acetyloxy-derivatives, six 5-hydroxy-
2Institute of Biosciences and Applications,
derivatives, and six 5-geranyloxy-derivatives—were synthesized, structurally charac-
National Centre for Scientific Research
“Demokritos”, Athens, Greece
terized and their antioxidant activity, lipoxygenase inhibitory ability, as well as their
cytotoxic activity against human neuroblastoma SK-N-SH and HeLa adenocarcinoma
cell lines were evaluated. The 5-acetyloxy-compounds 3a-3f were found to be the
best cytotoxic agents among all the compounds studied. The bromo-substituted cou-
marins 3a and 3b were remarkably active against HeLa cell line showing IC50 1.8 and
6.1 μM, respectively. Coumarin 5e possessing a geranyloxy-chain on position 5 of the
coumarin scaffold presented dual bioactivity, while 5-geranyloxy-coumarin 5f was
the most competent soybean lipoxygenase inhibitor of this series (IC50 10 μM). As
shown by in silico docking studies, the studied molecules present allosteric interac-
tions with soybean lipoxygenases.
3Laboratory of Pharmaceutical Chemistry,
Faculty of Health Sciences, School of
Pharmacy, Aristotle University of Thessaloniki,
Thessaloniki, Greece
Correspondence
Anastasia Detsi, Laboratory of Organic
Chemistry, School of Chemical Engineering,
National Technical University of Athens,
Heroon Polytechniou 9, Zografou Campus,
Athens 15773, Greece.
Email: adetsi@chemeng.ntua.gr
Funding information
European Regional Development Fund;
European Social Fund; State Scholarships
Foundation
K E Y W O R D S
acetyloxy-moiety, coumarins, cytotoxicity, lipoxygenase, molecular modeling, oxyprenylated
analogues
1
|
INTRODUCTION
considered just as biosynthetic intermediates of C-prenylated products
(Alhassan, Abdullahi, Uba, & Umar, 2014). Therefore, their biological
Coumarins are naturally plant-derived or synthetically obtained sub-
stances that belong to the benzopyrone family (Detsi, Kontogiorgis, &
Hadjipavlou-Litina, 2017). The wide bioactivity array, such as antioxi-
dant (Matos et al., 2017), anti-inflammatory (Melagraki et al., 2009;
Roussaki et al., 2014), anticancer (Kavetsou et al., 2017; Ricci et al.,
2018), antivirus (Atta-ur-Rahman., 2018), and neuroprotective
(Okuyama et al., 2013), renders coumarins attractive for further evalu-
ation as novel therapeutic agents. Research has shown that substitu-
tion on the coumarin scaffold strongly influences its pharmacological
profile. Thus, the structural modification of the coumarin core in the
pursuit of biologically active multi-targeting compounds is a con-
stantly intriguing research item (Garazd, Garazd, & Lesyk, 2017).
Oxyprenylated compounds (isopentenyloxy, geranyloxy, farnesyloxy
compounds, and their biosynthetic derivatives) have attracted the sci-
entific interest only in the last decade, while for years, they were
activities have recently started to be explored. Fiorito and coworkers
(Fiorito, Epifano, Taddeo, & Genovese, 2018) presented in 2018 the
key role of coumarins bearing a prenyloxy moiety, in the modulation
of melanogenesis using murine Melan-a cell line (Fiorito et al., 2018).
They have also reported the ability of plant isolated prenyloxy cou-
marins to inhibit RAW 264.7 proliferation (Genovese et al., 2017).
Moreover, it has lately been demonstrated that oxyprenylated cou-
marins were strong inhibitors of different types of lipoxygenases
(e.g., 5-LOX and 15-LOX) (Fiorito, Epifano, Preziuso, et al., 2018;
Kavetsou et al., 2017).
Lipoxygenases (LOX) are iron-containing enzymes that catalyze
the oxidation of polyunsaturated fatty acids such as linoleic acid
(in plants) and arachidonic acid (in mammals) at specific positions to
hydroperoxides (Detsi, Majdalani, Kontogiorgis, Hadjipavlou-Litina, &
ꢀ
Kefalas, 2009; Simijonovic et al., 2018). Inhibitors of LOX had initially
Drug Dev Res. 2020;1–14.
wileyonlinelibrary.com/journal/ddr
© 2020 Wiley Periodicals, Inc.
1

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KAVETSOU ET AL.
attracted attention as potential agents for the treatment of inflamma-
tory and allergic diseases. However, the association between chronic
and persistent inflammation and the development of malignant dis-
eases through certain pathways has now expanded their therapeutic
potential (Crusz & Balkwill, 2015; Genovese et al., 2017; Schneider &
Pozzi, 2011).
2
|
MATERIALS AND METHODS
| Chemicals and instruments
2.1
The chemicals used for synthesis, analysis, and evaluation were pur-
chased from Sigma-Aldrich and used without further purification.
NMR spectra were recorded on Varian 300 & 600 MHz spectrometer
and the HR-MS spectra on a UHPLC-MSn Orbitrap Velos-Thermo
mass spectrometer at the National Hellenic Research Foundation.
Melting points were obtained using a Gallenkamp MFB-595 melting
point apparatus and are uncorrected. Lipophilicity values were calcu-
lated in silico.
Oxidative stress has been associated with the inflammation pro-
cess. Reactive oxygen species are produced during the inflammation
process by phagocytic leukocytes that invade the tissue. Moreover,
these reactive species are involved in the cyclooxygenase (COX)- and
lipoxygenase (LOX)-mediated conversion of arachidonic acid into
proinflammatory intermediates. The implications of COXs and LOXs
have been discussed in numerous types of cancers, including colon,
pancreas, breast, lung, skin, urinary bladder, liver, and so on (Orafaie,
Matin, & Sadeghian, 2018).
2.2
|
Inhibition of linoleic acid lipid peroxidation
Although much research has been conducted and significant pro-
gress has been reported, cancer is still the second major cause of
death globally. It is noteworthy that current chemotherapeutic agents
present low effectiveness due to the development of multidrug resis-
tant cancers, while they display severe side effects (Bian et al., 2018;
The assay was performed according to the experimental procedure
from our previous publications (Melagraki et al., 2009; Roussaki et al.,
2014; Roussaki, Kontogiorgis, Hadjipavlou-Litina, Hamilakis, & Detsi,
2010). In an aqueous solution spectrophotometrically at 234 nm was
monitored and recorded the production of conjugated diene hydro
peroxide by oxidation of sodium linoleate. Ten microliters of the
16 mM sodium linoleate solution was added to the UV cuvette con-
taining 930 μL of 0.05 M phosphate buffer, pH 7.4 prethermostated
at 37ꢀC, under air by the addition of 50 μL of 40 mM AAPH solution
which was used as a free radical initiator. Ten microliters of the appro-
priate solutions of the tested compounds were added in the mixture,
whereas the same level of Dimethylsulfoxide (DMSO) was used as a
blanc for the measurement of lipid oxidation. The rate of oxidation at
37ꢀC was monitored by recording the increase in absorption at
234 nm caused by conjugated diene hydro peroxides. The results
were compared with the appropriate standard inhibitor Trolox (93%).
Mansoori, Mohammadi, Davudian, Shirjang,
& Baradaran, 2017;
Senapati, Mahanta, Kumar, & Maiti, 2018). Therefore, target identifi-
cation and searching for new anticancer drugs endowed with cyto-
toxic activity is of great importance, while plant-derived heterocyclic
compounds and their derivatives have always consisted an invaluable
source for the discovery of new therapeutic agents (Bathula et al.,
ꢁ
2015; Demir, Özen, Ünlüsoy, Öztürk, & Bozdag-Dündar, 2019; Hati
et al., 2016). Interestingly, heterocyclic scaffolds such as coumarins
have a tremendous ability to regulate diverse range of cellular path-
ways, thus they can be regarded as key structures for multi-targeting
purposes (Thakur, Singla, & Jaitak, 2015). Specifically, coumarins'
anti-tumor responses are related to various mechanisms such as apo-
ptosis induction, inhibition of cell growth, and of DNA-associated
enzymes, as topoisomerase (Majumdar et al., 2015). Characteristic
examples of natural coumarins possessing anticancer effects are
2.3
|
Soybean LOX inhibition study in vitro
auraptene (Hasan, Genovese, Fiorito, Epifano,
& Witt-Enderby,
2017; Lee et al., 2017), umbelliprenin (Kavetsou et al., 2017), and
umbelliferone (Mazimba, 2017).
The assay was performed according to the experimental procedure
from our previous publications (Melagraki et al., 2009; Roussaki et al.,
2010; Roussaki et al., 2014). The tested compounds as stock solutions
were dissolved in DMSO. Ten microliters were incubated at room
temperature with sodium linoleate (100 mM) and 0.2 ml of enzyme
solution (1/9 × 10−4 w/v in saline) in buffer pH 9 (tris) at room tem-
perature (final volume 1 ml). The conversion of sodium linoleate to
13-hydroperoxylinoleic acid at 234 nm was recorded. The results
were compared with the appropriate standard inhibitor Nor-
dihydroguaeretic acid NDGA (IC50 0.45 μM). Several concentrations
were used for the determination of IC50 values. The results are given
expressed as IC50 values.
Herein, in continuation to our previous studies (Kavetsou
et al., 2017), we present the synthesis, structural characterization,
and in vitro biological evaluation of 18 multi-substituted coumarin
analogues. The aim of the current research was to design and syn-
thesize novel coumarin derivatives with multi-targeting effects
against both inflammation and cancer cell viability based on our
previous work. Antioxidant activity, soybean LOX inhibitory activ-
ity, as well as cytotoxicity of the novel derivatives against human
neuroblastoma cell line SK-N-SH and cervical cancer cell line HeLa
were determined.
In addition, in silico docking studies were performed in soybean
LOX and provided useful interpretation of the experimental results
whereas the drug-likeness of the derivatives was determined from the
theoretical calculation of various molecular properties.
Experiments were repeated four times (experimental replicates).
The results were expressed as mean SD. Statistical comparisons
were made using the Kruskal Wallis test. Statistically significant differ-
ence was defined as p < .05.

KAVETSOU ET AL.
3
2.4
|
Cell culture
131.41, 130.16, 131.21, 128.45, 127.91, 124.35, 119.98, 115.45,
114.15, 21.52, 19.88.
Human neuroblastoma SK-N-SH cells (ATCC) were cultured in Mini-
mal Essential Medium (MEM) Earle's supplemented with 2 mM L-glu-
tamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,
1.5 g/L sodium bicarbonate, 10% Fetal Bovine Serum (FBS), and anti-
biotics. Human cervical adenocarcinoma HeLa cells (ATCC) were cul-
tured in Dulbecco's modified Eagle's medium (4500 mg/L glucose),
10% FBS, 1% nonessential amino acids, and antibiotics. The cell lines
were maintained in standard culture conditions (37ꢀC, and 5% CO2).
HRMS calcd for C18H14O4Br (M + H)+: m/z: 373.0070, found:
373.0069.
5-acetyloxy-3-(4-nitro-phenyl)-4-methyl-chromen-2-one (3c): brown
powder, yield: 42%, m.p.: 193–195ꢀC, 1H NMR (600 MHz, CDCl3): δ
(ppm) 8.32 (d, J = 8.4 Hz, 2H, H-30 & H-50), 7.57 (t, J = 8.4 Hz, 1H,
H-7), 7.48 (d, J = 9 Hz, 2H, H-20 & H-60), 7.32 (dd, J = 8.4 Hz,
J = 0.6 Hz, 1H, H-8), 7.02 (dd, J = 8.4 Hz, J = 0.6 Hz, 1H, H-6), 2.37
(s, 3H, 4-CH3), 2.36 (s, 3H, 5-CH3CO), 13C NMR (150 MHz, CDCl3):
δ (ppm) 169.19, 159.41, 153.92, 148.28, 147.89, 147.60, 141.36,
131.72, 131.48, 127.07, 123.91, 120.30, 115.50, 114.19, 21.55, 20.46.
HRMS calcd for C18H14O6N (M + H)+: m/z: 340.0816, found:
340.0814.
2.5
|
Cell viability assay
Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, MO)
assay (Berridge & Tan, 1993). SK-N-SH and HeLa cells were seeded in
96-well plates in complete culture medium at a density of 2.0 × 104
and 0.7 × 104, respectively. The assay was performed according to
the experimental procedure from our previous publication (Kavetsou
et al., 2017). Two independent experiments were done in triplicate for
each compound and the IC50 values were calculated using the Gra-
phPad Prism software.
5-acetyloxy-4-methyl-3-phenyl-chromen-2-one (3d): white powder,
yield: 46%, m.p.: 147–149 C (lit. m.p. 150–152ꢀC (Seshadri, 1952)),
1H NMR (600 MHz, CDCl3): δ (ppm) 7.50 (t, J = 8.4 Hz, 1H, H-7), 7.44
(t, J = 7.2 Hz, 2H, H-30 & H-50), 7.39 (t, J = 7.2 Hz, 1H, H-40), 7.29
(d, J = 8.4 Hz, 1H, H-8), 7.25 (d, J = 6.6 Hz, 2H, H-20 & 60), 6.97
(d, J = 8.4 Hz, 1H, H-6), 2.34 (s, 6H, 4-CH3 & 5-CH3CO).
ꢀ
5-acetyloxy-3-(2-methoxy-phenyl)-4-methyl-chromen-2-one (3e):
white powder, yield: 50%, m.p.: 150–152ꢀC, 1H NMR (600 MHz,
CDCl3): δ (ppm) 7.49 (t, J = 8.4 Hz, 1H, H-7), 7.39 (pseudotriplet,
H-40), 7.29 (d, J = 8.4 Hz, 1H, H-8), 7.14 (d, J = 7.2 Hz, 1H, H-6), 7.04
(t, J = 7.2 Hz, 1H, H-30), 6.98 (m, 2H, H-20 & H-50), 3.78 (s, 3H, 2'-
OCH3), 2.34 (s, 3H, 4-CH3), 2.29 (s, 3H, 5-CH3CO), 13C NMR
(75 MHz, CDCl3): δ (ppm) 169.34, 159.73, 157.23, 154.01, 147.90,
147.12, 131.22, 130.60, 130.17, 126.08, 123.39, 120.85, 119.72,
115.34, 114.63, 111.33, 55.73, 21.55, 19.99.
2.6
|
General procedure for the synthesis of
acetyloxy-coumarins 3a-3f
A mixture of the appropriate phenylacetic acid (3.72 mmol) and 20,60-
dihydroxyacetophenone (3.90 mmol) in the presence of triethylamine
(11.5 mmol) in acetic anhydride (4.1 ml) was refluxed for 3 hr. After
the completion of the reaction, water was added and the mixture was
extracted with dichloromethane. The organic phase was separated,
dried over anhydrous Na2SO4, filtered, and evaporated to give the
crude product. The products were purified by recrystallization from
methanol and dichloromethane.
HRMS calcd for C19H17O5 (M + H)+: m/z: 325.1071, found:
325.1072.
5-acetyloxy-3-(4-methoxy-phenyl)-4-methyl-chromen-2-one
(3f):
yellow powder, yield: 52%, m.p.: 153–155ꢀC, 1H NMR (600 MHz,
CDCl3): δ (ppm) 7.50 (t, J = 8.1 Hz, 1H, H-7), 7.29 (d, J = 7.8 Hz, 1H,
H-8), 7.21 (d, J = 8.7 Hz, 2H, H-2 & H-60), 6.99 (d, J = 8.4 Hz, 2H,
H-30 & H-50), 6.98 (d, J = 8.4 Hz, 1H, H-6), 3.88 (s, 3H, 4'-OCH3), 2.40
(s, 3H, 4-CH3), 2.39 (s, 3H, 5-CH3CO), 13C NMR (75 MHz, CDCl3):
δ (ppm) 169.35, 160.45, 159.67, 153.75, 147.95, 146.13, 131.41,
130.71, 128.82, 126.51, 119.86, 115.33, 114.82, 114.16, 55.44,
21.55, 20.43.
5-acetyloxy-3-(4-bromo-phenyl)-4-methyl-chromen-2-one
(3a):
white powder, yield: 50%, m.p.: 173–175 ꢀC, 1H NMR (600 MHz,
CDCl3): δ (ppm) 7.59 (d, J = 7.8 Hz, 2H, H-30, H-50), 7.53 (t, J = 8.4 Hz,
1H, H-7), 7.30 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H, H-8), 7.15 (d, J = 7.8 Hz,
2H, H-20 & H-60), 6.99 (dd, J = 8.4 Hz, J = 1.2 Hz, 1H, H-6), 2.36
(s, 3H, 4-CH3), 2.35(s, 3H, 5-CH3CO), 13C NMR (150 MHz, CDCl3)
δ (ppm) 169.28, 159.87, 153.84, 148.10, 146.80, 133.35, 131.96,
131.86, 131.17, 128.02, 122.84, 120.06, 115.42, 114.51, 21.55, 20.40.
HRMS calcd for C18H14O4Br (M + H)+: m/z: 373.0070, found:
373.0069.
HRMS calcd for C19H17O5 (M + H)+: m/z: 325.1071, found:
325.1073.
2.7
|
General procedure for the synthesis of
hydroxy-coumarins 4a-4f
5-acetyloxy-3-(2-bromo-phenyl)-4-methyl-chromen-2-one (3b): white
powder, yield: 43%, m.p.: 118–120ꢀC, 1H NMR (600 MHz, CDCl3):
δ (ppm) 7.69 (d, J = 7.8 Hz, 1H, H-30), 7.54 (t, J = 7.8 Hz, 1H, H-7), 7.41
(t, J = 7.8 Hz, 1H, H-50), 7.32 (d, J = 8.4 Hz, 1H, H-60), 7.28 (t, J = 7.8 Hz,
1H, H-40), 7.23 (d, J = 7.9 Hz, 1H, H-8), 7.01 (d, J = 8.4 Hz, 1H, H-6),
2.35 (s, 3H, 4-CH3), 2.28 (s, 1H, 5-CH3CO), 13C NMR (150 MHz, CDCl3):
δ (ppm) 169.22, 159.05, 154.10, 148.16, 147.84, 135.62, 133.09,
A mixture of the appropriate 5-acetyloxy-coumarin (1.50 mmol) and
hydrazine monohydrate (7.48 mmol) in methanol (24.3 ml) was stirred
at 42ꢀC for 1–2 hr. After the completion of the reaction, water was
added and the mixture was then extracted with ethyl acetate. The
organic phase was separated, dried over anhydrous Na2SO4, filtered,
and evaporated to give the crude product. The products were purified

4
KAVETSOU ET AL.
by recrystallization from dichloromethane and methanol. The yields
are reported by calculating the amount of the recrystallized product.
5-hydroxy-3-(4-bromo-phenyl)-4-methyl-chromen-2-one (4a): yellow
powder, yield: 40%, m.p.: 228ꢀC (decomp.), 1H NMR (600 MHz,
DMSO-d6): δ (ppm) 10.72 (br, 1H, 5-OH), 7.64 (d, J = 8.4 Hz, 2H,
H-30 & H-50), 7.39 (t, J = 8.4 Hz, 1H, H-7), 7.25 (d, J = 7.8 Hz, 2H, H-
20 & H-60), 6.82 (t, J = 8.4 Hz, 2H, H-6 & H-8), 2.40 (s, 3H, 4-CH3), 13C
NMR (150 MHz, DMSO-d6): δ (ppm) 159.51, 157.12, 153.77, 149.99,
134.50, 132.56, 131.88, 131.19, 123.67, 121.05, 111.63, 109.11,
107.09, 21.31, 21.29.
H-20 & H-60), 6.96 (d, J = 8.7 Hz, 2H, H-30 & H-50), 6.78, (dd,
J = 8.1 Hz, J = 1.5 Hz, 2H, H-6 & H-8), 3.79 (s, 3H, 4'-OCH3), 2.41 (s,
3H, 4-CH3), 13C NMR (75 MHz, DMSO-d6): δ (ppm) 161.64, 158.65,
156.90, 153.66, 149.35, 134.47, 131.51, 127.11, 124.51, 113.30,
111.51, 109.29, 107.02, 55.10, 21.33.
HRMS calcd for C17H15O4 (M + H)+: m/z: 283.0965, found:
283.0961.
2.8
|
General procedure for the synthesis of
5-hydroxy-3-(2-bromo-phenyl)-4-methyl-chromen-2-one (4b): yellow
powder, yield: 41%, m.p.: 264–265ꢀC, 1H NMR (600 MHz, CD3OD): δ
(ppm) 7.71 (d, J = 7.8 Hz, 1H, H-30), 7.45 (t, J = 7.2 Hz, 1H, H-7), 7.39
(t, J = 7.8 Hz, 1H, H-50), 7.32 (t, J = 7.8 Hz, 1H, H-40), 7.29 (d,
J = 7.8 Hz, 1H, H-60), 6.85 (d, J = 7.8 Hz, 1H, H-8), 6.78 (d, J = 8.4 Hz,
1H, H-6), 2.41 (s, 3H, 4-CH3), 13C NMR (150 MHz, DMSO-d6):
δ (ppm) 158.65, 157.23, 153.96, 150.71, 136.18, 132.38, 132.13,
131.98, 130.0, 127.99, 124.35, 124.08, 111.68, 108.66,
107.15, 20.74.
prenyloxy-coumarins 5a-5f
A mixture of the appropriate 5-hydroxy-coumarin (0.52 mmol) and
geranyl-bromide (0.62 mmol) in acetone (19.9 ml) in the presence of
potassium carbonate (K2CO3 0.52 mmol) was refluxed overnight.
After the completion of the reaction, potassium carbonate was fil-
tered off, washed with acetone and the filtrate was evaporated under
reduced pressure. The residue was then purified by flash column chro-
matography (petroleum ether/ethyl acetate 9:1).
HRMS calcd for C16H12O3Br (M + H)+: m/z: 390.9964, found:
5-geranyloxy-3-(4-bromo-phenyl)-4-methyl-chromen-2-one (5a): yel-
low powder, yield: 89%, m.p.: 91–94ꢀC, 1H NMR (600 MHz, CDCl3): δ
(ppm) 7.57 (d, J = 8.4 Hz, 2H, H-30 & H-50), 7.42 (t, J = 8.4 Hz, 1H,
H-7), 7.16 (d, J = 8.4 Hz, 2H, H-20 & H-60), 6.97 (d, J = 8.4 Hz, 1H,
H-8), 6.77 (d, J = 8.4 Hz, 1H, H-6), 5.49 (t, J = 6.6 Hz, 1H, H-200), 5.06
(t, J = 6.6 Hz, 1H, H-600), 4.63 (d, J = 6.6 Hz, 2H, H-100), 2.46 (s, 3H,
4-CH3), 2.10 (m, 4H, H-400 & H-500), 1.74 (s, 3H, 300-CH3), 1.65 (s,
3H, 800-CH3), 1.59 (s, 3H, 700-CH3), 13C NMR (150MHz, CDCl3): δ
(ppm) 160.68, 157.91, 154.39, 150.49, 142.14, 134.24, 132.27,
132.02, 131.87, 131.58, 131.42, 125.41, 123.89, 123.42, 122.27,
118.80, 111.35, 109.76, 107.72, 107.50, 66.12, 39.56, 26.35,
22.38, 22.03.
330.9961.
5-hydroxy-4-methyl-3-(4-nitro-phenyl)-chromen-2-one (4c): brown
powder, yield: 56%, m.p.: 282ꢀC (decomp.), 1H NMR (600 MHz,
DMSO-d6): δ (ppm) δ (ppm) 10.76 (br, 1H, 5-OH), 8.30 (d, J = 8.4 Hz,
2H, H-30 & H-50), 7.61 (d, J = 8.4 Hz, 2H, H-20 & H-60), 7.42 (t, J = 7.8 Hz,
1H, H-7), 6.84 (t, J = 9 Hz, 2H, H-6&H-8), 2.41 (s, 3H, 4-CH3), 13C
NMR (75 MHz, DMSO-d6): δ (ppm) 159.74, 157.05, 153.79, 149.62,
135.24, 131.71, 130.24, 128.23, 127.56, 124.85, 111.57, 109.20,
10,710, 21.32.
HRMS calcd for C16H10O5N (M-H)−: m/z: 296.0564, found:
296.0555.
5-hydroxy-4-methyl-3-phenyl-chromen-2-one (4d): white powder,
yield: 50%, m.p.: 277–278ꢀC (lit. m.p. 276–278ꢀC (Seshadri, 1952)),
1H NMR (600 MHz, DMSO-d6): δ (ppm) 10.65 (br, 1H, 5-OH), 7.44
(t, J = 7.8 Hz, 2H, H-30 & H-50), 7.38 (t, J = 7.8 Hz, 2H, H-7 & H-40),
7.27 (d, J = 7.2 Hz, 2H, H-20 & H-60), 6.82 (t, J = 7.8 Hz, 2H, H-6 &
H-8), 2.40 (s, 3H, 4-CH3), 13C NMR (75 MHz, DMSO-d6): δ (ppm)
159.21, 157.29, 153.86, 150.59, 146.90, 142.52, 132.22, 131.97,
123.29, 123.03, 111.71, 108.94, 107.13, 21.37.
HRMS calcd for C26H28O3Br (M + H)+: m/z: 467.1216, found:
467.1211.
5-geranyloxy-3-(2-bromo-phenyl)-4-methyl-chromen-2-one (5b): yel-
low oily product, yield: 85%, 1H NMR (600 MHz, CDCl3): δ (ppm) 7.69
(d, J = 7.8 Hz, 1H, H-30), 7.43 (t, J = 8.4 Hz, 1H, H-7), 7.39 (t, J = 7.8 Hz,
1H, H-50), 7.25 (m, 2H, H-40 & H-60), 6.99 (d, J = 8.4 Hz, 1H, H-8),
6.78 (d, J = 8.4 Hz, 1H, H-6), 5.49 (t, J = 6.0 Hz, 1H, H-200), 5.06 (t,
J = 6.6 Hz, 1H, H-600), 4.63 (d, J = 6.6 Hz, 2H, H-100), 2.38 (s, 3H,
4-CH3), 2.08 (m, 4H, H-400 & H-500), 1.74 (s, 3H, 300-CH3), 1.64 (s, 3H,
800-CH3), 1.58 (s, 3H, 700-CH3), 13C NMR (75MHz, CDCl3): δ (ppm)
159.94, 158.05, 154.70, 151.50, 142.12, 136.47, 133.01, 132.07,
131.73, 131.68, 129.79, 127.82, 125.94, 124.78, 123.66, 118.76,
111.03, 109.84, 107.57, 66.11, 39.55, 26.34, 25.80, 21.60,
17.84, 16.80.
5-hydroxy-3-(2-methoxy-phenyl)-4-methyl-chromen-2-one (4e): yel-
low powder, yield: 45%, m.p.: 252–255ꢀC, 1H NMR (600 MHz,
DMSO-d6): δ (ppm) 10.65 (br, 1H, 5-OH), 7.38 (m, 2H, H-20 & H-50),
7.14 (dd, J = 7.2 Hz, J = 1.2 Hz, 1H, H-6), 7.09 (d, J = 8.4 Hz, 1H, H-8),
7.01, (t, J = 7.2 Hz, 1H, H-7), 6.8 (t, J = 7.2 Hz, 2H, H-30 & H-40), 3.72
(s, 3H, 2'-OCH3), 2.32 (s, 3H, 4-CH3), 13C NMR (150 MHz, DMSO-d6):
δ (ppm) 159.14, 156.92, 156.86, 153.81, 150.07, 131.58, 131.23,
129.49, 123.80, 121.74, 120.32, 111.47, 111.28, 109.02, 107.03,
55.42, 20.86.
HRMS calcd for C26H28O3Br (M + H)+: m/z: 467.1216, found:
467.1216.
5-geranyloxy-4-methyl-3-(4-nitro-phenyl)-chromen-2-one (5c): yel-
low powder, yield: 64%, m.p.: 104–107ꢀC, 1H NMR (600 MHz,
CDCl3): δ (ppm) 8.30 (d, J = 8.4 Hz, 2H, H-30 & H-50), 7.46 (m, 3H, H-
20 & H-60 & H-7), 6.99 (d, J = 7.8 Hz, 1H, H-8), 6.80 (d, J = 8.4 Hz, 1H,
H-6), 5.49 (t, J = 6.6 Hz, 1H, H-200), 5.06 (br, 1H, H-600), 4.65 (d,
J = 6.6 Hz, 2H, H-100), 2.47 (s, 3H, 4-CH3), 2.10 (m, 4H, H-400 & H-500),
HRMS calcd for
281.0808.
C
17H13O4 (M-H)− m/z: 281.0819, found:
5-hydroxy-3-(4-methoxy-phenyl)-4-methyl-chromen-2-one (4f): yel-
low powder, yield: 62%, m.p.: 220–222ꢀC, 1H NMR (300 MHz,
DMSO-d6): δ (ppm) 7.33 (t, J = 8.9 Hz, 1H, H-7), 7.16 (d, J = 8.7, 2H,

KAVETSOU ET AL.
5
1.75 (s, 3H, 300-CH3), 1.65 (s, 3H, 800-CH3), 1.59 (s, 3H, 700-CH3), 13C
NMR (150 MHz, CDCl3) δ (ppm) 160.19, 158.09, 154.52, 151.24,
147.59, 142.42, 142.38, 132.22, 132.08, 131.69, 127.73, 124.45,
123.77, 123.65, 123.60, 118.60, 111.04, 109.78, 107.80, 66.22,
39.55, 26.33, 25.79, 22.27, 17.83, 16.80.
(e.g., auraptene) (Kavetsou et al., 2017; Roussaki et al., 2010; Roussaki
et al., 2014). The promising results of these works in combination with
the literature data (Dos Santos Nascimento et al., 2015; Ibrar,
Shehzadi, Saeed,
& Khan, 2018; Jabbari, Mousavian, Seyedi,
Bakavoli, & Sadeghian, 2017), which reveal the role of the phenyl
group at the prenylated coumarin's skeleton (Wiart, 2013), as well as
the role of different substitutions on the phenyl group of an acetyloxy
coumarin (Musa, Latinwo, Joseph, & Badisa, 2017), led us to design
new series of coumarin analogues which combine the 3-aryl substitu-
ent and acetyloxy-/hydroxy- or geranyloxy moiety in order to investi-
gate the effect of the presence of these groups on the biological
activity (Scheme 1).
HRMS calcd for C26H28O5N (M + H)+: m/z: 434.1962, found:
434.1962.
5-geranyloxy-4-methyl-3-phenyl-chromen-2-one (5d): yellow gummy
solid, yield: 95%, 1H NMR (600 MHz, CDCl3): δ (ppm) 7.40 (m, 4H, H-
7 & H-30& H-40 & H-50), 7.28 (br, 2H, H-20 & H-60), 6.98 (d, J = 8.4 Hz,
1H, H-8), 6.77 (d, J = 8.4 Hz, 1H, H-6), 5.49 (t, J = 6 Hz, 1H, H-200), 5.06
(br, 1H, H-600), 4.63 (d, J = 6.6 Hz, 2H, H-100), 2.46 (s, 3H, 4-CH3), 2.10
(m, 4H, H-400 & H-500), 1.74 (s, 3H, 300-CH3), 1.65 (s, 3H, 800-CH3), 1.59 (s,
3H, 700-CH3), 13C NMR (150 MHz, CDCl3): δ (ppm) 160.97, 157.89,
154.44, 150.10, 142.04, 135.36, 132.07, 131.34, 130.30, 128.57,
128.02, 127.75, 126.62, 123.70, 123.66, 118.87, 111.57, 109.77,
107.55, 66.10, 39.57, 26.36, 25.79, 22.17, 17.85, 16.79.
In order to efficiently synthesize the target compounds, a series of
three reactions was performed (Scheme 2): first, the 5-acetyloxy-
coumarins (3a-3f) were synthesized via a Perkin-Oglialoro condensation
reaction between an appropriately substituted phenylacetic acid (1a-1f)
and 20,60-dihydroxyacetophenone (2) in acetic anhydride in the presence
of a catalytic amount of triethylamine. Subsequently, the acetyl group
was removed using hydrazine monohydrate in methanol leading to
5-hydroxy-compounds (4a-4f). The final 5-geranyloxy-coumarins (5a-5f)
were obtained by alkylation of the 5-hydroxy-coumarins with geranyl
bromide and potassium carbonate (K2CO3) in acetone. All the products
were purified using flash column chromatography or recrystallization
and were structurally identified using 1H and 13C NMR spectroscopy
and HR-MS spectrometry. Lipophilicity values were calculated in silico.
HRMS calcd for C26H29O3 (M + H)+: m/z: 389.2111, found:
389.2117.
5-geranyloxy-3-(2-methoxy-phenyl)-4-methyl-chromen-2-one (5e):
white powder, yield: 96%, m.p.: 85–87ꢀC, 1H NMR (600 MHz, CDCl3):
δ (ppm) 7.37 (m, 2H, H-7 & H4'), 7.15 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H,
H-50), 7.03 (t, J = 7.8 Hz, 1H, H-30), 6.98 (m, 2H, H-6 & H-8), 6.75 (d,
J = 7.8 Hz, 1H, H-20), 5.49 (t, J = 6.6 Hz, 1H, H-200), 5.06 (t, J = 6.6 Hz,
1H, H-600), 4.62 (d, J = 6.6 Hz, 2H, H-100), 3.78 (s, 3H, 2'-OCH3), 2.09
(m, 4H, H-400 & H-500), 1.74 (s, 3H, 300-CH3), 1.65 (s, 3H, 800-CH3), 1.59
(s, 3H, 700-CH3), 13C NMR (75MHz, CDCl3): δ (ppm) 160.57, 157.79,
157.39, 154.58, 150.79, 141.83, 132.03, 131.49, 131.11, 129.74,
124.28, 123.70, 123.38, 120.82, 118.97, 11.55, 111.32, 109.77,
107.38, 66.04, 55.76, 39.55, 26.35, 25.80, 21.68, 17.85, 16.78.
HRMS calcd for C27H31O4 (M + H)+: m/z: 419.2217, found:
419.2216.
3.2
|
Antioxidant activity evaluation
The in vitro antioxidant activity of the synthesized molecules was
evaluated as their ability to inhibit lipid peroxidation (LP) (AAPH assay)
and the results are presented in Table 1.
5-geranyloxy-3-(4-methoxy-phenyl)-4-methyl-chromen-2-one (5f):
yellow powder, yield.: 56%, m.p.: 77ꢀC, 1H NMR (600 MHz, CDCl3): δ
(ppm) 7.39 (t, J = 6.6 Hz, 1H, H-7), 7.20 (d, J = 8.4 Hz, 2H, H-20 & H-60),
6.97 (d, J = 8.4 Hz, 2H, H-30 & H-50), 6.96 (d, J = 7.2 Hz, 1H, H-8), 6.76
(d, J = 8.4 Hz, 1H, H-6), 5.50 (t, J = 6.0 Hz, 1H, H-200), 5.06 (d, J = 6.0 Hz,
2H, H-600), 4.62 (d, J = 6.6 Hz, 2H, H-100), 3.84 (s, 3H, 4'-OCH3), 2.48 (s,
3H, 4-CH3), 2.10 (m, 4H, H-400 & H-500), 1.74 (s, 3H, 300-CH3), 1.65 (s, 3H,
700-CH3), 1.59 (s, 3H, 800-CH3), 13C NMR (75MHz, CDCl3): δ (ppm)
161.35, 159.32, 157.78, 154.26, 150.04, 141.97, 132.05, 131.55,
131.22, 127,40, 126.18, 118.92, 118.87, 114.04, 111.66, 109.70,
107.51, 66.07, 55.40, 39.55, 26.34, 25.80, 22.23, 17.84, 16.79.
HRMS calcd for C27H31O4: m/z: 419.2217, found: 419.2214.
3
|
RESULTS AND DISCUSSION
| Chemistry
3.1
Our latest research focuses on the design, synthesis, and bioactivity
evaluation of a series of 3-aryl coumarin analogues as well as a series
of prenyloxy derivatives of the natural compound umbelliferone
SCHE ME 1 Key structural features for the synthesis of the new
3-aryl-5-substituted coumarin analogues (Kavetsou et al., 2017;
Roussaki et al., 2014)

6
KAVETSOU ET AL.
SCHEME 2 Reagents and
conditions: (i) Et3N, AC2O, reflux,
3 hr, (ii) NH2NH2.H2O, MeOH,
42ꢀC, 1.5 hr, and (iii) K2CO3,
acetone, reflux, 24 hr
Among the 5-acetyloxy-coumarins (3a-3f), only compounds 3a
with the acetyloxy- and hydroxy- derivatives, but also compared
with the 5-unsubstituted analogue (4-methyl-3-(4-nitro-phenyl)-
coumarin) which was found to be inactive (Roussaki et al., 2010).
Lipophiicity, determined theoretically as c log p values does not
seem to influence the antioxidant activity. Compounds presenting
the highest activities do not show the highest c log p values (e.g., 3b,
4d, and 5c) (Biobyte Corp. C-QSAR Database, n.d.; Pontiki &
Hadjipavlou-Litina, 2008).
(87.7%) and 3b (100%) that contain a bromo-substituent on the
3-phenyl ring were found to be active. Removal of the acetyl group
(compounds 4a and 4b) as well as alkylation of the OH group (com-
pounds 5a and 5b) led to analogues with decreased activity. In addi-
tion, compound 3a, bearing an acetyloxy-group at position 5 of the
coumarin core, possesses enhanced antioxidant activity compared
with the 3-(4-bromo-phenyl)-4-methyl-coumarin analogue studied in
our previous work (36% LP inhibition) (Roussaki et al., 2010).
Moreover, as long as 5-hydroxy-coumarins (4a-4f) are concerned,
the most potent LP inhibitor was compound 4d (100%) indicating that
the absence of substitution on the phenyl group favors the bioactivity
in these series of compounds. Meanwhile, the presence of a hydroxyl
group at position 5 of the coumarin scaffold turned an inactive com-
pound (3d) into a strong LP inhibitor (4d).
3.3
evaluation
|
Soybean lipoxygenase inhibitory activity
It is well-known that lipoxygenases' activity, such as 15-LOX, is
closely related with inflammation responses, as previously described.
In addition, its metabolism products, such as 5-HETE and LTD4, pro-
mote cancer cell proliferation and growth, although the relationship
between lipoxygenases and cancer may depend on the tumor's micro-
environment. On this basis, scientific research focuses on the devel-
opment of 15-LOX inhibitors for the chemoprevention of different
Among the 5-geranyloxy-analogues, compound 5c, bearing a
nitro-substituent, exhibited significant LP inhibition (93.1%), higher
than the 5-hydroxy-analogue, 4c (24%), whereas the 5-acetyloxy-
analogue, 3c, showed no activity. In this case, the presence of a
geranyloxy moiety enhanced bioactivity not only in comparison

KAVETSOU ET AL.
7
TABLE 1 Inhibitory activity of coumarins 3a-5f against: lipid peroxidation induced by AAPH (%), soybean lipoxygenase activity (IC50), cell
growth of SK-N-SH and HeLa cell lines (IC50), and their theoretically calculated lipophilicity (C log P)
Inhibition of cell growth IC50 (μM)
C log P (biobyte Corp.
% inhibition of LP
Inhibition of soybean
Compound
C-QSAR database)
induced by AAPHa (100 μM)
lipoxygenase bIC50 (μM)
SK-N-SH
20.2
9.7
HeLa
1.8
3a
4.34
4.04
3.22
3.47
2.84
3.40
4.78
4.48
3.66
3.92
3.29
3.85
8.64
8.01
7.52
7.78
7.14
7.70
-
88 1.4*
100 2.3*
NA
57.5 0.7*
100 1.3**
NA
3b
6.1
3c
34.4
34.7
170.6
74.2
>500
60.5
>500
380.7
>500
No
55.4
24.5
47.8
17.9
79.1
12.8
104.6
37.4
116.8
>500
>500
222.0
>500
60.4
21.5
55.0
-
3d
NA
NA
3e
25 0.6*
NA
3f
5
0.3*
NA
4a
66 1.5**
48 1.1**
24 0.9*
100 3.2*
NA
NA
4b
NA
4c
40 1.0*
NA
4d
4e
21 0.3*
NA
4f
60 1.6**
38 1.1*
47 1.4*
93 2.8**
54 1.9*
25 0.5*
53 0.8*
93 2.6*
-
5a
64 0.8**
NA
366.1
13.6
NA
5b
5c
NA
5d
NA
35.8
9.7
5e
60 0.9**
10 0.1*
-
5f
94.8
-
Trolox
NDGA
-
0.45 0.1*
-
-
Note: NDGA: Nordihydroguaiaretic acid. NA: No Activity under the used experimental conditions.
aValues are means ( SD < 10%) of four different determinations.
bFor the IC50 determination of LOX inhibition six different concentrations were used.
*p < .05; **p < .01.
types of cancer, such as prostate and pancreatic. (Orafaie et al., 2018;
Yang et al., 2013).
highest LOX inhibitory activity (IC50 21 μM), whereas compound 4f
possessing a methoxy- group at position 4 was inactive. It is also
remarkable that the presence of a hydroxy- group at position 5 of the
4-nitro-substituted coumarin derivative (4c) significantly enhances
soybean LOX inhibitory activity in comparison with the non hydroxy-
analogue (Roussaki et al., 2010).
Soybean (Glycine max) LOX-1 is a 15-LOX, which has been used
widely as a prototype for studying the functional and structural prop-
erties of the homologous family of lipoxygenases. The crystal struc-
tures of soybean LOX-1 and that of a rabbit 15-LOX-1-inhibitor
complex in addition, to the three-dimensional models of the human
5-LOX indicated that the essential structural features of plant and ani-
mal LOXs are highly conserved (Brash, 1999; Funk, 2001).
Regarding the geranyloxy-analogues, the most potent LOX inhibi-
tory agent was compound 5f (IC50 10 μM), which is the best analogue
among the compounds studied in this work. Coumarins 5a and 5f
bearing a bromo or methoxy group, respectively, at position 4 on the
phenyl ring were more active than the corresponding 2-substituted
analogues (5b & 5e), indicating that the position of the substituent is
an important structural feature for LOX inhibition. As a general obser-
vation, the absence of substituents at the 3-phenyl ring leads to
complete loss of activity (3d, 4d, and 5d). Lipophilicity is a physico-
chemical property which can be related to LOX inhibition; however,
the results obtained in this study do not show this trend (Pontiki &
Hadjipavlou-Litina, 2008).
Soybean LOX was selected in this study and the inhibitory activ-
ity of the compounds was tested and is presented in Table 1.
The most active LOX inhibitors among the 5-acetyloxy-
derivatives were the bromo-substituted 3a (IC50 57.5 μM) and 3b
(IC50 100 μM) analogues, while deacetylation of these compounds led
to essentially lower bioactivity (4a and 4b). In our previous work,
3-(4-bromo-phenyl)-4-methyl-coumarin was found to be an inactive
anti-inflammatory agent. The recent results show that the insertion of
an acetyloxy- group at position 5 of the coumarin scaffold (3a) signifi-
cantly improves the activity (Roussaki et al., 2010).
Compounds 4e and 5f exhibited the higher IC50 values with low
interaction with AAPH, thus showing that LOX inhibition is not always
accompanied by antioxidant ability and vice versa (Curini et al., 2006).
Among the 5-hydroxy-coumarins, compound 4e bearing
a
methoxy- group at position 2 of the 3-aryl moiety, displayed the

8
KAVETSOU ET AL.
3.4
|
Cytotoxic activity evaluation
et al., 2019; Goswami et al., 2018; Park et al., 2019; Qi et al., 2019;
Swift, Zhang, Trippett, & Narendran, 2019; Yen et al., 2018).
Cytotoxicity of coumarin analogues against various types of cancer
cell lines stands out among their various bioactivities. In this study,
the potential cytotoxicity of 18 coumarin analogues via the MTT assay
using the human neuroblastoma cell line SK-N-SH and the human cer-
vical epithelioid cell line HeLa was investigated. SK-N-SH human neu-
roblastoma cell line was selected to allow comparisons on the
cytotoxic activity evaluation of hydroxy and oxyprenylated coumarin
derivatives studied previously by our group (Kavetsou et al., 2017).
Both SK-N-SH and HeLa cells have been widely used to examine the
anticancer effect of bioactive agents (Akram et al., 2018; Dickens
The results are presented in Table 1 and Figures 1 and 2.
Concerning the SK-N-SH neuroblastoma cell line, four of the
5-acetyloxy-coumarins presented significant activity, with IC50 rang-
ing from 9.7 to 34.7 μM. Coumarins 3a (IC50 20.2 μM) and 3b (IC50
9.7 μM) bearing a bromo-substituent were the most cytotoxic.
These results could be supported by an earlier publication, in
which it was recorded that the bromination of flavonoids has
improved their biological properties, such as antioxidant activity and
enhanced their lipophilicity and diffusion through membranes. Bromi-
nated flavonoids' metabolites may interact with signaling pathways
FIGURE 1 Effect of coumarin analogues 3a, 3b, 4b, 5b, and 5e on human neuroblastoma SK-N-SH cell line. Cell viability is expressed as
percent relative to the viability of control untreated cells (0 μM, incubation with the compounds dilution medium) set to 100%. Data are the
means SD, *p < .05 versus control; **p < .005 versus control; ***p < .0001 versus control

KAVETSOU ET AL.
9
FIGURE 2 Effect of coumarin analogues 3a, 3b, 4b, 5b, and 5e on cervical epithelioid HeLa cell line. Cell viability is expressed as percent
relative to the viability of control untreated cells (0 μM, incubation with the compounds dilution medium) set to 100%. Data are the means SD,
*p < .05 versus control; **p < .005 versus control; ***p < .0001 versus control
(involving cytokines and so on), closely related with inflammation and
the regulation of immune response, while hypobromous acid consti-
tutes a strong oxidant metabolite crucial for efficient cytotoxicity
(Justino, Rodrigues, Florêncio, & Mira, 2009).
favored the biological response. In all cases the 5-hydroxy-derivatives
were found to be less active, although compounds 4b (IC50 12.8 μM) and
4d (IC50 37.4 μM) still presented important bioactivity. It is also notewor-
thy that all the 5-hydroxy-compounds were more cytotoxic against HeLa
than SK-N-SH cell line. Concerning the 5-geranyloxy-analogues, couma-
rin 5e was the most cytotoxic (IC50 21.5 μM), while compounds 5a and
5b were completely inactive. It is remarkable that compound 5b,
exhibited preferential cytotoxicity against SK-N-SH (IC50 13.6 μM) than
HeLa line (IC50 222.0 μM).
The presence of a methoxy substituent (3e & 3f) resulted to
decreased cytotoxicity. The 5-hydroxy-derivatives were found to be
inactive, except for the 5-hydroxy-3-(2-bromo-phenyl)-4-methyl-
coumarin (4b) which exhibited moderate activity (IC50 60.5 μM).
The influence of substitution at position 2 of the 3-aryl moiety of the
5-geranyloxy-derivatives in cytotoxicity was remarkable, since the
presence of either a bromo- (5b, IC50 13.6 μM) or a methoxy- (5e,
IC50 9.7 μM) group enhanced the cytotoxicity against SK-N-SH cells.
In regard to HeLa adenocarcinoma cell line, all the 5-acetyloxy-
derivatives were significantly active (IC50 1.8–55.4 μM), with the best
effect presented again by compounds 3a (IC50 1.8 μM) and 3b (IC50
6.1 μM). In this cell line, it was noted that substitution at position 4 with
a bromo or methoxy group on the phenyl ring of the acetyloxy-analogues
Overall, the 5-acetyloxy-coumarins, and especially the bromo-
substituted derivatives (3a & 3b), were found to be the most active
agents against both carcinoma cell lines. The importance of the
acetyloxy moiety on the cytotoxicity of coumarins has been also men-
tioned by other research groups (Burgess, Shah, Hough, & Hynynen,
2016; Musa et al., 2017). More specifically, in the work of Raj et al.,
the presence of the acetyloxy group at positions 7 and 8 on the
4-methyl-coumarin skeleton has been shown to significantly inhibit

10
KAVETSOU ET AL.
the AFB1-induced apoptosis (Raj et al., 2001). In the work of Musiliyu
A. Musa et al. it was found that 7,8-diacetyloxy-3-(4-nitrophenyl)-
coumarin and 7,8-diacetyloxy-3-(4-methoxyphenyl)-coumarin pres-
ented remarkable cytotoxicity against various cancer cell lines (PC 3,
MDA-MB-231) (Musa et al., 2017; Musa, Latinwo, Virgile, Badisa, &
Gbadebo, 2015).
Recent research shows that treatment of cervical carcinoma cells
with a LOX inhibitor also inhibits their invasion and migration capaci-
ties (Yang et al., 2013). Such observations are in accordance with the
combined anti-inflammatory and cytotoxic activity exhibited by com-
pounds 3a and 3b against HeLa cancer cells, rendering them powerful
and promising multi-targeting agents.
All in all, substitution of the coumarin scaffold with a bromine as
well as an acetyloxy-moiety seems to enhance the combined bioactiv-
ity of coumarins.
FIGURE 3 Docking orientation of 5f (depicted in turquoise)
bound to soybean lipoxygenase (LOX-1)
3.5
|
Molecular docking simulations for soybean
lipoxygenase
study, it can be concluded that the novel derivatives present allosteric
interactions with the enzyme. Additionally, it seems that 5f accommo-
dates the extensively hydrophobic cavity close to the active site with
possible hydrophobic interactions (π-π stacking). The inhibitory activ-
ity of 5f is possible due to the obstruction of substrates to reach the
active site of LOX based on the fact that lipoxygenation, occurs via a
carbon-centered radical on a lipid chain and the majority of strong
LOX-inhibitors are antioxidants or free radical scavengers (Denisov &
Afanas0ev, 2005).
For the docking studies of soybean lipoxygenase (SLOX-1), 3PZW
was selected. UCSF Chimera was used for the visualization of the
protein (PDB code: 3PZW) (Pettersen et al., 2004).The above protein
was prepared for docking studies by removing water molecules,
adding missing residues with Modeler (Fiser & Šali, 2003), adding
hydrogen atoms and AMBER99SB-ILDN charges and setting the iron
charge to +2.0, with no restraint applied to the iron atom and the
ligands.
OpenBabel was used to generate and minimize Ligand 3D coordi-
nates (O'Boyle et al., 2011) using the MMFF94 force field (Halgren,
1996) while ACPYPE (AnteChamberPYthon Parser interfacE) (Sousa
da Silva & Vranken, 2012) was used to generate ligand topologies and
parameters using Antechamber (Wang, Wang, Kollman, & Case,
2006). Energy minimizations were accomplished using the
AMBER99SB-ILDN force field (Lindorff-Larsen et al., 2010) with
GROMACS 4.6.5 (Hess, Kutzner, van der Spoel, & Lindahl, 2008) as
the molecular dynamics simulation toolkit. Docking studies were exe-
cuted with AutoDockVina (1.1.2) (Trott & Olson, 2010) using a grid
box of size 100 Å, 70 Å, and 70 Å in X, Y, and Z dimensions for SLOX-
1. UCSF-Chimera was applied for the generation of docking input files
and analysis of the obtained docking results. Docking was carried out
with an exhaustiveness value of 10 and a maximum output of 20 dock-
ing modes SLOX-1.
3.7
|
Drug-likeness studies
The drug likeness of the derivatives was determined from the theoret-
ical calculation of various molecular properties, for example, partition
coefficient (log P), topological polar surface area (TPSA), hydrogen
bond donors and acceptors, rotatable bonds, number of atoms,
and molecular weight. The violations of Lipinski's rule of five were
also considered (Lipinski, Lombardo, Dominy,
& Feeney, 1997;
Molinspiration (http://www.molinspiration.com//cgi-bin/properties)
(Table 2). All the compounds have a molecular weight less than 500.
Thus, these molecules could be easily transported, diffused, and
absorbed. The theoretically calculated lipophilicity log P values of
series 3a-3f and 4a-4f were found to be less than 5 and are in agree-
ment to Lipinski's rule of five, suggesting satisfactory permeability
across the cell membrane. This was not found for compounds of series
5a-5f which have log P values more than 5. Counting the number of
hydrogen bond acceptors (O and N atoms) and the number of hydro-
gen bond donors (NH and OH) in the synthesized compounds, it
seems that both properties follow the Lipinski's rule of five (less than
10 and 5, respectively). The examined derivatives seem to be orally
active in accordance to Lipinski's rule of five. The hydrogen bonding
of the compounds is highly correlated to the TPSA. This property is
used as a significant indicator of the bioavailability of a bioactive mol-
ecule. The TPSA of the derivatives was observed in the range of
3.6
|
Molecular modeling of the synthesized
derivatives in soybean LOX
All the synthesized compounds have been studied with in silico dock-
ing. The molecular modeling evaluation provided useful interpretation
of the experimental results. The preferred docking pose in soybean
LOX (PDB code: 3PZW) for the most potent derivative 5f, shown in
Figure 3, has a high AutoDockVina score (−10.5) (Figure 3). From this

KAVETSOU ET AL.
11
TABLE 2 Molecular properties prediction-Lipinski “Rule of five”
No. of
atoms
No. of
No. of OH
and NHd
No. of
No. of
Compd.
3a
mi log Pa
4.34
4.38
3.58
3.62
3.63
3.67
4.66
4.61
3.81
3.85
3.85
3.90
8.37
8.34
7.60
7.64
7.65
7.70
TPSAb
56.52
56.52
102.34
56.52
65.75
65.75
50.44
50.44
96.26
50.44
59.67
59.67
39.45
39.45
85.27
39.45
48.68
48.68
O and Nc
violations
rotational bondse
Volumef
278.97
278.97
284.42
261.08
286.63
286.63
242.46
242.46
247.91
224.57
250.12
250.12
398.35
398.35
403.80
380.46
406.01
406.01
MWg
log BBh
0.2715
0.2777
−0.3045
0.1599
0.06915
0.07535
0.3819
0.37415
23
23
25
22
24
24
20
20
22
19
21
21
30
30
32
29
31
31
4
4
7
4
5
5
3
3
6
3
4
4
3
3
6
3
4
4
0
0
0
0
0
0
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
3
3
4
3
4
4
1
1
2
1
2
2
7
7
8
7
8
8
373.20
373.20
339.30
294.31
324.33
324.33
331.17
331.17
297.27
252.27
282.30
282.30
467.40
467.40
433.50
388.51
418.53
418.53
3b
3c
3d
3e
3f
4a
4b
4c
−0.20805
0.25635
0.16405
0.1718
4d
4e
4f
5a
1.06685
1.0622
5b
5c
0.4893
5d
5e
5f
0.9537
0.86295
0.8707
Note: Drug likeness of the synthesized compounds.
Abbreviations: MW, molecular weight; TPSA, topological polar surface area.
aLogarithm of partition coefficient between n-octanol and water (mi log P).
bTopological polar surface area (TPSA).
cNumber of hydrogen bond acceptors (n-ON).
dNumber of hydrogen bond donors (n-OHNH).
eNumber of rotatable bonds (n-rotb).
fMolecular volume.
gMolecular weight.
hBlood brain barrier.
39.45–102.34 Å and is well below the limit of 160 Å, indicating good
oral bioavailability. The upper limit for TPSA for a molecule to pene-
trate the brain is around 90 Å. The in silico predicted values (Fiser &
Šali, 2003) point that compounds with log BB values more than 0.3
are considered to be highly absorbed through BBB whereas values
between 0.3 and −0.1 and less than −0.1 are considered to be limited
transported through BBB. All compounds of series 5a-5f have log BB
more than 0.3. On the contrary, compounds from series 3 and 4 pres-
ented log BB values between 0.3 and − 0.1 except for 3c and 4c,
which have log BB values less than −0.1 and they are considered to
be limited transported through BBB. Our findings do not support the
permeability of them through BBB.
and 5c) are strong antioxidant scavengers, while the 5-geranyloxy-
compound 5f is the best soybean lipoxygenase inhibitor. All the novel
derivatives present allosteric interactions with the enzyme as it can be
concluded from the modeling studies.
In regard to the cytotoxic activity, it was observed that among
the three different types of substituents at position 5 of the coumarin
core, the acetyloxy-group led to the most effective anticancer agents,
especially for the compounds 3a and 3b bearing a bromo-substitution
on the phenyl ring. The theoretical calculations for drug likeness of
the derivatives showed that these molecules could be easily trans-
ported, diffused, and absorbed. Considering the calculated lipophilicity
log P values, the most potent in cytotoxicity tests compounds 3a
and 3b were found to be less than 5 and are in agreement with
Lipinski's rule of five, suggesting satisfactory permeability across the
cell membrane.
4
|
CONCLUSIONS
Overall, the presence of a bromo-substituent on the phenyl ring
as well as of the acetyloxy moiety at position 5 on the coumarin core
can be considered as key structural features for future design and
development of new therapeutic anticancer and anti-inflammatory
agents.
In conclusion, 18 multi-substituted coumarins—six acetyloxy-deriva-
tives, six hydroxy-derivatives, and six geranyloxy-derivatives—were
synthesized, structurally characterized, and evaluated for their bioac-
tivity. The results reveal that four coumarin analogues (3a, 3b, 4d,

12
KAVETSOU ET AL.
Biobyte Corp. C-QSAR Database. (n.d.). 201 West 4th Str., Suite
204, Claremont, CA 91711, USA.
ACKNOWLEDGMENTS
E. K. gratefully acknowledges State Scholarships Foundation (IκΥ).
This research is co-financed by Greece and the European Union
(European Social Fund [ESF]) through the Operational Programme
(Human Resources Development, Education and Lifelong Learning) in
the context of the project “Scholarships Programme for post-graduate
studies—2nd Study Cycle” (MIS-5003404), implemented by the State
Scholarships Foundation (IκΥ).
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& Balkwill, F. R. (2015). Inflammation and cancer:
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… Bellachio, E. (2006). Lipoxygenase inhibitory activity of Boropinic
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A. K. gratefully acknowledges State Scholarships Foundation
(IκΥ). This research is co-financed by Greece and the European Union
(ESF) through the Operational Programme (Human Resources Devel-
opment, Education and Lifelong Learning) in the context of the project
“Strengthening Human Resources Research Potential via Doctorate
Research” (MIS-5000432), implemented by the State Scholarships
Foundation (IκΥ).
ꢁ
Demir, S., Özen, C., Ünlüsoy, M. C., Öztürk, M., & Bozdag-Dündar, O.
(2019). Novel furochromone derivatives: Synthesis and anticancer
activity studies. Journal of Heterocyclic Chemistry, 56(4), 1341–1351.
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We are thankful to Biobyte and Dr. A. Leo for free use of C-QSAR
and support.
organic chemistry and biology. Boca Raton: CRC Press: Taylor
&
Francis.
Detsi, A., Kontogiorgis, C., & Hadjipavlou-Litina, D. (2017). Coumarin
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Meredith, M. P., … Smith, J. A. (2019). Enhanced glacial discharge from
the eastern Antarctic peninsula since the 1700s associated with a pos-
itive southern annular mode. Scientific Reports, 9, 14606.
We acknowledge support of this work by the project “An Open-
Access Research Infrastructure of Chemical Biology and Target-
Based Screening Technologies for Human and Animal Health,
Agriculture and the Environment (OPENSCREEN-GR)” (MIS 5002691)
which is implemented under the Action “Reinforcement of the
Research and Innovation Infrastructure,” funded by the Operational
Programme “Competitiveness, Entrepreneurship and Innovation”
(NSRF 2014-2020) and co-financed by Greece and the European
Union (European Regional Development Fund).
Dos Santos Nascimento, M. V., Arruda-Silva, F., Luz, A. B., Venzke, D.,
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6-Methoxycoumarin from polygala Sabulosa A.W. Bennett regulates
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Taddeo, V. A., … Genovese, S. (2018). Natural oxyprenylated couma-
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CONFLICT OF INTEREST
There are no conflicts to declare.
ORCID
Anastasia Detsi
https://orcid.org/0000-0001-8325-2980
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How to cite this article: Kavetsou E, Katopodi A, Argyri L,
et al. Novel 3-aryl-5-substituted-coumarin analogues:
Synthesis and bioactivity profile. Drug Dev Res. 2020;1–14.
https://doi.org/10.1002/ddr.21639